[Identification of a fibrinolytic enzyme producing Bacillus pseudomycoides and purification and characterization of the enzyme].
OBJECTIVE
To purify a single fibrinolytic enzyme from Bacillus pseudomycoides B-60 and to determine its N-terminal sequence and to characterize the fibrinolytic enzyme.


METHODS
We examined the fibrinolytic enzyme activity by fibrin plate and purified fibrinolytic enzyme by ammonium sulfate fractional precipitation and DEAE anion exchange chromatography.


RESULTS
Obtained a single protein fraction with fibrinolytic activity (BpFE) from B. pseudomycoides B-60. It appeared as a single band in the SDS-PAGE with a relative molecular weight of 34 kDa. The fibrinolytic activity of the protein was stable at 4-50 degrees C and at pH 5-10. The activity sharply decreased above 50 degrees C, and the total loss of activity at pH 3.0. The enzymatic activity was slightly enhanced by the ions of Ca2+, Mg2+, Mn2+, whereas strongly inhibited by Cu2+ ion. Phenylmethyl sulfonyl fluoride (PMSF) could completely inhibit its activity. In addition, the activity improved when the protein was enzymatically hydrolyzed using trypsin and pepsin. The first 15 amino acids of the N-terminal sequence of the enzyme were determined to be VTGTNAVGTGKGVLG. The partial amino acids sequence alignment study of the enzyme from B-60 strain with bacillolysin, neutral protease and hydrolase which were from B. cereus, B. thuringiensis, B. anthracis and Lactobacillus sp. was carried out, and there is a 100% homogeneity between them.


CONCLUSIONS
We obtained a single fibrinolytic enzyme. Through its N-terminal sequence alignment study, a plasmin with high homogeneity to this protein was not found yet. This provided a basis for further study of new thrombolytic drugs.
Gu
Changling
C
College of Life Science, Agricultural University of Hebei, Baoding 071001, China. guchl2000@yahoo.com
Guo
Xiaojun
X
Li
Jia
J
Zhao
Xiaoyu
X
Zhu
Baocheng
B
D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D004275:DNA, Ribosomal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid
D004275:DNA, Ribosomal; D005343:Fibrinolytic Agents