Abstract

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC 18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the beta-glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 degrees C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate beta-nitrophenyl-beta-D-glucoside were 0.19mM and 0.25mM, respectively.