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

Background: Lytic 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 HO is a more efficient cosubstrate for the enzyme than O, which could greatly affect the utilization of LPMOs in industrial settings.

Results: We probe the reactivity of LPMO9C from the cellulose-degrading fungus with a turbidimetric assay using phosphoric acid-swollen cellulose (PASC) as substrate and HO as a cosubstrate. The measurements were also followed by continuous electrochemical HO detection and LPMO reaction products were analysed by mass spectrometry. Different systems for the in situ generation of HO and for the reduction of LPMO's active-site copper were employed, including glucose oxidase, cellobiose dehydrogenase, and the routinely used reductant ascorbate.

Conclusions: We found for all systems that the supply of HO limited LPMO's cellulose depolymerization activity, which supports the function of HO 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.