【作者】 孙勇；

【导师】 王忠彦；

【作者基本信息】 四川大学 ， 微生物学， 2005， 硕士

【摘要】 本文利用糖胺聚糖可与氯化十六烷基吡啶（cetylpyridinium chloride,CPC）反应生成白色混浊的性质,从不同来源的500株菌种中筛选的到4株菌种,再通过琼脂糖凝胶电泳,筛选得到一株糖胺聚糖产生菌,编号为GS36。其发酵产物纯化后测得的红外光谱图与硫酸软骨素标准红外光谱图比较基本一致,可初步确定产物为硫酸软骨素。经形态观察和生理生化实验,初步鉴定该菌株为坚实芽孢杆菌（B.firmus）。 通过单因素实验和正交实验,进一步对该菌株的发酵条件进行了优化:以1.5%葡萄糖为碳源,0.3%牛肉膏为氮源,加入0.1%Na₂SO₄,0.1%K₂HPO₄,0.02%MgSO₄,培养基起始pH=7,30℃下摇瓶培养24h,发酵液中产物浓度最高为0.133mg/ml。 经过质粒提取和琼脂糖凝胶电泳,检测到有质粒存在,经过质粒消除,该菌发酵液中的产物浓度大幅度降低,推测该菌株调节酶活的基因可能存在于质粒上。 菌株活化后接种至发酵培养基培养24h后,对胞内胞外产物更多还原

【Abstract】 Utilize glycosaminoglycan’s character of producing white turbidity through reacting with quaternary ammonium salt, for example, cetylpyridinium chloride （CPC）, to obtain 4 screened strains from 500 ones of various sources. A glycosaminoglycan product strain GS36 is obtained by agarose gel electrophoresis. The infra-red spectrum tested after the purification of the ferment product is identical with the standard infra-red spectrum of the chondroitin sulfate. Therefore, the product is proved to be chondroitin sulfate. Through the observance of its shape and the physiological and biochemical test, it is identified as B.firmus.Through the single factor test and the orthogonal test, the ferment condition of the strain is further optimized: take 1.5% glucose asthe carbon source, 0.3% beef extract as the nitrogen source, add 0.1% Na₂SO₄, 0.1% K₂HPO₄ and 0.02% MgSO₄, with culture medium starting with PH7. Cultivated for 24h under 30℃ through shaking the bottle, the concentration of the ferment product is 0.133mg/ml.Through the extraction and agarose gel electrophoresis, plasmid is found. After elimination of it, the concentration of the ferment product is largely decreased. Thus it is presumed that the gene which regulates the vitality of enzyme exists in the plasmid.Inoculate the activated strain to the ferment culture medium,cultivating for 24h. Compare intracellular and extra cellular concentration of the product. It is concluded that the synthetic product of the strain mainly exists extracellularly.更多还原