Marine bacterium Bacillus sp.4SP1, method for producing plasmin by using marine bacterium Bacillus sp.4SP1 and application of marine bacterium Bacillus sp.4SP1

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a marine bacterium Bacillus sp.4SP1 as well as a plasmin production method and application thereof, the strain is preserved in China General Microbiological Culture Collection Center on November 6, 2024, the preservation number is CGMCC No.32524, the preservation unit address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No.32524, and the preservation number is CGMCC No.32524. Institute of Microbiology, Chinese Academy of Sciences; the bacterial colony of the bacterium is white and oval, has a rough surface, a convex center and irregular edges, and is gram-positive bacterium; the optimal growth conditions of the strain 4SP1 are as follows: the yeast is 5g / L, the tryptone is 10g / L, the pH value is 7.0, and the culture temperature is 40 DEG C. The plasmin produced by the marine bacterium Bacillus sp.4SP1 disclosed by the invention has thrombolysis and anticoagulant activity, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a marine bacterium Bacillus sp.4SP1 and a method and application thereof for producing fibrinolytic enzyme. Background Art

[0002] my country has abundant marine resources with huge potential for development and utilization and broad prospects. However, the quality, efficiency and benefits of marine resource utilization are not high, and marine resources are the main untapped source of fibrinolytic enzymes.

[0003] Plasmin is a type of proteolytic enzyme that can degrade fibrin and plays an important role in dissolving thrombi and preventing thrombosis. Plasmin plays a key role in eliminating vascular embolism in the human body. Its main function is to degrade fibrin, dissolve thrombi, and keep blood vessels unobstructed. Every year, about 18 million people die from cardiovascular diseases worldwide, accounting for 31% of the total number of deaths. Among them, ischemic heart disease ranks first among the top ten causes of death in the world in 2019 released by the World Health Organization (WHO); currently, cardiovascular disease deaths account for the first place in the total cause of death among urban and rural residents in my country, higher than tumors and other diseases. And with the change of people's living habits, the age of onset of cardiovascular diseases tends to be younger. At present, the thrombolytic drugs on the market are expensive, some of them 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.

[0004] Commonly used thrombolytic drugs in clinical practice are mainly used for the treatment of thrombotic diseases such as acute myocardial infarction, pulmonary embolism and deep vein thrombosis. These drugs dissolve thrombi and restore vascular patency by activating the fibrinolytic system. Currently, 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. However, these treatment methods have been reported to cause adverse reactions such as bleeding. Due to these adverse side effects, a new generation of thrombolytic drugs should have the advantages of good safety, few side effects, long half-life, low price and high thrombolytic efficiency. In recent years, natural fibrinolytic enzymes have been widely studied and have become very important in thrombolytic therapy. However, there are few natural fibrinolytic enzymes, and there are problems such as high optimal action temperature of the enzyme, acidic pH of the action, and long thrombolytic time. To this end, a marine bacterium Bacillus sp.4SP1 and a method and application of producing fibrinolytic enzymes are provided. Summary of the invention

[0005] The purpose of the present invention is to provide a marine bacterium Bacillus sp.4SP1 and a method and application thereof for producing fibrinolytic enzyme in order to solve the problems raised by the above-mentioned background technology in view of the defects of the prior art.

[0006] To achieve the above-mentioned purpose, the present invention provides the following technical solution: A marine bacterium Bacillus sp.4SP1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on November 6, 2024, with the deposit number: CGMCC No.32524, and the address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0007] A method for producing fibrinolytic enzyme using marine bacteria Bacillus sp.4SP1, comprising the following specific steps:

[0008] S1: The strain 4SP1 screened from shrimp was stored in a slant at 4°C in a refrigerator;

[0009] S2: Inoculate the strain into the growth medium with an inoculum volume of 2% and a liquid volume of 10%, and culture at 180 r / min and 40°C for 12 h to obtain a seed solution;

[0010] S3: Inoculate the seed liquid into the enzyme production medium with an inoculation volume of 2% and a liquid volume of 10%, and culture at 180 r / min and 30°C for 10 h to obtain a bacterial suspension;

[0011] S4: The fermentation liquid was centrifuged at 4°C and 10,000×g for 5 min to obtain the supernatant as the crude enzyme solution. The crude enzyme solution was filtered through a 0.22 μm filter membrane for sterilization, and the filtrate was concentrated and purified by centrifugation at 4°C and 4,000×g for 10 min using a 10 kDa ultrafiltration tube.

[0012] As a preferred technical solution of the present invention, the growth medium in step S2 is prepared with 5 g / L yeast, 10 g / L trypsin, and pure water at pH 7.0, and then sterilized at 121° C. for 20 minutes.

[0013] As a preferred technical solution of the present invention, the enzyme-producing culture medium in step S3 is prepared by using 5 g / L yeast, 10 g / L potassium nitrate, pure water to prepare pH 6.0, and high temperature sterilization at 121° C. for 20 minutes.

[0014] An application of the plasmin produced by the above method in an in vitro thrombolytic experiment comprises the following specific steps:

[0015] Step 1: Inoculate the strain onto the growth medium, fill the liquid volume with 10%, and culture at 180r / min and 40°C for 12h to obtain the seed solution, inoculate the seed solution onto the enzyme production medium, with an inoculation amount of 2%, a liquid volume of 10%, and culture at 180r / min and 30°C for 14h to obtain the fermentation liquid; centrifuge the fermentation liquid at 4°C and 10000×g for 5min to obtain the supernatant as the crude enzyme liquid, filter the crude enzyme liquid with a 0.22μm filter membrane for sterilization, and concentrate and purify the filtrate with a molecular weight 10kDa ultrafiltration tube at 4°C and 4000×g for 10min; collect the enzyme liquid in a large beaker;

[0016] Step 2: Take fresh human blood and evenly dispense 200 μL into 2.0-1.5 mL EP tubes, let stand at room temperature, wait for it to coagulate, absorb the blood on the surface with filter paper, weigh W1, and put them into dry EP tubes respectively, and divide them into 2 groups, 3 tubes in each group; prepare 2.0 mg / mL concentration of plasminogen activator, take 400 μL enzyme solution and put it into EP tubes containing blood clots, put them into a constant temperature rotary shaker, and incubate them at 37°C and 50 r / min for 18 hours; replace the enzyme solution with physiological saline as a control; take out the EP tube from the constant temperature box, centrifuge, discard the supernatant, absorb the blood with filter paper, weigh W2, and calculate the blood clot dissolution rate;

[0017] Step 3: Prepare 2 mg / mL plasmin and dispense 400 μL into EP tubes. Use an equal amount of normal saline as a negative control. Take fresh human blood and quickly add 200 μL each into each EP tube pre-added with 2 mg / mL plasmin or normal saline. Shake slightly to mix well and place at room temperature 20-25°C to observe the in vitro anticoagulant effect of plasmin.

[0018] As a preferred technical solution of the present invention, the formula for calculating the blood clot dissolution rate in step 2 is:

[0019]

[0020] The invention relates to an application of the plasmin produced by the above method in the preparation of a thrombolytic drug.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1: The present invention screens enzyme-producing microorganisms from marine samples. Marine microorganisms can produce enzymes with novel characteristics in the special environment of the ocean, such as high salt, low temperature, and low nutrition;

[0023] 2: Marine bacteria Bacillus sp.4SP1 produces fibrinolytic enzyme. The growth and enzyme production conditions of this bacterium are easy to achieve, and the fermentation enzyme production time is 12-14h;

[0024] 3: The plasmin produced by this enzyme has thrombolytic and anticoagulant activities. The action temperature and pH of this enzyme are close to those of the human body, which is conducive to the action of the enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a transparent circle image formed on the screening plate of the strain 4SP1 of the present invention;

[0026] Figure 2 This is the Gram staining picture of the strain 4SP1 of the present invention;

[0027] Figure 3 This is the spore map of strain 4SP1 of the present invention

[0028] Figure 4 This is a scanning electron micrograph of the strain 4SP1 of the present invention;

[0029] Figure 5 It is a phylogenetic tree diagram of the strain 4SP116S rDNA of the present invention;

[0030] Figure 6 This is a graph showing the effect of time on the growth of strain 4SP1 of the present invention;

[0031] Figure 7 This is a graph showing the effect of temperature on the growth of strain 4SP1 of the present invention;

[0032] Figure 8 This is a graph showing the effect of NaCl on the growth of strain 4SP1 of the present invention;

[0033] Fig. 9 This is a graph showing the effect of initial pH on the growth of strain 4SP1 of the present invention;

[0034] Fig.10 This is a graph showing the effect of the carbon source of the present invention on the growth of strain 4SP1;

[0035] Fig.11 This is a graph showing the effect of the nitrogen source of the present invention on the growth of strain 4SP1;

[0036] Fig.12 This is a diagram showing the effect of time on enzyme production of strain 4SP1 of the present invention;

[0037] Fig.13 This is a diagram showing the effect of temperature on enzyme production by strain 4SP1 of the present invention;

[0038] Fig.14 This is a graph showing the effect of initial pH on enzyme production by strain 4SP1 of the present invention;

[0039] Fig.15 This is a diagram showing the effect of NaCl on enzyme production by strain 4SP1 of the present invention;

[0040] Fig.16This is a graph showing the effect of the carbon source of the present invention on the enzyme production of strain 4SP1;

[0041] Fig.17 This is a diagram showing the effect of the nitrogen source of the present invention on the enzyme production of strain 4SP1;

[0042] Fig.18 is the optimum action temperature diagram of the enzyme of the present invention;

[0043] Fig.19 is the temperature stability diagram of the enzyme of the present invention;

[0044] Fig. 20 is the optimum pH diagram of the enzyme of the present invention;

[0045] Fig.21 is the pH stability diagram of the enzyme of the present invention;

[0046] Fig. 22 This is the effect of metal ions on enzyme activity;

[0047] Fig.23 This is a diagram showing the effect of the enzyme in vitro thrombolysis experiment of the present invention;

[0048] Fig.24 This is a diagram showing the effect of the enzyme in vitro anticoagulation experiment of the present invention. DETAILED DESCRIPTION

[0049] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0050] Embodiment: A marine bacterium Bacillus sp.4SP1, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 6, 2024, with a deposit number of CGMCC No.32524, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences;

[0051] The colony of this bacterium is white and oval, with a rough surface, a convex center and irregular edges. It is a Gram-positive rod. The 16S rDNA of strain 4SP1 has a homology of 99.85% with Bacillus velezensis. Based on the comprehensive morphological characteristics, strain 4SP1 was identified as Bacillus sp.; The optimal growth conditions for strain 4SP1 are: yeast: 5 g / L, soy peptone: 10 g / L, pH 7.0, and culture at 40°C.

[0052] A method for producing fibrinolytic enzyme using marine bacteria Bacillus sp.4SP1, comprising the following specific steps:

[0053] S1: The strain 4SP1 screened from shrimp was stored in a slant at 4°C in a refrigerator;

[0054] S2: Inoculate the strain into the growth medium with an inoculum volume of 2% and a liquid volume of 10%, and culture at 180 r / min and 40°C for 12 h to obtain a seed solution;

[0055] S3: Inoculate the seed liquid into the enzyme production medium with an inoculation volume of 2% and a liquid volume of 10%, and culture at 180 r / min and 30°C for 14 h to obtain a fermentation liquid;

[0056] S4: The fermentation liquid was centrifuged at 4°C and 10,000×g for 5 min to obtain the supernatant as the crude enzyme solution. The crude enzyme solution was filtered through a 0.22 μm filter membrane for sterilization, and the filtrate was concentrated and purified by centrifugation at 4°C and 4,000×g for 10 min using a 10 kDa ultrafiltration tube.

[0057] Application of fibrinolytic enzyme produced by marine bacteria Bacillus sp.4SP1,

[0058] The specific steps are as follows: inoculate the strain into the growth medium, fill the flask with 10% of the liquid, and culture at 180r / min and 40°C for 12 hours to obtain the seed solution; inoculate the seed solution into the enzyme production medium with an inoculation amount of 2%, a liquid volume of 10%, and culture at 180r / min and 30°C for 10 hours to obtain the fermentation liquid; centrifuge the fermentation liquid at 4°C and 10000×g for 5 minutes to take the supernatant as the crude enzyme liquid, filter the crude enzyme liquid with a 0.22μm filter membrane for sterilization, and concentrate and purify the filtrate with a molecular weight 10kDa ultrafiltration tube at 4°C and 4000×g for 5 minutes; collect the enzyme liquid in a large beaker. Take fresh human blood and evenly distribute 200 μL into 2.0mL EP tubes, let it stand at room temperature, wait for it to coagulate, absorb the blood on the surface with filter paper, weigh it (W1), and then put it into dry EP tubes respectively, and divide it into 2 groups, 3 tubes in each group; prepare 2.0 mg / mL concentration of plasmin, take 400 μL enzyme solution and put it into EP tubes containing blood clots, put them in a constant temperature rotary shaker, and incubate them at 37°C and 50 r / min for 18 hours; replace the enzyme solution with physiological saline as a control; take out the EP tube from the constant temperature box, centrifuge it, discard the supernatant, absorb the blood with filter paper and weigh it (W2), and calculate the blood clot dissolution rate. Prepare 2 mg / mL plasmin and dispense 400 μL into EP tubes. Use an equal amount of normal saline as a negative control. Take fresh human blood and quickly add 200 μL each into each EP tube pre-added with 2 mg / mL plasmin or normal saline. Slightly shake, mix, and place at room temperature (20-25°C) to observe the in vitro anticoagulant effect of plasmin.

[0059] The marine strain Bacillus sp.4SP1 provided by the present invention is a marine bacterium isolated from shrimp in Lianyungang, Jiangsu, China. The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on November 6, 2024, with a deposit number of CGMCC No.32524, and the address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The present invention provides the strain and a method for producing fibrinolytic enzyme using the strain, as well as the possibility of using the fibrinolytic enzyme produced by the strain to treat thrombotic diseases.

[0060] 1. A screening method for the strain of the present invention;

[0061] 1.1. The culture medium involved in the present invention:

[0062] Screening medium: weigh 0.01g of fibrinogen, dissolve in 5.0mL of sterile physiological saline, 1mL of thrombin solution (containing 50U of thrombin), sterilize through 0.22um filter membrane, and preheat in a 37℃ water bath; 100mL of LB agar solid medium, sterilize at 121℃ for 20 minutes, cool to about 45℃, add preheated fibrinogen solution and thrombin solution, mix quickly and evenly, pour into a plate immediately (pour 15-20mL), place horizontally, and wait for solidification;

[0063] Growth medium: 5 g / L yeast powder, 10 g / L soy peptone, pH 7.0;

[0064] Enzyme production culture medium: 5g / L yeast powder, 10g / L potassium nitrate, pure water, pH 6.0.

[0065] 1.2. Screening method of strains:

[0066] The shrimp sample was diluted 10 times in gradient, and the -3 to -5 gradient was selected and applied to the screening medium, cultured at 37°C for 24 hours, and the colonies with transparent circles were picked and purified by three-zone lines on the screening medium, and the purified strains were stored on the slant. 2. Morphological characteristics of the strain 4SP1 of the present invention;

[0067] 2.1. Colony morphology characteristics:

[0068] The colonies of strain 4SP1 are white and oval, with a rough surface, a raised center, and irregular edges ( Figure 1 ), a Gram-positive bacillus ( Figure 2 ), and spores were observed ( Figure 3 ), electron microscopic morphology Figure 4 .

[0069] 2.2. Molecular biological identification of strain 4SP1;

[0070] Molecular biological identification, the gene of the strain was extracted by the kit, and the universal primers of prokaryotic microorganisms 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTACGACTT-3') were used for PCR amplification. The PCR amplification system was 50μL (including: template 1μL, PCR Mix 25μL, 27F 2μL, 1492R 2μL, MgCl22μL, dd H2O 17μL,) and the amplification conditions were: 94℃ denaturation for 2min; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, 34 cycles; 72℃ final extension for 5min, and storage at 4℃. The PCR product was sent to Shanghai Biotech for sequencing. The sequencing results were submitted to the National Center of Biotechnology Information (NCBI) database for comparison, and the phylogenetic tree was constructed by neighbor-joining method using MEGA7.0 software. The 16S rDNA sequence PCR amplification product of strain 4SP1 was detected by 2% agarose gel electrophoresis. The 16S rDNA base length was 1465bp. The sequence was analyzed by BLAST on NCBI. The homology between 16S rDNA of strain 4SP1 and Bacillus velezensis was 99.85%. Based on the morphological characteristics, strain 4SP1 was identified as Bacillus sp. The phylogenetic tree was constructed using the NJ method in MEGA7.0 software. The results are shown in Figure 5 .

[0071] 3. Growth characteristics of strain 4SP1 of the present invention;

[0072] The growth characteristics of the strain 4SP1 provided by the invention are studied, and the growth conditions of the strain are obtained.

[0073] 3.1. Effect of time on the growth of strain 4SP1;

[0074] Incubate at 37°C and 180 r / min, take samples and measure OD every 2 hours 600 , strain 4SP1 grew most actively at 0-8h and was in the logarithmic phase. After 12h, the growth rate slowed down and entered the stable growth phase ( Figure 6 ).

[0075] 3.2 Effect of temperature on the growth of strain 4SP1;

[0076] The strain 4SP1 can grow at 22-55℃, and the optimal growth temperature range is 40-45℃. The growth rate drops sharply when the temperature exceeds 45℃. Figure 7 ).

[0077] 3.3. Effect of NaCl concentration on the growth of strain 4SP1;

[0078] The strain 4SP1 can grow under 0-8% NaCl conditions, and the best growth effect is achieved under the condition of no NaCl ( Figure 8 ).

[0079] 3.4 Effect of initial pH on the growth of strain 4SP1;

[0080] The optimum pH range for strain 4SP1 is 6-8, and it grows best at pH 7 ( Fig. 9 ).

[0081] 3.5. Effects of carbon and nitrogen sources on the growth of strain 4SP1;

[0082] Yeast, sucrose, glucose, lactose, corn starch, cassava starch, soluble starch, and fructose were used as carbon sources to replace yeast. The culture was carried out at 180 r / min and 40 °C for 6 h, and the absorbance value (OD 600nm The optimal carbon source was selected, and then peptone, potassium nitrate, sodium nitrate, fish meal peptone, soybean meal powder, soy peptone, and ammonium chloride were used as nitrogen sources to replace peptone. The effects of the above factors on the growth of strain 4SP3 were studied. Strain 4SP3 had good adaptability to most carbon sources, and the best carbon source for growth was yeast ( Fig.10 ), the best nitrogen source is soy peptone ( Fig.11 ).

[0083] 4. Enzyme production characteristics of strain 4SP1

[0084] 4.1. Effect of time on enzyme production of strain 4SP1;

[0085] The strain 4SP1 was cultured at 40°C and 180 r / min for 40 h, and samples were taken after 0 h to measure the enzyme activity. Fig.12 The results showed that the enzyme activity was highest at 14h.

[0086] 4.2 Effect of fermentation temperature on enzyme production of strain 4SP1;

[0087] The optimum temperature for enzyme production is 30°C ( Fig.13 ).

[0088] 4.3 Effect of initial pH on enzyme production of strain 4SP1;

[0089] The optimal initial pH for enzyme production is 6.0 ( Fig.14 ).

[0090] 4.4. Effect of NaCl concentration on enzyme production of strain 4SP1;

[0091] The optimal NaCl concentration for enzyme production is 0% ( Fig.15 ).

[0092] 4.5. Effects of carbon and nitrogen sources on enzyme production of strain 4SP1;

[0093] The strain 4SP1 can produce enzymes on a variety of carbon and nitrogen sources, among which the most suitable carbon source is yeast ( Fig.16 ); the optimal nitrogen source is potassium nitrate ( Fig.17 ).

[0094] 5. Properties of plasmin produced by strain 4SP1;

[0095] 5.1. Enzyme activity determination;

[0096] The fibrinogen plate method was used to determine the activity of plasmin produced by strain 4SP1.

[0097] 5.2 Effect of temperature on enzyme activity and stability;

[0098] Depend on Fig.18 It can be seen that the optimal temperature for the enzyme is 37°C. Between 25 and 40°C, after being kept at 25°C for 5 hours, the relative enzyme activity still remains above 85%. After being kept at 40°C for 5 hours, the residual enzyme activity is 60% or more ( Fig.19 ).

[0099] 5.3 Effect of pH on enzyme activity and stability;

[0100] Depend on Fig. 20 It can be seen that the enzyme is most suitable for action at pH 7.4; the enzyme has good stability at pH 6.0-8.0, and the enzyme stability decreases when the pH is lower than 5.5 and higher than 9.0 ( Fig.21 ).

[0101] 5.3. Effects of metal ions on enzyme activity;

[0102] The results of the effects of different metal ions on the activity of plasmin were Fig. 22 shown. Na + , Mn 2+ It promotes the enzyme activity of the enzyme; Cu 2+ It has a significant inhibitory effect on this enzyme, and the enzyme activity is reduced by 31.13%. 6. Application of strain 4SP1 to produce fibrinolytic enzyme;

[0103] 6.1. In vitro thrombolysis experiment;

[0104] The strain was inoculated into the growth medium, cultured at 180r / min and 40°C for 12h to obtain the seed solution; the seed solution was inoculated into the enzyme production medium, the inoculation amount was 2%, the liquid volume was 10%, and the fermentation liquid was cultured at 180r / min and 30°C for 16h; the fermentation liquid was centrifuged at 4°C and 10000×g for 5min to obtain the supernatant as the crude enzyme solution, the crude enzyme solution was filtered and sterilized using a 0.22μm filter membrane, and the filtrate was concentrated and purified using a 10kDa ultrafiltration tube at 4°C and 4000×g for 10min. The enzyme solution was collected in a large beaker, fresh human blood was taken, and 200μL was evenly distributed into 2.0mL EP tubes, and it was left to stand at room temperature. After it solidified, the blood on the surface was absorbed by filter paper and weighed (W1), and then placed in dry EP tubes respectively, divided into 2 groups, 3 tubes in each group. Prepare 2.0mg / mL concentration of plasmin, take 400μL enzyme solution and put it into EP tubes containing blood clots, put it into a constant temperature rotary shaker, and incubate it for 18h at 37℃ and 50r / min. Replace the enzyme solution with physiological saline as a control. Take out the EP tube from the constant temperature box, centrifuge it, discard the supernatant, absorb the blood water with filter paper and weigh it (W2), calculate the blood clot dissolution rate, the formula for blood clot dissolution rate is: The effect of enzyme in vitro thrombolysis experiment is shown in Fig.23 The blood clot dissolution rate of the enzyme-added group reached 86.97%, and the results showed that the enzyme could dissolve the formed blood clots and prevent blood vessel embolism.

[0105] 6.2. In vitro anticoagulation experiment;

[0106] Inoculate the strain into the growth medium, culture at 180r / min and 40℃ for 12h to obtain the seed solution; inoculate the seed solution into the enzyme production medium with an inoculation amount of 2% and a liquid volume of 10%, and culture at 180r / min and 30℃ for 14h to obtain the fermentation liquid; centrifuge the fermentation liquid at 4℃ and 10000×g for 5min to take the supernatant as the crude enzyme liquid, filter the crude enzyme liquid with a 0.22μm filter membrane for sterilization, and concentrate and purify the filtrate with a 10kDa ultrafiltration tube at 4℃ and 4000×g for 10min; collect the enzyme liquid in a large beaker. Prepare 2mg / mL plasmin, dispense 400μL into EP tubes, and use an equal amount of saline as a negative control; take fresh human blood, and quickly add 200μL each to each EP tube pre-added with 2mg / mL plasmin or saline, shake slightly, mix well, and place at room temperature (20-25℃) to observe the in vitro anticoagulant effect of plasmin, such as Fig.24 As shown, the enzyme-added group maintained the normal shape of blood, while the control group formed blood clots. The results showed that the enzyme has a significant in vitro anticoagulant effect.

[0107] The above embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims

1. A marine bacterium Bacillus sp.4SP1, characterized in that: This strain was deposited at the General Microbiology Center of China Microorganism Culture Collection Administration on November 6, 2024, with the deposit number: CGMCCNo.32524. The address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A method for producing fibrinolytic enzyme using marine bacteria Bacillus sp.4SP1 as claimed in claim 1, characterized in that: The specific steps are as follows: S1: The strain 4SP1 screened from shrimp was stored in a slant at 4°C in a refrigerator; S2: Inoculate the strain into the growth medium with an inoculum volume of 2% and a liquid volume of 10%, and culture at 180 r / min and 40°C for 12 h to obtain a seed solution; S3: Inoculate the seed liquid into the enzyme production medium with an inoculation volume of 2% and a liquid volume of 10%, and culture at 180 r / min and 30°C for 10 h to obtain a bacterial suspension; S4: The fermentation liquid was centrifuged at 4°C and 10,000×g for 5 min to obtain the supernatant as the crude enzyme solution. The crude enzyme solution was filtered through a 0.22 μm filter membrane for sterilization, and the filtrate was concentrated and purified by centrifugation at 4°C and 4,000×g for 10 min using a 10 kDa ultrafiltration tube.

3. The method for producing fibrinolytic enzyme by using marine bacterium Bacillus sp.4SP1 according to claim 2, characterized in that: In step S2, the growth medium is prepared with 5 g / L yeast, 10 g / L tryptone, and pure water to a pH of 7.0, and then sterilized at a high temperature of 121° C. for 20 min.

4. The method for producing fibrinolytic enzyme by using marine bacteria Bacillus sp.4SP1 according to claim 2, characterized in that: In step S3, the enzyme production culture medium is prepared by using 5 g / L yeast, 10 g / L potassium nitrate, pure water to adjust the pH to 6.0, and sterilizing at high temperature of 121° C. for 20 min.

5. Use of plasmin produced by the method according to any one of claims 2 to 4 in an in vitro thrombolytic experiment, characterized in that: The specific steps are as follows: Step 1: Inoculate the strain onto the growth medium, fill the liquid volume with 10%, and culture at 180r / min and 40°C for 12h to obtain the seed solution, inoculate the seed solution onto the enzyme production medium, with an inoculation amount of 2%, a liquid volume of 10%, and culture at 180r / min and 30°C for 14h to obtain the fermentation liquid; centrifuge the fermentation liquid at 4°C and 10000×g for 5min to obtain the supernatant as the crude enzyme liquid, filter the crude enzyme liquid with a 0.22μm filter membrane for sterilization, and concentrate and purify the filtrate with a molecular weight 10kDa ultrafiltration tube at 4°C and 4000×g for 10min; collect the enzyme liquid in a large beaker; Step 2: Take fresh human blood and evenly dispense 200 μL into 2.0-1.5 mL EP tubes, let stand at room temperature, wait for it to coagulate, absorb the blood on the surface with filter paper, weigh W1, and put them into dry EP tubes respectively, and divide them into 2 groups, 3 tubes in each group; prepare 2.0 mg / mL concentration of plasminogen activator, take 400 μL enzyme solution and put it into EP tubes containing blood clots, put them into a constant temperature rotary shaker, and incubate them at 37°C and 50 r / min for 18 hours; replace the enzyme solution with physiological saline as a control; take out the EP tube from the constant temperature box, centrifuge, discard the supernatant, absorb the blood with filter paper, weigh W2, and calculate the blood clot dissolution rate; Step 3: Prepare 2 mg / mL plasmin and dispense 400 μL into EP tubes. Use an equal amount of normal saline as a negative control. Take fresh human blood and quickly add 200 μL each into each EP tube pre-added with 2 mg / mL plasmin or normal saline. Shake slightly to mix well and place at room temperature 20-25°C to observe the in vitro anticoagulant effect of plasmin.

6. The use according to claim 5, characterized in that: The formula for calculating the clot lysis rate in step 2 is:

7. Use of plasmin produced by the method according to any one of claims 2 to 4 in the preparation of a thrombolytic drug.

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