酶法生产1,4-环己烷二甲胺  

Enzymatic production of 1,4-cyclohexanedimethylamine

作　　者：韩业挺 何志震 魏婉清 宋伟 刘立明 朱萌[4] 吴静 HAN Yeting;HE Zhizhen;WEI Wanqing;SONG Wei;LIU Liming;ZHU Meng;WU Jing(School of Life Sciences and Health Engineering,Jiangnan University,Wuxi 214122,Jiangsu,China;State Key Laboratory of Food Science and Resources,Jiangnan University,Wuxi 214122,Jiangsu,China;School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China;Wuxi Acryl Technology Co.,Ltd.,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学生命科学与健康工程学院,江苏无锡214122 [2]江南大学食品科学与资源挖掘全国重点实验室,江苏无锡214122 [3]江南大学生物工程学院,江苏无锡214122 [4]无锡阿科力科技有限公司,江苏无锡214122

出　　处：《生物工程学报》2024年第6期1882-1894,共13页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2021YFC2102700);国家自然科学基金(22178146);国家特支计划青年拔尖人才;中央高校基本科研业务费专项资金(JUSRP123011)。

摘　　要：1,4-环己烷二甲胺(1,4-cyclohexanedimethylamine,1,4-BAC)作为重要的生物基材料单体,被广泛应用于有机合成、医药、化工和材料等多个领域。目前主要采用化学法合成,存在金属催化剂价格昂贵、反应条件苛刻和安全隐患等问题,亟须寻找更环保的合成替代方法。本研究利用大肠杆菌(Escherichia coli)来源的转氨酶(transaminase,Ec TA)、酿酒酵母(Saccharomyces cerevisiae)来源的谷氨酸脱氢酶(glutamate dehydrogenase,Sc Glu-DH)和博伊丁假丝酵母(Candida boidinii)来源的甲酸脱氢酶(formate dehydrogenase,Cb FDH)成功构建了双菌三酶级联转化1,4-环己烷二甲醛生成1,4-环己烷二甲胺的路径。基于结构指导下的蛋白质工程改造,获得了有益突变体Ec TAF91Y,其比酶活和kcat/Km较野生型分别提升了2.2倍和1.9倍。通过重组菌株的构建和反应条件的优化,在最优条件下40 g/L底物可生成(27.4±0.9)g/L产物,摩尔转化率为67.5%±2.1%。1,4-cyclohexanedimethylamine(1,4-BAC)is an important monomer for bio-based materials,it finds wide applications in various fields including organic synthesis,medicine,chemical industry,and materials.At present,its synthesis primarily relies on chemical method,which suffer from issues such as expensive metal catalyst,harsh reaction conditions,and safety risks.Therefore,it is necessary to explore greener alternatives for its synthesis.In this study,a two-bacterium three-enzyme cascade conversion pathway was successfully developed to convert 1,4-cyclohexanedicarboxaldehyde to 1,4-cyclohexanedimethylamine.This pathway used Escherichia coli derived aminotransferase(EcTA),Saccharomyces cerevisiae derived glutamate dehydrogenase(ScGlu-DH),and Candida boidinii derived formate dehydrogenase(CbFDH).Through structure-guided protein engineering,a beneficial mutant,EcTAF91Y,was obtained,exhibiting a 2.2-fold increase in specific activity and a 1.9-fold increase in kcat/Km compared to that of the wild type.By constructing recombinant strains and optimizing reaction conditions,it was found that under the optimal conditions,a substrate concentration of 40 g/L could produce(27.4±0.9)g/L of the product,corresponding to a molar conversion rate of 67.5%±2.1%.

关 键 词：1 4-环己烷二甲胺 三酶级联 蛋白质工程改造 全细胞转化 1 4-环己烷二甲醛 

分 类 号：Q78[生物学—分子生物学] TQ236[化学工程—有机化工] TQ426.97