Preparation of immobilized biocatalysts

Methods for immobilization of enzymes, microbial cells, animal cells and plant cells can be
classified into three categories, that is, carrier-binding, crosslinking and entrapping methods.
Among them, the carrier-binding method is mainly applied for immobilization of enzymes,
and the entrapping method has been the most extensively investigated for immobilization of
microbial, animal and plant cells.

The carrier-binding method is based on linking enzymes or cells directly to water-insoluble

matrices and can be further divided into three categories according to the binding mode of the
enzymes or cells; that is, physical adsorption, ionic binding and covalent binding. As
matrices, water-insoluble polysaccharides (cellulose, dextran and agarose derivatives),
protein (gelatin and albumin), synthetic polymers (ion-exchange resins and polyacrylamide
gel), inorganic materials (porous glass, silica and alkaline earth metal ions) and so on are
used. Immobilization of enzymes by physical adsorption and ionic binding methods can be
simply achieved under mild conditions. In these cases, preparations having relatively high
activity are obtained. However the binding forces between enzyme and matrix are weak in
comparison with those in the covalent binding method. Therefore, leakage of the enzyme
from the matrix may occur by a change in ionic strength or pH of the substrate or product
solution. These types of immobilized enzymes can be regenerated when the enzyme activity
decreases after prolonged operation. Thus, the physical adsorption and ionic binding methods
are advantageous in comparison with the covalent binding method, particularly when
expensive matrices or enzymes are used. In contrast, immobilization of enzymes by covalent
binding is carried out under relatively severe conditions in comparison with physical
adsorption or ionic binding. Accordingly, unless immobilization of an enzyme by covalent
binding is carried out under well-controlled conditions, immobilized enzyme having high
activity cannot be obtained. The binding forces between the enzyme and matrix are strong,
and the enzyme cannot easily leak out from matrices even in the presence of substrates or
salts at high concentration. However, when the activity of enzymes immobilized by covalent
binding decreases for long-term operation, regeneration is impossible.

The crosslinking method is based on the formation of chemical bonds, as in the covalant
binding method, but water-insoluble matrices are not used in this method. The
immobilization of enzymes or cells occurs by the formation of intermolecular crosslinkages
between the enzyme molecules or the cells by means of bi- or multi-functional reagents. As
crosslinking reagents, glutaraldehyde, bis-isocyanate derivatives, bis-diazobenzidine and so
on have been employed. A few papers have appeared on this method so far. The entrapping
method is based on confining enzymes or cells in the lattice of a polymer matrix or enclosing
them in semipermeable membranes. This method differs from the covalent binding and
crosslinking methods in that the enzyme or cell itself does not bind to the matrix or
membrane. Thus, this method may have wide applicability. For this method, the following
matrices are employed: collagen, gelatin, agar, alginate, carrageenan, cellulose triacetate,
polyacrylamide, photo-crosslinkable resins, polyurethanes and so on. Alginate, carrageenan
and polyacrylamide, in particular, have been extensively used.