A novel pullulanase from a fungus Hypocrea jecorina QM9414: production and biochemical characterization

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 300C. 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 degrees-65 degreesC, 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 deltaA/min for amylopectin, starch and pullulan, respectively. The enzyme was stable at 40-70 degreesC for 30 min, but lost about 33% of its activity at 80 degreesC and about 43% of activity at 90 degreesC and 100 degreesC for the same incubation period. Pullulanase activity was stimulated by CoC1(2), NiC1(2), KI, NaC1, MgC1(2), and LiSO4. The enzyme was slightly inhibited by urea, CaC1(2) and beta3-mercaptoethanol. The enyzmatic characteristics, substrate specificity and the products of hydrolysis indicated that the enzyme was similar to those of type II pullulanases.