Enzymatic Synthesis of Theanine with L-glutamine-Zn(II) Complexes

Theanine, a unique amino acid found in tea plants, has many important physiological functions. Theanine can be enzymatically synthesized via the gamma-glutamyltranspeptidation reaction. In this study, we described a new method of theanine synthesis using the L-glutamine-Zn(II) (Zn(Gln)(2)) complex instead of glutamine as the donor, which successfully reduced the side autotranspeptidation reaction and led to higher yield of theanine. We prepared the Zn(Gln)(2) complexes and showed that they are stable in liquid bulk under 9.0 pH. After using the Zn(Gln)(2) in the gamma-glutamyltranspeptidation reaction, we utilized HPLC and Mass spectrometry analysis to demonstrated that Zn(Gln)(2) was an more effective gamma-glutamyl donor than glutamine. The autotranspeptidation reaction was restrained effectively. As a result, the theanine yield and the conversion rate for glutamine were vastly improved. In a reaction mixture containing 48 mM of Zn(Gln)(2), 1.6 M ethylamine, and 0.5 U/mL GGT, the final concentration of theanine obtained was 61.3 mM after incubation at 37 degrees C for three hours. The conversion rate for glutamine was 63.8%, which showed a 16.9% increase as compared to when using glutamine alone as the donor substrate.