J Thromb Thrombolysis DOI 10.1007/s11239-014-1093-2

In vitro thrombolytic activity of purified streptokinase extracted from Streptococcus equinus VIT_VB2 isolated from bovine milk Vaishnavi Babu • C. Subathra Devi

Ó Springer Science+Business Media New York 2014

Abstract Streptokinase (SK) is an extracellular enzyme secreted by various strains of b-hemolytic Streptococci. The main focus of the current study is to evaluate the in vitro thrombolytic activity of purified SK extracted from Streptococcus equinus VIT_VB2 (Accession no. JX406835) isolated from milk sample. The growth rate of S. equinus VIT_VB2 strain was studied with pH and biomass content which has positive significant effect on enzyme yield. A temperature of 10 °C and pH of 6 was found to be optimum for maximum SK activity. The specific activity of the purified SK produced by VIT_VB2 strain was found to be 6,585 IU mg-1. The molecular mass of the enzyme was determined as 47 kDa by SDS-PAGE. In vitro thrombolytic activity of purified SK was determined using synthetic chromogenic substrate S-2251, the activity of the purified enzyme was found to be 6,330 ± 2.2 IU. The purity of SK was compared with standard SK by HPLC. This is the first report which reveals the SK activity of S. equinus isolated from milk sample. Keywords Clot buster  Streptokinase  Streptococcus equinus VIT_VB2  b-Hemolytic Streptococci  Thrombolytics  Myocardial infarction

Introduction The incidence of the acute myocardial infarction is increasing worldwide. The condition occurs due to subsequent thrombus (blood clot) formation in blood vessels which could lead to

V. Babu  C. Subathra Devi (&) Industrial Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore 632 014, Tamil Nadu, India e-mail: [email protected]; [email protected]

thrombosis, stroke, pulmonary embolism, deep vein thrombosis [4, 5]. According to the World Health Organization report, cardiovascular diseases causing fatal to 17 million people, every year [26]. Therefore thrombolytic therapy has become a conventional treatment for myocardial infarction (AMI), but currently clinically prescribed thrombolytic drugs have such problems as delayed action and other side effects like bleeding, re-occlusion etc. [22, 23]. Streptokinase (SK) remains the most common thrombolytic agent used globally and known as an important indirect plasminogen activator [25]. It is produced by many strains of beta-hemolytic Streptococci of Lancefield group A, C and G [24]. The group C is preferred for SK production, as they do not produce erythrogenic toxin. SK is a extracellular protein, composed of 414 amino acids with a molecular mass of 47 kDa [6, 8]. The increasing potential of SK application promoted us to screen for novel SK producing organisms. Also the exponential increase in the application of SK in various fields in the last few decades demands extension in both qualitative improvement and quantitative enhancement. Now it is the leading fibrinolytic agent, included in the World Health Organization Model List of Essential Medicines [1, 25]. The study progressively focuses on Streptococcus equinus VIT_VB2 growth kinetics, effect of pH and temperature on SK activity and yield. To the best of our knowledge, this is the first report on the use of the new variant as potential producer of SK which was isolated from bovine milk. This offers considerable advantages over the other reported Streptococcus species.

Materials and methods Bovine serum albumin, tyrosine, thrombin, S-2251 (Valleu-lys-p-nitroaniline2HCl) and standard SK were

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obtained from Sigma Aldrich (Bangalore, India).Todd Hewitt broth, Glucose, yeast extract, ammonium sulphate, manganese chloride, sodium carbonate, sodium bicarbonate, magnesium sulfate, sodium acetate, sodium chloride, sodium hydroxide, ferrous sulfate were purchased from Hi Media laboratories (Mumbai, India). Copper-sulfate, Folin–Ciocalteau reagent, potassium dihydrogen phosphate, Trichloroacetic acid (TCA), Hydrochloric acid (HCl), Ethylene-diaminetetraacetic acid (EDTA), Tris were from Sisco research laboratories (SRL, Mumbai, India). Growth kinetics of Streptococcus equinus VIT_VB2 Streptococcus equinus VIT_VB2 was inoculated in 15 mL of Todd Hewitt broth, incubated for 12 h at 37 °C. 1 % of exponential phase inoculum was transferred to 250 mL Erlenmeyer flask containing 50 mL of Todd Hewitt broth. The flasks were incubated at 37 °C for 48 h. The growth kinetics, pH, cell biomass, SK activity of the strain was observed at regular intervals for 4 h. The culture broth was then centrifuged at 8,000 rpm for 10 min and the clear supernatant was used as the crude enzyme for SK activity by casein digestion method [20]. The growth curves were plotted and the curves were used to determine the generation time, growth rate constant and mean generation time. Production of streptokinase The SK production was carried out in 500 mL Erlenmeyer flasks containing 200 mL of the production medium [3]. S. equinus VIT_VB2 was incubated at 37 °C for 12 h with agitation of 150–200 rpm. Effect of pH and temperature on SK activity The production of SK at small scale or at commercial scale depends on several process parameters. These parameters vary for different Streptococcus strains being employed for the production and hence the process parameters like pH and temperature have been optimized to ensure the maximum enzyme activity. For the optimization of pH, 50 mL of Todd Hewitt broth was used. The pH of the medium was varied from 4.0–11.0 and incubated at 37 °C for 24 h. Similarly for temperature optimization, 50 mL of Todd Hewitt broth was used and pH was maintained at 7. The flasks were incubated at varying temperatures of 10, 25, 37, 45 and 60 °C for 24 h. The crude filtrate was extracted and the enzyme activity was calculated.

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Purification Ion exchange chromatography The culture filtrate of S. equinus VIT_VB2 was collected. The enzyme in the cell free supernatant was precipitated between 40 and 70 % saturation of ammonium sulfate. The precipitate was then eluted using 20 mM Tris–HCl (pH 7.5). These elutions were dialyzed overnight against 500 mL of 20 mM Tris–HCl at 4 °C. Then the protein solution was loaded onto column containing anion exchanger (DEAE-cellulose, HiMedia) with a bed volume of 25 mL equilibrated with 20 mM Tris–HCl buffer, pH 7.0. The bound proteins were eluted with a linear gradient of 0.05–0.5 M NaCl in 20 mM Tris–HCl, pH 7.0. Positive SK elutes were collected according to the absorbance of the fractions by UV spectrophotometer. The SK activity and protein concentration were measured and the specific activity was calculated. Size exclusion chromatography (Gel filtration) Gel filtration was carried out with Sephadex G-100 (1.5 cm in dm, 30 cm in gel bed height, 2 mL sample volume) equilibrated with column buffer 20 mM Tris–HCl (pH7.0). 2 mL of the sample was loaded on to the column with 1 mL/min flow rate. Fractions were eluted (2 mL) and protein content was analyzed at 280 nm. Determination of molecular weight by SDS-PAGE and purity by HPLC The molecular weight of the obtained enzyme was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) [14, 15]. The protein standards are phosphorylase b (molecular mass, 97.4 kDa), albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa) were used as markers. The fractions obtained from gel filtration column chromatography were analyzed for protein purity by High Performance Liquid Chromatography (HPLC) using a Waters 2487 HPLC system consisting of a dual detector, a Waters 1525 binary pump, and equipped with a Waters Symmetry C18 column (5 mm, 4.6150 mm) and Waters Sentry TM universal guard column (5 mm, 4.620 mm) (Waters Corporation, Milford, MA, USA). The mobile phase was acetonitrile and water in the ratio of 1:1. The protein was compared with standard SK (10 KU, Sigma-Aldrich) and purity was authenticated at 250 nm. The amount of SK present in the peak area was calculated using the formula:

In vitro thrombolytic activity of purified streptokinase

Response factor ¼ Area of standard  Amount of test=Area of test  Amount of standard Amount of test present in the peak area ¼ Amount of standard  Area of test  response factor=Area of standard Enzyme activity: casein digestion method The SK activity was determined using casein digestion method, which is based on the determination of the liberated tyrosine from casein after plasminogen activation. The values obtained are converted to 1 lM of tyrosine released per minute [20]. Determination of total protein content The total protein content was estimated by Lowry’s method. The quantity of protein was determined by the concentration of standard BSA versus absorbance at 550 nm in a UV spectrophotometer [17]. Blood clot lysis assay Clot lysis activity of the enzyme was determined by modified Holmstrom method [9, 18]. Fibrin plate method The modified fibrin plate method was used to determine the activity of purified SK against the clot [2, 14]. Partial clot lysis assay Clot lysis activity of the purified enzyme was determined by the percentage of RBC’s released at 542 nm. The percentage of total clot lysis was calculated with reference to the calibrated curve of RBCs used in the clot, in which the 100 % was equivalent to all the RBCs in the clot being released into the surrounding fluid [4, 10]. Chromogenic assay of streptokinase The in vitro plasminogen activation of SK was assayed using synthetic substrate S-2251 [7].The chromogenic assay method is based on the difference in absorbance between the p-nitroanilide (pNA) formed and the original substrate (H-D-Val-Leu-Lys-OH). The assay was performed using 96-well plates. Control samples were prepared with 20 lL 0.1 M Tris–HCl buffer, pH 7.4, 30 lL substrate S-2251(0.6 mM) and 100 lL plasminogen solution (0.1 IU/mL). The SK activity of the test was

determined from the standard SK graph. (1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, and 5,000 IU from a stock solution of 10,000 IU SK). Absorbance of the wells was measured at 405 nm (Fig. 8). Each SK dilution was assayed a minimum of three times. One unit was defined as the amount of enzyme activity that converts 1 lM of substrate/min/L. The enzyme activity was calculated using a total reaction volume of 150 lL and an optical path of 0.4 cm.

Results The specific growth rate with pH, biomass, SK activity and protein content was calculated for S. equinus VIT_VB2. The production of SK was gradually increased from lag phase to exponential phase at 8 h and continued to maintain in stationary phase at 12 h of the growth period. pH of the medium was gradually decreased with increase in time, which indicates the neutral condition for its growth and enzyme production. It is assumed that the bacteria stabilizes and maintains the pH (Fig. 1). The SK activity and the protein content gradually increased from exponential phase at 8 h of the growth and declined from 16th h (Fig. 2). The generation time of the S. equinus VIT_VB2 was found to be 24 min and the growth rate constant (k) was 0.045 h-1 respectively. Therefore the mean generation time of S. equinus VIT_VB2 was found to be 22.22 h (Fig. 2). Optimization of the culture conditions ensures ideal growth and in vitro enzyme production. The SK activity gradually increased from pH 4 (310 IU mL-1) and reached maximum at pH 6 (408 IU mL-1).The optimum pH for maximum SK production was found to be 6. At pH 7, 8, 9, 10 and 11, there was a considerable decrease in enzyme activity (Fig. 3) and maximum enzyme activity was observed after 24 h of incubation. There was a considerable increase in the SK activity at 10 °C (437 IU mL-1) and 37 °C (393 IU mL-1). The optimum temperature for SK activity was found to be 10 °C. The activity was reduced at 45 °C (246 IU mL-1) and 60 °C -1 (250 IU mL ) (Fig. 4). However, increase or decrease in optimum conditions affects the enzyme production. The purified SK showed maximum zone of hydrolysis on Casein–plasminogen overlay along with control and partially purified SK (Fig. 5). Blood clot lysis activity was observed visually at different concentrations from 20–50 lL of purified SK. The enzyme volume of 50 lL was able to liquefy the clot completely within 6 h of incubation at room temperature (Fig. 6) The total activity of crude SK from S. equinus VIT_VB2 was found to be 13463 IU with the total protein content of 51.5 mg from 50 mL of the production medium (Table 1).

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V. Babu, C. Subathra Devi Fig. 1 Growth rate, biomass and pH of S. equinus VIT_VB2

Fig. 2 Protein content and streptokinase acivity of S. equinus VIT_VB2

Fig. 3 Effect of pH on streptokinase activity

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Fig. 4 Effect of temperature on streptokinase activity

In vitro thrombolytic activity of purified streptokinase Fig. 5 SK activity on Casein plasminogen agarose overlay: a Partially purified SK b Standard streptokinase c Purified streptokinase from S. equinus VIT_VB2

Fig. 6 Blood clot lysis of purified streptokinase

The fractions eluted from first DEAE-cellulose showed 9.7 fold of purity and 32.5 % of yield. The specific activity was found to be 2541 IU mg-1. The active fractions obtained from DEAE-cellulose column was concentrated and then applied into a second Sephadex G-100 column. The fractions eluted from Sephadex G-100 showed only single peak with 25.2 fold of purity and 29.3 % of yield (Table 1). However, the most active fractions from 10 to 19 of the peak were pooled together and then tested for SK activity. The specific activity of the purified SK extracted from S. equinus VIT_VB2 was found to be 6,585 IU mg-1. A clear zone of hydrolysis on fibrin degradation agar plate

Fig. 7 a Zone of hydrolysis by partially purified enzyme b Zone of hydrolysis by purified enzyme (30 mm) on fibrin plate

of 30 mm indicates the fibrinolytic activity of the enzyme (Fig. 7). In vitro clot lysis assay was also performed to confirm the activity of SK on blood clots. Based on the results it was found that the enzyme could lyse both natural clot as well as synthetic clot of fibrinogen, plasmin and thrombin. The release of RBC’s was 100 % with purified SK at 20th and 30th min of incubation, while the control clot indicated no release of RBC’s (Table 2). The fibrinolytic activity of purified SK using S-2251 was calculated

Table 1 Purification of streptokinase Purification steps

Relative activity (U mL-1)

Total activity (U)

Total protein content (mg)

Specific activity (U mg-1)

Fold purified

% Yield 100

Crude enzyme

269

13,463

51.5

261

1

Ammonium sulfate (%) precipitate

554

5,259

7.03

748

2.9

39.1

Dialysis

862

4,743

3.41

1,391

5.3

35.2

Ion exchange chromatography

1,093

4,371

1.72

2,541

9.7

32.5

Gel filtration chromatography

1,580

3,951

0.6

6,585

25.2

29.3

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using the calibration curve of standard SK. SK activity of the purified enzyme with chromogenic substrate S-2251 was found to be 6330 ± 2.2 IU. In SDS-PAGE, precipitated enzyme, partially purified enzyme and purified were electrophoresed. The band around 47 kDa confirms the

Table 2 Percentage of RBC’s released in partial clot lysis assay Time

10 min (%)

20 min (%)

30 min (%)

Purified

94

100

100

Partially purified

45

67

93

80–90 % precipitate

10

23

76

1

2

2

Control

presence of purified SK (Fig. 8). The fractions from size exclusion chromatography were analyzed for protein content and SK activity. The fractions eluted from 10 to 20 expressed maximum SK activity and protein content (Fig. 9), which was pooled together and analyzed for purity of the SK. The retention time and purity was analyzed and confirmed by HPLC with standard SK as reference. The retention time of purified SK was 2.08 min (Fig. 10b) which is merely equal to standard SK 2.14 min (Fig. 10a). The amount of protein present was calculated using the peak area from the HPLC profile of standard SK and purified SK from S. equinus VIT_VB2. The amount of SK present in the peak area (Fig. 10b) was determined as 199 lg/mL.

Discussion

Fig. 8 SDS-PAGE profile—SK from S. equinus VIT_VB2 L1 (Lane 1), Protein marker; L2 (Lane 2), 60 % ammonium sulfate precipitate; L3 (Lane 3), partially purified enzyme; L4 (Lane 4), purified streptokinase from DEAE-cellulose column chromatography; L5 (Lane 5), purified streptokinase from Sephadex G-100 gel filtration column chromatography

Fig. 9 Gel filtration on Sephadex G-200 column eluted with 0.020 M Tris–HCl buffer, pH 7.6. Symbols D—D OD at 280 nm (mg/mL), O–O Streptokinase activity (U/mL)

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According to the studies reported by Karush et al. [12] pH is one of the important limiting factor for the growth of bhemolytic Streptococci. Comparison of this study with other existing reports showed, simultaneous adjustment of pH and temperature with standard inoculum size of 1 % could produce high yield of SK. The influence of temperature on growth, SK production and activity of Streptococcus equisimilis H46A has been investigated over the temperature range of 28–43 °C [21]. When compared to other media, Baewald production medium was suitable for the efficient production of SK [3]. In comparison with study reported by Malke et al. the SK activity and specific activity was found to be 8 to 100 IU mL-1 and 55 IU mg-1, respectively [19]. SK activates the plasminogen, in non-enzymatic mode to plasmin which dissolves the fibrin clot formed in the agar and observed as clear zone of hydrolysis indicating the fibrinolytic activity. The fibrin degradation activity of SK from S. equinus VIT_VB2 showed similar activity on fibrin clot indicating clear zone

In vitro thrombolytic activity of purified streptokinase

Fig. 10 HPLC profile of a Standard streptokinase b Purified streptokinase from S. equinus VIT_VB2

of hydrolysis as described by Leigh et al. [16]. The apparent molecular weight of the bovine plasminogen activator, SK, from S. equinus VIT_VB2 was similar to the purified SK from group A S. pyogenes, group C S. equisimilis H46A and group G Streptococcus sp. strain G19909 [11, 13]. The SK activity of S. equinus VIT_VB2 was found to be 3,951 IU, which was 29.3 fold higher than the results obtained from the wild strains of S. equisimilis [1]. The increasing potential of SK application promoted us to produce SK from S. equinus VIT_VB2. The present study reports the production of SK from non enterococcal group D S. equinus isolated from bovine milk and has showed a new variance in the field of thrombolytic agents. The production of SK and its activity depends on the variation with production medium, pH, temperature and incubation time. The future study will be focused on analyzing the

media optimization, strain improvement and recombinant gene expression for enhanced production as well as enzyme kinetics and stability of SK. Acknowledgments The authors are thankful to Vellore Institute of Technology for the constant encouragement, help and support for extending necessary facilities. They also extended their appreciation to Technology Business Incubator (TBI), VIT University for the use of HPLC facility and technical assistance. Conflict of interest of Interest.

The authors declare that they have no conflict

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