TY - CHAP
T1 - Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry
AU - Westereng, Bjørge
AU - Arntzen, Magnus Ø.
AU - Wittrup Agger, Jane
AU - Vaaje-Kolstad, Gustav
AU - Eijsink, Vincent G. H.
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - Lytic polysaccharide monooxygenase
KW - High-performance anion-exchange chromatography
KW - Porous graphitized carbon
KW - Aldonic acid
KW - Gemdiol
KW - Hydrophilic interaction liquid chromatography
U2 - 10.1007/978-1-4939-6899-2_7
DO - 10.1007/978-1-4939-6899-2_7
M3 - Book chapter
C2 - 28417362
SN - 978-1-4939-6898-5
VL - 1588
T3 - Methods in Molecular Biology
SP - 71
EP - 92
BT - Protein-Carbohydrate Interactions
A2 - Abbott, D. Wade
A2 - van Bueren, Alicia Lammerts
PB - Springer
ER -