产碱假单胞菌碱性脂肪酶的克隆表达及酶学性质  

Gene Cloning,Expression and Characterization of An Alkaline Lipase from Pseudomonas alcaligenes PA-9

作　　者：刘滔滔[1,2] 刘明瑞[1,2] 刘恒嘉 杜丽琴[1,2] 梁智群[1,2] 韦宇拓[1,2] 

机构地区：[1]广西大学生命科学与技术学院,广西南宁530005 [2]亚热带农业资源保护与利用国家重点实验室,广西南宁530005

出　　处：《广西科学》2016年第3期248-254,共7页Guangxi Sciences

基　　金：国家自然科学基金项目（31460437）资助

摘　　要：【目的】为获得可应用于酯类水解及合成的脂肪酶资源,本研究通过筛选分离得到能够水解长链脂肪酸酯的脂肪酶产生菌,克隆表达其脂肪酶基因并研究脂肪酶的酶学性质。【方法】从环境中筛选分离出可水解三硬脂酸甘油酯的菌株,利用16S rDNA对其进行分子鉴定,并扩增其脂肪酶基因和脂肪酶分子伴侣基因。以 pET-22 b（＋）为表达载体,构建共表达重组质粒,转化Escherichia coli BL21（DE3）进行异源表达,并对重组酶进行酶学性质研究。【结果】经16 S rDNA鉴定该菌株为产碱假单胞菌Pseudomonas alcaligenes。通过 PCR 成功克隆到该菌的脂肪酶基因（lipPA-9A）和脂肪酶分子伴侣基因（lipPA-9B）,并构建共表达重组质粒 pET22b-lipPA-9A-9B ,实现脂肪酶 LIP-9 A的活性表达。酶学性质研究表明 LIP-9 A的最适反应温度为35℃,最适反应 pH值为10.5,最适反应底物为对硝基苯酚辛酸酯（pNPO）；同时,LIP-9A 还可以催化醇和羧酸发生酯化反应产生酯类物质。【结论】LIP-9 A在碱性条件下具有较高活力,且可以催化酯化反应,在洗涤行业和酯合成领域具有一定的应用价值。Objective]In order to obtain the lipase that can be applied to the hydrolysis and syn-thesis of esters,the alkaline lipase-producing strains that hydrolyze long chain fatty acid ester were screened and isolated.The related genes were cloned and expressed,and enzyme charac-terization was studied.[Methods]A strain,which degraded glycerol tristearate,was isolated from the environment,and identified based on 16S rDNA sequence analyses.Then the lipase gene and the lipase chaperone gene were amplified by PCR.The target genes lipPA-9A and lipPA-9B were introduced into expression vector pET-22b（＋）and induced for expression inEscherichiacoli BL21（DE3）.Finally,the char-acteristics of the recombinant enzyme were stud-ied in detail.[Results]The strain was identified as the genus of Pseudomonas alcaligenes by ana-lyzing of 16S rDNA sequence.And the lipase gene （lipPA-9A ） and lipase chaperone gene （lipPA-9B）were cloned.The co-expression re-combinant plasmid of pET22b-lipPA-9A-9B was successfully constructed and expressed in E. coli BL21（DE3）.The maximum activity of LIP-9A was obtained at 35℃,pH 10.5,and pNPO was the most suitable substrate.Meanwhile,the active LIP-9A could catalyze fatty alcohols and fatty acids to generate esters.[Conclusion]LIP-9A is a lipase with relatively high activity in al-kaline conditions and can catalyze esterification reaction.Its characteristics are of high value in the detergent industry and biocatalytic applications.

关 键 词：产碱假单胞菌 碱性脂肪酶 共表达 酯化 

分 类 号：Q78[生物学—分子生物学] TQ925.6[轻工技术与工程—发酵工程]