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

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 pl of the purified enzyme to be 29.93 kDa and 6.35, respectively. When N-succinyl-Ala-Ala-Pro-Phe-pNA was used as an enzyme substrate, the K-m, V-max, and optimal reaction pH and temperature were 0.59 mM, 79.4 mu mole pNA/min mg, 9 and 60 degrees C, respectively. Among the synthetic substrates, the most sensitive were N-succinyl-Ala-Ala-Pro-Phe-pNA, followed by N-benzoyl-Val-Gly-Arg-rho NA. Chemical modifiers, such as phenyl methyl 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. (C) 2012 Elsevier Ltd. All rights reserved.