The Construction Of Recombinamt Escherichia Coli Of Expression D-Mannose Ketol Isomerase And Study Of Enzymic Characterization

D-mannose is the raw material of the production of mannitol, which is widely used in pharmaceuticals and food industry and in the field of biochemistry. The present promising method of mannose production, which catalyzes fructose directly to mannose by D-mannose isomerase, has great advantages over traditional chemical method in its low cost and energy consumption as well as environmental protection.The article was committed to obtain the D-mannose isomerase gene and efficient recombinant genetic engineering bacteria with high activity by means of genetic engineering method. Then, detect methods to explore the nature of the relevant enzyme for further study.First, the gene was cloned from the E. coli D-mannose isomerase gene (emi), D-mannose isomerase inducible expression plasmid pET30-emi and constitutive expression system pGEMK-HP-emi was constructed and then transformed into the expression host. After induction and culture, constitutive expression system expressed very lowly and inducible expression system abundantly expressed, but much exited as inclusion body. Second, the fmi gene sequence was designed 1245 nucleotides based on amino acid sequence of Fmi in agrobacterium radiobactor, while the preference of codon frequency was also taken into consideration to express fmi efficiently in E.coli. The designed sequence was synthesized using the gene splicing method, and the recombinant plasmid pET30-fmi and pGEMK-HP-emi was obtained and then transformed into the expression host. After induction and culture, inducible expression system abundantly expressed proteins and the protein proved soluble.Finally, detected the method of exploration, used resorcinol hydrochloric acid method for rapid qualitative detection of D-mannose isomerase activity and used HPLC for quantification of D-mannose isomerase activity. The results are as follows:Emi showed an optimum pH at 7 and an optimum temperature at 45℃, Fmi had an optimum pH at 7.5 and an optimum temperature at 45℃, under which Emi activity could reach 1.57U/ml in1 h, Fmi activity could reach 5.29U/ml. The conversion raito could reach 25.13% in 2h by Emi catalysing 25% fructose as the initial substrate and Fmi could reach 29.4%.