Abstract

An extracellular proteinase of Candida albicans was purified by a combination of 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephacryl S-200 HR molecular sieve chromatography. Its mlecular weight was approximately 41 kDa on SDS-PAGE and isoelectric point was 4.4. The enzyme was inhibited by pepstain A. Optimum enzyme activity ranged from pH 2.0 to 3.5 with its maximum at pH 2.5 and a temperature of 45℃. The addition of divalent cations, Ca^2+, Zn^2+ and Mg^2+, resulted in no significant inhibition of enzymatic activity. However, some inhibitory effects were observed by Fe^2+, Ag^2+ and Cu^2+. With BSA as substrate, an apparent K_m was determined to be 7 × 10^7 M and K_I, using pepstatin A as an inhibitor, was 8.05 × 10^8 M. N-terminal amino acid sequence was QAVPVTLXNEQ. Degradation of BSA and fibronectin was shown but not collagen, hemoglobin, immunoglobulin G, or lysozyme. The enzyme preferred peptides with Glu and Leu at the P₁position, but the enzyme activity was highly reduced when the P₂position was Phe or Pro. This enzyme showed antigenicity against sera of patients with candidiasis.

• Keywords: Candida albicans; Aspartic proteinase; Purification; characterization