Expt 7: Enzyme immobolization – Lipase

Aim: 1. To study immobilisation of enzyme in calcium alginate beads.

2. To study entrapment of enzyme and its effect on activity and stability

Requirements: Sodium alginate, calcium chloride solution, Lipase solution.

Theory:
Enzyme immobilization: The use of enzyme has been widely established in agriculture and food
industry, pharmaceutical industry, chemical industry, analytical methods and medical research.
Although enzymes can catalyse reaction in different stages e.g. as individual molecules in
solution or in aggregates with other entities, it is technically very difficult to recover active
enzyme after the reaction. Other problems involving the use of enzyme include their poor
stability governed by even slight changes in pH and temperature and their high cost of isolation
These problems may be overcome by immobilising the enzymes onto specific surfaces.
Immobilised enzymes refer to enzymes physically confined or localised in a certain defined
region of space with retention of their catalytic activities,which can be used repeatedly and
continuously. Immobilisation of enzymes improves their technical performance in industrial
process and their economy.

Components of an immobilised enzyme:

1. The enzyme
2. The matrix also known as support or carrier
3. The mode of attachment of the enzyme to matrix

Ideal support properties:

1. Physical resistance to compression
2. Hydrophilicity
3. Inertness towards enzyme
4. Biocompatibility
5. Resistance to microbial attack
6. Availability at low cost

Classification of supports
A. Organic
1. Natural polymers
2. Polysaccharides - agarose, cellulose, chitin
3. Proteins-collagen, albumin
4. Carbon

Synthetic polymers
1. Polystyrene
2. Other polymers - polyacrylate, vinyl and allyl polymers, polyamides
B. Inorganic
A) Natural polymers - silica, bentonite
B) Processed materials - glass (non porous), metals and controlled poor metal oxides

Methods of enzyme immobilisation

1. Irreversible and reversible methods
2. Strength of binding is usually inversely related to reversibility

Methods of irreversible enzyme immobilisation:

1. Upon immobilisation enzyme cannot be detached without destroying either the biological
activity of enzyme or support
2. Methods:
A) Covalent coupling
B) Entrapping
C) Microencapsulation
D) Cross linking

Methods of reversible enzyme immobilisation:

1. Reversibly immobilised enzymes can be detached from support under gentle conditions
2. Advantages: economical. When enzymatic activity decays, the support can be regenerated and
reloaded with fresh enzyme
3. Support often dictates overall cost of immobilised catalyst
4. Important for immobilising labile enzymes and for application in bioanalytical systems
5. Methods: Adsorption, ionic bonding, affinity bonds, chelation or metal binding, disulphide
bonds.

Advantages:
1. Enzymes can be stabilised against heat or solvent effects.
2. Enzymes can be retained, reused, reducing overall cost
3. Enzymes can be easily added to or removed giving greater control over the reaction
4. Problems of separating the catalyst from the products are eliminated
5. Continuous processes using columns of immobilised enzymes becomes more practical and
automation is possible

Disadvantages:
1. Reduction in activity if active group is involved in immobilisation
2. Diffusional limitation: If substrate is unable to pass through pores which has enzyme
immobilised internally in the pores.
3. Increase in cost

procedure to be replaced by procedure stated in ppt

Procedure:
1. Prepare 5-10% CaCl2, add 2% sodium alginate gel.
2. Add enzyme in sodium alginate gel equal to 100 units
3. Titrate this against CaCl2 solution in the flask to form calcium alginate beads
4. Compare the results with standard preparation of pure enzyme solution for parameters such
as pure enzyme activity

Conclusion: