Purification and Characterization of a 43-Kilodalton Extracellular Serine Proteinase from Cryptococcus neoformans

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Abstract
An extracellular proteinase was purified from culture filtrates of Cryptococcus neoformans NHPY24 by DEAE ion-exchange chromatography and gelatin affinity column chromatography with azoalbumin as the substrate. The molecular mass of the purified enzyme was 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its pH optimum was 7.0 to 8.0, and maximal activity was obtained at pH 7.5 and 37°C. By isoelectric focusing, the purified enzyme had a pI of 4.77. Enzyme activity was inhibited by serine proteinase inhibitors such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate. The purified enzyme was thus a serine proteinase. It hydrolyzed natural substrates including hemoglobin, β-casein, and gamma globulin.