乙酸钙不动杆菌磷脂酶C在大肠杆菌中重组表达、纯化及酶学性质分析

Overexpression， purification and characterization of phospholipase C from Acinetobacter calcoaceticus

摘要:

摘要:【目的】构建乙酸钙不动杆菌(Acinetobacter calcoaceticus)ATCC17902磷脂酶C(Phospholipase C，PLC)的重组大肠杆菌菌株、纯化重组酶并进行酶学性质分析比较。【方法】以A.calcoaceticus ATCC17902基因组DNA为模板，PCR扩增得到两段磷脂酶C基因(PLC1、PLC2)，构建重组大肠杆菌表达质粒并转化大肠杆菌BL21(DE3)中，实现PLC1、PLC2基因的表达。IPTG诱导表达后，经镍柱亲和层析纯化重组蛋白。【结果】成功构建两株产磷脂酶C的重组大肠杆菌并纯化，样品经SDS-PAGE分析在80 kDa附近均出现显著的特异性条带。NPPC法测得PLC1、PLC2酶活分别为31160± 418 U/mg、13640±354 U/mg，最适反应温度分别为65、50℃，最适pH值分别为8、7.5。在低于30℃时，pH值7－8时，PLC1、PLC2重组酶较稳定，40℃处理30min，PLC1酶活稳定而PLC2残余酶活低于25%。Mg2+ 、Ca2+ 增强PLC1、PLC2 的活性，Zn2+ 增强PLC1酶活性却抑制PLC2酶活。底物特异性分析表明PLC1、PLC2 均水解磷脂酰肌醇(Phosphatidylinositol，PI)，对其他种类磷脂不能水解或水解程度很低。【结论】本文首次实现了A． calcoaceticus ATCC17902来源的磷脂酶C的重组表达与功能验证，为其它食品安全性微生物来源的磷脂酶C的研究提供了一定的借鉴意义。

Abstract:

Abstract:［Objective］In this study，we constructed two recombinant Escherichia coli strains to produce phospholipase C (PLC) from Acinetobacter calcoaceticus．The recombinant enzymes were purified to homogeneity and characterized．［Methods］ We cloned the PLC encoding gene plc1，plc2 from genome DNA of A. calcoaceticus ATCC17902．The amplified fragments were inserted into pET28a (+) to obtain expression plasmids．E．coli BL21 (DE3) harboring the above plasmids were cultivated and induced with isopropyl-β-D-thiogalactopyranoside to express PLCs．The recombinant PLCs were purified by affinity chromatography and their catalytic properties were characterized．［Results］Two PLCs from A．calcoaceticus were cloned and functional expressed in E．coli．The recombinant enzymes have activities of 31160 ± 418 U/mg for PLC1 and 13640 ± 354 U/mg for PLC2，when using p-nitrophenyl phosphorycholine as substrate．The purified PLC1 and PLC2 exhibited optimum temperature at 65o C and 50o C，respectively．Their optimal pH were 8 and 7. 5，respectively．PLC2 was stable under 40oC and pH at 8，whereas the residual activity of PLC1 was less than 25% in the same condition．Mg2 + and Ca2 + stimulated two enzymes activity，whereas Zn2 + stimulated PLC1 and inhibited PLC2． PLC1 and PLC2 hydrolyzed phosphatidylinositol．［Conclusion］It is the first time to express and characterize the PLC gene from A． calcoaceticus ATCC17902．These research results provide reference for the study of food-safety microbiological PLC．