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Abstract：
Nicotinamide adenine dinucleotide phosphate (NADPH), as a well-known cofactor, is widely used in the most of enzymatic redox reactions, playing an important role in industrial catalysis. However, the absence of a comparable method for efficient NADP+ to NADPH cofactor regeneration radically impairs efficient green chemical synthesis. Alcohol dehydrogenase (ADH) enzymes, allowing the in situ regeneration of the redox cofactor NADPH with high specific activity and easy by-product separation process, are provided with great industrial application potential and research attention. Accordingly, herein a NADP+-specific ADH from Clostridium beijerinckii was selected to be engineered for cofactor recycle, using an automated algorithm named Protein Repair One-stop Shop (PROSS). The mutant CbADH-6M (S24P/G182A/G196A/H222D/S250E/S254R) exhibited a favorable soluble and highly active expression with an activity of 46.3 U/mL, which was 16 times higher than the wild type (2.9 U/mL), and a more stable protein conformation with an enhanced thermal stability: Δ T 1 / 2 60 min = + 3.6 °C (temperature of 50% inactivation after incubation for 60 min). Furthermore, the activity of CbADH-6M was up-graded to 2401.8 U/mL by high cell density fermentation strategy using recombinant Escherichia coli, demonstrating its industrial potential. Finally, the superb efficiency for NADPH regeneration of the mutant enzyme was testified in the synthesis of some fine chiral aromatic alcohols coupling with another ADH from Lactobacillus kefir (LkADH).
摘要：
烟酰胺腺嘌呤二核苷酸磷酸（NADPH），作为一个众所周知的辅因子，广泛用于大多数酶促氧化还原反应，在工业催化中发挥着重要作用。然而,缺乏有效的NADP与NADPH辅因子再生的可比方法从根本上损害了有效的绿色化学合成。酒精脱氢酶（ADH）酶，允许氧化还原辅因子NADPH的原位再生，具有高比活性和易于副产物分离过程，具有巨大的工业应用潜力和研究关注。因此，本文选择了来自贝氏梭菌的NADP特异性ADH进行工程改造以进行辅因子回收，使用名为蛋白质修复一站式服务（PROSS）的自动算法。突变体CbADH-6M（S24P/G182A/G196A/H222D/S250E/S254R）表现出良好的可溶性和高活性表达，活性为46.3U/mL，比野生型（2.9U/mL）高16倍，和更稳定的蛋白质构象，具有增强的热稳定性：ΔT1/260分钟=3.6°C（孵育60分钟后50％失活的温度）。此外，通过重组大肠杆菌的高细胞密度发酵策略，将CbADH-6M的活性上调至2401.8U/mL，展示其工业潜力。最后,在一些精细的手性芳族醇与另一种从开菲尔乳杆菌（LkADH）中的ADH偶联的合成中，证明了突变酶的NADPH再生的出色效率。
