CN100340660C - Hainan micromonosporin with antibiotic generation - Google Patents

Abstract

The invention discloses a Micromonospora hainan strain producing antibiotics, and the preservation number is CGMCC No.1551. The present invention has the advantages that: the antibiotics produced by Micromonas hainanensis have strong inhibitory effect on multiple G ⁺ bacteria, especially on methicillin-resistant Staphylococcus aureus and Mycobacterium tuberculosis, G ^- bacteria also have some inhibitory effect and have good prospects for drug development.

Description

An Antibiotic-Producing Micromonospora hainanensis

technical field

The invention relates to a Micromonospora hainan strain producing antibiotics, belonging to the technical field of microbes.

Background technique

Antibiotics are the most widely used drugs in hospital clinics, and play an important role in the control, prevention and treatment of various infectious diseases. In recent years, the isolation rate of Methicillin-resistant Staphylococcus aureus (MRSA) has been increasing continuously, and at the same time, there are very few drugs to choose from clinically. Harmful to human health, people must constantly look for new drug sources.

Marine organisms, which account for 80% of the earth's biological resources, have rich metabolic pathways and can produce a large number of metabolites with special structures and unique functions. They are an important source of new active substances. The unique marine environment makes marine microorganisms present a high diversity in terms of species, genetic composition and ecological functions. It is known as the "treasure house" of natural medicine resources and is a new resource of natural medicines that is currently receiving much attention.

Contents of the invention

The technical problem to be solved by the present invention is to provide a new species of Micromonospora capable of producing antibiotics.

To achieve the above object, the present invention adopts the following technical solutions:

A strain of Micromonospora hainanensis sp.nov. AM105 capable of producing antibiotics, the preservation number is CGMCC No.1551.

Micromonospora hainanensis CGMCC No.1551 is a wild actinomycete isolated from the seabed sediments of the South China Sea and obtained after strain selection.

Micromonospora hainanensis CGMCC No.1551 has the following microbial characteristics:

(1) Morphology and culture characteristics

This strain can form well-developed basal hyphae on various media, and the basal filaments are orange (Table 1). When cultured on oat juice medium, there are spore chains formed by one or more spores, the sporophore is of different lengths, and the spore surface is smooth and irregular in shape (Fig. 1 and Fig. 2).

Table 1 Culture characteristics of Micromonospora hainan culture medium aerial hyphae base hyphae Soluble pigment GYM Agar JCM Agar None orange red orange red None

Sauton's Agar Potato Juice Agar Oat Juice Agar Glycerol Asparagine Agar Gaoshi No. 1 Synthetic Agar Little, white, nothing, nothing, yellowish white Orange yellow, light orange red orange, light brown gradient light orange yellow orange, light brown gradient No no no no no no

(2) Physiological and biochemical characteristics

Among the nine commonly used carbon sources, only glucose is used, and L-arabinose, D-xylose, D-fructose, L-rhamnose, D-mannitol, sucrose, raffinose and inositol are not used. Does not liquefy gelatin, does not produce H ₂ S and melanoid, is positive for starch hydrolysis, can utilize cellulose, and is positive for nitrate reduction. Milk does not coagulate and does not peptonize.

(3) Analysis of chemical components of cell wall

The whole cell hydrolyzate contains meso-DAP (meso-diaminopimelic acid, Diaminopimelic acid) and glycine, cell wall type II; contains xylose, arabinose, and glycoform D.

(4) Micromonospora hainan of the present invention, its 16S rDNA sequence is as follows:

AGAGTTTGATCATGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGT

CGAGCGGAAAGGCCCTTCGGGGTACTCGAGCGGCGAACGGGTGAGTAACACGTG

AGTAACCTGCCCTAGGCTTTGGGATAACCCTCGGAAACGGGGGCTAATACCGGAT

ATGACCTTCTTCCGCATGGTGGGGGGTGGAAAGTTTTTCGGCCTGGGATGGGCTC

GCGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGC

CGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTA

CGGGGGGCAGCAGTGGGGAATATTGCACAATGGGCGGAAGCTGATGCAGCGACG

CCGCGTGAGGGATGACGGCCTTCGGGTTGTAAAACCTCTTCCAGCAGGGACGAAG

CGCAAGTGACGGTACCTGCAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCG

CGGTAAGACGTAGGGTGCGAGCGTTGTCCGGATTTACTGGGCGTAAAGAGCTCGT

AGGCGGCTTGTCGCGTCGACCGTGAAAACTTGGGGCTCAACCCCAAGCCTGCGG

TCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGACTGGAATTCCTGGTGTAGCGG

TGAAATGCGCAGACATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCC

GATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTG

GTAGTCCACGCTGTAAACGTTGGGCGCTAGGTGTGGGGGGCCTCTCCGGTTTCCT

GTGCCCGCACTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAA

AACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATT

CGATGCAACGCGAAGAACCTTACCTGGGTTTGACATGGCCACAAAACTTCCAGAG

ATGGGAGGTCCTTCGGGGGCGGTCACAGGTGGTGCATGGCTGTCGTCAGCTCGTG

TCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCGATGTTGCC

AGCGCGTTATGGCGGGGACTCATCGAAGACTGCCGGGGTCAACTCGGAGGAAGG

TGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACGCATGCTACA

ATGGCCGGTACAATGGGTTGCGATGCCGTGAGGTGGAGCGAATCCCAAAAAAGCC

GGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAG

TAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCG

CCCGTCACGTCACGAAAGTCGGCAACACCCGAAGCCGGTGGCCCAACCCTTGTG

GAGGGAGCCGTCGAAGGTGGGGCTGGCGATTGGGACGAAGTCGTAACAAGGTAA

CC.

The Micromonospora hainanensis CGMCC No.1551 involved in the present invention, through multiple classification and identification, is determined to be a new species of the genus Micromonospora, named as Micromonospora hainanensis (Micromonospora hainanensis sp. On December 5, 2010, the culture was preserved in the General Microorganism Center (CGMCC) of the China Committee for Culture Collection of Microorganisms, and it was proved to be alive. Its preservation registration number is CGMCC No.1551. The storage address is the Institute of Microbiology, Chinese Academy of Sciences, No. 13, North Yiyi Road, Zhongguancun, Haidian District, Beijing.

The invention has the advantages that the antibiotic produced by the micromonospora hainan has strong inhibitory effect on various G ⁺ and G ^- bacteria, especially obvious inhibitory effect on methicillin-resistant staphylococcus aureus and mycobacterium tuberculosis.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Description of drawings

Fig. 1 is the hyphae and sporocydium morphology of the strain of the present invention, 1500×.

Fig. 2 is the hyphae and sporocydium morphology of the strain of the present invention, 10000×.

Detailed ways

The present invention will be further described below in conjunction with specific embodiment:

Embodiment 1: Breeding of Micromonospora hainan CGMCC No.1551

Naturally isolated Micromonospora hainanensis was naturally selected to obtain a strain with large and full colonies, and the color was orange. Using this strain as the starting strain, after ultraviolet mutagenesis for 10-60s, a good mutagenesis effect was obtained, and a strain CGMCC No.1551 was obtained. The diameter of the inhibition zone of its fermentation liquid against MRSA increased from 19mm to 40mm, which is the starting point. strain 2.1 times.

Embodiment 2: the cultivation of Micromonospora hainan CGMCC No.1551

Training conditions:

(1) Incline culture: culture for 6 days. Medium: 0.4% yeast powder, 1% malt powder, 0.4% glucose, 2% agar, pH 7.2, culture temperature 28°C.

(2) Seed culture: pick the activated single colony from the slant and put it into the seed solution, and culture it in a shaker flask at 28° C. and 200 rpm for 48 hours. Medium: 1% glucose, 1% soybean powder, 1% yeast extract, 0.5% soluble starch, 0.05% KH ₂ PO ₄ , prepared from natural aged sea water, pH 7.2.

(3) Fermentation culture: 10% of the inoculum amount was inserted into the seed medium, and the shake flask was cultured for 6 days. Medium: 0.5% glucose, 1.5% starch, 1% soybean flour, 0.5% yeast extract, 0.025% K ₂ HPO ₄ , prepared with 50% natural aged seawater (fresh seawater after standing for a week) and 50% distilled water, initial pH Value 7.2, 28°C, shaker speed 200r/min.

Embodiment 3: the cultivation of Micromonospora hainan CGMCC No.1551

The activated CGMCC No.1551 was placed in a shake flask for seed culture, and the corresponding medium was the same as in Example 2, with the conditions that the temperature was 30° C., the shaker speed was 180 rpm, and cultured for 45 hours. Insert the fermentation medium with an inoculum size of 8%, and the medium is prepared with natural pure sea water, and cultured at 28°C and 200rpm for 7 days.

Example 4. Pharmacodynamic verification

1. Acquisition of antibiotics:

Carry out fermentation culture by the method for embodiment 3, fermented liquid carries out following processing:

1. Collect the fermentation product that was fermented on day 6, centrifuge at 8000r/min for 10min, and collect the supernatant.

2. Using an appropriate amount of ethyl acetate to extract the fermentation supernatant 4 to 5 times, combine the extracts and concentrate under reduced pressure to obtain crude antibiotics.

3. Using silica gel column chromatography, the eluent is: petroleum ether: ethyl acetate 6:1 → 3:1 → 1:1 → 100% ethyl acetate gradient elution, and the eluent is collected in sections.

4. Concentrate under reduced pressure, collect the active peak, and obtain a relatively pure antibiotic crude product through Sephadex LH20 column chromatography.

2. Antibacterial test

The minimum inhibitory concentration (MIC) of the antibiotic samples obtained by the plate double dilution method was determined for multiple detection strains, and the results are shown in Table 2. It can be seen that the antibiotics produced by Micromonospora hainanensis have a strong inhibitory effect on a variety of G+ bacteria, especially on methicillin-resistant Staphylococcus aureus and Mycobacterium tuberculosis, and also on G- bacteria A certain restraint.

Table 2 Determination of antibacterial activity of antibiotics produced by Micromonospora hainan Tested strain AM105-II (μg/mL) MRSA1MRSA2MRSA3MRSA4MRSA5MRSA6MRSA7MRSA8Bacillus subtiliSarcina luteaE.coliPseudomonus aeruginosaMycobacterium tuberculosisCandida albicansAspergillus niger ≤0.030.06≤0.030.12580.250.1250.125≤0.03≤0.0332640.06--

Note: MRSA1: resistance to methicillin, erythromycin, ciprofloxacin, amikacin; MRSA2: resistance to methicillin, erythromycin, tetracycline; MRSA3: resistance to methicillin, tetracycline, erythromycin, cycline Profloxacin, amikacin; MRSA4: resistance to methicillin, ciprofloxacin, amikacin, erythromycin; MRSA5: resistance to methicillin, tetracycline, erythromycin, ciprofloxacin; MRSA6: Resistance to methicillin, erythromycin, and tetracycline; MRSA7: resistance to methicillin, erythromycin, and tetracycline; MRSA8: resistance to methicillin, tetracycline, erythromycin, ciprofloxacin, and amikacin.

Sequence Listing

<110> Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences

<120>An antibiotic-producing Micromonospora hainanensis

<130>

<160>1

<170>PatentIn version 3.3

<210>1

<211>1475

<212> DNA

<213>Micromonospora hainanensis sp.nov

<400>1

agagtttgat catggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgagc 60

ggaaaggccc ttcggggtac tcgagcggcg aacgggtgag taacacgtga gtaacctgcc 120

ctaggctttg ggataaccct cggaaacggg ggctaatacc ggatatgacc ttcttccgca 180

tggtgggggg tggaaagttt ttcggcctgg gatgggctcg cggcctatca gcttgttggt 240

ggggtgatgg cctaccaagg cgacgacggg tagccggcct gagagggcga ccggccacac 300

tgggactgag acacggccca gactcctacg gggggcagca gtggggaata ttgcacaatg 360

ggcggaagct gatgcagcga cgccgcgtga gggatgacgg ccttcgggtt gtaaacctct 420

tccagcaggg acgaagcgca agtgacggta cctgcagaag aagcaccggc caactacgtg 480

ccagcagccg cggtaagacg tagggtgcga gcgttgtccg gatttactgg gcgtaaagag 540

ctcgtaggcg gcttgtcgcg tcgaccgtga aaacttgggg ctcaacccca agcctgcggt 600

cgatacgggc aggctagagt tcggtagggg agactggaat tcctggtgta gcggtgaaat 660

gcgcagacat caggaggaac accggtggcg aaggcgggtc tctgggccga tactgacgct 720

gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca cgctgtaaac 780

gttgggcgct aggtgtgggg ggcctctccg gtttcctgtg cccgcactaa cgcattaagc 840

gccccgcctg gggagtacgg ccgcaaggct aaaactcaaa ggaattgacg ggggcccgca 900

caagcggcgg agcatgcgga ttaattcgat gcaacgcgaa gaaccttacc tgggtttgac 960

atggccacaa aacttccaga gatgggaggt ccttcggggg cggtcacagg tggtgcatgg 1020

ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caaccctcgt 1080

tcgatgttgc cagcgcgtta tggcggggac tcatcgaaga ctgccggggt caactcggag 1140

gaaggtgggg atgacgtcaa gtcatcatgc cccttatgtc cagggcttca cgcatgctac 1200

aatggccggt acaatgggtt gcgatgccgt gaggtggagc gaatcccaaa aagccggtct 1260

cagttcggat cggggtctgc aactcgaccc cgtgaagtcg gagtcgctag taatcgcaga 1320

tcagcaacgc tgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacgtcacga 1380

aagtcggcaa cacccgaagc cggtggccca acccttgtgg aggagccgt cgaaggtggg 1440

gctggcgatt gggacgaagt cgtaacaagg taacc 1475

Claims (1)

1. A strain of Micromonospora hainanensis sp.nov. producing antibiotics, the preservation number is CGMCC No.1551.

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