Identification and engineering production of thermostable fungal xylanases via site-directed mutagenesis guided by sequence and structural analysis

In this thesis work, I investigate xylanase enzymes from Piriformospora indica (PIIN), a root-colonizing filamentous fungus whose genome revealed several xylanase candidates (XynP1, XynP2, XynP3, and XynP4), identified here for the first time. This fungus was selected for analysis because of its role in promoting plant growth, leading to the hypothesis that it must contain enzymes capable of breaking down biomass to facilitate symbiosis with plants. These enzymes were heterologously expressed in Pichia pastoris, with XynP2 demonstrating the highest activity. To enhance its thermostability and suitability for industrial applications, protein engineering techniques involving targeted mutations yielded thermostable variants (XynP2Ev, XynP2ds, XynP2os, and XynP2ts), identifying XynP2ds as the most stable and active. Further analysis included homology modeling, molecular dynamics simulations, and molecular docking studies with xylohexaose, which highlighted structural and functional adaptations responsible for the improved properties of XynP2ds. These findings provide valuable insights into enzyme optimization for industrial processes.