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

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.