[Cloning of the gene encoding a thermostable alpha-amylase from bacillus licheniformis CICIM B0204 and functional identification of its promoter].
Thermostable alpha-amylase, catalyzing the hydrolyzation of starch to dextrin, maltose and glucose at higher temperature, is one of the most industrial important enzymes. Several species of Bacillus have been found and genetic improved to produce the thermostable alpha-amylases. In present study, a gene, amyL, coding for a thermostable alpha-amylase with its flanking sequences was cloned from an industrial Bacillus licheniformis CICIM B0204 by using a combination of routine polymerase chain reactions (PCR) and inverse PCR with a pair of initial primers derived from the highly conserved region of bacterial alpha-amylase genes and the functional identifications of the cloned amyL and the activities of its promoter and signal peptide in Escherichia coli were investigated. The amyL was composed of 1539 bp with 180 bp at upstream for its promoter and 160 bp at downstream for its terminator. The deduced mature peptide of the a-amylase was composed of 512 amino acid residues and its signal peptide 29 amino acid residues at N-terminal. The nucleotide and deduced amino acid sequences of amyL were extremely similarity to those from Bacillus species with three amino acid residues difference (Arg163-->Leu, Ser339-->Gly, Ala349-->Ser) comparison to that from a laboratory strain B. licheniformis 584. Under the control of T7 promoter, the structural region of amyL was functionally expressed in Escherichia coli. Additionally, the structural region of the gene coding for a beta-mannosidase from B. licheniformis CICIM B2004 was inframely inserted into the downstream of the promoter and signal sequence of amyL and expressed in E. coli. The amyL promoter and signal sequence was functionally directed the expression and secretion of the beta-mannosidase in E. coli cells with the expression level of 295 U/mL.
Niu
Dan-dan
DD
The Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Southern Yangtze University, Wuxi 214036, China.
Xu
Min
M
Ma
Jun-shuang
JS
Wang
Zheng-xiang
ZX
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D011401:Promoter Regions, Genetic / Q000235:genetics*; D000516:alpha-Amylases / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D000516:alpha-Amylases