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.

Keywords: Escherichia coli; Fermentation optimization; Glutamate decarboxylase; Induction strategy; Pyridoxine hydrochloride.