腐生葡萄球菌M36耐有机溶剂脂肪酶基因的克隆与原核表达  

Cloning and expression of organic solvent tolerant lipase gene from Staphylococcus saprophyticus M36

作　　者：汤彦翀[1] 卢亚萍[1] 吕凤霞[1] 别小妹[1] 郭瑶[1] 陆兆新[1] 

机构地区：[1]南京农业大学食品科学与技术学院酶工程研究室,南京210095

出　　处：《生物工程学报》2009年第12期1989-1995,共7页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2008AA10Z309);江苏省自然基金(No.BK2007160);江苏省高技术计划(No.BE2008308)资助~~

摘　　要：脂肪酶是重要的工业用酶,在食品加工、生物柴油的合成等领域具有广泛的应用。但是在应用中有机溶剂对脂肪酶具有一定的毒性,因此获得耐有机溶剂的脂肪酶基因并实现高效表达是脂肪酶规模化应用的前提。本研究应用PCR技术首次从耐有机溶剂脂肪酶产生菌腐生葡萄球菌M36基因组DNA中扩增得到脂肪酶Ⅲ基因lip3(GenBank AccessionNo.FJ979867),其编码区长度为741bp,编码247个氨基酸,推测蛋白分子量大小为31.6kD。它与腐生葡萄球菌lip3推测的基因(GenBank AccessionNo.AP008934)只有83%的同源性。将该基因与大肠杆菌表达载体pET-DsbA连接,转化大肠杆菌EscherichiacoliBL21(DE3)获得重组菌株BL21(DE3)/pET-DsbA-lip3,在pH8、25oC条件下,OD600为1.0时用0.4mmol/LIPTG诱导12h酶活达到25.8U/mL。重组酶在甲醇、正己烷、异辛烷、正庚烷等有机溶剂中具有较好的耐性。lip3基因的克隆及在大肠杆菌中有效表达的研究为进一步进行基因工程改造和脂肪酶应用奠定了基础。Lipases are important biocatalysts that are widely used in food processing and bio-diesel production.However,organic solvents could inactivate some lipases during applications.Therefore,the efficient cloning and expression of the organic solvent-tolerant lipase is important to its application.In this work,we first found out an organic solvent-tolerant lipase from Staphylococcus saprophyticus M36 and amplified the 741 bp Lipase gene lip3 （GenBank Accession No.FJ979867）,by PCR,which encoded a 31.6 kD polypeptied of 247 amino acid residues. But the lipase shared 83% identity with tentative lip3 gene of Staphylococcus saprophyticus （GenBank Accession No. AP008934）. We connected the gene with expression vector pET-DsbA, transformed it into Escherichia coli BL21 （DE3）, and obtained the recombinant pET-DsbA-lip3. With the induction by 0.4 mmol/L of isopropyl beta-D-thiogalactopyranoside at pH 8.0, OD600 1.0, 25℃ for 12 h, the lipase activity reached up to 25.8 U/mL. The lipase expressed was stable in the presence of methanol, n-hexane, and isooctane, n-heptane.

关 键 词：腐生葡萄球菌 lip3基因 耐有机溶剂脂肪酶 基因克隆 序列分析 原核表达 

分 类 号：Q78[生物学—分子生物学]