Identification and characterization of two AA9 lytic polysaccharide monooxygenases isolated from the enzymatic arsenal in Phanerochaete chrysosporium

White rot fungi including Phanerochaete chrysosporium are known for their ability to mineralize plant-derived materials, such as cellulose, hemicellulose, and lignin, into CO₂ and H₂O. This process is achieved through a diverse array of hydrolytic and oxidative enzymes. However, the mode of action and specific characteristics of lytic polysaccharide monooxygenases (LPMOs) from P. chrysosporium are not well understood. In this study, two auxiliary activity (AA) family 9 genes from P. chrysosporium, PchAA9C and PchAA9F, were heterologously expressed in Pichia pastoris and functionally characterized. The recombinant PchAA9C and PchAA9F exhibited optimal activity at 60 °C and pH 6.0, with their activity significantly enhanced by 0.5-3.0 mmol/L ascorbic acid (P < 0.05). Substrate specificity analysis revealed that both PchAA9C and PchAA9F displayed robust activity against Icelandic moss lichenan, phosphoric acid swollen cellulose, and microcrystalline cellulose, indicating a preference for breaking down β-(Glc1 → 4Glc)-linked substrates. Further analysis using HPAEC-PAD and MALDI-TOF-MS revealed that PchAA9C functioned as a C1-specific oxidizing enzyme, whereas PchAA9F targeted both C1 and C4 positions of sugar rings. Synergistic experiments involving an enzyme cocktail of xylanase, glucanase, and pectinase showed that PchAA9C and PchAA9F significantly enhanced the production of reducing sugars from corn and soybean straws. Notably, PchAA9F represents the first reported C1/C4-double-oxidizing LPMO isolated from P. chrysosporium. This discovery provides new insights into the molecular basis of the biodegradation capabilities of wood-decaying fungi and highlights PchAA9F as a promising candidate for applications in lignocellulosic biomass biorefinery.