A thermostable bacterial lytic polysaccharide monooxygenase with high operational stability in a wide temperature range

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are oxidative, copper-dependent enzymes that function as powerful tools in the turnover of various biomasses, including lignocellulosic plant biomass. While LPMOs are considered to be of great importance for biorefineries, little is known about industrial relevant properties such as the ability to operate at high temperatures. Here, we describe a thermostable, cellulose-active LPMO from a high-temperature compost metagenome (called mgLPMO10). Results MgLPMO10 was found to have the highest apparent melting temperature (83 °C) reported for an LPMO to date, and is catalytically active up to temperatures of at least 80 °C. Generally, mgLPMO10 showed good activity and operational stability over a wide temperature range. The LPMO boosted cellulose saccharification by recombinantly produced GH48 and GH6 cellobiohydrolases derived from the same metagenome, albeit to a minor extent. Cellulose saccharification studies with a commercial cellulase cocktail (Celluclast®) showed that the performance of this thermostable bacterial LPMO is comparable with that of a frequently utilized fungal LPMO from Thermoascus aurantiacus (TaLPMO9A). Conclusions The high activity and operational stability of mgLPMO10 are of both fundamental and applied interest. The ability of mgLPMO10 to perform oxidative cleavage of cellulose at 80 °C and the clear synergy with Celluclast® make this enzyme an interesting candidate in the development of thermostable enzyme cocktails for use in lignocellulosic biorefineries.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.006196 ◽

2018 ◽

Vol 294 (5) ◽

pp. 1516-1528 ◽

Cited By ~ 21

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.

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

Biofuel Research Journal ◽

10.18331/brj2021.8.3.5 ◽

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.

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

10.21203/rs.3.rs-40886/v1 ◽

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.

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

Biotechnology for Biofuels ◽

10.1186/s13068-020-01832-7 ◽

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.

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Synthesis of Glycoconjugates Utilizing the Regioselectivity of a Lytic Polysaccharide Monooxygenase

10.1101/2020.03.13.990838 ◽

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.

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

10.21203/rs.3.rs-40886/v2 ◽

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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Structure of a C1/C4-oxidizing AA9 lytic polysaccharide monooxygenase from the thermophilic fungus Malbranchea cinnamomea

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798321006628 ◽

2021 ◽

Vol 77 (8) ◽

Author(s):

Scott Mazurkewich ◽

Andrea Seveso ◽

Silvia Hüttner ◽

Gisela Brändén ◽

Johan Larsbrink

Keyword(s):

Plant Cell Wall ◽

Plant Biomass ◽

Industrial Applications ◽

Thermophilic Fungus ◽

Lytic Polysaccharide Monooxygenase ◽

Large Loop ◽

Small Loop ◽

Polysaccharide Monooxygenases ◽

Equivalent Residue ◽

Residue Substitution

The thermophilic fungus Malbranchea cinnamomea contains a host of enzymes that enable its ability as an efficient degrader of plant biomass and that could be mined for industrial applications. This thermophilic fungus has been studied and found to encode eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), which collectively possess different substrate specificities for a range of plant cell-wall-related polysaccharides and oligosaccharides. To gain greater insight into the molecular determinants defining the different specificities, structural studies were pursued and the structure of McAA9F was determined. The enzyme contains the immunoglobulin-like fold typical of previously solved AA9 LPMO structures, but contains prominent differences in the loop regions found on the surface of the substrate-binding site. Most significantly, McAA9F has a broad substrate specificity, with activity on both crystalline and soluble polysaccharides. Moreover, it contains a small loop in a region where a large loop has been proposed to govern specificity towards oligosaccharides. The presence of the small loop leads to a considerably flatter and more open surface that is likely to enable the broad specificity of the enzyme. The enzyme contains a succinimide residue substitution, arising from intramolecular cyclization of Asp10, at a position where several homologous members contain an equivalent residue but cyclization has not previously been observed. This first structure of an AA9 LPMO from M. cinnamomea aids both the understanding of this family of enzymes and the exploration of the repertoire of industrially relevant lignocellulolytic enzymes from this fungus.

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Group I lytic polysaccharide monooxygenase (LPMO1) is required for efficient chitinous cuticle turnover during insect molting

10.21203/rs.3.rs-877720/v1 ◽

2021 ◽

Author(s):

mingbo Qu ◽

Myeongjin Kim ◽

Xiaoxi Guo ◽

Seulgi Mun ◽

Shuang Tian ◽

...

Keyword(s):

Developmental Stages ◽

Catalytic Domain ◽

Locusta Migratoria ◽

Oxidative Cleavage ◽

Lytic Polysaccharide Monooxygenase ◽

Tem Analysis ◽

Physiological Importance ◽

Group I ◽

Catalytically Active ◽

Polysaccharide Monooxygenases

Abstract Microbial lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of crystalline polysaccharides including chitin and cellulose. The discovery of a large assortment of LPMO-like proteins widely distributed in insect genomes suggests that they could be involved in assisting chitin degradation in the exoskeleton, tracheae and peritrophic matrix during development. However, the physiological functions of insect LPMO-like proteins are still undetermined. To investigate the functions of insect LPMO subgroup I-like proteins, which contain an AA15 LPMO catalytic domain and a conserved C-terminal cysteine-rich motif, two evolutionarily distant species, Tribolium castaneum and Locusta migratoria, were chosen for study. RNAi for the T. castaneum protein, TcLPMO1, caused molting arrest at all developmental stages, whereas RNAi of the L. migratoria protein, LmLPMO1, prevented only adult eclosion. In both species, LPMO1-deficient animals were unable to shed their exuviae and died. TEM analysis revealed failure of turnover of chitinous cuticle, which is critical for completion of molting. Purified recombinant LPMO1-like protein from Ostrinia furnacalis (rOfLPMO1) exhibited oxidative cleavage activity and substrate preference for chitin. These results reveal for the first time the physiological importance of catalytically active LPMO1-like proteins from distant insect species and provide new insight into the enzymatic mechanism of chitin turnover during molting.

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