4. 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.
5. 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”
6.
7. 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.
8. 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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9. 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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10. This method is based on covalent cross linking of
enzymes with appropriate bifunctional agents.
11. 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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12. 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
14. 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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15. Pros :
Leak-proof binding between enzyme
and matrix
The enzyme is strongly bound to the
matrix and, in many cases, it is also
stabilized
Cons :
Low yield of immobilized activity
Nonreversible character
High cost
COVALENT BINDING CONTINUED…
17. This technique was 1st
used in 1916 by Nelson &
Griffin to produce immobilized invertase using
charcoal as adsorbent.
Pros :
Mild
Easy to perform process
Usually preserves the catalytic activity of the
enzyme
Economically attractive
Cons :
Enzyme leakage from matrix when the interactions
are relatively weak.
19. 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.
20. 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.
Me
Me
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23. 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.
24.
25. 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.
