亮氨酸脱氢酶偶联NADH再生体系合成L-2-氨基丁酸

Synthesis of L-2-aminobutyric acid by leucine dehydrogenase coupling with an NADH regeneration system

摘要:

文中以大肠杆菌BL21(DE3) 为宿主，构建两株分别共表达亮氨酸脱氢酶 (LDH，来源蜡样芽孢杆菌)/甲酸脱氢酶 (FDH，来源水生弯杆菌) 和亮氨酸脱氢酶 (LDH，来源蜡样芽孢杆菌)/醇脱氢酶 (ADH，来源红球菌)的重组大肠杆菌。通过偶联两种不同NADH再生体系，以L-苏氨酸为起始原料，利用苏氨酸脱氨酶 (L-TD) 与LDH-FDH或LDH-ADH一锅法合成L-2-氨基丁酸，并对LDH-FDH工艺和LDH-ADH工艺进行对比优化。LDH-FDH工艺的最适反应pH为7.5，最适反应温度为35 ℃，通过加入50 g/L甲酸铵、0.3 g/L NAD+、10% LDH-FDH粗酶液 (V/V) 和7 500 U/L的L-TD酶液，对L-苏氨酸进行分批补加，以便控制2-丁酮酸浓度小于15 g/L，反应28 h，实现了L-2-氨基丁酸的产量为161.8 g/L，产率97%。LDH-ADH工艺的最适pH为8.0，最适反应温度为35 ℃，通过加入0.3 g/L NAD+、10% LDH-ADH粗酶液 (V/V) 及7 500 U/L的L-TD酶液，分批补加L-苏氨酸及1.2倍摩尔量异丙醇，以便控制2-丁酮酸浓度小于15 g/L，且每生成约40 g/L的L-2-氨基丁酸，抽真空去除丙酮，反应24 h，实现了L-2-氨基丁酸的产量为119.6 g/L，产率98%。文中所采用的工艺及结果可为L-2-氨基丁酸的工业化提供一定的参考依据。

Abstract:

In this study, Escherichia coli BL21 (DE3) was used as the host to construct 2 recombinant E. coli strains that co-expressed leucine dehydrogenase (LDH, Bacillus cereus)/formate dehydrogenase (FDH, Ancylobacter aquaticus), or leucine dehydrogenase (LDH, Bacillus cereus)/alcohol dehydrogenase (ADH, Rhodococcus), respectively. L-2-aminobutyric acid was then synthesized by L-threonine deaminase (L-TD) with LDH-FDH or LDH-ADH by coupling with two different NADH regeneration systems. LDH-FDH process and LDH-ADH process were optimized and compared with each other. The optimum reaction pH of LDH-FDH process was 7.5, and the optimum reaction temperature was 35 °C. After 28 h, the concentration of L-2-aminobutyric acid was 161.8 g/L with a yield of 97%, when adding L-threonine in batches for controlling 2-ketobutyric acid concentration less than 15 g/L and using 50 g/L ammonium formate, 0.3 g/L NAD+, 10% LDH-FDH crude enzyme solution (V/V) and 7 500 U/L L-TD. The optimum reaction pH of LDH-ADH process was 8.0, and the optimum reaction temperature was 35 °C. After 24 h, the concentration of L-2-aminobutyric acid was 119.6 g/L with a yield of 98%, when adding L-threonine and isopropanol (1.2 times of L-threonine) in batches for controlling 2-ketobutyric acid concentration less than 15 g/L, removing acetone in time and using 0.3 g/L NAD+, 10% LDH-ADH crude enzyme solution (V/V) and 7 500 U/L L-TD. The process and results used in this paper provide a reference for the industrialization of L-2-aminobutyric acid.