Microorganism of high-yield immunomodulatory peptide Octaeptins and high-throughput screening method thereof
Through the chromogenic acid benzenesulfonate and methylene blue detection systems, the high-throughput screening of Octapeptins in microbial fermentation products is simplified, and the problem of cumbersome screening methods in the prior art is solved, and efficient and simple screening effect is achieved.
Patent Information
- Application Number
- CN202411849885.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-16
- Publication Date
- 2025-02-25
AI Technical Summary
In the prior art, the high-throughput screening method of microbial fermentation broth is complicated and it is difficult to provide a simple high-yield Octapeptins microbial screening method.
The microbial fermentation product contains Octapeptins by chromogenic reaction method. The detection system of sodium cetylbenzenesulfonate and methylene blue was used, and the standard curve was constructed to achieve high-throughput screening.
This method simplifies the microbial screening process, has high sensitivity and selectivity, is suitable for high-throughput strain screening, and does not require the use of toxic organic solvents, has low analysis cost, is simple to operate, and is environmentally friendly.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to a microorganism with high yield of immunomodulatory peptide Octapeptins and a high-throughput screening method thereof. Background Art
[0002] Octapeptins are a broad-spectrum lipoctapeptide antibiotic with multiple homologues, which are divided into four types according to different amino acids: octapeptin A, octapeptin B, octapeptin C, and octapeptin D. The chemical structure of octapeptins is similar to that of polymyxins, both of which contain a cyclic heptapeptide core composed of multiple amino acids, including multiple diaminobutyric acids (Dab), each representing a cationic part. Octapeptins are considered ideal candidates for the new generation of polymyxin-like lipopeptide antibiotics due to their activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacteria, especially for polymyxin-resistant "superbugs" such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. Octapeptins show a broad antibacterial spectrum against these resistant Gram-negative and Gram-positive bacteria. In addition, octapeptins have shown activity against a wide range of bacteria in both in vitro and in vivo studies and have shown a lower toxicity trend than polymyxin in mouse models. These properties make octapeptins promising drug candidates for combating drug-resistant "superbugs."
[0003] Octapeptins synthesis by microorganisms is a common production method, which requires high-throughput screening of microorganisms; however, the conventional screening method is to subject the fermentation broth of the microorganism to high-performance liquid chromatography or mass spectrometry detection. This method not only requires fermentation and cultivation of the microorganisms, but also the detection process is too cumbersome. How to provide a simple high-throughput screening method for high-yield Octapeptins microorganisms is one of the hot topics of concern to technicians in this field. Summary of the invention
[0004] The invention provides a microorganism with high yield of immunomodulatory peptide Octapeptins and a high-throughput screening method for the microorganism with high yield of Octapeptins.
[0005] In the first aspect, the present invention provides the use of a microorganism in the preparation of Octapeptins, wherein the microorganism is Paenibacillus elgii 219, which was deposited in the China General Microorganism Culture Collection Center (CGMCC) on November 1, 2024, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the registration number of the microorganism in the China General Microorganism Culture Collection Center is CGMCC No.32461.
[0006] In a second aspect, the present invention provides a method for preparing Octapeptins, comprising fermenting a microorganism, collecting a fermentation product, and extracting Octapeptins from the fermentation product; the microorganism is deposited in the General Microorganism Center of the China General Microorganism Culture Collection Administration, and the registration number of the microorganism in the General Microorganism Center of the China General Microorganism Culture Collection Administration is CGMCC No.32461.
[0007] The method described above can be used to ferment the microorganism according to conventional techniques in the art. Further, the microorganism can be fermented using a liquid culture medium conventionally used for Paenibacillus in the art.
[0008] In the above method, the Octapeptins include Octapeptin A with a chemical formula as shown in Formula 1 and Octapeptin B with a chemical formula as shown in Formula 2:
[0009]
[0010] In a third aspect, the present invention provides a method for identifying whether a biological fermentation product contains Octapeptins, comprising:
[0011] A detection system including sodium hexadecylbenzenesulfonate and methylene blue was prepared, and the absorbance value of the detection system at 663 nm was detected, which was recorded as A1;
[0012] Mixing the fermentation product of the microorganism to be identified with the detection system to obtain a mixed system, and detecting the absorbance value of the mixed system at 663 nm, which is recorded as A2;
[0013] When A2 is greater than A1, it indicates that the fermentation product of the microorganism to be identified contains Octapeptins, otherwise the fermentation product of the microorganism to be identified does not contain Octapeptins.
[0014] The method as described above, further comprising:
[0015] Prepare a series of standard solutions containing Octapeptins in a concentration gradient;
[0016] Mixing the standard solution containing octapeptins with the detection system to obtain a mixed system, and detecting the absorbance value of the mixed system at 663 nm;
[0017] A standard curve is constructed by taking the concentration of octapeptins in the standard solution containing octapeptins as the horizontal coordinate and the absorbance value as the vertical coordinate;
[0018] The absorbance value of the mixed system of the microbial fermentation product to be identified and the detection system at 663 nm is substituted into the standard curve to obtain the concentration of Octapeptins in the microbial fermentation product to be identified.
[0019] In the method as described above, the molar ratio of sodium hexadecylbenzenesulfonate to methylene blue in the detection system is 1:(1-1.5).
[0020] The method as described above further comprises: adjusting the pH of the mixed system to 5.5 and the temperature to 25°C.
[0021] In a fourth aspect, the present invention provides a method for screening microorganisms that produce high octapeptins, comprising:
[0022] A detection system is prepared, wherein the detection system includes sodium hexadecylbenzenesulfonate and methylene blue;
[0023] The fermentation products of N microorganisms to be screened are mixed with the detection system respectively to obtain a mixed system, and the absorbance value of the mixed system at 663 nm is detected to obtain N absorbance values; N is an integer not less than 2;
[0024] The value with the largest value is selected from the N absorbance values to obtain the microorganism with the highest octapeptins production.
[0025] In a fifth aspect, the present invention provides a composition for determining whether a microbial fermentation product contains Octapeptins, comprising sodium hexadecylbenzenesulfonate and methylene blue.
[0026] The composition as described above further comprises a PBS buffer solution.
[0027] The present invention screens microorganisms through a color development reaction, and can determine whether the microbial fermentation products contain Octapeptins and their content without high-performance liquid chromatography or mass spectrometry detection of the microbial fermentation products. The screening provided by the present invention has high sensitivity and good selectivity, and is suitable for high-throughput strain screening. In addition, the method provided by the present invention does not use toxic organic solvents, and has the advantages of low analysis cost, simple operation, and green environmental protection.
[0028] Based on the above method, the present invention also screens out a strain with high octapeptins production, which can be used in the preparation and production of octapeptins. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 The mass spectrometry results of the fermentation broth of the screened strain CGMCC No.32461; wherein A is the primary mass spectrometry result of octapeptin A; B is the secondary mass spectrometry result of octapeptin A; C is the primary mass spectrometry result of octapeptin B; and D is the secondary mass spectrometry result of octapeptin B.
[0030] Figure 2 It is the absorbance change of the mixed solution of the detection system and Octapeptins in the buffer system with different pH; wherein, A is the absorbance of the mixed solution in the PBS buffer system with pH of 5.5, 6.0 and 6.5 respectively; B is the relative absorbance of the mixed solution in the buffer system with different pH conditions in A at different reaction times; C is the absorbance of the mixed solution in the acetic acid-sodium acetate buffer system with pH of 3.6, 4.0, 4.6, 5.0 and 5.6 respectively; D is the relative absorbance of the mixed solution in the buffer system with different pH conditions in C at different reaction times; E is the absorbance of the mixed solution in the Na2HPO4-citric acid buffer system with pH of 2.2, 3.0, 4.0, 5.0 and 6.0 respectively; F is the relative absorbance of the mixed solution in the buffer system with different pH conditions in E at different reaction times;
[0031] Figure 3 It is the relative absorbance of the mixed solution of the detection system and Octapeptins at different reaction temperatures. DETAILED DESCRIPTION
[0032] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.
[0033] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0034] Example 1: Determination of Octapeptins Content by Colorimetric Reaction
[0035] 1. Determination of the testing system
[0036] This example uses basic dye methylene blue (MB) as a color indicator and anionic detergent sodium hexadecylbenzene sulfonate (SDBS) as a medium to form a (SDBS-MB) complex. With the addition of Octapeptins, due to its stronger binding ability with SDBS, the SDBS in the (SDBS-MB) complex is competitively captured by Octapeptins, releasing free MB molecules and deepening the color of the reaction system. The above color development reaction can be used to screen whether microbial fermentation products contain Octapeptins and their content in high throughput.
[0037] 2. Determination of the optimal ratio of MB and SDBS:
[0038] MB was dissolved in ultrapure water to obtain a concentration of 2.08*10 -5 mol / L MB solution, SDBS was added to the MB solution to obtain a mixed solution with a final SDBS concentration as shown in Table 1, and the absorbance of the mixed solution at 663 nm was tested after adjusting the pH of the mixed solution to 5.0. Three parallel samples were set in the experiment, and the relative error was within the range of ±5%. The data results were taken as the average value. The test results are shown in Table 1.
[0039] Table 1 Absorbance of the system at 663 nm at different SDBS concentrations
[0040]
[0041] According to Table 2, as the concentration of SDBS increases, the absorbance of the mixed solution at 663 nm gradually decreases. When the molar ratio of MB to SDBS is close to 1:1, its absorbance drops to the minimum, and then the absorbance is basically stable when the concentration of SDBS is increased.
[0042] Example 2: Screening of strains with high octapeptins production
[0043] 2.1. Strain screening
[0044] According to the screening method provided in Example 1, the strain was subjected to high-throughput screening to screen a strain of Paenibacillus elgii 219. The strain was deposited in the China General Microbiological Culture Collection Center (CGMCC) on November 1, 2024, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The registration number of the microorganism in the China General Microbiological Culture Collection Center is CGMCC No. 32461.
[0045] 2.2. Strain fermentation verification
[0046] The strain CGMCC No.32461 was fermented using a liquid culture medium conventionally used for Paenibacillus in the art, wherein the final concentration of the fermentation culture medium was as follows: 3 g / L peptone; 16 g / L yeast powder; 2 g / L sucrose; 0.1 g / L magnesium sulfate; 5 g / L potassium dihydrogen phosphate, and the initial pH was 6.8±0.1. The culture conditions were 30°C and 220 rpm.
[0047] After the fermentation, the pH of the fermentation liquid was adjusted to 4.0, and the bacteria were removed by centrifugation at 8000rpm for 5min. 15% ammonium sulfate (g / mL) was added to the supernatant, and after the ammonium sulfate was dissolved, it was allowed to stand for 30min. After centrifugation, the precipitate was washed 3 times with a little water and 2 times with an appropriate amount of anhydrous ethanol, and the crude product was obtained by rotary evaporation for standby use. The crude extract was prepared by dissolving it in ultrapure water and diluted to a 200mg / mL solution. The above 200mg / mL solution was separated and purified by preparative high performance liquid chromatography (RP-HPLC) after passing through a 0.22μm filter membrane. The chromatographic column was a C18 (10×250mm) reverse phase column, and the instrument parameters were set as follows: detection wavelength 220nm; column temperature 25℃; flow rate 5mL / min; mobile phase A: ultrapure water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile + 0.1% trifluoroacetic acid, and the mobile phase elution conditions were shown in Table 2.
[0048] Table 2 Mobile phase purification elution gradient
[0049] Time: min A(％) B(％) Flow rate: mL / min 0.00 90.0 10.0 1.000 4.00 90.0 10.0 1.000 8.00 65.0 35.0 1.000 23.00 65.0 35.0 1.000 30.00 53.0 47.0 1.000 32.00 0.0 100.0 1.000 40.00 0.0 100.0 1.000 45.00 90.0 10.0 1.000 47.00 90.0 10.0 1.000
[0050] After removing acetonitrile from the collected fractions by rotary evaporation, freeze-drying was performed to obtain two white powder compounds. The molecular weights of the two compounds were 1023.72 and 1057.71, and the molecular formulas were C49H93N13O10 and C52H91N13O10, respectively. The MS-MS spectra of the two compounds are shown in Figure 1 , it can be inferred that the two compounds are octapeptin A and octapeptin B, and their structural formulas are shown below:
[0051]
[0052] 2.3 Absorption spectra of MB, SDBS and Octapeptin
[0053] MB and SDBS were dissolved in ultrapure water at a molar ratio of 1:1 and mixed evenly to obtain a concentration of 1.2*10 -5 mol / L mixed solution, octapeptin A and octapeptin B with a molar ratio of 1:1 were added to the mixed solution to prepare a mixed solution with a final concentration of octapeptins as shown in Table 3, the pH of the mixed solution was adjusted to 5.0, and the absorbance of the mixed solution at 663nm was tested. Three parallel samples were set in the experiment, and the relative error was within the range of ±5%. The data results were taken as the average value. The test results are shown in Table 3.
[0054] Table 3 Absorbance of the system at 663 nm at different Octapeptin concentrations
[0055]
[0056] According to Table 3, as the concentration of Octapeptins increases, the absorbance of the system at 663nm gradually increases. However, when the concentration of Octapeptins exceeds a certain range, its absorbance begins to stop increasing. Only when Octapeptins is in a lower concentration range, the concentration and the absorbance of the system are linearly related. The reason may be that there are multiple Dab residues in the Octapeptins molecule, and the positively charged amino group (NH3+) on each residue can bind to the anionic surfactant SDBS. Therefore, a lower concentration can free the MB molecules in the system.
[0057] 2.4. Optimization of reaction conditions
[0058] According to the above experimental results, MB and SDBS were dissolved in ultrapure water to prepare a MB concentration of 1.05×10 -4 mol / L, SDBS concentration was 0.995×10 -4mol / L mixed solution, the total volume of the mixed solution is 200μL. 10μL of the fermentation supernatant of strain CGMCC No.32461 and buffer systems of different pH were added to the mixed solution, and samples were taken at 0min, 10min, 20min, 30min, 40min, 50min, 70min, 100min and 24h and the absorbance of the mixed system was tested at 663nm. The test results are as follows Figure 2 shown.
[0059] according to Figure 2 It can be seen that in the three different buffer systems, as the pH of the buffer system continues to increase, the absorbance of the mixed solution gradually decreases. Under the PBS buffer with a pH of 5.5, the absorbance of the system is relatively stable within 0-100min, and the detection system is determined to be a PBS buffer with a pH of 5.5.
[0060] 2.5. Reaction temperature optimization
[0061] According to the above experimental results, the reaction system was determined as follows: the volume of MB-SDBS detection reagent added to the 96-well plate was fixed at 200 μL, and the concentrations were 1.05*10 -4 mol / L, 0.995*10 -4 mol / L, the buffer was PBS buffer, and then 10 μL of the fermentation supernatant of strain CGMCC No.32461 was added. The reaction pH was 5.5 and the reaction time was 10 min. Subsequently, the absorbance of the system was detected at 22, 24, 25, 26, and 27 °C.
[0062] according to Figure 3 It can be seen that temperature has a significant effect on the color development reaction. For the accuracy of the experiment, 25°C was determined as the detection temperature.
[0063] 2.6. Relationship between absorbance and Octapeptins concentration:
[0064] Octapeptin A (50.0 mg) and Octapeptin B (50.0 mg) were mixed and dissolved in 50 mL ultrapure water to prepare Octapeptin stock solution. Octapeptin stock solution was diluted into a series of standard working solutions with different concentrations, 10 μL of standard working solution was selected and mixed with the detection system containing MB and SDBS, the absorbance of the mixed system at 663 nm was measured, and a standard curve was established, as shown in Table 4.
[0065] Each of the above standard working solutions was diluted 10 times and added to the detection system containing MB and SDBS at a sample volume of 50 μL and 100 μL respectively. The absorbance of the mixed system at 663 nm was measured, and a standard curve was established, as shown in Table 4.
[0066] Table 4 Standard curve, correlation coefficient and linear range of SDBS / MB-Octapeptins system
[0067] sample Sample volume (μL) Regression equation R-Value Linear range (μg / mL) Standard working fluid 10 Y=0.0028X+1.9917 0.9783 0-600 Dilute 10 times 100 Y=0.0018X+2.0043 0.9938 0-500 Dilute 10 times 50 Y=0.001X+2.0991 0.9967 0-800
[0068] As can be seen from Table 4, when the standard working solution was diluted 10 times and the sample volume was 50 μL, the correlation coefficient of the regression equation and the detection range were high, which were 0.9967 and 0-800 μg / mL, respectively.
[0069] 2.7 Method Validation
[0070] In order to investigate the stability and reproducibility of the 2.6 standard curve, the content of Octapeptins in the fermentation broth of strain CGMCC No.32461 was determined by the standard addition method, and the results are shown in Table 5. The RSD was between 2.4% and 3.2%, and the recovery rate was between 99% and 103%, indicating that this method was stable and reliable.
[0071] Table 5 Determination of Octapeptins content in fermentation broth of strain CGMCC No.32461
[0072]
[0073]
[0074] In summary, methylene blue is a widely used and inexpensive indicator. After combining with the anionic surfactant sodium hexadecylbenzenesulfonate, it can be used as a colorimetric agent for the spectrophotometric determination of cationic surfactants. The advantages of this method are high sensitivity, good selectivity, high throughput and low analytical cost, simple operation, and no use of toxic organic solvents. The detection system has a good specific binding effect with octapeptins, is not affected by other factors in the fermentation broth, and has good selectivity.
[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. The use of microorganisms in the preparation of Octapeptins, characterized in that: The microorganism is deposited in the General Microorganism Center of China General Microorganism Culture Collection Administration Committee, and the registration number of the microorganism is CGMCC No.32461.
2. A method for preparing Octapeptins, characterized in that: The method comprises fermenting microorganisms, collecting fermentation products, and extracting Octapeptins from the fermentation products; the microorganisms are preserved in the General Microorganism Center of China General Microorganism Culture Collection Administration Committee, and the registration number of the microorganisms in the General Microorganism Center of China General Microorganism Culture Collection Administration Committee is CGMCC No.32461.
3. The method according to claim 2, characterized in that The octapeptins include Octapeptin A with a chemical structure shown in Formula 1 and Octapeptin B with a chemical structure shown in Formula 2:
4. A method for identifying whether a microbial fermentation product contains octapeptins, characterized in that: include: A detection system including sodium hexadecylbenzenesulfonate and methylene blue was prepared, and the absorbance value of the detection system at 663 nm was detected, which was recorded as A1; Mixing the fermentation product of the microorganism to be identified with the detection system to obtain a mixed system, and detecting the absorbance value of the mixed system at 663 nm, which is recorded as A2; When A2 is greater than A1, it indicates that the fermentation product of the microorganism to be identified contains Octapeptins, otherwise the fermentation product of the microorganism to be identified does not contain Octapeptins.
5. The method according to claim 4, characterized in that The method further comprises: Prepare a series of standard solutions containing Octapeptins in a concentration gradient; Mixing the standard solution containing octapeptins with the detection system to obtain a mixed system, and detecting the absorbance value of the mixed system at 663 nm; A standard curve is constructed by taking the concentration of octapeptins in the standard solution containing octapeptins as the horizontal coordinate and the absorbance value as the vertical coordinate; The absorbance value of the mixed system of the microbial fermentation product to be identified and the detection system at 663 nm is substituted into the standard curve to obtain the concentration of Octapeptins in the microbial fermentation product to be identified.
6. The method according to claim 4 or 5, characterized in that: The molar ratio of sodium hexadecylbenzenesulfonate to methylene blue in the detection system is 1:(1-1.5).
7. The method according to claim 4 or 5, characterized in that: The method further comprises: adjusting the pH of the mixed system to 5.5 and the temperature to 25°C.
8. A method for screening microorganisms with high production of octapeptins, characterized in that: include: A detection system is prepared, wherein the detection system includes sodium hexadecylbenzenesulfonate and methylene blue; The fermentation products of N microorganisms to be screened are mixed with the detection system respectively to obtain a mixed system, and the absorbance value of the mixed system at 663 nm is detected to obtain N absorbance values; N is an integer not less than 2; The value with the largest value is selected from the N absorbance values to obtain the microorganism with the highest octapeptins production.
9. A composition for determining whether a microbial fermentation product contains octapeptins, characterized in that: Includes sodium hexadecylbenzene sulfonate and methylene blue.
10. The composition according to claim 9, characterized in that The composition also includes a PBS buffer solution.