Purification and characterization of recombinant nattokinase from Bacillus subtilis - Tran Quoc Tuan

TAP CHI SINH HOC 2015, 37(1se): 75-84 DOI: 10.15625/0866-7160/v37n1se. PURIFICATION AND CHARACTERIZATION OF RECOMBINANT NATTOKINASE FROM Bacillus subtilis Tran Quoc Tuan1*, Le Thi Thuy Ai1, Dinh Minh Hiep2, Tran Cat Dong3 1University of Science, VNU Hochiminh city, *trqtuan@hcmus.edu.vn 2Management Board of Agricultural Hi-Tech Park 3The University of Medicine & Pharmacy, Hochiminh city ABSTRACT: Nattokinase, a thrombosis-degradation serin protease, is obtained from Bacillus subtilis Natto. The enzyme has been clinically proven effective and safe in the treatment of thrombotic disease in humans orally. Nattokinase is produced by solid-state fermentation (SSF) of soybean by Bacillus subtilis Natto so yield and quality should be limited. In recent years, there have been initially researches recombinant nattokinase in bacterial systems (Escherichia coli, Bacillus subtilis, Lactobacillus). This study conducted purified recombinant nattokinase from Bacillus subtillis DB104 strain and surveyed some properties of nattokinase. The crude enzyme was precipitated at 70% saturation of (NH4)2SO4. The optimal pH value and temperature were 8.0 and 55oC, respectively and alkaline stability within the range of pH 6.0-10. The molecular weight of Nattokinase was approximately 27.7 kDa. The apparent Vmax and Km values were 0.306 µmol/min/ml and 6.778, respectively. The enzyme was activated with Mg2+, Zn2+, Ca2+ and obviously inhibited by Mn2+ and Hg2+. Keywords: Bacillus subtillis, characterization, nattokinase, purification, recombinant. INTRODUCTION Nattokinase identified as a serine alkaline protease, a potent fibrinolytic enzyme, was primarily isolated from a traditional Japanese fermented food ‘Natto’ by Sumi et al. (1987) [22]. It has 4 times greater fibrinolytic activity than plasmin [21]. The enzyme was reported not only to possess plasminogen activator activity [6] but also to directly digest fibrin by limited proteolysis [5]. Nattokinase has been shown to potentiate fibrinolysis by cleaving the t-PA inhibitor type I in to low molecular weight fragments, leading to efficient lysis of blood clot in the body [24]. In the process of nattokinase purification, the traditional protein separation and purification techniques, such as organic solvent fractionation, salting out and protein chromatography, have been tested with a number of disadvantages, such as long separation and purification time required, more operation units and less activity recovery [3, 7, 17, 24]. Nakanishi produced nattokinase by a procedure consisting of alcohol or ammonium sulfate precipitation, hydrophobic interaction chromatography, ion exchange chromatographyand gel filtration [17]. In the present study, we report on the purification and characterization of recombinant nattokinase produced from Bacillus subtilis DB104. The fibrinolytic enzyme was purified to electrophoretic homogeneity and its biochemical and kinetic properties were investigated. MATERIALS AND METHODS Microorganism and its maintenance B. subtilis pBG01-aprN/BD104, containing the gene coding for nattokinase under the control of the inducible promoter PSgrac was preserved in our laboratory. Bacteria were maintained as spores suspended in 50% (v/v) glycerol, stored at -25◦C. Bacteria was cultured at 37oC in Luria-Bertani (LB) medium for culture collection and in fermentation medium (yeast extract 10 g l-1, CaCl2 5 g l-1, glucose 0,2 g l-1 and NaCl 10 g l-1) for enzyme purification. Chloramphenicol (10 µg ml-1) was added to the growth medium when necessary. Chemicals and reagents Thrombin and fibrinogen from humanplasma were purchased from Sigma-Aldrich (Milan, Italy). Sephadex G-75 was purchased from Pharmacia Co. (Amersham Bioscienses, Uppsala, Sweden). All other reagents were of the highest quality commercially available. Cell growth and nattokinase production A 5% (v/v) spore suspension was added to seed medium composed of: 10 g l-1 glucose, 10 g l-1 yeast extract, 1 g l-1 K2HPO4.3H2O, 0.5 g l-1 MgSO4·7H2O, with a pH value of 7.0-7.2. The subsequent cultivation lasted for 12 h at 37◦C and 170 rpm in an orbital shaker to obtain seed culture with an OD578 nm value of 7-8. Then, a 5% (v/v) seed culture was added to the production medium consisting of: 8.28 g l-1 soy peptone, 0.64 g l-1 calcium chloride, and 0.74 g l-1 yeast extract, 20 g l-1 maltose, 2 g/l K2HPO4.3H2O and 1 g l-1 MgSO4.7H2O. Shake flask cultures were carried out at 37◦C and 170 rpm in an orbital shaker to obtain seed culture with an OD578 nm value of 0.8-1 was induced with 0.08 mM isopropyl-β-D-thiogalactoside (IPTG). After the IPTG induction for 14 h, the supernatant of crude enzyme was obtained by centrifugation at 7,000 rpm for 30 min, and the supernatant were used for nattokinase activity assay. Protein assay and SDS-PAGE The protein concentration in supernatant of fermentation broth and stripping solution was determined by Bradford method [2] using bovine serum albumin as the standard protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in Laemmli’s system to determine the enzyme’s purity [14] and the molecular mass of purified enzyme, using 4% acrylamide in the stacking gel and 12.5% acrylamide in the separating gel. Protein bands were visualized by staining with Coomassie R 250 brilliant blue. Standard proteins (Amersham Biosciences) were used for molecular mass determination as follows: myoglobulin (200 kDa), β-galactosidase (116 kDa), phosphorylase b (97.2 kDa), bovine serum albumin (66.4 kDa), albumin egg (44.3 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20.1 kDa), lysozyme (14.3 kDa), and aprotinin (6.5 kDa). Determination of the fibrinolytic nattokinase activity Nattokinase activity was measured using a fibrin degradation assay developed by Japan Bio Science Laboratory Co., Ltd. (JBSL) [10]. To 1.4 mL of 50 mM sodium borate buffer (pH 8.5), a 0.4 mL of 0.72% (w/v) fibrinogen solution was added and kept at 37oC for 5 min. To the resulting fibrinogen solution was then added 0.1 mL of thrombin (20 U/mL) and kept at 37oC for another 10 min. After the addition of 0.l mL of enzyme, the proteolytic reaction was performed at 37oC for 60 min. After the reaction was ceased by adding 2 mL of 0.2M trichloroacetic acid (TCA), samples were kept at ambient temperature for 20 min and then centrifuged at 14000 rpm for 5 min. In this assay, 1 unit (fibrin degradation unit, FU) of enzyme activity is defined as a 0.01-per-minute increase in absorbance at 275 nm of the reaction solution. Purification of nattokinase All steps in the purification procedure were operated at room temperature except for centrifugation, which was conducted at 4°C. The supernatant of crude enzyme was obtained by centrifugation at 7,000 rpm for 30 min, and then ammonium sulfate was added to the solution to make different saturations ranging from 40 to 90% (in increments of 10%). Each fraction was obtained by centrifugation at 13,000 rpm, 4°C for 15 min, and dissolved in 20 mM Tris-HCl (pH 7.8) buffer; the solution was dialyzed against the same buffer overnight. Enzyme activity and protein content were determined in the fraction. Ammonium sulfate treatment: Solid ammonium sulfate was added to the supernatant containing the enzyme to make 40% saturation. This mixture stood overnight at 4°C and was centrifuged at 7,000 rpm for 30 min at 4°C to remove particle material. The supernatant was adjusted to 70% ammonium sulfate saturation by further addition of solid ammonium sulfate and stayed overnight at 4°C. The precipitate was collected by centrifugation at 10,000 rpm for 20 min at 4°C for further purification. Sephadex G75 column chromatography: The precipitate was dissolved in 20 mM Tris-HCl buffer (pH 7.8) containing 0.15 mol/l NaCl, applied to a Sephadex G75 column (1.5 cm × 35 cm) (AKTA system) equilibrated with 20 mM Tris-HCl buffer (pH 7.8), and then eluted with the same buffer at a flow rate of 0.5 ml/min. Elution peaks were collected, and the enzyme activity and protein concentration in each fraction were determined, respectively. The fraction containing the enzyme with highest activity was collected and concentrated by ultrafiltration. The active fraction were pooled, concentrated, and analyzed for purity by SDS-PAGE. Effect of pH and temperature on enzyme activity The effect of pH on the catalytic activity was studied by the standard assay method. To investigate the optimal pH, nattokinase activity was assayed at 37oC at various pH values (4.0-11.0) of buffer. The buffer systems used were acetate (50 mM, pH 4-5), phosphate (50 mM, pH 6-8), and Na2CO3-NaHCO3 (50 mM, pH 9-11). The enzyme activity was measured in the range of 25-90oC using the standard activity assay procedure at related temperature. Effect of metal ions, surfactant and nattokinase inhibitors on the enzymatic activity The effects of metal ions (1 mM and 5 mM ) were investigated using Mg2+, Cu2+, Fe2+, Ca2+, Zn2+, Mn2+ and Ba2+. The effects of nattokinase inhibitors were studied using phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA). The effects of surfactant was studied using 1–5 mM SDS. Enzyme was pre-incubated with metal ions, surfactants or inhibitors in 50 mM sodium borate buffer (pH 8.5) for 30 min at 30oC and then the residual nattokinase activity was tested. Fibrinogenolytic activity analysis Fibrinogenolytic activity was measured by a modified fibrinogenolytic assay [12] The fibrinogen solution (200 μl of 1% human fibrinogen in 0.05 mol/l pH 7.8 Tris-HCl buffer) was mixed with the purified enzyme solution (60 μl of 0.2 mol/l) and incubated at 37°C for 15 min, 1, 2, 4, 7 and 9 h, respectively. After the indicated time intervals, aliquots were transferred to ice and separated by SDS-PAGE to examine the cleavage pattern of the fibrinogen chains. Determination of Km and Vmax Different concentrations of fibrin (0.036-0.36) mg l-1 prepared to calculate the initial velocity of enzyme reaction toward the substrate for each constant of substrate concentrations. Estimate the value of Km and Vmax of the enzymatic reaction from the plot the relationship between initial velocity and substrate concentration according to Lineweaver-Burk Reciprocal plot and Hans-Woolf Plot [20]. RESULTS AND DISCUSSION Purification of nattokinase Nattokinase produced by B. subtilis DB104 was purified by simple method consisting of precipitation (table 1). Most of the enzyme activity was achieved at 50-70% saturation ammonium sulfate fraction having 673.34 units, in which specific activity was 64.87 U/mg protein with a yield of 74% (table 1). It is noted that ammonium sulfate salts is the most widely used in the concentration of enzymes due to high solubility and low cost, compared with the other organic solvents and no effect on pH or the stability of the enzyme [28]. In other studys the activity of precipitates in the crude enzyme supernatants with 30-80% saturations (NH4)2SO4 was contrasted to preserve the fibrinolytic activity of fibrinolytic enzyme as much as possible, according to the activity assay, the precipitate formed in 30-60% saturation of (NH4)2SO4 was collected [25]. The fibrinolytic enzyme were also purified by ammonium sulfate saturation. The protein fraction was precipitate with 85% ammonium sulfate [4]. In the present study, the activity of the precipitates in the crude enzyme supernates with 30-90% saturations (NH4)2SO4 was contrasted. To preserve the fibrinolytic activity of nattokinase as much as possible, according to the activity assay, the precipitate formed in 50-70% saturation of (NH4)2SO4 was collected. Then the dialyzed precipitate was applied to Sephadex G-75 (fig. 2). As summarized in table 2, the fibrinolytic enzyme was easily and quickly purified by the combination of various steps. The finally eluted proteins were subjected to SDS-PAGE, and only one band was observed in the purified sample on SDS-PAGE (fig. 3, lanes 2, 3). The molecular weight of the enzyme was approximately 27,7 kDa. With nattokinase as a standard, the final specific activity of the fibrinolytic enzyme increased more than 4-fold with a 78.89% recovery based on the initial culture supernatant. Table 1. The ammonium sulphate precipitation of nattokinase produced by B. subtilis DB104 Ammonium sulphate (%) Nattokinase activity (FU/ml) Total activity (FU) Protein (mg/ml) Total protein (mg) Specific activity (FU/mg) Crude enzyme 18.16 907.95 0.26 13.10 69.30 40% 16.67 33.33 0.29 0.57 58.28 50% 125.93 251.85 2.03 4.06 69.97 60% 179.01 358.02 2.47 4.94 72.53 70% 31.69 63.37 0.60 1.22 52.12 80% 20.99 41.97 0.44 0.89 47.27 90% 8.85 17.69 0.24 0.49 35.97 Table 2. Purification stages of nattokinase from B. subtilis DB104. Steps Total protein (mg) Specific activity (FU/mg) Total activity (FU) Purification (fold) Yield (%) Culture supernatant 3.2 162.0 522.6 1.0 100 (NH4)2SO4 precipitation 2.5 199.1 490.6 1.2 94 Sephadex G-75 gel filtration 0.7 631.9 412.1 3.9 79 Figure 1. SDS-PAGE analysis of the ammonium sulphate precipitation of nattokinase produce by B. subtilis DB104 Lane 1, Protein molecular mass marker (12-225 kD); lane 2, ammonium sulphate 40%; lane 3, ammonium sulphate 50%; lane 4, ammonium sulphate 60%; lane 5, ammonium sulphate 70; lane 6, ammonium sulphate 80% and lane 7, ammonium sulphate 90%. Figure 2. Gel filtration chromatography for purified nattokinase from B. subtilis DB104 by using Sephadex G75 column (1.5x54) cm The column was calibrated with 20mM Tris-HCl buffer pH 7.8; flow rate 30 ml/hrs and 5 ml/fraction. Nattokinase activity (FU/ml) (u), Protein (mg/ml) (n) Figure 3. SDS-PAGE analysis of the purified nattokinase expressed in B. subtilis DB104. Lane 1, protein molecular weight marker; lane 2, proteins in the culture supernatant of B. subtilis DB104 harboring pBG01; lane 3, purified nattokinase. Figure 5. Degradation of fibrinogen by nattokinase Lane 1 molecular mass markers; Lane 2 fibrinogen control without enzyme; Lanes 3-10 degradation products after 1, 5, 10, 20, 30, 60 and 120 min incubation at 37oC, respectively Figure 4. Effect of temperature and pH (a). Temperature optimum; (b). pH optimum. Effect of pH and temperature on nattokinase activity The effect of pH on the fibrinolytic activity was determined with buffers at various pH values. Results (fig. 4b) showed that this enzyme was active at neutral and alkaline pH (7.0-9.0), and was most active at about pH 8.0, identical to that from B. subtilis natto B-12 [25], B. subtilis TKU007 [27] and B. subtilis [4] and near to optimum pH (9.0) for the subtilisins from B. amyloliquefaciens DC-4 [19] and B. stearothermophilus [9]. Above 80% of its activity was retained at pH values ranging from 7.0 to 9.0. Nattokinase lost 60% of the activity at pH 4.0, and nearly 30% of initial enzyme activity was retained at pH 12. The nattokinase from B. subtilis VTCC-DVN-12-01 [23] and subtilisins from B. amyloliquefaciens DC-4 [19] B. stearothermophilus [9] were observed at pH 9. The influence of temperature on the fibrinolytic activity was examined at pH 8.7, and the result showed that the enzyme was active between 37oC and 55oC with an optimum activity at 55oC (fig. 4a). The enzyme showed 83% and 62% residual activity at 60oC and 70oC, respectively, but at 75oC, the fibrinolytic enzyme was lost. This optimum temperature was consistence with those mentioned by Liu et al. (2005) [16]; Wu et al. (2009) [29] and was higher than those determined (40oC) by Wang et al. (2009) [25]; (50oC) by Li Jung Yin et al. (2010) [15] and Hsu et al. (2009)[8]. Nevertheless, it was lower than some of other Bacillus strains 60oC (B. subtilis KCK-7) [18], 65oC (B. subtilis VTCC-DVN-12-01) [23] and 70oC (Bacillus sp. strain CK 11-4) [11]. Effect of metal ions and protease inhibitors on nattokinase activity To further characterize the purified nattokinase, we examined the effects of some regular divalent and trivalent metal ions on NK activity. The results are summarized in table 3. Zn2+, Ca2+ activated the enzyme activity, Ba2+, K+ and Mg2+ did not obviously activate or inhibit the enzyme activity. Co2+, Cu2+ and Fe3+ inhibited the fibrinolytic enzyme, and the inhibitory effects of Hg2+, Mn2+ and Pb2+ were most obvious among these metal ions. The effects of Zn2+, Co2+, Cu2+, Fe3+ and on nattokinase activity were consistent with nattokinase from B. subtilis YJ1 [15] and B. subtilis natto B-12 [25]. Ion Zn2+ is known to play amajor role in the activity of serine protease [13], which may account for the positive effect of Zn2+ in the present study. Table 3. Effect of metal ions and inhibitors on the activity of nattokinase Metal ions or inhibitor Residual activity (%) Concentration (1 mM) Concentration (5 mM) None 100 ± 0.5 100 ± 0.5 Ba2+ 110.2 ± 0.2 104.0 ± 1.5 Ca2+ 121.5 ± 1.5 136.2 ± 0.5 Co2+ 97.0 ± 0.7 82.0 ± 1.0 Cu2+ 91.0 ± 0.2 80.2 ± 0.9 Fe3+ 88.1 ± 0.3 78.0 ± 0.3 Hg2+ 8.5 ± 0.3 9.0 ± 0.4 K+ 106.7 ± 1.2 119.2 ± 0.5 Mg2+ 105.6 ± 0.7 124.9 ± 0.4 Mn2+ 70.0 ± 0.3 15.8 ± 0.7 Pb2+ 62.2 ± 0.4 38.1 ± 0.2 Zn2+ 142.9 ± 0.5 172.9 ± 0.2 EDTA 71.2 ± 0.4 56.5 ± 0.5 SDS 37.3 ± 0.5 2.0 ± 0.5 PMSF 0.0 ± 0.0 0.0 ± 0.0 Results are presented as means ± SD (n=3). These results are different from another nattokinase from B. subtilis VTCC-DVN-12-01 [23] and Pseudomonas sp. TKU015 [26] except for the effects of Ca2+ and Mg2+. TKU015 nattokinase activity increased significantly in the presence of 5 mmol/L Fe2+ and was not affected by many metal ions, such as Zn2+, Ca2+, Mg2+, Ba2+, Cu2+ and Mn2+. Jin7 nattokinase activity increased significantly in the presence of 10mmol/L Ca2+ and Mg2+. It was partly inhibited by Fe2+, Ba2+ and Mn2+ and significantly inhibited by Cu2+ and Co2+ [1]. The different effects of metal ions on nattokinase are mainly attributed to the different sources. Nattokinase was strongly inhibited by a typical serine protease inhibitor phenylmethane sulphonyluoride - PMSF (1 mM) (100%). The fibrinolytic activity of nattokinase was also decreased by EDTA (1 mM and 5 mM). The effect of detergents on nattokinase from B. subtilis DB104 was in agreement with current observations that the addition of SDS at 1 and 5mM to nattokinase decreased the activity by 62 and 98%, respectively, B. subtilis VTCC-DVN-12-01 [23]. Hydrolysis of fibrinogen by the purified nattokinase To explore the fibrinogenolytic activity of nattokinase, SDS-PAGE was performed. Interestingly, the hydrolysis rate wassignificantly different with the chains of fibrinogen. The Aα-chain was completely degraded within 1 min, and the Bβ-chain was completely degraded within 5 min. The g-chain was mostly hydrolyzed in 2 h (fig. 5). Km and Vmax of the fibrinolytic enzyme For Km and Vmax determination, the fibrinolytic enzyme assays was carried out with synthesis substrate at a serial of concentrations. Kinetic parameters for this fibrinolytic enzyme were obtained from individual experiments after the addition of decreasing amount of substrate to the sample cuvette. Data were listed in table 4, resulting in a linear Lineweaver-Burk plot. The apparent Km and Vmax value was 6.778 mg/ml and 0.306 µmol//ml/min, respectively. Figure 6. Lineweaver-Burk plot for fibrin hydrolysis by nattokinase Table 4. The result of the kinetic parameter [S] (mg/ml) 0.036 0.072 0.108 0.144 0.180 0.216 0.252 0.288 0.324 0.360 1/[S] (L/mmol) 27.78 13.89 9.25 6.94 5.55 4.63 3.96 3.47 3.08 2.78 V (mmol/min) 0.008 0.014 0.025 0.030 0.037 0.047 0.052 0.053 0.053 0.054 1/V (min/mmol) 125.0 71.43 40.0 33.33 27.02 21.28 19.23 18.87 18.87 18.51 CONCLUSION In conclusion, we observed that the fibrinolytic enzyme which isolated from B.subtilis BB104 by several steps included ammonium sulphate salt precipitation, then gel filtration in Sephadex G-75. It is noted that the characterization of nattokinase were the optimum pH activity 8.0. The optimum temperature for fibrinolytic enzyme activity was 55oC, and have important effects on fibrinolytic activity of nattokinase. Further study will be conducted to investigate the biochemistry methods of improving the fibrinolytic activity of nattokinase, themolecular structure such as amino acid sequence, and secondary and tertiary structures. 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Hồ Chí Minh TÓM TẮT Nattokinase là một serine protease từ Bacillus subtilis Natto, có khả năng làm tan đặc hiệu fibrin, phân hủy huyết tụ nội sinh ở người, được sử dụng trong phòng ngừa và hỗ trợ điều trị bệnh huyết khối. Nattokinase được thu nhận chủ yếu bằng con đường lên men bán rắn chủng Bacillus subtilis Natto trên cơ chất đậu nành nên năng suất và chất luợng bị giới hạn. Những năm gần đây đã có những nghiên cứu bước đầu tạo nattokinase tái tổ hợp có hoạt tính bằng hệ thống vi khuẩn (Escherichia coli, Bacillus subtilis, Lactobacillus). Trong nghiên cứu này, chúng tôi tiến hành tinh sạch nattokinase tái tổ hợp từ chủng Bacillus subtillis DB104 và khảo sát một số đặc tính của enzyme. Kết quả nhận được, sử dụng (NH4)2S04 70% bão hòa để thu nhận chế phẩm enzyme. Enzyme hoạt động tốt nhất ở nhiệt độ 55oC, pH 8, enzyme hoạt động trong khoảng pH kiềm 6.0-8.0. Các ion Mg2+, Zn2+, Ca2+ có tác dụng tăng hoạt. Các ion Mn2+, Hg2+, có tác dụng ức chế. Nattokinase thu nhận được có trọng luợng 27,7kDa có Vmax = 0,306 µmol/phút/ml và Km = 6,778. Từ khóa: Bacillus subtillis, bệnh huyết khối, nattokinase, serine protease, tái tổ hợp. Ngày nhận bài: 22-10-2014