Immobilized Enzymes

Immobilization of Enzymes Molecules

One factor that usually impedes the development of wide industrial application of enzymes is high cost. Immobilization is a technique to retain enzyme molecules for repeated use. The method of immobilization can be adsorption, covalent bonding, or entrapment. Semipermeable membranes in the form of flat sheets or hollow fibers are one way to restrain the enzyme while allowing smaller molecules to pass. Polyacrylamide gel, silica gel, and other similar materials have been used for entrapment of biologically active materials including enzymes. Encapsulation is another means of capture by coating liquid droplets containing enzymes with some semipermeable materials formed in situ.

Generally speaking, entrapment does not involve a chemical or physical/chemical reaction directly with the enzyme molecules; and the enzyme molecules are not altered. Physical adsorption on active carbon particles and ionic adsorption on ion-exchange resins are important for enzyme immobilization.

A method with a myriad of possible variations is covalent bonding of the enzyme to a selected carrier. Materials such as glass particles, cellulose, silica, and so on, have been used as carriers for immobilization. Enzymes immobilized by entrapment and adsorption may be subject to loss due to leakage or desorption. On the other hand, the chemical treatment in forming the covalent bond between an enzyme and its carrier may permanently damage some enzyme molecules. In enzyme immobilization, two efficiency terms are often used. Immobilization yield can be used to describe the percent of enzyme activity that is immobilized,

% yield = 100 ´ (activity immobilized/starting activity)

Immobilization efficiency describes the percent of enzyme activity that is observed:

percent efficiency = 100 ´ (observed activity/activity immobilized)

When an enzyme molecule is attached to a carrier, its active site might be sterically blocked and thus its activity becomes unobservable (inactivated). One of the most important parameters of an immobilized-carrier complex is stability of its activity. Catalytic activity of the complex diminishes with time because of leakage, desorption, deactivation, and the like. The half-life of the complex is often used to describe the activity stability. Even though there may be frequent exceptions, linear decay is often assumed in treating the kinetics of activity decay of an immobilized complex.

Immobilization by adsorption or by covalent bonding often helps to stabilize the molecular configurations of an enzyme against alternations including those that may cause thermal deactivation. Immobilized enzymes tend to be less sensitive to pH changes than are free enzymes. Although careful choice of the immobilization chemistry can result in stabilized activity, there are some enzymes that are much less stable after immobilization. Most carriers are designed to have high porosity and large internal surface areas so that a relatively large amount of enzymes can be immobilized onto a given volume or given weight of the carrier. Therefore, in an immobilized enzyme-carrier complex, the enzyme molecules are subject to the effect of the micro-environment in the pores of the complex. Surface charges and other micro environmental effects can create a shift up or down of optimal pH of the enzyme activity.

An immobilized enzyme-carrier complex is a special case that can employ the methodology developed for evaluation of a heterogeneous catalytic system. The enzyme complex also has external diffusional effects, pore diffusional effects, and an effectiveness factor. When carried out in aqueous solutions, heat transfer is usually good, and it is safe to assume that isothermal conditions prevail for an immobilized enzyme complex.