Kinetics and Technology Functionality of Microbial Fucoidanase

Fucoidans are a structurally highly diverse class of polysaccharides consisting of several monosaccharides which vary in the degree of sulfation. Especially fucoidans are of high scientific interest as they role as bioactive polymers and material sources for food and cosmetic industry. The PhD thesis report was on kinetics and technological functionality of microbial fucoidanases.

In this study, the enzyme kinetics of endo-fucoidanases is assessed based on spectral evolution profiling of changes of both substrate and products during enzymatic conversion in real time by using Fourier Transform InfraRed spectroscopy (FTIR) combined with Parallel Factor Analysis (PARAFAC). By the FTIR-PARAFAC assay, we established a new quantitative assay for assessing three different microbial endo-fucoidanase actitivty, which FcnAΔ229, FFA2 and Fhf1Δ470, on two different fucoidan substrates (Fucus evanescens and Fucus vesiculosus). We also demonstrate 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 F. evanescens at 42 ⁰C, pH 7.4, 100 mM NaCl and 10 mM CaCl₂.

Furthermore, the functional characterization was reported of the novel endo-fucoidanase enzymes Mef1 and Mef2 from the marine bacteria Muricauda eckloniae isolated from the rhizosphere of Ecklonia kurome brown alga.

For the first endo-fucoidanase Mef1, I determined the protein structure and furthermore found the Mef1 cleaves the 1-4 linkage between 2 fucose residues sulfated on C2 in this substrate: (→3)-α-L-Fucp2S-(1→4)-α-L-Fucp2S-(1→)n and the optimal operating conditions were found to be pH 8, 37⁰C, 50 mM NaCl and 10 mM CaCl₂.

For the second endo-fucoidanase, Mef2, I combined biochemical characterization and structure determination of hydrolysis products and elucidated a new structure of fucoidan from the brown seaweed Saccharina latissima. Using Carbohydrate-PolyAcrylamide Gel Electrophoresis (C-PAGE) and Nuclear Magnetic Resonance (NMR) analyses, we showed that the Mef2 endo-fucoidanase is the first endo 1,3 fucoidanase of the glycoside hydrolase family 107 (GH107) family to be specifically used on fucoidans from S. latissima, a sulfated polysaccharide with extremely complex structure. An octasaccharide from S. latissima, the main hydrolysis product of the Mef2 endo-1,3-fucoidanase was structurally identified with a novel side chain. Kinetic analysis of this enzyme also showed that Mef2 cleaves 1,3-fucanase bonds in fucoidan from S. latissima more easily than in the fucoidan from the other brown seawed F. evanescens.

The research in the thesis dissertation provided molecular insights into the kinetic, function and structure of endo-1,4-fucoidanase and endo-1,3-fucoidanase. These findings will contribute to a greater understanding of the utilization of fucoidan by marine bacteria.