Abstract
A potent fibrinolytic enzyme from Bacillus megaterium KSK-07 isolated from an Egyptian fermented food, kishk, has been purified to electrophoretic homogeneity by gel filtration and anion-exchange chromatography. Purification increased its specific activity to 1124-fold with a recovery of 23%. The subunit molecular mass of the purified enzyme was estimated to be 28.5 kDa by SDS-PAGE. The optimal reaction temperature, pH and pI values for chymotrypsin from B. megaterium KSK-07 were 50°C, 8.0, and 10.0, respectively. Enzyme hydrolyzed not only fibrin but also several synthetic substrates, particularly 3-carbomethoxypropionyl-L-arginyl-L-prolyl-L-tyrosine p-nitroaniline hydrochloride (MeO-Suc-Arg-Pro-Tyr-pNA-HCl). In addition, PMSF can completely inhibit its fibrinolytic activity. These results indicated that chymotrypsin from B. megaterium KSK-07 is a chymotrypsin-family serine protease. Its apparent K M , V max and K cat for the synthetic substrate L-succinyl-L-phenylalanine p-nitroanilide (N-Suc-Phe-pNA) were 0.61 mM, 10.2 μmoles mg−1min−1, and 56.7 s−1, respectively. It demonstrated direct action upon blood clots in vitro and prolonged the blood clotting time to 1.9-fold. The enzyme could not degrade collagen suggesting this enzyme be an effective thrombolytic agent with high specificity to fibrin and non-specificity to other plasma proteins.
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Kotb, E. Purification and partial characterization of serine fibrinolytic enzyme from Bacillus megaterium KSK-07 isolated from kishk, a traditional Egyptian fermented food. Appl Biochem Microbiol 51, 34–43 (2015). https://doi.org/10.1134/S000368381501007X
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DOI: https://doi.org/10.1134/S000368381501007X