植物乳杆菌谷氨酸脱羧酶催化pH范围的理性改造及高效转化生产g-氨基丁酸

Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum

摘要:

γ-氨基丁酸可由谷氨酸脱羧酶(glutamate decarboxylase, GAD)催化谷氨酸一步合成，反应体系成分简单、环境友好。然而，绝大多数GAD酶催化pH偏酸性且反应范围狭小，需要加入无机盐维持最适催化环境，增加了生产附加成分。此外，随着产物γ-氨基丁酸的生成，溶液pH会逐渐上升，不利于GAD酶的持续转化。本研究首先从实验室保藏的一株高产γ-氨基丁酸的植物乳杆菌(Lactobacillus plantarum)中克隆得到谷氨酸脱羧酶LpGAD，基于酶蛋白表面电荷修饰，选择9个位点进行定点突变及组合突变，酶学性质表征结果显示三突变体LpGAD^{S24R/D88R/Y309K}在催化pH区间内酶活力整体提高，尤其拓宽了在偏中性pH 6.0下的酶活，为野生酶的1.68倍。接下来，通过分子动力学模拟解析了酶活提高的机理。此外，将Lpgad与Lpgad^{S24R/D88R/Y309K}突变基因分别在谷氨酸棒杆菌(Corynebacterium glutamicum) E01中过表达，通过优化确定了摇瓶最适转化条件为反应温度40 ℃，菌体量OD₆₀₀=20，底物L-谷氨酸100.0 g/L，5-磷酸吡哆醛添加量为100 μmol/L。5 L发酵罐中，不调节pH，通过分批投料底物L-谷氨酸，γ-氨基丁酸产量高达402.8 g/L，较对照菌株提高了1.63倍。本研究成功拓宽了LpGAD的pH催化范围及酶活，提高了γ氨基丁酸的转化效率，为实现其规模化工业生产奠定了基础。

关键词:γ-氨基丁酸;谷氨酸脱羧酶;pH改造;表面电荷;全细胞转化

Abstract:

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGAD^{S24R/D88R/Y309K} was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and Lpgad^{S24R/D88R/Y309K} genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD₆₀₀) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Key words:γ-aminobutyric acid;glutamic acid decarboxylase;pH transformation;surface charge;whole cell transformation