Zhang Likun, Xiao Yanming, Yang Weihua, Hua Chao, Wang Yun, Li Jingya, Yang Taowei
Zhejiang Engineering Research Center of Industrial Biocatalysis and Transformation, Changxing Pharmaceutical Co. Ltd., Changxing 313100, Zhejiang, China.
School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2020 May 25;36(5):992-1001. doi: 10.13345/j.cjb.190327.
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.
在本研究中，以大肠杆菌BL21(DE3)作为宿主，构建了2种分别共表达亮氨酸脱氢酶(LDH，蜡样芽孢杆菌)/甲酸脱氢酶(FDH，水生食烷菌)或亮氨酸脱氢酶(LDH，蜡样芽孢杆菌)/乙醇脱氢酶(ADH，红球菌)的重组大肠杆菌菌株。然后，通过L-苏氨酸脱氨酶(L-TD)与LDH-FDH或LDH-ADH偶联两种不同的NADH再生系统来合成L-2-氨基丁酸。对LDH-FDH工艺和LDH-ADH工艺进行了优化并相互比较。LDH-FDH工艺的最佳反应pH为7.5，最佳反应温度为35℃。分批添加L-苏氨酸以控制2-酮丁酸浓度低于15 g/L，并使用50 g/L甲酸铵、0.3 g/L NAD⁺、10%的LDH-FDH粗酶液(V/V)和7500 U/L L-TD，28 h后，L-2-氨基丁酸浓度为161.8 g/L，产率为97%。LDH-ADH工艺的最佳反应pH为8.0，最佳反应温度为35℃。分批添加L-苏氨酸和异丙醇(为L-苏氨酸的1.2倍)以控制2-酮丁酸浓度低于15 g/L，及时除去丙酮，并使用0.3 g/L NAD⁺、10%的LDH-ADH粗酶液(V/V)和7500 U/L L-TD，24 h后，L-2-氨基丁酸浓度为119.6 g/L，产率为98%。本文所采用的工艺和结果为L-2-氨基丁酸的工业化生产提供了参考。
