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TOPIC 4IMMOBILIZED BIOCATALYSTProperties and Characterization of Immobilized BiocatalystsProperties of Immobilized EnzymesThe properties of immobilized enzyme preparations are governed by the properties of both the enzyme and the carrier material.Due to immobilization, the properties of enzymes will be altered such as catalytic activity with respect to the support matrix. The change in the enzyme properties in the immobilized enzyme is due to the enzyme and the substrate reacts in the microenvironment which is different from the enzyme substrate reaction in the bulk solution environment. As far as manufacturing costs are concerned the yield of immobilized enzyme activity is mostly determined by the immobilization method and the amount of soluble enzyme used.Under process conditions, the resulting activity may be further reduced by mass transfer effects, lead to lowered efficiency. Properties of Immobilized EnzymesMore precisely, the yield of enzyme activity after immobilization depends not only on losses caused by the binding procedure but may be further reduced as a result of diminished availability of enzyme molecules within pores or from slowly diffusing substrate molecules.

Carrier materials can be divided into those of inorganic and organic originSupport selection Selection criteria differ among one another, depending on the biocatalyst of interest, but there are still few basic features that must be considered. Material used as a carrier should have chemical, physical and biological stability during processing, as well as in the reaction conditions; sufficient mechanical strength, especially for its utilization in reactors and industry; should be nontoxic both for the immobilized cell/bioparticle, as well for the product; also should have adequate function groups for binding biocatalyst and high loading capacity. Profitability of the material application and its processing costs always have to be taken upon consideration. Other criteria, such as physical characteristics (porosity, swelling, compression, material and mean particle behavior), as well as possibility for microbial growth, biodegradability, solubility, are more application specific. Characteristics of the CarrierFunctional groups:

The type of activation, presence, distribution and density of functional groups determines the activity yields of an immobilization reaction, and stability and operational stability of the carrier-fixed enzyme.

Most immobilization procedures proceed via a nucleophilic attack of amino groups on activated carrier functional groups.

This is in many cases the appropriate way to couple an enzyme on a carrier surface, because it avoids the enzyme getting into contact with highly reactive organic compounds of low molecular weight.

Permeability and surface area:

In most cases a large surface area (>100m2 g1) and high porosity are desirable, so that enzyme and substrate can easily penetrate.

A pore size of >30 nm seems to make the internal surface accessible for immobilization of most enzymes.Hydrophilicity/hydrophobicity of the carrier matrix:

It influences type and strength of non-covalent proteinmatrix interaction.

In addition, it can influence the adsorption, distribution and availability of the substrate and product.Characteristics of the CarrierInsolubility:

This is essential, not only for prevention of enzyme loss, but also to prevent contamination of the product by dissolved matrix and enzyme.Mechanical stability/rigidity:

These properties are dependent on the type of reactor. If used in a stirred tank reactor, the support should be stable against sheer forces to minimize abrasion. Production of fines (particles below 10050 mm) can lead to plugging of sieve plates and filters.Characteristics of the CarrierResistance to microbial attack:

During long term usage the support has to be stable against microbial degradation.Form and size of support:

The particle size will have an influence on filtration times from stirred tank reactors in repeated batch mode. Furthermore, this factor is important for the performance in column reactors regarding back pressure and flow rates, which of course are correlated. For this purpose a size of spherical particles in the range of 150300 mm is preferred.Characteristics of the CarrierRegenerability:

This property is of interest in case of expensive carrier materials.Mass Transfer EffectsIdentify the parameters which affect the mass transfer of immobilized enzymes/cells. Explain.Kinetic PropertiesThere is usually a decrease in specific activity of an enzyme upon insolubilization: denaturation caused by the coupling processMicroenvironment after immobilization may be drastically different from that existing in free solution: the physical and chemical character of the support matrix, or interactions of the matrix with substrates or products involved in the enzymatic reaction The Michaelis constant may decrease by more than one order of magnitude when substrate of opposite charge to the carrier matrix A reduced reaction rate may result from external diffusional restrictions on the surface of carrier materials.The diffusion of substrate can limit the rate of the enzyme reaction: the thickness of the diffusion film determines the concentration of substrate in the vicinity of the enzyme and hence the rate of reaction The effect of the molecular weight of the substrate can also be large. This may be an advantage in some cases, since the immobilized enzymes may be protected from attack by large inhibitor molecules

Mass Transfer EffectsEffects of Enzyme Immobilization on Activity

14Kinetics of immobilized enzymesPartitioning effect

The solution lying within a few molecular diameters (10 nm) from the surface of an immobilized enzyme will be influenced by both the charge and hydrophobicity of the surface

The Km of an enzyme for a substrate is apparently reduced if [S] in the vicinity of the enzyme's active site is higher than that measured in the bulk of the solution

If the surface is predominantly hydrophobic Hydrophobic molecules will partition into the microenvironment of the enzyme and hydrophilic molecules will be partitioned out into the bathing solutionPartition will affect the apparent kinetic constants of the enzyme

Diffusional Limitation in Immobilized Enzyme SystemImmobilized enzyme system normally includes- insoluble immobilized enzyme- soluble substrate, or product

They are heterogeneous systemsCONCENTRATION DIFFERENCEFILM TRANSFERDRIVING FORCEDIFFUSIONHIGHImmobilized EnzymeSbELECTRIC ATTRACTIONLow S concentrationSubstrateCONCENTRATION DIFFERENCEFILM TRANSFERDRIVING FORCEDIFFUSIONHIGHImmobilized EnzymeSbELECTRIC ATTRACTIONREACTION

PRODUCTFILM TRANSFERDRIVING FORCEDIFFUSIONHIGHImmobilized EnzymeSbINTRA-PARTICLE TRANSFER

FILM TRANSFERPRODUCTHIGH

Immobilized EnzymeSbINTRA-PARTICLE TRANSFERREACTION

Diffusional Limitation in Immobilized Enzyme SystemsIn immobilized enzyme systems, the overall production rate is determined by

- liquid film mass transfer (external diffusion)The transport of substrates towards the surface, and products away)

- intraparticle mass transfer (internal diffusion)The transport of the substrates and products, within the pores of immobilised enzyme particles - enzyme catalysis reactionDiffusional Limitation in Immobilized Enzyme SystemSs: substrate concentration at the surface;Sb: substrate concentration in bulk solution.EnzymeSsSbLiquid Film Thickness, L

E+SDiffusion Effects in Surface-bound Enzymes on Nonporous Support MaterialsAssume the enzyme catalyzed reaction rate follows Michaelis-Mententype kinetics.EnzymeSsSbLiquid Film Thickness, L No intraparticle diffusionAssume:

Enzyme are evenly distributed on the surface of a nonporous support material.

All enzyme molecules are equally active.

Substrate diffuses through a thin liquid film surrounding the support surface to reach the reactive surface.The process of immobilization has not altered the enzyme structure and the intrinsic parameters (Vm, Km) are unaltered.Diffusion Effects in Surface-bound Enzymes on Nonporous Support Materialsis the maximum reaction rate per unit of external surface area (e.g. g/cm2-s)To determine the significant effect of external diffusion resistance on the rate of enzyme catalytic reaction rate: Damkhler numbers (Da)

is the liquid mass transfer coefficient (cm/s)Is the substrate concentration in bulk solution (g/cm3)Diffusion Effects in Surface-bound Enzymes on Nonporous Support MaterialsWhen Da >> 1, the external diffusion rate is limiting; Da >1Diffusion Effects in Surface-bound Enzymes on Nonporous Support MaterialsTo increase the overall reaction rate with external diffusion limitationIncrease .

Increase .

Diffusion Effects in Surface-bound Enzymes on Nonporous Support MaterialsThe liquid film mass transfer coefficient kL:(H. Fogler, Elements of Chemical Reaction Engineering 1999, p705)DAB is mass diffusivity of the substrate in the liquid phase, a function of temperature and pressure (m2/s)

is the kinematic viscosity (m2/s), a function of temperature.

U is the free-system liquid velocity (velocity of the fluid flowing past the particle) (m/s).

dp is the size of immobilized enzyme particle (m).

At specific T and P, increasing U and decreasing dp increase the liquid film mass transfer coefficient andthe external diffusion rate.

Diffusion Effects in Surface-bound Enzymes on Nonporous Support MaterialsWhen the system is strongly reaction limited,[Sb] [Ss]

the overall reaction rate is equal to the rate:

Da