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- Title
Cloning, heterologous expression, and comparative characterization of a mesophilic α-amylase gene from Bacillus subtilis JN16 in Escherichia coli.
- Authors
Yang, Haiquan; Liu, Long; Li, Jianghua; Du, Guocheng; Chen, Jian
- Abstract
A gene encoding mesophilic α-amylase from Bacillus subtilis JN16 was identified and designated as AmyQ. The AmyQ gene was cloned, sequenced, and expressed in Escherichia coli. AmyQ is 1,980 bp in length and encodes a protein of 660 amino acids. AmyQ was cloned in plasmid pET20b (+) on an NcoI- BamHI fragment, and used to transform competent E. coli amylase-negative cells (BL21); ampicillin-resistant transformants were screened for the production of α-amylase. The recombinant α-amylase encoded in E. coli was designated AmyQ A, and α-amylase from wild-type B. subtilis strain JN16 was designated AmyQ B. AmyQ A was characterized biochemically and showed maximal activity at pH 7.0 and maximal stability at pH 5.5; the optimum temperature for enzymatic activity was close to 70°C. The optimal pH for purified AmyQ B was 7.5. With soluble starch as substrate, the K and V of AmyQ A were 3.40 g/L and 15.70 g/(L min), respectively, and the K and V of AmyQ B were 2.01 g/L and 6.95 g/(L min), respectively. The activity of AmyQ A was enhanced by K, Mn, Co and Ca, and the activity of AmyQ B was enhanced in the presence of K, Co and Ca.
- Publication
Annals of Microbiology, 2012, Vol 62, Issue 3, p1219
- ISSN
1590-4261
- Publication type
Article
- DOI
10.1007/s13213-011-0364-9