Solid State Fermentation Technique And Chracterization Of Cordyceps Sinensis Plasmin

In recent years,thrombotic diseases have seriously impacted people’s health.The incidence of thrombotic diseases is increasing year by year.The incidence of thrombotic diseases shows a trend of younger age.The treatment of thrombotic diseases is still the focus of current research.At present,the drugs used to treat thrombosis in clinic generally remain effects for a short time and have weak thrombolytic ability and poor specificity.They are easy to cause systemic hemorrhage in vivo,so there are great risks.Therefore,the research and development of efficient,safe and specific thrombolytic drugs will be of great significance to human health.Cordyceps sinensis is one of the traditional and precious medicinal fungi in China.It has antimicrobial,anti-inflammatory,anti-tumor,anti-oxidation and other pharmacological activities.Cordyceps sinensis fibrinolytic enzyme is a protease that degrades fibrin directly and has potential for thrombolytic drug development.Using solid-state fermentation technology,a large number of mycelia and their metabolites can be obtained in a short time,which is expected to achieve large-scale production.In this paper,the solid-state fermentation technology,purification and enzymatic properties of Cordyceps sinensis fibrinolytic enzyme were systematically studied.The main results are as follows:1.Starting from several edible and medicinal fungi preserved in the laboratory,Cordyceps sinensis was selected as the experimental object,and the solid-state fermentation technology for producing fibrinolytic enzyme was optimized.Based on the yield of fibrinolytic enzyme in solid-state fermentation of Cordyceps sinensis,the optimum medium was determined as: grain A 63%,grain B 27% and rice bran 10%,added soluble starch 2.5%(w/w)and water 25%(v/w),and the optimum fermentation conditions were as follows: inoculation 0.24 m L/g,incubation at 22 ℃ for 8 days.After fermentation,the activity of fibrinolytic enzyme in crude enzyme solution reached 109.60 UI/m L,which was 1.82 times higher than that before optimization.2.The purified fibrinolytic enzyme,termed as CSP-1,was obtained by ammonium sulfate fractionation,Hi Trap SP cation exchange chromatography and Superdex 75 gel filtration chromatography.The result of native-PAGE gel electrophoresis showed a single band with a specific activity of 4186.25 UI/mg.3.Subsequently,enzymatic properties of CSP-1 were analyzed.It was found that CSP-1 is composed of two subunits with molecular weight of 27.60 k Da and 23.83 k Da,respectively,by SDS-PAGE gel electrophoresis.The activity of CSP-1 is the highest at 40 ℃ and p H 4.00.CSP-1 has no high thermostability,and its activity decreased rapidly above 40 ℃.Typical serine protease inhibitors PMSF and protitin inhibited the enzymatic activity by 56.43% and 53.61%,suggesting that the enzyme might be a serine protease.Cu2+,Mg2+,Ca2+ could promote the activity,while Zn2+,Fe3+,Al3+ inhibited the activity.The sequence of hydrolysis of fibrinogen by CSP-1 was γ chain-A α chain-B β chain.It was also found that the enzyme not only displayed the ability of degrading fibrin,but also activated plasminogen,showing excellent potential for development of new thrombotic drug.