Mining of a novel thermostable glutamate decarboxylase by ancestral sequence reconstruction and application of this enzyme in biosynthesis of food-grade γ-aminobutyric acid

Abstract:

γ-aminobutyric acid (GABA) is a non-protein amino acid that has been used as a new functional factor in the fields of food, medicine, chemical engineering, and agriculture. Glutamate decarboxylase (GAD) is a key rate-limiting enzyme that catalyzes the formation of GABA from l-glutamic acid (l-Glu). However, GADs from different sources have a common problem of limited thermal stability, which affects their industrial applications. In order to obtain GADs with high activity and thermal stability, and reduce the production cost of GABA, herein, a novel thermostable GAD (EgGAD) derived from Enterococcus gallinarum was obtained through ancestral sequence reconstruction of a candidate gene directly mined with the GAD of Lactobacillus brevis (LbGAD) as the initial template. The recombinant EgGAD exhibited the maximal activity at 60 ℃ and pH 5.0. The Michaelis constant (K_m) and catalytic efficiency (k_cat/K_m) of EgGAD with l-Glu as the substrate were 10.94 mmol/L and 2.07 L/(s∙mmol), respectively. Notably, EgGAD exhibited a larger shift in thermostability, with about 4-fold improvement of half-life (t_1/2) at 55 ℃ and a 4.94 ℃ increase in semi-inactivation temperature (T₅₀¹⁵) compared with that of LbGAD. Furthermore, a high-efficiency synthesis system for GABA was developed with dormant engineered Escherichia coli Nissle 1917 cells as biocatalysts. When E. coli Nissle (T7)/pET28a- EggadB cells were concentrated to reach the optical density (OD)₆₀₀ of 20 in 3 mol/L l-Glu solution, the GABA yield reached 300.94 g/L, with more than 99.5% conversion ratio, after reaction at 40 ℃ and 150 r/min for 4 h. Overall, this study emphasizes the value of an ancestral sequence reconstruction technique for direct gene mining to improve the thermal stability of GAD, provides a functional component for the biosynthesis of GABA, and sheds light on improving the thermal stability of other enzymes.