Purification and Characterization of NADPH-Dependent Cr(VI) Reductase from Escherichia coli ATCC 33456

A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456. The molecular mass was estimated to be 84 and 42 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, indicating a dimeric structure. The pI was 4.66, and optimal enzyme activity was obtained at pH 6.5 and 37 o C. The most stable condition existed at pH 7.0. The purified enzyme used both NADPH and NADH as electron donors for Cr(VI) reduction, while NADPH was the better, conferring 61% higher activity than NADH. The K m values for NADPH and NADH were determined to be 47.5 and 17.2 µmol, and the V max values 322.2 and 130.7 µmol Cr(VI) min -1 mg -1 protein, respectively. The activity was strongly inhibited by N-ethylmalemide, Ag 2+ , Cd 2+ , Hg 2+ , and Zn 2+ . The antibody against the enzyme showed no immunological cross reaction with those of other Cr(VI) reducing strains.