Extracellular constitutive production of pullulan-hydrolysing activity by Sclerotium rolfsii

Extracellular constitutive pullulan-hydrolysing activity was detected in Sclerotium rolfsii (1.05 μmol glucose equivalents liberated∙mL−1∙min−1) when cultivated in a synthetic medium containing starch as the carbon source. The influence of various inorganic nitrogen and carbon sources, surfactants, and organic supplements on the production of pullulan-hydrolysing activity was studied. In an optimized medium supplemented with 1% corn steep liquor, increased yields of enzyme activity (1.8 μmol∙mL−1∙min−1) were obtained. The enzyme was most active at pH 4.2 and was stable in the pH range of 3.5–5.5. The optimum temperature for pullulan hydrolysis was 50 °C and the activity was stable in the temperature range of 25–60 °C at pH 4.2 for 30 min. The Ea for the native enzyme from the Arrhenius plot with pullulan as a substrate was 22.6 kJ∙mol−1. The Km value for the native enzyme with pullulan as a substrate was 8.33 mg∙mL−1. The enzyme did not produce glucose or maltose from pullulan. Inability of the enzyme to rapidly decrease the specific viscosity (ηsp) of a 1% pullulan solution indicated susceptibility of α-(1 → 6) and not α-(1 → 4) linkages.