Department of Chemical Engineering, IIT Kanpur, Kanpur, UP, 208016, India.
Folia Microbiol (Praha). 2021 Feb;66(1):79-85. doi: 10.1007/s12223-020-00824-2. Epub 2020 Sep 18.
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 and appV, 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.
从兼性肺炎克雷伯氏菌中异源表达和纯化的偶氮还原酶被用于降解磺化偶氮染料。甲基橙（MO）被用作模型染料，以研究在不同条件下纯化酶对偶氮染料的脱色潜力。该酶在 40°C 和 pH8.0 时具有最大活性。该酶表现出热稳定性，因为即使在 80°C 下仍保留了一些酶活性。使用 MO 作为底物时，酶的表观动力学参数，即 appK 和 appV，分别为 17.18 μM 和 0.08/min。在 NADH 的存在下，纯化酶能够在 10 分钟内将约 83%的 MO（20 μM）脱色。因此，观察到纯化酶有效地对 MO 进行脱色。在纯化过程的最后四步中，重组酶的纯化倍数约为 18 倍，产率为 46%。通过凝胶过滤层析中洗脱的蛋白质体积和酶的单体分子量为 23 kDa，确认酶呈四聚体结构。与其他细菌来源的偶氮还原酶的效率相比，从肺炎克雷伯氏菌克隆并从重组大肠杆菌中纯化的偶氮还原酶的染料降解效率要高得多。在本研究中，我们报告了从肺炎克雷伯氏菌中克隆并在大肠杆菌中表达的偶氮还原酶的纯化和特性。
