Winfried Hartmeier

Methods of Immobilization

Adsorption is the simplest and the oldest method of immobilizing an enzyme onto a water-insoluble carrier. It has already been mentioned (p. 18) that as early as 1916 Nelson and Griffin observed that invertase adsorbed on active charcoal retained its sucrose-splitting activity. Since this discovery, the adsorption method has been applied for innumerable enzymes and whole cells. That whole cells can be made to adhere to suitable solid bodies has been known for even longer than the adsorption of single enzymes: in the nineteenth century (see Sect. 1.6) bacteria attached to wood shavings were already used in the production of vinegar.

Characteristics of Immobilized Biocatalysts

The influence of temperature on the activity of native and immobilized biocatalysts is usually represented in the form of so-called optimum curves (see Fig. 32), in which activity is plotted against temperature. Often, the activity is not given in international units but as the relative activity, i.e., the quotient of the actual activity and the highest activity measured.

Uses in Medicine

Immobilized enzymes are already used in medical analysis and diagnosis. The principles of the analytical methods have been dealt with in Chap. 6. In addition, certain of the L-amino acids produced by the methods described in Sects. 5.2 and 5.3 are of therapeutic value, while the methods for producing derivatives of penicillin (see Sect. 5.5) are of the utmost importance in medicine.

Special Developments and Trends

What can be termed the almost classical fields of immobilization of single enzymes on the one hand, and whole microorganisms on the other, have recently been extended in a variety of ways to include plant and animal cells and even cell organelles. Although it is too soon to say what significance such advances will have for science and production technology, a few of these special developments and trends will be discussed in the following sections.

Construction and Use of a Biochemical Electrode for Assaying Glucose

Using glucose oxidase and catalase, and a commercially available glass electrode (pH electrode), a biochemical electrode for measuring glucose will be constructed. From a series of measurements in which the parameters are glucose concentration and measuring time, it is intended to obtain values indicating the range of sensitivity and the response time of the enzyme electrode.

Adsorptive Coupling of Invertase to Active Charcoal

This experiment is intended to familiarize the student with the oldest and simplest method of coupling an enzyme to a solid carrier (cf. Sect. 2.1). It will be shown that adsorptive binding is not only easy to bring about, but is equally easily reversed.

Spinning of ß-Galactosidase into Cellulose Acetate Fibers

This exercise is intended to familiarize the experimenter with the method of spinning enzymes into fibers, a special form of enzyme entrapment in a matrix (see Sect. 2.5). The example chosen is the entrapment of yeast s- galactosidase, a procedure that is also used industrially to a a small extent.

Alginate Entrapment of Yeast Cells and Their Co-entrapment with Immobilized ß-Galactosidase

This exercise is intended as an introduction to entrapment in polymer matrices, a technique widely used for immobilizing whole cells. In addition, in order to enhance their substrate spectrum, cells will be co-entrapped with an immobilized enzyme.

Immobilization of ß-Galactosidase by Cross-linking

This exercise demonstrates that, using the cross-linking method discussed in Sect. 2.4 it is possible to obtain immobilized preparations with a high activity toward low-molecular substrates. In addition, the student is introduced to the enzyme s-galactosidase (= lactase), for which a growing role in industrial processes is predicted (cf. Sect. 5.7).

Covalent Binding of Glucoamylase to a Carrier bearing Oxirane Groups

The very important method of coupling single enzymes to carriers by covalent bonds will be demonstrated in this exercise by coupling glucoamylase to an activated carrier. At the same time the experiment serves to acquaint the student with the enzyme glucoamylase (= amyloglucosidase) which, in its soluble form, is much used in industrial processes.