Production and properties of a fibrinolytic enzyme by Schizophyllum commune BL23

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 o 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 (35.12x10 4 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 x10 4 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 o C. Enzyme was stable in the temperature range of 30-50 o C for 48 h but its activity was progressively lost at 60 o C. Activity was retained by 70% over the pH range of 5.0-11.0 at 28 o 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 o C, its activity was retained for 60 days. The fibrinolytic activity was inhibited by 1,10-phenanth roline and EDTA and was completely inactivated by Hg 2+ . 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.