Isolation And Prufication Of Heparosan

Heparosan is a precursor substance produced in the process of catalytic synthesis of heparin by the vertebrates. It can synthetic heparin though a series process of sulfated and isomerization. In this dissertation, the extraction, separation, purifition, detection and structural identification of heparosan were systematically studied. The main results are shown as following:1. Heparosan extracted with organic solvent precipitation method looked like pale yellow powder, and its uronic acid content was23.9%. Peroxide hydrogen oxidation method and Sevage method were carried out to remove pigments and protein before the purification of crude polysaccharides. The optimum conditions of peroxide hydrogen oxidation method and Sevage method were obtained with the help of orthogonal tests. In the peroxide hydrogen oxidation experiment, the concentration of hydrogen peroxide in the polysaccharides solution was1.5%, the reaction temperature and time were25℃and3hours, respectively. The decolorization rare of the crude polysaccharides was67.35%. In the Sevage experiments, the ratio of chloroform and n-buty alcohol was 4:1(V/V), the ratio of sample and reagent is1:1(V/V), the ectracted time is10minutes, and extracte for3times.The deproteinization rate of the crude polysaccharides was76.59%.2. The effects of ethanol precipitation method on the purification of polysaccharide were studied. Results showed that, the uronic acid content of heparosan was44.5%(W/W) and the yield of polysaccharide was43.50%(W/W) under the condition that the concentration of heparosan solution, Nacl solution and ethanol solution were14%(V/V),3%(W/V),70%(V/V), respectively, and repeated4times.3. The effects of chitosan precipitation method on the purification of polysaccharide were studied. The recovery of precipitated polysaccharide was94%(W/W) under the condition of the amount of chitosan was4.0mg. We obtained the purified polysaccharide from precipitation, and its uronic acid content was45.46%, the yield was40.36%.4. The effects of DEAE Sepharose fast flow ion exchange method on the purification of polysaccharide were studied. We obtained the purified polysaccharide using this method, and its uronic acid content was49.35%, the yield was38.52%.5. The uronic acid content, purity and molecular weight, the physical and chemical properties of heparosan polysaccharides were detected and identified. The optimum conditions of Sulfuric acid-Carbazole method was obtained with the help of orthogonal tests. In the Sulfuric acid-Carbazole experiment, hydrolysis temperature was25℃, the dosage of borax-sulfuric acid and carbazole-ethanol were4mL and0.15mL, respectively. And the chromogenic time was10minutes. This method proved reliable and accurate through reproducibility experiment, stability experiment and the recovery experiment. The purity and molecular weight of polysaccharides were identified by HPGPC. Examination results showed that polysaccharides puritied by ion exchange method had the highest purity(nearly100%), and its molecular weight was4.5x104Da. The physical and chemical properties of polysaccharides were analyzed, and results were according to the relevant characteristics of the polysaccharide.6. The structure of heparosan was identified with the help of UV、 IR、NMR、HPLC and LC-MS. The results showed that the structure of heparosan was [�'4)β-D-glucuronic acid (G1cA)(1�'4) N-acetyl-a-D-glucosamine (G1cNAc)(1�']n。...