1. Enhanced L-theanine production through semi-rational design of γ-glutamylmethylamide synthetase from Methylovorus mays.
- Author
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Fan C, Qi J, Cong Y, and Zhang C
- Subjects
- Directed Molecular Evolution, Kinetics, Bacterial Proteins metabolism, Bacterial Proteins genetics, Bacterial Proteins chemistry, Temperature, Carbon-Nitrogen Ligases, Glutamates metabolism, Glutamates biosynthesis, Enzyme Stability, Peptide Synthases metabolism, Peptide Synthases genetics, Peptide Synthases chemistry
- Abstract
The thermal instability of γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays has imposed limitations on its industrial applications, affecting both stability and activity at reaction temperatures. In this study, disulfide bridges were introduced through a combination of directed evolution and rational design to enhance GMAS stability. Among the variants that we generated, M12 exhibited a 1.46-fold improvement in relative enzyme activity and a 6.23-fold increase in half-life at 40℃ compared to the wild-type GMAS. Employing variant M12 under optimal conditions, we achieved the production of 645.7 mM (112.49 g/L) L-theanine with a productivity of 29.3 mM/h, from 800 mM substrate in an ATP regeneration system. Our strategy significantly enhances the biosynthesis efficiency of L-theanine by preserving the structural stability of the enzyme during the catalysis process., Competing Interests: Declaration of Competing Interest The authors have no financial conflicts of interest to declare, (Copyright © 2024. Published by Elsevier Inc.)
- Published
- 2024
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