1.13.11.9 | Escherichia coli cells overexpressing NicX are harvested and disrupted by passage through a French press, supernatant applied to DEAE-cellulose column with 50 mM NaH2PO4 buffer, pH 7.5, washed with buffer (0.1-0.3 M) containing 0.1 M NaCl, fraction with 2,5-dihydroxypyridine deoxygenase activity pooled and dialyzed with 20 mM NaH2PO4 buffer, pH 7.5, loaded onto hydroxyapatite column, eluted with 20-100 mM NaH2PO4 buffer, pH 7.0, fractions with enzyme activity pooled, dialyzed in 20 mM NaH2PO4 buffer, pH 7.5, and concentrated |