谷氨酸脱羧酶(GAD)催化L-谷氨酸脱羧成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。在这项研究中,利用与乳酸菌GAD同源的酶序列，使用基于序列的蛋白质设计方法设计高度突变的GAD。两个变异的GAD,FcGAD和AncGAD,通过全共识设计和祖先序列重建产生，具有比天然GAD更理想的属性。关于热稳定性，设计的GAD的半衰期比天然GAD高约10°C。FcGAD的生产率明显高于已知的GAD;在大肠杆菌表达系统中可以产生超过250mg/L的纯化酶。在使用26.4gL-谷氨酸和3.0g静息细胞的生产测试中，17.2克GABA可以在一小时内制备，没有净化，在一锅法合成中。
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.
