Studies On The Fibrinolytic Enzyme From Two Mixed Aspergillus Fermenting Soybean Residue

Thrombotic disease has been a serious threat to people’s lives and health, but the findings of fibrinolytic enzyme solved this problem. Fibrinolytic enzyme which is a naturally occurring protein could dissolve the blood clot, and restore blood circulation smoothly, thus caused the concerns of many researchers.In this paper soybean residue was used as cultural medium, fibrinolytic enzyme activity and protein concentration were used as detection index, Na-4-tosyl-L-arginine methyl ester hydrochloride (TAME) was used as the substrate of fibrinolytic enzyme, and fibrinolytic enzyme was produced by two mixed Aspergillus fermenting soybean residue.Under the optimal conditions, the activity of fibrinolytic enzyme from Aspergillus oryzae3.800, Aspergillus niger3.4309and the two mixed Aspergillus were compared respectively. The author came to this conclusion that fibrinolytic enzyme activity produced by the two mixed Aspergillus reached1.10261TAME unit/mL, which was much higher than Aspergillus oryzae3.800and Aspergillus niger3.4309separately. The optimal time producing enzyme was for72h and12hours in advance. Therefore, the two mixed Aspergillus were chosen to ferment soybean residue producing fibrinolytic enzyme.The fermentation conditions were optimized by the single-factor experiment:inoculum concentration10%, Aspergillus oryzae3.800and Aspergillus niger3.4309ritio of1:4, temperature30℃, the initial water content75%and pH value7, wheat bran as the best C source, C/N0.25.By the Box-Behnken design to optimize medium, it was concluded that fibrinolytic enzyme activity reached1.2653TAME unit/mL under the following conditions:the proportion of bran and soybean residue0.22, the beginning pH value and the initial moisture content7.22and75.78%, respectively.After fermentation medium was extracted for24h by50mL0.9%NaCl at4℃, Crude enzyme solution which was obtained by6000r/min frozen centriufgation20min was purified by ammonium sulfate fractional precipitation, dialyse, DEAE-Sepharose FastFlow ion exchange chromatogram and gel filtration. A single protein fraction with fibrinolytic activity was obtained. The relative molecular weight of purifed protein was30KDa. The purification fold was9.49, the purification multiple was17.08%, and fibrinolytic enzyme specific activity was47.83.The optimum reaction temperature of purified fibrinolytic enzyme was40℃, and the purified fibrinolytic enzyme activity was kept above90%under25℃. Relatively, the optimum reaction pH value of the fibrinolytic enzyme was9.0, and the fibrinolytic enzyme activity kept above80%with the pH value was in the range of6.0～9.0. The metal ions K+, Ca2+showed activation, and Cu2+, Mn2+showed inhibition.The fibrinolytic enzyme activity didn’t show same on the fibrinplate and plasminogen-free fibrin plate which indicated that the enzyme might contain both a fibrinolytic enzyme which dissolved fibrin directly and a plasminogen activator which degraded fibrin by activating plasminogen. PMSF was inhibitor of the fibrinolytic enzyme.The subject systematically studied the fermentation conditions, purification and characters of fibrinolytic enzyme from the two mixed Aspergillus. Recently, most studies of fibrinolytic enzyme was about screening compatible strains, and testing enzyme in different fermentation conditions. The reserch about purification of fibrinolytic enzyme produced by mixed strains were very rare, and so did the mechanism. Therefore, this paper about fibrinolytic enzyme produced by two mixed Aspergillus provided a theoretical basis to a new thrombolytic.