CN111778172B

CN111778172B - Streptomyces for producing antibacterial active compound and separation method and application thereof

Info

Publication number: CN111778172B
Application number: CN202010236652.0A
Authority: CN
Inventors: 张嵩亚; 司同; 刘涛; 朱静; 陈红; 张振坤
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2022-12-13
Anticipated expiration: 2040-03-30
Also published as: CN111778172A

Abstract

The invention relates to a streptomycete for producing an antibacterial active compound, a separation method and an application thereof, wherein the streptomycete for producing the antibacterial active compound is named as a streptomycete (Streptomyces sp.) ZSYNO2 strain, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation time is 1 month and 7 days 2020, the preservation number is CCTCC M2020020, and the address is as follows: wuhan university in Wuhan City, china. The novel streptomyces can produce a compound rishirilide A, and the yield reaches considerable 12mg/L; the invention also discovers that the compound rishiride A has antibiotic activity and has the characteristic of being developed into a high-efficiency anti-infection candidate drug.

Description

Streptomyces for producing antibacterial active compound and separation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to streptomyces and a separation method and application thereof, in particular to streptomyces for producing an antibacterial active compound and a separation method and application thereof.

Background

In the face of increasingly abusive bacterial resistance problems, people will face the situation of no medicine available in the near future, and therefore, the development of novel antibiotics is urgently needed. The antibacterial active compound of microbial origin is one of the important sources of high-efficiency antibiotics, and the antibiotics developed by microbial metabolites are important components of anti-infective drugs.

Streptomyces is the highest actinomycetes, has well-developed branched hyphae, has no transverse septa, and is differentiated into vegetative hyphae, aerial hyphae and spore filaments. The spore silk forms conidiospore again, and the morphology and color of the spore silk and the spore are different from species to species, and are one of the main identification characters of the seed separation. There are thousands of species of Streptomyces reported, which are mainly distributed in soil. There have been some reports of isolation of antibacterial active compounds from streptomyces.

CN103965275A discloses a separation and extraction method of streptomyces fermentation metabolite neooxytocin, which comprises the following steps: firstly heating the fermentation liquor to 60 ℃, killing streptomycete living bacteria in the fermentation liquor, removing solid matters and bacteria impurities in a culture medium in neoaomycin fermentation liquor through a filter cloth or a ceramic microfiltration membrane, then carrying out adsorption treatment on the fermentation liquor from which the solid matters are removed through macroporous adsorption resin, then adsorbing and enriching neoaomycin in macroporous adsorption resin effluent liquid by using cation exchange resin, and finally adopting 0.5-5.0% ammonia water for resolution and concentration to prepare neoaomycin mother liquor, thereby being a novel method for preparing a novel biological pesticide neoaomycin mother medicine or preparation with low cost.

CN105010399A discloses a separation and purification method of antifungal active substances in Streptomyces lydicus fermentation liquor, comprising the following steps: (1) Centrifuging fermentation liquor of streptomyces lydicus S.lydicus E9 with the preservation number of CGMCC No.3075, adding a flocculating agent into supernatant, and performing suction filtration to obtain filtrate; (2) Passing the filtrate through macroporous resin chromatographic column, washing with methanol water solution, and eluting with ethanol water solution to obtain active component; (3) combining the active eluents and concentrating to obtain a concentrated solution; (4) Subjecting the concentrated solution to Sephadex chromatography column chromatography, eluting with ethanol water solution, and collecting antifungal active components.

However, there are limited reports on the isolation of antibacterial active compounds from Streptomyces and limited types of active compounds isolated, and it is of great interest to develop a novel Streptomyces that produces novel antibacterial active compounds.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a streptomycete and a separation method and application thereof, in particular to a streptomycete for producing an antibacterial active compound and a separation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

on one hand, the invention provides Streptomyces for producing an antibacterial active compound, which is named as Streptomyces sp ZSYNO2 strain, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation time is 1 month and 7 days 2020, the preservation number is CCTCC M2020020, and the address is as follows: wuhan university in Wuhan City, china.

The invention discovers a novel streptomycete for the first time, which can produce a compound rishirilide A, and the yield of the streptomycete can reach considerable 12mg/L; the invention also discovers for the first time that the compound rishiride A has antibiotic activity and has the characteristic of being developed into a high-efficiency anti-infection candidate drug.

Preferably, the streptomyces is isolated from soil.

The Streptomyces sp ZSYNO2 strain is separated from natural soil of An Shitian south town Chen Cunda city, yichun, jiangxi.

Preferably, the culture medium used for separation is an M2 solid culture medium, and the formulation thereof is: each liter of culture medium contains 4.0g of glucose, 4.0g of yeast extract, 10.0g of malt extract, 15.0g of agar, 2g of calcium carbonate and 1000mL of distilled water, and the pH value is 7.2.

Preferably, the culture temperature of the separation is 25-30 ℃, such as 25 ℃, 26 ℃, 27 ℃,28 ℃, 29 ℃ or 30 ℃ and the like, the time is 7-13 days, such as 7 days, 8 days, 10 days, 12 days or 13 days and the like, and other points in the range can be selected, which is not described in detail herein.

The Streptomyces sp ZYNO 2 strain is a gram-positive strain, aerobic actinomycetes, and soluble pigment is generated when the strain grows on a TSB solid culture medium. The methods in the references of genomic DNA extraction, PCR amplification of 16S rDNA, sequence alignment and phylogenetic tree construction and preliminary identification of physiology, chemistry and morphology (Pan H. -Q., zhang D. -F., li L., jiang Z., cheng J., zhang Y. -G., wang H. -F., hu J. -C., li W. -J., nocardia oceani sp.n.and Nocardia nanoensis sp.n., two novel enzyme isolated from culture segment of South China Sea. Int.J.Syst. Evol.Microbiol.3365-3391) involved in the identification test of Streptomyces are described in the following references (Pan H. -Q., zhang D. -F., zhang D., li., li.L., J..

In a second aspect, the present invention provides the use of a streptomyces as described above in the preparation of the compound rishiride a.

We find for the first time that the strain is a rare production strain capable of producing the compound risilide A, and that the strain has a good yield, thereby being beneficial to reducing the production cost of the compound and being beneficial to the later development and utilization of the active compound, and providing a new strategy for the preparation process of the compound risilide A.

In a third aspect, the invention provides a preparation method of a compound rishirilide A, wherein the compound is prepared and separated from a fermentation culture of the Streptomyces sp.

Preferably, the specific flow of the preparation method is shown in fig. 1, and specifically comprises the following steps:

(1) Preparing a fermentation culture of streptomyces ZSYNO 2;

(2) Separating the fermentation broth and the mycelium of the fermentation culture obtained in the step (1), and extracting and concentrating respectively to obtain an extract, namely a crude extract of the fermentation culture;

(3) Carrying out solid phase extraction on the crude extract obtained in the step (2), carrying out methanol/water gradient elution, and collecting elution fractions;

(4) Performing silica gel column chromatography on the elution fraction obtained in the step (3), performing gradient elution by using ethyl acetate/methanol, and collecting the elution fraction;

(5) And (3) carrying out liquid chromatography on the elution fraction obtained in the step (4), and carrying out acetonitrile/water gradient elution to obtain the compound rishiride A.

Preferably, the preparation method of the fermentation culture in the step (1) specifically comprises the following steps:

(a) Inoculating activated streptomyces ZSYNO2 into a seed culture medium, and culturing to obtain a seed solution;

(b) Inoculating the seed liquid into a fermentation culture medium, and culturing to obtain a fermentation culture;

preferably, the culturing in step (a) is at a temperature of 25-30 ℃, such as 25 ℃, 26 ℃, 27 ℃,28 ℃, 29 ℃ or 30 ℃ and the like, for a time of 70-75h, such as 70h, 71h, 72h, 73h, 74h or 75h and the like, and at an oscillation speed of 150-250rpm, such as 150rpm, 180rpm, 200rpm, 220rpm or 250rpm and the like. The specific point values within the above ranges can be selected, and are not described in detail herein.

Preferably, the inoculation amount of the seed liquid in the step (b) is 5-15%, specifically, the volume ratio of the seed liquid in the fermentation medium is 5-15%, for example, 5%, 8%, 9%, 10%, 12%, 14%, or 15%, and the specific values within the range can be selected, and are not repeated herein.

Preferably, the temperature of the culturing in step (b) is 25-30 ℃, such as 25 ℃, 26 ℃, 27 ℃,28 ℃, 29 ℃ or 30 ℃ and the like, for 6-8 days, such as 6 days, 7 days or 8 days and the like, and the shaking speed is 150-250rpm, such as 150rpm, 180rpm, 200rpm, 220rpm or 250rpm and the like. The specific point values within the above ranges can be selected, and are not described in detail herein.

The formula of the seed culture medium and the fermentation culture comprises the following components: calculated by 100 percent of mass fraction, comprises 20 percent of glucose, 10 percent of soybean peptone, 2.34 percent of valine and CaCO ₃ 2％，CoCl ₂ Solution (1 mg/mL) is 1mL, pH is 7.2, the components and contents are mixed uniformly, and then sterilized for 30min at 121 ℃.

Preferably, the fermentation liquor of step (2) is extracted with ethyl acetate;

preferably, the mycelium of step (2) is extracted with acetone to obtain an extract, and then the extract is extracted with ethyl acetate.

The specific operation of step (2) may exemplarily be: separating fermentation liquor and mycelium of the fermentation culture, extracting the fermentation liquor by using ethyl acetate with the volume 2 times of that of the fermentation liquor, combining extract liquor of the ethyl acetate and concentrating to obtain extract A; extracting the mycelium part of the fermentation product with acetone with the same volume, combining the extracting solutions, recovering, extracting with ethyl acetate with the same volume, concentrating the ethyl acetate extract to obtain an extract B, and combining the extracts A and B to obtain the complete crude extract of the fermentation culture of Streptomyces sp.ZSYNO 2.

Preferably, the elution fraction in step (3) is the fraction eluted when the methanol volume ratio is 60%.

Preferably, the elution fraction in the step (4) is the fraction eluted when the volume of the ethyl acetate accounts for 100%.

The specific operations of steps (3) - (5) may exemplarily be: the crude extract was dissolved in methanol, passed through a solid phase extraction column, eluted with a gradient concentration of methanol aqueous solution (30%, 60%,80%, 100%), the eluate was collected, and concentrated by rotary evaporation to obtain the corresponding fractions (A1, A2, A3, A4), wherein 60% of the eluted fractions were again subjected to silica gel column chromatography, an ethyl acetate/methanol elution system (1, 0, 1). Subjecting the obtained fraction B1 to preparative liquid chromatography, and performing gradient elution with acetonitrile/water system (30% -90% acetonitrile, 20min,2mL/min flow rate), and keeping for 15min to obtain the compound rishirilide A.

As a preferred technical scheme of the present invention, the preparation method of the compound rishiride a specifically comprises the following steps:

(1) Inoculating activated streptomyces ZSYNO2 into a seed culture medium, culturing at 25-30 ℃ and 150-250rpm for 70-75h to prepare a seed solution, inoculating the seed solution into a fermentation culture medium at the inoculation amount of 5-15% by volume, performing shake culture at 25-30 ℃ and 150-250rpm for 6-8 days to prepare a fermentation culture of streptomyces ZSYN 02;

(2) Separating the fermentation broth and the mycelium of the fermentation culture obtained in the step (1), extracting the fermentation broth with ethyl acetate, combining the extracts of the ethyl acetate, and concentrating to obtain an extract A; extracting mycelia of the fermentation product with acetone, mixing the extracting solutions, recovering, extracting with ethyl acetate, concentrating the ethyl acetate extract to obtain an extract B, and mixing the extracts A and B to obtain a complete crude extract of the fermentation culture of the streptomyces ZSYN 02;

(3) Dissolving the crude extract obtained in the step (2) with methanol, passing through a solid phase extraction column, eluting with methanol with gradient concentration (30%, 60%,80%, 100%), collecting eluent, and concentrating by rotary evaporation to obtain corresponding fractions (A1, A2, A3, A4), wherein 60% of the eluted fractions are subjected to silica gel column chromatography again, an ethyl acetate/methanol elution system (1, 0, 1); subjecting the obtained fraction B1 to preparative liquid chromatography, and performing gradient elution with an acetonitrile/water system (30% -90% acetonitrile, 20min,2mL/min flow rate) for 15min to obtain a monomer compound, namely rishirilide A.

In a fourth aspect, the present invention provides a compound rishirilide a prepared by the above preparation method.

In a fifth aspect, the present invention provides the use of the compound rishiride a for the preparation of an antibacterial medicament.

Preferably, the antibacterial agent is an anti-gram-positive bacterial agent.

The present inventors have found for the first time that the compound rishiride A has excellent antibacterial activity against various gram-positive bacteria including Enterococcus faecalis (Enterococcus faecalis ATCC 29212), staphylococcus aureus (Staphylococcus aureus ATCC 29213, staphylococcus aureus ATCC 25923, staphylococcus aureus ATCC 100910), methicillin-resistant Staphylococcus aureus (Methiocillin-resistant Staphylococcus aureus ATCC 43300), staphylococcus epidermidis (Staphylococcus epidermidis ATCC 35984), bacillus cereus (Bacillus ATCC 14579), enterococcus faecalis (Enterococcus faecalis ATCC 19433), listeria monocytogenes (Listeria monocytogenes ATCC 19115), micrococcus flavus (Micrococcus avus) and the like. The MIC of the recombinant human anti-Methicillin protein has the MIC as low as 16ug/mL (Methicillin-resistant Staphylococcus aureus ATCC 43300) and has stronger bacteriostatic activity than that of the antibiotic ampicillin (ampicillin) used by a positive control. Meanwhile, the compound rishiride A has no inhibition effect on Escherichia coli, pseudomonas aeruginosa and the like of gram-negative bacteria. Therefore, the compound rishiride A has good activity of selectively inhibiting gram-positive bacteria, and is expected to become a new antibacterial drug.

Compared with the prior art, the invention has the following beneficial effects:

the invention separates a novel streptomycete for the first time, which can produce a compound rishirilide A, and the yield of the compound rishirilide A reaches considerable 12mg/L; and the compound rishiride A has obvious antibiotic activity and has the characteristic of being developed into a candidate drug of a high-efficiency anti-infective compound.

Drawings

FIG. 1 is a schematic flow diagram of a process for the preparation of the compound rishirilide A according to the present invention;

FIG. 2 is a colony morphology of the isolated Streptomyces of example 1; the Streptomyces is named as Streptomyces sp ZYNO 2 strain, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation time is 2020, 1,7 days, the preservation number is CCTCC M2020020, and the address is as follows: wuhan university in Wuhan City, china;

FIG. 3 is a schematic representation of a phylogenetic tree of Streptomyces ZSYNO2 based on the adjacency of the 16s rDNA sequence for the most closely related species;

FIG. 4 is a schematic representation of compound Rishirilide A ¹ H NMR spectrum;

FIG. 5 is a graphic representation of compound Rishirilide A ¹³ A C NMR spectrum;

FIG. 6 is an HSQC spectrum of compound Rishirilide A;

FIG. 7 is an HMBC profile of compound Rishirilide A;

FIG. 8 is a ROESY spectrum of compound Rishirilide A;

fig. 9 is a high resolution mass spectrum of compound rishiride a;

FIG. 10 is a UV absorption spectrum of compound Rishirilide A;

FIG. 11 is a schematic of the structural formula and NMR signals of compound Rishirilide A.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

This example isolated a ZSYNO2 strain of Streptomyces sp. The detailed separation method is as follows:

(1) Taking soil in natural village of high An Shitian of Yichun city of Jiangxi province, namely Nanzhen great town as a separation source, sampling by an S-shaped sampling method, uniformly mixing, filling into a sterilized self-sealing bag, and refrigerating in a refrigerator at 4 ℃ for later use.

(2) Before the experiment, target soil is firstly dried in an oven at 60 ℃ for 4 hours, then 0.5g of soil is weighed by using tinfoil paper, 6 sterilized test tubes are taken, the soil is put into the test tube filled with 5mL of sterilized distilled water, the test tube is repeatedly pushed and beaten or shaken by using a gun to obtain soil suspension, then 500 mu L of soil is taken to the second test tube for repeated pushing and sucking, and gradient dilution is carried out in sequence.

(3) The soil suspension of six test tubes was smeared on an M2 solid medium plate (M2 formulation: glucose 4g, yeast extract 4g, malt extract 10g, agar 15g, calcium carbonate 2g,1L mono-distilled water) with a sterilized cotton swab, and the plate was then inverted in a constant temperature incubator and incubated at 28 ℃ in the dark for 7 days.

(4) Observing the flat plate, screening out a target bacterial colony according to the phenomena that a culture medium around the bacterial colony on the flat plate is sunken and the bacterial colony is hard, and further performing scribing and purification to obtain the streptomycete.

Example 2

This example carried out morphological characterization, physiological and biochemical analysis, and molecular biological characterization of the Streptomyces isolated in example 1.

The strain is a gram-positive strain and aerobic actinomycetes, a colony morphology diagram of the strain on a TSB solid culture medium is shown in figure 2, the strain has mud fishy smell, and the appearance morphology of the colony is as follows: from the appearance of cochain mold on the flat plate, the hyphae are firm, dry, wrinkled and threaded. The mycelium is sunk into the culture medium, the combination of the bacterial colony and the culture medium is tight, the bacterial colony is not easy to pick up, bright white spores are distributed on the surface, and soluble pigments grow on the TSB solid culture medium. According to the above colony morphology characteristics, ZSYNO2 is classified as Streptomyces.

Example 3

This example carried out extraction, PCR amplification of 16S rDNA and determination of 1428 bases, the sequence of which is shown below, for the streptomyces references isolated in example 1 (Pan h. -q., zhang d. -f., li l., jiang z., cheng j., zhang y., wang h. -f., hu j., li w.w. -j., nocardiopsis oceani sp.nov.and Nocardiopsis nanohaiensis sp.nov., two novel expression of genomic amplified from a marker of South China sea. Int.j.syst. Evol.microbal.2015, 65). The sequences were submitted to Genbank, aligned by BLAST analysis of the 16S rDNA nucleotide sequences, and analyzed for phylogenetic trees as shown in FIG. 3. This strain was identified as Streptomyces sp, designated zsyn 2:

GGCGTGCTTAACACATGCAAGTCGAACGATGAAGCCCTTCGGGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATACCACTCCTGCCTGCATGGGCGGGGGTTGAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCCGAGGCTTAACCTCGGGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATATACCGGAAAGCATTAGAGATAGTGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGTGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCGTAAGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATG。

example 4

The embodiment provides a preparation method of rishiride a, which specifically comprises the following steps:

(1) Inoculating activated streptomyces ZSYNO2 into a seed culture medium, culturing at 25-30 ℃ and 150-250rpm for 70-75h to prepare a seed solution, inoculating the seed solution into a fermentation culture medium at the inoculation amount of 5-15% by volume, performing shake culture at 25-30 ℃ and 150-250rpm for 6-8 days to prepare a fermentation culture of streptomyces ZSYN 02; the seed culture medium and the fermentation culture formula are as follows: calculated by 100 percent of mass fraction, comprises 20 percent of glucose, 10 percent of soybean peptone, 2.34 percent of valine and CaCO ₃ 2％，CoCl ₂ 1mL of solution (1 mg/mL) and pH 7.2, uniformly mixing the components according to the content, and then sterilizing at 121 ℃ for 30min;

(2) Separating the fermentation broth and the mycelium of the fermentation culture obtained in the step (1), extracting the fermentation broth with ethyl acetate of which the volume is 2 times that of the fermentation broth, combining the extracts of the ethyl acetate, and concentrating to obtain an extract A; extracting mycelia of the fermentation product with acetone with the same volume, combining extracting solutions, recovering, extracting with ethyl acetate with the same volume, concentrating an ethyl acetate extracting solution to obtain an extract B, and combining the extracts A and B to obtain a complete crude extract of the fermentation culture of the streptomyces ZSYNO 2;

(3) Dissolving the crude extract obtained in the step (2) with methanol, passing through a solid phase extraction column, eluting with methanol with gradient concentration (30%, 60%,80%, 100%), collecting eluent, and concentrating by rotary evaporation to obtain corresponding fractions (A1, A2, A3, A4), wherein the fraction A2 is subjected to silica gel column chromatography again, an ethyl acetate/methanol elution system (1, 0, 1, 0), collecting an ethyl acetate/methanol volume ratio 1:0 elution fraction to obtain fractions (B1, B2, B3, B4); subjecting the obtained fraction B1 to preparative liquid chromatography, gradient eluting with acetonitrile/water system (30% -90% acetonitrile, 20min,2mL/min flow rate), and keeping for 15min to obtain monomer compound as yellow solid powder. The calculation results show that 12mg of product can be obtained per liter of fermentation liquor, namely the yield is 12mg/L.

Example 5

This example identifies the chemical structure of the compound prepared in example 4.

(1) ¹ H-NMR spectroscopy, as shown in FIG. 4, and data as shown in Table 1, showed the following signals: 4 aromatic ring methine proton signals (δ H7.01,7.25,6.99,7.59), 3 methine proton signals (δ H2.77,5.51,1.39), two sets of methylene proton signals (δ H1.49-1.70,2.54) and 3 methyl proton signals (δ H0.89,0.87,1.22).

(2) ¹³ C-NMR spectroscopy, as shown in FIG. 5, and data as shown in Table 1, showed the following signals: 2 carbonyl carbons (δ C199.4, 177.5), 1 phenolic hydroxyl carbon (δ C156.7), 7 sp2 hybridized methine carbons (δ C120.7, 131.1, 123.8, 131.8, 139.6, 123.2, 131.9), 3 vicinal oxygen carbons (δ C82.1, 85.3, 81.8), 1 vicinal oxygen tertiary carbon (δ C64.5), and other 7 sp3 hybridized methylene or methyl carbons (δ C50.7, 32.6, 30.9, 29.9, 23.1, 22.7, 12.6).

(3) HSQC mapping was performed as shown in FIG. 6.

(4) HMBC mapping was performed as shown in fig. 7, and the data is shown in table 1.

(5) ROESY mapping was performed as shown in fig. 8, and the data are shown in table 1.

TABLE 1

In the context of table 1, the following, ¹ the H-NMR data were measured at 500MHz, ¹³ C-NMR data are measured at 125MHz, and a sample is dissolved in MeOD for detection; coupling constants (Hz) are shown in parentheses.

(6) High resolution Mass Spectrometry, m/z C ₂₁ H ₂₄ O ₇ (found value [ M-H)]387.1453, calculated 387.1449), unsaturation 10, pattern as shown in fig. 9.

(7) Ultraviolet absorption Spectroscopy, lambda _max (log ε): 208 (1.7), 222 (1.3) and 325 (1.6) nm, as shown in FIG. 10, and the ultraviolet absorption spectrum UV has the maximum absorption wavelength at 208nm and 325nm, respectively, which indicates that the compound has functional groups such as aromatic ring.

From the above data results, it can be seen that: the carbon spectrum hydrogen spectrum data of the compound rishirilide A is substantially consistent with that reported in the literature, and the chemical structure of the compound can be substantially determined to be rishirilide A as shown in FIG. 11, reference (Iwaki, H., nakayama, Y., takahashi, M., uetsuki, S., kido, M., & Fukuyama, Y.Structure of rishirilides A and B, α 2-macrologlobulin inhibitor product by y Streptomyces, rishiriliensis OFR-1056.The Journal of antibiotics,1984,37 (9), 1091-1093).

Example 6

This example identifies the antibacterial activity of the compound prepared in example 4.

The compounds rishiride A were tested against Enterococcus faecalis (Enterococcus faecalis ATCC 29212), pseudomonas aeruginosa ATCC 27853, staphylococcus aureus (Staphylococcus aureus ATCC 29213, staphylococcus aureus ATCC 25923, staphylococcus aureus ATCC 100910), methicillin-resistant Staphylococcus aureus (Methylococcus-resistant Staphylococcus aureus ATCC 43300), escherichia coli ATCC 25922, staphylococcus epidermidis (Staphylococcus epidermidis ATCC 35984), enterobacter Humulus (Enterobacter hominis ATCC 700323), salmonella typhimurium (Salmonella typhimurium ATCC 13311, salmonella typhimurium 7207), saccharomyces cerevisiae (Saccharomyces cerevisiae ATCC 19479), saccharomyces cerevisiae ATCC 14533, staphylococcus aureus (ATCC 14579), staphylococcus aureus (Escherichia coli ATCC 2933), staphylococcus aureus (ATCC 2935), staphylococcus aureus (Staphylococcus aureus ATCC 2932), staphylococcus aureus (ATCC 14579)Cerrevisiae CEN. PK2-1 c), nocardia terpene (Nocardia terpenica), the plant saprophytic pathogenic fungus Sarocladium kiliense, listeria monocytogenes (Listeria monocytogenes ATCC 19115), acinetobacter baumannii (Acinetobacter baumannii), minimal Inhibitory Concentration (MIC) of Micrococcus flavus, experimental methods reference reports (Engeldt, K.Degnes, K.F., kemmler, M.M., bredhardt, H.,

e.g., klinkenberg, g., sletta, h., ellingsen, t.e. and Zotchev, s.b., production of a new thiopeptide antibody, TP-1161, by a marine Nocardiopsis species.appl.environ. Microbiol, 2010,76 (15), 4969-4976). The results are shown in Table 2.

TABLE 2

As is apparent from the data in Table 2, the compound rishiride A has excellent antibacterial activity against a variety of gram-positive pathogenic bacteria including Enterococcus faecalis (Enterococcus faecalis ATCC 29212), staphylococcus aureus (Staphylococcus aureus ATCC 29213, staphylococcus aureus ATCC 25923, staphylococcus aureus ATCC 100910), methicillin-resistant Staphylococcus aureus (Methiocillin-resistant Staphylococcus aureus ATCC 43300), staphylococcus epidermidis (Staphylococcus epidermidis ATCC 35984), bacillus cereus (Bacillus cereus ATCC 14579), enterococcus faecalis (Enterococcus faecalis ATCC 19433), listeria monocytogenes (Listeria monocytogenes ATCC 19115), micrococcus xanthus (Micrococcus flavus). The combined data indicate that the compound rishiride a can be used for preparing medicaments for gram-positive bacteria.

The Applicant states that the present invention is illustrated by the examples given above, but that the invention is not limited to them, i.e.it is not intended to be limited to them, which means that the invention must be practiced. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute of Chinese academy of sciences

<120> streptomycete for producing antibacterial active compound, and separation method and application thereof

<130> 2020

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1428

<212> DNA

<213> Streptomyces ZSYNO2 strain

<400> 1

ggcgtgctta acacatgcaa gtcgaacgat gaagcccttc ggggtggatt agtggcgaac 60

gggtgagtaa cacgtgggca atctgccctt cactctggga caagccctgg aaacggggtc 120

taataccgga taccactcct gcctgcatgg gcgggggttg aaagctccgg cggtgaagga 180

tgagcccgcg gcctatcagc ttgttggtgg ggtaatggcc caccaaggcg acgacgggta 240

gccggcctga gagggcgacc ggccacactg ggactgagac acggcccaga ctcctacggg 300

aggcagcagt ggggaatatt gcacaatggg cgaaagcctg atgcagcgac gccgcgtgag 360

ggatgacggc cttcgggttg taaacctctt tcagcaggga agaagcgaaa gtgacggtac 420

ctgcagaaga agcgccggct aactacgtgc cagcagccgc ggtaatacgt agggcgcaag 480

cgttgtccgg aattattggg cgtaaagagc tcgtaggcgg cttgtcacgt cggatgtgaa 540

agcccgaggc ttaacctcgg gtctgcattc gatacgggct agctagagtg tggtagggga 600

gatcggaatt cctggtgtag cggtgaaatg cgcagatatc aggaggaaca ccggtggcga 660

aggcggatct ctgggccatt actgacgctg aggagcgaaa gcgtggggag cgaacaggat 720

tagataccct ggtagtccac gccgtaaacg ttgggaacta ggtgttggcg acattccacg 780

tcgtcggtgc cgcagctaac gcattaagtt ccccgcctgg ggagtacggc cgcaaggcta 840

aaactcaaag gaattgacgg gggcccgcac aagcggcgga gcatgtggct taattcgacg 900

caacgcgaag aaccttacca aggcttgaca tataccggaa agcattagag atagtgcccc 960

ccttgtggtc ggtatacagg tggtgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg 1020

gttaagtccc gcaacgagcg caacccttgt cctgtgttgc cagcatgccc ttcggggtga 1080

tggggactca caggagaccg ccggggtcaa ctcggaggaa ggtggggacg acgtcaagtc 1140

atcatgcccc ttatgtcttg ggctgcacac gtgctacaat ggccggtaca atgagctgcg 1200

ataccgtgag gtggagcgaa tctcaaaaag ccggtctcag ttcggattgg ggtctgcaac 1260

tcgaccccat gaagtcggag tcgctagtaa tcgcagatca gcattgctgc ggtgaatacg 1320

ttcccgggcc ttgtacacac cgcccgtcac gtcacgaaag tcggtaacac ccgaagccgg 1380

tggcccaacc cgtaagggag ggagctgtcg aaggtgggac tggcgatg 1428

Claims

1. The application of streptomycete for producing antibacterial active compound in preparing compound rishiride A is characterized in that the streptomycete for producing antibacterial active compound is named as streptomycete (A)Streptomyces sp.) The ZSYNO2 strain has the preservation unit of China center for type culture Collection, the preservation time of 2020, 1 month and 7 days, the preservation number of CCTCC M2020020 and the address of: wuhan university in Wuhan City, china.

2. A preparation method of a compound rishiride A is characterized in that the preparation method is that streptomyces (Streptomyces)Streptomyces sp.) Prepared and separated from a fermentation culture of ZSYNO2 strain, streptomyces (Streptomyces: (Streptomyces))Streptomyces sp.) The preservation unit of the ZSYNO2 strain is China center for type culture Collection, the preservation time is 1 month and 7 days 2020, the preservation number is CCTCC M2020020, and the address is as follows: wuhan university in Wuhan City, china.

3. The method according to claim 2, wherein the method comprises the steps of:

(1) Inoculating activated streptomyces ZSYNO2 into a seed culture medium, culturing at 25-30 ℃ and 150-250rpm for 70-75h to prepare a seed solution, inoculating the seed solution into a fermentation culture medium at a volume ratio of 5-15%, culturing at 25-30 ℃ and 150-250rpm for 6-8 days by shaking to prepare a fermentation culture of the streptomyces ZSYNO 2; the seed culture medium and the fermentation culture medium have the following formula: calculated by 100 percent of mass fraction, comprises 20 percent of glucose, 10 percent of soybean peptone, 2.34 percent of valine and CaCO ₃ 2% and CoCl ₂ Solution 1mL, coCl ₂ CoCl in solution ₂ The concentration of the seed culture medium is 1mg/mL, the pH of the seed culture medium and the fermentation culture medium is 7.2, the components and the contents are uniformly mixed, and then the mixture is sterilized for 30min at the temperature of 121 ℃;

(3) Dissolving the crude extract obtained in the step (2) by methanol, passing the crude extract through a solid phase extraction column, eluting by methanol with gradient concentration, wherein the gradient concentration of the methanol is 30%,60%,80% and 100%, collecting eluent, performing rotary evaporation and concentration to obtain corresponding fractions A1, A2, A3 and A4, wherein the fraction A2 is subjected to silica gel column chromatography again, the volume ratio of ethyl acetate to methanol in an ethyl acetate/methanol elution system is 1:0,2:1,1:1 and 0:1 to obtain fractions B1, B2, B3 and B4, and the obtained fraction B1 is subjected to preparative liquid chromatography, wherein the gradient elution of an acetonitrile/water system is 30-90% in acetonitrile concentration, the elution time is 20min, the flow rate is 2mL/min, and the retention time is 15min to obtain the compound risilide A.