The plasmid of WT or mutant OPH was transformed into the E. coli strain BL21. The cells were grown in 250 ml standard lysogeny broth (LB) at 37°C and optical density (OD) = 0.4, then induced by 1 mM Isopropyl -D-1-thiogalactopyranoside (IPTG) at 16 °C overnight. Cells were harvested by centrifugation at 4500 g for 15 min and resuspended in 5 ml of Ni-NTA lysis buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole and 0.1 M pepstatin at a pH of 7.4. The cells were lysed using Microfluidizer (Microfluidics, Westwood, MA) and then centrifuged at 12,000 g for 30 min. The collected supernatant was incubated with 2 ml of Ni-NTA resin under end-to-end shaking and loaded on a 10 mL HisTrap FF column. After washing with buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, the proteins were eluted with buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole.