Site‐directed mutagenesis improves the practical application of L‐glutamic acid decarboxylase inEscherichia coli

Abstract

Abstractγ‐Aminobutyric acid (GABA) is a kind of non‐proteinogenic amino acid which is highly soluble in water and widely used in the food and pharmaceutical industries. Enzymatic conversion is an efficient method to produce GABA, whereby glutamic acid decarboxylase (GAD) is the key enzyme that catalyzes the process. The activity of wild‐type GAD is usually limited by temperature, pH or biotin concentration, and hence directional modification is applied to improve its catalytic properties and practical application. GABA was produced using whole cell transformation of the recombinant strainsEscherichia coliBL21(DE3)‐Gad B,E. coliBL21(DE3)‐Gad B‐T62S andE. coliBL21(DE3)‐Gad B‐Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively. The results showed that Gad B‐T62S and Gad B‐Q309A are two effective mutation sites. These findings may contribute to ideas for constructing potent recombinant strains for GABA production.Practical Application: Enzymatic properties of the GAD fromEscherichia coliand GAD site‐specific mutants were examined by analyzing their conserved sequences, substrate contacts, contact between GAD amino acid residues and mutation energy (ΔΔG) of the GAD mutants. The enzyme activity and stability of Gad B‐T62S and Gad B‐Q309A mutants were improved compared to Gad B. The kinetic parameters Kmand Vmaxof Gad B, Gad B‐T62S, and Gad B‐Q309A mutants were 11.3 ± 2.1 mM and 32.1 ± 2.4 U/mg, 7.3 ± 2.5 mM and 76.1 ± 3.1 U/mg, and 7.2 ± 3.8 mM and 87.3 ± 1.1 U/mg, respectively. GABA was produced using whole cell transformation of the recombinant strainsE. coliBL21(DE3)‐Gad B,E. coliBL21(DE3)‐Gad B‐T62S, andE. coliBL21(DE3)‐Gad B‐Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively.