Description of four novel OYE enzymes from the fungi Aspergillus niger and Botryotinia fuckeliana

Asymmetric alkene hydrogenation in industrial processes exploits ene-reductases(ERs) from the family of the Old Yellow Enzyme (OYE; E.C. 1.6.99.1). These are nicotinamide-dependent flavoproteins catalyzing the asymmetric hydrogenation of a wide panel of activated alkenes (e.g., α,β-unsaturated ketones, aldehydes, nitroalkenes, carboxylic acids, and derivatives). In recent years, unexpected reactivities due to the chemical versatility of the flavin cofactor have also been reported for this class of enzymes [1-6]. As a result, the discovery and the characterization of new ERs is being driven not only by the interest for classical C=C-bond reductions but also the curiosity to better understand the potentialities of this class of biocatalysts. Not at least, the research aiming at expanding the toolbox of ERs is also fueled by the necessity of tackling some limitations still encountered in their application (low stability in the harsh industrial conditions, modest turnover numbers, poor substrate tolerance and, in some cases, low enantioselectivity) [7]. These enzymes are ubiquitous in nature and have been found in yeasts, bacteria, plants and parasitic eukaryotes [8]. More than 100 ERs have been already identified and their number is constantly increasing, causing an almost continuous revision of their phylogenetic classification [9,10]. To the protein family of OYEs, we add now four new members of fungal origin, identified in the ascomycete species Aspergillus niger and Botryotinia fuckeliana (better known as Botrytis cinerea). We report here on the strategies adopted to succeed in expression and purification, and on the biochemical and biocatalytic characterization of these enzymes, namely AnOYE2, AnOYE8, BfOYE1 and BfOYE4.