Fermentation Of γ-glutamyltranspeptidase From Bacillus Subtilis And Its Application In L-theanine Production

γ-glutamyltranspeptidase(GGT, EC 2.3.2.2) catalyzes the transfer of the γ-glutamyl group of glutathione, or other compounds containing the γ-glutamyl moiety, to acceptors such as amino acids, dipeptides, or water, resulting in the corresponding transpeptidation product or hydrolysate. For new valuable γ-glutamyl products generation, the special biochemical properties of GGT, which can transfer the γ-glutamyl moiety from γ-glutamyl donor to acceptors, could be utilised. It is safe to say that utilising ethylamine as the acceptor with L-glutamine(L-Gln) as the donor have been generally studied. Presenting in green tea, an unmatchable non-protein amino acid L-theanine have been recorded that it majorly imparted a special umami taste to the tea. Thousands of researches had revealed or expounded that ingesting L-theanine will bring about pharmacological and physiological effects on humans, such as controlling blood pressure, enhancing cognitive ability, shielding virus attack, strengthen immunity, preventing cancer and releasing menstrual syndrome. For this reason, L-theanine is assuming to be the best-selling healthy production, for that the market need consistently increased through these years.In this study, the high density fermentation conditions of the recombinant Escherichia coli were optimized. Then the enzyme which is recombined from supernatant will be purified and characterized in this processing. Bacillus brevis cloned and expressed the GGT from Bacillus subtilis 168. The production of the recombinant GGT in B. brevis was investigated and optimized in shake flasks. Different feeding strategies of substrate were studied in L-theanine production. Based on these, the purification process of L-theanine was studied. The main results were listed as follows:(1) Different fermentation conditions were investigated in 3 L fermentor. The optimal conditions were as follows. The recombinant E. coli BL21(DE3)/pET-24a(+)-ggt was cultured under 30% DO concentration, 37°C and pH 7.0. When OD600 reached 40, the induction was performed with 0.03 g?L-1?h-1 lactose, maintained the induction temperature at 30 oC, and the extracellular activity reached 868 U?ml-1 after 60 h of cultivation, which was 10.2-fold of that in shake flask fermentation. The recombinant enzyme was purified and was exhibited a specific activity(total activity) of 323.9 U?mg-1. Moreover, the characterization of recombinant GGT was studied. The recombinant GGT was purified and the optimum temperature and pH were 50°C and 10.0, respectively, which were the same as that in shake flask fermentation. The half-life of recombinant GGT was 105 h at 50°C, which was shorter than that in shake flask fermentation(130 h).(2) The optimized sucrose isomerase gene(ggt) was linked to B. brevis expression plasmid pSVEB and the recombinant plasmid pSVEB-ggt was structured. Then the recombinant plasmid was transformed into B. brevis. In shake flask, the extracellular enzyme activity of B. brevis/pSVEB-ggt was 29 U·mL-1 in TM medium after cultivation of 48 h at 30°C. To enhance the improvement of the production of the recombinant GGT in B. brevis, the medium compositions were optimized in shake flasks. The optimal fermentation medium was as follows: 15 g·L-1 soy peptone, 2 g·L-1 polypeptone, 15 g·L-1 beef peptone, and 10 g·L-1 glucose. Under the optimal conditions, the enzyme activity reached 41 U·mL-1, which was recognised as 1.4 times as high as what not optimized. The combinant B. brevis/pSVEB-ggt was cultured in 3 L fermentor fot 60 h, and the extracellular activity reached 46 U?ml-1.(3) The optimization of L-theanine production by GGT was investigated. The constant speed feeding strategy was performed using 200 mmol·L-1 L-glutamine and 2 mol·L-1 rimantadine hydrochloride as the initial substrate, under the condition of 37°C, pH 10.0, 100 mmol·L-1 L-glutamine was added every 2 hours and the reaction sustained for 16 hours. Eventually the total concentration of L-glutamine and L-theanine were 900 mmol·L-1 and 610.1 mmol·L-1 respectively. The conversion rate of L-theanine reached 67.8%。(4) The optimization of the purification process of L-theanine transformation solution. The L-theanine transformation solution was purified by pretreatment, ion exchange process and crystallization process and the final recovery rate was 68%.