多酶催化拆分DL-正缬氨酸生产L-正缬氨酸  

Multi-enzymatic resolution of DL-norvaline for L-norvaline production

作　　者：戚云龙 杨套伟[1] 周俊平 郑俊贤 徐美娟[1] 张显[1] 饶志明[1] 

机构地区：[1]江南大学生物工程学院,工业生物技术教育部重点实验室,无锡214122

出　　处：《应用与环境生物学报》2017年第6期1015-1021,共7页Chinese Journal of Applied and Environmental Biology

基　　金：国家高技术研究发展计划(863计划)(2015AA021004);江苏省杰出青年基金项目(BK20150002);江苏省自然科学基金项目(BK20161292);国家自然科学基金项目(31570085);中央高校基本科研业务费专项资金(JUSRP51708A)资助~~

摘　　要：L-正缬氨酸是合成抗高血压药物培垛普利的重要中间体,其合成主要通过化学方法,具有产品纯度低、环境污染等缺点.提出一种基于多酶介导拆分DL-正缬氨酸结合不对称还原生产高纯度L-正缬氨酸的方法.首先,将混合型DL-正缬氨酸中的D型经过D-氨基酸氧化酶氧化成相应酮酸,然后利用亮氨酸脱氢酶将酮酸不对称还原生成L-正缬氨酸,同时NADH被氧化成NAD^+,最后利用甲酸脱氢酶构建NADH循环再生系统.D-正缬氨酸被氧化过程中会产生副产物过氧化氢(H_2O_2),通过外源添加过氧化氢酶消除该副产物.基于前期最适转化温度、最适转化pH优化基础上,对过氧化氢酶添加量和单次底物添加量进行优化,利用分批补料策略提高L-正缬氨酸产量.结果显示,在最适条件下,L-正缬氨酸产量达到61.09 g/L对映体过量值(e.e.)和转化率分别为99%和96.1%.本研究合成L-正缬氨酸较之化学方法,具有环境污染少、产品纯度高等特点,为制药行业中光学纯L-正缬氨酸的生产提供了一种有效的方法.Perindopril is an effective and widely used antihypertensive drug. L-Norvaline is an important intermediate in the synthesis of perindopril, which is mainly produced using chemical synthesis, with low enantiomeric purity and adverse environmental impacts. This paper presents an environmental friendly method for producing high-purity L-norvaline based on a multi-enzymatic resolution process combined with asymmetric reduction. D-Norvaline was oxidized from the substrate DL-norvaline to the corresponding keto acid using D-amino acid oxidase （DAAO）. Next, leucine dehydrogenase （LeuDH） was used to catalyze the asymmetric reduction of keto acid to L-norvaline, with concomitant oxidation of NADH to NAD＋. Finally, formate dehydrogenase （FDH） was used to introduce an additional NADH regeneration system. The unwanted by-product hydrogen peroxide generated during D-norvaline oxidation could be removed by addition of catalase. Based on the previous optimization of conversion temperature and pH, the amount of catalase added and the proportions of DAAO, LeuDH, and FDH were optimized, and a fed-batch strategy was used to obtain a high yield of L-norvaline. Under optimal conditions, 61.09 g/L of L-norvaline was achieved, with an enantiomeric excess of 〉 99% and a conversion rate of 96.1%. In comparison with chemical synthesis, the newly developed method offers the advantages of environmental protection and high product purity; hence, it represents an efficient strategy for pharmaceutical production of enantiomerically pure L-norvaline.

关 键 词：酶促拆分 D-氨基酸氧化酶 亮氨酸脱氢酶 NADH循环再生系统 甲酸脱氢酶 L-正缬氨酸 

分 类 号：R91[医药卫生—药学]