Enzyme Immobilization (Inactivation)
Enzyme immobilization may be defined as confining the enzyme molecules to a distinct phase from the one in which the substrates and the products are present; this may be achieved by fixing the enzyme molecules to or within some suitable material. It is critical that the substrates and the products move freely in and out of the phase to which the enzyme molecules are confined. Immobilization of enzyme molecules does not necessarily render them immobile; in some methods of immobilization, e.g. entrapment and membrane confinement, the enzyme molecules move freely within their phase, while in cases of adsorption and covalent bonding they are, in fact, immobile.
The materials used for immobilization of enzymes, called carrier matrices, are usually inert polymers or inorganic materials. The ideal carrier matrix has the following properties: (i) low cost, (ii) inertness, (iii) physical strength, (iv) stability, (v) regenerability after the useful lifetime of the immobilized enzyme, (vi) enhancement of enzyme specificity, (vii) reduction in product inhibition, (viii) a shift in the pH optimum for enzyme action to the desired value for the process, and (ix) reduction in microbial contamination and non-specific adsorption. Clearly, most matrices possess only some of the above features. Therefore, carrier matrix for the immobilization of an enzyme must be chosen with care keeping in view the properties and limitations of various matrices.
Methods of Immobilization:
The various methods used for immobilization of enzymes may be grouped into the following four types:
(i) Adsorption
(ii) Covalent Bonding
(iii)Entrapment and
(iv) Membrane Confinement
(i) Adsorption:
In case of adsorption, the enzyme molecules adhere to the surface of carrier matrix due to a combination of hydrophobic effects and the formation of several salt links per enzyme molecule. The binding of enzyme molecules to the carrier matrix is usually very strong, but it may be weakened during use by many factors e.g. addition of substrate, pH or ionic strength.
(ii) Covalent Binding:
In this system the enzyme molecules are attached to the carrier matrix by formation of covalent bonds. As a result the strength formation occurs with the side chains of amino acids of the enzyme, their degree of reactivity being dependent on their charged status. Roughly the following relation is observed in reactivity.
-S > -SH > -0 > -NH2 >- COO > OH >> -NH3+
(iii) Entrapment:
In this approach, enzyme molecules are held or entrapped within suitable gels or fibers and there may or may not be covalent bond formation between the enzyme molecules and the matrix. A non-covalent entrapment may be viewed as putting the enzyme molecule in a molecular cage just as a caged bird / animal. When covalent binding is also to be generated, the enzyme molecules are usually treated with a suitable reagent.
(iv) Membrane Confinement:
Enzyme molecules, usually in an aqueous solution, may be confined within a semipermeable membrane which, ideally, allows a. free movement in either direction to the substrates and products but does not permit the enzyme molecules to escape
Effects of Immobilization on Enzyme:
Often kinetic behavior of an immobilized enzyme may differ significantly from that of its free molecules. Different enzymes respond differently to the same immobilization protocol. Therefore, a suitable immobilization protocol has to be worked out for a given enzyme. The effects on enzyme kinetics (i.e. activity) may be due to the influence of matrix per se or due to conformational changes in the enzyme molecules induced by the procedure of immobilization.
Advantage of Immobilization:
Enzymes are costly items, and can be used repeatedly only if they can be recovered from the reaction mixtures. Immobilization permits their repeated use since such enzyme preparations can be easily separated from the reaction system.
1. Immobilized enzymes can be used in non-aqueous systems as well, which may be highly desirable in some cases.
2. Continuous production systems can be used, which is not possible with free enzymes.
3. Thermo stability of some enzymes may be increased. For example, glucose isomerase denatures at 45°C in solution, but is stable for about 1 yr even at 65°C when suitably immobilized.
4. Recovery of enzyme may also reduce effluent handling problems.
5. Enzymes can be used at much higher concentrations than free enzyme.