Chromatographic analysis of oxidized cello-oligomers generated by lytic polysaccharide monooxygenases using dual electrolytic eluent generation

Østby, Heidi; Jameson, John-Kristian; Costa, Thales H. F.; Eijsink, Vincent G. H.; Arntzen, Magnus Ø.

doi:10.1016/j.chroma.2021.462691

Cited by 9 publications

(4 citation statements)

References 39 publications

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“…Standards for quantification of glucose and cellobiose were purchased from Megazyme (Wicklow, Ireland). Glc4gemGlc standards for quantification of the C4-oxidized dimer (the by far dominating LPMO product in the reaction mixtures; , ) were produced in-house as previously described …”

Section: Methodsmentioning

confidence: 99%

Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H₂O₂-Producing Glucose Oxidase

Østby

Várnai

Gabriel

et al. 2022

ACS Sustainable Chem. Eng.

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Understanding and improving the efficiency of enzymatic saccharification of lignocellulosic biomass will promote the use of this renewable material. Here, we have studied several process parameters (reaction temperature, type of enzyme blend, type of substrate, type of reductant, and in situ supply of hydrogen peroxide) to better understand how saccharification could be optimized, focusing on the role of lytic polysaccharide monooxygenases (LPMOs). Comparison of a simple, LPMO-rich cellulolytic secretome from the thermophilic fungus Thermoascus aurantiacus with the commercial cellulase preparation Cellic CTec2 showed that saccharification of (lignin-poor) sulfite-pulped spruce at 60 °C with the secretome was as efficient as saccharification with Cellic CTec2 at 50 °C. Quantification of LPMO products showed that while LPMO activity contributed to saccharification efficiency, high levels of LPMO activity were not necessarily beneficial. Reactions with steam-exploded birch, rich in redox-active lignin, highlighted a strong impact of the feedstock on enzyme performance. In this case, the reaction with Cellic CTec2 at 50 °C was clearly most efficient. At 60 °C, enzyme inactivation became apparent for both enzyme blends, likely due to detrimental redox processes. Addition of H 2 O 2 -generating glucose oxidase to reactions with Cellic CTec2 at 50 °C led to strongly increased LPMO activity and, only for reactions with the lignin-poor substrate, improved saccharification yields. These results underpin the potential of the T. aurantiacus secretome for hydrolysis of lignin-poor substrates, and the usefulness of glucose oxidase for optimizing their saccharification. They also show that the efficiency of LPMO-containing cellulase preparations is highly dependent on the nature of the reductant and the substrate.

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Section: Methodsmentioning

confidence: 99%

Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H₂O₂-Producing Glucose Oxidase

Østby

Várnai

Gabriel

et al. 2022

ACS Sustainable Chem. Eng.

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“…Quantification of C1-oxidized dimers and trimers (GlcGlc1A and Glc 2 Glc1A) was performed using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), using a 26 min gradient as described previously. 49 HPAEC-PAD was performed with a Dionex ICS6000 (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a 1 × 250 mm Dionex CarboPac PA-200 analytical column attached to 1 × 50 mm Dionex CarboPac PA-200 guard column. The operational flow was at 63 μL/min, and 4 μL samples were injected.…”

Section: Methodsmentioning

confidence: 99%

Impact of the Copper Second Coordination Sphere on Catalytic Performance and Substrate Specificity of a Bacterial Lytic Polysaccharide Monooxygenase

Hall,

Mollatt,

Forsberg

et al. 2024

ACS Omega

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Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, using a single copper cofactor bound in a conserved histidine brace with a more variable second coordination sphere. Cellulose-active LPMOs in the fungal AA9 family and in a subset of bacterial AA10 enzymes contain a His-Gln-Tyr second sphere motif, whereas other cellulose-active AA10s have an Arg–Glu–Phe motif. To shine a light on the impact of this variation, we generated single, double, and triple mutations changing the His216–Gln219–Tyr221 motif in cellulose- and chitin-oxidizing MaAA10B toward Arg–Glu–Phe. These mutations generally reduced enzyme performance due to rapid inactivation under turnover conditions, showing that catalytic fine-tuning of the histidine brace is complex and that the roles of these second sphere residues are strongly interconnected. Studies of copper reactivity showed remarkable effects, such as an increase in oxidase activity following the Q219E mutation and a strong dependence of this effect on the presence of Tyr at position 221. In reductant-driven reactions, differences in oxidase activity, which lead to different levels of in situ generated H2O2, correlated with differences in polysaccharide-degrading ability. The single Q219E mutant displayed a marked increase in activity on chitin in both reductant-driven reactions and reactions fueled by exogenously added H2O2. Thus, it seems that the evolution of substrate specificity in LPMOs involves both the extended substrate-binding surface and the second coordination sphere.

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“…A C1-oxidized monomeric standard, gluconic acid (Glc1A), was purchased from Sigma-Aldrich and diluted as appropriate. C4-oxidized standards, Glc4gemGlc and Glc4gemGlc2, were made as previously described , using Nc AA9C from Neurospora crassa (prepared in-house, as described elsewhere). The Chromeleon 7 software package (Dionex) was used to analyze the results.…”

Section: Methodsmentioning

confidence: 99%

Light Exposure of Lignin Affects the Saccharification Efficiency of LPMO-Containing Cellulolytic Enzyme Cocktails

Angeltveit,

Kommedal,

Stepnov

et al. 2024

ACS Sustainable Chem. Eng.

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Chromatographic analysis of oxidized cello-oligomers generated by lytic polysaccharide monooxygenases using dual electrolytic eluent generation

Cited by 9 publications

References 39 publications

Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H₂O₂-Producing Glucose Oxidase

Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H₂O₂-Producing Glucose Oxidase

Impact of the Copper Second Coordination Sphere on Catalytic Performance and Substrate Specificity of a Bacterial Lytic Polysaccharide Monooxygenase

Light Exposure of Lignin Affects the Saccharification Efficiency of LPMO-Containing Cellulolytic Enzyme Cocktails

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Chromatographic analysis of oxidized cello-oligomers generated by lytic polysaccharide monooxygenases using dual electrolytic eluent generation

Cited by 9 publications

References 39 publications

Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H2O2-Producing Glucose Oxidase

Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H2O2-Producing Glucose Oxidase

Impact of the Copper Second Coordination Sphere on Catalytic Performance and Substrate Specificity of a Bacterial Lytic Polysaccharide Monooxygenase

Light Exposure of Lignin Affects the Saccharification Efficiency of LPMO-Containing Cellulolytic Enzyme Cocktails

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Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H₂O₂-Producing Glucose Oxidase

Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H₂O₂-Producing Glucose Oxidase