Enzymatic Characteristics of Thermostable p-Nitrophenylphosphatase
FENG Cong-jing 1), 2) , FU Yuan-yuan 3) , JI Chao-neng 1) , GU Shao-hua 1) , FU Wen-jun 2) , XIE Yi 1) , MAO Yu-min 1)* ( 1) State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Sciences, Fudan University, Shanghai 200433,China; 2) Institute of Plant Physiology and Ecology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; 3) United Gene Holding Group Corporation, LTD, Shanghai 200092,China)
Thermostable p-nitrophenylphosphatase (TNPPase) had been expressed in E. coli M15, and purified rapidly. It is sensitive to metal chelator and Mg ion is necessary to its activity. TNPPase exhibited a moderate degree of thermostability, and the thermostability of TNPPase increased significantly when treated with 1 mmol/L Mg, Mn, Cu, Ba, Ca, Ni and Zn ions. TNPPase is stable within a wide range of pH (pH 7.0��12.0). It catalyzed the hydrolysis of p-nitrophenylphosphate with a Michaelis constant 3.031 mmol/L and a V max 313.5 ��mol��min -1 ��mg -1 . The ��-helix percentage and ��-turn percentages were 74.5 and 25.5 in the secondary structure of TNPPase, respectively. TNPPase is not stable in SDS solution, but stable in Triton X-100 and Tween 20 solutions.