Abstract
Baeyer–Villiger monooxygenases are recognized by their ability and high selectivity as oxidative biocatalysts for the generation of esters or lactones using ketones as starting materials. These enzymes represent valuable tools for biooxidative syntheses since they can catalyze reactions that otherwise involve strong oxidative reagents. In this work, we present a novel enzyme, the Type I Baeyer–Villiger monooxygenase from Leptospira biflexa. This protein is phylogenetically distant from other well-characterized BVMOs. In order to study this new enzyme, we cloned its gene, expressed it in Escherichia coli and characterized the substrate scope of the Baeyer–Villiger monooxygenase from L. biflexa as a whole-cell biocatalyst. For this purpose, we performed the screening of a collection of ketones with variable structures and sizes, namely acyclic ketones, aromatic ketones, cyclic ketones, and fused ketones. As a result, we observed that this biocatalyst readily oxidized linear- and branched- medium-chain ketones, alkyl levulinates and linear ketones with aromatic substituents with excellent regioselectivity. In addition, this enzyme catalyzed the oxidation of 2-substituted cycloketone derivatives but showed an unusual selection against substituents in positions 3 or 4 of the ring.
Highlights
Baeyer–Villiger monooxygenases (BVMOs) comprise a rapidly growing group of enzymes recognized by their ability as oxidative biocatalysts
Protein sequence analysis We investigated the presence of BVMOs in L. biflexa by bioinformatic approaches and detected only one sequence corresponding to a putative Type I BVMO
The B VMOLepto groups in a subclade together with the BVMO1 from M. tuberculosis H37Rv (Fig. 1a) and they share 44% sequence identity (Additional file 1: Table S1). Taking all this into account, the BVMOLepto is an interesting candidate to be functionally produced in E. coli and characterized as a biocatalyst in order to contribute to the knowledge of Type I BVMOs
Summary
Baeyer–Villiger monooxygenases (BVMOs) comprise a rapidly growing group of enzymes recognized by their ability as oxidative biocatalysts. They are known to catalyze the oxidation of ketones to esters or lactones mainly, other oxidation reactions have been described for these enzymes such as epoxidations on carbon–carbon double bonds, sulfoxidations, and oxidations of boron and selenium compounds. Type I BVMOs contain non-covalently bound flavin adenine dinucleotide (FAD) as coenzyme and strictly depend on reduced nicotinamide adenine dinucleotide phosphate (NADPH) as source of reducing power They are the best studied BVMOs and the ones proven in scale-up biotransformation assays, as reviewed in (Balke et al 2012; Bučko et al 2016; Ceccoli et al 2014; de Gonzalo et al 2010; Leisch et al 2011). BVMOs are capable for combination within chemo-enzymatic
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