Enzymatic degradation of sulphonated azo dye using purified azoreductase from facultative Klebsiella pneumoniae
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ORIGINAL ARTICLE
Enzymatic degradation of sulphonated azo dye using purified azoreductase from facultative Klebsiella pneumoniae Shweta Dixit 1 & Sanjeev Garg 1 Received: 25 May 2020 / Accepted: 15 September 2020 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2020
Abstract 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., appKm and appVmax, 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.
Introduction The use of azo dyes has been reported in different industries such as paper, food, cosmetics, and pharmaceuticals with the largest consumer being the textile industries (Fontenot et al. 2003; Padamavathy et al. 2003). Azo dyes are considered as one of the major environmental pollutants due to their xenobiotic nature (Carliell et al. 1995; Chung and Stevens 1993). Most of these dyes are toxic, and their biotransformation products are known to be carcinogenic and mutagenic (Pinheiro et al. 2004). Microbial degradation is considered as one of the
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12223-020-00824-2) contains supplementary material, which is available to authorized users. * Sanjeev Garg [email protected] Shweta Dixit [email protected] 1
Department of Chemical Engineering, IIT Kanpur, Kanpur, UP 208016, India
ecofriendly and cost-effective methods to treat wastewater containing azo dye contamination (Sudha et al. 2014). Klebsiella pneumoniae is reported to be present in toxic dye-contaminated soil and wastewater. The ability of Klebsiella pneumoniae to degrade these toxic azo dyes is also repor
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