| Popis: |
Fucoidans are heterologous sulfated fucose-containing polysaccharides primarily found in the cell wall of brown seaweeds (Bilan and Usov, 2008; Ale and Meyer, 2013). Fucoidans were reported to possess various biological activities, including anti-oxidatant, anti-tumor, anti-coagulation, and anti-inflammatory (Ale et al., 2011; Li et al., 2008; Lim et al., 2014). Unfortunately, the complicated structures and too high molecular weight of fucoidans is a disadvantage for their applications in food, cosmetic, and pharmaceutical industry (Chollet et al., 2016; Flórez-Fernández et al., 2018). The modification of native fucoidans via enzymatic treatments able to specifically degrade fucoidan to produce chemical homogenous and well-defined bioactive oligosaccharides would be a valuable process.This thesis describes the discovery, stabilization, and characterization of four glycosyl hydrolase 107 (GH107) endo-fucoidanases all from marine bacteria, including Fhf2 from Formosa haliotis, Psf1 from Pseudoalteromonas sp. S3178, and Mef3 and Mef4 from Muricauda eckloniae. The recombinant fucoidanase enzymes were expressed and purified. Various fucoidan substrates were initially tested to determine the enzyme activity and specificity.A truncation strategy was used where parts of the C-terminal was removed from the Fhf2 sequence, since this protein was degraded during the expression. The substrate specificity of the truncated fucoidanase Fhf2Δ484 containing only the catalytic D1 domain characteristic of GH107 family members was investigated and the enzyme was biochemically characterized. The enzymatic reaction of Fhf2Δ484 on fucoidan from Fucus evanescens was carried out under the determined optimal conditions for production of oligosaccharides. Analysis of the structure of these hydrolyzed products using Nuclear Magnetic Resonance Spectroscopy (NMR) indicated that this enzyme is an α(1→4)- specific endo-fucoidanase and in addition it releases oligosaccharides with a substantial amount of C2 sulfated and C2, C4 disulfated fucose residues.The Psf1 gene found in the genomic sequence of the marine bacteria Pseudoalteromonas sp. S3178 isolated from a shrimp caught in the Southern Ocean in 2007 during the Galathea 3 global expedition was successfully cloned, expressed, and purified. Psf1 catalyzes the hydrolysis of α(1→3)-linked fucose residues containing fucoidan from Saccharina latissima and Undaria pinnatifida, and galactofucan type fucoidan from Sargassum oligocystum. The temperature optimum for Psf1 is 10-30 °C and interestingly, Psf1 shows activity down to even 1 °C and is therefore the first psychrophilic endofucoidanase described to date. Psf1 could be an interesting candidate for biotechnological applications that require low temperature condition.In the last phase of this PhD project, I focused on the C-terminal deletion strategy for stabilization and identification of the functional domains in the fucoidanases Mef3 and Mef4, as well as the characterization and determination of the substrate specificity and biochemical properties of the truncated enzymes. A galactose binding-like domain predicted in Mef3 and Mef4, might play an important role in fucoidanase function. Mef3 and Mef4 are likely endo-α(1→3)-specific fucoidanases. Besides sharing some similar characteristics, they also have distinct characteristics such as optimal temperature, pH, the influence of metal ions, and thermostability properties.The achievement of this PhD project reflect the diversity of biological characterizations and substrate specificity of fucoidanases from the GH107 family and extends the general knowledge of fucoidanases, which could be used to prepare chemically homogenous and well-defined bioactive oligosaccharides, a key requisite for application in the pharmaceutical industry. |
