Liquid Fermentation Technology And Pegylation On A Fibrinolytic Enzyme From Bacillus Subtilis SBS

The optimum liquid fermentation technology,isolation and purification as well as pegylation of Bacillus Thrombolytic enzyme(BTE) produced by Bacillus subtilis SBS screened from rice straw were studied in this paper.The aim of BTE pegylation was to improve its characteristics and give some basis for application in the future.The results showed that the optimal fermentation technology were as follows(g/L): oat bran 7,soluble starch 3,soybean isolate protein 25,CaCl₂ 0.2,natural pH 8.0 at 37℃with inoculation age 10h,inoculation volume 5%.Under the above conditions the liquid culture was carried out for 70h in 15L bioreactor,The activity of BTE amounted to 542 IU/mL.The isolation and purification of the BTE were studied using ammonium sulfate fraction precipitation and ion-exchange chromatography on DEAE-52 and Sephadex G75 gel filtration.The specific activity of purified BTE was 11854 IU/mL,and purification fold was 12.27 and a yield of 80.66%.SDS-PAGE displayed only one band,and the molecular weight of BTE was estimated to be 32 KDa.Fibrin-SDS-PAGE showed only 32 KDa protein had the thrombolytic ability.Site-directed pegylation modification of BTE was studied with branched PEG synthesized with glutamic acid as the central molecule.The results indicated that BTE activity decreased rapidly,SDS-PAGE band blurred,so the aim not realized.