Characterization of a thermostable alkaline phosphatase from a novel species Thermus yunnanensis sp. nov. and investigation of its cobalt activation at high temperature.
A thermostable alkaline phosphatase with high specific activity and thermal resistance was purified from a novel species of Thermus sp. named as Thermus yunnanensis sp. nov. The enzyme contains a single peptide with a molecular mass of about 52 kDa on SDS-PAGE analysis and appears to be a homodimer in solution with the molecular mass of 104 kDa. The optimal pH and temperature for its activities are pH 8.0-10.0 and 70-80 degrees C, respectively. The catalytic activities of the enzyme are metal ion dependent, and Mg2+, Zn2+ and Co2+ are the main activators. Among these, Co2+ is the most active stimulator and has unique activation effect at high temperature. Metal binding analysis showed the binding of Mg2+ at the metal binding site was easy to loss in the thermoinactivation, and Co2+ was apt to bind at that site and kept the favorable configuration of catalysis, which would result high activation in the incubation with Co2+ at high temperature. According to this study, a model was proposed for the explanation of the activation and the results of actual experiments demonstrated the validity of the model.
Gong
Ningping
N
Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, China.
Chen
Chaoyin
C
Xie
Liping
L
Chen
Hongtao
H
Lin
Xianzhi
X
Zhang
Rongqing
R
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D003035:Cobalt / Q000378:metabolism / Q000494:pharmacology*; D004275:DNA, Ribosomal; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013824:Thermus / Q000145:classification / Q000201:enzymology*
D004275:DNA, Ribosomal; D003035:Cobalt; D000469:Alkaline Phosphatase