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Mutagenesis‐Independent Stabilization of Class B Flavin Monooxygenases in Operation
Author(s) -
Goncalves Leticia C. P.,
Kracher Daniel,
Milker Sofia,
Fink Michael J.,
Rudroff Florian,
Ludwig Roland,
Bommarius Andreas S.,
Mihovilovic Marko D.
Publication year - 2017
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201700585
Subject(s) - chemistry , monooxygenase , mutagenesis , protein engineering , biocatalysis , cofactor , flavin group , enzyme , biochemistry , directed evolution , catalase , combinatorial chemistry , stereochemistry , catalysis , mutation , cytochrome p450 , reaction mechanism , gene , mutant
This paper describes the stabilization of flavin‐dependent monooxygenases under reaction conditions, using an engineered formulation of additives (the natural cofactors NADPH and FAD, and superoxide dismutase and catalase as catalytic antioxidants). This way, a 10 3 ‐ to 10 4 ‐fold increase of the half‐life was reached without resource‐intensive directed evolution or structure‐dependent protein engineering methods. The stabilized enzymes are highly valued for their synthetic potential in biotechnology and medicinal chemistry (enantioselective sulfur, nitrogen and Baeyer–Villiger oxidations; oxidative human metabolism), but widespread application was so far hindered by their notorious fragility. Our technology immediately enables their use, does not require structural knowledge of the biocatalyst, and creates a strong basis for the targeted development of improved variants by mutagenesis.