초록
A Bacillus strain with high productivity of α-amylase isolated from a starch farm was identified as Bacillus amyloliquefaciens. The α-amylase encoding gene amy1 was cloned into pMD18-T vector and amplified in E. coli DH5α. Shuttle vector pP43MNX was reconstructed to obtain vector pP43X for heterologous expression of the α-amylase in B. subtilis WB800. Recombinant enzyme was sufficiently purified by precipitation, gel filtration and anion exchange with a specific activity of 5566U/mg. The α-amylase sequence contains an open reading frame of 1545bp, which encodes a protein of 514 amino acid residues with a predicted molecular mass of 58.4kDa. The enzyme exhibited maximal activity at pH 6.0 and 60<SUP>o</SUP>C. Catalytic efficiency of the recombinant α-amylase was inhibited by Hg<SUP>2+</SUP>, Pb<SUP>2+</SUP> and Cu<SUP>2+</SUP>, but stimulated by Li<SUP>+</SUP>, Mn<SUP>2+</SUP> and Ca<SUP>2+</SUP>. 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 α-amylase in recombinant B. subtilis WB800.