Purification and characterization of fibrinolytic enzyme from a bacterium isolated from soil

A novel extracellular enzyme with strong fibrinolytic activity, produced by Bacillus tequilensis, which was isolated from the soil of Zhuhai City (China) was purified and characterized. The enzyme was secreted by cultured B. tequilensis in solid state and purified at a high efficiency using the combination of salting out, ion exchange chromatography, and size exclusion chromatography. The enzyme was estimated to have a molecular weight of approximately 27 kDa, pI of 8.9 +/- 0.1, to stable at pH 5.0-12.0 and up to 50 degrees C; the optimum pH and temperature are 10.5 and 45 degrees C (2373.59 +/- 54.81 U/mg), respectively. The fibrinolytic activity was enhanced by K+, Na+, Mg2+, Mn2+, Ca2+, and Ba2+ and inhibited by Cu2+, Zn2+, and Fe3+. Moreover, the activity was slightly enhanced by PMSF and EDTA at low concentrations and inhibited by beta-mercaptoethanol. The N-terminal amino acid sequence is AQSVPYGISQI. The enzyme has a higher enzymatic activity than most other fibrinolytic enzymes. The high thermal stability indicated that it is easy to preserve and could be activated under high-temperature conditions.