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purification and characterization of a novel potent fibrinolytic enzyme from paecilomyces tenuipes

Process Biochemistry 46 (2011) 1545–1553

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Puri?cation and characterization of a novel, highly potent ?brinolytic enzyme from Paecilomyces tenuipes
Hoe Chang Kim a,1 , Bong-Suk Choi a,1 , Kumar Sapkota a,d , Seung Kim b , Hyo Jeong Lee b , Jin Cheol Yoo c , Sung-Jun Kim a,?

Department of Biotechnology, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, Republic of Korea Department of Alternative medicine, Gwangju University, Gwangju 503-703, Republic of Korea c Department of Pharmacy, Chosun University, Gwangju 501-759, Republic of Korea d Central Department of Zoology, Tribhuvan University, Kirtipur, Kathmandu, Nepal

a r t i c l e

i n f o

a b s t r a c t
A ?brinolytic enzyme (PTEFP) was puri?ed from the entomopathogenic fungus Paecilomyces tenuipes. Analysis of the puri?ed PTEFP by SDS–PAGE and ?brin zymography demonstrated a single protein band of approximately 14 kDa. Fibrinolysis pattern showed that PTEFP rapidly hydrolyzed ?-chain followed by ?-chain. PTEFP rapidly degraded A?-chain of human ?brinogen but did not hydrolyze B?- or ?-chain indicating that it is ?-?brinogenase. The N-terminal sequence was AQNIGAVVNLSPPKQ, which is different from that of other known ?brinolytic enzymes. The PTEFP displayed maximum activity at 35 ? C and pH 5.0, and was stable between pH 5.0–8.0 and below 40 ? C. Calcium ion enhanced the enzyme activity whereas Zn2+ inhibited it. The ?brinolytic activity was strongly inhibited by PMSF identifying it as a serine protease. PTEFP exhibited high speci?city for the substrate H-D-Val-Leu-Lys-pNA and Km and Vmax values for this substrate were 0.17 mM and 59 U/ml respectively. These results suggest that PTEFP is a novel ?brinolytic enzyme and may have potential applications in treating thrombosis. ? 2011 Elsevier Ltd. All rights reserved.

Article history: Received 4 December 2010 Received in revised form 7 April 2011 Accepted 8 April 2011 Keywords: Paecilomyces tenuipes Fibrinolysis Fungal enzymes Thrombosis

1. Introduction Cardiovascular disease is a main contributing cause of death around the world. Due to its prevalence cardiovascular disease is expected to impose an ever-increasing impact on our society emotionally, socially and ?nancially. Intravascular thrombosis, a consequence of ?brin aggregation in the arteries, is one of the main causes of cardiovascular disease. Fibrin is the primary protein component of blood clots, which are formed from ?brinogen by the enzyme thrombin. Fibrinogen, a glycoprotein contains two sets of three polypeptide chains (A?, B? and ?). During ?brinolysis the insoluble ?brin ?ber is hydrolyzed into ?brin degradation products by plasmin. Plasmin is generated from plasminogen by plasminogen activators such as tissue plasminogen activator (t-PA), vascular plasminogen activator, blood plasminogen activator, urokinase, Hageman factor, and streptokinase-plasminogen complex [1]. Fibrin clot formation and ?brinolysis are normally well balanced in biological systems. However, when ?brin is not hydrolyzed due to some disorder, thromboses can occur. Myocardial infarction is the

? Corresponding author. Tel.: +82 62 230 6664; fax: +82 62 230 6664. E-mail address: sjbkim@chosun.ac.kr (S.-J. Kim). 1 These authors contributed equally to this study. 1359-5113/$ – see front matter ? 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.04.005

most common of these thromboses. Although t-PA and urokinase are still commonly used in thrombolytic therapy currently, they have some major disadvantages such as high cost, allergic reactions, and risk for internal bleeding within the intestinal tract when orally administrated [2,3]. Therefore, cheaper and safer ?brinolytic enzymes from diverse sources are currently being sought. In recent years, mushrooms have become an attractive source of thrombolytic agents because of their ef?ciency and safety. Many ?brinolytic enzymes have been identi?ed and characterized from several edible or medicinal mushrooms including Grifola frondosa [4], Flammulina velutipes [5], Tricholoma sapoceum [6], Fomitella fraxinea [7], Cordyceps sinensis [8] and Pleurotus eryngii [9]. Due to the encouraging biological advantages of using food sources of ?brinolytic enzymes, we have also extensively explored new sources of ?brinolytic enzymes from various edible mushrooms such as P. fraxinea, F. velutipes, P. ostreatus, C. militaris, and A. mellea [10–14]. Paecilomyces tenuipes (also referred to as Isaria japonica) is one of the famous Chinese medicinal entomopathogenic fungi as are the fungi such as Cordyceps sinensis and Cordyceps militaris. It is a parasitic fungus on the larvae of Lepidoptera. P. tenuipes is traditionally used in time-honored tonics and for improvement of blood loss, fatigue and anorexia in Japan, Korea, and China [15]. The fruiting bodies of P. tenuipes are highly valued as medicinal herbs due to their various biological and pharmacological activities


H.C. Kim et al. / Process Biochemistry 46 (2011) 1545–1553 The molecular weight and homogeneity of the puri?ed enzyme was determined by sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS–PAGE) and ?brin zymography. SDS–PAGE was performed according to the method of Laemmli [21], using 12% polyacrylamide gel and stained with Coomassie Brilliant Blue R-250. Fibrin zymography was carried out according to the method of Leber and Balkwill [22] and Kim et al. [23]. Resolving gel solution (12%) contained 0.12% (w/v) ?brinogen prepared in a total volume of 10 ml and centrifuged to remove insoluble impurities which were induced when SDS stock solution was mixed. Thrombin solution (1 U/ml) and TEMED (N, N, N0 , N0 -tetramethylethylenediamine) were added to the gel solution in ?nal concentrations of 0.1 U/ml and 0.028% (v/v), respectively. Puri?ed ?brinolytic protease was electrophoresed into ?brin gel and subsequently washed in 2.5% Triton X-100 solution and then incubated in a bath containing reaction buffer (prepared 20 mM Tris–HCl (pH 7.5) containing 0.15 M NaCl, 0.02% NaNO3 ) at 37 ? C for ?brin.

including immuno-stimulating and anti-tumor activities [16,17]. A number of bioactive compounds such as cordycepin, tenuipesine, sterol, cyclopeptide, and polysaccharides have been reported from P. tenuipes [18]. Nevertheless, no information is available regarding the ?brinolytic protease from P. tenuipes. We therefore attempted to identify thrombolytic agents in this mushroom and describe herein the puri?cation and characterization of a novel ?brinolytic enzyme from the fruiting body of P. tenuipes.
2. Materials and methods 2.1. Materials P. tenuipes was obtained from the Culture Collection of DNA Bank of Mushrooms (CCDMB, IUM01135), Incheon, Republic of Korea. Human ?brinogen, thrombin, plasmin, acrylamide, Citrate monohydrate, Trizma Base, Trizma HCl, carboxymethyl (CM)-cellullose, phenylmethylsulphonyl ?uoride (PMSF), tosyllysine chloromethyl ketone (TLCK), aprotinin, tosylphenylalnine chloromethyl keptone (TPCK), ethyleneglycolbis-(2-aminoethyl)-N,N,N ,N tetraacetic acid (EGTA), ethylenediaminetetra acetic acid (EDTA), sodium chloride, and diethylaminoethyl (DEAE)-Sepharose CL-6B were purchased from Sigma–Aldrich (St. Louis, MO, USA). Agarose was obtained from Invitrogen (Carlsbad, CA, USA). PageRulerTM color protein marker was purchased from Fermentas (Hanover, MD, USA). Sephadex G-75 and CL-6B fast ?ow columns were purchased from Phamacia (Uppasala, Sweden) and Poros HQ was from Applied Biosystems (Foster city, CA, USA). All other chemicals and reagents used were of analytical grade. 2.2. Puri?cation of ?brinolytic enzyme Unless otherwise stated, all procedures were carried out at 4 ? C. Two hundred grams of P. tenuipes fruiting bodies were homogenized in a double volume of deionized water in a homogenizer for 2 min at maximum speed. After repeated freezing and thawing, the homogenate was centrifuged at 5000 × g for 30 min at 4 ? C. Supernatant was placed into a stainless steel 1-l bucket, which was partially immersed in a salt- ice bath. An equal volume of ?70 ? C pre-chilled ethanol was added, drop wise, with constant stirring after which the solution was stirred for 1 h. Precipitated protein was removed by centrifugation at 10,000 × g for 30 min at 4 ? C. The ethanolsoluble fraction was returned to the steel bucket in the salt–ice bath and its ethanol concentration was increased, drop wise, to 75% with constant stirring. Stirring continued for 1 h after which the precipitated protein was recovered by centrifugation at 10,000 × g for 30 min at 4 ? C. After removal of the supernatant, the pellets were air-dried in a laminar ?ow hood for 15 min and the protein was then resuspended in the appropriate buffer. Insoluble materials were removed by centrifugation at 10,000 × g for 10 min at 4 ? C. The re-suspended pellets were introduced into a CM-cellulose column (3 × 10 cm) which had been equilibrated with the 10 mM citrate–NaOH (pH 6.5) buffer. Elution was carried out at a ?ow rate of 0.1 ml/min. Fractions showing high degrees of ?brinolytic activity (active fractions) were loaded onto a DEAE-Sepharose CL-6B column (1.6 × 20 cm) which had been pre-equilibrated with 10 mM Tris–HCl (pH 7.0) buffer and eluted with linear gradient of 0–0.5 M KCl with ?ow rate of 0.1 ml/min at 4 ? C. Then, the active fractions were collected individually and concentrated using a Vivaspin 500 (Sartorius Stedim Biotech, Gottingen, Germany). To further purify the sample, gel ?ltration was performed with a Sephadex G-75 column (1.0 cm × 63 cm) equilibrated with 10 mM Tris–HCl buffer (pH 7.0) and containing 0.1 M KCl at a ?ow rate of 0.1 ml/min. The active fractions with high activities were pooled and concentrated and then further puri?ed using a POROS 20 HQ ion exchange column (1.0 cm × 10 cm) equilibrated with 10 mM Tris–HCl buffer (pH 8.0) and eluted with linear gradient of 0–1.0 M KCl with a ?ow rate of 0.5 ml/min at 4 ? C. In all the puri?cation steps, the elutes were monitored by VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 350 nm. The activity of the protease was assessed utilizing the ?brinolytic assay described below. The puri?cation process is summarized in Fig. 1. 2.3. Assay of enzyme activity Protease activity was measured using a method described by Xiao et al. [19] with some modi?cations. Brie?y, an enzyme sample/column fraction (10 ?l) was incubated with 40 ?l of 1% human ?brinogen (prepared in 20 mM Tris–HCl (pH 7.5) containing 0.15 M NaCl) and 10 ?l of 5 ?U/ml thrombin at 37 ? C for 10 min. The absorbance of all the samples and standards were measured at 350 nm by microplate reader (Molecular Devices, Sunnyvale, CA, USA). Plasmin was used as a standard. One enzymatic unit was de?ned as the amount of enzyme causing conversion of 1 ?M of p-Nitroanilide from D-Val-Leu-Lys-p-Nitroanilide per minute at 37 ? C. 2.4. Protein analysis Protein concentration was determined using the Bradford method [20] with bovine serum albumin as a standard curve.

2.5. Fibrinolytic and ?brinogenolytic activities Fibrinolytic activity was determined using the method described by Astrup and Mullertz [24] with minor modi?cations as follows. The ?brin agarose plate was made to a 1-mm thickness and contained 1.2% agarose, 0.4% human ?brinogen, and 20 U/ml of human thrombin. The clot was allowed to stand for 1 h at room temperature. Then, 1.0 ?g of sample solution was carefully placed onto the plate. The plate was incubated for 12 h at 37 ? C and the diameter of the lytic circle was measured. In the ?brin plate method, a clear transparent region is observed in which ?brin is hydrolyzed and its diameter is directly proportional to the potency of the ?brinolytic activity. Fibrinolysis was performed using a slightly modi?ed method of Datta et al. [25]. Brie?y, 10 ?l of 1% human ?brinogen (prepared in 20 mM Tris–HCl (pH 7.5) containing 0.15 M NaCl) was mixed with human thrombin (0.1 U). The ?brin clot was allowed to stand for 1 h at room temperature. Formed clots were mixed with puri?ed enzyme (1 ?g) and incubated at 37 ? C for various time intervals. The resulting peptides were analyzed using SDS–PAGE on a 7.5% gel [21]. Plasmin (1 ?g) was used as a positive control. Fibrinogenolysis was determined as described previously [26]. Brie?y, 10 ?l of 1% human ?brinogen (prepared in 20 mM Tris–HCl, pH 7.5, containing 0.15 M NaCl) was incubated with 1 ?g of a puri?ed enzyme at 37 ? C. At various intervals, a portion of the reaction solution was withdrawn and analyzed by SDS–PAGE according to the method of Laemmli [21]. Plasmin was used as a positive control.

2.6. Effect of pH and temperature on enzyme activity The optimal pH for the ?brinolytic activity of the enzyme was determined within a pH range of 2.0–10.0. One micro gram of the enzyme solution was added to 90 ?l of 10 mM glycine–HCl (pH 2.0–3.0), 10 mM citric–NaOH (pH 4.0–6.0), 10 mM Tris–HCl (pH 7.0–8.0), and 10 mM glycine–NaOH (pH 9.0–10.0) buffers. The reaction mixtures were incubated for 1 h at 25 ? C and the enzyme activities were measured by the enzyme activity assay as described above. The pH stability of the puri?ed enzyme was examined by measuring the remaining ?brinolytic activity of each enzyme solution after incubation for 12 h from pH 2.0 to 10.0. The optimal temperature for enzymatic activity was determined by measuring residual activity after the incubation of 1.0 ?g of ?brinolytic enzyme in 90 ?l of 10 mM sodium phosphate buffer (pH 5.0) at different temperatures (20–80 ? C) for 1 h. The thermal stability of the enzyme was measured after pre-incubating the enzyme in the same buffer at the same pH but at various temperatures for different incubation times. The relative and residual activities were determined in the same conditions used to determine the ?brinolytic activity.

2.7. Effect of metal ions and protease inhibitors on the enzyme activity The effects of metal ions on enzyme activity were investigated using CaCl2 , CoCl2 , ZnCl2 , MgCl2 , and MnCl2 . The puri?ed enzyme (1.0 ?g) was incubated in the absence and the presence of cations such as Ca2+ , Co2+ , Zn2+ , Mg2+ , and Mn2+ with a ?nal concentration of 1 mM in 10 mM Tris–HCl for 1 h at 37 ? C. Each enzyme solution (10 ?l) was then incubated with human ?brinogen and thrombin at 37 ? C for 10 min and the enzyme activity was measured by microplate reader (Molecular Devices, Sunnyvale, CA, USA). The effects of protease inhibitors were also assessed using EDTA, EGTA, PMSF, TLCK, TPCK, and aprotinin. The puri?ed enzyme was pre-incubated in a ?nal concentration of 100 mg/ml PMSF, and TPCK, 50 mg/ml TLCK, 1 mM EDTA and EGTA, 1 mg/ml aprotinin in 10 mM Tris–HCl for 1 h at 37 ? C. After incubation of each enzyme solution (10 ?l) with human ?brinogen and thrombin for 10 min at 37 ? C, the enzyme activity was measured by microplate reader (Molecular Devices, Sunnyvale, CA, USA). The level of inhibition was expressed as a percentage of the remaining activity (with either metal ion or inhibitor) as compared to the control activity (without metal ion or inhibitor).

H.C. Kim et al. / Process Biochemistry 46 (2011) 1545–1553


Fig. 1. Flow chart showing the puri?cation scheme for ?brinolytic enzyme from P. tenuipes.


H.C. Kim et al. / Process Biochemistry 46 (2011) 1545–1553

Table 1 Puri?cation summary of the ?brinolytic enzyme obtained from P. tenuipes. Puri?cation step Homogenate Crude extract CM cellulose DEAE-sepharose CL6B fast ?ow Sephadex G-75 POROS 20 HQ Total protein (mg) 2487.5 14.99 10.23 5.841 2.374 0.176 Total activity (U) 6480 5040 4401 3996 3139 252 Speci?c activity (U/mg) 2.60 336.22 430.24 684.12 1322.24 1431.81 Recovery (%) 100 77.7 67.3 61.7 48.4 3.8 Fold 1 129.31 165.48 263.12 508.55 550.70

Note: The enzyme activity was measured by the protease activity assay as described under Section 2. One enzymatic unit was de?ned as the amount of enzyme causing conversion of 1 ?M of p-Nitroanilide from D-Val-Leu-Lys-p-Nitroanilide/min at 37 ? C.

2.8. Amidolytic activity of the enzyme Amidolytic activities were measured spectrophotometrically using synthetic chromogenic substrates such as, S-2288 (H-D-Ile-Pro-Arg-pNA for t-PA), S-2238 (H-D-Phe-Pip-Arg-pNA for thrombin), S-2251 (H-D-Val-Leu-Lys-pNA for plasmin), and S-2444 (pyroGlu-Gly-Arg-pNA for urokinase). Activities were evaluated by the mixing of the puri?ed enzyme (1 ?g/175 ?l of 50 mM Tris–HCl (pH 7.4)) with 25 ?l of a 4 mM substrate. After continuous measurement for 5 min at 37 ? C with a temperature-regulated spectrophotometer, the amount of released p-nitro aniline was determined by measuring the change in absorbance at 405 nm. The Km and Vmax of PTEFP were determined with different concentrations (0.1–0.3 mM) of H-D-Val-Leu-Lys-pNA as a substrate. 2.9. Determination of N-terminal amino acid sequence of the enzyme After SDS–PAGE was performed, the puri?ed enzyme on the polyacrylamide gel was transferred to a polyvinylidene di?uoride membrane (PVDF) (Millipore, Watford, UK) using a Minitransblot electroblotting system (Bio-Rad, Hercules, CA, USA) and then stained with Ponceau S solution. The stained portion was excised and used directly for N-terminal sequencing via the automated Edman method. A blast search was performed using the deduced amino acid sequence.

3. Results and discussion 3.1. Puri?cation of ?brinolytic enzyme The ?brinolytic enzyme was puri?ed using a combination of chromatographic steps listed in Table 1 and shown in Fig. 2. The extract was ?rst subjected to ion-exchange chromatography on CM-cellulose, and active fractions were obtained (Fig. 2A). These fractions were collected and applied onto a DEAE-Sepharose CL-6B column (Fig. 2B). The active fractions were further separated via gel ?ltration chromatography on the Sephadex G-75 (Fig. 2C). The

Fig. 3. SDS–PAGE and ?brin-zymography analysis of PTEFP. Analysis was performed on 12% polyacrylamide gels stained with Coomassie Brilliant Blue R-250. Lanes: M, protein size marker (Fermentas, Hanover, MD, USA); 1, puri?ed PTEFP; 2, ?brinzymography of puri?ed PTEFP.

Fig. 2. Puri?cation of ?brinolytic enzyme from P. tenuipes. (A) Cation-exchange chromatography on a CM-cellulose column; (B) anion-exchange chromatography on a DEAESepharose CL-6B column; (C) gel ?ltration with Sephadex G-75 column; (D) ion-exchange with POROS 20 HQ column. Protein concentration (o) and the enzyme activity (?) of each fraction were measured at 595 and 350 nm respectively. The enzyme activity was expressed in terms of U/ml; (E). SDS–PAGE of each puri?cation steps. Lanes: M, protein marker; 1, crude sample; 2, fraction from CM cellulose column; 3, fraction from DEAE-sepharose CL-6B column; 4, fraction from Sephadex G-75 column; 5, fraction from POROS 20 HQ column.

H.C. Kim et al. / Process Biochemistry 46 (2011) 1545–1553 Table 2 Comparison of N-terminal amino acid sequence of PTEFP with fungi serine proteases, hypothetical protein and other ?brinolytic enzymes. Enzyme Perenniporia fraxinea (PTEFP) Aspergillus oryzae RIB40 Aspergillus fumigatus A1163 Cordyceps militaris (CMase) Cordyceps sinensis (CSP) Pleurotus eryngii Fomitella fraxinea (FFP1) Nattokinase (NK) Skipjack Shiokara (katsuwokinase) Subtilisin DJ-4 Subtilisin E N-terminal amino acid sequence AQNIGAVVNLSPPKQ AENIGAMVNSRPLFD GMGGRAVVNLSLGGP IVGGVSVAIE ALATQHGAPW AMDSQTDASYGLAND APXXPXGPWGPQRIS AQSVPYGISQIKAP IVGGYEQZAHSQPHQ AQSVPYGVSQIKAP AQSVPYGIPQIKAPAHS Identity (%) – 53.3 40.0 20.0 10.0 6.7 6.7 14.3 13.3 21.4 11.8 Molecular weight (kDa) 15 – – 27.3 31.0 14.0 32.0 27.7 38.0 29.0 28.0


Reference This work [37] [38] [39] [8] [9] [7] [40] [41] [42] [43]

major fractions with ?brinolytic activity were collected and applied onto a POROS 20 HQ ion exchange column (Fig. 2D), which yielded one major peak showing strong ?brinolytic activity (Fig. 2E). From a 200 g sample of P. tenuipes, 0.18 mg of the enzyme was puri?ed 550.7-fold, with a yield of 3.8% (Table 1). The puri?ed enzyme had a speci?c activity of 1431.81 U/mg, which represents approximately a four-fold increase over the crude extract. SDS–PAGE and ?brin zymography were employed to verify the purity of the isolated enzyme (Fig. 3). The puri?ed enzyme was designated as P. tenuipes ?brinolytic protease (PTEFP). The molecular mass of PTEFP was found to be 14 kDa, as estimated by SDS–PAGE, and ?brin zymography (Fig. 3). The molecular weight of the puri?ed enzyme was the same as the molecular weight of the enzyme from P. eryngii (14 kDa) [9]. Although, P. tenuipes is an entomopathogenic fungi together with C. sinensis and C. militaris, the ?brinolytic enzyme puri?ed from P. tenuipes was much smaller than the ?brinolytic enzyme (52 kDa) from C. militaris [13]. Interestingly, its molecular weight is lower than that of the ?brinolytic enzymes so far reported from fungi such as G. frondosa (20 kDa) [4], A. mellea (21 kDa) [14], T. saponaceum (18.1 and 17.9 kDa) [6], P. ostrestus (32 kDa) [12], F. velutipes (37 kDa) [11], P. fraxinea (42 kDa) [10]. These data indicate that PTEFP is in fact one of the smallest fungal proteases found to date.

secondary effects such as platelet activation related to plasmin formation can be avoided. This is a speci?c advantage of this puri?ed enzyme over clinically used plasminogen activators. Recently, it has been reported that direct acting ?brinolytic agents have impressive biochemical and preclinical foundations for ultimate clinical application [31–33]. 3.3. N-terminal sequence analysis After SDS–PAGE and electroblotting the N-terminal amino acid sequence of the puri?ed ?brinolytic enzyme was analyzed via the automated Edman method. The N-terminal sequence of the ?rst 15 residues was found to be AQNIGAVVNLSPPKQ (Table 2). This sequence exhibited a high degree of identity (53%) at the amino acid level with the hypothetical protein RIB40 (NCBI GenBank accession no. XP-001820515) from fungus Aspergillus oryzae. Similarly, the puri?ed enzyme had a 40% sequence homology to serine protease from A. fumigates. However, a comparison with other ?brinolytic enzymes showed that the observed sequence of PTEFP differs greatly (Table 2). 3.4. Effects of pH and temperature on ?brinolytic activity and stability of PTEFP The effects of pH on ?brinolytic activity and enzyme stability are shown in Fig. 5A and B. The activity and stability of PTEFP were both in?uenced by pH. The puri?ed protease was active over the pH range 4.0–7.0, but exhibited its maximum activity at pH 5.0. Although the activity was completely lost in highly acidic and basic environment, over 60% of the activity was found stable in pH ranges of 5.0–8.0. The optimum pH of the enzyme is similar to those of FFP2 from F. fraxinea [7], PoMEP from fruiting body of P. ostreatus [4], AMMP from A. mellea [14], and ?brinolytic enzyme from P. eryngii [9]. The optimum temperature and thermostability of PTEFP are shown in Fig. 5C and 5D. The optimum temperature for PTEFP activity was 35 ? C. The enzyme was relatively stable below 40 ? C. However, remarkable loss of activity was observed at 50 ? C. The enzyme was relatively stable between 20 and 35 ? C, and the enzyme activity decreased slowly with time. These results are consistent with results from previous reports [5,13,14]. 3.5. Effect of metal ions and protease inhibitors on the ?brinolytic activity The effects of various metal ions on ?brinolytic activity were investigated via the protease activity assay after incubation of PTEFP with different metal ions for 1 h at 37 ? C. The result is summarized in Fig. 6A. The proteolytic activity of PTEFP was highly decreased by Zn2+ . On the contrary, the proteolytic activity was enhanced by Ca2+ . This suggests that the proteolytic activity of PTEFP is dependent upon the presence of bivalent cations such as Ca2+ and provides the PTEFP with important structural stability.

3.2. Fibrinolytic and ?brinogeolytic activity To investigate the ?brinolysis mechanism of PTEFP, the hydrolysis pattern of ?brin by PTEFP was ?rst analyzed utilizing the ?brin plate method. As shown in Fig. 4A, PTEFP formed a bigger lysis zone than that produced by equal amount of plasmin, suggesting that ?brinolytic ef?ciency of PTEFP is relatively greater than that of plasmin. Next, to elucidate the mode of reaction of PTEFP, degradation products were separated using SDS–PAGE. As shown in Fig. 4B, the puri?ed enzyme rapidly hydrolyzed the ?-chain followed by the ?-chain. Moreover, to examine the ?brinogenolytic activity, the degradation pattern of ?brinogen by PTEFP was analyzed by SDS–PAGE. As shown in Fig. 4C, PTEFP rapidly hydrolyzed A?chains of ?brinogen while it did not hydrolyze the B? and ?-chains. Notably, as shown in Fig. 4, PTEFP had stronger ?brinolytic and ?brinogenolytic activity than human plasmin. The ?brinogenolysis pattern of PTEFP is similar to that of ?-?brinogenase from snake venom which preferentially hydrolyzed the A ? chain of ?brinogen rather than the B? and ?-chains [27,28]. Its hydrolysis pattern is also similar to ?-?brinogenase from Lampetra japonica [19], Allium tuberosum [29] and Codium divaricatum [26]. Additionally, PTEFP seems to be a direct-acting ?brinolytic agent as it acts via direct cleavage of ?brin and ?brinogen and not through plasminogen activators such as SK, UK, and tPA. This effect is in consistent with ?brinolytic enzymes from P. eryngii, C. militaris and Streptomyces sp.CS684 [9,13,30]. A direct acting ?brinolytic enzyme has speci?c advantage over clinically used plasminogen activators since


H.C. Kim et al. / Process Biochemistry 46 (2011) 1545–1553

Fig. 4. Fibrino(geno)lytic activity of PTEFP. (A) Fibrinolytic activity was assayed on ?brin plates. Samples were applied to the wells in the plate and incubated for 12 h at 35 ? C. (B) Fibrinolysis pattern exhibited by PTEFP. (C) Analysis of the pattern of ?brinogenolysis by PTEFP. Fibrin and ?brinogen were incubated with PTEFP for the various times indicated. Fibrin (B), and ?brinogen (C) were used as controls (con). Plasmin was used as a positive control.

Our data regarding the effect of these metal ions on the activity of enzyme were consistent with the ?brinolytic enzymes belonging to class of serine proteases from mushrooms such as F. fraxinea [7], C. militaris [13], P. eryngii [9], and C. sinensis [8]. To classify the puri?ed enzyme, the protease activity was assayed in the presence of various protease inhibitors such as PMSF, TLCK, TPCK, aprotinin, EDTA, and EGTA. As shown in Fig. 6B, the enzyme activity was strongly inhibited by PMSF, a serine protease inhibitor. In contrast, the metal chelator (EDTA) had no effect on the proteolytic activity. Thus PTEFP

was found to be a serine protease. Most ?-?brinogenases of snake venom are metalloproteinases, thus their activity is inhibited by EDTA. However, Siigur et al. [34] reported a serine ?-?brinogenase from Vipera lebetina venom that is basic in nature rather than acidic. Interestingly, most ?-?brinogenases from plant sources are found to be serine proteinases, such as ATFE-II from Allium tuberosum [29] and CDP from Codium divaricatum [26]. Therefore PTEFP could have similar properties to these serine ?-?brinogenases from plant sources.

H.C. Kim et al. / Process Biochemistry 46 (2011) 1545–1553


Fig. 5. Effects of pH and temperature on the activity and stability of PTEFP. (A) Optimum pH was determined by assessing the enzyme activity with various pH buffers: 10 mM glycine–HCl (pH 2.0–3.0), 10 mM citric–NaOH (pH 4.0–6.0), 10 mM Tris–HCl (pH 7.0–8.0), and 10 mM glycine–NaOH (pH 9.0–10.0). (B) pH stability was determined by measuring the residual activity after aliquots of the enzyme sample were incubated with the respective buffers for 12 h. (C) Optimum temperature of the enzyme was determined by assaying the activity at various temperatures after incubation for 1 h. (D) Thermal stability of the enzyme was assessed by measuring the residual activity after the enzyme was incubated in 10 mM sodium phosphate buffer (pH 5.0) at various temperatures for different incubation times. Each value represents the mean ± SD for three determinations.

Fig. 6. Effects of metal ions (A), and protease inhibitors (B) on the activity of PTEFP. Each value represents the mean ± SD for three determinations.

1552 Table 3 Amidolytic activity on synthetic protease substrates. Synthetic protease substrate H-D-Phe-Pip-Arg-p-nitroanilide H-D-Val leu-Lys-p-nitroanilide H-D-Ile-Pro-Arg-p-nitroanilide pyroGlu-Gly-Arg-p-nitroanilide

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Activity (?mol/min/mg) 11.3 ± 3.2 16.2 ± 3.9 10.5 ± 3.0 7.3 ± 1.4

Protease Thrombin Plasmin t-PA Urokinase

Each value represents the mean ± SD for three determinations.

3.6. Amidolytic activity The amidolytic activity of puri?ed protease was assessed using several chromogenic substrates, such as S-2238 for thrombin (HD-Phe-Pip-Arg-pNA), S-2251 for plasmin (H-D-Val-Leu-Lys-pNA), S-2444 for urokinase (pyroGlu-Gly-Arg-pNA), and S-2288 for tPA (H-D-Ile-Pro-Arg-PNA). As shown in Table 3, PTEFP exhibited a higher degree of speci?city toward H-D-Val-Leu-Lys-pNA for plasmin. The Km and Vmax values of PTEFP for H-D-Val-Leu-Lys-pNA were 0.17 mM and 59 U/ml respectively. The ?brinolytic enzyme from P. eryngii [9] also exhibited the highest activity for plasmin. It is well known that important ?brinolytic enzymes, t-PA and plasmin, are serine proteases. Serine proteases contain many members functioning in the same milieu (e.g. human plasma) that are exclusive in their reactivity toward both substrates and inhibitors [35]. Lysine and/or arginine are common sites for serine proteases involved in blood coagulation and ?brinolysis. This speci?city was also observed among the serine proteases isolated from microorganisms [36]. In conclusion, the ?brinolytic protease PTEFP obtained from the fruiting bodies of the edible and medicinal mushroom P. tenuipes exhibits a remarkably high degree of ?brinolytic activity. Biochemical features of the puri?ed enzyme indicate that it might be a novel serine type ?-?brinogenase based on its molecular mass, N-terminal amino acid sequence, proteolytic activity, thermostability, and pH. Moreover, PTEFP is a direct acting ?brinolytic enzyme that could have signi?cant clinical implications. Although clinical results are necessary for thrombolytic applications, PTEFP may become an important new resource for thrombolytic agents. Acknowledgements This study was supported by Technology Development Program for Fisheries (2010), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. References
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