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 selectively convert naphthalene into 1-naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene while quenching the undesired peroxidative activity on 1-naphthol (one-electron oxidation). The resulting double mutant (G241D-R257K) of 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 H2O2, this soluble and stable biocatalyst has total turnover numbers of 50,000, with high regioselectivity (97%) and reduced peroxidative activity.

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