JSMC Biochemistry and Molecular Research

Research Article © Atakisi O, 2019

Evaluation of the Fibrinolytic Potential of Recombinant Subtilisin from 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 was determined. The recombinant 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 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 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 . 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 activator of fibrinolytic enzymes have such been as extensivelytissue-type (t-PA), (u-PA), and bacterial The fundamental pathophysiological mechanism underlying system and fibrinolytic system is disturbed [2]. haveinvestigated been considered and used as as therapeutic thrombolytic agents agents for thrombosis. [4]. Serine Notably,, 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 adheres is activated to the from unbroken plasminogen wall of blood by tissue vessels. plasminogen The fibrin activatorcomprising [3]. fibrinogen by 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 the concentration purified enzyme was determined (2 μL, 0.5 mg/mL),using a Bradford and 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 tyrosine activity per was min defined under as the the performed amount experimentalof enzyme that conditions. hydrolysed The the results substrate represent and produced the average 1 μg of 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 an degradation 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 the concentrations 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 drying, differentsoaked with applications 2% formaldehyde were made foron each 45 minutes part of the and fabric the excesswhich was divided into four parts. The fabric pieces were separately distilledincubated water with with 1 mL detergent of the subtilisin and 1 mL (0.5 of sterilemg/mL), distilled 1 mL ofwater the subtilisin and detergent (5% (v/v) tween 80), 1 mL of sterile with water, dried and the potential to remove blood was observed at 37°C for 1 hour. Following incubation, the fabric was washed Figure 1 Fibrinogen degradation of recombinant protease. The [14].Substrate specificity assay The proteolytic activity was measured according to a fibrinogen degradation productions were separated by 12% SDS-PAGE. Lane 1,marker; Lane 2, control (fibrinogen without casein, bovine serum albumin, lactoferrin and immunoglobulin recombinant protease); Lanes 3 to 7, fibrinogen degradations by Peterson’s modified method in the presence of blood clot, recombinant protease after 30, 60, 90, 120 min and overnight of incubation, respectively at 37°C.

JSMC Biochem Mol Res 3: 5 2/5 Human blood plasma contains a substantial amount of

proteins (60-85 mg per mL) [20]. These include albumin (~55%), globulin (~38%), fibrinogen (~7%), (pro) enzymes and enzymeprotease activityinhibitors of recombinant[21,22]. As shown protease in Figure were examined.(4), the effects The of fibrin, casein, bovine serum albumin and lactoferrin on the and proteolytic activity of its had low specify to serum albumin andrecombinant lactoferrin enzyme which showed blood proteins.the highest Also, specificity the enzyme against did fibrin not show proteolytic activity against immunoglobulin. A similar Figure 2 alkaline protease from C. tentaculata Comparison of the fibrinolytic activity of recombinant effect for serum albumin was also demonstrated by the CTSP protease from B.subtilisB. subtilis and plasmin. The evaluation of fibrinolytic [23]. A fibrinolytic serine activity by fibrin plate assay. The white elliptical area represents the protease (Brevithrombolase) Brevibacillus brevis demonstrated samplefibrinolytic solution region in by certain concentrations and plasma in was the carefully fibrin plate placed region. on M: Marker, pS: Purified Subtilisin. In the fibrin plate assay, 25 μL of

the plate and the plate was incubated for overnight at 37°C. Data are expressed as mean±SD of three independent experiments. subtilis and plasmin was compared. The clear zones produced differenceby recombinant was observed enzyme in andall concentrations plasmin did of not both significantly enzymes. Thesediffer onresults the fibrin indicate plate that (Figure recombinant 3). Interestingly, protease nois asignificant plasmin- dissolving the thrombi. like protease which can importantly degrade the fibrin, thereby Fibrinolytic potential of the recombinant protease degree of blood removal from the cotton fabric was found in the orderThe of result recombinant of fibrinolytic protease activity with is detergent shown in >recombinant Figure 4. The protease > sterile distilled water with detergent > sterile distilled water. Substrate specificity Recombinant enzyme degraded various protein substrates,

Figure 3 including fibrin, casein, BSA and lactoferrin. When the relative The result of fibrinolytic activity of recombinant protease. 1: activity of fibrin as a substrate is considered 100%, those of Enzyme; 2: Detergent+enzyme; 3: Detergent+sterile distilled water; casein, BSA, lactoferrin and immunoglobulin were 78.5%, 21.8%, 4: sterile distilled water. (A) Dripping and drying of blood of fabric. 12.05%, 0% (Figure 5). The caseinolytic activity of the enzyme (B) Wetting the fabric with 2% formaldehyde and washing after 45 was relatively high; however, activity was quite low against minutes (C) The final form of fabric. andBSA highand lactoferrin interest of (p<0.001). recombinant Also, protease the enzyme from didB. subtilis not show to activity against immunoglobulin substrate. The high specificity immunoglobulin is an important feature. fibrinas a fibrinolytic agent comparing to BSA, lactoferrin and Discussion

In the present study, we report fibrinolytic potential of recombinantabout 40 kD proteaseare combinant was found subtilisin to be higherfrom culture than the supernatant molecular of Bacillus sp. strain PTTC 1023. The molecular mass of Bacillus [17]. Themolecular weight of recombinant subtilisin was mass of fibrinolytic enzymes (18-38 kDa) purified from the genus

Figure 4 Effects of some substrates on recombinant protease. Data are found to be lower than KA38 (41 kDa), a fibrinolytic protease [18], but higher than many microbial fibrinolyticserine proteases such as (28 kDa) [19] and TPase (27.5 kDa) [20]. expressed as mean±SD of three independent experiments (p<0.001).. JSMC Biochem Mol Res 3: 5 3/5 protease from B. subtilis optimum fibrinolytic activity at pH 7.4 and 37°C, and showed PTTC 1023 was determined to be α- hydrolyticThe optimum activity toward temperature globulin, casein and pHand fibrinogen requirement [24]. of β-and γ-, which was similar to N-V protease [29], NJP [30] and recombinant protease was comparable to previously reported CSP [31], but absolutely different from that of FP84 [32] and subtilisinConclusion FS33 [33], where β-chains got hydrolyzed first. fibrinolytic enzymes isolated from such as Bafibrinase The data obtained indicates that recombinant enzyme is activity[25], Nattokinase suggested that [19] former and DJ-4 could [26]. be developed The optimum as an effective pH and subtilisin from B. subtilis temperature of recombinant subtilisin and its strong fibrinolytic thrombolytic drug capable of functioning at physiological , which can efficiently and directly conditions. Optimum activity exhibition of the enzyme in hashydrolyze advantages fibrinogen. of possible Recombinant production protease in large is amounts, a plasmin-like at low physiological conditions is an important because this feature fibrinolytic serine protease. Furthermore, recombinant enzyme reinforces its future therapeutic application as a thrombolytic agent. procedurecost, and with are easequite of advantageous purification. Highin terms purification of lower yield costs and in fold displayed by purified protease using a one-step purification potential candidate for the development of an antithrombotic Fibrinogen, a fibrin precursor, is a glycoprotein (about drugfibrinolytic and it therapy is being and considered nutraceutical for preclinical applications. studies It cans using a 340 kDa) and contains two sets of disulfide-bridged α-, β- and animal model to assess its in vivo thrombolytic potential. ϒ- chains. The recombinant subtilisin α, β and ϒ-ϒ chains of fibrinogen were totally degraded by the enzyme at 37°C References for 30 min. The fibrinolytic and fibrinogenolytic studiesalso 1. confirmed the plasmin-like activity of recombinant protease. disease from 2002 to 2 Fibrinolytic enzymes are typically classified as α-fibrinogenases Mathers CD, Loncar D. Projections of global mortality and burden of or β-fibrinogenases on the basis of specificity towards either 2. 030. PLoS Med. 2006; 3: 442. biochemical properties and antithrombotic effect of a novel the α -chain or β -chain of fibrinogen, respectively [27]. The Simkhada JR, Cho SS, Mander P, Choi YH, Yoo JC. Purification, recombinant protease can be classified as a αβ-fibrinogenase. The fibrinolytic pattern by recombinant subtilisin was similar to Streptomyces enzyme on carrageenan induced mice tail thrombosis the fibrin/fibrinogen degradation by Bafibrinase, a fibrinolytic 3. model. Thromb. Res. 2012; 129: 176-182. enzyme from Bacillus sp. strain AS-S20-I which was also interferingChang AK, Kimwith HY, blood Park homeostasis JE, Acharya throughP, Park IS, prothrombin Yoon SM, et activation al. Vibrio classified as a αβ-fibrinogenase [25]. In other study, although the vulnificus secretes a broad-specificity metalloprotease capable of α and β chains of fibrinogen were degraded by Brevithrombolase degradedafter 120 minutes, by Brevithrombolase α and β chains andwere plasmin degraded after by plasmin 120 and after 60 andMukhametova . LI, J. Bacteriol. AÄ 2005; 187: 6909-6916. 60 and 180 min, respectively. The ϒ-ϒ chains of fibrinogen were 4. -sina RB,Varfolomeev SD. Characterization of B. pumilus urokinase type plasminogen activator modified by phenylglyoxal. min, respectively [24]. Recombinant enzyme (AprEBS15) from 5. Bioorg. Khim. 2002; 28: 308-314. BS15 quickly degraded α and β chains of fibrinogen. The chain was hydrolyzed in 10 min and β chain was completely Nakamura T, Yamagata Y, Ichishima E. Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis (natto). Biosci Biotechnol degraded in 3 h. But as in this study, the γ-chain was not degraded 6. Biochem. 1992; 56: 1869-1871. even after 12 h [28]. The fibrinogenolysis pattern of recombinant gene from Bacillus stearothermophilus and its expression in Bacillus Jang JS, Kang DO, Chun MJ, Byun SM. Molecular cloning of a subtilisin J

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