Streptomyces pithecellobium clypearia, glycine cyclic peptide compound Tryptorubin A as well as preparation method and application of glycine cyclic peptide compound Tryptorubin A

Abstract

The invention discloses streptomyces clypearia, which is named as Streptomyces sp.HEHY, is preserved in the China General Microbiological Culture Collection Center (CGMCC), has the registration number of CGMCC No.26800, and is preserved on March 14, 2023. The streptomyces clypearia is named as Streptomyces sp.HEHY. The streptomyces clypearia is named as Streptomyces sp.HEHY and is preserved in the China General Microbiological Culture Collection Center (CGMCC). Meanwhile, the invention provides the glycine cyclic peptide compound Tryptorubin A separated by utilizing the streptomyces pithecellobium clypearia and application of the glycine cyclic peptide compound Tryptorubin A. A strain of actinomycete is separated from plant pithecellobium clypearia, specifically streptomyces pithecellobium clypearia, the taxonomic name is Streptomyces sp.HEHY, the streptomyces pithecellobium clypearia is used for producing a yellow solid Tryptorubin A, and the Tryptorubin A has antibacterial, antiviral, antitumor and other biological activities, can effectively inhibit a variety of gram-positive and gram-negative bacteria, and has a good application prospect. Particularly, the killing capability on drug-resistant bacteria is more obvious.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to Streptomyces auris, an amino acid cyclic peptide compound Tryptorubin A, and a preparation method and application thereof. Background Art

[0002] Antibiotics are a class of biologically active compounds that can inhibit or kill microorganisms such as bacteria, fungi and viruses. In clinical practice, antibiotics are widely used to treat infectious diseases. However, due to the overuse and abuse of antibiotics, the drug resistance of many strains has gradually increased, and even super strains resistant to multiple antibiotics have emerged, which seriously threatens public health and human health. Therefore, it is urgent to discover new antibiotics to meet this challenge.

[0003] Monkey earring is a plant unique to the south. It grows in mountains and jungles. Its roots, branches and leaves contain a large amount of biologically active substances. It is widely used in traditional Chinese medicine preparations for clearing heat and detoxifying, removing dampness and healing sores. It has great potential for discovering new antibacterial and antiviral compounds.

[0004] Bicyclic Tryptorubin A is a small molecule cyclic peptide composed of six amino acids, which has atypical rotational enantiomerism. For a long time, the structure of small molecules was considered to be defined only by atomic connectivity and central chirality or axial chirality. For example, steroid hormones all have the same basic carbon atom skeleton, and their different biological effects depend on the modification of the periphery of this basic skeleton. Unlike small molecules, biological macromolecules such as proteins need not only the correct atomic connectivity (i.e., primary structure) to achieve biological functions, but also often need to fold into a biologically active molecular shape (i.e., tertiary structure) without breaking or forming new covalent bonds. However, for some macromolecules, the main factor determining the molecular shape is atomic connectivity, not conformational changes. For example, DNA topoisomerase can switch the helical state and unwinding state of circular DNA by cutting and reconnecting phosphodiester bonds. This phenomenon is called topoisomerism. Topological isomerism also exists in catenane molecules. Without breaking and reforming chemical bonds, topoisomers cannot be converted into each other. The unique topological isomerism of Tryptorubin A determines that it is a natural product with potential medicinal value, with antibacterial, antiviral, antitumor and other biological activities. Due to its unique chemical structure and good pharmacological activity, Tryptorubin A has become one of the hot spots of current research. At present, there are many methods for preparing Tryptorubin A, such as chemical synthesis and natural product fermentation extraction. However, these existing methods have the problems of high cost, low yield and serious pollution. Therefore, it is of great significance to study a method for preparing Tryptorubin A with high efficiency, low cost and environmental protection. Summary of the invention

[0005] The present invention aims to provide a Streptomyces auriculata, and also to provide a peptide cyclic peptide compound Tryptorubin A and a preparation method and application thereof.

[0006] Based on the above purpose, the present invention adopts the following technical solutions:

[0007] A monkey ear-ring Streptomyces, named Streptomyces sp.HEHY, is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the registration number CGMCC No.26800 and the preservation date of March 14, 2023.

[0008] The Streptomyces cyprinifolia is isolated, cultured and purified from pretreated cyprinifolia, which is taken from the medicinal plant cyprinifolia from Wuzhi Mountain, Hainan Province.

[0009] Isolation and culture method: add a certain amount of distilled water to the pretreated monkey earrings, grind them, spread them on the culture medium, and culture them at room temperature until the colonies grow gray-brown colonies with a diameter of more than 1 mm. Purification method: adopt the plate streak method, place the colonies on the culture medium for purification culture, and repeat the purification culture steps; the culture medium is ISP2 culture medium.

[0010] A peptide cyclic peptide compound Tryptorubin A is prepared by culturing and purifying Streptomyces auris, and has a molecular formula of C 26 H 25 N2O5, molecular weight is 897.48.

[0011] The preparation method of the peptide cyclic peptide compound Tryptorubin A comprises the following steps:

[0012] 1) Cultivation: inoculating Streptomyces simianus into a culture medium for culturing to obtain a culture;

[0013] 2) Culture collection: filtering the culture in step 1) to obtain bacterial liquid and mycelium, soaking the mycelium in soaking liquid, evaporating and concentrating, extracting with an extractant, evaporating and concentrating to obtain extract A; extracting the bacterial liquid with an extractant, evaporating and concentrating to obtain extract B, combining extract A and extract B to obtain a crude extract of the amino acid cyclic peptide compound Tryptorubin A;

[0014] 3) Purifying the crude extract of the peptide cyclic peptide compound Tryptorubin A in step 2) to obtain the peptide cyclic peptide compound Tryptorubin A.

[0015] In step 1), the culture medium is ISP2 liquid culture medium, the culture temperature is 28° C., the rotation speed is 180 rpm / min, and the culture time is 7 days.

[0016] In step 2), the soaking liquid is ethanol, the extractant is ethyl acetate, the mycelium is soaked in ethanol, and the mycelium is taken out and soaked again, soaking for 3-4 times, each time for 6-7 days, and then concentrated by rotary evaporation, extracted with ethyl acetate for 3-4 times, and concentrated by rotary evaporation to obtain extract A; the bacterial solution is directly extracted with ethyl acetate for 3-4 times, and concentrated by rotary evaporation to obtain extract B, and the evaporation is carried out by a rotary evaporator.

[0017] In step 3), purification method: the crude extract of Tryptorubin A in step 2) is subjected to ultrafiltration, silica gel column chromatography, and preparative HPLC separation to obtain a pure product (high performance liquid chromatography).

[0018] Application of Tryptorubin A, a peptide cyclic peptide compound, in pharmaceutical products, cosmetics and food.

[0019] The pharmaceutical product is an antibacterial, antiviral or antitumor drug.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The invention separates an actinomycete from the plant monkey earring, specifically monkey earring Streptomyces, whose taxonomic name is Streptomyces sp.HEHY, and utilizes the monkey earring Streptomyces to produce a yellow solid Tryptorubin A, thereby solving the problems of high cost, complicated operation, low yield and the like in a traditional method, and having high production efficiency and economic benefit. Meanwhile, Tryptorubin A has biological activities such as antibacterial, antiviral and antitumor, can effectively inhibit a variety of Gram-positive and Gram-negative bacteria, and in particular, can achieve a killing ability of 85% for multi-drug resistant bacteria. The invention can be used for preparing antibacterial, antiviral and antitumor drugs, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 Phylogenetic tree constructed for the 16S rRNA sequence of Streptomyces sp.HEHY;

[0024] Figure 2 is the mass spectrum of Tryptorubin A;

[0025] Figure 3 This is the NMR spectrum of Tryptorubin A. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention is described in detail below, but the following embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by those skilled in the art without making creative work belong to the scope of protection of the present invention.

[0027] Example 1

[0028] A monkey ear-ring Streptomyces, named Streptomyces sp.HEHY, is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the registration number CGMCC No.26800 and the preservation date of March 14, 2023.

[0029] Streptomyces cyprinifolia was isolated, cultured and purified from pretreated cyprinifolia, which was obtained from the medicinal plant cyprinifolia in Wuzhi Mountain, Hainan Province.

[0030] The specific method for isolating Streptomyces sp.HEHY from monkey earrings is as follows:

[0031] 1) Monkey earring pretreatment

[0032] Rinse the soil on the surface of the plant monkey earrings with running water, then rinse with distilled water, absorb the surface moisture with absorbent paper, separate the branches, bark and leaves of the monkey earrings, process them into about 5.0mm×5.0mm, and then disinfect the surface, place them in a sterile bag for use, and the disinfection method is as follows: first soak in 1% Tween 20 for 30 seconds → rinse with distilled water 3 times → soak in 0.5% sodium hypochlorite solution for 5 minutes → rinse with distilled water 3 times → soak in 2.5% sodium thiosulfate solution for 10 minutes → rinse with distilled water 3 times → soak in 75% alcohol for 5 minutes → rinse with distilled water 3 times → soak in 10% sodium bicarbonate solution for 10 minutes → rinse with distilled water 3 times.

[0033] 2) Isolation and culture: 0.5 g of the pretreated branches, bark and leaves of the monkey earring were placed in a sterilized grinding mortar, and 5 ml of distilled water was added to fully grind to obtain a homogenate. 200 μl of the homogenate was evenly spread on ISP2 medium, and cultured at room temperature for 7 days. The growth of the colonies was observed, and the colonies were isolated when they grew to 1-3 mm.

[0034] 3) Purification: By observing the characteristics of the growing colonies and the morphology of the colonies under the microscope, the colonies suspected of being actinomycetes were picked using the plate streak method. The picked colonies were placed on ISP2 medium for purification culture using the plate streak method. The purification culture steps were repeated 3 times until a single target colony without any other bacteria was cultured.

[0035] The identification method of actinomycetes strains in the present invention is:

[0036] 1. Morphological characteristics: The colonies are yellow, with a diameter of 1.5-2.0 mm, a smooth surface, neat edges, soft texture and glossy.

[0037] 2. Physiological and biochemical characteristics: The strain is sensitive to antibiotics such as penicillin, streptomycin, chloramphenicol, tetracycline, rifampicin, cephalosporin, gentamicin, erythromycin, ampicillin, ampicillin, ampicillin thiazole, ampicillin, azithromycin, metronidazole, minocycline, clindamycin, florfenicol, tilmicosin, levofloxacin, ciprofloxacin, moxifloxacin, ofloxacin, meropenem, ceftriaxone, cefuroxime, cefradine, cefaclor, cefoperazone, and cefazolin, and is resistant to antibiotics such as rifampicin, cefotaxime, ceftriaxone, cefuroxime, cefradine, cefaclor, cefoperazone, and cefazolin.

[0038] 3. 16S rRNA gene sequence analysis: The 16S rRNA gene of the strain was amplified by PCR and sequenced. The sequencing results were compared with the 16S rRNA sequences in the GenBank database.

[0039] The strains of the target colony were subjected to 16S rRNA sequence analysis, and the phylogenetic tree constructed was as follows Figure 1 As shown, it has a high homology with known actinomycete strains. The 16S rRNA sequence of this actinomycete is 95.4% similar to that of Streptomyces, proving that it is a new actinomycete, named Streptomyces sp.HEHY. The specific sequence is shown in Sequence Table 1.

[0040] Example 2

[0041] A peptide cyclic peptide compound Tryptorubin A, which is obtained by culturing and purifying the Streptomyces auris of Example 1, is a yellow solid with a molecular formula of C 26 H 25 N2O5, molecular weight is 897.48.

[0042] The preparation method of the peptide cyclic peptide compound Tryptorubin A comprises the following steps:

[0043] 1) Cultivation: inoculate 10 mL of Streptomyces simianus into 500 mL of ISP2 liquid culture medium, culture at a temperature of 28° C. and a rotation speed of 180 rpm / min for 7 days to obtain a culture;

[0044] 2) Culture collection: Filter the culture of step 1) with gauze to obtain two parts: bacterial liquid and mycelium. Soak the mycelium part with ethanol (the soaking liquid covers the mycelium), take out the mycelium and soak it again, soak it 4 times, soak it for 7 days each time, then concentrate it by rotary evaporation at 50°C for 5 hours, extract it with an equal volume (the amount of ethanol used for each soaking) of ethyl acetate 4 times, and concentrate it by rotary evaporation at 50°C for 5 hours to obtain extract A; the bacterial liquid is directly extracted with an equal volume (the amount of ethanol used for each soaking) of ethyl acetate 4 times, and concentrated it by rotary evaporation at 50°C for 5 hours to obtain extract B, and extract A and extract B are combined to obtain a crude extract of the amino acid cyclic peptide compound Tryptorubin A;

[0045] 3) The crude extract of the peptide cyclic peptide compound Tryptorubin A in step 2) was first filtered with a 0.1 μm filter membrane, and then initially separated by silica gel column chromatography, with the mobile phase being ethyl acetate-methanol (55:45). Preparative HPLC was performed using a Phenomenex Luna C18 column, with the mobile phase being (acetonitrile-water containing 0.1% acetic acid), with a concentration gradient of 10 / 90% to 100 / 0% within 25 minutes), and the third peak was collected and dried to obtain the peptide cyclic peptide compound Tryptorubin A.

[0046] The application of the peptide cyclic peptide compound Tryptorubin A in pharmaceutical products, cosmetics and foods, wherein the pharmaceutical products are antibacterial, antiviral or antitumor drugs.

[0047] Example 3 Structural analysis and biological activity test of Tryptorubin A

[0048] RP HPLC was performed using an Agilent 6530B qTOF mass spectrometer. HRMS [M+H]+ m / z 897.3928 ( Figure 2 ) to obtain purified Tryptorubin A. The structure of Tryptorubin A was analyzed by Bruker AVANCE II 600MHz nuclear magnetic resonance, mass spectrometry and other methods. The nuclear magnetic resonance spectrum is shown in Figure 3 shown.

[0049] Depend on Figure 2 and 3 It is known that Tryptorubin A with a purity exceeding 95% can be obtained by the method of the present invention.

[0050] The microdilution method was used to test the biological activity of Tryptorubin A. The operation process is as follows:

[0051] 1. Inoculate the clinically isolated multidrug-resistant strains to be tested onto the corresponding solid culture plates and culture the revived strains in a 37°C bacterial incubator overnight.

[0052] 2. Weigh 10.0 mg of pure Tryptorubin A powder, add methanol to fully dissolve it, and prepare a storage solution for later use.

[0053] 3. Use nutrient broth medium to dilute the stock solution to the highest concentration of the drug to be tested. Take a sterile 96-well plate and dilute the drug in a biosafety cabinet. The specific steps are as follows:

[0054] Add 200 μL of the highest concentration of the drug to be tested to the first well (A1), add 100 μL of nutrient broth liquid culture medium to wells A2 to A12, then aspirate 100 μL from well A1 and add to well A2, mix well, aspirate 100 μL from well A2 and add to well A3, and so on, perform gradient dilution to well A12, and discard the last 100 μL of diluted liquid.

[0055] 4. Add 1 mL of sterile saline to a transparent plastic test tube; place it on a turbidity meter and adjust it to zero. Then pick the strain to be tested and fully dissolve it in the saline. Vortex to mix well. Adjust the turbidity to between 0.4 and 0.6 McFarland turbidity (MCF). Continue to dilute it 20 times with sterile saline for later use.

[0056] 5. Add 10 μL of the diluted bacterial suspension to each drug concentration well (A1-A12) in turn; place the 96-well plate in a 37°C bacterial incubator and culture for 16-18 hours.

[0057] 6. Read the lowest drug concentration at which no bacteria grow, which is the minimum inhibitory concentration (MIC) of the drug for the bacteria.

[0058] 7. The drug sensitivity test uses standard strains such as Escherichia coli ATCC25922 as quality control strains.

[0059] The results are shown in Table 1.

[0060] As shown in Table 1, Tryptorubin A has significant inhibitory effect on various bacteria such as Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, etc., and its minimum inhibitory concentration (MIC) is between 0.1-1 μg / mL, indicating that Tryptorubin A has antibacterial and antiviral biological activities. Tryptorubin A can be used in antibacterial, antiviral or antitumor drugs.

[0061] Table 1 Inhibitory effect of Tryptorubin A on multidrug-resistant bacteria isolated from clinical sites

[0062] Species name Minimum inhibitory concentration μg / mL Multidrug-resistant bacteria-Escherichia coli 0.25 Multidrug-resistant bacteria-Acinetobacter baumannii 0.5 Multidrug-resistant bacteria - Klebsiella pneumoniae 0.25 Multidrug-resistant bacteria - Pseudomonas aeruginosa 0.5 Methicillin-resistant Staphylococcus aureus 0.25 Multidrug-resistant bacteria - Enterococci 0.5

[0063] Sequence Listing 1

[0064] <110> PLA General Hospital Hainan Hospital

[0065] <120> A kind of Streptomyces auris, peptide cyclic peptide compound Tryptorubin A and preparation method and application thereof

[0066] <160> 1

[0067] <170> SIPOSequenceListing

[0068] <210> 1

[0069] <211> 1521

[0070] 212〉DNA

[0071] 〈213〉Endophytic actinomycetes in monkey earrings (Streptomyces p. HEHY)

[0072] <400> 1

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[0074] ggattagtggcgaacgggtgagtaacacgtgggcaatctgccctgcactctgggacaagccctggaaacggggtctaata

[0075] ccggatactcactgccttgggcatccaaggtggtggaaagctccggcggtgcaggatgagcccgcggcctatcagctagt

[0076] tggtgaggtaatggctcaccaaggcgacgacgggtagccggcctgagagggcgaccggccacactgggactgagaca

[0077] cggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcgaaagcctgatgcagcgacgccgcgtga

[0078] gggatgacggccttcgggttgtaaacctctttcagcagggaagaagcgagagtgacggtacctgcagaagaagcgccgg

[0079] ctaactacgtgccagcagccgcggtaatacgtagggcgcaagcgttgtccggaattattgggcgtaaagagctcgtaggc

[0080] ggcttgtcacgtcggttgtgaaagcccggggcttaaccccgggtctgcagtcgatacgggcaggctagagttcggtaggg

[0081] gagatcggaattcctggtgtagcggtgaaatgcgcagatatcaggaggaacaccggtggcgaaggcggatctctgggcc

[0082] gatactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacggtggg

[0083] cactaggtgtgggcaacattccacgttgtccgtgccgcagctaacgcattaagtgccccgcctggggagtacggccgcaa

[0084] ggctaaaactcaaaggaattgacgggggcccgcacaagcagcggagcatgtggcttaattcgacgcaacgcgaagaac

[0085] cttaccaaggcttgacatacaccggaaagcattagagatagtgccccccttgtggtcggtgtacaggtggtgcatggctgtc

[0086] gtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcccgtgttgccagcaagcccttcggg

[0087] gtgttggggactcacgggagaccgccggggtcaactcggaggaaggtggggacgacgtcaagtcatcatgccccttatg

[0088] tcttgggctgcacacgtgctacaatggccggtacaatgagctgcgataccgtgaggtggagcgaatctcaaaaagccggt

[0089] ctcagttcggattggggtctgcaactcgaccccatgaagtcggagttgctagtaatcgcagatcagcattgctgcggtgaat

[0090] acgttcccgggccttgtacacaccgcccgtcacgtcacgaaagtcggtaacacccgaagccggtggcccaaccccttgtg

[0091] ggagggagctgtcgaaggtgggaccagcgattgggacgaagtcgtaacaaggtagccgtaccggaaggtgcggctgg

[0092] atcacctcctt

Claims

1. A monkey ear-ring Streptomyces, named Streptomyces sp.HEHY, is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the registration number CGMCC No.26800 and the deposit date March 14, 2023.

2. The Streptomyces auriculata according to claim 1, wherein The monkey earring streptomyces is isolated, cultured and purified from pretreated monkey earrings.

3. The Streptomyces auriculata according to claim 2, characterized in that Isolation and culture method: add a certain amount of distilled water to the pretreated monkey earring tissue, grind it, spread it on the culture medium, and culture it at room temperature until gray-brown colonies with a diameter of more than 1 mm grow; purification method: adopt the plate streak method, place the colonies on the culture medium for purification culture, and repeat the purification culture steps; the culture medium is ISP2 culture medium.

4. A peptide cyclic peptide compound Tryptorubin A, characterized in that: It is obtained by culturing and purifying the Streptomyces auriculatus described in any one of claims 1 to 3, and has a molecular formula of C 26 H 25 N2O5, molecular weight is 897.

48.

5. A method for preparing a peptide cyclic peptide compound Tryptorubin A, characterized in that: The following steps are involved: 1) Cultivation: inoculating Streptomyces simianus into a culture medium for culturing to obtain a culture; 2) Culture collection: filtering the culture in step 1) to obtain bacterial liquid and mycelium, soaking the mycelium in soaking liquid, evaporating and concentrating, extracting with an extractant, evaporating and concentrating to obtain extract A; extracting the bacterial liquid with an extractant, evaporating and concentrating to obtain extract B, combining extract A and extract B to obtain a crude extract of the amino acid cyclic peptide compound Tryptorubin A; 3) Purifying the crude extract of the peptide cyclic peptide compound Tryptorubin A in step 2) to obtain the peptide cyclic peptide compound Tryptorubin A.

6. The method for preparing the peptide cyclic peptide compound Tryptorubin A according to claim 5, characterized in that: In step 1), the culture medium is ISP2 liquid culture medium, the culture temperature is 28° C., the rotation speed is 180 rpm / min, and the culture time is 7 days.

7. The method for preparing the peptide cyclic peptide compound Tryptorubin A according to claim 5, characterized in that: The soaking liquid is ethanol, the extracting agent is ethyl acetate, the mycelium is soaked in ethanol, and then the mycelium is taken out and soaked again, the soaking is performed for 3-4 times, each soaking lasts for 6-7 days, and then the extract is concentrated by rotary evaporation, and then the extract is extracted with ethyl acetate for 3-4 times and concentrated by rotary evaporation to obtain extract A; the bacterial liquid is directly extracted with ethyl acetate for 3-4 times and concentrated by rotary evaporation to obtain extract B.

8. The method for preparing the peptide cyclic peptide compound Tryptorubin A according to claim 5, characterized in that: In step 3), purification method: the crude extract of the amino acid cyclic peptide compound Tryptorubin A in step 2) is subjected to ultrafiltration, silica gel column chromatography, and preparative HPLC separation to obtain a pure product.

9. Application of Tryptorubin A, a cyclic peptide compound, in pharmaceutical products, cosmetics and food.

10. The use according to claim 9, characterized in that The pharmaceutical product is an antibacterial, antiviral or antitumor drug.

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