Engineering a Bacterial DyP-Type Peroxidase for Enhanced Oxidation of Lignin-Related Phenolics at Alkaline pH

Dye-decolorizing peroxidases (DyPs) are a family of microbial heme-containing peroxidases that show important properties for lignocellulose biorefineries due to their ability to oxidize lignin-related compounds. Directed evolution was used to improve the efficiency of the bacterial PpDyP from Pseudomonas putida MET94 for phenolic compounds. Three rounds of random mutagenesis by error-prone PCR of the ppDyP gene followed by high-throughput screening allow identification of the 6E10 variant showing a 100-fold enhanced catalytic efficiency (kcat/Km) for 2,6-dimethoxyphenol (DMP), similar to that exhibited by fungal lignin peroxidases (∼105 M–1 s–1). The evolved variant showed additional improved efficiency for a number of syringyl-type phenolics, guaiacol, aromatic amines, Kraft lignin, and the lignin phenolic model dimer guaiacylglycerol-β-guaiacyl ether. Importantly, variant 6E10 displayed optimal pH at 8.5, an upshift of 4 units in comparison to the wild type, showed resistance to hydrogen peroxide inacti...