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A new FTIR assay for quantitative measurement of endo-fucoidanase activity.
- Source :
-
Enzyme & Microbial Technology . Aug2022, Vol. 158, pN.PAG-N.PAG. 1p. - Publication Year :
- 2022
-
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 Fucus evanescens and Fucus vesiculosus , 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−1 are in agreement 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, U f , 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 min) on 20 g/L pure fucoidan from F. evanescens at 42 °C, pH 7.4, 100 mM NaCl and 10 mM CaCl 2. This new quantitative endo-fucoidanase assay can pave the way for better kinetic characterizations as well as novel explorations of endo-fucoidanases. • Quantitative endo-fucoidanase activity determination by FTIR and PARAFAC analysis. • Spectral evolution profiling of endo-fucoidanase action on fucoidans in real time. • First endo-fucoidanase activity unit established. • Specific activity comparison of three microbial endo.fucoidanases. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 01410229
- Volume :
- 158
- Database :
- Academic Search Index
- Journal :
- Enzyme & Microbial Technology
- Publication Type :
- Academic Journal
- Accession number :
- 156733666
- Full Text :
- https://doi.org/10.1016/j.enzmictec.2022.110035