Purification and characterization of a fibrinolytic enzyme from the food-grade fungus, Neurospora sitophila

Liu, Xiao Lan; Kopparapu, Narasimha Kumar; Zheng, Hong; Katrolia, Priti; Deng, Yu-Feng; Zheng, Xi

doi:10.1016/j.molcatb.2016.10.006

Cited by 16 publications

(6 citation statements)

References 31 publications

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“…A broad spectrum pH stability profile (3)(4)(5)(6)(7)(8)(9)(10)(11) retaining >90% was reported for fibrinolytic protease produced by Cordyceps militaris [41]. A similar pH stability profile (4-11) was reported for fibrinolytic protease with more than 70% of its activity by Neurospora sitophila [63].…”

Section: Stability Of Fibrinolytic Protease At Different Temperaturessupporting

confidence: 52%

“…Maximum stability (100%) was observed at 20 • C and retained 76.59% of its activity up to 80 • C after 2 h of incubation ( Figure 6C). The obtained fibrinolytic enzyme has more stability than those reported earlier, which showed thermo-stability below 40 • C [62] and up to 52 • C [63].…”

Section: Stability Of Fibrinolytic Protease At Different Temperaturesmentioning

confidence: 49%

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Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

et al. 2019

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The present study demonstrates the production and thrombolytic potential of a novel thermostable thiol-dependent fibrinolytic protease by Bacillus cereus RSA1. Statistical optimization of different parameters was accomplished with Plackett-Burman design and validated further by central composite design with 30.75 U/mL protease production. Precipitation and chromatographic approaches resulted in 33.11% recovery with 2.32-fold purification. The molecular weight of fibrinolytic protease was 40 KDa and it exhibited a broad temperature and pH stability range of 20-80 • C and pH 5-10 with utmost activity at 50 • C and pH 8, respectively. The protease retained its fibrinolytic activity in organic solvents and enhanced the activity in solutions with divalent cations (Mn 2+ , Zn 2+ , and Cu 2+ ). The enzyme kinetics revealed K m and V max values of 1.093 mg/mL and 52.39 µg/mL/min, respectively, indicating higher affinity of fibrinolytic activity towards fibrin. Also, complete inhibition of fibrinolytic activity with DFP and a 2-fold increase with DTT and β-mercaptoethanol indicates its thiol-dependent serine protease nature. MALDI-TOF analysis showed 56% amino acid sequence homology with Subtilisin NAT OS = Bacillus subtilis subsp. natto. The fibrinolysis activity was compared with a commercial thrombolytic agent for its therapeutic applicability, and fibrinolytic protease was found highly significant with absolute blood clot dissolution within 4 h in in vitro conditions. The isolated fibrinolytic protease of Bacillus cereus RSA1 is novel and different from other known fibrinolytic proteases with high stability and efficacy, which might have wide medicinal and industrial application as a thrombolytic agent and in blood stain removal, respectively.Biomolecules 2020, 10, 3 2 of 23 (active protease), converting soluble fibrinogen (glycoprotein) into fibrin polymer (insoluble blood clot) [5]. Fibrin clots are hydrolyzed by plasmin [12], which is stimulated from plasminogen by plasminogen activators (PAs) [13,14]. This natural dynamic equilibrium is disturbed when the process of natural fibrin clot hydrolysis undergoes pathophysiological shambles, leading to formation of fibrin clots. Such clots may cause hindrance in the circulation of blood, causing blockage in blood vessels, ultimately leading to cardiac disorders which are life-threatening [15,16].Approaches such as use of anti-coagulant agents, anti-platelet drugs, fibrinolytic enzymes, and surgical operations are employed for the treatment of thrombosis and to dissolve the blood clots [17]. Further, there are numerous side effects which may occur following the administration of the available anti-thrombotic strategies, as well as the high expense, which limit their scope. The effects of reperfusion, urticaria (allergic reaction), and hemorrhage are the major inimical consequences of such thrombolytic approaches on human health [18,19]. Other after effects include headache, dizziness, ulcers, increased clotting time, nausea and vomiting, etc. Management of thrombosis usin...

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Section: Stability Of Fibrinolytic Protease At Different Temperaturessupporting

confidence: 52%

Section: Stability Of Fibrinolytic Protease At Different Temperaturesmentioning

confidence: 49%

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

et al. 2019

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“…Zhang et al (2015) showed the highest specific activity using ALPF to measure the fibrinolytic activity produced by Rhizopus microsporus (43.2 U/mg) in FS. Cordyceps militaris, also produced under FS, had a similar activity of 40.7 U/mg (Liu et al, 2015;Liu et al, 2016) Of the 5 studies (23.8%) that used spectrophotometric methodology, 4 were performed with the fungus Mucor subtilissimus, which was capable of producing up to 57.1 U/mg of fibrinolytic enzyme in FES (Nascimento et al, 2020). The use of spectrophotometric techniques implies a more assertive quantification of the biomolecule of interest since it is a sensitive and automated technique (Galo and Colombo, 2009).…”

Section: Fibrinolytic Activitymentioning

confidence: 99%

“…Inhibition of fibrinolytic proteases is another relevant segment of the characterization; inhibitors can act on enzymes by modifying structural characteristics that determine their mechanism of action and consequently inhibit enzymatic activity (Vargas, 2020). According to the studies analyzed, several compounds were able to inhibit the activity of the enzymes studied, these were PMSF (serine protease inhibitor) (Kim et al, 2011;Choi et al, 2011;Liu et al, 2017;Moon et al, 2014;Liu et al, 2015;Liu et al, 2016;Nascimento et al, 2016;Nascimento et al, 2017;Shirasaka et al, 2012;Cleentino et al, 2011); EDTA (metalloprotease inhibitor) (Guggisberg et al, 2011;Xiao-Ian et al, 2014;Andrade et al, 2018;Lins et al, 2019); SBTI (serine protease inhibitor) (Liu et al, 2016;Liu et al, 2017;Deng et al, 2018;Li et al, 2020); Aprotinin (Inhibitor of serine and cysteine protease) (Liu et al, 2016); TPCK (serine protease inhibitor) (Shirasaka et al, 2021); EGTA (metalloprotease inhibitor) (Choi et al, 2013;Meschram et al, 2016;Andrade et al, 2018;Lins et al, 2019;Choi et al, 2016) and β-mercaptoethanol (cysteine protease inhibitor) (Clementino et al, 2019).…”

Section: Inhibitorsmentioning

confidence: 99%

Systematic analysis on the obtaining of fibrinolytic fungi enzymes

Cardoso

Nascimento

Batista

et al. 2022

RSD

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Fibrinolytic proteases operate directly on fibrin clot and are able to maintain blood flow. Fungi show up as viable sources for obtaining this enzyme. The purpose of this systematic review is to is to unveil all the information concerning production, purification and characterization of fibrinolytic proteases by fungi. The search was conducted in ScienceDirect, PubMed and Scopus databases, using as keywords “(Fibrinolytic enzyme) OR (Fibrinolytic protease) AND (Fungal or Fungus or Fungi)”. Delimiting period of 10 years (2011-2021). The results obtained were filtered by selection criteria, and review articles or articles outside the scope of this work were excluded. The articles were evaluated and scored (0-10) according to pre-established criteria. None of the studies obtained the score 10, however the study with the highest score (9) presented relevant data in all criteria analyzed, obtaining fibrinolytic enzyme from Xylaria curta. Among the 21 selected articles, 12 different genera appear and Submerged Fermentation and purification of Serino-proteases were more described. This work also observed a greater representation of purification and characterization steps, indicating the need for attention to cultivation process and enzymatic application. It is clear that the production of these enzymes by fungi is pertinent towards the high recovery observed even after purification and its tendency for pharmaceutical application.

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“…Fermented soybeans have been consumed as traditional foods in many Asian countries, and indeed, they are sources of nattokinase and other fibrinolytic enzymes. These includes natto, a Japanese traditional fermented soybean obtained from fermentation with Bacillus subtilis G8 and Bacillus subtilis (Chang et al, 2012;Lucy et al, 2019), moromi, oncom and gambus (Indonesian traditional fermented soybean obtained from fermentation with Bacillus cereus, Bacillus subtilus, Bacillus pumilus, and Stenotrophomonas sp, respectively) (Afifah et al, 2014;Nailufar et al, 2016;Stephani et al, 2017;Syahbanu et al, 2020) as well as douchi and doufuru (Chinese traditional fermented soybean obtained from fermentation with Bacillus (Ok and Choi, 2005;Wang et al, 2006;Jo et al, 2011;Wei et al, 2011;Yuan et al, 2012;Chen et al, 2013;Heo et al, 2013;Park et al, 2013;Gad et al, 2014;Thokchom and Joshi, 2014;Bi et al, 2015;Huy et al, 2016;Liu et al, 2016;Nailufar et al, 2016;Hu et al, 2019;Ito, 2020;Syahbanu et al, 2020 (Shirasaka et al, 2012;Biji et al, 2016;Vijayaraghavan et al, 2016a;Vijayaraghavan et al, 2016b;Vijayaraghavan et al, 2019), seafood fermented with Bacillus sp, Bacillus. velezensis BS2, Bacillus pumilus BS15 and Bacillus coagulans (Anh et al, 2015;Prihanto and Firdaus, 2019;Yao et al, 2019;Kim et al, 2020) and liquors obtained from fermentation with Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus-Genus (Lapsongphon et al, 2013;Johnson e...…”

Section: Microorganisms As Sources Of Fibrinolytic Enzymesmentioning

confidence: 99%

Role of Fibrinolytic Enzymes in Anti-Thrombosis Therapy

Altaf

Kasim

2021

Front. Mol. Biosci.

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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. Furthermore, they could be produced with high-yield and in a cost-effective manner from microorganisms as well as other sources. Hence, they have been considered as potential compounds for thrombosis therapy. Herein, we will discuss about natural mechanism of fibrinolysis and thrombus formation, the production of fibrinolytic enzymes from different sources and their application as drugs for thrombosis therapy.

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Purification and characterization of a fibrinolytic enzyme from the food-grade fungus, Neurospora sitophila

Cited by 16 publications

References 31 publications

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

Systematic analysis on the obtaining of fibrinolytic fungi enzymes

Role of Fibrinolytic Enzymes in Anti-Thrombosis Therapy

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