Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A. Winters, J. Gallagher, N. Barron, A. Rollan, A. P. McHale

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Abstract

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.
Original languageEnglish
Pages (from-to)1387-1390
Number of pages4
JournalBiotechnology Letters
Volume18
Issue number12
DOIs
Publication statusPublished - Dec 1996

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