Salt-tolerant bacillus for producing plasmin and application thereof

Abstract

The invention discloses a salt-tolerant bacillus halodurans N1 for producing plasmin, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO: 31939, and the preservation date is September 11, 2024. The salt-tolerant bacillus provided by the invention is separated and screened from Inner Mongolia soil, and can tolerate the salt concentration of 13% at most. The strain is subjected to liquid fermentation to obtain crude enzyme liquid, the fibrinolytic activity of the crude enzyme liquid is 325.38 U / mL, and the crude enzyme liquid has obvious anticoagulant activity and thrombolytic activity in vitro. The salt-tolerant bacillus and the fermentation liquor thereof can be used for the development of anticoagulant drugs and thrombolytic drugs, and have wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of halotolerant bacillus producing fibrinolytic enzyme and an application of the thrombolytic function thereof. Background Art

[0002] Cardiovascular disease (CVD), such as acute myocardial infarction, ischemic heart disease, valvular heart disease, and peripheral vascular disease, is the leading cause of death worldwide, accounting for about 31% of the total mortality rate. A report provided by the World Health Organization shows that 17 million people die from cardiovascular disease each year, and the number may exceed 23.6 million by 2030.

[0003] Treatments for thrombosis include surgery, anticoagulant therapy and thrombolytic therapy, among which thrombolytic therapy is the most commonly used in clinical practice. Thrombolytic drugs are mainly divided into two categories, namely plasminogen activators and plasmin. The former activates the body's plasminogen to convert into plasmin, exerting an indirect fibrinolytic effect, such as urokinase, streptokinase and alteplase. However, due to their limited affinity for fibrin and short half-life, they must be injected continuously to be effective, and they can also cause side effects such as internal bleeding and allergic reactions. In comparison, plasmin, which can specifically act on fibrin and dissolve blood clots, has more advantages. Therefore, most current research focuses on finding plasmin with strong fibrinolytic activity, long half-life, few side effects and low production cost.

[0004] Existing studies have found fibrinolytic enzymes from different sources, such as snakes, earthworms, insects, mushrooms, leeks, and various microorganisms. Microbial fibrinolytic enzymes are considered to be the most promising enzyme-producing organisms due to their strong specificity, low production cost, relatively high yield, and the possibility of genetic modification through recombinant DNA technology and protein engineering methods. Therefore, screening strains that efficiently produce fibrinolytic enzymes and using microorganisms to develop thrombolytic drugs or functional health foods are of great significance to human life. Summary of the invention

[0005] The existing thrombolytic drugs in clinical practice have the limitations of short half-life, low specificity, and high cost. The present invention aims to provide a fibrinolytic enzyme-producing Bacillus, specifically to the salt tolerance of a strain of haloperidol-tolerant Bacillus producing fibrinolytic enzyme, and the effect of its fermentation liquid on anticoagulation and in vitro thrombolysis. The present invention isolates and screens the haloperidol-tolerant Bacillus N1 ( Bacillus halodurans N1), the strain can grow in a salt concentration of up to 13%, and its liquid fermentation supernatant has 325.38 U / mL fibrinolytic activity, and has significant anticoagulant and thrombolytic activity in vitro. The salt-tolerant Bacillus and its fermentation broth can be used in the development of anticoagulant and thrombolytic drugs, and have broad application prospects.

[0006] In order to achieve the above technical effects, the present invention is implemented through the following technical solutions: A fibrinolytic enzyme-producing halodurans Bacillus halodurans N1, wherein the halodurans is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC NO: 31939 and a deposit date of September 11, 2024.

[0007] The method for producing fibrinolytic enzyme by the above-mentioned halodurable Bacillus N1 is as follows: the halodurable Bacillus N1 is inoculated into a culture medium at an inoculation rate of 2% for fermentation culture, and a crude enzyme liquid is separated from the fermentation product to obtain the fibrinolytic enzyme.

[0008] Furthermore, the components of the culture medium are peptone 12 g / L, glucose 17.5 g / L, yeast powder 0.8 g / L, and FeCl3 0.05 g / L; the fermentation conditions are: temperature 35°C, rotation speed 150 rpm, and fermentation time 18 hours.

[0009] Furthermore, the above separation method is to centrifuge the fermentation product at 4°C, 10,000 rpm for 5 min, and take the supernatant as the crude enzyme solution.

[0010] Furthermore, before fermentation culture, the halotolerant Bacillus N1 is activated and a seed solution is prepared.

[0011] Furthermore, the activation method is to inoculate the halotolerant Bacillus N1 into LB liquid culture medium and culture it at 35° C. for 16-18 h to obtain a seed solution.

[0012] The application of the above-mentioned halotolerant Bacillus N1 in the preparation of thrombolytic drugs.

[0013] The beneficial effects of the present invention are: The salt-tolerant Bacillus N1 provided by the present invention ( Bacillus halodurans N1) can tolerate a maximum salinity of 13% and can be fermented to produce fibrinolytic enzyme. After liquid fermentation, its crude enzyme solution has 325.38 U / mL fibrinolytic activity. In addition, the crude enzyme solution also has a certain anticoagulant effect and in vitro thrombolytic ability, and can be used for the development of anticoagulant and thrombolytic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Fibrinolytic enzyme-producing halodurable Bacillus N1 ( Bacillus halodurans N1) screening, a is skimmed milk powder-LB primary screening plate; b is fibrin secondary screening plate.

[0015] Figure 2 : Fibrinolytic enzyme-producing halodurable Bacillus N1 ( Bacillus haloduransN1), a is the colony morphology on the LB plate; b is the electron micrograph of the strain N1.

[0016] Figure 3 :Halobacterium-tolerant Bacillus N1 producing fibrinolytic enzyme ( Bacillus halodurans N1) phylogenetic tree.

[0017] Figure 4 :Halobacterium-tolerant Bacillus N1 producing fibrinolytic enzyme ( Bacillus halodurans N1) salt tolerance analysis.

[0018] Figure 5 :Halobacterium-tolerant Bacillus N1 producing fibrinolytic enzyme ( Bacillus halodurans Figure 3. Fibrinolytic activity analysis of crude enzyme solution of strain N1. a is the standard curve of urokinase; b is the transparent circle formed by crude enzyme solution of strain N1 on the fibrin plate.

[0019] Figure 6 :Halobacterium-tolerant Bacillus N1 producing fibrinolytic enzyme ( Bacillus halodurans Analysis of the anticoagulant effect of crude enzyme solution of N1). ad is the coagulation status of fresh blood in different solutions for 5 min, 1 h, 3 h and 7 h (the solutions from left to right are normal saline, crude enzyme solution of N1, 10-fold dilution of crude enzyme solution of N1 and urokinase).

[0020] Figure 7 :Halobacterium-tolerant Bacillus N1 producing fibrinolytic enzyme ( Bacillus halodurans Figure 3 In vitro thrombolytic activity analysis of crude enzyme solution of N1, a is the state of blood clot in different solutions before reaction; b is the state of blood clot in different solutions after 12 hours of reaction (the solutions in the tubes from left to right are normal saline, crude enzyme solution of N1, 5-fold dilution of crude enzyme solution of N1 and urokinase). DETAILED DESCRIPTION

[0021] The following examples are further elaborations of the present invention. It should be noted that the descriptions of these embodiments are used to help understand the present invention, but do not constitute limitations of the present invention.

[0022] In this patent, unless otherwise specified, "strain N1" refers to Bacillus halodurans N1 (CGMCC NO: 31939).

[0023] 1. Materials and Methods 1.1 The materials involved in the following examples are as follows: Urokinase and bovine fibrinogen were purchased from Solebao Company, thrombin was purchased from Shanghai Yuanye Company, pMD19-T vector was purchased from Novozymes Biopharmaceuticals, and ampicillin was purchased from Beijing Solebao Technology Co., Ltd.

[0024] 1.2 The culture medium and solutions involved in the following examples are as follows: Skim milk powder solid culture medium: weigh 1.0 g skim milk powder, add pure water to 50 mL, and sterilize at 115 ºC for 15 min; weigh 0.5 g yeast powder, 1.0 g peptone, 1.0 g NaCl, and 1.75 g agar, add 50 mL water, sterilize at 121 ºC for 20 min, cool to about 50 ºC, mix the two culture media in a volume ratio of 1:1, pour into a culture dish, and solidify at room temperature to obtain a skim milk powder-LB plate, which should be placed at room temperature for use.

[0025] LB liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, dissolve in pure water, sterilize with high-temperature steam at 121 °C for 20 min and set aside.

[0026] LB plate: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, then add 1.5% agar, sterilize with high-temperature steam at 121 °C for 20 min and set aside.

[0027] Preparation of 100 mg / mL ampicillin stock solution: Dissolve 0.1 g of ampicillin powder in 1 mL of sterile dd H2O and pass through a water membrane (purchased from Changde Beekman Biotechnology Co., Ltd., product number: 130209001) for later use.

[0028] Fibrin plate: ① 0.05 M boric acid-physiological saline solution (BSB): weigh 1.9070 g of Na2B4O7·10 H2O and 0.9000 g of NaCl; adjust the pH to 7.8 with 0.2 M NaOH, and finally make up to 100 mL with ultrapure water; ② Fibrinogen solution: weigh 0.02 g of bovine fibrinogen and dissolve it in BSB solution preheated at 37 ºC, and keep it warm in a 42 ºC water bath; ③ 1% agar solution: weigh 0.1 g of agar powder, add 10 mL of ultrapure water, heat to dissolve, and cool to about 42 ºC; ④ Fibrin plate: mix the fibrinogen solution with the 1% agar solution, add 10 μL of thrombin (1000 U / mL), mix well, pour on a plate, let it stand at room temperature to solidify, and punch holes for later use.

[0029] Liquid fermentation medium: peptone 12 g / L, glucose 17.5 g / L, yeast powder 0.8 g / L, FeCl3 0.05 g / L, sterilized with high-temperature steam at 115 °C for 30 min before use.

[0030] 1.3 Screening of halotolerant Bacillus producing fibrinolytic enzyme (1) Skim milk powder - LB plate initial screening Take 10 g of soil sample from Inner Mongolia, remove the branches, put it into a conical flask containing 90 mL of sterile water, mix it, shake it at 180 rpm for 1 h at 30 ºC, let it stand for 10 min, take the soil supernatant and dilute it 10 times, then spread it on a skimmed milk powder-LB plate. After incubation at 30 ºC for 14 h, pick the colonies that produce transparent circles for streak purification.

[0031] (2) Fibrin plate rescreening Pick a single bacterium and put it into 5 mL of LB liquid culture medium, culture it at 30 ℃, 150 rpm for 18 h, take 30 μL of bacterial solution and add it to the wells of the fibrin plate, incubate it at 37 ℃ for 18 h, the bacteria that produce transparent circles are the fibrinolytic enzyme-producing halotolerant Bacillus, and select the strain with the strongest fibrinolytic activity for the next experiment.

[0032] 1.4 Identification of halodurable Bacillus producing fibrinolytic enzyme (1) Morphological identification Streak the strain obtained in step (2) of 1.3 on an LB plate, culture it at 37°C overnight, and observe its colony morphology; the single bacterial morphology of the strain was photographed using a scanning electron microscope.

[0033] (2) Molecular biological identification of haloperidol-tolerant Bacillus producing fibrinolytic enzyme ① Extract bacterial genome: inoculate the strain into LB liquid culture medium, culture overnight at 30°C and 150 rpm, and then extract genomic DNA from 3 mL of bacterial solution (Sangong Ezup Column Bacterial Genomic DNA Extraction Kit).

[0034] ② 16S rRNA amplification: Using genomic DNA as a template, PCR amplification of the 16S rRNA gene of the strain was performed using universal primers for bacterial 16S rRNA genes (forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3', as shown in SEQ ID NO: 2; reverse primer: 5'-GGTTACCTTGTTACGACTT-3', as shown in SEQ ID NO: 3). Reaction system: 2×Taq PCR Master Mix 12.5 μL, upstream and downstream primers 1 μL each, DNA template 1 μL, ddH2O 9.5 μL; reaction program: 94 ℃, 3 min; 94 ℃, 30 s; 50 ℃, 45 s; 72 ℃, 1 min40 s (35 cycles); 72 ℃, 7min.

[0035] ③ Gel excision and recovery: The obtained PCR products were subjected to agarose gel electrophoresis and gel excision and recovery (SanPrepColumn DNA Gel Extraction Kit). The recovered products were verified by 1% agarose gel electrophoresis and concentration detection, and then temporarily stored at -20 ℃.

[0036] ④ TA cloning: The recovered PCR product was connected to the pMD19-T Vector at 16°C, and then the connection product was transformed into Escherichia coli DH5α cells and spread on LB plates containing 100 ug / mL ampicillin for 16 h. After the culture was completed, a single bacterium was picked into 1 mL LB liquid medium (containing 100 ug / mL ampicillin), cultured at 37 ℃, 180 rpm for 12 hours, and the bacterial solution was taken for PCR verification. Reaction system: 2×Taq PCRMaster Mix 12.5 μL, upstream and downstream primers 1 μL each, DNA template 1 μL, ddH2O 9.5 μL; reaction program: 94 ℃, 5 min; 94 ℃, 30 s; 56 ℃, 30 s; 72 ℃, 1min (30 cycles); 72 ℃, 7 min. The PCR product was verified by 1% agarose gel electrophoresis, and the bacterial solution containing the target band was sent to Shanghai Biotech for sequencing.

[0037] ⑤ Construction of phylogenetic tree: The measured 16S rRNA gene sequences were compared in the EzBioCloud database, and the sequences with higher similarity were selected to construct the phylogenetic tree using MEGA 11.0 software.

[0038] 1.5 Salt tolerance test of halophilic Bacillus The isolated pure cultured Bacillus was cultured in LB liquid medium at 35°C for 16 h, and then the bacterial solution was inoculated with 1% inoculum into LB liquid medium with a salinity of 1% to 19%. After culturing at 35°C and 150 rpm for 12 h, the OD of the culture solution was measured. 600 , and analyzed the salt tolerance of the strains.

[0039] 1.6 Determination of fibrinolytic activity of crude enzyme solution from halodurous Bacillus sp. (1) Preparation of urokinase standard song The 1000 U / mL urokinase stock solution was diluted with 0.9% saline to 30 U / mL, 50 U / mL, 100 U / mL, 150 U / mL, and 200 U / mL standards. 30 μL of each standard was added to the fibrin plate. After incubation at 37 °C for 18 h, the inner and outer diameters of the transparent circles formed by different concentrations of urokinase were measured. The experiment was repeated three times and the average value was taken. Finally, the urokinase standard curve was drawn with the urokinase concentration as the x-axis and the square difference of the inner and outer diameters as the y-axis.

[0040] (2) Analysis of fibrinolytic activity of crude enzyme solution The strains that grew transparent circles on the fibrin plate were inoculated into 5 mL LB liquid medium for activation at 35°C; then inoculated into liquid fermentation medium at 2% inoculation volume, cultured at 35°C 150 rpm for 18 h, and the bacterial solution was aliquoted and centrifuged at 4°C, 10,000 rpm for 5 min, and the supernatant was taken as the crude enzyme solution. 30 μL of the crude enzyme solution was added to the fibrin plate, incubated at 37°C for 18 h, and the inner and outer diameters of the transparent circles formed by each strain were measured. The fibrinolytic activity was calculated based on the urokinase standard curve, and the strain with the strongest activity was selected for the next experiment.

[0041] 1.7 Anticoagulant effect test of crude enzyme solution of haloduronic Bacillus sp. producing fibrinolytic enzyme Take 500 μL of physiological saline, crude enzyme solution, 10-fold dilution of crude enzyme solution (diluted with 0.9% physiological saline), and urokinase (200 U / mL), add each to 1 mL of fresh chicken blood, place in a 37 ℃ water bath, and record the coagulation or dissolution status of the blood in each tube at 5 min, 1 h, 3 h, and 7 h.

[0042] 1.8 Analysis of in vitro thrombolytic activity of crude enzyme solution from halodurous Bacillus sp. producing fibrinolytic enzyme After fresh chicken blood was stored at 4 °C and coagulated, it was divided into several 2 mL centrifuge tubes with a volume of about 1.0 g each, and the initial weight was recorded. Physiological saline, crude enzyme solution, 5-fold dilution of crude enzyme solution (diluted with 0.9% physiological saline) and urokinase (200 U / mL) were added to each tube, respectively. After incubation at 37 °C for 12 h, the liquid was discarded, the water on the surface of the blood clot was dried, and the final weight of the blood clot after reaction was weighed. The relative dissolution rate and absolute dissolution rate of the blood clot in different solutions were calculated. The relative dissolution rate = the weight of the blood clot after reaction / the initial weight of the blood clot; the absolute dissolution rate = the relative dissolution rate of the blood clot-the relative dissolution rate of the blood clot in physiological saline.

[0043] 2 Results and Analysis 2.1 Screening of halotolerant Bacillus producing fibrinolytic enzyme The supernatant dilution of the Inner Mongolia soil sample was spread on a skimmed milk powder-LB plate for initial screening. The results after incubation were as follows Figure 1 a; 8 strains with different colony morphologies that produced transparent circles were selected from 1a, cultured in LB liquid medium, and then the bacterial solution was added to the perforated fibrin plate and incubated at 37 °C for 18 h. Figure 1 As shown in b, the transparent zone formed by strain N1 is the largest.

[0044] 2.2 Identification of the halodurogenic Bacillus species producing fibrinolytic enzyme The colony morphology of strain N1 on LB plate is as follows Figure 2a, It is a round colony with a raised center, neat edges, wrinkled surface, milky white, translucent and sticky. Figure 2 b is the single bacterial morphology of strain N1 under electron microscope, which is cylindrical rod-shaped and without flagella. The 16s rRNA gene sequence was sequenced and the sequencing results were compared on NCBI, and it was found that it was Bacillus halotolerans (Bacillus halodurans) has a coverage rate of 94% and a consistency of 99.60%. Then, Ez BioCloud was used to compare and construct a phylogenetic tree. The results are as follows Figure 3 As shown, strain N1 and Bacillus halotolerans In the same branch, the bootstrap value is 100, so strain N1 is identified as Bacillus halotolerans The accession number of the 16S rRNA gene sequence of the strain in the GenBank database is PQ135177, and the nucleic acid sequence of the strain is shown in SEQ ID NO: 1.

[0045] 2.3 Analysis of salt tolerance of strain N1 The growth of strain N1 in LB liquid medium with a salt concentration of 1% to 19% can be measured by OD 600 To detect, such as Figure 4 As shown in the figure, strain N1 grows normally at a salt concentration of 1% to 3%; when the salt concentration is higher than 7%, the growth of the strain is greatly affected; when the salt concentration is higher than 13%, the growth of the strain approaches 0. Based on this analysis, the highest salt tolerance of strain N1 is 13%.

[0046] 2.4 Analysis of fibrinolytic activity of crude enzyme solution of strain N1 according to Figure 5 aThe standard curve of urokinase can be calculated Figure 5 The fibrinolytic activity of the crude enzyme solution of strain N1 in b was 325.38 U / mL.

[0047] 2.5 Analysis of the anticoagulant effect of crude enzyme solution of strain N1 according to Figure 6 The coagulation and hemolysis of blood in physiological saline, N1 crude enzyme solution, 10-fold dilution of N1 crude enzyme solution and 200 U / mL urokinase were recorded and statistically analyzed in Table 1. It was found that fresh blood in strain N1 crude enzyme solution and its 10-fold dilution showed only slight coagulation phenomenon at 1 h, while fresh blood in physiological saline and 200 U / mL urokinase had completely coagulated at 1 h, indicating that strain N1 crude enzyme solution has a certain anticoagulant effect.

[0048] Table 1 Coagulation state of fresh blood in different solutions at different time points (+++: completely solidified; ++: partially solidified; +: slightly solidified; -: not solidified; d-: slightly dissolved; d--: partially dissolved; d---: completely dissolved) 2.6 Analysis of the in vitro thrombolytic effect of crude enzyme solution of strain N1 By comparison Figure 7 a and Figure 7 b, it can be found that after the blood clot reacted in the crude enzyme solution for 12 hours, many red blood cells were released into the liquid, forming a dark red liquid, which was darker than the liquid in other tubes. The relative dissolution rate and absolute dissolution rate of the blood clots in each tube were calculated. The results are shown in Table 2. The relative dissolution rate and absolute dissolution rate of the N1 crude enzyme solution were 21.00% and 16.56%, respectively, and the relative dissolution rate and absolute dissolution rate of the 5-fold dilution of the N1 crude enzyme solution were 19.20% and 14.76%, respectively. It was preliminarily judged that it had a good thrombolytic effect.

[0049] Table 2 Relative and absolute dissolution rates of blood clots in different solutions The above description is only a preferred specific implementation method of the invention, but the protection scope of the invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and inventive concepts of the invention within the technical scope disclosed by the invention, which should be covered by the protection scope of the invention.

Claims

1. A strain of halophilic Bacillus ( Bacillus halodurans N1, characterized by: The halotolerant Bacillus is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO: 31939 and the deposit date September 11, 2024.

2. The method for producing fibrinolytic enzyme by halodurable Bacillus N1 according to claim 1, characterized in that: The halodurous Bacillus N1 is inoculated into a culture medium at an inoculation rate of 2% for fermentation culture, and a crude enzyme liquid is separated from the fermentation product to obtain fibrinolytic enzyme.

3. The method according to claim 2, characterized in that: The components of the culture medium are peptone 12 g / L, glucose 17.5 g / L, yeast powder 0.8 g / L, and FeCl3 0.05 g / L; the fermentation conditions are: temperature 35°C, rotation speed 150 rpm, and fermentation time 18 hours.

4. The method according to claim 2, characterized in that: The separation method is to centrifuge the fermentation product at 4°C and 10,000 rpm for 5 min, and take the supernatant as the crude enzyme solution.

5. The method according to claim 2, characterized in that: Before fermentation, the halotolerant Bacillus N1 was activated and seed solution was prepared.

6. The method according to claim 4, characterized in that: The activation method is to inoculate the halotolerant Bacillus N1 into LB liquid culture medium and culture it at 35° C. for 16-18 hours to obtain a seed solution.

7. Use of the halotolerant Bacillus N1 as claimed in claim 1 in the preparation of thrombolytic drugs.

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