Immobilization of enzymes

1. Immobilization of enzymes:
By
Dr. Harinatha Reddy A
ASSISTANT PROFESSOR
Department of Biotechnology
biohari14@gmail.com

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3. Introduction:
What is immobilization :
 An immobilized enzyme is an enzyme attached to an inert
insoluble material such as calcium alginate.
 Immobilized enzyme is a golden egg in Enzyme technology

4. There are several advantages of immobilized enzymes:
 a. Stable and more efficient in function.
 b. Can be reused again and again.
 c. Products are enzyme-free.
 d. Minimum reaction time (Rate of reaction is very fast)
 e. Control of enzyme function is easy.
 f. Suitable for industrial and medical use.
 g. Resistant changes in pH and temperature.
 Product is pure form the Contamination.
 Product purification is very easy.

5. Disadvantages:
 The possibility of loss of biological activity of an enzyme during
immobilization or while it is in use.
 Immobilization is an expensive. (isolation and purification )

6. Immobilized Enzymes Immobilized Whole cells
An enzyme catalyses only a
specific reaction, and there is no
risk of by-product formation
By-Products are formed
Often purification of the product
is much easier.
Often purification of the
product is difficult because
bi-products produced by the
whole cells.
Immobilized enzymes are generally preferred over immobilized
cells due to specificity to yield the products in pure form.

7. Immobilized enzymes are generally preferred over immobilized
cells due to specificity to yield the products in pure form.

8. Enzymes Whole cells
However, purified enzymes have
limitations of
(i) high cost and
(ii) less stability when compare to whole
cells.
(i) Low cost
(ii) High stability.

9. Methods of Immobilization:
 The commonly employed techniques for immobilization of
enzymes are:
 Adsorption,
 Entrapment,
 Covalent binding
 Cross-linking.

10. Adsorption:
 Adsorption involves the
physical binding of enzymes
(or cells) on the surface of an
inert support.
 The support materials may be
inorganic (e.g. alumina, silica
gel, calcium phosphate ) or
 Organic (starch,
carboxymethyl cellulose,
DEAE-cellulose, DEAE-
sephadex).

11. Adsorption:
 The support materials may be inorganic (e.g. alumina, silica gel,
calcium phosphate gel, glass) or organic (starch, carboxymethyl
cellulose, DEAE-cellulose, DEAE-sephadex).
 Adsorption of enzyme molecules (on the inert support) involves
weak forces such as ionic bonds, Van der Waals forces and
hydrogen bonds.
 Therefore, the adsorbed enzymes can be easily removed by
minor changes in pH, ionic strength or temperature.
 This is a disadvantage for industrial use of enzymes

12. Entrapment:
Collagen, gelatin, starch, cellulose, and silicone.
Enzymes are entrapping inside a polymer or a gel matrix.

13. Enzymes can be entrapped by several ways.
 Enzyme inclusion in fibres:::
 Enzyme inclusion in gels:
 Enzyme inclusion in
microcapsules:

14. Entrapment:
 Enzymes can be immobilized by physical entrapment inside a
polymer or a gel matrix.
 The matrices used for entrapping of enzymes include
polyacrylamide gel, collagen, gelatin, starch, cellulose, silicone and
rubber.

15.  Enzyme inclusion in gels:
 This is an entrapment of enzymes inside the gels.
 Enzyme inclusion in fibres:
 The enzymes are trapped in a fibre format of the matrix.
Enzymes can be entrapped by several ways.

16. Enzyme inclusion in microcapsules:
 In this case, the enzymes are trapped inside a microcapsule matrix.
 The major limitation for entrapment of enzymes is their leakage
from the matrix.
 Most workers prefer to use the technique of entrapment for
immobilization of whole cells.
 Entrapped cells are in use for industrial production of amino acids
(L-isoleucine, L-aspartic acid), L-malic acid and hydroquinone.

17. Microencapsulation:
Microencapsulation is a type of entrapment.

18. Microencapsulation:
 Microencapsulation is a type of entrapment.
 It refers to the process of spherical particle formation wherein a
liquid or suspension is enclosed in a semipermeable membrane.
 The membrane may be polymeric, lipoidal, phospholipids,
lipoprotein-based or non-ionic in nature.
 Microencapsulation is recently being used for immobilization of
enzymes and mammalian cells.

19. Microencapsulation:
 For instance, pancreatic cells grown in cultures can be immobilized
by microencapsulation.
 Hybridoma cells have also been immobilized successfully by this
technique.

20. Covalent Binding :
Amino group
Carboxylic group
Indole group
Imidazole group
Activation of functional groups in support
material require.
Pre treatment with CNBr,
glutaraldehyde and HCI.

21. Covalent Binding:
 Immobilization of the enzymes can be achieved by creation of
covalent bonds between the chemical groups of enzymes and the
chemical groups of the support.
 This technique is widely used.
 However, covalent binding is often associated with loss of some
enzyme activity.
 The inert support usually requires pretreatment (to form pre-
activated support) before it binds to enzyme.

22. Cyanogen bromide activation:
 The inert support materials (cellulose, sepharose, sephadex)
containing glycol groups are activated by CNBr, which then
bind to enzymes and immobilize.
 Activation of functional groups in support meterial require Pre
treatment with CNBr, glutaraldehyde NaNO2 and HCI.

23. Cross-Linking:
Glutaraldehyde,
Hexa-methylene,
Di-isocyanate
Poly-functional reagents.

24. Cross-Linking:
 The absence of a solid support is a characteristic 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
reagents.
 These reagents in fact react with the enzyme molecules and
create bridges which form the backbone to hold enzyme
molecules.
 There are several reagents in use for cross-linking. These include
glutaraldehyde, diazobenzidine, hexamethylene diisocyanate and
toluene di- isothiocyanate.

25.  Glutaraldehyde cross linking has been successfully used to
immobilize several industrial enzymes.
 Example: Glucose isomerase, Penicillin amidase.

26. Genetic Engineering for Microbial Enzyme
Production:

27. Introduction:
 Recombinant DNA technology have certainly helped for increasing
the microbial production of commercial enzymes.
 It is now possible to transfer the desired enzyme genes from one
organism to the other.

28. There are two different methods for improvement of strains:
 Mutation.
 Genetic recombination.
 *** Refer strain improvement ppt.

29. For example:
 The enzyme lipolase, found in the fungus Humicola sp. is very
effective to remove fat stains in fabrics.
 However, industrial production of lipolase by this organism is not
possible due to a very low level of synthesis.
 The gene responsible for lipolase was isolated, cloned and inserted
into Aspergillus oryzae.

30. Rennet (chymosin):
 Rennet (chymosin) is an enzyme widely used in making cheese.
 It is mainly obtained from the stomachs of young calves.
 Consequently, there is a shortage in its supply.
 The gene for the synthesis of chymosin has been cloned for its
large scale production.

31. THANK YOU