Enzyme immoblization

1. A SEMINAR ON
ENZYME IMMOBLIZATION
PRESENTED BY
ADITYA
M.Sc. II SEM
BIOTECHNOLOGY

2. INTRODUCTION
Enzyme are large biological molecules responsible for the thousands
of metabolic processes that sustain life.
Traditionally Enzyme in free solutions react with subtracts to result
in products. such use of Enzyme is west ful particularly for Industrial
purposes, since enzyme are not stable and they can not be
recovered for reuse.
DEFINATION :-
“Immobilization of enzyme (or cells) refer to the technique of
confining anchoring the enzyme in on an inert support for their
stability and function reuse”

3. PROPERTIES
 The material used for enzyme immobilization called carrier
matrices .
 Are usually inert polymer or inorganic material.
 The ideal carrier matrix has the following properties are:-
 Low costs
 Inert less
 Physical strength
 Regenerability after use
 Reduction product inhibition
 Enhancement of enzyme specificity

4.  ADSORPTION
 ENTRAPMENT
 MICROCAPSULATION
 COVALENT BINDING
 CROSS LINKING
METHODS

5. Adsorption involves the physical binding of enzyme (or cells) on the
surface of an inert support.
The support material may be inorganic (e.g. alumina ,silica gel ,calcium
phosphate gel glass) or organic ( starch, carboxymethyl, cellulose ,
DEAE cellulose).
Adsorption of enzyme molecules involves weak forces such as Vander
Waals forces and hydrogen bonds.
Therefore ,the adsorbed enzyme can be easily removed by minor
changes in pH, ionic strength or Temperature.
ADSORPTION

6. FIG:1 ADSORPTION
Enzymes are held on to a solid
support (matrix) by weak
forces
such as hydrogen bonding

7. Enzyme can be immobilized physical Entrapment inside
a polymer or gel.
In this technique, commonly referred to as lattice
entrapment ,the enzyme (or cells ) is not subjected to
strong binding forces and structural distortions.
Some deactivation may however ,occur during
immobilization process due to changes in pH or tem.
The materices used for entraping of emzyme include
polyacrylmide gel collgen,gelatin,starch cellulose
silicone and rubber.
ENTRAPMENT

8. enzyme can be entrapped by several ways :-
1.Enzyme inclusion in gels.
2.Enzyme inclusion in fibers.
3.Enzyme inclusion in microcapsules.
FIG:2 a) Entrapment ,b) micro capsulation

9. MICROCAPULATION
 Microcapulation is a type of entrapment.itrefer to the
process of spherial particle for mation where in a liquid or
suspension in enclosed in a semipermeable membrene.
 The mem. May be polymeric lipodial,lipoprotein besed or
distinct way of microencapsulation.
 1.Building of special mem reactors
 2.Stabilization of emulsion to from microcapsules.
 3.Formation of emulsions.

10. COVALENT
BINDING
 Immobilization of the enzyme can be achived by
crectionof covalent bonds between the chemical groups
of enzyme and chemical groups of the support.
 The following are the common methods of covalent
binding:-
 1.cyanogen bromide activation
 2.Diazotation
 3.Peptide bonds formation
 4.Activation by Bi or polyfunctional regents.

11. FIG : COVALENT BINDING
Enzymes are covalently
bonded
to a matrix such as
cellulose
or collagen

12. CROSS –LINKING
 The absence of a solid support is a characteristics feature of
immobilization of enzymes by cross-linking.
 The enzyme molecules are immobilized by creating cross-links
between them,through the involvement of poly-functional
reagent.These reagent in fact react with the enzyme molecules
and create brides which form the backbone to hold enzyme
molecules.
 Glutaraldehyde is the most extensively used cross-linking reagent
.


13. FIG:6 CROSS LINKING
Another more expensive
method involves
enzymes which are both
covalently bonded
to, and cross-linked within,
a matrix
Cross-linking and covalent
bonding may
cause some enzymes to
lose their catalytic
activity especially if the
active site is involved
in forming the linkages

14. EFFECT OF IMMOBILIZATION ON
ENZYME PROPERTIES
 Enzyme immobilization is frequently associated with
alterations in enzyme properties ,particularly the kinetic
properties of enzyme some are listed below:-
 There is substantial decrease in the enzyme specificity .
This may be due to conformational changes that occur
when the enzyme gets immobilized.
 The kinetic constants km and V max of an immobilization
enzyme differ from that of the native enzyme .This is
because the conformational changes of the enzyme will
affect the affinity between enzyme and substrate.

15. IMMOBILIZED ENZYME REACTORS
 The immobilized enzyme or cells are utilized in the
Industrial process in the form of enzyme reactors :-
 They are broadly of three types :-
 BATCH REACTORS
 CONTINUOUS REACTORS
 MEMBRANE REACTORS

16. BATCH REACTORS & CONTINUOUS REACTORS
FIG : 9 a) fluidized bed, b) continuous stirred tank reactors

17. MEMBRENE REACTOR
FIG:8 RECYCLE REACTOR

18. APPLICAION OF IMMOBILIZED
ENZYMES AND CELLS
 INDUSTRIAL USES
 ANALYTICAL
 THERAPEUTIC

19. TABLE No.1 A Selected list of important immobilized
enzymes and their industrial applications

20. ADVANTGES
 Stable and more efficient in function.
 Can be reused again and again.
 Minimize effluent disposal problems.
 Ideal for multiple enzyme function system.
 Suitable for industrial and medical uses.
 Product are enzyme free.

21. DISADVANTAGES
 There are however certain disadvatas also associaed with
immobilization :-
 Immobilization mean additional cost.
 This approch can not be used when one of the substrats is
insoluble
 When live cells are immobilized ,they may proliferate .The
fresh cells formed on the surface would cause diffusion
problems and they may cotaminationate products.