Method for efficiently separating and purifying Borreelidin from streptomyces rochei PPcase activated bacteria

Abstract

The invention discloses a method for efficiently separating and purifying Borreelidin from streptomyces rochei PPcase activated bacteria. In particular, the invention relates to the efficient separation of the Borreelidin from the fermentation broth of a strain of Streptomyces rochei TRM46813 / pWHU2449, wherein the Streptomyces rochei TRM46813 / pWHU2449 is activated by the strain of Streptomyces rochei. The invention further provides a high-recovery-rate separation and purification method of the Borridin, which comprises the following steps: after large-scale fermentation of a strain, firstly, carrying out extraction, rotary evaporation and concentration on fermentation liquor by using ethyl acetate, redissolving an ethyl acetate extract by using methanol, and purifying by using gel column chromatography and prepared liquid phase to finally obtain a target compound. The invention provides an efficient purification methodology for separation of actinomycetes metabolites, and the recovery rate is improved and can reach 10mg / L culture medium.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification of actinomycete natural products, and relates to a method for efficiently separating and purifying Borrelidin from Streptomycete rochei PPtase-activated bacteria; specifically, it relates to a method for efficiently separating Borrelidin from a fermentation broth of a strain of Streptomycete rochei PPtase-activated bacteria, namely, Streptomycete rochei TRM46813 / pWHU2449. Background Art

[0002] Borrelidin is a nitrogen-containing macrolide antibiotic isolated from the fermentation broth of a PPtase-activated strain of Streptomyces rocheri from the arid region of Xinjiang. It is an inhibitor of bacterial (Gram-positive bacteria) and eukaryotic threonyl-tRNA synthetase, and can also lead to the accumulation of cell wall precursors of clinically resistant bacteria. The literature also reported that the screening of various aaRS inhibitors for anti-chain Plasmodium found that Borrelidin has an outstanding antimalarial effect. The results of the study showed that Borrelidin has broad-spectrum antibiotic properties against clinically resistant bacteria and plant pathogenic fungi, especially against Verticillium dahliae, which causes cotton wilt disease, and has the potential to be developed into an antibacterial pesticide.

[0003] At present, the recovery rate of Borrelidin separation using traditional silica gel column chromatography under laboratory conditions is low, basically maintained at about 1 mg / L. It not only uses a large amount of organic solvent for elution, but also takes a long time. If the yield of the target compound of the strain fermentation is not high, it is difficult to obtain high-quality Borrelidin using traditional separation and purification methods. For example, Li Zhou and Zhao Shengyin optimized the fermentation conditions of the 18-membered macrolide antibiotic Borrelidin produced by solid fermentation of Streptomyces rocheri ATCC10739 in "Optimization of Fermentation Conditions and Separation and Purification of Borrelidin Antibiotics by Streptomyces rocheri ATCC10739"; the fermentation product was purified by separation techniques such as organic solvent extraction, silica gel chromatography and semi-preparative high-performance liquid chromatography (HPLC) to obtain Borrelidin; it also has the problem of low yield, which is only about 1 mg / L culture medium. At the same time, the separation and purification technology used is 2 times of silica gel chromatography, which is time-consuming, has strong adsorption, and has large sample loss and poor purification effect.

[0004] Therefore, it is necessary to develop an effective method to separate Borrelidin from the fermentation broth of Streptomyces rocheri PPtase-activated bacteria to reduce losses and experimental cycles. Summary of the invention

[0005] The purpose of the present invention is to provide a method for efficiently separating and purifying Borrelidin from Streptomyces rocheri PPtase-activated bacteria in order to solve the problems of long separation period, high cost and low recovery rate of Borrelidin in the prior art.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] <First aspect>

[0008] The invention relates to a method for separating and purifying Borrelidin from a fermentation liquid of Streptomyces rocherii PPtase activated bacteria.

[0009] As an embodiment of the present invention, the Streptomycete rochei PPtase activating bacteria is Streptomycete rochei TRM46813 / pWHU2449, and its deposit number is CCTCC NO: M2025265.

[0010] As an embodiment of the present invention, the method comprises the following steps:

[0011] S1, subjecting the Streptomyces rocherii to liquid fermentation to obtain a fermentation product;

[0012] S2, extracting the fermentation product (bacteria and fermentation liquid) obtained in step S1 with ethyl acetate, combining and recovering the solvent by rotary evaporation under reduced pressure to obtain a crude extract; resuspending the crude extract in methanol to obtain a methanol-soluble substance, and recovering the solvent by rotary evaporation under reduced pressure to obtain a methanol-soluble portion;

[0013] S3, purifying the methanol soluble portion obtained in step S2 by gel column chromatography to obtain various fractions;

[0014] S4. Collect the fractions obtained in step S3 using a preparative liquid chromatograph to finally obtain the target compound.

[0015] In some embodiments, gel column chromatography (Sephadex LH-20) is used.

[0016] In some implementation examples, a liquid chromatograph (1260 Infinity, Agilent) is used.

[0017] As an embodiment of the present invention, step S1 comprises: taking 80-100 μL of bacterial solution stored at -80°C (refrigerator) and applying it to solid culture medium, culturing at 30°C for 5-7 days; scraping about 1-2 cm 2The bacteria were inoculated into 50 mL of seed culture medium, cultured at 30°C, 200 r.pm for 48 h, and then 5 mL of seed solution was added to 500 mL of fermentation medium and cultured at 30°C, 200 r.pm for 7 days.

[0018] As an embodiment of the present invention, the solid culture medium for growing the strain is formulated as follows: soluble starch 2% (w / v), KNO 3 0.1% (w / v), FeSO 4 0.001%(w / v),K 2 HPO 4 0.05%(w / v),MgSO 4 7H2O 0.05% (w / v), agar 1.8% (w / v), water 1L, PH=7.2-7.4, sterilize at 121℃ for 20min.

[0019] As one embodiment of the present invention, the fermentation seed culture medium formula is: tryptone 1.5% (w / v), soy peptone 0.5% (w / v), sodium chloride 0.5% (w / v), water 1L, pH = 7.2-7.4, sterilization at 121°C for 20min.

[0020] As an embodiment of the present invention, the fermentation medium formula is: sucrose 10% (w / v), glucose 1.0% (w / v), yeast extract 0.5% (w / v), MgCl 2 6H 2 O1.0%(w / v), K 2 SO 4 0.025% (w / v), casamino acid 0.01% (w / v), 3-morpholinepropanesulfonic acid 2.1% (w / v), NaOH 0.2% (w / v), agar 1.8% (w / v), water 1L, PH=7.2-7.4, sterilize at 121°C for 20min.

[0021] As an embodiment of the present invention, in step S2, an equal volume of ethyl acetate is used for extraction (twice), centrifuged at 5000 rpm for 10 min, and the supernatant organic phase is concentrated by rotary evaporation under reduced pressure to obtain a crude extract extracted with ethyl acetate.

[0022] As an embodiment of the present invention, in step S3, the amount of sample loaded on the gel column is 500 mg to 1000 mg of methanol-soluble matter, and the sample needs to be centrifuged at 12000 rpm for 10 min before loading.

[0023] In some implementation examples, in step S3, the amount of sample loaded on the gel column is 1000 mg soluble in methanol, and the sample needs to be centrifuged at 12000 rpm for 10 min before loading.

[0024] In some implementation examples, in step S3, the gel column filler is Sephadex LH-20, the glass chromatography column model: inner and outer diameters are 24 mm / 29 mm, the effective column length of the gel filler is 1.2 m to 1.5 m, the elution solvent is methanol, and the gel column fraction is collected at 5 to 7 mL / tube, and 80 to 120 tubes are collected.

[0025] As an embodiment of the present invention, in step S3, after the fractions are collected, liquid chromatography is required to track and analyze the target compound. The sample is detected by liquid chromatography, the detection wavelength is 193nm, the mobile phase is acetonitrile (chromatographic grade), water (deionized double distilled water). In some implementation examples, the sample is detected by LC / MS, and the chromatographic column is XDB-C18 (4.6×150mm, 5μm, Agilent). The detection wavelength is 193nm, and the mobile phase is water (H 2 O), B is acetonitrile (CH 3 CN), the mobile phase parameters are: A=H 2 O(0.1%v / v formic acid), B=CH 3 CN (0.1% v / v formic acid). Gradient elution time parameters: t = 0-1min, 5% solvent B; t = 1-2min, 5-30% solvent B; t = 2-20min, 30-95% solvent B; t = 20-25min, 95% solvent B; t = 25-26min, 95-5% solvent B; t = 26-30min, 5% solvent B. Flow rate: 0.4mL / min, 25℃. Sample volume: 20μL.

[0026] As an embodiment of the present invention, in step S4, the target compound is collected in the preparation liquid phase according to the retention time of ultraviolet absorption at a wavelength of 193nm, the flow rate is 2mL / min, the collection time period is 16min~18.5min, one tube is collected every 30s, the same components are combined, and the solvent is recovered by reduced pressure rotary evaporation / vacuum recovery to obtain the target compound Borrelidin.

[0027] In some implementation examples, the preparation liquid phase parameters are: the chromatographic column is C18 (10×150 mm, 5 μm, The detection wavelength was 193 nm, and the mobile phase was water (H 2 O), B is acetonitrile (CH 3 CN). The mobile phase parameters are: A = H 2 O(0.1%v / v formic acid), B=CH 3CN (0.1% v / v formic acid). Gradient elution time parameters: t = 0-2min, 30% solvent B; t = 2-35min, 30-60% solvent B; t = 35-37min, 95% solvent B; t = 37-39min, 95-30% solvent B; t = 39-40min, 30% solvent B. Flow rate: 2mL / min, 25℃, sample volume 100μL.

[0028] As an embodiment of the present invention, based on the molecular weight of the target compound, m / z 488.3017 [MH] - , to track the target compound.

[0029] <Second Aspect>

[0030] The invention also provides a Streptomycete rochei PPtase activated bacterium Streptomycete rocheiTRM46813 / pWHU2449, whose deposit number is CCTCC NO:M2025265.

[0031] <Third Aspect>

[0032] The Streptomycete rochei PPtase activated bacteria Streptomycete rochei TRM46813 / pWHU2449 of the present invention has a deposit number of CCTCC NO: M2025265, and its application in the preparation of nitrogen-containing macrolide antibiotic Borrelidin also belongs to the protection scope of the present invention.

[0033] The Streptomycete rochei PPtase activated bacteria (Streptomycete rochei TRM46813 / pWHU2449) of the present invention has a deposit number of CCTCC NO: M2025265, a deposit date of February 20, 2025, a deposit unit of China Center for Type Culture Collection, and a deposit address of Wuhan University, Wuhan, China.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The original strain (Streptomycete rochei TRM46813) used in the present invention was isolated from the Taklimakan Desert in Xinjiang, China, and after activation with PPtase, the PPtase-activated Streptomycete rochei strain (Streptomyceterochei TRM46813 / pWHU2449) was obtained, thus realizing the initiative of independent research and development.

[0036] (2) The present invention obtains a PPtase activated strain by adopting the technical feature of specific gene activation, with a higher yield of about 10 mg / L culture medium, which is increased by about 10 times.

[0037] (3) The separation and purification steps of Borrelidin in the present invention are simpler and more efficient, and only three steps are required: first, ethyl acetate extraction and concentration; second, Sephadex LH-20 gel chromatography to obtain components; and third, liquid phase preparation to efficiently obtain the target product. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments with reference to the following figures / tables:

[0039] Figure 1 This is a schematic diagram of the efficient separation and purification process of Borrelidin;

[0040] Figure 2 is the HRMS mass spectrum of Borrelidin;

[0041] Figure 3 is the HPLC chromatogram of Borrelidin;

[0042] Figure 4 is the chemical structure of Borrelidin;

[0043] Figure 5 is the infrared spectrum of Borrelidin;

[0044] Figure 6 The figure shows the UV and mass spectrometry comparison of the ethyl acetate extraction products of wild bacteria and activated bacteria fermentation broth; (a) is the UV comparison, and (b) is the mass spectrometry comparison; the red line is the result of activated bacteria, and the black line is the result of wild bacteria.

[0045] Figure 7 The process of isolating and purifying 142 mg of Borrelidin from 14 L of fermentation broth. DETAILED DESCRIPTION

[0046] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0047] Example

[0048] This example relates to the efficient separation of Borrelidin (Borrelidin) from the fermentation broth of a PPtase-activated strain of Streptomycete rochei TRM46813 / pWHU2449. Figure 4 Chemical structure) methods, such as Figure 1 , the specific steps are as follows:

[0049] S1. Perform liquid fermentation of Streptomyces to obtain fermentation products: take 100 μL of bacterial solution stored in a -80℃ refrigerator with a pipette and apply it to the solid culture medium, and activate and culture at 30℃ for 7 days; scrape about 1-2 cm with a sterile bamboo stick 2 The bacteria were inoculated into 50 mL of seed culture medium and cultured in a constant temperature shaker at 30°C, 200 r.pm for 2 days; 5 mL of seed solution was added to 500 mL of fermentation medium and cultured in a constant temperature shaker at 30°C, 200 r.pm for 7 days.

[0050] The solid medium formula for strain growth is: soluble starch 2% (w / v), KNO 3 0.1% (w / v), FeSO 4 0.001%(w / v),K 2 HPO 4 0.05%(w / v),MgSO 4 7H2O 0.05% (w / v), agar 1.8% (w / v), water 1L, PH=7.2-7.4, sterilize at 121℃ for 20min.

[0051] Formula of fermentation seed culture medium: tryptone 1.5% (w / v), soy peptone 0.5% (w / v), sodium chloride 0.5% (w / v), water 1L, PH=7.2-7.4, sterilization at 121°C for 20min.

[0052] Fermentation medium formula: sucrose 10% (w / v), glucose 1.0% (w / v), yeast extract 0.5% (w / v), MgCl 2 6H 2 O1.0%(w / v), K 2 SO 4 0.025% (w / v), casamino acid 0.01% (w / v), 3-morpholinepropanesulfonic acid 2.1% (w / v), NaOH 0.2% (w / v), agar 1.8% (w / v), water 1L, PH=7.2-7.4, sterilize at 121°C for 20min.

[0053] S2, collect the fermentation broth, extract it twice with one volume of ethyl acetate, centrifuge it at 5000r.pm for 10min, take the supernatant organic phase and concentrate it by rotary evaporation under reduced pressure to obtain a crude extract extracted with ethyl acetate, then re-dissolve the crude extract with methanol, centrifuge it at 12000r.pm for 5min, take the supernatant which is methanol-soluble, concentrate it by rotary evaporation under reduced pressure to obtain a methanol-soluble

[0054] S3, gel purification, the sample loading amount on the gel column is 1000 mg of methanol-soluble material, and the sample needs to be centrifuged at 12000 rpm for 10 min before loading.

[0055] The gel column filler is Sephadex LH-20, the glass chromatography column model: the inner and outer diameters are 24 mm / 29 mm, the effective column length of the gel filler is 1.2 m to 1.5 m, the elution solvent is methanol, the gel column fraction is collected in 5 mL / tube, and 120 tubes are collected.

[0056] After the fractions were collected, liquid chromatography-mass spectrometry (LC / MS) was used to track and analyze the target compounds. Samples were detected using LC / MS, with a chromatographic column of XDB-C18 (4.6×150 mm, 5 μm, Agilent), a detection wavelength of 193 nm, and a mobile phase of water (H 2 O), B is acetonitrile (CH 3 CN), the mobile phase parameters are: A=H 2 O(0.1%v / v formic acid), B=CH 3 CN (0.1% v / v formic acid); gradient elution time parameters: t = 0-1min, 5% solvent B; t = 1-2min, 5-30% solvent B; t = 2-20min, 30-95% solvent B; t = 20-25min, 95% solvent B; t = 25-26min, 95-5% solvent B; t = 26-30min, 5% solvent B; flow rate: 0.4mL / min, 25℃. Sample volume: 20μL.

[0057] S4, the target compound Borreidin was collected and purified by preparative liquid phase, and fractions were collected according to the retention time of ultraviolet absorption at a wavelength of 193nm, flow rate: 2mL / min, collection time period: 16min~18.5min, one tube every 30s, and the same components were combined, and the solvent was recovered by reduced pressure rotary evaporation / vacuum recovery to obtain the target compound Borrelidin.

[0058] Preparation liquid phase parameters: Chromatographic column is C18 (10×150mm, 5μm, Waters, detection wavelength 193nm, mobile phase, A is water (H2 O), B is acetonitrile (CH 3 CN), the mobile phase parameters are: A=H 2 O(0.1%v / v formic acid), B=CH 3 CN (0.1% v / v formic acid); gradient elution time parameters: t=0-2min, 30% solvent B; t=2-35min, 30-60% solvent B; t=35-37min, 95% solvent B; t=37-39min, 95-30% solvent B; t=39-40min, 30% solvent B; flow rate: 2mL / min, 25℃, sample volume 100μL.

[0059] S5. Collect and merge the components. The molecular weight of the target compound is m / z 488.3017 [MH] - ,m / z 977.6169[2M-H] - To track the target compound ( Figure 2 ). The molecular signals contained in each fraction, those with matching molecular weights are the target compound fractions, the same components are combined, and the solvent is recovered by vacuum rotary evaporation / vacuum recovery to obtain the target compound Borrelidin.

[0060] Table 1 Optical rotation values ​​of Borrelidin

[0061] Parallel detection Optical rotation(α) 1 -0.119 2 -0.12 3 -0.12 4 -0.127 5 -0.122 6 -0.114 7 -0.129 8 -0.131 9 -0.126 10 -0.124 Average value -0.123±0.005

[0062] Figure 2 is the HRMS mass spectrum of Borrelidin, Figure 2 It can be seen that the obtained component is Borrelidin. Figure 3 is the HPLC chromatogram of Borrelidin, Figure 5 is the infrared spectrum of Borrelidin, Figure 3 , Figure 5 It can be seen that the purity of the obtained components is relatively high. It has been verified by experiments that 142 mg of pure Borrelidin can be obtained from 14 L of fermentation broth, with an average of 10 mg / L. Figure 7 .

[0063] Figure 6 The UV and mass spectrometry comparison results of the ethyl acetate extraction products of wild bacteria and activated bacteria fermentation broth; activated bacteria ( Figure 6 (a) red line) has obvious UV absorption at 17.5min. Figure 6 (a) black line) has no absorption peak; activated bacteria ( Figure 6 (b) Red line) Mass spectrum shows the molecular weight of Borrelidin, wild bacteria ( Figure 6 (b) black line) No. It can be seen that the present invention activates Boreeliden in wild bacteria through PPtase enzyme, and obtains activated strains, making them more productive.

[0064] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A method for separating and purifying Borrelidin, characterized in that: Borrelidin was isolated and purified from the fermentation broth of Streptomyces rocheri PPtase activated bacteria.

2. The method for separating and purifying Borrelidin according to claim 1, characterized in that: The Streptomycete rochei PPtase activating bacteria is Streptomycete rochei TRM46813 / pWHU2449, and its deposit number is CCTCC NO: M2025265.

3. The method for separating and purifying Borrelidin according to claim 1 or 2, characterized in that: The method comprises the following steps: S1, subjecting the Streptomyces rocherii to liquid fermentation to obtain a fermentation product; S2, extracting the fermentation product obtained in step S1 with ethyl acetate, combining and recovering the solvent by rotary evaporation under reduced pressure to obtain a crude extract; resuspending the crude extract in methanol to obtain a methanol-soluble substance, and recovering the solvent by rotary evaporation under reduced pressure to obtain a methanol-soluble portion; S3, purifying the methanol soluble portion obtained in step S2 by gel column chromatography to obtain various fractions; S4. Collect the fractions obtained in step S3 using a preparative liquid chromatograph to finally obtain the target compound.

4. The method for separating and purifying Borrelidin according to claim 3, characterized in that: In step S1, the liquid fermentation comprises: S1-1, take 80-100 μL of bacterial solution stored at -80℃ and apply it to solid culture medium, and activate and culture at 30℃ for 5-7 days; S1-2, take 1 to 2 cm 2 The solid plate bacteria were inoculated into 50 mL of seed culture medium and cultured at 30°C, 200 rpm for 48 h. S1-3. Take 5 mL of seed solution and add it to 500 mL of fermentation medium. Culture at 30°C and 200 rpm for 7 days.

5. The method for separating and purifying Borrelidin according to claim 4, characterized in that: Include at least one of the following technical features: A. The solid culture medium is formulated as follows: soluble starch 2% (w / v), KNO3 0.1% (w / v), FeSO4 0.001% (w / v), K2HPO4 0.05% (w / v), MgSO4·7H2O 0.05% (w / v), agar 1.8% (w / v), water 1L, PH = 7.2-7.4, sterilized at 121°C for 20min; B. The seed culture medium formula: tryptone 1.5% (w / v), soy peptone 0.5% (w / v), sodium chloride 0.5% (w / v), water 1L, pH = 7.2-7.4, sterilized at 121°C for 20min; C. Formula of the fermentation medium: sucrose 10% (w / v), glucose 1.0% (w / v), yeast extract 0.5% (w / v), MgCl2·6H2O 1.0% (w / v), K2SO4 0.025% (w / v), casamino acid 0.01% (w / v), 3-morpholinepropanesulfonic acid 2.1% (w / v), NaOH 0.2% (w / v), agar 1.8% (w / v), water 1 L, pH = 7.2-7.4, sterilized at 121°C for 20 min.

6. The method for separating and purifying Borrelidin according to claim 3, characterized in that: In step S2, one volume of ethyl acetate is used for extraction, and the mixture is centrifuged at 5000 rpm for 10 min. The supernatant organic phase is concentrated by rotary evaporation under reduced pressure.

7. The method for separating and purifying Borrelidin according to claim 3, characterized in that: In step S4, the target compound is collected in the preparation liquid phase according to the retention time of ultraviolet absorption at a wavelength of 193nm, the flow rate is 2mL / min, the collection time period is 16min~18.5min, one tube is collected every 30s, the same components are combined, and the solvent is recovered by reduced pressure rotary evaporation / vacuum recovery to obtain the target compound Borrelidin.

8. The method for separating and purifying Borrelidin according to claim 7, characterized in that: Based on the molecular weight of the target compound, m / z 488.3035[MH] - , to track the target compound.

9. A Streptomycete rochei PPtase activated bacterium, Streptomycete rochei TRM46813 / pWHU2449, whose deposit number is CCTCC NO: M2025265.

10. Use of the Streptomycete rochei PPtase activated bacteria Streptomycete rochei TRM46813 / pWHU2449 as claimed in claim 9 in the preparation of the nitrogen-containing macrolide antibiotic Borrelidin.

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