ENZYME TECHNOLOGY

Overview• Enzyme definition• Enzyme classification &

nomenclature• The nature of enzymes• Enzyme preparation & source of

commercial food enzyme• Enzyme applications in food industry• Enzyme immobilization

• Biocatalysts include any enzymatically-catalyzed reaction, whether the enzyme has been purified or is part of a whole (microbial) cell– Amylases, glucanases, proteinases –

widely used in the brewing process– Baker’s yeast (Saccharomyces cerevisiae)

to convert sugars to CO2 to make leavened bread

What is an enzyme?

Enzymes are usually proteins of high molecular weight

(15 000<MW<several million daltons) that catalyze biologically important reactions

Enzyme classification & nomenclature

• The accepted system for classification & nomenclature of enzymes embodies 3 general principles:

1. Enzyme names, ending in -ase should be used only for single enzymes i.e. single catalytic entities

2. Enzyme is named & classified according to the reaction it catalyzed i.e the observed chemical changes produced by the enzyme

3. Based on the type of reaction catalysed, and together with the name(s) of the substrate(s) this provides a basis for naming individual enzymes. It is also the basis for classification and code numbers.

EC 1.1.3.4,

• glucose oxidase – trivial/working

• systematic name, -D-glucose:oxygen 1-oxidoreductase

• These code numbers contain four elements separated by points, with the following meaning:

(i) the first number shows to which of the six main divisions (classes) the enzyme belongs,

(ii) the second figure indicates the subclass,

(iii) the third figure gives the sub-subclass,

(iv) the fourth figure is the serial number of the enzyme in its sub-subclass.

• EC 2.1.1 Methyltransferases• EC 2.1.2 Hydroxymethyl-, Formyl- and Related

Transferases• EC 2.1.3 Carboxyl- and Carbamoyltransferases• EC 2.1.4 Amidinotransferases

• A given enzyme often has 2 names:

1. Systematicformed in accordance with definite rules, showed the reaction it catalyzed, thus identifying the enzyme precisely.

2. Working or trivial sufficiently short for general use, but not systematic

Enzyme classes

1. Oxidoreductases – oxidation-reduction reactions

– Acting on the CH-OH group of donors– Acting on the aldehyde or oxo group of donors

2. Transferases – transfer of functional groups– One C-groups– Aldehyde or ketonic groups

3. Hydrolases (hydrolysis reactions)– Esters– Glycosidic bonds– Peptide bonds

4. Lyases (addition of double bonds)– Carbon-carbon lyases– Carbon-oxygen

5. Isomerases (isomerization reactions)– Racemases & epimerases– Cis-trans isomerases

6. Ligases (formation of bonds with ATP cleavage)

– Forming carbon-oxygen bonds– Forming carbon-sulfur bonds

CLASSESCLASSES E.G. INDUSTRIAL ENZYMESE.G. INDUSTRIAL ENZYMES

11 OxidoreductasesOxidoreductases PeroxidasesPeroxidases

CatalasesCatalases

Glucose oxidasesGlucose oxidases

22 Transferases Transferases Fructosyl-transferasesFructosyl-transferases

Glucosyl-transferasesGlucosyl-transferases

33 Hydrolases Hydrolases Amylases, Lipases, PectinasesAmylases, Lipases, Pectinases

Cellulase, ProteasesCellulase, Proteases

44 Lyases Lyases Pectate lyasesPectate lyases

-acetolactate decarboxylases-acetolactate decarboxylases

55 IsomerasesIsomerases Glucose isomeraseGlucose isomerase

66 Ligases Ligases DNA ligase (Not use at present-DNA ligase (Not use at present-exclusive for biological purposes)exclusive for biological purposes)

The nature of enzymes

1. The enzyme remains unaltered at the end of reaction

S + E SE P + E Substrate Enzyme Enzyme Product Enzyme

substrate complex

• Enzyme is not used up in the reaction but can be used over & over again.

• Implication: small amount of enzyme can catalyze the bioconversion of a large amount of substrate, thus save cost

2. Chemical reactions take place under mild conditions Chemical catalysts often require organic

solvents, high temperature, extreme pH & high pressure

Most enzyme operate in aqueous solution, at mild temperature & pH, and at atmospheric pressure.

– Lower energy & materials cost

E.g. Maltose is usually synthesised by hydrolysis of starch

• Non-enzymatic

Maltose + H2O boil, HCl 2 Glucose

• Enzymatic Maltose + H2O maltase, 25oC 2 Glucose

3. Highly specific action

• Requirement for complementarily in the configuration of substrate and enzyme explains the specificity of most enzymes.

Analogy that a substrate molecule binds to the enzyme is like a key in a lock.

4. Fast reaction rates• Enzymes reduce the activation energy -

less energy for each molecule of substrate converted to product.

• More molecules of substrate would be converted when the enzyme is present than when it is absent The reaction is faster in a given period of time.

Advantages of enzymes

• Enzyme

1. Higher product quality– Produce consistent quality products

– Improve the conversion of raw material to its constituent parts e.g. hydrolysis of starch to glucose

– Acid hydrolysis gives limited conversion whereas enzyme can improve yield

2. Lower manufacturing cost– Enzyme can be reused

– Better use of raw materials

3. Less waste– Effluent from enzyme hydrolysis is less toxic,

therefore cheaper in term of waste disposal – environmental benefit

4. Reduced energy consumption

• Chemical treatment– Generally non-specific– Not always easily controlled – May create harsh conditions– Produce undesirable side effects – waste disposal

problems

Enzyme preparations used in food processing

• Definition– consist of biologically active proteins, at times combined with

metals, carbohydrates and/or lipids. – They are obtained from animal, plant or microbial sources and

may consist of whole cells, parts of cells, or cell-free extracts– May contain one or more active components as well as

carriers, solvents, preservatives, antioxidants and other substances consistent with good manufacturing practice.

– They may be liquid, semi liquid, dry or in an immobilized form (immobilized enzyme preparations are preparations which have been made insoluble in their intended food matrix by physical and/or chemical means).

– Their colour may vary from virtually colourless to dark brown. 

Material used in the production of enzyme preparations:

• Plant - must consist of components that leave no residues harmful to health in the processed finished food under normal conditions of use. 

• Animal tissues - must comply with meat inspection requirements and be handled in accordance with good hygienic practice. 

• Microbial sources - may be native or variants strains of microorganisms, or by the processes of selective serial culture or genetic modification.

Source of commercial food enzyme

3 primary sources:1. Plants• Malt amylase (malted barley), papain

(papaya), bromelain (pineapple), ficin (figs)

2. Animal tissue• Pepsin & rennet (stomach mucosa),

catalase (liver), proteases, amylase, lipases (pancreas)

3. Microorganisms • Yeast (Aspergillus), mold, bacteria

(Bacillus)

Selection of microorganisms• Strain able to give high yield of enzyme at

short fermentation time• Produce extracellular enzyme for easier

isolation• A food-grade m/o (GRAS) that does not

produce any toxic substances• Able to grow on inexpensive medium

containing cheap substrate• Low amount of interfering by-products (i.e.

pigments, slime, proteases)

Advantages of enzyme production from microbes

1. Fast & ease of growth Large volumes can be produced with a uniform quality and

high purity Supply of ingredients extracted from

animals/plants/humans is limited by the availability of a potentially unsuitable range of raw materials that can vary in quality

2. Can be easily controlled during growth can be made consistently to recognised quality standards

3. Produce enzymes that are easy to extract esp. extracellular enzymes

Safety of microbial enzyme preparations used in food

• Primary consideration in evaluating safety of a production strain:

1. Toxigenic potential

– Possible synthesis of toxins that are active

2. Pathogenic potential

– Not usually an area of concern for consumer safety but important to worker safety

• Enzyme preparation rarely contain contain viable organisms

Production of enzymes

• Naturally occurring enzymes are quite often not readily available in sufficient quantities for food applications or industrial use

• Isolating microbial strains that produce the desired enzyme & optimizing the conditions for growth obtain commercial quantities

Production of enzymes

1. Cultivate the organisms producing the desired enzyme via fermentation

– Surface cultures– Submerged cultures

2. Cell separated from the media – filtration, centrifugation

– Extracellular enzyme – fermentation broth – Intracellular enzyme – cells biomass

• Recovery involves disruption of cells & removal of cell debris & nucleic acid

Enzyme in starch industry

• Starch – polysaccharide of plants

• Commercial source – the seed of cereal grains (rice, corn, wheat, sorghum), roots (tapioca) and tubers (potato)

• Most starch contain 2 types of glucose polymer– Amylose- linear polymer with -1,4 linkage– Amylopectin- branched polymer at -1,6

branch point

Representative partial structure of amylose

Representative partial structure of amylopectin

• Starches from different sources differ from each other in terms of– Length of the chains– Degree of branching

the amounts & combinations of enzymes used to hydrolyze starch are different in different operations

• Traditionally, starch was, and still is, hydrolyzed to low-molecular-weight dextrins and glucose using acid, but enzymes have several advantages.– First, the specificity of enzymes allows the

production of sugar syrups with well-defined physical and chemical properties.

– Second, the milder enzymatic hydrolysis results in few side reactions and less “browning.”

• Production of High Fructose Corn Syrup (HFCS) – Closely mimicked the sweetness of sucrose (table

sugar).– Converts large quantities of corn & other botanical

starches to HFSC & other useful sweeteners– High sweetening property – can be used to

replace sucrose syrups in foods & beverages– Sweeteners – soft drinks, candies, baking, jams,

jellies, etc.

Major steps in production of sweeteners

Starch

Slurry preparation

Liquefaction

Saccharification

Purification

Isomeration

Refining

-amylase Maltodextrins

Glucoamylase/pullulanase

Glucose isomerase

Maltose syrupsGlucose syrupsMixed syrups

Frutose syrups

1. Hydrolysis with -amylase (liquefaction)– Starch is gelatinized at temperature 105-110oC & liquefied to

reduce viscosity using thermostable -amylase, pH7• To make starch chains shorter • To make more chain ends

2. Hydrolysis with glucoamylase (saccharification)– Glucoamylase or -amylase enzymes are used to produce

glucose & maltose syrups from the dextrin respectively, pH 3.5-5.0 & 4.8-6.5 respectively; 60oC

– Pullulanase added along with glucoamylase to improve enzyme efficiency to evolve glucose syrups containing 95-96% glucose in shorter periods of time

3. Isomerization with glucose isomerase – Glucose isomerase converts glucose to fructose by

isomerization– Since glucose & fructose have a roughly equimolar equilibrium,

the product is a mixture of about 50-53% glucose, 42-45% fructose & 5% other products

– Finishing steps – ion exchange, decolourization & evaporation to give HFCS 42, or enrich it to increase its fructose content

– 3 major types of amylase:

1. -amylase (starch liquefying enzyme; endo-enzyme)

• breaks -1,4 glycosidic bonds randomly on the amylose chain & solubilizes amylose to give maltose & short oligosaccharides

2. -amylase (saccharifying enzyme, exo-enzyme) • hydrolyzed -1,4 glycosidic bonds on the non-reducing

ends of amylose to yield maltose

3. Glucoamylase (saccharifying enzyme)• capable of cleaving both -1,4 and -1,6 glucose

linkages, which releases glucose• Pullulanase hydrolyzes -1,6 glycosidic linkages in

branched polysaccharides e.g. amylopectin

Enzyme in brewing industry

• Beer production involve 2 biological process – malting & fermentation

• Starch present in cereal grain (usually barley) is broken down

• Yeasts cannot metabolise starch & in order to yield alcohol (brewing), the starch needs to be hydrolyzed

• Bacterial -amylase in mashing – Splits insoluble & soluble starch into shorter

chains -amylase: starch maltose & dextrins– glucoamylase: dextrins glucose

-glucanase in mashing– Assist in mashing of grits, reduce wort

viscosity & improve beer finability & filterability – reduce haze formation

• Glucoamylase in mashing– Cleavage of terminal -1,6 glycosidic bonds

of oligosaccharides, thus an additional amount of fermentable glucose in the wort.

• Fungal -amylase in fermentation– Increase fermentable CHO in the wort

extract produce more alcohol

• Proteases– Hydrolyzed high molecular weight

proteins into simpler peptides reduce haze & better foam stability

• Special brewing process– Low calorie beer (diet/light lagers) –

highly carbonated fermentation products with almost complete conversion of all CHO into alcohol & CO2 assisted by bacterial & fungal enzymes e.g. glucoamylase

Enzyme in fruit juice processing

• The fruit cell wall– The most important characteristic affecting the

extraction of juice – The composition of the cell wall can vary

significantly for different types of fruit, but mainly consists of pectin, hemicellulose, cellulose, lignin and other components.

• 1st application of enzymes in the fruit juice industry. 1930s – pectinases for fruit juice clarification

• Juices extracted from ripe fruit contain significant amount of pectin– Pectins contribute to fruit juice viscosity & turbidity

Fruit juice extraction process

• Mixture of pectinases & amylases is used to clarify fruit juices– Degrade pectin & starch during clarification stage

prevents post-bottling haze formation– Decreases filtration time up to 50%

• Pectinases in combination with other enzymes (hemicellulases, arabinases, cellulases & xylanases)– Lowering the viscosity of pulp– Preventing araban haze formation after concentration

of the juice– Increase the pressing efficiency of the fruits for fruit

extraction increase juice yield– Better colour extraction

Enzyme in dairy industry

• Milk itself contains a large number of enzymes, some of which are important in milk processing:

– naturally occuring proteases - contribute for the flavour characteristics of the cheese

– naturally occuring lipases - frequently lead to the development of rancidity in products containing milk fat.

• Main category of enzyme involved in dairy industry:1.Lactase – hydrolyse lactose in milk & whey

-galactosidase/lactase: lactose glucose + galactose

– Potential use of hydrolysed lactose:• Nutritional sweet syrup – dairy, confectionery,

baking, beverage• An accelerating fermentation medium in yogurt,

cheese• Lactose free products for lactose intolerance ppl

2. Milk clotting enzymes – Initially, unpurified crude preparations

extracted from the stomachs of ruminants were used to coagulate milk

a. Rennet – milk clotting enzymes derived from animal source• Bovine rennet consists of 2 main proteinases, the

milk clotting enzyme chymosin & pepsin

b. Coagulants – milk clotting enzymes derived from microbes• Derived from fungal sources• Produced by fermentation where the proenzyme

is converted to its active form during production by the slightly acidic environment

c. Fermentation produced chymosin – milk clotting enzymes produced using genetic engineering

• Produced by fermentation brought about by an organism genetically modified with a gene for chymosin

4. Lipase– Milk fat consists mainly of triglycerides

• Lipase: triacylglycerols di- and mono-acylglycerols, free fatty acids & glycerol

– Contribute to distinctive flavor development during ripening stage of cheese production

Enzyme in baking industry

• Introduction– Ancient Egyptians made use of enzymes present

endogenously in the flour– 20th century – enzymes used as flour improvers– 1st application in baked goods- supplementation

of -amylase by addition of malt to correct the concentration of endogenous -amylase in the flour

– Malt substituted by microbial -amylase – more suitable thermostability for baking

Main component of wheat flour is starch, Main component of wheat flour is starch, gluten , non-starch polysaccharides, lipids & gluten , non-starch polysaccharides, lipids & trace amount of mineralstrace amount of minerals– Amylase: degrade starch & produce small dextrins Amylase: degrade starch & produce small dextrins

for the yeast to act uponfor the yeast to act upon

Gluten – combination of proteins which forms Gluten – combination of proteins which forms a large network during dough formationa large network during dough formation– Xylanases (hemicellulases), lipases & oxidases – Xylanases (hemicellulases), lipases & oxidases –

directly or indirectly improve the strength of the directly or indirectly improve the strength of the gluten network – improve the quality of the gluten network – improve the quality of the finished breadfinished bread

Enzyme Enzyme Effect Effect

AmylaseAmylase

Glucose oxidaseGlucose oxidase

LipaseLipase

Maximizes the fermentation Maximizes the fermentation process to obtain an even process to obtain an even crumb structure & a high loaf crumb structure & a high loaf volumevolume

Oxidizes free sulphydryl Oxidizes free sulphydryl groups in gluten to make groups in gluten to make weak doughs stronger & weak doughs stronger & more elasticmore elastic

Dough conditioning by Dough conditioning by producing more uniform, producing more uniform, smaller crumb cells & a smaller crumb cells & a silkier texture & whiter crumb silkier texture & whiter crumb colourcolour

Enzyme Enzyme Effect Effect

Lipoxygenase Lipoxygenase

XylanaseXylanase

ProteaseProtease

Bleaching & strengthening Bleaching & strengthening doughdough

Dough conditioning. Easier Dough conditioning. Easier dough handling & improved dough handling & improved crumb structurecrumb structure

Weakens the gluten to Weakens the gluten to provide the plastic properties provide the plastic properties required in doughs for required in doughs for biscuitsbiscuits

Enzyme immobilisation

Definitions• Immobilized enzymes have been

defined as enzymes that are physically confined or localized, with retention of their catalytic activity, and which can be used repeatedly and continuously– Applicable to enzymes, cellular organelles,

microbial cells, plant cells & animal cells, that is, to all types of biocatalysts

Advantages of immobilisation

1. Reusability• Easily recovered & separated from the product• Suitable for continuous processes Lowering processing costs2. Stability • Increase if the carrier provides a micro environment

capable of stabilizing the enzyme

3. Product is not contaminated with the enzyme  • Enzyme can be readily removed from the

reaction mixture • Useful especially in the food and pharmaceutical

industries4. Lower reaction time• Due to higher enzyme to substrate ratios5. Reduce effluent problems• Downstream processing is easier

Disadvantages

1. Diffusion of substrates & products may be hampered by partitioning of the enzyme in immobilised layer

2. The enzyme may have a more constrained conformation in the immobilised state, giving it a lower catalytic activity

3. High initial investment compared to free enzyme

– Cost of supports & reagents

Type of support - 3 categories:1. Hydrophilic biopolymers based on

natural polysaccharides such as agarose, dextran and cellulose

2. Lipophilic synthetic organic polymers such as polyacrylamide, polystyrene and nylon

3. Inorganic materials such as controlled pore glass and iron oxide.

• Selection of support material– The binding capacity

• Charge density, functional groups, porosity & hydrophobicity of the support surface

– Stability & retention of enzymatic activity• Functional groups on support material &

microenvironment conditions• If immobilization causes some conformational

changes on the enzyme, or if reactive groups on the active site of the enzyme involved in binding, a loss in enzyme activity can take place upon immobilization

Desirable enzyme carrier possesses: • large surface area • permeable• insolubility • chemical, mechanical and thermal stability • high rigidity • suitable shape and particle size • resistance to microbial attack • regenerability

Immobilisation techniques

2 major methods of immobilization– Surface immobilization

• Adsorption• Covalent bonding• Cross-linking

– Entrapment• Matrix entrapped• Membrane entrapped

1. Adsorption• Attachment of enzymes on the surfaces of

support particles by weak physical forces i.e. van der Waals, hydrogen bonding, hydrophobic interaction, or combined action

• Support materials – Inorganic – alumina, silica, porous glass,

ceramics, diatomaceous earth, clay, bentonite– Organic – cellulose (CMC, DEAE-cellulose),

starch, activated carbon, ion-exchange resins (Amberlite, Sephadex, Dowex)

• Common problem – desorption of enzymes esp. in the presence of strong hydrodynamic forces

– Stabilized by cross-linking with glutaraldehyde

2. Covalent binding• Retention of enzymes on support surfaces by

covalent bond formation• Bind to support material via certain functional

groups i.e. amino, carboxyl, hydroxyl & sulfhydryl groups

• Functional groups on support material usually activated using chemical reagents i.e. cyanogen bromide, carboiimide & glutaraldehyde

3. Cross-linking • Intermolecular cross-linking of enzyme

molecules using bi- and multifunctional compounds reagents i.e. glutaraldehyde, bis-diazobenzidine & 2,2-disulfonic acid

• Cause changes in the active site of enzymes, severe diffusion limitations

4. Entrapment • Physical enclosure of enzyme in a small

space• Methods

– Matrix entrapment– Membrane entrapment – macroscopic

membrane, microencapsulation

• Matrices used – polymeric materials e.g. Ca-alginate, agar, -carragenan, polyacrylmide, collagen; solid matrices e.g. activated carbon, porous ceramic, diatomaceous earth

• Membrane used – nylon, cellulose, polysulfone, polyacrylate

• Disadvantages– Enzyme leakage into solution– Diffusional limitations e.g. substrates of high

molecular mass– Lack of control of microenvironment

conditions reduced enzyme activity & stability

QUIZ 1

1. Name 3 main vectors used in recombinant DNA? (3 marks)

2. Name 5 basic media composition for the fermentation process. (5 marks)

3. What is the purpose of downstream processing? (2 marks)

• Plasmid, bacteriophage, cosmids

• Water, carbon, nitrogen, vitamin & mineral

• To obtain the product with requisite concentration & purity