地衣芽孢杆菌α-淀粉酶基因的克隆和及其启动子功能鉴定

根据已知α-淀粉酶编码基因保守区核苷酸序列,通过PCR和反向PCR技术克隆出Bacillus licheniformisCICIM B0204α-淀粉酶编码基因amyL全长序列及其上下游序列。B.licheniformisCICIM B0204amyL由1539bp组成,其上游180bp为启动子序列,下游160bp为终止子序列;成熟肽由512个氨基酸残基组成,氨基端的29个氨基酸残基为α-淀粉酶的信号肽。通过基因及其氨基酸序列比对发现,amyL及其编码产物与芽孢杆菌来源的α-淀粉酶具有高度相似性。将amyL的结构基因在PT7介导下于大肠杆菌中诱导表达,获得具有α-淀粉酶活性的表达产物。将amyL的启动子序列和信号肽序列与B.licheniformisCICIM B2004的β-甘露聚糖酶结构基因进行读框内重组,在大肠杆菌中获得了β-甘露聚糖酶的分泌表达,重组大肠杆菌表达295U/mL的β-甘露聚糖酶酶活。

Cloning of the gene encoding a thermostable a-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.