A GENE ENCODING FOR AN ALPHA-AMYLASE FROM THERMOPHILIC BACILLUS SP STRAIN TS-23 AND ITS EXPRESSION IN ESCHERICHIA-COLI

An alpha-amylase gene from Bacillus sp. strain TS-23 was cloned and ex pressed by using its own promoter on the recombinant plasmid pTS917 in Escherichia coli. A cell fractionation experiment revealed that appro ximately 60% of the amylase activity was in the periplasmic space. Ana lysis and activity staining of the concentrated supernatant fraction b y SDS-polyacrylamide gel electrophoresis showed an apparent protein ba nd with a mel. wt of approximately 65 000. The amylase gene (amyA) con sisted of an open reading frame of 1845 bp encoding a protein of 613 a mino acids with a calculated mel. wt of 69543. The predicted amino aci d sequence showed high homology with Bacillus species, E. cell and Sal monella typhimurium alpha-amylases. Deletion of 96 amino acids from th e C-terminal portion of the amylase did not result in the loss of amyl olytic activity. The truncated amylase, deletion of the first 50 amino acids from the N-terminus, was overexpressed in E. coli system and re folded to yield an activable enzyme.