Discovery of the combined oxidative cleavage of plant xylan and cellulose by a new fungal polysaccharide monooxygenase

Frommhagen, Matthias; Sforza, Stefano; Westphal, Adrie H.; Visser, Jaap; Hinz, S.W.A.; Koetsier, Martijn J.; Berkel, Willem J.H. van; Gruppen, Harry; Kabel, Mirjam A.

doi:10.1186/s13068-015-0284-1

Cited by 198 publications

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“…Oligosaccharides released and not released refers to + and −, respectively a Data from Frommhagen et al [ 2 ] b Gluco-oligosaccharides oxidized at the C1 (GlcOS n # ) or C4 position (GlcOS n *) c Xylo-oligosaccharides oxidized at the C1 (XOS n # ) or C4 position (XOS n *) d Regenerated amorphous cellulose (RAC) e Xyloglucan from tamarind seed, β-(1 → 3, 1 → 4)-glucan from barley and β-(1 → 3, 1 → 4)-glucan from oat spelt f Oat spelt xylan (OSX), birchwood xylan (BiWX), wheat arabinoxylan (WAX) g β-(1 → 4)-linked gluco- and xylo-oligosaccharides, degree of polymerization 2–5…”

Section: Resultsmentioning

confidence: 99%

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Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1 differ in substrate preference and reducing agent specificity

Frommhagen

Koetsier

Westphal

et al. 2016

Biotechnol Biofuels

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142

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BackgroundLytic polysaccharide monooxgygenases (LPMOs) are known to boost the hydrolytic breakdown of lignocellulosic biomass, especially cellulose, due to their oxidative mechanism. For their activity, LPMOs require an electron donor for reducing the divalent copper cofactor. LPMO activities are mainly investigated with ascorbic acid as a reducing agent, but little is known about the effect of plant-derived reducing agents on LPMOs activity.ResultsHere, we show that three LPMOs from the fungus Myceliophthora thermophila C1, MtLPMO9A, MtLPMO9B and MtLPMO9C, differ in their substrate preference, C1-/C4-regioselectivity and reducing agent specificity. MtLPMO9A generated C1- and C4-oxidized, MtLPMO9B C1-oxidized and MtLPMO9C C4-oxidized gluco-oligosaccharides from cellulose. The recently published MtLPMO9A oxidized, next to cellulose, xylan, β-(1 → 3, 1 → 4)-glucan and xyloglucan. In addition, MtLPMO9C oxidized, to a minor extent, xyloglucan and β-(1 → 3, 1 → 4)-glucan from oat spelt at the C4 position. In total, 34 reducing agents, mainly plant-derived flavonoids and lignin-building blocks, were studied for their ability to promote LPMO activity. Reducing agents with a 1,2-benzenediol or 1,2,3-benzenetriol moiety gave the highest release of oxidized and non-oxidized gluco-oligosaccharides from cellulose for all three MtLPMOs. Low activities toward cellulose were observed in the presence of monophenols and sulfur-containing compounds.ConclusionsSeveral of the most powerful LPMO reducing agents of this study serve as lignin building blocks or protective flavonoids in plant biomass. Our findings support the hypothesis that LPMOs do not only vary in their C1-/C4-regioselectivity and substrate specificity, but also in their reducing agent specificity. This work strongly supports the idea that the activity of LPMOs toward lignocellulosic biomass does not only depend on the ability to degrade plant polysaccharides like cellulose, but also on their specificity toward plant-derived reducing agents in situ.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0594-y) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

“…Masses of lithium-adducted C1- or C4-oxidized gluco-oligosaccharides for RAC incubated with Mt LPMO9B or Mt LPMO9C, respectively, were determined and assigned as described previously [2]. …”

Section: Methodsmentioning

confidence: 99%

Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1 differ in substrate preference and reducing agent specificity

Frommhagen

Koetsier

Westphal

et al. 2016

Biotechnol Biofuels

Self Cite

142

133

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“…It has previously been shown that certain LPMOs can cleave xylan, only if the xylan is complexed with cellulose [23]. In order to gain further insight into the potential role of FgLPMO9A in plant cell wall degradation, we mixed cellulose (PASC) with different hemicelluloses and treated the resulting polysaccharide mixtures with FgLPMO9A, in the presence or absence of ascorbic acid as electron donor.…”

Section: Activity On Mixtures Of Hemicelluloses and Pascmentioning

confidence: 99%

“…LPMOs cleave polysaccharides while oxidizing one of the new chain ends at the C1 or C4 position, thus contributing to substrate depolymerization while increasing accessibility of the substrate to conventional GHs [12,15,17,18]. Since their discovery in 2010 [12], LPMOs with preference for various plant polysaccharides, such as cellulose [19,20], xyloglucan and other (1,4)-linked β-glucans [21], starch [22] and xylan [23] have been identified. LPMOs have become an…”

Section: Introductionmentioning

confidence: 99%

FgLPMO9A from Fusarium graminearum cleaves xyloglucan independently of the backbone substitution pattern

et al. 2016

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Lytic polysaccharide monooxygenases (LPMOs) are important for the enzymatic conversion of biomass and seem to play a key role in degradation of the plant cell wall. In this study, we characterize an LPMO from the fungal plant pathogen Fusarium graminearum (FgLPMO9A) that catalyzes the mixed C1/C4 oxidative cleavage of cellulose and xyloglucan, but is inactive towards other (1,4)-linked β-glucans. Our findings indicate that FgLPMO9A has unprecedented broad specificity on xyloglucan, cleaving any glycosidic bond in the β-glucan main chain, regardless of xylosyl substitutions. Interestingly, we found that when incubated with a mixture of xyloglucan and cellulose, FgLPMO9A efficiently attacks the xyloglucan, whereas cellulose conversion is inhibited. This suggests that removal of hemicellulose may be the true function of this LPMO during biomass conversion.

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“…Lytic polysaccharide monooxygenases are copperdependent enzymes that use molecular oxygen and an external electron donor to cleave glycosidic bonds in various polysaccharides, such as cellulose [26,27], hemicelluloses [28,29], and chitin [30]. These enzymes are assigned into auxiliary activity (AA) families 9, 10, 11, and 13 in the CAZy database.…”

Section: Abstract: Aa13;mentioning

confidence: 99%

Fungal lytic polysaccharide monooxygenases bind starch and β‐cyclodextrin similarly to amylolytic hydrolases

et al. 2016

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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...

show abstract

Discovery of the combined oxidative cleavage of plant xylan and cellulose by a new fungal polysaccharide monooxygenase

Cited by 198 publications

References 41 publications

Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1 differ in substrate preference and reducing agent specificity

Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1 differ in substrate preference and reducing agent specificity

FgLPMO9A from Fusarium graminearum cleaves xyloglucan independently of the backbone substitution pattern

Fungal lytic polysaccharide monooxygenases bind starch and β‐cyclodextrin similarly to amylolytic hydrolases

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