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SiO₂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@SiO₂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.SiO₂ 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@SiO₂ 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@SiO₂ immobilization enzyme shows good catalytic performance for cyclic, chain and aromatic ketenes.

宋浩雷(1976-),男,博士,高级工程师,研究方向为生物医药,songhl_yanuo@163.com