Molecular Cloning and Expression of an α-Amylase Gene from an α-Amylase Extrahyper Producing Bacillus subtilis

Abstract

Bacillus subtilis 2633 hyperproduced α-amylase (about 3000-fold amount of the parental strain, B.subtilis 6160). An α-amylase gene of B. subtilis 2633 was cloned into Escherichia coli using a B. subtilis–E. coli shuttle vector, pHY300PLK. When the recombinant plasmid pAM26 containing the α-amylase gene was introduced into B. subtilis 6160, a transformant produced about 40,000 U/ml of α-amylase, which is 4000 times that of B. subtilis 6160. This indicates that the α-amylase gene (amyR-amyE) of 2633 has hyperproducing activity in itself. The nucleotide sequencing of the promotor region and comparison with those of other B. subtilis strains suggested that several differences in the nucleotide sequences upstream from the transcription initiation site could be responsible for the α-amylase production. Southern blot analysis showed that B. subtilis 2633 contains several copies of the α-amylase gene, which should also contribute to the hyperproductivity.