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Production and purification of nattokinase from Bacillus subtilis

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Nguyễn Gia Hào

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Food Biotechnology

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Production and purification of nattokinase from Bacillus subtilis

Nguyen Hoang Minh, Huynh Thi Quynh Trang, Tran Bao Van & Nguyen Hoang Loc

To cite this article: Nguyen Hoang Minh, Huynh Thi Quynh Trang, Tran Bao Van & Nguyen Hoang Loc (2022) Production and purification of nattokinase from Bacillus�subtilis, Food Biotechnology, 36:1, 1-21, DOI: 10.1080/08905436.2021.2005622

To link to this article: https://doi.org/10.1080/08905436.2021.2005622

Published online: 09 Jan 2022.

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Production and purification of nattokinase from Bacillus subtilis

Nguyen Hoang Minha, Huynh Thi Quynh Trangb, Tran Bao Vanb, and Nguyen Hoang Locb

aClinical Skills Laboratory, University of Medicine and Pharmacy, Hue University, Hue, Vietnam;

bDepartment of Biotechnology, University of Sciences, Hue University, Hue, Vietnam

ABSTRACT

The present study is focused on the production and purification of nattokinase, a fibrinolytic protease, from B. subtilis TH9 (NatTH9) based on an aqueous two-phase system (ATPS) tech- nique. The results showed that the optimal ATPS for NatTH9 recovery was 20% (w/v) polyethylene glycol 6000 and 15% (w/v) potassium phosphate at pH 8. The partitioning coefficient, the partitioning yield, and the activity of NatTH9 were 6.25, 76.7%, and 547.02 U/mg, respectively. The purified NatTH9 demon- strated the ability to degrade fibrin and dissolve the clot.

Fibrin zymography showed three clear zones on the gel with molecular weights of approximately 37, 27, and 21 kDa. The optimal pH and temperature of purified NatTH9 were 8 and 39°

C, respectively.

KEYWORDS

Aqueous two-phase system;

Bacillus subtilis; nattokinase;

polyethylene glycol;

potassium phosphate

1. Introduction

Nattokinase (also known as fibrinolytic protease) belongs to the serine pro- tease group that is generally active at neutral and alkaline pH, with an optimal pH between 8 and 10 (Raju and Divakar 2014) and is capable of degrading fibrin which is normally formed from fibrinogen by the action of thrombin (Fujita et al. 1993). The enzyme not only directly cleaves cross-linked fibrin but also activates the production of tissue plasminogen activator (tPA), result- ing in the transformation of inactive plasminogen to activate plasmin for slicing fibrin to avoid thrombosis in blood vessels (Nagai et al. 2004; Peng, Yang, and Zhang 2005). Furthermore, nattokinase enhances its fibrinolysis through cleavage and inactivation of plasminogen activator inhibitor-1 (PAI- 1), which is the primary inhibitor of fibrinolysis and regulates total fibrinolytic activity by its relative ratio with tPA (Peng, Yang, and Zhang 2005). In addition to its potent fibrinolytic and thrombolytic effects, nattokinase is also known to have antihypertensive, anti-atherosclerotic, lipid-lowering, antiplatelet/anticoagulant, and neuroprotective effects. All these pharmacolo- gical activities of nattokinase are relevant for the prevention and treatment of

CONTACT Nguyen Hoang Loc nhloc@hueuni.edu.vn University of Sciences, Hue University, 77 Nguyen Hue St, Hue, Vietnam

2022, VOL. 36, NO. 1, 1–21

https://doi.org/10.1080/08905436.2021.2005622

© 2022 Taylor & Francis Group, LLC

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cardiovascular diseases (Chen et al. 2018). According to Weng et al. (2017), both nattokinase and lumbrokinase (derived from earthworms), unlike most proteins, are more resistant to the highly acidic gastric fluids in the stomach and can be absorbed in the later sections of the digestive tract. Previously, Fujita et al. (1995) demonstrated that nattokinase could be absorbed from the rat intestinal tract in an intact form and fibrinogen was degraded in plasma blood samples.

On the basis of the catalytic mechanism, fibrinolytic proteases from micro- organisms are classified into three types, serine protease (e.g. nattokinase from Bacillus), metalloprotease (e.g. Armillaria mellea metalloprotease), and a mix- ture of both types of proteases above (e.g. protease from Streptomyces) (Raju and Divakar 2014). Recently, some types of nattokinase with fibrinolytic and clot lysis activities have been obtained from strains of various B. subtilis isolated from fermented soybean foods which proved to be effective in attenu- ating the effects of diabetes mellitus, blood pressure, and cardiac disorders (Frias et al. 2021; Hu et al. 2019; Jayachandran and Xu 2019; Ju et al. 2019;

Lucy et al. 2019; Pinontoan, Elvina, and Jo 2021; Sahoo et al. 2020; Sharma et al. 2020). However, most of these studies did not use aqueous two-phase system (ATPS) for nattokinase purification, a technique that could easily be applied to industrial-scale enzyme production.

ATPS is widely used in biotechnology applications as a cost-effective and environment-friendly technique for the purification of proteins/enzymes from intracellular and extracellular extracts (Xu, He, and Li 2005). ATPS is formed by mixing solutions of two polymers (e.g. polyethylene glycol (PEG)/dextran) or a polymer (PEG) and a certain salt (e.g. sulfate, phosphate, or citrate) to form two immiscible phases (Iqbal et al. 2016). Proteins and cellular debris display differential solubility between the two phases so that this technique can be used both for the separation of proteins from cellular debris and for the partitioning of enzymes during protein purification (Scawen and Hammond 2002). Most often, this technique is employed in the production of enzymes on an industrial or laboratory scale. Therefore, ATPS has significant potential for the purification of fermented products carried out in downstream processing (Vázquez-Villegas et al. 2018).

This study was aimed to produce nattokinase from B. subtilis TH9 (NatTH9), a bacterial isolate that can highly biosynthesize NatTH9 with suitable characteristics (e.g. acts at 37–39°C) for application as an additive in functional foods to prevent cardiovascular diseases. Besides, ATPS technique formed by mixing PEG and potassium phosphate (PP) with the appropriate molecular weight (MW) and concentration was also applied to purify the enzyme. B. subtilis is considered a benign organism as it does not possess traits that cause disease (EPA 1997), therefore, the results of the present research promise potential applications in the biotechnological production of enzymes.

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2. Materials and methods

The experimental scheme is shown in Figure 1.

2.1. Bacillus subtilis culture and protease production

B. subtilis TH9 isolated from Vietnamese traditional fermented soybean food on skim milk- Luria-Bertani (LB) agar plate (per 1 L: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar, and 10 g skim milk) at 37°C after 48 h of culture.

A clear zone surrounding the colony confirmed the protease secretion to the medium. Molecular identification of isolates was performed by 16S rRNA sequencing and a BLAST search in the database of Genbank. Among the isolates, B. subtilis TH9 with high protease activity was chosen for determining fibrinolytic activity. B. subtilis TH9 culture and extracellular protease produc- tion were carried out based on previous research with a slight modification (Loc, Mien, and Thuy 2010). Bacteria were cultured in a medium consisting of (per L) 3 g beef extract, 10 g peptone, and 5 g sodium chloride (pH 7) at 37°C for 36 h on the rotary shaker with a speed of 200 rpm. The doubling time for cell density (Td) was calculated using the following formula (Roth 2006):

Td ¼ t�log 2 log a log b

Where: t is the duration of culture, a is final cell biomass, and b is initial cell biomass.

Figure 1. Scheme of the experimental design.

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The extracellular matrix proteases were produced in the same conditions by using a medium containing (per L) 1.8 g beef extract, 6 g peptone, 5 g sodium chloride, and 10 g skim milk (Merck) as a substrate (pH 7). After 16 h of fermentation, the culture medium was centrifuged at 15,500 × g at 4°C for 5 min to harvest the broth for use as the crude enzyme.

2.2. Preparation of aqueous two-phase systems

Aqueous two-phase systems consisting of primary and back extraction were essentially prepared as described in our previous study (Loc, Mien, and Thuy 2010). Various ATPSs were formed from 60% (w/v) polyethylene glycol (PEG) and 30% (w/v) potassium phosphate (PP) at room temperature (RT) with a final volume of 10 mL. The pH of ATPSs was adjusted to 8 by using an appropriate ratio of dipotassium/monopotassium phosphate. Primary extrac- tion was carried out by adding 4 mL of the crude enzyme from B. subtilis TH9 culture to each ATPS. These ATPS components were lightly mixed and then centrifuged at 385 × g for 10 min at RT to separate into two phases. The phase volume ratios were measured in 15 mL graduated Falcon tubes. Nattokinase activity was also determined for the top and bottom phases. The nattokinase partitioning coefficient (KNatTH9) is defined as the ratio of nattokinase activity in the top and bottom phases. The volume ratio (V) was the ratio of the volume in the top phase (VT) to the volume in the bottom phase (VB). The partitioning yield of nattokinase (%) in the top phase, Y, is given by the following equation (Albertson 1958):

Y ¼ 100

VVB

T

� �

K 1

NatP09

� �

Back extraction was conducted to recover NatTH9 which partitioned selec- tively to the PEG-rich top phase in the primary extraction. 25% (w/v) fresh PP buffer was added to the PEG-rich top phase of the primary extraction at a ratio of 3:7. Low-speed centrifugation of 385 × g at RT for 10 min was performed to separate the phases. The precipitate of NatTH9 and PP in the bottom phase was dialyzed at 4°C overnight to remove the salt and used for further experiments.

The purification factor (PF) and recovery yield (RY) of the enzyme was calculated according to the following formulas:

PF¼EC EP

RY¼VC

VP�100

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Where: EC is the activity of crude enzyme and EP is the activity of partially purified crude enzyme or purified NatTH9, VC is the total volume of crude enzyme solution and VP is the total volume of partially purified crude enzyme solution or purified NatTH9 solution.

2.3. Protease activity assay

Protease activity was assayed by incubating 1 mL of the partially purified crude enzyme and 2 mL of 2% (w/v) casein substrate (Sigma-Aldrich) in 50 mM Tris-HCl (pH 7) at 50°C for 15 min. The reaction was then stopped by the addition of 5 mL of 5% (w/v) trichloroacetic acid (TCA) (Anson 1938). For partial purification, the crude enzyme was precipitated by ammonium sulfate (70% saturation) at 4°C for 2 h and then centrifuged at 17,800 × g at 4°C for 10 min. The pellet was resuspended and dialyzed for further use.

Tyrosine released from casein was measured at a wavelength of 750 nm.

One unit of protease activity is defined as the amount of enzyme required to release 1 µmol of tyrosine from substrate per 1 mL per min under the standard conditions.

2.4. Nattokinase activity assay

Nattokinase activity was determined as described by Deepak et al. (2008). The reaction mixture consists of 140 µL of 50 mM Tris-HCl (pH 7) and 40 µL of 0.72% fibrinogen (Sigma-Aldrich) was incubated at 37°C for 10 min, followed by the addition of 0.2 NIH U thrombin (Sigma-Aldrich) and 10 µL of purified NatTH9, and left at 37°C for 60 min. The reaction was terminated with 200 µL of 0.2 M TCA, the supernatant was recovered by centrifugation at 17,800 × g at 4°C for 5 min to measure the absorbance at 275 nm. One unit of nattokinase activity is defined as the amount of enzyme required to increase the absor- bance by 0.01 at 275 nm after 1 h of incubation. The nattokinase activity is expressed as units of enzyme per milligram of total protein (TP).

TP content from culture broth was determined by Bradford’s method (1976) with bovine serum albumin (Sigma-Aldrich) as standard. The absor- bance was measured at 595 nm against the blank.

2.5. Characterization of NatTH9

The optimal temperature and pH for nattokinase activity were investigated in a range of 31–47°C and pH 3–10, respectively. Buffers for optimal pH deter- mination were 20 mM citrate solution (pH 3–6), 20 mM phosphate solution (pH 7–8), and 20 mM glycine-sodium hydroxide solution (pH 9–10). The thermal and pH stability of NatTH9 was determined by incubating the enzyme

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for 30 min at the same temperatures and pHs of the optimal investigations without substrate, enzyme solution was then kept at 4°C before activity assay (Thu et al. 2020).

Effect of some metal ions (Mg2+, Cu2+, Ca2+, Zn2+, and Mn2+) and reagents (phenylmethylsulfonyl fluoride – PMSF, sodium dodecyl sulfate – SDS, Triton X-100, and ethylenediaminetetraacetic acid – EDTA) on nattokinase activity were investigated by incubating enzyme at the optimal temperature and pH for 30 min with 5 mM of a metal ion or 5% of a reagent (Thu et al. 2020).

The nattokinase activity after incubation was determined as described above. The relative activity of NatTH9 is the ratio of the nattokinase activity at different temperatures and pHs to that at 37°C and pH 7 (control) and expressed as a percentage.

2.6. Fibrin zymography

Fibrin zymography was performed as described by Choi et al. (2005) with a slight modification. 12% (w/v) polyacrylamide gel was prepared in the pre- sence of 0.12% (w/v) fibrinogen and 1 NIH U thrombin. 2 µg of purified NatTH9 dissolved in sample buffer (0.5 M Tris-HCl pH 6.8, 10% SDS, 20%

glycerol, and 0.03% bromophenol blue) was loaded into each well of the gel.

Electrophoresis was conducted at 4°C and the gel was then incubated in the solution consisting of 50 mM Tris-HCl (pH 7.4) and 2.5% Triton X-100 for 30 min at RT to remove SDS. In the next step, the gel was washed with double distilled water (ddH2O) for 30 min to remove Triton X-100 and then incu- bated in a reaction buffer (30 mM Tris-HCl pH 7.4 and 0.02% sodium azide) at 37°C for 12 h. Finally, the gel was stained with Coomassie Blue R-250 for 1 h to reveal the zones where fibrin was digested and visualized as clear zones (unstained zones) of the zymogram gel.

2.7. Fibrin plate and clot lysis assay

The fibrin plate test was employed for evaluating the fibrinolytic activity of the purified NatTH9. Fibrinogen level 0.6% (w/v) in a 50 mM sodium phosphate buffer (pH 7.4) was mixed with 2% (w/v) agarose and 1 NIH U thrombin. The mixture was poured into a Petri dish and was left for 1 h at RT to form a fibrin clot layer. 20 μL (2 μg) of the enzyme was then dropped on the surface of the fibrin plate, and the plate was incubated at 37°C for 8–12 h. Fibrinolytic activity was estimated by measuring the diameter of the clear zone (Choi et al. 2004).

A volume of 500 μL of rabbit blood was transferred to the Eppendorf tube and incubated at 37°C for 30 min. After clot formation, the serum was completely removed, and the clot was washed with physiological salt solution

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four times. The clot was then transferred into a new tube containing 5 mL of physiological salt solution and 500 μL of NatTH9. Finally, the tube was incubated at 37°C for 12 h for clot lysis (Sahoo et al. 2020).

2.8. Statistical analaysis

Each experiment was repeated 3 times and the means were compared by one- way ANOVA (Duncan’s test at a confidence coefficient of 0.05) by using the SPSS program.

3. Results and discussion 3.1. Enzyme production

B. subtilis TH9 displayed a simple growth curve pattern and showed the changes in the size of a bacterial population over time during culture. Data in Figure 2 shows a time course of cell growth and TP production of B. subtilis TH9 during 32 h of growth phase. Samples were taken periodically for the determination of cell density and TP content. Cell growth and protein content increased continuously from the beginning to 20 h of culture with the density reaching a maximum value of approximately 1.2 × 1010 cells/mL (correspond- ing to Td of cell density was about 5.6 h) and TP content was 0.4 mg/mL.

During the period of 20–24 h, the cells appeared to enter the stationary stage, so the differences in cell density were non-significant (p > .05). After 24 h, the

Figure 2. Time course of cell growth in terms of density and total protein production of B. subtilis TH9 during 32 h of culture. Different letters on a curve represent statistically significant differences with p < .05 (Duncan’s test), uppercase letter: cell density, lowercase letters: total protein.

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cells were already in the death phase. The profile of protease activity during growth of B. subtilis TH9 is shown in Figure 3. The activity peaked after 16 h of culture, 4 h before the end of the log phase, with a value of about 84 U/mg.

Figure 3. Profile of protease activity (U/mg) during 32 h of B. subtilis TH9 culture. Different letters represent statistically significant differences with p < .05 (Duncan’s test).

Figure 4. The partition yield (Y%) of NatTH9 in the top phase of different ATPSs was formed by combining PP from 9–15% with 25% PEG 2000, 20% PEG 6000, and 15% PEG 10000. Different letters represent statistically significant differences with p < .05 (Duncan’s test).

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3.2. Partitioning of nattokinase

The partitioning of a protein depends on its MW and charge, the MW and concentration of the polymer, the presence of polyvalent salts, temperature, pH, and the ionic strength of ATPS (Johansson 1985). The present study investigated the effects of PEG and PP on the partitioning of NatTH9. The results indicated that NatTH9 exhibited a high affinity for the top phase (PEG- rich phase).

3.2.1. Effect of PEG on partition coefficient

Effect of PEG (MW from 2000 to 10000 and concentration from 15 to 25%) on the partition coefficient of NatTH9 was investigated in combination with 12%

PP. The results in Table 1 showed that NatTH9 tends to move up to the top

Figure 5. The specific activity (U/mg) of NatTH9 in the top phase of different ATPSs was formed by combining PP from 9–15% with 25% PEG 2000, 20% PEG 6000, and 15% PEG 10000. Different letters represent statistically significant differences with p < .05 (Duncan’s test).

Table 1. Effect of PEG on the partitioning of NatTH9.

PEG

V KNatTH9

MW Concentration (%)

2000 15 0.29 ± 0.01 0.92 ± 0.03

20 0.41 ± 0.03 1.63 ± 0.12

25 0.49 ± 0.02 1.79 ± 0.14

6000 15 0.39 ± 0.01 1.81 ± 0.16

20 1.04 ± 0.11 3.74 ± 0.31

25 0.55 ± 0.02 1.91 ± 0.12

10000 15 1.85 ± 0.13 5.75 ± 0.46

20 0.68 ± 0.03 2.16 ± 0.14

25 0.41 ± 0.02 1.68 ± 0.11

Note: Concentration of PP in systems was 12% (w/v). The values are expressed as the means of three replicates ± the standard errors, this note is also used for Table 2.

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phase of ATPSs (KNatTH9 from 0.92 to 5.75). KNatTH9 increased with the MW of PEG, from 2000 to 6000 for 20–25% PEG or from 2000 to 10000 for 15%

PEG and reached a maximum value of 5.75 at 15% PEG 10000.

The polymerization increase (20–25% PEG 10000) resulted in less space for proteins to partition into the top phase. The fewer hydroxyl groups increased hydrophobicity in the top phase, and the size-exclusion effect was suggested as the main cause for the reduction of partitioning coefficient (Zhi et al. 2004).

Partitioning of NatTH9 in ATPSs with PEG 2000 (lower MW) increased slightly. However, in ATPSs with PEG 6000 and 10000 (higher MW), NatTH9 was strongly partitioned to the top phase in 20% PEG 6000 or 15%

PEG 10000. It is possible that the large size of the PEG molecules had caused the size exclusion effect.

3.2.2. Effect of PP on partition coefficient

In this study, two concentrations of 9 and 15% PP were combined with the appropriate concentrations and MWs of PEG compared to the previous investigation which employed 12% PP to determine the partitioning coeffi- cient for NatTH9. The data in Table 2 shows that KNatTH9 increased with PP concentration from 9 to 15% when combined with PEG 2000 and 6000 and peaked at a value of 6.52 in the 20% PEG 6000 and 15% PP system. The partitioning coefficient of NatTH9 was also relatively high in the system of 15% PEG 10000 and 9–15% PP with K reaching from 2.34 to 5.75.

In ATPS using PEG and PP, the PEG-rich phase is the top phase, while the PP-rich phase is the bottom phase. Protein solubility in the top phase was determined by interaction between the ethylene group of PEG and the hydro- phobic residue of protein (Baskir, Hantton, and Sutter 1989; Lee and Sandler 1990). Reducing the protein solubility in the bottom phase by increasing the

Table 2. Effect of PP on the partitioning of NatTH9.

PEG PP (%) V KNatTH9

25% PEG 2000 9 0.29 ± 0.02 0.98 ± 0.08

12 0.49 ± 0.02 1.79 ± 0.14

15 0.61 ± 0.04 1.92 ± 0.14

20% PEG 6000 9 0.49 ± 0.03 1.87 ± 0.15

12 1.04 ± 0.11 3.74 ± 0.31

15 2.32 ± 0.24 6.52 ± 0.55

15% PEG 10000 9 0.76 ± 0.3 2.34 ± 0.18

12 1.85 ± 0.13 5.75 ± 0.46

15 1.31 ± 0.10 4.21 ± 0.40

Table 3. Summary of extracellular enzymes from B. subtilis TH9.

Enzyme

Total volume (mL)

Total protein (mg/mL)

Nattokinase activity (U/mg)

Purification factor

Recovery yield (%)

Crude enzyme 4 0.53 121.14 1 100

Partially purified crude enzyme

3.48 0.42 401.24 3.31 87.2

Purified NatTH9 3.1 0.06 547.02 4.52 76.7

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concentration of PP can lead to an increase in the partitioning coefficient of the enzyme. The present study also showed an effective partition of NatTH9 from B. subtilis TH9 in 20% PEG 6000 and 15% PP system.

3.2.3. Partitioning yield and purification factor

The partitioning yield (Y) of an enzyme depends on its VB/VT and K values. In the majority of ATPSs investigated in this study, the partitioning of NatTH9 was mainly directed toward the top phase, so the volume values (VT) were relatively high, resulting in a high Y value. The highest Y of NatTH9 was 63.12% (specific activity of NatTH9 was about 547 U/mg) with KNatTH9

coefficient of 6.52, VB/VT ratio of 0.43 (with VT/VB ratio of 2.32), and pur- ification factor (PF) of 4.52 in 20% PEG 6000 and 15% PP system (Figs. 4 and 5, Tables 2 and 3).

Some previous studies reported on the partitioning of enzymes from the various ATPSs, especially fibrinolytic proteases. Nascimento et al.

(2016) obtained the K of fibrinolytic enzymes from Mucor subtilissimus UCP 1262 as 0.2 in the 30% PEG 6000 and 13.2% sodium sulfate system.

Cruz Filho et al. (2020) obtained the K of fibrinolytic enzymes from B.

stearothermophilus DPUA 1729 as 1.45 in the 10% PEG 1000 and 25% PP system. Values for PF, K, and Y reached 6.26, 1.32, and 141.57%, respec- tively, found in the system of 12.5% PEG 8000 and 15% sodium phos- phate at pH 8 of fibrinolytic proteases from Gliricidia sepium seed (Silva et al. 2020). A study by Souza et al. (2012) indicated that in all ATPS studied, fibrinolytic proteases from Bacillus sp. UFPEDA 485 partitioned to the top phase and the highest extraction was obtained in the 24% PEG 6000 and 11.6% sodium sulfate system with PF = 3.30, K = 5.03, Y

= 91.40%, and fibrinolytic activity in the top phase reached 821 U/mL.

In Streptomyces sp. DPUA1576, fibrinolytic protease partitioned preferen- tially to the PEG-rich phase with the highest partition coefficient (K = 37) obtained in the system of 20% PEG 1500 and 14% phosphate at pH 8. The system that allowed for the highest extraction consisted of 15% PEG 3350 and 12% phosphate at pH 7. In these conditions, a yield of 155% and a purification factor of 1.51 was obtained for a partition coefficient of 6.41 with the fibrinolytic activity retained in the top phase (Silva et al. 2013).

ATPS is a cost effective method, easy to do and can be scaled up for purification of enzyme in industrial production for commercial purposes.

The purity of enzyme recovered from this method is high and especially, ATPS does not influence the secondary structure of the enzyme. Thus, the enzyme activity was still maintained at a high level after purification (Iqbal et al. 2016). The production of nattokinase has been carried out extensively in Bacillus sp. and other bacterial species. However, B. subtilis species is considered a benign organism by WHO as it does not possess traits that cause disease. It is not considered pathogenic or toxigenic to

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humans, animals, or plants. The potential risk associated with the use of this bacterium in fermentation facilities, e.g. nattokinase production, is low (VKM 2016). Currently the studies on the production of nattokinase from B. subtilis and the application of ATPS are limited. Moreover, nattokinase from B. subtilis strains is very diverse in molecular weight and characteristics, so the potential applications are also very different.

NatTH9 has high fibrinolytic and thrombolytic activity, so it is convenient for application as an additive of functional foods to potentially prevent cardiovascular disease in humans.

3.3. Fibrinolytic activity

Analysis of the SDS-PAGE (sodium dodecyl sulfate – polyacrylamide gel electrophoresis) and fibrin zymography of the purified NatTH9 found fibrinolytic activity on the gel with three clear zones which have MWs of approximately 37, 27, and 21 kDa with the strongest activity found at 37 kDa (Fig. 6). A study by Lucy et al. (2019) also found 4 clear zones corresponding with 4 nattokinase bands from fibrin zymogram of B.

subtilis G8 with molecular weights of 19.1, 22.5, 27.5, and 29.3 kDa.

Figure 6. SDS-PAGE (a) and fibrin zymography (b) of enzyme from B. subtilis TH9. M: PageRuler Prestained Protein Ladder (Thermo Fisher Scientific). TH9(a): partially purified crude enzyme. TH9 (b): purified NatTH9.

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Whereas, Pinontoan, Elvina, and Jo (2021) observed six zymogram bands of approximately 42, 35.5, 30.8, 26.7, 20, and 13.7 kDa also from B.

subtilis G8, with the strongest fibrinolytic activity at 20 kDa.

Figure 7 reinforced the evidence of the fibrinolytic activity of the targeted enzyme, NatTH9, as seen on the fibrin plate assay and clot lysis assay.

Fibrinolytic activity was highest of all, with the widest clear zone diameter of about 1 cm on the fibrin plate (Fig. 7a). Enzyme NatTH9 completely dissolved the thrombus, while the crude enzyme (after partial purification) had poorer results (Fig. 7b). Nattokinase has the property to degrade the blood clots by degrading fibrin (Singh et al. 2008). Therefore, the purified enzyme from B.

subtilis TH9 was identified and characterized as a type of nattokinase and these results were similar to previous reports on the fibrinolytic activity and throm- bolysis (clot lysis) of enzymes from Bacillus sp. CK 11–4 (Kim et al. 1996), B.

subtilis BK-17 (Lee et al. 1999), and B. subtilis (Sharma et al. 2020).

3.4. Characteristics of NatTH9

The activity of NatTH9 at pH 7 and 37°C (547 U/mg) was used as the control to evaluate the effect of pH and temperature on the catalytic efficiency of the enzyme. The present study found the optimal tempera- ture of NatTH9 was 39°C at pH 7 and the thermal stability was retained in the range of 31–39°C with the relative activities achieved was about 115% and 85%, respectively (Fig. 8). Since the effect of pH on nattokinases from B. subtilis is very variable (Lin et al. 2015; Man and Xiang 2019;

Nguyen, Quyen, and Le 2013; Wang et al. 2009), this study chose a pH range of 3–10 to investigate for NatTH9. The observations at 39°C showed

Figure 7. Fibrinolytic activity on fibrin plate (a) and in vitro clot lysis (b) of enzyme from B. subtilis TH9. NC: negative control (enzyme buffer solution). 1–5: purified NatTH9 treatment times from 8–

12 h, in increments of 1 h. S1: partially purified crude enzyme. S2: purified NatTH9.

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that the enzyme achieved the highest relative activity of approximately 125% at pH 8. The pH stability ranged from pH 7–10 with relative activities of 97% or more (Fig. 9).

In general, types of nattokinase from B. subtilis strains have very different optimal pH. Some of them have the optimal pH around 7 such as nattokinase of strains DC-2 (Ashipala and He 2008), DC27 (Hu et al.

2019), WTC016 (Ju et al. 2019), and S127e (Frias et al. 2021). However, others are active at alkaline or acidic pHs such as nattokinase of strains CK 11–4 (Kim et al. 1996), BK-17 (Lee et al. 1999), B-12 (Wang et al.

2009), and B060 (Suwanmanon and Hsieh 2014).

Similar to NatTH9, nattokinase from B. subtilis C10 has potent fibri- nolytic activity at 37°C (Thu et al. 2020). However, Nguyen, Quyen, and Le (2013) found a nattokinase from B. subtilis VTCC-DVN-12-01 with an optimal temperature up to 65°C. Many other Bacillus strains also have higher optimal temperatures than NatTH9 such as 40°C of strain B-12 and KH-4 (Kim et al. 2002; Wang et al. 2009), 45°C of strain DC27 (Hu et al. 2019), 48°C of S127e (Frias et al. 2021), or lower such as 30°C of strains B060 (Suwanmanon and Hsieh 2014) and WTC016 (Ju et al. 2019), even as little as 25°C of BK-17 (Lee et al. 1999).

Figure 8. The optimal temperature and thermal stability of purified NatTH9. Different letters on a curve represent statistically significant differences with p < .05 (Duncan’s test), uppercase letter:

optimal temperature, lowercase letters: thermal stability.

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As is known, some enzymes require the participation of metal ions as cofactors to enhance biocatalysis (Hernick and Fierke. 2010). However, the effects of metal ions on enzymes are also varied. They can activate or inhibit the biocatalysis of enzymes through interaction with the amine or carboxylic acid groups of amino acids, thereby increasing or decreasing the amount of product produced from a biochemical reaction (Ishida et al. 1980). Based on the literature, several metal ions with enhanced effects on the activity of nattokinase were investigated in this study (Frias et al.

2021; Hu et al. 2019; Jeong et al. 2015; Peng et al. 2003; Urano et al.

2001).

Except for Mg2+ which increased NatTH9 activity (relative activity of approximately 120%), other tested metal ions negatively affected the enzyme (relative activity varied from 22% (Cu2+) to 92% (Mn2+)). In a study by Hu et al. (2019), tested metal ions, except for Cu2+, have an effect on fibrinolytic proteases from B. subtilis DC27 in contrast to NatTH9. Ions such as Mn2+ and Ca2+ increased the relative activity of fibrinolytic protease, while Mg2+ and Cu2+ inhibited enzyme; especially Cu2+ (relative activity of the enzyme was only 18%) and Zn2+ does not affect enzyme activity.

Figure 9. The optimal pH and pH stability of purified NatTH9. Different letters on a curve represent statistically significant differences with p < .05 (Duncan’s test), uppercase letter: optimal pH, lowercase letters: pH stability.

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The catalytic activity of a nattokinase, AprE127, from B. subtilis S127e, increased by 10% and 80% in the presence of Mg2+ and Ca2+, respectively (Frias et al. 2021). These results are different from other fibrinolytic enzymes like NAT (Urano et al. 2001), DC-4 (Peng et al. 2003), and AprE176 (Jeong et al. 2015) which are activated by Ca2+ but not by Mg2

+; or NatTH9 which is activated by Mg2+ but not by Ca2+. On the other hand, AprE127 was also inhibited by Cu2+ (80%) and Zn2+ (80%) similar to NatTH9.

In tested reagents, NatTH9 was inhibited almost completely (over 90%) by inhibitors such as PMSF and SDS. Other studies on nattokinase from B. subtilis also obtained similar results to this study (Hu et al. 2019; Kim et al. 1996). While Triton X-100, a nonionic detergent, significantly increased enzyme activity up to 160%. Some reports suggested that Triton X-100 can increase the flexibility of protein structure thereby enhancing enzyme activity, for example as in nattokinase from B. subtilis (Nguyen, Quyen, and Le 2013; Thu et al. 2020). EDTA and Tween 20 also partially inhibited NatTH9 activity, with a relative activity of only about 77 and 87% (Fig. 10). Kim et al. (1996), Borah et al. (2012) and Frias et al.

(2021) also found the partial inhibition by EDTA on nattokinase from B.

subtilis.

Figure 10. Effect of some metal ions and reagents on purified NatTH9 activity. The activity of purified NatTH9 at 39°C and pH 8 was used as the control. Different letters represent statistically significant differences with p < .05 (Duncan’s test).

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4. Conclusions

This study demonstrated that ATPS consisting of 20% (w/v) polyethylene glycol 6000 and 15% (w/v) potassium phosphate was highly effective in purifying NatTH9, a nattokinase of B. subtilis TH9. This enzyme is highly active at 37–39°C in a mildly alkaline medium (pH 8) with peak activity of 684 U/mg, has strong fibrinolytic activity and can completely dissolve blood clots after 12 h at 37°C. Therefore, NatTH9 may be a valuable nutritional supplement to support healthy blood flow and circulation to prevent cardio- vascular diseases.

Acknowledgments

We would like to thank Hue University for supporting facilities for this study.

Authors’ contribution

NHM: managed the literature searches, performed the experiments and analyzed the results, HTQT and TBV: performed the experiments, NHL: designed study, analyzed the results, and prepared the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

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