Design of Artificial Alcohol Oxidases: Alcohol Dehydrogenase-NADPH Oxidase Fusions for Continuous Oxidations

Abstract

To expand the arsenal of industrially applicable oxidative enzymes, fusions of alcohol dehydrogenases with an NADPH-oxidase were designed. Three different alcohol dehydrogenases (LbADH, TbADH, ADHA) were expressed with a thermostable NADPH-oxidase fusion partner (PAMO C65D) and purified. The resulting bifunctional biocatalysts retained the catalytic properties of the individual enzymes, and acted essentially like alcohol oxidases: transforming alcohols to ketones by using dioxygen as mild oxidant, while merely requiring a catalytic amount of NADP⁺ . In small-scale reactions, the purified fusion enzymes show good performances, with 69-99 % conversion, 99 % ee with a racemic substrate, and high cofactor and enzyme total turnover numbers. As the fusion enzymes essentially act as oxidases, we found that commonly used high-throughput oxidase-activity screening methods can be used. Therefore, if needed, the fusion enzymes could be easily engineered to tune their properties.

Keywords: alcohol dehydrogenases; biocatalysis; enzyme engineering; fusion enzymes; oxidases.

Scheme 1

Alcohol dehydrogenases (ADHs) can catalyze alcohol oxidations and ketone reductions. By fusing an ADH with a NOX enzyme, which can oxidize the reduced nicotinamide cofactor NADPH by using oxygen, the equilibrium is driven toward catalyzing alcohol oxidations. In essence, the fusion of the two enzymes acts like an alcohol oxidase: an alcohol substrate is converted at the cost of oxygen, and hydrogen peroxide is produced.

Scheme 2

Kinetic resolution of rac‐1‐phenylethanol with the NOX‐ADH fusions. The reaction would ideally yield 50 % acetophenone, and 50 % of 99 % ee (R)‐ or (S)‐1‐phenylethanol.

Figure 1

With an HRP‐coupled assay, the alcohol oxidation activity of the NOX‐A fusion can be detected without any addition of NADP⁺ (3). The reaction mixture included buffer (50 mm Tris⋅HCl pH 7.5), HRP (0.8 U), AAP (0.1 mm) and DCHBS (1 mm), cell‐free extract containing NOX‐A (10 % v/v) and 30 mm cyclohexanol. Controls: 1) no substrate and 2) no cell‐free extract.

Figure 2

The fusion of an ADH with a NOX enables the detection of alcohol oxidation activity in colonies. The plates contain colonies that expressed A) NOX‐A or B) NOX. Only the colonies that produce the fusion enzyme turn dark blue after addition of the assay mix.