Microbial degradation of azo dye

偶氮染料的微生物降解

• 批准号: 13480174
• 负责人: KINOSHITA Shinichi
• 金额: $ 9.34万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480174/
• 关键词: Azo Dye; Microbial Decolorisation; Azoreductase; アゾレダクターゼ; Bacillus sp.; azo dye; azoreductase; gene; enzyme; 脱色分解; 脱色酵素; Aeromonas sp.; Trichoderma sp.; オレンジII; コンゴーレッド

Orange16 was efficiently decolorized by five kinds of bacterial mixed culture, isolated from the effluents of dyeing company, up to the 120 ppm and repeated decolorization was achieved under the oxygen limiting condition. A bacterium was isolated from this mixed culture and identified as Aeromonas sp. Aer2-l. Optimum conditions for Orange 16 decolorization were determined. Orange 16 was efficiently decolorized up to 1200 ppm and completely decolorized at 750 ppm on 50 h.Azoreductase from isolated bacterium, identified as Bacillus sp. B29 was purified using several column chromatographies. Purified enzyme showed homodimer having MW of 68 kDa. and stable at pH 6.0 and less than 60oC. Optimal conditions for Orange II decolorization were pH 6.5 at 55oC. The enzyme was required NADH for decolozation. The enzyme showed highly activity to Orange 16 and Now cossin but no activity to diazo compounds.Azoreductase genes from Bacillus sp. B29 were cloned. Three kinds of DNA fragment were amplified by PCR and sequenced. Predicted ORF from DNA sequences had MW of about 24 kDa. which different from purified enzyme described above. The azr genes (azr6, azr8, and azr18) were expressed using Escherichia coli expression system. Azr8 was purified and characterized. MW was calculated as 24 kDa. and the enzyme showed monomer protein. Azr8 required NADH for activity but not NADPH and FMN and FAD were stimulated the activity The enzyme had highly activity to Methyl Red and Ethyl Red but not active Orange G and Orange II. Diazo compounds such as Trypan Blue and Conge Red were decolorized slightly by the enzyme.

从印染厂废水中分离得到5种细菌混合培养物，对Orange 16进行高效脱色，脱色率可达120 ppm，并在限氧条件下实现了重复脱色。从该混合培养物中分离到一株细菌，经鉴定为气单胞菌Aer 2 - 1。确定了橙子16脱色的最佳条件。橙子16在1200 ppm时有效脱色，在750 ppm时50 h完全脱色。使用几种柱色谱法纯化来自分离的细菌（鉴定为芽孢杆菌属B29）的偶氮还原酶。纯化的酶显示具有68 kDa的MW的同源二聚体。在pH6.0和低于60 ℃下稳定。橙子Ⅱ脱色的最佳条件为pH 6.5，温度55 ℃。该酶需要NADH进行脱色。该酶对橙子16和Now cossin具有较高的活性，但对重氮化合物无活性。用PCR方法扩增了3种DNA片段，并进行了序列测定。从DNA序列预测的ORF具有约24 kDa的MW。其不同于上述纯化的酶。使用大肠杆菌表达系统表达azr基因（azr 6、azr 8和azr 18）。对Azr 8进行了纯化和表征。MW计算为24 kDa。酶为单体蛋白。Azr 8对甲基红和乙基红有较高的活性，但对橙子G和橙子II没有活性。台盼蓝和康吉红等重氮化合物被酶轻微脱色。