The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay

Frantisek Filandr,Petr Halada,Hucheng Chang,Daniel Kracher,Petr Man,Roland Ludwig

doi:10.1186/s13068-020-01673-4

Abstract

BackgroundLytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that H2O2 is a more efficient cosubstrate for the enzyme than O2, which could greatly affect the utilization of LPMOs in industrial settings.ResultsWe probe the reactivity of LPMO9C from the cellulose-degrading fungus Neurospora crassa with a turbidimetric assay using phosphoric acid-swollen cellulose (PASC) as substrate and H2O2 as a cosubstrate. The measurements were also followed by continuous electrochemical H2O2 detection and LPMO reaction products were analysed by mass spectrometry. Different systems for the in situ generation of H2O2 and for the reduction of LPMO’s active-site copper were employed, including glucose oxidase, cellobiose dehydrogenase, and the routinely used reductant ascorbate.ConclusionsWe found for all systems that the supply of H2O2 limited LPMO’s cellulose depolymerization activity, which supports the function of H2O2 as the relevant cosubstrate. The turbidimetric assay allowed rapid determination of LPMO activity on a cellulosic substrate without the need for time-consuming and instrumentally elaborate analysis methods.

Highlights

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses
LPMO activity monitored by a turbidimetric assay Turbidimetry has been recently employed to screen the cellulolytic activity of a fungal LPMO towards phosphoric acid-swollen cellulose (PASC), which represents a disordered, amorphous form of cellulose
We adapt this procedure into a continuous, turbidimetric assay to measure the time-dependent conversion of PASC by a celluloseactive LPMO

Summary

Introduction

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. LPMO activity has been demonstrated in biomass-degrading bacteria [3], fungi [4] and, as of recently, in firebrat (Thermobia domestica) [5], insect poxvirus [6] and. Since their discovery in 2010 [3], LPMOs have received ample attention in basic and applied research due to their synergistic interaction with hydrolytic enzymes [14, 15]. Key questions on the LPMO catalytic cycle and kinetics, including the cosubstrate preference, await experimental clarification [16]. Potential electron-donating systems in other organisms, e.g. in bacteria or insects, await identification

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