XII Imobilisasi Enzim(Enzyme Immobilization)

Departemen Teknologi Industri PertanianFATETA - IPB

2011

mk SATUAN PROSES

ENZYME

bull The use of enzymes in industrial applications has been limited by several factors - high cost of the enzymes - instability - availability in small amounts - soluble in aqueous media and it is difficult and expensive to recover them from reactor effluents at the end of the catalytic process

bull The technological developments in the field of immobilized biocatalysts can offer the possibility of a wider and more economical exploitation of biocatalysts in industry waste treatment medicine and in the development of bioprocess monitoring devices like the biosensor

IMMOBILIZATION

Definition bdquoImmobilization means that the biocatalysts are limited in moving due to chemically or physically treatmentldquo

transformation of enzyme to insoluble form or inclusion to definite space

method for reuse and stabilisation of enzyme one-step reactions - domain of immobilized

enzymes

The attractions of immobilized enzymes from an analytical standpoint are primarily their reuseabilityand hence cost saving and the greater efficiency and control of their catalytic activity (eg potentially longer half-lives predictable decay rates and more efficient multi-step reactions)

Immobilized enzyme

An immobilized enzyme is enzyme an that is attached to an inert insoluble material such as calcium alginate (produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride)

This can provide increased resistance to changes in conditions such as pH or temperature

It also allows enzymes to be held in place throughout the reaction following which they are easily separated from the products and may be used again - a far more efficient process and so is widely used in industry for reactions

An alternative to enzyme immobilization is whole cell immobilization

httpenwikipediaorgwikiImmobilized_enzyme

ADVANTAGES OF IMMOBILIZED ENZYME

- Development of continuous processes allowing more economic organization of the operations automation decrease of labour and investmentcapacity ratio -Availability of the product in greater purity Purity of the product is very crucial in food processing and pharmaceutical industry since contamination could cause serious toxicological sensory or immunological problems - Greater control over enzymatic reaction as well as high volumetric productivity with lower residence time which are of great significance in the food industry specially in the treatment of perishable commodities as well as in other applications involving labile substrates intermediates or products

Commercial use

Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the reaction of which include

Convenience Minuscule amounts of protein dissolve in the reaction so workup can be much easier Upon completion reaction mixtures typically contain only solventt and reaction products

Economical The immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst

Stability Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme

ENZYME

bull The use of enzymes in industrial applications has been limited by several factors - high cost of the enzymes - instability - availability in small amounts - soluble in aqueous media and it is difficult and expensive to recover them from reactor effluents at the end of the catalytic process

bull The technological developments in the field of immobilized biocatalysts can offer the possibility of a wider and more economical exploitation of biocatalysts in industry waste treatment medicine and in the development of bioprocess monitoring devices like the biosensor

IMMOBILIZATION

Definition bdquoImmobilization means that the biocatalysts are limited in moving due to chemically or physically treatmentldquo

transformation of enzyme to insoluble form or inclusion to definite space

method for reuse and stabilisation of enzyme one-step reactions - domain of immobilized

enzymes

The attractions of immobilized enzymes from an analytical standpoint are primarily their reuseabilityand hence cost saving and the greater efficiency and control of their catalytic activity (eg potentially longer half-lives predictable decay rates and more efficient multi-step reactions)

Immobilized enzyme

An immobilized enzyme is enzyme an that is attached to an inert insoluble material such as calcium alginate (produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride)

This can provide increased resistance to changes in conditions such as pH or temperature

It also allows enzymes to be held in place throughout the reaction following which they are easily separated from the products and may be used again - a far more efficient process and so is widely used in industry for reactions

An alternative to enzyme immobilization is whole cell immobilization

httpenwikipediaorgwikiImmobilized_enzyme

ADVANTAGES OF IMMOBILIZED ENZYME

- Development of continuous processes allowing more economic organization of the operations automation decrease of labour and investmentcapacity ratio -Availability of the product in greater purity Purity of the product is very crucial in food processing and pharmaceutical industry since contamination could cause serious toxicological sensory or immunological problems - Greater control over enzymatic reaction as well as high volumetric productivity with lower residence time which are of great significance in the food industry specially in the treatment of perishable commodities as well as in other applications involving labile substrates intermediates or products

Commercial use

Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the reaction of which include

Convenience Minuscule amounts of protein dissolve in the reaction so workup can be much easier Upon completion reaction mixtures typically contain only solventt and reaction products

Economical The immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst

Stability Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme

IMMOBILIZATION

Definition bdquoImmobilization means that the biocatalysts are limited in moving due to chemically or physically treatmentldquo

transformation of enzyme to insoluble form or inclusion to definite space

method for reuse and stabilisation of enzyme one-step reactions - domain of immobilized

enzymes

The attractions of immobilized enzymes from an analytical standpoint are primarily their reuseabilityand hence cost saving and the greater efficiency and control of their catalytic activity (eg potentially longer half-lives predictable decay rates and more efficient multi-step reactions)

Immobilized enzyme

An immobilized enzyme is enzyme an that is attached to an inert insoluble material such as calcium alginate (produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride)

This can provide increased resistance to changes in conditions such as pH or temperature

It also allows enzymes to be held in place throughout the reaction following which they are easily separated from the products and may be used again - a far more efficient process and so is widely used in industry for reactions

An alternative to enzyme immobilization is whole cell immobilization

httpenwikipediaorgwikiImmobilized_enzyme

ADVANTAGES OF IMMOBILIZED ENZYME

- Development of continuous processes allowing more economic organization of the operations automation decrease of labour and investmentcapacity ratio -Availability of the product in greater purity Purity of the product is very crucial in food processing and pharmaceutical industry since contamination could cause serious toxicological sensory or immunological problems - Greater control over enzymatic reaction as well as high volumetric productivity with lower residence time which are of great significance in the food industry specially in the treatment of perishable commodities as well as in other applications involving labile substrates intermediates or products

Commercial use

Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the reaction of which include

Convenience Minuscule amounts of protein dissolve in the reaction so workup can be much easier Upon completion reaction mixtures typically contain only solventt and reaction products

Economical The immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst

Stability Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme

Immobilized enzyme

An immobilized enzyme is enzyme an that is attached to an inert insoluble material such as calcium alginate (produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride)

This can provide increased resistance to changes in conditions such as pH or temperature

It also allows enzymes to be held in place throughout the reaction following which they are easily separated from the products and may be used again - a far more efficient process and so is widely used in industry for reactions

An alternative to enzyme immobilization is whole cell immobilization

httpenwikipediaorgwikiImmobilized_enzyme

ADVANTAGES OF IMMOBILIZED ENZYME

- Development of continuous processes allowing more economic organization of the operations automation decrease of labour and investmentcapacity ratio -Availability of the product in greater purity Purity of the product is very crucial in food processing and pharmaceutical industry since contamination could cause serious toxicological sensory or immunological problems - Greater control over enzymatic reaction as well as high volumetric productivity with lower residence time which are of great significance in the food industry specially in the treatment of perishable commodities as well as in other applications involving labile substrates intermediates or products

Commercial use

Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the reaction of which include

Convenience Minuscule amounts of protein dissolve in the reaction so workup can be much easier Upon completion reaction mixtures typically contain only solventt and reaction products

Economical The immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst

Stability Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme

ADVANTAGES OF IMMOBILIZED ENZYME

- Development of continuous processes allowing more economic organization of the operations automation decrease of labour and investmentcapacity ratio -Availability of the product in greater purity Purity of the product is very crucial in food processing and pharmaceutical industry since contamination could cause serious toxicological sensory or immunological problems - Greater control over enzymatic reaction as well as high volumetric productivity with lower residence time which are of great significance in the food industry specially in the treatment of perishable commodities as well as in other applications involving labile substrates intermediates or products

Commercial use

Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the reaction of which include

Convenience Minuscule amounts of protein dissolve in the reaction so workup can be much easier Upon completion reaction mixtures typically contain only solventt and reaction products

Economical The immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst

Stability Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme

Commercial use

Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the reaction of which include

Convenience Minuscule amounts of protein dissolve in the reaction so workup can be much easier Upon completion reaction mixtures typically contain only solventt and reaction products

Economical The immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst

Stability Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme

ENZYME IMMOBILIZATION METHOD

ldquoCarrier ndashbindingrdquo ldquoCross-linkingrdquo ldquoEntrapmentrdquo

Adsorpsi Fisik

Ikatan Kovalen

Ikatan Ionik

Jenis Mikrokapsul

Jenis Kisi

Aspects of the immobilization procedure

1 The properties of the free enzyme2 The type of support used3 The methods of support activation and enzyme attachment

0 1995 IUPAC Pure and Applied Chemistry

1 Properties of the Free Enzyme

Source of the enzyme

Purity (and method of purification)

Catalytic activity and details of other constituents - - etc

The above information permits direct comparison of enzymes from different sources

2 Enzyme Support

The support material can have a critical effect on the stability of the enzyme and the efficiency of enzyme immobilization

The most important requirements for a support material are that it must be insoluble in water have a high capacity to bind enzyme be chemically inert and be mechanically stable

-The enzyme binding capacity is determined by the available surface area both internal (pore size) and external (bead size or tube diameter) the ease with which the support can be activated and the resultant density of enzyme binding sites

-The surface charge and hydrophilicity must be considered

Parameters of Enzyme Immobilization

- Effective easy cheap acceptable (non-toxic in food and medical applications)

- Rate and yield dependent on the parameters involved (eg type of carrier concentrations pH temperature method reaction time)- Empirical optimization- External protein surface properties (eg hydrophobicity

ionic groups functional groups for covalent binding)- Protein surface engineering- Introduction of functional groups increases binding

interactions stability (eg nanoparticles protecting molecules) and activity (eg cofactors)

Method Immobilization Enzyme

1 Adsorption on glass alginate beads or matrix Enzyme is attached to the outside of an inert material In general this method is the slowest among those listed here As adsorption is not a chemical reaction the active site of the immobilized enzyme may be blocked by the matrix or bead greatly reducing the activity of the enzyme

Adsorption

- Binding onto silica clay or ion-exchange materials by weak interactions

(eg ionic electrostatic hydrophobic) - Dependent on process conditions (eg pH

temperature ionic strength hydrophobicity) - Simple and cost-effective reversible (stabilized

by cross linking) but may cause enzyme unfolding

Karbon aktif

-amilase

Aduk 10 0C 1 jam Saring

Enzim Imobil

Lar Pati

Amilase imobil

Gula

I n o r g a n i c Ca r r i e r s

1048713 High pressure stability1048713 May undergo abrasion in stirred vessels1048713 SiO2 based carriers functionalized by introduction of amino groups (eg treating with aminopropyl triethoxysilane)1048713 Porous glass (Corning Waters Schuller)1048713 Silica (Grace Solvay Degussa)1048713 Mineral materials (clays)1048713 Celite - adsorption and stabilisation of enzyme in organic media

1048713 Bentonite - excellent adsorption capacity (up to 15 g protein per g bentonit) used for enzyme isolation by dsorptiondesorption

Crosslinking with glutaraldehyde prevents desorption

O r g a n i c C a r r i e r s f r o m Na t u r a l S o u r c e s

1048713 Favorable compatibility with proteins1048713 High range of polysacharides and derivatives used for immobilization1048713 Wide network structure1048713 Hydrophilic properties - weak interactions with proteins1048713 Cellulose derivatives 1048713 DEAE-cellulose (diethylaminoethyl-cellulose) 1048713 CM-cellulose (carboxymethyl-cellulose)1048713 Dextran 1048713 widely used for enzyme immobilization 1048713 activated by cyanogen bromide 1048713 mechanical stability limited1048713 Other polysacharides 1048713 agarose starch pectine and chitosan1048713 Proteins (gelatine)

O r g a n i c S y n t h e t i c C a r r i e r s

1048713 High chemical and mechanical stability1048713 Wide range of carriers with good capacity and

simple manipulation1048713 (ion-exchange) resins1048713 copolymerization with functional groups (eg nitration sulfonation carboxylation

epoxydation)

Example 1048713 polystyrene1048713 polyvinylacetate1048713 acrylic polymers

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

2 Covalent Binding

1048713 Better stabilization of enzyme on carrier 1048713 Introduction of functional group (eg amino epoxy thiol cyanide)

1048713 Principle 1 activation 2 derivatization 3 binding of enzyme

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Aspects of the immobilization procedure

1 The properties of the free enzyme2 The type of support used3 The methods of support activation and enzyme attachment

0 1995 IUPAC Pure and Applied Chemistry

1 Properties of the Free Enzyme

Source of the enzyme

Purity (and method of purification)

Catalytic activity and details of other constituents - - etc

The above information permits direct comparison of enzymes from different sources

2 Enzyme Support

The support material can have a critical effect on the stability of the enzyme and the efficiency of enzyme immobilization

The most important requirements for a support material are that it must be insoluble in water have a high capacity to bind enzyme be chemically inert and be mechanically stable

-The enzyme binding capacity is determined by the available surface area both internal (pore size) and external (bead size or tube diameter) the ease with which the support can be activated and the resultant density of enzyme binding sites

-The surface charge and hydrophilicity must be considered

Parameters of Enzyme Immobilization

- Effective easy cheap acceptable (non-toxic in food and medical applications)

- Rate and yield dependent on the parameters involved (eg type of carrier concentrations pH temperature method reaction time)- Empirical optimization- External protein surface properties (eg hydrophobicity

ionic groups functional groups for covalent binding)- Protein surface engineering- Introduction of functional groups increases binding

interactions stability (eg nanoparticles protecting molecules) and activity (eg cofactors)

Method Immobilization Enzyme

1 Adsorption on glass alginate beads or matrix Enzyme is attached to the outside of an inert material In general this method is the slowest among those listed here As adsorption is not a chemical reaction the active site of the immobilized enzyme may be blocked by the matrix or bead greatly reducing the activity of the enzyme

Adsorption

- Binding onto silica clay or ion-exchange materials by weak interactions

(eg ionic electrostatic hydrophobic) - Dependent on process conditions (eg pH

temperature ionic strength hydrophobicity) - Simple and cost-effective reversible (stabilized

by cross linking) but may cause enzyme unfolding

Karbon aktif

-amilase

Aduk 10 0C 1 jam Saring

Enzim Imobil

Lar Pati

Amilase imobil

Gula

I n o r g a n i c Ca r r i e r s

1048713 High pressure stability1048713 May undergo abrasion in stirred vessels1048713 SiO2 based carriers functionalized by introduction of amino groups (eg treating with aminopropyl triethoxysilane)1048713 Porous glass (Corning Waters Schuller)1048713 Silica (Grace Solvay Degussa)1048713 Mineral materials (clays)1048713 Celite - adsorption and stabilisation of enzyme in organic media

1048713 Bentonite - excellent adsorption capacity (up to 15 g protein per g bentonit) used for enzyme isolation by dsorptiondesorption

Crosslinking with glutaraldehyde prevents desorption

O r g a n i c C a r r i e r s f r o m Na t u r a l S o u r c e s

1048713 Favorable compatibility with proteins1048713 High range of polysacharides and derivatives used for immobilization1048713 Wide network structure1048713 Hydrophilic properties - weak interactions with proteins1048713 Cellulose derivatives 1048713 DEAE-cellulose (diethylaminoethyl-cellulose) 1048713 CM-cellulose (carboxymethyl-cellulose)1048713 Dextran 1048713 widely used for enzyme immobilization 1048713 activated by cyanogen bromide 1048713 mechanical stability limited1048713 Other polysacharides 1048713 agarose starch pectine and chitosan1048713 Proteins (gelatine)

O r g a n i c S y n t h e t i c C a r r i e r s

1048713 High chemical and mechanical stability1048713 Wide range of carriers with good capacity and

simple manipulation1048713 (ion-exchange) resins1048713 copolymerization with functional groups (eg nitration sulfonation carboxylation

epoxydation)

Example 1048713 polystyrene1048713 polyvinylacetate1048713 acrylic polymers

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

2 Covalent Binding

1048713 Better stabilization of enzyme on carrier 1048713 Introduction of functional group (eg amino epoxy thiol cyanide)

1048713 Principle 1 activation 2 derivatization 3 binding of enzyme

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

1 Properties of the Free Enzyme

Source of the enzyme

Purity (and method of purification)

Catalytic activity and details of other constituents - - etc

The above information permits direct comparison of enzymes from different sources

2 Enzyme Support

The support material can have a critical effect on the stability of the enzyme and the efficiency of enzyme immobilization

The most important requirements for a support material are that it must be insoluble in water have a high capacity to bind enzyme be chemically inert and be mechanically stable

-The enzyme binding capacity is determined by the available surface area both internal (pore size) and external (bead size or tube diameter) the ease with which the support can be activated and the resultant density of enzyme binding sites

-The surface charge and hydrophilicity must be considered

Parameters of Enzyme Immobilization

- Effective easy cheap acceptable (non-toxic in food and medical applications)

- Rate and yield dependent on the parameters involved (eg type of carrier concentrations pH temperature method reaction time)- Empirical optimization- External protein surface properties (eg hydrophobicity

ionic groups functional groups for covalent binding)- Protein surface engineering- Introduction of functional groups increases binding

interactions stability (eg nanoparticles protecting molecules) and activity (eg cofactors)

Method Immobilization Enzyme

1 Adsorption on glass alginate beads or matrix Enzyme is attached to the outside of an inert material In general this method is the slowest among those listed here As adsorption is not a chemical reaction the active site of the immobilized enzyme may be blocked by the matrix or bead greatly reducing the activity of the enzyme

Adsorption

- Binding onto silica clay or ion-exchange materials by weak interactions

(eg ionic electrostatic hydrophobic) - Dependent on process conditions (eg pH

temperature ionic strength hydrophobicity) - Simple and cost-effective reversible (stabilized

by cross linking) but may cause enzyme unfolding

Karbon aktif

-amilase

Aduk 10 0C 1 jam Saring

Enzim Imobil

Lar Pati

Amilase imobil

Gula

I n o r g a n i c Ca r r i e r s

1048713 High pressure stability1048713 May undergo abrasion in stirred vessels1048713 SiO2 based carriers functionalized by introduction of amino groups (eg treating with aminopropyl triethoxysilane)1048713 Porous glass (Corning Waters Schuller)1048713 Silica (Grace Solvay Degussa)1048713 Mineral materials (clays)1048713 Celite - adsorption and stabilisation of enzyme in organic media

1048713 Bentonite - excellent adsorption capacity (up to 15 g protein per g bentonit) used for enzyme isolation by dsorptiondesorption

Crosslinking with glutaraldehyde prevents desorption

O r g a n i c C a r r i e r s f r o m Na t u r a l S o u r c e s

1048713 Favorable compatibility with proteins1048713 High range of polysacharides and derivatives used for immobilization1048713 Wide network structure1048713 Hydrophilic properties - weak interactions with proteins1048713 Cellulose derivatives 1048713 DEAE-cellulose (diethylaminoethyl-cellulose) 1048713 CM-cellulose (carboxymethyl-cellulose)1048713 Dextran 1048713 widely used for enzyme immobilization 1048713 activated by cyanogen bromide 1048713 mechanical stability limited1048713 Other polysacharides 1048713 agarose starch pectine and chitosan1048713 Proteins (gelatine)

O r g a n i c S y n t h e t i c C a r r i e r s

1048713 High chemical and mechanical stability1048713 Wide range of carriers with good capacity and

simple manipulation1048713 (ion-exchange) resins1048713 copolymerization with functional groups (eg nitration sulfonation carboxylation

epoxydation)

Example 1048713 polystyrene1048713 polyvinylacetate1048713 acrylic polymers

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

2 Covalent Binding

1048713 Better stabilization of enzyme on carrier 1048713 Introduction of functional group (eg amino epoxy thiol cyanide)

1048713 Principle 1 activation 2 derivatization 3 binding of enzyme

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

2 Enzyme Support

The support material can have a critical effect on the stability of the enzyme and the efficiency of enzyme immobilization

The most important requirements for a support material are that it must be insoluble in water have a high capacity to bind enzyme be chemically inert and be mechanically stable

-The enzyme binding capacity is determined by the available surface area both internal (pore size) and external (bead size or tube diameter) the ease with which the support can be activated and the resultant density of enzyme binding sites

-The surface charge and hydrophilicity must be considered

Parameters of Enzyme Immobilization

- Effective easy cheap acceptable (non-toxic in food and medical applications)

- Rate and yield dependent on the parameters involved (eg type of carrier concentrations pH temperature method reaction time)- Empirical optimization- External protein surface properties (eg hydrophobicity

ionic groups functional groups for covalent binding)- Protein surface engineering- Introduction of functional groups increases binding

interactions stability (eg nanoparticles protecting molecules) and activity (eg cofactors)

Method Immobilization Enzyme

1 Adsorption on glass alginate beads or matrix Enzyme is attached to the outside of an inert material In general this method is the slowest among those listed here As adsorption is not a chemical reaction the active site of the immobilized enzyme may be blocked by the matrix or bead greatly reducing the activity of the enzyme

Adsorption

- Binding onto silica clay or ion-exchange materials by weak interactions

(eg ionic electrostatic hydrophobic) - Dependent on process conditions (eg pH

temperature ionic strength hydrophobicity) - Simple and cost-effective reversible (stabilized

by cross linking) but may cause enzyme unfolding

Karbon aktif

-amilase

Aduk 10 0C 1 jam Saring

Enzim Imobil

Lar Pati

Amilase imobil

Gula

I n o r g a n i c Ca r r i e r s

1048713 High pressure stability1048713 May undergo abrasion in stirred vessels1048713 SiO2 based carriers functionalized by introduction of amino groups (eg treating with aminopropyl triethoxysilane)1048713 Porous glass (Corning Waters Schuller)1048713 Silica (Grace Solvay Degussa)1048713 Mineral materials (clays)1048713 Celite - adsorption and stabilisation of enzyme in organic media

1048713 Bentonite - excellent adsorption capacity (up to 15 g protein per g bentonit) used for enzyme isolation by dsorptiondesorption

Crosslinking with glutaraldehyde prevents desorption

O r g a n i c C a r r i e r s f r o m Na t u r a l S o u r c e s

1048713 Favorable compatibility with proteins1048713 High range of polysacharides and derivatives used for immobilization1048713 Wide network structure1048713 Hydrophilic properties - weak interactions with proteins1048713 Cellulose derivatives 1048713 DEAE-cellulose (diethylaminoethyl-cellulose) 1048713 CM-cellulose (carboxymethyl-cellulose)1048713 Dextran 1048713 widely used for enzyme immobilization 1048713 activated by cyanogen bromide 1048713 mechanical stability limited1048713 Other polysacharides 1048713 agarose starch pectine and chitosan1048713 Proteins (gelatine)

O r g a n i c S y n t h e t i c C a r r i e r s

1048713 High chemical and mechanical stability1048713 Wide range of carriers with good capacity and

simple manipulation1048713 (ion-exchange) resins1048713 copolymerization with functional groups (eg nitration sulfonation carboxylation

epoxydation)

Example 1048713 polystyrene1048713 polyvinylacetate1048713 acrylic polymers

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

2 Covalent Binding

1048713 Better stabilization of enzyme on carrier 1048713 Introduction of functional group (eg amino epoxy thiol cyanide)

1048713 Principle 1 activation 2 derivatization 3 binding of enzyme

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Parameters of Enzyme Immobilization

- Effective easy cheap acceptable (non-toxic in food and medical applications)

- Rate and yield dependent on the parameters involved (eg type of carrier concentrations pH temperature method reaction time)- Empirical optimization- External protein surface properties (eg hydrophobicity

ionic groups functional groups for covalent binding)- Protein surface engineering- Introduction of functional groups increases binding

interactions stability (eg nanoparticles protecting molecules) and activity (eg cofactors)

Method Immobilization Enzyme

1 Adsorption on glass alginate beads or matrix Enzyme is attached to the outside of an inert material In general this method is the slowest among those listed here As adsorption is not a chemical reaction the active site of the immobilized enzyme may be blocked by the matrix or bead greatly reducing the activity of the enzyme

Adsorption

- Binding onto silica clay or ion-exchange materials by weak interactions

(eg ionic electrostatic hydrophobic) - Dependent on process conditions (eg pH

temperature ionic strength hydrophobicity) - Simple and cost-effective reversible (stabilized

by cross linking) but may cause enzyme unfolding

Karbon aktif

-amilase

Aduk 10 0C 1 jam Saring

Enzim Imobil

Lar Pati

Amilase imobil

Gula

I n o r g a n i c Ca r r i e r s

1048713 High pressure stability1048713 May undergo abrasion in stirred vessels1048713 SiO2 based carriers functionalized by introduction of amino groups (eg treating with aminopropyl triethoxysilane)1048713 Porous glass (Corning Waters Schuller)1048713 Silica (Grace Solvay Degussa)1048713 Mineral materials (clays)1048713 Celite - adsorption and stabilisation of enzyme in organic media

1048713 Bentonite - excellent adsorption capacity (up to 15 g protein per g bentonit) used for enzyme isolation by dsorptiondesorption

Crosslinking with glutaraldehyde prevents desorption

O r g a n i c C a r r i e r s f r o m Na t u r a l S o u r c e s

1048713 Favorable compatibility with proteins1048713 High range of polysacharides and derivatives used for immobilization1048713 Wide network structure1048713 Hydrophilic properties - weak interactions with proteins1048713 Cellulose derivatives 1048713 DEAE-cellulose (diethylaminoethyl-cellulose) 1048713 CM-cellulose (carboxymethyl-cellulose)1048713 Dextran 1048713 widely used for enzyme immobilization 1048713 activated by cyanogen bromide 1048713 mechanical stability limited1048713 Other polysacharides 1048713 agarose starch pectine and chitosan1048713 Proteins (gelatine)

O r g a n i c S y n t h e t i c C a r r i e r s

1048713 High chemical and mechanical stability1048713 Wide range of carriers with good capacity and

simple manipulation1048713 (ion-exchange) resins1048713 copolymerization with functional groups (eg nitration sulfonation carboxylation

epoxydation)

Example 1048713 polystyrene1048713 polyvinylacetate1048713 acrylic polymers

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

2 Covalent Binding

1048713 Better stabilization of enzyme on carrier 1048713 Introduction of functional group (eg amino epoxy thiol cyanide)

1048713 Principle 1 activation 2 derivatization 3 binding of enzyme

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Method Immobilization Enzyme

1 Adsorption on glass alginate beads or matrix Enzyme is attached to the outside of an inert material In general this method is the slowest among those listed here As adsorption is not a chemical reaction the active site of the immobilized enzyme may be blocked by the matrix or bead greatly reducing the activity of the enzyme

Adsorption

- Binding onto silica clay or ion-exchange materials by weak interactions

(eg ionic electrostatic hydrophobic) - Dependent on process conditions (eg pH

temperature ionic strength hydrophobicity) - Simple and cost-effective reversible (stabilized

by cross linking) but may cause enzyme unfolding

Karbon aktif

-amilase

Aduk 10 0C 1 jam Saring

Enzim Imobil

Lar Pati

Amilase imobil

Gula

I n o r g a n i c Ca r r i e r s

1048713 High pressure stability1048713 May undergo abrasion in stirred vessels1048713 SiO2 based carriers functionalized by introduction of amino groups (eg treating with aminopropyl triethoxysilane)1048713 Porous glass (Corning Waters Schuller)1048713 Silica (Grace Solvay Degussa)1048713 Mineral materials (clays)1048713 Celite - adsorption and stabilisation of enzyme in organic media

1048713 Bentonite - excellent adsorption capacity (up to 15 g protein per g bentonit) used for enzyme isolation by dsorptiondesorption

Crosslinking with glutaraldehyde prevents desorption

O r g a n i c C a r r i e r s f r o m Na t u r a l S o u r c e s

1048713 Favorable compatibility with proteins1048713 High range of polysacharides and derivatives used for immobilization1048713 Wide network structure1048713 Hydrophilic properties - weak interactions with proteins1048713 Cellulose derivatives 1048713 DEAE-cellulose (diethylaminoethyl-cellulose) 1048713 CM-cellulose (carboxymethyl-cellulose)1048713 Dextran 1048713 widely used for enzyme immobilization 1048713 activated by cyanogen bromide 1048713 mechanical stability limited1048713 Other polysacharides 1048713 agarose starch pectine and chitosan1048713 Proteins (gelatine)

O r g a n i c S y n t h e t i c C a r r i e r s

1048713 High chemical and mechanical stability1048713 Wide range of carriers with good capacity and

simple manipulation1048713 (ion-exchange) resins1048713 copolymerization with functional groups (eg nitration sulfonation carboxylation

epoxydation)

Example 1048713 polystyrene1048713 polyvinylacetate1048713 acrylic polymers

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

2 Covalent Binding

1048713 Better stabilization of enzyme on carrier 1048713 Introduction of functional group (eg amino epoxy thiol cyanide)

1048713 Principle 1 activation 2 derivatization 3 binding of enzyme

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Adsorption

- Binding onto silica clay or ion-exchange materials by weak interactions

(eg ionic electrostatic hydrophobic) - Dependent on process conditions (eg pH

temperature ionic strength hydrophobicity) - Simple and cost-effective reversible (stabilized

by cross linking) but may cause enzyme unfolding

Karbon aktif

-amilase

Aduk 10 0C 1 jam Saring

Enzim Imobil

Lar Pati

Amilase imobil

Gula

I n o r g a n i c Ca r r i e r s

1048713 High pressure stability1048713 May undergo abrasion in stirred vessels1048713 SiO2 based carriers functionalized by introduction of amino groups (eg treating with aminopropyl triethoxysilane)1048713 Porous glass (Corning Waters Schuller)1048713 Silica (Grace Solvay Degussa)1048713 Mineral materials (clays)1048713 Celite - adsorption and stabilisation of enzyme in organic media

1048713 Bentonite - excellent adsorption capacity (up to 15 g protein per g bentonit) used for enzyme isolation by dsorptiondesorption

Crosslinking with glutaraldehyde prevents desorption

O r g a n i c C a r r i e r s f r o m Na t u r a l S o u r c e s

1048713 Favorable compatibility with proteins1048713 High range of polysacharides and derivatives used for immobilization1048713 Wide network structure1048713 Hydrophilic properties - weak interactions with proteins1048713 Cellulose derivatives 1048713 DEAE-cellulose (diethylaminoethyl-cellulose) 1048713 CM-cellulose (carboxymethyl-cellulose)1048713 Dextran 1048713 widely used for enzyme immobilization 1048713 activated by cyanogen bromide 1048713 mechanical stability limited1048713 Other polysacharides 1048713 agarose starch pectine and chitosan1048713 Proteins (gelatine)

O r g a n i c S y n t h e t i c C a r r i e r s

1048713 High chemical and mechanical stability1048713 Wide range of carriers with good capacity and

simple manipulation1048713 (ion-exchange) resins1048713 copolymerization with functional groups (eg nitration sulfonation carboxylation

epoxydation)

Example 1048713 polystyrene1048713 polyvinylacetate1048713 acrylic polymers

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

2 Covalent Binding

1048713 Better stabilization of enzyme on carrier 1048713 Introduction of functional group (eg amino epoxy thiol cyanide)

1048713 Principle 1 activation 2 derivatization 3 binding of enzyme

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

I n o r g a n i c Ca r r i e r s

1048713 High pressure stability1048713 May undergo abrasion in stirred vessels1048713 SiO2 based carriers functionalized by introduction of amino groups (eg treating with aminopropyl triethoxysilane)1048713 Porous glass (Corning Waters Schuller)1048713 Silica (Grace Solvay Degussa)1048713 Mineral materials (clays)1048713 Celite - adsorption and stabilisation of enzyme in organic media

1048713 Bentonite - excellent adsorption capacity (up to 15 g protein per g bentonit) used for enzyme isolation by dsorptiondesorption

Crosslinking with glutaraldehyde prevents desorption

O r g a n i c C a r r i e r s f r o m Na t u r a l S o u r c e s

1048713 Favorable compatibility with proteins1048713 High range of polysacharides and derivatives used for immobilization1048713 Wide network structure1048713 Hydrophilic properties - weak interactions with proteins1048713 Cellulose derivatives 1048713 DEAE-cellulose (diethylaminoethyl-cellulose) 1048713 CM-cellulose (carboxymethyl-cellulose)1048713 Dextran 1048713 widely used for enzyme immobilization 1048713 activated by cyanogen bromide 1048713 mechanical stability limited1048713 Other polysacharides 1048713 agarose starch pectine and chitosan1048713 Proteins (gelatine)

O r g a n i c S y n t h e t i c C a r r i e r s

1048713 High chemical and mechanical stability1048713 Wide range of carriers with good capacity and

simple manipulation1048713 (ion-exchange) resins1048713 copolymerization with functional groups (eg nitration sulfonation carboxylation

epoxydation)

Example 1048713 polystyrene1048713 polyvinylacetate1048713 acrylic polymers

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

2 Covalent Binding

1048713 Better stabilization of enzyme on carrier 1048713 Introduction of functional group (eg amino epoxy thiol cyanide)

1048713 Principle 1 activation 2 derivatization 3 binding of enzyme

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

O r g a n i c C a r r i e r s f r o m Na t u r a l S o u r c e s

1048713 Favorable compatibility with proteins1048713 High range of polysacharides and derivatives used for immobilization1048713 Wide network structure1048713 Hydrophilic properties - weak interactions with proteins1048713 Cellulose derivatives 1048713 DEAE-cellulose (diethylaminoethyl-cellulose) 1048713 CM-cellulose (carboxymethyl-cellulose)1048713 Dextran 1048713 widely used for enzyme immobilization 1048713 activated by cyanogen bromide 1048713 mechanical stability limited1048713 Other polysacharides 1048713 agarose starch pectine and chitosan1048713 Proteins (gelatine)

O r g a n i c S y n t h e t i c C a r r i e r s

1048713 High chemical and mechanical stability1048713 Wide range of carriers with good capacity and

simple manipulation1048713 (ion-exchange) resins1048713 copolymerization with functional groups (eg nitration sulfonation carboxylation

epoxydation)

Example 1048713 polystyrene1048713 polyvinylacetate1048713 acrylic polymers

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

2 Covalent Binding

1048713 Better stabilization of enzyme on carrier 1048713 Introduction of functional group (eg amino epoxy thiol cyanide)

1048713 Principle 1 activation 2 derivatization 3 binding of enzyme

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

O r g a n i c S y n t h e t i c C a r r i e r s

1048713 High chemical and mechanical stability1048713 Wide range of carriers with good capacity and

simple manipulation1048713 (ion-exchange) resins1048713 copolymerization with functional groups (eg nitration sulfonation carboxylation

epoxydation)

Example 1048713 polystyrene1048713 polyvinylacetate1048713 acrylic polymers

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

2 Covalent Binding

1048713 Better stabilization of enzyme on carrier 1048713 Introduction of functional group (eg amino epoxy thiol cyanide)

1048713 Principle 1 activation 2 derivatization 3 binding of enzyme

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

2 Covalent Binding

1048713 Better stabilization of enzyme on carrier 1048713 Introduction of functional group (eg amino epoxy thiol cyanide)

1048713 Principle 1 activation 2 derivatization 3 binding of enzyme

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

3 Crosslinked

- The enzyme is covalent bonded to a matrix through a chemical reaction

- This method is by far the most effective method

- As the chemical reaction ensures that the binding site does not cover the enzymes active site the activity of the enzyme is only affected by immobility

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Use reagent which usually has 2 identical functional groups reacted with amino acid residue of the enzyme

Crosslinked

Diisocyanate

Glutaraldehyde

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

4 Entrapment The enzyme is trapped in insoluble beads or microspheres such as

calcium alginate beads However this insoluble substances hinders the arrival of the substrate and the exit of products

lattice type (alginat k-caragenan

Poliacrylamide )

bull microcapsule type1 ndash 300 m

Permanently polymer Membran

Nopermanently

bull Nilonbull Poliureabull Etil selulosabull Polistirenbull Kolodionbull Nitroselulosabull Butil asetat selulosa

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Poliacrylamide Gel

Immobilization by Entrapment

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

I n c l u s i o n i n t o Polymeric Network

1048713 One of the most convenient method for whole cell immobilization

1048713 Problems with enzyme diameter and leak out of the particle

1048713 Combination with cross linking

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Techniques and supports for immobilization

A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports The choice of the support as well as the technique depends on the nature of the enzyme nature of the substrate and its ultimate application

Therefore it will not be possible to suggest any universal means of immobilization It can only be said that the search must continue for matrices which provide facile secure immobilization with good interaction with substrates and which conform in shape size density and so on to the use for which they are intended

httpwwwiasacincurrscijul10articles15htm

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Techniques and supports for immobilization

Care has to be taken to select the support materials as well as the reagents used for immobilization which have GRAS status particularly when their ultimate applications are in the food processing and pharmaceutical industries

Macromolecular colloidal viscous sticky dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries

httpwwwiasacincurrscijul10articles15htm

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Conclusion

Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation diagnostics pharmaceutical and food industries

Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase lipases proteases invertase etc and are being currently used as catalysts in various large scale processes

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Perubahan Sifat Enzim Terimobilisasi

1 Aktivitas

V1 tidak deaktivasi enzim akibat imobilisasiV2 kemungkinan untuk mengimobilisasi enzim lebih banyaksedikit per unit volume katalis

Penyebab penurunan aktivitas bull Konfigurasi menghalangi substratbull Grup reaktif pada sisi aktif ikut terikat pada matriksbull Terbentuk konfigurasi tidak aktifbull Kondisi reaksi denaturasi

V1(aktivitas relatif) Perbandingan aktivitas enzim imobil vs enzim

larut dalam jumlah samaV2 (aktivitas spesifik absolut) Kecepatan reaksi per unit berat atau unit volume

seluruh katalis

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

2 pH optimum enzim imobilPenyebab perubahan pH distribusi yang tidak seragam dari ion H+ ion OH- dan substrat bermuatan

Carrier bermuatan negatif pH optimum bersifat basaCMCMaleac anhydrideetilenAsam galakturonatAsam poliaspartat

Carrier bermuatan positif pH lebih asamDEAE-selulosaPolimer polyornithyl

c a b

Akt

ivita

s R

elat

if (

)

rarr pH4 7

a enzim chymotripsin larutb kopolimer chym ndash anhydride ethylene (-)c chym ndash polyornithyl (+)

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

3 Stabilitas Stabilitas operasi = t12 (half-life) = waktu dimana terjadi kehilangan 50 dari aktivitas enzim

semula

EElog

t2303k

k0693t 0

21

k = konstanta kerusakan enzimt = waktu operasiE0 = aktivitas enzim mula-mulaE = aktivitas enzim pada wktu t

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis reaktor

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Stabilitas operasi ditentukan oleh bull Jenis enzimbull Cara imobilisasibull Jenis bioreaktor

EnzimNIlai t12 pada gelas berpori)

Substrat Suhu (0C) T12(hari)

L-asam amino aksidase

L-leusin 37 43

Alkalin fosfatase P-nitrofenil fosfat

23 55

Papain Kasein 45 35Laktase Laktosa 50 20Glukoamilase Pati 45 645) gelas berpori dilapisi ZrO2 40 ndash 80 mesh = 550 Aring

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Enzim Substrat Km (milimolar)Larut Imobil

Invertase Sukrosa 0448 0448Arilsulfatase P-nitrofenil-fosfat 185 157Glukoamilase Pati 122 030Alkalin-fosfatase P-nitrofenil-fosfat 010 290Urease Urea 100 760Gluoksidase Glukosa 770 680L-asam amino oksidase

L-leusin 100 400

Kinetika Enzim Imobil

Nilai Km (konstanta Michaelis-Menten)

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Bioreaktor Enzim Imobil

Reaktor Curah (Batch) bull Sederhanabull Viskositas tinggi amp aktivitas enzim rendah

CSTR bull Pengontrolan lbh mudahbull Cocok untuk kasus inhibisi

(penghambatan) substratbull Menghindari kontak enzim oleh

substrat dan produk yang terlalu lama

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Fixed-bed PFR (Unggun DiamTerkemas) - Sinambung paling sering digunakan- Aliran substrat dpt dari atas bawah atau daur-ulang

Fluidized-bed (Unggun Terfluidisasi) - Untuk viskositas tinggi amp terbentuk gas- Laju fluidisasi perlu diatur agar enzim imobil tak rusak

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Recycle Packed Column Reactor - allow the reactor to operate at high fluid velocities- a substrate that cannot be completely processed on a single pass

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Immobilization of Microorganism Cells

1048713 First example in 1823 Acetobacter adsorbed to wood chips (acetic production)

1048713 Multienzyme systems (eg alcohol production)1048713 Applicable if enzyme(s) difficult to isolate or show low

stabilityactivity outside cell (eg nitrile hydratase in acrylamide production)

1048713 Continous processing with (re)synthesis of enzyme in immobilized living cells

1048713 Mostly resting cells - limited in growth by controlling C- N- or P- sources

1048713 Industrial applications of immobilized viable cells 1 Beer maturation with yeast cells 2 Anchorage-dependent mammalian cell (production of vaccines) 3 Environmental technologies using mixed cultures

httploschmidtchemimuniczpeglecturebiocat_lecture02pdf

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Immobilization of Microorganism Cells

Advantages1048713 no enzyme isolation and purification1048713 multienzyme complex reactions1048713 cofactor regeneration in native system1048713 synthrophic mixed cultures

Limitations1048713 insufficient stability low resistance1048713 mass transfer limitation1048713 side reactions degradation of product1048713 byproducts from lysis of cell or toxic metabolites 1048713 low productivity

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

E x a m p l e s i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Applications of immobilized enzymes

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

L Amino Acid Resolution

The first industrial use of an immobilized enzyme is amino acid acylase for the resolution of racemic mixtures of chemically synthesized amino acids

Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled

Some of the methods of enzyme immobilization used for this purpose - ionic binding to DEAE-sephadex- entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate - immobilized on macroporous beads made of flexiglass-like material

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Example L-Methionine Production

Amino acid needed by the body but not manufactured naturally by it L-Methionine can be acquired through proper diet and supplements

Methionine improves the bodyrsquos ability to synthesize muscle protein It is a source of sulfur required for the synthesis of other substances important for energy production like choline creatine and carnitine

Comercially methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine separated by immobilized aminoacylase (using DEAE-Sephadex) deacylated of L-methionine L-methionine

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

High Fructose Syrups Production

The most important application of immobilized enzymes in industry

The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase the most of the commercial preparations use either the adsorption or the cross- linking technique

Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Low Lactose Milk amp Whey Production

Preparation of lactose-hydrolysed milk and whey usingβ ndashgalactosidase (lactase)

Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products

Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient

A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

L-Aspartic Acid Production

L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener )

The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid

The enzymes have been immobilized using the whole cells of Escherichia coli onto eg phenolformal-dehyde resin for adsorbing aspartase

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

Enzyme carrier Fixation Examples of immobilized Enzymes

Amberlite FPA54 Anionic Maltose phosphorylase trehalose phosphorylase

Amberlitetrade FPC3500 Cationic Lysozyme (recovery) Cytochrome C Acylase

Amberlitetrade XAD7 HP Adsorption Thermolysin Penicillin acylase Lipase szlig-amylase

Amberlitetrade XAD761 Adsorptionszlig-amylase szlig-Galactosidase Lactase Papain Chymotrypsin Glucoseoxidase Lipase

Duolite A568 Anionic Glucose isomerase Lipase

Duolite A7 Adsorption Trypsin Aspartase Aminocylase RNase Lactase

httpwwwrohmhaascomionexchangepharmaceuticalsenzymeshtm

Enzyme Immobilization on Polymeric Resins - Amberlitetrade and Duolitetrade Strive to Improve Catalysis Economics Through Reuse

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

Malic Acid Production

- The immobilized fumarase is used for the production of malic acid (for pharmaceutical use)

- These processes make use of immobilized nonviable cells of B flavus as a source of fumarase

- An active biocatalyst for the synthesis of L-malic acid from fumaric acid was obtained based on cells immobilized in carrageenan

ABSTRACT

The yeast Saccharomyces cerevisiae was entrapped within polyacrylamide gel beads by employing a procedure that uses sodium dodecylsulfate as a detergent to improve the spherical configuration of the beads The resulting preparation showed a rate of fumarate bioconversion to L-malic acid about 60 times higher than that found forthe free cells Almost all fumarate was converted in 30 min of incubation (Oliveira et al)

httpwwwspringerlinkcomcontentn6655r1x12451534

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

ANTIBIOTIC PRODUCTION

Example 6-aminopenicillanic acid (6-APA) Production

Production of 6-aminopenicillanic acid (6-APA) by the deacylation of the side chain in either penicillin G or V using penicillin acylase (penicillin amidase) One of the major reasons for its success is in obtaining a purer product thereby minimizing the purification costs

A number of immobilized systems have been patented or commercially produced for penicillin acylase which make use of a variety of techniques either using the isolated enzyme or the whole cells

Lecture 14 Application of Immobilized Enzyme

wwwyoutubecomwatchv=_hM8I-yzOAo28 Nov 2008 - 47 menit - Diunggah oleh nptelhrdLecture Series on Enzyme Science and Engineering by ProfSubhash Chand Department of Biochemical

Lecture - 26 Applications

BIOSENSOR (Zhao amp Jiang 2010)

A biosensor can be defined as a device incorporating a biological sensing element connected to a transducer to convert an observed response into a measurable signal whose magnitude is proportional to the concentration of a specific chemical or set of chemcials (Eggins 1996)

Biosensor Type-According to the receptor type biosensors can be classified as enzymatic biosensors genosensors immunosensors etc -Biosensors can be also divided into several categoriesbased on the transduction process such as electrochemical optical piezoelectric and thermalcalorimetric biosensors Among these various kinds of biosensors electrochemical biosensors are a class of the most widespread numerous and successfully commercialized devices of biomolecular electronics (Dzyadevych et al 2008)

Sumber Pustaka

1995 IUPAC Pure and Applied Chemistry

httpwwwspringerlinkcomcontentn6655r1x12451534

httpwwwiasacincurrscijul10articles15htm

httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes

httpwwwbiotecharticlescom

• XII Imobilisasi Enzim (Enzyme Immobilization)
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
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• Bioreaktor Enzim Imobil
• Slide 33
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• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Slide 49
• Slide 50
• Slide 51
• Slide 52
• Slide 53

ABSTRACT

The yeast Saccharomyces cerevisiae was entrapped within polyacrylamide gel beads by employing a procedure that uses sodium dodecylsulfate as a detergent to improve the spherical configuration of the beads The resulting preparation showed a rate of fumarate bioconversion to L-malic acid about 60 times higher than that found forthe free cells Almost all fumarate was converted in 30 min of incubation (Oliveira et al)

httpwwwspringerlinkcomcontentn6655r1x12451534

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

ANTIBIOTIC PRODUCTION

Example 6-aminopenicillanic acid (6-APA) Production

Production of 6-aminopenicillanic acid (6-APA) by the deacylation of the side chain in either penicillin G or V using penicillin acylase (penicillin amidase) One of the major reasons for its success is in obtaining a purer product thereby minimizing the purification costs

A number of immobilized systems have been patented or commercially produced for penicillin acylase which make use of a variety of techniques either using the isolated enzyme or the whole cells

Lecture 14 Application of Immobilized Enzyme

wwwyoutubecomwatchv=_hM8I-yzOAo28 Nov 2008 - 47 menit - Diunggah oleh nptelhrdLecture Series on Enzyme Science and Engineering by ProfSubhash Chand Department of Biochemical

Lecture - 26 Applications

BIOSENSOR (Zhao amp Jiang 2010)

A biosensor can be defined as a device incorporating a biological sensing element connected to a transducer to convert an observed response into a measurable signal whose magnitude is proportional to the concentration of a specific chemical or set of chemcials (Eggins 1996)

Biosensor Type-According to the receptor type biosensors can be classified as enzymatic biosensors genosensors immunosensors etc -Biosensors can be also divided into several categoriesbased on the transduction process such as electrochemical optical piezoelectric and thermalcalorimetric biosensors Among these various kinds of biosensors electrochemical biosensors are a class of the most widespread numerous and successfully commercialized devices of biomolecular electronics (Dzyadevych et al 2008)

Sumber Pustaka

1995 IUPAC Pure and Applied Chemistry

httpwwwspringerlinkcomcontentn6655r1x12451534

httpwwwiasacincurrscijul10articles15htm

httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes

httpwwwbiotecharticlescom

• XII Imobilisasi Enzim (Enzyme Immobilization)
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21
• Slide 22
• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Bioreaktor Enzim Imobil
• Slide 33
• Slide 34
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
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• Slide 47
• Slide 48
• Slide 49
• Slide 50
• Slide 51
• Slide 52
• Slide 53

(httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes)

ANTIBIOTIC PRODUCTION

Example 6-aminopenicillanic acid (6-APA) Production

Production of 6-aminopenicillanic acid (6-APA) by the deacylation of the side chain in either penicillin G or V using penicillin acylase (penicillin amidase) One of the major reasons for its success is in obtaining a purer product thereby minimizing the purification costs

A number of immobilized systems have been patented or commercially produced for penicillin acylase which make use of a variety of techniques either using the isolated enzyme or the whole cells

Lecture 14 Application of Immobilized Enzyme

wwwyoutubecomwatchv=_hM8I-yzOAo28 Nov 2008 - 47 menit - Diunggah oleh nptelhrdLecture Series on Enzyme Science and Engineering by ProfSubhash Chand Department of Biochemical

Lecture - 26 Applications

BIOSENSOR (Zhao amp Jiang 2010)

A biosensor can be defined as a device incorporating a biological sensing element connected to a transducer to convert an observed response into a measurable signal whose magnitude is proportional to the concentration of a specific chemical or set of chemcials (Eggins 1996)

Biosensor Type-According to the receptor type biosensors can be classified as enzymatic biosensors genosensors immunosensors etc -Biosensors can be also divided into several categoriesbased on the transduction process such as electrochemical optical piezoelectric and thermalcalorimetric biosensors Among these various kinds of biosensors electrochemical biosensors are a class of the most widespread numerous and successfully commercialized devices of biomolecular electronics (Dzyadevych et al 2008)

Sumber Pustaka

1995 IUPAC Pure and Applied Chemistry

httpwwwspringerlinkcomcontentn6655r1x12451534

httpwwwiasacincurrscijul10articles15htm

httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes

httpwwwbiotecharticlescom

• XII Imobilisasi Enzim (Enzyme Immobilization)
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
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• Slide 23
• Slide 24
• Slide 25
• Slide 26
• Slide 27
• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Bioreaktor Enzim Imobil
• Slide 33
• Slide 34
• Slide 35
• Slide 36
• Slide 37
• Slide 38
• Slide 39
• Slide 40
• Slide 41
• Slide 42
• Slide 43
• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Slide 49
• Slide 50
• Slide 51
• Slide 52
• Slide 53

Lecture 14 Application of Immobilized Enzyme

wwwyoutubecomwatchv=_hM8I-yzOAo28 Nov 2008 - 47 menit - Diunggah oleh nptelhrdLecture Series on Enzyme Science and Engineering by ProfSubhash Chand Department of Biochemical

Lecture - 26 Applications

BIOSENSOR (Zhao amp Jiang 2010)

A biosensor can be defined as a device incorporating a biological sensing element connected to a transducer to convert an observed response into a measurable signal whose magnitude is proportional to the concentration of a specific chemical or set of chemcials (Eggins 1996)

Biosensor Type-According to the receptor type biosensors can be classified as enzymatic biosensors genosensors immunosensors etc -Biosensors can be also divided into several categoriesbased on the transduction process such as electrochemical optical piezoelectric and thermalcalorimetric biosensors Among these various kinds of biosensors electrochemical biosensors are a class of the most widespread numerous and successfully commercialized devices of biomolecular electronics (Dzyadevych et al 2008)

Sumber Pustaka

1995 IUPAC Pure and Applied Chemistry

httpwwwspringerlinkcomcontentn6655r1x12451534

httpwwwiasacincurrscijul10articles15htm

httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes

httpwwwbiotecharticlescom

• XII Imobilisasi Enzim (Enzyme Immobilization)
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
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• Slide 26
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• Slide 28
• Slide 29
• Slide 30
• Slide 31
• Bioreaktor Enzim Imobil
• Slide 33
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• Slide 36
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• Slide 44
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Slide 49
• Slide 50
• Slide 51
• Slide 52
• Slide 53

BIOSENSOR (Zhao amp Jiang 2010)

A biosensor can be defined as a device incorporating a biological sensing element connected to a transducer to convert an observed response into a measurable signal whose magnitude is proportional to the concentration of a specific chemical or set of chemcials (Eggins 1996)

Biosensor Type-According to the receptor type biosensors can be classified as enzymatic biosensors genosensors immunosensors etc -Biosensors can be also divided into several categoriesbased on the transduction process such as electrochemical optical piezoelectric and thermalcalorimetric biosensors Among these various kinds of biosensors electrochemical biosensors are a class of the most widespread numerous and successfully commercialized devices of biomolecular electronics (Dzyadevych et al 2008)

Sumber Pustaka

1995 IUPAC Pure and Applied Chemistry

httpwwwspringerlinkcomcontentn6655r1x12451534

httpwwwiasacincurrscijul10articles15htm

httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes

httpwwwbiotecharticlescom

• XII Imobilisasi Enzim (Enzyme Immobilization)
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
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• Slide 31
• Bioreaktor Enzim Imobil
• Slide 33
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Sumber Pustaka

1995 IUPAC Pure and Applied Chemistry

httpwwwspringerlinkcomcontentn6655r1x12451534

httpwwwiasacincurrscijul10articles15htm

httpwwwscribdcomdoc22862853Applications-of-Immobilized-Enzymes

httpwwwbiotecharticlescom

• XII Imobilisasi Enzim (Enzyme Immobilization)
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
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• Bioreaktor Enzim Imobil
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• XII Imobilisasi Enzim (Enzyme Immobilization)
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• Bioreaktor Enzim Imobil
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