Screening Of Microbe Which Synthesize Theanine As A Catalyst And The Construction Of Recombinant Strain

Theanine, which was first separated from green tea in 1950, is not only the main free amino acid component of tea, but also the major umami component of tea. It possesses high physiological and medical benefits. The preparation of theanine is a research hot point, that It is very difficult and more expensive to extract a high purity theanine directly from tea leaves. The technics of chemosynthesis theanine is complicated;The yield of theanine by tissue culture is low. So, the biosynthesis of theanine is getting more and more attention. In this study, we screened the microbe and construct the recombinant strain, taking the biotechnology to the preparation of natural matter. In this paper, the screening of microbe was done, the ability of synthesis theanine as a catalyst of nine kinds microbe were compared, it was found that the ability of nine kinds microbe is low. We constructed a recombinant strain, the conditions of biosynthesis theanine were optimized. The activity of recombinant strain is about 15 times higher than the Escherichia coli DH5a (E.coli DH5a). The main results are as follows:1, The ability of synthesis theanine as a catalyst of nine kinds microbe were compared, It was found that nine kinds microbe have a low ability of synthesis theanine. The most yield of theanine is only 7.9mg/L.2, We got the gene of γ-glutamyltranspeptidase from E.coli DH5a by PCR. The recombinant plasmid pET-GGT was constructed by the gene of γ-glutamyltranspeptidase and pET-32a. The recombinant plasmid pET-GGT was transformed to E.coli BL21. γ-Glutamyltranspeptidase was produced with 0.05mM IPTG in 32℃ incubation. The activity of 1g fresh cells of recombinant strain is usually 2.0 units/ml, it is about 15 times higher than the E.coli DH5α.3, The reaction condition for synthesis theanine of recombinant strain were investigated. The recombinant strain was incubated at 37℃for 2h, then incubated at 32℃ with 0.05mM IPTG for 5h. In 1ml reaction system, when 0.35M L-Gln was used, 2.0M ethylamin hydrochlorid, 70mg/ml fresh cells, pH9.5, and incubation at 30℃ for 4h were the optimum condition. The yield of theanine was 29.40g/L, equivalent to lg fresh cells, can produce 420g/L theanine. The conversion rate of L-Gln as to theanine being 48.22%. Compared with Suzuki' report, 20.90g/L theanine was synthesized by recombinant microbe, the conversion rate of L-Gln as to theanine being 60%.The recombinant strain has a good ability of synthesis theanine as a catalyst. For the future, the experimentation of synthesis theanine utilizing the recombinant strain should be done, The recombinant plasmid pET-GGT should be transformed to different carrier system, looking for the carrier system of high expression Y-glutamyltranspeptidase. This study has a better instruction and utilizing value for produceing theanine of microbe ferment.