摘 要：金橙II染料在现代生产中被广泛应用，因其具有强的致毒性和致癌性，数量种类多，长时间难降解，严重污染环境并且威胁人类健康。虽然目前处理方法有很多种，如吸附法、电化学法，但微生物降解法是目前研究的热点。本文以金橙II高效脱色菌株AO7-3为材料，通过生物信息学分析和PCR扩增，获得了该菌株中一个偶氮还原酶编码基因，命名为azo888。通过载体构建方式成功地构建了该基因的重组表达载体pET29a-azo888，并在大肠杆菌BL21菌株中实现了功能表达。采用Ni-IDA亲和层析方法从重组表达菌株中纯化了金橙II降解酶，并初步研究了该酶的催化活性。本论文以该基因为研究对象，对其进行序列分析和克隆、表达及酶学特性研究，从基因和酶的水平上初步阐明了AO7-3菌株脱色偶氮染料金橙II的分子机理。94759

毕业论文关键词：金橙Ⅱ，偶氮还原酶，融合表达

Abstract: Acid orange II is widely used in in the contemporary production, which will eventually cause severe environmental contaminations and threaten humans’ health due to its pathogenicity and carcinogenicity。 Presently, there are a host of processing methods like adsorption method, electrochemical method。 However, microbial degradation that is relatively direct and has few side effects is a hotspot of current researches。 Microbial degradation of environmental pollutants is a hotspot of the current research。 In this work, a carbendazim degrading strain AO7-3 was used as the research material。 The key enzyme encoding gene named azo888 that is responsible for the first step in carbendazim degradation reaction was cloned from strain AO7-3。 The gene recombinant expression vector pET29a-azo888 has been constructed successfully。 The function expression of the enzyme azo888 was realized in E。 coli BL21 strain。 Acid orange II degradation enzyme was purified from recombinant expression strains by the method of Ni-IDA affinity chromatography and the catalytic activity of the enzyme was also studied preliminarily。 This work clarified the first step hydrolysis reaction mechanism of Acid orange II degrading by strain AO7-3 on the gene and enzyme level。 

Keywords: Acid orange II; azo reductase; fusion expression

目  录

1  前言 4

2  材料与方法 5

2。1  供试菌株和试剂 5

2。2  培养基 5

2。3  研究方法 5

2。3。1  金橙II降解酶基因的克隆 5

2。3。2  PCR产物TA克隆验证 6

2。3。3  金橙II降解酶基因序列分析 7

2。3。4  金橙II降解酶基因重组表达载体pET29a-azo888构建 7

2。3。5  表达载体pET29a-azo888转化到感受态E。coil BL21细胞 7

2。3。6  重组表达载体pET29a-azo888的诱导表达 8

2。3。7  SDS聚丙烯酰胺（SDS-PAGE）凝胶电泳分析 8

2。3。8  Azo888纯化 8

2。3。9  酶活性检测 9

3  结果与分析 9

3。1  金橙II降解菌株总DNA的提取 9

3。2  疑似基因的PCR扩增 9

3。3  PCR产物TA克隆验证