天然高产脂肪酶细菌的分离鉴定及脂肪酶基因的分析.docVIP

天然高产脂肪酶细菌的分离鉴定及脂肪酶基因的分析 作者：张迎光，施晓聪，林全思，王震，吕建新 【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。 【关键词】 脂肪酶基因；PCR；TA克隆 　　Abstract: 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 DH5αto 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 endonuclease digesting and sequencing. standard lipase gene registered on NCBI (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. 　　Key words: lipase gene；PCR；TA cloning 　　脂肪酶是一类酯键水解酶，广泛存在于微生物、植物种子及动物组织中，能在油水界面上催化脂类物质分解、合成和酯交换反应。作为重要的工业用酶，脂肪酶在医药、食品、制革、饲料、洗涤、油酯化工等传统工业领域有着广泛的应用，如可添加在洗涤剂增加去油脂能力，添加在高脂食物以增加口感，而近年来其在生物能源、有机合成、药物手性拆分和工具酶等新领域也展现了良好的应用前景[1]。以上领域应用的脂肪酶，或为天然菌分离或为基因重组来源，但其微生物来源脂肪酶应用最广泛[2-5]。据统计，细菌有28个属，放线菌有4个属，酵母菌有10个属，其他真菌有23个属，共计达65个属的微生物产脂肪酶。而动物脂肪酶、植物脂肪酶由于其作用底物谱窄，体外表达活性、产量低，该类基因很少作为基因工程的脂肪酶来源基因[6-8]。 　　本课题拟通过从高含油脂的环境中分离到产高活力脂肪酶的微生物并克隆高活力脂肪酶基因，为后续的获得工程菌清除高油脂污水、获得较高活力脂肪酶基因并通过