Nattokinase Research Articles

Probing the Role of Catalytic Triad on the Cleavage between Intramolecular Chaperone and NK Mature Peptide

Many proteases require the assistance of an intramolecular chaperone (IMC) that is essential for protein folding. Subtilisin is produced as a precursor that requires its N-terminal propeptide to act as an IMC to chaperone the folding of its subtilisin domain. During the precursor folding, the cleavage of the peptide bond between the IMC and the subtilisin domain is the most important and rate-limiting step, which leads to the structural reorganization of the subtilisin domain and IMC's degradation. It is speculated that the cleavage is fulfilled by the nucleophilic attack of Ser221, with the assistance of Asp32 positioning the correct tautomer of His64 and His64 accepting a proton from Ser221. In this study, our results suggested that there was a different mechanism of cleavage of the peptide bond between the IMC and the subtilisin domain in nattokinase (NK), and the role of the NK catalytic triad on the cleavage was not consistent with the classical theory. This finding suggested that members of the subtilisin family had evolved different mechanisms to acquire their own active subtilisin efficiently.

Subtilisin NAT, a subtilisin-like serine protease present in fermented soybean “natto” extract, inhibits <i>Streptococcus mutans</i> biofilm formation

Our previous study indicated that the water-soluble extract obtained from commercially available natto products inhibited the sucrose-dependent cariogenic biofilm formation of Streptococcus mutans. This inhibitory effect correlated with the protease activity levels of the extract. Subtilisin NAT (nattokinase), a subtilisin-like serine protease, is a dominant protease found in natto products. In this study, we investigated the effect of natto products produced by a subtilisin NAT-deficient mutant (aprN−) of the Bacillus subtilis strain NBRC16449 on S. mutans biofilm formation. The mutation decreased the γ-poly glutamic acid (γ-PGA) levels and reduced protease activity in natto products. A significant reduction of the inhibitory effect was observed in the extract from natto products prepared with the aprN− strain. The γ-PGA obtained from the natto products did not affect biofilm formation. We propose that subtilisin NAT plays a major role in biofilm inhibition induced by natto extract.

Expression of a novel dual-functional polypeptide and its pharmacological action research

AimsTo develop a dual-functional medicine for hypoglycemic and anti-thrombus. Main methodsThe long-acting glucagon like peptide-1 (5×GLP-1) and nattokinase (NK) were cloned by SOE PCR and gained the GLP-1 and NK fusion polypeptide after transformed into E. coli. Use of mice models for the hypoglycemic and anti-thrombus activity of the fusion polypeptide. Balb/C mice were given the carrageenan by intraperitoneal injection to induce tail thrombus models. Type 2 diabetes mellitus mice model was used to research the hypoglycemic function of the fusion polypeptide. Key findingsResults showed that the fusion polypeptide could significantly prevent thrombus formation after oral administration. Continuous administration for 15 days, fasting blood glucose levels of the experimental group decreased to nearly normal levels. SignificanceThe present study investigated the expression, purification and functional activity of the rolGLP-1 and NK fusion polypeptide, which provided a foundation for further studying the detailed pharmaceutical mechanism and drug development.

Nattokinase mitigated dextran sulfate sodium-induced chronic colitis by regulating microbiota and suppressing tryptophan metabolism via inhibiting IDO-1

• NK relieved DSS-induced chronic colitis. • NK maintained mucosal integrity by inhibiting apoptosis. • NK regulated gut microbiota of DSS-induced chronic colitis. • NK suppressed Trp metabolism via inhibiting IDO-1 in DSS-induced chronic colitis. In this study, we investigated the protective effect of Nattokinase (NK), the most active ingredient in natto used for healthcare, on dextran sulfate sodium (DSS)-induced chronic colitis and unraveled the potential mechanisms. NK dramatically attenuated clinical symptoms and pathological damage of DSS-induced chronic colitis. NK also inhibited proinflammatory cytokines production in DSS-induced colonic tissues. Mucosal integrity was maintained by NK. Moreover, nattokinase reversed DSS-induced decline of bacteria community diversity and intestinal microbial imbalance by decreasing levels of Firmicutes and increasing levels of Bacteroidetes . Tryptophan (Trp) metabolism analysis demonstrated that NK suppressed Trp catabolism especially the Kynurenine (Kyn) pathway in chronic colitis. Furthermore, NK strikingly down-regulated the protein expression of IDO-1 in chronic colitis mice. Taken together, the study demonstrated that NK relieved DSS-induced chronic colitis by regulating gut microbiota and suppressing Trp metabolism via inhibiting IDO-1. This study indicated that NK might be a promising and effective agent for IBD.

Nattokinase reduces brain infarction, fibrinogen and activated partial thromboplastin time against cerebral ischemia-reperfusion injury

Natto is a traditional Japanese food prepared from fermented soybeans. nattokinase (NK) is considered one of the most active functional ingredients in natto. The present study evaluated the neuroprotective effects of NK in gerbils subjected to focal cerebral ischemia. The gerbils were separated into four groups: control group, ischemic group and two NK groups (4 and 40 mg/day i.g. for 7 days). All gerbils were fed standard chow with or without NK prior to cerebral ischemia. Focal cerebral ischemia was induced by the occlusion of the right common carotid artery and middle cerebral artery for 60 min, followed by reperfusion. Infarct volumes were determined by 2, 3, 5-triphenyltetrazolium chloride staining. Anti-thrombotic effects of NK, such as fibrinogen, prothrombin time and activated partial thromboplastin time, were also determined. Our data showed that NK significantly reduced infarct volumes by 54-68% (p ＜ 0.01), compared to the ischemic group. Prothrombin time was not significantly different in all groups. However, low dosage NK (4 mg/day) reversed ischemic-induced increase in fibrinogen concentrations and activated partial thromboplastin time. In summary, the data indicated that oral intake of NK for 7 days may alter fibrinolytic activity and provide neuroprotective effects against focal cerebral ischemia in gerbils.

Unsaturated fatty acids enhance the fibrinolytic activity of subtilisin NAT (nattokinase).

Subtilisin NAT (STN), alternatively designated nattokinase, is a serine protease with potent fibrinolytic activity. In this study, we screened several foods to enhance the fibrinolytic potential of STN and identified unsaturated fatty acid-rich ones as candidates. We isolated linoleic acid as a major active compound from one of the most active foods, red pepper. Linoleic acid promoted the STN-mediated fibrin/fibrinogen degradation at >20μg/ml. STN cleaved three of the fibrinogen polypeptide chains, among which linoleic acid accelerated Bβ-chain and γ-chain degradations, but slightly suppressed the degradation of α-chain fragments. Linoleic acid failed to affect small synthetic peptide degradation, suggesting a conformational modulation of fibrin/fibrinogen for the linoleic acid promotion of STN activity. Of the various fatty acids tested, unsaturated ones were active but saturated ones were rather inhibitory to STN-mediated fibrinolysis. Thus, our data shed new light on the dietary promotion of STN activity. PRACTICAL APPLICATIONS: Subtilisin NAT (STN) is a serine protease abundantly contained in natto, a soybean food fermented with Bacillus subtilis var. natto. The use of STN as functional foods to improve blood circulation is getting attention because STN actively degrades fibrin. Our results demonstrate that widely occurring unsaturated fatty acids such as linoleic, eicosapentaenoic, and docosahexaenoic acids enhance the fibrinolytic activity of STN. Thus, the intake of natto or STN supplements in combination with unsaturated fatty acid-containing oil can be a novel way to gain cardiovascular benefits.

Molecular Characterization of Bacterial Fibrinolytic Proteins from Indonesian Traditional Fermented Foods.

Previously, the crude extracts of recombinant Nattokinase (NK) variants i.e. NatTK and NatOC and one wild type Douchi Fibrinolytic Enzyme (DFE) from Indonesian traditional fermented foods has been shown to demonstrate fibrinolytic activity. Both NKs contain substitutions of D41N, V192A and 252-RLQHTLEALSTM-263 but NatOC has additional V4F. In the present study, the effects of amino acid substitutions in NK variants and G169A in DFE on their enzyme characteristics were evaluated. Pure proteins were obtained using two sequential steps chromatography using ion exchange and a gel filtration columns. Their activities were determined with fibrin plate, fibrin zymography, fibrinogen hydrolysis, and chromogenic assays. The fibrinogen degradation profile of the wild type NK (NatWT) was different to the NK variants but similar to DFEs. Optimum activity of all the NKs and DFEs was achieved at 50°C while the optimum pH for NatWT/DFEs and NK variants were 8 and 7, respectively. DFEG169A exhibited higher fibrinogen degradation rate and fibrin specific activity than DFE. PMSF inhibited all the NKs and DFEs while SDS and EDTA caused lower activity. The NK variants were more resistant towards Na+ and Ca2+ but more sensitive to K+. The amino acid substitutions in NK variants alter their fibrinogen degradation profile, optimum working pH, working pH range, and resistance to some ions. Substitutions in NK variants likely promote structural changes, particularly with the binding mode of the calcium ion cofactor. The results provide a beneficial basis for future development of fibrino(gen)olytic proteins with improved properties for cardiovascular diseases therapy.

Breaking the vicious loop between inflammation, oxidative stress and coagulation, a novel anti-thrombus insight of nattokinase by inhibiting LPS-induced inflammation and oxidative stress

Thrombosis is a principle cause of cardiovascular disease, the leading cause of morbidity and mortality worldwide; however, the conventional anti-thrombotic approach often leads to bleeding complications despite extensive clinical management and monitoring. In view of the intense crosstalk between inflammation and coagulation, plus the contributing role of ROS to both inflammation and coagulation, it is highly desirable to develop safer anti-thrombotic agent with preserved anti-inflammatory and anti-oxidative stress activities. Nattokinase (NK) possesses many beneficial effects on cardiovascular system due to its strong thrombolytic and anticoagulant activities. Herein, we demonstrated that NK not only effectively prevented xylene-induced ear oedema in mice, but also remarkably protected against LPS-induced acute kidney injury in mice through restraining inflammation and oxidative stress, a central player in the initiation and progression of inflammation. Fascinatingly, in line with our in vivo data, NK elicited prominent anti-inflammatory activity in RAW264.7 macrophages via suppressing the LPS-induced TLR4 and NOX2 activation, thereby repressing the corresponding ROS production, MAPKs activation, and NF-κB translocation from the cytoplasm to the nucleus, where it mediates the expression of pro-inflammatory mediators, such as TNF-α, IL-6, NO, and PAI-1 in activated macrophage cells. In particular, consistent with the macrophage studies, NK markedly inhibited serum PAI-1 levels induced by LPS, thereby blocking the deposition of fibrin in the glomeruli of endotoxin-treated animals. In summary, we extended the anti-thrombus mechanism of NK by demonstrating the anti-inflammatory and anti-oxidative stress effects of NK in ameliorating LPS-activated macrophage signaling and protecting against LPS-stimulated AKI as well as glomeruler thrombus in mice, opening a comprehensive anti-thrombus strategy by breaking the vicious cycle between inflammation, oxidative stress and thrombosis.

Development of a nattokinase–polysialic acid complex for advanced tumor treatment

Cancer-associated thrombus (CAT) impedes delivery of nanoparticles to tumor sites and also inhibits the ability of immune cells to detect and attack these tumors, particularly in advanced tumors with old thrombi. Nattokinase (NK) is an extract from a popular Japanese food, natto, which consists of boiled soybeans fermented with bacteria. Nattokinase exerts strong fibrinolytic and thrombolytic activities and can unblock blood vessels. To deliver NK to thrombus sites in tumors, we modified the surface of NK with polysialic acid (PSA), which formed complexes via electrostatic interactions, resulting in NK–PSA. Particle size and zeta potential of NK–PSA were evaluated, and differential scanning calorimetry, Fourier-transform infrared spectroscopy, and morphological analyses of NK–PSA were performed. To determine the efficacy of the NK–PSA complex on delivery of nanoparticulate drugs, sialic acid-modified doxorubicin liposomes (DOX-SAL) were used as a model drug. In vivo pharmacokinetic and tissue distribution analyses showed that the blood clearance rate of DOX-SAL was significantly enhanced by NK–PSA, and NK–PSA increased accumulation of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) labeled SAL (DiR-SAL) in tumors. Analysis of anti-tumor efficacy showed that the combination of NK–PSA and DOX-SAL enhanced anti-tumor activity. These results suggested that NK–PSA combined with DOX-SAL may be an effective strategy to clear CAT and increase the ability of nanoparticles and immune cells to reach tumors.

A nano polymer conjugate for dual drugs sequential release and combined treatment of colon cancer and thrombotic complications.

Thrombotic complications turn into the second leading cause of death in colon cancer patients due to the hypercoagulable state caused by malignancy. Therefore, it is necessary to treat colon cancer and its thrombosis complications simultaneously. Herein, a nano polymer conjugate based on disulfide cross-linked low-generation peptide dendrimers was developed to treat colon cancer and its thrombotic complications. First, two-generation polyglutamic acid dendrimer was bonded to nattokinase (NK) and then cross-linkers containing disulfide linkages were used to obtain polymer conjugates (NK-G2)n. Then doxorubicin (Dox) was encapsulated. The system can release drugs sequentially due to the dissociation of the polymer conjugates. In vitro thrombolytic experiments exhibited a significant thrombolysis ability of (NK-G2)n. The toxicity and cellular uptake tests on HCT116 cells showed that Dox loaded polymer conjugates had good endocytosis ability and anti-cancer effect. Therefore, this drug delivery system will be a promising strategy to the combined treatment of colon cancer and thrombotic complications.

The Potential of Serratiopetidase and Lumbrokinase for the Degradation of Prion Peptide 106-126 - an In Vitro and In Silico Perspective.

PrPC is a host-encoded prion protein, which gets post translationally modified into a transmissible, β-sheet rich disease associated protein called PrPSc, responsible for the Prion disease including mad cow disease in cattle and CJD in humans. The PrP 106-126 region in PrPSc peptide initiates the conformational change in that protein leading to fibrillation. Any agent that can destabilize or disintegrate such proteins can be served as a potential drug candidate for Prion diseases. In the present study, an enzyme Lumbrokinase (LK) was isolated from earthworm and its activity was exploited towards PrP 106-126 amyloids in vitro along with another enzyme Serratiopeptidase (SP) taking Nattokinase (NK) as a standard. The results showed that PrP 106-126 amyloid formation was inhibited by both LK and SP, as evidenced from Thioflavin T fluorescence assay. Further, the size of fibrils as estimated by dynamic light scattering, was also found to be lower at different time intervals after incubation of the prion amyloids with LK and SP. Additionally, the molecular dynamics simulation revealed the thermodynamically favorable interaction of PrP 106-126 with LK as well as with SP with high affinity. Finally, the toxicity of the disintegrated amyloids was assessed using PC12 cell lines which showed higher cell viability in case of LK and SP treated amyloids compared to only PrP 106- 126 amyloid treatment. Altogether, the study concluded that the serine proteases like LK and SP have the potential to disintegrate PrP 106-126 amyloids with improved cell viability. The in vivo studies are needed to be executed in future.

A nattokinase carrier bonding with polyglutamic acid peptide dendrimer for improved thrombolysis

Thrombosis has become one of the major diseases harmful to human health. The new‐generation thrombolytic drug nattokinase (NK) has been used in the treatment of thrombosis because of its high safety, small side effects, and good thrombolytic effect. However, its application is limited because of its short half‐life and inactivation in external environments. Therefore, it is necessary to retain the enzyme activity of NK to improve its environmental stability. In this study, NK was decorated by bonding peptide dendrimers to improve its enzymatic activity. Polyglutamic acid dendrimers of generation 2 were firstly synthesized, and then the targeting molecule arginine‐glycine‐aspartic acid (RGD) was linked to dendrimers by reaction of carboxyl and amino groups. Subsequently, polyglutamic dendrimer particles were bonded on the periphery of NK by chemical reaction and were used as carriers for NK. The carriers were characterized by Fourier transform infrared (FTIR) and thermogravimetric (TG) analysis, and their size and morphology were evaluated. Their enzyme activity at different pH and temperature was determined, and improved NK activity was achieved after bonding with dendrimers under acid and alkaline conditions and high temperature. Their thrombolytic properties were evaluated by in vitro and in vivo thrombolytic experiments. Effective thrombolytic effect was observed. This study indicated that the NK carrier prepared by chemical bonding may have applications in the treatment of thrombosis.

Synthesis of mesoporous silica/polyglutamic acid peptide dendrimer with dual targeting and its application in dissolving thrombus.

With the frequent occurrence of thrombus diseases, thrombus has become a factor endangering human health. Nattokinase (NK) is a new generation of thrombolytic drug with efficient thrombolytic effect and no major side effects. However, it is easily inactivated in external environment due to its sensitivity, which is still a challenge for its generalized application. Herein, a mesoporous silica/polyglutamic acid peptide dendrimer (M-MSNs-G3 -RGD) nanoparticle was prepared to protect and transport NK. First, magnetic mesoporous silica nanoparticles (M-MSNs) were prepared as the core of the whole nanoparticle, then polyglutamic acid peptide dendrimer (G3 ) was bonded to form M-MSNs-G3 . At last, arginine-glycine-aspartic peptide (RGD) was grafted onto the M-MSNs-G3 to obtain M-MSNs-G3 -RGD. The physical and chemical characteristics and biological toxicity of M-MSNs-G3 -RGD were studied. Thrombus-targeting nanocomposites M-MSNs-G3 -RGD/NK were prepared by loading the thrombolytic drug NK via electrostatic interaction. In vitro and in vivo targeted thrombolytic experiments showed that the nanoparticles exhibited significant thrombolysis ability. These results suggested the potential application of M-MSNs-G3 -RGD/NK in dual targeted thrombolysis.

Improvement of the acid resistance, catalytic efficiency, and thermostability of nattokinase by multisite‐directed mutagenesis

Nattokinase (NK) is a serine protease of the subtilisin family; asa potent fibrinolytic enzyme, it is potentially useful for thrombosis therapy. For NK to be applied as an oral medicine for the treatment of cardiovascular diseases, it must overcome the extremely acidic environments of the gastrointestinal tract despite its limited acidic stability. In this study, three strategies were adopted to improve the acid resistance of NK: (a) Surface charge engineering, (b) sequence alignment, and (c) mutation based on the literature. Eleven variants were constructed and four single-point mutations were screened out for their distinctive catalytic properties: Q59E increased the specific activity; S78T improved the acid stability; Y217K enhanced the acid and thermal stabilities; and N218D improved the thermostability. Based on these observations, multipoint variants were constructed and characterized, and one variant with better acid stability, catalytic efficiency, and thermostability was obtained. Molecular dynamics simulation was carried out to clarify the molecular mechanism of the increased stability of S78T and Y217K mutants under acidic conditions. This study explored effective strategies to engineer acid resistance of NK; moreover, the NK variants with better catalytic properties found in this study have potential applications for the medical industry.

Nattokinase-heparin exhibits beneficial efficacy and safety-an optimal strategy for CKD patients on hemodialysis.

Heparin is a widely used anticoagulant in hemodialysis (HD) for patients with chronic kidney disease (CKD); however, it entails the risk of thrombus formation due to heparin-induced thrombocytopenia. Indeed, CKD patients on HD are associated with excessive mortality from cardiovascular disease due to their prothrombotic profile. Therefore, it would be a significant breakthrough to develop a thrombolytic adjuvant that facilitates heparin to achieve its proper anticoagulant efficiency at a much lower dose for greater safety. Nattokinase (NK), a valuable dietary supplement possessing strong fibrinolytic and thrombolytic activity, was reported to interact with heparin and thereby the beneficial efficacy of NK-heparin was investigated herein. NK-heparin induced a synergistic enhancement of clotting time both in vitro and in vivo evaluations, whereas the overall fibrinolytic activity was only marginally enhanced. Moreover, it was demonstrated for the first time that NK induced potent degradation of all three chains of fibrinogen. In particular, NK-heparin markedly reinforced the fibrinolysis activity of NK, which may underlie, at least in part,the mechanism by which NK-heparin benefited their overall thrombolytic and anticoagulant activity. Collectively, we clarified the beneficial combination efficacy of NK and heparin for greater safety, providing a powerful impetus for physicians to administer heparin to a larger portion of patients with CKD.

Acute toxicity and genotoxicity evaluations of Nattokinase, a promising agent for cardiovascular diseases prevention

Cardiovascular diseases (CVDs) are the leading cause of death in the world; however, current agents for CVDs prevention are still limited. Owing to the serious bleeding risk of Aspirin, FDA recently recommended against it from preventing first heart attacks. Nattokinase (NK), a serine protease possessing many key beneficial effects on cardiovascular system, is being pursued as a promising alternative agent. In light of this, the safety profile of NK, in particular its potential genotoxicity, need to be characterized. The present study is therefore aimed to evaluate the toxicological profile of NK. To assess acute safety, mice were orally administrated with NK at its maximum concentration and the maximum feeding volume twice in a single day, no mortality or toxicological signs were observed. Hence, the maximum daily tolerant dose of NK in mice is up to 480000 FU/kg, which is 1000 times more compared to the recommended daily dose for human. In the genotoxicity studies, NK showed no mutagenic activity as tested by both Ames test and in vivo micronucleus assay. Moreover, NK demonstrated no evidence of potential to induce chromosome aberrations in CHL cells. These results indicate that there is no safety concern for NK in the present preclinical safety studies, supporting the safety of NK as an agent for CVDs prevention.

Coproduction of menaquinone-7 and nattokinase by Bacillus subtilis using soybean curd residue as a renewable substrate combined with a dissolved oxygen control strategy

Numerous physiological functions of menaquinone-7 (MK-7) act to reduce vascular calcification, suggesting that MK-7 may be a potential therapy for Alzheimer’s and Parkinson’s disease, and in this study, we attempted to increase the concentration of MK-7 synthesized by Bacillus subtilis natto, a standard nattokinase (NK) producing strain. Different Bacillus subtilis isolates demonstrated positive correlations between MK-7 and NK concentrations. Response surface methodology (RSM) was employed to optimize a culture medium for the simultaneous production of these molecules; the optimized medium contained the following components (%, w/v): soybean curd residue, 12.2; soya peptone, 5.7; lactose, 2.6; and K2HPO4, 0.6. The fermentation process was subsequently optimized based on online feedback control of fermentation process parameters. The dissolved oxygen (DO) concentration played an important role in the production of MK-7 and NK. With increased DO concentrations, the cell growth rate and NK activity increased. In contrast, at low DO concentrations, the concentration of MK-7 rapidly increased during the late fermentation stage. Thus, in this study, the production of MK-7 and NK by Bacillus subtilis was accomplished using soybean curd residue through medium optimization and DO control. This novel coproduction strategy was developed by controlling the aeration rate during the fermentation process. The concentrations of MK-7 and NK achieved in this study reached 91.25 mg/L and 2675.73 U/mL, respectively.

Multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer: Design, synthesis, and dissolving thrombus.

Thrombotic events affect many individuals in a number of ways, all of which can cause significant morbidity and mortality. Nattokinase (NK), as a novel thrombolytic drug, has been used for thrombolytic therapy. It not only possesses plasminogen activator activity, but also directly digests fibrin through limited proteolysis. However, it may undergo inactivation and denaturation in the harsh external environment. In this study, a multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer was fabricated and used as a carrier for NK protection and delivery. Different arm numbers of polyethylene glycol-polyglutamic acid peptide dendrimers (x-PEG(G3 )x , x=2, 4, 6, 8) were designed, prepared, and characterized by 1 H NMR and FTIR. Then, x-PEG(G3 )x were loaded with NK to form nanocomposites. Their size and morphology were determined by dynamic light scattering and transmission electron microscopy. Enzyme activity was evaluated via UV-Vis absorbance spectra, fluorescence spectra, circular dichroism spectra, and zeta potential measurements. The study reveals that the obtained x-PEG(G3 )x /NK nanocomposites possess high enzyme activity. In addition, the nanocomposites show increased viability of rat macrophage cells, and excellent thrombolysis ability in vitro and in vivo. This work establishes a multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer with potential application in NK carrier and thrombolytic therapy. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1687-1696, 2018.

Bacterial Fibrinolytic Enzyme Coding Sequences from Indonesian Traditional Fermented Foods Isolated Using Metagenomic Approach and Their Expression in Escherichia Coli

ABSTRACTFibrinolytic proteins play an important role in treatment of cardiovascular disease. This study aimed to express coding sequences of new bacterial fibrinolytic protein from Indonesian traditional fermented foods obtained by a PCR-based metagenomic approach and to test their biological activity. New amino acid variations were found in nattokinase (NAT) at several positions i.e. D41N and V192A from dried Tauco, V4F, D41N, and V192A from yellow Oncom but not in Douchy Fibrinolytic Enzyme (DFE) from Terasi. The NAT and DFE recombinant versions were overproduced in Escherichia coli BL21(DE3) using pET16b(+). Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis analysis showed all clones produced mature soluble fibrinolytic proteins of 28 kDa. All recombinant proteins displayed caseinolytic and fibrinolytic activities. In conclusion, metagenomic approach can be applied to obtain and to express coding sequences of new variant bacterial fibrinolytic protein as active soluble mature form in E. coli.

Nattokinase: A Promising Alternative in Prevention and Treatment of Cardiovascular Diseases.

Cardiovascular disease (CVD) is the leading cause of death in the world and our approach to the control and management of CVD mortality is limited. Nattokinase (NK), the most active ingredient of natto, possesses a variety of favourable cardiovascular effects and the consumption of Natto has been linked to a reduction in CVD mortality. Recent research has demonstrated that NK has potent fibrinolytic activity, antihypertensive, anti-atherosclerotic, and lipid-lowering, antiplatelet, and neuroprotective effects. This review covers the major pharmacologic effects of NK with a focus on its clinical relevance to CVD. It outlines the advantages of NK and the outstanding issues pertaining to NK pharmacokinetics. Available evidence suggests that NK is a unique natural compound that possesses several key cardiovascular beneficial effects for patients with CVD and is therefore an ideal drug candidate for the prevention and treatment of CVD. Nattokinase is a promising alternative in the management of CVD.