Studies On The Fermentation For Fibrinolytic Enzyme From Pichia Pastoris Pk53

Thrombus often lead to serious thrombotic cardiovascular disease,sudden onset,high death rate,fibrinolytic therapy is a common means . Fibrinolytic enzyme is able to break down fibrin clots and thus dissolve clots to restore blood flow and promote tissue recovery. the thrombolytic enzyme do not hydrolyze plasma protein fibers,so unlikely to cause bleeding tendency. Therefore, fibrinolytic enzyme has a broad development prospect.Pichia pastoris expression system is one of the best and most widely used foreign gene expression system. Exogenous eukaryotic gene can be corrected translation and post-translational processing,it is able to product many proteins,what more the product easy to be purified. Fermentation medium of Pichia Yeast is expensive,we has been constructed a genetically engineered strain in the early . This study found a cheap culture medium, reducing the production cost of fibrinolytic enzyme,and studies on fermentation conditions for the liquid and the 10L fermenter amplification test.The fermentation conditions of pichia pastoris pk53,including carbon source,nitrogen source,inorganic salt,metal iron,surfactants and so on,were investigated in shake flask level. It was found that the concentrations of glucose,Soybean powder,KH2PO4 had the more obvious influences. The fermentation medium were studied via single factor test and orthogonal design of the three factors.The optimal fermentation medium was determined as follows:wheat bran 1.5%,Soybean powder 2.0%,KH₂PO₄,0.5%,MgSO₄·7H₂O 0.05%,On the process of optimizing fermentation condition,impact of inoculation amount, methanol supply,initial pH,induce pH, dissolution oxygen on fermentation have been studied in shake flask level. And the best fermentation conditions were:inoculating time 14 h,inoculating amount 1%,methanol supply 1.5%,medium volume 30 mL in 250 mL flask,initial pH 5.5,induce pH 6.0.orthogonal test of the four factors was designed to studying the methanol supply,induce pH,medium with initial pH,flask liquid volume in the four factors on the impact of enzyme production. The results showed that R flask liquid volume > R methanol supply > R induced pH> R initial pH,Under such conditions,the activity of fibrinolytic enzyme was 1841.28 U/mL,which was 3.02 times of the original strain. and the peak enzyme production a day ahead.Based on the results at shake flask level, scale up studies were carried out in 10 L fermentor. The metabolizing parameters including dissolution oxygen,pH,total sugar, deoxidize sugar, protein and fibrinolytic enzyme activity were tested.It laid a foundation for large scale fermentation.