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 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 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 Mechanism of hydrogen peroxide formation by lytic polysaccharide monooxygenase

2019 ◽  
Vol 10 (2) ◽  
pp. 576-586 ◽  
Author(s):  
Octav Caldararu ◽  
Esko Oksanen ◽  
Ulf Ryde ◽  
Erik D. Hedegård
Keyword(s):  
Hydrogen Peroxide ◽  
Peroxide Formation ◽  
Lytic Polysaccharide Monooxygenase ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Hydrogen Peroxide Formation ◽  
Polysaccharide Monooxygenase

A mechanism for the formation of hydrogen peroxide by lytic polysaccharide monooxygenases (LPMOs) in the absence of substrate is proposed.

Combination of MALDI-TOF MS and UHPLC-ESI-MS for the characterization of lytic polysaccharide monooxygenase activity

2020 ◽  
Vol 12 (2) ◽  
pp. 149-161 ◽  
Author(s):  
Caio de Oliveira Gorgulho Silva ◽  
Tallyta Santos Teixeira ◽  
Kelly Barreto Rodrigues ◽  
Amanda Araújo Souza ◽  
Antonielle Vieira Monclaro ◽  
...  
Keyword(s):  
Lytic Polysaccharide Monooxygenase ◽  
Monooxygenase Activity ◽  
Maldi Tof Ms ◽  
Esi Ms ◽  
Maldi Tof ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Tof Ms ◽  
Polysaccharide Monooxygenase

Two new mass spectrometry methods, MALDI-TOF MS and hydrophilic interaction UHPLC-ESI-MS, were developed for the characterization of cellulose-active lytic polysaccharide monooxygenases, expanding the analytical toolbox for the study of these enzymes.

scholarly journals Further structural studies of the lytic polysaccharide monooxygenase AoAA13 belonging to the starch-active AA13 family

Amylase ◽  
2019 ◽  
Vol 3 (1) ◽  
pp. 41-54 ◽  
Author(s):  
Sebastian J. Muderspach ◽  
Tobias Tandrup ◽  
Kristian E. H. Frandsen ◽  
Gianluca Santoni ◽  
Jens-Christian N. Poulsen ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Metal Binding ◽  
Binding Studies ◽  
Orthorhombic Crystal ◽  
Lytic Polysaccharide Monooxygenase ◽  
X Ray ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Triclinic Structure ◽  
Polysaccharide Monooxygenase

Abstract Lytic polysaccharide monooxygenases (LPMOs) are recently discovered copper enzymes that cleave recalcitrant polysaccharides by oxidation. The structure of an Aspergillus oryzae LPMO from the starch degrading family AA13 (AoAA13) has previously been determined from an orthorhombic crystal grown in the presence of copper, which is photoreduced in the structure. Here we describe how crystals reliably grown in presence of Zn can be Cu-loaded post crystallization. A partly photoreduced structure was obtained by severely limiting the X-ray dose, showing that this LPMO is much more prone to photoreduction than others. A serial synchrotron crystallography structure was also obtained, showing that this technique may be promising for further studies, to reduce even further photoreduction. We additionally present a triclinic structure of AoAA13, which has less occluded ligand binding site than the orthorhombic one. The availability of the triclinic crystals prompted new ligand binding studies, which lead us to the conclusion that small starch analogues do not bind to AoAA13 to an appreciable extent. A number of disordered conformations of the metal binding histidine brace have been encountered in this and other studies, and we have previously hypothesized that this disorder may be a consequence of loss of copper. We performed molecular dynamics in the absence of active site metal, and showed that the dynamics in solution differ somewhat from the disorder observed in the crystal, though the extent is equally dramatic.

scholarly journals Is density functional theory accurate for lytic polysaccharide monooxygenase enzymes?

2020 ◽  
Vol 49 (5) ◽  
pp. 1501-1512 ◽  
Author(s):  
Ernst D. Larsson ◽  
Geng Dong ◽  
Valera Veryazov ◽  
Ulf Ryde ◽  
Erik D. Hedegård
Keyword(s):  
Density Functional Theory ◽  
Energy Efficient ◽  
Density Functional ◽  
Efficient Production ◽  
Lytic Polysaccharide Monooxygenase ◽  
Functional Theory ◽  
Polysaccharide Monooxygenase

The lytic polysaccharide monooxygenase (LPMO) enzymes boost polysaccharide depolymerization through oxidative chemistry, which has fueled the hope for more energy-efficient production of biofuel.

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 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 pH-Dependent Relationship between Catalytic Activity and Hydrogen Peroxide Production Shown via Characterization of a Lytic Polysaccharide Monooxygenase from Gloeophyllum trabeum

2018 ◽  
Vol 85 (5) ◽  
Author(s):  
Olav A. Hegnar ◽  
Dejan M. Petrovic ◽  
Bastien Bissaro ◽  
Gry Alfredsen ◽  
Anikó Várnai ◽  
...  
Keyword(s):  
Enzyme Activation ◽  
Gloeophyllum Trabeum ◽  
Dihydroxybenzoic Acid ◽  
Lytic Polysaccharide Monooxygenase ◽  
Aerobic Conditions ◽  
Dependent Relationship ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Catalyzed Oxidation

ABSTRACT Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that perform oxidative cleavage of recalcitrant polysaccharides. We have purified and characterized a recombinant family AA9 LPMO, LPMO9B, from Gloeophyllum trabeum (GtLPMO9B) which is active on both cellulose and xyloglucan. Activity of the enzyme was tested in the presence of three different reductants: ascorbic acid, gallic acid, and 2,3-dihydroxybenzoic acid (2,3-DHBA). Under standard aerobic conditions typically used in LPMO experiments, the first two reductants could drive LPMO catalysis whereas 2,3-DHBA could not. In agreement with the recent discovery that H2O2 can drive LPMO catalysis, we show that gradual addition of H2O2 allowed LPMO activity at very low, substoichiometric (relative to products formed) reductant concentrations. Most importantly, we found that while 2,3-DHBA is not capable of driving the LPMO reaction under standard aerobic conditions, it can do so in the presence of externally added H2O2. At alkaline pH, 2,3-DHBA is able to drive the LPMO reaction without externally added H2O2, and this ability overlaps entirely the endogenous generation of H2O2 by GtLPMO9B-catalyzed oxidation of 2,3-DHBA. These findings support the notion that H2O2 is a cosubstrate of LPMOs and provide insight into how LPMO reactions depend on, and may be controlled by, the choice of pH and reductant. IMPORTANCE Lytic polysaccharide monooxygenases promote enzymatic depolymerization of lignocellulosic materials by microorganisms due to their ability to oxidatively cleave recalcitrant polysaccharides. The properties of these copper-dependent enzymes are currently of high scientific and industrial interest. We describe a previously uncharacterized fungal LPMO and show how reductants, which are needed to prime the LPMO by reducing Cu(II) to Cu(I) and to supply electrons during catalysis, affect enzyme efficiency and stability. The results support claims that H2O2 is a natural cosubstrate for LPMOs by demonstrating that when certain reductants are used, catalysis can be driven only by H2O2 and not by O2. Furthermore, we show how auto-inactivation resulting from endogenous generation of H2O2 in the LPMO-reductant system may be prevented. Finally, we identified a reductant that leads to enzyme activation without any endogenous H2O2 generation, allowing for improved control of LPMO reactivity and providing a valuable tool for future LPMO research.

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