This slide was prepared using the book 'Introduction to Biotechnology' by Giriraj Kulkarni.

1. Amrita Sutradhar
B. Pharm, M. Pharm (JU)
Lecturer
Department of Pharmacy
Primeasia University

2. What is enzyme?
Enzymes are biological catalysts which
enhance the rate of biochemical reactions
from 106 - 1012 times when compared to
those of uncatalyzed reactions.
All enzymes are proteins and contain an
active site where substrates are converted to
products. They possess both speed of action
and precision.

3. Mechanism of enzyme action
In biochemical reaction, the molecules possessing
high internal energy can go to a reactive form
instantly. This reactive form is called transition
state. To reach transition state, all the molecules
must possess activation energy.
The enzyme can accelerate the reaction by
combining with substrate. The substrate-enzyme
complex is then converted to products and enzyme
is liberated. The transition state of this enzyme
substrate complex has much lower activation
energy than transition state of substrate alone.

4. Mechanism of enzyme action
Due to this reduction in activation energy, more
substrate molecules participate in the reaction and
hence reaction rate will be increased.
The enzyme recognizes substrate which fits into its
active site perfectly. Once enzyme-substrate
complex is formed, making and breaking of
chemical bond occurs which converts the
substrates into products and the enzyme is set free.
Thus the enzyme and substrate complex can be
compared with a lock and key model.

5. Sources of enzymes
Enzymes are usually obtained from three major
sources-
Plants
Animals
Micro-organisms
The advent of fermentation technology led to the
preparation of purer form of enzymes from
microbes. Today, most of the industrially used
enzymes are from microbial source.

6. Advantaged of microbes over other sources of
enzymes:
1. Microbes have a short generation time and
hence the times required for enzyme production
in large quantities is less compared to plants
and animals.
2. Enzymes can be easily extracted from microbes.
If the enzymes are extracellular, they will be
secreted into the medium and can be purified
easily.
3. Micro-organisms produce enzymes throughout
the year and no seasonal variation is found
which is common in case of plants and animals.

7. Advantaged of microbes over other sources of
enzymes:
4. The enzymes obtained from microbes are
stable under extreme environmental conditions.
5. Micro-organisms can produce a large varieties
of enzymes which is not possible with plants and
animals. They can be manipulated easily by
mutation or by genetic engineering to give high
yield of enzymes.

8. Enzyme immobilization
Immobilization is defined as the imprisonment of
an enzyme in a distinct phase that allows exchange
with, but is separated from the bulk phase in which
the substrate and product molecules are dispersed.
Imprisonment refers to the physical or chemical
trapping of the enzyme either onto or into a
polymer matrix.

9. Why is enzyme immobilization necessary?
Most of the enzymes are expensive and hence they should
be used as efficiently as possible. An important function of
enzyme is their capacity to perform same function again
and again.
But if the enzymes are used in pure or crude form, it is not
possible to separate them from the products after
completion of catalysis since both of them are in dissolved
state. If they are separated, the process becomes very costly
and difficult.
Some enzymes are too costly and the process of enzymatic
catalysis becomes cost effective only if they are reused.
Hence, immobilization techniques have been developed.
The immobilization involves the conversion of water
soluble enzymes into a solid form of catalyst which is
insoluble.

10. Advantages of immobilized enzymes:
1. Reuse: The same enzyme can be used
repeatedly.
2. Continuous use: Continuous production system
can be designed easily.
3. Cost effectiveness: If the enzymes are
immobilized, they can be used continuously or
repeatedly and hence cost effectiveness is
achieved.
4. Less contamination: Since the immobilized
enzymes remain within the polymer matrix, it
will not contaminate the final product.

11. Advantages of immobilized enzymes:
5. Stability: Immobilization increases the thermal
stability of the enzymes. For example,
immobilized glucose isomerase is stable at 650C
for almost one year whereas the pure enzyme is
denatured within few hours at a temperature of
450C.
6. Process control: Since the immobilized enzymes
have a standardized activity, the process control
becomes very easy.
7. Enzyme Substrate ratio: It is very high in
immobilized enzymes and this increases cost
effectiveness.

12. Immobilization ‘in’ a support
This technique involves-
Entrapment
Microencapsulation

13. Entrapment
In this technique, the enzyme is entrapped within a
cross linked polymer matrix. Enzyme is dissolved in
a solution of precursors of the polymer and then
polymerization is initiated.
The enzyme is physically entrapped within the matrix
and it can’t escape by permeation. However,
substrate molecules can diffuse in, can be acted upon
by the enzyme, and product molecules can diffuse out
of the matrix.
The common polymers used include- Polyacrylamide
gel, Cellulose triacetate, Gelatin etc.

14. Drawbacks of entrapment
Immobilization of enzymes by entrapment
technique has the following problems-
Due to wide pore size distribution in the gel,
there will be continuous leakage of enzyme.
Since enzyme is bound within the matrix,
substrate accessibility is reduced.
Some of the enzyme activity is lost or reduced
due to free radicals produced during
polymerization.

15. Microencapsulation
In this method, the enzyme is immobilized by
enclosing a droplet of enzyme in a
semipermeable membrane capsule. Once
encapsulation is done, the enzyme can not escape
whereas the low molecular weight substrate and
products can diffuse through the membrane. The
capsule can be made up of either permanent
materials like nylons or biodegradable materials
like poly-lactic acid.

16. There are two general methods of encapsulating
an enzyme –
1.The membrane are prepared by phase
separation, just like emulsification. Both
phases are immiscible and form coacervate
when mixed. The enzyme is entrapped in this
coacervate.
2.Water insoluble membrane can be prepared by
chemical polymerization and the enzyme can
be entrapped during polymerization.

17. Immobilization ‘On’ a support
Three techniques-
1.Adsorption
2.Covalent binding
3.Cross-linking

18. Adsorption
The adsorption occurs due to nonspecific
bonding like electrostatic, hydrophobic or
affinity bonding to a specific ligand. Some
adsorbents used in this technique are
Carboxy methyl Cellulose (CMC), Silica gel
etc. During immobilization the enzyme
concentration and PH should be controlled
carefully because a change in PH may lead to
desorption.

19. Adsorption
In the process of immobilization by
adsorption, the adsorbent is packed in a
water jacketed column and washed with a
preconditioning solution. The enzyme
solution is prepared and buffered fairly.
Such solution is then circulated through the
column for several hours. Then the solution
is drained and column is washed with water,
then with 0.5 M NaCl and again with water.
The immobilized enzyme column is now
ready for use.

20. Covalent binding
In this process the enzyme forms a covalent bond
with the active groups on a polymer support. This
can be done by two ways-
1. Through the reactive groups on the side chains
of its amino acids such as lysine, arginine and
tyrosine.
2. Through the terminal amino and carboxyl
groups of polypeptide chains.

21. Cross linking
Three techniques are used for immobilization of
enzymes by cross linking-
1. Cross linking of the enzyme with
gluteraldehyde to form an insoluble aggregate.
2. Adsorption of the enzyme onto a surface
followed by cross linking. For example, the
enzyme can be cross linked and adsorbed onto
the surface of particles like colloidal silica.
3. Impregnation of porous matrix materials with
the enzyme and cross linking of the enzyme
within the pores. The common porous matrix
used is collodion membrane.

23. The enzyme applications are broadly classified
into four categories-
1.Medical use
2.Analytical use
3.Manipulative use
4.Industrial use

24. Medical use:
Treatment of leukemia can be done by administering
asparaginase from bacteria. Leukemic cell require
exogenous asparagine for their growth and this will be
destroyed by the enzyme.
Lysozyme, a protective body fluid e.g. nasal mucosal
fluid, lacrimal fluid etc. is used as antibacterial agent.
Hyaluronidase, an enzyme is used in dental work to
facilitate drug penetration.
Penicillinase is used to stop Penicillin
hypersensitivity.
Glucose oxidase is used to diagnose the presence of
glucose in urine.

25. Analytical use:
Enzymes can be used to analyze the blood
concentration of D-glucose, D-alanine, D-
galactose, Urea etc.

26. Manipulative use
Restriction enzymes are used in recombinant
DNA technology to cut DNA.
DNA ligase is used to join foreign genes to
plasmid DNA.
Alkaline phosphatase is used to prevent the
recirculation of cut plasmid.
Reverse transcriptase is used to produce DNA
from RNA.
DNA Polymerase is used in the formation of
DNA

27. Industrial use
Invertase is used to convert glucose to fructose.
Penicillin acylase is used to produce
semisynthetic Penicillin.
Proteolytic enzymes are used to soften skin in
leather industry.