Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry

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

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

Abstract

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 activeon 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 reducingbut instead has an acidic functionality, whereas C4 oxidation leads to products that are inherently labileat high and low pH and that exist in a keto-gemdiol equilibrium that is strongly shifted toward the gemdiolin 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 glycosidehydrolases 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
EditorsD. Wade Abbott, Alicia Lammerts van Bueren
Volume1588
PublisherSpringer
Publication date2017
Pages 71-92
Chapter7
ISBN (Print)978-1-4939-6898-5
ISBN (Electronic)978-1-4939-6899-2
DOIs
Publication statusPublished - 2017
SeriesMethods in Molecular Biology
ISSN1064-3745

Keywords

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

Cite this

Westereng, B., Arntzen, M. Ø., Wittrup Agger, J., Vaaje-Kolstad, G., & Eijsink, V. G. H. (2017). Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry. In D. W. Abbott, & A. L. van Bueren (Eds.), Protein-Carbohydrate Interactions: Methods and Protocols (Vol. 1588, pp. 71-92). Springer. Methods in Molecular Biology https://doi.org/10.1007/978-1-4939-6899-2_7