Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan.
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan.
Int J Biol Macromol. 2021 Nov 1;190:113-119. doi: 10.1016/j.ijbiomac.2021.08.166. Epub 2021 Sep 1.
Glutamate decarboxylase B (GadB) from Escherichia coli, an intrinsic pyridoxal 5'-phosphate (PLP)-dependent enzyme has been employed for 4-aminobutyric acid (GABA) biosynthesis, which involves the glutamate import and GABA export via a transporter located in the inner membrane as rate determined step of whole-cell (WC) biotransformation. Herein, GadB was cloned and overexpressed in E. coli under a constitutive promoter in a high copy number plasmid, and 46.9 g/L GABA was produced. It was observed that GadB migrated to the periplasm when the WC were subjected to -20 °C cold treatment for 24 h prior to the biotransformation. Kinetic studies indicated that the enzymatic turnover rate of WC increased 2-fold after cold treatment, which was correlated with the migration rate of GadB, and up to 88.6% of GadB. The export or possible migration of GadB mitigated the rate-limiting step of WC biotransformation, and a 100% conversion of substrate to GABA was obtained. Finally, we launched a promising strategy for GABA production of 850 g/L from cost-effective monosodium glutamate (MSG) by using WC biocatalysts with 10-times recycling.
大肠杆菌谷氨酸脱羧酶 B（GadB）是一种内在的吡哆醛 5′-磷酸（PLP）依赖性酶，已被用于 4-氨基丁酸（GABA）的生物合成，其中涉及谷氨酸的导入和 GABA 的通过位于内膜中的转运体导出，这是整个细胞（WC）生物转化的限速步骤。在此，GadB 在高拷贝数质粒的组成型启动子下在大肠杆菌中克隆和过表达，可生产 46.9 g/L GABA。观察到 WC 在生物转化前经过 24 小时的 -20°C 冷处理后，GadB 迁移到周质空间。动力学研究表明，冷处理后 WC 的酶周转速率增加了 2 倍，这与 GadB 的迁移率相关，GadB 的迁移率高达 88.6%。GadB 的外排或可能的迁移减轻了 WC 生物转化的限速步骤，使底物 100%转化为 GABA。最后，我们使用具有 10 倍循环利用的 WC 生物催化剂，从具有成本效益的谷氨酸单钠（MSG）生产 850 g/L GABA，推出了一项很有前途的策略。
