In this study, the gene of mesophilic α-amylase(amyL) from a mesophilic α-amylase-producing strain (Bacillus subtilis 264) has been cloned into an expression plasmid pAX01 at Bam HI and SacII sites, forming an integrative recombinant plasmid pAX01-amyL.Following its transformation into B.subtilis 264, the recombinant plasmid pAX01-amyL mediates chromosomal integration of the amyL expression cassette by homologous recombination at lacA locus, and therefore, increases the copy number of the amyL genein B.subtilis 264. As a proof of concept, a high-level expression transformant, designated as B. subtilis ZHWY, was isolated and verified by Southern blot. It was shown that the secreted α-amylase activity of B. subtilis ZHWY in a 500mL shake flask reached 730U/mL (156U/mg protein) which was an improvement of 70% when compared with the wildtype strain B.subtilis 264. Moreover, in a 5L fermenter, B.subtilis ZHWY afforded a high α-amylase activity of 1450U/mL, demonstrating its good potential for the industrial production of mesophilic α-amylase.
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