Study On Preparation, Enzymology Characters And Applications In The Peptides Synthesize Of γ-glutamyltranspeptidase

γ-glutamyltranspeptidase(γ-GTP) can catalyze the hydrolyze of γ-glutamyl from γ-glutamyl peptide and transfer it to the other amino acids or peptides. So it can be use to synthesize all kind of γ-glutamyl peptide, and it have a good foreground in industry. Nowadays many of people in the world study it, but almost no one in the nation.In this research, Bacillus subtilis NX-2, a bacillus with high product of γ-GTP be used to get γ-GTP. After optimizing culture medium and conditions, it was observed that the optimum culture medium for the shaking incubator and fermentation tank is: 2% sucrose, 3% maize slurry, 1% peptone, 1.5% K2HPO4, 0.05% MgSO4. culture condition(shaking incubator):originate pH is pH 7.0, capacity is 60mL/500mL, 32℃ and ferment for 36h. In this condition product of γ-GTP is 3.2U/L higher than the best reported value in the world. Process curves in the shaking incubator and fermentation tank were get and compared.The distribution of γ-GTP in the ferment system was get: γ-GTP was produced in the periplasm and secrete to the other place, and 60% in the medium. A method of purification from medium was establish: deposit γ-GTP with same volume of acetone, use 60-90% grade (NH4)2SO4 separate the solution of the deposition, and with the buffer of pH8.0, 0.05M Tris-HCL, hydrophobic chromatography column was used. γ-GTP been washed with the concentration of 30% (NH4)2SO4. At last with same butter DEAE chromatography column was used. γ-GTP been collected in the first pink of grade wash with 0.1-0.4M NaCL (30min). SDS-PAGE analysis of γ-GTP purification show that the two subunits of γ-GTP is 43,000Da and 32,000Da.γ-GTP can endure the temperature lower than 60℃, in the normal temperature it can be preserve 30min without change of enzyme activity. It steady in the basic condition and lose its activity fast in acid condition. Its optimum reaction temperature is 40℃, and optimum reaction pH is pH8.0. In the low temperature it show a high transpeptidase rate and highest transpeptidase activity is in the 40℃, highest hydrolyze activity is in the 60℃. With improvement of pH the transpeptidase rate is also improved. The highest transpeptidase and hydrolyze activity is in the condition of pH9.0. It can be known that hydrolyze and transpeptidase reactions were cooperated by two subunit of γ-GTP. But not each of them own the complete activity. The reaction is process as pingpong mechanism.Chromatogram and mass spectrum were used to prove that γ-GTP which is produced by Bacillus subtilis NX-2 can catalyze the γ-glutamyl transfer reactions.