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.

1. Prepared By
Ms. Ashvini V. Soyam
(Assistant Professor)
Dr. Rajendra Gode Institute of Pharmacy, Amravati
Enzyme Immobilization

2. What is Enzyme Immobilization?????
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.
What Is An Immobilized Enzyme????
An immobilized enzyme is the one whose movement in
space has been restricted either completely or to a small
limited region.

3. Why Immobilize Enzymes?????
Protection from degradation and deactivation.
Re-use of enzyme for many reaction cycles, lowering the total
production cost of enzyme mediated reactions.
Easy separation of the enzyme from the product.
Ability to stop the reaction rapidly by removing the enzymes from
the reaction.
Product is not contaminated with the enzyme.
Enhanced stability.

4. Advantages of enzyme immobilization…
 Recovered at the end of the reaction thereby can be
reused.
 Easy separation of enzyme from the products occurs
 Stability of immobilized enzyme increases.
 Enhanced enzyme properties.
 Efficiency of the catalytic reaction is better in a few
cases.
 Better control of reaction can be achieved.
 Catalytic process can be operated continuously.
 Multi enzyme reaction possible.
 Potential in industrial & medicinal use.

5. Disadvantages of enzyme immobilization….
 It invariably affects the stability and activity of enzymes.
 Certain immobilization protocols offer serious problems with
respect to the diffusion of the substrate to have an access to the
enzyme.
 It gives rise to an additional bearing on cost.
 The technique may not prove to be one of the substrate is found
to be insoluble.

6. CLASSIFICATION OF CARRIERS…..
Inorganic
Carriers
• May undergo
abrasion
• High
pressure
stability.
Examples:
Porous glass,
Silica, Celite
Organic
Natural
Carriers
• Favourable
compatibility
with proteins.
Examples:
Pectin, Starch,
Chitosan
Organic
Synthetic
Carriers
• High
chemical
stability.
Example:
Acrylic
polymers,
Polystyrene

7. Ideal Characteristics of the Carrier:-
• Low Cost & of optimum quality
• Inertness
• Physical Strength
• Stability
• Regenerability
• Enhancement of enzyme specificity
• Reduction of product inhibition
Carrier For Immobilized Enzyme…..

8. Types of Carriers….
 Naturally occurring
Structural proteins (Ex: Ceratin, Collagen)
Globular proteins (Ex: Albumin)
Carbohydrates (Ex: Dextran)
 Synthetic organic
Ex: polyvinyls, epoxide, etc.
 Inorganic
Ex: glass, silica gel, bentonite, etc.

10. Adsorption Covalent
Cross-Linking Encapsulation
Entrapment

11. Chemical Methods
a) Covalent Bonding
b) Cross Linking
c) Complexation & Chelation
Physical Methods
a) Adsorption
b) Entrapping
c) Membrane confinement
Methods of Immobilization…

12. Parameters for Method Selection…..
 Overall catalytic activity.
 Effectiveness of the catalytic utilization.
 Deactivation & Regeneration characteristics.
 Cost effective.
 Intended application of immobilized enzyme.
 Toxicity of immobilized enzyme.
 Waste disposal (of immobilization process).

13. ADSORPTION
 Involves the physical binding of the enzyme on the surface of carrier
matrix.
 Carrier may be organic or inorganic.
 The process of adsorption involves the weak interactions like Vander
Waal or hydrogen bonds.
 Carriers:- silica, bentonite, cellulose, etc.
 e.g. catalase & invertase

14. Advantages:
 Simple & Economical
 Limited Loss of activity
 Can be Recycled, Regenerated & Reused (R3)
Disadvantages:
 Relatively Low surface area for binding
 Exposure of enzyme to microbial attack.
 Smaller particles cause high Pressure drop in continuous packed bed
reactor.
 Yield are often low due to inactivation & desorption.

15. ENTRAPMENT…..
• Enzymes are held or entrapped within suitable gels or fibres.
• In a gel it may causes:
 Matrix polymerization or
 Precipitation or
 Coagulation
• Entrapment in calcium alginate is the most widely used for entrapment
for:
 Microbial
 Animal&
 Plant enzymes/cells
Ex: Glucose oxidase + Polyacrylamide (gel entrapment)
• NOTE: Adsorbent (mostly commonly used)
Ex: polyacrylamides, collagen, gel, alginates, etc.

16. Advantages:
 No chemical modification
 Relatively stable forms.
 Easy handling & reusage.
Disadvantages:
 May diffusion of substrate & product occurs.
 Substrate accessibility may reduced due to free radical polymerization of
gel.
 Enzyme in-activation.
 Loss of enzyme content.
NOTE: Sometimes covalent bonding may forms between the entrapped
enzyme & the matrix.

17. CHEMICAL BONDING…..
• Enzyme forms co-valent link with active group of the matrix (like terminal
NH2 COOH, etc).
• Support with groups like:
 -OH : support activation covalently by CNBr.
 -COOH : supports ( like CMC ) activation covalently by azide derivatives.
 -NH2 : supports activation covalently by forming diazonium chlorides on
treatment with NaNO2 + HCI.
NOTE: The functional group of enzyme which is involved in the linkage,
should not affect the active properties of the said enzyme.

18. Advantages:
 Not affected by pH
 lonic Strength
Disadvantages:
 Active site may be modified
 Not cost effective
NOTE: Adsorbent
Ex:
Agarose, Cellulose, sepharose,
Polyacrlamide, etc.

19. CROSS LINKING…..
 It involves cross linking of enzyme to a multifunctional reagent
without use of any solid support.
 Alternatively, chemical bridge of some other molecule between
& with the chemical support(i.e., reaction of enzyme with
reagent bridge or chemical bridge).
 Activated carriers are used.
Ex: Sepharose by CNBr (most commonly used)
Ex: Sepharose by ethyl chloroformate

20. Advantages:
 Strong linkage leads to low enzyme leakage while use.
 Higher stability (i.e., pH, ionic & substrate concentration.
Disadvantages:
 Partially or wholly inactivation by active site modification.
 Not cost effective.

21. Uses of Immobilized Enzymes……
Biotransformation
Secondary metabolite production
Biosensors
Enzyme-linked immunosorbent assays (ELISAs)
Biological washing Powders
Food Industry
Seed Germination

22. THANK YOU…..