PROPERTIES OF THE BETA-GLUCOSIDASE FROM CELLULOMONAS-FLAVIGENA AND FROM ESCHERICHIA-COLI HARBORING THE RECOMBINANT PLASMID PJS']JS3

Plasmid-coded beta-glucosidase produced by Escherichia coli was characterized and compared to the enzyme produced by Cellulomonas flavigena. Cell-free extracts, non-denaturing PAGE and 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside (X-glu) as substrate were used to compare both enzymes. The beta-glucosidase was assayed for cellobiose and p-nitrophenyl-glucopyranoside (PNPG). Cellobiose hydrolysis was performed at 50-degrees-C for the enzyme from C. flavigena and at 37-degrees-C for that from E. coli pJS3, both with an optimal pH of 6.5. For PNPG hydrolysis, the optimal conditions were pH 5.5 and 37-degrees-C for both cell extracts. Most of the beta-glucosidase activity was intracellular. When cultures of C. flavigena were grown with cellobiose or carboxymethylcellulose (CMC) as inducers, the expression of beta-glucosidase was increased considerably. E. coli pJS3 produces a cellobiase which hydrolyzes cellobiose and PNPG. The K(m) values for cellobiose and PNPG indicated that the beta-glucosidase activity of C.flavigena had a higher affinity for cellobiose as substrate, whereas the beta-glucosidase from E. coli pJS3 showed higher affinity for PNPG.