产碱假单胞菌碱性脂肪酶的克隆表达及酶学性质

摘要

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.%【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。