Purification, Properties Analysis And Gene Cloning Of Bilirubin Oxidase From Myrothecium

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.