Patcharaporn Pandee, Aran H-Kittikul, Ohsugi, Masahiro, and Yaowaluk Dissara

pp. 447 - 453

Abstract

Schizophyllum commune BL23 was cultivated for the production of a fibrinolytic enzyme under submerged culture conditions. Maximum growth (8.93 g/l) with fibrinolytic enzyme activity (576.73 units) was achieved when S. commune BL23 were cultured in a peptone yeast extract dextrose broth with an initial pH of 6.0, a temperature of 35°C and a shaking speed of 150 rpm for 7 days. The protein fraction precipitated with 80% ammonium sulfate saturation had the highest fibrinolytic activity (35.12x10⁴ units/mg protein). Ammonium sulfate was found to activate the fibrinolytic activity after dialysis. Fibrinolytic enzyme was partially purified using anion exchange chromatography (DEAE-Sephacel). Purity was increased 86 fold and specific activity of 39.31 x10⁴ units/mg protein was obtained. A single protein band after native polyacrylamide gel electrophoresis (Native PAGE) exhibited fibrinolytic enzyme activity. The maximum activity of the partially purified
enzyme was found at 50°C. Enzyme was stable in the temperature range of 30-50°C for 48 h but its activity was progressively lost at 60°C. Activity was retained by 70% over the pH range of 5.0-11.0 at 28°C for 20 min. After prolonged incubation (48 h), it was stable only in a narrow pH range (6.0-9.0). At pH 7.0 and 30°C, its activity was retained for 60 days. The
fibrinolytic activity was inhibited by 1,10-phenanthroline and EDTA and was completely inactivated by Hg²⁺. Increasing concentrations of EDTA progressively decreased the enzyme activity. However, its activity was not affected by PMSF and SBTI. The results indicate that the enzyme was most likely a metalloprotease.