【作者】 乔环宇；

【导师】 赵晓瑜；

【作者基本信息】 河北大学 ， 植物学， 2004， 硕士

【摘要】 胆红素氧化酶（BOD）是一种催化胆红素生成胆绿素和其它物质的一种酶。一些胆红素氧化酶已从疣孢漆斑菌和微紫青霉等生物体中被分离纯化出来。 本研究对漆斑菌属的疣孢漆斑菌和三种露湿漆斑菌进行培养，培养过程中发现，加入26×10^-4％的诱导剂（CuSO₄）可很大程度地提高这四种菌BOD的活性。我们从漆斑菌属的四种菌中分离纯化出了胆红素氧化酶，纯化过程中阳离子交换树脂代替活性碳、低温、甘油都有助于酶活的保持。 与疣孢漆斑菌BOD相比较，露湿漆斑菌3.1967BOD的分子量、胆红素-聚丙烯酰胺凝胶电泳和聚丙烯酰胺凝胶电泳条带、糖染结果、等电点、纯化条件等都不相同。这说明露湿漆斑菌3.1967BOD是不同于疣孢漆斑菌BOD的一种酶。在BOD的鉴定过程中胆红素平板、胆红素-聚丙烯酰胺凝胶电泳、糖染与银染结合法是未被报导过的新方法，这些方法的应用对于BOD的鉴定很有帮助。 本实验对BOD的测定方法也有所改进，以露湿漆斑菌3.1967BOD各种性质作根据，在酶的最适反应条件下（37℃、pH8.1）进行反应，胆红素有最大吸收峰的波长440nm下测定OD值。以抗坏血酸和EDTA作为反应终止剂将反应终止在特定的时间，使酶活测定不用恒温分光光度计，而只用普通的分光光度计就可测定，这也将有助于胆红素酶法测定的推广和应用。 根据Genebank中疣孢漆斑菌胆红素氧化酶（MV BOD）基因的编码序列设计引物，利用PCR从疣孢漆斑菌的染色体DNA中扩增得到1937bp的基因，将其克隆到T载体中，构建成重组质粒，并对其测序，通过测序验证了本实验所合成引物的正确性，为下一步的基因工程操作打下了基础。更多还原

【Abstract】 Bilirubin oxidases (BOD) are enzymes catalyzing the oxidation of bilirubin to biliverdin and other substrates. Some kinds of bilirubin oxidases have been purified from Myrothecium uerrucuria and Peniciilium jan thinellum.In this study, we cultured Myrothecium verrucaria and three kinds of Myrothecium roridum. During the culture, we observed that adding 26 X 10-4% CuS04 into the medium can greatly promote the enzymatic activity of BOD. We have purified BOD from all of them and some factors, such as low temperature, glycerin and so on, are helpful to remain the enzymatic activity.Comparing with BOD from Myrothecium verrucaria(MV BOD), BOD from Myrothecium roridum (MR BOD) shows difference in the molecular weight, the result of electrophoresis, isoelectric point and the condition of purification. It can be concluded that.BOD from Myrothecium roridum is a new type of BOD which is different from MV BOD. Agarose plate with bilirubin, Bilirubin-PAGE and Double Staining of schiff agent and sliver, are new methods that will be used in identification of BOD activities.Based on the properties of MR BOD, the measure method is developed. The ordinary methods of measuring the BOD’ s activity have to be performed under constant temperature. We improved the method by using Vitamin C and EDTA as a terminating agent, which made the method more convenient to carryout.According to the encoding sequence of the MV BOD gene in the Genebank,the primers is designed and the 1937bp DNA fragment is obtained by PCR amplification. Then, this fragment is cloned into the plasmid pGEM-T-easy vector and sequenced. The result shows that the primers that I designedAbstractare corrected.更多还原