A kind of high-fibrinolytic enzyme-producing actinomycete and its application

Abstract

The present invention relates to a high-yield fibrinolytic enzyme actinomycete, and specifically to a high-yield fibrinolytic enzyme actinomycete new strain Streptomyces (Streptomyces p.) 214L11 and its application. The present invention obtains a new strain of actinomycete Streptomyces (Streptomyces p.) 214L11 by separating, screening and identifying soil samples from Huoyan Mountain area in Xinjiang, and the strain is subjected to stress resistance experiments and metabolic fermentation to obtain high-yield fibrinolytic enzyme, and is verified by characteristic tests such as fibrinolytic enzyme production, thrombolytic ability, anticoagulant ability, functional enzyme and antibacterial performance. The present invention provides a strain with good salt and alkali resistance, and its fermentation liquid has thrombolytic, anticoagulant and antibacterial properties, etc. The produced fibrinolytic enzyme activity is 939.9FU / mL, and the fibrinolytic enzyme produced by its fermentation liquid has in vitro thrombolytic activity, indicating that Streptomyces (Streptomyces p.) 214L11 has good application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microbial strains and their applications, and specifically to the technical field of application of a new actinomycete, Streptomyces sp. 214L11, and its fermentation liquid. Background Art

[0002] Plasmin is a proteolytic enzyme that can degrade fibrin and dissolve blood clots. It is an effective ingredient in drugs for treating thrombosis. Thrombosis is closely related to most cardiovascular diseases. According to a survey conducted by the World Health Organization (WHO), 17.9 million people died of cardiovascular diseases in 2016, accounting for 31% of the total number of deaths worldwide. In addition, as people's lifestyles change, the incidence of cardiovascular diseases is gradually getting younger. Currently, thrombolytic drugs on the market are expensive, some have low specificity for fibrin, and are prone to side effects such as gastrointestinal bleeding. Therefore, finding plasmin with good efficacy and strong specificity has always been one of the research focuses.

[0003] Commonly used thrombolytic agents in clinical practice include tissue plasminogen activator (t-PA), streptokinase (SPK) and urokinase (UK). In addition, nattokinase is also commonly used in the prevention of thrombotic diseases. It is mainly derived from microorganisms or genetically engineered bacteria. A variety of strains that produce fibrinolytic enzymes, such as Bacillus subtilis, lactic acid bacteria, Enterococcus faecalis, etc., also include Streptomyces in actinomycetes can produce fibrinolytic enzymes. In recent years, with the gradual excavation of rich microbial resources bred in special environments, the functional enzymes of their metabolism often show excellent functions, and have also become one of the important sources for the screening of fibrinolytic enzyme-producing strains. At present, there are many reports on the fibrinolytic enzymes produced by Streptomyces strains from oceans, hot springs, etc. The fibrinolytic enzymes metabolized by some strains can effectively degrade fibrin in a short time, and have strong thrombolytic properties, providing a new choice for the development of thrombolytic drugs. Existing reports indicate that extremely hot and arid regions are rich in actinomycete resources, and the functional enzymes of their metabolism often have characteristics such as good thermal stability, bioavailability and solubility, which provide a theoretical basis for the exploration and development of relevant high-performance fibrinolytic enzymes. However, there are few reports at home and abroad on the exploration and development of relevant fibrinolytic enzyme strains from extremely hot and arid regions. It is necessary to continuously explore and find better strains or strains with better properties and applications. Summary of the invention

[0004] The current technical situation is that thrombolytic drugs are expensive, have low specificity for fibrin, are prone to side effects such as gastrointestinal bleeding, and there are few strains for producing plasmin. The present invention aims to provide a new actinomycete with high fibrinolytic enzyme production, and specifically relates to a new actinomycete with high fibrinolytic enzyme production, Streptomyces sp.214L11, and an application thereof. The present invention separates and screens a new actinomycete with high fibrinolytic enzyme production, obtains the new actinomycete Streptomyces sp.214L11 with high fibrinolytic enzyme production, optimizes the conditions for the fermentation broth of the strain to produce fibrinolytic enzyme, and further conducts characteristic tests such as fibrinolytic enzyme thrombolytic ability, anticoagulant ability, functional enzyme experiment, stress resistance experiment and antibacterial performance to verify the production of fibrinolytic enzyme. The strain has fibrinolytic enzyme activity, thrombolytic and anticoagulant functions, as well as good salt resistance, alkali resistance and antibacterial performance. The fibrinolytic enzyme activity of the fermentation broth of the strain Streptomyces sp.214L11 is 939.9FU / mL, and the produced fibrinolytic enzyme has in vitro thrombolytic activity; this indicates that the new actinomycete Streptomyces sp.214L11 with high fibrinolytic enzyme production and the fermentation broth thereof provided by the present invention are applied to the pharmaceutical industry.

[0005] In order to achieve the above technical effects, the present invention is implemented through the following technical solutions.

[0006] The invention provides a new strain of actinomycete Streptomyces sp.214L11 with high fibrinolytic enzyme production. The strain Streptomyces sp.214L11 is preserved in China Center for Type Culture Collection (CCTCC), and the strain preservation number is CCTCCNO: M 20221543.

[0007] The new high-yield fibrinolytic enzyme actinomycete Streptomyces sp.214L11 provided by the present invention is screened and separated from soil samples in Huoyan Mountain area of ​​Xinjiang. Through 16S rRNA gene sequence phylogenetic analysis and morphological analysis, Streptomyces sp.214L11 belongs to the genus Streptomyces. By sequencing the gene of the strain, the obtained sequence is subjected to BLAST comparison analysis on the NCBI website. The 16S rRNA gene sequence of the strain Streptomyces sp.214L11 is consistent with the standard model bacteria of the genus Streptomycesfumanus NBRC 5154 T The highest homology was 98.85%, which was similar to that of other adjacent model strains, Streptomyces fimbriatus NBRC 15411. T The homology was 98.58%. Sequences with higher homology were selected to construct the phylogenetic tree of 16SrRNA gene sequences.T After polyphasic taxonomic identification, Streptomyces sp.214L11 is closely related to Streptomycesfumanus NBRC 5154T and Streptomycesfimbriatus NBRC 15411. T There are obvious differences, and it can be determined that the strain Streptomyces sp.214L11 is a new species of the genus Streptomyces, with typical characteristics of new species.

[0008] The new actinomycete Streptomyces sp.214L11 with high fibrinolytic enzyme production provided by the present invention is identified at the molecular level by the above-mentioned strain system well-known and recognized in the art, combined with morphological identification and identification and analysis of physiological and biochemical characteristics. The strain Streptomyces sp.214L11 involved in the present invention is Gram-positive, aerobic, motile, and can form white aerial mycelium. The mycelium is twisted and differentiated into spiral spores. After culturing for 3 days, the mycelium begins to appear blue-green.

[0009] The physiological and biochemical characteristics of the strain showed that the activities of protease, amylase and cellulase of the strain Streptomyces sp.214L11 were all positive. The sugar carbon sources that could be utilized were xylose, sucrose, inositol, L-arabinose, D-fructose, D-galactose, D-glucose, D-mannitol, raffinose and L-rhamnose. The physiological and biochemical characteristics of the strain showed that it was similar to the neighboring strain Streptomyces fumanus NBRC 5154. T and Streptomyces fimbriatus NBRC 15411 T There are clear differences.

[0010] The above-mentioned strain identification, molecular level identification of the strain system well-known and recognized in the art, and physiological and biochemical system test verification of the strain confirmed that the obtained strain number 214L11 is a typical new strain Streptomyces sp. 214L11 within the category of Streptomyces. The strain has been deposited with the Budapest Treaty International Microbiological Depository: China Type Culture Collection (CCTCC) before the application date, address: Wuhan University, Wuhan, China, Postal Code: 430072, Deposit Number: CCTCC NO: M 20221543, and the deposit date is October 7, 2022.

[0011] The gene sequence of the strain Streptomyces sp. 214L11 is shown in SEQ ID NO: 1.

[0012] The isolation medium of the strain Streptomyces sp.214L11 of the present invention is: blood powder Gao's No. 1 agar (fresh pig blood clots purchased from the market, crushed, dried, ground, sieved, and added to Gao's No. 1 medium at a mass volume ratio of 1.0%, Gao's No. 1 medium is soluble starch 20g / L, KNO 3 1.0 g / L, K 2 HPO 4 0.5 g / L, MgSO 4 7H 2 O0.5g / L, NaCl 0.5g / L, FeSO 4 7H 2 O 0.01g / L, agar 15g / L, 1000mL pure water, pH7.4-7.6).

[0013] The purification medium of the strain Streptomyces sp.214L11 of the present invention is: Gao's medium No. 1 uses 20g / L soluble starch, KNO 3 1.0 g / L, K 2 HPO 4 0.5 g / L, MgSO 4 7H 2 O 0.5g / L, NaCl 0.5g / L, FeSO 4 7H 2 O 0.01g / L, agar 15g / L, 1000mL pure water, pH 7.4-7.6.

[0014] At the same time, the present invention provides a method for preparing a fermentation broth of a new strain of high-yielding fibrinolytic enzyme actinomycete Streptomyces sp.214L11, and the fermentation broth is prepared by the following steps: a single colony of the strain Streptomyces sp.214L11 is inoculated into Gao's No. 1 liquid culture medium, and cultured at 37°C and 150rpm for 2 days to obtain a Streptomyces sp.214L11 seed liquid; the Streptomyces sp.214L11 seed liquid is inoculated into Gao's No. 1 liquid culture medium at an inoculation rate of 2% by volume, and fermented and cultured at 37°C and 150rpm for 4 days to obtain a Streptomyces sp.214L11 fermentation broth.

[0015] Furthermore, the present invention provides an application of a new actinomycete, Streptomyces sp. 214L11, in the preparation of thrombolytic and anticoagulant drugs.

[0016] By implementing the above specific technical solutions provided by the present invention to implement the content of the present invention, the following can be obtained:

[0017] Beneficial effects:

[0018] (1) The present invention provides a strain of Streptomyces sp.214L11, which has been systematically and scientifically identified as a new actinomycete, and it is necessary to preserve it in accordance with legal requirements. The strain Streptomyces sp.214L11 was isolated and screened from soil samples in the Huoyan Mountain area of ​​Xinjiang to obtain a high-producing fibrinolytic enzyme actinomycete. The strain was verified by optimizing the fibrinolytic enzyme production conditions, fibrinolytic enzyme thrombolytic ability, anticoagulant ability, functional enzyme experiments, stress resistance experiments and antibacterial properties. The strain has fibrinolytic enzyme activity, thrombolytic and anticoagulant functions, as well as good salt resistance, alkali resistance and antibacterial properties. The fermentation broth of the strain has high fibrinolytic enzyme activity, and the produced fibrinolytic enzyme has in vitro thrombolytic activity; indicating that the strain Streptomyces sp.214L11 and its fermentation broth provided by the present invention have good application prospects in the pharmaceutical industry.

[0019] (2) The novel actinomycete Streptomyces sp. 214L11 provided by the present invention is verified by its properties such as fibrinolytic enzyme production, thrombolytic ability, anticoagulant ability, functional enzyme experiment, stress resistance experiment and antibacterial performance. The novel actinomycete Streptomyces sp. 214L11 has the ability to produce active fibrinolytic enzyme, and the fibrinolytic enzyme produced has the functions of thrombolysis and anticoagulation, and has good salt resistance, alkali resistance and antibacterial performance. The fibrinolytic enzyme activity of the fermentation broth of Streptomyces sp. 214L11 is 939.9 FU / mL. After the strain is fermented, the fibrinolytic enzyme produced by the fermentation broth has in vitro thrombolytic activity, which provides a new choice for the development of thrombolytic drugs.

[0020] (3) The new actinomycete Streptomyces sp. 214L11 provided by the present invention has the activity of producing fibrinolytic enzyme. When the strain culture temperature is 35°C and the buffer pH value is 8.0, the relative activity of the fibrinolytic enzyme is the largest. The fibrinolytic enzyme produced can dissolve blood clots and resist blood coagulation. It grows well under low temperature or high temperature and high pH conditions, and has good cellulose and starch hydrolysis ability and strong antibacterial ability. It can effectively inhibit pathogenic bacteria Escherichia coli and Staphylococcus aureus in food. Therefore, the present invention can not only be used as a thrombolytic agent to treat thrombosis, but also can inhibit pathogens in the body. The fibrinolytic enzyme produced by the strain Streptomyces sp. 214L11 can also be added to food. Compared with the fibrinolytic enzyme produced by some bacterial pathogens, the fibrinolytic enzyme produced by the new actinomycete Streptomyces sp. 214L11 provided by the present application is safer and more reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown is the phylogenetic tree constructed based on 16S rRNA gene sequence for Streptomyces p.214L11.

[0022] Figure 2 Shown are the colony morphological characteristics of the strain Streptomyces sp.214L11, where A is the spore morphology of the strain under a microscope; B is the spore morphology after 3 days of culture; and C is the spore morphology after 5 days of culture.

[0023] Figure 3 Shown is the graph of plasmin activity and protease activity of Streptomyces p.214L11 fermentation broth.

[0024] Figure 4 It shows the anticoagulant activity of Streptomyces p.214L11 fermentation broth, where A is the blood sample with added physiological saline; B is the blood sample with added fermentation broth.

[0025] Figure 5 Shown is the in vitro thrombolytic activity of Streptomyces p.214L11 fermentation liquid, where A represents the time immediately after the solvent is added, B represents the time after the addition of normal saline, C represents the time after the addition of liquid culture medium, and D represents the time after the addition of fermentation liquid for 24 hours.

[0026] Figure 6 Shown is the effect of temperature on the activity of plasmin produced by the fermentation broth of Streptomyces p.214L11.

[0027] Figure 7 Shown is the effect of pH on the activity of plasmin produced by the fermentation broth of Streptomyces sp.214L11.

[0028] Figure 8 Shown is the effect of metal ions on the activity of plasmin produced by the fermentation broth of Streptomyces sp.214L11. DETAILED DESCRIPTION

[0029] The present invention is described below with reference to the following examples, but the present invention is not limited to the following examples. All raw and auxiliary materials used in the present invention, as well as the selected bacterial strain culture methods are well known in the art, and the % involved in the present invention are all mass volume percentages, unless otherwise specified.

[0030] In order to better explain the present invention, the main contents of the present invention are further illustrated below in conjunction with specific examples, but the contents of the present invention are not limited to the following examples. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available commodities.

[0031] The blood powder Gao's No. 1 agar of the present invention is made of commercially available fresh pig blood clots, which are crushed, air-dried, ground, sieved, and added to Gao's No. 1 culture medium (soluble starch 20g / L, KNO 31.0 g / L, K 2 HPO 4 0.5 g / L, MgSO 4 7H 2 O 0.5g / L, NaCl 0.5g / L, FeSO 4 7H 2 O 0.01g / L, agar 15g / L, 1000mL pure water, pH 7.4-7.6.

[0032] The Gao's No. 1 culture medium of the present invention adopts 20 g / L soluble starch, KNO 3 1.0 g / L, K 2 HPO 4 0.5g / L, MgSO 4 7H 2 O 0.5g / L, NaCl 0.5g / L, FeSO 4 7H 2 O 0.01g / L, agar 15g / L, 1000mL pure water, pH 7.4-7.6.

[0033] The protein skimmed milk powder culture medium of the present invention adopts 20 g / L skimmed milk powder and 20 g / L agar.

[0034] The blood powder agar plate of the invention adopts commercially available fresh pig blood, which is crushed, air-dried, ground, sieved, and added into a 1.2% agarose solution which is heated in advance and water-bathed at 60° C. in a ratio of 1.0%, and mixed to prepare the plate.

[0035] The fibrin plate of the present invention adopts the following methods: A: 0.022 g of fibrinogen is dissolved in 10 mL of normal saline and the mixture is heated in a water bath at 37° C. for 5-10 min; B: 0.0005 g of thrombin (with a concentration of 40 U / mg) is added into 2 mL of normal saline and the mixture is heated in a water bath at 37° C. for 5-10 min; C: 0.1 g of agarose is weighed and dissolved in 8 mL of normal saline and the mixture is heated to fully dissolve the agarose; B is added into C and then A is added, the mixture is quickly mixed and evenly poured into a plate and holes are punched after solidification.

[0036] Example 1 Isolation, purification and identification of Streptomyces sp. 214L11

[0037] (I) Separation and purification

[0038] The plate dilution method was used to separate actinomycetes. The soil samples collected from Huoyan Mountain in Turpan, Xinjiang were separated and purified by gradient dilution. 10 g of soil sample was weighed and placed in 100 mL of sterile water. Sterilized glass beads were added and shaken at 200 rpm and 30 °C for 20 min to fully disperse the sample. Standard gradient dilution was used to dilute with sterile water to make 10-1 -10 -3 Concentration dilution; take 100μL of each concentration dilution, spread it on the blood meal Gao's No. 1 medium plate by conventional spreading method, and place it in a 37℃ constant temperature incubator for culture; after one week, when the colonies grow on the plate, pick the single colony on the plate for purification culture. The purified single colony is transferred to the Gao's No. 1 medium slant for storage.

[0039] (II) 16S rRNA gene identification

[0040] 1. Extraction of PCR Template DNA

[0041] The purified Streptomyces sp. 214L11 was inoculated into Gao's No. 1 liquid culture medium and cultured at 37°C in a shaking incubator for 2 days. The bacteria were collected and the total genomic DNA was extracted using a DNA extraction kit.

[0042] 2. PCR Amplification

[0043] 16S rRNA gene sequence primers:

[0044] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0045] 1492R: 5'-GGTTACCTTGTTACGACTT-3';

[0046] The total volume of the PCR reaction system was 25 μL, and the PCR amplification conditions were: 94°C for 5 min; 94°C for 30 s, 54°C for 30 s, 72°C for 1 min 30 s, 35 cycles; 72°C for 10 min.

[0047] 3. Sequence Determination

[0048] The PCR amplification product was sequenced after electrophoresis detection and purification, and the sequence length was 1339 bp. The test results are shown in SEQ ID No: 1. BLAST homologous sequence search was performed on NCBI, and the phylogenetic tree was established by the Neighbor-Joining method using MEGA 7.0 software commonly used in the field (repeated sampling 1000 times). The results are shown in the attached Figure 1 As shown, the obtained sequence was compared and analyzed on the NCBI website and it was found that Streptomyces sp.214L11 was similar to the standard model bacteria of the genus Streptomycesfumanus NBRC 5154 T The highest homology was 98.85%, and it was similar to other adjacent model strains Streptomyces fimbriatus 15411 TThe homology is 98.58%. In the phylogenetic tree constructed with 16S rRNA gene sequences, the 16S rRNA gene sequence of Streptomyces sp.214L11 is similar to that of strain Streptomyces fimbriatusNBRC 15411. T (AB184659) clustered in one branch, indicating that Streptomyces sp.214L11 may be related to Streptomycesfimbriatus NBRC 15411 in evolution. T Closer, through the comprehensive judgment of the similarity and homology of the strains, it is consistent with the conclusion of the typical analysis of the molecular level identification of new strains in the field of strain identification, confirming that the obtained strain number 214L11 within the genus Streptomyces is a typical new strain.

[0049] Example 2: Physiological and biochemical characteristics of Streptomyces sp. 214L11

[0050] (I) Colony morphology of Streptomyces sp.214L11

[0051] Inoculate the Streptomyces sp. 214L11 to be observed onto Gao's No. 1 plate and culture at 37°C for 4 days. When the colonies have grown all over the plate, observe and record the characteristics of the colonies and take photos for preservation. At the same time, record the scanning electron microscope photos of the colonies. The recording results are shown in the attached Figure 2 shown.

[0052] By the attached Figure 2 The results show that the Streptomyces sp. 214L11 provided by the present invention is a Gram-positive bacterium, aerobic, motile, and can form white aerial mycelium. The mycelium is twisted and differentiated into spiral spores. After 3 days of cultivation, the mycelium begins to appear blue-green.

[0053] Based on the above biological characteristics, the above Streptomyces sp. 214L11 was identified as Streptomyces sp. The strain has been deposited in the Budapest Treaty International Microbiological Depository: China Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, postal code: 430072, deposit number: CCTCC NO: M 20221543, deposit date is October 7, 2022.

[0054] (II) Physiological and biochemical characteristics of Streptomyces sp. 214L11

[0055] Streptomyces sp.214L11 was inoculated on Gao's medium No. 1, and the physiological and biochemical characteristics were detected respectively. The characteristics of growth temperature, salinity, pH range, gelatin liquefaction, nitrate reduction, hydrogen peroxide production, urease, amylase, esterase, cellulose decomposition, carbon source utilization, etc. were mainly analyzed with reference to the "Common Bacterial System Identification Manual". Streptomyces sp.214L11 can grow at 20-45℃, pH5.5-11.0, and can tolerate more than 5% NaCl. Its metabolic protease, amylase and cellulase activities are all positive; the sugar carbon sources that Streptomyces sp.214L11 can utilize are xylose, sucrose, inositol, L-arabinose, D-fructose, D-galactose, D-glucose, D-mannitol, raffinose and L-rhamnose. In the antibacterial assay against pathogens, the strain showed a certain resistance to Escherichia coli and Staphylococcus aureus.

[0056] The 16S rRNA gene sequence homology comparison analysis, morphological identification, physiological and biochemical characteristics comparison and other aspects provided in Example 1 are clearly different from the standard model strain, which is consistent with the conclusion of the typical analysis of new strains identified in the field. It is determined that Streptomyces sp.214L11 is a new strain in the genus Streptomyces among actinomycetes, and has the significant characteristics of a new strain in the same genus Streptomyces.

[0057] Example 3: Streptomyces sp.214L11 fermentation broth

[0058] In this example, based on Example 1-2, the fermentation liquid of Streptomyces sp.214L11 was obtained by the following steps: a single colony of Streptomyces sp.214L11 was inoculated into Gaodi No. 1 liquid culture medium, and cultured at 37°C and 150rpm for 2 days to obtain Streptomyces sp.214L11 seed liquid; the Streptomyces sp.214L11 seed liquid was inoculated into Gao's No. 1 liquid culture medium at a volume ratio of 2%, and fermented and cultured at 37°C and 150rpm for 4 days to obtain Streptomyces sp.214L11 fermentation liquid.

[0059] Example 4: Application of Streptomyces sp.214L11 fermentation broth

[0060] In this example, based on Examples 1-3, the application of Streptomyces sp. 214L11 and its fermentation broth was tested.

[0061] 1. Test of fibrinolytic enzyme activity in fermentation broth of Streptomyces sp.214L11

[0062] The supernatant of Streptomyces sp.214L11 fermentation broth was used for relevant fibrinolytic enzyme activity detection test. The specific results are shown in the attached Figure 3 shown.

[0063] By the attached Figure 3 The results show that the new bacterium Streptomyces sp. 214L11 provided by the present invention has good plasminogen activitiy and protease activities. It is calculated that the plasminogen activitiy of the fermentation broth of Streptomyces sp. 214L11 is 939.9 FU / mL.

[0064] 2. In vitro anticoagulant activity test of Streptomyces sp.214L11 fermentation broth

[0065] 200 μL of saline, Streptomyces sp.214L11 fermentation broth and 1 mL of rabbit blood were placed in a sterile tube and incubated at 37°C. The blood was tilted every 1 min to observe whether it was always fluid. The rabbit blood was reacted with the enzyme solution and saline in a centrifuge tube for 5 min, and its anticoagulant activity was observed. The specific results are shown in the attached Figure 4 shown.

[0066] By the attached Figure 4 The results show that no coagulation was observed in the sample added with the fermentation broth of Streptomyces sp.214L11, while flow retardation occurred in the tube added with physiological saline. After 24 hours, the centrifuge tube added with the fermentation broth sample of Streptomyces sp.214L11 still showed a flowing state, while the blood in the sample added with physiological saline was completely coagulated, and the blood was clearly separated from the physiological saline. After inversion, the blood coagulated into blocks and could not flow. The results show that the fermentation broth of Streptomyces sp.214L11 provided by the present invention has an anticoagulant effect.

[0067] 3. Determination of thrombolytic activity of Streptomyces sp.214L11 fermentation broth

[0068] Take several 8mm wide ampoule tubes, draw a scale line at a height of 1cm, add the freshly collected rabbit blood that has not yet coagulated to the scale line, and keep it vertical until the rabbit blood naturally coagulates into a thrombus-like shape. Add 3mL of fermentation liquid to the tube, and observe the thrombus dissolution with normal saline as a control. Observe the thrombus dissolution just after adding the Streptomyces sp.214L11 fermentation liquid and 12 hours after adding the Streptomyces sp.214L11 fermentation liquid. For specific results, see the attached Figure 5 shown.

[0069] Depend on Figure 5 The data show that after standing for 12 hours, the liquid in the tube to which the Streptomyces sp.214L11 fermentation broth was added had the darkest color. When shaken, the blood clots were loose and the volume was significantly reduced compared with the control. The color of the tube to which the culture medium was added in the control group was slightly darker than that of the colorimetric tube to which physiological saline was added. Like the physiological saline tube, the tube maintained its original morphology, and only a small amount of red blood cells were lysed. This indicates that the plasmin produced by the Streptomyces sp.214L11 fermentation broth provided by the present invention has in vitro thrombolytic activity.

[0070] 4. Determination of fibrinolytic enzyme activity in Streptomyces sp.214L11 fermentation broth

[0071] (1) Optimal temperature of plasmin

[0072] Take 50 μL of Streptomyces sp.214L11 fermentation broth and inoculate it into a fibrin plate. Place it in an incubator at 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃ for 18 hours. Then calculate the hydrolysis zone area of ​​the enzyme solution to investigate the effect of temperature change on the fibrinolytic enzyme produced by Streptomyces sp.214L11 fermentation broth. For specific results, see the attached Figure 6 shown.

[0073] Depend on Figure 6 The data show that the relative activity of plasmin in the fermentation broth of Streptomyces sp. 214L11 provided by the present invention increases first and then decreases with increasing temperature, and the optimal culture temperature is 35° C., after which the plasmin activity begins to decrease.

[0074] (2) Optimal pH for plasmin action

[0075] Prepare buffer systems with different pH values ​​(4, 5, 6, 7, 8, 9, 10), take 25 μL of buffer, add 25 μL of Streptomyces sp.214L11 fermentation broth and mix evenly, then inoculate into fibrin plate and incubate in 37℃ incubator for 18h, calculate the hydrolysis zone area of ​​bacterial solution, and investigate the effect of buffer pH value on plasmin activity of Streptomyces sp.214L11 fermentation broth. For specific results, see Appendix Figure 7 shown.

[0076] Depend on Figure 7The data show that the pH value of the enzyme reaction also has a significant effect on the relative activity of plasmin. The pH affects the enzymatic efficiency by affecting the groups on the enzyme-related active sites. Plasmin is inactivated by either too high or too low a pH value. The optimal pH for plasmin in the fermentation broth of Streptomyces sp. 214L11 is 8.0, and it has a wide pH range, showing good enzyme activity at pH 6.0-9.0. However, when the pH further decreases or increases, its enzyme activity decreases rapidly. At pH 4.0 and pH 10.0, its enzyme activity is only about 20%.

[0077] (3) Effects of metal ions on plasmin

[0078] When the ion concentration was 5.0 mM, the effects of different metal ions on the fibrinolytic enzyme activity of Streptomyces sp. 214L11 fermentation broth were investigated. The metal ions were Mg 2+ , Cu 2+ , Fe 2+ , Ca 2+ , K + 、Na + , Fe 3+ With Mn 2+ The fermentation broth of Streptomyces sp. 214L11 was incubated with metal ions at 37°C for 60 min. The degree of inhibition was expressed as the percentage of the residual activity (with metal ions) to the control activity (without metal ions). The specific statistical results are shown in the attached Figure 8 shown.

[0079] By the attached Figure 8 The data show that when the fermentation broth of Streptomyces sp.214L11 was treated with different metal ions at the same concentration, the metal ion Mn 2+ It has a significant promoting effect on the activity of plasmin produced by the fermentation broth of Streptomyces sp.214L11, and can be used as an activator of plasmin produced by the fermentation broth of Streptomyces sp.214L11; K + , Cu 2+ Can slightly promote enzyme activity, Ca 2+ With Mg 2+ It has a certain inhibitory effect on the production of fibrinolytic enzyme in the fermentation broth of Streptomyces sp.214L11.

[0080] (4) Effects of enzyme inhibitors on plasmin

[0081] In this study, the effects of different protease inhibitors on the fibrinolytic activity of Streptomyces sp. 214L11 fermentation broth were investigated. Common enzyme inhibitors such as soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF), 2-mercaptoethanol (2-ME), chymotrypsin inhibitor (TPCK), and EDTA were added at a concentration of 10 mM, respectively. The cultures were incubated at 37°C for 60 min. The degree of inhibition was expressed as the percentage of the remaining activity (with inhibitor) to the control activity (without inhibitor). The specific statistical results are shown in Table 1.

[0082] Table 1: Effects of different inhibitors on the fibrinolytic activity of Streptomyces p.214L11 fermentation broth

[0083] Reagents <![CDATA[Concentration / (mmol·L -1 )]]> Relative enzyme activity / (%) Control 0 100 SBTI 10 102.94±2.64 PMSF 10 101.36±1.42 2-ME 10 100.60±3.27 TPCK 10 104.47±1.31 EDTA 10 0

[0084] From the data in Table 1, it can be seen that in the presence of different enzyme inhibitors, the activity of plasmin in the fermentation broth of Streptomyces sp. 214L11 was completely inhibited by 10 mM EDTA inhibitor, which indicates that the plasmin in the fermentation broth of Streptomyces sp. 214L11 is a metalloproteinase with a metal ion binding site.

[0085] The above experiments show that the present invention isolates and screens a new bacterium Streptomyces sp.214L11 from soil samples in the Huoyan Mountain area of ​​Xinjiang. The strain and its fermentation broth have fibrinolytic ability, thrombolytic and anticoagulant functions, and good salt resistance, alkali resistance and antibacterial properties. The fibrinolytic activity of the fermentation broth of the new bacterium Streptomyces sp.214L11 is 939.9FU / mL, and the produced fibrinolytic enzyme has unique and significant in vitro thrombolytic activity, indicating that the new actinomycete Streptomyces sp.214L11 and its fermentation broth provided by the present invention are used in the pharmaceutical industry.

[0086] The above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A strain of actinomycete Streptomyces ( Streptomyces sp. )214L11, It is characterized in that It is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20221543. The actinomycete Streptomyces ( Streptomyces sp. ) The gene sequence of 214L11 is shown in SEQ ID NO:

1.

2. A strain of actinomycetes Streptomyces as claimed in claim 1 ( Streptomyces sp. ) Preparation method of 214L11 fermentation broth, It is characterized in that The fermentation broth is prepared by the following steps: Streptomyces A single colony of sp. 214L11 was inoculated into Gao's liquid medium No. 1 and cultured at 37°C and 150 rpm for 2 days. Streptomyces sp. 214L11 seed solution; Will Streptomyces sp. 214L11 seed solution was inoculated into Gao's No. 1 liquid medium at a volume ratio of 2%, and fermented at 37°C and 150 rpm for 4 days to obtain Streptomyces sp. 214L11 fermentation broth.

3. The actinomycete Streptomyces prepared by the preparation method according to claim 2 ( Streptomyces sp. )Application of 214L11 fermentation broth in the preparation of thrombolytic and anticoagulant drugs.

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