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. 2017 Dec;7(1):87.
doi: 10.1186/s13568-017-0390-5. Epub 2017 Apr 27.

Cloning and characterization of the Type I Baeyer-Villiger monooxygenase from Leptospira biflexa

Romina D Ceccoli  1 , Dario A Bianchi  2 , Michael J Fink  3   4 , Marko D Mihovilovic  3 , Daniela V Rial  5
Affiliations

Cloning and characterization of the Type I Baeyer-Villiger monooxygenase from Leptospira biflexa

Romina D Ceccoli et al. AMB Express. 2017 Dec.

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.

Keywords: Baeyer–Villiger monooxygenase; Biocatalysis; Ketones; Leptospira.

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Figures

Fig. 1
Fig. 1
Sequence homology analysis of BVMOLepto. a Maximum-likelihood phylogenetic tree of recombinant BVMOs. The scale bars indicate the number of substitutions per site per unit of branch length. The aLRT values are shown at the nodes: >0.75 (black) and <0.75 (grey). The colors of branches indicate the groups I (pink), II (orange), III (blue), IV (red), V (cyan), VI (violet) and VII (gray) as previously defined (Ferroni et al. ; Szolkowy et al. 2009). Blue circles indicate recombinant BVMOs that were insoluble or their activity was not detected. BVMO from L. biflexa is indicated in bold. Protein sequences with their corresponding accession numbers are listed in Additional file 1: Table S1. b Multiple sequence alignment of six representative BVMOs belonging to different clades. The partial alignment of PAMO from T. fusca (Q47PU3), CHMO from Acinetobacter sp. NCIMB 9871 (BAA86293), HAPMO from P. fluorescens ACB (AAK54073), CPMO from Comamonas sp. NCIMB 9872 (BAC22652), CDMO from R. ruber SC1 (AAL14233) and BVMO from L. biflexa (ABZ97795) is shown. The two Rossmann-fold motifs (GxGxxG/A) and the two consensus sequences of Type I BVMOs (G/AGxWxxxxF/YPG/MxxxD and FxGxxxHxxxWP/D) are written in bold
Fig. 2
Fig. 2
SDS-PAGE of recombinant BVMOLepto. Samples corresponding to the soluble (lane 1) and insoluble (lane 2) fractions of protein extracts of E. coli BL21(DE3)/pHLb01 and the soluble (lane 3) and insoluble (lane 4) fractions of BL21(DE3) protein extract were subjected to 12% SDS-PAGE followed by Coomassie Blue staining. Lane 5 molecular marker. The arrow indicates the protein band corresponding to the recombinant BVMOLepto
Fig. 3
Fig. 3
General Baeyer–Villiger oxidation catalyzed by BVMOs
Fig. 4
Fig. 4
Baeyer–Villiger biooxidation of racemic fused ketones 34, 35 and 36 to normal and abnormal lactones
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