Microbial degradation of azo dye

从印染废水中分离得到的5种细菌混合培养物对橙色16有较高的脱色效果，可达120ppm，在限氧条件下可实现反复脱色。从混合培养物中分离到一株细菌，经鉴定为气单胞菌属(Aeromonas sp.)航空二号--L。确定了橙色16的最佳脱色条件。橙色16的脱色率可达1200ppm，在750ppm的条件下50h即可完全脱色。用几种柱层析对B29进行了纯化。纯化的酶显示均二聚体的相对分子质量为68 kDa。在pH 6.0和低于60oC的条件下稳定。橙色II脱色的最佳条件为：pH 6.5，温度55℃。该酶的脱色需要NADH。该酶对橙色16和现在的Cossin表现出很高的活性，但对重氮化合物没有活性。克隆了B29。用聚合酶链式反应扩增了3种DNA片段，并进行了测序。从DNA序列预测的ORF的相对分子质量约为24 kDa。这与上述纯化的酶不同。利用大肠杆菌表达系统对azr基因(azr6、azr8和azr18)进行了表达。对Azr8进行了纯化和鉴定。相对分子质量为24 kDa。该酶为单体蛋白。Azr8的活性需要NADH，但不需要NADPH，FMN和FAD被激活，该酶对甲基红和乙基红有很高的活性，但对橙G和橙II没有活性。

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.