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Screening of a high fibrinolytic enzyme producing strain and characterization of the fibrinolytic enzyme produced from Bacillus subtilis LD-8547

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Abstract

A fibrinolytic enzyme producing strain Bacillus subtilis LD-8 was isolated from douchi, a traditional Chinese soybean-fermented food. After mutagenesis treatments by UV, NTG (N-methyl-N′-nitroso-N-nitroso-guanidine) and γ-radiation, a high fibrinolytic enzyme producing strain B. subtilis LD-8547 was obtained. Under optimum condition, LD-8547 was able to yield the average fibrinolytic activity of 4220 U/mL in 15 L fermenter. The strong fibrin-specific enzyme was purified from supernatant of B. subtilis LD-8547 culture broth using the combination of various steps. The optimal temperature and pH value of this fibrinolytic enzyme were 50 °C and 8.0, respectively. The molecular weight was about 30 kDa measured by SDS-PAGE. The amidolytic activity of this fibrinolytic enzyme was inhibited completely by 1 mmol/L phenylmethanesulfonyl fluoride (PMSF), but EDTA and EGTA did not affect the enzyme activity. The apparent K m and V max values were 0.521 mmol/L and 0.049 mmol/min, respectively. In vitro assays revealed that the enzyme could catalyze blood clot lysis effectively, indicating that this enzyme could be a useful thrombolytic agent.

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Acknowledgement

This work was supported by the Education Office of Fujian Province of China (No. JA05240, JB06115), the Natural Sciences Foundation of Fujian Province of China (No. B0610008, 2006J0054), and by the Science and Technology Department of Fujian Province of China (2005J017). Prof. Yi-Zheng Zhang and Dr. Yong Peng (Sichuang University, Chengdu, China) were greatly appreciated for technology discussion and reference donation.

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Correspondence to Shi H. Wang.

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Wang, S.H., Zhang, C., Yang, Y.L. et al. Screening of a high fibrinolytic enzyme producing strain and characterization of the fibrinolytic enzyme produced from Bacillus subtilis LD-8547. World J Microbiol Biotechnol 24, 475–482 (2008). https://doi.org/10.1007/s11274-007-9496-2

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Keywords

  • Fibrinolytic enzyme
  • Isolation
  • Purification
  • Activity
  • Thrombolytic agent