After IPTG introduction and the ultrasonication of the recombinant E. coli BL21 cells, supernatant was brought to 70% saturation with (NH4) 2 SO 4 and stored overnight at 4uC, and then again centrifuged. The enzymes expressed by pET28a(+) vector were fused to an N-terminal six-histidine tag and purified via nickel chelate affinity chromatography (GE, U.S.), which was eluted with 150 mM imidazol. The quality of the purified protein was evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and coomassie blue staining.