Purification and Characterization of Hexavalent Chromate Reductase Activity in Cell Free Extract of Bacillus subtilis Strain Isolated From Treated Tannery Effluent

Abstract

A Bacillus subtilis strain previously isolated from treated tannery effluent was tolerant to a maximum of 1400 mg/L Cr(VI) and reduced 77% toxic Cr(VI) to less toxic Cr(III) within 24 hrs. at pH 8.5 and 35°C temperature under shaking condition (120 rpm). The chromate reductase enzyme was found to be membrane bound and it has been purified using ammonium sulphate fractionation, anionexchange chromatography and gel filtration chromatography on Sephadex G-75 by cell free extract of the bacterium. The enzyme was characterized based on optimal temperature, pH, metal ions and initial Cr(VI) concentration in the reaction mixture. Maximum chromate reductase activity was observed at pH 8.5 and 35ºC temperature. The enzyme was significantly active between pH 6.5-10.0 and was found to be stable between 30ºC-40ºC. The chromate reductase activity was slightly decreased with increasing “the concentration of Cr(VI). The enzymatic activity was completely inhibited by Hg(II) and As(III) while Ni(II), Cd(II), Mn(II), Zn(II), Pb (II) and Co (II) showed moderate effect on chromate reductase activity of Bacillus subtilis and the chromate reduction was enhanced by Cu(II) and Fe(III). Keywords: Bacillus subtilis, chromate reductase, Cr(VI), tannery.