Engineering Cellobiose Dehydrogenase for Enhanced Electron Transfer Efficiency and Minimized Oxygen Interference

Abstract Cellobiose dehydrogenase (CDH) is a typical biomass catalyst with significant potential for applications such as lignocellulose degradation, bioremediation, and bioelectrocatalysis. Its two-domain flavoheme structure enables the construction of direct electron transfer (DET)-based bioelectrochemical devices. However, the natural enzyme suffers from low electron transfer efficiency and oxygen reactivity that leads to H2O2 formation, hindering the performance of CDH. Here, we established a region-specific engineering strategy to enhance electron transfer efficiency and minimize oxygen interference in CDH. Through structure-based site-directed mutagenesis, molecular docking, and molecular dynamics (MD) simulations, the G1 variant (A165C-W316H-S560V) achieved a 10-fold increase in electron transfer rate and a 4.5-fold increase in catalytic activity, with H2O2 formation below the detection limit under the tested conditions. Rapid kinetics and electrochemical characterization further revealed the apparent kinetic behaviors of the mutants. The G1 variant displayed a 20-fold increase in catalytic current density compared to WT. The region-specific rational engineering strategies presented here may provide valuable insights into interdomain electron transfer, CYT domain surface electron transfer, and oxygen-dependent side reactivity simultaneously within CDH and offer a possible approach for improving the DET efficiency of other multi-domain oxidoreductases.