INTRODUCTION:

Nattokinase is a naturally occurring protrolytic enzyme that has been used in pharmaceutical industry, health care and medicine. It exhibits a strong fibrinolytic activity [1-3] and it maintains the blood flow and cardiovascular system. The substrate for the enzyme nattokinase production is a cheese like food (Natto) made from fermented soybeans [4, 5] using the bacterium Bacillus natto. The botanical source for Nattokinase is Glycine max (L.) Merr. It appears as a yellow-white fine powder [6]. This enzyme used as thrombolytic agent but it is high cost [7]. Nattokinase was formerly known as subtilisin NAT that possesses fibrinolytic (anti-clotting) activities [8-12].

The enzyme nattokinase was orally administered and used for prevention and treatment of CVDs as anti-cloting agent [13, 14]. Moreover, this enzyme is used as an effective alternative medicine for heart disease, and Alzheimer's disease which is associated with diabetes[15]. It is also used for pain, stroke, chest pain, chronic fatigue syndrome, endometriosis, uterine fibroids, muscle spasms, infertility, cancer, and a vitamin-deficiency disease called beriberi [16]. Likewise it enhances the body’s production of plasmin and urokinase by a direct cleavage of plasminogen activator inhibitor [1, 13].

Nattokinase enzyme was generally produced by bacterial fermentation techniques. The sources of medium for fermentation are agricultural wastes such soya bean, wheat bran, rice husk etc. type of substrate is one of the crucial factors for enhanced production. The fermentation process of enzyme production was achieved maximum at optimum conditions like pH, temperature, agitation, incubation time etc. The optimum temperature for fermentation process is increased from 40°C to 43°C. The maximum temperature reached during the fermentation process should be is 50°C or above which the fermentation stops as the bacteria die.

Several studies reported that fibrinolytic enzymes were identified and studied among many organisms including snakes, earthworms, and bacteria: Streptococcus pyogenes, Aeromonas hydrophila, Serratia E15, B. natto, Bacillus amyloliquefacens, Actinomycetes and fungi: Fusarium oxysporum; Mucor sp, Armillaria mellea [17].

B. subtilis is a rod-shaped Gram-positive catalase-positive bacterium. Its ability to form a tough protective endospore allows the organism to tolerate environments of extreme conditions. The present study aims to produce nattokinase from Bacillus subtilis using soyabeans as a cheaper substrate and to get high yield. The production parameters are optimized and the enzyme is extracted by liquid-liquid extraction method.

MATERIALS AND METHODS:

Isolation and identification of B. subtilis from soil samples:

Soil samples were collected from 5 different sites from different locations in Thiruvanamalai district. Soil samples were serially diluted and plated on nutrient agar plate and incubated for 37ºC for 24 hrs. The bacterial colonies were screened and subculture on nutrient agar slants. The bacterial strains identified as Bacillus subtilis based on different staining techniques and biochemical characterization tests followed by Bergey’s Manual of systematic bacteriology [18].

Screening using casein agar medium:

The bacterial strains were subjected to casein hydrolysis assay for screening of nattokinase production by using casein agar medium. About 8 mm diameter well was made in petriplates containing Casein agar medium then 50 µL of bacterial culture was inoculated into the well. The plates incubated at 37°C for 24 hrs. Colorless zone of casein hydrolysis around the well was an indication of nattokinase secretion.

Optimization of medium composition and culture condition for nattokinase production:

Effect of solid substrate:

In this investigation the effect of substrate on on nattokinase production was assayed by using four types of substrates are included rice husk, and wheat bran and soybean meal. Ten grams of each agricultural waste were prepared, and then inoculated with 1.0 ml of the B. subtilis and incubated at 37 ºC for 48 hrs. After filtration, the volume of crude extract (clear supernatant) of each flask was measured and it was applied for nattokinase productivity according to the following equation:-

Enzyme activity (µ/ml) X Vol. of crude sample

Enzyme Productivity =----------------------------------------------------------

Amount of substrate (gm)

The specific activity of the enzyme was calculated as follow:

Specific activity (µ/mg protein) = Enzyme activity (µ/ml)/ Protein concentration (mg/ml)

Protein concentration was estimated by Lowry’s method.

Effect of carbon and nitrogen source:

To study the effect of carbon source on nottokinase enzyme production was performed by using four different carbon sources such as Glucose, Fructose, dextrin and sucrose in different concentrations such 20, 40, 60, 80, 100, 120 g/L. Effect of nitrogen sources was studied in different concentrations like 1, 2, 3, 4, 5 and 6 g/l for fibrinolytic nottokinase enzyme production in Bacillus subtilis.

Effect of pH and temperature:

In this investigation, different pH (2, 4, 5, 6, 7, 8, and 10) were adjusted by following the above method using 0.1M carbonate buffer and the enzyme activity was monitored using spectrophotometer. The effect of temperature on the enzyme activity was determined at the temperature of 20, 30, 35, 40 and 45°C in 50 mM Tris buffer (pH 7.8).

Effect of activator and inhibitor:

The various activators like 10% of KCl, NaCl, MnCl₂, FeSO₄, CaCl₂, and ZnCl₂ were added to the above mentioned method and the enzyme assay was performed to analyze the activation of enzyme production. The inhibitor 10% IAA, EDTA, AgNO₃, HgCl₂ and SDS were added to the enzyme by following the above method and the enzyme assay was carried out to check the activity of inhibitor in enzyme production.

Optimum incubation period:

The suitable incubation time for nattokinase production was carried out with different incubation times of the reaction mixture containing optimum pH, inoculated with 1.0 ml of 24 hrs old culture isolate and incubated at 35 ºC for 24, 48, 72, 96, 120 and 144 hrs.

Purification of nattokinase enzyme by liquid-liquid extraction method:

The extraction of an enzyme from the crude sample was carried out by using liquid-liquid separation technique. In this method 30 ml of pure hexane was added with 30 ml of crude enzyme sample in a separating funnel and shake it thoroughly for 30 minutes and incubate the mixture for 30 minutes without disturbances.

RESULTS AND DISCUSSION:

Isolation of nattokinase producing bacteria:

Nattokinase producing bacteria was isolated from various soil samples from different sources. The Bacillus sp was screened based on their nattokinase producing capability. Screening was carried out from the 15 isolates of Bacillus sp in order to identify the nattokinase activity form zone around the well in the screening medium. The seven Bacillus sp strains were secreted considerable amount of nattokinase from the 15 isolates. These seven strains were biochemically and cultural characters were analyzed. Among them one strain was identified as Bacillus subtilis. This strain was used for further optimization and production of the enzyme nattokinase.

Identification of bacteria:

The isolated bacterium tested positive for Voges–Proskauer tests, citrate utilization, TSI test (acid form glucose), Gelatin hydrolysis, Starch hydrolysis, Nitrate hydrolysis, and Catalase test. The results were negative for growth on MacConkey agar, Indole test, Methyl red test, H₂S production, gas from glucose, TSI test (acid from lactose), Urea hydrolysis, and Phosphatase test. Then the bacterial culture were maintained in nutrient agar slant and used for further studies Table 1.

Table 1: Biochemical characters of the isolates

S.NO	Tests	*Bacillus subtilis*
1	Gram staining	Positive (+) ve rod
2	Motility	Motile
3	Spore staining	Positive
4	Catalase	Positive
5	Oxidase	Variable
6	Urease	Negative
7	Coagulase	Negative
8	Indole	Negative
9	Methyl red	Negative
10	Voges proskauer	Positive
11	Citrate utilization	Positive
12	TSI	A/A,G+
13	Glucose	Positive
14	Sucrose	Positive
15	Lactose	Variable
16	Maltose	Positive
17	Nitrate reduction	Positive
18	Gelatin hydrolysis	Positive
19	Starch hydrolysis	Positive
20	H2S production	Negative

Screening the Bacillus isolates for nattokinase production:

All Bacillus isolates were screened; eleven isolates were nattokinase producer when clear zone of inhibition was formed around the casein agar plate well. The diameter of clear zone were different from one isolates to another, two Bacillus isolates were gave the highest clearance zone ratios (2.3 - 3.5 mm in diameter), while the others were ranged from1.5 to 2.5 mm in diameter.

Optimization of conditions for nattokinase (NK) production:

Effect of solid substrate:

Effect of solid substrates included rice husk, wheat bran and soybean meal was examined for their efficiency in nattokinase production. Among all the substrates, soya bean was the best substrate for production of nattokinase using Bacillus subilis. The specific enzyme activity of nattokinase using soya bean meal was calculated as 37.55U/mg protein with productivity is 195.9U/gm. However, specific activity of NK using rice husk, and wheat bran were 6.43, and 26.14U/mg protein with productivity 163.65, and 180.3U/gm substrate, respectively (Figure 1).

Figure 1: Effect of different types of substrate on nattokinase production from Bacillus subtilis

Figure 2: Effect of carbon source on nattokinase production using Bacillus subtilis

Carbon sources are very essential for the growth of B. subtilis. In this study, the effect of carbon sources on nattokinase production was analyzed using glucose, fructose, dextrin and sucrose, which are utilized by Bacillus subtilis and produce nattokinase enzyme. Among the carbon sources; dextrin influences a maximum nattokinase enzyme activity of 112.0 U/ml (Figure 2).

Three different nitrogen sources such as peptone, yeast extract and beef extract were tested for extracellular protein and Fibrinolytic enzyme production in Bacillus subtilis. Among them peptone supported a maximum extracellular protein content of 5 mg/ml at 36 h and fibrinolytic enzyme production of 12.5 U/ml at 36 h (Figure 3).

Figure 3: Effect of nitrogen source on nattokinase production using Bacillus subtilis

Figure 4: Effect of pH on nattokinase production from Bacillus subtilis

pH strongly influences the nattokinase enzyme production it supports the cell growth and enzymatic processes. To determine the effect of pH, different values of pH 2, 4, 5, 6, 7, 8 and 10 were maintained and flasks were incubated for 24 hours. The results thus obtained for different values of pH are shown in Figure 4. The highest enzyme activity was recorded at pH 7.0 and it was found to be 198 U/ml which is more suitable condition for enzyme production.

Figure 5- Effect of temperature on nattokinase production from Bacillus subtilis

Figure 6- Effect of inhibitors on nattokinase production from Bacillus subtilis

The effect of temperature on the enzyme activity was also studied in a range of 20 to 85°C at pH 7.0, as shown in Fig. 5. The optimum temperature for enzyme activity was approximately at 35°C. Chang et al [8] and Lin et al [19] reported that the optimum temperature for the enzyme is 55 ºC and 60 ºC respectively.

When the enzyme was incubated at room temperature, for 10 min in phosphate buffer (1mM) with 10% KCl, NaCl, MnCl₂, FeSO₄, CaCl₂ and ZnCl₂, the enzyme activity was completely inhibited (Figure 6). Enzyme activity was partially inhibited by ZnCl₂ and CaCl₂ but no inhibition was shown by the others. The nattokinase enzyme was incubated with activators in phosphate buffer, EDTA shows enzyme activity. Others not have activation property shown in Figure 7. Similar results has been reported by Paik et al [10] and Lin et al [19]

The optimum incubation time for production of nattokinase enzyme was studied by detecting the bacterial growth phase at different incubation times (6, 12, 18, 24, 36, 48, 60, 72 and 96 hrs). Figure 8 shows the result of maximum specific activity at 36 h of incubation time,, which was found to be 45.68U/mg protein.

Figure 7- Effect of activators on nattokinase production from Bacillus subtilis

Figure 8: Effect of incubation time on nattokinase production

Whereas after 60 hrs of incubation the specific activity was decreased with increasing the incubation, this may be due to the change in the conditions of culture along these periods such as diminishing of oxygen, nutrients and accumulating of toxic metabolites which inhibit the bacterial growth [20, 21].

Liquid - Liquid extraction of enzyme:

In liquid – liquid extraction method, after 1 h the incubation two separated layers were formed in the separating funnel. The concentration of Nattokinase in extract and raffinate phase was found to be 321 and 190 U/ml respectively.

CONCLUSION:

In the present study, soya bean powder is used as substrate for nattokinase production by the bacterium Bacillus subtilis, which was isolated form soil. Other parameters like carbon source, nitrogen source, inhibitor, activator, incubation time, temperature and pH were studied for enhanced production of NK enzyme. Maximum Nattokinase was observed at the using of soya bean meal, dextrin, and peptone and incubation time 36hrs, pH-7.0, temperature is 35°C and it was found to be 220 U/ml. Hence Bacillus subtilis is a potent producer of Nattokinase enzyme when soybean is used as substrate.

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Received on 13.04.2017 Modified on 21.04.2017

Research J. Pharm. and Tech. 2017; 10(6): 1934-1938.

DOI: 10.5958/0974-360X.2017.00339.0