Design of a Full-Consensus Glutamate Decarboxylase and Its Application to GABA Biosynthesis |
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Authors: | Hiroshi Takagi Kohei Kozuka Kenta Mimura Prof Dr Shogo Nakano Prof Dr Sohei Ito |
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Affiliation: | 1. Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan
Numazu Technical Support Center, Industrial Research Institute of Shizuoka Prefecture, Shizuoka, Japan;2. Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan |
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Abstract: | Glutamate decarboxylase (GAD) catalyses the decarboxylation of L-glutamate to gamma-aminobutyric acid (GABA). Improvement of the enzymatic properties of GAD is important for the low-cost synthesis of GABA. In this study, utilizing sequences of enzymes homologous with GAD from lactic acid bacteria, highly mutated GADs were designed using sequence-based protein design methods. Two mutated GADs, FcGAD and AncGAD, generated by full-consensus design and ancestral sequence reconstruction, had more desirable properties than native GADs. With respect to thermal stability, the half-life of the designed GADs was about 10 °C higher than that of native GAD. The productivity of FcGAD was considerably higher than those of known GADs; more than 250 mg/L of purified enzyme could be produced in the E. coli expression system. In a production test using 26.4 g of l -glutamate and 3.0 g of resting cells, 17.2 g of GABA could be prepared within one hour, without purification, in a one-pot synthesis. |
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Keywords: | biosynthesis full-consensus design gamma-aminobutyric acid glutamate decarboxylase lactic acid bacteria |
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