Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry: Methods and Protocols: Second Edition

Bjørge Westereng*, Magnus Ø. Arntzen, Heidi Østby, Jane Wittrup Agger, Gustav Vaaje-Kolstad, Vincent G. H. Eijsink

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review


Lytic polysaccharide monooxygenases perform oxidative cleavage of glycosidic bonds in various polysaccharides. The majority of LMPOs studied so far possess activity on either cellulose or chitin and analysis of these activities is therefore the main focus of this review. Notably, however, the number of LPMOs that are active on other polysaccharides is increasing. The products generated by LPMOs from cellulose are either oxidized in the downstream end (at C1) or upstream end (at C4), or at both ends. These modifications only result in small structural changes, which makes both chromatographic separation and product identification by mass spectrometry challenging. The changes in physicochemical properties that are associated with oxidation need to be considered when choosing analytical approaches. C1 oxidation leads to a sugar that is no longer reducing but instead has an acidic functionality, whereas C4 oxidation leads to products that are inherently labile at high and low pH and that exist in a keto-gemdiol equilibrium that is strongly shifted towards the gemdiol in aqueous solutions. Partial degradation of C4-oxidized products leads to the formation of native products, which could explain why some authors claim to have observed glycoside hydrolase activity for LPMOs. Notably, apparent glycoside hydrolase activity may also be due to small amounts of contaminating glycoside hydrolases since these normally have much higher catalytic rates than LPMOs. The low catalytic turnover rates of LPMOs necessitate the use of sensitive product detection methods, which limits the analytical possibilities considerably. Modern liquid chromatography and mass spectrometry have become essential tools for evaluating LPMO activity and this chapter provides an overview of available methods together with a few novel tools. The methods described constitute a suite of techniques for analyzing oxidized carbohydrate products, which can be applied to LPMOs as well as other carbohydrate-active redox enzymes.
Original languageEnglish
Title of host publicationProtein-Carbohydrate Interactions : Methods and Protocols
EditorsWade Abbott, Wesley Zandberg
Place of PublicationNew York
PublisherHumana Press
Publication date2023
ISBN (Print)978-1-0716-3150-8
ISBN (Electronic)978-1-0716-3151-5
Publication statusPublished - 2023
SeriesMethods in Molecular Biology


  • Lytic polysaccharide monooxygenase
  • High-performance anion-exchange chromatography
  • Porous graphitized carbon
  • Aldonic acid
  • Gemdiol
  • Hydrophilic interaction liquid chromatography


Dive into the research topics of 'Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry: Methods and Protocols: Second Edition'. Together they form a unique fingerprint.

Cite this