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Highlights • Laccase oxidation reaction can be monitored using FTIR spectral profiling. • FTIR coupled to PARAFAC can be used to assess and quantify laccase activity. • Laccases can be distinguished on the basis of their origin by assessing the oxidation reaction pattern. • FTIR combined with multiway analysis can create a reference system for comparison of laccase action across large data sets. Abstract Laccases (EC 1.10.3.2) are enzymes known for their ability to catalyze the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Laccase activity is commonly determined by monitoring spectrophotometric changes (absorbance) of the product or substrate during the enzymatic reaction. Fourier Transform Infrared Spectroscopy (FTIR) is a fast and versatile technique where spectral evolution profiling, i.e. assessment of the spectral changes of both substrate and products during enzymatic conversion in real time, can be used to assess enzymatic activity when combined with multivariate data analysis. We employed FTIR to monitor enzymatic oxidation of monolignols (sinapyl, coniferyl and p -coumaryl alcohol), sinapic acid, and sinapic aldehyde by four different laccases: three fungal laccases from Trametes versicolor , Trametes villosa and Ganoderma lucidum , respectively, and one bacterial laccase from Meiothermus ruber. By coupling the FTIR measurements with Parallel Factor Analysis (PARAFAC) we established a quantitative assay for assessing laccase activity. By combining PARAFAC modelling with Principal Component Analysis we show the usefulness of this technology as a multivariate tool able to compare and distinguish different laccase reaction patterns. We also demonstrate how the FTIR approach can be used to create a reference system for laccase activity comparison based on a relatively low number of measurements. Such a reference system has potential to function as a high-throughput method for comparing reaction pattern similarities and differences between laccases and hereby identify new and interesting enzyme candidates in large sampling pools. [ABSTRACT FROM AUTHOR]

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]