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刘滔滔,刘明瑞,刘恒嘉,杜丽琴,梁智群,韦宇拓.产碱假单胞菌碱性脂肪酶的克隆表达及酶学性质[J].广西科学,2016,23(3):248-254. [点击复制]
- LIU Taotao,LIU Mingrui,LIU Hengjia,DU Liqin,LIANG Zhiqun,WEI Yutuo.Gene Cloning,Expression and Characterization of An Alkaline Lipase from Pseudomonas alcaligenes PA-9[J].Guangxi Sciences,2016,23(3):248-254. [点击复制]
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| 产碱假单胞菌碱性脂肪酶的克隆表达及酶学性质 |
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刘滔滔1,2, 刘明瑞1,2, 刘恒嘉1,2, 杜丽琴1,2, 梁智群1,2, 韦宇拓1,2
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| (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) |
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| 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-22b (+)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16S 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-9A的活性表达。酶学性质研究表明LIP-9A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-9A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。 |
| 关键词: 产碱假单胞菌 碱性脂肪酶 共表达 酯化 |
| DOI：10.13656/j.cnki.gxkx.20160713.008 |
| 投稿时间：2016-05-08修订日期：2016-06-19 |
| 基金项目:国家自然科学基金项目(31460437)资助。 |
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| Gene Cloning,Expression and Characterization of An Alkaline Lipase from Pseudomonas alcaligenes PA-9 |
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LIU Taotao1,2, LIU Mingrui1,2, LIU Hengjia1,2, DU Liqin1,2, LIANG Zhiqun1,2, WEI Yutuo1,2
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| (1.College of Life Science & Technology of Guangxi University, Nanning, Guangxi, 530005, China;2.State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Nanning, Guangxi, 530005, China) |
| Abstract: |
| [Objective] In order to obtain the lipase that can be applied to the hydrolysis and synthesis 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 characterization 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 in Escherichia coli BL21(DE3).Finally,the characteristics of the recombinant enzyme were studied in detail.[Results] The strain was identified as the genus of Pseudomonas alcaligenes by analyzing of 16S rDNA sequence.And the lipase gene (lipPA-9A) and lipase chaperone gene (lipPA-9B) were cloned.The co-expression recombinant 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 alkaline conditions and can catalyze esterification reaction.Its characteristics are of high value in the detergent industry and biocatalytic applications. |
| Key words: Pseudomonas alcaligenes alkaline lipase co-expression esterification |
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