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Purification and Characterization of a Novel Fibrinolytic Enzyme from Culture Supernatant of Pleurotus ostreatus
Purification and Characterization of a Novel Fibrinolytic Enzyme from Culture Supernatant of Pleurotus ostreatus
Journal of Microbiology and Biotechnology. 2014. Feb, 24(2): 245-253
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : July 22, 2013
  • Accepted : November 19, 2013
  • Published : February 28, 2014
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About the Authors
Xiao-lan Liu
Heilongjiang Provincial Key University Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China
liuxiaolan001@126.com
Xi-qun Zheng
Heilongjiang Provincial Key University Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China
Peng-zhi Qian
Heilongjiang Provincial Key University Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China
Narasimha-kumar Kopparapu
Heilongjiang Provincial Key University Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China
Yong-ping Deng
Heilongjiang Provincial Key University Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China
Masanori Nonaka
Faculty of Agriculture, Niigata University, Niigata, Japan
Naoki Harada
Faculty of Agriculture, Niigata University, Niigata, Japan

Abstract
A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDS-PAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the α and β chains of fibrinogen followed by the γ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at 45℃ and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea . The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.
Keywords
Introduction
Fibrin is the major protein component of blood clots, which is formed from fibrinogen by thrombin (E.C. 3.4.21.5). The insoluble fibrin fiber is hydrolyzed into its degradation products by plasmin, a serine protease that is activated from plasminogen by plasminogen activators, such as tissue plasminogen activator (t-PA). Fibrin formation and fibrinolysis are normally well balanced in biological systems. When fibrin is not hydrolyzed because of some disorder, thrombosis occurs, which leads to acute myocardial infarction and other cardiovascular diseases, including high blood pressure, ischemic heart, and stroke. Cardiovascular diseases are a leading cause of death throughout the world [21] .
Current fibrinolytic enzymes available for clinical use are mostly plasminogen activators such as a tissue-type plasminogen activator, a urokinase-type plasminogen activator (u-PA), and the bacterial plasminogen activator streptokinase. All these agents are not only expensive but also have limitations, such as rapid degradation, allergic reactions, resistance to reperfusion, and hemorrhage [8 , 23] . Therefore, researchers are actively searching for novel fibrinolytic enzymes from various sources. Sumi et al . [30] have reported nattokinase (NK), from a traditional Japanese fermented soybean food, “ natto ”, and its oral administration enhanced fibrinolysis in canine plasma [6 , 31] . These results triggered the search for novel fibrinolytic enzymes from foods. Over the past decade, fibrinolytic enzymes have been reported from various microorganisms isolated from traditional fermented foods [7 , 21 , 27] . The fibrinolytic enzymes derived from food-grade microorganisms have great potential to be developed as functional food additives and drugs to prevent or cure thrombotic diseases [27] . Therefore, research on fibrinolytic enzymes from non-toxic mushrooms has received wide attention for thrombolytic therapy.
Mushrooms are commonly consumed as food and also used in traditional oriental medicine. In recent years, mushrooms have become an attractive source of various bioactive compounds. Mushroom extracts have been reported to show apoptotic effects on human neuroblastoma cells, as well as antiviral, antitumorigenic, hypotensive, and hepatoprotective properties [13 , 32] . Mushrooms constitute an important source of fibrinolytic enzymes and can be used in the prevention and treatment of thrombosis [14] . Several fibrinolytic enzymes have been reported from various mushrooms such as Tricholoma saponaceum [9] , Armillaria mellea [14] , Fomitella fraxinea [15] , Cordyceps militaris [4 , 10] , Cordyceps sinensis [17] , Flammulina velutipes [24] , and Schizophyllum commune [20 , 23 , 25] .
Pleurotus ostreatus , known as oyster mushroom, is a white rot basidiomycete. Researchers have reported various proteases from fruiting bodies, mycelia, and culture fluid of this mushroom [5 , 26 , 28] . In our preliminary studies, we found that when cultured by submerged method, the Pleurotus ostreatus mycelia can secrete fibrinolytic enzyme into the culture medium (unpublished data). Although artificial culturing of some mushrooms had been solved in some specific solid media, the growth time is too long to satisfy economic efficiency. Submerged culturing has potential advantages, and the productivity of the product could be increased by optimizing the culture conditions [25] . In the present study, we report the purification and characterization of a fibrinolytic enzyme obtained from submerged culture supernatant of Pleurotus ostreatus . To the best of our knowledge, this is the first report describing a fibrinolytic enzyme from such P. ostreatus culture.
Materials and Methods
- Chemicals and Reagents
Octyl-Sepharose Fast Flow media, Phenyl-Sepharose High Performance media, a Superdex 75 16/60 pre-packed column, a Gel Filtration LMW Calibration Kit, and an isoelectric focusing (IEF) calibration kit were purchased from GE Life sciences (Pittsburgh, PA, USA). Bovine fibrinogen, sodium dodecyl sulfate (SDS), agarose, N -tosyl-L-phenylalanine chloromethyl ketone (TPCK), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Thrombin was obtained from the Institute of Blood Products (Tianjin, China). The low-molecular-weight marker for SDS-PAGE was obtained from Sangon Co. (Shanghai, China). All other reagents used were of analytical grade unless otherwise stated.
- Strain and Conditions for Enzyme Production
Slants of Pleurotus ostreatus , No. 4241 of China General Microbiological Culture Collection Center (CGMCC), stored at -80℃ in 20% glycerol, were used to inoculate potato dextrose agar (PDA) plates. After 5 days of incubation at 23℃, the mycelia were inoculated by punching out approximately 1 cm 2 of cultures with a sterilized cutter into 250 ml flasks containing 80 ml of synthetic medium (pH 7.0): 4 g glucose, 0.3 g KH 2 PO 4 , 0.15 g MgSO 4 , 70 ml water, and 10 ml soy milk (obtained by filtering the boiled mixture of 5 g soybean and 100 ml water). The liquid culture was fermented at 25℃ on a r otary shaker at 150 rpm for 6days.
- Purification of Fibrinolytic Enzyme
Pleurotus ostreatus culture supernatant was collected by centrifugation (4,500 × g for 25 min at 4℃). The fibrinolytic enzyme from the culture supernatant was purified by a five-step process as follows. The supernatant collected after centrifugation was kept at -20℃ for 24 h. Frozen supernatant containing the enzyme was thawed at room temperature, and then centrifuged at 10,000 × g for 15 min at 4℃ to remove flocculated particles. The clear supernatant was taken for ammonium sulfate precipitation.
Solid ammonium sulfate was added to the supernatant to make 0-20% saturation. This mixture was kept at 4℃ for overnight and centrifuged at 7,000 × g for 30 min at 4℃. The pellet was discarded and the supernatant was adjusted to a final concentration of 45% ammonium sulfate by addition of solid ammonium sulfate. The mixture was allowed to stand overnight at 4℃ with constant stirring. The precipitated proteins were removed by centrifugation (10,000 × g , 15 min, and 4℃) and the clear supernatant was taken for further purification.
The supernatant containing 45% saturation of ammonium sulfate was loaded onto an Octyl-Sepharose FF column (2.6× 20 cm) equilibrated with 20 mM phosphate buffer (pH 7.4) containing 45% ammonium sulfate. The bound proteins were eluted with a step-wise decreasing gradient of 45%, 30%, and 0% of ammonium sulfate in the same buffer at a flow rate of 2 ml/min. The active fractions were pooled, brought to a concentration of 35% ammonium sulfate saturation, kept overnight at 4℃, and then centrifuged.
The active supernatant obtained by centrifugation was applied onto a Phenyl Sepharose HP column (1.6× 20 cm) previously equilibrated with 20 mM phosphate buffer (pH 7.4) containing 35% saturation of ammonium sulfate. The elution was performed with a decreasing linear gradient of 35-0% saturation of ammonium sulfate in the same buffer at a flow rate of 2 ml/min. The active fractions were pooled, dialyzed against deionized water, and lyophilized.
The lyophilized powder was dissolved in 20 mM phosphate buffer (pH 7.4) containing 300 mM NaCl and loaded onto a Superdex 75 (16× 60) pre-packed gel filtration column, which was previously equilibrated with the same buffer. The proteins were eluted at a flow rate of 1 ml/min. The active fractions were pooled, dialyzed against deionized water, and lyophilized. The purified enzyme was used for further characterization.
- Fibrinolytic Activity Assay and Determination of Protein Concentration
Fibrinolytic activity was determined by the fibrin-plate method, as described by Astrup and Mullertz [2] , with some modifications. In a Petri dish, 5 ml of a 0.4% ( w /v) fibrinogen solution in 100 mM barbital sodium-chlorhydric acid buffer (pH 7.8) was mixed with 5 ml of a 0.5% (w/v) agarose solution along with 1 ml of a thrombin solution (200 U/ml). The Petri plates were left for 1 h at room temperature to form a fibrin clot layer. Usually, commercially available fibrinogen contains some plasminogen, so plasminogen-free plates were prepared by using a similar method, but were heated at 85℃ for 30 min to inactivate plasminogen. Then, a 10 μl sample was carefully placed onto a fibrin plate. The plates were incubated at 37℃ for 6h and the activity was quantified by measuring the area of lysis on the plate.
The protein concentration of the samples at every step of chromatography was determined spectrophotometrically at 280 nm. The protein concentration of pooled enzyme solution from each purification step was measured according to the Lowry method [18] , using bovine serum albumin (BSA) as standard.
- Determination of Molecular Mass by SDS-PAGE and Gel Filtration
The molecular mass of the purified enzyme under reducing denaturing conditions was determined by SDS-PAGE as described by Laemmli [12] , using a 12% polyacrylamide gel. After electrophoresis, the protein bands were visualized by staining with Coomassie brilliant blue R-250. For calibration, an SDS-PAGE LMW standards kit comprising rabbit phosphorylase B (97.4 kDa), BSA (66.2 kDa), rabbit actin (43 kDa), bovine carbonic anhydrase (31 kDa), trypsin inhibitor (20.1 kDa), and hen egg white lysozyme (14.4 kDa) was used.
The native molecular mass of the enzyme was determined by gel filtration chromatography using a Superdex 75 (16× 60 cm) column (ÄKTA purifier 100). The column was operated at room temperature and the buffer used was 20 mM phosphate buffer (pH 7.4) containing 300 mM NaCl. A gel filtration LMW calibration kit comprising BSA (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) was used. The void volume of the column was calculated by using blue dextran 2000.
- Determination of Isoelectric Point
The isoelectric point (pI) of the purified fibrinolytic enzyme was determined by isoelectric focussing (IEF). The samples were loaded onto a prefocused ampholyte polyacrylamide gel (7.5% polyacrylamide with 3% cross-linkage). IEF conditions include prefocusing of carrier ampholytes at 60 V for 15 min and focusing of the sample at 550 V, 8 mA, and 10℃. The pI of the sample was calculated by comparing with standards (pI 3.5-9.3) from an IEF calibration kit (GE Life Sciences).
- Fibrinogenolytic Activity Analysis
Fibrinogenolytic activity was measured by a modified fibrinogenolytic assay [11] . The fibrinogen solution (45 μl of 2% human fibrinogen in 0.05 mol/l Tris-HCl buffer (pH 7.6)) was mixed with the purified enzyme solution (45 μl of 0.102 mg/ml, 29.46U/m l) and incubated at 37℃ for different time intervals: 3, 15, and 45 min, and 2, 4, 6, 10, 14, and 24 h, respectively. After the indicated time intervals, aliquots were transferred onto ice and analyzed by SDS-PAGE to examine the cleavage pattern of the fibrinogen chains.
- Effects of pH and Temperature on Enzyme Activity
The optimum pH of the fibrinolytic enzyme activity was determined by incubating the enzyme on fibrin plates with different pH buffers. The concentration of each buffer solution was 20 mM and the buffers used were as follows: citrate-phosphate (pH 3.0-7.0), barbital sodium-chlorhydric acid buffer (pH 7.0-9.5), Na 2 CO 3 /NaHCO 3 (pH 9.5-10.4), and Na 2 HPO 4 /NaOH (pH 11.0). For pH stability, the enzyme was incubated in different pH buffers for 24 h and the residual activities were analyzed by the standard method.
The optimum temperature of the enzyme activity was measured at pH 7.6 over a temperature range of 18-60℃. For temperature stability, the enzyme was incubated at 23-55℃ for 4 h and the residual activities were determined by the standard method.
- Effects of Metal Ions and Protease Inhibitors on the Enzyme Activity
Six protease inhibitors including EDTA, TPCK, soybean trypsin inhibitor (SBTI), PMSF, aprotinin, and pepstatin, were used to check their effect on the activity of purified fibrinolytic enzyme. EDTA, SBTI, PMSF, and aprotinin were dissolved in deionized water, pepstatin was dissolved in methanol, and TPCK was dissolved in DMSO. Each inhibitor was mixed with purified enzyme at two different concentrations (2 and 10 mM) and incubated at 37℃ for 15 and 30 min. Residual enzyme activities were determined.
Metal chlorides were dissolved in deionized water, mixed with purified enzyme, and incubated at 37℃ for 12 h. The final concentration of metal ions in the reaction mixture was 1, 2, 8, 10, or 100 mM; the enzyme activity was determined by fibrin plate assay. The relative activities were calculated on the basis of the activities of the purified enzyme without any additives under the same experimental conditions.
- Identification of Partial Amino Acid Sequence
For determining the partial amino acid sequences, purified fibrinolytic enzyme was subjected to SDS-PAGE and the gel was stained with Coomassie brilliant blue R250. The stained portion was used for analysis of amino acid sequence by Q-TOF2. The protein band was excised from the gel and submitted for amino acid sequencing using high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) at the National Center of Biomedical Analysis, Beijing (China). Mass spectral sequencing was performed using a Q-TOF II mass analyzer (Q-TOF2; Micromass Ltd., Manchester, UK). Peptide sequencing was performed using a palladium-coated borosilicate electrospray needle (Protana, Denmark). The mass spectrometer was used in positive-ion mode with a source temperature of 80℃, and a potential of 800 V was applied to the nanospray probe. MS/MS spectra were transformed using MaxEnt3 (MassLynx, Micromass), and amino acid sequences were interpreted manually using PepSeq (BioLynx, Micromass).
Results and Discussion
- Purification of the Fibrinolytic Enzyme
When Pleurotus ostreatus was cultured by the submerged method, the mycelia could secrete fibrinolytic enzyme to the culture liquid. Under the optimized culture conditions, the activity of fibrinolytic enzyme produced by Pleurotus ostreatus reached to 8.10 ± 1.56 (U/mg). In the present study, soybean milk was used as a major component of the submerged culture medium, which was different from that of other mushrooms [17 , 24] . Soybean protein in soybean milk not only acts as an effective inducer and nitrogen source, but also has an advantage of low price. Fibrinolytic enzymes have been purified and characterized from submerged culture broth of Formitella fraxinea , Cordyceps sinensis , and S. commune [15 , 17 , 23 , 25] .
The fibrinolytic enzyme was purified to electrophoretic homogeneity by the steps listed in Table 1 . The supernatant, after removal of mycelia of Pleurotus ostreatus , was frozen at -20℃, and then thawed at room temperature. During this process, flocculation occurred and some particles including polysaccharides produced during fermentation were flocculated. It is known that during submerged culture fermentation of mushrooms, some polysaccharides are produced and secreted into the culture medium by mycelia. In general, it is difficult to separate the polysaccharides and proteins of the culture medium. Precipitation of polysaccharides by addition of ethanol to the culture fluid is a commonly used method [13 , 16 , 29] . However, in our present study, addition of ethanol caused inactivation of the fibrinolytic enzyme. During the process development for the purification of the fibrinolytic enzyme, it was observed that freeze-thaw treatment of the culture fluid could flocculate some polysaccharides and some unwanted proteins from the culture fluid, while the fibrinolytic activity of the fluid showed no significant change (data not shown). The enzyme was salted-out by ammonium sulfate for further purification.
Summary of purification steps for the fibrinolytic enzyme fromPleurotus ostreatus.
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aProtein was measured by the method of Lowry et al. [18]. bActivity was measured in 100 mM barbital sodium-chlorhydric acid buffer (pH 7.8) at 37℃ for 6 h.
After ammonium sulfate precipitation, an Octyl-Sepharose FF column, a Phenyl-Sepharose HP column, and Superdex 75 columns were used to purify the enzyme to homogeneity. The Octyl-Sepharose FF gave two fibrinolytic enzyme peaks ( Fig. 1 A). The major fraction with fibrinolytic activity was eluted in 30% saturation of ammonium sulfate. This fraction was collected and applied onto the Phenyl-Sepharose HP column, which also yielded two peaks showing fibrinolytic activity in the linear gradient fraction of 35-0% and 0% of ammonium sulfate, respectively ( Fig. 1 B). The maximum activity was found in the 35-0% fraction. Two steps of hydrophobic interaction chromatography using Octyl-Sepharose FF and Phenyl-Sepharose HP increased the purity of the enzyme by 6.95-fold ( Table 1 ). The enzyme was further purified by a Superdex 75 gel filtration column. During this, a clear single peak with a high specific activity was obtained at 77.57 ml ( Fig. 1 C). As summarized in Table 1 , the enzyme was purified by 147-fold with a final yield of 7.54%. The specific activity of the purified enzyme was 1,199.75 U/mg of protein.
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Purification of the fibrinolytic enzyme from Pleurotus ostreatus. (A) Hydrophobic interaction chromatography (HIC) on an Octyl-Sepharose FF column; (B) HIC on a Phenyl-Sepharose HP column; (C) Gel filtration chromatography on a Superdex 75 column.
- Determination of Molecular Mass and Isoelectric Point of Purified Enzyme
The molecular mass of the purified fibrinolytic enzyme was determined by both gel filtration chromatography and SDS-PAGE ( Figs. 2 A and 2 B). The molecular mass was calculated according to the method of Andrews [1] . The elution curve and co-relation between lgMr and K av of the markers on a prepacked Superdex 75 (16 × 60 cm) column are shown in Fig. 2 A. The elution volume and K av of the fibrinolytic enzyme were 77.57 ml and 0.4343, respectively, so the native molecular mass of the enzyme was 13.6 kDa as estimated by gel filtration, while under reducing denaturing conditions the molecular mass was 18 kDa as estimated by SDS-PAGE ( Fig. 2 B). It indicates that the enzyme is a monomeric protein.
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Determination of molecular mass and isoelectric point of the purified enzyme. (A) The elution curve and relation between lg Mr and Kav of the standards on a Superdex 75 (16 × 60 cm) column. The void volume of the column was calculated by using blue dextran (2,000 kDa, (Peak 1). The standard proteins used for calibration are BSA (67 kDa, Peak 2), ovalbumin (43 kDa, Peak 3), chymotrypsinogen A (25 kDa, Peak 4), and ribonuclease A (13.7 kDa, Peak 5). (B) SDS-PAGE under reducing denaturing conditions: Lane 1, Molecular weight marker; lane 2, purified enzyme. (C) Isoelectric focusing: Lane 1, pI marker; lane 2, purified enzyme.
The molecular mass of fibrinolyic enzymes from mushrooms are reported in the range of 17-100 kDa [19] . The MW of the purified enzyme was smaller than that of fibrinolytic enzymes from Cordyceps militaris (52 kDa), Pleurotus ostreatus (32 kDa), and Flammulina velutipes (37 kDa) [10 , 24 , 28] but somewhat similar to fibrinolytic enzymes from Armillaria mellea (18.5k Da), Tricholoma saponaceum (18.1 kDa), and Schizophyllum commune (17 kDa) [8 , 9 , 23] . Because of its small size, the fibrinolytic enzyme from Pleurotus ostreatus culture fluid may result in less antigenicity. The isoelectric point of the fibrinolytic enzyme was 8.52 as determined by IEF ( Fig. 2 C).
- Biochemical Characterization of the Purified Enzyme
The optimum pH of the enzyme for the fibrinolytic activity was found to be 7.4; however, the relative activity was also higher in the pH range of 6.8-8.2 ( Fig. 3 A). The pH stability of the enzyme was studied in the range of pH 3-11 by measuring the residual activities after incubation at each pH for 24 h ( Fig. 3 B). The enzyme was very stable in the pH range of 6-9 at 37℃ for 24 h, but the residual activity decreased sharply above pH 9 ( Fig. 3 B).
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Effects of pH and temperature on fibrinolytic activity of the enzyme. (A) The optimum pH of the purified enzyme was checked at 37℃ in 20 mM of different buffers: citrate-phosphate for pH 3.0-7.0, barbital sodium-chlorhydric acid buffer for pH 7.0-9.5, Na2CO3/NaHCO3 for pH 9.5-10.4, and Na2HPO4/NaOH for pH 11.0. (B) For pH stability, the enzyme was incubated at 37℃ for 24 h over a range of pH buffers and the residual activities were determined. (C) The optimum temperature of the purified enzyme was determined at different temperatures in pH 7.8 buffer. (D) For temperature stability, the enzyme was incubated at different temperatures for 0.5 h (◆), 1 h (▲), 2 h (−), 3 h (■) and 4 h (●), , and the residual activities were measured at standard conditions.
The optimum pH for mushroom fibrinolytic enzymes is pH 5.0-10.0 [19] . The optimum pH of the purified enzyme for the fibrinolytic activity was 7.4, which is the physiological pH of humans. Its optimum pH is similar to fibrinolytic enzymes from Cordyceps militaris , Armillaria mellea , and Tricholoma saponaceum , but higher than that of Formitella faxinea , Flammulina velutipes , and Schizophyllum commune enzymes [8 , 9 , 10 , 15 , 23 , 24] . It showed a higher pH stability than enzymes from Schizophyllum commune (pH 4.0-6.0) and Pleurotus ostreatus (pH 6.0-7.0) [23 , 28] .
The effect of temperature on the fibrinolytic activity of the enzyme was studied at pH 7.8 ( Fig. 3 C). The enzyme exhibited maximum activity at 45℃. The enzyme was very stable below 45℃, and showed 78%, 76%, and 72% of the initial activity after incubating for 4 h at 31℃, 37℃, and 45℃, respectively ( Fig. 3 D). Mushroom fibrinolytic enzymes have an optimum temperature of 20-60℃ [19] . The optimal temperature of the enzyme and its thermal stability are comparable to MsK of Schizophyllum commune [23] , but higher than most of the reported mushroom fibrinolytic enzymes from Cordyceps sp., Formitella fraxinea , Tricholoma saponaceum , and Armillaria mellea [10 , 14 , 17] . The enzyme FVP-1 from Flammulina velutipes was stable only upto 30℃ [24] . The present fibrinolytic enzyme has good characteristics such as physiological optimum pH, and high thermal and pH stability, which makes it unique and can be useful in fibrinolytic therapy.
The effect of various metal ions and reagents on the enzyme activity was studied. The enzyme activity was enhanced when incubated with metal ions Ca 2+ (111%) and K + (120%) at a final concentration of 1 mM; but its activity was inhibited by Fe 2+ (58%) and Fe 3+ (0%) ions at a higher concentration of 100 mM. Moreover, the enzyme activity was completely inhibited by the metal chelator EDTA (2.5m M). These results indicate that the metal ions are located at or near the active site of the enzyme and are necessary for catalytic activity. Protease inhibitors (at 2 mM final concentration) TPCK, SBTI, PMSF, aprotinin, and pepstatin did not show any significant effect on the activity of the enzyme. However, at higher concentration (10 mM), SBTI and pepstatin showed inhibition on enzyme activity with residual activities of 0% and 64%, respectively. This kind of behavior was earlier observed in the case of MsK from Schizophyllum commune , PoFE from Pleurotus ostreatus mycelia, and AMMP from Armillaria mellea [14 , 23 , 28] .
Comparison of internal peptide fragments of fibrinolytic enzyme fromPleurotus ostreatuswith earlier reported metalloproteases.
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aIdentical amino acid sequences are underlined.
Based on their inhibitor specificities, mushroom fibrinolytic enzymes can be classified into two groups as serine proteases (those inhibited by serine protease inhibitors such as PMSF, TPCK, aprotinin) and metalloproteases (those inhibited by metalloprotease inhibitors like EDTA). Our results are consistent with view that the fibrinolytic enzymes belonging to metalloproteases require divalent metal ions for their activities. Hence, the enzyme activity is inhibited by EDTA and enhanced by metal ions Ca 2+ , Mg 2+ , and Zn 2+ [27 , 28] . These results suggest that the purified fibrinolytic enzyme is a metalloprotease.
The partial amino acid sequence of the purified enzyme was determined by Q-TOF2. Three internal peptide sequences were obtained. The amino acid sequences of the three peptide fragments are ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. All three peptides were analyzed by using the NCBI-BLAST database for sequence similarity with earlier reported fibrinolytic enzymes ( Table 2 ). The sequences of these peptide fragments showed a high degree of homology with a metallo-endopeptidase (PoMEP) from P. ostreatus fruiting body ( Table 2 ) [22] . It is noteworthy that the peptide II showed 100% similarity with metalloendopeptidases from P. ostreatus and Armillaria mellea ( Table 2 ).
- Fibrinogenolytic Activity of the Purified Enzyme
The enzyme also exhibited fibrinogenolytic activity, and the degradation patterns of fibrinogen by the purified enzyme were analyzed by SDS-PAGE ( Fig. 4 A). On control run, reduced fibrinogen separated into α, β, and γ chains. When fibrinogen was incubated with the purified enzyme, the α band disappeared first, followed by the β band and then the γ band. This indicates that the enzyme degraded the α chain first, followed by the β chain and the γ chain. The α chain was degraded within 3 min and the β chain in 45mi n, while the γ chain was initially resistant but degraded slowly by 10 h. The α and β chains were degraded without the formation of fibrin. This implied that the cleavage sites of α and β chains were different from thrombin.
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Analysis of fibrinogenolysis and plasminogen activation by the purified enzyme. (A) SDS-PAGE analysis of reduced human fibrinogen after digestion by the purified enzyme; lane C, control sample containing reduced α, β, and γ chains of fibrinogen; lanes 1-9, degradation pattern of fibrinogen at different time intervals of 3 min, 15 min, 45 min, 2, 4, 6, 10, 14, and 24 h, respectively. (B) Activation of plasminogen on the plasminogen-free fibrin plate. (C) Activation of plasminogen on the plasminogen-rich fibrin plate. The purified enzyme (10 μl) was placed at four spots (1-4) on each plate and incubated at 37℃ for 6 h. Activity was quantified by measuring the area of lysis (1-4 circles) and compared between plasminogen-free and -rich plates.
These results are similar to the degradation patterns of PoFE from Pleurotus ostreatus mycelia, FVP-1 from Flammulina velutipes , and CSP from Cordyceps sinensis [17 , 24 , 28] . However, some fibrinolytic enzymes cannot hydrolyze all three chains of fibrinogen; AJ from Staphylococcus sp. and AMMP from Armillaria mellea can hydrolyze only the α chain [3 , 14] . The fibrinolytic enzyme from fermented shrimp paste cannot hydrolyze the α chain of fibrinogen [7] .
The area and clarity of the lytic circles produced by the fibrinolytic enzyme were significantly different between plasminogen-free ( Fig. 4 B) and plasminogen-rich plates ( Fig. 4 C). These results indicate that the fibrinolytic enzyme is capable of degrading fibrin directly (direct lysis), and can also activate plasminogen to plasmin (plasminogen activator type). Fibrinolytic enzymes exhibiting dual functions of direct lysis and plasminogen activator types are not common and are rarely reported [23] .
Oral administration of the fibrinolytic enzyme nattokinase (NK) has been reported to enhance fibrinolytic activity in plasma and the production of endogenous plasminogen activators t-PA [31] . NK has already been developed as an oral drug in the market. Pleurotus ostreatus is an edible and medicinal mushroom. Taking into account of these studies and facts, the fibrinolytic enzyme from Pleurotus ostreatus might also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis. Further investigation of the enzyme is under way.
Acknowledgements
This work was supported by the National Natural Science Foundation of P. R. China (NSFC) (No. 31171744) and the Natural Science foundation of Heilongjiang province (No. ZD201305), China.
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