Development of an efficient recombinant bacterium and its application in the degradation of environmentally hazardous azo dyes

Abstract

In the present work, azoreductase gene, azoK, from a bacterium Klebsiella pneumoniae, isolated from the sludge of a local dyeing industry was cloned and expressed in Escherichia coli. The wild-type K. pneumoniae was previously observed to efficiently decolorize mono-, di- and tri-substituted azo dyes. The recombinant E. coli was utilized for the degradation of a model azo dye, methyl orange. azoK was amplified through polymerase chain reaction and subsequently cloned in pGEM-T vector followed by sequencing of a cloned gene to check its integrity. Further, the gene was overexpressed in E. coli BL21 (DE3) using recombinant pET-28b-azoK to produce the protein of interest. A strong band with an approximate molecular mass of 23 kDa was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recombinant azoK gene encoded a protein consisting of 201 amino acids with azoreductase activity. The cell fractionation method was used to determine the location of azoreductase within the periplasmic fraction of the cells and, thus, involved in the extracellular reduction pathway of azo dyes. Some amount of the enzyme was also present in the cytoplasmic fraction. The enzyme from recombinant strain could successfully decolorize the model azo dye. The decolorization efficiency of the enzyme was observed to increase in the presence of a redox mediator. Thus, the viability of the recombinant azoK for the abatement of azo dyes from industrial wastewater was established. It was noted that azoK was the first azoreductase gene to be cloned from K. pneumoniae and expressed in E. coli.