Purification and biochemical characterization of a fibrinolytic enzyme from Bacillus subtilis BK-17 |
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Authors: | Jeong Yong Kee Park Jeong Uck Baek Hyun Park Sung Hoon Kong In Soo Kim Dong Wan Joo Woo Hong |
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Institution: | (1) Department of Microbiology, Dong-Eui University, Pusan, 614-714, Korea;(2) Department of Microbiology, College of Medicine, Gyeongsang National University, Chinju, 660-280, Korea;(3) Department of Chemical Engineering, Pusan National University, Pusan, 609-735, Korea;(4) Department of Biotechnology and Bioengineering, Pukyong National University, Pusan, 608-737, Korea;(5) Department of Microbiology, Changwon National University, Changwon, 641-773, Korea;(6) Department of Biology, Changwon National University, Changwon, 641-773, Korea |
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Abstract: | A fibrinolytic enzyme from Bacillus subtilis BK-17 has been purified to homogeneity by gel-filtration and ion-exchange chromatography. Compared to the crude enzyme extract, the specific activity of the enzyme increased 929-fold with a recovery of 29%. The subunit molecular mass of the purified enzyme was estimated to be 31 kDa by SDS–PAGE. The N-terminal amino acid sequence of the purified fibrinolytic enzyme was: A-Q-S-V-P-Y-G-V-S-Q-I-K-A-P-A-A-H-N. The sequence was highly homologous to the fibrinolytic enzymes nattokinase, subtilisin J and subtilisin E from Bacillus spp. However, there was a substitution of three amino acid residues in the N-terminal sequence. The amidolytic activity of the purified enzyme for several substrates was assessed. In comparison with nattokinase and CK (fibrinolytic enzyme from a Bacillus spp.), which showed strong fibrinolytic activity, the amidolytic activity of the enzyme for the synthetic substrate, kallikrein (H-D-Val-Leu-Arg-pNA, S-2266) increased 2.4- and 11.8-fold, respectively. |
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Keywords: | Amidolytic activity Bacillus subtilis BK-17 fibrinolytic enzyme homologous proteins N-terminal amino acid sequencing protease inhibitor thrombolytic agent |
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