JSMC Biochemistry and Molecular Research Research Article © Atakisi O, 2019 Evaluation of the Fibrinolytic Potential of Recombinant Subtilisin from Bacillus subtilis Onur Atakisi1, Canan Gulmez2*, and Melek Ozturkler1 1Department of Chemistry, Kafkas University, Turkey 2Department of Pharmacy Services, Igdir University, Turkey Abstract The aim of this study was to investigate the potential for the treatment of thromboembolic diseases by determining the fibrinolytic, fibrinogenolytic activities and substrate specificity of the recombinant subtilisin from Bacillus subtilis strain PTTC 1023. The subtilisin gene from Bacillus sp. was synthesized, cloned into the vector pD441-NH and expressed in E. coli BL21 (DE3). Recombinant subtilisin (~40 kDa) was purified in a single-step procedure by affinity chromatography. The fibrinolytic, fibrinogenolytic activities and substrate specificity of the pure recombinant enzyme was determined. The recombinant protease of B. subtilis displayed an optimum fibrinolytic activity at pH 7.5 and 37°C.The α, β and ϒ-ϒ chains of fibrinogen were totally degraded by the recombinant enzymes at 37°C for 30 min. However, the ϒ chains were more resistant to enzyme digestion in all times. The clear zones produced by recombinant enzyme and plasmin on the fibrin plate did not significant difference The enzyme showed the highest proteolytic activity against fibrin, while it showed low activity against serum albumin and lactoferrin. The enzyme did not show activity against immunoglobulin. The data obtained indicates that recombinant enzyme is a plasmin-like fibrinolytic serine protease. The recombinant protease may have potential applications in the prevention and treatment of thrombosis and other cardiovascular diseases. Keywords: Fibrinolytic enzyme; Recombinant; Bacillus serine protease; Bacillus subtilis Introduction Thromboembolic diseases, including cardiovascular disease, new fibrinolytic enzymes produced from natural sources for cerebrovascular disease and venous thromboembolism, are low cost, no side effects and safety, specificity and efficacy of within the leading causes of death as exposited in the global fibrinolytic treatment [4]. mortality projections to 2030 by WHO [1]. Thromboembolism often consists when the physiological equilibrium between plasminogenA variety activatorof fibrinolytic streptokinase enzymes have such been as extensivelytissue-type plasminogen activator (t-PA), urokinase (u-PA), and bacterial The fundamental pathophysiological mechanism underlying coagulation system and fibrinolytic system is disturbed [2]. haveinvestigated been considered and used asas therapeutic thrombolytic agents agents for thrombosis.[4]. Serine Notably,proteases, the which genus showBacillus fibrinolytic/fibrinogenolytic has been well regarded for activity,its role cardiovascular diseases is the formation of fibrin (blood clots), in the production of a variety of industrially important enzymes. which adheresis activated to the from unbroken plasminogen wall of bloodby tissue vessels. plasminogen The fibrin activatorcomprising [3]. fibrinogen by thrombin activation is lysed by plasmin, The fibrinolytic enzymes from Bacillus sp. such as subtilisin NAT The uptake of thrombolytic drugs that dissolve blood clots characterized for the development of strong thrombolytic drugs. [5], subtilisin J [6] and subtilisin E [7], have been purified and for treatment is only an alternative to surgical interventions to The recombinant subtilisin from B. subtilis remove or by pass the blockage [2]. Fibrinolytic enzymes, which are widely used in the traditional treatment of thromboembolism, et al., showed that the recombinant enzyme strain encoded PTTC by 1023 the aprE was have vital disadvantages such as high cost, short half-life, high identified as fibnoloytic enzyme by Ghasemi et al., [8]. Ghasemi reported subtilisins and the E. coli transformants. In a later study, and allergic reactions. Therefore, it is important to investigate proteolytic(Accession No.activity HQ699519.1) of subtilisin has from higher B. subtilis subtilisin activity other therapeutic dose, low specificity to fibrin, bleeding complications it was carried out a comprehensive analysis of strain the recombinant PTTC 1023 Submitted: 22 May 2019 | Accepted: 16 June 2019 | Published: 20 June was studied for use in detergent applications [9]. In this study, 2019 enzyme produced for use in pharmaceutical purposes. The aim of the study was to investigate the potential of the enzyme for *Corresponding author: Canan Gulmez, Department of Pharmacy treatment of thromboembolic diseases determining the activities Services, Tuzluca Vocational High School, Igdir University, Igdir-Turkey, Tel: 90-4762230010/3406; Email: [email protected] subtilisin from Bacillus subtilis. Copyright: © 2019 Atakisi O, et al. This is an open-access article of fibrinolytic, fibrinogenolytic and proteolytic of the recombinant distributed under the terms of the Creative Commons Attribution Materials and Methods License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chemicals Citation: Atakisi O, Gulmez C, Ozturkler M (2019) Evaluation of the Fi- brinolytic Potential of Recombinant Subtilisin from Bacillus subtilis. JSMC with the analytical grade or the highest available purity were Biochem Mol Res 3: 5. Unless specified, all chemicals, substrates, and reagents purchased from Sigma Chemical Co. (St. Louis, MO, USA). JSMC Biochem Mol Res 3: 5 1/5 1147. 1(1): Res Clin Cardiol J Production of recombinant enzyme The recombinant enzyme was produced as described in our substrates [15]. One mL of a 10 mg/mL substrate solution was inmixed substrate with theconcentration purified enzyme was determined (2 μL, 0.5 mg/mL),using a Bradfordand the was optimized for E. coli reaction mixtures were incubated at 37°C for 20 min. The change previous study [9]. Briefly, the coding sequence of subtilisin protein assay [16]. There action was stopped by adding 1 mL and 0.5 codon usage. The subtilisin gene (NCBI 2 3 ® accession no. HQ699519.1) from Bacillus subtilis PTTC 1023 was phenol reagent was added to the 0.5 mL of 2% (w/v) trichloroaceticacid. 1.5 mL of 0.5 M Na CO directly synthesized and cloned in expression vector pD441-NH were transformed into competent cells E.coli mL of Folin-Ciocalteu by DNA 2.0 (Atum, Newark, CA, USA). After, recombinant vectors absorbance was calculated at 660 nm spectrophotometrically supernant. The solution was incubated at 37°C for 30 min. The BL21 (DE3). The against a blank control. In the activity measurement, no enzyme C-terminal 6xHis-tagged enzyme was purified in a single-step was added to the medium while preparing the control and all procedure by affinity chromatography using Ni-NTA agarose other steps were performed according to standard protocol. resinDetermination (Qiagen GmbH, of Hilden,fibrinogenolytic Germany). activity Fibrinogenolytic activity was determinated as follows: aminoOne unit acid of equivalent proteolytic to tyrosineactivity perwas min defined under as the the performed amount experimentalof enzyme that conditions. hydrolysed The the results substrate represent and producedthe average 1 μgof three experiments. In this assay, enzyme activity was expressed 80 mL of 1% human fibrinogen prepared in 50 mMTris/HCl as a relative activity, compared to that of non-enzyme treated buffer containing 0.1 M NaCl was incubated with 2μg of purified control. subtilisin at various periods (30, 60, 90, 120 minutes and toovernight) Laemmli’smethod at 37°C [10]. [11]. Then, 20 μL samples taken from the Results reaction medium were analyzed using 12% SDS-PAGE according Determination of fibrinolytic activity-I Fibrinogenolytic activity Fibrinolytic activity was determined according to Astrup and The molecular weight of the enzyme analyzed by 10% SDS- Mullertz’s method (1952) [12] with slight modification. The fibrin recombinantPAGE was about subtilisin 40 kDa of (FigureB. subtilis 1). Fibrinogendisplayed andegradation optimum plate was prepared as follows: 5 mL bovine plasma fibrinogen of recombinant protease was analyzed 12% SDS-PAGE. The solution (0.6% in 50 mMTris-HCl buffer, pH 7.5) was mixed with 200 μL thrombin solution (10 U/mL) and 5 mL calcium chloride fibrinolytic activity at pH 7.5 and 37°C (data not shown). solution (0.7%) in petri dish. After clot formation was observed Recombinant protease exhibited strong fibrinogenolytic (about 2 hours), 25μL of recombinant enzyme and plasmin activities. As shown in Figure 2, the ϒ-ϒ, α and β chains of addedprepared separately in different on theconcentrations same plate. (2.0,The plate1.0, 0.5, was 0.25, incubated 0.125, digestionfibrinogen in were all times. totally degraded by the enzymes at 37°C for 0.0625 µg/mL) was carefully placedon a plate. The enzymes were 30 min. However, the ϒ chains were more resistant to enzyme Fibrinolytic activity areaovernight of the at clear 37°C. zone The wasfibrinolytic determinated enzyme by activity the Image was Jcalculated [13], and theby measuring number of the units area was of calculatedthe clear zone using on the the standard fibrin plate. curve The of B. plasmin. The fibrinolytic activity was examined by fibrin plate assay and the fibrinolytic activities of recombinant protease from Determination of fibrinolytic activity-II blood was dripped and allowed to dry. Then the fabric was A cotton cloth was divided into 4 sections and 200 μL of formaldehyde was removed by washing with water. After