Abstract

Fibrinolytic microorganisms were screened from the Chinese soy cheese doufuru as well as Japanese natto and Chinese douchi. Initially, 16 isolates with fibrinolytic activity were selected based on activity and cultural characteristics for further characterization. Anexponential standard curve, covering 39 to 2500 IU/mL of urokinase activity, was well established with urokinase through the fibrin plate method. With fermentation and preliminary purification, the fibrinolytic activities of the crude enzymes of these isolates were estimated to range across 142 to 1833 IU/mL of urokinase through the standard curve using the thrombolytic area on the fibrin plate. The strain FR-33 from doufuru was chosen based on the maximum activity 1833 IU/mL and better reproducibility. Further purification of its crude enzyme through Sephadex G-50 gel filtration increased theenzyme activity to 2474 IU/mL, and the SDS-PAGE performance indicated that its molecular weight is approximately 30 kDa. The 16S rDNA of the isolate FR-33 was sequenced and analyzed, and the strain was identified as Bacillus subtilis based on the sequence combined with morphological, cultural, physiological and biochemical characteristics. However, in contrast to the typical B. subtilis, the strain FR-33 could grow in 9 and 12% NaCl, but could not secret pyruvate dehydrogenase and urease, nor ferment lactose, sucrose and maltose. The crude enzyme of the isolate showed stronger activity than those reported from natto and douchi, and this makes it an attractive agent as a biomaterial for health-producing foods.

Key words: Doufuru, Bacillus subtilis, fibrinolytic enzyme, isolation, identification.