Solid-State Fermentation,Purification And Characterization Of Fibrinolytic Enzyme From Neurospora Crassa

In recent years,thrombotic diseases have gradually increased,and have seriously endangered human health,and the global mortality rate of cardiovascular diseases remains high.Therefore,it is urgent to prevent and treat thrombotic diseases.Fibrinolytic enzyme can specifically hydrolyze fibrin,and microbial-derived fibrinolytic enzyme is the focus of current research.Neurospora crassa is an outstanding filamentous fungus with the advantages of short growth cycle,low production cost,fast enzyme production and high enzyme safety.It has been used in fermented products.Proteins with fibrinolytic activity in metabolites,and fibrinolytic enzyme from this source has not been reported.Thus,in this paper,the solid-state fermentation of Neurospora crassa was used to produce fibrinolytic enzyme,and the following studies were carried out on the enzyme:optimization of solid-state fermentation conditions,isolation and purification,enzymatic characterization and application of the fibrinolytic enzyme.Below are key research findings.（1）Firstly,agricultural and industrial by-products were used for solid-state fermentation to produce fibrinolytic enzyme by Neurospora crassa,and the fermentation process was optimized by single factor experiment,Plackett-Burman design,path of steepest ascent method and Box-Behnken design.The results were:the ratio of sunflower meal to wheat bran was 10:1,and the water content was 96%（v/w）,natural p H of the medium,15%（v/w）inoculum,cultured at 28℃for 5 d,the fibrinolytic activity of the crude enzyme reached 403.59 U/m L,which was nearly 2times higher than that of the unoptimized one.（2）Fibrinolytic enzyme was separated and purified by 40%-60%saturation ammonium sulfate precipitation,dialysis,SP-Sepharose Fast Flow cation exchange chromatography method.Specific activity of the purified enzyme reached to 3614.94U/mg,the recovery rate was 4.91%,and the purification fold was 17.89 times.The target enzyme was identified by SDS-PAGE to achieve electrophoresis purity,and the apparent molecular weight of the enzyme was determined to be about 32 k Da.（3）The optimum temperature and p H of the purified enzyme was 40℃and 7.4,which was close to the physiological environment of the human body,and it had a certain thermal stability in the range of 25-50℃,and it was stable at p H 5.0-9.0.Metal ions Na⁺（119%）,Zn²⁺（119%）and Cu²⁺（132%）could activate the fibrinolytic activity,K⁺,Mg²⁺and Mn²⁺had little effect on the enzyme activity,while the enzyme activity was inhibited by Ca²⁺（77%）and Fe²⁺（28%）.The protease inhibitors all showed different degrees of inhibition on the activity of the target enzyme,and the enzyme activity was slightly inhibited by N-p-Tosyl-L-phenylalanine chloromethyl ketone（TPCK）and ethylenediaminetetraacetic acid（EDTA）,strongly inhibited by pepstatin（68%）and completely inhibited by phenylmethylsulfonyl fluoride（0%）,soybean trypsin inhibitor（0%）and aprotinin（0%）.The sulfhydryl reagentβ-mercaptoethanol（130%）had a significant activation effect on the purified enzyme,while the reduced glutathione（67%）and oxidized glutathione（71%）strongly inhibited the activity;the denaturant urea（54%）and sodium lauryl sulfate（0%）and organic reagent acetone（85%）showed significant inhibition;the fibribolytic activity was activated by peptone（119%）,and sucrose,glycerol,bovine serum albumin（BSA）and lysine had no significant effect on fibrinolytic activity.In conclusion,the active center of the target enzyme may contain disulfide bonds and serine,and it belongs to the serine protease class.（4）The enzyme activity of the target enzyme on plasminogen-rich and plasminogen-free fibrin plates differed by 0.35%,indicating that the enzyme could not activate plasminogen and could directly dissolve fibrin.The target enzyme could effectively degrade the three polypeptide chains of fibrinogen,and the degradation rate was as follows:αchain（5 min）>βchain（30 min）>γchain（4 h）,and it did not have thrombin-like activity.The anticoagulant effect of the fibrinolytic enzyme on black goat blood was investigated in vitro,and it was found that the blood group containing the target enzyme only formed a small amount of clots after being incubated at 37°C for 24 h,and still had good fluidity,while the heparin sodium group（positive control）and normal saline group（negative control）was completely coagulated,indicating that the target enzyme had strong anticoagulant activity in vitro and had the potential to be developed as an anticoagulant drug.At the same time,the dissolution of black goat blood clot by the fibrinolytic enzyme was studied.After 4 h,the dissolution rates of100 U nattokinase,100 U purified enzyme and normal saline group were 1.34%,3.54%,0.06%,respectively,and reached 8%and 14%,0.13%after 24 h incubation,and the same dose of the purified enzyme to blood clot dissolution rate is higher than nattokinase,indicating that the purified enzyme has a certain ability to dissolve thrombosis.The tolerance of the fibrinolytic enzyme to the blood and gastric juice environment was simulated in vitro,and the enzyme activity did not change significantly after incubation at 37°C for 4 h in the simulated blood environment,indicating that the target enzyme could withstand the blood environment in a short time;The relative enzyme activities were 0%,0%and 82%,respectively,when the purified enzyme was incubated at 37°C for 4 h before simulated eating,in gastric juice after consuming sucrose and after consuming protein,showed the enzyme could be protected by protein substances.It is speculated that the fibrinolytic enzyme may have the potential to be developed into intravenous and oral drugs.