Purification and characterization of an extracellular alkaline phosphatase from Penicillium chrysogenum

Preparative Biochemistry & Biotechnology
M PolitinoJ J Usher

Abstract

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...Continue Reading

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