天然高产脂肪酶细菌的分离鉴定及脂肪酶基因的分析  

Isolation and identification of high-yield lipase producing bacteria and analysis of lipase gene

作　　者：张迎光[1] 施晓聪[1] 林全思[1] 王震[1] 吕建新[1] 

机构地区：[1]温州医学院检验与生命科学学院浙江省医学遗传学重点实验室,浙江温州325035

出　　处：《温州医学院学报》2009年第6期541-545,共5页Journal of Wenzhou Medical College

基　　金：温州市科技局科研基金资助项目(Y20060123)

摘　　要：目的:从环境中分离高产脂肪酶的细菌,并克隆其脂肪酶基因。方法:从天然高油脂含量土壤筛选高产脂肪酶的细菌,鉴定菌种后,设计相应引物PCR扩增脂肪酶基因并进行TA克隆,重组载体转入E.coli DH5α构建工程菌,双酶切及测序鉴定脂肪酶基因。结果:经过微生物菌种鉴定获得4株高活力菌,其中一株金葡菌产酶最高,利用PCR技术成功扩增脂肪酶基因,该基因经过克隆测序及其双酶切鉴定证实为新发现的突变脂肪酶基因,其二级结构与标准基因(EU310372)有较大不同。结论:成功分离一株高产脂肪酶的菌株——金黄色葡萄球菌,克隆的脂肪酶基因为新发现的突变型。Objective：To separate natural bacteria producing high activities lipase from environment,and clone its lipase gene.Methods：High activities lipase producing bacteria was screened from soil which contained plenty of oils and fats.After identifying bacteria,PCR amplified lipase gene which was cloned to pMD18-T Vector.The recombint plasmid was transformed into E.Coli DH5α to build the engineering bacteria.At last,the lipase gene was identified with two restriction endonuclease digesting and sequencing.Results： After identifiying, 4 producing high activitie lipases bacteria strains were obtained, one of which was a Staphylococcus aureus producing highest activities lipase,its lipase gene was amplified by PCR,then separated and identified as a new mutant lipase gene by restriction endonuciease digesting and sequencing, standard lipase gene registered on NLOBI （EU310372）, The secondary structure of the new mutant lipase gene was more different from standard lipase. Conclusion： A strain of high activities lipase producing bacteria-Staphylococcus aureus is successfully isolated and the cloned lipase gene is a new discovered mutant type.

关 键 词：脂肪酶基因 PCR TA克隆 

分 类 号：Q785[生物学—分子生物学]