Optimization of the reaction conditions for GABA production using purified GAD.

<p>(A) Effect of pH on GABA production. (B) Effect of temperature on GABA production. (C) Effect of enzyme dosage on GABA production. (D) Effect of substrate concentration on GABA production. Error bars represent the standard deviations from three independent determinations. The figure was generated using Origin 9.0.</p

Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

<div><p>Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from <i>Escherichia coli</i> K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in <i>E</i>. <i>coli</i>. The GAD produced in media supplemented with 0.05 mM soluble vitamin B₆ analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL^-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg^-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher <i>k</i>_cat/<i>K</i>_m (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L^-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L^-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA.</p></div

Effects of PN on the production of GAD.

<p>(A) Effects of PN on the activity of GAD. (B) SDS-PAGE analysis of GAD expression in <i>E</i>.<i>coli</i>. Lane M, molecular weight markers; lane1, Intracellular soluble fraction from culture in TB medium; lane 2, Intracellular soluble fraction from culture in TB medium supplemented with PN. (C) Effects of different concentration of pyridoxine hydrochloride on GAD production. Error bars represent the standard deviations from three independent measurements. The figure was generated using Origin 9.0.</p

Summary of the purification of GAD.

<p>Summary of the purification of GAD.</p

CD spectra of GAD in 20 mM citric acid-Na₂HPO₄ buffer (pH 6.5, containing 0.15 mM PLP).

<p>CD spectra of GAD in 20 mM citric acid-Na₂HPO₄ buffer (pH 6.5, containing 0.15 mM PLP).</p

The purification of GAD.

<p>Lane M, molecular weight marker; lane 1, GAD-C; lane 2, GAD-V.</p

Comparison of enzymatic properties.

<p>(A) pH optimum. The activities of GAD-C and GAD-V were determined in 50 mM citric acid-Na₂HPO₄ buffer (pH 3.0–7.0). The activity at optimal pH was taken as 100%. (B) pH stability. Samples were assayed after incubation for 24 h at 4°C in 50 mM citric acid-Na₂HPO₄ buffer (pH 3.0–7.0). The activity without preincubation was taken as 100%. (C) Optimum temperature. The activities of the GAD-C and GAD-V were determined at 30–60°C. The activity at optimal temperature was taken as 100%. (D) Thermostability. Both of the GAD-C and GAD-V were incubated at 37°C in 50 mM citric acid-Na₂HPO₄ buffer (pH 4.8). The activity without preincubation was taken as 100%. Error bars correspond to the standard deviation of three independent determinations. The figure was generated using Origin 9.0.</p

Gel filtration chromatogram of enzyme.

<p>Calibration curve of molecular weight versus elution volume. Standard: 1, Blue Dextran; 2, Apoferritin from horse spleen; 3, β-amylase; 4, Alcohol Dehydrogenase from yeast; 5, Carbonic Anhydrase from bovine.</p

Content of secondary structures predicted by CD.

<p>Content of secondary structures predicted by CD.</p

Gel filtration chromatograms of GAD-C(A) and GAD-V(B).

<p>The blue line represents the absorbance peak at 280 nm. Both the elution volumes of GAD were 11.2 mL.</p