腾冲嗜热厌氧菌6-磷酸果糖激酶的克隆、表达及其生物活性  

Cloning,expression and characterization of the 6-phosphofructokinase from Thermoanaerobacter tengcongensis

作　　者：周传奇[1] 王敬强[1] 钱忠[1] 马延和[2] 刘斯奇[1] 

机构地区：[1]中国科学院北京基因组研究所,北京101318 [2]中国科学院微生物研究所,北京100080

出　　处：《微生物学报》2006年第2期249-254,共6页Acta Microbiologica Sinica

基　　金：国家"973项目"(2004CB719605)~~

摘　　要：6_磷酸果糖激酶(PFK)是糖酵解途径一个关键酶。基于腾冲嗜热厌氧菌基因组中的注释,基因TTE1816可能是PFK的一种,但是,它是否确有生物活性还必须有实验数据的支持。腾冲嗜热厌氧菌在最适温度培养后,提取细菌全蛋白,并采用双向电泳将可溶性蛋白质分离,然后运用质谱鉴定若干染色斑点。实验表明,TTE1816在高温条件下能够表达蛋白质。将TTE1816基因体外克隆至细菌表达载体,并在BL_21大肠杆菌中表达为可溶性蛋白。酶动力学实验表明,重组蛋白TTE1816具有PFK的催化活性,最适反应温度在60℃。它还能够催化葡萄糖、果糖、甘露糖和6_磷酸葡萄糖的磷酸化反应。另外,在高底物浓度和酶浓度的条件下,TTE1816还表现果糖二磷酸酶的特性。结果证明,TTE1816是腾冲嗜热厌氧菌中PFK家族的一个新成员。The enzyme of 6-phosphofructokinase （PFK） is a key element in glycolysis, widely distributed in most eukaryote and prokaryote. Although the gene TTE1816 from Thermoanaerobacter tengcongenisis was annotated as a PFK based upon genomic analysis, its catalytic properties have to be examined experimentally. The cells of T. tengcongenisis were cultured at optimal temperature followed by the separation of the bacterial proteins with two-dimensional electrophoresis （2-DE）. These 2-DE spots located around the theoretical values of pl and MW for TTE1816 were picked up and identified by mass spectrometry, suggesting that TTE1816 indeed expressed at such high temperature. Furthermore, TTE1816 was cloned into an expression vector and expressed soluble protein in E. coli BL-21 strain. The kinetic data revealed that the recombinant TTE1816 exhibited the catalysis to phosphorylate fructose-6-phosphate （F-6-P）. Not only converting F-6-P to F-1,6-BP, does TTE1816 also catalyze the phosphorylation of glucose, fructose, mannose and glucose-6-phosphate（G-6-P） with optimal temperature at 60℃. Interestingly, TTE1816 is capable to catalyze the reverse reaction as a bisphosphatase for dephosphorylation of F-1,6-BP under the reaction conditions with high concentrations of enzyme as well as substrates. The data reported herein demonstrate that a new member of PFK family has been identified in T. tengcongenisis.

关 键 词：腾冲嗜热厌氧菌 嗜热菌 6-磷酸果糖激酶 糖酵解 

分 类 号：Q78[生物学—分子生物学] Q936