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 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 1,845 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.
Lin
L L
LL
Department of Food Nutrition, Hung Kuang Institute of Medical and Nursing Technology, Shalu, Taichung, Taiwan.
Hsu
W H
WH
Chu
W S
WS
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D000516:alpha-Amylases