Computational Design of Stable, Soluble and Highly Active Alcohol Dehydrogenase for NADPH Regeneration

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 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 = +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, 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 anther ADH from Lactobacillus kefir (LkADH). 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 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: ΔT60 min 1/2 = +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, 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 anther ADH from Lactobacillus kefir (LkADH).