Enzyme Immobilization

ENZYME IMMOBILIZATION

Immobilization means imprisonment of an enzyme in a distinct phase. Immobilized enzymes are enzymes which are attached in or onto the surface of an insoluble support. The immobilized enzymes have several advantages over the soluble enzyme:

Convenience: Miniscule amounts of protein dissolve in the reaction, so workup can be much easier. Upon completion, reaction mixtures typically contain only solvent and reaction products.

Economical: Immobilized enzymes can be easily removed from the reaction. There is no loss of enzymes. So the enzymes can be repeatedly reused.

Stability: Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme

Less contamination: There is less chance of contamination in products while immobilized enzymes are used.

There are a number of requirements to achieve a successful immobilization:

—  The biological component must retain substantial biological activity after attachment

—  It must have a long-term stability

—  The sensitivity of the enzyme must be preserved after attachment

—  Overloading can block or inactivate the active site of the immobilized biomaterial, therefore, must be avoided

Methods of Enzyme Immobilization:

There are many different methods of immobilizing enzymes. The following methods are commonly used;

1. adsorption
2. entrapment
3. encapsulation
4. covalent binding

  

Figure 1: Different methods of enzyme immobilization

1. Adsorption:

It is the simplest immobilization method in which the enzyme and the support are mixed in suitable conditions. The first immobilized enzyme model: invertase on the activated charcoal was developed by Nelson and Griffin, 1916. The forces are weak so leakage is generally a problem in adsorption method. Supports such as alluminium hydroxide are often utilized. With a suitable charged matrix, ionic interactions may also be promoted. This technique is technically undemanding and economically attractive. The regeneration is also easy in this technique. The best known industrial example: amino acylase immobilized on DEAE-Sephadex in the production of amino acids is an example of adsortion method.

2. Entrapment

Enzymes may be entrapped within the matrix of a polymeric gel such as polyacrylamide type gels naturally derived gels e.g. cellulose triacetate, agar, gelatin carrageenan, alginate etc. The form and nature of matrix vary. The pore size of matrix should be adjusted to prevent the loss of enzyme from the matrix due to diffusion.

In order to immobilize the enzymes by this technique, the enzyme together with the gel monomers are incubated. Then gel polymerization is promoted by adding few catalysts. Once there is complete polymerization of gels, the enzymes are immobilized in to the net working of the polymerized gels. Polyacrylamide and polymethacrylamide gels are examples of gels used in  this technique. Gel pore size is a crucial factor in this technique.

Figure 2: Enzyme immobilization by entrapment and encapsulation techniques

3. Encapsulation

Encapsulation involves entrapping the enzymes within a semipermeable membrane capsule such as cellulose nitrate and nylon-based membranes. The method of encapsulation is cheap and simple but its effectiveness largely depends on the stability of enzyme although the catalyst is very effectively retained wiithin the capsule. The main disadvantage of this technique is that only small amount of substrate molecule is utilized with the intact membrane.

4. Covalent binding

The most widely used method for enzyme immobilization is the covalent binding method. It is technically more complex and requires a variety of often expensive chemicals. It is time-consuming. But immobilized enzyme preparations are stable and leaching is minimal. Enzymes are immobilized by a suitable group in the surface such as hydroxyl groups in supports (e.g cellulose, dextran, agarose) and amino, carboxyl and sulfhydryl groups in amino acids. The conditions for immobilization by covalent binding are much more complicated and less mild than in the cases of physical adsorption and ionic binding. Therefore, 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. Covalent attachment to a support matrix must involve only functional groups of the enzyme that are not essential for catalytic action. Higher activities result from prevention of inactivation reactions with amino acid residues of the active sites. A number of protective methods have been devised such as covalent attachment of the enzyme in the presence of a competitive inhibitor or substrate . Many factors influence on the activity of enzymes while immmobilizing by this technique. 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 should be 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)

—  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