title: Enhanced production of recombinant Escherichia coli glutamate decarboxylase through optimization of induction strategy and addition of pyridoxine.
abstract: This report describes the optimization of recombinant Escherichia coli glutamate decarboxylase (GAD) production from engineered E. coli BL21(DE3) in a 3-L fermentor. Investigation of different induction strategies revealed that induction was optimal when the temperature was maintained at 30°C, the inducer (lactose) was fed at a rate of 0.2 g L(-1)h(-1), and protein expression was induced when the cell density (OD600) reached 50. Under these conditions, the GAD activity of 1273.8 U mL(-1) was achieved. Because GAD is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, the effect of supplementing the medium with pyridoxine hydrochloride (PN), a cheap and stable PLP precursor, on GAD production was also investigated. When the culture medium was supplemented with PN to a concentration of 2mM at the initiation of protein expression, and then again 10h later, the GAD activity reached 3193.4 U mL(-1), which represented the highest GAD production ever reported.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors.forename: Lingqia
authors.initials: L
authors.lastname: Su
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Electronic address: jingwu@jiangnan.edu.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D003470:Culture Media / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development*; D005968:Glutamate Decarboxylase / Q000096:biosynthesis*; D007785:Lactose / Q000378:metabolism; D011736:Pyridoxine / Q000494:pharmacology*; D013696:Temperature; D014803:Vitamin B Complex / Q000378:metabolism*
chemical_list: D003470:Culture Media; D014803:Vitamin B Complex; D005968:Glutamate Decarboxylase; D007785:Lactose; D011736:Pyridoxine
keywords: Escherichia coli; Fermentation optimization; Glutamate decarboxylase; Induction strategy; Pyridoxine hydrochloride