Engineering a Binding Peptide for Oriented Immobilization and Efficient Bioelectrocatalytic Oxygen Reduction of Multicopper Oxidases

Enzymatic fuel cells (EFCs) are emerging as promising technologies in renewable energy and biomedical applications, utilizing enzyme catalysts to convert the chemical energy of renewable biomass into electrical energy, known for their high energy conversion efficiency and excellent biocompatibility. Currently, EFCs face challenges of poor stability and catalytic efficiency at the cathodes, necessitating solutions to enhance the oriented immobilization of multicopper oxidases for improved heterogeneous electron transfer efficiency. This study successfully identified a surface-binding peptide (SBP, 13 amino acids) derived from a methionine-rich fragment (MetRich, 53 amino acids) in <i>E. coli</i> CueO through semirational design. The first phase of engineering focused on the structural characteristics of MetRich, pinpointing fragment N394-H406 (SBP 1.0, corresponding to variant CueO-M12) as the key region dominating the binding. Subsequent site-saturation mutagenesis, combined with electrochemical screening, yielded three variants, and among them, the variant CueO-M12-1 (CueO-M12 H398I) exhibited a more uniform favorable orientation with a 1.38-fold increase in current density. Further electrocatalytic kinetics analysis revealed a significant 21.2-fold improvement in kinetics current density (<i>J</i>_<i>k</i>) compared with that of CueO-WT, leading to the development of SBP 2.0. When SBPs were fused 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 adsorption process into a rapid and oriented one. In addition, compared with SBP 1.0, SBP 2.0 endows <i>Bp</i>L and <i>Mv</i>BOD with enhanced electrocatalytic capabilities for oxygen reduction and glucose/O₂ EFC performance. The engineered SBPs are promising for serving as a versatile "glue" to enable the immobilization of oxidoreductases in an oriented manner, which leads to a breakthrough in bioelectrocatalysis and thereby overcoming the current bottleneck of EFCs.