Fibrinolytic Serine Protease Isolation from Bacillus amyloliquefaciens An6 Grown on Mirabilis jalapa Tuber Powders

• Published in: Applied biochemistry and biotechnology Vol. 162; no. 1; pp. 75 - 88
• Main Authors: Agrebi, Rym, Hmidet, Noomen, Hajji, Mohamed, Ktari, Nawrez, Haddar, Anissa, Fakhfakh-Zouari, Nahed, Nasri, Moncef
• Format: Journal Article
• Language: English
• Published: New York New York : Humana Press Inc 01-09-2010; Humana Press Inc; Springer; Springer Nature B.V
• Subjects: Animals; Bacillus - enzymology; Bacillus - growth & development; Bacillus amyloliquefaciens; Bacteria; Biochemistry; Biological and medical sciences; Biotechnology; Blood clots; carbon; casein; Cattle; Chemistry; Chemistry and Materials Science; cost effectiveness; culture media; Culture Media - metabolism; enzyme inactivation; Enzyme Stability; enzyme substrates; Enzymes; Fibrinolysis; Fundamental and applied biological sciences. Psychology; High temperature; Hydrogen-Ion Concentration; industrial microbiology; Mirabilis - metabolism; Mirabilis jalapa; molecular weight; nitrogen; Plant Tubers - metabolism; polyacrylamide gel electrophoresis; Powders; Proteases; Serine Proteases - chemistry; Serine Proteases - isolation & purification; Serine Proteases - metabolism; Serine Proteinase Inhibitors - pharmacology; serine proteinases; Subtilisin - isolation & purification; Subtilisin - metabolism; Temperature; tubers; Yeasts; Protease; Fibrinase; Serine endopeptidases; Mirabilis jalapa; Enzyme; Bacillus amyloliquefaciens; Bacillaceae; Plasmin; Peptidases; Bacillales; Tuber; Bacteria; Hydrolases; Isolation
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-009-8800-z

In this study, Mirabilis jalapa tuber powder (MJTP) was used as a new complex organic substrate for the growth and production of fibrinolytic enzymes by a newly isolated Bacillus amyloliquefaciens An6. Maximum protease activity (1,057 U/ml) with casein as a substrate was obtained when the strain was grown in medium containing (grams per liter) MJTP 30, yeast extract 6, CaCl₂ 1, K₂HPO₄ 0.1, and K₂HPO₄ 0.1. The strain was also found to grow and produce extracellular proteases in a medium containing only MJTP, indicating that it can obtain its carbon, nitrogen, and salts requirements directly from MJTP. The B. amyloliquefaciens An6 fibrinase (BAF1) was partially purified, and fibrinolytic activity was assayed in a test tube with an artificial fibrin clot. The molecular weight of the partially purified BAF1 fibrinolytic protease was estimated to be 30 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for the caseinolytic activity were 60 °C and 9.0, respectively. The enzyme was highly stable from pH 6.0 to 11.0 and retained 62% of its initial activity after 1 h incubation at 50 °C. However, the enzyme was inactivated at higher temperatures. The activity of the enzyme was totally lost in the presence of phenylmethylsulfonyl fluoride, suggesting that BAF1 is a serine protease.