Directed evolution of glutamate decarboxylase B for enhancing its enzyme activity towards nearly neutral pHs based on error-prone PCR

Abstract

Glutamate decarboxylases (GADs) can catalyze the conversion of l-glutamate to γ-aminobutyric acid (GABA), while consuming one H⁺. However, the GADs found so far are catalytically active in the pHs of 3.8-5.0, and are inactivated at nearly neutral pHs (>5.0). In order to obtain GADs with a high activity at nearly neutral pHs, the directed evolution of GadB was performed. The gadB gene was amplified by error-prone PCR, and was transformed into E. coli BL21(DE3) to establish a random mutagenesis library. A high throughput screening based on the changes in the color of bromothymol blue was used to screen the mutated strain whose GadB was active at nearly neutral pHs. The mutated GadB was purified to investigate enzymatic properties and the mechanism of pH adaption by molecular docking. The results indicated that the mutated GadB^D304G/F433L was screened and its activities were respectively increased by 935.90 % and 984.31 % at pHs 5.8 and 6.6 as compared to those of GadB(WT). By simulating the molecular docking, GadB^D304G/F433L could form more hydrogen bonds with the substrate and had a lower binding energy, thus increasing the affinity for the substrate. This study contributes to a basis for the use of GadB at nearly neutral pHs.

Keywords: Directed evolution; Enzymatic properties; Glutamate decarboxylase; Kinetic parameters; Molecular docking.