Purification and biochemical properties of a fibrinolytic enzyme from Bacillus subtilis-fermented red bean

• Published in: Food chemistry 2012-08, Vol.133 (4), p.1611-1617
• Main Authors: Chang, Chen-Tien, Wang, Pei-Ming, Hung, Ya-Fang, Chung, Yun-Chin
• Format: Article
• Language: English
• Subjects: Ammonium sulfate; Bacillus; Bacillus subtilis; Biochemical properties; Biological and medical sciences; Fibrinolytic enzyme; Food industries; Food microbiology; Fruit and vegetable industries; Fundamental and applied biological sciences. Psychology; Red bean
• ISSN: 0308-8146; 1873-7072
• DOI: 10.1016/j.foodchem.2012.02.061

► Bacillus subtilis fermented red bean with fibrinolytic activity was prepared. ► The purified fibrinolytic enzyme was a subtilisin-like serine protease with 29.93kDa. ► This enzyme showed amidolytic activity for the hydrolysis of several synthetic substrates. Natto-red bean with fibrinolytic activity was prepared by fermenting red beans with Bacillus subtilis. A fibrinolytic enzyme was purified from fermented natto-red bean by sequential steps of ammonium sulfate fractionation, Sephacryl S-200 HR gel filtration and PBE 94 chromatofocusing. Through these steps, the purity of the enzyme increased 291-fold with 1.5% activity recovery. SDS–PAGE and isoelectric focusing electrophoresis showed the molecular mass and pI of the purified enzyme to be 29.93kDa and 6.35, respectively. When N-succinyl-Ala-Ala-Pro-Phe-ρNA was used as an enzyme substrate, the Km, Vmax, and optimal reaction pH and temperature were 0.59mM, 79.4μmole ρNA/minmg, 9 and 60°C, respectively. Among the synthetic substrates, the most sensitive were N-succinyl-Ala-Ala-Pro-Phe-ρNA, followed by N-benzoyl-Val-Gly-Arg-ρNA. Chemical modifiers, such as phenylmethyl sulfonyfluoride, N-bromosuccinimide and N-ethyl-5-phenylisoxazolium-3′-sulfonate, almost completely inhibited the activity of the purified enzyme. These results indicated that the purified fibrinolytic enzyme was a subtilisin-like serine protease.