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. 2013 Jun 14;3(1):33.
doi: 10.1186/2191-0855-3-33.

Cloning, overexpression and biocatalytic exploration of a novel Baeyer-Villiger monooxygenase from Aspergillus fumigatus Af293

Maria Laura Mascotti  1 , Maximiliano Juri Ayub, Hanna Dudek, Marcela Kurina Sanz, Marco W Fraaije
Affiliations

Cloning, overexpression and biocatalytic exploration of a novel Baeyer-Villiger monooxygenase from Aspergillus fumigatus Af293

Maria Laura Mascotti et al. AMB Express. .

Erratum in

Abstract

The presence of several putative Baeyer-Villiger Monooxygenases (BVMOs) encoding genes in Aspergillus fumigatus Af293 was demonstrated for the first time. One of the identified BVMO-encoding genes was cloned and successfully overexpressed fused to the cofactor regenerating enzyme phosphite dehydrogenase (PTDH). The enzyme named BVMOAf1 was extensively characterized in terms of its substrate scope and essential kinetic features. It showed high chemo-, regio- and stereoselectivity not only in the oxidation of asymmetric sulfides, (S)-sulfoxides were obtained with 99% ee, but also in the kinetic resolution of bicyclo[3.2.0]hept-2-en-6-one. This kinetic resolution process led to the production of (1S,5R) normal lactone and (1R,5S) abnormal lactone with a regioisomeric ratio of 1:1 and 99% ee each. Besides, different reaction conditions, such as pH, temperature and the presence of organic solvents, have been tested, revealing that BVMOAf1 is a relatively robust biocatalyst.

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Figures

Figure 1
Figure 1
Multiple sequence alignment of some BVMOs sequences. Sequences are: BVMOAf1 (XP_747160), BVMOAf2 (XP_746949) and BVMOAf3 (XP_755274) from A. fumigatus Af293, PAMO (YP_289549) from Thermobifida fusca, CHMO (AAG10021) from Acinetobacter sp., HAPMO (AAK54073) from Pseudomonas fluorescens, CPDMO (BAE93346) from Pseudomonas sp. HI70 and CAMO (AET80001.1) from Cylindrocarpon radicicola. The two Rossmann folds (GxGxxG) and the BVMO fingerprint (FxGxxxHxxxWP/D) are highlighted.
Figure 2
Figure 2
Spectral characterization of BVMOAf1. Visible spectra of native BVMOAf1 (solid line) and BVMOAf1 after unfolding by 1% SDS and incubation at 80°C (dotted line). Spectral changes observed upon reduction of BVMOAf1 by excess of NADPH (dashed line).
Figure 3
Figure 3
Oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one catalyzed by BVMOAf1. (A) Time course BVMOAf1-catalyzed oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one (1) to 2-oxabicyclo[3.3.0]oct-6-en-3-one (2) and 3-oxabicyclo[3.3.0]oct-6-en-2-one (3). (B) Structural formulas of bicyclo [3.2.0]hept-2-en-6-one isomers and lactones formed by BVMOAf1 activity.
Figure 4
Figure 4
Optimal conditions for activity. (A) temperature-dependent activity profile, (B) temperature-dependent stability, for which the remaining activity was measured after 1.5 h incubation at the indicated temperatures, (C) pH-dependent activity profile, and (D) pH-dependent stability profile, by measuring the activity after 1 h incubation at different pH values. The error bars indicate standard deviations.
Figure 5
Figure 5
BVMOAf1 stability in organic solvents. Activity was measured after 1.0 h incubation in the presence of 5% (v/v) of organic solvents at room temperature. As control, no solvent was added to the buffer. The error bars indicate standard deviations.
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