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

: l-Theanine (l-Th) was synthesized by simply mixing the reactants (l-Gln and ethylamine in water) at 25 °C and Bacillus subtilis-GGT (γ-glutamyl transferase) covalently immobilized on glyoxyl-agarose according to a methodology previously reported by our research group; neither buffers, nor other additives were needed. l-Gln (donor)/ethylamine (acceptor) ratio, pH, enzymatic units (IU), and reaction time were optimized (1:8 molar ratio donor:acceptor, pH 11.6, 1 IU mL-1, 6 h), furnishing l-Th in 93% isolated yield (485 mg, 32.3 g L-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 l-Th preparation, besides having a favorable green chemistry metrics.