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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)
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ժҪ ͨ����봿����øѧ���ʲⶨ ,ȷ�����ȶ���������ø (p nitrophenylphosphatase)�ǽ���ø���� ,���Բ�λ����þ���� ;��ø���ж����ȵ����� ,����������������������ȶ��� .��ø��Km Ϊ 3 0 31mmol L ,VmaxΪ 313 5 ��mol��min-1��mg-1;�����ṹ�д�Լ�� 74 5 %������ ,2 5 5 % ��ת�� .��pH 7 0�� 12 0��Χ���൱�ȶ� .��ø��SDS��Һ���ȶ��Խϲ� ,��TritonX 10 0��Һ�н�Ϊ�ȶ� ,��Tween 2 0��Һ�к��ȶ� .
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Abstract ��
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.
Keywords ��
thermostable nitrophenylphosphatase
enzymatic characteristics
thermostability
metal ion
secondary structure
Received 2003-10-06;
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