Characterization of a Novel Fibrinolytic Enzyme, BsfA, from Bacillus subtilis ZA400 in Kimchi Reveals Its Pertinence to Thrombosis Treatment

Abstract

Recently, the cardiovascular disease has been widely problematic in humans probably due to fibrin formation via the unbalanced Western style diet. Although direct (human plasmin) and indirect methods (plasminogen activators) have been available, bacterial enzyme methods have been studied because of their cheap and mass production. To detect a novel bacterial fibrinolytic enzyme, 111 bacterial strains with fibrinolytic activity were selected from kimchi. Among them, 14 strains were selected because of their stronger activity than 0.02 U of plasmin. Their 16S rRNA sequence analysis revealed that they belong to Bacillus, Leuconostoc, Propionibacterium, Weissella, Staphylococcus, and Bifidobacterium. The strain B. subtilis ZA400, with the highest fibrinolytic activity, was selected and the gene encoding fibrinolytic enzyme (bsfA) was cloned and expressed in the E. coli overexpression system. The purified enzyme was analyzed with SDS-PAGE, western blot, and MALDI-TOF analyses, showing to be 28.4 kDa. Subsequently, the BsfA was characterized to be stable under various stress conditions such as temperature (4-40oC), metal ions (Mn²⁺, Ca²⁺, K²⁺, and Mg²⁺), and inhibitors (EDTA and SDS), suggesting that BsfA could be a good candidate for development of a novel fibrinolytic enzyme for thrombosis treatment and may even be useful as a new bacterial starter for manufacturing functional fermented foods.

Introduction

Thrombus is a known risk factor for cardiovascular disease [27], and it is generally formed via various routes, including fibrinogen, von Willebrand factor, tissue plasminogen activator inhibitor, fibrin D-dimer, etc. [20]. In the United States (2010), 235.5 of 100,000 people are dead owing to cardiovascular disease every year, indicating that it is one of the most popular causes of death by human diseases [6]. In the healthy condition, the homeostatic balance between coagulation and anticoagulation is maintained. However, once this balance is abolished owing to stress or disease, the thrombus is excessively formed, possibly causing the cardiovascular diseases [23]. To remove this thrombus in humans, two types of thrombolysis mechanisms have been identified: the indirect mechanism for bioconversion of plasminogen to plasmin using plasminogen activators, including urokinase, human tissue plasminogen activator (tPA), DSPA α1 from human tissues, and streptokinase from Streptococcus [2]; and the direct mechanism for lysis of thrombus using plasmin and bacterial fibrinolytic enzymes (nattokinase) [19,21]. However, although these plasminogen activators really work for bioconversion of plasminogen to plasmin, they are relatively expensive, cannot be used for oral administration, and could even cause side effect of bleeding in human tissues [31]. To overcome these weak points, bacterial fibrinolytic enzymes have been screened and tested [5,9,14,19]. Among them, the nattokinase from Japanese natto fermented by Bacillus subtilis natto is a well-studied fibrinolytic enzyme [32]. This enzyme can be orally administrated without side effect and can be obtained with cheap production cost using the E. coli overexpression and purification system or mutated nattokinase-producing strains [24,29]. In addition, other bacteria, including Bacillus subtilis, Bacillus amyloliquefaciens, Lactobacillus delbrueckii subsp. bulgaricus, Leuconostoc mesenteroides subsp. mesenteroides, and even Bifidobacterium sp., showed fibrinolytic activity [8]. Furthermore, earlier studies about fibrinolytic enzymes from Korean traditionally fermented foods have been reported. A thermostable fibrinolytic enzyme originated from Staphylococcus sp. strain AJ in an anchovy fermented food was screened and characterized [5]. However, Staphylococcus is generally recognized as a pathogenic microorganism and its fibrinolytic enzyme activity decreases at pH range 6–8. In addition, the other fibrinolytic enzymes from Bacillus sp. CK-11 (70℃) and Bacillus sp. KDO-13 (50℃) showed that the optimum temperatures are 70℃ and 50℃, respectively [13,16], implying that there may be limitation for human treatment. Therefore, these results suggest that a novel fibrinolytic enzyme with optimum fibrinolytic activity in human conditions needs to be developed.

In this study, bacterial strains with strong fibrinolytic activity were screened from the bacterial culture collection containing 1,589 strains isolated from eight Korean traditionally fermented foods containing kimchi, jeotgal, and Korean block of fermented soybean (meju) families [15]. Among them, B. subtilis ZA400 with the strongest fibrinolytic activity was selected. This strain was studied to produce a fibrinolytic enzyme (BsfA) applicable for human applications using the E. coli expression system and to further elucidate its biological characteristics for further application in fermented functional foods. This purified BsfA could be useful for treatment of cardiovascular disease as well as for development of a new-type functional foods with strong fibrinolytic activity.

 

Materials and Methods

Bacterial Strains, Plasmids, Primers, and Growth Conditions

The bacterial strain, plasmids, and primers used in this study are listed in Table 1. E. coli strains were routinely cultivated with shaking at 37℃ in Luria-Bertani (LB) medium (Difco, Detroit, MI, USA). B. subtilis ZA400 was routinely incubated with shaking at 37℃ in de Man-Rogosa-Sharpe (MRS) medium (Difco). Ampicillin sulfate (Sigma Aldrich, St. Louis, MO, USA) and kanamycin sulfate (Carl Roth, Essen, Germany) were added to the growth medium of E. coli for selection at the same concentration of 50 μg/ml (final concentration). The agar medium was prepared with supplementation of 1.8% Bacto agar (Difco). The kimchi sample was purchased from Daedokimchi, Inc. (Kwangju, South Korea).

Table 1.a, Ampr and Kanr, resistance to ampicillin and kanamycin, respectively. Underlined sequences in primers indicate the specific restriction enzyme sites (BamHI and XhoI).

Fibrinolytic Activity Assay

Fibrinolytic activity was basically determined using the standard fibrin plate method developed by Astrup and Müllertz [3]; the standard method was modified for optimal fibrinolytic activity assay as follows: The plasminogen-free fibrinogen solution was prepared with the mixture of 10 ml of 1× PBS buffer (pH 7.4) and 0.3% (w/v) bovine fibrinogen (Sigma Aldrich). In addition to this solution, 20 units of bovine thrombin (Lee biosolution, St. Louis, MO, USA) was rapidly mixed in. The mixture was poured onto a new disposable petri dish and solidified at room temperature for preparation of fibrin plate. On this fibrin plate, 20 μl of each sample was spotted and formation of a clear zone on the spot was visually confirmed. Additionally, 0.02 U of plasmin (Roche, Mannheim, Germany) was used as a positive control.

Cloning and Sequence Analysis of the Gene Encoding BsfA

The total DNA extract of B. subtilis ZA400 was obtained from bead-beating of 200 μl of culture for 30 sec using MiniBeadBeater-16 (BioSpec Products, Bartlesville, OK, USA), according to the manufacturer’s protocol. After brief centrifugation, its supernatant was used as a template DNA for PCR amplification of the specific gene encoding the fibrinolytic enzyme, BsfA. For the PCR, ZA400-F and ZA400-R (Table 1) were designed and chemically synthesized, based on the DNA sequence of the aprN gene encoding subtilisin in B. subtilis LSSE-22 (GenBank Accession No. JN392072.1). The reaction mixture (final volume, 25 μl) consisted of 1 μl of DNA template from the bead-beat supernatant, 0.6 μM of each primer set, 0.2mM of dNTP mix (Toyobo, Osaka, Japan), 25mM of MgSO4, and 0.5 U of KOD–plus– DNA polymerase (Toyobo). The PCR conditions were as follows: 1 cycle of 94℃ for 2 min, 20 cycles of 94℃ for 15 sec, at a specific annealing temperature of each primer set for 40 sec, 68℃ for 1 min 20 sec and 1 cycle of 68℃ for 5 min. The PCR product was purified using an AxyPrep DNA Gel Extraction Kit (Axygen, Tewksbury, MA, USA). The purified PCR product was A-tailed by Taq DNA polymerase (New England Biolabs (NEB), Ipswich, MA, USA) with 0.2 mM dATP at 72℃ for 20 min. The final PCR product was ligated into pGEM-T Easy Vector System (Promega, Madison, WI, USA) and the cloned vector (pGEM T-ZA400) was recovered from the selected colonies using LB agar medium containing 50 μg/ml ampicillin (final concentration). Then, the cloned PCR insert was sequenced. The DNA sequence of the cloned PCR insert was analyzed by the BLASTN program [1].

Construction of Expression Vectors and Overexpression of bsfA Gene

To construct the expression vector, the insert DNA containing gene bsfA was obtained from the pGEM T-ZA400 cloning vector by BamHI/XhoI double digestion and cloned into the pET26b(+) vector (Novagen, Madison, WI, USA), resulting in pET26b-ZA400. The constructed expression vector pET26b-ZA400 was introduced into E. coli BL21(DE3)pLysS (Novagen) using the standard heat-shock transformation method [7]. When the optical density at 600 nm wavelength (OD600nm) of the culture reached to 0.6, 1 mM of IPTG was added for bsfA gene expression.

Total RNA Isolation and Quantitative Real-Time PCR

After bsfA gene expression using IPTG, the culture of B. subtilis ZA400 (OD600nm = 0.9) was obtained and treated with RNA protect solution (Qiagen, Valencia, CA, USA) for protection of total RNA degradation. Total RNA was isolated and purified using the RiboPure Bacteria kit (Life Technology, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Primers and a probe for TaqMan real-time PCR targeting the bsfA gene were designed using the GenScript Real-Time PCR (TaqMan) Primer Design site (https://www.genscript.com/ssl-bin/app/primer) (Table 1). All real-time PCRs were performed using a CFX Connect Real-Time PCR Detection System (BioRad) with a TaqMan PrimeScript reverse transcriptase (Takara, Shiga, Japan) and Prime Ex Taq (Takara). The composition of the Real- Time PCR mixture was according to the manufacturer’s instructions. The Real-Time PCR condition for the bsfA-F/bsfA-R primer set and bsfA-probe was as follows: 1 cycle of 48℃ for 15 min, 1 cycle of 95℃ for 30 sec, 40 cycles of 95℃ for 5 sec and 57.5℃ for 33 sec. The collected data were statistically analyzed using the Microsoft Excel program (Microsoft, Redmond, WA, USA).

Production and Purification of BsfA

After IPTG induction, BsfA enzyme containing His·tag was produced in the culture supernatant. Therefore, the supernatant was collected and concentrated with polyethylene glycol (PEG) 6000 (Samchun, Seoul, Korea) and Spectra/Por 4 Dialysis Tubing (12-14 kDa) (Spectrum Labs, Laguna Hills, CA, USA). The BsfA enzyme in the concentrated supernatant was purified using the HisPur Ni-NTA Chromatography Cartridge (Thermo Scientific, Rockford, IL, USA) and concentrated again with Amicon Ultra-4 (EMD Millipore, Billerica, MA, USA). The fibrinolytic activity of purified BsfA was determined using the modified fibrin plate method previously used in this study.

SDS-PAGE, Western Blot, and MALDI-TOF Analyses

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a precast-TGX gel (Bio-Rad, Hercules, CA, USA) was conducted to fractionate the protein samples, and the bands were transferred onto a polyvinylidene difluoride membrane (Bio-Rad). For detection of specific protein band, western blot analysis was performed using His·Tag AP Western Reagents (Novagen, Madison, WI, USA) and His·Tag Monoclonal Antibody (Novagen). To confirm the purity and the molecular mass of the produced BsfA enzyme, matrix-assisted laser desorption and ionization-time of flight (MALDI-TOF) was conducted by the Korea Basic Science Institute (KBSI, Seoul, Korea).

Stress Stability Test of BsfA

For determination of the stability of BsfA fibrinolytic activity, it was treated under various stress conditions such as temperature (4℃, 20℃, 30℃, 37℃, 40℃, 50℃, 60℃, and 70℃ for 30 min) and pH (2.0, 4.0, 6.0, 7.0, 8.0, 10.0, and 12.0 at 37℃ for 30min) according to Kim’s method [12]. In addition, it was also treated to various inhibitors (EDTA and SDS) and 5 mM metal ions (CoCl2, CuSO4, CaCl2, MnCl2, KCl, and MgCl2) at 37℃ for 30 min [9]. After stress treatment, the BsfA fibrinolytic activity in each specific condition was determined using the modified fibrin plate method previously used in this study.

 

Results

Isolation and Selection of B. subtilis ZA400

A total of 204 strains associated with kimchi fermentation were isolated from the purchased kimchi sample according to the BAM of US FDA (http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm). Subsequent fibrinolytic activity assay revealed that 111 strains showed fibrin lysis. Among them, 14 strains showed stronger fibrin lysis activity than that of 0.02 U of plasmin, and the strain ZA400 was selected for further characterization study of its fibrinolytic enzyme (Fig. 1). The 16S rRNA sequence analysis confirmed that this strain belongs to Bacillus subtilis. In addition, morphological observation revealed that it has typical morphology of B. subtilis-like rod shape, supporting this identification.

Fig. 1.Fibrinolytic activity assay of the culture supernatants of 14 selected bacteria from kimchi.

Cloning and Sequence Analysis of bsfA Gene

Because the complete genome sequence of B. subtilis ZA400 was not available at this time, the B. subtilis LSSE-22 aprN gene-based PCR primer set (ZA400-F/ZA400-R) was used for PCR amplification of gene bsfA, encoding the fibrinolytic enzyme (Fig. 2). DNA sequence analysis of the 1.2 kb PCR product showed it had 1,155 bp with the start codon ATG. Further sequence analysis of the bsfA gene revealed that the deduced protein of the ORF contained 381 amino acids, and this gene contained a signal peptide (26 amino acids; a.a.), a propeptide (80 a.a.), and a mature enzyme (275 a.a.) (Fig. 3). The cloning vector pGEM T-ZA400 with 4,017 bp length containing the bsfA gene was constructed with the pGEM-T Easy vector (Fig. 2). The insert DNA including bsfA gene was obtained by double restriction enzyme digestion with BamHI and XhoI with pGEM T-ZA400 and purified using the agarose gel extraction method for further construction of the bsfA gene expression vector (Fig. 2).

Fig. 2.Construction of expression vector pET26b-ZA400 for production of BsfA enzyme.

Fig. 3.Conformation of fibrinolytic enzyme activity of the purified BsfA enzyme.

Construction of Expression Vector and Gene Expression of bsfA Gene

The purified insert DNA including the bsfA gene was cloned into the pET26b(+) gene expression vector, resulting in pET26b-ZA400 for production of the BsfA enzyme in the E. coli BL21(DE3)pLysS host system (Fig. 2). Interestingly, this pET26b(+) vector has a pelB leader sequence just before the multiple cloning site for extracellular secretion of the produced protein from the cloned gene [17]. Therefore, this pET26b-ZA400 can express the bsfA gene with IPTG induction to produce BsfA fibrinolytic enzyme, and the produced BsfA can be secreted to the culture medium without formation of any inclusion body. Surprisingly, the fibrinolytic activity assay showed that only the culture medium had the activity to lyse fibrin, indicating that the BsfA enzyme is secreted to the culture medium after transcription and translation of the bsfA gene (data not shown).

The E. coli BL21(DE3)pLysS host was used for production of BsfA enzyme via IPTG induction. The advantage of this host system is tight repression of specific gene expression in the insert DNA without IPTG [22]. To confirm the efficiency of gene expression in this E. coli host system, real-time PCR using the TaqMan probe was used for monitoring of expression of gene bsfA with or without IPTG. As previously explained, expression of gene bsfA was tightly repressed without IPTG but was highly induced with IPTG (Fig. 3). The IPTG-induced E. coli expression host expressed the bsfA gene up to 132-fold compared with expression of the bsfA gene in the original B. subtilis ZA400 (Fig. 4). This tight regulation of fibrinolytic enzyme gene expression in E. coli may be a key point, because only one successful result was reported for production of fibrinolytic enzyme using the same host system, E. coli BL21(DE3)pLysS [17]. Furthermore, the bsfA gene expression using pET26b-ZA400 in E. coli BL21(DE3) was not successful, supporting this.

Fig. 4.Comparison of expression levels of the bsfA gene encoding BsfA enzyme, using quantitative real-time PCR.

Production, Purification, and Concentration

Production of BsfA fibrinolytic enzyme using pET26b-ZA400 in the E. coli BL21(DE3)pLysS host system was successful. For purification and concentration of the BsfA enzyme containing His·tag, three purification and concentration steps were conducted: PEG concentration, Ni-NTA chromatography purification, and Amicon concentration. After these purification/concentration steps, one arbitrary unit (1 AU) of the BsfA enzyme was defined to the same fibrinolytic activity of 1 U of plasmin. In each step, the amount and the arbitrary unit of BsfA enzyme was monitored (Table 2). After the final step, only 0.02% of total proteins was recovered and the specific activity increased from 0.02 AU/mg to 4.48 AU/mg, indicating that the BsfA enzyme was successfully concentrated and purified (Table 2). This purified BsfA enzyme was used for further characterization studies.

Table 2.a, AU, arbitrary unit. 1 AU of BsfA enzyme was defined to the same fibrinolytic activity as with 1 U of human plasmin.

Analyses of SDS-PAGE, Western Blot, and MALDI-TOF

To confirm the production of BsfA fibrinolytic enzyme from the E. coli expression and secretion system, SDS-PAGE (for protein size), western blot (for immune-detection), and MALDI-TOF (for protein size and purification) analyses were performed. SDS-PAGE analysis showed a 28.4 kDa size band, which was identical to the predicted protein size of original BsfA enzyme (Fig. 5A). For further confirmation of BsfA enzyme produced from E. coli system, western blot analysis with the commercial His·Tag monoclonal antibody was conducted and the result showed the 28.4 kDa size band (Fig. 5B). In addition, MALDI-TOF analysis revealed that only one apparent peak with the 28.4 kDa molecular weight was present, confirming that it is the BsfA enzyme (Fig. 6). These three different confirmation experiments substantiated that the BsfA fibrinolytic enzyme was indeed produced from the E. coli expression and secretion system.

Fig. 5.Confirmation of BsfA enzyme production using SDS-PAGE (A) and western blot (B) analyses.

Fig. 6.Confirmation of production and purification of the BsfA enzyme, using MALDI-TOF.

Fibrin Degradation Analysis of BsfA and Plasmin

To determine the fibrinolysis efficiency of BsfA enzyme, it was compared with human plasmin. After fibrin lysis by BsfA and plasmin, the degraded samples were analyzed using SDS-PAGE (Figs. 7A and 7B). Interestingly, BsfA had higher activity to break down the γ-bond and γ- γ bond in the fibrin. The structure of fibrin indicates that the γ-bond or γ-γ bond is important for formation of fibrin clot [25]. Therefore, this strong degradation activity of BsfA is more important to remove fibrin clot to protect from cardiovascular disease.

Fig. 7.Comparative fibrin degradation analysis of human plasmin (A) and BsfA enzyme (B).

Stability of BsfA Fibrinolytic Activity Under Various Temperatures, pH, Metal Ions, and Protease Inhibitors

For various industrial production and applications of BsfA enzyme, it should be stable under various stress conditions. To evaluate this enzyme for industrial applications, the stability of BsfA fibrinolytic activity was tested under various temperatures, pH, metal ions, and protease inhibitors. Interestingly, BsfA enzyme was stable in the wide range of temperature (4℃ to 40℃) (Fig. 8A) and pH conditions (pH 6.0 to 10.0) (Fig. 8B). The optimum temperature and pH of the BsfA enzyme was 30-37℃ and pH 6.0. Interestingly, this optimum temperature of BsfA is quite different from optimum temperatures of the previously reported fibrinolytic enzymes from Bacillus sp. CK-11 (70℃) [13] and Bacillus sp. KDO-13 (50℃) [16], but Bacillus sp. KA38 has a similar optimum temperature (40℃) [10] to that of BsfA, suggesting that BsfA is better for human applications.

Fig. 8.Stress stability evaluation of the BsfA enzyme under various temperatures (A), pH (B), and protease inhibitors and metal ions (C).

In addition to conditions of temperature and pH, the protease inhibitors/metal ions study is also required for applications of BsfA in an optimum condition. The inhibition study of BsfA using metal ions revealed that the fibrinolytic activity of BsfA enzyme was highly inhibited by Cu2+ and Zn2+ ions, but not by other metal ions (Ca2+, Mn2+, Zn2+, Mg2+, and K+). In addition, protease inhibitors (SDS and EDTA) only slightly inhibited the activity, suggesting that the BsfA enzyme is generally stable against many metal ions and protease inhibitors (Fig. 8C).

 

Discussion

Whereas plasmin is a natural fibrin lysis enzyme, which is converted from plasminogen by plasminogen activators such as urokinase and streptokinase [2], BsfA is a bacterial fibrinolytic enzyme, which was characterized in this study. Although most bacterial fibrinolytic enzymes have been detected and isolated from the soy-fermentation-associated bacteria [11,26,30], the BsfA enzyme produced by B. subtilis ZA400 is a fibrinolytic enzyme derived from kimchi. This fibrinolytic enzyme was obtained by several experimental steps; cloning and expression of the bsfA gene in the E. coli expression system, concentration and purification of BsfA enzyme, and confirmation of the enzyme using SDS-PAGE, western blot, and MALDI-TOF analyses. Interestingly, this BsfA enzyme is highly stable under various stress conditions, such as temperature (4-40℃), pH (6-10), metal ions (Ca2+, Mn2+, Zn2+, Mg2+, and K+), and protease inhibitors (SDS and EDTA), suggesting that this fibrinolytic enzyme may be a good candidate for human applications.

This bsfA gene consists of a signal peptide sequence, propeptide sequence, and mature enzyme sequence (Fig. 9). The signal peptide and propeptide are generally associated with protein secretion and regulation of the protein activity in the host strain before secretion, respectively [18]. During secretion of the protein, the signal peptide and propeptide are normally removed by the host protease, which is the so-called post-translational modification [4]. However, expression of the bsfA gene and production of the BsfA enzyme were conducted in the E. coli expression and secretion system, not in the B. subtilis system. Because the BsfA enzyme was successfully produced with fibrinolytic activity in the E. coli expression and secretion system, this system should have the post-translational modification mechanism to mature the BsfA enzyme during secretion. However, the E. coli pET26b(+) expression vector system does not have a specific protease gene for maturation of BsfA enzyme via this post-translational modification, suggesting that the post-translational modification system in this E. coli host may be compatible to the B. subtilis post-translational modification system for maturation of BsfA enzyme. Prior to successful production of the BsfA enzyme in the E. coli pET26b(+) expression and secretion system, the E. coli pET21a expression system was used for production of the BsfA enzyme. Before cloning of the bsfA gene into this expression vector, it was cloned into vector pET21a without protein secretion, resulting in pET21-ZA400. However, the fibrinolytic activity assay of the produced BsfA enzyme in the cell extract showed no activity for fibrin lysis (data not shown). Even although the bsfA gene was successfully expressed by IPTG, the BsfA enzyme probably still has a signal peptide and propeptide, resulting in no fibrinolytic activity. Therefore, production of BsfA enzyme from expression of the bsfA gene containing signal peptide and propeptide sequences may have to be conducted with a bacterial expression and secretion system with a post-translational modification system for protein maturation.

Fig. 9.Nucleotide sequence of the cloned bsfA gene region in the pET26b-ZA400 expression vector.

As mentioned previously, the BsfA enzyme has optimum conditions (37℃, pH 6.0, and even various metal ions) that are similar to human conditions (Figs. 8A-8C). However, some fibrinolytic enzymes favor the acidic condition (50℃) for optimum activity [13,16], suggesting that the BsfA enzyme may be useful for human trials. In addition, B. subtilis ZA400 is a food-fermenting bacterium, so it could be used as a functional food additive for improving the fibrinolysis function of fermented foods. In addition, the BsfA enzyme has strong fibrinolytic activity by breakdown of the γ-bond or γ- γ bond of fibrin, because these bonds are important to maintain the structure of fibrin [28]. However, human plasmin has weak lysis activity of the γ-bond or γ- γ bond but it could break down α- or β-bonds, suggesting that the BsfA enzyme may be better for efficient fibrin lysis in humans (Figs. 7A and 7B).

In this study, a novel fibrinolytic enzyme, BsfA, was produced and characterized using the E. coli expression and secretion system. This BsfA enzyme and its production host would be useful for direct application for thrombosis treatment using the purified enzyme, or for indirect application for development of functionally improved fermented foods for thrombosis alleviation using the strain ZA400.