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Research Article
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Production, Optimization and Characterization of Fibrinolytic Enzyme by Bacillus subtilis RJAS19 |
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D.J. Mukesh Kumar,
R. Rakshitha,
M. Annu Vidhya,
P. Sharon Jennifer,
Sandip Prasad,
M. Ravi Kumar
and
P.T. Kalaichelvan
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ABSTRACT
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The present study aimed at the production, purification and characterization of fibrinolytic nattokinase enzyme from the bacteria isolated from natto food. For the purpose, a fibrinolytic bacterium was isolated and identified as Bacillus subtilis based on 16S rDNA sequence analysis. The strain was employed for the production and optimization of fibrinolytic enzyme. The strain showed better enzyme production during 72nd h of incubation time with 50°C at the pH 9. The lactose and peptone were found to be increasing the enzyme production rate. The enzyme produced was purified and also characterized with the help of SDS-PAGE analysis. The activity and stability profile of the purified enzyme was tested against different temperature and pH. The observations suggesting that the potential of fibrinolytic enzyme produced by Bacillus subtilis RJAS19 for its applications in preventive medicines.
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How
to cite this article:
D.J. Mukesh Kumar, R. Rakshitha, M. Annu Vidhya, P. Sharon Jennifer, Sandip Prasad, M. Ravi Kumar and P.T. Kalaichelvan, 2014. Production, Optimization and Characterization of Fibrinolytic Enzyme by Bacillus subtilis RJAS19. Pakistan Journal of Biological Sciences, 17: 529-534. DOI: 10.3923/pjbs.2014.529.534 URL: https://scialert.net/abstract/?doi=pjbs.2014.529.534
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Received: March 19, 2013;
Accepted: April 04, 2013;
Published: November 26, 2013
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INTRODUCTION
Nattokinase is considered as an active fibrinolytic enzyme synthesized by bacteria
belongs to the Bacillus sp. and was identified in natto, a Japanese traditional
food (Sumi et al., 1987). Natto is rich in
protein and was made by fermenting the soyabeans with Bacillus subtilis.
Nattokinase is a serine protease, belongs to the subtilisin family which exhibits
increased fibrinolytic activity when compared to plasmin (Fujita
et al., 1995; Peng et al., 2003).
The increased fibrinolytic activity of nattokinase makes it feasible for the
thrombolytic therapy.
The nattokinase possess worldwide attention due to its various therapeutic
applications. The breakdown of fibrin by nattokinase can be carried out by promoting
the activity of urokinase and tissue plasminogen activator, which in turn promotes
the conversion of plasminogen into plasmin and degrades fibrin (Milner
and Makise, 2002). Fibrin is a soluble plasma glycoprotein made by fibrinogen
in liver. Nattokinase possess various applications including anti-hypertension,
blood thinning, thrombolytic and digestive capability (Yokota
et al., 1996). They were also used for cardiovascular diseases.
The nattokinase is a food based dietary supplement being marketed worldwide
and found to be stable in the gastrointestinal tract, despite of changes in
pH and temperature. Nattokinase is produced by bacteria of genus Bacillus
especially Bacillus subtilis which is Generally Recognized As Safe (GRAS)
organism. During the past decades, considerable progresses have been made to
engineer B. subtilis for the production of nattokinase (Milner
and Makise, 2002).
With the help of the above points, the present study was aimed at the isolation
of a fibrinolytic nattokinase producing bacteria from natto. The production
of fibrinolytic enzyme was carried out followed by its optimization. The enzyme
produced was then characterized further.
MATERIALS AND METHODS
Chemicals and sample: All the chemicals used in the present study were
of analytical grade and bought from Sigma Alrich and SRL.
Isolation of fibrinolytic bacteria: The Bacillus subtilis RJAS19
(Genbank Accession No. KC527834), producing fibrinolytic enzyme was isolated
from fermented soya food. The strain was characterized biochemically and identified
based on 16S rDNA sequence analysis and was stored at -4°C for further analytical
procedures.
Production of fibrinolytic enzyme: For the production of fibrinolytic
enzyme, the bacterial inoculum was prepared with the help of LB broth. The overnight
grown bacteria culture was inoculated on 100 mL of production medium (Galactose
1 g, NaCl 0.5 g, CaCO3 0.5 g, Tryptone 0.5 g, Soybean 0.3 g, MgSO4.7H2O,
KH2PO4 0.08 g, K2HPO4 0.02 g, CuSO4
0.005 g, FeSO4 0.001 g, Distilled water 100 mL) (Hassanein
et al., 2011). The production of enzyme was facilitated by incubating
the production medium in shaken condition for 48 h at 30°C. The production
medium was centrifuged and the supernatant was used as enzyme source.
Assay procedures
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Biomass: The biomass was determined by reading the
absorbance of production medium at 600 nm (Henriette
et al., 1993) |
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Total protein content: The amount of protein present in the culture
filtrate was measured by Bradfords method (Bradford,
1976) |
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Fibrinolytic enzyme: The fibrinolytic activity was assayed with
the help of fibrin as a substrate (Hassanein et al.,
2011). The reaction mixture consists of 2 mL of fibrin, 7 mL of sodium
phosphate buffer and 1 mL of culture filtrate was incubated at 37°C
for 5 min. the reaction was stopped by adding 1 mL of 5% Trichloroacetic
acid. The reaction mixture was incubated for 30 mins at 4°C and centrifuged
to collect the supernatant. The absorbance was read at 750 nm. One unit
of fibrinolytic activity was defined as the amount of fibrin that hydrolyzed
by the enzyme during standard incubation conditions |
Parameter optimization studies: For the production of fibrinolytic enzyme
the cultural conditions like incubation time, pH and temperature were optimized.
The parameters optimization was done by cultivating the bacteria at different
incubation time (24-144 h), different pH (5-11) and different temperature (35-65°C).
Apart from the environmental factors, medium components such as carbon (Sucrose,
Lactose, Glucose, Starch, Fructose, Mannose) and nitrogen (Yeast extract, Beef
extract, Peptone, Ammonium chloride, Sodium nitrate, Ammonium carbonate) sources
were also optimized.
Extraction and purification of fibrinolytic enzyme: The enzyme was allowed
to produce under optimized production conditions. The enzyme from the culture
filtrate was extracted with the help of ammonium sulphate precipitation with
40-60% saturation. The extracted protein was dialyzed overnight with sodium
phosphate buffer with three buffer transfers. This partially purified fibrinolytic
enzyme was then purified with the help of ion exchange chromatography with CM-Sepharose
column (Yin et al., 2010).
Characterization of purified fibrinolytic enzyme:
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SDS-PAGE analysis: The molecular weight (kDa) of the
purified fibrinolytic enzyme was analyzed using SDS-PAGE analysis (LaemmLi,
1970) |
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Effect of temperature and pH on enzyme stability: The effect of
temperature and pH on the stability of purified fibrinolytic activity was
determined with the help of fibrin as the substrate. The pH stability of
the purified enzyme was determined by incubating the purified enzyme in
buffers with different pH from 8.0 to 12.0 at 50°C for 40 mins (pH 5.0-7.0
Sodium phosphate buffer, pH 7.0-9.0 Tris Hcl buffer, pH 9.0-12.0 Sodium
carbonate buffer). Followed by the incubation the relative activity of the
enzyme was determined according to Chang et al.
(2000). Similarly, the temperature stability of the purified enzyme
was determined by incubating the enzyme at different temperatures ranging
from 45-95°C at the pH 9.0 |
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Effect of temperature and pH on enzyme activity: The pH dependence
of the enzyme was determined by using fibrin as substrate in a series of
buffers of different pH values ranging from 8.0-2.0 (buffers mentioned above).
The fibrinolytic activity of the purified enzyme was measured at different
pH after 30 min incubation. The effect of temperature was determined by
incubating the samples at different temperatures ranging from 45-95°C
for 30 min and the activity of the enzyme was measured by standard conditions |
Application of purified fibrinolytic enzyme: The application of fibrinolytic
enzyme was evaluated with the help of the method followed by Omura
et al. (2005) by degrading the artificial blood clot. For the same,
fresh blood collected from the healthy volunteer was allowed to coagulate in
glass test tube. The artificial blood clot was rinsed with phosphate buffer
in prior to the treatment with purified enzyme and incubated for 60 min. For
control, the blood clot was treated with saline. The observation was made for
the degradation of blood clot.
RESULTS AND DISCUSSION
In current scenario, enzymes play as active ingredients in various applications
in the fields of food, detergent, medicine, etc. Since enzymes overcomes difficulties
of the conventional methods in the breakdown of complex matters (Negi
and Banerjee, 2010). Nattokinase is one of the fibrinolytic enzymes which
possess applications in medical field. The fibrinolytic activity of the nattokinase
made it one of the life saving drug. Since the enzyme having vast applications
in medical, the present study was aimed at the isolation, production and purification
of fibrinolytic enzyme.
The Bacillus subtilis strain RJAS19 was selected as potent fibrinolytic
organism and was used for the present study.
For the production of enzyme, the culture medium plays an important role, as
the culture medium should provide all the essential nutrients required by the
organism for enzyme production (Al-Juamily and Al-Zaidy,
2012). In order to minimize the production cost, production medium must
possess cheap source of carbon, nitrogen, growth promoters and trace elements.
The growth conditions such as pH, incubation time and temperature were also
maintained at optimal conditions for the enhanced enzyme production. In prior
to the inoculation, the cultural and medium conditions were to be optimized.
At very first, the incubation time was optimized by harvesting the production
medium at different intervals and assayed for enzyme production. The production
of the enzyme by the strain was observed in exponential phase and showed linearity
with the cell growth. The maximum production of nattokinase was observed during
72nd h of incubation (Fig. 1). The production of fibrinolytic
enzymes usually lies between 48 to 96 h (Krishnaveni and
Rani, 2011). The extra cellular enzymes are usually produced during log
phase but within the log phase the fermentation time may be vary.
The incubation temperature is the next most critical factor that has to be
optimized for enzyme production. The optimization of temperature is very important
as it determines the velocity of the enzyme reaction. All enzymes have an optimal
temperature at which reaction rates go fastest without denaturing the enzyme
(Al-Juamily and Al-Zaidy, 2012). The optimum temperature
for the fibrinolytic enzyme production by Bacillus subtilis was found
to be 50°C and beyond which the enzyme activity was reduced gradually (Fig.
2). From the observed results, the isolated strain was found to be thermophillic.
When come to the pH, considerably less production of enzyme was observed at
acidic pH and increased enzyme production was obtained at weak alkaline pH.
The maximum production of fibrinolytic enzyme was noticed at pH of 9.0 (Fig.
3). The results indicated that the isolated Bacillus subtilis was
an alkaline organism. Changes in pH can change the shape of the active site
and during high or low pH concentrations result in loss of enzyme activity due
to denaturation.
The medium components play a significant role in enhancing the enzyme production
(Gupta et al., 2002). Various carbon sources were
used for the production of fibrinolytic enzyme by the isolated Bacillus subtilis.
Lactose was found to be the best carbon source for fibrinolytic enzyme production
(Fig. 4).
Bacillus subtilis RJAS19 was allowed to grow in the presence of different
nitrogen sources. Interestingly increased fibrinolytic activity was achieved,
when the cells were grown in a medium containing peptone (Fig.
5).
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Fig. 1: |
Enzyme activity and growth profile with respect to different
incubation time |
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Fig. 2: |
Enzyme activity and growth profile with respect to different
temperature |
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Fig. 3: |
Enzyme activity and growth profile with respect to different
pH |
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Fig. 4: |
Enzyme activity and growth profile with respect to different
carbon sources |
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Fig. 5: |
Enzyme activity and growth profile with respect to different
nitrogen sources |
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Fig. 6: |
Effect of pH on enzyme activity and stability |
In contrast, a medium containing casein and soyabean meal enhanced the production
of similar proteolytic enzyme by bacteria (Kiran et al.,
2008; Saurabh et al., 2007).
The enzyme produced under optimal conditions was precipitated by ammonium sulphate
precipitation. The further purification was done using dialysis. The dialysed
enzyme samples were subjected to SDS-PAGE analysis.
The molecular weight of the enzyme produced was analyzed by SDS-PAGE. The purified
fibrinolytic enzyme was checked for its homogeneity and molecular weight was
determined by SDS-PAGE. The purified fibrinolytic enzyme migrated as a single
band corresponding to molecular mass of 35 kDa. The findings are in accordance
with Chang et al. (2000) and Borah
et al. (2012).
While carrying out stability test, the purified fibrinolytic enzyme was found
to be stable at pH 9.5 to 11.0 (Fig. 6). The pH stability
of the fibrinolytic enzyme was found to be similar to that of similar enzyme
produced by B. amyloliquefaciens DC-4 (Peng et
al., 2003).
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Fig. 7: |
Effect of temperature on enzyme activity and stability |
The thermostability of the enzyme was found to be high at 65 to 85°C (Fig.
7). The similar thermal stability profile was also observed in the strains
such as Bacillus subtilis YJ1 (Yin et al.,
2010), B. amyloliquefaciens DC-4 (Peng et
al., 2003) and Bacillus sp. KCK-7 (Kim et
al., 1996).
In further to the stability test, the enzyme activity profile was tested against
the broad range of temperature and pH. From the observations, the enzyme activity
was found to be maximum at 65°C and at the pH of 9.5. The similar enzyme
produced by Bacillus subtilis YJ1 (Yin et al.,
2010) and Bacillus subtilis (Dubey et al.,
2011) were also possess similar optimal conditions. The ability of fibrinolytic
enzyme to degrade the blood clot was experimented as the method followed by
Omura et al. (2005). While comparing to the
control, the enzyme degraded the clot completely which indicated the degrading
ability of the enzyme. The theory was previously supported by Sumi
et al. (1987).
CONCLUSION
From the results obtained in the present investigation, the isolated bacterial
strain Bacillus subtilis RJAS19 was found to be having ability to synthesize
economically important fibrinolytic enzyme. Furthermore, the activity and stability
profile of the fibrinolytic enzyme from Bacillus subtilis RJAS19 suggesting
its potential for the further applications in preventive medicines.
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