Bioaffinity Based Immobilization of Enzymes - Advances in Biochemical Engineering Biotechnology - page 218

4.2
Immobilization of Chimeric Enzymes

Ability to bind to appropriate ligands can be conferred on enzymes,that lack
affinity for the ligand,by linking them to polypeptides that exhibit high affinity
for the ligand. Such chimeric enzymes may be affinity bound on supports bearing
appropriate ligands. Fusion proteins and enzymes have been widely used in the
purification of cloned gene products [158,159] and their remarkable potential in
the enzyme immobilization is clearly evident from some recent investigations.
The exoglucanase (Cex) and endoglucanase (CenA) of Cellulomonas fimi
bind strongly to microcrystalline cellulose through their cellulose binding
domain (CBD)[160]. The CBD functions quite independently of the catalytic
domain from which it is separated by a 22-aminoacid long Pro-Thr box [161].
Ong et al. [162] constructed a fusion protein by genetically linking the genes of
b

-glucosidase of Agrobacterium sp .and Cellulomonas fimi . The fusion protein
retained 42 % of -glucosidase activity,bound strongly to cellulose supports and

facilitated efficient substrate hydrolysis. The hybrid enzyme remained stably
adsorbed to Avicel for upto 7 weeks at 4 °C and 37 °C,retained activity upto 70 °C
and in upto 1.0 M salt solution. The enzyme could be readily eluted with distilled
water [163]. More recently Phelps et al. [164] chemically coupled CBD to glucose
oxidase with the help of glutaraldehyde and demonstrated the concept and
feasibility of a reloadable biosensor based on reversible immobilization of
enzyme using CBD technology. While genetic fusion ensures uniformity and
homogeneity of the conjugate as well as simplification of its production,size and
ratio of the two proteins may be more easy to control in chemical coupling
procedures.
A hybrid protein comprising of protein A and

b

-lactamase has also been con-
structed using recombinant DNA technology [165]. The hybrid enzyme bound
specifically and strongly to IgG-Sepharose and the immobilized preparation was
superior in its ability to hydrolyse penicillin G as compared to the covalently
immobilized enzyme.
Neusted et al. [166] in an interesting recent study have shown that resistance
to trypsin inactivation can be engineered in L -asparaginase through production
of chimeric protein between the enzyme and a protective single chain antibody.
Although the objective of the investigation was not to immobilize the enzyme
the work clearly indicates the potential antibody-enzyme conjugates in the
stabilization and immobilization of enzymes.

b

Acknowledgements. Parts of the author's work cited in the review were supported by the
Indian Council for Medical Research,Department of Science and Technology,CSIR India and
the Volkswagen Foundation,Germany. The author is grateful to Prof. Dr. T. Scheper of Insti-
tute fuer Technische Chemie der Universitat Hannover for many helpful discussions and
suggestions.

Note added in proof. It has been shown in a recent study that differences between affinity towards
metal ions chelated by Sepharose-linked immunodiacetic acid groups could be made use of to
bind two enzymes in a manner that facilated their selective elution and reloading (P. Sosnitza,
M. Farooqui,M. Saleemuddin,R. Ulber and T. Scheper 1988. Anal. Chim. Acta 368 : 197).