不同抑制剂对谷氨酸棒状杆菌磷酸烯醇式丙酮酸羧化酶酶活的影响  

Phosphoenolpyruvate Carboxylase from Corynebacterium glutamicum Inhibited by Diverse Biochemicals

作　　者：吴新阳[1,2] 裴广胜[1,2] 郑小梅[3,2] 曹国强 刘娇[3,2] 郑平[3,2] 王敏[1] 孙际宾[3,2] 

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,天津300308 [3]中国科学院系统微生物工程重点实验室,天津300308

出　　处：《生物技术通报》2013年第7期172-178,共7页Biotechnology Bulletin

基　　金：国家重点基础研究发展计划(973计划)(2009CB724703);中科院知识创新方向性项目(KSCX2-EW-Q-13);天津市应用基础与前沿技术研究计划重点项目(10JCZDJC16200;11ZCZDSY08400;11ZCZDSY08500)

摘　　要：磷酸烯醇式丙酮酸羧化酶(Phosphoenolpyruvate carboxylase,PPC)是生物体碳代谢四碳回补途径的重要酶,是很多生物基化学品合成途径中的关键反应。为揭示谷氨酸棒状杆菌的PPC酶受体内代谢物影响的情况,对谷氨酸棒状杆菌Corynebacterium glutamicumATCC13032的ppc基因进行了PCR克隆,成功地在大肠杆菌中诱导表达为可溶性蛋白。利用Ni柱亲和层析纯化了该蛋白,酶活性测定表明其具有磷酸烯醇式丙酮酸羧化酶活性。细致研究了各种抑制剂,如柠檬酸、异柠檬酸、富马酸、琥珀酸、苹果酸、谷氨酸、天冬氨酸等对PPC酶活的影响,证实天冬氨酸和苹果酸是谷氨酸棒杆菌PPC的强抑制剂,并首次发现PPC也受到N-乙酰谷氨酰胺的抑制作用。Phosphoenolpyruvate earboxylase （ PPC ） , an enzyme involved in replenishment of the four-carbon diearboxylie acid intermediate pool of the TCA cycle, plays an.important role in both biological and biotechnological synthesis pathways. To discover the effect of diverse inhibitors on the activity of PPC, the full-length sequence of the ppc gene from Corynebacterium glutamicum ATCC13032 was cloned and expressed as soluble form in Escherichia coll. The recombinant PPC was purified with Ni-Column. The purified enzyme showed high phosphoenolpyruvate carboxylase activity, and was inhibited by diverse inhibitors such as citrate, isocitrate, fumarate, glutamate, aspartate, malate etc. Aspartate and malate were the strongest inhibitors. For the first time, the inhibition of PPC activity by N-acetyl glutamine was clearly demonstrated in a glutamate-producing strain of C. glutamicum.

关 键 词：磷酸烯醇式丙酮酸羧化酶 谷氨酸棒状杆菌 抑制剂 N-乙酰谷氨酰胺 

分 类 号：Q936[生物学—微生物学]