INTRODUCTION

All enzymes are proteins and therefore exhibit sensitivity towards

temperature, pH and ionic concentration in the medium they function. In order to

study the in vitro enzyme-catalyzed reaction, the enzyme has to be isolated from
its source and the course of reaction to be followed over a period of time under
optimal conditions. Though the enzyme remains unconsumed after the reaction,
it cannot be recovered since it is water soluble and difficult to separate from
reaction mixture. This problem has been overcome by enzyme immobilization

Enzyme immobilization can be defined as “the imprisonment of a

biocatalyst in a distinct phase that allows exchange with, but separated from bulk
phase in which substrate, effector or inhibitor molecules are dispersed and
monitored”. Thus, an immobilized enzyme is physically entrapped or covalently
bonded by chemical means to an inert, insoluble material such as calcium
alginate, where it can act upon its natural substrate. This can provide increased
resistance to changes in conditions such as pH or temperature.

In 1916, Nelson and Griffin discovered that invertase “exhibited the same
activity when absorbed on a solid (charcoal or aluminium hydroxide) at the
bottom of the reaction vessel as when uniformly distributed throughout the
solution”. This discovery was the first of various enzyme immobilization
techniques currently available.

Besides adsorption, different covalent methods of enzyme immobilization

were developed in the 1950s and 1960s. Several hundred enzymes have been
immobilized in different forms and approximately a dozen immobilized enzymes,
for example penicillin-G acylase, lipases, proteases, invertase, etc. have been
used as catalysts in various large scale processes.
TECHNIQUES FOR ENZYME IMMOBILIZATION

There are various methods for enzyme immobilization. These methods are as
follows:-

1. Physical adsorption.

2. Entrapment in microcapsules.

3. Ionic bonding.

4. Covalent bonding

5. Cross –linking method

Combination of these techniques may also be used.

Immobilization of enzyme should be thought of if:-

 Proper enzyme activity is available.

 Cost wise it is cheap.

 Large-scale exploitation is possible.

For immobilization to be effective the important points are:-

• Selection of appropriate strain to organism/enzyme.

• Selection of appropriate immobilization procedure.

• No leakage of cells or enzymes should occur.

• There should be no diffusion restrictions for substrates.

• No compression or deterioration in reactor should occur.

• Selection of conditions should such that the maximize activity and stability
of enzyme occurs.
 1. PHYSICAL ADSORPTION

In this method immobilization of an enzyme is based on the physical

adsorption of enzyme protein on the surface of water-insoluble carriers. Hence,
the method causes little or no conformational change of the enzyme or
destruction of its active center. If a suitable carrier is found, this method can be
both simple and cheap. The binding is being mainly by electrostatic forces,
hydrophobic interaction, hydrogen bonds, multiple salt linkages, and Van der
Waal's forces between the atoms and ions of the carrier and the enzyme
molecules.

Figure: Physical adsorption

Commonly used adsorbents are:-

Bentinite, alumina, collodian, calcium carbonate, clay etc. Some ion-exchangers

like DEAE-cellulose, carboxymethyl cellulose and sephadex are also used.
Advantage

(1) No reagents are used and only a minimum of activation steps are required.

(2) Adsorption is cheap, easily carried out, and tends to be less disruptive to
the enzymic protein than chemical means of attachment.

(3) Stabilization of enzymes temporarily adsorbed onto a matrix can be achieved

by cross-linking the protein in a chemical reaction subsequent to its physical
adsorption.

2. ENZYME ENTRAPMENT IN POLYMER MATRIX

In this method, the enzyme molecules are entrapped in a cross polymer.

Entrapment is carried out by mixing enzyme molecule in monomer solution,
followed by chemical reaction initiated by chemical reaction or change in
temperature. Polymer can be formed in particulate form or block.

Figure: Entrapment in matrix

Commonly used matrix is as follows:-

Polyacrylamide, collagen, cellulose acetate, calcium alginate, carrageenan, etc.

Advantage

(1) This method is simple.

(2) Conditions of preparation are mild.

(3) There are minimum constraints on enzymes.

The modification of this technique is spinning fibres of cellulose triacetate or

calcium alginate within which enzymes are trapped. Advantage of this method is
high surface area to volume ratio and reduced diffusion limitations.

a) ENCAPSULATION

In this method the enzyme molecules are trapped in a semi-permeable

membrane which acts as barrier to prevent the free movement of enzyme
molecules. Thus small sacs of a definite size and porosity are formed. This
process is called as encapsulation.

Figure: MICROCAPSULES

The small membrane capsules containing the enzyme molecules are

called microcapsules, and the process of formation of microcapsules is called as
microencapsulation. This method has been practiced to immobilize the enzymes,
such as urease, asparaginase, carbonic anhydrase etc.

b) LIPOSOMAL ENTRAPMENT

Liposomes are lipid bodies consisting of two layers of lipids. Phospholipids are
generally used in the preparation of liposomes. The prepared enzymes are
inserted into the lipid layers or membranes. The enzyme and the lipid are mixed
together in a flask or in a tube and shaken well to induce the enzymes for being
inserted into the lipid bodies. As a result of proper mixing, the lipid completely
surrounds the enzymes and forms liposomes.
 3. IONIC BINDING

The ionic binding method relies on the ionic binding of the enzyme protein to
water-insoluble carriers containing ion-exchange residues.

The binding of an enzyme to the carrier is easily carried out, and the
conditions are much milder than those needed for the covalent binding method.
Hence, the ionic binding method causes little changes in the conformation and
the active site of the enzyme. Therefore, this method yields immobilized enzymes
with high activity in most cases.

Leakage of enzymes from the carrier may occur in substrate solutions of

high ionic strength or upon variation of pH. This is because the binding forces
between enzyme proteins and carriers are weaker than those in covalent binding.

4. COVALENT BONDING

The covalent binding method is based on the binding of enzymes and

water-insoluble carriers by covalent bonds. The functional groups that may take
part in this binding are listed below:

 Amino group

 Carboxyl group

 Sulfhydryl group

 Hydroxyl group

 Imidazole group

 Phenolic group

 Thiol group

 Threonine group
  Indole group

This method can be further classified into diazo, peptide and alkylation
methods according to the mode of linkage. The conditions for immobilization by
covalent binding are much more complicated and less mild than in the cases of
physical adsorption and ionic binding. Therefore, covalent binding may alter the
conformational structure and active center of the enzyme, resulting in major loss
of activity and/or changes of the substrate. However, the binding force between
enzyme and carrier is so strong that no leakage of the enzymes occurs, even in
the presence of substrate or solution of high ionic strength.

Covalent attachment to a support matrix must involve only functional groups

of the enzyme that are not essential for catalytic action. Higher activities result
from prevention of inactivation reactions with amino acid residues of the active
sites.

A number of protective methods have been devised:

 Covalent attachment of the enzyme in the presence of a competitive inhibitor or

substrate.

 A reversible, covalently linked enzyme-inhibitor complex.

 A chemically modified soluble enzyme whose covalent linkage to the matrix is

achieved by newly incorporated residues.

 A zymogen precursor.
Covalent binding can be brought about by the following:

 Diazotization ( SUPPORT--N=N—ENZYME)

 Amide bond formation (SUPPORT--CO-NH—ENZYME)

 Schiff's base formation (SUPPORT--CH=N—ENZYME)

 Amidation reaction (SUPPORT--CNH-NH—ENZYME)

 Thiol-Disulfide interchange (SUPPORT--S-S—ENZYME)

 Alkylation and arylation

(SUPPORT-CH2-NH-ENZYME, SUPPORT-CH2-S-ENZYME)

 Mercury-Enzyme interchange

 Gamma-Irradiation induced coupling

 Carrier binding with bifunctional reagent

(SUPPORT-O (CH2)2 N=CH (CH2)3-CH=N-ENZYME)

The active site of the enzyme must not be hindered. There must be ample
space between the enzyme and the backbone. It is possible in some cases to
increase the number of reactive residues of an enzyme in order to increase the
yield of the immobilized enzyme. This provides alternative reaction sites to those
essential for enzymatic activity. The wide variety of binding reactions and
insoluble carriers (with functional groups capable of covalent coupling or being
activated to give such groups) makes this a generally applicable method of
immobilization. This is true even if very little is known about the protein structure
or active site of the enzyme to be coupled.

5. CROSS LINKING

Immobilization of enzymes has been achieved by intermolecular cross-linking of

the protein, either to other protein molecules or to functional groups on an insoluble
support matrix. Cross-linking an enzyme to it is both expensive and insufficient, as
some of the protein material will inevitably be acting mainly as a support. This will result
in relatively low enzymatic activity. Generally, cross-linking is best used in conjunction
with one of the other methods. It is used mostly as a means of stabilizing adsorbed
enzymes and also for preventing leakage from Polyacrylamide gels.

Figure: Cross Linking

Since the enzyme is covalently linked to the support matrix, very little desorption
is likely using this method. Marshall (1973), for example, reported that carbamy
phosphokinase cross-linked to alkyl amine glass with glutaraldehyde lost only 16% of its
activity after continuous use in a column at room temperature for fourteen days.
The most common reagent used for cross-linking is glutaraldehyde. Cross-linking
reactions are carried out under relatively severe conditions. These harsh conditions can
change the conformation of active center of the enzyme; and so may lead to significant
loss of activity.

ADVANTAGES OF IMMOBILIZED ENZYME:-

1. The immobilized enzymes have more stability than the free enzymes. So
the turnover of substrate into products is higher.

2. The immobilized enzymes are firmly attached to the solid support. So the
recovery of the enzyme after the completion of reaction is very easy. The
wastage of enzymes during the extraction of the product is avoided.

3. The reaction potential of the immobilized enzymes is more as compared to

free enzymes. So they catalyze the production of maximum amount of
product within a unit time.
 4. As the enzymes of the desired species are immobilized onto the solid
support, so the chances of contamination are very low. Hence the
enzymes produce only desired products with nearly cent percent purity.

5. The solid materials freely allow the substrate to reach the immobilized
enzyme.

6. The cell free reaction system behaves as a model system for studying the
enzyme action of living cells.

7. Immobilization method increase the adsorptive area o the enzymes. So the

immobilized enzyme show high rate of binding with the substrate.

8. Immobilized enzymes do not need any modification during the catalytic

reaction.

9. Immobilized enzymes strictly obey the kinetic character of the free

enzymes as explained by Michaeli’s and Menton.

DISADVANTAGE OF IMMOBILIZED ENZYME:-

1. Some amount of enzyme may undergo denaturation during the

immobilization of enzymes.

2. Sometimes denatured enzymes may be immobilized in solid carrier during

the immobilization.

3. Enzyme immobilization is an exothermic process which denatures some

amount of enzymes during the immobilization.

4. Some carriers are very weak in the immobilization of enzymes. Such

carriers readily dissociate enzyme molecules in the solution or reaction
mixture. These dissociated enzyme molecules is extracted along with the
product, hence the quality of the enzymatic reaction becomes very low.
 5. The diffusion of the larger enzyme molecules into the carrier is a
somewhat difficult process. Hence the rate of immobilization of these
molecules is very low.

6. Sometimes the entrapped enzymes are released in the reaction mixture.

This leakage of enzymes causes the wastage of enzymes.

7. Sometimes the immobilization reduces the reaction kinetics of the

enzymes.

8. The polymers need proper chemical treatment before being used for
immobilizing enzyme.

APPLICATION OF IMMOBILIZED ENZYME:-

The important applications are as follows:-

1. Immobilized enzymes are used an analytical agents in enzyme electrodes.

The electrode is made up of a glass electrode surrounded by a thin film of
immobilized enzyme .The enzyme electrode is very sensitive ; it is used to
detect the presence of certain substances in the solution. It determines the
presence of these compounds even when they are present in mild dosage.
Now-a-days commercial immobilized enzymes are available for
determining the presence of a small amount of drugs, pesticides and
antitoxins in a solution. For example, alcohol dehydrogenase enzyme is
used in an enzyme electrode. This enzyme electrode is used to determine
the presence of morphine and other drugs containing barbiturate in the
blood stream, these drugs block (inhibit) the enzyme.

2. In food industry, the immobilized enzymes play the following important

roles:

 Immobilized enzymes are used to convert starch into glucose.

 Milk whey contains lactase; the immobilized enzymes are used to convert
the whey into simple sugars.

 Immobilized enzymes are used in the preparation of cottage cheese.

 Immobilized glucose isomerase enzyme is used in the manufacture of

fructose syrup.

3. In dairy industry, the immobilized enzymes are used to coagulate the milk
protein during cheese-making and are also used to treat the waste whey.

4. Immobilized aminoacylase enzyme is used in pharmaceutical industry.

This enzyme converts D, L-acyl amino acids into L-amino acids. The L-
amino acids are used as ingredients in food stuffs.

5. Immobilized enzyme is used in enzyme therapy. The enzyme

asparaginase is used in the treatment of leukaemias. This disease
develops in human beings owing to the deficiency of asparaginase
enzyme. So this disease can be cured by using enzyme therapy. Insulin is
treated with a small dose of zinc ions before it is marketed; the zinc ions
increase the survivability of the enzyme and also increase the reaction
potential of the insulin.

6. They are used in the study of biochemical reactions of organisms under

different physiological conditions. Here the immobilized enzyme reaction is
used as a model system to determine the characteristic cellular
metabolism in living cells.
CONCLUSION

The immobilized enzyme technology may help in the future for integrating
bioprocess with downstream processing with an effort to increase productivity
while minimizing the product recovery cost. Immobilized enzyme technology may
also be useful in non-aqueous enzymology, not only in terms of stabilization of
the biocatalysts but also in the development of continuous bioreactors. One of
the rapidly emerging areas in the field of sensors is the development of
biosensors for industrial process control wherein immobilized enzyme technology
play a pivotal role. Stabilization of enzymes (heat) is crucial for some of the
applications. On the other hand, cold active enzymes also may gain potentials in
certain biocoversions. It is most likely that the future developments will come
from within the industry and the role of academic researchers will probably
remain to some extent as the laying of the theoretical foundations for this work
and development of new approaches. Thus, there are interesting possibilities
within the field of immobilized enzymes and it is imminent that in the future many
applications will be replaced by immobilized systems and many more new
systems will become technically as well as commercially feasible
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1. Biotechnology: Principles and Application, S.C. Rastogi, Narosa

Publishing House.

2. Zoology, volume-II, S.S.Bhattacharya, A.S.Ansari, Sheth Publications.

3. http://www.wikipedia.org

4. K.Opwis.D.Knittel, T.Bahners, and E.Schollmeyer, 2005. Photochemical

Enzyme Immobilization on Textile Carrier Materials:

5. Enzymes – Biochemistry, Biotechnology and Clinical chemistry by Trever

Palmer

6. Fundamentals of Enzymology: Price and Stevens

7. Lehninger- Principles of biochemistry by David. L. Nelson and Michael .M.

Cox