【作者】 何峰；

【导师】 余龙江；

【作者基本信息】 华中科技大学 ， 生物化工， 2005， 硕士

【摘要】 链霉菌以产生抗生素而著名,而关于链霉菌产多糖的研究目前鲜见报道。本论文从数千株链霉菌中筛选到一株能产胞外多糖的菌株,并对该链霉菌多糖（streptomyces polysaccharide,SPS）进行了分离纯化与鉴定; 研究了该菌株产胞外多糖的发酵工艺及其抗菌、抗氧化与抗肿瘤的生物活性。主要内容包括: 1)多糖的分离纯化（1）对多糖进行分离纯化采用95%乙醇醇析从发酵液中分离出粗多糖,再用分段盐析法去处蛋白质,透析除去小分子物质得到多糖半纯品; 然后用Sephadex G-75 分离纯化。（2） 紫外光谱分析表明多糖样品不含蛋白质和核酸,硫酸-蒽酮法检测表明多糖样品纯度为96.2%。（3）红外光谱对多糖样品进行分析结果表明,多糖样品在4000 cm^-1～700 cm^-1具有明显的糖吸收峰; 单糖是以吡喃糖苷的形式存在,可能有甘露糖,不含α-糖苷键,而含β-糖苷键。（4）利用GC-MS 分析多糖的单糖组分进行分析结果表明:多糖是由甘露糖、葡萄糖、半乳糖三种单糖组成的,其组成比例约为2:1:1。（5）利用Sephadex G-150 凝胶柱层析法测得多糖相对分子质量约为68,000 D。（6）高碘酸氧化、Smith 降解对多糖结构分析表明,糖苷键的连接方式可能有（1→3） 和（1→6）。2)通过单因子试验和正交试验,确定了链霉菌的发酵培养基成分和最佳发酵条件发酵培养基成分为:葡萄糖3%,淀粉4%,豆饼粉4%,硫酸铵0.8%,硫酸镁0.1%,磷酸氢二钾0.4%,氯化钠1.0%,碳酸钙0.03%,初始pH7.4～7.6。发酵条件为:种子的种龄为48h,接种量为10%,培养温度为30℃,发酵时间为168h,摇床转速为200rpm 以上,此时,多糖的产量可达25.5 g/L。3)采用药理学的试验方法,研究多糖的抗菌、抗氧化、抗肿瘤等生物活性。结果表明,该多糖具有较广的抗菌谱、抗菌能力强; 对邻苯三酚自氧化过程中产生的超氧阴离子有清除作用; 对过氧化氢诱导的红细胞溶血有一定的抑制作用,能抑制CCl₄引发的小鼠肝脂质过氧化; 在一定的浓度范围内能抑制S 180 在小鼠体内生长。更多还原

【Abstract】 Streptomyce is famous for its capability of producing many kinds of antibiotics, Few report related to SPS （Streptomyces Polysaccharide） has been found so far. In this study, the exopolysaccharide producing strain-Stretomyces was selected from thousands of strains, and the progresss of isolation, purifyication of SPS from fermentation broth was studied. The structure of the purifyied SPS was analysed; the culture medium and conditions of SPSfermentation was optimized; and the biological activity of SPS on antibacterium, anti-oxidant and antitumor was discussed. The main content were as following: 1) Isolation, purification and structure of SPS （1） The crude SPS was precipitated with volume 95% ethanol in method of ethanol precipitation and then the protein was removed by Savage method and salted out by （NH4）2SO4, respectively, followed by being dialysed by dialysis tubing. The purified SPS was purified from this product by column chromatography on Sephadex G-75. （2） From the ultraviolet spectrum of SPS, the sample of SPS did not have protein and nucleic acid,the purity of the sample was 96.2% by Sulfuric acid-anthrone coloremetric methd. （3） SPS was analyzed for infrared spectra by FT-IR infrared spectrometer scanning from 4800to 700cm-1.The results showed that SPS had typical infrared spectra of polysaccharide, and the monosaccharides was linked with β-glycoside bond. （4） The results of GC-MS showed that the sample was made of mannose, glucose, galactose, which was 2:1:1 by proportion. （5） Its molecular weight was 68000dal determined by Sephadex G-150 chromatography . （6） periodate oxidation, Smith degeradation showed that SPS has （1 →3） and （1→6）-linked glycoside bond. 2) The optimal culture medium selected by single factor experiment was as following :Glucose and amylum were the optimal carbon source and the fitting concentration was 3% and 4% respectively; soybean meal and （NH₄）₂SO₄ were the optimal nitrogen source and the fitting concentration were 4%and 0.8% respectively; salts such as MgSO4（0.1%）, K2HPO4（ 0.4%）,NaCl（1.0%） could promote the SPS yield; CaCO3 （0.03%） is necessary to keep pH in range of 7.2⁷.6; the initial pH was 7.4⁷.6. The fermentation condition obtained by orthogonal experiment was as following: the inoculum age and inoculum size were 48h and 10%（v/v） respectively; the optimum charge quantity was 50ml culture medium in 500ml flask, and the optimum temperature 30 ℃; and shaker rotation speed 200rpm; fermentation time was 168h. The average production of SPS could reach 25.5g/L. 3) The biological activity on antibacterium,anti-oxidant and antitumor of SPS was studied. The results showed that SPS could inhibit the growth of bacterium including Cercospora beticola, Magnaporthe grisea, Escherichia coli, Bacillus Subtilis, Pseudomonas aeruginosa and Staphylococcus aureus; And SPS could scavenge O2·-generated by the self-oxidation of pyrogallic acid, alleviate H2O2-induced hemolysis of RBC, inhibit the microsomal lipid peroxidation in the damaged liver homogenate of mice induced by CCl4; Further more, SPS had obvious inhibition on S180 in the body .更多还原