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 fibri- nolitik 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, mush- room, 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 throm- bolytic 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