E. Coli Ggt Cloning, Expression, And The Use Of Recombinant Ggt Synthesis Of L-theanine

Theanine,which was first extracted from green tea in 1950,is not only the main amino acid marker,but also the major umami component of tea,thereby possessing high physiological and commercial values.The preparation of theanine is very hot.The biosynthesis of theanine is getting more and more attention because of the high cost of extracting theanine directly from tea leaves with high purity,the complexity of chemo-synthesizing theanine,the low yield of theanine by tissue culture.In this study, we constructed the recombinant bacterial cell strain for the preparation of natural substances.In this paper,we optimized the conditions of inducing recombinant GGT and biosynthesis of theanine.The enzymatic activity of recombinant bacterial cell strain is about 35 times higher than the one of initial Escherichia coli K-12(E.coli K-12).The main results are as follows:1,We amplified the gene coding for r-glutamyltranspeptidase from the genome of E.coli K-12 by PCR.The recombinant plasmid pET28a-ggt was constructed,and then transformed to E.coli BL21.2,To optimize the induction conditions,the initial OD₆₀₀of the microbe mixture, the induction time,and the concentration of IPTG were studied via the central composite design and their interactions on the yield of recombinant GGT were also investigated.The predictive polynomial quadratic equations model was developed by SAS software.Optimal conditions of reducing the recombinant bacterial cell strain could be concluded as follows:the initial OD₆₀₀of the microbe mixture was at 0.52,the concentration of IPTG was up to 0.42mM and the induction time was up to 7h.On these conditions,the enzymatic activity of crude extract was usually 81.8625 units/ml,which was about 35 times higher than the one of crude extract of E.coli K-12.3,With the help of PCR amplification,construction and transformation of expression vectors,we got recombinant vectors containing the large subunit or small subunit of ggt.Enzymatic.activity analysis showed that the transforming activity of crude extract of recombinant large subunit had no significant improvements compared with the one of E.coli K-12,neither did the transforming activity of crude extract of recombinant small subunit.4,The reaction conditions for synthesizing recombinant theanine were also investigated.The recombinant bacterial cell strain was incubated at 37℃until the OD₆₀₀of the microbe mixture reached 0.52,then incubated at 20℃with 0.42mM IPTG forth,finally incubated at37℃for 4h in lml reaction system(0.267M L-Gln,2.0M ethylamine,16U crude extract,pH9.5).The yield of theanine was 26.9g/L,and the conversion rate of L-Gln as to theanine was 48.22%.Compared with Suzuki' report, we increased the yield from 20.90g/L to 26.9g/L,though the conversion rate of L-Gln as to theanine slightly reduced.The crude extract of recombinant bacterial cell strain showed a good capacity of synthesizing theanine.In the future,we can enlarge the production of recombinant theanine by this crude extract.Meanwhile,the possibilities of using other plasmids or different hosts,expression vectors should also be considered to pursue a higher expression of r-glutamyltranspeptidase.Therefore,our study had a profound value for the instruction of producing theanine via bioconversion.