[Cloning and expression of the alpha-amylase gene from a Bacillus sp. WS06, and characterization of the enzyme].
A Bacillus sp. WS06, which produces an extracellular alpha-amylase, was isolated from the cecum in a piglet. An amyF gene from this Bacillus strain was cloned and its nucleotide sequence was determined. An open reading frame composed of 1581 bases, which encodes 526 amino acid residues was found. The amyF gene shows high sequence homologies with other microbial amylase genes, such as Bacillus megaterium and Bacillus polymyxa (93% and 53% identity). The deduced amino acid sequence revealed that four highly conserved regions of the alpha-amylase family. The amyF gene was overepressed using the pET21a vector and Escherichia coli BL21 (DE3). The recombinant enzyme was purified 22.2 fold to electrophoretic homogeneity and had a molecular mass of 57kD (by SDS-PAGE). The enzyme was optimally active at pH 7 and 55 approximately 60 degrees C and showed stability at the temperature below 55 degrees C. This enzyme efficiently hydrolyzed various types of starch to yield a series of malto-oligosaccharides by endo-cleavage mode.
Peng
Ping
P
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China. pengping02@163.com
Wu
Jin
J
Cheng
An-Chun
AC
Gao
Qi-Yu
QY
Zhang
Shu-Zheng
SZ
D000818:Animals; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013552:Swine / Q000382:microbiology; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D000516:alpha-Amylases