Characterization and Production of Thermostable and Acid-stable Extracellular Fibrinolytic Enzymes from Cordyceps militaris

Abstract

Biochemical and enzymatic characterization for extracellular protease isolated from Cordyceps militaris cultivated on rice bran medium was investigated. C militaris produced proteolytic enzymes from 10 days after inoculation, maximum enzyme production was found at 25 days. The optimum temperature and pH of proteases production was at <TEX>$25^{\circ}C$</TEX> and pH 7.0, respectively. The protease activity was observed in the four peaks (Pro-I, Pro-II, Pro-III, and Pro-IV) separated through Sephadex G-100 column chromatography. The separated protease was optimally active at <TEX>$25^{\circ}C$</TEX>. Optimum pH of the protease was between 7 and 8. Enzyme was also stable over at <TEX>$30-80^{\circ}C$</TEX>. The enzyme was highly stable in a pH range of 4-9. Protease activity was found to be slightly decreased by the addition of <TEX>$Mg^{2+}$</TEX>, <TEX>$Mn^{2+}$</TEX>, <TEX>$Zn^{2+}$</TEX>, <TEX>$Fe^{2+}$</TEX> and <TEX>$Cu^{2+}$</TEX>, whereas inhibited by the addition of <TEX>$Ca^{2+}$</TEX> and <TEX>$Co^{2+}$</TEX> Protease activity was inhibited by protease inhibitor PMSF. On the other hand, the partially purified protease was investigated on proteolytic protease activity by zymogram gel electrophoresis using three substances (casein, gelatin and fibrin). Four active bands (F-I, FII, F-III, and F-IV) of fibrin degradation were revealed on fibrin zymogram gels. Both of F-II and FIII showed caseinolytic, fibrinolytic and gelatinolytic activities in three gels. Thermostability, pH stability, and pH-thermostability of the enzyme determined the residual fibrinolytic activity also displayed on fibrin zymogram gel. The only one enzyme (F-II) displayed over a broad range of temperature at <TEX>$30-90^{\circ}C$</TEX>. The FII displayed fibrinolytic activity in the pH range 3-5, but was inactivated in the range of pH 6-11. The F-I and F-III showed enzyme activity in the pH range of 6-11. In the pH-thermostability, the F-II only kept fibrinolytic activity after heating at <TEX>$100^{\circ}C$</TEX> for 10, 20 and 30 min at pH 3 and pH 7, respectively. On the other hand, the F-II was retained activity until heating for 10 min under pH 11 condition. By using fibrin zymogram gel electrophoresis, extracellular fibrinolytic enzyme F-II from C. militaris showed unusual thermostable under acid and neutral conditions.