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

Silva, Caio de Oliveira Gorgulho; Teixeira, Tallyta Santos; Rodrigues, Kelly Barreto; Souza, Amanda Araújo; Monclaro, Antonielle Vieira; Mendes, T. D.; Ribeiro, José Antônio de Aquino; Siqueira, Félix Gonçalves de; Fávaro, Léia Cecília de Lima; Abdelnur, Patrícia Verardi

doi:10.1039/c9ay01774g

Cited by 9 publications

(4 citation statements)

References 25 publications

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“…Mt LPMO belongs to the auxiliary activity 9 (AA9) family of the CAZy database () and exhibits a double regioselectivity mode of action; the enzyme releases a mix of C1, C4, and C1/C4 double oxidized oligosaccharides from OS-pretreated tunic. LC-ESI MS/MS analysis (Figure S1, Supporting Information) occurred based on reports in the literature, and it reveals the formation of C1 and C4-oxidized products in hydrated form (aldonic acids and gemdiols) with a DP = 2–5, in nonhydrated form (lactones and 4-ketoaldoses) with a DP = 3–6, as well as nonoxidized cello-oligosaccharides with varying size (DP = 3–8), as shown in Table . Traces of oxidized pentose oligosaccharides were also detected, which probably originate from the activity of Mt LPMO on sulfated xylans that have been observed in tunic of C.…”

Section: Resultssupporting

confidence: 82%

“…37 This LPMO exhibits a double C1/C4-oxidizing activity, although it is the C1oxidizing effect that is the most important in the context of this study. The enzymatic treatment of tunic took place at 50 acetate, pH 6.0, and 50 °C, for 18 h. Ascorbic acid was used as the electron donor at a final concentration of 5 mM. The initial solid loading was 4.5% w/v, and the enzyme loading was 30 mg/g of substrate.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

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Lytic Polysaccharide Monooxygenase-Assisted Preparation of Oxidized-Cellulose Nanocrystals with a High Carboxyl Content from the Tunic of Marine Invertebrate Ciona intestinalis

Karnaouri

Jalvo

Moritz

et al. 2020

ACS Sustainable Chem. Eng.

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The tunicate species Ciona intestinalis is a fast-growing marine invertebrate animal that contains cellulose in its outer partthe tunic. The high crystallinity and microfibril aspect ratio of tunicate cellulose make it an excellent starting material for the isolation of nanocellulose. In the present work, tunic from C. intestinalis was subjected to organosolv pretreatment followed by bleaching and acid-hydrolysis steps for the isolation of nanocrystals. Applying an intermediate enzymatic treatment step with a lytic polysaccharide monooxygenase (LPMO) from the thermophilic fungus Thermothelomyces thermophila was proved to facilitate the isolation of nanocellulose and to improve the overall process yield, even when the bleaching step was omitted. LPMOs are able to oxidatively cleave the glycosidic bonds of a polysaccharide substrate, either at the C1 and/or C4 position, with the former leading to introduction of carboxylate moieties. X-ray photoelectron spectroscopy analysis showed a significant increase in the atomic percentage of the CO/O−C−O and O−CO bonds upon the addition of LPMO, while the obtained nanocrystals exhibited higher thermal stability compared to the untreated ones. Moreover, an enzymatic post-treatment with LPMOs was performed to additionally functionalize the cellulose nanocrystals. Our results demonstrate that LPMOs are promising candidates for the enzymatic modification of cellulose fibers, including the preparation of oxidized-nanocellulose, and offer great perspectives for the production of novel biobased nanomaterials.

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Section: Resultssupporting

confidence: 82%

Section: ■ Experimental Sectionmentioning

confidence: 99%

Lytic Polysaccharide Monooxygenase-Assisted Preparation of Oxidized-Cellulose Nanocrystals with a High Carboxyl Content from the Tunic of Marine Invertebrate Ciona intestinalis

Karnaouri

Jalvo

Moritz

et al. 2020

ACS Sustainable Chem. Eng.

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“…The molecular weight of the polysaccharide product was characterized by MALDI-TOF-MS. − As shown in Figure S3a (Supporting Information), the maximum DP of the polysaccharide product is 26. Obviously, a series of separated molecular ion peaks can be clearly observed, and the difference in molecular weight between two neighboring peaks is 162 Da, corresponding to one glucose unit.…”

Section: Resultsmentioning

confidence: 99%

Solid-Phase Synthesis of Polysaccharides from Unprotected Glucose Catalyzed by Hβ Zeolites

Dong

Xiao

Zhang

et al. 2022

ACS Sustainable Chem. Eng.

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Polysaccharide is a kind of biological macromolecule with a complex structure, which plays an irreplaceable role in the organism. Due to the complex purification process of natural polysaccharide, chemical synthesis is an important means to obtain it. In this work, a green and efficient solid-phase polymerization method was used to synthesize glucan polysaccharide from unprotected glucose in one step under mild solid-phase conditions, using Hβ zeolites as acidic catalyst. The final polysaccharide product was characterized by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with a conversion of 35.0% and a maximum degree of polymerization of 26. More importantly, it was speculated that the glycosylation of unprotected glucose catalyzed by Hβ zeolites conforms to the Fischer glycosylation mechanism. In addition, the results of methylation analysis and one-dimensional, twodimensional nuclear magnetic resonance (1D, 2D NMR) showed that the resulting polysaccharides were mainly linear, containing 10 different glycosidic linkages, and (1,6-Glcp), (1-Glcp), and (1,4-Glcp) were the most abundant linkage types among them.

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“…Since the 2,5-dihydroxybenzoic acid (DHB) matrix generates signals in the low molecular weight region of the spectrum, the optimal detection range for MALDI-TOF MS includes longer oligosaccharides (DP between 3 and 10). By combining the high sensitivity offered by ESI-MS with the efficiency of hydrophilic interaction chromatography to analyze polar compounds, De Oliveira Gorgulho Silva et al developed a UHPLC-ESI-MS method that can detect smaller oligosaccharides (DP 1-5) [64]. Additionally, this method is compatible with online mass-spectrometry detection.…”

Section: Uhplc-esi-msmentioning

confidence: 99%

Oxidative Power: Tools for Assessing LPMO Activity on Cellulose

2021

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Lytic polysaccharide monooxygenases (LPMOs) have sparked a lot of research regarding their fascinating mode-of-action. Particularly, their boosting effect on top of the well-known cellulolytic enzymes in lignocellulosic hydrolysis makes them industrially relevant targets. As more characteristics of LPMO and its key role have been elucidated, the need for fast and reliable methods to assess its activity have become clear. Several aspects such as its co-substrates, electron donors, inhibiting factors, and the inhomogeneity of lignocellulose had to be considered during experimental design and data interpretation, as they can impact and often hamper outcomes. This review provides an overview of the currently available methods to measure LPMO activity, including their potential and limitations, and it is illustrated with practical examples.

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

Abstract: 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.

Cited by 9 publications

References 25 publications

Lytic Polysaccharide Monooxygenase-Assisted Preparation of Oxidized-Cellulose Nanocrystals with a High Carboxyl Content from the Tunic of Marine Invertebrate Ciona intestinalis

Lytic Polysaccharide Monooxygenase-Assisted Preparation of Oxidized-Cellulose Nanocrystals with a High Carboxyl Content from the Tunic of Marine Invertebrate Ciona intestinalis

Solid-Phase Synthesis of Polysaccharides from Unprotected Glucose Catalyzed by Hβ Zeolites

Oxidative Power: Tools for Assessing LPMO Activity on Cellulose

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