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

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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 H2O2 is a more efficient cosubstrate for the enzyme than O2, which could greatly affect the utilization of LPMOs in industrial settings. Results: We 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. Conclusions: We 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.

Original languageEnglish
Article number37
JournalBiotechnology for Biofuels
Volume13
Issue number1
DOIs
Publication statusPublished - 5 Mar 2020
Externally publishedYes

Keywords

  • Cellobiose dehydrogenase
  • Cellulose
  • Glucose oxidase
  • Hydrogen peroxide
  • Lytic polysaccharide monooxygenase
  • Neurospora crassa

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Renewable Energy, Sustainability and the Environment
  • Energy(all)
  • Management, Monitoring, Policy and Law

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