Abstract:The effects of the addition of relatively inexpensive coenzyme precursors on glutamate decarboxylase(GAD) enzyme activity and catalytic efficiency during the fermentation were studied. The recombinant strain B.subtilis/pHY300PLK-gadB which contained the gadB gene deriving from Escherichia coli was constructed. The effects of coenzyme pyridoxal phosphate(PLP), coenzyme precursors pyridoxal(PL) and pyridoxine(PN) on the production of GAD were studied. The rotating speed of shaking flask was 200 r/min. The fermentation temperature was 33 ℃. The initial pH of fermentation medium was 7.0, while PLP, PL and PN were added to the final concentration of 0.5 mmol/L. The enzyme activity reached 25.40, 28.14 and 15.55 U/mL after 48 h induction, respectively. Compared with the control group, the enzyme activity was increased by 1.55, 1.72 and 0.95 times. Based on the above results, the effects of PL concentration on the growth of recombinant bacteria and the expression of GAD protein were further studied. The results showed that the production of GAD in the fermentation broth increased with the increase of PL concentration. When PL concentration was 0.1 mmol/L, the maximum activity of GAD was 28.28 U/mL. The preparation of γ-aminobutyric acid(GABA) by whole cells of recombinant bacteria showed that the optimum pH was 5.0, the optimum temperature was 40 ℃, and the optimum amounts of the recombinant bacteria with PL (GAD-PL) and the recombinant bacteria without coenzyme precursor (GAD-0) were 40 U/g (in glutamic acid) and 50 U/g(in glutamic acid), respectively. With the concentration of 400 g/L glutamic acid, the GABA yield reached 275.60 g/L and 273.61 g/L, respectively.
