CN112899199B - Micromonospora TMD166 and application thereof - Google Patents

Abstract

The invention relates to the fields of microbiology and pharmacy, discloses micromonospora TMD166 and application thereof, and more particularly discloses micromonospora TMD166 and a preparation method and application of a novel thiopeptide antibiotic produced by the micromonospora TMD 166. Micromonospora sp TMD166 has a preservation number of CGMCC NO. 21622. The fermentation liquor of Micromonospora sp contains a novel thiopeptide antibiotic named Xuanzangmicin, and the compound has excellent antibacterial activity and good application prospect as a new anti-infective medicine for human or livestock.

Description

Micromonospora TMD166 and application thereof

Technical Field

The invention relates to the fields of microbiology and pharmacy, in particular to micromonospora TMD166 and a preparation method and application of a novel thiopeptide antibiotic produced by the micromonospora TMD 166.

Background

Pathogenic bacteria and antibiotic-resistant bacteria thereof, including multidrug-resistant or pan-resistant bacteria, represented by 'ESKAPE' (enterococcus, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter) and Mycobacterium tuberculosis seriously threaten the health of human and livestock. At present, with the resistance of these pathogenic bacteria to therapeutic antibacterial drugs, the effective available antibiotics are gradually reduced, and therefore, the discovery of antibiotics with novel structures and unique mechanisms of action, particularly the discovery of novel compound molecules effective against the drug-resistant bacteria of "ESKAPE" and mycobacterium tuberculosis, is the focus of the research and development of current anti-infective drugs.

The thiopeptide antibiotics are secondary metabolites of structurally complex polypeptides produced by microorganisms, which contain multiple thiazole rings. The first thiopeptide antibiotic was micrococcin (micrococin), which was discovered in 1948, and to date, hundreds of such antibiotics have been reported. The antibiotics have a characteristic nitrogen six-membered heterocyclic ring, and can be divided into five types according to the oxidation degree and the substitution mode of the nitrogen six-membered heterocyclic ring: piperidines, dehydropiperidines, dihydroimidazopyridines, trisubstituted pyridines, and hydroxypyridines. The thiopeptide antibiotics have various activities, including antibacterial, antitumor, antimalarial, immunosuppressive activity, etc., and among them, have strong antibacterial activity against gram-positive bacteria, and have attracted extensive attention in academia and pharmaceutical industry. In addition to thiostrepton and nosiheptide, which have been used as animal feed additives, several derivatives of thiostrepton antibiotics have entered clinical research phase, such as GE2270A analog LFF571, completed phase II clinical trials as a drug for the treatment of intestinal infections caused by clostridium difficile (clostridium difficile). Another GE2270A derivative, NA1003, has a narrow spectrum of antibacterial activity against propionibacterium acnes (propionibacterium acnes), and has also been studied clinically in phase II as a new drug for the treatment of acne. Although the antibacterial activity of the thiopeptide antibiotics is mainly to prevent the synthesis of bacterial proteins through bacterial ribosomes, different types of thiopeptide antibiotics have different acting targets, and the discovery of the new structure of thiopeptide antibiotics has the high possibility of developing novel therapeutic drugs for treating infection related to human and livestock and the problem of bacterial drug resistance.

Disclosure of Invention

The invention aims to provide a micromonospora strain capable of producing a novel antibiotic and application thereof.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

the Micromonospora sp is an endophytic actinomycete with strong antibacterial activity, namely a Micromonospora strain TMD166 which is separated from a dry desert plant camel thorn of Xinjiang Takara Ma and is preserved in China general microbiological culture Collection center (CGMCC for short, the address: West Lu No. 1 of the sunward area of Beijing, China academy of sciences, microbiological research institute, postal code 100101) in 13 months at 2021 and 13 days in 2021, and the classification name is the Micromonospora sp with the number of CGMCC No. 21622.

The method for obtaining the micromonospora TMD166 comprises the following steps: the micromonospora strain TMD166 provided by the invention is separated from the stems of desert plant alhagi sparsifolia with a strict surface disinfection. Crushing the surface-sterilized alhagi sparsifolia with a sterilized crusher, spreading on a separation culture medium, and separating after culturing to obtain the purified strain.

Further, the present invention provides a fermentation product of the Micromonospora sp TMD 166.

The fermentation product contains xuzaniangmicin (Xuanzangmicin), and the chemical structural formula of xuzaniangxing is shown as a formula I:

the invention researches the active secondary metabolite of micromonospora TMD166 by liquid fermentation and changing the components of the culture medium, applies a plurality of chromatographic separation technologies to separate a compound with strong antibacterial activity from the active secondary metabolite, and performs the separation according to UV, IR, HR-ESI-MS,¹H-NMR、¹³C-NMR, DEPT data testing and¹H-¹h correlation spectrum (¹H-¹H COSY)、¹H-¹³C correlation spectroscopy (HSQC), reverse detection remote¹H-¹³The structure of the C heteronuclear multiple bond correlation spectrum (HMBC), the two-dimensional NOE spectrum (NOESY), the rotational coordinate system NOE spectrum (ROESY) and the homonuclear chemical shift full correlation spectrum (TOCSY) is determined by analyzing. The compound has a core skeleton of the thiopeptide antibiotics: multiple thiazole rings and characteristic nitrogen six-membered heterocyclic rings, pyridine hydroxide; furthermore, it was first found that a dimethoxy-substituted pyranose is attached to the hydroxyl group of the pyridine ring; it is also the first time to find thiopeptide antibiotics containing four glycosyl side chains. Further passing the Minimal Inhibitory Concentration (MIC) (as NCCLS Ready-to-the-Meter)Micro broth dilution recommended by the national committee for standardization in clinical laboratories) was tested for its antibacterial activity. The experimental result shows that the xuanzanamicin has the bacterial inhibitory activity: the antibacterial activity in vitro against gram-positive bacteria such as staphylococcus (beta-lactam antibiotic resistant strain) and enterococcus (vancomycin resistant strain) is strong, the MIC value is in the range of 0.125-0.5 mug/ml, and the results show that the xuanzanamicin has broader-spectrum gram-positive bacteria resistance; in addition, xuanzang Mixing to Mycobacterium tuberculosis H₃₇Rv (ATCC27294) showed moderate inhibitory activity with MIC values of 16. mu.g/ml. The invention provides a new resource and a new drug candidate for developing antibacterial drugs.

The invention provides a microbial inoculum, which contains the Micromonospora sp TMD166 or the fermentation product.

Preferably, the microbial inoculum is a solid microbial inoculum or a liquid microbial inoculum.

The compound of the formula also comprises isomers, racemates, enantiomers and diastereomers.

The invention also provides application of the Micromonospora sp TMD166 or the fermentation product or the microbial inoculum in inhibiting bacterial growth or preparing an antibacterial product.

The bacteria are gram positive bacteria.

The gram-positive bacteria are staphylococcus or enterococcus or mycobacterium.

In particular, Micromonospora sp TMD166 or fermentation products or microbial agents of the present invention are suitable for inhibition of beta-lactam antibiotic-resistant gram-positive bacteria staphylococcus, vancomycin-resistant enterococcus and mycobacterium tuberculosis.

The invention also provides the application of the Micromonospora sp TMD166 in the preparation of the xuanzanamicin, which is described above.

The present invention also provides a method for preparing xuanzanamixing, the xuanzanamixing is as described above, the method comprises: fermenting and culturing the Micromonospora sp TMD166 to obtain fermentation liquor; then extracting and separating the xuanzanamicin from the fermentation liquor.

The fermentation culture comprises the following steps: inoculating the Micromonospora sp TMD166 on a seed culture medium for culture, and then transferring the culture medium into a fermentation culture medium for shake culture;

wherein the culture conditions on the seed culture medium are 28-30 ℃, 150-; the culture condition on the fermentation culture medium is 28-30 ℃, 150-;

the seed culture medium and the fermentation culture medium have the same formula (No. 38 culture medium), and specifically comprise the following components: 4g of glucose, 4g of yeast powder, 5g of malt extract powder and ZnSO₄.7H₂O 0.001g，MnCl₂.4H₂O 0.001g，FeSO₄.7H₂0.002g of O, 0.001g of phenylalanine, 0.0003g of alanine, 0.001g of vitamin B10.001g, 20.001g of vitamin B, 60.001g of vitamin B, 0.001g of nicotinic acid, 0.001g of biotin and 1.0L of sterile distilled water; the pH of the seed culture medium and the fermentation culture medium is 8.5.

The extraction and separation steps comprise: firstly, centrifuging the fermentation liquor, taking supernatant fluid to carry out HP-20 macroporous adsorption resin column chromatography, eluting with acetone water (30%, 50%, 80%), collecting 80% acetone part eluent, carrying out low-temperature reduced-pressure rotary concentration to remove organic solvent, and obtaining crude extract. Carrying out Sephadex LH-20 gel column chromatography on the crude extract, collecting according to volume, and separating to obtain a semi-pure product; dissolving the semi-pure product in methanol, preparing by HPLC, and freeze-drying to obtain pure compound.

The specific extraction and separation steps are as follows: centrifuging the fermentation liquid by a large-capacity centrifuge at 4500rpm, taking the supernatant, performing HP-20 macroporous adsorption resin column chromatography, and discarding the effluent liquid; eluting with deionized water twice the column volume, removing salt and water soluble components, and discarding the eluate; eluting with 30% acetone water eluent with twice column volume, and discarding eluent; eluting with 50% acetone water eluent with twice column volume, and discarding the eluent; and finally eluting with 80% acetone water eluent with the volume twice that of the column, collecting the eluent, carrying out rotary evaporation at low temperature under reduced pressure, removing the organic solvent, and carrying out freeze drying on the aqueous solution. Dissolving the freeze-dried product in methanol, carrying out Sephadex LH-20 gel column chromatography, eluting with methanol, collecting a light yellow band at the front end, and carrying out reduced pressure rotary evaporation to dryness to obtain a semi-pure product; dissolving the semi-pure product in methanol, preparing by HPLC, removing methanol by rotary evaporation under reduced pressure at low temperature, and freeze-drying the aqueous solution to obtain light yellow powder pure product compound.

As a preferred embodiment, the xuanzanamicin is prepared by the following method:

fermentation and culture of xuanzanamicin producing bacteria: firstly, inoculating a micromonospora strain TMD166 growing on a slope to a seed culture medium, culturing for 72 hours on a rotary shaking table, then transferring to a fermentation culture medium and culturing on a shaking table, and harvesting fermentation liquor for 96 hours;

extracting and separating xuanzang micaceous: centrifuging the fermentation liquor, taking the supernatant, carrying out HP-20 macroporous adsorption resin column chromatography, carrying out gradient elution by using acetone water as an eluent, collecting 80% acetone partial eluent, carrying out rotary evaporation and concentration at low temperature under reduced pressure to remove an organic solvent to obtain a reddish brown crude extract solution, freeze-drying the crude extract, dissolving the crude extract in methanol, carrying out Sephadex LH-20 gel column chromatography, collecting a light yellow ribbon at the front end by using the methanol as the eluent, and carrying out rotary evaporation under reduced pressure; dissolving the semi-pure product in methanol, preparing by HPLC, and freeze-drying to obtain light yellow powder of the target compound.

The invention has the beneficial effects that:

the invention discovers a micromonospora strain TMD166 derived from Takrama drydesert plants, separates thiopeptides antibiotics-xuanzanamicin from secondary metabolites thereof, and determines that xuanzanamicin is a novel thiopeptides antibiotic through the careful analysis of spectral data such as ultraviolet spectrum, infrared spectrum, mass spectrum, nuclear magnetic resonance and the like, the structure of the thiopeptides antibiotic is different from all thiopeptides antibiotics discovered so far, and the hydroxyl group of hydroxylated pyridine with a parent nucleus is connected with dimethoxy substituted pyranose. Xuanzanamicin is the first antibiotic with four glycosyl side chains. The evaluation of antibacterial activity of xuanzanamicin shows that xuanzanamicin has strong inhibitory activity in vitro on gram-positive bacteria such as staphylococcus, enterococcus faecalis, enterococcus faecium (including beta-lactam antibiotic and vancomycin resistant strains) and the like; the zanamicin also showed moderate inhibitory activity against Mycobacterium tuberculosis H37Rv (ATCC 27294); has potential good development prospect as a new anti-infection lead compound.

Drawings

FIG. 1 shows the colony morphology of Micromonospora strain TMD166(Micromonospora sp.TMD 166).

FIG. 2 shows a N-J phylogenetic tree constructed based on the 16S rRNA gene sequence of Micromonospora strain TMD 166.

FIG. 3 is a schematic diagram showing the relationship among COSY, TOCSY, HMBC, NOE and ROE of xuanzanamixing.

FIG. 4 shows UV spectrum of xuanzanamixing.

FIG. 5 shows an IR spectrum (FT-IR Microscope Transmission; scan number 100; resolution 8.000) of xuanzanamicin.

FIG. 6 is a high-resolution mass spectrum (HR-ESI-MS) of xuanzanamicin.

FIG. 7 shows the zang-mi-xing in CDCl₃+CD₃In OD (9:1)¹H-NMR spectrum.

FIG. 8 shows the zang-mi-xing in CDCl₃+CD₃In OD (9:1)¹³C-NMR spectrum.

FIG. 9 shows the zang-mi-xing in CDCl₃+CD₃DEPT spectra in OD (9: 1).

FIG. 10 shows the zang-mi-xing in CDCl₃+CD₃In OD (9:1)¹H-¹H COSY spectra.

FIG. 11 shows the zang-mi-xing in CDCl₃+CD₃In OD (9:1)¹HSQC spectra.

FIG. 12 shows the zang-mi-xing in CDCl₃+CD₃In OD (9:1)¹HMBC spectra.

FIG. 13 shows the zang-mi-xing in CDCl₃+CD₃NOESY spectra in OD (9: 1).

FIG. 14 shows the zang-mi-xing in CDCl₃+CD₃ROESY spectra in OD (9: 1).

FIG. 15 shows the zang-mi-xing in CDCl₃+CD₃TOCSY spectra in OD (9: 1).

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. In the examples, the starting materials were all of analytical grade commercially available unless otherwise specified, except that the HPLC preparation of methanol was pure chromatographic grade.

Example 1 isolation of Micromonospora strain TMD166

Cleaning the stems of alhagi sparsifolia collected from the tacrolimus dry desert with clear water, ultrasonically removing surface impurities, sequentially soaking the sample in 1% tween-20 for 1 minute, in a sodium hypochlorite aqueous solution containing 0.4% of available chlorine for 7 minutes, in 2.5% of sodium thiosulfate for 9 minutes, in 75% of ethanol for 2 minutes, in 10% of sodium bicarbonate for 10 minutes, and cleaning with sterile water for 3 times after each soaking. After the sample WAs dried in the air, it WAs pulverized into a powder by a pulverizer and uniformly scattered on an M-WA medium (10 g of glycerin, 0.5g of yeast, 1.0g of proline, 1.0g of aspartic acid, 0.5g of potassium nitrate, 1.25g of sodium pyruvate, 1.25g of betaine, 20g of agar, 1L of sterile water, pH8.0, sterilized at 121 ℃ for 30 minutes), and cultured at a constant temperature of 28 ℃. After taking out the surface simultaneously for sterilization, 200. mu.l of distilled water from the third washing was spread on an ISP2 plate and incubated at 28 ℃ for 2 weeks to check whether the surface was sterilized completely. Selecting single colony on a purification plate taking ISP-2 as a culture medium under aseptic condition, carrying out three-zone streaking, purifying for 3 times to obtain a pure culture colony, and naming the strain as TMD 166.

Example 2 identification of Micromonospora strain TMD166

(1) And (3) morphological identification:

micromonospora species TMD166 of the present invention is shown in FIG. 1. Colonies on ISP-2 medium showed a light orange color, covered with a layer of powdered spores, and grew rapidly at a culture temperature of 28-30 ℃.

(2) And (3) molecular identification:

extracting the genome DNA, and carrying out amplification analysis by using universal primers 27f and 1492r to obtain a single DNA band. Performing Blast alignment on the strain in GenBank database, screening typical strain sequences with highest similarity, performing cluster analysis by MEGA5.2 software (Neighbor-join) and constructing phylogenetic tree, as shown in FIG. 2, TMD166 and Micromonospora polyrhachis NEAU-ycm2^TClustered in the same branch with a similarity of 99.48%, finally identified M.camelina strain TMD166 as a Micromonospora sp.

Example 3 fermentation of Micromonospora Strain TMD166

Inoculating Micromonospora strain TMD166 growing in slant culture medium into seed culture medium of No. 38 culture medium (glucose 4.0g, yeast powder 4.0g, malt extract powder 5.0g, ZnSO)₄.7H₂O 0.001g，MnCl₂.4H₂O 0.001g，FeSO₄.7H₂0.002g of O, 0.001g of phenylalanine, 0.0003g of alanine, 10.001g of vitamin B, 20.001g of vitamin B, 60.001g of vitamin B, 0.001g of nicotinic acid, 0.001g of biotin and 1.0L of sterile distilled water, and the pH value is 8.5). Every 500mL of shake flask is filled with 100mL of No. 38 culture medium, the culture is carried out for 72h on a rotary shaking table at 28-30 ℃ and 180rpm, then the culture is transferred into a fermentation culture medium (homozygote culture medium) with 10 percent of inoculation amount, every 5.0L of shake flask is filled with 1.0L of fermentation culture medium, the culture is carried out for 96h on the rotary shaking table at 180rpm and 28-30 ℃, and then the fermentation broth is harvested.

Example 4 extraction of xuanzanamicin

The fermentation broth obtained in example 3 was centrifuged (4500rpm, 20min), and the supernatant (18L) was subjected to HP-20(1.5L) macroporous adsorbent resin column chromatography, gradient elution was performed with acetone water (30%, 50%, 80% acetone each 3L as eluent), and the 80% acetone fraction eluate was collected and evaporated under reduced pressure at low temperature to remove the organic solvent, to obtain a reddish brown crude extract (320 mg).

EXAMPLE 5 separation of xuanzanamixing

The crude extract obtained in example 4 was dissolved in 8mL of methanol, and then applied to Sephadex LH-20(500mL) gel column, and eluted with methanolThe yellow band at the tip was collected, and the residue was evaporated to dryness under reduced pressure to give a semi-pure product (42mg) containing the objective compound. The semi-pure product was dissolved in 3.0mL of methanol and prepared by HPLC using a Shimadzu shi-pack PREP-ODS (H) column (20X 250 mm)²5 μm); the mobile phase is 80% methanol water solution; the flow rate was 3 mL/min. Collecting components with retention time of 45min (ultraviolet detection wavelength of 230nm), mixing, rotary evaporating to remove methanol, and freeze drying to obtain pure product of xuanzanamicin as light yellow powder (20 mg).

Example 6 Structure identification of xuanzanamixing

The physical and chemical properties of the purified product obtained in example 5 were determined and shown in Table 1. According to UV spectrum (FIG. 4), IR spectrum (FIG. 5), HR-ESI-MS (FIG. 6), and in CDCl₃+CD₃Nuclear magnetic resonance spectrum with OD (9:1) as solvent:¹H-NMR (FIG. 7),¹³C-NMR spectrum (FIG. 8), DEPT spectrum (FIG. 9),¹H-¹H COSY correlation spectra (FIG. 10),¹H-¹³C correlation spectra HSQC (FIG. 11), reverse detection remote¹H-¹³The analysis results of the C heteronuclear multiple bond correlation spectrum HMBC (FIG. 12), NOESY spectrum (FIG. 13), ROESY spectrum (FIG. 14) and TOCSY spectrum (FIG. 15) confirm that the structure of xuanzanamixing is shown in the formula I.

TABLE 1 physicochemical Properties of xuanzang Mixing

Through the opposite xuanzang rice star¹H-NMR、¹³Comprehensive analysis of C-NMR, HSQC and DEPT spectra revealed that the molecule contained 83 carbon atoms, 31 quaternary carbons (delta)_C:180.8，170.4，170.2，168.1，168.0，166.8，164.7，162.1，161.5，161.4，160.8，160.4，160.1，159.7，155.0，154.3，151.7，150.4，148.4，145.9，144.7，138.8，137.5，133.3, 131.8, 131.5, 129.7, 129.3, 124.9, 117.8, 110.5), 32 tertiary carbons (δ)_C128.0, 126.5, 125.6, 125.5, 124.4, 124.4, 121.3, 114.6, 102.1, 100.2, 98.5, 96.7, 88.4, 83.1, 81.6, 81.0, 80.3, 80.3, 77.2, 75.9, 70.5, 70.2, 69.9, 69.9, 69.2, 68.9, 56.8, 66.9, 53.8, 71.7, 71.4, 49.5), 6 secondary carbons (delta_C104.6, 66.9, 64.5, 40.1, 38.3, 29.2) 14 primary carbons (. delta.)_C:60.9，60.8，59.9，59.4，59.0，57.8，57.8，55.9，17.7，17.7，17.7，17.7，17.6，13.7)。

The chemical shift of each C directly linked to H was determined from HSQC (see table 2).

TABLE 2 xuanzangmixin in CDCl₃+CD₃NMR data on OD (9:1)

¹H-NMR：600MHz；¹³C-NMR：150MHz。

Further analysis of the hydrogen and carbon spectra may conclude that xuanzanamicin is a thiopeptide antibiotic containing 5 thiazole rings, including 3 carbonylthiazole rings (Thz1, Thz2, Thz5), 1 fully substituted carbonylthiazole ring (Thz3), 1 thiazole ring (Thz 4); 1 tetra-substituted hydroxypyridine (Pyr), 1 threonine (Thr), 1 methoxy-substituted dehydrothreonine (Dht), 1 dehydroalanine (Deala), 12, 3, 4-trisubstituted indole ring (Ind), 1 hydroxyglutamic acid (Glu) and one cysteine (Cys).

In that¹H-¹In HCOSY, δ 5.73(1H, d, J ═ 1.8Hz, S1-1H) and δ 4.12(1H, dd, J ═ 3.6, 1.8Hz, S1-2H)(ii) a δ 4.30(1H, dd, 10.2, 3.6Hz, S1-3H) and δ 4.12(1H, dd, J ═ 3.6, 1.8Hz, S1-2H) and δ 3.20(1H, m, S1-4H); δ 3.20(1H, m, S1-4H) and δ 3.57(1H, m, S1-5H); δ 3.57(1H, m, S1-5H) and δ 1.32(3H, d, J ═ 6.0, S1-C5-Me-H). In 2D TOCSY, S1-1H and S1-2H, S1-3H are shown; S1-2H is related to S1-1H, S1-3H, S1-4H, S1-5H, S1-C5-Me-H. Bound in HMBC, two methoxyhydrogens, delta 3.57(3H, S, S1-C2-OMe) and delta 3.52(3H, S, S1-C4-OMe) are associated with C-2 and C-4, respectively. From these information, it was confirmed that S1 was 2, 4 dimethoxy substituted rhamnopyranoside. The coupling constants for S1-2H and S1-1H, S1-3H were 1.8Hz and 3.6Hz, respectively, and for S1-3H and S1-4H were 10.2Hz, indicating that S1-1H and S1-2H are in the equatorial bond orientation on the pyran ring, while S1-3H and S1-4H are in the homeotropic bond orientation. Of NOESY and ROESY, S1-4H and S1-C5-Me-H are related, indicating that S1-5H is also located in the upright bond orientation. Thus, glycoside S1 was determined to be 2, 4 dimethoxy substituted α -L-rhamnopyranoside. In addition, in HMBC, S1-1H and Pyr-C3 are related, and the connection position of the glycoside S1 and the pyridine ring is determined.

Same general analysis¹H-¹HCOSY, 2D TOCSY, identified S2 as 2, 3 dimethoxy substituted rhamnopyranoside, S3 as 2-deoxyhexoside, and S4 as 2, 4 dimethoxy substituted rhamnopyranoside (related COSY, TOCSY is shown in FIG. 3). The coupling constants of S2-2H and S2-1H, and S2-3H are 1.8Hz and 3.0Hz respectively, and the chemical shifts of carbon and hydrogen of S1 combined with S2 and philippimycin are almost consistent, which shows that S2 and S1 of philippimycin have the same configuration and are 2, 3-dimethoxy substituted alpha-L-rhamnopyranoside. S3-1H and S3-2H₁、S3-2H₂Has coupling constants of 9.6Hz and 1.8Hz, indicating that S3-1H is located in the direction of the upright bond; S3-2H₁Has coupling constants of 13.2, 5.4 and 1.8Hz, and shows S3-2H₁The double doublet with the coupling constant of 9.0Hz for S3-4H, located in the equatorial bond direction and S3-3H in the axial bond direction, indicates that S3-4H and S3-5H are also located in the axial bond direction on the pyran ring, thus determining that the glycoside S3 is deoxyβ -D-pyranoside. The coupling constants of S4-2H were 3.0Hz and 1.8Hz, and the coupling constant of S4-4H was a doublet of 9.0Hz, indicating that S4-3H, S4-4H, S4-5H is located in the direction of the orthobond on the pyran ring,S4-2H is located in the direction of equatorial bond, combined in NOESY and ROESY, S4-1H and S4-4H, S4-5H have no related peaks, and S4 is determined to be 2, 4 dimethoxy substituted alpha-L-rhamnopyranoside. In HMBC, S2-1H and Thz3-C5, S2-4H and S3-C1, S3-4H and S4-C1 are related, and the connection positions of the glycoside S2 and thiazole ring-5 and the glycoside S2 and S3, S3 and S4 are determined.

Example 7 determination of antibacterial Activity of xuanzang Mixing against bacteria

The activity assay was performed using the compound obtained in example 5. The Minimum Inhibitory Concentration (MIC) was determined according to the broth microdilution method recommended by the american committee for clinical laboratory standardization (NCCLS). The specific implementation steps are as follows: the xuzanamicin is dissolved in DMSO with the concentration of mother liquor of 2.0mg/ml, and the drug is half-diluted with the culture medium solution. After dilution by half, the antimicrobial solutions of different concentrations were added to sterilized 96-well polystyrene plates, 100. mu.l of each of the 1 st to 11 th wells were dosed, and the 12 th well was not dosed as a growth control. Determination of the Activity of bacteria culture medium Mueller-Hinton (MH) broth recommended by NCCLS, pH7.2-7.4, was used. Directly taking the colony cultured for 24h, and blending with normal saline to obtain 0.5 McLeod turbidimetric standard (2 × 10)⁸CFU/ml). The bacterial suspension to be tested is diluted by MH broth at a ratio of 1:1000, 100 mul of the diluted bacterial suspension is added into each hole, and the mixed solution is placed in a constant temperature incubator at 35 ℃ for 20h to judge the result. And (4) judging a result: the lowest drug concentration that completely inhibited bacterial growth in the wells was the MIC and the assay was repeated three times.

The results of the activities of xuanzanifloxacin against common pathogenic bacteria are shown in Table 3. Xuanzaniflozin has strong inhibitory activity on gram-positive bacteria: including staphylococci and enterococci, and has MIC value in the range of 0.125-0.5 μ g/ml.

TABLE 3 antibacterial Activity of xuanzang Mixing

Among them, the strain Staphylococcus epidermidis 16-5 is shown in published papers: beilunycin, a new viral microorganisms from cultured-derived Streptomyces sp.2BBP-J2 and the antigenic activity by inhibition transfer journal of antibiotic Products research.2020, Sep 14; 1-9(https:// doi.org/10.1080/10286020.2020.1810669); the strain Enterococcus faecalium 12-1 is shown in published papers: xiaokemycin A, alpha novel pyranosylanthraquinone antioxidant, produced by the Streptomyces sp.CC8-201 from the soil of a karst cave the Journal of Antibiotics 2015, 68, 771-774.

EXAMPLE 8 determination of antibacterial Activity of xuanzang Mixing against Mycobacterium tuberculosis H37Rv

The activity assay was performed using the compound obtained in example 5. Selecting Mycobacterium tuberculosis H37Rv (ATCC27294), culturing for 3 weeks to obtain culture, making into bacterial suspension, inoculating into Middlebrook 7H9 liquid culture medium (Middlebrook 7H9 broth), standing and culturing at 37 deg.C for 2 weeks until the turbidity is 10⁷CFU/mL, and then diluted to OD with Mie's 7H9 liquid medium₅₈₀The value was 0.01, and was added to a 96-well plate at 100. mu.L per well. The compound was dissolved in DMSO to give a starting solution at a concentration of 10mg/mL, and diluted in half with Mie's 7H9 liquid medium to give a solution at a concentration of 0.125-64. mu.g/mL. The compound solution was then added to a 96-well plate containing the bacterial suspension to a final concentration of 0.0625-32 μ g/mL using an isonicotinus trap (INH) as a positive control. Incubation of 96-well plates at 37 ℃ for 10 days, OD determination₅₈₀Values, MIC values are the lowest compound concentration without bacterial growth and the assay is repeated three times.

The zanamicin showed moderate inhibitory activity against Mycobacterium tuberculosis H37Rv (ATCC27294) and the MIC value was 16. mu.g/mL.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. Micromonospora bacterium (A), (B), (CMicromonosporasp.) TMD166, characterized in that Micromonospora bacterium (A), (B)Micromonospora sp.) The preservation number of (2) is CGMCC number 21622.

2. Micromonospora bacterium (A) according to claim 1Micromonosporasp.) Fermentation product of TMD 166.

3. The fermentation product of claim 2, comprising zanamicin, the chemical formula of zanamicin being as follows:

。

4. a microbial preparation comprising the Micromonospora bacterium (Micromonospora bacterium) according to claim 1Micromonosporasp.) TMD166 or a fermentation product according to claim 2 or 3.

5. Micromonospora bacterium (A) according to claim 1Micromonosporasp.) Use of TMD166 or a fermentation product according to claim 2 or 3 or a bacterial agent according to claim 4 for the preparation of an antibacterial product.

6. Use according to claim 5, wherein the bacteria are gram-positive bacteria.

7. Use according to claim 6, wherein the gram-positive bacterium is a staphylococci, an enterococci or a mycobacteria.

8. Micromonospora bacterium (A) according to claim 1Micromonosporasp.) Use of TMD166 in the preparation of xuanzanamicin, which is as claimed in claim 3.

9. A method for preparing xuanzanamixing as claimed in claim 3, which comprises: the method of claim1 Micromonospora bacterium (1)Micromonosporasp.) TMD166, harvesting fermentation liquor; then extracting and separating the xuanzanamicin from the fermentation liquor.

10. The method of claim 9, wherein the fermentation culture comprises: micromonospora bacterium (A) according to claim 1Micromonosporasp.) TMD166 is inoculated on a seed culture medium for culture, and then transferred into a fermentation culture medium for shake culture;

wherein the culture conditions on the seed culture medium are 28-30 ℃, 150-; the culture condition on the fermentation culture medium is 28-30 ℃, 150-;

the seed culture medium and the fermentation culture medium have the same formula, and specifically comprise the following components: 4g of glucose, 4g of yeast powder, 5g of malt extract powder and ZnSO₄.7H₂O 0.001 g，MnCl₂.4H₂O 0.001g，FeSO₄.7H₂0.002g of O, 0.001g of phenylalanine, 0.0003g of alanine, 10.001 g of vitamin B, 20.001 g of vitamin B, 60.001 g of vitamin B, 0.001g of nicotinic acid, 0.001g of biotin and 1.0L of sterile distilled water; the pH of the seed culture medium and the fermentation culture medium is 8.5.

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