Engineering\na Binding\nPeptide for Oriented Immobilization\nand Efficient Bioelectrocatalytic Oxygen Reduction of Multicopper\nOxidases

Enzymatic fuel cells (EFCs) are emerging\nas promising\ntechnologies\nin renewable energy and biomedical applications, utilizing enzyme\ncatalysts to convert the chemical energy of renewable biomass into\nelectrical energy, known for their high energy conversion efficiency\nand excellent biocompatibility. Currently, EFCs face challenges of\npoor stability and catalytic efficiency at the cathodes, necessitating\nsolutions to enhance the oriented immobilization of multicopper oxidases\nfor improved heterogeneous electron transfer efficiency. This study\nsuccessfully identified a surface-binding peptide (SBP, 13 amino acids)\nderived from a methionine-rich fragment (MetRich, 53 amino acids)\nin <i>E. coli</i> CueO through semirational design. The\nfirst phase of engineering focused on the structural characteristics\nof MetRich, pinpointing fragment N394-H406 (SBP 1.0, corresponding\nto variant CueO-M12) as the key region dominating the binding. Subsequent\nsite-saturation mutagenesis, combined with electrochemical screening,\nyielded three variants, and among them, the variant CueO-M12-1 (CueO-M12\nH398I) exhibited a more uniform favorable orientation with a 1.38-fold\nincrease in current density. Further electrocatalytic kinetics analysis\nrevealed a significant 21.2-fold improvement in kinetics current density\n(<i>J</i><sub><i>k</i></sub>) compared with that\nof CueO-WT, leading to the development of SBP 2.0. When SBPs were\nfused to laccase from <i>Bacillus pumilus</i> (<i>Bp</i>L) and fungal bilirubin oxidase from <i>Myrothecium verrucaria</i> (<i>Mv</i>BOD), respectively, they transformed a sluggish\nadsorption process into a rapid and oriented one. In addition, compared\nwith SBP 1.0, SBP 2.0 endows <i>Bp</i>L and <i>Mv</i>BOD with enhanced electrocatalytic capabilities for oxygen reduction\nand glucose/O<sub>2</sub> EFC performance. The engineered SBPs are\npromising for serving as a versatile “glue” to enable\nthe immobilization of oxidoreductases in an oriented manner, which\nleads to a breakthrough in bioelectrocatalysis and thereby overcoming\nthe current bottleneck of EFCs.