Abstract
Nattokinase (NK, also known as subtilisin NAT) (EC 3.4.21.62) is one of the most considerable extracellular enzymes produced by Bacillus subtilis natto. The main interest about this enzyme is due to its direct fibrinolytic activity. Being stable enough in the gastrointestinal tract makes this enzyme a useful agent for the oral thrombolytic therapy. Thus, NK is regarded as a valuable dietary supplement or nutraceutical. Proven safety and ease of mass production are other advantages of this enzyme. In addition to these valuable advantages, there are other applications attributed to NK including treatment of hypertension, Alzheimer’s disease, and vitreoretinal disorders. This review tends to bring a brief description about this valuable enzyme and summarizes the various biotechnological approaches used in its production, recovery, and purification. Some of the most important applications of NK, as well as its future prospects, are also discussed.
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Introduction
In 1987, Sumi et al. (1987) introduced a novel fibrinolytic enzyme from Natto (a typical and popular fermented soybean food in the Japanese diet) and named it nattokinase. Nattokinase (NK, also known as subtilisin NAT) (EC 3.4.21.62) is among the most considerable extracellular enzymes being produced by Bacillus subtilis natto (Hsieh et al. 2009; Liu et al. 2005; Unrean and Nguyen 2013). The present review aims to detail the current knowledge of the NK enzyme. This aim is followed by the critical review regarding its production, extraction, formulation, and applications. SCOPUS, Web of Science, and PubMed databases were reviewed until July 2014 using the “nattokinase” and “subtilisin NAT” keywords as the search terms. The lists of articles identified by this search strategy were studied and the relevant articles were selected.
Nattokinase structure
Natto is a traditional soybean food produced by B. subtilis natto fermentation. Natto is considered a rich source of valuable products including NK and menaquinone-7 (Berenjian et al. 2014b). We previously demonstrated this strain as the dominant microorganism for the industrial production of these health-promoting compounds (Berenjian et al. 2012, 2013).
NK is a serine protease composed of 275 amino acids (molecular weight 27.7 kDa and pI 8.6) which belongs to the subtilisin family. The enzyme is a cysteine-free protease; thus, no disulfide bond is observed in its structure. Inhibition of NK by phenylmethylsulfonyl fluoride (PMSF) indicates its membership to the serine protease family of enzymes (Fujita et al. 1993; Yanagisawa et al. 2013; Zheng et al. 2005). NK is encoded by the aprN gene. The protein is synthesized in a precursor form, in which a signal peptide and a propeptide are joined to the N-terminus of the mature polypeptide (Nakamura et al. 1992). The three-dimensional structure of NK at 1.74-Ǻ resolution is resolved recently. The structure is almost identical with that of subtilisin E from Bacillus subtilis DB104. There are differences in residues at positions 85 and 192 between subtilisin E and NK. The finding was confirmed by the analysis of both the DNA sequence of subtilisin NAT gene (Nakamura et al. 1992) and the protein sequence of purified nattokinase (Fujita et al. 1993). Although NK is highly homologous to many subtilisins in serine protease family, only this enzyme shows high substrate specificity for fibrin (Peng et al. 2005). The enzyme shows considerable fibrinolytic activity at pH 6–12 but lacks functional and structural stability in alkaline environments. The enzyme activity also slows down at temperatures beyond 60 °C (Fujita et al. 1993; Maeda et al. 2001). NK retains more than 95 % of its activity after five rounds of freezing and thawing (Sumi et al. 1987). The NK characteristics are summarized in Table 1.
The catalytic triad of the NK enzyme is Ser-His-Asp (D32, H64, S221) situated in a shallow groove on the surface of globular protein (Nguyen et al. 2013; Zheng et al. 2005). Hydrogen bonds occurring in the catalytic triad and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond. The four residues of Ser33, Asp60, Ser62, and Thr220 form several hydrogen bonds which function to stabilize the transition state of the hydrolysis reaction (Zheng et al. 2006). A new nucleophilic catalytic mechanism for NK is proposed, and the action of hydroxyl in the catalytic environment as well as the action of S221 for locating the active peptide bond is clarified (Zheng et al. 2005). Residues of the S3 binding site of NK, namely Gly100, Ser101, and Leu126, are critical for protease and fibrinolytic activities. Introducing amino acids with longer side chains at position Gly100 that project into the active site likely decreases substrate binding and catalytic activity of this enzyme. In opposition to the Gly100, introduction of bulky side chains at position Ser101 increases protease activity. Leu126 is considered an essential structure component of the active cleft of NK which is buried in the active cleft and close to the catalytic triad. This residue is conserved and very important for the function among all subtilisins (Wu et al. 2007).
Fibrinolytic and antithrombotic activity
Several reports indicate the various fibrinolytic enzymes produced by Bacillus spp. (Chang et al. 2000, 2012; Ghasemi et al. 2012; Kim et al. 1996; Peng et al. 2003; Wang et al. 2009a; Yin et al. 2010). Among these enzymes, NK is assumed an interesting one. This considerable interest is due to the direct fibrinolytic activity of this enzyme, both in vitro and in vivo (Fujita et al. 1995a; Kamiya et al. 2010; Xu et al. 2014). It not only degrades fibrin directly but also increases the release of tissue plasminogen activator from cells to degrade fibrin (Yatagai et al. 2008). Moreover, it also enhances the body’s production of clot-dissolving agents including plasmin and pro-urokinase. As compared to plasmin, NK is less sensitive on the cleavage of fibrinogen but more sensitive on the cleavage of cross-linked fibrin (Fujita et al. 1995c). Furthermore, NK inactivates plasminogen activator inhibitor type 1 and is capable of platelet aggregation inhibition by blocking thromboxane formation (Jang et al. 2013; Urano et al. 2001). The fibrinolytic activity of NK is retained in blood longer than 3 h (Tai and Sweet 2006). Decreasing the plasma levels of fibrinogen, factor VII, and factor VIII is another feature which makes NK a nutraceutical used in cardiovascular diseases (Hsia et al. 2009).
Thus, NK is currently considered an efficient, secure, and economic enzyme and also the focus of thrombolytic drug studies (Huang et al. 2013; Unrean and Nguyen 2013). Several strategies to minimize the side effects of NK to improve its stability and lengthen its circulation time for the treatment of thrombotic diseases hold great promise (Huang et al. 2013). As such, by means of site-directed mutagenesis experiments, the oxidative stability of NK was substantially increased by optimizing the amino acid residues Thr220 and Met222, which were in the vicinity of the catalytic residue Ser221 of the enzyme (Weng et al. 2009). However, attempts for increasing thermostability of NK by introducing cysteine residues and therefore forming disulfide bonds were not successful. In this regard, Weng et al. (2014) constructed three double mutants of NK (G61C/S98C, T22C/S87C, and S24C/S87C) which contained two cysteines and determined their thermostability. The results demonstrated that disulfide bond was not formed within two cysteine residues and the three double mutants did not show increment in thermostability as compared to the wild-type NK.
Nattokinase production and formulation
Traditionally, most nattokinase-producing strains were isolated from the Japanese food Natto. Resembling fibrinolytic enzymes are also obtained from other traditional fermented foods such as Chinese douche, Korean doen-jang, Korean Chungkook-jang soy sauce, and Thua nao from northern Thailand (Inatsu et al. 2006; Kim et al. 1996; Wei et al. 2012). Besides Bacillus spp., alternative sources of obtaining this enzyme are Pseudomonas sp. and marine creatures (Mahajan et al. 2012; Sumi et al. 1992; Wang et al. 2009c). There are comprehensive studies regarding the optimal fermentation conditions for NK production (Berenjian et al. 2014a; Chen and Chao 2006; Chen et al. 2007a; Cho et al. 2010; Deepak et al. 2008; Ku et al. 2009; Kwon et al. 2011; Liu et al. 2005; Rasagnya and Vangalapati 2013; Unrean et al. 2012; Wang et al. 2009b; Mahajan et al. 2010). Concentration adjustment and feeding strategy of essential nutrients are critical for enhancing NK production. As such, it has been observed that yeast extract, soy peptone, and glycerol are the most effective nutrients to enhance the NK production rate. Moreover, we recently demonstrated that compared to the batch fermentation, the fed-batch addition of glycerol during the cell growth phase increases NK production significantly (Berenjian et al. 2014a). Ku et al. (2009) optimized the cultivation medium composed of defatted soybean and glucose. Defatted soybean is a by-product formed in salad oil manufacturing, and glucose is regarded a very common and cheap carbon source. The most important advantage is the low cost of substrates used to produce valuable NK. Interestingly, Pseudomonas sp. TKU015 and B. subtilis TKU007 are able to produce NK using shrimp shell wastes as the sole carbon/nitrogen source. Shrimp shell powder is a fishery waste; thus, the production procedure using this substrate would be very cost effective and favors producing inexpensive fibrinolytic enzymes (Wang et al. 2009c, 2011). Fibrinogen and fibrin have also been used as substrates in culture media for NK production. Park et al. (2013) used fibrin- or fibrinogen-containing tryptic soy broth media to produce and purify NK from B. subtilis WRL101. It was observed that in spite of increased fibrinolytic activity obtained in the fibrinogen-containing medium, fibrin-containing medium decreased the activity.
NK gene from B. subtilis natto is also cloned and expressed in a variety of hosts including B. subtilis, Escherichia coli, Lactococcus lactis, and Spodoptera frugiperda insect cells (Chiang et al. 2005; Li et al. 2007; Liang et al. 2007a, b; Liu and Song 2002; Nguyen et al. 2013). Many efforts have been invested to increase the recombinant NK production. For instance, by altering sequences of the -10 or -35 elements of the NK gene promoter (PaprN), especially -10 element, the extracellular expression of recombinant NK was improved (Wu et al. 2011). Employing expression vectors with high structure stability also led to higher production of recombinant NK (Chen et al. 2007b). DNA family shuffling is another mean to improve the fibrinolytic activity of NK. Using this method, it is feasible to generate a mutant library of NK to obtain a mutant with enhanced catalytic efficiency (Yongjun et al. 2011). Another promising procedure to enhance the extracellular production of NK in recombinant B. subtilis is assumed to be nutrient supplementation strategy. Using this technique, fourfold increase in recombinant NK production is reported with the medium supplemented with metal ions and glutamate (Chen and Chao 2006).
During downstream processing in the NK purification procedure, the traditional protein separation and purification methods, including organic solvent fractionation, salting out, and protein chromatography, have been utilized. However, some drawbacks such as lengthy separation and purification time, more operation units, and less activity recovery make these methods less useful (Chang et al. 2000; Deepak et al. 2009; Fujita et al. 1993; Rasagnya and Vangalapati 2013; Tonova and Lazarova 2008; Urano et al. 2001). Recently, novel methods for extraction and purification of NK are considered. For instance, reverse micelle extraction of this enzyme from fermentation broth using AOT/isooctane reverse micelles is reported for the first time, and the effects of temperature as well as phase volume ratio were examined (Liu et al. 2004, 2006). Three-phase partitioning (TPP), an efficient bioseparation technique, has been used to purify NK from fermentation broth of Bacillus natto NRRL-3666. In this technique, a combination of ammonium sulfate and t-butanol is exploited to precipitate protein between the lower aqueous layer and the upper organic layer (Garg and Thorat 2014). Alternative effective method of NK purification which is claimed to be faster compared to other methods is using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine (Yang et al. 2006). In a study performed by Chiang et al. (2005) either NK or pro-NK could be overexpressed in E. coli as a recombinant protein fused to the C-terminus of olesin, a unique structural protein of seed oil bodies, by means of a linker polypeptide called intein. Active NK was released through self-splicing of intein, after reconstitution of artificial oil bodies. This action was induced by alteration in temperature and spontaneous cleavage of the propeptide.
In addition to the site-directed mutagenesis and protein engineering techniques for improvement of NK stability, other methods have been tried. NK immobilization is conducted with polyhydroxybutyrate (PHB) nanoparticles which increases the stability of the enzyme. Accordingly, activity was completely retained on storage at 4 °C for 25 days (Deepak et al. 2009). Microencapsulation of NK using γ-polyglutamic acid as a coating material can improve temperature and pH stability of microencapsulated NK compared to those of the free form (Hsieh et al. 2009).
In order to protect NK from being denatured in the gastric juice, a new formulation was designed to control its release rate when it passes through the human gastrointestinal tract. Hence, NK powder was first compacted into a tablet, which was then coated with a mixture of an enteric material, called Eudragit L100-55, and hydroxypropylcellulose by direct compression (Law and Zhang 2007).
Nattokinase as a dietary supplement and functional food
As a functional food, NK owns several advantages compared to the available clinical thrombolytic drugs, such as safety, low cost, confirmed efficacy, prolonged effects, preventative use, and easy oral administration (Peng et al. 2005; Zheng et al. 2005). NK is absorbed from the intestinal tract and has pH and temperature stability so that it can remain stable in the gastrointestinal system (Fujita et al. 1995b; Sumi et al. 1987). The effectiveness of B. subtilis NK capsules in dissolving thrombi in dogs is reported by Sumi et al. (1990). In a study performed by Suzuki et al. (2003b), dietary natto-extract supplementation caused suppression of intimal thickening produced by endothelial injury in rat femoral artery. Similarly, it has been observed that dietary supplementation with natto extract containing NK, suppresses intimal thickening after vascular injury (Suzuki et al. 2003a). As Ero et al. (2013) demonstrated, when administering oral single dose of NK supplement, peak serum levels were observed at approximately 13.3 h ± 2.5 h post-dose. In order to create Lactococcus lactis probiotic strain that is capable of producing or even secreting NK, Lee et al. (2010) combined the functions of L. lactis and the NK from B. subtilis natto and further assessed the safety and the risk of oral administration of this genetically modified microorganism. Murine models were also used to detect the allergenicity of this genetically modified lactic acid bacterium which showed to be regarded as safe to use (Chiang et al. 2011). Orally administrated NK combined with red yeast rice showed lipid-lowering effect. However, this effect was not associated with NK consumption alone, suggesting that combined NK and red yeast rice will be a better neutraceutical for patients with hyperlipidemia (Yang et al. 2009).
Effects on blood pressure
In a randomized double-blinded placebo-controlled trial, the effects of NK supplementation on blood pressure in pre-hypertension or stage 1 hypertension subjects were examined. In this regard, NK supplementation resulted in systolic and diastolic blood pressure reduction which is suggestive of its role in the prevention and treatment of hypertension (Kim et al. 2008). Inhibition of angiotensin I-converting enzyme (ACE) by NK and its degradation peptides may be attributed to suppression of blood pressure (Murakami et al. 2012). Therefore, consumption of functional foods containing NK with antihypertensive activity is a successful strategy for the treatment of hypertension (Suwanmanon and Hsieh 2014). A research was conducted to elucidate the mechanism by which NK prevents hypertension and clarify whether the protease activity of this enzyme is needed or not. The results demonstrated that both intact form of NK and its fragments are absorbed from the intestine and reduce hypertension in spontaneously hypertensive rats. Nevertheless, depending on the form of oral administration, different mechanisms are suggested for antihypertensive effect. When intact NK (having protease activity) is administered, blood pressure may decrease through the reduction of blood viscosity by cleaving plasma fibrinogen. On the other hand, the fragments obtained from NK suppress hypertension via downregulation of plasma angiotensin II level (Fujita et al. 2011).
Further therapeutic applications of NK
When medical devices are in contact with blood, thrombogenesis is likely to occur immediately. After days to weeks without careful antithrombotic therapy, the thrombus complications may be evident. Therefore, considerations should be made to inhibit implant-associated thrombosis. To prevent this complication, NK is used as an innovative strategy for coating medical devices with antithrombotic activity. Local application of NK as novel composite particles has also been indicated for inhibition of implant-associated thrombosis (Wei et al. 2014). Oral administration of NK in the rat model of Alzheimer’s disease has been demonstrated a remarkable effect in modulation of certain factors of this disease (Fadl et al. 2013). NK is also earning interest as a novel enzyme for pharmacologic vitreolysis in patients with proliferative vitreoretinal disorders. This action is due to its efficacy in inducing posterior vitreous detachment (PVD) (Takano et al. 2006). A significant, dose-dependent decrease of red blood cell aggregation and low shear viscosity of blood have been observed in NK administration. This fact indicates positive hemorheological effects of this enzyme which suggests its potential value as a therapeutic enzyme (Pais et al. 2006).
Deep venous thrombosis (DVT) and pulmonary embolism (PE) are matters of concern associated with prolonged air travel. Flite Tabs (Aidan, AZ, USA) displayed meaningful promotion of vein health and reduction of leg swelling in a randomized controlled study of subjects on international long-haul flights. This medication contains a new pharmacologic compound, pinokinase (a combination of pycnogenol and nattokinase). This mixture improves fibrinolysis and controls edema. Thus, administering NK can be considered an effective preventive strategy in subjects at high risk for DVT during long-haul flights (7–8 h) (Cesarone et al. 2003).
Miscellaneous applications of NK
Prion diseases are transmissible through contaminated surgical instruments. NK is able to tolerate a temperature of 50 °C and functions under basic conditions, such as pH 10, suggesting that it might be useful in instrument decontamination. Hsu et al. (2009) demonstrated that purified NK is capable of decreasing amyloid structure of recombinant human PrP fibrils (human prion peptide sequence 108–144) after 48 h of exposure (40 °C, pH 7). This method is also a valuable mean of enzymatic inactivation of prions in soil environments (Booth et al. 2013). An aqueous solution of NK provides lubricity of poly(etheretherketone), reducing the wear of the polymer. It can also be considered an effective lubricant in the presence of NaCl in the solution (Minn and Sinha 2012). Furthermore, NK is being used as the key ingredient of extraction method to recover encapsulated mesenchymal stem cells from 3D fibrin gels (Carrion et al. 2013). NK is gaining attention as a valuable enzyme in dairy processing too. During cheese ripening process, a variety of biochemical alterations occur. Proteolysis is one of the most significant of these biochemical changes which harden cheese varieties such as cheddar. Addition of NK as an exogenous proteolytic enzyme can accelerate this process. In addition, by using NK, higher levels of free amino acids is observed, which serve as substrates for many catabolic reactions and in turn improve the flavor of cheese (Upadhyay et al. 2006).
Conclusion and perspectives
Since NK is derived from food-grade microorganisms, it has the potential to be developed as functional food additive. NK also represents a promising drug candidate for use in both prevention and treatment of thrombotic disorders. Being stable enough in the gastrointestinal tract makes this enzyme a useful agent for oral thrombolytic therapy. Thus, NK is also regarded as a valuable food dietary supplement or nutraceutical. Proven safety and ease of mass production are among the advantageous factors. This enzyme has attracted more attention for its application in various industries. Besides nattokinase’s effectiveness and safety for managing a wide range of thrombovascular diseases, it has a significant potential for the treatment of hypertension, Alzheimer’s disease, and vitreoretinal disorders. The current retail prices for nattokinase in 100 mg quantities (equivalent to 2,000 fibrinolytic units) have a market price of US$50 to US$75. Therefore, the current deal for nattokinase production is to decrease the cost of product by increasing the yield or integrated processes to reduce the processing cost and make the product available to a wide range of consumers.
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Dabbagh, F., Negahdaripour, M., Berenjian, A. et al. Nattokinase: production and application. Appl Microbiol Biotechnol 98, 9199–9206 (2014). https://doi.org/10.1007/s00253-014-6135-3
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DOI: https://doi.org/10.1007/s00253-014-6135-3