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

作　　者：张利坤 肖延铭 杨卫华 华超 王云 李敬亚 杨套伟[2] Likun Zhang;Yanming Xiao;Weihua Yang;Chao Hua;Yun Wang;Jingya Li;Taowei Yang(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)

机构地区：[1]长兴制药股份有限公司,工业生物催化与转化浙江省工程研究中心,浙江长兴313100 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《生物工程学报》2020年第5期992-1001,共10页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2015AA021004)资助。

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7500 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7500 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。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℃.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 7500 U/L L-TD.The optimum reaction pH of LDH-ADH process was 8.0,and the optimum reaction temperature was 35℃.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 7500 U/L L-TD.The process and results used in this paper provide a reference for the industrialization of L-2-aminobutyric acid.

关 键 词：共表达 L-2-氨基丁酸 生物催化 NADH再生 

分 类 号：TQ922[轻工技术与工程—发酵工程]