【作者】 张宗武；

【导师】 武波；

【作者基本信息】 广西大学 ， 微生物学， 2006， 硕士

【摘要】 苯胺用途广，危害大，对它的生物法处理潜力很大。在好氧菌中，苯胺双加氧酶基因簇是降解苯胺的关键基因，编码苯胺降解途径的第一个酶，负责将苯胺转化成为邻苯二酚。而邻苯二酚是一种重要的化工产品，用途广泛，需求量大，对其的生物法生产与许多传统方法相比具有更大的优势。 用实验室保存的一株能高效降解苯胺的不动杆菌ANA5构建了XbaⅠ完全酶切的基因文库，通过菌落原位杂交，结合功能筛选等方法从中筛选到了阳性克隆，经亚克隆测序拼接得到了完整的苯胺双加氧酶基因簇。序列分析表明该基因簇包含6个完整的ORF，有启动子特征序列，全序列与已报道的不动杆菌YAA的苯胺双加氧酶基因簇在氨基酸水平上有较高的同源性。经实验发现，该基因簇在连接于无启动子的pLAFR6载体上时表达效果较好，用此重组质粒构建了基因工程菌。经发酵条件优化，发现该工程菌在苯胺浓度0.5mg／mL，采用LB培养基，E．Coli DH5α为宿主菌，于37℃培养，接种量为3％的条件下，邻苯二酚产量在20h达到0.546mg／mL，底物分子水平转化率可达92.4％。更多还原

【Abstract】 Aniline is a widely used and poisonous compound, and its biodegrade has a huge industrial interest. In aerobic micoorganisms, aniline dioxygenase gene cluster plays an essential role in degradeing aniline by encoding the first enzyme in this metabolism, and catalyzes aniline to catechol. Catechol is an important compound and is used widely in industry. Producing catechol by microorganism has more advantages than by traditional methords.A gene library was constructed with the chromosomal DNA isolated from Acinetobacter sp. strain ANA5, this strain is able to utilize aniline as the sole carbon, nitrogen and energy source. A positive clone was selected by clone in situ hybridization and functional screening. A complete aniline dioxygenase gene cluster was obtained through subcloning and jointing. Sequence analysis showed that the gene cluster had six intact ORFs, ahead of which there was a promoter, and the whole sequence had high similarity with that of Acinetobacter sp. YAA at amino acid level. The gene cluster was cloned into a cosmid vector pLAFR6 which has no promoter to form更多还原