产碱假单胞菌脂肪酶的克隆表达及酶学性质研究

Cloning, expression and characterization of lipase from Pseudomonas alcaligenes

【目的】克隆产碱假单胞菌的脂肪酶基因,实现其在大肠杆菌中异源表达并进行酶学性质研究。【方法】通过基因文库构建和PCR,获得脂肪酶基因,并以pET30a(+)为表达载体、E.coli BL21(DE3)为宿主菌,在大肠杆菌中进行异源表达,表达产物经HisTrapTM亲和层析柱纯化后进行酶学性质研究。【结果】从产碱假单胞菌中克隆得到一个脂肪酶基因,大小为1 575 bp(GenBank登录号为JN674069)。该酶分子量为55 kD,最适底物为p-NPO,最适反应温度和pH分别为35°C、pH 9.0。重组酶经1 mmol/L的Cu2+处理30 min可使酶活提高至156%。在最适反应条件下重组酶的比活力为275 U/mg,Km和Vmax分别为80μmol/L和290 mmol/(min.g protein)。【结论】产碱假单胞菌脂肪酶基因的克隆与表达不仅积累了脂肪酶基因的资源,并为其在手性拆分中的应用奠定基础。

[Objective] The lipase gene of P. alcaligenes was cloned and expressed in Es- cherichia coli, the enzymatic properties were characterized. [Methods] The lipase gene was obtained by constructing the genomic library of P. alcaligenes and PCR. Then the gene over- expressed in E. coli BL21 （DE3） with plasmid pET30a（＋）. The recombinant lipase was purified with HisTrapTM affinity chromatography and the enzymatic properties were determined. [Re- sults] A 1 575 bp lipase gene was attained （GenBank assession number： JN674069）. The mo- lecular weight of lipase was 55 kD, the optimal substrate of the lipase was p-NPO, the optimal temperature and pH were 35 ~C and pH 9.0. The lipase activity was increased to 156% after treated by 1 mmol/L Cu2＋ for 30 min. Under the optimum conditions, the specific activity, Km and Vmax of the enzyme were 275 U/mg, 80μmol/L and 290 mmol/（min.g protein）, respectively. [Conelusion] The cloning and expressing of lipase provided the fundamental to the application in chiral resolution.