Expression Of B.amyloliquefaciens GGT In B.subtilis And Its Application For The Synthesis Of L-Theanine

?-Glutamyltranspeptidase?GGT?is widely present in organisms,which can catalyze the transfer of?-glutamyl compound to amino acids or peptides.Bacterial GGT have wider substrate specificity,which means that a variety of?-glutamyl amino acid derivatives or peptides can be synthesized under their catalysis.These enzymatically synthesized products have important application value in medicine and food.For example,GGT can use L-glutamine?L-Gln?and ethylamine as substrates to synthesis of L-theanine.L-theanine is a major amino acid in tea.It can not only be used as a food additive to remove bitterness and improve the flavor of food,but also has high development value in the field of medicine and health products.The demand for L-theanine in the market is increasing rapidly,and people are paying more attention to the preparation of high-quality GGT.In this study,Bacillus amyloliquefaciens BH072 GGT was used as the research object.The ggt gene was introduced into Bacillus subtilis,and the efficient secretion expression and purification of recombinant GGT in B.subtilis 168 were successfully achieved.Finally,the production of L-theanine was achieved by using recombinase.The main experimental results are as follows:?1?Extract the genomic DNA of B.amyloliquefaciens BH072 and use it as a template to amplify the ggt-6his/ggt?_sp-6his gene.The gene fragment was then ligated to the shuttle plasmid p HT43 and placed downstream of the inducible promoter P_lac and the signal peptide sp _{Amy Q}.The ligated product was introduced into Escherichia coli to construct the recombinant strain,and the plasmid was extracted to obtain the recombinant plasmid p HT43-sp _{Amy Q}-ggt-6his/p HT43-sp _{Amy Q}-ggt?sp-6his.The recombinant plasmid was introduced into B.subtilis 168 separately by electrotransformation to obtain the recombinant strain B.subtilis 168(p HT43-sp _{Amy Q}-ggt-6his/p HT43-sp _{Amy Q}-ggt?sp-6his).Recombinant bacteria were induced to express recombinant GGT by IPTG,and the recombinant protein was collected and purified by nickel column affinity chromatography.SDS-PAGE electrophoresis showed that both the dual signal peptide(sp _{Amy Q}-sp _GGT)and the single signal peptide(sp _{Amy Q})can successfully guide the extracellular secretion expression of the recombinase.Under the same conditions,the expression of recombinase guided by the dual signal peptide is higher,which indicates that the dual signal peptide can more efficiently guide the secretory expression of the recombinase.The activity test showed that the recombinase was active,indicating that after guiding the secretion and expression of the recombinase,its N-terminal signal peptide was degraded by the extracellular protease or peptidase secreted by B.subtilis 168.?2?Optimizing the induction conditions of the recombinant strain.After induced expression at a final concentration of 80?M IPTG for 24 h,the recombinant strain obtained the highest yield of recombinase.After purification,the concentration of the recombinase was 90±0.2 mg/L,and the specific enzyme activity was 55±0.5 U/mg.At the same time,the expression of the recombinase would not affect the host bacteria.Collecting and purifying recombinase for enzymatic properties research.Recombinase had the highest transpeptide activity at 40?and p H 9.0;the optimal storage conditions were 40?and p H 8.0,in which recombinase can be stored stably.The determination of kinetic parameters showed that when?-glutamyl-p-nitroaniline?Gp NA?was used as the substrate,the Mie constant?K_m?was 0.214 m M,and the maximum reaction rate?V_m?was 88.13?mol/min/mg.?3?Recombinase catalyzes the synthesis of L-theanine from the substrates L-glutamine and ethylamine hydrochloride.According to UPLC-MS detection and analysis,it was confirmed that the product contained L-theanine.The reaction substrate and products were detected by O-phthalaldehyde?OPA?precolumn derivatization,and the peak time of the main substances was successfully determined.Then,the enzymatic reaction conditions were optimized by detecting the conversion of L-theanine under different conditions.The optimal conditions for enzymatic synthesis were as follows:the final concentration of the recombinant enzyme was 0.5 U/m L,the final concentration of L-glutamine was 20 m M,and the final concentration of ethylamine hydrochloride was 100 m M?substrate ratio was 1:5?,the response time was 6 h.Under these conditions,the conversion rate of L-theanine catalyzed by the recombinant enzyme was the highest,with a conversion rate of 48.2%.