African Journal of Biotechnology Vol. 10(8), pp. 1408-1420, 21 February, 2011 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB10.1268 ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper Isolation, production, purification, assay and characterization of fibrinolytic enzymes (Nattokinase, Streptokinase and Urokinase) from bacterial sources R. Dubey*, J. Kumar, D. Agrawala, T. Char and P. Pusp Biotechnology centre, Galaxy Concepts Pvt. Limited. Kolkata, India. Accepted 10 January, 2011 Nattokinase, Streptokinase and Urokinase are novel fibrinolytic enzymes which are isolated from Bacillus subtilis, β-haemolytic Streptococci and urine sample . The fibrinolytic enzyme Nattokinase, Streptokinase and Urokinase was purified from supernatant of Bacillus subtilis, β-haemolytic Streptococci and recombinant E.coli containing short fragment genomic DNA of Pseudomonas sp. Culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH of Nattokinase, Streptokinase and Urokinase were 37-55°C and 9, 27-37°C and 7 and 55°C and 9, respectively. The molecular weight of Nattokinase, Streptokinase and Urokinase was approximately 28 kDa, 47 kDa and 34 kDa, respectively, as determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The caseinolytic activity of Nattokinase, Streptokinase and Urokinase were 576.73 U, 467.73 U and 785.73 U, respectively, while fibrinolytic activity achieved by fibrin plate method were 10 U, 5 U and 15 U, respectively. Key words: Anticoagulant activity, submerge fermentation, fibrinolytic enzyme activity, protein fraction precipitation, casein, serum and plasminogen plate technique, enzyme thermodynamics, haemolytic activity, enzyme screening, expression system, zymography, Edman degradation. INTRODUCTION Fibrinolytic enzymes were identified and studied among physiochemical properties have been characterized. many organisms including snakes, earthworms, and bac- Fermented shrimp paste, a popular Asian seasoning, was teria: Streptococcus pyogenes, Aeromonas hydrophila, shown to have strong fibrinolytic activity. These novel Serratia E15, B. natto , Bacillus amyloliquefacens , Actino- fibrinolytic enzymes derived from traditional Asian foods mycetes and fungi: Fusarium oxysporum ; Mucor sp, are useful for thrombolytic therapy. They will provide an Armillaria mellea (Jian Sha et al, 2003). adjunct to the costly fibrinolytic enzymes that are Fibrinolytic enzymes can be found in a variety of foods, currently used in managing heart disease, since large such as Japanese Natto, Tofuyo, Korean Chungkook- quantities of enzyme can be conveniently and efficiently Jang soy sauce and edible honey mushroom. Fibrinolytic produced. In addition, these enzymes have significant enzymes have been purified from these foods and their potential for food fortification and nutraceutical applica- tions, such that their use could effectively prevent cardio- vascular diseases (Yoshinori et al., 2005). Accounts of cardiovascular diseases have become the *Corresponding author. E-mail: [email protected]. leading cause of death in the Western world (Viles et al., 2004). Many blood clot-dissolving agents, such as uro- Abbreviations: t-PA, Tissue plasminogen activator; SK, kinase, streptokinase, and tissue plasminogen activator streptokinase; NAT, nattokinase; UK, urokinase; CU, caseinolytic activity; PTC, phenyl thiocarbamoyl; TLC, thin layer (t-PA), have been utilized in clinical treatments for cardio- chromatography; PMSF, phenyl methyl sulfonyl fluoride; EDTA, vascular diseases. Hemostasis is a complex process ethylene diamine tetraacetic acid; SDS-PAGE, sodium obtained through an optimal balance between bleeding deodecyl sulphate polyacrylamide electrophoresis; BSA, bovine and blood clot formation. In an unbalanced state, fibrin serum albumin; PTH, phenylthiohydantoins. clots may not be lysed resulting in thrombosis . Thrombolytic Dubey et al. 1409 agents from various sources have been extensively nogen to form the active enzyme plasmin. It specifically investigated. Enzymes, such as urokinase, streptokinase catalyzes the cleavage of the Arg-Val bond in plasmi- and tissues plasminogen activators have been widely nogen. The active plasmin is then able to break down the used in the treatment of thrombosis. However, these fibrin polymers of blood clots. Clinically, UK is given to enzymes are often expensive, thermolabile and can patients suffering from thrombolytic disorders. Among the produce undesirable side effects (Chitte and Dey, 2000). plasminogen activators, UK provides a superior alterna- Subtilisin nattokinase (NAT) (formerly designated Sub- tive for the simple reasons of its being more potent as tilisin BSP), produced by Bacillus subtilis natto, is a compared to tissue-plasminogen activator and non- serine protease and is reported to have potent fibrinolytic antigenic by virtue of its human origin unlike strepto- activity (Nakamura, et al., 1992). Besides in vitro tests of kinase. Based on these observations, UK is a strong fibrinolytic activity, many in vivo studies had been plasminogen activator. Hence, UK, as one of the most reported (Sumi et al. 1990). Fujita et al. (1995) treated potent plasminogen activators is attracting a great deal of dogs with nattokinase by oral administration, and the attention. The mechanism of action, physico-chemical fibrinolytic activity in plasma increased and showed that properties, in vitro production, cloning and expression, subtilisin NAT could pass the rat intestinal tract and and clinical applications of UK are shown in the present dissolve the chemically induced thrombosis. Suzuki et al. study (Adinarayana et al., 2008). (1990) found that dietary supplementation of natto suppressed intimal thickening and modulated the lysis of mural thrombi after endothelial injury in rat femoral artery. MATERIALS AND METHODS Sumi et al. (1990) also reported a similar effect of dietary Bacillus natto productive protein on in vivo endogenous Isolation and Identification of microorganisms thrombolysis. Similar fibrinolytic enzyme-producing B. subtilis bacteria have also been isolated from Japanese shio- kara, Korea chungkook-jang (Banerjee et al., 2004), and B. subtilis producing Nattokinase (NK) was isolated from soil Chinese douchi. Nevertheless, it is still the most stable sample collected from various regions in Kolkata, India and and economic way to obtain protein with fibrinolytic identified by colony morphology, Gram’s staining, biochemical test B. subtilis and selective media. The identified microorganisms were stored at - activity by natto. On the basis of its food origin, 20°C. relatively strong fibrinolytic activity, stability in the gastrointestinal tract, and convenient oral administration, subtilisin NAT has advantages for commercially used β-haemolytic streptococci medicine for preventative and prolonged effects (Uversky et al., 2004). β-haemolytic streptococci with haemolytic activity was isolated from different samples of blood and biomass from infected throat. The Streptokinase is an extra cellular protein, extracted bacteria was identified by blood agar selective media, Gram’s from certain strains of beta hemolytic streptococcus. It is staining and biochemical tests. The three isolates exhibited a non-protease plasminogen activator that activates plas- streptokinase activity (SK) and stored at -20°C. minogen to plasmin, the enzyme that degrades fibrin cloth through its specific lysine binding site; it is used therefore as a drug in thrombolytic therapy (Mohammad Pseudomonas sp . et al., 2009). Streptokinase is currently used in clinical Pseudomonas sp producing Urokinase activity was isolated from medicine as a therapeutic agent in the treatment of human urine sample and identified by cetrimide agar selective thromboembolic blockages, including coronary throm- media, Gram’s staining and biochemical tests. The identified bosis (Banerjee et al., 2004; Endrogan et al., 2006). isolates were stored at -20°C. Streptokinase (SK) a group of extracellular proteins produced by a variety of streptococci beta-hemolytic Screening of enzymes strains, and is a plasminogen activator composed of 414 amino acids with a molecular mass of 47 kDa. Unlike uro- Screenings of fibrinolytic enzymes was done using nutrient agar kinase or tissue-type plasminogen activator that performs medium containing 2% casein and 2 ml human serum. direct proteolysis, SK forms a high affinity equimolar complex with a plasminogen (Kim et al., 2000). Urokinase (UK) is given to patients suffering from Enzyme production thrombolyticdisorders like deep vein thrombosis, Nattokinase thrombosis of the eye, pulmonary embolism, and myo- cardial infarction. This enzyme is a strong plasminogen B. subtilis was grown on basal medium containing (g/lit.) Soya activator which specifically cleaves the proenzyme/ Peptone, 10. K 2HPO 4, 2. MgSO 4, 1. Maltose, 20. Yeast extract, 10. zymogen plasminogen to form the active enzyme plasmin Glucose, 2. and 1000 ml distilled water. The pH was adjusted to 7.2 with 2 M acetic acid and 2 M NaOH. Medium was sterilized by (Kunamneni et al., 2008). UK is a serine protease, which autoclaving at 121°C for 35 min and cooled to room temperature. specifically cleaves the proenzyme/zymogen plasmi- One ml of uniformly prepared suspension of B. subtilis was used as 1410 Afr. J. Biotechnol. an inoculum; incubated at 37°C and 150 rpm in an orbital shaker. saturation. The protein fraction was precipitated with 85% ammo-