Studies On Breeding Of Xanthine Oxidase Producing Strains And Cloning Of Xanthine Oxidase Gene

Xanthine oxidase (XOD, EC1.2.3.22) is a key enzyme which plays an important partin purine metabolic processes. It has been used for the production of immune antibodiesand anti-tumor clinical agents, as well as diagnostic reagents for detection of diseases. So,the development and researches on XOD may have very important theoretical researchsignificance and broad application outlooks. The XOD used nowadays is mainly extractedfrom milk. Because there are lack of sources, high production costs, and its content andquality under the influence of the different regions and different kinds of cows, which leadto significant limitations in its applications.This research mainly focuses on the following respects: the selecting and breeding ofthe XOD-producing microorganism, and cloning the gene encoding XOD.1. Using xanthine as sole carbon, nitrogen, and energy source，two xanthine oxidaseproducers were isolated from the soil. Meanwhile, we bought the strain ATCC21606ascontrol, referring to others’ reports. Comparing the characters of XOD from these threestrains, we decided to use the ATCC21606as the original strain.2. ATCC21606genomic DNA library was constructed for screening theXOD-encoding gene by the sign of transparent hydrolysis circle. We used DH5α as therecipient strain. By overcoming the difficulty of large fragment gene connection with itscarrier, and the low transformation efficiency, we found a certain percentage oftransformants secret alkaline substance when they carried the exogenous DNA fragments.The alkaline substance dissolved xanthine around the strain, and false positive clonesappeared. This increased the difficulty of the ATCC21606genomic DNA libraryconstruction for screening the XOD-encoding gene.3. Parts of XOD A and XOD B from ATCC21606, encoding the heterodimericmolybdo-iron-sulfur-protein xanthine oxidase, were cloned and sequenced. Because thehost genome has a high level of GC, we probed the condition of PCR, using the genomicDNA with a high level of GC as a template. We amplificated two fragments successfullyby adding8%dimethyl sulfoxide in PCR system, refining the cycle parameters, and usingthe GC Buffer. The preliminary analysis and alignment results revealed that two fragmentsbelong to XOD encoding sequences.4. A fusant was obtained with high xanthine oxidase activity by genome shufflingmethod. Mutants with high XOD activity were obtained by treating ATCC21606withphysical and chemical mutagenesis. Treating the mutants by cell fusion to get a fusant with higher XOD activity. The yield of xanthine oxidase in the fusant was50.1%higher thanthat of the original strain, and it also has good genetic stability.