Cloning, enhanced expression and characterization of an alpha-amylase gene from a wild strain in B. subtilis WB800

摘要

A Bacillus strain with high productivity of alpha-amylase isolated from a starch farm was identified as Bacillus amyloliquefaciens. The alpha-amylase encoding gene amyl was cloned into pMD18-T vector and amplified in E. coli DH5 alpha. Shuttle vector pP43MNX was reconstructed to obtain vector pP43X for heterologous expression of the alpha-amylase in B. subtilis WB800. Recombinant enzyme was sufficiently purified by precipitation, gel filtration and anion exchange with a specific activity of 5566 U/mg. The alpha-amylase sequence contains an open reading frame of 1545 bp, which encodes a protein of 514 amino acid residues with a predicted molecular mass of 58.4 kDa. The enzyme exhibited maximal activity at pH 6.0 and 60 degrees C. Catalytic efficiency of the recombinant alpha-amylase was inhibited by Hg2+, Pb2+ and Cu2+, but stimulated by Li+, Mn2+ and Ca2+. The purified enzyme showed decreased activity toward detergents (SDS, Tween 20 and Triton X-100). Compared with production by the wild strain, there was a 1.48-fold increase in the productivity of alpha-amylase in recombinant B. subtilis WB800. (C) 2015 Elsevier B.V. All rights reserved.