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Journal of Clinical Microbiology, February 2004, p. 722-726, Vol. 42, No. 2
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.2.722-726.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Laboratory of Antimicrobial Resistant Pathogens, Department of Bacteriology, National Institute of Health ,¹ Department of Biology, Faculty of Natural Science, Chung-Ang University, Seoul, Korea²

Received 7 February 2003/ Returned for modification 17 March 2003/ Accepted 3 November 2003

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, ß-casein, and gamma globulin.