1/5 J Cardiol Clin Res 1(1): 1147.JSMC Biochem Mol Res 3: 5

JSMC Biochemistry and Molecular Research

Submitted: 22 May 2019 | Accepted: 16 June 2019 | Published: 20 June 2019

*Corresponding author: Canan Gulmez, Department of Pharmacy Services, Tuzluca Vocational High School, Igdir University, Igdir-Turkey, Tel: 90-4762230010/3406; Email: canan_glm@hotmail.com

Copyright: © 2019 Atakisi O, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Atakisi O, Gulmez C, Ozturkler M (2019) Evaluation of the Fi-brinolytic Potential of Recombinant Subtilisin from Bacillus subtilis. JSMC Biochem Mol Res 3: 5.

Evaluation of the Fibrinolytic Potential of Recombinant Subtilisin from Bacillus subtilis

Onur Atakisi1, Canan Gulmez2*, and Melek Ozturkler1

1Department of Chemistry, Kafkas University, Turkey2Department of Pharmacy Services, Igdir University, Turkey

AbstractThe 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

IntroductionThromboembolic diseases, including cardiovascular disease,

cerebrovascular disease and venous thromboembolism, are within the leading causes of death as exposited in the global mortality projections to 2030 by WHO [1]. Thromboembolism often consists when the physiological equilibrium between coagulation system and fibrinolytic system is disturbed [2]. The fundamental pathophysiological mechanism underlying cardiovascular diseases is the formation of fibrin (blood clots), which adheres to the unbroken wall of blood vessels. The fibrin comprising fibrinogen by thrombin activation is lysed by plasmin, which is activated from plasminogen by tissue plasminogen activator [3].

The uptake of thrombolytic drugs that dissolve blood clots for treatment is only an alternative to surgical interventions to remove or by pass the blockage [2]. Fibrinolytic enzymes, which are widely used in the traditional treatment of thromboembolism, have vital disadvantages such as high cost, short half-life, high therapeutic dose, low specificity to fibrin, bleeding complications and allergic reactions. Therefore, it is important to investigate

new fibrinolytic enzymes produced from natural sources for low cost, no side effects and safety, specificity and efficacy of fibrinolytic treatment [4].

A variety of fibrinolytic enzymes such as tissue-type plasminogen activator (t-PA), urokinase (u-PA), and bacterial plasminogen activator streptokinase have been extensively investigated and used as thrombolytic agents [4]. Serine proteases, which show fibrinolytic/fibrinogenolytic activity, have been considered as therapeutic agents for thrombosis. Notably, the genus Bacillus has been well regarded for its role in the production of a variety of industrially important enzymes. The fibrinolytic enzymes from Bacillus sp. such as subtilisin NAT [5], subtilisin J [6] and subtilisin E [7], have been purified and characterized for the development of strong thrombolytic drugs. The recombinant subtilisin from B. subtilis strain PTTC 1023 was identified as fibnoloytic enzyme by Ghasemi et al., [8]. Ghasemi et al., showed that the recombinant enzyme encoded by the aprE (Accession No. HQ699519.1) has higher subtilisin activity other reported subtilisins and the E. coli transformants. In a later study, proteolytic activity of subtilisin from B. subtilis strain PTTC 1023 was studied for use in detergent applications [9]. In this study, it was carried out a comprehensive analysis of the recombinant enzyme produced for use in pharmaceutical purposes. The aim of the study was to investigate the potential of the enzyme for treatment of thromboembolic diseases determining the activities of fibrinolytic, fibrinogenolytic and proteolytic of the recombinant subtilisin from Bacillus subtilis.

Materials and MethodsChemicals

Unless specified, all chemicals, substrates, and reagents with the analytical grade or the highest available purity were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Research Article © Atakisi O, 2019

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Production of recombinant enzyme

The recombinant enzyme was produced as described in our previous study [9]. Briefly, the coding sequence of subtilisin was optimized for E. coli codon usage. The subtilisin gene (NCBI accession no. HQ699519.1) from Bacillus subtilis PTTC 1023 was directly synthesized and cloned in expression vector pD441-NH by DNA 2.0 (Atum, Newark, CA, USA). After, recombinant vectors were transformed into competent cells E.coli BL21 (DE3). The C-terminal 6xHis-tagged enzyme was purified in a single-step procedure by affinity chromatography using Ni-NTA agarose resin (Qiagen GmbH, Hilden, Germany).

Determination of fibrinogenolytic activity

Fibrinogenolytic activity was determinated as follows: 80 mL of 1% human fibrinogen prepared in 50 mMTris/HCl buffer containing 0.1 M NaCl was incubated with 2μg of purified subtilisin at various periods (30, 60, 90, 120 minutes and overnight) at 37°C [10]. Then, 20 μL samples taken from the reaction medium were analyzed using 12% SDS-PAGE according to Laemmli’smethod [11].

Determination of fibrinolytic activity-I

Fibrinolytic activity was determined according to Astrup and Mullertz’s method (1952) [12] with slight modification. The fibrin plate was prepared as follows: 5 mL bovine plasma fibrinogen 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 solution (0.7%) in petri dish. After clot formation was observed (about 2 hours), 25μL of recombinant enzyme and plasmin prepared in different concentrations (2.0, 1.0, 0.5, 0.25, 0.125, 0.0625 µg/mL) was carefully placedon a plate. The enzymes were added separately on the same plate. The plate was incubated overnight at 37°C. The fibrinolytic enzyme activity was calculated by measuring the area of the clear zone on the fibrin plate. The area of the clear zone was determinated by the Image J [13], and the number of units was calculated using the standard curve of plasmin.

Determination of fibrinolytic activity-II

A cotton cloth was divided into 4 sections and 200 μL of blood was dripped and allowed to dry. Then the fabric was soaked with 2% formaldehyde for 45 minutes and the excess formaldehyde was removed by washing with water. After drying, different applications were made on each part of the fabric which was divided into four parts. The fabric pieces were separately incubated with 1 mL of the subtilisin (0.5 mg/mL), 1 mL of the subtilisin and detergent (5% (v/v) tween 80), 1 mL of sterile distilled water with detergent and 1 mL of sterile distilled water at 37°C for 1 hour. Following incubation, the fabric was washed with water, dried and the potential to remove blood was observed [14].

Substrate specificity assay

The proteolytic activity was measured according to a Peterson’s modified method in the presence of blood clot, casein, bovine serum albumin, lactoferrin and immunoglobulin

substrates [15]. One mL of a 10 mg/mL substrate solution was mixed with the purified enzyme (2 μL, 0.5 mg/mL), and the reaction mixtures were incubated at 37°C for 20 min. The change in substrate concentration was determined using a Bradford protein assay [16]. There action was stopped by adding 1 mL of 2% (w/v) trichloroaceticacid. 1.5 mL of 0.5 M Na2CO3 and 0.5 mL of Folin-Ciocalteu® phenol reagent was added to the 0.5 mL supernant. The solution was incubated at 37°C for 30 min. The absorbance was calculated at 660 nm spectrophotometrically against a blank control. In the activity measurement, no enzyme was added to the medium while preparing the control and all other steps were performed according to standard protocol. One unit of proteolytic activity was defined as the amount of enzyme that hydrolysed the substrate and produced 1 μg amino acid equivalent to tyrosine per min under the performed experimental conditions. The results represent the average of three experiments. In this assay, enzyme activity was expressed as a relative activity, compared to that of non-enzyme treated control.

Results

Fibrinogenolytic activity

The molecular weight of the enzyme analyzed by 10% SDS-PAGE was about 40 kDa (Figure 1). Fibrinogen degradation of recombinant protease was analyzed 12% SDS-PAGE. The recombinant subtilisin of B. subtilis displayed an optimum fibrinolytic activity at pH 7.5 and 37°C (data not shown). Recombinant protease exhibited strong fibrinogenolytic activities. As shown in Figure 2, the ϒ-ϒ, α and β chains of fibrinogen were totally degraded by the enzymes at 37°C for 30 min. However, the ϒ chains were more resistant to enzyme digestion in all times.

Fibrinolytic activity

The fibrinolytic activity was examined by fibrin plate assay and the fibrinolytic activities of recombinant protease from B.

Figure 1 Fibrinogen degradation of recombinant protease. The fibrinogen degradation productions were separated by 12% SDS-PAGE. Lane 1,marker; Lane 2, control (fibrinogen without recombinant protease); Lanes 3 to 7, fibrinogen degradations by recombinant protease after 30, 60, 90, 120 min and overnight of incubation, respectively at 37°C.

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Figure 2 Comparison of the fibrinolytic activity of recombinant protease from B.subtilis and plasmin. The evaluation of fibrinolytic activity by fibrin plate assay. The white elliptical area represents the fibrinolytic region by B. subtilis and plasma in the fibrin plate region. M: Marker, pS: Purified Subtilisin. In the fibrin plate assay, 25 μL of sample solution in certain concentrations was carefully placed on 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 by recombinant enzyme and plasmin did not significantly differ on the fibrin plate (Figure 3). Interestingly, no significant difference was observed in all concentrations of both enzymes. These results indicate that recombinant protease is a plasmin-like protease which can importantly degrade the fibrin, thereby dissolving the thrombi.

Fibrinolytic potential of the recombinant protease

The result of fibrinolytic activity is shown in Figure 4. The degree of blood removal from the cotton fabric was found in the order of recombinant protease with detergent >recombinant protease > sterile distilled water with detergent > sterile distilled water.

Substrate specificity

Recombinant enzyme degraded various protein substrates, including fibrin, casein, BSA and lactoferrin. When the relative activity of fibrin as a substrate is considered 100%, those of casein, BSA, lactoferrin and immunoglobulin were 78.5%, 21.8%, 12.05%, 0% (Figure 5). The caseinolytic activity of the enzyme was relatively high; however, activity was quite low against BSA and lactoferrin (p<0.001). Also, the enzyme did not show activity against immunoglobulin substrate. The high specificity and high interest of recombinant protease from B. subtilis to fibrinas a fibrinolytic agent comparing to BSA, lactoferrin and immunoglobulin is an important feature.

DiscussionIn the present study, we report fibrinolytic potential of

about 40 kD are combinant subtilisin from culture supernatant of Bacillus sp. strain PTTC 1023. The molecular mass of recombinant protease was found to be higher than the molecular mass of fibrinolytic enzymes (18-38 kDa) purified from the genus Bacillus [17]. Themolecular weight of recombinant subtilisin was found to be lower than KA38 (41 kDa), a fibrinolytic protease [18], but higher than many microbial fibrinolyticserine proteases such as nattokinase (28 kDa) [19] and TPase (27.5 kDa) [20].

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 protease inhibitors [21,22]. As shown in Figure (4), the effects of fibrin, casein, bovine serum albumin and lactoferrin on the enzyme activity of recombinant protease were examined. The recombinant enzyme showed the highest specificity against fibrin and proteolytic activity of its had low specify to serum albumin and lactoferrin which blood proteins. Also, the enzyme did not show proteolytic activity against immunoglobulin. A similar effect for serum albumin was also demonstrated by the CTSP alkaline protease from C. tentaculata [23]. A fibrinolytic serine protease (Brevithrombolase) Brevibacillus brevis demonstrated

Figure 3 The result of fibrinolytic activity of recombinant protease. 1: Enzyme; 2: Detergent+enzyme; 3: Detergent+sterile distilled water; 4: sterile distilled water. (A) Dripping and drying of blood of fabric. (B) Wetting the fabric with 2% formaldehyde and washing after 45 minutes (C) The final form of fabric.

Figure 4 Effects of some substrates on recombinant protease. Data are expressed as mean±SD of three independent experiments (p<0.001)..

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optimum fibrinolytic activity at pH 7.4 and 37°C, and showed hydrolytic activity toward globulin, casein and fibrinogen [24].

The optimum temperature and pH requirement of recombinant protease was comparable to previously reported fibrinolytic enzymes isolated from bacteria such as Bafibrinase [25], Nattokinase [19] and DJ-4 [26]. The optimum pH and temperature of recombinant subtilisin and its strong fibrinolytic activity suggested that former could be developed as an effective thrombolytic drug capable of functioning at physiological conditions. Optimum activity exhibition of the enzyme in physiological conditions is an important because this feature reinforces its future therapeutic application as a thrombolytic agent.

Fibrinogen, a fibrin precursor, is a glycoprotein (about 340 kDa) and contains two sets of disulfide-bridged α-, β- and ϒ- chains. The recombinant subtilisin α, β and ϒ-ϒ chains of fibrinogen were totally degraded by the enzyme at 37°C for 30 min. The fibrinolytic and fibrinogenolytic studiesalso confirmed the plasmin-like activity of recombinant protease. Fibrinolytic enzymes are typically classified as α-fibrinogenases or β-fibrinogenases on the basis of specificity towards either the α -chain or β -chain of fibrinogen, respectively [27]. The recombinant protease can be classified as a αβ-fibrinogenase. The fibrinolytic pattern by recombinant subtilisin was similar to the fibrin/fibrinogen degradation by Bafibrinase, a fibrinolytic enzyme from Bacillus sp. strain AS-S20-I which was also classified as a αβ-fibrinogenase [25]. In other study, although the α and β chains of fibrinogen were degraded by Brevithrombolase after 120 minutes, α and β chains were degraded by plasmin after 60 and 180 min, respectively. The ϒ-ϒ chains of fibrinogen were degraded by Brevithrombolase and plasmin after 120 and 60 min, respectively [24]. Recombinant enzyme (AprEBS15) from B. pumilus BS15 quickly degraded α and β chains of fibrinogen. The chain was hydrolyzed in 10 min and β chain was completely degraded in 3 h. But as in this study, the γ-chain was not degraded even after 12 h [28]. The fibrinogenolysis pattern of recombinant

protease from B. subtilis PTTC 1023 was determined to be α- β-and γ-, which was similar to N-V protease [29], NJP [30] and CSP [31], but absolutely different from that of FP84 [32] and subtilisin FS33 [33], where β-chains got hydrolyzed first.

ConclusionThe data obtained indicates that recombinant enzyme is

subtilisin from B. subtilis, which can efficiently and directly hydrolyze fibrinogen. Recombinant protease is a plasmin-like fibrinolytic serine protease. Furthermore, recombinant enzyme has advantages of possible production in large amounts, at low cost, and with ease of purification. High purification yield and fold displayed by purified protease using a one-step purification procedure are quite advantageous in terms of lower costs in fibrinolytic therapy and nutraceutical applications. It cans a potential candidate for the development of an antithrombotic drug and it is being considered for preclinical studies using animal model to assess its in vivo thrombolytic potential.

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