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| Naji, M. |
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| Motta, Antonella |
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| Aletan, Dirar |
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| Mohamed, Tarek |
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| Ertürk, Emre |
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| Taccardi, Nicola |
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| Kononenko, Denys |
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| Petrov, R. H. | Madrid |
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| Alshaaer, Mazen | Brussels |
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| Bih, L. |
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| Casati, R. |
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| Muller, Hermance |
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| Kočí, Jan | Prague |
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| Šuljagić, Marija |
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| Kalteremidou, Kalliopi-Artemi | Brussels |
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| Azam, Siraj |
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| Ospanova, Alyiya |
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| Blanpain, Bart |
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| Ali, M. A. |
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| Popa, V. |
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| Rančić, M. |
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| Ollier, Nadège |
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| Azevedo, Nuno Monteiro |
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| Landes, Michael |
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| Rignanese, Gian-Marco |
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Nguyen, Thuan Thi
in Cooperation with on an Cooperation-Score of 37%
Topics
Publications (4/4 displayed)
- 2023Structural Characterization and Cytotoxic Activity Evaluation of Ulvan Polysaccharides Extracted from the Green Algae Ulva papenfussiicitations
- 2023Structural and functional characterization of the novel endo-α(1,4)-fucoidanase Mef1 from the marine bacterium Muricauda eckloniaecitations
- 2022The Endo-α(1,3)-Fucoidanase Mef2 Releases Uniquely Branched Oligosaccharides from Saccharina latissima Fucoidanscitations
- 2022A new FTIR assay for quantitative measurement of endo-fucoidanase activitycitations
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article
A new FTIR assay for quantitative measurement of endo-fucoidanase activity
Abstract
Endo-fucoidanases, including EC 3.2.1.211 endo-α-1,3-L-fucanase and EC 3.2.1.212 endo-α-1,4-L-fucanase activities, catalyze depolymerization of fucoidans – a group of bioactive, sulfated fucosyl-polysaccharides found primarily in brown macroalgae (brown seaweeds). Quantitative assessment of endo-fucoidanase activity is critical for characterizing endo-fucoidanase kinetics and for comparing the action of different endo-fucoidanases on different types of fucoidans. However, the current state-of-the-art endo-fucoidanase assay consists of a qualitative assessment based on Carbohydrate–Polyacrylamide Gel Electrophoresis. Here, we report a new quantitative endo-fucoidanase assay based on real time spectral evolution profiling of changes in substrate and product during endo-fucoidanase action using Fourier Transform InfraRed spectroscopy (FTIR) combined with Parallel Factor Analysis (PARAFAC). The FTIR-PARAFAC assay was validated by monitoring the reaction progress of three different microbial endo-fucoidanase enzymes, FcnAΔ229, FFA2 and Fhf1Δ470, on two different fucoidan substrates. The substrates were purified from the brown macroalgae <i>Fucus evanescens</i> and <i>Fucus vesiculosus</i>, respectively. The evolution profiling showed that the strongest spectral change of the fucoidans during enzymatic depolymerization occurred in the spectral range 1220–1260 cm−1, but the profiles differed depending on the substrate and the enzyme used. Spectral changes within 1220–1260 cm<sup>−1</sup> agree with the enzymatic depolymerization inducing signature changes in the mid-infrared absorption of sulfated fucosyls as sulfate ester bonds and C-O stretching vibrations absorb in this spectral region. Based on the data obtained, we also introduce an activity unit for endo-fucoidanases: One endo-fucoidanase Unit, Uf, is the amount of enzyme able to catalyze a change in the FTIR-PARAFAC score by 0.01 during 498 s of reaction (8.3 minutes) on 20 g/L pure fucoidan from <i>F. evanescens</i> at 42 °C, pH 7.4, 100 mM NaCl and 10 mM CaCl<sub>2</sub>. This new quantitative endo-fucoidanase assay can pave the way for better kinetic characterizations as well as novel explorations of endo-fucoidanases.