An immobilized enzyme is an enzyme attached to an inert, insoluble material—such as calcium alginate (produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride). This can provide increased resistance to changes in conditions such as pH or temperature. Traditionally, four methods are used for enzyme immobilization, namely (1) non-covalent adsorption and deposition, (2) physical entrapment, (3) covalent attachment, and (4) bio-conjugation . Support binding can be physical or chemical, involving weak or covalent bonds.

1. IMMOBILIZATION OF
ENZYMES
BY : ANJALI SHARMA (M.Sc.)
DEARTMENT OF MICROBIOLOGY

2. IMMOBILIZATION OF MICROBIAL ENZYMES (whole cells ):
 IMMOBILIZED ENZYMES
 ENZYME IMMOBILIZATION
 PRINCIPLE
 METHODS OF IMMOBILIZATION OF ENZYMES
1. PHYSICAL
2. CHEMICAL

3. IMMOBILIZED ENZYMES
 An immobilized enzyme is one whose movement inn space has
been restricted either completely or to small limited region by
attaching to inert insoluble support .

4. ENZYME IMMOBILIZATION :
 ENZYME IMMOBILIZATION is defined as a process of confining (limiting) the
enzyme molecule to a solid support over which a substrate is passed and
converted to products.

5. PRINCIPLE:
 BASED on the difference of size of enzyme and substrate or product molecule in
comparison with membrane pore size .
 The membrane allows small sized molecule such as substrate / product to diffuse
in and out of membrane while refrains larger size enzyme molecule .

6. METHODS OF IMMOBILIZATION OF
ENZYMES :
PHYSICAL
• ADSORPTION
• ENTRAPMENT
• ENCASPULATION
CHEMICAL
• COVALENT BONDING
• SUPPORT
• CROSS-LINKING

7. ADSORPTION:
 Oldest technique.
 In this , physical binding of enzymes take place on the surface of carrier matrix.
 The process of adsorption involving the weak interaction like –
 Van der wall forces
 Hydrogen bond .
 CARRIER : silica , cellulose , SiO2 .
 E.g. : catalase and invertase .

8.  ADVANTAGES :
1. Simple and economical .
2. Limited loss of activity .
3. Can be recycled , regenerated , reused .
 DISADVANTAGES :
1. Exposure of enzymes to microbial attack.
2. Yield are low due to inactivation.

9. ENTRAPMENT:
 In this the enzyme or cell are not directly attached to the support surface but
simply trapped inside the polymer matrix .
 Enzymes are held on entrapped within the suitable gels or fibres .
 Entrapment is done in such a way to retain protein while allowing penetration of
substrate .
 IT CAN BE CLASSIFIED INTO:
1. LATTICE TYPE
2. MICROCASPULAR TYPE

10.  LATTICE TYPE : entrapment involves entraooing enzymes within the intertisial
space of a cross –linked water –soluble polymer.
 E.g. : polyvinylalcohol , starch etc .
 MICROCASPULAR TYPE: entrapping involved the enzymes within the semi-
permeable membrane .

12. ENCASPULATION:
 This is the process of spherical particles formation where a liquid or suspension is
enclosed in a dense but semi-permeable membrane .
 There are 3 techniques for the formation of semi-permeable membrane for
immobilization.
1. Membran reactors
2. Emulsion
3. Microcapsules

14. COVALENT BONDING :
 It is based on the binding of enzymes and water insoluble matrix carriers by
covalent bonds.
 The functional group that may take part in this binding :
1. AMINO group [NH2]
2. CARBOXYL group [COOH]
3. HYDROXYL group [OH]
4. PHENOLIC group ]

15. CROSS-BINDING METHOD:
 Involves intermolecular cross-linking of enzymes molecule in the present or
absence of solid supports .
 This method produces a 3-D cross linked enzymes by keans of multi-functional
reagents that links covalently to the enzyme molecule.
 It is highly soluble.

16. ADVANTAGES OF IMMOBILIZATION OF
ENZYMES:
 Increased functional efficiency of enzymes .
 Enhanced reproducibility of the process they are undertaking .
 Reuse of enzyme .
 Continuous use of enzyme .
 Less labour input in the processes.
 Saving in capital cost and investment of the process.
 Minimum reaction time.
 Less change of contamination in products .

17.  More stability of products .
 Stable supply of products in the market.
 Improved process control.
 High enzyme substarte ratio.

18. DISADVANTAGES OF IMMOBIIZATION OF
ENZYMES :
 Even though there are many advantages of immobilized enzymes, there are some
disadvantages also.
 High cost for the isolation, purification and recovery of active enzyme.
 Industrial applicationa are limited and only very few industries are using
immobilized enzymes or immobilized whole cells .
 Catalytic properties of some enzymes are reduced or completely lost after their
immobilization or support or carrier .
 Some enzymes become unstable after immobilization .
 Enzymes are inactivated by the heat generated in the system.

19. APPLICATION OF IMMOBILIZATION OF
ENZYMES:
 INDUSTRIAL PRODUCTION:
Industrial production of antibiotics , beverages , amino acids , etc. uses immobilized
enzymes or whole cells.
 BIOMEDICAL APPLICATONS:
Immobilized enzymes are widely used in diagnosis and treatment of many diseases .
Immobilized enzymes can be used to overcome inborn metabolic disorders by the supply
of immobilized enzymes .
Immobilization techniques are effectively used in drug delivery systems especially to
oncogenic sites.

20.  FOOD INDUSTRY:
Enzymes like pectinases and cellulases immobilized on suitable carriers re
successfully used in the production of jams , jellies , and syrups from fruits and
vegetables.
 RESEARCH:
A research activity extensively uses many enzymes.
The use of immobilized enzymes allow researcher to increase the efficiency of
different enzymes such as HORSE RADISH PEROXIDASE (HRP) in blotting
experiments and different proteases for cell or organelle lysis .

21. REFERENCES:
 https://www.easybiologyclass.com/enzyme-cell-immobilization-techniques/
 https://www.google.com/search?q=immobilization+of&client=firefox-b-
d&source=lnms&tbm=isch&sa=X&ved=2ahUKEwiph66m39rvAhU4wTgGHbFtBf4
Q_AUoAXoECAEQAw&biw=1536&bih=750
 https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-
biology/immobilization