Heterogenous Synthesis And Activity Analysis Of Theanine

Theanine is a free amino acid that does not participate in protein composition.It has a wide range of medicinal and health benefits such as lowering blood pressure,improving immunity,inhibiting excitement,reducing body weight,relieving mental stress,improving learning ability and memory.A biologically active substance that is difficult to replace.The target product of microbial heterologous expression synthesis has the advantages of short cycle,large scale,low cost and good product quality.It is an important supplement to the traditional production methods of direct separation and extraction and chemical synthesis,and is also the main direction of biopharmaceutical development..In this study,we further studied the probiotic lactic acid bacteria based on the expression of ?-glutamine transpeptidase(GGT)heterologously synthesized by the industrial model microorganism Escherichia coli.The feasibility of synthesizing theanine provides a basis for efficient,safe and safe production of theanine.The main findings are as follows:1.Screening and functional prediction of the E.coli ?-GGT gene.A ?-GGT gene(NCBI no.EG10374;E.coli K-12)was screened from the NCBI public database according to homology.Bioinformatics analysis indicated that it encoded 580 amino acids,and the enzyme protein was acidic unstable hydrophilic.The protein,with a molecular weight of 61.7 kDa,has a signal peptide sequence and a conserved functional domain,plays a role in the periplasm and is evolutionarily conserved.2.Construction of E.coli engineered bacteria with ?-GGT gene.The artificially synthesized sequence of the lactic acid bacteria partial transformation of the E.coli ?-GGT gene,The E.coli?-GGT expression vector was constructed by double digestion of XbaI and BamHI with the same digested pET28 a plasmid.The E.coli BL21 strain was transformed by CaCl2 method,and the engineering strain E.coli/pET28a-GGT was obtained by colony PCR and double enzyme digestion.3.Establishment of a heterologous synthetic theanine system from E.coli engineeringbacteria.The single-factor experiment and three-factor three-level orthogonal experiment were used to optimize the induction temperature,inducer concentration,induction time and different substrate concentrations to establish a heterologous synthetic theanine system of E.coli engineering bacteria:(1)recombinant protein Induction.The temperature was 30?,and the IPTG concentration was 0.2 mmol/L for 4 h;(2)the catalytic reaction system of GGT enzyme.160 rmp,30?,1 ml reaction system(bacteria amount 70 mg / ml,pH 9.5),glutamine(L-Gln)concentration of 0.25 M,ethylamine hydrochloride concentration of 2.0M,reaction for 8h.The content of theanine synthesized under this optimized condition was 2932.02 nmol/mL.4.Analysis of the activity of the theanine of heterologous synthesis of E.coli engineered bacteria.The effect of heterologous synthetic theanine on cells was studied by using theanine standard and 0.2% DMSO as control.It was found that the theanine(crude extract)catalyzed by E.coli engineering bacteria had a significant inhibitory effect on the proliferation of human prostate PC3 cancer cells,which was most significant when the crude extract was diluted 50 times.This inhibition is more pronounced with time.5.Construction of L.lactis engineered bacteria with ?-GGT gene.The E.coli ?-GGT gene modified by lactic acid bacteria was digested with Xba I and SacI,and ligated with pNZ804 plasmid to construct lactic acid bacteria expression plasmid pNZ8048-GGT.The L.lactis NZ3900 strain was transformed by electroporation,and the L.lactis NZ3900/pNZ8048-GGT engineering strain was obtained by colony PCR and double enzyme digestion.At the same time,a high-copy strain of the target gene was screened out by Real-time PCR technology.6.Establishment of heterologous synthetic theanine system of L.lactis engineering bacteria.The concentration and induction time of the inducer Nisin were optimized by single factor and enzyme-linked immunosorbent assay(ELISA).The results showed that the?-glutamine transpeptidase activity encoded by the ?-GGT gene was the highest when induced by3 ng/ml of Nisin for 4h.