Purification and characterization of recombinant nattokinase from Bacillus subtilis R0H1

Abstract

Nattokinase (NK) is a fibrinolytic enzyme with the potential for fighting cardiovascular diseases (CVD) thanks to its antithrombotic, antihypertensive, anticoagulant, anti-atherosclerotic, and neuroprotective effects. Nattokinase was first discovered and purified from soybean fermented food by Bacillus subtilis natto. To enhance NK's activity and simplify downstream processes, production of recombinant NK using several microbial expression systems such as Escherichia coli, B. subtilis, and Lactococcus lactic has been studied. Among all of them, B. subtilis is a prominent host for overproduction of functional proteins which can be secreted directly into the culture medium. In this study, recombinant NK from B. subtilis R0H1 was purified using two-step membrane filtration. Results showed 3.2-fold increase in activity and a recovery rate of more than 80%. Molecular weight of NK was approximately 28 kDa and its fibrinolytic degradation capacity was proved according to SDS-PAGE. The optimal pH and temperature of this NK were 8.5 and 55°C, respectively. The enzyme activity was boosted by Mg 2+ , Ca 2+ and obviously inhibited by Co 2+ , Zn +2 , Fe 2+ , and SDS. The apparent Km and Vmax with fibrin as the substrate were 3.08 mM and 6.7 nmol/min, respectively. The results suggested that membrane filtration is a useful method for purification of recombinant NK from B. subtilis R0H1. Therefore, application of membrane system is proposed to purify NK at the pilot scale. In addition, our findings indicated that recombinant NK produced in B. subtilis R0H1 showed high and stable proteolytic activity in slightly alkaline pH and at high temperature. It also exhibited strong fibrinolytic activity again both substrates: fibrinogen and fibrin.

Full text

Vietnam Journal of Biotechnology 20(2): 369-377, 2022
369
PURIFICATION AND CHARACTERIZATION OF RECOMBINANT
NATTOKINASE FROM BACILLUS SUBTILIS R0H1
Nguyen Thi Thuy Ngan, Le Tuan, Nguyen Lan Huong
School of Biotechnology and Food Technology, Hanoi University of Science and Technology, 1 Dai
Co Viet Road, Hai Ba Trung District, Hanoi, Vietnam
To whom correspondence should be addressed. E-mail: huong.nguyenlan@hust.edu.vn
Received: 22.4.2021
Accepted: 30.8.2021
SUMMARY
Nattokinase (NK) is a fibrinolytic enzyme with the potential for fighting cardiovascular diseases
(CVD) thanks to its antithrombotic, antihypertensive, anticoagulant, anti-atherosclerotic, and
neuroprotective effects. Nattokinase was first discovered and purified from soybean fermented food
by Bacillus subtilis natto. To enhance NK’s activity and simplify downstream processes, production
of recombinant NK using several microbial expression systems such as Escherichia coli, B. subtilis,
and Lactococcus lactic has been studied. Among all of them, B. subtilis is a prominent host for
overproduction of functional proteins which can be secreted directly into the culture medium. In this
study, recombinant NK from B. subtilis R0H1 was purified using two-step membrane filtration.
Results showed 3.2-fold increase in activity and a recovery rate of more than 80%. Molecular weight
of NK was approximately 28 kDa and its fibrinolytic degradation capacity was proved according to
SDS-PAGE. The optimal pH and temperature of this NK were 8.5 and 55°C, respectively. The
enzyme activity was boosted by Mg2+, Ca2+ and obviously inhibited by Co2+, Zn+2, Fe2+, and SDS.
The apparent Km and Vmax with fibrin as the substrate were 3.08 mM and 6.7 nmol/min,
respectively. The results suggested that membrane filtration is a useful method for purification of
recombinant NK from B. subtilis R0H1. Therefore, application of membrane system is proposed to
purify NK at the pilot scale. In addition, our findings indicated that recombinant NK produced in B.
subtilis R0H1 showed high and stable proteolytic activity in slightly alkaline pH and at high
temperature. It also exhibited strong fibrinolytic activity again both substrates: fibrinogen and fibrin.
Keywords: Bacillus subtilis, characterization, nattokinase, purification, recombinant
INTRODUCTION
Nattokinase (NK) is a serine protease which
belongs to the subtilisin family. Historically, it is
extracted and purified from a Japanese food
called natto (Sumi et al., 1987). It can dissolve
fibrin fibers in blood clots that is known as the
main cause of cardiovascular diseases (CVD)
(Chen et al., 2018). The fibrinolytic activity of
NK was shown to be more stable and effective
than that of plasmin (Sumi et al., 1990; Fujita et
al., 1995). In 2016, around 17.9 million people
died from CVD, accounting for 31% of all
registered premature deaths (Kaptoge et al.,
2019). This number may reach 23.6 million
annually by 2030, mostly due to stroke and heart
disease (Deepak et al., 2010). In this context, NK
might be a brilliant potential product for
prevention and treatment of CVD.
Downstream processing is the key and
bottle-neck step in the production of NK due to
the presence of impurities proteins and high
viscosity of culture broth. In order to reduce the

Nguyen Thi Thuy Ngan et al.
370
complexity of the purification process, NK was
over-expressed in different hosts such as Bacillus
subtilis (Cui et al., 2018; Liu et al., 2019; Tian et
al., 2019), Escherichia coli (Bora et al., 2018),
Lactococcus lactis (Liang et al., 2007). From
previous works, salt precipitation was frequently
used for purification of both wild type and
recombinant NK. This was combined with gel
filtration chromatography (GFC) (Tuan et al.,
2015; Hu et al., 2019) and/or ultrafiltration (Tian
et al., 2019; Xin et al., 2019). Recovery yield of
recombinant NK may reach up to 80% (Tuan et
al., 2015; Tian et al., 2019), which is
significantly higher than that of from wild
enzymes. Other methods for purification of
recombinant NK such as Ni-NTA and GFC was
used but its final recovery yield attained only
16.8% (Bora et al., 2018). From these
publications, it seems that the selection of
purification methods strongly determines the
efficiency of the NK recovery process.
Ultrafiltration may be an alternative method to
achieve high purification efficiency when
maintaining a high recovery yield.
In the present study, the purification of
recombinant NK from B. subtilis R0H1 using a
two-steps filtration was determined.
Biochemical and kinetics properties of purified
enzyme were then investigated.
MATERIALS AND METHODS
Microorganism and media
Recombinant strain R0H1 (B. subtilis 3NA
carrying aprN gene with inducible promoter Pveg)
was maintained in 50% glycerol and stored at -
80°C. The bacterium was activated on Luria-
Bertani skim milk (LBS) agar (g.L-1) (yeast
extract 5, tryptone 10, NaCl 5, skim milk 10 and
agar 15) supplemented with 5 μg.mL-1
chloramphenicol at 37°C. Colony with clear halo
was selected for cultivation.
Chemicals and reagents
Fibrin bovine blood was provided by MP
Biomedicals (France). Analytical chemicals and
reagents were purchased from Sigma Aldrich
(USA). Media nutrients were purchased from
Himedia (India) and Oxoid (England).
Enzyme production and purification
One colony was grown overnight in Luria-
Bertani (LB) medium containing
chloramphenicol (5 μg.mL-1) at 37°C and 150
rpm until its OD600 nm reached 4.5. Cells from the
seed culture was then transferred into
fermentation medium (g.L-1) (yeast extract 5,
CaCl2 0.15, tryptone 35 and NaCl 5) at initial
OD600 nm value of 0.2. Shake flask cultures were
carried out at 37°C and 150 rpm. After 14 hours
of fermentation, the crude enzyme was obtained
by centrifugation the culture broth at 10,000 rpm
and 4°C for 15 min.
To purify the crude enzyme, a two-steps-
filtration was applied. The supernatant obtained
from centrifugation was first filtered through a
0.2 µm cut-off to eliminate cells and large
impurities. The permeate obtained from 0.2 µm
filtration was then subjected to a 10 kDa cut-off
and the purified enzyme was remained in the
retentate. In each purification step, protein
concentration was determined by Bradford
method (Bradford, 1976) using bovine serum
albumin (BSA) as standard protein.
The yield and purification fold were
calculated as the follow:
Yield (%) = Total activity in purified sample x 100
Total initial activity
Purification (fold) = Specific activity of purified sample FU
mg
Specific activity of initial sample FU
mg
SDS-PAGE analysis
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) analysis was
carried out using 12% polyacrylamide gel in the
separating gel and 4% polyacrylamide gel in the
stacking gel (Laemmli, 1970). A fixed volume

Vietnam Journal of Biotechnology 20(2): 369-377, 2022
371
(10 μL) of samples was loaded and the
electrophoresis was carried out at 15 mA. Protein
bands were visualized by staining with
Coomassie brilliant blue R 250. Gangnam stain
protein ladder (Amersham Biosciences
CAT.NO.24052) was used as standard.
Enzyme assay
The fibrinolytic activity was determined
according to the method of Lin et al. (2015) with
minor modifications. Firstly, 150 µL of fibrin
solution (4 g.L-1) was added to 420 µL 0.1 M
Tris-HCl buffer (containing CaCl2 0.01 M, pH
7.4) and kept at 37°C for 5 min. Then, 30 µL of
enzyme was added and the reaction mixture was
incubated at 37°C for 30 min after which, the
reaction was ceased by 300 µL of trichloroacetic
acid (TCA). The samples were kept at 37°C for
20 min and then centrifuged at 10,000 rpm for 15
min. The absorbance of the supernatant was then
measured at 275 nm.
One unit of fibrinolytic activity (FU) is
defined as the amount of enzyme required to
produce an increase in absorbance at 275 nm
equal to 0.01 in 1 min.
Fibrinogen degradation by nattokinase
The degradation of fibrinogen by NK from
the recombinant strain R0H1 was investigated
using fibrinogen 10 g.L-1 as a substrate and
incubated at 37°C for different reaction times (up
to 60 min). Hydrolysis products were visualized
on SDS-PAGE.
Effect of pH and temperature on enzyme
activity
The effect of pH on NK activity was
determined at 37°C and in various pH (from 6 to
10). A various of buffers (0.1 M Tris-HCl buffer
for pH 6 - 9, and 0.05 M Na2CO3 - NaHCO3
buffer for pH 9 - 10) were used accordingly.
To determine the effect of temperature on NK
activity, the enzyme activity was measured in 0.1
M Tris-HCl buffer (pH 7.4) at temperatures
ranging from 30 - 75°C.
The relative activity of NK was measured,
and the highest activity was defined as 100%.
Effect of pH and temperature on enzyme
stability
The effect of pH on enzyme stability was
evaluated based on its residual activity after pre-
incubating the enzyme at pH 2.5, 7.4 and 8.5 for
30 to 300 min at 37°C. Whereas residual activity
of NK as measured, the highest activity was
defined as 100%.
The thermal stability of NK was examined by
measuring the residual activity after incubating
enzyme at various temperatures (30 - 80°C) for
60 to 420 min.
Effect of metal ions and inhibitors on enzyme
activity
The effect of metal ions (Na+, Mg2+, Mn2+,
K2+, Zn2+, Co2+, Fe2+, Ca2+, and Cu2+) and
inhibitors (EDTA and SDS) at concentrations of
1 and 5 mM on enzyme activity were examined
by performing the enzyme assay in the presence
of these ions or inhibitors. The relative activity
of enzyme was calculated as the percentage of
the treated enzyme activity compared with that
of the untreated enzyme.
Enzyme kinetics
Enzymatic reactions were performed by using
purified NK and different concentrations of
fibrin (0.25 - 4 g.L-1). The Lineaver-Burk
reciprocal plot was generated for 1/S versus 1/V.
The Michaelis-Menten constant (Km) and
maximum velocity (Vmax) were calculated based
on the intercept value and slop of this plot
(Lineweaver and Burk, 1934).
All measurements were carried out in
duplicate with the resulting values being the
mean of the cumulative data obtained.
RESULTS AND DISCUSSION
Purification of nattokinase
Nattokinase produced by B. subtilis R0H1
was purified by two-step membrane filtration
(Table 1). It has been observed that the specific

Nguyen Thi Thuy Ngan et al.
372
activity in the retentate of 10 kDa increased more
than 3-fold with a yield of 89.7 ± 8% based on
the crude enzyme. It suggested that the
membrane filtration is one of the useful methods
to obtain the high recovery yield for recombinant
NK. Recently, Tian et al. (2019) reported a yield
of 80% when purifying of NK from B. subtilis
WB800N/pHT43-pro-aprN via salt precipitation
and ultrafiltration. Tuan et al. (2015) also
achieved a similar recover yield of 79% when
purifying NK from B. subtilis pBG01-
aprN/BD104 using ammonium sulfate
precipitation combined with gel filtration
chromatography. While Xiao-Lan et al. (2005)
and Xin et al. (2019) only obtained recovery
yields of 42.6 and 48.3%, respectively after
purifying NK from wild type NK producing
strains using ammonium sulfate precipitation
and gel filtration chromatography. It is noted that
the recovery rate of purification process is
strongly dependent on initial state of the crude
enzyme. The high recovery yield obtained in this
study might related to the original characteristic
of host strain B. subtilis 3NA, which was known
as low protease producing strain (Reuß et al.,
2015).
Table 1. Purification of Nattokinase from B. subtilis R0H1.
Sample
Total
activity
(FU)
Total protein
(mg)
Specific activity
(FU/mg)
Purification
(fold)
Yield
(%)
Crude enzyme
585.0 ± 5.0
0.911 ± 0.050
642.2 ± 41.07
1 ± 0
100 ± 1
Permeate 0.2 µm
556.9 ± 26.3
0.816 ± 0.007
682.5 ± 38.0
1.10 ± 0.02
95.2 ± 5.0
Retentate10 KDa
524.6 ± 39.7
0.252 ± 0.005
2082.2 ± 36.4
3.20 ± 0.19
89.7 ± 8.0
Figure 1. SDS-PAGE analysis of
Nattokinase produce by B. subtilis R0H1. 1:
Crude enzyme; 2: Permeate of 0.2 µm cut-
off; 3: Retentate of 10 kDa cut-off; 4:
Gangnam stain protein ladder.
Figure 2. Degradation of fibrinogen by Nattokinase. 1:
Gangnam stain protein ladder; 2: Fibrinogen control without
enzyme; 3-8: Degradation products after 5, 10, 30 sec and
1, 10, 60 min incubation at 37oC, respectively. α, β, and γ
denotes the alpha, beta and gamma fragments of fibrinogen
from bovine plasma, respectively.

Vietnam Journal of Biotechnology 20(2): 369-377, 2022
373
SDS-PAGE analysis indicated single band
with similar molecular weight for both crude and
purified enzyme (Figure 1). The molecular
weight of NK from strain R0H1 was estimated at
approximately 28 kDa, which showed good
correlation with reported the molecular weight of
NK expressed in recombinant strains such as E.
coli BL21 (DE3) (Yongjun et al., 2011), B.
subtilis pBG01-aprN/BD104 (Tuan et al., 2015),
B. subtilis BSN01 (Cui et al., 2018), and B.
subtilis WB800N/pHT43-pro-aprN (Tian et al.,
2019).
Fibrinogen degradation by nattokinase
Fibrinogen is a 340-kDa soluble plasma
protein consisting of three pairs of disulfide
bonded α-, β-, and γ-chains (Walker, Nesheim,
1999). These chains of fibrinogen from bovine
plasma have molecular weight of 63.5, 56, and
47 kDa, respectively. The degradation of
fibrinogen into several lower molecular weight
fragments and the profile of the hydrolysis
products were strongly dependent on the reaction
time (Figure 2). After 5 sec (lane 3), bands
corresponding to α- and β-chains were clearly
broken, and several bands appeared between 11
and 48 kDa. It showed that the α-chain was
completely degraded within 5 sec, the β-chain
was degraded within 10 min, and most of γ-chain
was hydrolyzed in 60 min. These results were
consistent with previous reports of the
degradation of fibrinogen by NK from B. subtilis
BD104 (Tuan et al., 2015) and B. subtilis (Zen et
al., 2018). It suggested that NK degraded α-chain
first, and then followed by the β-chain and γ-
chain of fibrinogen to smaller products (Tuan et
al., 2015; Ren et al., 2018).
Effect of pH and temperature on enzyme
activity
The effects of pH and temperature on NK
activity were illustrated in Figure 3. Nattokinase
from strain R0H1 retained above 60% of its
activity at pH values ranging from 7 to 9 and
tended to rapidly lose its activity when further
decrease pH (Figure 3a). The pH for optimal
activity of this enzyme was 8.5 which was close
to the values report for recombinant NK from B.
subtilis BD104 (Tuan et al., 2015), and wild type
NK from B. subtilis natto B-12 (Wang et al.,
2009), B. subtilis TKU007 (Wang et al., 2011),
and Bacillus sp. B24 (Hmood, Aziz, 2016).
Nattokinase activity was significantly
enhanced by increasing reaction temperature
from 30 to 55°C. At temperature above 55°C,
enzyme activity showed a strong decreasing
trend, and it was completely inactivated at 75°C
(Figure 3b). The optimum temperature of this
enzyme was 55oC that was similar with those
mentioned by Wu et al. (2009); Tuan et al.
(2015) and was higher than those determined
(40oC) by Wang et al. (2009). However, its
optimum temperature was lower than those
recovered from some other Bacillus strains, i.e.,
60oC (Bacillus sp. B24) (Hmood, Aziz, 2018),
65oC (B. subtilis VTCC-DVN-12-01) (Thao et
al., 2013).
Figure 3. Effect of pH (a) and temperature (b) on Nattokinase activity (the highest activity was taken as 100%).

Nguyen Thi Thuy Ngan et al.
374
Effect of pH and temperature on enzyme
stability
To investigate the potential use of NK for
CVD treatment, enzyme stability at pH of gastric
(2.5), blood (7.4) and gut (8.5) was evaluated.
Figure 4a indicated the rapid inactivation of NK
from strain R0H1 at pH 2.5 after only 0.5 h. The
complete loss of NK activity at low pH (2 - 4)
was also reported for NK from Rhizopus
chinensis 12 (Xiao-Lan et al., 2004), B. subtilis
natto B-12 (Wang et al., 2009), B. subtilis natto
(Chang et al., 2012). However, the loss of
enzyme activity at pHs 7.4 and 8.5 was
negligible after 5 h at 37°C. Our results
suggested that NK from strain R0H1 may
perform the best action in human blood or gut.
Figure 4. pH (a) and thermal stability (b) of Nattokinase from B. subtilis R0H1 (the highest activity was illustrated
as 100%).
The NK from strain R0H1 was stable up to
60°C and retained more than 80% of its activity
after incubation at this temperature for 4 hours.
Further increase in temperature negatively
affects enzyme activity. At 70°C, less than 40%
of the enzyme activity was remained after 5 h
incubation and the enzyme was completely
inactivated after 1 h at 80°C (Figure 4b). Lin et
al. (2015) reported that NK from B. subtilis N1
incubated at 55C for 2 h remained a relative
activity of higher than 40% while its relative
activity dropped down to less than 30% at 65C
within 20 min. As for NK from B. subtilis natto
B-12, the enzyme was almost inactivated after 60
min at 60°C (Wang et al., 2009). Besides, NK
from B. subtilis BSN1 showed 52% of its initial
activity at 70oC (Wang et al., 2011). Obtained
results suggested that NK from strain R0H1 may
be considered a thermophilic protease.
Effect of metal ions and protease inhibitors on
NK activity
The enzyme activity was boosted up to 109,
115 and 116% in the presence of 1 mM Na+, Ca2+
and Mg2+, respectively. However, further increase
of the ion’s concentrations to 5 mM did not
improve NK activity. Other agents such as Mn2+
(1 mM), K+ (1 & 5 mM), EDTA (1 & 5 mM) and
Na+ (5 mM) showed no significant effect on NK
activity. On the contrary, NK from strain R0H1
was inhibited by Co2+, Zn2+, Fe2+, Cu+2, and SDS.
The presences of these ions or inhibitors led to a
drop of enzyme activity by 13 – 78%, depending
on effector’s concentrations (Table 2). The effects
of ions and inhibitors on NK activity were
partially consistent with previous works. Xin et al.
(2018) reported 86% of residual activity of NK
from Bacillus tequilensis (No. 11462) in the
presence of 5 mM Cu2+. Chang et al. (2012) also
claimed no significant effects of K+, Na+, Ca2+,
Mg2+ and Zn2+ on the activity of NK from B.
subtillis fermented red bean. Across the literature,
it seems that effects of different ions on NK
activity were not unified. For example, Fe2+ at 5
mM was a booster for NK from B. subtilis
TKU007 (Wang et al., 2011) but opposite
conclusion was witnessed from others works
(Wang et al., 2009; Hu et al., 2019). Similar

Vietnam Journal of Biotechnology 20(2): 369-377, 2022
375
contradiction statement about effect of Zn2+ and
Cu2+ on enzyme activity was reported. Garg and
Thorat (2014) observed an 8% increase in the
activity of purified NK from B. natto (NRRL B-
3666) in presence of 5 mM Zn2+ or Cu2+. In the
other hand, a loss of more than 15% activity in the
presence of neither Zn2+ or Cu2+ at 5 mM was
reported for NK from Bacillus tequilensis (No.
11462) (Xin et al., 2018). For NK from B. subtilis
BD104, Zn2+ (5 mM) boosted the NK activity by
1.72-fold while Cu2+ (5 mM) led to a 20% activity
loss (Tuan et al., 2015).
Table 2. Effect of metal ions and inhibitors on the activity of Nattokinase.
Metal ions and inhibitor
Relative activity (%)
Concentration (1 mM)
Concentration (5 mM)
None
100 ± 1
100 ± 1
Na+
109 ± 6
98 ± 6
Mg2+
116 ± 4
103 ± 4
Mn2+
100 ± 2
53 ± 2
K+
100 ± 0
95 ± 0
Zn2+
74 ± 5
38 ± 5
Co2+
73 ± 2
0
Fe2+
46 ± 0
0
Ca2+
115 ± 4
113 ± 4
Cu2+
87 ± 2
85 ± 1
EDTA
101 ± 4
88 ± 4
SDS
49 ± 6
22 ± 0
Kinetics of nattokinase
Figure 5. Lineweaver-Burk plot for fibrin hydrolysis by
Nattokinase.
Kinetic parameters of NK from strain R0H1
were determined from initial velocity at various
substrate concentrations. The Lineweaver-Burk
plot showed that the y-intercept at 1/V was
149.04 (min/µmol) and the x-intercept at 1/S was
-0.33 (L/mmol) (Figure 5). The values of Km and
Vmax devised from this data were 3.08 mM and
6.7 nmol/min, respectively. As the obtained Km
was low, it showed that the NK had high affinity
for fibrin. Garg and Thorat (2014) reported that
the values of Km and Vmax of NK from Bacillus
natto NRRL B-366 for the substrate N-Succinyl-
Ala-Ala-Pro-Phe-p-nitro-anilide (S-7388) were
3.5 mM and 1250 nmol/min, respectively.
CONCLUSION
It was found that NK from the recombinant
strain B. subtilis R0H1 could be purified using
two-steps membrane filtration with yield of more
than 80% and purification fold of 3.2. The
purified NK showed a single protein band of
approximately 28 kDa which possessed strong
fibrinolytic degradation activity according to
SDS-PAGE analysis. The recombinant enzyme
exhibited significant stabilities for pH and

Nguyen Thi Thuy Ngan et al.
376
temperature. Its maximum activities were
reported at pH of 8.5 and temperature of 55°C.
This NK’s activity was fairly increased in
presence of Mg2+, Ca2+ but strongly inhibited by
Co2+, Fe2+ and SDS. Further study should be
focused on the fermentation strategy to improve
NK production and the application of membrane
system to purify NK at the pilot scale.
Acknowledgements: This research was funded
by the program on hi-tech research, training and
infrastructure construction of the Ministry of
Science and Technology under grant number
CNC.10.DAPT/17.
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