immobilized ennzymes.pptx

7/27/2019 Immobilized ennzymes.pptx

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IMMOBILIZEDENZYMES

Module 2


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Introduction

Restriction of enzyme mobility

Advantages

1. Enzyme reutilization

2. Elimination of enzyme recovery and

purification

3. Improve enzyme activity


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Immobilization techniques

Two major types

1. Entrapment

2. Surface immobilization


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Entrapment

Physical enclosure of enzyme

1. Matrix entrapment

2. Membrane entrapment

3. Microencapsulation


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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 anddiatomaceous earth

The matrix can be a particle, a membrane or a

fiber


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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


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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


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Disadvantages of entrapment

1. Enzyme leakage into solution

2. Diffusional limitation

3. Reduced enzyme activity and stability

4. Lack of control of micro environmental

conditions


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Surface immobilization

Two types

1. Adsorption

2. Covalent binding


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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


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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 usuallyactivated by using cyanogen bromide,

carbodiimide and glutaraldehyde


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Cross-linking of enzyme

Done using glutaraldehyde, 2,2-disulfonic acid

Done in different ways

1. Enzyme can cross-linked with glutaraldehyde to

form an insoluble aggregate

2. Adsorbed enzyme is cross-linked

3. Impregnation of porous support with enzyme

solution


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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 micro-environmental conditions


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Diffusional limitations in

immobilized enzyme systems


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Diffusion resistance depends on nature of

supporting material, hydrodynamic conditions

surrounding the support material

Damkohler number determines diffusion resistancehave 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)


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Damkohler no of CSTR

For non-ideal reactor


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If Da>>1 diffusion rate is limiting

If Da


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us on e ec s n sur ace- ounenzymes on nonporous support

materialsAssumptions

1. Enzymes are bound and evenly distributed on

the surface

2. All enzyme molecules are equally active

3. Substrate diffuses through a thin liquid film

surrounding the support surface

4. Immobilization has not altered protein structureand intrinsic kinetic parameters (Vm, Km)


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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


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If Da


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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 thiscase

Tortuousity:- Ratio of actual distance substrate

travelled to minimum distance between thepoints


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Assumptions

1. Enzyme is uniformly distributed in a spherical

support particle

2. Reaction kinetics are expressed by Michaelis-Menten kinetics

3. No partition of the substrate between the

exterior and interior of the support


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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


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Previous equation can be written in

dimensionless form


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Where

With boundary conditions at and

at


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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


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Effectiveness factor, Ratio of the reaction

rate with diffusion limitation to the reaction rate

with no diffusion limitation

Value ofis a measure of the extend ofdiffusion limitation

When


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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)

1. Enzyme loading should be less than

10mg/cm

2. Particle size


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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


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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 weightsubstrate is reduced by immobilization

Due to steric hinderance by the support


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Immobilization results in increase in thermal

stability

Thermal stability due to the presence of

thermal diffusion barriers and constraints onprotein folding

L l d ti f


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Large scale production of

enzymes

Eg:- proteases (subtilism, rennet), hydroleases

(pectinase, lipase, lactase), isomerases

(glucose isomerase) and oxidases (glucose

oxidase) Steps in production of enzymes

1. Overproducing strains of certain organisms

2. Separation and purification of an enzymefrom organism by cell disruption, removal of

cell debris and nucleic acid

3. Precipitation of protein


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Uses of enzymes


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immobilized ennzymes.pptx

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