Computational design of highly stable and soluble alcohol dehydrogenase for NADPH regeneration

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).

Keywords: Alcohol dehydrogenase; Chiral alcohols; Computational design; NADPH regeneration; Soluble expression.

Fig. 1

Biosynthetic reactions using NADPH

Fig. 2

Characterization of alcohol dehydrogenases. a Enzyme activity and soluble expression of alcohol dehydrogenases in shake-flask fermentation at 25 ℃ with 0.5 mM IPTG dosage. b Stability of the crude extract enzymes in atmosphere at 25 °C

Fig. 3

Effects of induction conditions on the wild CbADH activity in shake-flask fermentation. a Induced by different IPTG concentrations (0.1–1.0 mM) at 25 ℃. b Induced at different temperatures (16–30 ℃) with 0.5 mM IPTG dosage

Fig. 4

Characterization of CbADH mutants compared with the wild type. a Enzyme activity in shake-flask fermentation at 25 ℃ with 0.5 mM IPTG dosage. The green column represents the crude enzyme activity, while the beige column represents OD₆₀₀. The red square represents the proportion of soluble protein to total protein. b Thermal stability of the wild CbADH and the mutants. c SDS-PAGE analysis of protein expression of the wild-type CbADH-WT and mutant CbADH-6M: Lane M: molecular weight marker; Lane W: whole cell protein; Lane S: supernatant; Lane P: precipitation

Fig. 5

Protein structure of CbADH-6M (red backbone) compared with the wild CbADH (gray backbone). a The mutant S24P located in the loop region. b The mutant G182A located in the α-helix. c The mutant G196A located at β-sheets. d New salt bridge Glu64–Arg80 formed by CbADH-6M. e Schematic diagram of new salt bridge network centered on Arg254

Fig. 6

Fermentation process of recombinant Escherichia coli-pET-28a-CbADH-6M in a 15.0-L mechanically stirred fermentor. a Fermentation parameter control. b Cell growth and CbADH-6M expression during the fermentation. c SDS-PAGE analysis of protein expression of samples at different time: Lane M: molecular weight marker; Lane W: whole cell protein; Lane S: supernatant; Lane P: precipitation

Fig. 7

Characterization of the recombinant CbADH-6M. a Temperature optimum: the enzyme activity was measured at various temperatures (15–75 ℃) in phosphate buffer (pH 7.5). b Thermostability: 1.0 μg/mL purified enzyme was incubated at the specific temperature (50, 60, 70) for different times, and the residual activity was measured by standard assay. c pH optimum: the enzyme activity was assayed in various buffers (6.0–12.5) at 35 ℃. d pH stability: pH stability was measured by incubation of purified enzyme in different buffers (pH 6–10) for 24 h at 35 ℃, and the residual activity was measured by standard assay

Fig. 8

The reduction of acetophenone for synthesis of (S)-1-phenylethanol. a LkADH mono-enzymatic reaction. b LkADH and CbADH (CbADH-6M) dual enzyme catalyzed reaction