Biosynthesis Of L-Theanine Using γ-Glutamylmethylamide Synthetase

L-Theanine(γ-glutamylethylamide)is a unique non-protein amino acid naturally present in tea trees.As researchers continue to deepen their research into the nutritional value,physiological effects,and medicinal functions of L-glutamine,a series of functions of L-theanine have also become familiar with people.It was found to be effective in regulating loosening of the human brain and lowering blood pressure.A series of products based on L-theanine further promote the development of the pharmaceutical and healthcare industries.Gamma-glutamine synthetase(GMAS)is a class of enzymes found in the bacteriophthora that catalyze the synthesis of gamma-glutamine by L-glutamic acid and methylamine.Some studies have shown that certain microbial-derived GMAS have a higher affinity for ethylamine.In the presence of ATP and mg2+,L-glutamic acid and ethylamine can be catalyzed to synthesize L-theanine.Polyphosphate kinase 2(PPK2)is an enzyme for ATP re gene ration.It is capable of catalyzing the direct conversion of polyp ho sphoric acid and ADP to ATP.In this paper,GMAS and PPK2 were heterologously expressed by E.coli BL21(DE3).The recombinant enzymes were used to catalyze the production of L-theanine.Under the condition of pH 8.0 and 32℃ for 8h,20.68 g/L L-theanine was formed by the above double enzyme coupling system,and the molar conversion was 79.24%.Based on the verification of the enzyme function,a genetic engineering strain of Escherichia coli was constructed in this paper to further reduce the cost of the reaction,and L-theanine is obtained by the fermentation with the addition of the precursor ethylamine.A single copy of the T7RNA polymerase gene derived from T7 phage was intergrated in the genome of E.coli W3110 under the control of xylose promoter.Double copies of the glutamylmethylamine synthase gene gmas derived from Methylovorus mays were then intergrated in the genome of E.coli W3 110 under the control of T7 promoter.Therefore a plasmid-free and genetically stable engineered strain of E.coli gmas2 was constructed for L-theanine production.By optimizing the fermentation conditions of the engineered bacteria,the yield of L-theanine was 30.45 g/L in 20 h fermentation,and the conversion rate was 20.17%.According to the physicochemical properties of L-theanine,a suitable separation and extraction route was proposed.Firstly,the microbial membrane and the ultrafiltration were combined to remove the bacteria,proteins and pigments in the fermentation broth.Secondly,the activated carbon is used to further remove the pigment of the concentrated liquid,so that the transmittance reaches the refining requirement.Finally,L-theanine crystals were obtained by concentration and cold ethanol crystallization.Qualification of final product was detected by nuclear magnetic resonance.The extraction yield of the whole process was 70.34%,and the purity of the finished product was 98.51%.