Purification and Characterization of Extracellular Cr(VI) Reductase from Bacillus amyloliquefaciens (CSB 9) isolated from Chromite Mine Soil for Detoxification of Hexavalent Chromium

Abstract

Chromate is widely used industrial pollutant which can be detoxified effectively using chromate reductase from chromium resistant bacteria. A soluble Cr(VI) reductase from Bacillus amyloliquefaciens (CSB 9) was extracted and purified to electrophoretic homogeneity through (NH4)2SO4 precipitation followed by dialysis and gel filtration chromatography with specific activity of 0.167 U/mg and 6% yield with 30.36-fold increase in purity. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the purified enzyme was estimated to be ~ 116 KDa. Further, functional characterization with respect to pH, temperature and storage stability showed optimum Cr(VI) activity at 35 °C and pH 7.0 on standard analysis conditions. Using potassium dichromate as substrate, the enzyme kinetics showed maximum activity (Vmax) of 3.5 U/mL with its corresponding KM value of 27.78 μM and the second-order constant (Vmax/Km) 0.125 U/μM mL. The purified enzyme exhibited higher stability (μM/mg/min) when treated with additives such as metal ions K+ (1.3 ± 0.056), surfactant tween 80 (2.33 ± 0.52), inhibitor β-mercaptoethanol (0.67 ± 0.15) and organic solvent glycerol (1.33 ± 0.011). These remarkable qualities found with the chromate reductase produced by B. amyloliquefaciens (CSB 9) could make it an ideal candidate for bioremediation of hexavalent chromium under a wide range of environmental conditions.