[Cloning, expression and characterization of the 6-phosphofructokinase from Thermoanaerobacter tengcongensis].
摘要
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 pI 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 degrees C . 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.