IMMOBILIZED ENZYMES

Module 2

Introduction

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Restriction of enzyme mobility Advantages Enzyme reutilization Elimination of enzyme recovery and purification Improve enzyme activity

Immobilization techniques

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Two major types Entrapment Surface immobilization

Entrapment

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Physical enclosure of enzyme Matrix entrapment Membrane entrapment Microencapsulation

Matrix entrapment

Physical enclosure of enzyme by using matrices Matrices are usually polymeric material Polymer materials like Ca-alginate, agar, collagen, polyacrylamide and k-carrageein Solid matrices also used E.g. activated carbon, porous ceramic and diatomaceous earth The matrix can be a particle, a membrane or a fiber

Immobilization in polymer matrix

Enzyme solution is mixed with polymer solution Mixing should be before polymerization Polymerized gel contain enzyme Enzyme is extruded or a template is used to shape the particle from mixture

Membrane entrapment

Semi permeable membrane is used Membrane of nylon, cellulose, polysulfone and polyacrylate Hollow fiber arrangement is commonly used Semi permeable membrane retains high molecular weight compounds (enzymes) while allows low molecular weight compounds (substrate and product) to access enzyme

Microencapsulation

Form of membrane entrapment Microscopic hollow spheres are formed Spheres contain the enzyme solution Sphere is enclosed in porous membrane Membrane can be polymeric or an enriched interfacial phase formed around a micro drop

Disadvantages of entrapment
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Enzyme leakage into solution Diffusional limitation Reduced enzyme activity and stability Lack of control of micro environmental conditions

Surface immobilization

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Two types Adsorption Covalent binding

Adsorption

Attachment of enzymes on the surface of supporting particle Weak physical force of attachments Van der Waals or dispersion forces Active site of enzymes are unaffected Adsorption is stabilized by cross-linking with glutaraldehyde Supporting materials are alumina, silica, porous glass, ceramics, cellulose (CMC, DEAE cellulose), starch etc

Covalent binding

Retention of enzymes on support surface by covalent bond Enzyme molecule bind to support material via certain functional group Functional groups are amino, carboxyl, hydroxyl and sulfhydryl Functional group on support material are usually activated by using cyanogen bromide, carbodiimide and glutaraldehyde

Cross-linking of enzyme

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Done using glutaraldehyde, 2,2-disulfonic acid Done in different ways Enzyme can cross-linked with glutaraldehyde to form an insoluble aggregate Adsorbed enzyme is cross-linked Impregnation of porous support with enzyme solution

Two major criteria for support selection 1. Binding capacity of support material Function of charge density, functional groups, porosity and hydrophobicity of support surface 2. Stability and retention of enzymatic activity Function of functional group and microenvironmental conditions

Diffusional limitations in immobilized enzyme systems

Diffusion resistance depends on nature of supporting material, hydrodynamic conditions surrounding the support material Damkohler number determines diffusion resistance have significant effect on the rate of enzymatic reaction rate

Sb is substrate concentration in bulk liquid (g/cc) and kL is mass transfer coefficient (cm/s)

Damkohler no of CSTR

For non-ideal reactor

If Da>>1 diffusion rate is limiting If Da<<1 the reaction rate is limiting If Da 1 diffusion and reaction resistance are comparable

Diffusion effects in surface-bound enzymes on nonporous support materials

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Assumptions Enzymes are bound and evenly distributed on the surface All enzyme molecules are equally active Substrate diffuses through a thin liquid film surrounding the support surface Immobilization has not altered protein structure and intrinsic kinetic parameters (Vm, Km)

At steady state

Quadratic equation w.r.t Ss When Da>>1 system is mass transfer limited Ss =0 and system behaves pseudo first order

If Da <<1, reaction rate becomes

where

Diffusion effects in enzymes immobilized in a porous matrix

Substrate diffuses through tortuous pathway React with enzyme immobilized on the pore surface Diffusion and reaction are simultaneous in this case Tortuousity:- Ratio of actual distance substrate travelled to minimum distance between the points

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Assumptions Enzyme is uniformly distributed in a spherical support particle Reaction kinetics are expressed by MichaelisMenten kinetics No partition of the substrate between the exterior and interior of the support

At steady state:-

With boundary conditions [S] = [Ss] at r = R and d[S]/dr =0 at r = 0 De is the effective diffusivity of substrate within the porous matrix

Previous equation can be written in dimensionless form

Where

With boundary conditions at

at

and

The rate of substrate consumption is equal to the rate of substrate transfer through external surface of the support particle at steady state into sphere

Under diffusion limitation, the rate per unit volume is expressed as

Effectiveness factor, Ratio of the reaction rate with diffusion limitation to the reaction rate with no diffusion limitation Value of is a measure of the extend of diffusion limitation When <1 conversion is diffusion limited If 1 conversion is limited by reaction rate Factor, =f(,)

For a zero order reaction rate (0), 1 for a large Thiele modulus values (1<<100) For a 1st order reaction rate( ),
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Internal diffusion limits the enzymatic reaction rate, the constant Vm,app and Km,app values are not true intrinsic values, but apparent values

To eliminate diffusion resistance use small particle sizes, a high degree of turbulence around particle and high substrate concentration Main variable in designing immobilized enzyme systems are Vm and R Km and De are fixed Particle size should be small as possible

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Higher enzyme content higher enzyme activity per unit of reactor volume But lower effectiveness factor For higher (0.8) Enzyme loading should be less than 10mg/cm Particle size <10m

Electrostatic and steric effects in Immobilized enzyme systems

Immobilized enzyme in charged matrix experience shift in pH Bulk pH of immobilized will shift to that of soluble enzyme Charged matrix will repel or attract substrate, product, H+ ions depending on type and quantity of surface charged Shift in pH activity profile is given by

F is Faraday constant (96500 coulomb/eq.g) is electrostatic potential pHi and pHe are internal and external pH values z is charge on the substrate R is gas constant Intrinsic activity of enzyme is altered Alteration of apparent kinetics due change in pH Enzyme activity towards high molecular weight substrate is reduced by immobilization Due to steric hinderance by the support

Immobilization results in increase in thermal stability Thermal stability due to the presence of thermal diffusion barriers and constraints on protein folding

Large scale production of enzymes

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Eg:- proteases (subtilism, rennet), hydroleases (pectinase, lipase, lactase), isomerases (glucose isomerase) and oxidases (glucose oxidase) Steps in production of enzymes Overproducing strains of certain organisms Separation and purification of an enzyme from organism by cell disruption, removal of cell debris and nucleic acid Precipitation of protein

Uses of enzymes