A Coprinus comatus fibrinolytic enzyme, its preparation method and application

Abstract

The present invention provides a Coprinus comatus fibrinolytic enzyme, a preparation method thereof and an application thereof, relating to the technical field of bioengineering. The Coprinus comatus fibrinolytic enzyme of the present invention is obtained by liquid fermentation using the Coprinus comatus strain YY-17 as a strain. The optimal action temperature of the Coprinus comatus fibrinolytic enzyme is 42 °C, and it can maintain good activity at the physiological pH of the human body; Mn 2+ , Ca 2+ and Zn 2+ have obvious activation effects on the enzyme, while different concentrations of Fe 3+ , Fe 2+ , Al 2+ show obvious inhibitory effects on the Coprinus comatus fibrinolytic enzyme. It is confirmed by experiments that the Coprinus comatus fibrinolytic enzyme of the present invention can not only directly degrade fibrin, but also activate plasminogen to indirectly degrade fibrin, and can sequentially degrade the α, β and γ chains of bovine fibrinogen, indicating that the Coprinus comatus fibrinolytic enzyme of the present invention can not only independently thrombolyze, but also activate plasminogen to indirectly thrombolyze, has no obvious acute toxicity, and has good thrombolytic and antithrombotic effects.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a Coprinus comatus fibrinolytic enzyme and a preparation method and application thereof. Background Art

[0002] Thromboembolic diseases have become the number one killer affecting human health, with morbidity and mortality rates increasing year by year. The main cause is the abnormal fibrinolytic system, which causes fibrinogen to form fibrin under the action of thrombin and deposit in blood vessels to form thrombi, which is one of the main causes of human death. Thrombolytic therapy is currently the main means of treating and preventing thrombotic diseases. Although the currently used thrombolytic products such as lumbrokinase, urokinase, and tissue plasminogen activator have definite efficacy, they still have disadvantages such as poor specificity, short half-life, and high price. Therefore, the development of safe and reliable thrombolytic agents with few side effects is currently an urgent need, and natural biological resources are an important resource for the development of new thrombolytic agents.

[0003] Coprinus comatus, scientifically known as Coprinus comatus, belongs to Fungi, Agaricales, Copriniaceae, Coprinus genus. Coprinus comatus is delicious, nutritious, high in protein, rich in 20 kinds of amino acids including 8 essential amino acids for the human body and a variety of biologically active factors. It is beneficial to the stomach, clears the mind, aids digestion, and increases appetite. It has been identified by the Food and Agriculture Organization of the United Nations and the World Health Organization as one of the rare edible fungi that integrates "natural, nutritious, and health-care" functions. Studies have found that it has antioxidant, blood sugar-lowering, liver damage prevention, anti-tumor, immunity-enhancing, and antibacterial effects. It is a traditional medicinal and edible fungus with important medical value. However, the current development of Coprinus comatus is insufficient, and the functional factors are not fully explored. Therefore, Coprinus comatus is an important biological resource that needs to be developed urgently.

[0004] For nearly a decade, the inventor team has been committed to the preparation, separation and purification of microbial fibrinolytic enzymes and their functional research. They have isolated 5 new fibrinolytic enzymes from Cordyceps militaris, Neurospora edulis and Agrocybe chinensis, and authorized 5 related invention patents, with the authorization numbers ZL201510159992.7, ZL201010204473.5, ZL200910072630.9, ZL200810137564.4 and ZL201910822101.X. The first 4 fibrinolytic enzymes were obtained by fungal fermentation, and the fifth fibrinolytic enzyme was isolated from the fruiting body. However, it is worth noting that there has been no report on the extraction of fibrinolytic enzymes from Coprinus comatus in related research at home and abroad. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a Coprinus comatus fibrinolytic enzyme, which not only has good thrombolytic performance, but also has good safety in use, a relatively small molecular weight, relatively low immunogenicity, is easy to be utilized by the human body, has a low preparation cost, and has the prospect of development and application.

[0006] To solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides a Coprinus comatus fibrinolytic enzyme, which is a single-subunit protein, and the molecular weight of the Coprinus comatus fibrinolytic enzyme is 19.5 kDa; the 9 amino acid sequences at the N-terminus of the Coprinus comatus fibrinolytic enzyme are shown in SEQ ID NO.1.

[0008] Preferably, the Coprinus comatus fibrinolytic enzyme is prepared from the Coprinus comatus strain YY-17; the preservation number of the Coprinus comatus strain YY-17 is CGMCC NO.40393.

[0009] The present invention provides a preparation method of the above Coprinus comatus fibrinolytic enzyme, and the preparation method includes the following steps:

[0010] Using the seed liquid of the Coprinus comatus strain YY-17 for liquid fermentation culture, and obtaining the crude enzyme liquid of the Coprinus comatus fibrinolytic enzyme after centrifuging the obtained fermentation liquid;

[0011] Subjecting the crude enzyme liquid to salting out, first hydrophobic interaction chromatography, gel chromatography, strong cation exchange chromatography, and second hydrophobic interaction chromatography in sequence, and then the Coprinus comatus fibrinolytic enzyme can be obtained.

[0012] Preferably, the culture medium for the liquid fermentation includes the following components in mass percentage: fructose 1-5%, wheat bran 0.5-2.5%, soybean cake powder 1.5-5.5%, NaCl 0.15-0.25%, KH2PO4 0.15-0.25%, MgSO4 0.15-0.25%.

[0013] Preferably, the conditions for the liquid fermentation are: inoculation amount 3-20%, temperature 20-25 °C, time 4-8 days.

[0014] Preferably, the environmental temperature for the salting out is 3-5 °C, and the time for the salting out is 10-14 h.

[0015] Preferably, both the first hydrophobic interaction chromatography and the second hydrophobic interaction chromatography adopt linear elution; the starting buffer solution for the first hydrophobic interaction chromatography is a 0.01 - 0.03 mol / L PBS buffer solution with (NH4)2SO4 saturation of 25 - 35%, the eluent is a 0.01 - 0.03 mol / L PBS buffer solution, and the flow rate is 3 - 5 mL / min; the starting buffer solution for the second hydrophobic interaction chromatography is a 0.01 - 0.03 mol / L PBS buffer solution with (NH4)2SO4 saturation of 20 - 30%, the eluent is a 0.01 - 0.03 mol / L PBS buffer solution, and the flow rate is 1 - 3 mL / min.

[0016] Preferably, the eluent for the gel chromatography is a 0.01 - 0.03 mol / L NaH2PO4 - HCl buffer solution; the pH value of the NaH2PO4 - HCl buffer solution is 4.0 - 5.0, and the flow rate is 5 - 7 mL / min.

[0017] Preferably, the strong cation exchange chromatography adopts linear elution. The starting buffer solution for the strong cation exchange chromatography is a NaH2PO4 - HCl buffer solution, and the eluent is a NaH2PO4 - HCl buffer solution containing 0.1 - 0.2 mol / L NaCl; the concentration of the NaH2PO4 - HCl buffer solution is 0.01 - 0.03 mol / L, and the pH is 4.0 - 5.0; the flow rate of the elution is 3 - 5 mL / min.

[0018] The present invention also provides the application of the above - mentioned Coprinus comatus fibrinolytic enzyme or the above - mentioned preparation method in the preparation of thrombolytic and antithrombotic drugs.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The present invention provides a Coprinus comatus fibrinolytic enzyme, which is obtained by liquid fermentation using the Coprinus comatus strain YY - 17 as the strain. The optimal action temperature of the Coprinus comatus fibrinolytic enzyme is 42°C, and it can maintain good activity at the physiological pH of the human body; Mn 2+ 、Ca 2+ and Zn 2+ have obvious activation effects on this enzyme, while different concentrations of Fe 3+ 、Fe 2+ 、Al 2+It shows an obvious inhibitory effect on Coprinus comatus fibrinolytic enzyme. It can be confirmed through experiments that the Coprinus comatus fibrinolytic enzyme described in the present invention can not only directly degrade fibrin, but also activate plasminogen to indirectly degrade fibrin, and can sequentially degrade the α, β, and γ chains of bovine fibrinogen, indicating that the Coprinus comatus fibrinolytic enzyme described in the present invention can not only thrombolyze independently, but also activate plasminogen to indirectly thrombolyze, has no obvious acute toxicity, and has good thrombolytic and antithrombotic effects.

[0021] Biological deposit description

[0022] The Coprinus comatus strain YY-17 described in the present invention, taxonomic name: Coprinus comatus, is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC NO.40393, the deposit date is March 17, 2023, and the deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Description of the drawings

[0023] Figure 1 SDS-PAGE electrophoresis map of Coprinus comatus fibrinolytic enzyme; among them, 1 represents the standard protein Marker, and 2 represents Coprinus comatus fibrinolytic enzyme.

[0024] Figure 2 is the optimal action temperature of Coprinus comatus fibrinolytic enzyme.

[0025] Figure 3 is the optimal action pH of Coprinus comatus fibrinolytic enzyme.

[0026] Figure 4 is the action diagram of Coprinus comatus fibrinolytic enzyme dissolving fibrin in Example 1.

[0027] Figure 5 is the degradation diagram of Coprinus comatus fibrinolytic enzyme on bovine fibrinogen verified by SDS-PAGE method. Detailed implementation manners

[0028] The present invention provides a Coprinus comatus fibrinolytic enzyme, the Coprinus comatus fibrinolytic enzyme is a single-subunit protein, and the molecular weight of the Coprinus comatus fibrinolytic enzyme is 19.5 kDa; the 9 amino acid sequences at the N-terminus of the Coprinus comatus fibrinolytic enzyme are shown in SEQ ID NO.1.

[0029] In the present invention, the 9 amino acid sequences at the N-terminus of the Coprinus comatus fibrinolytic enzyme are A-T-Y-T-G-G-S-Q-T; the Coprinus comatus fibrinolytic enzyme described in the present invention is preferably prepared from the Coprinus comatus strain YY-17; the deposit number of the Coprinus comatus strain YY-17 is CGMCC NO.40393. The Coprinus comatus strain YY-17 described in the present invention is collected from the Daxing'anling region, and through biological identification, its taxonomic name is Coprinus comatus.

[0030] The present invention provides the above-mentioned preparation method of Coprinus comatus fibrinolytic enzyme, and the preparation method comprises the following steps:

[0031] Performing liquid fermentation culture by using the seed liquid of Coprinus comatus strain YY-17, and centrifuging the obtained fermentation broth to obtain the crude enzyme solution of Coprinus comatus fibrinolytic enzyme;

[0032] Subjecting the crude enzyme solution to salting out, first hydrophobic interaction chromatography, gel chromatography, strong cation exchange chromatography, and second hydrophobic interaction chromatography in sequence, and then the Coprinus comatus fibrinolytic enzyme can be obtained.

[0033] In the present invention, the seed liquid of Coprinus comatus strain YY-17 is preferably obtained by slant activation culture and plate subculture of Coprinus comatus strain YY-17. The present invention has no special requirements for the specific methods of the slant activation culture and plate subculture, and the conventional slant activation culture and plate subculture methods in the art can be adopted. In the present invention, the culture medium for the liquid fermentation preferably comprises the following components in mass percentage: fructose 1-5%, wheat bran 0.5-2.5%, soybean cake powder 1.5-5.5%, NaCl 0.15-0.25%, KH2PO4 0.15-0.25%, MgSO4 0.15-0.25%; more preferably, it comprises the following components in mass percentage: fructose 3%, wheat bran 1%, soybean cake powder 3.5%, NaCl 0.15%, KH2PO4 0.25%, MgSO4 0.2%. In the present invention, the conditions for the liquid fermentation are preferably: inoculum size 3-20%, temperature 20-25 °C, time 4-8 days; more preferably: inoculum size 10%, temperature 24 °C, time 6 days. In the liquid fermentation of the present invention, the seed age of the seed liquid of Coprinus comatus strain YY-17 is preferably 7-9 days, more preferably 8 days; the filling volume of the liquid fermentation is preferably 25-150 mL, more preferably 50 mL; the rotation speed of the liquid fermentation is preferably 160 r / min. The present invention has no special requirements for the pH value during the liquid fermentation, and it can be kept natural.

[0034] In the present invention, the salting out preferably uses ammonium sulfate for salting out; during the salting out, the saturation of (NH4)2SO4 is preferably 50%-70%, more preferably 60%. In the present invention, the environmental temperature for the salting out is preferably 3-5 °C, more preferably 4 °C; the time for the salting out is preferably 10-14 h, more preferably 12 h. The salting out in the present invention is mainly to remove the miscellaneous proteins in the crude enzyme solution, which is beneficial to the obtaining of Coprinus comatus fibrinolytic enzyme.

[0035] In the present invention, after the crude enzyme solution is subjected to ammonium sulfate salting out, solid-liquid separation is carried out. The obtained precipitate is dissolved in PBS buffer solution, and the first hydrophobic interaction chromatography separation is carried out. In the present invention, the first hydrophobic interaction chromatography is preferably Octyl-Sepharose Fast Flow hydrophobic interaction chromatography; the starting buffer solution of the first hydrophobic interaction chromatography is preferably a 0.01-0.03 mol / L PBS buffer solution with a (NH4)2SO4 saturation of 25-35%, more preferably a 0.02 mol / L PBS buffer solution with an ammonium sulfate saturation of 30%; the eluent of the first hydrophobic interaction chromatography is preferably a 0.01-0.03 mol / L PBS buffer solution, more preferably a 0.02 mol / L PBS buffer solution. In the present invention, the first hydrophobic interaction chromatography preferably adopts linear elution; the flow rate is preferably 3-5 mL / min, more preferably 4 mL / min; the independent pH of the buffer solution is preferably 7.4.

[0036] In the present invention, the chromatographic column of the gel chromatography is preferably a Sephadex G-25 gel chromatographic column; the eluent of the gel chromatography is preferably a 0.01-0.03 mol / L NaH2PO4-HCl buffer solution, more preferably a 0.02 mol / L NaH2PO4-HCl buffer solution; the pH value of the NaH2PO4-HCl buffer solution is preferably 4.0-5.0; the flow rate is preferably 5-7 mL / min, more preferably 6 mL / min. In the present invention, the purpose of the gel chromatography separation is to decolorize, remove excess ammonium sulfate salt, and exchange buffer solution.

[0037] In the present invention, the active ingredient obtained by the gel chromatography separation is further subjected to strong cation exchange chromatography separation. In the present invention, the strong cation exchange chromatography preferably adopts linear elution, and the chromatographic column is preferably an SP-Sepharose High Performance strong cation exchange chromatographic column. In the present invention, the starting buffer solution of the strong cation exchange chromatography is preferably a NaH2PO4-HCl buffer solution, and the eluent is preferably a NaH2PO4-HCl buffer solution containing 0.1-0.2 mol / L NaCl, more preferably a NaH2PO4-HCl buffer solution containing 0.1 mol / L NaCl. In the present invention, the concentration of the NaH2PO4-HCl buffer solution is preferably 0.01-0.03 mol / L, more preferably 0.02 mol / L; the pH of the NaH2PO4-HCl buffer solution is preferably 4.0-5.0, more preferably pH 4.0; the elution flow rate is preferably 3-5 mL / min, more preferably 4 mL / min.

[0038] The active components obtained by strong cation exchange chromatography in the present invention are subjected to a second hydrophobic interaction chromatography. In the present invention, the second hydrophobic interaction chromatography preferably adopts linear elution; the chromatographic column is preferably a Source 15PHE hydrophobic interaction chromatographic column. In the present invention, the starting buffer solution for the second hydrophobic interaction chromatography is preferably a 0.01 - 0.03 mol / L PBS buffer solution with a (NH4)2SO4 saturation of 20 - 30%, more preferably a 0.02 mol / L PBS buffer solution with a ammonium sulfate saturation of 20%; the eluent is preferably a 0.01 - 0.03 mol / L PBS buffer solution, more preferably a 0.02 mol / L PBS buffer solution; the pH value of the buffer solution is preferably 7.4 independently; the elution flow rate is preferably 1 - 3 mL / min, more preferably 2 mL / min.

[0039] The present invention also provides the use of the above Coprinus comatus fibrinolytic enzyme or the above preparation method in the preparation of thrombolytic and antithrombotic drugs.

[0040] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be described in detail below in conjunction with embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0041] In the following embodiments, unless otherwise specified, they are all conventional methods.

[0042] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0043] Example 1

[0044] Preparation of Coprinus comatus fibrinolytic enzyme

[0045] (1) Preparation of crude enzyme solution of fermentation broth: Using fructose 3%, bran 1%, soybean cake powder 3.5%, NaCl 0.15%, KH2PO4 0.25%, MgSO4 0.2% as the culture medium, inoculating the liquid seeds of Coprinus comatus with an inoculation age of 8 days for liquid fermentation, with an inoculation amount of 10%, a liquid loading amount of 50 mL / 250 mL Erlenmeyer flask, natural pH, and fermenting and culturing at 24°C and a rotation speed of 160 r / min for 6 days to obtain the fermentation broth; centrifuging the fermentation broth at 4°C and 10000 r / min for 20 min, and the obtained supernatant is the crude enzyme solution of Coprinus comatus fibrinolytic enzyme;

[0046] (2) Adjust the ammonium sulfate saturation of the crude enzyme solution of Coprinus comatus fibrinolytic enzyme to 60%, salt out overnight at 4°C, centrifuge at 4°C and 10000 r / min for 20 min, discard the supernatant, dissolve the precipitate with 0.02 mol / L PBS (pH 7.4) buffer solution, and keep it at 4°C for standby;

[0047] (3) First hydrophobic interaction chromatography: The enzyme solution after salting-out and reconstitution in step (2) was separated by an Octyl-Sepharose Fast Flow hydrophobic chromatography column. The starting buffer was 0.02 mol / L PBS buffer with 30% ammonium sulfate saturation; the eluent was 0.02 mol / L PBS buffer, with a flow rate of 4 mL / min, and the active fractions were collected.

[0048] (4) Gel chromatography for desalting and decolorization separation: The active fractions obtained in step (3) were separated by a Sephadex G-25 gel chromatography column. The eluent was 0.02 mol / L NaH2PO4-HCl (pH 4.0) buffer solution, with a flow rate of 6 mL / min, and the active fractions were collected.

[0049] (5) Strong cation exchange chromatography separation: The active fractions in step (4) were separated by an SP-Sepharose High Performance strong cation exchange chromatography column. The starting buffer solution was 0.02 mol / L NaH2PO4-HCl (pH 4.0) buffer solution, and the eluent was 0.02 mol / L NaH2PO4-HCl (pH 4.0) containing 0.1 mol / L NaCl. Then, linear elution was performed with 4 column volumes of the starting buffer solution and 4 column volumes of the eluent in the direction of increasing NaCl concentration, with a flow rate of 4 mL / min, and the active fractions were collected.

[0050] (6) Second hydrophobic interaction chromatography separation: The active fractions in step (5) were separated by a Source 15PHE hydrophobic interaction chromatography using a linear elution method; the starting buffer solution was 0.02 mol / L PBS (pH 7.4) buffer solution with 20% ammonium sulfate saturation, and the eluent was 0.02 mol / L PBS (pH 7.4) buffer solution. After sample loading, linear elution was performed with 2 column volumes of the starting buffer solution and 2 column volumes of the eluent from 20% - 0 ammonium sulfate saturation, and the collected active fractions were the Coprinus comatus fibrinolytic enzyme of the present invention.

[0051] Example 2

[0052] Preparation of Coprinus comatus fibrinolytic enzyme

[0053] (1) Preparation of crude enzyme solution from fermentation broth: Using fructose 1%, wheat bran 2.5%, soybean cake powder 1.5%, NaCl 0.15%, KH2PO4 0.25%, and MgSO4 0.25% as the medium, inoculate the liquid seeds of Coprinus comatus with a seed age of 7 days for liquid fermentation. The inoculation amount is 5%, the liquid filling amount is 150 mL / 250 mL Erlenmeyer flask, the pH is natural, and ferment and culture at 24°C and a rotation speed of 160 r / min for 8 days to obtain the fermentation broth; centrifuge the fermentation broth at 4°C and 10,000 r / min for 20 min, and the obtained supernatant is the crude enzyme solution of Coprinus comatus fibrinolytic enzyme;

[0054] (2) Adjust the ammonium sulfate saturation of the crude enzyme solution of Coprinus comatus fibrinolytic enzyme to 50%, salt out overnight at 4°C, centrifuge at 4°C and 10,000 r / min for 20 min, discard the supernatant, and dissolve the precipitate with 0.01 mol / L PBS (pH 7.4) buffer solution for standby at 4°C;

[0055] (3) First hydrophobic interaction chromatography: Separate the enzyme solution after salting out and redissolving in step (2) through an Octyl-Sepharose Fast Flow hydrophobic chromatography column. The starting buffer solution is 0.03 mol / L PBS buffer solution containing an ammonium sulfate saturation of 25%; the eluent is 0.03 mol / L PBS buffer solution, with a flow rate of 4 mL / min, and collect the active components;

[0056] (4) Gel chromatography for desalting and decolorization separation: Separate the active components obtained in step (3) through a Sephadex G-25 gel chromatography column. The eluent is 0.01 mol / L NaH2PO4-HCl (pH 4.0) buffer solution, with a flow rate of 7 mL / min, and collect the active components;

[0057] (5) Strong cation exchange chromatography separation: Separate the active components in step (4) through an SP-Sepharose High Performance strong cation exchange chromatography column. The starting buffer solution is 0.01 mol / L NaH2PO4-HCl (pH 4.0) buffer solution, and the eluent is 0.01 mol / L NaH2PO4-HCl (pH 4.0) containing 0.1 mol / L NaCl. Then, perform linear elution with 4 column volumes of the starting buffer solution and 4 column volumes of the eluent in the direction of increasing NaCl concentration, with a flow rate of 3 mL / min, and collect the active components;

[0058] (6) Second hydrophobic interaction chromatography separation: The active component obtained in step (5) is separated by Source 15PHE hydrophobic interaction chromatography using a linear elution method; the starting buffer solution is a 0.01 mol / L PBS (pH 7.4) buffer solution containing ammonium sulfate at a saturation of 30%, and the eluent is a 0.01 mol / L PBS (pH 7.4) buffer solution. After sample loading, linear elution is carried out with 2 column volumes of the starting buffer solution and 2 column volumes of the eluent according to an ammonium sulfate saturation of 25% - 0. The collected active component is the Coprinus comatus fibrinolytic enzyme of the present invention.

[0059] Example 3

[0060] Preparation of Coprinus comatus fibrinolytic enzyme

[0061] (1) Preparation of crude enzyme solution from fermentation broth: Using fructose 5%, wheat bran 0.5%, soybean cake powder 5.5%, NaCl 0.25%, KH2PO4 0.15%, MgSO4 0.15% as the culture medium, inoculating the Coprinus comatus liquid seeds with a seed age of 9 days for liquid fermentation, with an inoculation amount of 20%, a liquid loading of 25 mL / 250 mL Erlenmeyer flask, natural pH, and fermenting and culturing at 24°C and a rotation speed of 160 r / min for 4 days to obtain the fermentation broth; centrifuging the fermentation broth at 4°C and 10,000 r / min for 20 min, and the obtained supernatant is the crude enzyme solution of Coprinus comatus fibrinolytic enzyme;

[0062] (2) Adjust the ammonium sulfate saturation of the crude enzyme solution of Coprinus comatus fibrinolytic enzyme to 70%, carry out salting out overnight at 4°C, centrifuge at 4°C and 10,000 r / min for 20 min, discard the supernatant, dissolve the precipitate with a 0.03 mol / L PBS (pH 7.4) buffer solution, and keep it at 4°C for standby;

[0063] (3) First hydrophobic interaction chromatography: Separate the enzyme solution after salting out and re-dissolution in step (2) through an Octyl-Sepharose Fast Flow hydrophobic chromatography column. The starting buffer solution is a 0.01 mol / L PBS buffer solution containing ammonium sulfate at a saturation of 35%; the eluent is a 0.01 mol / L PBS buffer solution, with a flow rate of 4 mL / min, and collect the active component;

[0064] (4) Gel chromatography desalting and decolorization separation: Separate the active component obtained in step (3) through a Sephadex G-25 gel chromatography column. The eluent is a 0.03 mol / L NaH2PO4-HCl (pH 4.0) buffer solution, with a flow rate of 5 mL / min, and collect the active component;

[0065] (5) Strong cation exchange chromatography separation: The active component from step (4) is separated by an SP-Sepharose High Performance strong cation exchange chromatography column. The starting buffer solution is 0.03 mol / L NaH2PO4-HCl (pH 4.0) buffer solution, and the eluent is 0.03 mol / L NaH2PO4-HCl (pH 4.0) containing 0.1 mol / L NaCl. Then, linear elution is carried out with 4 column volumes of the starting buffer solution and 4 column volumes of the eluent in the direction of increasing NaCl concentration at a flow rate of 5 mL / min, and the active component is collected;

[0066] (6) Second hydrophobic interaction chromatography separation: The active component from step (5) is separated by Source 15PHE hydrophobic interaction chromatography using a linear elution method; The starting buffer solution is 0.03 mol / L PBS (pH 7.4) buffer solution with 20% ammonium sulfate saturation, and the eluent is 0.03 mol / L PBS (pH 7.4) buffer solution. After sample loading, linear elution is carried out with 2 column volumes of the starting buffer solution and 2 column volumes of the eluent from 25% - 0 ammonium sulfate saturation, and the collected active component is the Coprinus comatus fibrinolytic enzyme of the present invention.

[0067] Example 4

[0068] Determine the properties of the Coprinus comatus fibrinolytic enzyme separated and purified in Example 1

[0069] The enzyme activity is determined by the fibrin plate method, and the specific steps are as follows:

[0070] (1) The Coprinus comatus fibrinolytic enzyme separated and purified in Example 1 is subjected to SDS-PAGE electrophoresis to verify the purity and analyze its molecular weight, and the results are as Figure 1 .

[0071] It can be seen that the Coprinus comatus fibrinolytic enzyme prepared by the present invention is a single-subunit protein with a molecular weight of 19.5 kDa and has reached electrophoresis purity. The N-terminal 9 amino acid sequence of the subunit measured by Edman degradation method is:

[0072] A-T-Y-T-G-G-S-Q-T.

[0073] (2) Take 10 μL of the above Coprinus comatus fibrinolytic enzyme and spot it on the fibrin plate, place it at different temperatures for reaction for 6 h, calculate the relative enzyme activity by measuring the diameter of the lysed circle, and thus determine the optimal action of the Coprinus comatus fibrinolytic enzyme, and the results are as Figure 2 .

[0074] It can be seen that the optimal temperature of the Coprinus comatus fibrinolytic enzyme of the present invention is 42 °C.

[0075] (3) Prepare fibrin plates with different pH values. Take 10 μL of the Coprinus comatus fibrinolytic enzyme prepared in Example 1 and spot it on the fibrin plates with different pH values. React at 37 °C for 6 h. By measuring the diameter of the lysis circle and calculating the relative enzyme activity, the optimal pH of the Coprinus comatus fibrinolytic enzyme is determined, and the results are as Figure 3 shown.

[0076] It can be seen that the optimal pH for the Coprinus comatus fibrinolytic enzyme of the present invention is 7.6, indicating that the Coprinus comatus fibrinolytic enzyme can maintain good activity at the physiological pH of the human body.

[0077] (4) Mix different concentrations of metal ions Cu 2+ , Ca 2+ , Mg 2+ , Ba 2+ , Na + , Al 2+ , Fe 2+ , Fe 3+ , Mn 2+ and Zn 2+ with the Coprinus comatus fibrinolytic enzyme in a 1:1 ratio, place it at 4 °C and react for 12 h. Then take 10 μL of the mixed solution and spot it on the plate, react at 37 °C for 6 h, measure the diameter of the lysis circle, and calculate the relative enzyme activity. The results are shown in Table 1.

[0078] Table 1 shows the effects of metal ions on the Coprinus comatus fibrinolytic enzyme

[0079]

[0080]

[0081]

[0082] It can be seen that Mn 2+ , Ca 2+ and Zn 2+ have obvious activating effects on the enzyme, while different concentrations of Fe 2+ , Fe 3+ , Al 2+ show obvious inhibitory effects on the Coprinus comatus fibrinolytic enzyme.

[0083] Example 5

[0084] Use the fibrin plate method to test the fibrinolytic activity of the Coprinus comatus fibrinolytic enzyme prepared in Example 1. The specific steps are as follows:

[0085] The fibrin plate contains fibrinogen (fibrin may be contained in commercially available fibrinogen) and thrombin. Soluble fibrinogen forms fibrin monomers under the action of thrombin, and the fibrin monomers spontaneously associate and polymerize to form visible fibrin gels. Add the Coprinus comatus fibrinolytic enzyme solution to the surface of this plate gel. After a short period of incubation, the Coprinus comatus fibrinolytic enzyme can dissolve fibrin, forming a visible transparent circle on the surface of the plate gel. The results are shown in Figure 4 .

[0086] Example 6

[0087] Use SDS-PAGE electrophoresis to test the solubility of this enzyme in fibrinogen. The specific steps are as follows:

[0088] Mix the Coprinus comatus fibrinolytic enzyme prepared in Example 1 and bovine fibrinogen in equal volumes and mix well. React in a water bath at 37°C. Use SDS-PAGE to detect the degradation of bovine fibrinogen by the Coprinus comatus fibrinolytic enzyme. The degradation pattern is shown in Figure 5 ; From left to right, the lanes are standard protein, bovine fibrinogen, the mixture of Coprinus comatus fibrinolytic enzyme and bovine fibrinogen after reacting for 30 s, 1 min, 5 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, and 4 h respectively.

[0089] It can be seen that the α-chain is completely degraded after 30 s, the β-chain is completely degraded after 15 min, and although the γ-chain is not completely degraded after 4 h, its structure has been damaged and the degradation products increase. This degradation sequence is the same as that of human fibrinolytic enzyme degrading fibrinogen. This shows that the Coprinus comatus fibrinolytic enzyme of the present invention can dissolve fibrinogen and can sequentially degrade the α, β, and γ chains of bovine fibrinogen.

[0090] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structural or equivalent process transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall be included in the patent protection scope of the present invention by the same token.

Claims

1. A Coprinus comatus fibrinolytic enzyme, characterized in that, The Coprinus comatus fibrinolytic enzyme is a single-subunit protein, and the molecular weight of the Coprinus comatus fibrinolytic enzyme is 19.5 kDa; the 9 amino acid sequences at the N-terminus of the Coprinus comatus fibrinolytic enzyme are as shown in SEQ ID NO.1; The Coprinus comatus fibrinolytic enzyme is prepared from the Coprinus comatus strain YY-17; the preservation number of the Coprinus comatus strain YY-17 is CGMCC NO.40393; The preparation method of the Coprinus comatus fibrinolytic enzyme comprises the following steps: Performing liquid fermentation culture with the Coprinus comatus strain YY-17 seed liquid, and centrifuging the obtained fermentation liquid to obtain the crude enzyme liquid of the Coprinus comatus fibrinolytic enzyme; Subjecting the crude enzyme liquid to salting out, the first hydrophobic interaction chromatography, gel chromatography, strong cation exchange chromatography, and the second hydrophobic interaction chromatography in sequence, and then the Coprinus comatus fibrinolytic enzyme can be obtained; The culture medium for the liquid fermentation comprises the following components in mass percentage: fructose 1-5%, wheat bran 0.5-2.5%, soybean cake powder 1.5-5.5%, NaCl 0.15-0.25%, KH2PO4 0.15-0.25%, MgSO4 0.15-0.25%; The conditions for the liquid fermentation are: inoculum amount 3-20%, temperature 20-25 °C, time 4-8 days; The environmental temperature for the salting out is 3-5 °C, and the time for the salting out is 10-14 h; Both the first hydrophobic interaction chromatography and the second hydrophobic interaction chromatography adopt linear elution; the starting buffer solution for the first hydrophobic interaction chromatography is a 0.01-0.03 mol / L PBS buffer solution with (NH4)2SO4 saturation of 25-35%, the eluent is a 0.01-0.03 mol / L PBS buffer solution, and the flow rate is 3-5 mL / min; the starting buffer solution for the second hydrophobic interaction chromatography is a 0.01-0.03 mol / L PBS buffer solution with (NH4)2SO4 saturation of 20-30%, the eluent is a 0.01-0.03 mol / L PBS buffer solution, and the flow rate is 1-3 mL / min; The eluent for the gel chromatography is a 0.01-0.03 mol / L NaH2PO4-HCl buffer solution; the pH value of the NaH2PO4-HCl buffer solution is 4.0-5.0, and the flow rate is 5-7 mL / min; The strong cation exchange chromatography adopts linear elution, the starting buffer solution for the strong cation exchange chromatography is a NaH2PO4-HCl buffer solution, and the eluent is a NaH2PO4-HCl buffer solution containing 0.1-0.2 mol / L NaCl; the concentration of the NaH2PO4-HCl buffer solution is 0.01-0.03 mol / L, pH is 4.0-5.0; the flow rate of the elution is 3-5 mL / min.

2. The preparation method of the coprinus comatus fibrinolytic enzyme according to claim 1, characterized in that, The preparation method comprises the following steps: Performing liquid fermentation culture with the Coprinus comatus strain YY-17 seed liquid, and centrifuging the obtained fermentation liquid to obtain the crude enzyme liquid of the Coprinus comatus fibrinolytic enzyme; The crude enzyme solution is successively subjected to salting out, the first hydrophobic interaction chromatography, gel chromatography, strong cation exchange chromatography, and the second hydrophobic interaction chromatography to obtain the Coprinus comatus fibrinolytic enzyme; The liquid fermentation medium comprises the following components in mass percentage: fructose 1-5%, wheat bran 0.5-2.5%, soybean cake powder 1.5-5.5%, NaCl 0.15-0.25%, KH2PO4 0.15-0.25%, MgSO4 0.15-0.25%; The conditions for the liquid fermentation are: inoculum amount 3-20%, temperature 20-25°C, time 4-8 days; The environmental temperature for the salting out is 3-5°C, and the time for the salting out is 10-14 h; Both the first hydrophobic interaction chromatography and the second hydrophobic interaction chromatography adopt linear elution; the starting buffer solution for the first hydrophobic interaction chromatography is a 0.01-0.03 mol / L PBS buffer solution with (NH4)2SO4 saturation of 25-35%, the eluent is a 0.01-0.03 mol / L PBS buffer solution, and the flow rate is 3-5 mL / min; the starting buffer solution for the second hydrophobic interaction chromatography is a 0.01-0.03 mol / L PBS buffer solution with (NH4)2SO4 saturation of 20-30%, the eluent is a 0.01-0.03 mol / L PBS buffer solution, and the flow rate is 1-3 mL / min; The eluent for the gel chromatography is a 0.01-0.03 mol / L NaH2PO4-HCl buffer solution; the pH value of the NaH2PO4-HCl buffer solution is 4.0-5.0, and the flow rate is 5-7 mL / min; The strong cation exchange chromatography adopts linear elution, the starting buffer solution for the strong cation exchange chromatography is a NaH2PO4-HCl buffer solution, and the eluent is a NaH2PO4-HCl buffer solution containing 0.1-0.2 mol / L NaCl; the concentration of the NaH2PO4-HCl buffer solution is 0.01-0.03 mol / L, and the pH is 4.0-5.0; the flow rate of the elution is 3-5 mL / min.

3. Use of the Coprinus comatus fibrinolytic enzyme according to claim 1 or the preparation method according to claim 2 in the preparation of thrombolytic and antithrombotic drugs.

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