Food Biochemistry and Food Processing

210 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking

Various types of beaded supports have been used
successfully: for example, natural polymers (e.g.,
agarose, dextran, and cellulose), synthetic polymers
(e.g., polyacrylamide, polyacryloyl trihydroxymeth-
ylacrylamide, polymethacrylate), inorganic (e.g.,
silica, metal oxides, and controlled pore glass), and
microporous flat membranes (Calleri et al. 2004).
The immobilization procedure consists of three
steps (Calleri et al. 2004): (1) activation of the sup-
port, (2) coupling of ligand, and (3) blocking of re-
sidual functional groups in the matrix. The choice of
coupling chemistry depends on the enzyme to be
immobilized and its stability. A number of methods
are available in the literature for efficient immobiliza-
tion of enzyme through a chosen particular function-
al side chain’s group by employing glutaraldehyde,
oxirane, cyanogen bromide, 1,1-carbonyldiimida-

zole, cyanuric chloride, trialkoxysilane to derivatize

glass, and so on. Some of them are illustrated in Fig-

ure 8.21.

Cross-linking

This type of immobilization is achieved by cross-

linking the enzymes to each other to form complex

structures as shown in Figure 8.20C. It is therefore a

support-free method and less costly than covalent

linkage. Methods of cross-linking involve covalent

bond formation between the enzymes using bi- or

multifunctional reagent. Cross-linking is frequently

carried out using glutaraldehyde, which is of low

cost and is available in industrial quantities. To min-

imize close proximity problems associated with

the cross-linking of a single enzyme, albumin and

Figure 8.21.Commonly used methods for the covalent immobilization of enzymes. (A) Activation of hydroxyl support

by cyanogen bromide. (B) Carbodiimides may be used to attach amino groups on the enzyme to carboxylate groups

on the support or carboxylate groups on the enzyme to amino groups on the support. (C) Glutaraldehyde is used to

cross-link enzymes or link them to supports. The product of the condensation of enzyme and glutaraldehyde may be

stabilized against dissociation by reduction with sodium borohydride.