Purification and characterization of alkaline phosphatase from Thermotoga neapolitana.
A hyperthermophilic alkaline phosphatase was purified from Thermotoga neapolitana by heat treatment at 100 degrees C in the presence of Co2+ followed by ion-exchange and affinity chromatographies. The enzyme was purified 2,880-fold with 44% yield. The purified enzyme showed a single protein band of M(r) 45,000 on SDS-PAGE and an apparent molecular weight of 87,000 estimated by gel filtration chromatography. This suggested a homogenous dimer structure. The optimal pH and temperature for enzyme activity were 9.9 and 85 degrees C, respectively. Under optimal conditions, T. neapolitana alkaline phosphatase displayed 30% higher activity than calf intestine alkaline phosphatase did with p-nitrophenyl-phosphate as substrate. The hyperthermostable enzyme had a half-life of 238 min at 90 degrees C and K(m) and Vmax values of 183 microM and 1,352 U mg-1, respectively. Co2+ enhanced the enzyme activity, thermostability, and ligand affinity during column chromatography. The alkaline phosphatase was twice as active with Co2+ than with either Zn2+ or Mn2+ as the metal cofactor.
Dong
G
G
Department of Biochemistry, Michigan State University, East Lansing, USA.
Zeikus
J G
JG
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013379:Substrate Specificity; D013997:Time Factors
D002413:Cations, Divalent; D000469:Alkaline Phosphatase