A new FTIR assay for quantitative measurement of endo-fucoidanase activity

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 CaCl₂. This new quantitative endo-fucoidanase assay can pave the way for better kinetic characterizations as well as novel explorations of endo-fucoidanases.