Killer toxin of Pichia anomala NCYC 432; purification, characterization and its exo-β-1,3-glucanase activity

Pichia anomala NCYC 432 secretes a killer toxin which is inhibitory to a variety of yeasts including pathogenic Candida spp. The killer toxin in the culture supernatant was concentrated by ultrafiltration and purified to homogenity by two successive gel filtration chromatographies with a TSK G2000SW column. Biochemical characterization of the toxin showed that it is a glycosylated protein with a molecular mass of 47 kDa and pI values of 3.4 and 3.7. The toxin showed high stability at pH values between 3 and 5.5 and up to 37 °C. Its N-terminal amino acid sequencing yielded the sequence GDYWDYQNDKIR which is 100% identical with that of mature exo-β-1,3-glucanase (accession no. AJ222862) of P. anomala strain K. The toxin displayed high activity against laminarin thus showing a β-glucanase activity. The Michaelis constants Km and Vmax for laminarin hydrolysis were 0.3 mg ml−1 and 350 μmol min−1 mg−1.