Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark; NhaTrang Institute of Technology Research and Application, Vietnam Academy of Science and Technology, 02 Hung Vuong Street, Nhatrang 650000, Vietnam.
Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
Enzyme Microb Technol. 2022 Aug;158:110035. doi: 10.1016/j.enzmictec.2022.110035. Epub 2022 Mar 26.
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, but the profiles differed depending on the substrate and the enzyme used. Spectral changes within 1220-1260 cm 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, 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. This new quantitative endo-fucoidanase assay can pave the way for better kinetic characterizations as well as novel explorations of endo-fucoidanases.
内切岩藻聚糖酶，包括 EC 3.2.1.211 内切-α-1,3-L-岩藻聚糖酶和 EC 3.2.1.212 内切-α-1,4-L-岩藻聚糖酶活性，催化岩藻聚糖的解聚-一组主要存在于褐藻（褐藻）中的生物活性、硫酸化岩藻糖基多糖。内切岩藻聚糖酶活性的定量评估对于表征内切岩藻聚糖酶动力学以及比较不同内切岩藻聚糖酶对不同类型岩藻聚糖的作用至关重要。然而，目前最先进的内切岩藻聚糖酶测定方法是基于基于碳水化合物-聚丙烯酰胺凝胶电泳的定性评估。在这里，我们报告了一种新的基于实时光谱演变的定量内切岩藻聚糖酶测定法，该方法基于傅里叶变换红外光谱（FTIR）与平行因子分析（PARAFAC）结合使用，在岩藻聚糖酶作用过程中监测底物和产物的变化。FTIR-PARAFAC 测定法通过监测三种不同微生物内切岩藻聚糖酶 FcnAΔ229、FFA2 和 Fhf1Δ470 在两种不同岩藻聚糖底物上的反应进展来验证。两种底物均从褐藻 Fucus evanescens 和 Fucus vesiculosus 中纯化得到。演化分析表明，在酶解聚过程中，岩藻聚糖发生的最强光谱变化发生在 1220-1260cm 的光谱范围内，但谱图因底物和使用的酶而异。1220-1260cm 范围内的光谱变化与诱导硫酸化岩藻糖基中中红外吸收特征变化的酶解聚一致，因为硫酸酯键和 C-O 伸缩振动在此光谱区域吸收。基于获得的数据，我们还为内切岩藻聚糖酶引入了一个活性单位：一个内切岩藻聚糖酶单位，U，是能够在 42°C、pH7.4、100mMNaCl 和 10mM CaCl 下，在 20g/L 纯 F.evanescens 岩藻聚糖上反应 498s（8.3min）时，使傅里叶变换红外光谱-平行因子分析（FTIR-PARAFAC）得分变化 0.01 的酶量。这种新的定量内切岩藻聚糖酶测定法可以为更好的动力学特性以及内切岩藻聚糖酶的新探索铺平道路。
