240 Journal of Marine Science and Technology, Vol. 23, No. 2, pp. 240-248 (2015) DOI: 10.6119/JMST-014-0617-1 PURIFICATION AND CHARACTERIZATION OF NATTOKINASE FROM CULTURAL FILTRATE OF RED ALGA PORPHYRA DENTATA FERMENTED BY BACILLUS SUBTILIS N1 Hong-Ting Victor Lin1, 3, Guan-James Wu2, Meng-Chien Hsieh1, Shun-Hsien Chang1, and Guo-Jane Tsai1, 3 Key words: Bacillus subtilis, fermentation, nattokinase, Porphyra is formed from fibrinogen via the proteolytic action of throm- dentata. bin. To avoid thrombosis, a process called fibrinolysis, which involves the dissolution of insoluble fibrin clots by the hy- drolytic action of plasmin, is activated by tissue plasminogen ABSTRACT activators (Cesarman-Maus and Hajjar, 2005). If the fibrin Fibrinolytic enzyme nattokinase was first extracted from a clots fail to be hydrolyzed because of some disorders, throm- traditional Japanese fermented soybean food, Natto. Edible bosis can occur and lead to myocardial infarction and other red alga Porphyra dentata shares similar high protein content CVDs (Meade et al., 1993; Chandler et al., 1997). Further- with soybean. In this study, we successfully purify and char- more, tissue-type plasminogen activator (t-PA) (Collen and acterize nattokinase from the cultural filtrate of P. dentata Lijnen, 2004), streptokinase (Reed et al., 1999), and urokinase fermented by Bacillus subtilis N1. The crude enzyme was pu- (Duffy, 2002), which are widely used in the treatment of rified by ion-exchange and gel filtration to reach electropho- thrombosis, can activate plasminogen into active plasmin for retic homogeneity. The nattokinase, which has a molecular degrading fibrin. However, these enzymes are expensive or weight of 46.5 kDa and an isoelectric point of 8.35, was stable have side effects, prompting scientists to look for safer and from pH 5 to 9 and at temperatures up to 55C, and it showed cheaper alternatives. optimum enzyme activity at pH 8 and at 55C. This enzyme is Nattokinase (formerly called subtilisin NAT) is a well- characterized as a serine-protease, and its activity can be studied protease of microbial origin that possesses fibrinolytic stimulated by adding CuSO4 or FeCl3. Our results identified (anti-clotting) activities (Sumi et al., 1987; Chang et al., 2000; the fibrinolytic nattokinase in the cultural filtrate of P. dentata Lee et al., 2001; Ko et al., 2004; Paik et al., 2004; Wang et al., fermented by Bacillus subtilis N1 and provided affecting fac- 2009; Yin et al., 2010). It was first extracted and purified from tors to its fibrinolytic activity. a traditional Japanese soybean-fermented food, natto (Sumi et al., 1987), and sources and properties of nattokinase are de- scribed well by Peng et al. (2005). It has been shown that I. INTRODUCTION nattokinase has a greater fibrinolytic activity than plasmin, Intravascular thrombosis, the formation of blood clots in and it supports the body in degrading and dissolving the un- blood vessel, is one of the main causes of cardiovascular dis- healthy coagulation of blood (Chang et al., 2000; Suzuki, 2003). ease (CVD), a major reason for disability and premature death Moreover, nattokinase hydrolyzes active recombinant pro- worldwide. Fibrin, the major protein component of blood clots, karyotic plasminogen activator inhibitor-1 (PAI-1), indicating that fibrin clot lysis by nattokinase also involves the cleavage and inactivation of PAI-1 (Urano, 2001). These findings, Paper submitted 11/22/12; revised 01/22/14; accepted 06/17/14. Author for along with the observation that nattokinase can be absorbed correspondence: Prof. Guo-Jane Tsai (e-mail: [email protected]). across the intestinal tract after oral administration (Sumi et al., 1 Department of Food Science, National Taiwan Ocean University, Keelung, 1990; Fujita et al., 1995), make it a promising anti-clotting Taiwan, R.O.C. agent for the prevention and treatment of CVDs. Furthermore, 2 Department of Food Science, National Penghu University, Makung, Penghu, a recent study has revealed that nattokinase can degrade Taiwan, R.O.C. 3 Center of Excellence for the Oceans, National Taiwan Ocean University, amyloid (Hsu et al., 2009), which is believed to be associated Keelung, Taiwan, R.O.C. with various neurodegenerative diseases. H.-T. V. Lin et al.: Nattokinase from B. subtilis-Fermented P. dentata 241 The marine red alga Porphyra spp. are widely distributed The proximate composition including moisture, crude and are traditionally used as food in East Asian countries (Wei protein, crude lipid, crude fiber and ash of P. dentata was et al., 2003). They are attracting attention as a valuable food analyzed according to AOAC official methods of analysis source in Western societies because of their high mineral, (AOAC, 1998). Moisture content was determined using a vitamin, and protein contents (MacArtain et al., 2007; Smith et hot-air oven (Sanyo Electric Co., Ltd., Kaizuka, Osaka, Ja- al., 2010). Antioxidant phenolic compounds, such as rutin, pan) at 100C for 24 h. Nitrogen content was measured using catechol, epigallocatechin gallate (EGCG), hesperidin, morin, a Kjeltec TM 2100 (Foss, Sweden) and the crude protein caffeic acid, and catechin, have been identified in Porphyra content was calculated by multiplying nitrogen content by a spp. (Yoshie et al., 2000; Yoshie-stark et al., 2003; Kazłowska factor of 6.25. Crude lipids were extracted using a Soxtex et al., 2010). Furthermore, P. dentata powder is a folk medi- System HT 1043 Extraction Unit (Tecator, Sweden) and cine used for treating various types of allergies (Kimiya et al., the crude lipid content was determined after oven-drying 2008) and inflammatory diseases, such as lymphadenitis and (100C) the extract for 2 h. Crude fiber content was deter- bronchitis. Several ethnopharmacological studies have sur- mined by filtering with a Fibertec system (Fibertec Inc., veyed various Porphyra spp. and have identified their poten- Scotland). Ash content was measured by heating the dried tial for treating aging (Zhao et al., 2008), diarrhea, abdominal powder of P. dentata in a Furnace (Risen Instruments Co., cramps, vomiting (Kim et al., 2006), hypersensitivity (Kimiya Ltd., Taiwan) at 550C for 12 h. et al., 2008), acute liver injury (Guo et al., 2007), and cancers (Ichihara et al., 1999). In addition to the high contents of bio- 4. Microorganism and Cultivation for Nattokinase active compounds in Porphyra spp., the alga is exempt from Production the negativity associated with terrestrial biomass resources, B. subtilis N1 was isolated from a commercial natto product which is said to be responsible for the rising food prices. In (Typhula natto, Dounanhiratsukashokuhin Co., Ltd., Nobori- this study, Bacillus subtilis N1 was grown on a medium con- betsu, Hokkaido, Japan) by our group. Its potential for natto- taining only P. dentate powder for the production of natto- kinase production was identified by the presence of clear zone kinase. The nattokinase in the culture filtrate was purified and on the fibrin plate, based on the method described by Ko et al. characterized to determine its fibrinolytic activity and bio- (2004). This strain was cultivated in tryptic soy broth (TSB, logical properties. Becton, Dickinson and Company, Sparks, MD, USA) at 37C for 24 h and added with equal volume of sterile glycerol for II. MATERIALS AND METHODS storage at -80C. It was sub-cultured twice in TSB at 37C for 24 h before the use as an inoculum for the production of 1. Source of Porphyra dentata nattokinase. Dried P. dentata was purchased from Don-Xie-Chen Manu- A 5-L-fermentor containing 2 L P. dentata suspension (3%, facturing Factory (Makung city, Penghu Hsien, Taiwan). The w/v) was inoculated with the culture of B. subtilis N1 to have 6 dried P. dentata was ground, screened through a mesh (0.42 an initial cell density of 10 CFU/mL and fermented at 37C m/m) and stored in a descanter for use. with an agitation rate of 150 rpm and an aeration rate of 2.0 vvm for 48 h. After centrifugation (9,600 g, 15 min) the 2. Analytical Chemicals supernatant was collected and defined as crude enzyme solu- All chemicals used in this research were of analytical grade. tion for following experiments. Trichloroacetic acid (TCA) and sodium carbonate were ob- 5. Enzyme Assay tained from Merck Chemical Industries, Ltd. (Darmstadt, Germany). Acrylamide, Coomassie Brilliant Blue R 250, fi- The nattokinase activity was determined by measuring its brinogen, inhibitors of iodoacetic acid (IAA), ethylenediamine fibrinolytic activity, according to the method described by tetraacetic acid (EDTA), N-ethylmaleimide (NEM), phenyl- Chang et al. (2000) with some modifications. This assay was methane sulfonyl fluoride (PMSF) and tosylphenylalanine carried out by incubating the reaction mixture containing 2.5 chloromethyl ketone (TPCK), resins of CM-Sepharose Fast mL of 1.2% (w/v) fibrin solution, 2.5 mL of 0.1 M Tris buffer Flow and Sephadex G-50, salts of calcium chloride, copper (pH 7.8) and 1 mL of suitable diluted crude or purified enzyme sulfate, ferric chloride, magnesium chloride, potassium chlo- solution at 37C for 15 min before adding 5 mL of 0.1 M TCA ride, sodium chloride and zinc chloride, sodium dodecyl sul- to stop reaction. The reaction mixture was kept at room tem- fate (SDS), thrombin, tris (aminomethane) hydrochloride, perature for 20 min. After centrifugation (12,000 g, 10 min), tyrosine, protein markers and protein assay kit were purchased the supernatant of reaction mixture was collected and meas- from Sigma Chemical Industries, Ltd. (Saint Louis, MO, ured for the absorbance at 275 nm. A standard curve was U.S.A.). PhastGel IEF 3-9, IEF buffer and IEF calibration kit obtained by measuring the absorbance at 275 nm of 0-100 were purchased from GE Healthcare Life Sciences, Ltd. g/mL tyrosine solution. One unit of the enzyme activity was (Uppsala, Sweden). defined as the amount of enzyme that gave an increase in ab- sorbance at 275 nm equivalent to 1 g of tyrosine per minute 3.