Purification and characterization of Ophiocordyceps sinensis plasmin from solid culture mycelia

Abstract:

[Background] In recent years, thrombotic diseases have seriously affected people’s health even at younger age. The research of efficient, safe and specific thrombolytic drugs will be of great significance to human health. [Objective] To establish a method for separation and purification of plasmin produced by Ophiocordyceps sinensis from solid culture, and to analyze the enzymatic properties of purified plasmin. [Methods] Ophiocordyceps sinensis plasmin was separated by ammonium sulfate salting-out, HiTrap SP cation exchange chromatography and Superdex 75 gel filtration chromatography. The protein concentration was determined by Bradford method, the plasmin activity was determined by the fibrin plate method, the purity was determined by Native-PAGE, and the relative molecular weight was determined by SDS-PAGE. [Results] In solid culture, Ophiocordyceps sinensis mycelia could produce at least two plasmins, wherein the purified OSP-1 specific activity reached 4 186.25 U/mg, the purification factor was 41.69 times. OSP-1, a serine protease, is composed of two subunits with relative molecular weights of 27.60 kD and 23.83 kD respectively. The optimum temperature and pH of the enzyme were 40 °C and 4.0, respectively. Cu2+ promotes OSP-1 activity, while Zn2+ inhibits enzyme activity. It was also found that OSP-1 not only displayed the ability to degrade fibrin but also activated plasminogen. The enzyme sequentially degrades the γ chain, the Aα chain, and the Bβ chain in the process of hydrolyzing fibrin. [Conclusion] The above results provide a theoretical basis for the development of the enzyme into a new thrombolytic drug.