Subtilisin Nattokinase Research Articles

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.

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.

Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation

Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu31 for Ile31 resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent.

Effects of Bacillus subtilis var. natto products on symptoms caused by blood flow disturbance in female patients with lifestyle diseases.

The fermented soybean product natto is a popular traditional food in Japan and is considered a health supplement. NKCP®, a natto-derived dietary food supplement whose main component is bacillopeptidase F, has antithrombotic, fibrinolytic, and blood pressure-lowering effects. We examined whether daily intake of NKCP® effectively improves subjective symptoms in patients with lifestyle diseases in this cross-over, double-blind study. Fermented soya extract with subtilisin NAT (nattokinase) as the main component was used as an active placebo. A 4-week course of NKCP® significantly decreased the visual analog scale (VAS) score for shoulder stiffness from 42.3 to 32.4 (P=0.009), the VAS score for low back pain from 25.5 to 18.8 (P=0.02), and the VAS score for coldness of the extremities from 33.1 to 25.7 (P=0.002). However, no significant difference was found in the VAS score for headache. After a 4-week course of active placebo, no significant changes in the VAS score were found for any symptoms. The significant improvement in the symptoms secondary to blood flow disturbance was caused by the improvement in blood flow by NKCP®. The use of dietary supplements based on the Japanese traditional food natto helps to relieve subjective symptoms for patients with lifestyle diseases receiving medical care.

Thermostability of subtilisin nattokinase obtained by site-directed mutagenesis

To study the thermostability of Nattokinase (subtilisin NAT, NK), three double mutant plasmids (pET-28a-NKG61C/S98C, pET-28a-NKT22C/S87C, pET-28a-NKS24C/S87C) were constructed by site-directed mutagenesis. Target enzymes were detected using SDS-PAGE and disulfide bond formation was detected using Western blotting analysis. Thermostability was tested by rates of inactivation at certain temperature. The results showed that disulfide bond was not formed within two cysteines and the thermostability of three double mutants was not increased compared with the wild-type NK. The thermostability of NK performed in Ca2+ was stronger than in ethylenediaminetetraacetic acid (EDTA). But when the temperature reached 62 °C, the enzymes rapidly denatured and inactivated even in the presence of Ca2+. Although the thermostability of mutants was not increased, this study shows a tendency of improving thermostability of NK in protein engineering.

Inhibition of angiotensin I converting enzyme by subtilisin NAT (nattokinase) in natto, a Japanese traditional fermented food

Angiotensin I converting enzyme (ACE) was inhibited by the culture medium of Bacillus subtilis subsp. natto, which ferments boiled soy beans to natto, a Japanese traditional food. Subtilisin NAT (nattokinase) produced by B. subtilis also inhibited ACE, and the inhibition was markedly stimulated by heat treatment of subtilisin at 120 °C for 15 min. Inhibition of ACE by subtilisin was of a mixed type: the decrease in V(max) and the increase in K(m) value. SDS-polyacrylamide gel electrophoresis showed that heat treatment of subtilisin caused inactivation with fragmentation of the enzyme protein into small peptides. The inhibitory action of subtilisin was not due to an enzymatic action of protease, but may be ascribed to the potent ACE-inhibitory peptides such as LY and FY, amino acid sequences in subtilisin. HPLC-MS analysis of heat-inactivated subtilisin confirmed that LY and FY were liberated by fragmentation of the enzyme. Inhibition of ACE by subtilisin and its degradation peptides such as LY and FY may participate in the suppression of blood pressure by ingestion of natto.

Functional analysis of propeptide as an intramolecular chaperone for in vivo folding of subtilisin nattokinase

Here, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to β-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model.

Safety and risk assessment of the genetically modified Lactococci on rats intestinal bacterial flora

The interaction between Lactococcus lactis NZ9000/pNZPNK and intestinal microflora was evaluated as a method to assess safety of genetically modified microorganisms (GMMs). L. lactis NZ9000/pNZPNK is one kind of GMM and able to produce the intracellular subtilisin NAT (nattokinase) under induction with nisin. The host strain L. lactis NZ9000 was a generally recognized as safe (GRAS) microorganism. Six groups of Wistar rats were orally administered with L. lactis NZ9000/pNZPNK and L. lactis NZ9000 for 6 weeks. Fecal and cecal contents were collected to determine the number of L. lactis NZ9000, L. lactis NZ9000/pNZPNK, Lactobacillus, coliform bacteria, beneficial bacteria Bifidobacterium and harmful bacteria Clostridium perfringens. The liver, spleen, kidney and blood were evaluated for the bacterial translocation. After 6 weeks consumption with GM and non-GM Lactococcus, no adverse effects were observed on the rat's body weight, hematological or serum biochemical parameters, or intestinal microflora. The bacterial translocation test showed that L. lactis NZ9000/pNZPNK did not translocate to any organ or blood. Bifidobacterium was significantly increased in feces after administration of both Lactococcus strains ( L. lactis NZ9000 and L. lactis NZ9000/pNZPNK), while C. perfringens remained undetectable during the experiment. These results suggested that L. lactis NZ9000/pNZPNK could be safe in animal experiments and monitoring of the interaction between test strains and intestinal microflora might be applied as a method for other GMM safety assessments.

Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli

Nattokinase (subtilisin NAT, NK) is a bacterial serine protease with strong fibrinolytic activity and it is a potent cardiovascular drug. In medical and commercial applications, however, it is susceptible to chemical oxidation, and subsequent inactivation or denaturation. Here we show that the oxidative stability of NK was substantially increased by optimizing the amino acid residues Thr 220 and Met 222, which were in the vicinity of the catalytic residue Ser 221 of the enzyme. Two nonoxidative amino acids (Ser and Ala) were introduced at these sites using site-directed mutagenesis. Active enzymes were successfully expressed in Escherichia coli with periplasmic secretion and enzymes were purified to homogeneity. The purified enzymes were analyzed with respect to oxidative stability, kinetic parameters, fibrinolytic activity and thermal stability. M222A mutant was found to have a greatly increased oxidative stability compared with wild-type enzyme and it was resistant to inactivation by more than 1 M H 2O 2, whereas the wild-type enzyme was inactivated by 0.1 M H 2O 2 ( t 1/2 ∼ 11.6 min). The other mutant (T220S) also showed an obvious increase in antioxidative ability. Molecular dynamic simulations on wild-type and T220S mutant proteins suggested that a hydrogen bond was formed between Ser 220 and Asn 155, and the spatial structure of Met 222 was changed compared with the wild-type. The present study demonstrates the feasibility of improving oxidative stability of NK by site-directed mutagenesis and shows successful protein engineering cases to improve stability of NK as a potent therapeutic agent.

Characterization of Bacillus subtilis strains in Thua nao, a traditional fermented soybean food in northern Thailand

To clarify the diversity of Bacillus subtilis strains in Thua nao that produce high concentrations of products useful in food manufacturing and in health-promoting compounds. Production of amylase, protease, subtilisin NAT (nattokinase), and gamma-polyglutamic acid (PGA) by the Bacillus subtilis strains in Thua nao was measured. Productivity of protease NAT by these strains tended to be higher than by Japanese commercial natto-producing strains. Molecular diversity of isolated strains was analysed via randomly amplified polymorphic DNA-PCR fingerprinting. The strains were divided into 19 types, including a type with the same pattern as a Japanese natto-producing strain. B. subtilis strains that could be a resource for effective production of protease, amylase, subtilisin NAT, or PGA were evident in Thua nao produced in various regions in northern Thailand. This study clearly demonstrated the value of Thua nao as a potential resource of food-processing enzymes and health-promoting compounds.

Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation

Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33-->Ala33, Asp60-->Ala60, Ser62-->Ala62, and Thr220-->Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (DeltaDeltaG(T)). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the K(m) values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations.

The Profibrinolytic Enzyme Subtilisin NAT Purified fromBacillus subtilis Cleaves and Inactivates Plasminogen Activator Inhibitor Type 1

In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1). Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B. subtilis (natto). Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments. Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1': Arg(346)-Met(347)). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02-1.0 nm; half-maximal effect at approximately 0.1 nm). Subtilisin NAT dose dependently (0.06-1 nm) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 +/- 1.4% at 1 nm) and especially in the presence of rpPAI-1 (78 +/- 2.0% at 1 nm). The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT. The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1. This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis.