5530
Nora Nabil Abdel aleem Elgamal
Production And Biochemical Studies On Thrombolytic Enzyme From Some Indigenous Bacilli Of Egyptian Environments
Bacillus subtilis, blood clots, fibrinolytic enzyme, solidstate fermentation, purification
This work included the isolation of strains of Bacillus spp. from different Egyptian Governorates. These isolates were screened for their ability to produce fibrinolytic enzyme. Bacillus isolate No. achieved the highest fibrinolytic enzyme activity and it was identified by S rRNA sequencing and data analysis as Bacillus subtilis strain with similarity. Central composite design was employed to optimize and investigate interactions between critical variables needed for enzyme production. In submerged fermentation the practical application of the optimal concentrations of each parameter produced U/ml of the enzyme under study. But in solid state fermentation, the fibrinolytic enzyme activity was U/g. Purification of fibrinolytic enzymes from Bacillus subtilis Egy. was carried out by three-steps purification scheme. Partial purifiation by salting out with ammonium sulphate ( - saturation). Under these conditions, specific activity reached U/mg protein and yield recovery . Then, after dialysis against mM TrisHCl buffer pH over night, this partially purified enzyme was applied to ion exchange column on DEAE cellulose, third purification step (Gel filtration on Sephadex G- ), the specific activity of U/mg protein and yield recovery of . Accordingly, the fibrinolytic enzyme of Bacillus subtilis Egy. was purified times with final yield recovery of . The molecular weight of the purified enzyme is KDa by SDS PAGE electrophoresis. Purified enzyme is heat stable upon heating to oC its activity increased progressively with incubation reaction temperature up to oC at pH . Upon using different reaction incubation times on purified enzyme activity shows increasing with increase the reaction incubation time till min. The enzyme activity responded linearly to the increase in substrate concentration up to mg/mL. Thrombolytic activity of purified fibrinolytic enzyme of Bacillus subtilis Egy. showed significant degradation of blood clot and it is safe to use because it exhibited no mortality after days of subcutaneous administration of rats.
2018
Ph.d
Ain Shams
Science