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 cultureconditions. Maximum growth (8.93 g/l) with fibrinolytic enzyme activity (576.73 units) was achieved when S. communeBL23 were cultured in a peptone yeast extract dextrose broth with an initial pH of 6.0, a temperature of 35oC and a shakingspeed of 150 rpm for 7 days. The protein fraction precipitated with 80% ammonium sulfate saturation had the highest fibrinolyticactivity (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 increased86 fold and specific activity of 39.31 x104 units/mg protein was obtained. A single protein band after native polyacrylamidegel electrophoresis (Native PAGE) exhibited fibrinolytic enzyme activity. The maximum activity of the partially purifiedenzyme was found at 50oC. Enzyme was stable in the temperature range of 30-50oC for 48 h but its activity was progressivelylost at 60oC. Activity was retained by 70% over the pH range of 5.0-11.0 at 28oC 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 30oC, its activity was retained for 60 days. Thefibrinolytic activity was inhibited by 1,10-phenanthroline and EDTA and was completely inactivated by Hg2+. Increasingconcentrations of EDTA progressively decreased the enzyme activity. However, its activity was not affected by PMSF andSBTI. The results indicate that the enzyme was most likely a metalloprotease.

Keywords

• Schizophyllum commune
• mycelial culture
• fibrinolytic enzyme