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}^{60mathrm{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).
Highlights
The ability of enzymes to operate in aqueous systems in a highly efficient manner makes them attractive environmentally benign synthetic reagents
Synthetic genes encoding wild-type An alcohol dehydrogenase from Clostridium beijerinckii (CbADH) (CbADH-Wild type (WT)) and the five designs were expressed in E. coli BL21 (DE3), and the results showed that mutations except CbADH24M were expressed with higher solubility and activity than the wild-type CbADH, and the soluble enzyme protein ratio and activity dramatically decreased with the number of mutation sites increased (Fig. 4a and Additional file 1: Fig. S8), that was different from Goldenzweig’s work
In summary, CbADH with high stability and specific activity was identified for Nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and a computational protein design named Protein Repair One-Stop Shop (PROSS) was applied for the soluble modification of this enzyme
Summary
The ability of enzymes to operate in aqueous systems in a highly efficient manner makes them attractive environmentally benign synthetic reagents. 30% of the biotransformations in industry (Straathof et al 2002), and the vast majority are dependent on two nicotinamide cofactors NADH or NADPH. These two cofactors differ only by the 2′-phosphate group that is attached to the adenine ribose in NADPH, they play a completely different role in nature. NADH is used almost exclusively for oxidative degradations that eventually lead to production of ATP, whereas NADPH is confined with few exceptions to the biosynthetic reactions (Carugo and Argos 1997), involving a spectrum of over 300 known, repeatedly used reaction types (Woodyer et al 2005), e.g., C–H oxygenation (Landwehr et al 2006), regioselective halogenation (Mori et al 2019), Baeyer–Villiger oxidation (Schmidt et al 2015), stereoselective reduction (Zhu and Hua 2006) and reductive amination
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