This study explores the entrapment, immobilization, and direct electron transfer-type bioelectrocatalysis mediated by TthLPMO9G on carbon-based electrode materials, focusing on carbon felt (CF) due to its high conductivity, chemical stability, and large surface area. At first, entrapment of an LPMO from Thermohelomyces thermophila,TthLPMO9G was achieved using Nafion-coated carbon fibers. At the next step, CF electrodes were chemically oxidized to introduce carboxyl groups, quantified by conductometric titration, and used for covalent immobilization of the enzyme. The immobilization process for TthLPMO9G was optimized, and the catalytic activity was assessed based on cellulose oxidation. The success of the immobilization process was evaluated using three parameters: yield (%), efficiency (%), and %recovery (%). Electrochemical studies, including cyclic voltammetry (CV) and Fourier-transform alternating current voltammetry (FTacV), were performed to evaluate TthLPMO9G's electrochemical activity. Immobilized LPMO activity was detectable only through the more sensitive FTacV. Direct electron transfer (DET) from the electrode to the enzyme active site remains a challenge. This work provides insight into the limitations of the studied strategies in LPMO-based electrocatalysis, thus offering guidance for improving immobilization process and electrochemical integration in future applications targeting DET bioelectrocatalysis.
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