Preparation, Isolation And Structure Elucidation Of Alginate Oligomers Degraded By The Alginate Lyase

This thesis mainly involved the producing technics and preparation of alginate oligomers degraded by an alginate lyase, which was produced by a stain Vibrio 510 selected from algae, as markers. Another aim of preparation is enrichment of library of marine oligosaccharides. At the same time isolation purification and structure confirming of these blocks is the first stage of determination of action pattern of the lyase. The alginate lyase has higher specific activity of 22.5unit/mL. Analysis of the degree of degradation by UV scan, HPGPC and PAGE, analysis of samples' molecular and percent of every fraction by HPGPC, in which series TSK G3000PWxL and TSK G2500PWxL was applied, are the foundation of producing technics of alginate oligomers. Isolation and purification of alginate oligomers: Gel filter chromatography (Bio-Gel P-4, P-6) and anion exchange chromatography(Q-sepharose Fast. Flow) were applied, in which fast performance liquid chromatography (FPLC) was applied and there are good results. Four blocks (dp: 2-5) were got from the end products of enzyme hydrolysis. Each of the four blocks showed one peak on perfusion chromatography with HQ20-poros with the purity at least 85%-97%. And the molecular were confirmed by ESI-MS. The structure of the block of dp2 was confirmed by 1H-NMR, 13C-NMR, 1H-13C HQMC and 1H-1H COSY. But the block of dp3 showed two or three bands in fluorophore-assisted carbohydrate electrophoresis (FACE). And both other two blocks show not only one peak in HPCE. It's the infections of isomers confirmed by NMR. This will be one of the most important information for determination of action pattern of the alginate lyase.