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Design of Artificial Alcohol Oxidases: Alcohol Dehydrogenase–NADPH Oxidase Fusions for Continuous Oxidations
Author(s) -
Aalbers Friso S.,
Fraaije Marco W.
Publication year - 2019
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201800421
Subject(s) - alcohol oxidase , alcohol dehydrogenase , cofactor , enzyme , chemistry , alcohol , alcohol oxidoreductase , biochemistry , oxidative phosphorylation , oxidase test , phosphofructokinase 2 , biocatalysis , bifunctional , catalysis , substrate (aquarium) , combinatorial chemistry , nad+ kinase , biology , gene , reaction mechanism , recombinant dna , pichia pastoris , ecology
Abstract To expand the arsenal of industrially applicable oxidative enzymes, fusions of alcohol dehydrogenases with an NADPH‐oxidase were designed. Three different alcohol dehydrogenases ( Lb ADH, Tb ADH, 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.