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A gene encoding for an alpha-amylase from thermophilic Bacillus sp. strain TS-23 and its expression in Escherichia coli

Lin, L.L., Hsu, W.H., Chu, W.S.
Journal of applied microbiology 1997 v.82 no.3 pp. 325-334
Bacillus (bacteria), Escherichia coli, Salmonella Typhimurium, alpha-amylase, amino acid sequences, amino acids, cell fractionation, enzyme activity, genes, open reading frames, plasmids, polyacrylamide gel electrophoresis
An alpha-amylase gene from Bacillus sp. strain TS-23 was cloned and expressed by using its own promoter on the recombinant plasmid pTS917 in Escherichia coli. A cell fractionation experiment revealed that approximately 60% of the amylase activity was in the periplasmic space. Analysis and activity staining of the concentrated supernatant fraction by SDS-polyacrylamide gel electrophoresis showed an apparent protein band with a mol. wt of approximately 65 000. The amylase gene (amyA) consisted of an open reading frame of 1845 bp encoding a protein of 613 amino acids with a calculated mol. wt of 69 543. The predicted amino acid sequence showed high homology with Bacillus species, E. coli and Salmonella typhimurium alpha-amylases. Deletion of 96 amino acids from the C-terminal portion of the amylase did not result in the loss of amylolytic activity. The truncated amylase, deletion of the first 50 amino acids from the N-terminus, was overexpressed in E. coli system and refolded to yield an activable enzyme.