产耐高温纤维素酶细菌的筛选及酶学性质研究

【作者】 贺芸；

【导师】 李大力； 杨树林；

【作者基本信息】 南京理工大学 ， 生物化工， 2004， 硕士

【摘要】 纤维素酶的研究对利用具有巨大潜力的可再生资源——纤维素有重要意义。实验筛选出了产耐高温纤维素酶的细菌菌株，获得了有良好耐热性的纤维素酶，并对筛得的菌株的生理生化特性及所产酶的酶学特性进行了研究。实验利用柴草进行高温堆肥，采样后用半限制性培养基筛选，筛选过程中，用3，5—二硝基水杨酸比色法测量所产纤维素酶活，并通过测不含菌体的发酵液酶活，来判断所产是否为胞外酶，结果筛得一株能产胞外耐高温纤维素酶的菌株。通过菌体观察实验、菌体运动性实验、V-P实验、甲基红实验、淀粉水解实验、过氧化氢酶生成测定实验、碳源利用实验等，对筛得的菌株进行分类鉴定，初步判定其属于嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)。利用盐析、凝胶过滤层析等方法对纤维素酶进行分离纯化，用SDS-聚丙烯酰胺凝胶电泳检验，电泳结果中出现多条带，而Native—聚丙烯酰胺凝胶电泳结果只显示一条带，初步判定其为多亚基结构，至少有八个亚基。论文研究了该菌所产纤维素酶的最适温度、pH值、稳定性、酶活影响因子及Km值，确定其最适温度在60℃～70℃之间，最适pH值为7.0。用薄层层析的方法，将酶促反应产物与标准样进行比较，鉴定酶促反应产物，结果显示产物主要为葡萄糖。更多还原

【Abstract】 A thermophil bacterial strain was isolated, in order to gain thermostable cellulase that can convert cellulose, the most abundant renewable source in the world, into glucose and soluble sugars. Physiological and biochemical characteristics of the strain and characteristics of the cellulase were studied. The diluted samples selected from hot compost were cultured on the hemi-restrictive culture medium. The cellulase activities of different strains were measured and compared by the method of DNS. Besides, the cellulase activity of the ferment liquid with bacterium and that of clear liquid without bacterium were compared to determine if the cellulase was extracellular. Physiological and biochemical characteristics of the strain were studied. The results of tests show that the target strain is Bacillus stearothermophilus. Cellulase was purified from ferment liquid by salt out and gel-filteration. The purified cellulase was resolved into at least 8 subunits by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. But, PAGE without SDS didn’t resolve it. The characteristics of the cellulase were studied. These characteristics include optimum temperature, optimum pH, and effect of various factors and the value of Km. The results show that the optimum temperature of cellulase reaction process rangs from 60℃~70℃ and the optimum pH is 7.0. The cellulase product and standard samples were compared by thin layer chromatography (TLC) to determine what the product was. The results show that the cellulase product is mainly glucose.更多还原