Protein Engineering of a Dye Decolorizing Peroxidase from Pleurotus ostreatus For Efficient Lignocellulose Degradation

Dye decolorizing peroxidases (DyPs) have received extensive attention due to their biotechnological importance and potential use in the biological treatment of lignocellulosic biomass. DyPs are haem-containing peroxidases which utilize hydrogen peroxide (H2O2) to catalyse the oxidation of a wide range of substrates. Similar to naturally occurring peroxidases, DyPs are not optimized for industrial utilization owing to their inactivation induced by excess amounts of H2O2. Furthermore, DyPs are active only under acidic conditions and typically lose activity at neutral or alkaline pH. A dye decolorizing peroxidase from the Pleurotus ostreatus (Pleos-DyP4) was identified recently as a first fungal DyP oxidizing Mn2+ to Mn3+ similar to other fungal peroxidases. However, despite its unique pH and thermal stability, similar to other DyPs, it is not suited for industrial applications. Protein engineering methods are widely used to enhance the stability and catalytic efficiency of biocatalysts to render them suitable for industrial purposes. Different directed evolution approaches (namely, error-prone PCR and saturation mutagenesis) were used to construct mutant libraries of DyP4. For protein expression studies, the mutant enzymes were co-expressed with OsmY protein (a novel secretion-enhancing protein) in order to secrete intracellular protein into the media and hence facilitate the screening of mutants. ABTS assay was used to screen for mutants with improved activities in 96-well microtiter plates. Four rounds of error-prone PCR (epPCR) and saturation mutagenesis led to the identification of a mutant with an approximately 10-fold improvement in total activity and resistance to H2O2 inactivation in comparison with the wild type (WT). This study showcases the usefulness of the OsmY-based secretion mechanism of protein in E. coli as a tool in facilitating the screening of DyP4 mutants, and potentially of other heterologous protein variants in E. coli – the preferred host for expression and directed evolution studies.