Arginine Biosynthetic Genes Cluster Of Corynebacterium Crenatum

L-arginine is an important amino acid for the human body's metabolism. It is widelyapplied in the fields of food and medicine with the deep study. This research mainly focusedon the exploitation of the clustered-gene information of arginine biosynthesis ofCorynebacterium crenatum A.S.1.542 whose quantity of producing arginine could not beprobed and its mutant treated by NTG, named Corynebacterium crenatum A.S.M2 whichcould produce 20g/L arginine, on the comparison between C. crenatum A.S 1.542 and C.crenatum A.S.M2, on the analysis the function of argR which is a regulation gene in argininebiosynthesis via the methods of gene knock-out technology ,and on the espial the infectionof argR gene to the yield of arginine by the construction of expression vector pC2-P-argR-Telectransferred into C. crenatum A.S.M2. In the research, the key enzymes of argininebiosynthesis were investigated by the method of affinity and SDS-PAGE. The main resultsare illustrated as following:1. With the probe of the argB gene from Corynebacterium glutacium ATCC13032, itwas made sure that the argB genes between C. crenatum A.S 1.542 and C. glutaciumATCC13032 had the upper homology by Southern Blotting. Four pairs of differential primerswere designed according to the above mentioned result and compared to the conservativesequences of the cluster-gene argCJBDFR from Corynebacterium diphtheriae gravisNCTC13129,Corynebacterium efficiens YS-314 and Mycobacterium tuberculosis CDC1551.A 6080bp sequence, which contained five intact open-reading frames(ORF):argJ,argB,argF,argD and argR,and one partial ORF:argC, was obtained by PCR. These five intactORFs were analyzed by Blastp and the characteristic information was obtained.2. A 6083bp sequence which contained the same five intact ORFs was obtained from C.crenatum A.S.M2 by using the same primers and PCR technique. The possible reason of theelevated arginine of C. crenatum A.S.M2 was analyzed in the level of bio-informatics viacomparison the distinctness of the five genes between C. crenatum A.S.M2 and C. crenatumA.S 1.542.3. Whereas the argR gene being activity,a knock-out plasmid pGEM-T-RKR wasconstructed for analyzing the function of argR gene from C. crenatum. By assaying the yieldof arginine and acetylglutamate kinase,it was concluded that the argR gene of C. crenatumacted as a positive control factor in arginine biosynthesis. An expression plasmidpC2-P-argR-T was constructed and electransferred into C. crenatum A.S.M2 for observingthe infection of the positive control factor to the yield of arginine. The result showed that theaim of improvement the yield of arginine was not achieved4. The key enzymes producing arginine from C. crenatum were investigated by themethods of affinity chromatography and SDS-PAGE. The result indicated that one of theenzyme was acetylglutamate kinase,MW 34 kD,and another protein,MW 49kD, showedaffinity to arginine would be acetylglutamate sythesase, but it was uncertain. The charateristicstudy on acetylglutamate kinase showed that the optimum temperature was 49℃,theoptimum pH was 8.0,the activity was strongly inhibited by metal ion Pb2+,Cu2+,Mn2+andFe2+,and effected by DTT.