Design of a Full‐Consensus Glutamate Decarboxylase and Its Application to GABA Biosynthesis
Design of a Full‐Consensus Glutamate Decarboxylase and Its Application to GABA Biosynthesis
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全一致性谷氨酸脱羧酶的设计及其在 GABA 生物合成中的应用
DOI：
10.1002/cbic.202100447
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发表时间：
2021
期刊：
影响因子：
3.2
通讯作者：
Ito Sohei
中科院分区：
文献类型：
--
作者：
Takagi Hiroshi;Kozuka Kohei;Mimura Kenta;Nakano Shogo;Ito Sohei
谷氨酸脱羧酶（GAD）催化L -谷氨酸脱羧生成γ -氨基丁酸（GABA）。改善GAD的酶学性质对于低成本合成GABA具有重要意义。本研究利用乳酸菌中与GAD同源的酶序列，利用基于序列的蛋白设计方法设计高度突变的GAD。通过完全共识设计和祖先序列重建产生的两个突变GADs FcGAD和AncGAD比原生GADs具有更理想的特性。在热稳定性方面，设计的GAD的半衰期比天然GAD高约10°C。FcGAD的产率明显高于已知的gad；纯化酶可达250 mg/L以上。coliexpression系统。在生产试验中，使用26.4 g谷氨酸和3.0 g静息细胞，在一锅合成中，无需纯化，可以在1小时内制备17.2 g GABA。
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 theE. coliexpression system. In a production test using 26.4 g ofl‐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.