[Purification and characterization of fibrinolytic enzyme from Bacillus subtilis BS-26].
OBJECTIVE
Thrombolytic therapy is a safe and effective treatment for thrombotic diseases. Microorganisms are possible sources of thrombolytic drugs. We purified and characterized fibrinolytic enzyme produced by Bacillus subtilis strain BS-26.


METHODS
We examined the fibrinolytic enzyme activity by fibrin plate and purified fibrinolytic enzyme by ammonium sulfate fractional precipitation, anion-exchange chromatography on DEAE-Sepharose Fast Flow and preparative PAGE.


RESULTS
The fibrinolytic enzyme of the strain BS-26 was stable blow 50 degrees C and pH5.0-11.0, the optimal temperature was 42 degrees C and optimal pH was 9.0. Ca2+ and Mg2+ ions enhanced the fibrinolytic activity, whereas Cu2+ completely inhibited the enzyme. Phenylmethylsulfonyl fluoride (174.2 microg/mL), chicken ovomucoid (1000 microg/mL) and soybean trypsin inhibitor (1000 microg/mL) could inhibit enzyme activity, which indicated that the enzyme belonged to serine protease group. On plasminogen-free fibrin plates and plasminogen fibrin plates, the fibrinolytic activity had no obvious difference, indicating that the enzyme was a fibrinolytic enzyme which degraded fibrin directly, but not a plasminogen activator which degraded fibrin by activating plasminogen. A fibrinolytic enzyme was purified from the fermentation broth with recovery yield of 3.2%, purification factor of 41.0 fold and the specific activity 8750.0 U/mg. SDS-PAGE analysis of the purified protein showed only one band with molecular mass of 32 kDa.


CONCLUSIONS
A single fibrinolytic enzyme was purified, which provided the basis for large-scale production of fibrinolytic enzyme.
Niu
Shumin
S
College of Life Science, Agriculture University of Hebei, Baoding 071001, China.
Guo
Xiaojun
X
Li
Shuna
S
Yuan
Hongshui
H
Zhu
Baocheng
B
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D005285:Fermentation; D005341:Fibrinolysin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D005341:Fibrinolysin