RESUMO
Pullulanase production from a fungus Hypocrea jecorina QM9414 that produces native extracellular hydrolases having industrial applications was carried out in a shaking flask culture containing 0.5% amylopectin at a pH of 6.50 at 30°C. The enzyme was purified 11-fold by ammonium sulfate fractionation, anion-exchange and gel-filtration chromatographies with a yield of 10.12% and a specific activity of 1.36 ± 0.14 U/mg protein. The molecular mass of pullulanase was estimated to be 130.56 kDa by PAGE and SDS-PAGE, indicating that the native enzyme was a monomer. The optimum pH and temperature for purified enzyme was 6.5 and between 35°-65°C, respectively. The Km values for amylopectin, starch and pullulan as substrates were 10.7, 15.5 and 38.4 mg/mL, respectively. The Vmax values were found to be 3.32, 3.32 and 3.82 ΔA/min for amylopectin, starch and pullulan, respectively. The enzyme was stable at 40-70°C for 30 min, but lost about 33% of its activity at 80°C and about 43% of activity at 90°C and 100°C for the same incubation period. Pullulanase activity was stimulated by CoCl2, NiCl2, KI, NaCl, MgCl2, and LiSO4. The enzyme was slightly inhibited by urea, CaCl2 and b-mercaptoethanol. The enyzmatic characteristics, substrate specificity and the products of hydrolysis indicated that the enzyme was similar to those of type II pullulanases.