Arun IEM

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INDUSTRIAL AND ENVIRONMENTAL MICROBIOLOGY

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What is enzyme immobilization?

Immobilization is defined as the imprisonment of cell or enzyme in a distinct support or matrix.The
support or matrix on which the enzymes are immobilized allows the exchange of mediumcontaining
substrate or effector or inhibitor molecules. The practice of immobilization of cells isvery old and the
first immobilized enzyme was amino acylase of Aspergillusoryzaefor theproduction of L-amino acids in
Japan.
Advantages of immobilized enzymes:
(1). Increased functional efficiency of enzyme
(2). Enhanced reproducibility of the process they are undertaking
(3). Reuse of enzyme
(4). Continuous use of enzyme
(5). Less labour input in the processes
(6). Saving in capital cost and investment of the process
(7). Minimum reaction time
(8). Less chance of contamination in products
(9). More stability of products
(10). Stable supply of products in the market
(11). Improved process control
(12). High enzyme substrate ratio
Disadvantages of enzyme immobilization:
(1). Even though there are many advantages ofimmobilized enzymes, there are some disadvantages
also.
(2). High cost for the isolation, purification andrecovery of active enzyme (most importantdisadvantage)
(3). Industrial applications are limited and onlyvery few industries are using immobilized enzymesor
immobilized whole cells.
(4). Catalytic properties of some
enzymes arereduced or completely lost
after theirimmobilization on support or
carrier.
(5). Some enzymes become unstable

afterimmobilization.
(6). Enzymes are inactivated by the

heatgenerated in the system
Applications of enzyme immobilization:
(1). Industrial production: Industrial

production of antibiotics, beverages,
aminoacids etc. uses immobilized enzymes or whole cells.
(2). Biomedical applications: Immobilized enzymes are widely used in thediagnosis and treatment of
many diseases. Immobilized enzymes can be used toovercome inborn metabolic disorders by the supply
of immobilized enzymes.Immobilization techniques are effectively used in drug delivery systems
especially tooncogenic sites.
(3). Food industry: Enzymes like pectinases and cellulases immobilized on suitablecarriers are
successfully used in the production of jams, jellies and syrups from fruitsand vegetables.
(4). Research: A Research activity extensively uses many enzymes. The use ofimmobilized enzyme allow
researcher to increase the efficiency of different enzymessuch as Horse Radish Peroxidase (HRP) in
blotting experiments and differentProteases for cell or organelle lysis.
(5). Production of bio-diesel from vegetable oils.
(6). Waste water management: treatment of sewage and industrial effluents.
(7). Textile industry: scouring, bio-polishing and desizing of fabrics.
(8). Detergent industry: immobilization of lipase enzyme for effective dirt removalfrom cloths.
Methods of Immobilization:
Based on support or matrix and the type of bonds involved, there are five different methods of
immobilization of enzyme or whole cells.
(1). Adsorption
(2). Covalent bonding
(3). Entrapment
(4). Copolymerization
(5). Encapsulation
(1). Adsorption
Adsorption is the oldest and simplest method of

enzymeimmobilization. Nelson & Griffin used charcoal to
adsorbinvertase for the first time in 1916. In this method enzyme
isadsorbed to external surface of the support. The support
orcarrier used may be of different types such as:
(1). Mineral support (Eg. aluminum oxide, clay)
(2). Organic support (Eg. starch)
(3). Modified sepharose and ion exchangeresins
There is no permanent bond formation between carrier and

theenzyme in adsorption method. Only weak bonds stabilize
theenzymes to the support or carrier. The weak bonds (low
energybonds) involved are mainly:
(a). Ionic interaction
(b). Hydrogen bonds
(c). Van der Waal forces
For significant surface bonding the carrier particle size must be small (500 Å to 1 mmdiameter). The
greatest advantage of adsorption method is that there will not be “porediffusion limitations” since
enzymes are immobilized externally on the support or the carrier.
Methods of adsorption:
(1). Static process: Immobilization to carrier by allowing the solution containing enzyme tocontact the
carrier without stirring.
(2). Dynamic batch process: Carrier is placed in the enzyme solution and mixed by stirringor agitation.
(3). Reactor loading process: Carrier is placed in the reactor, and then the enzymesolution is transferred
to the reactor with continuous agitation.
(4). Electrode position process: Carrier is placed near to an electrode in an enzyme bathand then the
current is put on, under the electric field the enzyme migrates to the carrier anddeposited on its surface.
(2). Covalent bonding:
This method involves the formation of covalent bonds between

the chemical groups in enzymeand to the chemical groups on
the support or carrier. It is one ofthe widely used methods of
enzyme immobilization. Hydroxylgroups and amino groups of
support or enzyme form covalentbonds more easily. Chemical
groups in the support or carrierthat can form covalent bonds
with support are amino groups,imino groups, hydroxyl groups,
carboxyl groups, thiol groups,methylthiol groups, guanidyl
groups, imidazole groups andphenol ring.
Important functional groups of the enzyme that providechemical

groups to form covalent bonds with support or carrierare:
1. Alpha carboxyl group at ‘C’ terminal of enzyme
2. Alpha amino group at ‘N’ terminal of enzyme
3. Epsilon amino groups of Lysine and Arginine in the enzyme
4. β and γ carboxyl groups of Aspartate and Glutamate
5. Phenol ring of Tyrosine
6. Thiol group of Cysteine
7. Hydroxyl groups of Serine and Threonine
8. Imidazole group of Histidine
9. Indole ring of Tryptophan

(3). Entrapment:
In this method enzymes are

physically entrapped inside a
porous matrix. Bonds involved in
stabilizing the enzyme to the

matrix may be covalent or non-
covalent. The matrix used will be
a water soluble polymer. The

form and nature of matrix varies
with different enzymes. Pore
size of matrix is adjusted to prevent the loss of enzyme. Pore size of the matrix can be adjusted
with the concentration of the polymer used. Agar-agar and carrageenan have comparatively
large pore sizes. The greatest disadvantage of this method is that there is a possibility of
leakage of low molecular weight enzymes from the matrix.
Examples of commonly used matrixes for entrapment are:
(1). Polyacrylamide gels
(2). Cellulose triacetate
(3). Agar
(4). Gelatin
(5). Carrageenan
(6). Alginate
Methods of entrapment:
(a). Inclusion in the gels: enzymes trapped inside the gels.
(b). Inclusion in fibers: enzymes supported on fibers made of matrix material.
(c). Inclusion in microcapsules: Enzymes entrapped in microcapsules formed by

monomer mixtures such as
polyamine and calcium
alginate.
4). Cross linking

(copolymerization):
This method is also called

ascopolymerization. In
thismethod of
immobilizationenzymes are
directly linked bycovalent
bonds between
variousgroups of enzymes
viapolyfunctional reagents.
Unlikeother methods, there
is nomatrix or support
involved inthis method. Commonly usedpolyfunctional reagents areglutaraldehyde and diazoniumsalt.
This technique is cheap andsimple but not often used withpure enzymes. This method iswidely used in
commercialpreparations and industrialapplications. The greatestdisadvantage or demerit of this method
is that the polyfunctional reagents used for crosscatalytic properties.
(5). Encapsulation:
This type of immobilization is done by enclosing the enzymes in a membrane capsule. Thecapsule will be
made up of semi permeable
membrane like nitro
cellulose or nylon. In
thismethod the effectiveness
depends upon the stability
of enzymes inside the
capsule.

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ASSIGNMENT OF

INDUSTRIAL AND ENVIRONMENTAL MICROBIOLOGY

What is enzyme immobilization?

Advantages of immobilized enzymes:

(1). Increased functional efficiency of enzyme

(2). Enhanced reproducibility of the process they are undertaking

(3). Reuse of enzyme

(4). Continuous use of enzyme

(5). Less labour input in the processes

(6). Saving in capital cost and investment of the process

(7). Minimum reaction time

(8). Less chance of contamination in products

(9). More stability of products

(10). Stable supply of products in the market

(11). Improved process control

(12). High enzyme substrate ratio

Disadvantages of enzyme immobilization:

(5). Some enzymes become unstable

(6). Enzymes are inactivated by the

Applications of enzyme immobilization:

(1). Industrial production: Industrial

(5). Production of bio-diesel from vegetable oils.

(6). Waste water management: treatment of sewage and industrial effluents.

(7). Textile industry: scouring, bio-polishing and desizing of fabrics.

immobilization of enzyme or whole cells.

(2). Covalent bonding

Adsorption is the oldest and simplest method of

(1). Mineral support (Eg. aluminum oxide, clay)

(2). Organic support (Eg. starch)

(3). Modified sepharose and ion exchangeresins

There is no permanent bond formation between carrier and

(a). Ionic interaction

(b). Hydrogen bonds

(c). Van der Waal forces

This method involves the formation of covalent bonds between

Important functional groups of the enzyme that providechemical

1. Alpha carboxyl group at ‘C’ terminal of enzyme

2. Alpha amino group at ‘N’ terminal of enzyme

3. Epsilon amino groups of Lysine and Arginine in the enzyme

4. β and γ carboxyl groups of Aspartate and Glutamate

5. Phenol ring of Tyrosine

6. Thiol group of Cysteine

7. Hydroxyl groups of Serine and Threonine

8. Imidazole group of Histidine

9. Indole ring of Tryptophan

In this method enzymes are

stabilizing the enzyme to the

a water soluble polymer. The

leakage of low molecular weight enzymes from the matrix.

Examples of commonly used matrixes for entrapment are:

(1). Polyacrylamide gels

(2). Cellulose triacetate

(a). Inclusion in the gels: enzymes trapped inside the gels.

(b). Inclusion in fibers: enzymes supported on fibers made of matrix material.

(c). Inclusion in microcapsules: Enzymes entrapped in microcapsules formed by

4). Cross linking

This method is also called

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