A novel alkaline serine protease with fibrinolytic activity from the polychaete, Neanthes japonica

SummaryProtease-like activity which split plasminogen-free fibrin was demonstrated in 2 M KSCN extracts of the lung and spleen of conventional rats. The activity was virtually undetectable in tissue extracts from germ-free rats. The extracts from the conventional rat tissues split fibrin and fibrinogen remarkably at neutral pH, but not casein, when examined using fibrin, fibrinogen-agar and casein-agar plates. The fibrinolytic activity was inhibited by STI and DFP, indicating a serine protease nature. The activity was not inhibited by TLCK, t-AMCHA or dansyl-L-arginine-methylpiperidine amide (a selective synthetic thrombin inhibitor, OM189). It was neither activated nor inhibited by cysteine, KCN or iodoacetic acid. The results obtained indicate that the protease-like activity of the lung and spleen extracted with 2 M KSCN from conventional rats has properties which differ from those of trypsin, plasmin, plasminogen-activator, thrombin, and cathepsin A, B and C.

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Molecular Cloning, Nucleotide Sequence, and Expression of the Structural Gene for Alkaline Serine Protease from AlkaliphilicBacillussp. 221

Bioscience Biotechnology and Biochemistry ◽

10.1271/bbb.56.1455 ◽

1992 ◽

Vol 56 (9) ◽

pp. 1455-1460 ◽

Cited By ~ 12

Author(s):

Hideto Takami ◽

Tetsuo Kobayashi ◽

Masato Kobayashi ◽

Mami Yamamoto ◽

Satoshi Nakamura ◽

...

Keyword(s):

Nucleotide Sequence ◽

Molecular Cloning ◽

Serine Protease ◽

Structural Gene ◽

Alkaline Serine Protease

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Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea

Canadian Journal of Microbiology ◽

10.1139/m76-023 ◽

1976 ◽

Vol 22 (2) ◽

pp. 165-176 ◽

Cited By ~ 34

Author(s):

Poh Seng Ong ◽

G. Maurice Gaucher

Keyword(s):

Serine Protease ◽

Thermophilic Fungus ◽

Amino Acid Residues ◽

Submerged Cultures ◽

Ph Optimum ◽

Alkaline Serine Protease ◽

Associated Production ◽

Specificity Study ◽

Malbranchea Pulchella ◽

The Stability

The thermophilic fungus Malbranchea pulchella produces a single extracellular, alkaline, serine protease when grown at 45 °C, on 2% casein as sole carbon source. The growth-associated production of protease in submerged cultures was inhibited by addition of glucose, amino acids, or yeast extract. A simple four-step purification which yields homogeneous protease in 78% yield is described. The protease has an isoelectric point of 6.0, a pH optimum of 8.5, and is completely inhibited by serine protease inhibitors. A specificity study with small synthetic ester substrates indicated that the protease preferentially hydrolyzed bonds situated on the carboxyl side of aromatic or apolar amino acid residues which are not β-branched, positively charged or of the D configuration. Peptidase substrates and others such as N-acetyl-L-tyrosine-ethyl ester were not hydrolyzed. The protease was stable over a broad range of pH (6.5–9.5 at 30 °C, 20 h), and was particularly thermostable (t1/2 = 110 min at 73 °C, pH 7.4) in the presence of Ca2+ (10 mM). Macromolecules and Ca2+ also provide protection against the significant autolysis which occurs at pure protease concentrations greater than 0.01 mg/ml, as well as against surface denaturation which is enhanced by the presence of a silicone antifoam agent. Hence the stability of protease in submerged cultures is rationalized.

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