Biocatalytic Synthesis of γ-glutamyl-L-leucine, a Kokumi-Imparting Dipeptide, by Bacillus licheniformis γ-Glutamyltranspeptidase

ABSTRACT

Several γ-glutamyl compounds are known to have favorable features for a variety of biotechnological and pharmaceutical applications. However, so far only a limited number of γ-glutamyl compounds have been synthesized through γ-glutamyltranspeptidase (GGT). With the help of thin layer chromatography and computer-assisted image analysis techniques, we performed an enzymatic method involving Bacillus licheniformis GGT (BlGGT) for the synthesis of γ-glutamyl-L-leucine (γ-Glu-Leu). The optimum conditions for a biocatalytic synthesis of γ-Glu-Leu were determined to be 200 mM glutamine, 200 mM leucine, 50 mM Tris-HCl buffer (pH 9.0), and BlGGT at a working concentration of 2.0 U/mL. Upon a 5-h incubation of the reaction mixture at 50°C, the product yield could reach approximately 52%. The preparative synthesis of γ-Glu-Leu by BlGGT was done under optimal conditions at millimol scale level. The reaction products were subjected to mass spectrometry/nuclear magnetic resonance analyses to ensure the successful synthesis of γ-Glu-Leu. Taken together, the present study demonstrates the high potentiality of BlGGT-catalyzed reaction in the synthesis of γ-glutamyl compounds.

KEYWORDS:

• γ-glutamyl-L-leucine
• γ-Glutamyltranspeptidase
• biocatalytic synthesis
• bacillus licheniformis
• thin-layer charomatography

Acknowledgments

We thank Prof. Yu-Jang Li, Department of Applied Chemistry, National Chiayi University, for ESI-MS and NMR analyses and for fruitful discussions.

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Funding

This work was supported by a grant-in-aid for scientific research (MOST-103-2313-B-415-008-MY3) from the Ministry of Science and Technology of Taiwan.