蜡样芽胞杆菌磷脂酶C在乳酸克鲁维酵母中重组表达、纯化及酶学性质分析

Heterologous expression, purification and characterization of phospholipase C from Bacillus cereus in Kluyveromyces lactis

摘要:

摘要：【目的】构建蜡样芽胞杆菌(Bacillus cereus)磷脂酶C (Phospholipase C，PLC)的重组乳酸克鲁维酵母(Kluyveromyces lactis)菌株、纯化重组蛋白并对其进行酶学性质分析。【方法】以B. cereus基因组DNA为模板，PCR扩增得到磷脂酶C基因(bcplc)，构建重组乳酸克鲁维酵母表达质粒并转化到乳酸克鲁维酵母中，实现bcplc基因的表达。利用镍柱亲和层析纯化和脱盐柱得到电泳纯的重组磷脂酶C (rbcPLC)。【结果】成功构建产磷脂酶C的重组乳酸克鲁维酵母并纯化了重组磷脂酶C，纯化后rbcPLC经SDS-PAGE分析在40 kDa附近出现显性条带。NPPC法测得rbcPLC酶活为19251 U/mg，最适反应温度为80 °C，最适pH为9.0。在低于40 °C时，pH 7.0?8.0时，rbcPLC重组酶较稳定。Cu2+和Co2+对其有明显的抑制作用；Zn2+、Mn2+、Ca2+、Mg2+对其有明显的促进作用。【结论】首次实现了对蜡样芽胞杆菌来源的磷脂酶C在乳酸克鲁维酵母中的重组表达、纯化及其酶学性质分析，为其它食品安全性微生物来源的磷脂酶C的研究提供了借鉴意义。

关键词:关键词:蜡样芽胞杆菌，乳酸克鲁维酵母，重组表达，纯化，酶学性质

Abstract:

Abstract: [Objective] In this study, we constructed recombinant Kluyveromyces lactis strains to produce phospholipase C (PLC) of Bacillus cereus. The recombinant enzymes were purified and characterized. [Methods] We cloned the PLC encoding gene bcplc of Bacillus cereus. And the amplified fragments were inserted into pKLAC1 to obtain expression plasmids. K. lactis harboring the above plasmids was cultivated to express PLC that was purified by HisTrapTM affinity chromatography and characterized. [Results] PLC of B. cereus was cloned and expressed in K. lactis. The recombinant enzyme had shown activity of 19251 U/mg when using p-nitrophenyl phosphorycholine as substrate. Purified PLC exhibited optimum temperature at 80 °C and optimal pH at 9.0. The recombinant enzyme was stable below 40 °C and pH between 7.0 and 8.0. Cu2+ and Co2+ inhibited its activity whereas Zn2+, Mn2+, Ca2+ and Mg2+ stimulated its activity. [Conclusion] It is the first time to express and characterize the PLC gene in K. lactis. These research results provide reference for the study of recombinant PLC.

Key words:Keywords: Bacillus cereus, Kluyveromyces lactis, recombinant, purification, characterization