Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli
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- Winters, A., Gallagher, J., Barron, N. et al. Biotechnol Lett (1996) 18: 1387. doi:10.1007/BF00129340
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A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-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 β-glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl-β-D-glucoside were 0.19mM and 0.25mM, respectively.