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.

SUBMITTER: Aalbers FS 

PROVIDER: S-EPMC6899577 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature