DOCSLIB.ORG

Purification and Characterization of Nattokinase from Cultural Filtrate of Red Alga Porphyra Dentata Fermented by Bacillus Subtilis N1

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Purification and Characterization of Nattokinase from Cultural Filtrate of Red Alga Porphyra Dentata Fermented by Bacillus Subtilis N1 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.
Load more

Recommended publications
  • Bacterial Proteases As Thrombolytics and Fibrinolytics
    - Review paper Bacterial Proteases as Thrombolytics and Fibrinolytics Taqiyah Akhtar1, Md. Mozammel Hoq2 and Md. Abdul Mazid1 1Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka-1000, Bangladesh 2Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh (Received: September 05, 2017; Accepted: November 07, 2017; Published (web): December 23, 2017) Abstract: Proteases regulate important pathophysiological processes in human body such as homeostasis, blood coagulation, fibrinolysis, tumor progression, etc. These biological effects of proteases largely attribute to their applicability as therapeutic agents. Imbalance in blood coagulation and fibrinolysis, two important physiological processes in human body, leads to thrombosis, a leading cause of cardiovascular complications including myocardial infarction, stroke, etc. The enzymes used to dissolve thrombus (blood clot) are known as thrombolytic agents and among them, the enzymes involving hydrolysis of fibrin called fibrinolytic agents. Thrombolytic agents can be classified according to generation, mechanism of action, source and active site of the enzymes. Among the commercially available thrombolytic agents, uPA and tPA are generally safe but are very expensive. On the other hand, the bacterial streptokinase is a relatively cheap thrombolytic agent but causes undesirable side effects such as bleeding complications. For this reason, worldwide research for potent thrombolytic agents to prevent and treat cardiovascular diseases have been continuing. Microbes are considered as a potential source of as well as safe vectors for expressing thrombolytic and fibrinolytic enzymes. Bacilli are one of the largest groups for this purpose. They have been collected from different traditional fermented foods or have been produced by solid state fermentation using appropriate nutrient substrates including different agro-industrial wastes such as rice straw, molasses, soybean curd residues, etc.
    [Show full text]
  • Nattokinase: an Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease
    International Journal of Molecular Sciences Review Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease Yunqi Weng 1, Jian Yao 1, Sawyer Sparks 2 and Kevin Yueju Wang 2,* 1 Emergency Department, The Affiliated Hospital of Qingdao University, Qingdao 266005, China; [email protected] (Y.W.); [email protected] (J.Y.) 2 Department of Natural Sciences, Northeastern State University at Broken Arrow, Broken Arrow, OK 74014, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-918-449-6479; Fax: +1-918-449-6473 Academic Editor: David Arráez-Román Received: 24 January 2017; Accepted: 26 February 2017; Published: 28 February 2017 Abstract: Natto, a fermented soybean product, has been consumed as a traditional food in Japan for thousands of years. Nattokinase (NK), a potent blood-clot dissolving protein used for the treatment of cardiovascular diseases, is produced by the bacterium Bacillus subtilis during the fermentation of soybeans to produce Natto. NK has been extensively studied in Japan, Korea, and China. Recently, the fibrinolytic (anti-clotting) capacity of NK has been recognized by Western medicine. The National Science Foundation in the United States has investigated and evaluated the safety of NK. NK is currently undergoing a clinical trial study (Phase II) in the USA for atherothrombotic prevention. Multiple NK genes have been cloned, characterized, and produced in various expression system studies. Recombinant technology represents a promising approach for the production of NK with high purity for its use in antithrombotic applications. This review covers the history, benefit, safety, and production of NK. Opportunities for utilizing plant systems for the large-scale production of NK, or for the production of edible plants that can be used to provide oral delivery of NK without extraction and purification are also discussed.
    [Show full text]
  • Heterologous Expression of Nattokinase from B. Subtilis Natto
    Guangbo et al. BMC Biotechnology (2021) 21:49 https://doi.org/10.1186/s12896-021-00708-4 RESEARCH Open Access Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity Yan Guangbo1, Shu Min1, Shen Wei1, Ma Lixin1, Zhai Chao1*†, Wang Yaping1*† and Huang Zunxi2 Abstract Background: Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. Results: This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. Conclusions: This study suggests that the P.
    [Show full text]
  • Nattokinase, Streptokinase and Urokinase) from Bacterial Sources
    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.
    [Show full text]
  • Interactions Between Nattokinase and Heparin/Gags
    Glycoconj J (2015) 32:695–702 DOI 10.1007/s10719-015-9620-8 ORIGINAL ARTICLE Interactions between nattokinase and heparin/GAGs 1 2,5 1,2,3,4 Fuming Zhang & Jianhua Zhang & Robert J. Linhardt Received: 9 July 2015 /Revised: 30 August 2015 /Accepted: 4 September 2015 /Published online: 28 September 2015 # Springer Science+Business Media New York 2015 Abstract Nattokinase (NK) is a serine protease extracted examine NK interacting with heparin as well as other from a traditional Japanese food called natto. Due to its strong glycosaminoglycans (GAGs). These studies showed that fibrinolytic and thrombolytic activity, NK is regarded as NK is a heparin binding protein with an affinity of avaluabledietarysupplementornutraceuticalforthe ~250 nM. Examination with differently sized heparin oral thrombolytic therapy. In addition, NK has been in- oligosaccharides indicated that the interaction between vestigated for some other medical applications including NK and heparin is chain-length dependent and the min- treatment of hypertension, Alzheimer’sdisease,and imum size for heparin binding is a hexasaccharide. vitreoretinal disorders. The most widely used clinical Studies using chemically modified heparin showed the anticoagulants are heparin and low molecular weight 6-O-sulfo as well as the N-sulfo groups but not the 2-O- heparins. The interactions between heparin and proteins sulfo groups within heparin, are essential for heparin’s modulate diverse patho-physiological processes and hep- interaction with NK. Other GAGs (including HS, DS, arin modifies the activity of serine proteases. Indeed, and CSE) displayed modest binding affinity to NK. heparin plays important roles in almost all of NK’spo- NK also interfered with other heparin-protein interac- tential therapeutically applications.
    [Show full text]
  • A Single-Dose of Oral Nattokinase Potentiates Thrombolysis and Anti
    www.nature.com/scientificreports OPEN A single-dose of oral nattokinase potentiates thrombolysis and anti- coagulation profiles Received: 28 August 2014 1 1 1 2 3 Accepted: 29 May 2015 Yuko Kurosawa , Shinsuke Nirengi , Toshiyuki Homma , Kazuki Esaki , Mitsuhiro Ohta , 4 1 Published: 25 June 2015 Joseph F. Clark & Takafumi Hamaoka Our aim was to determine the quantitative effects of a single-dose of Nattokinase (NK) administration on coagulation/fibrinolysis parameters comprehensively in healthy male subjects. A double-blind, placebo-controlled cross-over NK intervention study was carried out in 12 healthy young males. Following the baseline blood draw, each subject was randomized to receive either a single-dose of 2,000 FU NK (NSK-SD, Japan Bio Science Laboratory Co., Ltd) or placebo with subsequent cross-over of the groups. Subjects donated blood samples at 2, 4, 6 and 8 hours following administration for analysis of coagulation/fibrinolysis parameters. As a result, D-dimer concentrations at 6, and 8 hours, and blood fibrin/fibrinogen degradation products at 4hours after NK administration elevated significantly (p < 0.05, respectively). Factor VIII activity declined at 4 and 6 hours (p < 0.05, respectively), blood antithrombin concentration was higher at 2 and 4 hours (p < 0.05, respectively), and the activated partial thromboplastin time prolonged significantly at 2 and 4 hours following NK administration (p < 0.05 and p < 0.01, respectively). All the changes, however, were within the normal range. In conclusion, thus, a single-dose of NK administration appears enhancing fibrinolysis and anti-coagulation via several different pathways simultaneously. Fibrinolytic enzymes have been derived from different microorganisms, including the genus bacillus which occurs in traditional fermented foods.
    [Show full text]
  • Construction of a Genetically Engineered Strain of Nattokinase and Assessment of Its Fibrinolytic Activity
    Vol. 13(27), pp. 488-499, September, 2019 DOI: 10.5897/AJMR2019.9181 Article Number: 68623CA61924 ISSN: 1996-0808 Copyright ©2019 Author(s) retain the copyright of this article African Journal of Microbiology Research http://www.academicjournals.org/AJMR Full Length Research Paper Construction of a genetically engineered strain of nattokinase and assessment of its fibrinolytic activity Li Tian, Wenke Zhou and Youhong Zhang* School of Environmental Ecology and Biological Engineering, Wuhan Institute of Technology, Wuhan 430205, China. Received 25 July, 2019; Accepted 3 September, 2019. A strain of Bacillus natto was isolated from natto and found to have a high yield of nattokinase. The aprN and pro-aprN gene fragments, encoding nattokinase from Bacillus natto, were amplified using two pair of primers and were expressed in Bacillus subtilis WB800N host cells using the pHT43 plasmid as a vector. In this system, the effect of leader peptides on nattokinase activity, revealing that these leader peptides mediate the folding function of nattokinase was explored. After optimizing the expression conditions (1 mmol/L IPTG inducer at 37°C, pH 7.5, cell culture OD600 of 0.6), maximal nattokinase enzymatic activity of 848.52 IU/mL after induction of fermentation for 4 h, was achieved, at which time maximal extracellular protein had been produced. The fermentation medium of the engineering strain was optimized, and purified nattokinase via salt precipitation and ultrafiltration was isolated. Relative to fermentation supernatants, the purification ratio of nattokinase reached 6.63, with a total recovery of 80%, and a specific enzyme activity of 11507.92 IU/mg. These results indicate that the nattokinase overexpression using the pHT43 vector in WB800N cells is an effective means of achieving efficient nattokinase production, and the engineered strains constructed herein have great promise as potential industrial strains for nattokinase production.
    [Show full text]
  • Screening and Characterization of Thermostable Fibrinolytic Enzyme from Bacillus Amyloliquefaciens EGE-B-2D.1
    Turkish Journal of Biochemistry – Türk Biyokimya Dergisi 2016; 41(3): 167–176 Biotechnology Research Article – 20053 Birkan Slem, Yüksel Gezgin, Rengin Eltem* Screening and characterization of thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 Bacillus amyloliquefaciens EGE-B-2d.1’den termostabil fibrinolitik enzimin taranması ve karakterizasyonu DOI 10.1515/tjb-2016-0027 molecular weight of the purified enzyme were estimated Received August 19, 2015; accepted February 3, 2016 to be 44.46 units/mg protein and 30 kD respectively. Abstract: Objective: To screen fibrinolytic enzyme-produc- Conclusion: The thermostable fibrinolytic enzyme from ing Bacillus isolates (n=210) and to characterize of thermo- Bacillus amyloliquefaciens EGE-B-2d.1 was purified and stable fibrinolytic enzyme from Bacillus amyloliquefaciens characterized. This enzyme might also be used as a natural EGE-B-2d.1 that had the highest level of fibrinolytic activ- agent for oral fibrinolytic therapy or thrombosis prevention. ity together with the highest thermostability. Keywords: Fibrinolytic enzyme, Bacillus amyloliquefa- Methods: Firstly, a total of 210 isolates were screened for ciens, production, purification, plasminogen activator, their fibrinolytic enzyme production. The potent fibrino- thrombolytic agent lytic enzyme producing isolates were evaluated for the thermostability of their fibrinolytic enzymes and one isolate showing prominent fibrinolytic activity was iden- Özet: Amaç: Fibrinolitik enzim-üreten Bacillus izolatları- tified as molecular. Fermentation process was carried out nın (n=210) taranması ile Bacillus amyloliquefaciens EGE- on the isolate that had both the highest level of fibrinolytic B-2d.1’den elde edilen en yüksek termostabilite ve fibrino- activity and enzyme thermostability. The thermostable litik aktiviteye sahip enzimi karakterize etmektir.
    [Show full text]
  • Anticoagulant Mechanism, Pharmacological Activity
    www.nature.com/scientificreports OPEN Anticoagulant mechanism, pharmacological activity, and assessment of preclinical safety Received: 18 September 2017 Accepted: 26 March 2018 of a novel fbrin(ogen)olytic serine Published: xx xx xxxx protease from leaves of Leucas indica Debananda Gogoi1, Neha Arora2, Bhargab Kalita1, Rahul Sarma3, Taufkul Islam1, Sidhhartha S. Ghosh2, Rajlakshmi Devi3 & Ashis K. Mukherjee1 The harnessing of medicinal plants containing a plethora of bioactive molecules may lead to the discovery of novel, potent and safe therapeutic agents to treat thrombosis-associated cardiovascular diseases. A 35 kDa (m/z 34747.5230) serine protease (lunathrombase) showing fbrin(ogen)olytic activity and devoid of N- and O- linked oligosaccharides was purifed from an extract of aqueous leaves from L. indica. The LC-MS/MS analysis, de novo sequencing, secondary structure, and amino acid composition determination suggested the enzyme’s novel characteristic. Lunathrombase is an αβ-fbrinogenase, demonstrating anticoagulant activity with its dual inhibition of thrombin and FXa by a non-enzymatic mechanism. Spectrofuorometric and isothermal calorimetric analyses revealed the binding of lunathrombase to fbrinogen, thrombin, and/or FXa with the generation of endothermic heat. It inhibited collagen/ADP/arachidonic acid-induced mammalian platelet aggregation, and demonstrated antiplatelet activity via COX-1 inhibition and the upregulation of the cAMP level. Lunathrombase showed in vitro thrombolytic activity and was not inhibited by endogenous protease inhibitors α2 macroglobulin and antiplasmin. Lunathrombase was non-cytotoxic to mammalian cells, non-hemolytic, and demonstrated dose-dependent (0.125–0.5 mg/kg) in vivo anticoagulant and plasma defbrinogenation activities in a rodent model. Lunathrombase (10 mg/kg) did not show toxicity or adverse pharmacological efects in treated animals.
    [Show full text]
  • Role of Fibrinolytic Enzymes in Anti-Thrombosis Therapy
    REVIEW published: 28 May 2021 doi: 10.3389/fmolb.2021.680397 Role of Fibrinolytic Enzymes in Anti-Thrombosis Therapy Farwa Altaf 1, Shourong Wu 1,2* and Vivi Kasim 1,2* 1The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, China, 2The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing, China Thrombosis, a major cause of deaths in this modern era responsible for 31% of all global deaths reported by WHO in 2017, is due to the aggregation of fibrin in blood vessels which leads to myocardial infarction or other cardiovascular diseases (CVDs). Classical agents such as anti-platelet, anti-coagulant drugs or other enzymes used for thrombosis treatment at present could leads to unwanted side effects including bleeding complication, hemorrhage and allergy. Furthermore, their high cost is a burden for patients, especially for those from low and middle-income countries. Hence, there is an urgent need to develop novel and low-cost drugs for thrombosis treatment. Fibrinolytic enzymes, including plasmin like proteins such as proteases, nattokinase, and lumbrokinase, as well as plasminogen activators such as urokinase plasminogen activator, and tissue-type plasminogen activator, could eliminate thrombi with high efficacy rate and do not have significant drawbacks by directly degrading the fibrin. Edited by: Wangsa Tirta Ismaya, Furthermore, they could be produced with high-yield and in a cost-effective manner from Dexa Medica, Indonesia microorganisms as well as other sources. Hence, they have been considered as potential Reviewed by: compounds for thrombosis therapy. Herein, we will discuss about natural mechanism of Nikhat Jamal Siddiqi, fibrinolysis and thrombus formation, the production of fibrinolytic enzymes from different King Saud University, Saudi Arabia Md.
    [Show full text]
  • Data Recorded in Real Life Support the Safety of Nattokinase in Patients with Vascular Diseases
    nutrients Article Data Recorded in Real Life Support the Safety of Nattokinase in Patients with Vascular Diseases Giuseppe Gallelli 1,†, Giulio Di Mizio 2,*,† , Caterina Palleria 3, Antonio Siniscalchi 4, Paolo Rubino 1, Lucia Muraca 5, Erika Cione 6 , Monica Salerno 7, Giovambattista De Sarro 3,8 and Luca Gallelli 3,8 1 Department of Vascular Surgery, Pugliese Ciaccio Hospital, 88100 Catanzaro, Italy; [email protected] (G.G.); [email protected] (P.R.) 2 Forensic Medicine, Department of Law, Magna Graecia University of Catanzaro, 88100 Catanzaro, Italy 3 Department of Health Science, School of Medicine, University of Catanzaro, Clinical Pharmacology and Pharmacovigilance Unit, Mater Domini University Hospital, 88100 Catanzaro, Italy; [email protected] (C.P.); [email protected] (G.D.S.); [email protected] (L.G.) 4 Department of Neurology and Stroke Unit, Annunziata Hospital of Cosenza, 87100 Cosenza, Italy; [email protected] 5 Department of General Medicine, ASP 7 Catanzaro, 88100 Catanzaro, Italy; [email protected] 6 Department of Pharmacy Helath and Nutritional Sciences, Department of Excellence 2018-2022, University of Calabria, 87036 Rende, Italy; [email protected] 7 Department of Medical, Surgical and Advanced Technologies “G.F. Ingrassia”, University of Catania, 95041 Catania, Italy; [email protected] 8 Department of Health Science, School of Medicine, Clinical Pharmacology Research Center FAS@UMG, University of Catanzaro, 88100 Catanzaro, Italy * Correspondence: [email protected] † Both authors contributed equally to this manuscript. Citation: Gallelli, G.; Di Mizio, G.; Abstract: Nattokinase (NK) is a serine protease enzyme with fibrinolytic activity. Even if it could be Palleria, C.; Siniscalchi, A.; Rubino, P.; Muraca, L.; Cione, E.; Salerno, M.; De used for the treatment of several diseases, no data have been published supporting its use patients Sarro, G.; Gallelli, L.
    [Show full text]
  • Nattokinase Decreases Plasma Levels of Fibrinogen, Factor VII, and Factor
    Available online at www.sciencedirect.com Nutrition Research 29 (2009) 190–196 www.nrjournal.com Nattokinase decreases plasma levels of fibrinogen, factor VII, and factor VIII in human subjects☆ Chien-Hsun Hsiaa, Ming-Ching Shenb, Jen-Shiou Linc, Yao-Ke Wend, Kai-Lin Hwange, ⁎ Thau-Ming Chamf, Nae-Cherng Yangg, aDivision of Cardiovascular Medicine, Changhua Christian Hospital, Changhua 500, Taiwan bDivision of Hematology-Oncology, Changhua Christian Hospital, Changhua 500, Taiwan cDepartment of Laboratory Medicine, Changhua Christian Hospital, Changhua 500, Taiwan dDivision of Nephrology, Changhua Christian Hospital, Changhua 500, Taiwan eDepartment of Public Health, Chung Shan Medical University, Taichung 402, Taiwan fGraduate Institute of Pharmaceutical Sciences, Kaohsiung Medical University, Kaohsiung 807, Taiwan gDepartment of Nutrition and Health Science, Chungchou Institute of Technology, Changhua 510, Taiwan Received 17 December 2008; revised 21 January 2009; accepted 28 January 2009 Abstract Nattokinase, a serine proteinase from Bacillus subtilis, is considered to be one of the most active functional ingredients found in natto. In this study, we hypothesized that nattokinase could reduce certain factors of blood clotting and lipids that are associated with an increase risk for cardiovascular disease (CVD). Thus, an open-label, self-controlled clinical trial was conducted on subjects of the following groups: healthy volunteers (Healthy Group), patients with cardiovascular risk factors (Cardiovascular Group), and patients undergoing dialysis (Dialysis Group). All subjects ingested 2 capsules of nattokinase (2000 fibrinolysis units per capsule) daily orally for 2 months. The laboratory measurements were performed on the screening visit and, subsequently, regularly after the initiation of the study. The intent-to-treat analysis was performed on all 45 enrolled subjects.
    [Show full text]