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

作　　者：汪艳红[1] 张梁[1] 顾正华[1] 丁重阳[1] 石贵阳[1] 

机构地区：[1]江南大学,粮食发酵工艺与技术国家工程实验室,工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《微生物学报》2014年第10期1221-1227,共7页Acta Microbiologica Sinica

基　　金：国家“863计划”(2011AA100905);教育部“新世纪优秀人才支持计划”(NCET-11-0665);江南大学食品科学与技术国家重点实验室自由探索资助课题(SKLF-ZZA-201201)~~

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。[ 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 plcl, 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 65℃ and 50℃, respectively. Their optimal pH were 8 and 7.5, respectively. PLC2 was stable under 40℃ and pH at 8, whereas the residual activity of PLC1 was less than 25% in the same condition. Mg^2＋ and Ca^2＋ stimulated two enzymes activity, whereas Zn^2＋ 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.

关 键 词：乙酸钙不动杆菌 磷脂酶C 纯化 重组表达 

分 类 号：Q78[生物学—分子生物学]