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A gene encoding for an α‐amylase from thermophilic Bacillus sp. strain TS‐23 and its expression in Escherichia coli
Author(s) -
Lin L.L.,
Hsu W.H.,
Chu W.S.
Publication year - 1997
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1046/j.1365-2672.1997.00364.x
Subject(s) - escherichia coli , biology , amylase , microbiology and biotechnology , amino acid , biochemistry , peptide sequence , enzyme , gene
An α‐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 ( amy A) 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 α‐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.