IMMOBILIZED ENZYME SYSTEMS

NEW ERA OF HETEROGENOUS CATALYSIS

PRODUCTION &

APPLICATIONSOF

PRESENTED BYRASHMI

REGN. NO. 10T48BCOLLEGE OF FOOD TECHNOLOGY

CONTENTS...

Enzyme immobilization can be broadly described as the process by which a homogeneous catalyst can be

converted into a heterogeneous catalyst.

According to Trevan (1980) , “It is a process by which an enzyme is seperated into a distinct

phase (usually water soluble and often of high molecular weight) from a bulk substrate

containing phase.

The term “immobilized enzymes” refers to “enzymes physically confined or localised in a certain defined region or

space with retention of their catalytic activities and which can be used continuously and repeatedly”

The entrapment method is based on the occlusion of an enzyme within a polymeric network that allows the substrate and products to pass through but retains the enzyme.

Gel & fibre entrapment :

Active preparations of enzymes are entrapped in gel like structure eg. Starch gel & polyacrylamide gel.

Enzyme activity recovered is from 1.9-6.6%.

Limitations : Enzymes tend to leak Cannot be used for trapping hydrolytic enzymes that degrade the gel Hydrolytic enzymes are not suitable for entrapping because the substrates they attack are large in molecular weight and cant penetrate the gel matrix.

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Microencapsulation :

Semi-permeable microencapsules made of synthetic polymers (collodion , silicone resin , nylon etc.) are used for encapsulation of enzymes.

Encapsulation of enzymes into semi permeable membranes may be very useful in medical field.

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This method is based on covalent cross linking of enzymes with appropriate bifunctional agents.

Covalent cross linking gives rise to thin enzymes membranes or a 3-D network of enzyme molecules.

Bifunctional agents used are glutaraldehyde and bis-diazobenzidine.

Enzyme activity recovered ranges from 17% in case of trypsin to 80% in esterase & 99% in bovine trypsin.

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Pros : Leak proof binding between enzyme & matrix. Substrate molecules have easier access to enzyme molecules as the immobilized enzyme is in the form of an envelope around the particle & thus the particle is in direct contact with the substrate solution.

Cons : High cost Irreversible nature

Immobilized enzymes are covalently bound to stationary carriers.

Coupling methods in general can be divided in two main classes:

(1) activation of the matrix by addition of a reactive function to a polymer and (2) modification of the polymer backbone to produce an activated group

Retention of enzyme activity is around 25%.

The variety of carriers used is extensive. It uses materials such as glass , silica , sephadex , cellulose , nylon & polystyrene.

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Pros :Leak-proof binding between enzyme and matrixThe enzyme is strongly bound to the matrix and, in many cases, it is also stabilized Cons :Low yield of immobilized activityNonreversible characterHigh cost 

COVALENT BINDING CONTINUED…

Adsorption is the adhesion of atoms, ions, or molecules from a gas, liquid, or dissolved solid to

a surface. This process creates a film of the adsorbate on the surface of the adsorbent. 

This technique was 1st used in 1916 by Nelson & Griffin to produce immobilized invertase using charcoal as adsorbent.

Pros :MildEasy to perform processUsually preserves the catalytic activity of the enzymeEconomically attractive

Cons :Enzyme leakage from matrix when the interactions are relatively weak.

Adsorption can be classified further into 3 categories :

Transition metal salts or hydroxides deposited on the surface of organic carriers become bound by coordination with nucleophilic groups on the matrix. The metal salt or hydroxide is precipitated onto the support (e.g., cellulose, chitin, alginic acid, and silica-based carriers) by heating or neutralization. Because of steric factors, it is impossible for the matrix to occupy all coordination positions of the metal; therefore some of the positions remain free to coordinate with groups from the enzymes.

Enzymatic activity recovered ranges from 30-80%.This method is also known as metal link immobilization and employs the use of mainly titanium & zirconium salts.

Pros :Relatively high enzymatic activity recovery.Simple & easy procedure.

Cons :Operational stabilities are variable.Results are not easily reproducible.

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APPLICATIONS CONTINUED…

1. Medical fieldThe development of immobilized

enzymes & enzymes enased in semi permeable microspheres offers promise in the treatment of specific diseases in which the inherited abnormality of a single enzyme has profound metabolic effects, usually produced by the accumulation of a substrate for the involved enzyme.

It is now feasible to think in terms of removal of these accumulated materials by means of specific enzymes of exogenous origin.

eg. Galactose phosphate in galactosemia.

APPLICATIONS CONTINUED…

The analytical use of immobilized enzymes is in the form of enzyme electrodes.

It has been used for the following probes :

1.Glucose oxidase – oxygen probe system to measure glucose concentration upto 10-3 mol/l.2.Urease – ammonium ion probe to detect urea concentration.3.Trace toxic metals & pesticide probes.

If specificity of an enzyme can be coupled with some fluorometric systems , then one can achieve sensitive analyzes , possibly as low as 10-12 molar concentration.

4. MISCELLANEOUS

APPLICATIONS CONTINUED…