Front Matter

weak interaction forces the application of enzymes immobilized in this way often

suffers from a considerable protein leakage, limiting their repeated use. The loss of

protein can be reduced by additional crosslinking. Exceptions to this behavior are

known however, and some companies offer immobilized enzymes. For example, an

immobilized triacylglycerol hydrolase (EC 3.1.1.3) which catalyzes esterification

and interesterification reactions has been obtained by adsorption of the enzyme

onto macroporous anion-exchange resins (LipozymeÒRM; Novo Nordisk). Some

examples of the adsorptive immobilization of PLA 2 to a SiO 2 -matrix from different

reaction media are shown in Figure 2. No significant protein release was observed

after washing with water; however, the enzyme was removed in time from the carrier

during PC-hydrolysis (see Section 13.4.1).

By contrast,covalent bindingleads to tight fixation of a protein molecule, but this

is often accompanied by a heavy loss of catalytic activity. The carrier surface must

bear functional groups in order to enable covalent linkage. The activity of the im-

mobilized catalyst obtained is influenced by many experimental parameters that

must be optimized; these include the concentration of functional groups, the reac-

tion time between the enzyme and the chemically modified carrier, the reaction

temperature, the pH, ionic strength, and polarity of the reaction milieu, the amount

of enzyme present during the immobilization procedure, the porosity of the carrier

material and its particle size (see Sections 13.3.2 and 13.3.3). In many cases the

additional insertion of a spacer (the chain length of which must also be opti-

mized) between the enzyme molecule and the carrier surface improves the catalytic

activity significantly (Manecke et al., 1979).

Glutaraldehyde is a bifunctional compound that is often used to bind the enzyme to

a carrier with terminal amino groups on its surface. The coupling reaction occurs

predominantly via amino groups of the biocatalyst, and this can result in low activ-

ities of the bound enzyme when the corresponding amino acids are essential for the

catalytic process, as has been shown for example with the immobilization of PLA 2

(see Section 13.4.1). Other bifunctional reagents applied for this purpose are car-

boxylic acid dichlorides or carbodiimides. As in these cases side reactions are pos-

sible, it may be advantageous to use heterobifunctional spacers with one functional

group modified for enzyme binding after it has been bound to the surface. Carriers

are available commercially which are activated with epoxy residues (Table 2) and

allow direct coupling of the biocatalyst by the formation of highly stable C–S–, C–

N– or C–O– bonds. One disadvantage of these materials is that in some cases the

reaction time required to reach high enzyme loading and sufficient activity (see

corresponding product information), together with a high storage stability, is very

long (Tischler and Wedekind, 1999).

Immobilization may also be achieved by means of biologically active compounds.

Solomon et al. (1987) prepared highly active immobilized enzymes by binding them

via corresponding monoclonal antibodies to a suitable carrier. Farooqi et al. (1997)

recently reported a method which uses the carbohydrate recognition site of lectins to

bind glycoproteins such as glucose oxidase. Interesting biocatalysts were also ob-

tained by co-immobilization of enzymes and cells or cell fragments, the aim being

to combine different catalytic properties (Hartmeier, 1983; Hartmeier et al., 1987). A

significant progress in the field of fine chemicals production in the presence of en-

zymes was the development of enzyme membrane reactors with simultaneous co-

268 13 Preparation and Application of Immobilized Phospholipases