Rational Engineering of Cellobiose Dehydrogenase via Substrate Pocket Redesign and Electron Transfer Optimization for Efficient Bioelectrosynthesis of Lactobionic Acid

Abstract

Cellobiose dehydrogenase (CDH) is a fungal extracellular flavocytochrome crucial for biomass degradation and promising for bioelectrocatalytic applications. However, its practical application remains limited by suboptimal catalytic efficiency and inefficient electron transfer. Here, an integrated engineering strategy was developed that couples substrate‐pocket modification with electron‐transfer optimization to enhance overall catalytic cycle simultaneously. Structure‐guided engineering identified beneficial substrate‐pocket mutations, which were subsequently combined with the previously validated electron‐transfer‐enhancing mutations (W316H and A165C), yielding the triple mutant A165C‐N313D‐W316H. This variant exhibited a fourfold increase in catalytic specific activity and an 8.5‐fold enhancement in electron transfer rate relative to the WT. Molecular dynamics simulations further revealed that the mutation improves the hydrogen‐bonding network within the substrate‐binding pocket. Moreover, the mutant retained high thermostability, displayed enhanced specificity toward lactose, and reduced hydrogen peroxide generation by 82% compared with the WT. When immobilized on electrodes, A165C‐N313D‐W316H showed substantially improved electrocatalytic performance. Under optimized conditions, the MET‐based bioelectrocatalytic system achieved a 94% conversion yield of lactobionic acid within 18 h. This study demonstrates that the integration of substrate‐pocket and electron‐transfer engineering effectively improves the overall catalytic cycle of CDH and provides a promising approach for engineering multidomain redox enzymes.