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Synthesis of 1‐Naphthol by a Natural Peroxygenase Engineered by Directed Evolution
Author(s) -
MolinaEspeja Patricia,
Cañellas Marina,
Plou Francisco J.,
Hofrichter Martin,
Lucas Fatima,
Guallar Victor,
Alcalde Miguel
Publication year - 2016
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.201500493
Subject(s) - naphthalene , chemistry , hydroxylation , biocatalysis , directed evolution , mutagenesis , quenching (fluorescence) , catalysis , protein engineering , substrate (aquarium) , mutant , enzyme , turnover number , combinatorial chemistry , stereochemistry , regioselectivity , biochemistry , organic chemistry , reaction mechanism , fluorescence , biology , physics , quantum mechanics , gene , ecology
There is an increasing interest in enzymes that catalyze the hydroxylation of naphthalene under mild conditions and with minimal requirements. To address this challenge, an extracellular fungal aromatic peroxygenase with mono(per)oxygenase activity was engineered to convert naphthalene selectively into 1‐naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene together with quenching of the undesired peroxidative activity on 1‐naphthol (one‐electron oxidation). The resulting double mutant (G241D‐R257K) obtained from this process was characterized biochemically and computationally. The conformational changes produced by directed evolution improved the substrate's catalytic position. Powered exclusively by catalytic concentrations of H 2 O 2 , this soluble and stable biocatalyst has a total turnover number of 50 000, with high regioselectivity (97 %) and reduced peroxidative activity.