PHYSICAL METHODS OF IMMOBILIZATION

Weak interactions like hydrogen bonding or Vandeer Waals forces and containment of the enzyme with in a matrix of membrane are used in immobilizing the enzymes through physical methods.
Physical methods are again of three types depending on the way we immobilize the enzyme. They are:

• Adsorption
• Entrapment
• Encapsulation
  

ADSORPTION:
The enzyme molecules are adsorbed onto solid suppourt. Adsorption is due to weak interactions like Vandeer Waals forces or Hydrogen bonds.
Commonly used supports for adsorption are : Allumina, Silica, Charcoal, Clay, Glass, CaCo3 and modified materials like tannins, aminohexocellulose.
Surface properties of the solid supports should be taken into account before selecting one. The surface properties may be hydrophobicity, hydrophilicity, charge etc. Ideally the solid support should not interact with the enzyme which when occurs leads to reduced enzyme activity.

Advantages

• The native structure of the enzyme is less disturbed.
• Pore diffusion limitations are absent i.e., no IMTR, only EMTR is encountered by the substrate.
• Preperation is easy.
• Regeneration of enzyme is possible in adsorption, while it isn't possible in cross linking, covalent bonding and entrapment.
• Cost of immobilization is low.

Disadvantages

• Substrate specificity is unchangeable.
• The overall enzyme activity is low.
• The general applicability of the enzyme is low.
• Hydrophobicity, Hydrophilicity of the suppourt may influence the enzyme thereby decrease its efficiency.

ENTRAPMENT:

Entrapment involves the containment of the enzyme within a physical matrix of fibres or gels.

Water soluble gels like polyacrylamide gels, agar, gelatin, sodiumalginate, agarose, corragheenan cellulose triacetate etc are commonly used.

The fibrous materials used for entrapping within fibre matrix include Silica, nulon, PVC etc.

The form and nature of the matrix depends upon the material used.

When an enzyme is immobilized through entrapment the substrates encounter both EMTR and IMTR.

The pore diffusion limitations are very important in the process of entrapment.

The pore size should be larger enough that the substrate can easily pass through the matrix and that enzyme shouldnt leak out into the bulk solution.

Pore sizes obtained using agar and agarose are large (10 microns) and thus they are not used for entrapment.

Methods of Entrapment:

• Entrapment in a matrix of gel; Water soluble gels r used. Pore diffusion is a major limitation
• Entrapment in spun fibres; nylon silica, PVC are usually used
• Entrapment in a porus holloe fibre
• Entrapment in a microcapsule

Case study

Entrapment of Lipase in Alginate - Gelatin Matrix

• Mix 5 gm of sodium alginate and 2.5gm gelatin in 100ml water and heat the solution.
• Then add 1gm of Lipase powder and 0.5ml of glutaraldehyde.
• Prepare 2% CaCl2 solution seperately.
• Take the gel with enzyme into a seperating funnel and allow the beeds to fall into the 2% CaCl2 solution.
• Then upon falling into solution, Sodium alginate converts into calcium alginate and thus solidify to give strong beeds. The mechanical strength of these beeds is very high.
• During beed formation the enzyme is entrapped in the matrix of Alginate, Gelatin and Glutaraldehyde.

Advantages

• Enzyme native structure is not disturbed. Infact Entrapment helps in stabilizing the enzyme and reduce the enzyme deactivation rates (Prevents unfolding)
• Reuasbility is high i.e beeds can be reused mant times
• Cost is low
• General applicability is high i.e the method can be used for immobilizing many enzymes.
• Industrially useful

Disadvantages

• EMTR and IMTR
• Regeneration of enzyme is impossible.
• Preperation is difficult.
• Lack of control on microenvironmental conditions.
• The enzyme may leak from the pores.

Remedial steps to overcome some disadvantages

• Reducing pore size using a combination of gels to prevent enzyme leakage.
• Reduce beed size to overcome diffusional limitations.

ENCAPSULATION:

Encapsulation is the containment of the enzyme solution in a capsule of semipermeable membrane.

The membranes can be made from PLa, cellulose nitrate and even Liposomes (Phospholipids) can be used.

This is similar to entrapment.

Pore diffusion limitations are significant even in encapsulation process.

Control over microenvironmental conditions is less.

Stability of the enzyme with in the capsules is very important.

This method is mostly restricted to medical sector.

Advantages

• Enzyme native structure is not disturbed.
• This method is suitable for medical and health care sectors.
• Enzyme capsules can be reused many times.
• General applicability is high.

Disadvantages

• Diffcult to prepare.
• Cost is high.
• Pore diffusional limitations are present.
• Chance for enzyme leakage.
• Lack of control on microenvironmental conditions