Background: Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that cleave polysaccharides. Results: We describe a novel LPMO and use a range of analytical methods to characterize its activity. Conclusion: Cellulose and cello-oligosaccharides are cleaved by oxidizing the sugar at the nonreducing end in the C4 position. Significance: This study provides unequivocal evidence for C4 oxidation of the nonreducing end sugar and demonstrates a novel LPMO substrate specificity.

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa, NcLPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by NcLPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that NcLPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures.biorefinery | metallo enzymes | GH61 | CBM33

Background:The recently discovered lytic polysaccharide monooxygenases (LPMOs) are important in enzymatic conversion of lignocellulosic biomass. Results: We describe structural and functional studies of NcLPMO9C, which cleaves both cellulose and certain hemicelluloses. Conclusion: NcLPMO9C has structural and functional features that correlate with the enzyme's catalytic capabilities. Significance: This study shows how LPMO active sites are tailored to varying functionalities and adds to a growing LPMO knowledge base.

Edited by Judit Ov adiStarch-binding modules of family 20 (CBM20) are present in 60% of lytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative breakdown of starch, which highlights functional importance in LPMO activity. The substrate-binding properties of starch-active LMPOs, however, are currently unexplored. Affinities and binding-thermodynamics of two recombinant fungal LPMOs toward starch and b-cyclodextrin were shown to be similar to fungal CBM20s. Amplex Red assays showed ascorbate and Cu-dependent activity, which was inhibited in the presence of b-cylodextrin and amylose. Phylogenetically, the clustering of CBM20s from starch-targeting LPMOs and hydrolases was in accord with taxonomy and did not correlate to appended catalytic activity. Altogether, these results demonstrate that the CBM20-binding scaffold is retained in the evolution of hydrolytic and oxidative starch-degrading activities. Keywords: AA13;carbohydrate-binding module; CBM20; lytic polysaccharide monooxygenase; starch binding; b-cyclodextrin Starch is a major renewable energy storage polysaccharide in plants and an important resource not only as a food but also as an industrial feedstock in biofuels, pharmaceuticals, detergents, and cosmetics [1][2][3]. Starch consists of two types of homo-glucose polymers: the mainly linear a-1,4-linked amylose and amylopectin, constituting 65-82% (w/w) of the starch granule and differing from amylose by having a larger molecular mass and roughly 5% a-1,6-branches of 12-15 glucosyl units long on average [2,4,5]. Starch is biosynthesized as insoluble granules, varying in size, morphology, crystal packing, and crystallinity that ranges from 15 to 45% depending on botanical origin [6][7][8]. Radially alternating amorphous and semicrystalline layers in the starch granule arise from the packing of double helices formed by adjacent branches in amylopectin, with the semicrystalline regions contributing to resistance of starch to enzymatic degradation [9,10]. Many industrial applications require the disruption of starch granules through hydrothermal, harsh chemical, or enzymatic treatments [11][12][13]. Despite development of relatively efficient a-amylases and other starch-degrading enzymes, there is still a significant margin for improving starch hydrolysis yields and shortening processing time, which would significantly reduce energy and costs of the process [14,15].Typically, glycoside hydrolases (GHs) that degrade complex polysaccharides possess carbohydrate-binding modules (CBMs) that promote enzyme-substrate proximity and thereby enhance catalytic efficiency [16,17]. Moreover, CBMs can also modulate the specificity and activity of cognate enzymes against plant cell wall Abbreviations AA, auxiliary activity; CAZy, carbohydrate-active enzymes; CBM, carbohydrate-binding module; DSC, differential scanning calorimetry; GH, glycoside hydrolase; ITC, isothermal titration calorimetry; LPMO, lytic polysaccharide monooxygenase; SBS, starch-binding site; SDS/PAGE, sodium dodecyl sulfate-polyacrylamide ge...

Abstract:The article suggests to replace the conventional unit of thermodynamic temperature with one based on the energy unit joule by including the gas constant into the temperature definition. The suggestion may be seen as a contribution to the ongoing efforts to redefine base units. The gas constant includes the Boltzmann constant which both will be abrogated and molar heat capacity and entropy will become dimensionless pure numbers. The suggestion has no impact on thermodynamic theory, but will make thermodynamic relations more translucent and easier to grab for students.

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