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dc.contributor.authorWestereng, Bjørge
dc.contributor.authorArntzen, Magnus Øverlie
dc.contributor.authorAachmann, Finn Lillelund
dc.contributor.authorVarnai, Aniko
dc.contributor.authorEijsink, Vincent
dc.contributor.authorWittrup Agger, Jane
dc.date.accessioned2017-11-14T09:25:07Z
dc.date.available2017-11-14T09:25:07Z
dc.date.created2016-05-31T08:24:44Z
dc.date.issued2016
dc.identifier.citationJournal of Chromatography A. 2016, 1445 46-54.nb_NO
dc.identifier.issn0021-9673
dc.identifier.urihttp://hdl.handle.net/11250/2466080
dc.description.abstractLytic polysaccharide monooxygenases play a pivotal role in enzymatic deconstruction of plant cell wall material due to their ability to catalyze oxidative cleavage of glycosidic bonds. LPMOs may release different products, often in small amounts, with various oxidation patterns (C1 or C4) and with varying stabilities, making accurate analysis of product profiles a major challenge. So far, HPAEC has been the method of choice but it has limitations with respect to analysis of C4-oxidized products. Here, we compare various HPLC methods and present procedures that allow efficient separation of intact C1- and C4-oxidized products. We demonstrate that both PGC and HILIC (in WAX-mode) can separate C1- and C4-oxidized products and that PGC gives superior chromatographic performance. In contrast to HPAEC, these methods are directly compatible with mass spectroscopy and charged aerosol detection (CAD), which enables online peak validation and quantification with LOD levels in the low ng range. While the novel methods show lower resolution than HPAEC, this is compensated by easy peak identification, allowing, for example, discrimination between chromatographically highly similar native and C4-oxidized cello-oligomers. HPAEC-MS studies revealed chemical oxidation of C4-geminal diol products, which implies that peaks commonly believed to be C4-oxidized cello-oligomers, in fact are on-column generated derivatives. Non-destructive separation of C4-oxidized cello-oligosaccharides on the PGC column allowed us, for the first time, to isolate C4-oxidized standards. HPAEC fractionation of a purified C4-oxidized tetramer revealed that on-column decomposition leads to formation of the native trimer, which may explain why product mixtures generated by C4-oxidizing LPMOs seem to be rich in native oligosaccharides when analyzed by HPAEC. The findings and methods described here will aid in future studies in the emerging LPMO field.nb_NO
dc.language.isoengnb_NO
dc.publisherElseviernb_NO
dc.rightsAttribution-NonCommercial-NoDerivatives 4.0 Internasjonal*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/deed.no*
dc.titleSimultaneous analysis of C1 and C4 oxidized oligosaccharides, the products of lytic polysaccharide monooxygenases acting on cellulosenb_NO
dc.typeJournal articlenb_NO
dc.typePeer reviewednb_NO
dc.description.versionacceptedVersionnb_NO
dc.source.pagenumber46-54nb_NO
dc.source.volume1445nb_NO
dc.source.journalJournal of Chromatography Anb_NO
dc.identifier.doi10.1016/j.chroma.2016.03.064
dc.identifier.cristin1358547
dc.relation.projectNorges forskningsråd: 226247nb_NO
dc.relation.projectNorges forskningsråd: 239003nb_NO
dc.relation.projectNorges forskningsråd: 214613nb_NO
dc.relation.projectNorges forskningsråd: 244259nb_NO
dc.relation.projectNorges forskningsråd: 237841nb_NO
dc.description.localcode© 2016. This is the authors’ accepted and refereed manuscript to the article. LOCKED until 25.3.2018 due to copyright restrictions. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/nb_NO
cristin.unitcode194,66,15,0
cristin.unitnameInstitutt for bioteknologi og matvitenskap
cristin.ispublishedtrue
cristin.fulltextoriginal
cristin.qualitycode2


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Attribution-NonCommercial-NoDerivatives 4.0 Internasjonal
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