Design of artificial alcohol oxidases:alcohol dehydrogenase - NADPH oxidase fusions for continuous oxidations

With the aim of expanding the arsenal of industrially-applicable oxidative enzymes, fusions of alcohol dehydrogenases with an NADPH-oxidase were designed. In total 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 act essentially like alcohol oxidases: transforming alcohols to ketones 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% conversions, 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 can be easily engineered to tune their properties