This document discusses immobilized enzymes and their advantages. It describes how enzymes are attached to an inert support material to prevent loss of activity while allowing easy separation from products. Some key benefits of immobilized enzymes are their economic reuse in continuous processes, convenience of separation, and improved stability. The document then covers various immobilization methods like adsorption, covalent binding, cross-linking, entrapment, and encapsulation as well as their properties and applications in industries like food and detergents.

1.  Immobilized enzymes are enzymes which are attached to
an inert , material, that will prevent loss of enzyme
activity by not changing chemical nature of the reactive
groups in the binding site of the enzyme.
– Economical: continuous use of biocatalyst is possible
– Convenience: separation of biocatalyst and product is much
easier than conventional batch process.
– Stability: Immobilized enzymes typically have greater
thermal and operational stability than the soluble form of the
enzyme
Need for Immobilization
Enzyme immobilization

2. Immobilization methods
a) adsorption
b) covalent binding
c) Cross- linking
d) Entrapment
e) Encapsulation

3. Adsorption and Ionic binding
 Simplest immobilization method
 Mix the enzyme and support in suitable conditions
 First immobilized enzyme model: invertase on the activated charcoal
(Nelson and Griffin, 1916)
 Forces are weak so leakage is generally a problem
 With a suitable charged matrix, ionic interactions may also be
promoted
 This technique is economically attractive
 Regeneration is easy
 Best known industrial example: amino acylase immobilized on
DEAE-Sephadex in the production of amino acids
 Ex, alumina , calcium carbonate , clay
 Ion – exchangers , DEAE- cellulose ,CM – cellulose , Sephadex.

4. Covalent immobilization
 The most widely used method for enzyme
immobilization
 It is technically more complex
 It requires a variety of often expensive chemicals
 But immobilized enzyme preparations are stable and
leaching is minimal
 Enzymes are immobilized by a suitable group in the
surface:
 Hydroxyl groups in supports (e.g cellulose, dextran,
agarose)
 Amino, carboxyl and sulfhydryl groups in amino acids

5. Immobilized Enzyme Systems
Cross-linking is to cross link enzyme
molecules with each other using agents
such as glutaraldehyde.
Features: similar to covalent binding.
Several methods are combined.

6. Immobilized Enzyme Systems
Enzyme Immobilization:
To restrict enzyme mobility in a fixed space.

7. Entrapment Immobilization is based on
the localization of an enzyme within the
lattice of a polymer matrix or membrane.
- retain enzyme
- allow the penetration of substrate.
It can be classified into matrix and micro
capsule types.
Immobilized Enzyme Systems

8. Gel-fibre entrapment and
encapsulation
Entrapment
 Enzymes may be entrapped within the matrix of a polymeric
gel
 Incubate the enzyme together with the gel monomers
 Promote gel polymerization
 Polyacrylamide and polymethacrylamide gels are examples
 Gel pore size is a crucial factor
Encapsulation
 Encapsulation involves entrapping the enzymes within a
semipermeable membrane such as cellulose nitrate and
nylon-based membranes

9. Properties of support material
 The form, shape, density, porosity, pore size distribution,
operational stability and particle size distribution of the supporting
matrix will influence the result
 The ideal support is cheap, inert, physically strong and stable
 Ideally, it should:
 increase the enzyme specificity (kcat/Km)
 shift the pH optimum to the desired value for the process
 discourage microbial growth and non-specific adsorption
 Some matrices may possess other properties which are useful for
particular purposes such as
 ferromagnetism (e.g. magnetic iron oxide, enabling transfer of the
biocatalyst by means of magnetic fields)
 a catalytic surface (e.g. manganese dioxide, which catalytically removes
the inactivating hydrogen peroxide produced by most oxidases)

10. Kinetic Properties
 There is usually a decrease in specific activity of an enzyme upon
insolubilization: denaturation caused by the coupling process
 Microenvironment after immobilization may be drastically
different from that existing in free solution: the physical and
chemical character of the support matrix, or interactions of the
matrix with substrates or products involved in the enzymatic
reaction
 The Michaelis constant may decrease by more than one order of
magnitude when substrate of opposite charge to the carrier matrix
 The diffusion of substrate can limit the rate of the enzyme
reaction: the thickness of the diffusion film determines the
concentration of substrate in the vicinity of the enzyme and hence
the rate of reaction
 The effect of the molecular weight of the substrate can also be
large.High mol wt substrates shows lower activity than low mol wt
substrates

11. Kinetics of immobilized enzymes
 It is also a useful method for protecting oxygen-labile enzymes by
'salting out' the oxygen from the vicinity of the enzyme
 Partition of hydrogen ions  The pH of the microenvironment may
differ considerably from the pH of the bulk solution
• Enzyme immobilised on charged
supports:
free enzyme
enzyme bound to a (+)ly
charged support; a bulk pH
of 5 is needed to produce a
pH of 7 within the
microenvironment
enzyme bound to a (-)ly
charged support; a pH of 7
within the microenvironment
is produced by a bulk pH of
9

12. Types of reactors
Stirred tank batch reactor
Batch membrane reactor
Packed bed reactor
Continuous flow stirred tank reactor
Fluidized bed reactor

13. TYPES OF REACTORS

15. Applications of immobilized enzymes in
Industries
Industries - Food Industry
Starch Hydrolysis
Production of HFCS
Use of proteases
Production of Amino Acids
Antibiotics Production
OTHER INDUSTRIAL APPLICATIONS
Detergent industry

16. REFERENCES
 Rastogi,S.C.2007.Biotechnology,Principles
and applications
 Palmer,Trevor.Enzymes:Biochemistry,
Biotechnology, Clinical chemistry,3rd
edition
 David. L.Nelson,Michael M
.Cox,Lehninger,principles of Biochemistry,
4th
edition
 M.F.Chaplin,C.Bucke, Enzyme
technology,1990,cambridge

17. THANK YOU