Purification And Characteristics Of The Fibrinolytic Enzyme From Cordyceps Militaris

As the main drug to prevent and treat thromboembolism disease, thrombolytic agents ofmicrobial origin show a wide prospect. Our project group found that Cordyceps militarisproduced at least two kinds of fibrinolytic enzymes by submerged cultivation when sucroseand bean cake as major materials. In this paper, the purification methods, resistant coagulationand thrombolysis properties of the fibrinolytic enzymes from Cordyceps militaris wereinvestigated, and established a hyperlipaemia model with the tendency of thrombus, and thentesting the influence of these enzymes to ibrinolytic ability, which are very help for developefficient, safe, economical and novel thrombolytics agents and provide a theoretical basis.The fibrinolytic enzymes were purified by20%ammonium sulfate precipitation,Sephadex G-25gel filtration chromatography, Phenyl-Sepharose HP hydrophobic interactionchromatography, CM-Sepharose FF ion exchange chromatography and Superdex75gelfiltration chromatography. The purified enzymes were homogeneous, as examined by gelfiltration chromatography and SDS-PAGE. The specific activity of fibrinolytic enzyme I was1467.44U/mg, the total purification fold was36.07, and recovery yield was5.79%. Thespecific activity of purified fibrinolytic enzyme II was1681.58U/mg, the total purificationfold was41.33, and recovery yield was4.00%.Eight peptide sequences of the enzyme were determined by Q-TOF2mass spectrometry,and the results of peptide sequences were: I-E-D-F-P-Y-Q-V-D-L-R, A-N-C-G-G-T-V-I-S-E-K, Y-V-L-T-A-G-H-C-A-E-G-Y-T-G-L-N-I-R, T-N-Y-A-S-V-T-P-I-T-A-D-M-I-C-A-G-F-P-E-G-K, K-D-S-C-S-G-D-S-G-G-P-L-V-T-G-G-K, D-S-C-S-G-D-S-G-G-P-L-V-T-G-G-K, V-V-G-I-V-S-F-G-T-G-C-A-R, A-N-K-P-G-V-Y-S-S-V-A-S-A-E-I-R, and had100%homologywith trypsin-like serine protease CM01from Cordyceps militaris.Cordyceps militaris fibrinolytic enzyme could inhibit the formation of thrombus block atany stage of the coagulation process; the effect in vitro thrombolysis was notable, it coulddissolve blood clots mainly within4h; The order that the fibrinolytic enzyme act with bloodprotein fiber (former) was that decomposition rate of α-subunit was larger than β-andγ-subunits. The fibrinolytic enzyme didn’t have kinds of blood coagulation enzyme activity.And it could hydrolyse the immune globulin and serum albumin partly, which indicate thisenzyme will exist certain effects in actual drug use process, but the influence was not sinificant. At the same time, the fibrinolytic enzyme can make the thrombin degrade partly.From these results we can conclude that, the enzyme could also have certain effect inthrombosis preventative treatment. The fibrinolytic enzyme had no effct on the behavior andcoordination movement of mices, and the mices were in good health, without allergicphenomenon.The fribrinolytic enzyme can significantly reduce the TC, TG, LDL-C, and increase thelevel of HDL-C of mice with Hyperlipidemia. There were significan differences between highand low groups (p<0.01) at TC and TG level, respectively; The high group showedsignificantly differences at HDL-C (p<0.01); The two groups show significantly differencesat LDL-C (p<0.01or p<0.05). The heart, spleen, kidney weigh indices of low and high dosageof fibrinolytic enzyme was higher than the hyperlipidemia, and the liver weigh index waslower, which can protect the heart, spleen, kidney and liver.