Fermentation Production Of γ-glutamyltranspeptidase And Enzymatic Synthesis Of γ-glutamylmethylamide

Gamma-glutamyltranspeptidase （GGT, EC2.3.2.2）, a heterodimeric enzyme, is found from bacteria to mammals and is involved in the metabolism ofγ-glutamyl compounds. It catalyzes the cleavage of theγ-glutamyl linkage ofγ-glutamyl compounds such as glutathione, and the transfer of theirγ-glutamyl moiety to the other amino acids and peptides. The transpeptidation was widely used in the fields of pharmaceutical and food, a lot of functional ingredients could be synthesized by transpeptidation, such as glutathione, theanine andγ-glutamyltaurine. Bacterial GGT was superior to GGT from other resources because it had broad substrate specificity and needed no additional energy in transpeptidation; furthermore theγ-glutamyl donor could be obtained easily. However, the most challenge of the application of transpeptidation is to improve the production of bacterial GGT.Gamma-glutamylmethylamide （N-methyl-L-glutamine,γ-GMA） is a non-protein amino acid; it has been reported to have a hypotensive effect to mammals. However, there were a lot of problems in previous methods for synthesizingγ-GMA, such as the difficulty of enzyme purification, additional energy requirement in reaction. An efficient method for producingγ-GMA is necessary to be obtained.In this study, a strain named Bacillus subtilis SK11.004 was used to produce GGT. The effects of nutrients in medium and environmental factors in fermentation on GGT synthesis were analyzed, and the strategy for improving GGT production was confirmed. Furthermore, the function of GGT in cell metabolism was deduced from the effect of glutamate on GGT production. Finally, the efficient method of synthesizingγ-GMA by GGT was validated, and the effect of factors in enzymatic reaction was investigated. The main results are as follows:1. Fractional factorial design was used to screen the important factors. Peptone and yeast extract were identified as the most significant ingredients. The path of steepest ascent was employed to approach near the optimal region. Central composite design and response surface analysis were applied to determine the optimal value. The final optimal media conditions were （g/L）: glucose 15.0, peptone 15.1, yeast extract 27.85, K₂HPO₄·3H₂O 1.0 and MgSO₄·7H₂O 0.6. Under the optimal nutritional condition, the maximum GGT actvity could reach 3.46 U/mL. Ca²⁺ and Zn²⁺ could repress the synthesis of GGT, and 100μmol/L Mn²⁺ could mostly stimulate GGT synthesis.2. The synthesis of GGT could be at both exponential phase and stationary phase, and reached the maximum at later period of stationary phase. Glucose could repress the synthesis of GGT.3. The effect of different temperature control on GGT synthesis was studied. Lower temperature had no effect on GGT synthesis, and higher temperature led to GGT degradation at later period of stationary phase. The suitable temperature for GGT production was 37℃. Constant pH control indicated specific GGT synthesis rate at acidic condition was higher than at non-acidic condition. Results of dissolved oxygen level control showed single level control was unfavorable for GGT synthesis. Higher level was fit for cell growth at the beginning, while induced the unstability of GGT at stationary phase; lower level was propitious for GGT synthesis at stationary phase, nonetheless resulted in oxygen limitation at the beginning. A combined strategy of constant pH （6.5） and stage dissolved oxygen control （0-4 h, 40%; 4-6 h, 30%; 6-18 h, 10%） was proposed. Combined strategy attained higher average specific cell growth rate and average specific GGT synthesis rate than those of none or single control process.4. The addition of glutamate to medium repressed GGT synthesis. As the concentration of glutamate in medium was observed to decrease after addition of GGT inhibitor, and the molecular weight of extracelluar poly-glutamate decreased with GGT activity increased, we deduced that GGT hydrolyzed poly-glutamate and provided glutamate for cell metabolism.5.γ-GMA was successfully synthesized using GGT with glutamine and methylamine as substrates. It was identified and characterized by data of HPLC, MS, IR and NMR. Central composite design and response surface analysis were applied to determine the optimal values for the production ofγ-glutamylmethylamide. The optimal conditions were pH 10.6, 0.34 mol/L glutamine, 1.5 mol/L methylamine, 0.56 U/mL GGT and incubation at 37℃for 5 h. Under this optimal reaction condition, the maximum production ofγ-glutamylmethylamide could be attained with 23.95 g/L.