l-Theanine Goes Greener: A Highly Efficient Bioprocess Catalyzed by the Immobilized γ-Glutamyl Transferase from Bacillus subtilis

t-Theanine (t-Th) was synthesized by simply mixing the reactants (t-glutamine and ethylamine in water) at 25 degrees C and Bacillus subtilis gamma-glutamyl transferase (BsGGT) covalently immo-bilized on glyoxyl-agarose according to a methodology pre-viously reported by our research group; neither buffers, nor other additives were needed. Ratio of t-glutamine (donor) to ethylamine (acceptor), pH, enzymatic units (IU), and reaction time were optimized (molar ratio of donor/acceptor=1 : 8, pH 11.6, 1 IU mL(-1), 6 h), furnishing t-Th in 93% isolated yield (485 mg, 32.3 gL(-1)) and high purity (99 %), after a simple filtration of the immobilized biocatalyst, distillation of the volatiles (unreacted ethylamine) and direct lyophilization. Immobilized BsGGT was re-used (four reaction cycles) with 100% activity retention. This enzymatic synthesis represents a straightforward, fast, high-yielding, and easily scalable approach to t-Th preparation, besides having a favorable green chemistry metrics.

Automated summary

In this study, t-Theanine (t-Th) was synthesized by simply mixing t-glutamine and ethylamine in water at 25 degrees C using Bacillus subtilis gamma-glutamyl transferase (BsGGT) immobilized on glyoxyl-agarose. The optimal conditions for the reaction were a molar ratio of donor/acceptor of 1:8, pH 11.6, 1 IU mL(-1) of enzymatic units, and a reaction time of 6 hours, resulting in a 93% isolated yield of t-Th with high purity (99%). The immobilized BsGGT could be reused for four reaction cycles with 100% activity retention. This enzymatic synthesis provides a straightforward, fast, high-yielding, and easily scalable approach to t-Th preparation with favorable green chemistry metrics.