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Purification and characterization of a fibrinolytic enzyme


World J Microbiol Biotechnol (2012) 28:2479–2486 DOI 10.1007/s11274-012-1055-9

ORIGINAL PAPER

Puri?cation and characterization of a ?brinolytic enzyme from Streptomyces sp. XZNUM 00004
Xiuyun Ju ? Xiaoying Cao ? Yong Sun ? Zhe Wang ? Chengliang Cao ? Jinjuan Liu Jihong Jiang
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Received: 21 December 2011 / Accepted: 9 April 2012 / Published online: 17 April 2012 ? Springer Science+Business Media B.V. 2012

Abstract A ?brinolytic enzyme (SFE1) from Streptomyces sp. XZNUM 00004 was puri?ed to electrophoretic homogeneity with the methods including ammonium sulfate precipitation, polyacrylamide gel, DEAE-Sepharose Fast Flow anion exchange and gel-?ltration chromatography. The molecular weight of SFE1 was estimated to be 20 kDa by SDS-PAGE, ?brin zymography, and gel ?ltration chromatography. The isoelectric point was 4.9. Km and Vmax values were 0.96 mg/ml and 181.8 unit/ml, respectively. It was very stable at pH 5.0–8.0 and below 65 °C. The optimum pH for enzyme activity was 7.8. The optimum temperature was 35 °C. The ?brinolytic activity of SFE1 was enhanced by Na?, K?, Mn2?, Mg2?, Zn2? and Co2?. Conversely, Cu2? showed strong inhibition. Furthermore, the ?brinolytic activity was strongly inhibited by PMSF, and partly inhibited by EDTA and EGTA. SFE1 rapidly hydrolyzed the Aa-chain of ?brinogen, followed by the Bb-chain and ?nally the c-chain. The ?rst 15 amino acids of the N-terminal sequence were APITLSQGHVDVVDI. Additionally, SFE1 directly digested ?brin and not by plasminogen activators in vitro. SFE1 can be further developed as a potential candidate for thrombolytic therapy. Keywords Fibrinolytic enzyme ? Puri?cation ? Streptomyces ? Thrombolytic therapy

Introduction It is well known that thrombotic disease is regarded as a major cause of human death in the worldwide. And thrombolytic therapy is still the best way to achieve recanalization in these diseases nowadays (Marder 2009). Thrombolytic drugs used for clinical applications are divided into three generations (Wang et al. 2010). The ?rst is composed of streptokinase (SK) (Banerjee et al. 2004), urokinase (UK) (Blasi and Sidenius 2010), etc. The second generation consists of tissue plasminogen activator (t-PA) (Kent et al. 2004), single-chain urokinase-type plasminogen activator (scu-PA, or pro-urokinase, pro-UK) (Blasi and Sidenius 2010), etc. The third generation are novel agents derived from the ?rst or the second generations of thrombolytic agents by modern molecular biological ? techniques (mutants of pro-UK and t-PA) (Mar?n et al. 2009; Killer et al. 2010). Despite widespread used, these ?brinolytic agents suffer important shortcomings including bleeding complications, short half-life, high cost, the risk of allergic reactions and large therapeutic doses (Killer et al. 2010). Therefore, it is necessary to search for novel ?brinolytic agents from other sources. In the last decade, ?brinolytic enzymes have been identi?ed from various sources including actinomycetes (Simkhada et al. 2010), bacteria (Wang et al. 2008; Agrebi et al. 2009), fungi (Kim et al. 2008), marine polychaete worms (Deng et al. 2010), and so on. Actinomycetes are a special group of microorganisms, which have been demonstrated to be excellent producers of ? bioactive and structurally novel metabolites (Berdy 2005; He et al. 2010; Kavitha et al. 2010). Therefore, we decided to investigate a thrombolytic agent from actinomycetes. During a screening programme on actinomycetes from rhizosphere soil of Chinese Polygonatum sibiricum Red.,

X. Ju ? X. Cao ? Y. Sun ? Z. Wang ? C. Cao ? J. Liu ? J. Jiang (&) The Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, Jiangsu Normal University, Xuzhou 221116, China e-mail: wjwxwy@126.com

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Streptomyces sp. XZNUM 00004 producing a strong ?brinolytic enzyme was selected. In this paper, puri?cation and characterization of a ?brinolytic enzyme, SFE1, from Streptomyces sp. XZNUM 00004 were investigated.

Materials and methods Materials Thrombin was purchased from Hangzhou Medicine Co. (China). Bovine serum albumin (BSA), azocasein, trichloroacetic acid (TCA), L-tyrosine, ethylene diamine tetraacetic acid (EDTA), phenylmethylsulfonyl ?uoride (PMSF), and ethylene glycol-bis (b-aminoethyl ether)N,N,N0 ,N0 -tetraacetic acid (EGTA) were obtained from Sigma-Aldrich Co. (USA). Pepstatin, aprotinin, and soybean trypsin inhibitor b-mercaptoethanol were purchased from Shanghai Sangon Co. (China). DEAE-Sepharose Fast Flow and Bio-gel Polyacrylamide Gel were acquired from BIO-RAD (USA). Fibrinogen (Human) was from FIBRORAAS (Shanghai, China). The protein molecular weight markers (SM0671 and SM0431) for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Fermentas (Canada). Urokinase was acquired from Chinese Medicine Testing Institute. All other reagents were of analytical grade. Strain and culture conditions Strain XZNUM 00004 was isolated from rhizosphere soil of P. sibiricum Red., a Chinese traditional medicinal plant. It was identi?ed as a strain related to Streptomyces based on morphological and 16S rDNA sequences in our previous study (Ju et al. 2010) (Accession no. GU211008 in GenBank). A loopful of spores were scraped from the plate and inoculated into seed medium containing 2 % beef extract, 0.1 % yeast extract, 0.05 % peptone, 0.05 % glucose and 0.05 %NaCl. It was cultivated in shaking incubator (180 r/min) at 28 °C. After 24 h cultivation, a 5 % (v/v) inoculum was transferred into fermentation medium (2 % soluble starch, 0.1 % KNO3, 0.05 % NaCl, 0.05 % K2HPO4, 0.05 % MgSO4, 0.001 % FeSO4, pH 7.2) and fermented at 28 °C at 180 r/min for 5 days. Culture supernatant of the strain was obtained via centrifugation (1,7539g, 10 min). Enzyme puri?cation Solid (NH4)2SO4 was slowly added to the supernatant to 80 % saturation. The mixture was stored at 4 °C overnight. The precipitate was collected by centrifugation (7,0129g, 10 min, 4 °C) and resuspended in 10 mM Tris–HCl buffer

(pH 8.0). After removal of insoluble materials by centrifugation (7,0129g, 5 min, 4 °C), the suspension was applied to a 2.6 cm 9 30 cm Polyacrylamide Gel column (Bio-gel P60) equilibrated with 10 mM Tris–HCl buffer (pH 8.0) at a ?ow rate of 1 ml/min. The active fractions were pooled and further puri?ed using DEAE-Sepharose Fast Flow anion exchange column (2.0 cm 9 6 cm) equilibrated with 10 mM Tris–HCl buffer (pH 8.0). The bound proteins were eluted with a linear gradient of 0–1 mol/l NaCl in the same buffer at a ?ow rate of 2 ml/ min. The active fractions were pooled, lyophilized and further puri?ed by a 1.5 cm 9 75 cm Polyacrylamide Gel column (Bio-gel P30) equilibrated with 10 mM Tris–HCl buffer (pH 8.0) at a ?ow rate of 0.5 ml/min. The fractions with strong ?brinolytic activity were pooled, lyophilized and used as the puri?ed enzyme preparation. Protein concentration Protein concentration was determined by the method of Bradford (Bradford 1976) using bovine serum albumin as the standard protein, measuring the absorbance at 595 nm. Assay of enzyme activity on ?brin plate Fibrinolytic activity was measured according to the ?brin plate method with some modi?cations (Astrup and Mullertz 1952). Brie?y, 5 ml ?brinogen solution [5 mg/ml ?brinogen (Human) in 0.9 % NaCl, standing for 5 min at 45 °C] was mixed with 5 ml agar solution [0.5 % agar in 0.9 % NaCl, standing for 30 min at 45 °C]. The mixture was poured into the plate (9 cm diameter) with 50 ll thrombin solution [200 IU/ml thrombin in 0.9 % NaCl]. The plate was left for 30 min at room temperature to form a ?brin clot layer. 10 ll sample solution was placed on the plate. The plate was incubated at 37 °C for 18 h. The ?brinolytic activity was quanti?ed by measuring the diameter of the clear zone according to the standard curve using urokinase (Zheng et al. 2000). The activity was expressed in the unit of clear zone area per lg of the protein (mm2/lg) (Mander et al. 2011). Native PAGE Native PAGE was performed at room temperature with the use of a 5 % stacking gel and a 12 % separating gel in a BIO-RAD Mini-Cell electrophoresis system. Electrophoresis was run at constant voltage of 90 V in 37 mM Tris–glycine buffer (pH 8.9) until the tracking dye reached the bottom of the gel. The gel was stained with 0.25 % Coomassie blue R-250 for 20 min; and destined with 10 % methanol, 10 % acetic acid until the background was clear.

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Determination of molecular weight and ?brin zymography The molecular weight of SFE1 was determined by the method of SDS-PAGE with a 5 % stacking gel and a 12 % separating gel. Protein molecular weight markers were used as reference proteins. Gel was stained with 1 % (w/v) Coomassie Brilliant Blue R-250 and destained with destaining solution (ethanol: acetic acid: distilled water = 7:1:12, v/v/v). The molecular mass of SFE1 was also analyzed by gel ?ltration chromatography using a 1.0 cm 9 90 cm column (Sephadex G-100) at a ?ow rate of 0.4 ml/min. Molecular weight markers for gel ?ltration chromatography included b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12 kDa). Fibrin zymography has been shown to be a sensitive quantitative technique. The gel used for ?brin zymography contained a protease substrate, ?brin. Samples were unheated in SDS-loading buffer lacking reducing agent. Following electrophoresis, SDS was removed in the gel by exchange in Triton X-100. This allowed the ?brinolytic enzyme in the sample to renature and activate. By overnight incubation in an appropriate buffer, the activity was revealed by an absence of protein staining in the region where ?brin had been digested. Speci?cally, Fibrin zymography was performed out according to Kim et al. (1998) with slight modi?cations. Fibrinogen (2.0 mg/ml) and thrombin (1 U/ml) were mixed with 12 % polyacrylamide gel solution. Following electrophoresis of the puri?ed enzyme on the ?brin gel, the gel was soaked in 2.5 % Triton X-100 containing Tris–HCl (50 mM) buffer (pH 7.8) and distillation water for 30 min at room temperature, respectively. Subsequently, the gel was incubated in 30 mM Tris–HCl buffer (pH 7.8) containing 200 mM NaCl at 35 °C for 16 h. The gel was stained with Coomassie blue for 1 h and then destained. Determination of isoelectric point Isoelectric focusing analysis of SFE1 was carried out using disc electrophoresis in 7.5 % polyacrylamide gel containing 3 % ampholyte with pH range 3.0-10.0. The cylindrical gels with and without 1 lg SFE1 were run using 5 % (v/v) H3PO4 as anolyte and 2 % (w/v) NaOH as catholyte. Focusing was carried out at a constant voltage of 100 V until the current was close to zero. The gel containing sample was stained with 1 % (w/v) Coomassie Brilliant Blue R-250 and destained with destaining solution (ethanol: acetic acid: distilled water = 7:1:12, v/v/v). The blank gel was cut into segments of 5 mm length from anolyte to catholyte. The pH was determined after the gel segments

eluted in 1 ml bidistilled water for 24 h. The standard curve was made according to pH and the gel length. The isoelectric point (pI) of SFE1 was estimated by the length of the protein band to anolyte according to the standard curve (Li 1998). Effect of pH on ?brinolytic enzyme activity and stability The optimal pH for SFE1 was determined through measuring the enzyme activity within a pH range of 4.0–10.0 at 37 °C for 18 h. The pH stability of SFE1 was estimated by measuring the remaining ?brinolytic activity after incubating the enzyme for 1 h at 37 °C with different buffers. The following buffer systems were used: 100 mM sodium acetate buffer (pH 4.0–5.0), 100 mM phosphate buffer (pH 6.0–8.0), and 100 mM glycine-NaOH buffer (pH 9.0– 10.0).The enzyme activity was measured according to the ?brin plate method. Maximum activity was represented 100 % and other sample activities were expressed as a relative percentage to the maximum. Effect of temperature on ?brinolytic enzyme activity and stability The optimal temperature of SFE1 was determined by measuring the enzyme activity at different temperatures (25–85 °C) for 18 h. The thermal stability of SFE1 was evaluated by measuring the remaining ?brinolytic activity after incubating the enzyme in various temperatures (25–85 °C) for 1 h at pH 7.8. The ?brinolytic activity was assessed by the ?brin plate method. Effect of metal ions and protease inhibitors on ?brinolytic activity The effect of seven protease inhibitors on the activity of SFE1 was studied using pepstatin (0.1 mg/ml), aprotinin (0.1 mg/ml), soybean trypsin inhibitor (0.1 mg/ml), PMSF (1 mM), EGTA (1 mM), EDTA (1 mM) and b-mercaptoethanol (1 mM). SFE1 was incubated with these inhibitors at corresponding concentration for 30 min at 37 °C and the residual activity was measured by the ?brin plate method. The activity of SFE1 measured in the absence of inhibitors was taken as 100 %. The effect of various metal ions on the activity of SFE1 was investigated using NaCl, KCl, CaCl2, MnCl2, MgCl2, ZnCl2, CuCl2, CoCl2 and FeSO4. SFE1 was incubated with different metal ion saline solutions (5 mM) for 2 h at 37 °C. The residual activity was measured by the ?brin plate method. The activity of SFE1 assayed in the absence of metal ions was taken as 100 %.

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Assay of ?brinogenolytic activity Fibrinogenolytic activity was detected via a modi?ed ?brinogenolytic assay (Cha et al. 2010). 180 ll ?brinogen (2.0 % human ?brinogen in 50 mM Tris–HCl buffer, pH 7.8) was mixed with 20 ll puri?ed enzyme (0.2 mg/ml) and incubated at 37 °C. 20 ll mixture solution was withdrawn at various intervals (5 min, 15 min, 30 min, 1 h, 2 h, 3 h and 4 h) and boiled for 5 min to terminate the reaction. The samples were analyzed by SDS-PAGE. Determination of kinetic constants The kinetic constants, Km and Vmax of SFE1 were calculated by the method of Lineweaver–Burk double-reciprocal plot (Lineweaver and Burk 1934) with azocasein as a substrate. Puri?ed enzyme (10 ll) was mixed with 240 ll of azocasein solution (0.1–4.0 mg/ml azocasein in 50 mM Tris–HCl buffer pH7.8). After incubation at 37 °C for 10 min, the reaction was terminated by adding 500 ll 10 % (w/v) trichloroacetic acid, followed by standing in ice water for 10 min and centrifuged (10,000 r/min, 10 min, 4 °C). The absorbance of the supernatant was measured at 340 nm. One unit (IU) of enzyme activity was expressed as the amount of the enzyme causing an increase in absorbance of 0.001 per minute at the assay condition. Analysis of N-terminal amino acid sequence SFE1 was applied to SDS-PAGE, transferred to a polyvinylidene di?uoride (PVDF) membrane and stained with Coomassie Blue. The stained band was used directly for analysis of N-terminal amino acid sequence by a Procise 491 amino acid sequencer (Applied Biosystems, USA) at College of Life Sciences, Peking University. Evaluation of the anticoagulant effect of SFE1 in vitro The anticoagulant effect of SFE1 in vitro was estimated by the method of Lu et al. (2010) with minor modi?cations. In brief, 1 ml fresh whole blood was added to 1 ml 200 IU/ml of SFE1 solution (0.9 % NaCl, pH 7.4) in a glass test tube. The mixture was incubated at 37 °C for 3 h. The fresh whole blood was obtained from healthy male rat. As positive and negative controls, 200 IU/ml urokinase (0.9 % NaCl, pH 7.4) and normal saline were used instead of SEF1, respectively. Analysis of thrombolysis mechanism of SFE1 in vitro The fresh whole blood was placed to form blood clot at 4 °C. 1 g blood clots were heated at 85 °C for 30 min to

deactivate the plasminogen. 1 ml 200 IU/ml of SFE1 solution (0.9 % NaCl, pH 7.4) was added to heated and unheated blood clots in a glass test tube, respectively. The mixtures were incubated at 37 °C. Thrombolysis mechanism was analyzed according to the dissolution rates of heated and unheated blood clots.

Results Enzyme puri?cation SFE1 ?brinolytic enzyme was puri?ed by the four-step procedure described in the section ‘Enzyme puri?cation’. After the ?nal puri?cation step, the enzyme showed a single band on SDS-PAGE (Lane 1 of Fig. 1a) and nature PAGE (Fig. 1b), which indicated that the enzyme was a monomeric protein. 1.1 mg ?brinolytic enzyme was obtained from 3 liters culture supernatant. It was puri?ed 8.8-fold with a recovery of 2.2 % and a speci?c activity of 2750.6 IU/mg of protein (Table 1). Molecular weight and isoelectric point The molecular weight of SFE1 was estimated to be 20 kDa by SDS-PAGE (Lane 1 of Fig. 1a) and ?brin zymography (Lane 2 of Fig. 1a), corresponding with that determined by gel ?ltration. The isoelectric focusing electrophoresis of SFE1 was shown in Fig. 2b. The pI of SFE1 was measured about 4.9 according to the standard curve of pH-Gel length (Fig. 2).

Fig. 1 SDS-PAGE and nature PAGE of SFE1. a Lane M molecular mass markers; Lane 1 puri?ed enzyme; Lane 2 ?brin zymography of puri?ed enzyme; b nature PAGE of SFE1

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World J Microbiol Biotechnol (2012) 28:2479–2486 Table 1 Summary of SFE1 puri?cation
Step Total protein (mg) 429.0 81.4 11.7 4.7 1.1 Total activity (IU) 134,768.0 43,144.3 13,198.8 6,176.1 3,025.6 Speci?c activity (IU/mg) 314.1 530.0 1,132.9 1,311.3 2,750.6 Yield (%) Puri?cation factor

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Culture supernatant 80 % (NH4)2SO4 Bio-gel P60 DEAE-Sepharose FF Bio-gel P30

100.0 32.0 9.8 4.6 2.2

1.0 1.7 3.6 4.2 8.8

Effect of pH and temperature on ?brinolytic enzyme activity and stability To determine whether pH had effect on the activity and stability, SFE1 was treated in different pH from 4.0 to 10.0. As indicated in Fig. 3, pH had a great in?uence on activity and stability of SFE1. The optimum pH was 7.8, but in the range of pH 5.0–8.0, SFE1 was also relatively stable. To further investigate the role of temperature in controlling the activity and stability, SFE1 was put in different temperature from 25 to 85 °C at pH 7.8. As displayed by the results, it was active below 65 °C, and the maximum activity was observed at 35 °C (Fig. 4). Effect of metal ions and protease inhibitors on the ?brinolytic enzyme activity To test whether metal ions and protease inhibitors could participate in the regulation of ?brinolytic enzyme activity, nine metal cations (Na?, K?, Mn2?, Mg2?, Zn2?, Co2? and Cu2?) and seven protease inhibitors (pepstatin,
Fig. 2 Isoelectric focusing analysis of SFE1. a The standard curve of pH-Gel length; b disc electrophoresis was carried out in 7.5 % polyacrylamide gel containing ampholyte with pH range 3.0–10.0. The gel was stained for proteins with 0.25 % Coomassie brilliant blue R-250 solution Fig. 3 Effect of pH on the activity and stability of SFE1. The optimal pH for SFE1 was determined by measuring the ?brinolytic activity within a pH range of 4.0–10.0 at 37 °C for 18 h. The pH stability of SFE1 was estimated by measuring the remaining ?brinolytic activity after incubating the enzyme for 1 h at 37 °C with different buffers: 100 mM sodium acetate buffer (pH 4.0–5.0), 100 mM phosphate buffer (pH 6.0–8.0), and 100 mM glycine-NaOH buffer (pH 9.0–10.0)

aprotinin, soybean trypsin inhibitor, PMSF, EGTA, EDTA and b-mercaptoethanol) were introduced into our study. As shown in Table 2, Na?, K?, Mn2?, Mg2?, Zn2?and Co2? enhanced the activity, while Cu2? showed strong inhibition. In addition, the ?brinolytic activity of SFE1 was strongly inhibited by a typical serine protease inhibitor PMSF (1 mM), but the other serine protease inhibitors mentioned above (aprotinin, soybean trypsin inhibitor) had no obvious effects on the enzyme activity. The ?brinolytic activity of SFE1 was also decreased by EDTA (1 mM) and EGTA (1 mM) (Table 2).

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Fig. 5 Degradation of ?brinogen by SFE1. Lane M molecular mass markers; Lane 0 ?brinogen control without SFE1; Lanes 1–7 degradation products after 5, 15, 30, 60, 120, 180 and 240 min incubation at 37 °C, respectively Fig. 4 Effect of temperature on the activity and stability of SFE1. The optimal temperature of SFE1 was determined by measuring the ?brinolytic activity at different temperatures for 18 h. The thermal stability of SFE1 was evaluated by measuring the remaining ?brinolytic activity after incubating the enzyme in various temperatures for 1 h at pH 7.8

Table 2 Effect of metal ions and protease inhibitors Metal ion or inhibitor None NaCl KCl CaCl2 MnCl2 MgCl2 ZnCl2 CuCl2 CoCl2 FeSO4 Pepstatin Aprotinin Soybean trypsin inhibitor PMSF EGTA EDTA b-mercaptoethanol 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 5 mM 0.1 mg/ml 0.1 mg/ml 0.1 mg/ml 1 mM 1 mM 1 mM 1 mM Concentration Residual activity % 100.0 ± 0.5 124.2 ± 2.6 112.3 ± 2.2 101.3 ± 2.3 134.6 ± 2.9 122.6 ± 2.8 111.5 ± 1.2 39.3 ± 2.5 131.3 ± 2.9 100.7 ± 1.1 97.5 ± 0.8 100.9 ± 1.8 99.9 ± 0.3 16.4 ± 2.0 59.9 ± 0.8 62.6 ± 1.0 97.8 ± 1.6 Fig. 6 Lineweaver-Burk double-reciprocal plot of SFE1

Kinetic parameters of the ?brinolytic enzyme To evaluate the kinetic constants of SFE1 for the proteolytic reaction, the initial velocities of enzyme reactions were determined at various concentrations of the azocasein substrate. The Km and Vmax values of SFE1 for azocasein were 0.96 mg/ml and 181.8 unit/ml, respectively (Fig. 6).

Results are presented as means ± SD (n = 3)

N-terminal amino acid sequences of the ?brinolytic enzyme The N-terminal amino acid sequence of SFE1 was analyzed via the automated Edman method after SDS-PAGE and electroplating. The N-terminal 15 amino acids sequence was APITLSQGHVDVVDI. The amino acid sequence was analyzed using standard protein–protein BLAST (blastp) in NCBI protein databases (http://www. ncbi.nlm.nih.gov/BLAST) and revealed similarity only with hypothetical protein.

Hydrolysis of ?brinogen by the puri?ed ?brinolytic enzyme To explore the ?brinogenolytic activity of SFE1, SDSPAGE was performed. Interestingly, the hydrolysis rate was signi?cantly different with the chains of ?brinogen. The Aa-chain was completely degraded within 5 min, and the Bb-chain was completely degraded within 60 min. The c-chain was mostly hydrolyzed in 2 h (Fig. 5).

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Fig. 7 Effect on the anticoagulant of SFE1 in vitro. 1. 0.9 % (w/v) NaCl (as a negative control); 2. 200 IU/ml UK (as a positive control); 3. 200 IU/ml SFE1

The anticoagulant effect of SFE1 in vitro As shown in Fig. 7, no blood clots were observed in the test tube of SFE1 after 3 h (Fig. 7-3). In the test tube of UK, the blood clots were partly formed after 3 h (Fig. 7-2). Meanwhile, clotting had occurred in the test tube of normal saline (Fig. 7-1). The result indicated that the enzyme exhibited an ef?cient anticoagulant effect in vitro. Thrombolysis mechanism of SFE1 in vitro To estimate thrombolysis mechanism of SFE1 in vitro, the rates of heated and unheated blood clots digested by SFE1 were compared. The result showed that the rate of unheated blood clots digested by SFE1 was similar to that of heated blood clots. Therefore, SFE1 directly digested ?brin and not by plasminogen activators such as streptokinase, urokinase and tissue plasminogen activator. Discussion Thrombolytic therapy is still the best way to achieve recanalization in thrombosis diseases nowadays. Despite some thrombolytic agents’ widespread use, all of them have drawbacks (Killer et al. 2010). Therefore, it is indispensable to screen new thrombolytic agents from diverse sources. In this study, we described puri?cation and characterization of a ?brinolytic enzyme, designated as SFE1, from Streptomyces sp. XZNUM 00004. The enzyme was puri?ed to electrophoretic homogeneity by combination of chromatographic steps on DEAE-Sepharose Fast Flow anion exchange and gel-?ltration chromatography.

Molecular mass of the puri?ed enzyme was estimated to be approximately 20 kDa by SDS-PAGE and gel ?ltration chromatography. The molecular mass of SFE1 was lower than that of the ?brinolytic enzymes from Streptomyces sp. CS684 (35 kDa) (Simkhada et al. 2010), a marine bacterium Bacillus subtilis A26 (28 kDa) (Agrebi et al. 2009), Bacillus subtilis LD-8547 (30 kDa) (Wang et al. 2008) and Perenniporia fraxinea mycelia (42 kDa) (Kim et al. 2008). With respect to the effect of pH and temperature, the optimum pH (7.8) of SFE1 was similar to FP84 (7.5) from Streptomyces sp. CS684, but its optimum temperature, 35 °C, was different to FP84 (45 °C) (Simkhada et al. 2010). The pI was about 4.9, which was similar to that of lumbrokinase (F-I-0) (4.85) (Nakajima et al. 1993)and scolonase (4.8) (You et al. 2004). The pI of SFE1 was higher than that of PM-1 (4.4) (Ahn et al. 2003), N–V protease (4.5) (Zhang et al. 2007), and NJF (4.4) (Deng et al. 2010); but lower than subtilisin DFE (8.0) (Peng et al. 2003) and NJP (9.2) (Wang et al. 2011). The ?rst ?fteen N-terminal amino acid residues were Ala-Pro-Ile-Thr-Leu-Ser-Gln-Gly-His-Val-Asp-Val-ValAsp-Ile. In NCBI Blast searches, it revealed similarity only with hypothetical protein. However, except this, it did not show any homology with other ?brinolytic enzyme reported so far. Furthermore, SFE1 was strongly inhibited by a typical serine protease inhibitor PMSF (1 mM). SFE1 was also decreased 40.1 % by EDTA (1 mM) and 37.4 % by EGTA (1 mM). Taken together, we suggest that SFE1 can be a ?brinolytic enzyme with both serine and metalloprotease activity as reported by Simkhada et al. (2010) and Wang et al. (1999). To investigate the thrombolysis mechanism of SFE1 in vitro, the ?brinolytic activity of SFE1 on heated and unheated blood clots was examined. SFE1 formed a similar-rate for both blood clots. SFE1 also exhibited ?brinogenolytic activity by rapidly hydrolyzing the Aa-chain, then the Bb-chain, and the last c-chain. Furthermore, we also found that SFE1 showed an ef?cient anticoagulant effect, which was similar to PPFE-I (Lu et al. 2010). These results indicated that SFE1 was a direct-acting ?brinolytic and ?brinogenolytic agent and not by plasminogen activators such as streptokinase, urokinase and tissue plasminogen activator. In conclusion, the ?brinolytic enzyme SFE1, obtained from Streptomyces sp. XZNUM 00004, exhibits a profound ?brinolytic activity. Therefore, Streptomyces may become a source for thrombolytic agents to treat thrombosis. Further studies on the physiological function of SFE1 are proceeding.
Acknowledgments This work was supported by the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), National Natural Science Foundation

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2486 of China (No. 31000005, 31170605), the Project of Outstanding Scienti?c and Technological Innovation Team for Higher Education Institutions in Jiangsu Province (Pre-development of medical microbiology) and University-Industry Cooperation Program of Jiangsu Province (No. BY2009116).

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