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

A Winters, J Gallagher, N Barron, A Rollan, AP McHale

Research output: Contribution to journalArticle

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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.
LanguageEnglish
Pages1387-1390
JournalBiotechnology Letters
Volume18
Issue number12
DOIs
Publication statusPublished - Dec 1996

Fingerprint

Micromonospora
Restriction Mapping
beta-Glucosidase
Molecular Cloning
Escherichia coli
Chromogenic Compounds
Genomic Library
Actinobacteria
DNA
Glucosides
Enzymes
Genes
Plasmids
Hot Temperature
Phenotype

Cite this

Winters, A ; Gallagher, J ; Barron, N ; Rollan, A ; McHale, AP. / Molecular cloning and expression of a Micromonospora chalcae beta-glucosidase encoding gene in Escherichia coli. In: Biotechnology Letters. 1996 ; Vol. 18, No. 12. pp. 1387-1390.
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Molecular cloning and expression of a Micromonospora chalcae beta-glucosidase encoding gene in Escherichia coli. / Winters, A; Gallagher, J; Barron, N; Rollan, A; McHale, AP.

In: Biotechnology Letters, Vol. 18, No. 12, 12.1996, p. 1387-1390.

Research output: Contribution to journalArticle

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AB - 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.

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