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摘要：偶氮染料废水引起的环境污染问题日渐引起人们重视。环境友好的生物降解法是处理偶氮染料废水一条重要的途径。一些微生物体内含有偶氮还原酶，这类酶不仅作用条件温和，而且可以再生物体内大量生产，在降解染料废水方面有着巨大的应用前景。本课题的前期研究克隆得到Bacilluscereus偶氮还原酶BC的基因，初步研究显示该酶对多种偶氮染料有较强的脱色能力，但对其在不同条件下的脱色性能研究较少，因此本研究探究了在不同pH、温度及盐浓度下，偶氮还原酶BC对染料的脱色性能。

本研究通过大肠杆菌表达菌株BL21将带有His-tag偶氮还原酶BC基因（azoBC）进行表达，后经Ni-IMAC分离纯化得到偶氮还原酶BC纯酶液。在pH为3～11的条件下，确定pH7为偶氮还原酶BC对丽春红脱色降解的最适pH（比活力=1.07U/mg）。在温度为0～90℃的条件下，得到60℃为偶氮还原酶BC对丽春红脱色反应的最适温度(比活力=1.48U/mg)。在加入0～1%浓度NaCl时，偶氮还原酶BC在0.2%NaCl中对丽春红脱色能力最强(比活力=5.17U/mg)，但在0.2%～1%NaCl浓度范围内，随着NaCl浓度的增加，脱色性能减弱。考察偶氮还原酶BC对丽春红、诱惑红、日落黄、直接蓝、甲基红这五种染料的酶促反应动力学常数，发现偶氮还原酶BC对几种偶氮染料均有脱色作用，其中和丽春红的亲和性最好（Km=44.87μmol/L）。关键字：偶氮染料；偶氮还原酶BC；脱色；丽春红

Abstract：The environmental problems caused by azo dye wastewater have attracted more and more attention. Eco-friendly biodegradation is one of the important methods to treat the azo dye wastewater. Azo reductases, existed in some microorganisms, can degrade azo dyes under mild conditions. Since they can be produced in large quantities by microorganisms, azo reductases have great application prospect in treatment of the azo dye wastewater. The previous research had cloned and expressed the gene of azo reductase BC (azo BC) from Bacillus cereus. The preliminary study showed that the enzyme had a strong decoloring ability on various azo dyes, but the research on its decoloration performance under different conditions was very rare. Therefore in present study we intended to explore the decoloration performance of azo BC on azo dyes, under different pH, temperature and salinity.

In this study, the gene of azo reductase BC (azo BC, with His-tag) was expressed by Escherichia coli expressing strain BL21, and the expression products were then purified by Ni-IMAC to obtain pure enzyme solution. Under the condition of pH3 ~ 11, it was determined that pH 7 was the optimum pH for decoloration of ponceau S (Specific activity = 1.07 U/mg). At the temperature of 0~ 90℃, under the conditions of 60℃ azo BC showed the strongest decoloring ability (Specific activity = 1.48 U/mg). When adding 0 ~ 1% NaCl, azo BC exhibited the most potent decoloring activity on ponceau S with adding of 0.2% NaCl (Specific activity = 5.17 U/mg), however the decoloring ability weakened in higher salinity condition. The kinetics of the enzymatic reaction of azo BC against five dyes, ponceau S, temptation red, sunset yellow, direct blue and methyl red, was also studied. The results implied that azo BC works on all of the tested azo dyes, and it showed the best affinity with ponceau S (Km = 44.87μmol/L).

Keywords：Azo dyes; azo reductase BC; decoloration; ponceau S

目 录

第一章 绪论 1

1.1染料概述 1

1.2偶氮染料废水的危害 2

1.3偶氮染料废水的特点 2

1.4染料废水的处理方法 3