Lignin-IB-Peroxidase

This record contains a research manuscript developed within the framework of a Marie Skłodowska-Curie Postdoctoral Fellowship project focused on the production and engineering of ligninolytic peroxidases for sustainable lignin valorization. Lignin is an abundant yet underutilized component of lignocellulosic biomass, and its efficient conversion remains a major challenge for the development of circular bioeconomy processes. This work investigates biotechnological strategies for the heterologous production of key lignin-degrading enzymes, including lignin peroxidase (LiP) and versatile peroxidase (VP). The study explores recombinant expression in bacterial systems, with particular emphasis on inclusion body formation, solubilization, and refolding approaches to recover bioactive enzymes. Process optimization strategies, including buffer conditions and cofactor incorporation, are discussed in relation to enzyme activity and stability. In addition, alternative expression strategies using the yeast Pichia pastoris are considered to enable secretion-based production and improved protein folding. The manuscript also provides a critical assessment of methodological challenges encountered during enzyme production and engineering. These include limitations associated with protein refolding from inclusion bodies, difficulties in functional evaluation of engineered variants, and constraints related to the application of data-driven and AI-based protein design approaches in cases of limited sequence diversity. Overall, this work contributes to the development of robust workflows for recombinant production of ligninolytic enzymes and provides insights into practical considerations for enzyme engineering and process scalability. The findings are relevant for applications in lignin valorization, industrial biotechnology, and sustainable bioprocess development.