Enzyme immobilization involves confining enzyme molecules to a solid support to facilitate reuse. Common carrier matrices include inorganic materials and polymers. Key immobilization methods are adsorption, entrapment in gels or fibers, covalent binding to functional groups, and encapsulation in semi-permeable membranes. Advantages include repeated use, stability, and easier product separation. Disadvantages can include added costs and loss of enzyme activity or specificity. Immobilized enzymes have applications in industry, medicine, food processing, and environmental management.

1. Enzyme immobilization
By :- Abhishek singh rana
Rkgit(Pharmacy)

2. Index
• Definition
• Features
• Sources of enzymes
• Carrier matrices
• Method of enzyme immobilization
• Advantages
• Disadvantages
• Applications

3. Definition
• Enzyme immobilization may be defined as a process
of confining the enzyme molecules to a solid support
over which a substrate is passed and converted to
products.

4. Salient Features.…
• They may be recovered from the reaction mixture and used
again.
• It may be accomplished by fixing the enzyme molecules to
appropriate substance.
• It should be absolutely critical that both the substrates and the
products migrate quite freely in and out of the phase to which
the specific enzymes molecules are actually confined.
• The enzyme phase is called as carrier phase which is water
insoluble but hydrophilic porous polymeric matrix, e.g.
agarose, cellulose, etc.
• The enzyme phase may be in the form of fine particulate,
membranous, or microcapsule.

5. Contd…
• The enzyme in turn may be bound to another enzyme via
cross linking.
• A special module is produced employing immobilization
techniques through which fluid can pass easily,
transforming substrate into product and at the same
time facilitating the easy removal of catalyst from the
product as it leaves the reactor.
• The support or carrier utilized in immobilization
technique is not stable at particular pH, ionic strength, or
solvent conditions. Hence, may be disrupted or dissolved
releasing the enzyme component after the reaction.

6. Sources of Enzymes
• The sources of enzymes are:-
1. Animal based
2. Plant based
3. Fungal based

7. contd...
• Animal-Based Enzymes - Animal-based enzymes also function
exclusively within a limited pH level range, which renders them
fairly ineffective in the gut. They become unstable in a low pH level
(acidic) environment, resulting in the enzyme being destroyed
before it can perform its function.
• Plant-Based Enzymes- These enzymes are effective over a broad
scope of pH levels. This range is generally believed to be between
3.0 and 9.0, which is highly compatible with the human
gastrointestinal environment.
• Fungal Enzymes- Fungal enzymes are commonly produced from a
fungal source called Aspergillus. For example, Aspergillus oryzae is
used in the preparation of sake and soy sauce, while Aspergillus so
jae is also used in soy sauce preparation, as well as in miso soup.

8. Carrier matrices
The substances that are solely employed for the immobilization
of enzymes are known as carrier matrices e.g., inorganic
materials (salts), and inert polymers.
An ideal carrier matrix has the following characteristic features,
namely :
(a) cost effectiveness,
(b) inertness,
(c) reasonable physical strength,
(d) adequate stability,
(e) regenerability after the gainful lifespan of the immobilized
enzyme,
(f) enhancement in specificity of enzyme,
(g) reduction in product inhibition,

9. Methods of Enzyme immobilization

10. Adsorption
• The appropriate enzyme is adequately mixed with a
right adsorbent usually under appropriate pH
parameters as well as the desired ionic strength.
• After incubation for a stipulated duration, the carrier
matrix is washed thoroughly to get rid of the entire
unabsorbed enzyme molecules, whereby the
‘immobilized enzyme’ is ready for actual usage.
• Interestingly, this specific method invariably gives
rise to a high loading (nearly 1 g enzyme per g
matrix) of the enzyme.

11. Contd..
• ADVANTAGES
1. Simple and economical
2. Limited loss of activity
3. Can be Recycled,Regenerated & Reused.
• DISADVANTAGES
1. Relatively low surfacearea for binding
2. Exposure of enzyme tomicrobial attack.
3. Yield are often low due to inactivation.

12. Entrapment
• In entrapment, the enzymes or cells are not directly
attached to the support surface, but simply trapped
inside the polymer matrix.
• Enzymes are held or entrapped within the suitable gels
or fibres.
• It is done in such a way as to retain protein while
allowing penetration of substrate.
• It can be classified into lattice and micro capsule types.
 Inclusion in gels: Poly acryl amide gel, Poly vinyl
alcohol gels.
 Inclusion in fibers: Cellulose and Poly -acryl amide gels.
 Inclusion in micro capsules: Polyamine, Polybasic -acid
chloride monomers.

13. Contd..
• ADVANTAGES
1. No chemical modification.
2. Relatively stable forms
3. Easy handling and re-usage.
DISADVANTAGES
The enzyme may leakfrom the pores.

14. Covalent Binding
• Based on the binding of enzymes and water-insoluble
carriers by covalent bonds
• The functional groups that may take part in this binding are
Amino group, Carboxyl group, Sulfydryl group, Hydroxyl
group, Imidazole group, Phenolic group, Thiol group, etc
• Advantages : the binding force between enzyme and
carrier is so strong that no leakage of the enzymes occurs,
even in the presence of substrate or solution of high ionic
strength
• Disadvantages : covalent binding may alter the
conformational structure and active center of the enzyme,
resulting in major loss of activity and/or changes of the
substrate

15. Encapsulation
• It involves enclosing the enzymes within semi-
permeable polymer membranes e.g. semi
permeable collodion or nylon membranes in the
shape of spheres.
• ADVANTAGES
1. No chemicalmodification.
2. Relatively stableforms.
3. Easy handling and re-usage.
• DISADVANTAGES
The enzyme may leakfrom the pores.

16. Advantages of Enzyme Immobilization
• Repeated usage
• Immobilized enzymes may be used predominenty
in most continuous production systems
• may exhibit thermostability of the highest order
• Importantly, the ultimate recovery of
‘immobilized enzyme’ would drastically minimise
the high effluent disposable problems
• Immobilized enzymes may be employed at a
much higher concentration range in comparison
to the corresponding free enzyme.

17. Disadvantages of Enzyme
Immobilization
• Gives rise to an additional bearing on cost.
• Immobilization of enzymes invariably affects the
stability.
• Practical utilization of the ‘immobilized enzymes’
may not prove to be of any use or advantage
when one of the substrates is found to be
insoluble.
• Certain immobilization protocols do offer a good
number of serious problems with respect to the
diffusion of the ensuing substrate to have an
access to the corresponding enzyme.

18. Applications
• Industrial production of antibiotics, beverages,
amino acids uses immobilized enzymes.
• Biomedical application – immobilized enzymes
are widely used in diagnosis and treatment of
diseases.
• Food industry – Enzymes like peptinase and
cellulase immobilized on suitablr careers are used
in the production of jams, jellies etc.
• Production of biodiesel from vegetable oils.
• Waste water management : treatment of sewage
and industrial effluents

19. .