Assessing iron reductase activity: Methodologies for fungal degradation of lignocellulose

Lignocellulosic biomass (LCB) represents a critical renewable resource for bioenergy and bioproducts, yet its efficient degradation is constrained by the recalcitrance of lignin. This study evaluates the methodologies for assessing iron reductase activity of recombinant proteins IR1 and IR2 from brown rot fungi Serpula lacrymans, emphasizing their capacity to degrade lignocellulose. Iron plays a crucial role in wood decay, by functioning within the active sites of ligninolytic enzymes and fostering the production of reactive oxygen species (ROS) through iron-binding compounds. The study explores the expression, purification, and characterization of IR1 and IR2, in addition to assays quantifying Fe³⁺ reduction and phenolic compound release. Results indicate that both proteins effectively degrade substrates such as purified cellulose (Avicel) and wheat straw powder, with IR1 showing significantly higher activity in reducing nitrated lignin. The examination of the phenolic chelator 2,3-dihydroxybenzoic acid (DHBA) highlights its role in enhancing the reduction of ferric iron and promoting Fenton chemistry. Overall, this research enhances our understanding of enzymatic mechanisms in lignocellulose degradation and provides valuable insights for optimizing pre-treatment strategies that are essential for biorefinery development and sustainable use of lignocellulosic resources for bioenergy production.