CN113528388A - Streptomyces corallini symbiosis, method for producing actinomycin D by fermentation and application - Google Patents
Streptomyces corallini symbiosis, method for producing actinomycin D by fermentation and application Download PDFInfo
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- CN113528388A CN113528388A CN202110811350.6A CN202110811350A CN113528388A CN 113528388 A CN113528388 A CN 113528388A CN 202110811350 A CN202110811350 A CN 202110811350A CN 113528388 A CN113528388 A CN 113528388A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses a coral streptomyces symbiosis HNM0560, belongs to the field of fermentation of coral streptomyces symbiosis, and has a preservation number of CCTCCM 2019966. The invention also discloses a method for producing actinomycin D by fermenting the coral streptomyces symbiosis, which is obtained by fermenting streptomyces HNM 0560. The actinomycin D can be used for preventing and treating tilapia streptococcus agalactiae, and the minimum inhibitory concentration for preventing and treating tilapia streptococcus agalactiae is 0.25 mu g/ml. The yield of actinomycin D of the streptomyces HNM0560 provided by the invention can reach 58mg/l without any optimization of culture medium and fermentation conditions. Actinomycin D has stronger inhibitory activity to the activity of tilapia streptococcus agalactiae, and the streptomycete HNM0560 has good industrialization potential and application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a strain, in particular to a method for producing actinomycin D by fermentation of coral streptomyces symbiosis and application
Background
Coral is the first large invertebrate of the tropical sea. Coral surfaces and internal tissues contain dense and diverse microbial communities, and coral-associated microorganisms can produce a variety of structurally diverse compounds with a wide range of biological activities, including antibacterial compounds against a variety of pathogens, and are becoming an important source for antimicrobial drug screening.
Actinomycin D (Actinomycin D) is a polypeptide antibiotic found and isolated from Streptomyces parvulus (Streptomyces parvulus) in Waksman et al in the 40 th century in 19 th century. Thereafter, actinomycin D was found from various species of Streptomyces. Examples thereof include Streptomyces griseus (S.griseus), Streptomyces fradiae (S.fradiae), Streptomyces antibioticus (S.antibioticus), and Streptomyces aureobasicus (S.chrysomolus). Actinomycin D initially showed antibacterial activity. However, in the further development process of research, actinomycin D shows excellent antitumor activity and has good effect on the clinical medicine aspect of treating certain malignant tumors, so that actinomycin D is mainly applied as an anticancer drug at present.
In recent years, the antibacterial activity of actinomycin D has attracted attention, and it has been reported that actinomycin D has a relatively strong resistance to some pathogenic bacteria such as gram-positive bacteria, and has a wide application prospect in the research of antibacterial drugs. Streptococcus agalactiae (Streptococcus agalactiae) is a gram-positive pathogen of Streptococcus, can cause meningitis, pneumonia, septicemia and other diseases of human bodies, various terrestrial and aquatic organisms, and has high pathogenic and lethal rates. In recent years, streptococcus agalactiae diseases frequently outbreak in the field of aquatic products, wherein the streptococcus agalactiae diseases of tilapia are the most serious, and the streptococcus agalactiae diseases of tilapia form serious threats to the healthy culture of tilapia and the sustainable development of the tilapia industry. Therefore, the development of antibacterial drugs or probiotics aiming at the bacteria has important application prospect.
Disclosure of Invention
The invention aims to provide coral symbiotic streptomyces.
In order to achieve the purpose, the technical scheme of the invention is as follows: providing a coral Streptomyces which is Streptomyces (Streptomyces sp) HNM0560, being preserved in China Center for Type Culture Collection (CCTCC) in 11 months and 22 days in 2019, wherein the preservation number of the strain is CCTCC M2019966, and the preservation addresses are as follows: eight-path Lojia mountain in Wuchang region of Wuhan city, Hubei province.
The invention also aims to provide a method for producing actinomycin D by fermentation of coral streptomyces symbiosis, which comprises the following steps:
s1, culturing HNM0560 seed liquid;
s2, inoculating the seed liquid of HNM0560 obtained by culture into a fermentation culture medium for fermentation culture to obtain fermentation liquid;
s3, separating actinomycin D from the fermentation liquor.
Further, in step S2, the fermentation medium includes soluble starch, yeast extract, peptone, tap water, and seawater, and has a pH of 7.0.
Further, 8-15 g of soluble starch, 2-8 g of yeast extract powder, 1-5 g of peptone, 400-600 ml of tap water and 400-600 ml of seawater.
Further, in step S2, the fermentation conditions are 26-30 ℃, 150-180 rpm, and 5-7 days.
Further, the fermentation conditions were 28 ℃, 180rpm, 7 days.
Further, the method for producing actinomycin D by fermenting the coral streptomyces symbiosis also comprises the following steps:
s3-1, filtering the fermentation liquor to obtain thalli;
s3-2, leaching the thalli with acetone, and evaporating to dryness under reduced pressure to obtain actinomycin D crude extract;
s3-3, separating the actinomycin D crude extract by LH-20 gel column chromatography (eluting with 80% methanol water solution) to obtain actinomycin D.
Further, the maximum yield of the actinomycin D reaches 58 mg/L.
The invention further aims to provide application of actinomycin D in controlling tilapia streptococcus agalactiae.
Furthermore, the minimum inhibitory concentration for preventing and treating tilapia streptococcus agalactiae is 0.25 mug/ml, which is better than that of positive drug tobramycin (32 mug/ml).
The yield of actinomycin D of the streptomyces HNM0560 provided by the invention can reach 58mg/l without any optimization of culture medium and fermentation conditions. The method for producing actinomycin D provided by the invention is streptomyces fermentans HNM0560, the fermentation is stable, and the compound separation process is simple and convenient. The actinomycin D has stronger inhibitory activity to the activity of tilapia streptococcus agalactiae, and provides new activity of the compound. The streptomycete HNM0560 has good industrialization potential and application prospect.
Detailed Description
The following is a detailed description of the embodiments of the present invention, which is implemented on the premise of the technical solution of the present invention, and the detailed implementation and specific operation procedures are given, but the protection scope of the present invention is not limited to the following embodiments. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Isolation and identification of Streptomyces (Streptomyces) HNM0560
1. Isolation of Streptomyces HNM0560
The strain separation culture medium comprises 10g of soluble starch, 0.3g of casein, 2g of potassium nitrate, 0.05g of magnesium sulfate heptahydrate, 2g of sodium chloride, 2g of dipotassium phosphate, 0.02g of calcium carbonate, 0.01g of ferrous sulfate heptahydrate, 0.1g of potassium dichromate, 500ml of distilled water, 500ml of aged seawater, 20g of agar and pH 7.2-7.4.
Weighing 1g of coral sample (collected from Wenchang Hainan sea area), grinding with 10 ml of sterile seawater, diluting with 10 times, 100 times and 1000 times of sterile seawater, respectively sucking 100 mul of diluent, coating on a separation culture medium plate, and inversely culturing in a constant temperature incubator at 28 ℃ for 1-4 weeks. And (4) picking out the actinomycete colonies on the plate by using a sterile bamboo stick according to the morphological characteristics of the colonies on the plate for purification. A strain is obtained and named as strain HNM 0560.
A single colony of the purified strain HNM0560 was picked and stored in sterile 20% glycerol in a refrigerator at-20 ℃.
2. Identification of Streptomyces HNM0560
The 16S rRNA gene sequence is amplified by adopting the universal primers 27F and 1492R PCR, and the length of the effective fragment of the 16S rRNA gene sequence of the strain HNM0560 is measured to be 1491 bp. Sequence similarity search of the available species was performed in the EzTaxon database, and the strain HNM0560 was found to be highly related to the strain of streptomyces, indicating that the strain HNM0560 is an actinomycete of streptomyces.
The strain HNM0560 was identified as Streptomyces according to 16S rRNA gene sequence analysis.
The streptomyces HNM0560 has been preserved in China Center for Type Culture Collection (CCTCC) in 2019 at 11 and 22 months, and the preservation number is CCTCC M2019966.
Production of actinomycin D by using streptomycete HNM0560
Secondly, producing actinomycin D by applying streptomycete HNM0560
The preparation method of the seed culture medium is as follows (pH7.5): 4g of yeast extract powder, 10g of malt extract powder, 4g of glucose, 500mL of tap water and 500mL of old seawater are added to the solution until the volume is IL. Sterilizing at 121 deg.C for 20 min.
The preparation method of the fermentation medium is as follows (pH9.0): 10g of soluble starch, 4g of yeast extract powder, 2g of peptone, 500ml of tap water, 500ml of seawater and pH 7.0. The volume is fixed to IL. Sterilizing at 121 deg.C for 20 min.
1. Fermentation of bacterial strains
1.1 seed culture
Streptomyces HNM0560 was inoculated into 100ml of seed medium. Culturing at 28 deg.C and 180rpm for 96 hr to obtain seed solution.
1.2 fermentation culture
(1.2-1) 200ml of fermentation medium was placed in a 500ml Erlenmeyer flask.
(1.2-2) inoculating 2ml of seed solution into the fermentation medium, culturing at 28 ℃ and 180rpm for 7 days, and harvesting the fermentation broth.
2. Separation and purification of effective components
2.1, filtering 1L of the fermentation solution by using 3 layers of gauze to obtain the thalli.
2.2, leaching the bacteria with 200ml of acetone (standing overnight at room temperature), filtering with 3 layers of gauze, collecting filtrate, and evaporating to dryness under reduced pressure to obtain a crude extract, namely the crude extract of actinomycin D.
2.3, separating the actinomycin D crude extract by LH-20 gel column chromatography (eluting by 80% methanol aqueous solution) to obtain actinomycin D. HPLC analysis is carried out.
HPLC analysis parameters adopted YMC-Pack C18, 150X 4.6mmL.D., S-5 μm.12nm. The mobile phase is methanol water solution gradient elution, and the flow rate is 1.0 ml/min; the detection wavelength was 240 nm.
The retention time of the HPLC main active ingredient was about 17.8 min.
3. Identification of active ingredients
3.1, appearance
Red powder.
3.2 solubility
Is easily soluble in methanol, ethanol, acetone and DMSO, and is insoluble in water.
3.3 Mass Spectrometry
ESI-MS m/z:1255.6302[M-H]-Molecular formula C62H86N12O6Consistent with the results reported in the literature for actinomycin D.
3.4 ultraviolet spectrum
The ultraviolet spectrum testing instrument is Mariner System5304 instruments.
The active component has maximum absorption peaks at 201, 240 and 445nm of ultraviolet spectrum. Consistent with the ultraviolet characteristics of actinomycin D reported in the literature.
3.5 Nuclear magnetic map Hydrocarbon Signal attribution
The NMR analyzer was Varian nova 600MHZ and the solvent used was DMSO-d 6. The active component hydrocarbon signal assignments are shown in tables 1 and 2. Consistent with literature reports.
Tables 1 and 2 nuclear magnetic signal assignments for the hydrocarbon spectra of the active components:
combining the above results, the structural formula of the active component is shown as formula (I), and the active component is actinomycin D.
Formula (I)
Thirdly, detecting the activity of the compound against tilapia streptococcus agalactiae
Bovine brain culture medium: 38.5 g of brain-heart infusion broth, 1000ml of tap water and pH 7.4-7.6. After dissolution, sterilization was carried out at 121 ℃ for 20 minutes.
1. Preparation of bacterial liquid
Counting by a blood counting plate, and preparing tilapia streptococcus agalactiae into (3-6) multiplied by 10 by using a bovine brain culture medium6CFU/mL of bacterial liquid.
2. Preparation of solutions to be tested
The compound and the positive drug tobramycin are prepared into a mother solution of 1280 mu g/ml by taking sterile DMSO as a solvent. Then, the mixture was sequentially diluted with sterile DMSO to give dilutions having concentrations of 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5, and 1.25. mu.g/ml.
3. Determination of minimal inhibitory concentration of compound in inhibiting tilapia streptococcus agalactiae
3.1, taking a sterile 96-well cell culture plate, adding into each well: 80ul bovine brain medium.
3.2, taking the 96-well cell culture plate which completes the step 3.1, and grouping the 96-well cell culture plates as follows:
positive control group (11 wells): respectively adding 20ul of 11 dilutions of the positive control drug diluent prepared in step 2;
experimental group (11 wells): respectively adding 20ul of 11 dilutions of the compound prepared in step 2;
negative control (11 wells): 20ul of sterile DMSO was added separately.
3.3, taking a 96-well cell culture plate which finishes the step 3.2, adding 100ul of the bacterial liquid obtained in the step 1 into each well, culturing at 28 ℃ for 48 hours, and observing the growth condition of the bacteria in each well: if the hole is turbid, the corresponding concentration of the compound does not have the anti-tilapia streptococcus agalactiae activity; if the well is clear, the corresponding concentration of the compound is proved to have the anti-tilapia streptococcus agalactiae activity. The minimum final concentration of the compound corresponding to the hole in which the growth of the tilapia streptococcus agalactiae is completely inhibited is the minimum inhibitory concentration and MIC value of the compound to the tilapia streptococcus agalactiae.
The MIC of actinomycin D for resisting the activity of tilapia streptococcus agalactiae is 0.25 mu g/ml, and the MIC of the positive drug tobramycin is 32 mu g/ml. The activity of the tilapia streptococcus agalactiae of actinomycin D is better than that of a positive medicine.
TABLE 1 carbon spectrum NMR Signal assignment of active Components
TABLE 2 hydrogen spectrum NMR Signal attribution of active Components
The scope of the invention is defined by the appended claims, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. A coral symbiotic streptomyces is characterized in that: the Streptomyces corallinus is Streptomyces corallinus sp HNM0560, which is preserved in China center for type culture collection (CCTCC M2019966) in 2019, 11 months and 22 days.
2. A method for producing actinomycin D by fermentation of Streptomyces corallinus according to claim 1, comprising the steps of:
s1, culturing HNM0560 seed liquid;
s2, inoculating the seed liquid of HNM0560 obtained by culture into a fermentation culture medium for fermentation culture to obtain fermentation liquid;
s3, separating actinomycin D from the fermentation liquor.
3. The method for producing actinomycin D by fermentation of Streptomyces corallinus according to claim 2, characterized in that: in step S2, the fermentation medium includes soluble starch, yeast extract, peptone, tap water, and seawater, and has a pH of 7.0.
4. The method for producing actinomycin D by fermentation of Streptomyces corallinus according to claim 3, characterized in that: 8-15 g of soluble starch, 2-8 g of yeast extract powder, 1-5 g of peptone, 400-600 ml of tap water and 400-600 ml of seawater.
5. The method for producing actinomycin D by fermentation of Streptomyces corallinus according to claim 2, characterized in that: in step S2, the fermentation conditions are 26-30 ℃, 150-180 rpm, 5-7 days.
6. The method for producing actinomycin D by fermentation of Streptomyces corallinus according to claim 5, characterized in that: the fermentation conditions were 28 ℃, 180rpm, 7 days.
7. The method for producing actinomycin D by fermentation of Streptomyces corallinus according to claim 2, further comprising the steps of:
s3-1, filtering the fermentation liquor to obtain thalli;
s3-2, leaching the thalli with acetone, and evaporating to dryness under reduced pressure to obtain actinomycin D crude extract;
s3-3, separating the actinomycin D crude extract by LH-20 gel column chromatography to obtain actinomycin D.
8. The method for producing actinomycin D by fermentation of Streptomyces corallinus according to claim 2 or 7, characterized in that: the maximum yield of the actinomycin D reaches 58 mg/L.
9. The use of actinomycin D according to claim 2 for the control of tilapia streptococcus agalactiae.
10. The use of actinomycin D according to claim 9 for the control of tilapia streptococcus agalactiae characterised in that: the minimum inhibitory concentration for preventing and treating tilapia streptococcus agalactiae is 0.25 mug/ml.
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