Cloning, heterologous expression, and comparative characterization of a mesophilic α-amylase gene from Bacillus subtilis JN16 in Escherichia coli

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 Km and Vmax of AmyQ A were 3.40 g/L and 15.70 g/(L min-1), respectively, and the Km and Vmax 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+, Co 2+ and Ca2+. © Springer-Verlag and the University of Milan 2011.