Exploitation of Enzymes for the Production of Biofuels: Electrochemical Determination of Kinetic Parameters of LPMOs

Abstract

Lytic polysaccharide monooxygenases (LPMOs) consist of a class of enzymes that boost the release of oxidised products from plant biomass, in an approach that is more eco-friendly than the traditional ones, employing harsh chemicals. Since LPMOs are redox enzymes, they could possibly be exploited by immobilisation on electrode surfaces. Such an approach requires knowledge of kinetic and thermodynamic information for the interaction of the enzyme with the electrode surface. In this work, a novel methodology is applied for the determination of such parameters for an LPMO from the filamentous fungus Thermothelomyces thermophila, MtLPMO9H.

Integrated Experimental and Theoretical Investigation of Copper Active Site Properties of a Lytic Polysaccharide Monooxygenase from Serratia marcescens

“…These results significantly differ from previously reported values obtained by the indirect method using TMP as a redox indicator. There are few direct electrochemical measurements of redox potentials for LPMO enzymes. − The use of indirect methods can lead to errors or imprecisions as the choice of the redox indicator as well as the strict control of anaerobicity can induce non-negligible bias on the measurements. Indeed, Hemsworth et al have previously reported that the choice of the redox indicator could lead to discrepancies in the determination of accurate redox potentials .…”

Immobilization of TthLPMO9G on Carbon Felt for Potential Electrochemical Applications

“…In a previous study, LPMOs were successfully entrapped in a Nafion matrix on glassy carbon electrodes, demonstrating evidence of direct electron transfer between the enzyme and the electrode. 9 Notably, no substrate was present in this system, as the setup was designed solely to observe the electrochemical activity of the enzyme, without involving the oxidation of cellulose. Building on this, the current study evaluated the entrapment and electrochemical activity of TthLPMO9G on various carbon-based electrode substrates, including glassy carbon, carbon fiber bundles and CF, where electrochemical activity refers to the enzyme's ability to accept and donate electrons.…”

“…23 Nafion was selected as the initial entrapment substrate to provide a literature-based reference point, as it is the only method previously reported for electron transfer studies involving AA9 LPMOs in electrochemical setups. 9 Building on these findings, we focused at the following step on TthLPMO9G immobilization on modified CF, which is able to facilitate electron transfer to the enzyme's copper-active site. This study addresses key challenges in LPMO immobilization, including enzyme entrapment, optimization of immobilization conditions, and interactions with carbon-based conductive materials.…”

Fourier-Transformed Alternating Current Voltammetry (FTacV) for Analysis of Electrocatalysts

“…By conducting multiple FTacV measurements at different frequencies and identifying the range where the reaction exhibits quasi-reversibility, one can extract an estimate of the rate constant from a simulated plot of peak current vs the normalized rate constant (Figure 7a). Zouraris et al 95 used this methodology to estimate the rate constant of a redox enzyme, lytic polysaccharide monooxygenase, that was immobilized on an electrode. This study demonstrated that this methodology is applicable for the initial estimation of the kinetic parameters of redox reactions.…”