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Analytical Tools for Characterizing Cellulose-Active Lytic Polysaccharide Monooxygenases (LPMOs)

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Book cover Cellulases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1796))

Abstract

Lytic polysaccharide monooxygenases are copper-dependent enzymes that perform oxidative cleavage of glycosidic bonds in cellulose and various other polysaccharides. LPMOs acting on cellulose use a reactive oxygen species to abstract a hydrogen from the C1 or C4, followed by hydroxylation of the resulting substrate radical. The resulting hydroxylated species is unstable, resulting in glycoside bond scission and formation of an oxidized new chain end. These oxidized chain ends are spontaneously hydrated at neutral pH, leading to formation of an aldonic acid or a gemdiol, respectively. LPMO activity may be characterized using a variety of analytic tools, the most common of which are high-performance anion exchange chromatography system with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry (MALDI-MS). NMR may be used to increase the certainty of product identifications, in particular the site of oxidation. Kinetic studies of LPMOs have several pitfalls and to avoid these, it is important to secure copper saturation, avoid the presence of free transition metals in solution, and control the amount of reductant (i.e., electron supply to the LPMO). Further insight into LPMO properties may be obtained by determining the redox potential and by determining the affinity for copper. In some cases, substrate affinity can be assessed using isothermal titration calorimetry. These methods are described in this chapter.

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Acknowledgments

This work was supported by the Norwegian Research Council through grants 214613, 216162, 214138, 226244, 221576, 226247, and 244259.

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Authors and Affiliations

  1. Biotechnology and Food Science, Faculty of Chemistry, Norwegian University of Life Sciences, Ås, Norway

    Bjørge Westereng, Jennifer S. M. Loose, Gustav Vaaje-Kolstad, Morten Sørlie & Vincent G. H. Eijsink

  2. Department of Biotechnology and Food Science, NOBIPOL, NTNU Norwegian University of Science and Technology, Trondheim, Norway

    Finn L. Aachmann

Authors
  1. Bjørge Westereng
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  2. Jennifer S. M. Loose
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  3. Gustav Vaaje-Kolstad
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  4. Finn L. Aachmann
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  5. Morten Sørlie
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  6. Vincent G. H. Eijsink
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Corresponding author

Correspondence to Vincent G. H. Eijsink .

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Editors and Affiliations

  1. Section of Sustainable Biotechnology, Department of Chemistry and Bioscience, Aalborg University, Copenhagen, Denmark

    Mette Lübeck

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Westereng, B., Loose, J.S.M., Vaaje-Kolstad, G., Aachmann, F.L., Sørlie, M., Eijsink, V.G.H. (2018). Analytical Tools for Characterizing Cellulose-Active Lytic Polysaccharide Monooxygenases (LPMOs). In: Lübeck, M. (eds) Cellulases. Methods in Molecular Biology, vol 1796. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7877-9_16

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  • DOI: https://doi.org/10.1007/978-1-4939-7877-9_16

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