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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
Part of the Methods in Molecular Biology book series (MIMB, volume 1588)

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 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 toward 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.

Key words

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

Notes

Acknowledgments

This work was supported by the Norwegian Research Council through grants and 193817, 214138, 214613, 216162, 243663, and 244259.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Bjørge Westereng
    • 1
  • Magnus Ø. Arntzen
    • 1
  • Jane Wittrup Agger
    • 2
  • Gustav Vaaje-Kolstad
    • 1
  • Vincent G. H. Eijsink
    • 1
  1. 1.Department of Chemistry, Biotechnology and Food ScienceNorwegian University of Life SciencesÅs AkershusNorway
  2. 2.Center for BioProcess Engineering, Department of Chemical and Biochemical EngineeringTechnical University of DenmarkLyngbyDenmark

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