KR20050021146A - Fibrinolytic Enzyme from Cordyceps militaris - Google Patents

Abstract

PURPOSE: A fibrinolytic enzyme from Cordyceps militaris is provided, which enzyme has different substrate specificity from plasmin, that is, the enzyme has improved fibrin and fibrinogen dissociating activity, so that it can be useful for prevention and treatment of thrombosis. CONSTITUTION: The fibrinolytic enzyme is isolated from Cordyceps militaris, and has the properties of: molecular weight of 52 kDa, optimal temperature of 37 deg. C, optimal hydrogen concentration of pH 7, increased activity by Ca2+ and Mg22+, decreased activity by Co2+, Cu2+, Fe2+ and Zn2+, a serine protease of which activity is inhibited by penylmethylsulfonylfluoride(PMSF) and not by EDTA, a chymotripsin-like protease which exhibits the highest activity at a substrate of chimotrypsin, hydrolysis of alpha-, beta- and gamma-chains of fibrin, and Aalpha- and Bbeta-chains of fibrinogen, and nonhydrolysis of gamma-chain of fibrinogen.

Description

Fibrinolytic Enzyme from Cordyceps militaris from Cordyceps sinensis Extract

The present invention relates to a thrombolytic enzyme extracted from Militaris cordyceps sinensis and a liquid culture comprising the enzyme, and more particularly, to a thrombolytic enzyme which is distinguished in the substrate specificity from the conventional thrombolytic enzyme. Liquid cultures, their use in the treatment and prevention of thrombi and methods for their purification.

Blood clots are formed through the process of converting fibrinogen to fibrin by the protein lytic action of thrombin, followed by the formation of insoluble fibrin clots (thrombosis). Fibrin clots are dissolved by hydrolysis of plasmin which is activated from plasminogen by tissue plasminogen activator. In general, the hydrolysis of fibrin is called fibrinolysis (thrombolytic). The formation of blood clots and the dissolution of blood clots are well balanced in living organisms. However, when fibrin (fibrin) is not hydrolyzed due to other abnormalities, blood clots such as myocardial infarction occur. Intravenous administration of urokinase and streptokinase, which can degrade fibrin, is widely used in the treatment of such thrombosis. Unfortunately, these enzymes have low specificity for fibrin and are expensive. Tissue plasminogen activator (tPA) has been continuously developed as a therapeutic agent for thrombosis due to its good efficacy and strong affinity for fibrin.

Mushrooms, on the other hand, are well known for their nutritional value and pharmacological value. Recent studies on the biochemistry of mushrooms have been carried out around proteases, ribonucleases, antifungal proteins, anti-inflammatory proteins and fibrin lytic enzymes. In addition, some mushroom proteases are known to exhibit fibrinolytic properties, and proteases of Armillaria mellea , Tricholoma saponaceum , Daedaleopsis styracina , Trichaptum abietium , Coriolus versicolor , Pisolithus tinctorius, and Tricholomopsis decora have been actively studied. Therefore, the search for a substance having a useful pharmacological effect on such a natural substance is constantly being requested.

The present inventors have recently isolated the enzyme having a thrombolytic ability from the extract of the parasitic fungus Militaris cordyceps, and examined its properties, not only has the characteristics different from the conventional enzymes, but also shows strong thrombolytic activity. The present invention has been found and the present invention has been completed.

Accordingly, it is an object of the present invention to provide a thrombolytic enzyme isolated from Militaris cordyceps sinensis and liquid cultures comprising the same, uses for the prophylaxis and prevention of their thrombosis and methods for their purification.

In one aspect, the present invention provides a thrombolytic enzyme purified from Militaris cordyceps sinensis and a liquid culture comprising the same.

Militaris cordyceps ( C. militaris ) is a fungi parasitic to the larvae of the species Lepidoptera, one of the promising herbs that attract attention as a herbal medicine. This mushroom has been widely used as a tonic to enhance longevity, endurance and vitality, and its various pharmacological effects have been reported in relation to anti-inflammatory action, elevated natural killer activity, metastasis suppression activity and immunosuppression.

Recently, studies have been actively conducted on the isolation of fibrin and fibrinogen lytic enzymes for use as thrombolytic agents for traditional mushrooms, fermented foods and other natural extracts, and the present inventors have identified candidates for malitaris cordyceps as a candidate material having such function. It was selected and used as.

Millitaris Cordyceps Sinensis Extract and Isolated Thrombolytic Enzyme According to the Present Invention Have a Substrate Specificity Different from Previously Known Plasmins, Appear as Single Band by Zymography and SDS-PAGE, Molecular Weight of about 52 kDa, optimum activity temperature of about 37 ° C., belonging to the family of neutral proteases which exhibit optimal enzyme activity around pH 7, and enhanced by Ca ²⁺ , Mg2 ²⁺ in relation to enzyme activity against metal ions, Is a serine protease that is inhibited by, Co ²⁺ , Cu ²⁺ , Fe ²⁺ and Zn ²⁺ , and whose activity is markedly inhibited by phenylmethylsulfonylfluoride (PMSF) in relation to enzyme inhibitors, but not affected by EDTA, It is a chymotrypsin-like protease that exhibits the highest activity against the substrate for chymotrypsin, and hydrolyzes α-chain, β-chain and γ-γ-chains of fibrin, and While it rapidly hydrolyzes the Aα-chain, Bβ-chain of nogen, the γ-chains are characterized by having a function that does not hydrolyze rapidly.

The liquid culture of the present invention can be obtained by shaking culture of Millitaris cordyceps mycelium in a flask of SDAY medium (40 g of dextrose in 1 liter, 10 g of peptone, 10 g of yeast extract). The liquid culture medium may be lyophilized and then powdered to be used for necessary purposes such as food raw materials.

In a further aspect, the present invention provides a use for the treatment and prevention of thrombosis of the Militaris cordyceps and liquid culture medium comprising the same. Thrombolytic enzymes contained in the culture have different substrate specificities than plasmin and can therefore be used as a new form of thrombolytic agent.

In still another aspect, the present invention provides a method for homogenizing the fruiting body of liquid cultured C. militaris , precipitating the homogenate with pre-cooled ethanol, ion exchange chromatography on DEAE-sephacel and Provided are a method for purifying fibrin lytic enzymes, which is purified by gel permeation chromatography of Sephadex G-75.

<Example>

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are described to aid the understanding of the present invention and do not limit the content and scope of the invention.

Example 1 Culture of Militaris Cordyceps Sinensis

Millitaris Cordyceps militaris was obtained from the American Type Culture Collection, Rockville Md., USA (ATCC 26848). Millitaris Cordyceps mycelium was grown at 150 rpm in a 500 ml Ehrlenmeyer flask containing 100 ml of SDAY medium (40 g of dextrose in 1 liter, 10 g of peptone, 10 g of yeast extract) in 900 ml of water. Fruiting bodies were obtained on medium containing lepidopteran pupae at 18 ° C.

Example 2: Purification of Fibrin and Fibrinogen Lysing Enzymes

Unless stated otherwise, all purification procedures were performed at 4 ° C. Protein concentration was determined using the BCA Protein Essay Kit (Pierce Co.). The millitaris cordyceps fruiting body obtained in Example 1 was immediately stored in a -70 ° C freezer. 100 g of frozen mushrooms were thawed and homogenized for 2 minutes at maximum speed in a homogenizer using 100 ml of the same volume of water. After homogenization was centrifuged at 600 × g for 30 minutes at 4 ° C., the crude extract was placed in 1 liter bucket stainless steel contained in a salt-ice bath. An equal volume of pre-frozen (-70 ° C.) ethanol was added dropwise under continuous stirring, then the solution was stirred for an additional hour. The precipitated protein was removed by centrifugation at 600 x g for 30 minutes at 0 ° C. The clear ethanol-soluble fractions were transferred to a steel bucket contained on salt-ice and then added dropwise by increasing ethanol to 75% under constant stirring. After further stirring for 1 hour, the precipitated protein was recovered by centrifugation at 25 ° C. for 30 minutes. The supernatant was removed, the pellet was dried and the proteins suspended in 20 mM Tris-HCl, pH 7.5. Insoluble materials were removed by centrifugation at 10,000 xg for 10 minutes at 4 ° C. Suspended protein pellets containing enzyme were added to a DEAE-Sephacel column (5 cm x 10 cm) pre-equilibrated with the same 20 mM Tris-HCl buffer (pH 7.5) and 0.5 to 4 ° C. with a linear gradient of 0 to 0.5 M NaCl. Eluted at a flow rate of ml / min.

The results of fractionation of fibrinolytic enzymes by ion exchange chromatography on DEAE-Sephacel are shown in FIG. 1. Fraction volume was 5 ml. FIG. 1 shows ion exchange chromatography fractions of an electrolytic enzyme derived from Militaris cordyceps on DEAE-sephacel, with active protein peaks eluted from the column by increasing the linear gradient of NaCl. Bands showing thrombolytic activity were detected in relatively large protein peaks eluted with a 0.2 M NaCl gradient.

Active fractions from ion exchange chromatography on DEAE-sephacel were collected and concentrated using centricon. For further purification, gel permeation was performed at 4 ° C. using a Sephadex G-75 column (1.5 cm × 100 cm) equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl. And eluted at a flow rate of 0.5 ml / min. Active fractions were collected by lyophilization. The gel permeation profile of the protein eluate through the Sephadex G-75 column is shown in FIG. Fraction volume was 5 ml. Only one peak with fibrin lytic activity could be isolated.

Example 3: Protease Essay

Protease activity was determined by measuring the release of acid-soluble material from azocasein (Sigma Co.). 50 μl of enzyme sample / column fraction was added to 300 μl of 1% (W / V) azocasein (50 mM Tris-HCl, pH 7.0). After incubation at 37 ° C. for 20 minutes, 600 μl of ice-cold 10% (W / V) trichloroacetic acid was added with immediate vortexing. Samples were left on ice for 10 minutes and then centrifuged at 15,000 g for 15 minutes. The amount of acid-soluble material in the supernatant was measured by absorbance at 600 nm. One unit of protease activity is defined as the amount necessary to sufficiently produce an acid-soluble substance that exhibits an absorbance of 0.1 at 366 nm after incubation for 1 hour at 37 ° C. from azocasein. The results are summarized in Table 1 below.

Example 4: Molecular Weight Measurement

The molecular weight of the enzyme was determined using the standard marker protein, ProSieve Protein Markers (BMA). Determined by SDS-PAGE and fibrin-Zymography.

SDS-PAGE was performed according to Laemmli's method using a 12% polyacrylamide gel, and the gel was stained with Coomassie Brilliant Blue R 250. Fibrin-Zymography was performed according to the method of Kim et al. A marine gel solution (12%) containing 0.12% (W / V) fibrinogen was prepared at a total volume of 10 ml to remove insoluble impurities caused when the SDS stock was mixed by centrifugation. Thrombin (1 unit / ml) solution and TEMED (N, N, N ', N'-tetramethylethylenediamine) were added to the gel solution at final concentrations of 0.1 unit / ml and 0.028% (V / V), respectively. Purified CMF enzyme was electrophoresed into fibrin gel and then washed in 2.5% Triton X-100 solution and 37 ° C. in a bath containing reaction buffer (20 mM Tris HCl, pH 7.5 0.15 M NaCl, 0.02% NaN ₃ ). Incubate for 12-15 hours at. In fibrin-gymography, thrombolytic enzymes appear as a clear single band of thrombolysis against the blue background of the non-hydrolyzed fibrin substrate.

The electrophoretic pattern of the protein eluted from the peaks of the active protein for each chromatographic isolate according to Example 2 was investigated on an SDS-PAGE gel. Figure 3 is a photograph showing the SDS-PAGE and fibrin-zymography of the CMF enzyme, lane 1, protein molecular weight markers; Lane 2, crude protein from Militaris cordyceps sinensis; Lane 3, purified CMF enzyme; Lane 4 shows purified CMF enzymes on fibrin-Zymograph respectively.

Active protein peaks from the ion exchange column showed multiple protein bands on the SDS-PAGE gel. However, the electrophoretic pattern of active protein peaks from gel permeation chromatography showed only one protein band. This result indicates that proteins of different molecular weights do not contain fibrinolytic activity so these active peaks could be removed during gel permeation. Zymography also showed a clear band with fibrinolytic activity in the purified enzyme preparation, and the molecular weight of the CMF enzyme was found to be about 52 kDa. Purified fibrin lytic enzyme was found to have a single band by zymography and SDS-PAGE, and its apparent molecular weight was determined to be 52 kDa (FIG. 3, lane 3-lane 4).

After the final purification step, the purified enzymes had a specific activity of 846 units / mg protein, meaning that this enzyme was 483-fold purified in approximately 11.2% yield.

The separation characteristics of the fibrinolytic enzymes identified in the above examples are summarized in Table 1 below.

Purification Characteristics of Fibrinolytic Enzymes from Militaris Cordyceps Sinensis step Volume (ml) Protein (mg) Protein activity Specific activity (units / mg) % Recovery Fold Homogenate 624 n.d. n.d. - - - Crude extract 400 4,141 7,247 1.75 (100) (One) Pellet 56 9.6 4,124 430 57 246 CM23 Elution 15.6 4.08 1,848 453 25.5 259 Sepadex G-75 Elution 3 0.96 812 846 11.2 483 Note: n.d. not detected

Example 5: Effect of Temperature and pH on Enzyme Activity

The optimal temperature of the enzyme activity was determined by measuring protease activity at various temperatures. To test the effect of temperature, 10 μg of the thrombolytic enzyme in 90 μl of 20 mM Tris-HCl (pH 7.5) was incubated at various temperatures for 1 hour, followed by evaluation of residual activity in the supernatant.

To determine the optimal pH of thrombolytic enzyme activity, fibrin lytic activity was evaluated at various pHs from pH 2 to 10. To determine the pH stability of the enzyme, 10 μg of enzyme solution was added to 0.5 M glycerin-HCl (pH 2.0 to 3.0), 0.5 M acetate (pH 4.0 to 5.0), 0.5 M Tris-HCl (pH 6.0 to 8.0), and 0.5 90 μl M glycine-NaOH (pH 9.0-10.0) buffer was added. After incubation at room temperature for 1 hour, the residual protease activity was measured with 1% azocasein of each enzyme solution and the results are shown in FIG. 4. The enzyme showed optimal activity at pH 7.0, and the thrombolytic activity was relatively higher in neutral than in the alkaline and acidic regions. Thus, the purified CMF enzyme was found to be a neutral protease.

The effect of temperature on the activity of thrombolytic enzymes was examined within the range of 30-80 ° C. in 20 mM Tris-HCl (pH 7.5) buffer. 5 is a graph showing the effect of temperature on the activity of the CMF enzyme, the enzyme activity was evaluated by maintaining it at various temperatures for 1 hour, and azocasein assay was performed. As shown in FIG. 5, the optimum temperature for enzyme activity was about 37 ° C. This optimum temperature is determined by Bacillus sp. By other fibrinolytic enzymes, such as Lee et al. 50 ° C. from KDO-13 strain, 65 ° C. from Bacillus substilis K-54 by Yoo et al and Kim et al. [Jun-Ho Kim and Yang-Sun Kim. Purification and characterization of fibrinolytic enzyme from Armillaria mellea . Kor. J. Mycology 1998; 26 (4): 583-588], lower than 55 ° C from Armillaria mellea . However, purified CMF enzymes were stable between 30-40 ° C. and rapidly decreased activity above 50 ° C.

Example 6: Influence of Metal Ions and Proteases on Enzyme Activity

The effect of metal ions was investigated using MgCl ₂ , ZnCl ₂ , CoCl ₂ , FeCl ₃ , CaCl ₂ , LiCl ₂ , CsCl ₂ and CuSO ₄ . Purified enzymes may be used in the presence or absence of divalent cations such as Mg ²⁺ , Ca ²⁺ , Co ²⁺ , Cu ²⁺ , Li ²⁺ , Zn ²⁺ , Fe ²⁺ , and Cs ²⁺ and other inhibitors. Pre-incubation at 37 ° C. for 1 hour at 1 mM final concentration in 20 mM Tris-HCl. After incubation for 1 hour at room temperature, the residual protease activity was measured by 1% azocaine of each enzyme solution. The results are expressed as the ratio of relative activity to that not shown. The effect of metal ions on fibrinolytic activity is shown in Table 2.

Effect of Metal Ions on the Activity of CMF Enzymes metal Relative activity (%) 1 mM absence 100 CuSO ₄ 77.5 CoCl ₂ 79.5 CaCl ₂ 102.5 ZnCl ₂ 86.1 FeCl ₂ 88.2 MgCl ₂ 104.1

Residual enzyme activity was analyzed after incubating the enzyme with 1 mM metal ion for 1 hour at 37 ° C., while the enzyme activity was enhanced by Ca ²⁺ , Mg2 ²⁺ , while Co ²⁺ , Cu ²⁺ , Fe ²⁺ And Zn ²⁺ .

The effect of protease inhibitors on enzyme activity was also tested using EDTA, phenylmethyl sulfonylfluoride (PMSF), and mercaptoethanol. To study the effects of various inhibitors, enzymes were previously incubated for 1 hour in 20 mM Tris-HCl (pH 7.5) at 37 ° C. using different inhibitors. After incubation, the residual protease activity of the enzyme was analyzed by 1% azocasein assay. The results are expressed as the ratio of relative activity to that not shown. The effect of protease inhibitors on thrombolytic activity is shown in Table 2.

Effect of Protease Inhibitors on the Activity of CMF Proteins Protease inhibitors Relative activity (%) 1 mM PMSF 65.1 EDTA 101.8 TLCK 76.7 TPCK 78.2

The enzyme activity was markedly inhibited by 1 mM phenylmethylsulfonylfluoride (PMSF), but not by EDTA, which means that the CMF enzyme of the present invention is a serine protease.

Specific inhibition by PMSF is a phenomenon seen in known thrombolytic enzymes such as nattokinase and shiokara enzyme.

Example 7: Fibrin and Fibrinogen Dissolution Activity Assays

80 μl of 1% human fibrinogen (20 mM Tris-HCl, pH 7.5, 0.15 M NaCl) was incubated with 10 μg purified CMF enzyme at 37 ° C. In addition, 10 μg of human fibrinogen solution (20 mM Tris-HCl, pH 7.5, 0.15 M NaCl) was added to human thrombin (0.1 NIH unit) and left at room temperature for 1 hour. The formed clots were added to purified CMF enzymes and incubated at 37 ° C. at various reaction time intervals. Subsequently, thrombolytic assays were performed according to the method of SDS-PAGE (Laemmli, 1970).

Hydrolysis of fibrin by purified CMF enzyme is shown in Figure 6 the results analyzed by SDS-PAGE. Purified enzymes rapidly hydrolyzed α-chain, β-chain and γ-γ-chains. The hydrolysis pattern due to the purified CMF enzyme was not the same as that caused by plasmin, which shows that the CMF enzyme had a substrate specificity distinct from the known fibrin lytic enzymes.

In addition, the CMF enzyme had fibrinogen degrading activity, and the pattern of fibrinogen degradation was analyzed by SDS-PAGE after incubating the enzyme with 10 μg of fibrinogen in 20 mM Tris-HCl buffer (pH 7.5) for 1 hour at 37 ° C. And, the results are shown in the electrophoresis picture of FIG.

As shown in Figure 7, CMF enzyme treated human fibrinogen rapidly hydrolyzed the Aα-chain, Bβ chain, whereas the γ-chains did not hydrolyze rapidly within 1 hour. However, all the chains disintegrated gradually within two hours.

Example 8: Amidolytic Activity on Chromogenic Substrates

Chromogenic substrates for measuring amidolytic activity include S-2222 (Bz-Ile-Glu- (OR) -Gly-Arg-pNA for factor Xa), S-2288 (HD-Ile-Pro for t-PA). -Arg-pNA), S-2238 (HD-Phe-Pip-Arg-pNA for thrombin), S-2251 (HD-Val-Leu-Lys-pNA for plasmin and streptokinase activated plasminogen), S-2444 (pyroGlu-Gly-Arg-pNA for eurokinase), S-2586 (MeO-Suc-Arg-Pro-Tyr-pNAHCl for chymotrypsin) and S-2765 (ZD-Arg- for factor Xa) Chromogenic protease substrates such as Gly-Arg-pNA2HCl) were used and were purchased from Chromogenix Co. Activity was measured by mixing purified CMF enzyme (200 μl of 1 μg / 50 mM Tris-HCl, pH 7.4) with 300 μl of 0.5 mM substrate. After continuous measurement at 37 ° C. for 5 minutes with a temperature controlled spectrophotometer, the amount of p-nitroaniline released was determined by measuring the change in absorbance at 405 nm and the results are shown in FIG. 8.

The CMF enzyme showed the highest activity against S-2586 (MeO-Suc-Arg-Pro-Tyr-pNAHCl, the substrate for chymotrypsin, meaning that this CMF enzyme is a chymotrypsin-like protease.

As described above, it was confirmed that the CMF enzyme according to the present invention is a novel thrombolytic enzyme that specifically degrades fibrin and fibrinogen having different substrate specificities from plasmin. Therefore, other enzymes and fermentation extracts comprising the same can be effectively used for the treatment and prevention of thrombosis.

1 is an ion exchange chromatography diagram of fibrinolytic enzyme derived from Militaris cordyceps on DEAE-sephacel.

Fig. 2 is a gel permeation chromatography diagram of fibrinolytic enzyme derived from Militaris cordyceps on Sephadex G-75.

Figure 3 is a photograph showing the SDS-PAGE and fibrin-zymography of the CMF enzyme.

Lane 1, protein molecular weight marker; Lane 2, crude protein from Militaris cordyceps sinensis; Lane 3, purified CMF enzyme; Purified CMF enzyme on lane 4 fibrin-Zymography.

4 is a graph showing the effect of pH on the activity of CMF enzymes.

5 is a graph showing the effect of temperature on the activity of CMF enzymes.

Figure 6 is an electrophoresis picture showing a fibrin dissolution pattern by CMF enzyme.

Lane 1, control; Lane 2, after 5 min reaction; Lane 3, after 10 min reaction; Lane 4, after 15 min reaction; Lane 5, after 30 min reaction; Lane 6, after 1 hour reaction.

Figure 7 is an electrophoresis picture showing the dissolution pattern of fibrinogen by CMF enzyme. Lane 1, control; Lane 2, after 5 min reaction; Lane 3, after 10 min reaction; Lane 4, after 15 min reaction; Lane 5, after 30 min reaction; Lane 6, after 1 hour reaction.

8 is a graph showing Aminolytic activity on chromogenic substrates.

Claims (4)

Neutral protease, isolated from C. militaris , shown as a single band by zymography and SDS-PAGE, having a molecular weight of about 52 kDa and optimal enzyme activity around about 37 ° C. and pH 7 Belonging to the family and enhanced by Ca ²⁺ , Mg2 ²⁺ with respect to enzymatic activity to metal ions, while inhibited by Co ²⁺ , Cu ²⁺ , Fe ²⁺ and Zn ²⁺ , and with enzyme inhibitors In this context, it is a serine protease whose activity is markedly inhibited by phenylmethylsulfonylfluoride (PMSF) but unaffected by EDTA, is a chymotrypsin-like protease with the highest activity against substrates for chymotrypsin, and α-chain of fibrin , hydrolyzes β-chain and γ-γ-chains and rapidly hydrolyzes the Aα- and Bβ-chains of fibrinogen, whereas γ-chains have a function that does not hydrolyze rapidly Ms other less milli other less fungus Cordyceps protease derived from the gong (C. militaris protease). A liquid culture medium comprising the enzyme of claim 1. A thrombolytic composition comprising an enzyme or liquid culture medium derived from Cordyceps sinensis according to claim 1 as an active ingredient. The fruiting body of C. militaris was homogenized, and this homogenate was precipitated with ethanol precooled to −70 ° C., ion exchange chromatography on DEAE-sephacel and gel permeation chromatography of Sephadex G-75. The purification method of the fibrin soluble enzyme characterized by the above-mentioned.