Expression Of γ-glutamyltranspeptidase From E. Coli And B. Subtilis And Its Application For L-thenine Synthesis

L-theanine is a unique amino acid that is naturally found in tea plants. L-theanine has many favorable physiological effects, such as lowering blood pressure, anti-cancer, neuroprotective and so on. Based on these promising advantages, L-theanine is widely used in food and pharmacy industry.γ-glutamyltranspeptidase (GGT, EC 2.3.2.2) can catalyze L-glutamine and ethylamine to produce L-theanine. The reaction process does not require ATP. So GGT’s application in catalysis of L-theanine has become a research focus. In this study, γ-glutamyltranspeptidase genes from E. coli K12 and B. subtilis SX were cloned and expressed in E.coli BL21 (DE3). Then the recombinant E. coli was used as catalyst to produce L-theanine. Finally, L-theanine was purified from the reaction solution by using the cation-exchange resin. The main research contents in this paper are listed as follows:Firstly, the genes of GGT from E.coli K12 and B.subtilis SX were cloned and ligated into pET28a to obtain the rencombinant plasmids pET28a-EGGT and pET28a-BGGT. The effects of different expression conditions of recombinant E. coli BL21 (DE3)/pET28a-BGGT were investigated systematically to improve the expression level of recombinant enzyme. Then, the recombinant EGGT and BGGT were purified by Ni-NTA affinity chromatography separately and the enzymatic properties were also studied.Secondly, the byproducts in this reaction were analyzed, there are three side reactions:(a) the irreversible degradation of L-glutamine to L-pyroglutamic acid; (b) the auto-transpeptiadation to form glu-gln catalyzed by GGT; (c) the transpeptiadation to form glu-theanine catalyzed by GGT. The effects of pH, temperature, concentration of recombinant cells and substrate on the enzymatic synthesis of L-theanine were also investigated. The results showed the optimal reaction was:300 mM L-glutamine,1.8 M ethylamine and 20 mg/mL recombinant cells in 100 mM Na2CO3-NaHCO3 (pH10.0) solution at 30℃. The highest titer of L-theanine was obtained about 28.2 g/L after 24 h and the conversion ratio was 55.2%.Then, the conditions of L-theanine production which using the whole cell of recombinant E.coli BL21(DE3)/pET28a-BGGT was optimized.The results showed the optimal reaction contained 300 mM L-glutamine,3 M ethylamine and 30 mg/mL recombinant cells in 100 mM Na2CO3-NaHCO3 (pH 10.5) solution at 30℃. The highest yield of L-theanine was obtained about 34.65 g/L after 6 h and the conversion ratio was 64.88%. By feeding of L-glutamine to this reaction, the titer of L-theanine could reach 63.2 g/L.Finally, L-theanine was purified from the reaction solution by using the HZ016 cation-exchange resin and the purity of L-theanine was 94.6%. The yield of L-theanine was 40.4%.