Cloning And Expression Of N-acetylglutamate Kinase Gene From Corynebacterium Crenatum

L-arginine is one kind of nonessential semi-essential amino acids in human and animal bodies,and plays a major role of intermediate metabolite in urea circulation, which has been widely applied on medicine industry. Besides, L-arginine is also an important material on nutrition,food and cosmetics industry.The strain Corynebacterium crenatum SYPA was investigated in our study,and the acetylglutamate kinase(NAGK) encoded by gene argB is a key enzyme which is feedback inhibited in its activity by arginine.By genetic engineering we study the character and effect of NAGK as a key enzyme producing L-arginine and the affect on producing L-arginine if the gene argB was expressed strongly.C. crenatum SYPA is a gram-positive strain, which is used in industry area.There is no system used to express C. crenatum especially in the literature we had consulted.In this work,three expression vectors pET-28a-argB, pC2-argB and pJC1-tac-argB,which carries argB gene from C. crenatum SYPA has been constructed in our research successfully and transducted to Escherichia coli.The SDS-PAGE and specific activity of NAGK showed that the gene argB was expressed successfully in recombinant E.coli.But,we found that the expression vector pET-28a-argB had higher expression,the pC2-argB was at last and the pJC1-tac-argB was in middle. Then NAGK was purified by Ni-NTA affinity chromatography.The results showed that a single band was about 34kD on SDS-PAGE gel, the specific activity was about 0.73U/mg, and the purification fold is 6.61 times. The optimum activity of NAGK was at pH 9.0 and 30℃.Km of N-acetylglutamate was 3.35mmol/L.Its activity was obviously inhibited by Cu²⁺,Mn²⁺ and EDTA.The expression vector pC2-argB and were transducted to C. crenatum SYPA.After the research in activity of NAGK in C. crenatum SYPA (pJC1-tac-argB),we saw that gene argB was expressed well C. crenatum SYPA (pJC1-tac-argB). The fermentive character of C.c.(pJC1-tac-argB) was also primary analysed. The result shows that the acid producing ability of recombinant strain is improved by 23.4%,and also the specific activity is improved by 42.6%,but we found that the excessive expression of NAGK made cells grow slowly. Quantitative determination of the amino acid proved that the excessive expression of NAGK can promoted the utililization of glutamate and skewed the metabolic flux towards the formation of L-Arginine.