苯酚降解细菌GXP04及其苯酚羟化酶基因的研究

【作者】 吴培诚；

【导师】 武波；

【作者基本信息】 广西大学 ， 微生物学， 2002， 硕士

【摘要】 苯酚是造纸、炼焦、炼油、塑料、农药、医药合成等行业生产的原料或中间体。随着经济的发展，未经处理的含酚废水对人类的生存环境已经造成了严重的威胁。利用微生物降解的方法处理含酚废水是一种经济有效且无二次污染的方法。 本研究利用传统微生物学的方法，从南宁市郊被含酚废水污染的土样中筛选到一株高效的苯酚降解细菌GXP04，能够在以苯酚为唯一碳源和能源的无机培养基上生长，通过形态观察及16SrRNA编码序列同源性比较分析，我们初步鉴定该菌株为琼氏不动杆菌。通过对GXP04菌株在不同温度、pH值，以及培养基中不同苯酚浓度条件下的生长和降解苯酚的情况，我们初步确定了GXP04的最适生长和降解苯酚的温度为34℃，最适pH值为7.0，苯酚最高的耐受浓度为800mg／L． 根据苯酚羟化酶所共有的保守序列设计合成了一对引物，以GXP04总DNA为模板，通过PCR反应，扩增出683bp的同源片段。对该DNA序列测序，并通过序列分析表明，该片段与已报道的醋酸不动杆菌苯酚羟化酶有84％的同源性。以此DNA片段为探针，通过Southern blot的方法，从用不同酶切的GXP04总DNA中获得阳性印迹，确认其苯酚羟化酶基因的位置，进而构建了GXP04苯酚羟化酶的部分文库。通过菌落原位杂交，找到一个含有苯酚羟化酶基因的阳性克隆，为进一步研究苯酚羟化酶和构建高效苯酚降解基因工程菌打下基础。更多还原

【Abstract】 Phenol is the important raw material or midst substance of paper making- coking plant- oil refining- plastic- pesticide and medicine composing etc. More and more phenol wastewater without treatment is threatening the environment heavily with the development of economy. It is economic and effective that using the method of microorganism biodegradation to treat with phenol wastewater. And it has not second pollution too.A strain that can grow with phenol as the sole source of carbon and energy, GXP04, was isolated from the soil samples where was polluted by wastewater of chemical plant at Nanning suburban. On the basis of observing of modality and the analysis of the 16SrDNA sequence, GXP04 was identified as Acinetobacter junii primarily. About 600bp fragment which homologous to phenol hydroxylase gene of Acinetobacter calcoaceticus , was amplified by PCR, using conservative sequence of PH gene as primers. The fragment was sequenced and indicated that it has 84% homology to the PH gene of A.calcoaceticus which reported before. The radioactively labeled PCR product was used as probe to hybridize with the total DNA of GXP04 which cut by serveral tyres of restriction endonucleases, the result showed the PH gene’s location. After constructed a PH gene’s partial library, positive clone which including PH gene had been found by in situ hybridization at last. Our work laid a funcdation for reseaching PH gene.更多还原