scholarly journals Synthesis of Glycoconjugates Utilizing the Regioselectivity of a Lytic Polysaccharide Monooxygenase

2020 ◽  
Author(s):  
Bjørge Westereng ◽  
Stjepan K. Kračun ◽  
Shaun Leivers ◽  
Magnus Ø. Arntzen ◽  
Finn L. Aachmann ◽  
...  
Keyword(s):  
Plant Biomass ◽  
Cellulose Degradation ◽  
Hydroxyl Groups ◽  
Carbonyl Groups ◽  
Lytic Polysaccharide Monooxygenase ◽  
Renewable Chemicals ◽  
Polysaccharide Monooxygenases ◽  
Chemical Coupling ◽  
Potential Biodegradability ◽  
Polysaccharide Monooxygenase

ABSTRACTPolysaccharides from plant biomass are the most abundant renewable chemicals on Earth and can potentially be converted to a wide variety of useful glycoconjugates. While anomeric hydroxyl groups of carbohydrates are amenable to a variety of useful chemical modifications, selective cross-coupling to non-reducing ends has remained challenging. Several lytic polysaccharide monooxygenases (LPMOs), powerful enzymes known for their application in cellulose degradation, specifically oxidize non-reducing ends, introducing carbonyl groups that can be utilized for chemical coupling. This study provides a simple and highly specific approach to produce oxime-based glycoconjugates from LPMO-functionalized oligosaccharides. The products are evaluated by HPLC, mass spectrometry and NMR. Furthermore, we demonstrate potential biodegradability of these glycoconjugates using selective enzymes.

scholarly journals Recent Advances in Screening Methods for the Functional Investigation of Lytic Polysaccharide Monooxygenases

2021 ◽  
Vol 9 ◽  
Author(s):  
Damao Wang ◽  
Yanping Li ◽  
Yuting Zheng ◽  
Yves S. Y. Hsieh
Keyword(s):  
Copper Ions ◽  
Biomass Conversion ◽  
Screening Methods ◽  
Lytic Polysaccharide Monooxygenase ◽  
Indirect Measurements ◽  
Glycosidic Bonds ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Functional Investigation ◽  
Polysaccharide Monooxygenase

Lytic polysaccharide monooxygenase (LPMO) is a newly discovered and widely studied enzyme in recent years. These enzymes play a key role in the depolymerization of sugar-based biopolymers (including cellulose, hemicellulose, chitin and starch), and have a positive significance for biomass conversion. LPMO is a copper-dependent enzyme that can oxidize and cleave glycosidic bonds in cellulose and other polysaccharides. Their mechanism of action depends on the correct coordination of copper ions in the active site. There are still difficulties in the analysis of LPMO activity, which often requires multiple methods to be used in concert. In this review, we discussed various LPMO activity analysis methods reported so far, including mature mass spectrometry, chromatography, labeling, and indirect measurements, and summarized the advantages, disadvantages and applicability of different methods.

scholarly journals Kinetic insights into the role of the reductant in H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase

2018 ◽  
Vol 294 (5) ◽  
pp. 1516-1528 ◽  
Author(s):  
Silja Kuusk ◽  
Riin Kont ◽  
Piret Kuusk ◽  
Agnes Heering ◽  
Morten Sørlie ◽  
...  
Keyword(s):  
Reduction Reaction ◽  
Reaction Conditions ◽  
Lytic Polysaccharide Monooxygenase ◽  
Glycosidic Bonds ◽  
Initial Reduction ◽  
Catalytically Active ◽  
Polysaccharide Monooxygenases ◽  
Theoretical Analyses ◽  
Polysaccharide Monooxygenase

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of an external electron donor (reductant). In the classical O2-driven monooxygenase reaction, the reductant is needed in stoichiometric amounts. In a recently discovered, more efficient H2O2-driven reaction, the reductant would be needed only for the initial reduction (priming) of the LPMO to its catalytically active Cu(I) form. However, the influence of the reductant on reducing the LPMO or on H2O2 production in the reaction remains undefined. Here, we conducted a detailed kinetic characterization to investigate how the reductant affects H2O2-driven degradation of 14C-labeled chitin by a bacterial LPMO, SmLPMO10A (formerly CBP21). Sensitive detection of 14C-labeled products and careful experimental set-ups enabled discrimination between the effects of the reductant on LPMO priming and other effects, in particular enzyme-independent production of H2O2 through reactions with O2. When supplied with H2O2, SmLPMO10A catalyzed 18 oxidative cleavages per molecule of ascorbic acid, suggesting a “priming reduction” reaction. The dependence of initial rates of chitin degradation on reductant concentration followed hyperbolic saturation kinetics, and differences between the reductants were manifested in large variations in their half-saturating concentrations (KmRapp). Theoretical analyses revealed that KmRapp decreases with a decreasing rate of polysaccharide-independent LPMO reoxidation (by either O2 or H2O2). We conclude that the efficiency of LPMO priming depends on the relative contributions of reductant reactivity, on the LPMO's polysaccharide monooxygenase/peroxygenase and reductant oxidase/peroxidase activities, and on reaction conditions, such as O2, H2O2, and polysaccharide concentrations.

scholarly journals Lytic polysaccharide monooxygenase (LPMO) mediated production of ultra-fine cellulose nanofibres from delignified softwood fibres

2019 ◽  
Vol 21 (21) ◽  
pp. 5924-5933 ◽  
Author(s):  
Salla Koskela ◽  
Shennan Wang ◽  
Dingfeng Xu ◽  
Xuan Yang ◽  
Kai Li ◽  
...  
Keyword(s):  
Efficient Method ◽  
Energy Efficient ◽  
Environmentally Friendly ◽  
Oxidative Enzymes ◽  
Lytic Polysaccharide Monooxygenase ◽  
Lytic Polysaccharide Monooxygenases ◽  
Cellulose Nanofibres ◽  
Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenase

An environmentally friendly, energy-efficient method for cellulose nanofibre (CNF) production from softwood holocellulose utilising oxidative enzymes, lytic polysaccharide monooxygenases (LPMOs).

scholarly journals LyGo: A platform for rapid screening of lytic polysaccharide monooxygenase production

2020 ◽  
Author(s):  
Cristina Hernández-Rollán ◽  
Kristoffer B. Falkenberg ◽  
Maja Rennig ◽  
Andreas B. Bertelsen ◽  
Johan Ø. Ipsen ◽  
...  
Keyword(s):  
Critical Role ◽  
Optimal Strategies ◽  
Building Blocks ◽  
Cloning And Expression ◽  
Rapid Screening ◽  
Expression Vectors ◽  
Lytic Polysaccharide Monooxygenase ◽  
Polysaccharide Monooxygenases ◽  
Combine Gene ◽  
Polysaccharide Monooxygenase

AbstractEnvironmentally friendly sources of energy and chemicals are essential constituents of a sustainable society. An important step towards this goal is the utilization of non-edible biomass as supply of building blocks for future biorefineries. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that play a critical role in breaking the chemical bonds in the most abundant polymers found in recalcitrant biomass, such as cellulose and chitin. Predicting optimal strategies for producing LPMOs is often non-trivial, and methods allowing for screening several strategies simultaneously are therefore needed. Here, we present a standardized platform for cloning LPMOs. The platform allows users to combine gene fragments with different expression vectors in a simple 15-minute reaction, thus enabling rapid exploration of several gene contexts, hosts and expression strategies in parallel. The open-source LyGo platform is accompanied by easy-to-follow online protocols for both cloning and expression. As a demonstration, we utilize the LyGo platform to explore different strategies for expressing several different LPMOs in Escherichia coli, Bacillus subtilis, and Komagataella phaffii.

scholarly journals Combining pieces: a thorough analysis of light activation boosting power and co-substrate preferences for the catalytic efficiency of lytic polysaccharide monooxygenase MtLPMO9A

2021 ◽  
Vol 8 (3) ◽  
pp. 1454-1464
Author(s):  
Ana Gabriela V. Sepulchro ◽  
Vanessa O.A. Pellegrini ◽  
Lucas D. Dias ◽  
Marco A.S. Kadowaki ◽  
David Cannella ◽  
...  
Keyword(s):  
Plant Biomass ◽  
Biomass Conversion ◽  
Direct Electron Transfer ◽  
Catalytic Efficiency ◽  
Reaction Products ◽  
Lytic Polysaccharide Monooxygenase ◽  
Substrate Preferences ◽  
Polysaccharide Monooxygenases ◽  
Significant Release ◽  
Cost Efficient

Cost-efficient plant biomass conversion using biochemical and/or chemical routes is essential for transitioning to sustainable chemical technologies and renewable biofuels. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that make part of modern hydrolytic cocktails destined for plant biomass degradation. Here, we characterized MtLPMO9A from Thermothelomyces thermophilus M77 (formerly Myceliophthora thermophila) and demonstrated that it could be efficiently driven by chlorophyllin excited by light in the presence of a reductant agent. However, in the absence of chemical reductant, chlorophyllin and light alone do not lead to a significant release of the reaction products by the LPMO, indicating a low capacity of MtLPMO9A reduction (either via direct electron transfer or via superoxide ion, O2•-). We showed that photocatalysis could significantly increase the LPMO activity against highly crystalline and recalcitrant cellulosic substrates, which are poorly degraded in the absence of chlorophyllin and light. We also evaluated the use of co-substrates by MtLPMO9A, revealing that the enzyme can use both hydrogen peroxide (H2O2) and molecular oxygen (O2) as co-substrates for cellulose catalytic oxidation.

scholarly journals Water-soluble chlorophyll-binding proteins from Brassica oleracea allow for stable photobiocatalytic oxidation of cellulose by a lytic polysaccharide monooxygenase

2020 ◽  
Author(s):  
N. Dodge ◽  
D. A. Russo ◽  
B.M. Blossom ◽  
R.K. Singh ◽  
B. van Oort ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Plant Biomass ◽  
Light Exposure ◽  
Water Soluble ◽  
Lytic Polysaccharide Monooxygenase ◽  
Cellulose Oxidation ◽  
Biomass Processing ◽  
Polysaccharide Monooxygenases ◽  
Cellulose Depolymerization

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are indispensable redox enzymes used in industry for the saccharification of plant biomass. LPMO-driven cellulose oxidation can be enhanced considerably through photobiocatalysis using chlorophyll derivatives and light. Water soluble chlorophyll binding proteins (WSCPs) make it is possible to stabilize and solubilize chlorophyll in aqueous solution, allowing for in vitro studies on photostability and ROS production. Here we aim apply a WSCP-Chl a as a photosensitizing complex for photobiocatalysis with the LPMO, TtAA9. Results We have in this study demonstrated how WSCP reconstituted with chlorophyll a (WSCP-Chl a) can create a stable photosensitizing complex which produces controlled amounts of H2O2 in the presence of ascorbic acid and light. WSCP-Chl a is highly reactive and allows for tightly controlled formation of H2O2 by regulating light intensity. TtAA9 together with WSCP-Chl a shows increased cellulose oxidation under low light conditions, and the WSCP-Chl a complex remains stable after 24 hours of light exposure. Additionally, the WSCP-Chl a complex demonstrates stability over a range of temperatures and pH conditions relevant for enzyme activity in industrial settings. Conclusion With WSCP-Chl a as the photosensitizer, the need to replenish Chl is greatly reduced, enhancing the catalytic lifetime of light-driven LPMOs and increasing the efficiency of cellulose depolymerization. WSCP-Chl a allows for stable photobiocatalysis providing a sustainable solution for biomass processing.

scholarly journals Water-soluble chlorophyll-binding proteins from Brassica oleracea allow for stable photobiocatalytic oxidation of cellulose by a lytic polysaccharide monooxygenase

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
N. Dodge ◽  
D. A. Russo ◽  
B. M. Blossom ◽  
R. K. Singh ◽  
B. van Oort ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Plant Biomass ◽  
Light Exposure ◽  
Water Soluble ◽  
Lytic Polysaccharide Monooxygenase ◽  
Cellulose Oxidation ◽  
Biomass Processing ◽  
Polysaccharide Monooxygenases ◽  
Cellulose Depolymerization

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are indispensable redox enzymes used in industry for the saccharification of plant biomass. LPMO-driven cellulose oxidation can be enhanced considerably through photobiocatalysis using chlorophyll derivatives and light. Water soluble chlorophyll binding proteins (WSCPs) make it is possible to stabilize and solubilize chlorophyll in aqueous solution, allowing for in vitro studies on photostability and ROS production. Here we aim to apply WSCP–Chl a as a photosensitizing complex for photobiocatalysis with the LPMO, TtAA9. Results We have in this study demonstrated how WSCP reconstituted with chlorophyll a (WSCP–Chl a) can create a stable photosensitizing complex which produces controlled amounts of H2O2 in the presence of ascorbic acid and light. WSCP–Chl a is highly reactive and allows for tightly controlled formation of H2O2 by regulating light intensity. TtAA9 together with WSCP–Chl a shows increased cellulose oxidation under low light conditions, and the WSCP–Chl a complex remains stable after 24 h of light exposure. Additionally, the WSCP–Chl a complex demonstrates stability over a range of temperatures and pH conditions relevant for enzyme activity in industrial settings. Conclusion With WSCP–Chl a as the photosensitizer, the need to replenish Chl is greatly reduced, enhancing the catalytic lifetime of light-driven LPMOs and increasing the efficiency of cellulose depolymerization. WSCP–Chl a allows for stable photobiocatalysis providing a sustainable solution for biomass processing.

scholarly journals Temporal Alterations in the Secretome of the Selective Ligninolytic Fungus Ceriporiopsis subvermispora during Growth on Aspen Wood Reveal This Organism's Strategy for Degrading Lignocellulose

2014 ◽  
Vol 80 (7) ◽  
pp. 2062-2070 ◽  
Author(s):  
Chiaki Hori ◽  
Jill Gaskell ◽  
Kiyohiko Igarashi ◽  
Phil Kersten ◽  
Michael Mozuch ◽  
...  
Keyword(s):  
Cellulose Degradation ◽  
Alcohol Oxidase ◽  
White Rot ◽  
Protein Abundance ◽  
Lytic Polysaccharide Monooxygenase ◽  
Content Type ◽  
Ceriporiopsis Subvermispora ◽  
Liquid Chromatography Tandem Mass ◽  
Nano Liquid Chromatography ◽  
Polysaccharide Monooxygenase

ABSTRACTThe white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such asCeriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue,C. subvermisporawas grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis byC. subvermispora.

scholarly journals The role of the active site tyrosine in the mechanism of lytic polysaccharide monooxygenase

2021 ◽  
Vol 12 (1) ◽  
pp. 352-362
Author(s):  
Aina McEvoy ◽  
Joel Creutzberg ◽  
Raushan K. Singh ◽  
Morten J. Bjerrum ◽  
Erik D. Hedegård
Keyword(s):  
Active Site ◽  
Lytic Polysaccharide Monooxygenase ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Active Site Tyrosine ◽  
Polysaccharide Monooxygenase

With QM/MM, we investigate the mechanism of tyrosine deprotonation in lytic polysaccharide monooxygenases. Our results support deprotonation and our calculated UV-vis spectra show that two isomers must be formed to match recent experiments.

scholarly journals Water-soluble chlorophyll-binding proteins from Brassica oleracea allow for stable photobiocatalytic oxidation of cellulose by a lytic polysaccharide monooxygenase.

2020 ◽  
Author(s):  
N. Dodge ◽  
D. A. Russo ◽  
B.M. Blossom ◽  
R.K. Singh ◽  
B. van Oort ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Plant Biomass ◽  
Light Exposure ◽  
Water Soluble ◽  
Lytic Polysaccharide Monooxygenase ◽  
Cellulose Oxidation ◽  
Biomass Processing ◽  
Polysaccharide Monooxygenases ◽  
Cellulose Depolymerization

Abstract Background: Lytic polysaccharide monooxygenases (LPMOs) are indispensable redox enzymes used in industry for the saccharification of plant biomass. LPMO-driven cellulose oxidation can be enhanced considerably through photobiocatalysis using chlorophyll derivatives and light. Water soluble chlorophyll binding proteins (WSCPs) make it is possible to stabilize and solubilize chlorophyll in aqueous solution, allowing for in vitro studies on photostability and ROS production. Here we aim apply a WSCP-Chl α as a photosensitizing complex for photobiocatalysis with the LPMO, TtAA9. Results: We have in this study demonstrated how WSCP reconstituted with chlorophyll a (WSCP-Chl α) can create a stable photosensitizing complex which produces controlled amounts of H2O2 in the presence of ascorbic acid and light. WSCP-Chl α is highly reactive and allows for tightly controlled formation of H2O2 by regulating light intensity. TtAA9 together with WSCP-Chl α shows increased cellulose oxidation under low light conditions, and the WSCP-Chl α complex remains stable after 24 hours of light exposure. Additionally, the WSCP-Chl α complex demonstrates stability over a range of temperatures and pH conditions relevant for enzyme activity in industrial settings.Conclusion: With WSCP-Chl α as the photosensitizer, the need to replenish Chl is greatly reduced, enhancing the catalytic lifetime of light-driven LPMOs and increasing the efficiency of cellulose depolymerization. WSCP-Chl α allows for stable photobiocatalysis providing a sustainable solution for biomass processing.

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