Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency

Eibinger, Manuel; Ganner, Thomas; Bubner, Patricia; Roŝker, Stephanie; Kracher, Daniel; Haltrich, Dietmar; Ludwig, Roland; Plank, Harald; Nidetzky, Bernd

doi:10.1074/jbc.m114.602227

Cited by 255 publications

(203 citation statements)

References 48 publications

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“…In the light of obstacle model, any auxiliary activity that is able to remove obstacles should work synergistically with processive enzymes. Indeed, the results of several studies have pointed to the fact that synergism between processive enzymes and non-processive endo-enzymes (23,63,75,76) or lytic polysaccharide monooxygenases (77,78) is mediated by the removal of obstacles. Apparently, the molecular nature of the obstacles depends on the source and pre-treatment conditions of the chitin/cellulose substrate.…”

Section: Discussionmentioning

confidence: 99%

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases

Kurašin

Kuusk

et al. 2015

Journal of Biological Chemistry

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Background:The role of slow off-rates of processive enzymes acting on recalcitrant polysaccharides is poorly understood. Results: Chitinase variants with high off-rates and weak product binding were inefficient in degradation of recalcitrant chitin. Conclusion: Slow off-rates and strong product binding are required for high efficiency and processivity. Significance: Knowledge of determinants of processivity aids to design better enzymes.

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Section: Discussionmentioning

confidence: 99%

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases

Kurašin

Kuusk

et al. 2015

Journal of Biological Chemistry

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“…Recently, a semiquantitative high-throughput method for screening activity toward water-soluble substrates has been reported (35), which is suitable for a wide variety of carbohydrate-acting enzymes and can be adapted for LPMOs (13). LPMO action on solid substrates may also be quantified using confocal laser scanning microscopy after labeling C-1-oxidized cellulose chain ends with a fluorescence dye that is specific for carboxylic acids (36). Unlike for endo-acting glycoside hydrolases, the use of gel permeation chromatography or viscosity measurements for detecting LPMO-generated decreases in the molecular weight of carbohydrate polymers has not yet been reported.…”

Section: Introductionmentioning

confidence: 99%

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

Kojima

Várnai

Ishida

et al. 2016

Appl Environ Microbiol

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Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity.IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.

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“…These enzymes introduced a new, oxidative mechanism to polysaccharide degradation. In cellulose degradation, it is assumed that LPMOs act on the surface of crystalline cellulose fibrils, thereby rendering them more accessible to cellulases (Harris et al, 2010; Levasseur et al, 2013;Eibinger et al, 2014). Intriguingly, these enzymes can derive the electrons needed for this process from lignin via long-range electron transfer (Westereng et al, 2015).…”

Section: Auxiliary Enzymesmentioning

confidence: 99%

Genetic modification: a tool for enhancing cellulase secretion

et al. 2017

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Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency

Cited by 255 publications

References 48 publications

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

Genetic modification: a tool for enhancing cellulase secretion

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