Paenibacillus for producing lipopeptide and preparation method of lipopeptide
By providing a Bacillus egicia that can efficiently separate and purify permetin A in fermentation broth, the problem of research and development of new antibacterial drugs is solved, and the production of permetin A with high purity and high yield is achieved, with significant antibacterial activity and environmental protection advantages.
Patent Information
- Application Number
- CN202311553071.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-21
- Publication Date
- 2025-05-30
AI Technical Summary
In recent years, the research and development of new antibacterial drugs has become increasingly difficult, and microorganisms that can produce new antibacterial drugs have become increasingly difficult to isolate, resulting in insufficient supply of antibacterial drugs.
A strain of Paenibacillus elgii was provided, with the storage number CGMCC No. 19830, which can separate and purify the antibacterial active substance lipopeptide permetin A in its fermentation broth. This strain can efficiently produce high-purity permetin A through specific fermentation and extraction methods.
The Bacillus egicia can produce permetin A with high yield and high purity. The purity of the prepared permetin A single product can reach 98.31%, which has significant antibacterial activity against a variety of Gram-positive and Gram-negative bacteria, and is harmless to the environment.
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Figure CN120059988A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology and relates to Paenibacillus. Background Art
[0002] Permetin A is a lipopeptide secreted by Bacillus circulans, which contains 2,4-diaminobutyric acid (Dab), leucine, isoleucine, phenylalanine, valine, serine (molar ratio 3:2:1:1:1:1) and 1 β-hydroxyheptanoic acid. It has activities against Gram-positive bacteria, Gram-negative bacteria and some anaerobic bacteria (Takahara, Yoshiyuki, 1979, 32(2):115-120). In 1979, YOSHIYUKI TAKAHARA et al. first isolated and purified the lipopeptide permetin A from the fermentation broth of Bacillus circalans AJ 3902 (Takahara, Yoshiyuki, 1979, 32(2):115-120).
[0003] However, in recent years, the research and development of new antibacterial drugs have become increasingly difficult, and it has also become more and more difficult to isolate microorganisms that can produce new antibacterial drugs. Summary of the Invention
[0004] The present invention provides a Paenibacillus elgii, from the fermentation broth of which an antibacterial active substance, lipopeptide permetin A, can be isolated and purified.
[0005] The object of the present invention is achieved by the following measures:
[0006] A Paenibacillus elgii, with the preservation number of CGMCC No. 19830, was preserved in the China General Microbiological Culture Collection Center on May 15, 2020, and registered and numbered in this preservation center. It is self-named Z121.
[0007] The Paenibacillus elgii is a rod-shaped bacterium that is thermophilic, aerobic, spore-forming, Gram-positive, widely exists in nature, non-toxic and harmless to humans and animals, and does not pollute the environment. During the growth process, it can produce a variety of secondary metabolites and enzymes, and has the ability to inhibit the activities of pathogenic fungi, bacteria and viruses.
[0008] Another object of the present invention is to provide the application of the above-mentioned Paenibacillus elgii.
[0009] The application of the above-mentioned Paenibacillus elgii in the preparation of lipopeptides.
[0010] The application of the above-mentioned Paenibacillus elgii in bacteriostatic agents and fungicides.
[0011] The third object of the present invention is to provide a method for preparing lipopeptides.
[0012] A method for preparing lipopeptides, using the above-mentioned Paenibacillus ehimensis.
[0013] The above method for preparing lipopeptides includes the following steps:
[0014] (1) Fermentation: Fermentation is carried out using the above-mentioned Paenibacillus ehimensis. The fermentation medium formula is soybean meal powder 10±5 g / L, yeast powder 5±2 g / L, peptone 10±5 g / L, glucose 2±1 g / L, sodium chloride 5±2 g / L.
[0015] (2) Preparation of permetin A crude extract: The fermentation broth of Paenibacillus ehimensis Z121 is centrifuged at 4°C and 8000 g for 30 min, and the obtained supernatant is freeze-dried to obtain a freeze-dried powder; Immunomodulatory peptides are extracted according to the ratio of 1 g of supernatant freeze-dried powder to 5 mL of acetonitrile, and the acetonitrile after extraction is removed in a rotary evaporator to obtain the permetin A crude extract.
[0016] (3) Separation and purification of Permetin A
[0017] The permetin A crude extract is dissolved in pure water and diluted into a solution of 200 mg / mL. The above 200 mg / mL solution is passed through a 0.22 μm filter membrane; Separation and purification are carried out by preparative reversed-phase high-performance liquid chromatography. The chromatographic column is a C18 (10×250 mm) reversed-phase column, and the instrument parameters are set as follows: detection wavelength 220 nm; column temperature 25°C; flow rate 5 mL / min; mobile phase A: ultrapure water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile + 0.1% trifluoroacetic acid; 500 μL of each sample is loaded, the fractions corresponding to the chromatographic peaks are collected, and the acetonitrile is removed by a rotary evaporator and then freeze-dried to obtain the pure lipopeptide Permetin A.
[0018] Beneficial effects
[0019] 1. The Paenibacillus ehimensis provided by the present invention can produce lipopeptides with high yield and high purity. The purity of the single lipopeptide Permetin A prepared can reach 98.31%, and the yield can reach 1049.54 mg / L.
[0020] 2. The Paenibacillus ehimensis of the present invention and the lipopeptide Permetin A produced thereby have significant antibacterial activities against both Gram-positive and Gram-negative bacteria, especially Staphylococcus aureus CMCC26003, Clostridium perfringens ATCC13124, Salmonella CVCC534, Escherichia coli CVCC1515, Escherichia coli CVCC1518, Escherichia coli CVCC1557, Escherichia coli CVCC1569, Escherichia coli ATCC25922, Salmonella ATCC13076, Pseudomonas aeruginosa ATCC27853, Shigella dysenteriae CMCC(B)51252, Shigella flexneri ATCC29903, and Shigella sonnei CICC21679, and the MIC values against these bacteria are all no higher than 8 μg / ml.
[0021] 3. The Paenibacillus ehimensis of the present invention can directly promote crop growth by biological nitrogen fixation, phosphorus solubilization, production of the phytohormone indole-3-acetic acid (IAA), and release of siderophores. They can also provide protection against insects, herbivores, and plant pathogens, including bacteria, fungi, nematodes, and viruses. This is achieved by producing various antibacterial agents and insecticides and by triggering the hypersensitive defense response of plants, namely induced systemic resistance (ISR). The antibacterial agents produced by the Paenibacillus ehimensis of the present invention also have medical applications, including not only the production of Permetin A, but also polymyxin and fusaric acid. Others include exopolysaccharides (EPS) and enzymes, such as amylase, cellulase, hemicellulase, lipase, pectinase, oxygenase, dehydrogenase, lignin-modifying enzyme, and mutant enzyme, which may have applications in detergents, food and feed, textiles, paper, biofuels, and healthcare. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Cultivation and screening of Paenibacillus ehimensis Z121
[0023] Figure 2 Annotation diagram of Paenibacillus ehimensis Z121 species classification in the NR database
[0024] Figure 3 High performance liquid chromatography peak diagram of the isolation and purification of lipopeptide Permetin A
[0025] Figure 4 High performance liquid chromatography peak diagram of the purity detection of lipopeptide Permetin A DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention will be further described in detail below with reference to the drawings and examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
[0027] Example 1
[0028] Screening of Paenibacillus elgii Z121, the strain producing permetin A
[0029] Soil samples were collected from Lanfeng Forest Farm in Rongchang. After mixing, 5 g of the soil sample was dissolved in 50 mL of sterile physiological saline, shaken at room temperature for 30 min, settled for 2 - 3 min, and then used as a standby. 0.5 mL of the supernatant of the soil eluate was diluted in 4.5 mL of sterile physiological saline and serially diluted 10-fold, diluted 10 4 / 5 times. Using Staphylococcus aureus ATCC 29213 as the indicator bacterium, an indicator bacterium plate was prepared. 100 μL of the soil dilution was spread and cultured at 37 °C. A single colony with an inhibition zone was found ( Figure 1 ). The single colony was picked out and streaked on an NB plate for isolation. A single colony was picked from the isolated and purified plate, inoculated into 5 ml of NB culture medium, and cultured in a test tube at 37 °C and 200 rpm / min until the culture medium became turbid. The bacterial cells were collected by centrifugation, and the strain was preserved by resuspending the bacterial cells with glycerol at a final concentration of 25% and NB culture medium, named Z121.
[0030] Z121 was cultured in NB at 33 °C / 180 rpm until the stationary phase. An appropriate amount of bacterial cells was collected. The genomic DNA of the sample was extracted by the SDS method. Then, the purity and integrity of the DNA were detected by agarose gel electrophoresis, and quantitative analysis was performed using Qubit. The qualified DNA sample by electrophoresis was randomly fragmented into fragments with a length of about 350 bp using a Covaris ultrasonic disruptor. After the treatment of the DNA fragments, the Ultra TM DNA Library Prep Kit for Illumina (NEB, USA) was used. The entire library construction was completed through steps such as end repair, adding A-tail, adding sequencing adapters, purification, and PCR amplification. The library was first preliminarily quantified using Qubit 2.0, and the library was diluted to 2 ng / μL. Subsequently, Agilent 2100 was used to detect the inserted fragments of the library. After the insert size met the expectations, the Q-PCR method was used to accurately quantify the effective concentration of the library to ensure the library quality. After passing the library inspection, different libraries were sequenced on an Illumina NovaSeq PE150 according to the effective concentration and the target output data volume, and NR (Non-Redundant Protein Database) annotation was performed. It was found that Z121 belongs to Paenibacillus elgii ( Figure 2 ), and the preservation number is CGMCC No. 19830.
[0031] Example 2
[0032] 1) Extraction of Permetin A
[0033] The fermentation broth of Paenibacillus ehimensis Z121 was centrifuged at 4 °C and 8000 g for 30 min, and the obtained supernatant was freeze-dried to obtain a freeze-dried powder. Immunomodulatory peptides were extracted according to the ratio of 1 g of the freeze-dried supernatant powder to 5 mL of acetonitrile. The acetonitrile after extraction was removed in a rotary evaporator to obtain a crude extract of permetin A. The crude extract was dissolved in ultrapure water and diluted to a solution of 200 mg / mL for high performance liquid chromatography separation and purification.
[0034] 2) High performance liquid separation and purification of Permetin A
[0035] The above 200 mg / mL solution was filtered through a 0.22 μm filter membrane and then separated and purified by preparative reverse phase high performance liquid chromatography. The chromatographic column was a C18 (10×250 mm) reverse phase column, and the instrument parameters were set as follows: detection wavelength 220 nm; column temperature 25 °C; flow rate 5 mL / min; mobile phase A: ultrapure water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile + 0.1% trifluoroacetic acid. The elution conditions of the purification mobile phase are shown in Table 1. Figure 3 The chromatographic peak corresponding to lipopeptide Permetin A is numbered 51. The fractions corresponding to the chromatographic peak were collected and freeze-dried to obtain a single product of lipopeptide Permetin A. The purity was detected by high performance liquid chromatography, and the results were as Figure 4 . Each sample injection was 500 μL, and the instrument automatically collected the fractions into glass test tubes according to the set fraction collection trigger conditions. The collected fractions were evaporated to remove acetonitrile in a rotary evaporator and then freeze-dried to obtain a pure product of lipopeptide Permetin A. The pure product was dissolved in ultrapure water for high performance liquid chromatography purity detection. The molecular formula of the lipopeptide Permetin A compound obtained by mass spectrometry detection is C 54 H 92 O 12 N 12 The structural formula is It was found by high performance liquid chromatography detection that the purity of the single product of lipopeptide Permetin A prepared by high performance liquid chromatography was 98.31%.
[0036] Table 1 Elution gradient for purification of lipopeptide Permetin A
[0037]
[0038]
[0039] 3) Minimum inhibitory concentration
[0040] Refer to the National Committee for Clinical Laboratory Standards, and serially dilute the concentration of lipopeptide Permetin A with a mixed solution of 0.01% acetic acid and 0.2% BSA to 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / mL. Add 10 μL of the diluted lipopeptide Permetin A to each well of a 96-well plate, and add 100 μL of the indicator bacteria solution with a concentration of 2 - 7×105 CFU / mL resuspended in MH to each well. Incubate with shaking for at least 18 h, and read the OD value at 600 nm. The MIC is the minimum concentration of the immunomodulatory peptide that inhibits the growth rate by more than 50%. All experiments are repeated three times. As shown in Table 2, lipopeptide Permetin A has good antibacterial activity against both Gram-positive and Gram-negative bacteria, especially Staphylococcus aureus CMCC26003, Clostridium perfringens ATCC13124, Salmonella CVCC534, Escherichia coli CVCC1515, Escherichia coli CVCC1518, Escherichia coli CVCC1557, Escherichia coli CVCC1569, Escherichia coli ATCC25922, Salmonella ATCC13076, Pseudomonas aeruginosa ATCC27853, Shigella dysenteriae CMCC(B)51252, Shigella flexneri ATCC29903, and Shigella sonnei CICC21679. The MIC values of lipopeptide Permetin A against these bacteria are all not higher than 8 μg / ml.
[0041] Table 2 Minimum Inhibitory Concentrations of Lipopeptide Permetin A against Various Indicator Bacteria
[0042]
[0043]
Claims
1. A Paenibacillus elgii with the preservation number of CGMCC No. 19830 was preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on May 15, 2020.
2. Use of the Paenibacillus elgii according to claim 1 in the preparation of lipopeptides.
3. Use of the Paenibacillus elgii according to claim 1 in bacteriostatic agents and fungicides.
4. A method for preparing lipopeptides using the above-mentioned Paenibacillus elgii.
5. The method for preparing lipopeptides according to claim 4, comprising the following steps: (1) Fermentation: Fermentation is carried out using the above-mentioned Paenibacillus elgii. The fermentation medium formula is soybean meal powder 10 ± 5 g / L, yeast powder 5 ± 2 g / L, peptone 10 ± 5 g / L, glucose 2 ± 1 g / L, sodium chloride 5 ± 2 g / L; (2) Preparation of permetin A crude extract: The fermentation broth of Paenibacillus elgii Z121 is centrifuged at 8000 g for 30 min at 4°C, and the obtained supernatant is freeze-dried to obtain a freeze-dried powder; Immunomodulatory peptides are extracted according to the ratio of adding 5 mL of acetonitrile to 1 g of the supernatant freeze-dried powder, and the acetonitrile after extraction is removed in a rotary evaporator to obtain the permetin A crude extract; (3) Separation and purification of Permetin A: The permetin A crude extract is dissolved in pure water and diluted into a solution of 200 mg / mL. The above 200 mg / mL solution is filtered through a 0.22 μm filter membrane; Separation and purification are carried out by preparative reverse-phase high-performance liquid chromatography. The chromatographic column is a C18 (10X250 mm) reverse-phase column, and the instrument parameters are set as follows: detection wavelength 220 nm; column temperature 25°C; flow rate 5 mL / min; mobile phase A: ultrapure water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile + 0.1% trifluoroacetic acid; Each time 500 μL of the sample is loaded, the fractions corresponding to the chromatographic peaks are collected, and the acetonitrile is removed by a rotary evaporator and then freeze-dried to obtain the pure lipopeptide Permetin A.