Heterologously expressed and purified azoreductase enzyme from facultative Klebsiella pneumoniae was used to degrade sulphonated azo dye. Methyl orange (MO) was used as the model dye to study the azo dye decolorization potential of the purified enzyme at different conditions. The enzyme had maximum activity at 40 °C and pH 8.0. The enzyme was observed to be thermo-stable as some enzyme activity was retained even at 80 °C. The apparent kinetic parameters, i.e., appK_m and appV_max, for azoreductase using MO as a substrate were found to be 17.18 μM and 0.08/min, respectively. The purified enzyme was able to decolorize approximately 83% of MO (20 μM) within 10 min in the presence of NADH. Thus, efficient decolorization of MO was observed by the purified enzyme. The recombinant enzyme was purified approximately 18-fold with 46% yield at the end of four steps of the purification process. Enzyme was present in a tetrameric structure as confirmed by the volume at which protein was eluted in gel filtration chromatography, and the monomeric molecular mass of enzyme was found to be 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The dye degradation efficiency of azoreductase cloned from Klebsiella pneumoniae and purified from recombinant Escherichia coli was observed to be much higher as compared with the efficiencies of the reported azoreductases from other bacterial strains. In the present study, we report the purification and characterization of the azoreductase cloned from Klebsiella pneumoniae and expressed in Escherichia coli.

来自兼性肺炎克雷伯菌的异源表达和纯化的偶氮还原酶用于降解磺化偶氮染料。以甲基橙（MO）为模型染料，研究纯化酶在不同条件下的偶氮染料脱色潜力。该酶在 40 °C 和 pH 8.0 时具有最大活性。观察到该酶是热稳定的，因为即使在 80°C 下也保留了一些酶活性。表观动力学参数，即appK _m和appV _max，对于使用 MO 作为底物的偶​​氮还原酶，发现分别为 17.18 μM 和 0.08/min。在 NADH 存在下，纯化的酶能够在 10 分钟内使大约 83% 的 MO（20 μM）脱色。因此，通过纯化的酶观察到 MO 的有效脱色。在纯化过程的四个步骤结束时，重组酶被纯化了大约 18 倍，产率为 46%。酶以四聚体结构存在，这由凝胶过滤色谱中蛋白质洗脱的体积证实，并且在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 上发现酶的单体分子量为 23 kDa。克隆自肺炎克雷伯菌并重组纯化的偶氮还原酶的染料降解效率与报道的来自其他细菌菌株的偶氮还原酶的效率相比，观察到大肠杆菌的效率要高得多。在本研究中，我们报告了从肺炎克雷伯菌克隆并在大肠杆菌中表达的偶氮还原酶的纯化和表征。