Extracellular Production of a Potent and Chemical Resistant Nattokinase in Immobilized Escherichia coli Using Response Surface Methodology

Abstract

Nattokinase is a potent fibrinolytic protease, besides various industrial applications used as a drug for treatment or a supplement for preventing thrombosis. Generally, most of native extracellular proteins such as nattokinase from B.subtilis are lysed by some secretory proteases. Therefore, one way for solving this problem, is to apply other hosts for nattokinase production. There are different factors for producing secretory form of nattokinase from B.subtilis, such as suitable host, optimized media for the maximum activity and easy purification which are studied in this investigation. E.coli BL21 (DE3), as a reliable host was selected for production of a high yield of an ectracellular recombinant nattokinase. A mature nattokinase gene from Bacillus subtilis 1023, was cloned in the expression vector pET22b and transformed in the host. The recombinant nattokinase was expressed by 1mM IPTG induction for 4 hours. The expressed protein was confirmed by SDS-PAGE, and its fibrinolytic activity was assayed on the fibrin plates. Afterwards, the enzyme was purified by Ni-NTA native affinity column. Evaluating different media components for maximum nattokinase production and activity, the most enzyme activity reached to 883.371 U/ml when the media consisting of yeast extract (4.3804 g/L), tryptone (3.9899 g/L), K2HPO4 (1.6107 g/L) and CaCl2 (0.01 g/L). Entrapping the transformant cell in calcium alginate could gain more activity and lowering the media cost. The present study aim to produce the soluble nattokinase with high activity in the low costs of media that having high resistance against metal ions, detergents and organic solvents, which can easily use in medicines and detergents.

Keywords:

Nattokinase, Fibrinolytic, E.coli BL21(DE3), Calcium alginate, Industrial applications.

Introduction:

Nattokinase (EC 3.4.21.62) or subtilisin NAT is a serine protease produced by Bacillus subtilis strain, firstly was introduced from natto (a Japanese food) (Sumi, Hamada et al. 1987). In spite of other thrombolytic agents, nattokinase can hydrolyze fibrin in both directly and indirectly paths; therefore, nattokinase is more

effective than other thrombolytic drugs (Peng, Huang et al. 2003). Nattokinase has different therapeutical and industrial applications. Three forms of therapeutic nattokinase is available; the one type is a pure enzyme as a potent drug for cardiovascular diseases. The two other forms are not pure and are used as supplements for prevention of thrombosis; the second type is along with other extracellular enzymes without bacteria, and the third type, is a complex of nattokinase, other enzymes and B.subtilis which use as probiotics and drug together (Dubey, Kumar et al. 2011, Dabbagh, Negahdaripour et al. 2014). Nattokinase passes securely through the digestive tract; therefore, different forms of nattokinase are suitable for oral administration. However, for fast acting, pure nattokinase is injected in vessels (Peng, Yang et al. 2005, Gad, Nirmala et al. 2014). Industrial applications of nattokinase are more widespread than the clinical uses, especially because of incomplete and partial purification (Weng, Yao et al. 2017). Therefore, providing optimal situations for nattokinase production, such as suitable host, best low-cost media component and proper environmental situations, are important factors. To improve production of recombinant nattokinase and make the downstream manipulation easier, several microbial hosts have been used in different studies (Wu, Feng et al. 2011, Wei, Zhou et al. 2015). Recombinant nattokinase is highly expressed in E. coli, but after refolding of inclusion bodies and the purifying processes, the end yield is negligible (Liang, Jia et al. 2007, Han, Guo et al. 2009). E.coli BL21 (DE3) is a good selection for producing a recombinant B.subtilis enzyme when its other extracellular enzymes (especially proteases) are absent. Because of extracellular proteases in a media of B.subtilis, the yield of proteins production is usually lower than E.coli (Westers, Westers et al. 2004). So to produce a high yield of active nattokinase in E. coli, a secretory expression system was used with the help of the N-terminal signal sequence PelB that was embedded in pET22b, and because of C-terminal His-Tag sequence the nattokinase purification is simplified by Ni NTA column (Liang, Jia et al. 2007, McIntosh, Renfro et al. 2011). To date, different studies are performed for nattokinase production (Liu, Xing et al. 2005, Deepak, Kalishwaralal et al. 2008, Wang, Chiu et al. 2009, Wu, Feng et al. 2011, Ghasemi, Dabbagh et al. 2012, Nguyen, Quyen et al. 2013, Gad, Nirmala et al. 2014, Vignesh, Eajas Basha et al. 2014). However, most of the studies have some problems, which in this study was tried to solve some of them such as host system, potent enzyme, suitable media culture, bacterial immobilization to decrease costs and easier purification

Materials and Methods:

Strains and Plasmid

pET22b (Pasteur Institute, Iran) were used for cloning of mature nattokinase gene from B.subtilis 1023 (PTCC, Iran) Afterwards, the construct was transformed to E.coli BL21 (DE3) (Novagen, Germany).

Culture Growth Conditions

Recombinant cells were cultivated at 37˚C in Luria-Bertani (LB) broth, which is made of 10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl, with the concentration of 100 g/ml ampicillin and 200 rpm shaking.

Cloning of nattokinase

A mature nattokinase gene (825-bp) from B. subtilis PTCC 1023 (Pasteur Institute, Iran) that encode 276 aa-mature nattokinase with 28 kDa, was amplified by PCR with specific primers (NdeI-linked forward primer, 5'-GTACATATGGCGCAATCTGTTCCTT-3' and XhoI-linked reverse primer, 5'-GTCTCGAGTTGTGCAGCTGCTTG3') and sequenced (Nielsen, Engelbrecht et al. 1997). With the help of restriction endonucleases NdeI and XhoI (Fermentas, EU), both the desired gene and the bacterial secretory-expression vector (pET-22b) were digested, after that the gene was directly ligated into the vector (Rapid DNA Ligation Kit, Fermentas, EU). The ligation product was transformed to competent E. coil BL21(DE3) cell using Gene Pulser XcellTM electroporation system Bio-Rad bacterial. Recombinant vector was extracted from transformed cell using high pure plasmid isolation kit (Roche, Germany). Colony PCR analysis was applied to check the proper insertion of the gene in the recombinant plasmid using specific gene primers. The nucleotide sequence of the recombinant gene was confirmed by automated DNA sequencing (CinnaGen Co., Iran).

Expression and purification of nattokinase

The nattokinase construct was transformed into E. coli BL21 (DE3). All transformants were grown in LB medium with 100µg/ml ampicillin at 37˚C and shaking (200 rpm), until the cell concentration achieved to OD600= 0.5–0.7, and induced with 1 mM isopropyl-b-d-thiogalactopyranoside (IPTG) (purchased from Fermentas, EU). . After 4 h induction, the cells were collected by centrifugation at 6,000 g for 10 min, and the culture supernatant was used for nattokinase activity. Expression of recombinant nattokinase, intra and extracellular form, was respectively evaluated by sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) (Laemmli 1970), and protein bands were stained by Coomassie Brilliant Blue G-250.

Medium Composition and Statistical Experimental

In order to choose the significant effective media factors, a full factorial central composite design (CCD) was applied. Via MODDE software version 9 (Umetrics, Sweden) statistical analysis was performed, and the variance (ANOVA) test determined the significant differences among the clusters. The p-value less than 0.05 was considered statistically significant, and all data were calculated as means ± standard deviations.

Optimization of IPTG induction:

To optimize the concentration of IPTG, the transformant cells were cultured at the optimized media with 100µg/ml ampicillin at 37˚C with shaking (200 rpm), until the cell density were reached to OD600= 0.5–0.7, then induced by different concentrations of IPTG ( 0.2, 0.4, 0.6, 0.8, 1 ,1.2 and 1.4 Mm) for 4hours (Sadeghi, Rabbani et al. 2011, Mualif, Teow et al. 2015, Margawati, Fuad et al. 2017). To optimize the time of IPTG induction, after culturing the transformant cells at the optimized media with 100µg/ml ampicillin at 37˚C with shaking (200 rpm), the induction was done by 1mM IPTG for 2, 4. 6, 8, 12, 24 hours (Margawati, Fuad et al. 2017).

Purification of nattokinase

C-terminal 6xHis tag in the recombinant nattokinase simplifies the purification of it by Ni2+-ProBond™ resin (Invitrogen Corp., Carlsbad, USA) affinity chromatography under native conditions. A volume of 8 ml culture supernatant from the optimized medium was harvested by centrifugation at 3000 g and 4°C for 5 min, afterward the supernatant was added to a 2 ml resin column, which was equilibrated with native binding buffer and incubated for 60 min at room temperature. The resin in the column was settled by gravity, and the supernatant was carefully extracted to remove all non-6xHis-tagged proteins and washed three times with native wash buffer. The bound protein was eluted with 8 ml of native elution buffer, and 1 ml per fraction was collected.

Determining fibrinolytic assay

Fresh frozen human plasma, which had been mixed with citrate phosphate dextrose adenine (CPDA-1) (as an anticoagulant), was provided by the Iranian Blood Transfusion Organization. One milliliter of the plasma was added 50 µL of CaCl2

concentrations stock 0.2 mol on a plate and incubated for 20 minutes at 37°C in 5% CO2 to complete coagulation and formation of fibrin plate (Banihashemi, Mohkam et al. 2015, Banihashemi, Safari et al. 2017). 50µl of transformant cells was added to fibrin plate and incubated 30 minutes at 37 ˚C.

Nattokinase activity assay

For 200 μl reaction mixture, 1 mM Suc-AAPF-pNA as a substrate, and 50 mM Tris– HCl (pH 8.0), 5 mM CaCl2 were mixed . 3 μl of the enzyme was added and then incubated at 37°C for 10 min; the enzyme activation was arrested by adding 50 μl of 30% (v/v) acetic acid. The nattokinase activity was quantified by cleavage of p-nitroanilide from Suc-AAPF-pNA and measured by the absorption at 405 nm on a microplate spectrophotometer (Powerwave X2 from BioTek). One unit of nattokinase activity was expressed as nmol of p-nitroaniline released due to substrate hydrolysis/min/mL by the enzyme (Ko, Yan et al. 2004, Mahajan, Gokhale et al. 2010, Berenjian, Mahanama et al. 2014).

Immobilization of transformant cells in Sodium Alginate, and in polyvinyl alcohol (PVA) alginate beads

Firstly, sodium alginate (3 g) and distilled water (90 ml) were heated to dissolve. Afterward the transformant cells which were cultivated in LB medium. Then the cell pellets were mixed with sterilized agitated (25˚C) carefully in a sterilized glass rod for an equal diffusion of cells. The mixture was splashed to sterilized chilled calcium chloride solution (0.2 M) by a sterile syringe from the 20 cm height. For fixation of the formed beads they were maintained in sterile CaCl2 solution at 4°C for one hour, then they washed three times in sterile distilled water and kept at 4°C (Divatar, Sandhya et al. 2015).

After centrifugation (according to above method), the cell pellets were added to a mixture of sodium alginate solution 1% and PVA solution 7%, on a shaker. The hemogenous mixture was splashed in a sterile calcium chloride solution 1%.with a sterile syringe The gel beads were kept in CaCl2 solution for one hour, , and the beads were washed three times in sterile distilled water and kept at 4°C (Lorenzetti, Moro et al. 2015).

Optimization of temperature and pH

Optimization of temperature and pH of the nattokinases (in Suc-AAPF-pNA mixture) were performed, by using 50 mM Tri-HCl buffer (pH 8) at the temperature from 30 to 80°C and using 50 mM acetate buffer (pH 4-5), 50 mM phosphate buffer (pH 6-8), and 50 mM Tris-HCl buffer (pH8-9) at 45°C, separately (Doddapaneni, Tatineni et al. 2009, Nguyen, Quyen et al. 2013).

Temperature and pH stability

To determine temperature and pH stability, the purified nattokinases, were incubated at the temperature range of 30 to 80°C and pH 7 for 60 min, and under different pH ranges (with 50 mM acetate at pH 4-5, 50 mM phosphate at pH 6-8, and 50 mM Tris buffer at pH 8-9 separately) at 30°C for 1 hour. The changing in enzyme activity after 60 min was measured (Rachadech, Navacharoen et al. 2010, Nguyen, Quyen et al. 2013).

Effect of metal ions, detergents, and organic solvents

Changing in the nattokinases activity in the presence of metal ions, EDTA, detergents, and organic solvents were measured by preincubating the nattokinases, in Na2HPO4 (50 Mm, pH 7) and exposed to 1, 5, and 10 mM of metal ions (Ca2+, Fe2+, Mg2+, Mn2+, Zn2+) and EDTA, or exposed to 0.2, 0.5, 1% (w/v) of SDS and TritonX-100 as detergents, or exposed to 10 and 30% (v/v) of ethanol, methanol and acetone as organic solvents at 30°C for 1 hour (Doddapaneni, Tatineni et al. 2009, Nguyen, Quyen et al. 2013).All the above experiments were performed in three times, and the results were the mean of the obtained data.

Results and Discussion

Gene analysis

The gene sequence of mature nattokinase from B. subtilis PTCC 1023 is 100% identical with aprE gene from B. subtilis strains T30, NRS231 and W23, and 94% with sequences from B.subtilis strain (KK-2B10 and MTCC-7264 nattokinase (aprN) gene.

Cloning of a nattokinase gene from B. subtilis 1023

A partial gene of mature nattokinase from B.subtilis PTTC1023 was amplified by pfu DNA polymerase and ligated into pET22b secretory expression vector. The recombinant vector was applied for transformation of E. coli BL21 (DE3). Colony PCR analysis and double digestion on the plasmid of transformant cells were applied by nattokinase specific primers to check the correct insertions in both E. coli strains (Fig. 1 and 2).

Fig. 1: Colony PCR analysis of 1, 2. Transformant E.coli BL21 (DE3).

Fig. 2. pET22b plasmid 2. Nattokinase gene. 3. Double digestion of plasmid from transformant E.coli BL21 (DE3).

The nattokinase gene was cloned in E.coli BL21 (DE3), and then the recombinant cell was cultured at 37 ˚C in LB medium containing 100 µg/ml ampicillin and induced by 1 mM IPTG for 4 h. The result of the nattokinase expression was displayed approximately at 35kDa (Fig. 3a). The His-tag at the C-terminal of the

pET22b was made protein heavier and simplified the purification of recombinant nattokinase by Ni-NTA native affinity column (Janknecht and Nordheim 1992). The recombinant His-tagged nattokinases was purified, and the single band from the supernatant was displayed in Fig. 3b. The media pH became highly acidic after protein expression which can justify by erratic mobility of nattokinase in SDS-PAGE and interference between lines. Because SDS cannot bind to acidic proteins in standard situations of SDS-PAGE (García‐Ortega, De los Ríos et al. 2005, Fattahian, Rasooli et al. 2011).

Fig3: SDS-PAGEe analysis, (a) 1. Intracellular expression of nattokinase. 2. Intracellular preoteins before cloning (b) Extracellular expression of nattokinase in E. coli BL21 (DE3).

Fibrinolytic activity of nattokinases

Purified nattokinase or supernatant from E. coli (10 µl) were added to fibrin plate (Banihashemi, Safari et al. 2017). and incubated at 37˚C for 10 min The results indicate good enzyme activity for both products (Fig. 4), but the purified products lysed the fibrin 10 min later than supernatant products. Enzymes in the supernatants have a natural condition, so immediately response to fibrin, but purified enzyme needs more time to complete the action.

Fig. 4: Fibrinolytic activity of nattokinase, (A, D) supernatant of blank E. coli BL21 (DE3). (C, F) Supernatant of transformed E. coli BL21 (DE3). (B, E) Purified nattokinase from E. coli BL21 (DE3).

Medium Composition and Statistical Experimental Design

The Condition of cells growth has an important role to enhance protein production. Therefore, media culture optimization is a noticeable method to improve production and enzyme activity of the recombinant proteins (Vásquez-Bahena, Vega-Estrada et al. 2006, Ghoshoon, Berenjian et al. 2015, Zamani, Berenjian et al. 2015). In our study by means of media optimization, production and activity of the nattokinase in E. coli BL21 (DE3) has been enhanced. At first some theoretical and experimental preliminary studies were done on media components and according to the results four various factors (yeast extract, tryptone, K2HPO4 and CaCl2 ) were defined for optimization methods that performed by a fractional factorial design (Huy and Huong , Liu, Xing et al. 2005, Deepak, Kalishwaralal et al. 2008, Mahajan, Gokhale et al. 2010, Al-Juamily and Al-Zaidy 2012, Ghoshoon, Berenjian et al. 2015, Zamani, Berenjian et al. 2015). Applying response surface methodology (RSM) the four media ingredients were selected for optimizing nattokinase production and activity. To investigate the relations of effective ingredients and the most values of them central composite face design (CCF) was used. In various concentrations of four media ingredients the enzyme activity were ranging from 20.1 U/ml to 788.7 U/ml in E. coli BL21 (DE3), and 0.421 to 2.581 for the cell density (OD600). The experimental results for two responses are shown in Table. 1.

Table. 1: Experimental design and optimization results of nutritional factors on extracellular nattokinase activity and cell density in E. coli BL21 (DE3).Run Experimental factorsa Responses

Tryptone

Yeast Extract

CaCl2 K2HPO4 Activity(U/ml)

Cell density (OD600)

1 0.5 0.5 0.01 0.2 28.1 0.421

2 5 0.5 0.01 0.2 194.3 1.312

3 0.5 5 0.01 0.2 203.5 1.209

4 5 5 0.01 0.2 457.2 2.39

5 0.5 0.5 0.1 0.2 31 0.611

6 5 0.5 0.1 0.2 211.3 1.406

7 0.5 5 0.1 0.2 231.2 1.428

8 5 5 0.1 0.2 466 2.581

9 0.5 0.5 0.01 2 109.3 0.529

10 5 0.5 0.01 2 611.1 1.419

11 0.5 5 0.01 2 571.8 1.266

12 5 5 0.01 2 792.6 2.351

13 0.5 0.5 0.1 2 121.5 0.664

14 5 0.5 0.1 2 596.3 1.217

15 0.5 5 0.1 2 568 1.502

16 5 5 0.1 2 788.7 2.195

17 0.5 2.75 0.055 1.1 493.5 1.005

18 5 2.75 0.055 1.1 610.2 2.111

19 2.75 0.5 0.055 1.1 418.9 1.154

20 2.75 5 0.055 1.1 745.7 2.219

21 2.75 2.75 0.01 1.1 719.2 2.416

22 2.75 2.75 0.1 1.1 738.2 2.318

23 2.75 2.75 0.055 0.2 286.1 2.504

24 2.75 2.75 0.055 2 728.4 2.19

25 2.75 2.75 0.055 1.1 716.5 2.411

26 2.75 2.75 0.055 1.1 752.3 2.352

27 2.75 2.75 0.055 1.1 739 2.259

a Real values are expressed in g/L

There is an opposite association between the model and the experimental data which was shown by predicted results against observed ones. All the statistical analysis of the experiments of CCD were listed in Table. 2.

Table. 2: Central composite face (CCF) design matrix for the significant variables and observed and predicted enzyme activity and cell density in E.coli BL21 (DE3)

ActivityObserved

ActivityPredicted

Cell densityObserved

Cell densityPredicted

1 28.1 -26.6669 0.421 0.374609

2 194.3 216.882 1.312 1.385683 203.5 250.421 1.209 1.198464 457.2 395.694 2.39 2.455285 31 -8.54599 0.611 0.5999016 211.3 227.028 1.406 1.397727 231.2 271.416 1.428 1.4928 466 408.715 2.581 2.535579 109.3 168.848 0.529 0.547456

10 611.1 558.172 1.419 1.3587811 571.8 543.361 1.266 1.2780512 792.6 834.41 2.351 2.3351213 121.5 170.294 0.664 0.60249814 596.3 551.643 1.217 1.2005715 568 547.682 1.502 1.4013516 788.7 830.755 2.195 2.2451617 493.5 441.09 1.005 1.1406818 610.2 704.401 2.111 2.0681219 418.9 464.146 1.154 1.2657920 745.7 742.246 2.219 2.2000121 719.2 745.979 2.416 2.3795722 738.2 753.212 2.318 2.4472323 286.1 373.757 2.504 2.4227924 728.4 682.535 2.19 2.3640125 716.5 694.142 2.411 2.2478626 752.3 694.142 2.352 2.2478627 739 694.142 2.259 2.24786

According to the media ingredients, cell density of E.coli BL21 (DE3) was estimated by Eq1, and the enzyme activity estimation of E.coli BL21 (DE3) was measured by Eq2.Yeast extract (X1), Tryptone (X2), K2HPO4 (X3) and CaCl2 (X4)

Eq1: Y1= 2.24786 + 0.467111 X1 + 0.463722 X2 + 0.165537 X4 X4 -0.514963 X1

X1 -0.643463 X2 X2 Eq2: Y3= 694.142 + 139.05 X1 + 131.656 X2 + 154.389 X3 -121.396 X2 X2 -165.996 X3 X3

Results of regression for enzyme activity and cell density in E.coli BL21 confirm the significant (p>0.05) effects of the quadratic model Table. 3, Table. 4. X1 and X2

are significant effective single factors on cell density and enzyme activity. Also, X3

has significant effect on enzyme activity, but all significant interactions have minus effects on both responses.

Table. 3: Statistical analysis and coefficients from the central composite face design experiments for extracellular nattokinase activity in E. coli BL21 (DE3).

Activity Coefficient

Standard Error p value

Constant 694.142 26.482 5.81464e-012

try 131.656 16.9379 5.04332e-006

yea 139.05 16.9379 2.88319e-006

CaC 3.61667 16.9379 0.834503K2H 154.389 16.9379 9.65371e-

007try*try -121.396 44.8135 0.0189932

yea*yea -90.9463 44.8135 0.0651928CaC*CaC 55.4537 44.8135 0.239594

K2H*K2H -165.996 44.8135 0.00301279try*yea -24.5688 17.9654 0.196517

try*CaC -1.99377 17.9654 0.913467try*K2H 36.4438 17.9654 0.0652941yea*CaC 0.718732 17.9654 0.968749yea*K2H 24.3562 17.9654 0.20015

CaC*K2H -4.16874 17.9654 0.820415

R2 =0.964 R2 Adj. =0.922

p value less than 0.05 indicates that the term was significant.

Table. 4: Statistical analysis and coefficients from the central composite face design experiments for cell density of nattokinase in E. coli BL21 (DE3).

Cell density

Coefficient Standard Error p value

Constant 2.24786 0.0432068 1.67141e-015try 0.463722 0.0276352 1.07115e-009

yea 0.467111 0.0276352 9.84773e-010CaC 0.0338333 0.0276352 0.244338K2H -0.0293889 0.0276352 0.308507

try*try -0.643463 0.0731158 1.39792e-006yea*yea -0.514963 0.0731158 1.35047e-005

CaC*CaC 0.165537 0.0731158 0.0428972K2H*K2H 0.145537 0.0731158 0.0698087

try*yea 0.0614374 0.0293115 0.0579535try*CaC -0.0533125 0.0293115 0.0939664try*K2H -0.0499375 0.0293115 0.114175yea*CaC 0.0170625 0.0293115 0.571269yea*K2H -0.0233126 0.0293115 0.441866

CaC*K2H -0.0425625 0.0293115 0.172121R2 =0.987 R2 Adj. = 0.971

p value less than 0.05 indicates that the term was significant.

The R2 value of 0.987 for cell density, and R2 value of 0.964 for enzyme activity, confirms an acceptable model fit that can show more than 96% of the variability (Fig. 5 and 6).

0 100 200 300 400 500 600 700 800 900-200

0

200

400

600

800

1000

f(x) = 0.963894501869819 x + 17.2903922323492R² = 0.963894379109486

obse

rved

act

ivity

(U/m

l)

predicted activity(U/ml)

Fig5: Plot of observed versus predicted values for nattokinase activity in E. coli BL21 (DE3).

0 0.5 1 1.5 2 2.5 30

0.51

1.52

2.53

f(x) = 0.986817797459293 x + 0.0221845660536937R² = 0.986819066189916

obse

rved

(OD

600)

predicted (OD600)

Fig6: Plot of observed versus predicted values for cell density in E. coli BL21 (DE3).

Table5 and Table6 show the results of ANOVA tests for enzyme activity and cell density in E. coli BL21 (DE3), respectively. Collectively these results confirm the correctness of the model.

Table5: ANOVA for the fitted quadratic model for nattokinase activity in E. coli BL21 (DE3).Activity Degree

of FreeSum of Square

Mean Square

(variance)

F-value Probability (P)>F

Total Corrected

26 1.71632e+006 66012.4    

Regression 14 1.65435e+006 118168 22.8827 0.000Residual 12 61968.9 5164.08    

Table6: ANOVA for the fitted quadratic model for cell density in E. coli BL21 (DE3).

Cell density Degree of Free

Sum of Square

Mean Square (variance) F-value Probability(P)>F

Total Corrected 26 12.515 0.481347    

Regression 14 12.3501 0.882148 64.1718 0.000Residual 12 0.16496 0.0137467    

The important goal for nattokinase production is to achieve the most amount of activity, so at the end stage of optimization process the cell density was excluded and maximized enzyme activity was considered as the response. Interactions and optimal points of the variables for achieving optimum nattokinase activity were shown on surface plots in Fig. 7. According to the plots, along with increasing in the concentration of all factors except CaCl2, the increase in enzyme activity was observed, however, the enzyme activity increased in the maximum and minimum amounts of CaCl2. Therefore, the most amount of nattokinase activity that the model was predicted for E. coli BL21 (DE3) was 883.371 U/ml when the media was supplemented with yeast extract (4.3804 g/L), tryptone (3.9899 g/L), K2HPO4

(1.6107 g/L) and CaCl2 (0.01 g/L). Observed enzyme activities with three times repetition in the optimized media were very close to predicted terms.

Fig7: Response surface plot of nattokinase activity by recombinant E. coli BL21 (DE3) showing the effect of (A) yeast extract and tryptone, (B) tryptone and K2HPO4,(C) yeast extract and K2HPO4., (D) Tryptone and CaCl2.

Optimization of IPTG induction and determine the durability of nattokinase activity

Generally, pET expression systems are induced by IPTG, here to achieve maximum activity and determine its durability the concentration of IPTG and time of induction was measured. The maximum activity was obtained by applying 1mM of IPTG. IPTG concentrations less than 1mM had no significant activity, and values higher than 1mM could not increase the activity. The highest obtained activity was 4 hours after IPTG induction, after that time, the activity did not change until 72 hours (Fig. 8). The nattokinase in the supernatant was maintained at 4˚C, and after 30 days it had retained their activity.

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.60

100200300400500600700800900

1000

Act

ivity

(U

/ml)

IPTG (mM)

0 20 40 60 80 100 1200

100200300400500600700800900

1000

Act

ivity

(U

/ml)

Time (hours)B

Fig8: (A) Effects of IPTG concentration and (B) Time of induction and durability for nattokinase activity.

Immobilization of transformant cells

The maximum activity in both supernatants of E.coli strains was less than their maximum activity from media with whole cells. It showed that enzyme could not release to the media completely. Therefore, transformant cells were immobilized by two methods to gain more amounts of enzyme in the media. Immobilized cells in calcium alginate, which was cultivated in optimized media at 37˚C and pH 6.5 with 1Mm IPTG, were stable until 120 hours but after 72 hours the nattokinase activity did not change and needed fresh media. Therefore immobilized cells in calcium, PVA and alginate were not stable at 12 to 24 hours after cultivated cells were gone out from the entrapment. For entrapped transformant cells in calcium

A

alginate every 24 hours the enzyme activity was measured (Fig. 9). The results showed that after 72 hours calcium alginate entrapment could produce active nattokinase with high activity and trace media. According to the immobilization results, for an economical and efficient production of nattokinase batch fermentation of the immobilized transformant cells could be used.

0 20 40 60 80 100 120 1400

200

400

600

800

1000

1200

1400

1600Chart Title

Time (hours)

Fig. 9: Nattokinase activity from both immobilized E.coli BL21 (DE3).

pH and Temperature stabilityThe highest activity of nattokinase was obtained at 55˚C and 60˚C, but was stable up to 50˚C (Fig. 10a). The enzyme was completely stable from pH=4 to 9, and their maximum activities were at pH= 5.5 to 6.5 (Fig. 10b). According to the results the best temperature and pH for the function of the nattokinase, especially at the industrial functions, are 45˚C to 50˚C and pH=5.5 to 7.

25 30 35 40 45 50 55 60 65 70 750

200

400

600

800

1000

1200 activity

Act

ivity

(U

/ml)

Temperature ˚Ca

4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 90

100200300400500600700800900

1000 activity stability

Act

ivity

(U/m

l)

pHb

Fig. 10: Effects of temperature and pH on activity and stability of nattokinase.

Effects of metal ions and chelating agents

By increasing the concentrations of metal ions could increase the nattokinase activity, except Mg2+ which at the 5mM had a maximum activity.1mM of EDTA could increase the enzymes activity, but in the other concentrations the activity was blocked. According to the results (Fig. 11), EDTA as a chelating agent (1mM) envelop by Mg2+ (5mM), Ca2+, Zn2+, Fe2+ and Mn2+ (10mM) showing a great effect on nattokinase activity.

Ca2+ Mg2+ Mn2+ Zn2+ Fe2+ EDTA0

200

400

600

800

1000

1200

1400

1600

1 5 10

Act

ivity

(U/m

l)

Concentration (mM)Fig. 11: Effects of metal ions and EDTA on nattokinase activity.

Detergents effects on nattokinase activity

Some chemicals especially detergents in low concentrations increase the enzyme activity (Handbook of Detergents:Properties- page 674), therefore, SDS as ionic and Tween X-100 as nonionic detergents were tested on activation of nattokinase. The nattokinase activity in the presence of 0.2, 0.5, and 1% (w/v) concentration of Tween-X100 increase, but SDS mostly arrested the activity enzyme (Fig. 12). Therefore, to improve the enzymes activity addition of 0.5% (w/v) of Tween X-100 could be a viable choice.

0.2 0.5 10

200

400

600

800

1000

1200TritonX100 SDS

Act

ivity

(U/m

l)

Concentration (%w/v)

Fig. 12: Effects of detergents on nattokinase activity.

Organic solvents effects on nattokinase activity

There are many investigations on the effects of organic solvents on enzyme activity (Wiggers, Cheleski et al. 2007, Nguyen, Quyen et al. 2013). Therefore, in this study the concentration of 10 and 30% (v/v) of acetone, ethanol, and methanol on the nattokinase was examined. Based on the results methanol do not change the nattokinase activity, however, 10% (v/v) of ethanol, and both 10% and 30% (v/v) of acetone increased the enzymes activity (Fig. 13). According to the results, it is better to add 10% (v/v) of ethanol and acetone for achieving the highest activity.

Methanol Ethanol Acetone0

200

400

600

800

1000

1200

10 30

Act

ivity

(U/m

l)

Concentration (%v/v)

Fig. 13: Effects of organic solvents on nattokinase activity

Nattokinases and subtilisins have the same functions as serine proteolytics, but they are so different in properties, especially in stability, temperature, pH, pI, and facing chemicals and materials (Chantawannakul, Oncharoen et al. 2002, Hwang, Choi et al. 2007, Dubey, Kumar et al. 2011, Wang, Wu et al. 2011). Here, in the

present study, the secretory potent nattokinase was produced by E.coli BL21 (DE3) in an optimized media included of yeast extract and tryptone as the nitrogen and carbon sources, which are essential for cells growth and also included CaCl2 and K2HPO4 as the calcium and phosphate sources which is used for protein secretion in the gram negative bacteria (Pines and Inouye 1999, Mergulhao, Summers et al. 2005, Rosano and Ceccarelli 2014). To save the cells energy, for protein production, immobilizing the transformant cells in calcium alginate the highest activity reached to 1480 U/ml at 72 hours, and can keep the activity for a long time in the supernatant and after purification. The activity of the obtained enzyme was higher than some other studies either on production of non-recombinant nattokinase (Deepak, Kalishwaralal et al. 2008, Wang, Chiu et al. 2009, Berenjian, Mahanama et al. 2014) or recombinant nattokinase production (Ikram, Naz et al. 2009, Nguyen, Quyen et al. 2013). However, some studies on non-recombinant nattokinase gained more activity as compared to the present work (Liu, Xing et al. 2005, Unrean 2017). Therefore, high extracellular production of the potent nattokinase in E.coli omitted renaturation and refolding process of intracellular production (Ni, Guo et al. 2016), and decreased the troubles of B.subtilis secreted proteases (Wang, Du et al. 2009, Gad, Nirmala et al. 2014, Garg and Thorat 2014). Generally, most the nattokinases and subtilisins are stable in different pH and until to 60 ˚C (Fujita, Nomura et al. 1993, Kim, Choi et al. 1996, Weng, Yao et al. 2017), and their activity do not decrease against meatal ions, some detergents and organic solvents (Wang, Du et al. 2009, Wang, Wu et al. 2011, Borah, Shahin et al. 2012). The stability and resistant results of the nattokinase were nearly match to the other nattokinases. However, according to the results, the product of this study could be a promising candiate for medical and industrial applications.

Conclusions

Due to extensive benefits of nattokinase products, such as a potent thrombolytic drug, a dietary supplement for thrombolytic therapy and industrial consumptions as a proteolytic enzyme in detergents, we tried to produce a recombinant nattokinase in a suitable host. The nattokinase from Bacillus subtilis PTTC 1023 was expressed in E.coli BL21 (DE3) extracellularly by pET22b. The results of media optimization showed yeast extract and tryptone are effective on cell growth and CaCl2 and K2HPO4 are effective on enzyme secretion. Therefore, E.coli DE3 (BL21) and pET22b are respectively suitable host and vector for producing secretory

nattokinase. By entrapping transformant E.coli DE3 (BL21) in calcium alginate could obtain the maximum activity with the minimum amount of media. The nattokinase was purified by Ni-NTA native affinity column, and the obtained nattokinase was stable against environmental conditions such as pH, temperature, metal ions, detergent and organic; therefore it could be suggested for industrial applications.

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