多酶一步纯化共固定系统的构建及催化不对称还原合成手性醇的研究  

Construction of a multi-enzyme one-step purification co-immobilization system to catalyze asymmetric reduction for synthesis of chiral alcohols

作　　者：宋浩雷 欧阳亚萍 原泉 王天尧 马丽[2] SONG Hao-le;OUYANG Ya-ping;YUAN Quan;WANG Tian-yao;MA Li(Hebei Yanuo Bioscience Co.,Ltd.,Shijiazhuang 050035,China;School of Chemical Engineering and Technology,Hebei University of Technology,Tianjin 300401,China)

机构地区：[1]河北亚诺生物科技股份有限公司,河北石家庄050035 [2]河北工业大学化工学院,天津300401

出　　处：《现代化工》2023年第11期104-110,共7页Modern Chemical Industry

基　　金：河北省自然科学基金(B2020202036);河北省省级科技计划资助(22372802D)。

摘　　要：开发了一步纯化共固定老黄酶(OYE1)、醇脱氢酶(ADH-A)和葡萄糖脱氢酶(GDH)系统并一锅级联催化烯酮转化为手性醇类化合物,同时实现辅酶的原位再生。以镍离子金属螯合亲和层析介质(Ni-NTA)修饰的SiO_(2)纳米花为载体,对带有His-tag的目的蛋白ADH-A、OYE1和GDH粗酶液进行一步分离纯化及固定化,并在优化的反应条件下进行反应,对手性醇产物的产率最高可达92.37%。固定化酶相比于游离酶的稳定性更加出色。在连续5次循环使用后,固定化酶在使用时仍有较高的转化率。同时,固定化酶OYE1&ADH-A&GDH@Ni-NTA@SiO_(2)对环状、链状及芳香族烯酮类化合物均具有良好的催化性能。A system is developed for one-step purification co-immobilization of old yellow enzyme(OYE1),alcohol dehydrogenase(ADH-A)and glucose dehydrogenase(GDH),and a one-pot cascade catalytic system is developed to catalyze ketene to high value-added chiral alcohol compounds while the in situ regeneration of coenzymes(NADPH and NADH)is achieved.SiO_(2) nanoflower modified by nickel ion metal chelation affinity chromatography medium(Ni-NTA)is taken as carrier,crude enzyme solutions containing ADH-A,OYE1,and GDH,targeted proteins with His-tag,are separated,purified and immobilized in one-step,and the reaction is performed under the optimized reaction conditions.The highest yield of chiral alcohol products can reach 92.37%.Compared with free enzyme,OYE1&ADH-A&GDH@Ni-NTA@SiO_(2) immobilization enzyme has better stability.After it has been used for 5 consecutive cycles,the immobilization enzyme still delivers a high conversion.OYE1&ADH-A&GDH@Ni-NTA@SiO_(2) immobilization enzyme shows good catalytic performance for cyclic,chain and aromatic ketenes.

关 键 词：老黄酶 不对称合成 一步纯化固定化 多酶级联 手性化合物 

分 类 号：TQ46[化学工程—制药化工]