Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1

A mutant of Bacillus subtilis IMR-NK1, which is used for the production of domestic "natto" in Taiwan, produced high fibrinolytic enzyme activity by s olid-state fermentation using wheat bran as medium. In addition, a strong f ibrinolytic enzyme was purified from the cultivation media. The purified en zyme was almost homogeneous, as examined by SDS-PAGE and capillary electrop horesis. The enzyme had an optimal pH of 7.8, an optimal temperature of 55 degrees C, and a K-m of 0.15% for fibrin hydrolysis. The molecular mass est imated by gel filtration was 31.5 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 8.3. The enzyme also showed act ivity for hydrolysis of fibrinogen, casein, and several synthetic substrate s. Among the synthetic substrates, the most sensitive substrate was N-succi nyl-Ala-Ala-Pro-Phe-pNA. PMSF and NBS almost completely inhibited the activ ity of the enzyme. These results indicate that the enzyme is a subtilisin-l ike serine protease, similar to nattokinase from Bacillus natto.