A recombinant glutamate decarboxylase from metagenome: enzymatic properties and whole-cell catalytic synthesis of γ-aminobutyric acid

Abstract:

[Background] Glutamate decarboxylase is widely present in organisms. γ-aminobutyric acid, the catalytic product of glutamate decarboxylase, is an inhibitory neurotransmitter in mammals. At present, many glutamate decarboxylases from microorganisms have poor thermal stability and acid-base stability. [Objective] To heterologously express a glutamate decarboxylase gene mined from gut microbiota and study the enzymatic properties of the recombinant protein, so as to provide an enzyme source for the biosynthesis of γ-aminobutyric acid. [Methods] The glutamate decarboxylase gene was amplified from the fecal microbial metagenome of Pygmy loris and expressed in Escherichia coli BL21(DE3). The enzymatic properties of the recombinant enzyme were characterized, and the enzyme was then used for the whole-cell synthesis of γ-aminobutyric acid. [Results] The glutamate decarboxylase gene Xby1_8 was obtained, and the molecular weight of the recombinant enzyme Xby1_8 was 54.46 kDa. Under optimal reaction conditions of pH 5.0 and 55℃, the K_m andV_max values of the enzyme were (10.2±1.5) mmol/L and (3 574.0±198.3) μmol/(min·mg), respectively. Xby1_8 had the highest specific activity of 3 106.2 U/mg compared with other glutamate decarboxylases from microbial sources. Xby1_8 had good pH and thermal stability. The residual activity of Xby1_8 was still more than 100% after incubation at pH 4.0–8.0 or 30–50℃ for 1 h. The whole-cell recombinant E. coli/Xby1_8 (OD₆₀₀ of 3.5) reacted with 260 mmol/L l-glutamic acid at 55℃ for 2.5 h showed the conversion rate of 100% for the production of γ-aminobutyric acid. [Conclusion] The glutamate decarboxylase gene Xby1_8 was obtained from the fecal microbial metagenome and successfully expressed in E. coli BL21(DE3). This enzyme has good pH and thermal stability. The whole cell preparation of γ-aminobutyric acid from recombinant E. coli/Xby1_8 has a high conversion rate, thereby demonstrating promising potential for application in food and industrial fields.