Purification and characterization of an extracellular alkaline phosphatase from Penicillium chrysogenum.
An extracellular alkaline phosphatase from Penicillium chrysogenum was purified to homogeneity using DEAE ion-exchange chromatography and size exclusion chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of 58,000. The mobility of the native enzyme on a Superose 12 column suggests that the active form of the enzyme is a monomer. The enzyme catalyzes the hydrolysis of phosphate from a variety of substrates including p-nitrophenyl phosphate, alpha-naphthyl phosphate and the anti-tumor compound etoposide phosphate. The apparent K(m) for the substrate p-nitrophenyl phosphate is 1.3 mM and the enzyme is inhibited by inorganic phosphate. The pH optimum of the enzyme is 9.0 with a broad optimal temperature range between 40 and 50 degrees C. The isoelectric point of the enzyme is approximately 5.5. The enzyme is a glycoprotein; digestion with endoglycosidase H indicates that the protein consists primarily of N-linked carbohydrates. Enzymatic activity is enhanced by the addition of divalent cations such as Mg+2 and Mn+2 and inhibited by addition of a chelator such as EDTA suggesting a metal ion requirement. The enzyme was found to be an inexpensive catalyst for the conversion of etoposide phosphate to etoposide in the manufacture of this anti-tumor compound.
Politino
M
M
Biotechnology Development Laboratories, Bristol-Myers Squibb Company, Syracuse, New York 13221-4755, USA.
Brown
J
J
Usher
J J
JJ
D000469:Alkaline Phosphatase / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005047:Etoposide / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000378:metabolism; D010408:Penicillium chrysogenum / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism
D009943:Organophosphorus Compounds; D008345:Manganese; C061400:etoposide phosphate; D005047:Etoposide; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc