大肠杆菌中青霉素G酰化酶成熟的限制性因素  

The Bottleneck Steps Limiting Maturation of Penicillin G Acylase in Escherichia coli

作　　者：徐京宁[1] 杨运桂[2] 龚毅[2] 杨胜利[2] 俞俊棠[1] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]中国科学院上海生物工程研究中心,上海200233

出　　处：《生物工程学报》2001年第5期570-574,共5页Chinese Journal of Biotechnology

摘　　要：为了研究大肠杆菌中青霉素G酰化酶 (PenicillinGacylase ,PAC)成熟的限制性步骤 ,分别构建了PAC表达质粒pKKpacSP ,pETpacSP ,并将它们在大肠杆菌宿主中表达。通过酶活性的测定及Westernblotting分析 ,分别在PAC自身表达系统 ,Tac ,T7及氧调控表达系统中 ,研究PAC前体蛋白加工成α亚基、β亚基的效率及亚基折叠组装形成活性酶的能力。结果表明 :PAC成熟过程中的限制性步骤因宿主 载体系统而异 ;PAC本身表达系统中 ,前体肽加工成α亚基、β亚基的效率为 5 7 2 % ,亚基组装能力为 0 72 ;Tac启动子表达调控系统中α亚基的折叠和稳定成为限制性步骤 ;T7及氧调控表达系统中 ,PAC前体肽加工成α亚基、β亚基的效率达 90 %左右 ,亚基组装成活性酶的能力分别为 1 82 ,2 ;低氧调控系统表达PAC时 ,成熟的效率最高 ,PAC表达的单位重量活性提高We have identified the bottleneck steps limiting maturation of penicillin G acylase (PAC) through comparison of the maturation performance for various PAC\|expression systems (Pac,Tac,T7,Vgb+T7)with different efficiencies of proteolysis,subunit folding and assembly.The maturation of PAC could be limited by various steps,such as translocation,periplasmic proteolysis,subunit folding and assembly depending on the host/vector systems.In BL21(pPA6)cells,maturation of PAC were limited by proteolysis and folding steps;the efficiency of proteolysis was 57.2%;the subunit folding and assembly capacity was 0.72.In BL21(pKKpacSP)cells,the stability and folding of α subunit was bottleneck steps.In T7 and dissolved\|oxygen regulation expression systems,PAC proprecursor could be maturated efficiently.Results also indicate that the folding of α peptide plays a key role in folding of precursor for PAC in \%E.coli.\%Developing proper host/vector systems and fermentation technology with superior abilities on subunit folding and assembly of precursor for PAC could be plausible for enhancing production of PAC.In this study,pac could be expressed (transcribed,translated and maturated) efficiently under the control of T7 promoter.

关 键 词：青霉素G酰化酶 成熟过程 宿主/载体系统 大肠杆菌 限制性因素 

分 类 号：Q78[生物学—分子生物学]