scholarly journals Exploitation of Enzymes for the Production of Biofuels: Electrochemical Determination of Kinetic Parameters of LPMOs

2021 ◽  
Vol 11 (11) ◽  
pp. 4715
Author(s):  
Dimitrios Zouraris ◽  
Anthi Karnaouri ◽  
Raphaela Xydou ◽  
Evangelos Topakas ◽  
Antonis Karantonis
Keyword(s):  
Kinetic Parameters ◽  
Filamentous Fungus ◽  
Electrode Surface ◽  
Plant Biomass ◽  
Electrochemical Determination ◽  
Redox Enzymes ◽  
Electrode Surfaces ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) consist of a class of enzymes that boost the release of oxidised products from plant biomass, in an approach that is more eco-friendly than the traditional ones, employing harsh chemicals. Since LPMOs are redox enzymes, they could possibly be exploited by immobilisation on electrode surfaces. Such an approach requires knowledge of kinetic and thermodynamic information for the interaction of the enzyme with the electrode surface. In this work, a novel methodology is applied for the determination of such parameters for an LPMO from the filamentous fungus Thermothelomyces thermophila, MtLPMO9H.

Mono-6-thio-β-cyclodextrin-functionalized AuNP/two-dimensional TiO2 nanosheet nanocomposite for the electrochemical determination of trace methyl parathion in water

2019 ◽  
Vol 11 (37) ◽  
pp. 4751-4760 ◽  
Author(s):  
Xu-Cheng Fu ◽  
Chao Zhang ◽  
Xuan-Hua Li ◽  
Jian Zhang ◽  
Gan Wei
Keyword(s):  
Electrode Surface ◽  
Methyl Parathion ◽  
Au Nanoparticles ◽  
Electrochemical Determination ◽  
Two Dimensional

In this work, two-dimensional TiO2 nanosheets material composited with Au nanoparticles and mono-6-thio-β-cyclodextrin was prepared on electrode surface (SH-β-CD/AuNPs/TiO2NSs/GCE).

scholarly journals Deciphering the Regulatory Network between the SREBP Pathway and Protein Secretion in Neurospora crassa

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Lina Qin ◽  
Vincent W. Wu ◽  
N. Louise Glass
Keyword(s):  
Neurospora Crassa ◽  
Unfolded Protein Response ◽  
Molecular Oxygen ◽  
Protein Secretion ◽  
Plant Biomass ◽  
Unfolded Protein ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACT Sterol regulatory element binding proteins (SREBPs) are conserved from yeast to mammalian cells and function in the regulation of sterol homeostasis. In fungi, the SREBP pathway has been implicated in the adaptation to hypoxia and in virulence. In Neurospora crassa and Trichoderma reesei, the SREBP pathway also negatively regulates protein secretion under lignocellulolytic conditions. Here we utilized global transcriptional profiling combined with genetic and physiological analyses to address the regulatory link between the SREBP pathway and protein secretion in N. crassa. Our results demonstrated that the function of the SREBP pathway in ergosterol biosynthesis and adaptation to hypoxia was conserved in N. crassa. Under lignocellulolytic conditions, the SREBP pathway was highly activated, resulting in the reduced expression of lytic polysaccharide monooxygenases, which require molecular oxygen for catalytic activity. Additionally, activation of the SREBP pathway under lignocellulolytic conditions repressed a set of genes predicted to be involved in the endoplasmic reticulum stress response. Here we show that the inability of a hac-1 mutant, which bears a deletion of the major regulator of the unfolded protein response (UPR), to efficiently produce cellulases and utilize cellulose was suppressed by mutations in the SREBP pathway. The analyses presented here demonstrated new SREBP pathway functions, including linkages to the UPR, and provide new clues for genetic engineering of filamentous fungi to improve their production of extracellular proteins. IMPORTANCE The role of SREBP transcription factors in the regulation of sterol biosynthesis is conserved from humans to yeast. In filamentous fungi, this pathway regulates the secretion of lignocellulolytic enzymes during plant biomass deconstruction. Here we show that the SREBP pathway in Neurospora crassa regulates the production of specific cellulases, lytic polysaccharide monooxygenases that utilize molecular oxygen. Via global transcriptional profile and genetic analyses, a relationship between the SREBP pathway and the unfolded protein response (UPR) pathway was revealed, suggesting a regulatory interplay of these two pathways in the trafficking of plant biomass-degrading enzymes. These findings have implications for our understanding of the cross talk of the SREBP and UPR pathways in other organisms and will guide the rational engineering of fungal strains to improve cellulolytic enzyme production. IMPORTANCE The role of SREBP transcription factors in the regulation of sterol biosynthesis is conserved from humans to yeast. In filamentous fungi, this pathway regulates the secretion of lignocellulolytic enzymes during plant biomass deconstruction. Here we show that the SREBP pathway in Neurospora crassa regulates the production of specific cellulases, lytic polysaccharide monooxygenases that utilize molecular oxygen. Via global transcriptional profile and genetic analyses, a relationship between the SREBP pathway and the unfolded protein response (UPR) pathway was revealed, suggesting a regulatory interplay of these two pathways in the trafficking of plant biomass-degrading enzymes. These findings have implications for our understanding of the cross talk of the SREBP and UPR pathways in other organisms and will guide the rational engineering of fungal strains to improve cellulolytic enzyme production.

New combination between chitosan, single walled carbon nanotubes and neodymium(iii) oxide found to be useful in the electrochemical determination of rutin in the presence of morin and quercetin

2017 ◽  
Vol 9 (46) ◽  
pp. 6474-6481 ◽  
Author(s):  
Jorge A. Calderón ◽  
María Cardozo-Pérez ◽  
Alfredo Torres-Benítez ◽  
Olimpo García-Beltrán ◽  
Edgar Nagles
Keyword(s):  
Carbon Nanotubes ◽  
Glassy Carbon ◽  
Electrode Surface ◽  
Single Walled Carbon Nanotubes ◽  
Electrochemical Determination ◽  
New Combination ◽  
Carbon Electrode ◽  
Single Walled Carbon ◽  
Walled Carbon Nanotubes

A glassy carbon electrode surface was modified with a new combination of chitosan, single walled carbon nanotubes (SWCNT) and neodymium(iii) oxide (NdOX) and was found to be effective in the detection of rutin (RU).

scholarly journals Fenton-type chemistry by a copper enzyme: molecular mechanism of polysaccharide oxidative cleavage

2016 ◽  
Author(s):  
Bastien Bissaro ◽  
Asmund K Rohr ◽  
Morten Skaugen ◽  
Zarah Forsberg ◽  
Svein J Horn ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Reaction Kinetics ◽  
Mode Of Action ◽  
Process Conditions ◽  
Enzyme Assays ◽  
Enzymatic Conversion ◽  
Redox Enzymes ◽  
Anaerobic Conditions ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

The discovery of Lytic Polysaccharide Monooxygenases (LPMOs) has been instrumental for the development of economically sustainable lignocellulose biorefineries. Despite the obvious importance of these exceptionally powerful redox enzymes, their mode of action remains enigmatic and their activity and stability under process conditions are hard to control. By using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show that H2O2, and not O2 as previously thought, is the co-substrate of LPMOs. By controlling H2O2 supply, stable reaction kinetics and high enzymatic rates are achieved, the LPMOs work under anaerobic conditions, and the need for adding stoichiometric amounts of reductants is alleviated. These results offer completely new perspectives regarding the mode of action of these unique mono-copper enzymes, the enzymatic conversion of biomass in Nature, and industrial biorefining.

scholarly journals Structure determination of lytic polysaccharide monooxygenases interactions with substrate

2018 ◽  
Vol 74 (a2) ◽  
pp. e36-e36
Author(s):  
Tobias Tandrup ◽  
Kristian E. H. Frandsen ◽  
Thomas J. Simmons ◽  
Jens-Christian N. Poulsen ◽  
Louis F. L. Wilson ◽  
...  
Keyword(s):  
Structure Determination ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

scholarly journals Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs)

2020 ◽  
pp. jbc.RA120.015545
Author(s):  
Kristian E. H. Frandsen ◽  
Mireille Haon ◽  
Sacha Grisel ◽  
Bernard Henrissat ◽  
Leila Lo Leggio ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Time Course ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Substrate Binding ◽  
Glycoside Hydrolases ◽  
Sequence Alignments ◽  
Lytic Polysaccharide Monooxygenases ◽  
Molecular Determinants ◽  
Polysaccharide Monooxygenases

Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall-derived hemicellulosic polysaccharides and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharides degradation suggests that the subtle differences in amino acids sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO sub-clusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)-linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries.

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