Front Matter

covalent binding; a surface area of about 100 m^2 g–1in order to guarantee a sufficient

protein capacity with a pore size of approximately 100 nm (depending on the size of

the protein molecule); and mechanical and chemical stability. A selection of com-

mercially available carrier materials, together with some of their properties is listed

in Table 2. Guidelines for the characterization of immobilized biocatalysts have been

elucidated by the Working Party on Immobilized Biocatalysts within The European

Federation of Biotechnology, and are illustrated by an article concerning the immo-

bilization ofNitrosomonas europaeain Ca-alginate (van Ginkel et al., 1983).

13.3.3 Kinetics of immobilized enzymes

The attachment of an enzyme to the surface of a water-insoluble carrier or an en-

trapment of the biocatalyst within a polymer matrix is often accompanied by a

change of its kinetic behavior which may be classified as follows.

Conformational changes

These are often caused by a covalent binding of an enzyme to a support. As a con-

sequence of rigid fixation, the enzyme molecules may lose their ability to undergo

the necessary conformational changes during the interaction with the substrate with-

in the region of the active site. An immobilization via amino or carboxylic groups is

comparable with a chemical modification of the amino acids of the enzyme. A short

distance between the enzyme molecules and the carrier – and especially an orienta-

tion of its active site towards the surface after the binding process – aggravates or

even prevents access of the substrate and/or effectors to the enzyme. A high protein

loading can also create steric hindrance. Amino acids in, or near to, the active site

may be involved in the immobilization reaction. All these constraints may lead to

changes in the kinetic parameters of the bound enzyme compared to the correspond-

ing data of the biocatalyst in solution.

Partitioning effects

As the reaction medium and the insoluble carrier are two different phases, then de-

pletion as well as enrichment of substrate, buffer constituents, etc. in one of the

phases is possible. As a result, the immediate surrounding – the microenvironment

• of the enzyme on the surface of the carrier material is different from that of the bulk

solution. This means that the enzyme-specific data calculated with concentrations

measured in the solution are apparent values and must (if possible) be corrected

by the corresponding partitioning coefficients. Especially if the carrier is a poly-

meric matrix (e.g. an ion-exchange resin), this partitioning effect causes an increase

or a decrease of the pH-optimum and the situation becomes more complex when the

substrate and/or the products are charged. In addition to ionic partitioning, hydro-

phobic as well as specific interactions between a given solute and the matrix, are also

observed.

13.3 Immobilization of biocatalysts 271