宏基因组来源重组谷氨酸脱羧酶的酶学特性及全细胞合成γ-氨基丁酸

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

摘要:

【背景】 谷氨酸脱羧酶广泛存在于生物体内，其催化产物γ-氨基丁酸是哺乳动物重要的抑制性神经递质。目前，微生物来源的谷氨酸脱羧酶在热和酸碱条件下仍不够稳定。【目的】 挖掘肠道微生物的谷氨酸脱羧酶基因，异源表达并研究其酶学性质，为γ-氨基丁酸的生物合成提供酶原。【方法】 从倭蜂猴粪便微生物宏基因组扩增谷氨酸脱羧酶基因，在大肠杆菌(Escherichia coli) BL21(DE3)中进行异源表达、酶学性质研究及全细胞合成γ-氨基丁酸的应用。【结果】 获得谷氨酸脱羧酶基因Xby1_8，重组酶Xby1_8分子量为54.46 kDa，最适作用条件为pH 5.0、55 ℃，K_m和V_max分别为(10.2±1.5) mmol/L和(3 574.0±198.3) μmol/(min·mg)，较其他微生物来源的谷氨酸脱羧酶具有最高比活3 106.2 U/mg。Xby1_8具有较好的pH和温度稳定性，pH 4.0–8.0或30–50 ℃处理1 h，剩余酶活仍高于100%。在l-谷氨酸浓度260 mmol/L，反应温度55 ℃，细胞浓度OD₆₀₀为3.5条件下反应2.5 h，重组菌E. coli/Xby1_8全细胞催化制备γ-氨基丁酸的转化率为100%。【结论】 从粪便微生物宏基因组中获得谷氨酸脱羧酶基因Xby1_8，并成功在大肠杆菌BL21(DE3)中表达。重组酶Xby1_8的pH和温度稳定性较好，重组菌E. coli/Xby1_8全细胞催化制备γ-氨基丁酸具有较高的转化率，在食品、工业等领域具有潜在的应用价值。

关键词:宏基因组;粪便微生物;谷氨酸脱羧酶;γ-氨基丁酸;全细胞催化

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.

Key words:metagenome;fecal microorganism;glutamate decarboxylase;γ-aminobutyric acid;whole-cell catalysis