13.3.1 Immobilization techniques
The immobilization of single enzymes for one-step reactions or whole cells for more
complex reactions (for example, the production of ethanol in the presence of yeast
cells) can be performed in different ways:
* Fixation onto the surface of a water-insoluble carrier (adsorption, adherence, bind-
ing via ionic or metal complex interaction, covalent binding).
* Entrapment within microspheres (Chang, 1998), hollow-fiber modules, enzyme
membrane reactors, etc., where the biocatalysts are still dissolved but prevented
from direct contact with the bulk solution by semipermeable membranes.
* Entrapment within a gel matrix; this method is used preferentially for the immo-
bilization of cells (Guiselly, 1989; Rehm and Omar, 1993).
* Crosslinking of enzyme molecules with bifunctional reagents to an insoluble
‘macromolecule’; in this process, no carrier material is needed.
If an enzyme acts in organic medium it may be regarded as immobilized in so far as it
is insoluble and thus easy to separate from the reaction mixture for re-use. In addi-
tion, several features that result from the immobilization of an enzyme to a solid
support, including diffusion limitation, conformational changes, or reduced molecu-
lar flexibility (see Section 13.3.3) are also observed to a greater or lesser extent for
enzymes applied in organic media (Klibanov, 1997). Therefore, and due to the fact
that the presence of organic solvents is closely linked to phospholipase activity, some
examples of phospholipase-catalyzed reactions in near-water-free organic solvents
are included in Sections 13.4.1 and 13.4.2.
Immobilization by adsorptionis normally a very mild and simple method, and the
residual activity of the adsorbed biocatalyst will remain high. However, due to the
13.3 Immobilization of biocatalysts 267
Figure 2. Immobilization of phospholipase A 2 by adsorption to Cab-osilÒM-5 (Fluka) from aqueous
solutions and different organic solvents.