Candida olea 148 secretes two distinct proteolytic enzymes whose production is pH-dependent. In acidic conditions, the organism produces a single acid protease with molecular weight 30,900 Da, optimal activity at pH 3.3 and 42°C, and containing 11% carbohydrate. In alkaline conditions, a single alkaline protease is produced with molecular weight 23,400 Da, optimal activity at pH 8.0-9.0 and 40°C, containing 17% carbohydrate. Both enzymes were purified to homogeneity using gel filtration and ion-exchange chromatography. The acid protease is a carboxyl protease inhibited by pepstatin and diazo compounds but resistant to serine, thiol, and metalloprotease inhibitors. The alkaline protease is a serine protease inhibited by PMSF and EDTA, with activity dependent on disulfide linkages. Both enzymes are thermolabile above 42-46°C. The acid protease's retention of activity at low temperatures makes it suitable for beer chill-proofing applications, which was the original application driving this characterization work.

Key findings

• Candida olea 148 produces pH-regulated proteases: acid protease at low pH and alkaline protease at high pH
• The acid protease (Mr 30,900) is a carboxyl protease with optimal activity at pH 3.3 and 42°C
• The alkaline protease (Mr 23,400) is a serine protease with optimal activity at pH 8.0-9.0 and 40°C
• Both purified enzymes are glycoproteins with distinct inhibition patterns and isoelectric points
• The acid protease retains significant activity at 0°C, explaining its utility in chill-proofing beer