J. Gen. Appl. Microbiol., 61, 241–247 (2015)doi 10.2323/jgam.61.2412015 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation

Full Paper

*Corresponding author: Palaniyandi Velusamy, Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Chennai-603 203, Tamil Nadu, India.Tel: +91-8939731191 Fax: + 91-442357824 E-mail: velusamy.p@ktr.srmuniv.ac.in

None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriatelyinfluence their work.

ease, blood clotting, peripheral vascular disease, and cer-ebrovascular disease (Lim, 2013). Among these diseases,blood clotting is an emerging problem that seriously threat-ens the health of human beings. There are more than twentyenzymes in the body that assist in the clotting of blood,while there is only one capable of breaking clots down.Fibrin is an important protein constituent of blood clotsthat is normally formed from fibrinogen via the action ofthrombin (EC. 3.4.21.5) following injury (Wolberg, 2007).Therefore, the accumulation of fibrin in blood vessels usu-ally results in thrombosis, leading to myocardial infarc-tion and other cardiovascular diseases (Collen and Lijnen,1991). Owing to the increased incidence of thrombosis,the search for novel thrombolytic agents is of the utmostimportance.

A variety of thrombolytic agents, including tissue plas-minogen activator (t-PA) urokinase (EC 3.4.21.73), strep-tokinase (EC 3.4.99.0), and staphylokinase (EC 3.4.99.22),have been investigated (He et al., 2008). Fibrinolytic en-zymes have attracted attention as thrombolytic agentsowing to their widespread applications. Several studieshave demonstrated that fibrinolytic enzymes can be ob-tained from different sources, including animals (Geng etal., 2014), plants (Siritapetawee et al., 2012), foods(Montriwong et al., 2012), and microbes (Huang et al.,2013). Soils have been extensively investigated for thepresence of microorganisms that produce useful biologi-cally-active molecules. However, soil microorganismshave only recently been investigated as potential bio-fac-tories for the synthesis of various enzymes. Currently, useof bacteria in this field is rapidly gaining importance ow-ing to a growing success, ease of handling and possibili-ties for genetic modification. Enzymes produced by soilbacteria can provide numerous advantages over traditional

Isolation and identification of a novel fibrinolytic Bacillus tequilensis CWD-67from dumping soils enriched with poultry wastes

(Received December 26, 2014; Accepted August 18, 2015)

Palaniyandi Velusamy,1,∗ Raman Pachaiappan,1 Meera Christopher,1 Baskaralingam Vaseeharan,2

Periasamy Anbu,3 and Jae-Seong So3

1 Department of Biotechnology, School of Bioengineering, SRM University, Chennai-603 203, Tamil Nadu, India2 Department of Animal Health and Management, Alagappa University, Karaikudi-630 003, Tamil Nadu, India

3 Department of Biological Engineering, Inha University, Incheon 402-751, South Korea

A newly isolated strain, CWD-67, which exhib-ited high fibrinolytic activity, was screened fromdumping soils enriched with poultry wastes. Thestrain was identified as Bacillus tequilensis(KF897935) by 16Sr RNA gene sequence analysisand biochemical characterization. A fibrinolytic en-zyme was purified to homogeneity from the culturesupernatant using ammonium sulfate precipitation,membrane concentration, dialysis, ion-exchange,and gel filtration chromatography. SDS-PAGEanalysis showed that the purified enzyme was amonomeric protein with an apparent molecularweight of 22 kDa, which is the lowest among Bacil-lus fibrinolytic enzymes reported to date. The pu-rified enzyme was confirmed to have fibrinolyticactivity by a fibrin zymogram. The optimal pH andtemperature values of the enzyme were 8.0 and45°C, respectively. The enzyme was completely in-hibited by PMSF and significantly inhibited byEDTA, TPCK, Co2+, Zn2+, and Cu2+, suggesting achymotrypsin-like serine metalloprotease. In vitroassays revealed that the purified enzyme couldcatalyze fibrin lysis effectively, indicating that thisenzyme could be a useful fibrinolytic agent.

Key Words: Bacillus tequilensis; fibrinolytic bac-teria; serine metalloprotease; zymogram

Introduction

There has recently been increased attention to life-threat-ening cardiovascular diseases, such as coronary heart dis-

242 VELUSAMY et al.

enzymes owing to the wide range of environments fromwhich they are recovered (Mander et al., 2011).

The first fibrinolytic enzyme isolated from Bacillusnatto, nattokinase (EC 3.4.21.62), has been widely appliedas a thrombolytic agent (Cao et al., 2009). Additionally,Pseudomonas sp. TKU015 (Wang et al., 2009), B. subtilis(Chang et al., 2012), and Streptomyces sp. CS624 (Manderet al., 2011) have recently been reported to synthesize fi-brinolytic enzymes. Enzyme synthesis methods can becategorized into intracellular and extracellular synthesisaccording to the place at which the enzymes are formed.Investigations of extracellular enzyme synthesis are cur-rently being conducted to elucidate the mechanisms ofsynthesis to enable easy downstream processing and rapidscale-up processing. For these reasons, bacterial systemshave the potential for use in the extracellular synthesis ofsuch enzymes. Here, we report a newly isolated Bacillustequilensis CWD-64 from dumping soils that possess fi-brinolytic activity. Further, extraction, purification, andcharacterization of chymotrypsin-like serinemetalloprotease from the culture supernatant of B.tequilensis CWD-64 are reported, and their fibrinolyticactivity is described.

Materials and Methods

Reagents and media. Culture media was purchased fromHiMedia (HiMedia Ltd., Mumbai, India). Fibrinogen,thrombin, and plasmin were procured from Sigma (SigmaAldrich, CA, USA). Sephadex G-75 FF was purchasedfrom GE Healthcare (GE Healthcare BioSciences AB,Uppsala, Sweden). DEAE-Cellulose FF was acquired fromGE (GE Healthcare Life Sciences, UK). Phenyl methylsulfony fluorides (PMSF), ethylene diamine tetraaceticacid (EDTA), and N-α-tosyl-L-phenylalanine chloromethylketone (TPCK) were obtained from Merck (Merck,Mumbai, India). Other reagents and chemicals used wereof analytical grade.Isolation and screening of bacteria. Samples were col-lected from dumping soils enriched with broiler wastescontaining muscles, blood, and skin, from different sitesin Potheri, Chennai, India. The soils were serially dilutedwith sterile water until a dilution containing 106 coloniesforming units (CFU) g–1 of soil was obtained, after whichthey were inoculated onto skim milk agar plates and incu-bated at 30°C for 24 h. Strains exhibiting a clear zone (deg-radation of casein) around the colony were selected andsub-cultured several times on nutrient agar medium untilpurity, after which they were stored until further analysis.Fibrin plate assay. Fibrinolytic activity was investigatedby a fibrin plate assay as previously described (Astrup andMullertz, 1952), with minor modifications. The fibrinagarose gel (5-mm thick) contained 2.0% agarose, 0.12%(w/v) fibrinogen, 0.5 U/ml thrombin and 0.1 M phosphatebuffer (pH 7.4) containing 0.15 M NaCl. The clot was al-lowed to set for 30 min at room temperature, after whichcell-free bacterial culture supernatant of the selected pro-teolytic strain was carefully loaded onto the fibrin agarplate. Un-inoculated broth medium was used as a nega-tive control. The loaded plates were incubated at 37°C for24 h, after which the clear zones around the paper discs

indicating fibrinolytic activity were measured.Bacterial identification. To identify the bacterium, apolymerase chain reaction (PCR) was conducted to am-plify the 16S rRNA gene from the genomic DNA of se-lected fibrinolytic strains as previously described(Weisburg et al., 1991) using the following primers: for-ward 5′-TGGCTCAGAACGAACGCTGGCGGC-3′, re-verse 5′-CCCACTGCCTCCCGTAAGGAGT-3′. The tem-perature cycle for the reaction consisted of 30 cycles of94°C for 30 s, 55°C for 1 min, and 72°C for 1 min 30 sfollowed by final extension for 5 min at 72°C. The PCRproduct was cloned into pGEM-T easy vector (Promega,Madison, WI, USA), after which the nucleotide sequenceof the 16S rRNA gene was sequenced using an ABI Prism377 DNA sequencer (PE Applied Biosystems, Foster City,CA, USA). The obtained sequence was then subjected toa BLAST search of the GenBank database (NCBI,Bethesda, MD, USA) to identify the bacterial isolates.Identification of the strain was confirmed by biochemicaltests according to Bergey’s Manual of Systemic Bacteri-ology.Purification of extracellular enzyme. The selected bacte-rial strain was cultured in nutrient broth medium (pep-tone, 5 g/L; beef extract, 1.5 g/L; yeast extract, 1.5 g/L;sodium chloride, 5 g/L) and incubated at 30°C in a shaker(180 rpm) for 96 h under aerobic conditions. Cell-freesupernatants were then collected by centrifugation at 8,000rpm for 20 min, after which ammonium sulfate was addedto the supernatant at 50% saturation and the mixture wasincubated overnight. The precipitate was then centrifugedat 8,000 rpm for 20 min, after which the pellet wasresuspended in 50 mM Tris-HCl buffer (pH 8.0) anddialyzed against the same buffer overnight. Next, the dia-lysate was loaded onto a DEAE-Cellulose column (2.4 ×45 cm) that had previously been equilibrated with Tris-HCl at pH 8.0 by fast protein liquid chromatography(FPLC Amersham Bioscience). Elution with a linear gra-dient of 0.0 to 1.0 M NaCl resulted in a single peak offibrinolytic activity. Peaks that exhibited higher fibrino-lytic activity were pooled, concentrated and then polishedusing a Sephadex G-75 FF column (1.5 × 25 cm) that hadbeen equilibrated with Tris-HCl at pH 8.0 by FPLC. Allfractions were collected separately and the absorbance at280 nm was measured with a spectrophotometer. All puri-fication steps were conducted at 4°C.Assay of enzyme activity. Enzyme activity was estimatedby measurement of the acid-soluble material released fromazocasein (Huang et al., 2013) using the following buffersystems: 0.1 M glycine-HCl (pH 3.0), 0.1 M disodiumhydrogen phosphate-sodium citrate (pH 4.0–7.0), 0.1 MTris-HCl (pH 8.0–9.0), 0.1 M glycine-NaOH (pH 10.0),0.1 M disodium hydrogen phosphate-NaOH (pH 11.0), and0.1 M KCl-NaOH (pH 12.0–13.0). The reaction mixture,which was composed of 0.6 mL of 1% (w/v) azocaseinsolution and a 20-µL enzyme sample, was incubated at37°C for 20 min, after which it was supplemented with1.2 mL of 10% (w/v) trichloroacetic acid. Next, the sam-ple was placed on ice for 10 min, then centrifuged at 4°Cand 12,000 rpm for 10 min. The concentration of acid-soluble material in the supernatant was measured basedon the absorbance at 360 nm. One unit of protease activ-

Fibrinolytic enzyme from Bacillus tequilensis 243

ity was defined as the amount of enzyme needed to in-crease the absorbance at 360 nm by 0.1 units.SDS-PAGE and fibrin zymography. The molecular weightof the purified enzyme was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)as described by Laemmli (1970) using 5% (w/v) stackingand 12% (w/v) polyacrylamide separating gels. Follow-ing electrophoresis, the gel was stained with CoomassieBrilliant Blue (CBB) R-250, then destained with a metha-nol: glacial acetic acid: distilled water solution (4:1:5 byvolume). A low-range protein marker kit (Genei, India)was used for calibration. Fibrin zymography was con-ducted as described by Choi and Kim (2000) with minormodification. Briefly, a separating gel (12% w/v) was pre-pared in the presence of 0.12% fibrinogen (w/v) and 150µL of thrombin (100 U/mL). The enzyme samples weresubsequently mixed in a sample buffer, loaded onto amodified fibrin gel and subjected to electrophoresis usinga Mini-Protean II system (BioRad Laboratories, Hercules,CA, USA). Electrophoresis was conducted at 100 V atroom temperature for 1.5 h. Subsequently, the gel wassoaked in 2.5% Triton X-100 containing Tris-HCl (50 mM)buffer (pH 7.4) for 30 min at room temperature. Afterwashing in distilled water for 30 min, the gel was incu-bated in 30 mM Tris-HCl buffer (pH 7.4) containing 200mM NaCl, 10 mM CaCl2, and 0.02% NaN3 at 37°C for 16h. Finally, the gel was stained with CBB-R250 for 30 minand then destained.Effect of pH and temperature on enzyme activity. The ef-fect of pH on fibrinolytic activity was assayed from pH2.0–11.0 using several buffer systems with the followingpH ranges: 100 mM glycine-HCl buffer (pH 2.0–3.0), 50mM sodium acetate buffer (pH 4.0–6.0), 100 mM Tris-HCl (pH 7.0–9.0), and 100 mM sodium hydroxide buffer(pH 10.0–11.0). The reaction mixtures were incubated for1 h at 37°C, after which the enzyme activities were mea-sured by the fibrin plate method. The maximum activitywas represented as 100%. Similarly, the effect of tempera-ture on fibrinolytic activity was measured from 20 to 60°Cfor 60 min.Effect of inhibitors and metal ions on enzyme activity.Several enzyme inhibitors including PMSF, Bestatin A,Pepstatin, EDTA, and TPCK were used to characterize thepurified fibrinolytic enzyme. Purified enzyme (2 µg) wasincubated in 50 mM Tris-HCl buffer (pH 8.0) with vari-ous enzyme inhibitors for 1 h. To investigate the effect ofmetal ions on enzyme activity, the following metal ionswere used (5 mM concentration): Ca2+, Co2+, Mg2+, Zn2+,Cu2+, and Fe2+. The samples were incubated for 1 h withvarious metal ions and the residual enzyme activity wasdetermined.Fibrinolytic activity of purified enzyme. Quantitativeanalyses of fibrinolytic activity were conducted by fibrinagar plate assay, with minor modification (Astrup andMullertz, 1952). Briefly, 15 mL of 0.8 mg/mL bovine fi-brinogen solution (10 mM Tris-HCl buffer, pH 7.4) wasmixed with 1 mL of thrombin solution (7.5 U/mL) and 20mL of 1.0% agarose solution in a Petri dish. Next, 10 µlof the enzyme sample was placed in a paper disc (7 mm indiameter). The loaded plate was incubated at 37°C for 24h and then stained with 0.1% CBB R-250, 40% methanol,

and 10% acetic acid for 1 h, after which it was destainedin 25% ethanol and 10% acetic acid. After measuring thediameter of the clear zone, the units (U) of the enzymeactivities were determined based on comparison with astandard curve generated using urokinase.

Results and Discussion

Isolation and identification of fibrinolytic bacteriumA total of 23 soil samples were collected from broiler

waste dumping sites. Pilot scale screening revealed 137strains that exhibited proteolytic activity on skim milk agarmedia (Fig. 1a). Among these, 12 were able to degradefibrin in a fibrin agarose medium after 24 h of incubationusing culture supernatant. Strain CWD-67, which exhib-ited the maximum halo zone (4.3 cm in diameter) aroundthe colony, was selected from among the 12 positive strains(Fig. 1b). Biochemical characterization revealed that strainCWD-67 was Gram-positive, endospore-forming, motile,and round in shape. In addition, the bacterium was cata-lase and oxidase positive and capable of growth at 20°Cto 40°C, with optimum growth occurring at 30°C. Thegenomic DNA of strain CWD-67 was amplified using uni-versal primers, and the 16S rRNA gene sequence wasanalyzed. Alignment of the sequence (819 bp) of strainCWD-67 with similar 16S rRNA gene sequences in theGenBank database revealed a high similarity (99%) toBacillus tequilensis. Based on these results, strain CWD-67 was identified as a member of Bacillus tequilensis, anddesignated as B. tequilensis CWD-67 (GenBank accessionnumber KF897935). Other Bacillus species have beenshown to produce fibrinolytic enzymes. For example, Ba-cillus natto is a well-known fibrinolytic bacteria isolatedfrom natto, a Japanese fermented soybean. Natto has beenextensively studied and shown to produce a strong fibri-nolytic enzyme known as nattokinase, which is currentlyused as a natural alternative blood thinner and blood clotdissolver (Cao et al., 2009; He et al., 2008). Many otherstudies have reported strains of bacilli that produce fibrino-lytic activity, including Bacillus subtilis HQS-3 and Ba-cillus amyloliquefaciens DC-4 isolated from douchi, a tra-ditional Chinese soybean (Huang et al., 2013; Peng et al.,2003), Bacillus sp. KA38 isolated from the Korean saltyfermented fish, jeot-gal (Kim et al., 1997), and Bacillussp. CK11-4 from the Korean fermented-soybean sauce,Chungkook-Jang (Kim et al., 1996). However, to the best

Fig. 1. Screening of proteolytic bacteria on skim milk agar medium at30°C for 24 h (a), and clear zone due to hydrolysis of fibrin bythe culture supernatant of B. tequilensis CWD-64 (b).

244 VELUSAMY et al.

of our knowledge, this is the first report of the isolationand identification of B. tequilensis with fibrinolytic ac-tivity from broiler waste soils.

Purification of extracellular fibrinolytic enzymeFibrinolytic enzyme was purified from a culture

supernatant of B. tequilensis CWD-67 using ammoniumsulfate precipitation followed by DEAE-Cellulose andSephadex G-75 FF chromatography. Following ammoniumsulfate precipitation, the dialyzed sample exhibited fibri-nolytic enzyme activity. DEAE-Cellulose chromatographyyielded several protein peaks that could be detected at 280nm. The active fraction of fibrinolytic peak was collectedand applied to a Sephadex G-75 FF column that had beenequilibrated with 50 mM Tris-HCl buffer (pH 8.0) at aflow rate of 0.5 ml/min. Only one protein peak with fi-brinolytic activity could be purified (Fig. 2). Based on thepurification scheme, the fibrinolytic enzyme produced byB. tequilensis CWD-67 was purified 34.7-fold with anoverall yield and specific activity of 8.2% and 58.5 units/mg, respectively (Table 1).

SDS-fibrin zymogramSDS-PAGE and fibrin-zymography of the purified en-

zyme were conducted to verify the enzyme purity and es-timate its molecular mass. The molecular weight of thepurified enzyme was estimated to be 22 by SDS-PAGEusing a standard marker (Genei, India). Fibrin-zymographyindicated that the protein purified from the culturesupernatant of B. tequilensis CWD-67 showed fibrinolyticenzyme activity (Fig. 3). Taken together, these results in-

dicated that the enzyme is a monomeric protein that dif-fers from other fibrinolytic enzymes produced by the Ba-cillus species. The molecular mass of nattokinase from B.natto, which is known to be a direct fibrinolytic enzyme,is 28 kDa (Cao et al., 2009). However, fibrinolytic en-zymes produced by Bacillus species have molecularmasses varying from 20 to 41 kDa (Chang et al., 2012;Huang et al., 2013), while those produced by Pseudomonassp. and Streptomyces sp. are 21 kDa, and 18 kDa, respec-tively (Mander et al., 2011; Wang et al., 2009). In con-trast, the fibrinolytic enzyme isolated in this study wasmuch less than that from Bacillus subtilis ICTF-1 (28 kDa)(Mahajan et al., 2012), while it was greater than that fromA. mellea (20 kDa) (Lee et al., 2005) and (14 kDa) from P.eryngii (Cha et al., 2010).

Effects of pH and temperature on enzyme activity andstability

The effects of pH and temperature of the purified en-zyme from B. tequilensis CWD-67 are presented in Fig.4. The results showed that the fibrinolytic enzyme wasactive at pH 6.0–8.0, with the optimum activity occurringat pH 8.0 (Fig. 4a). The activity decreased greatly in theacidic and alkaline range, indicating that it is a neutralenzyme. The pH optimum of B. tequilensis CWD-67 co-incides with that of subtilisin-like protease from Bacillussubtilis (Kim et al., 2006) and chymotrypsin-like serinemetalloprotease from Cordyceps militaris (Choi et al.,

Steps Total protein (mg) Total activity (U)* Specific activity (U/mg) Purification fold Yield (%)

Crude extract 329.6 1582.1 1.8 1.0 100.0(NH4)2SO4 precipitation 106.2 930.9 5.0 5.8 77.2DEAE-Cellulose 37.1 744.7 22.0 19.1 35.3Sephadex G-75 FF 9.7 363.8 58.5 34.7 8.2

Fig. 2. Elution profile of the fibrinolytic enzyme from B. tequilensisCWD-64.

Gel filtration chromatography with a Sephadex G-75 FF column. Theproteins were eluted at a flow rate of 0.5 mL min–1 by FPLC.

Table 1. Purification of fibrinolytic enzyme from B. tequilensis CWD-64.

*Enzyme activity was measured using the synthetic substrate azocasein (see Section “Materials and Methods”).

Fig. 3. SDS-PAGE and zymogram of the purified fibrinolytic enzymefrom Bacillus tequilensis CWD-67.

Fibrinolytic enzyme from Bacillus tequilensis 245

2011), while its pH stability was wider than of B. subtilis(Kim et al., 2006). As shown in Fig. 4b, the enzyme wasactive at 35–50°C, exhibiting maximum activity at 45°Cand showing a rapid decrease in activity above 50°C,mainly due to thermal denaturation (Choi et al., 2011).The enzyme was stable and retained more than 80% of itsinitial activity between 45°C and 50°C, but rapidly lostactivity when the temperature increased above 55°C. Theoptimal temperature for the purified enzyme coincides withthat of serine proteases from B. subtilis A26 (Agrebi etal., 2009). The thermal stability range for the reportedenzyme was comparatively wider than that of chymot-rypsin-like serine protease from Paenibacillus polymyxaEJS-3 (Lu et al., 2010) and chymotrypsin-like serinemetalloprotease from C. militaris (Choi et al., 2011).

Effects of inhibitors and metal ions on enzyme activityThe effects of various inhibitors on enzyme activity were

investigated using PMSF, Bestatin A, Pepstatin, EDTA,and TPCK. The purified enzyme was completely inacti-vated by PMSF, which is considered a serine protease in-hibitor. Moreover, the purified enzymes were significantlyinhibited by metalloprotease (EDTA) and chymotrypsin-

like proteinase (TPCK) inhibitors, whereas acid proteaseinhibitor (pepstatin) and amidopeptidase inhibitor(Bestatin) did not exert an inhibitory effect (Table 2). Metalions also exerted varied effects on the purified enzyme.Specifically, the activity was significantly inhibited byCo2+, Zn2+, and Cu2+, while it was slightly enhanced byCa2+, Mg2+, and Fe2+. These findings suggest that the pu-rified enzyme might belong to the chymotrypsin-like ser-ine metalloprotease family, which is known to be involvedin the treatment of thrombosis (Choi et al., 2011). Theenzyme was similar to the fibrinolytic enzyme fromFusarium sp. CPCC 480097 (Wu et al., 2009) and A.mellea metalloprotease (AMMP), a fibrinolytic enzymefrom cultured mycelia of A. mellea (Wu et al., 2009), whichare known to be a chymotrypsin-like serinemetalloprotease and chymotrypsin-like metalloprotease,respectively.

Hydrolysis of fibrinogen by the purified enzymeTo investigate the degradation patterns of fibrinogen,

purified enzyme was assayed using the fibrin plate method.After incubation at 37°C, the enzyme formed a larger clearzone around the disc than that generated by an equalamount of plasmin, as illustrated in the photograph takenat 12 h (Fig. 5). Since the area of the clear zone is directly

Metal ions or inhibitors Concentration (mM) Relative activity (%)*

None 100

PMSF 2 0Pepstatin A 2 97 ± 1.5

Bestatin 2 94 ± 0.7

EDTA 2 9 ± 2.5

TPCK 2 4 ± 1.4

Ca2+ 5 92 ± 5

Co2+ 5 34 ± 1.3

Mg2+ 5 100 ± 0.6

Zn2+ 5 39 ± 0.3

Cu2+ 5 27 ± 0.4

Fe3+ 5 99 ± 3.7

Fig. 4. (a) Effect of pH on the activity and stability of the fibrinolyticenzyme from B. tequilensis CWD-64. (b) Effect of tempera-ture on the activity and stability of the fibrinolytic enzyme fromB. tequilensis CWD-64.

Fig. 5. Analysis of fibrinolysis on fibrin-agarose plate: (1) Plasmin usedas a standard, and (2) purified enzyme from B. tequilensis CWD-64.

*Values are the mean ±SD of three experiments.

Table 2. Effect of metal ions and inhibitors on the activity of fibri-nolytic enzyme.

246 VELUSAMY et al.

proportional to the enzymatic activity, it can be assumedthat the purified enzyme has a greater fibrinolytic activitythan that of plasmin. Subsequently, the fibrinolytic activ-ity of the enzyme was compared in the presence, and ab-sence, of plasminogen. Additional plasminogen did notcontribute to the enhancement of the clear zone, implyingthat the enzyme is a plasmin-like protease that directlydegrades fibrin (data not shown). Fibrinogen is cross-linked by thrombin into fibrin, which has a structure thatincludes γ-, α-, and β-chains, while a dimer γ-γ chain offibrin is formed through Ca2+ cross-linking (Shen et al.,1975). The addition of the enzyme caused a clear hollowon the plasminogen-free plate, while the size of the clearhollow did not change obviously in the presence of plas-minogen, suggesting that the purified enzyme was a plas-min-like, direct-acting, fibrinolytic protease that did notrequire endogenous fibrinolytic factors to lyse the thrombi.Hence, secondary effects, such as platelet activation re-lated plasmin formation caused by plasminogen activa-tors, could be avoided (Wu et al., 2009). The degradationpattern of fibrin by the purified enzyme showed that theenzyme preferentially hydrolyzed the α chain of fibrin,followed by the β chain, and finally the γ-γ chain, with apattern similar to those of the enzymes from C. militaris(Choi et al., 2011) and Armillaria mellea (Lee et al., 2005).

Conclusion

In this study, a unique fibrinolytic enzyme was purifiedto homogeneity from Bacillus tequilensis CWD-67 via athree-step procedure with a 35-fold increase in specificactivity and 8% recovery. The molecular weight of thepurified enzyme was estimated to be 22 kDa by SDS-PAGE. The purified enzyme was homogenous on SDS-PAGE, forming a single band, suggesting that it was amonomer. The purified enzyme activity was completelyinhibited by PMSF and significantly inhibited by EDTA,TPCK, Co2+, Zn2+, and Cu2+, indicating that the enzymeis a chymotrypsin-like serine metalloprotease. In addition,the enzyme can directly degrade fibrin with highersubstrate specificity than the positive control (plasmin).Taking together, these findings indicate that the fibrino-lytic enzyme from B. tequilensis CWD-67 is a potentialcandidate for the prevention and treatment of thrombosis.Microbes have the innate potential for synthesis of pri-mary metabolites and can be regarded as potential bio-factories for the synthesis of fibrinolytic enzymes. Fur-ther studies are needed to elucidate the effectiveness ofthrombolysis by this enzyme in vivo.

Acknowledgments

The authors are grateful to the Management and School of Bioengi-neering, SRM University, for providing the necessary facilities and en-couragement. The author (Periasamy Anbu) would like to thank InhaUniversity Research Grant, Inha University.

References

Agrebi, R., Haddar, A., Hmidet, N., Jellouli, K., Manni, L. et al. (2009)BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilisA26: purification, biochemical and molecular characterization.Process Biochem., 44, 1252–1259.

Astrup, T. and Mullertz, S. (1952) The fibrin plate method for estimat-ing fibrinolytic activity. Arch. Biochem. Biophys., 40, 346–351.

Cao, X. H., Liao, Z. Y., Wang, C. L., Cai, P., Yang, W. Y. et al. (2009)Purification and antitumour activity of a lipopeptide biosurfactantproduced by Bacillus natto TK-1. Biotechnol. Appl. Biochem., 52,97–96.

Cha, W. S., Park, S. S., Kim, S. J., and Choi, D. (2010) Biochemicaland enzymatic properties of a fibrinolytic enzyme from Pleurotuseryngii cultivated under solid-state conditions using corn cob.Bioresour. Technol., 101, 6475–6481.

Chang, C. T., Wang, P. M., Hung, Y. F., and Chung, Y. C. (2012) Purifi-cation and biochemical properties of a fibrinolytic enzyme fromBacillus subtilis fermented red bean. Food Chem., 133, 1611–1617.

Choi, D. B., Cha, W. S., Park, N., Kim, H. W., Lee, J. H. et al. (2011)Purification and characterization of a novel fibrinolytic enzyme fromfruiting bodies of Korean Cordyceps militaris. Bioresour. Technol.,101, 6475–6481.

Choi, N. S. and Kim, S. H. (2000) Purification and characterization ofsubtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened fromDoen-Jang. Biosci. Biotechnol. Biochem., 64, 1722–1725.

Collen, D. and Lijnen, H. R. (1991) Basic and clinical aspects of fibri-nolysis and thrombolysis. Blood, 78, 3114–3124.

Geng, P., Lin, L., Li, Y., Fan, Q., Wang, N. et al. (2014) A novel fibrin(ogen) olytic trypsin-like protease from Chinese oak silkworm(Antheraea pernyi): Purification and characterization. Biochem.Biophys. Res. Commun., 445, 64–70.

He, J., Di, J., Xu, R., and Zhao, B. (2008) Novel recombinant thrombo-lytic and antithrombotic staphylokinase variants with RGD motifat their N-terminus. Biotechnol. Appl. Biochem., 50, 17–23.

Huang, S., Pan, S., Chen, G., Huang, S., Zhang, Z. et al. (2013) Bio-chemical characteristics of a fibrinolytic enzyme purified from amarine bacterium, Bacillus subtilis HQS-3. Int. J. Biol. Macromol.,62, 124–130.

Kim, H. K., Kim, G. T., Kim, D. K., Choi, W. A., Park, S. H. et al.(1997) Purification and characterization of a novel fibrinolytic en-zyme from Bacillus sp. KA38 originated from fermented fish. J.Ferment. Bioeng., 84, 307–312.

Kim, S. B., Lee, D. W., Cheigh, C. I., Choe, E. A., Lee, S. J. et al.(2006) Purification and characterization of a fibrinolytic subtili-sin-like protease of Bacillus subtilis TP-6 from an Indonesian fer-mented soybean, Tempeh. J. Ind. Microbiol. Biotechnol., 33, 436–444.

Kim, W., Choi, K., Kim, Y., Park, H., Choi, J. et al. (1996) Purificationand characterization of fibrinolytic enzyme produced from Bacil-lus sp. strain CK11-4 screened from Chungkook-Jang. Appl.Environ. Microbiol., 62, 2482–2488.

Laemmli, U. K. (1970) Cleavage of structural proteins during the as-sembly of the head of bacteriophage T4. Nature, 277, 680–685.

Lee, S. Y., Kim, J. S., Kim, J. E., Sapkota, K., Shen, M. H. et al. (2005)Purification and characterization of fibrinolytic enzyme from cul-tured mycelia of Armillaria mellea. Protein Exp. Purif., 43, 1–7.

Lim, G. B. (2013) Public health: Global burden of cardiovascular dis-ease. Nat. Rev. Cardiol., 10, 59.

Lu, F., Lu, Z., Bie, X., Yao, Z., Wang, Y. et al. (2010) Purification andcharacterization of a novel anticoagulant and fibrinolytic enzymeproduced by endophytic bacterium Paenibacillus polymyxa EJS-3.Thromb. Res., 126, 349–355.

Mahajan, P. M., Nayak, S., and Lele, S. S. (2012) Fibrinolytic enzymefrom newly isolated marine bacterium Bacillus subtilis ICTF-1:Media optimization, purification and characterization. J. Biosci.Bioeng., 113, 307–314.

Mander, P., Cho, S. S., Simkhada, J. R., Choi, Y. H., and Yoo, J. C.(2011) A low molecular weight chymotrypsin-like novel fibrino-lytic enzyme from Streptomyces sp. CS624. Process Biochem., 46,1449–1455.

Montriwong, A., Kaewphuak, S., Rodtong, S., Roytrakul, S., andYongsawatdigul, J. (2012) Novel fibrinolytic enzymes fromVirgibacillus halodenitrificans SK1-3-7 isolated from fish saucefermentation. Process Biochem., 47, 2379–2387.

Peng, Y., Huang, Q., Zhange, R. H., and Zhang, Y. Z. (2003) Purifica-tion and characterization of a fibrinolytic enzyme produced by Ba-cillus amyloliquefaciens DC-4 screened from douchi, a traditionalChinese soybean food. Comp. Biochem. Physiol. B, 134, 45–42.

Fibrinolytic enzyme from Bacillus tequilensis 247

Shen, L. L., Hermans, J., McDonagh, J., McDonagh, R. P., and Carr, M.(1975) Effects of calcium ion and covalent cross linking on forma-tion and elasticity of fibrin gels. Thromb. Res., 6, 255–265.

Siritapetawee, J., Thumanu, K., Sojikul, P., and Thammasirirak, S.(2012) A novel serine protease with human fibrino (geno) lytic ac-tivities from Artocarpus heterophyllus latex. Biochem. Biophys.Acta, 1824, 907–912.

Wang, S. L., Chen, H. J., Liang, T. W., and Lin, D. L. (2009) A novelnattokinase produced by Pseudomonas sp. TKU015 using shrimp

shells as substrate. Process Biochem., 44, 70–76.Weisburg, W. G., Barns, S. M., and Lane, D. J. (1991) 16S ribosomal

DNA amplification for phylogenetic study. J. Bacteriol., 173, 697–703.

Wolberg, A. S. (2007) Thrombin generation and fibrin clot structure.Blood Rev., 21, 131–142.

Wu, B., Wu, L., Chen, D. Y. Z., and Luo, M. (2009) Purification andcharacterization of a novel fibrinolytic protease from Fusarium sp.CPCC480097. J. Indust. Microbiol. Biotechnol., 36, 451–459.