Enhanced production of recombinant Escherichia coli glutamate decarboxylase through optimization of induction strategy and addition of pyridoxine

• Recombinant E. coli GAD was produced in a 3-L fermentor.
• Induction strategy and addition of pyridoxine hydrochloride were optimized.
• The GAD activity obtained (3193.4 U mL−1) is the highest reported to date.

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 2 mM at the initiation of protein expression, and then again 10 h later, the GAD activity reached 3193.4 U mL−1, which represented the highest GAD production ever reported.