亚胺还原酶与甲酸脱氢酶的融合表达及其固定化

Fusion expression and immobilization of an imine reductase and a formate dehydrogenase

摘要:

手性胺作为关键的有机合成中间体，广泛应用于药物、精细化学品及生物活性分子的合成，具有重要的工业价值。手性胺的不对称合成一直备受关注，NAD(P)H依赖型亚胺还原酶(imine reductase, IRED)具有底物范围广、高活性和立体选择性的特点，可用于不对称还原合成手性胺。本研究旨在通过构建一种高效、稳定的IRED与甲酸脱氢酶(formate dehydrogenase, FDH)双酶融合表达与固定化体系，解决辅酶再生难题并提升催化效率，为手性胺的绿色合成提供新策略。该体系用于催化1-甲基-3,4-二氢异喹啉不对称还原合成(S)-1-甲基-1,2,3,4-四氢异喹啉。通过对比发现，融合表达体系的催化效果优于共表达和单独表达双酶的体系。其次，利用介孔氧化硅纳米花(mesoporous silica nanoflowers, MSN)作为载体，共价法固定化融合表达双酶，固定化时间为1.5 h且初始酶浓度为2.5 mg/mL时，蛋白负载量为193.2 mg/g。固定化酶展现出良好的pH稳定性、热稳定性和储藏稳定性。将制备的固定化酶应用于催化其他环状亚胺如1-乙基-3,4-二氢异喹啉、5-苯基-3,4-二氢-2H-吡咯、2,3,3-三甲基-3H-吲哚、2,3,3,5-四甲基吲哚和麦斯明等的不对称还原反应，转化率高于95%，对映体过量值(enantiomeric excess, e.e.)高于96%，验证了固定化融合酶催化体系在手性胺绿色高效合成中的应用潜力。本研究为手性胺的工业化生物合成提供了新思路，所开发的融合酶固定化策略对解决辅酶依赖型生物催化过程中的共性技术难题具有重要的理论与应用价值。

Abstract:

Chiral amines as pivotal intermediates in organic synthesis are widely utilized in the production of pharmaceuticals, fine chemicals, and bioactive molecules, holding significant industrial value. The asymmetric synthesis of chiral amines has always gained great attention. NAD(P)H-dependent imine reductases (IREDs) with wide substrate ranges, high activity, and high enantiomeric selectivity can be used for asymmetric reduction of imines to chiral amines. Our study aims to develop an efficient and stable immobilized dual-enzyme system through the fusion expression of imine reductase and formate dehydrogenase, in order to address the challenge of coenzyme regeneration and enhance catalytic efficiency, thereby providing a novel strategy for the green synthesis of chiral amines. The constructed system was applied to catalyze the asymmetric reduction of 1-methyl-3,4-dihydroisoquinoline to synthesize (S)-1-methyl-1,2,3,4-tetrahydroisoquinoline. Comparative analysis indicated that the catalytic efficiency of this fusion expression system exceeded that of both co-expression and standalone dual-enzyme systems. Furthermore, mesoporous silica nanoflowers were utilized as carriers to immobilize the fusion-expressed dual enzymes through a covalent method. In the case of covalent binding duration of 1.5 h and an initial enzyme concentration of 2.5 mg/mL, the protein loading achieved 193.2 mg/g. The immobilized enzymes demonstrated excellent pH, thermal, and storage stability. When the immobilized enzymes were employed to catalyze asymmetric reduction reactions of other cyclic imines, such as 1-ethyl-3,4-dihydroisoquinoline, 5-phenyl-3,4-dihydro-2H-pyrrole, 2,3,3-trimethyl-3H-indole, 2,3,3,5-tetramethylindole, and myosmine, the conversion rates exceeded 95%, and the values of e.e. surpassed 96%. The data confirm the application potential of the immobilized fusion enzymes in the green and efficient synthesis of chiral amines. Our study provides a novel strategy for the industrial biosynthesis of chiral amines, and the developed fused-enzyme immobilization approach holds significant theoretical and practical value for addressing common technical challenges in cofactor-dependent biocatalytic processes.