Solid State Fermentation,Purification And Characteristics Of A Fibrinolytic Enzyme Of Rhizopus Microsporus

Cardiovascular diseases, including acute myocardial infarction (MI), ischemic heart disease and high blood pressure are the leading causes of death in the world. The insoluble fibrin fiber is hydrolyzed into fibrin degradation products by plasmin, which is generated from plasminogen by plasminogen activators (PA), such as tissue plasminogen activator (t-PA), reconstructive tissue plasminogen activator (rt-PA) and urokinase. Thrombosis occurs when the clots are not lysed. A variety of plasminogen activator have been widely studied and used as thrombolytic agents. However, these agents are very expensive and have some side effects. Therefore the searches for new fibrinolytic enzymes from various sources are being continued. Fibrinolytic enzyme starting strain (identified as Rhizopus microsporus var. tuberosus) isolated by our laboratory was confirmed as high fibrinolytic activity. The dissertation focused on the optimization of solid fermentation condition for fibrinolytic enzyme production, purification and partial characteristics of the fibrinolytic enzyme.The results show that the appropriate medium are as follows:carbon (bran) nitrogen (bean pulp) ratio 2:1, moisture of culture 1 mL/g dry basis, initial pH 5.0, MgSO4 1.2 g/kg, 26.4 g/kg and FeSO40.456g/kg. The optimized inoculum size, fermentation time and temperature are 6×106 spores/g solid substrate,72 h and 29℃, respectively. Under the optimized conditions the average fibrinolytic enzyme activity reached 684.39± 34.22 U/g. It is to 8.2 times that of the original enzyme activity, indicating that solid-state fermentation of rhizopus microsporus var. tuberosus for production of fibrinolytic enzyme is provided with industrial potential, and providing the base data foundation of industrialization.The fibrinolytic enzyme from Rhizopus microsporus var. tuberosus was purified through extraction, ammonium sulfate precipitation, DEAE-Sepharose FF weak anion exchange chromatography, Sephadex G-75 gel chromatography and HiTrap Capto Q strong anion exchange chromatography with a final activity 2329.74 U/mg. The purification protocol resulted in a 70.81-fold purification of the enzyme, with a final yield of 24.7%. The apparent molecular weight of the enzyme was 27.8 kDa, determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis.The enzyme not only degrades fibrin directly, but also activates plasminogen into plasmin to degrade fibrin. The purified enzyme exhibits maximum activity at 37℃ and pH 7.0, respectively. The enzyme is stable at less than 37℃ and retained more than 95% of its original activity for 4 h. The enzyme is stable at neutral and retained most of its original activity, but it lost all activity when pH< 3.4 and pH> 11.8. The enzyme activity is promoted in the presence of Na、Ca2+、Mg2+ and K+, and Mg2+ promoted the best; Whereas the presence of Zn2+、Fe2+、Cu2+ and Mn2+ inhibits the enzyme activity, and Cu2+ inhibited most of all the activity. Inhibitory effect of EDTA is more intense than the metal ions. Under the conditions of the presence of EDTA (10 mmol/L), the enzyme almost lost all activity.