| γ-glutamyltranspeptidase (GGT, EC2.3.2.2) catalyzes the transfer of the γ-glutamylgroup of glutathione, or other compounds containing the γ-glutamyl moiety, to acceptors suchas amino acids, dipeptides, or water, resulting in the corresponding transpeptidation productor hydrolysate. The biochemical properties of GGT to transfer the γ-glutamyl moiety from γ-glutamyl donor to acceptors for producing γ-glutamyl compounds can be exploited indifferent ways for biotechnological interests, such as producing new valuable γ-glutamylproducts. Among them, the enzymatic synthesis of L-theanine using L-glutamine (L-Gln) asthe donor and ethylamine as the acceptor was the most widely studied. L-theanine is a uniquenon-protein amino acid present in green tea that has been reported to impart an umami taste tothe tea. Several studies have shown or suggested that ingestion of L-theanine hasphysiological and pharmacological effects on humans, such as reducing psychological andphysiological stress cognitive, improving performance and learning ability, improvingimmune response, inhibiting the growth and survival of cancer cells and so on. Therefore, L-theanine has been increasingly used in the functional food industry and its market demandincreases year by year.In this study, the GGT from Bacillus subtilis168was cloned and expressed as a secretedprotein using Escherichia coli BL21(DE3). The production of the recombinant GGT in E.coli,were investigated and optimized in shake flasks. Then the recombinant enzyme fromsupertant was purified and the enzymatic properties of the GGT were studied. Based on these,the optimal conditions for the enzymatic synthesis of L-theanine were investigated in detail.The main results were listed as follows:(1) GGT gene, a1680bp fragment from B. subtilis168, was successful to achieve aheterologous expression in E. coli using chromosomal DNA as the template by PCR. Andthen the recombinant plasmid ggt/pET-20b(+) was constructed and transformed into E. coliBL21(DE3). The recombinant strain was cultured in TB medium and the secreted enzymeactivity (42.6U·mL-1) was reached after cultivation at25℃for48h.(2) The recombinant GGT was purified and the enzymatic properties were studied indetail. The recombinant GGT was purified by ammonium sulfate precipitation, DEAE-Sepharose anion exchange chromatography, and Superdex75gel filtration chromatography.The purified enzyme was determined to be homogeneous by SDS-PAGE analysis andexhibited a specific activity (total activity) of136.0U·mg-1using L-γ-glutamyl-p-nitroanilideas the substrate. The Kmwas2.15mmol·L-1for L-γ-glutamyl-p-nitroanilide, and0.93mmol·L-1for L-glutamine. The Vmaxwas191.3μmmol·min-1·mg-1using L-γ-glutamyl-p-nitroanilide and95.4μmmol·min-1·mg-1using L-Gln. Recombinant GGT exhibited optimaltransferase and hydrolase activities at pH10.0and alao proved to exhibit broad pH stability.The activity of the enzyme increased with temperature until it peaked at50℃and the enzymeshowed50%residual activity when incubated for130h at50℃. This thermostability of theenzyme is desirable for its application to the enzymatic production of L-theanine.(3) To further improve the production of the recombinant GGT in E. coli, the cultureconditions and medium compositions were investigated and optimized in shake flasks. The optimal fermentation medium was as follows:10g·L-1glycerol,21g·L-1yeast extract,7g·L-1peptone, and130mmol·L-1PO43-. In addition, the optimal culture condition was as follows:fermentation temperature25℃, induced by0.2mmol·L-1IPTG at4h of the fermentationcourse. Under these conditions, the maximal enzyme activity reached81.2U·mL-1, which was1.9times as high as that not optimized.(4) The optimal conditions for the enzymatic synthesis of L-theanine were investigatedin detail. The enzymatic synthesis of L-theanine can be carried out using the GGT with L-Glnand ethylamine as reactants. Several reaction conditions were studied and optimized for L-theanine production, including the reaction pH, the amount of Gln to ethylamine, thetemperature and the enzyme dosage. In5h at37℃, the enzyme converted200mmol·L-1L-glutamine and2.2mol·L-1ethylamine to L-theanine with a final yield of78%. To get furtherinformation about the ability of GGT to produce L-theanine, enzymatic reactions wereperformed using different concentrations of L-Gln. Finally, yields of L-theanine decreased to58%when using500mmol·L-1L-Gln and45%when using1mol·L-1L-Gln. The yield of L-theanine obtained at high substrate concentration provides the basis for the industrial-scaleproduction of L-theanine. |
