(545q) Identification and Enzymatic Characterization of a Novel NADH Dependent Azoreductase, Encoded By Azok in Klebsiella Pneumoniae

Sulphonated azo dyes, known xenobiotic, are extensively used in textile, plastic, food, pharmaceutical and cosmetic products. These are highly carcinogenic and mutagenic to humans and their discharge into the surface water adversely affects the ecosystem. The microbial degradation of azo dyes involves the reductive cleavage of azo bond which is catalyzed by the group of NADH or NADPH dependent enzymes called azoreductases, few of which are flavin dependent. In this study, we have identified an azoreductase enzyme which is dependent on NADH for its catalytic activity. Azoreductase was encoded by AzoK from the previously isolated Klebsiella pneumoniae C1 strain. We have cloned AzoK gene from Klebsiella pneumoniae C1 strain and was heterologously expressed in E. coli. The azoreductase sequence encoded a functional protein of 201 amino acids with a molecular mass of approximately 23 kDa. The expressed azoreductase had 99% protein sequence similarity with the azoreductase of Klebsiella pneumoniae subsp. pneumoniae MGH 78578. The protein had only one amino acid change, serine replaced by glycine, at the 176^th position. The recombinant azoreductase required NADH as electron donor for utilizing methyl orange as a substrate. The purification and characterization of recombinant enzyme for its optimal temperature, pH and thermostability is in progress. 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 was also present in the cytoplasmic fraction. The enzyme from recombinant strain could successfully decolorize the model azo dye, methyl orange within 12 h in an extracellular environment through a specific metabolic pathway. The decolorization efficiency of the enzyme was observed to increase in the presence of NADH and decolorized methyl orange within 4 h. Thus, the viability of the recombinant AzoK for the abatement of azo dyes from industrial wastewater was established. It was noted that AzoK represents the first azoreductase gene to be identified and cloned from K. pneumoniae and expressed in E. coli.