Screening and Characterization of Thermostable Fibrinolytic Enzyme from Bacillus Amyloliquefaciens EGE-B-2D.1

Turkish Journal of Biochemistry – Türk Biyokimya Dergisi 2016; 41(3): 167–176

Biotechnology Research Article – 20053

Birkan Slem, Yüksel Gezgin, Rengin Eltem* Screening and characterization of thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1

Bacillus amyloliquefaciens EGE-B-2d.1’den termostabil fibrinolitik enzimin taranması ve karakterizasyonu doi 10.1515/tjb-2016-0027 molecular weight of the purified enzyme were estimated Received August 19, 2015; accepted February 3, 2016 to be 44.46 units/mg protein and 30 kD respectively.

Abstract: Objective: To screen fibrinolytic enzyme-produc- Conclusion: The thermostable fibrinolytic enzyme from ing Bacillus isolates (n=210) and to characterize of thermo- Bacillus amyloliquefaciens EGE-B-2d.1 was purified and stable fibrinolytic enzyme from Bacillus amyloliquefaciens characterized. This enzyme might also be used as a natural EGE-B-2d.1 that had the highest level of fibrinolytic activ- agent for oral fibrinolytic therapy or thrombosis prevention. ity together with the highest thermostability.

Keywords: Fibrinolytic enzyme, Bacillus amyloliquefa- Methods: Firstly, a total of 210 isolates were screened for ciens, production, purification, plasminogen activator, their fibrinolytic enzyme production. The potent fibrino- thrombolytic agent lytic enzyme producing isolates were evaluated for the thermostability of their fibrinolytic enzymes and one isolate showing prominent fibrinolytic activity was iden- Özet: Amaç: Fibrinolitik enzim-üreten Bacillus izolatları- tified as molecular. Fermentation process was carried out nın (n=210) taranması ile Bacillus amyloliquefaciens EGE- on the isolate that had both the highest level of fibrinolytic B-2d.1’den elde edilen en yüksek termostabilite ve fibrino- activity and enzyme thermostability. The thermostable litik aktiviteye sahip enzimi karakterize etmektir. fibrinolytic enzyme from this isolate was then purified and characterized. Metod: İlk olarak, toplam 210 tane Bacillus izolatı fibrinolitik enzim üretimi açısından taranmıştır. Fibrinolitik Results: The fibrinolytic enzyme activities of 21 Bacillus sp. enzim üreten izolatların fibrinolitik enzimlerinin termos- isolates in Nutrient Yeast Salt Medium were found to be in tabiliteleri değerlendirilmiş ve belirgin fibrinolitik akti- the range of 0.176–1.734 U/ml. The fibrinolytic activity of vite gösteren bir izolat moleküler olarak tanımlanmıştır. the enzyme purified from the culture supernatant of Bacil- Fermentasyon prosesi hem en yüksek fibrinolitik aktivite lus amyloliquefaciens EGE-B-2d.1 was relatively stable at ve hem de enzimi termostabil olan bu izolat ile gerçekleş- pH 7.0–11.0 for 24 h and also showed stability at a tempera- tirilmiştir. Bu termostabil fibrinolitik enzim daha sonra ture of 60°C for 60 min. The enzyme degraded the fibrin saflaştırılmış ve karakterize edilmiştir. clots by direct fibrinolysis. The specific activity and the Bulgular: 21 tane Bacillus sp. izolatının Nutrient Maya Tuz Ortamındaki fibrinolitik aktivite değerleri 0.176–1.734 U/ *Corresponding author: Rengin Eltem: Ege University, Department ml aralığında bulunmuştur. Bacillus amyloliquefaciens of Bioengineering, İzmir, Turkey, e-mail: [email protected] Birkan Slem: Ege University, Department of Bioengineering, İzmir, EGE-B-2d.1’in kültür sıvısından saflaştırılan enzim 24 saat Turkey, e-mail: [email protected] süresince pH 7.0–11.0 aralığında ve 60 dakika boyunca Yüksel Gezgin: Ege University, Department of Bioengineering, İzmir, 60°C sıcaklıkta kararlılık göstermiştir. Enzim doğrudan Turkey, e-mail: [email protected] fibrinolizis ile fibrin pıhtılarını parçalamıştır. Saflaştırılan 168 Birkan Slem et al.: Thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens enzimin spesifik aktivitesi ve moleküler ağırlığı sırasıyla Various traditional fermented food products such as Jap- 44.46 unit/mg protein and 30 kD olarak belirlenmiştir. anese Natto [13], Korean Chungkook-Jang [9], natto-red bean [14], and Korean fermented food, Jeot-gal [15], have Sonuç: Bacillus amyloliquefaciens EGE-B-2d.1’den termos- been reported as sources for isolating the microorganisms tabil fibrinolitik enzim saflaştırılmiş ve karakterize edil- that demonstrate fibrinolytic activity. miştir. Bu enzimin ağız yoluyla fibrinolitik terapi ve trom- In this research, Bacillus spp. and endospor con- bozun önlenmesi için doğal bir ajan olarak kullanılabilme taining thermophilic isolates were isolated from original potansiyeline sahip olduğu belirlenmiştir. sources such as samples of soil of pine nut forest, mushroom, dried fig, soil of fig orchards and soil of vineyard Anahtar Kelimeler: Fibrinolitik enzim, Bacillus amyloliqu- and various geothermal sources of West Anatolia Region efaciens, üretim, saflaştırma, plasminojen aktivatör, trom- of Turkey. The isolates were initially screened on plates bolitik ajan for their fibrinolytic enzyme production. Strains display- ing relatively high fibrinolytic activity on these plates were cultured in flasks for quantification and to determine the Introduction thermostability of their fibrinolytic enzymes. Finally, the isolate giving the highest level of fibrinolytic activity, Blood clotting is characterised by the formation of insol- together with the highest enzyme thermostability, was uble fibrin polymers from fibrinogen via the proteolysis cultivated in a bioreactor and its thermostable fibrinolytic by thrombin (EC 3.4.21.5) to maintain haemostasis. Blood enzyme product was purified and characterized. clots are designed to be temporary, and when the process of vessel repair begins, fibrin polymers are removed by the hydrolytic action of serine protease plasmin (EC 3.4.21.7), derived from its circulating zymogen (plasminogen), by the Materials and Methods enzymatic action of tissue-plasminogen activator (t-PA). Normally fibrin formation and degradation is bal- Microorganisms and culture conditions anced in the metabolism and if this normal balance is disturbed, fibrin accumulates in the blood vessels causing A total of 210 isolates were supplied by the Bioengineering thrombosis, which can lead to myocardial infarction and Culture Collection of Ege University, Turkey (Table 1). The other cardiovascular diseases [1–3]. type culture also used was Geobacillus stearothermophilus Early methods of treatment were based on the use of NRRL B-1102. anticoagulants, such as warfarin (Coumadin) and heparin to inhibit the formation of fibrin clots [4]. As enzyme based treatments gained importance, urokinase (u-PA, Materials EC 3.4.21.73) and t-PA were widely used and they are still in use in thrombolytic therapy [5]. However, these agents Fibrin washed from human plasma (F5386), dextrin have some undesirable side effects [6–8]. (D2131), trizma base (T1503), thrombin from human Microorganisms are important resources for thrombolytic agents and fibrinolytic enzymes have been suc- Table 1: Source of isolates used in study. cessively discovered in many different types of organism, such as fungi, bacteria, actinomycetes, and algae [6]. Isolates Total number Based upon their working mechanisms, microbial 1 fibrinolytic enzymes can have indirect specificity to fibrin Soil of vineyard 58 Edible mushroom (Agaricus bisporus) 6 (plasminogen activators), can directly degrade the fibrin Edible mushroom (Pleurotus sp.) 8 in blood clots (plasmin-like proteins) or can utilise a com- Beach of Izmir Marine Solar Saltern 21 bination of mechanisms [6]. Dried fig 9 Currently, the microbial enzymes produced by Bacil- Soil of fig orchard2 26 3 lus spp. attract much more interest as thrombolytic Soil of forest 12 Municipal wastewater sludge4 3 agents because of their fibrinolytic process efficiency Soil of pine nut forest5 7 including plasmin activation [9]. Consequently, consid- Total 150 erable research has been carried out on potent fibrino- 1: Manisa; 2: Aydın; 3: Muğla; 4: Çiğli-İzmir; 5: Bergama-İzmir (Turkey). lytic enzyme production by various Bacillus spp. [10–12]. Birkan Slem et al.: Thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens 169 plasma (T7009), plasminogen from human plasma with cell free supernatant, and the released tyrosine was (P7999), N,N,N′,N′-tetramethyl-ethylenediamine (T7024), monitored. Fibrinolytic activity was measured by the ammonium sulfate (A4418), dialysis tubing cellulose hydrolysis of fibrin according to the procedure described membrane (D9527) from Sigma-Aldrich, CA, USA, elec- by Anson [17] with some modifications. The incubation trophoresis protein marker (P7702) from New England mixture contained 60 µl of 1.2% fibrin (0.1 g of fibrin was Biolabs (NEB), UK and L-tyrosine (108371) was purchased dissolved in 6.66 ml of 0.2 N NaOH and adjusted to pH 7.8 from Merck, Darmstadt, Germany. with 6 N HCl; the final volume was adjusted to 8.33 ml with

Fast protein liquid chromatography (FPLC) AKTA (18– water), 60 µl of 0.1 M Tris-HCl (10 mM CaCl2, pH 7.8), and 1140–45), Hiprep 26/60 Sephacryl S 200 HR (17–1195–01) 20 µl cell free supernatant. The incubation was carried out (GE Healthcare, formerly Amersham Biosciences, Pisca- at 30°C for 10 min. The reaction was stopped by adding taway, USA), Ultrospec 1100 pro UV/visible (18–1157–29) 120 µl of 0.11 M trichloroacetic acid containing 0.22 M were obtained from Pharmacia Co. (Amersham Biosci- sodium acetate and 0.33 M acetic acid. The final solution ences). Sartocon slice 200 system (17525–01) and Biostat® was centrifuged at 10.000 rpm for 20 min, then 200 µl of A plus bioreactor (G-11870) from Sartorius (Sartorius A this clear solution was mixed with 1.8 ml ultrapure water plus, Goettingen, Germany), centrifuge (4K15) were pur- in a Quartz cuvette. The absorbance at 275 nm of the tri- chased from Sigma-Aldrich, CA, USA and midi gel elec- chloroacetic acid soluble product was determined using a trophoresis system (PS 304) was obtained from Apelex, spectrophotometer. A fibrinolytic unit was defined as the Massy, France. amount of cell free supernatant that gave an increase in absorbance at 275 nm, equivalent to 1 µg of tyrosine per minute at 30°C. Assay of fibrinolytic enzyme activity

All patients provided written informed consent prior to Isolate identification the use of healthy human plasma as approved by the Ege University Medical School, Research Ethics Committee by A single colony showing prominent fibrinolytic activity was the protocol number 09–5.1/8. selected and subjected to taxonomic analysis as described Fibrinolytic activity of isolates were analysed firstly by in Bergey’s Manual of Systematic Bacteriology. For further the plasma plate method of Ko et al. [16] with some mod- identification of the bacterium, polymerase chain reaction ifications and then by the spectrophotometric method (PCR) was performed to amplify a part of the bacterial 16S described by Anson [17] with some modifications. In the rRNA gene. Genomic DNA from the isolated bacteria was plasma plate method (primary screening), the plate was prepared using a DNA isolation kit (Promega, Madison, prepared by the addition of 2 ml of healthy human plasma USA) following manufacturer’s protocol. The 16S rDNA (from Karsiyaka Park Polyclinic, Izmir) into 6 ml 20 mM was amplified using the universal eubacteria primers Tris–HCl buffer (including 0.75% agar, 0.15 M NaCl, pH 8.0) 27F, 5΄-AGAGTTTGATC(C/A)TGGCTCAG-3΄ and 1492R, at 50°C. This 8 ml solution was poured into a Petri dish 5΄-TACGG(C/T)TACCTTGTTACGACTT-3΄ [18–20]. Partial 16S (60 mm diameter) with 40 µl thrombin solution (100 NIH/ rDNA raw sequence data was imported into the BioEdit ml thrombin in 20 mM Tris-HCl buffer, pH 8.0). The plate Sequence Alignment Editor and a contiguous consensus was allowed to stand for 30 min at room temperature to sequence generated. Using BLAST (Basic Local Alignment form fibrin clots, and thirteen holes were then made on the Tool) against GenBank database, the closest relatives of fibrin plates using a capillary glass tube (3 mm in diame- these consensus sequences were identified. ter). The plate was heated at 85°C for 30 min to deactivate the plasminogen. Ten microliters of cell free supernatant was placed in each hole and the plates were incubated at Thermostability and pH stability 37°C for 18 h. After incubation, the clear zone was observed either directly or after washing with 0.11 M trichloroacetic In order to determine pH stability of fibrinolytic enzymes, acid. Areas of clear zones, formed as a result of the degra- 40 µl of supernatant was added to 120 ml acetate (0.1 M, dation of fibrin due to digestion, was considered as being pH 3.0, 4.0, 5.0), phosphate (0.1 M, pH 6.0, 7.0, 8.0) and a quantitative parameter for the primary screening of the glycine (0.1 M, pH 9.0, 10.0, 11.0), buffers and the solutions fibrinolytic enzyme-producing microorganisms. thus prepared were incubated at 4°C for 24 h. The resid- In the spectrophotometric method (secondary screen- ual fibrinolytic activities of these solutions were measured ing), fibrin clots in a liquid suspension were incubated using the spectrophotometric method. 170 Birkan Slem et al.: Thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens

The thermostability of fibrinolytic enzymes was The specific activity of the enzyme was calculated using determined by pre-incubating the enzyme preparations the following formula: at 35, 40, 45, 50, 55 and 60°C for 1h at pH 7.0 and then Specific activity (U/mg)=total activity (U)/total protein (mg). measuring the remaining activities spectrophotometri- cally and comparing the results with the activity of the supernatant kept at 4°C. Ultrafiltration

One liter of the cell free supernatant was subjected to a two Effect of plasminogen addition on fibrinolytic step ultrafiltration. At the first stage, the supernatant was enzyme activity subjected to ultrafiltration with a 30.000 nominal molecular weight cut-off limit (NMWL) polyethersulfone cassette The incubation mixture contained 150 µl of 1.2% fibrin (3081465902E-SG, Sigma). The filtrate obtained from the

(pH 7.8), 150 µl of 0.1 M Tris-HCl (10 mM CaCl2, pH 7.8), first step was further subjected to ultrafiltration with a the enzyme solution (50 µl), and 50 µl plasminogen 10.000 NMWL polyethersulfone cassette (3081463902E- (0.625 U). Fibrinolytic activity was measured by using SG, Sigma). the spectrophotometric method and was compared with the activity of the enzyme without plasminogen. In addition, the effect of plasminogen addition was con- Ammonium sulfate treatment trolled on the fibrin plate. The fibrin plate, which was prepared by the addition of 2 ml of 1.2% fibrin (pH 7.8) The retentate was subjected to ammonium sulfate (70% w/v) to 2 ml 20 mM Tris-HCL (including 0.75% agar, 0.15 M precipitation by stirring using a magnetic stirrer at 4°C for NaCl, pH 8.0) at 50°C. overninght. The precipitate formed was centrifuged at 8.000 rpm for 30 min and the precipitate obtained was dissolved in a minimum amount of 50 mM Tris-HCl buffer, pH 7.2. Enzyme production

Bacillus amyloliquefaciens EGE-B-2d.1 was cultivated at Dialysis 30°C for 18 h in a 500 ml Erlenmeyer flask containing 100 ml nutrient yeast salt broth (NYSM). The inoculum was The dark brown solution obtained was dialyzed at 4°C for then transferred into a 5 L bioreactor containing 2 L pro- 24–48 h against 10 mM Tris-HCl buffer, pH 7.2. duction medium (20 g dextrin, 20 g soybean meal, 5.0 g yeast extract, 0.2 g MgSO4.7H2O, 0.2 g CaCl2, 2 g K2HPO4, and 0.2 g Na2HPO4 per liter) and cultivation was carried Gel filtration with FPLC out at 30°C for 26 h. The pH and the dissolved O2 concentration were maintained at 7.0 with 30% air saturation, Hiprep 26/60 Sephacryl S 200 HR was used for gel filtra- respectively. Fermentation was stopped when the fibri- tion chromatography. A column (2.6x60 cm) was equili- nolytic activity reached the max level, after 26 h. Cell brated with 50 mM Tris-HCl buffer containing 0.1 M NaCl, free supernatant containing the fibrinolytic enzyme was pH 7.2. The active fraction was dissolved, dialyzed, and obtained from the culture broth by centrifugation at 9.000 loaded onto the column (3 ml/injection) and eluted with rpm for 10 min. the same buffer. Fractions were collected at a flow rate of 0.5 ml/min at 0.3 mPa.

Enzyme purification Gel electrophoresis The fibrinolytic enzyme was purified in four steps: ultrafiltration, (NH4)2SO4 precipitation, dialysis using a cellu- Sodium dodecyl sulfate-polyacrylamide gel electrophore- lose membrane and gel filtration chromatography with sis (SDS-PAGE) was performed on 12% gels at 4°C follow- Hiprep 26/60 Sephacryl S 200 HR. All purification steps ing the method of Laemmli [21]. Gels were stained with were performed at 4°C. At each purification step, the Coomassie Brilliant Blue R- 250. For molecular weight Bradford Method was used while monitoring the protein determinations, NEB electrophoresis protein marker concentration. (2–212 kD) was used. Birkan Slem et al.: Thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens 171

Table 2: Isolation sources, fibrinolytic activity and thermostability of Bacillus isolates.

Isolates Sources Enzyme activity (U/ml) Residual activity (%) at 1 hour

50°C 55°C 60°C

Bacillus sp. EGE-B-2.9 Soil of vineyard 0.38 – – – Bacillus sp. EGE-B-1.1.a Edible mushroom 0.95 – – – Bacillus sp. EGE-B-2.8 Soil of vineyard 1.09 46.5 15.9 0 Bacillus sp. EGE-B-1.d.10 Marine solar saltern 0.53 – – – Bacillus sp. EGE-B-2.6 Soil of vineyard 1.26 46.1 34.7 6.1 Bacillus sp. EGE-B-2d.1 Marine solar saltern 1.73 91.9 84.5 81.8 Bacillus sp. EGE-B-1d Marine solar saltern 1.12 71.2 32.2 0 Bacillus sp. EGE-B-1d.5 Marine solar saltern 0.88 – – – Bacillus sp. EGE-B-1d.6 Dried fig 0.18 – – – Bacillus sp. EGE-B-1d.7 Marine solar saltern 0.49 – – – Bacillus sp. EGE-B-1d.9 Marine solar saltern 0.61 – – – Bacillus sp. EGE-B-9.2 Soil of vineyard 0.65 – – – Bacillus sp. EGE-B-1.5 Soil of vineyard 0.83 51.5 35.6 15.5 Bacillus sp. EGE-B-12.2i Soil of fig orchard 0.56 – – – Bacillus sp. EGE-B-19.2i Soil of fig orchard 0.65 – – – Bacillus sp. EGE-B-27.4 Soil of vineyard 1.12 71.9 57.7 1.0 Bacillus sp. EGE-B-i.1.1 Dried fig 1.60 55.0 40.3 0 Bacillus sp. EGE-B-i.1.4 Dried fig 0.78 – – – Bacillus sp. EGE-B-i.1.5 Dried fig 1.19 73.1 43.3 0 Bacillus sp. EGE-B-i.1.6 Dried fig 1.15 75.6 52.3 29.7 Bacillus sp. EGE-B-i.1.7 Dried fig 1.03 48.9 36.0 0

–: not investigated.

pH large clear zones on plasma plates and they were therefore 3 4 5 6 7 8 9 10 11 selected. The fibrinolytic enzyme activities of 21 Bacillus iso- 0.2 1.8 lates in NYSM were determined between 0.176–1.734 U/ml. 1.6 In secondary screening, 10 isolates out of 21 were detected 1.4 1.2 that showed considerably high fibrinolytic activity (Table 2). 1 0.8 Only these isolates were tested for their enzyme thermostabil- 0.6 ities. As seen from the tabulated values in Table 2, the isolate, 0.4 0.2 Bacillus sp. EGE-B-2d.1 was found to have the highest level of Enzyme activity (U/ml) activity Enzyme 0 fibrinolytic activity together with the highest thermostability. 35 40 45 50 55 60 Thus this strain was chosen for further investigation. Temp (oC)

Figure 1: Stability of the fibrinolytic enzyme from Bacillus amyloliq- Identification of the fibrinolytic enzyme- uefaciens. (■) pH stability, (▲) temperature stability. producing Bacillus sp.

Bacillus sp. EGE-B-2d.1 was isolated from beach of Izmir Results Marine Solar Saltern, Turkey (Table 2). B. amyloliquefaciens EGE-B-2d.1 isolate is a gram-positive rod-shaped, Screening of fibrinolytic enzyme-producing endospore forming bacterium. The cells were 0.8–1.0 µm microorganisms wide and 2.0–3.0 µm long. It was form oval and 0.7–0.8 µm wide and 1.0–1.5 µm long spores. A total of 150 Bacillus spp. were isolated from various sources Bacillus sp. EGE-B-2d.1 isolate was identified based on (Table 1). These isolates together with the 60 endospor its 16S rRNA gene sequence. The nucleotide sequence is containing thermophilic isolates from various geothermal deposited in the GenBank under the accession number of sources of West Anatolia Region of Turkey were subjected JF926530. The BLAST analysis showed a high degree of simi- to primary screening and a total of 21 isolates gave relatively larity of Bacillus sp. EGE-B-2d.1 to Bacillus amyloliquefaciens. 172 Birkan Slem et al.: Thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens

incubation, the fibrinolytic activity was very stable and showed 94.4%, 94.1%, 93.0%, 91.9%, 84.5% and 81.8% residual activity at 35°C, 40°C, 45°C, 50°C, 55°C and 60°C respectively. As shown in Fig. 1, the optimum pH for the fibrinolytic activity of the enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 was pH 8.0 and which was relatively stable in the range of pH 7.0–11.0 at 4°C for 24 h.

Effect of plasminogen addition on fibrinolytic enzyme activity

The mixture of plasminogen and enzyme did not give a higher activity than the activity of the enzyme alone. A similar result was obtained from fibrin plate analysis (Fig. 2). This revealed that, the fibrinolytic enzyme degraded fibrin directly and it was not the activity of the plasmin- Figure 2: Plasminogen activator assay of the enzyme in the fibrin plate. Zone A1 and B1 represented 10 and 20 µl purified enyzme. Zone ogen activator which degraded fibrin by activating plas- A and B represented 10 µl purified enzyme containing 0.625 U plasminogen (Fig. 2). minogen and 20 µl purified enzyme containing 0.625 U plasminogen. Purification of the fibrinolytic enzyme from Thermostability and pH stability of fibrino- Bacillus amyloliquefaciens EGE-B-2d.1 lytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 The fibrinolytic enzyme activity measured at different hours during of growth, ranged from 0.872 U/ml to The thermostability profile of the fibrinolytic enzyme from 1.965 U/ml. It reached a maximum at 26 h after which it B. amyloliquefaciens EGE-B-2d.1 showed that the enzyme decreased due to sporulation (Data not shown). At the was stable at high temperatures (Fig. 1). After a 60-min end of the ultrafiltration, the recovery of the fibrinolytic

Retention time

Figure 3: Purification of fibrinolytic protease by gel filtration on Hiprep 26/60 Sephacryl S 200 HR column.*Peak showed stronger fibrinolytic activity. Birkan Slem et al.: Thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens 173

Table 3: Purification steps of fibrinolytic protease from B. amyloliquefaciens EGE-B-2d.1.

Step Volume Total activity Total protein Specific activity Yield Purification fold (ml) (U) (mg) (U/mg) (%)

Culture supernatant ultrafiltration 1000 1965 428.7 4.58 100 1 30.000 NMWL cut-off (filtrate) 900 1700.9 137.97 12.32 86.5 2.68 10.000 NMWL cut-off (filtrate) 90 159.93 5.796 27.59 8.1 6.02 Ammonium sulfate precipitation 10 16.54 0.556 29.74 0.84 6.48 Dialysis 15 15.78 0.475 33.22 0.80 7.24 Gel filtration (FPLC) 5 5.025 0.113 44.46 0.26 9.7

a b c c d Gel electrophrosesis 66.4 kDa 55.6 kDa The protein was eluted from gel filtration. After concentration, the purified protein was subjected to SDS-PAGE. A 42.7 kDa single band was observed in the purified sample, having a 34.6 kDa molecular mass of 30 kD on SDS-PAGE (Fig. 4).

27.0 kDa Discussion

In recent years, fibrinolytic enzymes have been discov- 20.0 kDa ered in some traditional soybean fermented foods such as natto, don-chi, chonggok-jang and shiokara. In this study, the twenty-one fibrinolytic enzyme producing Bacillus 14.3 kDa spp. were isolated from totally different sources, as tabulated in Table 1 and Table 2, and most of these sources are studied for the first time in literature. There is a recent study conducted using Bacillus spp. isolated from crab shells, fish scales, brine, sand, breathing roots of weeds, Figure 4: Results of SDS-PAGE. (a) Sample from ultrafiltration with clay, mud of marine environment, traditional egyptian fer- 30.000 and 10.000 NMWL membrane cassettes; (b) sample from mented food, corn husk, fermented shrimp paste [5,22–24], the last hour of production; (c) the fractions of tube 17 from Hiprep but as seen in Table 2, out of the twenty-one fibrinolytic 26/60 Sephacryl S 200 HR gel column; (d) protein size marker. enzyme producing Bacillus spp. studied in this research, six Bacillus isolates have beeen isolated from vineyard soil enzyme activity was 90.43% and there was 91% of reduc- and one isolate from edible mushroom, six isolates from tion in volume. The crude enzyme obtained after ammo- Beach of Izmir marine solar saltern, six isolates from dried nium sulphate precipitation and dialysis suggested that fig, and two isolates from fig orchard soil. This study high- the ultrafiltration could retain the specific molecular lights these sources as potent isolation sources for ther- weight proteins. mostable fibrinolytic enzyme producing Bacillus spp. and The fibrinolytically active protein fractions were col- should be further investigated in future studies. lected using the gel column. The sample in tube 17 showed Fibrinolytic enzyme production by a thermophilic the strongest fibrinolytic activity (1.005 U/ml) (Fig. 3). Also Streptomyces megasporus strain SD5 has already been three small protein peaks, without fibrinolytic activity, reported in the literature [25,26], but the fibrinolytic were observed in the fraction tubes (Fig. 3). As summa- enzyme activities of 60 endospor containing thermophilic rized in Table 3, the fibrinolytic enzyme was purified 9.7- isolates and the fibrinolytic activity of the control Geoba- fold via these purification steps. The specific activity of cillus stearothermophilus NRRL B-1102 were carried out for the final enzyme preparation was estimated to be 44.46 the first time in this study. The 60 endospor containing units/mg proteins. thermophilic isolates and Geobacillus stearothermophilus 174 Birkan Slem et al.: Thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens

Table 4: pH stability, optimal pH and temperature, thermostability and molecular weight of fibrinolytic enzymes obtained from Bacillus spp.

Some properties of fibrinolytic enzymes obtained from Bacillus spp

The fibrinolytic enzymes and Bacillus spp. The pH Optimal pH Optimal Termostability Molecular References stability temperature (°C) weight (kDa)

1. B. amyloliquefaciens EGE-B-2d.1 7.0–11.0 8.0 30 60oC; 60 min 30.0 This study

2. B. subtilis ICTF-1 5.0–11.0 9.0 50 25–37°C 28.0 [5]

3. CK from Bacillus sp. strain CK 11–4 7.0–10.5 10.5 70 50°C below 28.2 [10] (very stable at 40°C); 1h

4. Nattokinase from B. natto 7.0–12.0 – – 50°C below 27.7 [13]

5. Subtilisin QK-2 from B. subtilis QK02 3.0–12.0 8.5 55 40°C; 30 min 28.0 [16]

6. B. amyloliquefaciens FCF-11 5.0–9.0 Maximal Maximal below 45°C 18.2 [22] activity-8.0 activity-40

7. B. megaterium KSK-07 5.0–13.0 8.0 50 below 55°C 28.5 [23]

8. Jeot-gal from Bacillus sp. KA38 7.0–9.0 7.0 40 to 40°C 41.0 [27]

9. B. subtilis KCK-7 7.0–10 7.0 60 up to 60°C 44.0 [28]

10. Bacillus sp. KDO-13 7.0–9.0 8.0 50 50°C below 45.0 [29]

11. Subtilisin DFE from B. amyloliquefaciens DC-4 6.0–10 9.0 48 50°C below 28.0 [30]

12. Bacillus sp. nov. SK006 5.0–11.0 7.2 30 40°C below; 4 h 46.0 [31]

13. Bacillus sp. strain AS-S20-I – 7.4 37 – 32.3 [32]

14. B. amyloliquefaciens MJ5-41 6.0–7.0 7.0 45 45°C 27.0 [33] Most stable

15. B. subtilis – 8.0 30 – – [34]

16. Bacillokinase II from B. subtilis Strain A1 4.0–9.0 7.0 50 up to 50°C 31.4 [35]

17. B. pumilus 2.g 5.0–9.0 Highest 50 60°C below 20.0 [36] activity-7.0

18. B. amyloliquefaciens CB1 Highest Highest 45°C below 28.0 [37] activity-6.0 activity-40°C

NRRL B-1102 were found to show no fibrinolytic activity. fibrinolytic enzymes studied so far (Table 4). The fibrinolytic enzyme activity of 21 Bacillus isolates Comparing to other studies on the fibrinolytic enzyme were determined between 0.176–1.734 U/ml against 0.009 from B. amyloliquefaciens, this enzyme showed the high U/ml (for Bacillus sp. KA38 [27] and 1.84 U/ml (for Bacillus stability at a temperature of 60°C for 60 min (22, 30, 33, sp. CK 11–4 [10]. 37). Kotb [22], analyzed the maximal activity of the purified The enzyme from Bacillus amyloliquefaciens EGE- enzyme from B. amyloliquefaciens FCF-11, the maximum B-2d.1 was extracellular in nature, so its purification and temperature and pH were determined as 40°C and pH of yield would be economically feasible. Also, due to its 8.0, respectively. This enzyme lost 85% of its initial activ- resistance to a broad range of pH levels and temperature ity after incubation at 50°C for 60 min and at pH 6.0 [22]. its industrial production on a large scale would be easy Heo et al. [37] reported that no enzyme activity remained and cheap. This enzyme showed broad pH stability alka- at 55°C (Table 4). line environment and can be classified as an alkaline pro- As shown in Fig. 1, the fibrinolytic enzyme isolated from tease. Thermostability of this enzyme was very high and it B. amyloliquefaciens EGE-B-2d.1, was more stable in basic was close to that given for B. subtilis KCK-7 [28], which was pH environment, the similar result was found that Subtili- reported to be stable up to 60°C. sin DFE from B. amyloliquefaciens DC-4 was stable between The thermostability was higher than all the other pH 6.0–10 (30). These results indicate that the enzyme from Birkan Slem et al.: Thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens 175

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