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 m and V max of AmyQ A were 3.40 g/L and 15.70 g/(L min−1), respectively, and the K m and V max of AmyQ B were 2.01 g/L and 6.95 g/(L min−1), respectively. The activity of AmyQ A was enhanced by K+, Mn2+, Co2+ and Ca2+, and the activity of AmyQ B was enhanced in the presence of K+, Co2+ and Ca2+.
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This work was supported financially by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the National Natural Science Foundation of China (20836003), the Major State Basic Research Development Program of China (973 Program, 2012CB720806), and the National High Technology Research and Development Program of China (863 Program, 2011AA100905), and 111 Project (111-2-06).
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Yang, H., Liu, L., Li, J. et al. Cloning, heterologous expression, and comparative characterization of a mesophilic α-amylase gene from Bacillus subtilis JN16 in Escherichia coli . Ann Microbiol 62, 1219–1226 (2012). https://doi.org/10.1007/s13213-011-0364-9
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DOI: https://doi.org/10.1007/s13213-011-0364-9