关键词: 中度嗜盐菌, α-淀粉酶, 克隆, 表达

Abstract:

A strain of moderately halophilic Bacillus sp.XJ1-05 producing α-amylase was isolated from a salt lake, Aibi Lake in Xinjiang by our lab. With the prime designed on the basis of the published converse nucleotide sequence of α-amylase gene, DNA fragment of α- amylase gene was obtained from Bacillus sp.XJ1-05 and amplified by PCR, cloned into vector pGM-T and sequenced. The results showed that the fragment length of α- amylase gene is about 1500 bp and this α- amylase gene has a homology of 95% with the α-amylase gene sequence of Bacillus licheniformis. Then the gene fragment was directionally subcloned into prokaryotic expression vector pET-32a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) and then expressed under the induction of IPTG. SDS-PAGE pattern showed that the α-amylase gene was successfully expressed in prokaryotic expression vector E. coli BL21, and the relative molecular weight of the expressed protein is about 61 ku, which is consistent with that from theoretical derivation. Furthermore, the α-amylase produced by the constructed E. coli engineering bacteria was inclusion body. After smashing the inclusion boby with ultrasonic cell smash instrument, the releasedα-amylase activity was determined. It was calculated that the α-amylase activity was 1.8 times as much as that produced by original strain.

Key words: moderately halophilic bactrium, α- amylase, cloning, prokaryotic expression