祖先酶序列重构策略介导的耐热型谷氨酸脱羧酶挖掘及用于食品级γ-氨基丁酸高效合成

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

摘要:

γ-氨基丁酸(γ-aminobutyrate acid, GABA)是一种非蛋白质氨基酸，作为新型功能性因子，在食品、医药、化工及农业等领域有着广泛的应用价值。谷氨酸脱羧酶(glutamate decarboxylase, GAD)是生物法合成GABA的限速酶，具有良好热稳定性是实现其工业化应用的重要前提。为挖掘热稳定性好、催化活性高的GAD，降低GABA的制备成本，本研究以GABA工业生产菌株短促生乳杆菌(Levilactobacillus brevis) GAD氨基酸序列(LbGAD)为模板，利用FireProt^ASR系统获得其祖先酶蛋白序列，进一步通过“基因探矿”技术获得1个潜在的鹑鸡肠球菌(Enterococcus gallinarum)源耐热型GAD (EgGAD)。酶学性质分析显示，EgGAD的最适反应温度和pH值分别为60 ℃和pH 5.0，米氏常数(K_m)为10.94 mmol/L，催化效率(k_cat/K_m)为2.07 L/(s∙mmol)，半失活温度(T₅₀¹⁵)为61.14 ℃，较LbGAD高4.94 ℃，在55 ℃条件下的半衰期(t_1/2)为105.04 min，约为LbGAD的5倍，表明EgGAD具有良好的催化活性和热稳定性。在此基础上，通过代谢工程手段于益生菌Escherichia coli Nissle 1917细胞中实现了EgGAD的可溶性表达，并研究了工程菌株催化合成食品级GABA的性能。结果显示，将诱导后的E. coli Nissle (T7)/pET28a-EggadB细胞加入至含有3 mol/L l-谷氨酸(l-Glu)的200 mL纯水中(OD₆₀₀为20)，在40 ℃、150 r/min且非控制pH条件下反应4 h后，GABA产量高达300.94 g/L，转化率超过99.5%。本研究通过祖先酶序列重构策略介导的基因探矿技术挖掘了1个新的耐热型GAD，为生物法合成GABA提供了一个良好的功能元件，也为其他酶稳定性的改造提供了一个可供借鉴的方法。

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.