Purification and characterization of fibrinolytic alkaline protease from Fusarium sp. BLB.
Fusarium sp. BLB, which produces a strongly fibrinolytic enzyme, was isolated from plant leaf (Hibiscus). Fibrinolytic alkaline protease was purified from a culture filtrate of Fusarium sp. BLB by precipitation with (NH4)2(SO4) and column chromatography with CM-Toyopearl 650 M and Superdex 75. The purified enzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was 27,000 by SDS-PAGE. Maximum activity of protease was observed at pH 9.5 and 50 degrees C. Purified protease was active between pH 2.5 and 11.5 and was found to be stable up to 50 degrees C. The enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy because this enzyme showed pH resistance. The activity was inhibited by diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases from Fusarium sp., Streptomyces griseus, Bos taurus bovine, Katsuwo pelamis digestive tract, and Lumbricus rubellus.
Ueda
Mitsuhiro
M
Laboratory of Biocycle Engineering, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan. mueda@biochem.osakafu-u.ac.jp
Kubo
Toshihiro
T
Miyatake
Kazutaka
K
Nakamura
Takumi
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001709:Biotechnology; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D005337:Fibrin / Q000378:metabolism*; D005670:Fusarium / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D031584:Hibiscus / Q000382:microbiology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000382:microbiology; D011480:Protease Inhibitors / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D011480:Protease Inhibitors; D005337:Fibrin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C087822:alkaline protease