Functional Enhancement of Recalcitrant Peroxidase Kerl via Fusion Strategy and Active-Site Redesign

Limited functional solubility of peroxidases in Escherichia coli (E. coli) remains a pervasive bottleneck for their application in biocatalytic processes such as tryptophan hydroxylation. Here, a peroxidase (Kerl) with poor solubility derived from Candidatus Entotheonella factor was selected as the model enzyme to address this bottleneck. Fusion tag screening identified NusA as the optimal solubility enhancer, enabling soluble expression with preserved activity. Structure-guided mutagenesis was performed to identify residues involved in catalytic enhancement and substrate preference. Variant I284Q exhibited a 2.67-fold increase in catalytic efficiency, and residue R275 was identified as a key determinant of substrate discrimination. Molecular dynamics (MD) simulations were further employed to elucidate the structural basis the improved catalytic performance. This study presents an integrated framework for solubility enhancement and functional optimization of peroxidases.