CN104450580B - Preparation method and application of actinomycin D - Google Patents
Preparation method and application of actinomycin D Download PDFInfo
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Abstract
The invention discloses a novel actinomycete strain for producing actinomycin D, Streptomyces mutabilis Streptomyces mutabilis TRM45540 is preserved in U.S. department of Agricultural Culture resource (ARS) Culture Collection in 4-14 th month in 2014, the Collection number is NRR L B-59117, and simultaneously, a seed Culture medium and a fermentation Culture medium for efficiently producing actinomycin D by using the strain and a preparation process for extracting and separating the actinomycin D from fermentation liquor are also disclosed, and an antibacterial activity test shows that the actinomycin D produced by the strain and the fermentation thereof has broad-spectrum and high-efficient activity on pathogenic bacteria such as xanthomonas gossypii, pythium Juglandis, staphylococcus aureus, staphylococcus epidermidis, candida albicans and the like, and has important application value and good application and development prospect on prevention and control of diseases such as cow mastitis, vaginitis and the like of crops such as cotton and walnut.
Description
Technical Field
The invention relates to the field of agriculture and medicine, in particular to a new application of actinomycin D, a new strain capable of producing actinomycin D and a preparation method for producing actinomycin D by using the strain.
Background
The two strains of Smelanochromogens No.1779 and Streptomyces parvulus can produce actinomycete D, and the main action mechanism is to block the function of RNA polymerase, inhibit the synthesis of RNA, especially mRNA, to block the synthesis of protein and inhibit the growth of tumor, and to combine with radiotherapy to raise the sensitivity of tumor to radiotherapy.
Cotton wilt and cotton verticillium wilt are the most main diseases damaging cotton production, and in recent years, cotton wilt and cotton verticillium wilt are becoming more and more serious in cotton areas of China. However, no effective agent for controlling cotton wilt and cotton verticillium wilt exists at present. The search for biopesticides from microbial resources is one of the effective ways, and agricultural antibiotics produced by actinomycetes are the most important sources. The research and the popularization of biological pesticides such as validamycin, abamectin, Qinling mycin and the like fully prove the rationality and the superiority of screening economic and efficient agricultural antibiotics from metabolites of actinomycetes. At present, registered antibiotics for preventing and treating cotton verticillium wilt are not reported.
Therefore, it is the main object of the present invention to screen high-efficiency antibiotics for prevention and treatment of cotton wilt and cotton verticillium wilt from actinomycetes.
Disclosure of Invention
The invention aims to solve the technical problem of providing an actinomycin D producing actinomycete, which is Streptomyces mutabilis (TRM 45540) and is preserved in U.S. department of Agricultural Research Service (ARS) Culture Collection (address: U.S. department of Agricultural, Pelora, I L61604 USA) on 4-14 th month in 2014, wherein the actinomycete is NRR L B-59117 and survives through detection.
The actinomycete is applied to antibiosis, and is used for preventing and treating cotton fusarium wilt and verticillium wilt and walnut pythium rot plant pathogenic bacteria, or used for preventing and treating staphylococcus aureus and staphylococcus epidermidis as cow mastitis pathogenic bacteria, or used for preventing and treating candida albicans as female vaginitis pathogenic bacteria.
The application of the actinomycete in producing actinomycin D. The application is characterized by comprising the following steps: culturing the actinomycetes under a proper condition to obtain a fermentation liquid; separating and purifying actinomycin D from the fermentation liquor.
The culture medium formula and culture conditions for culturing the actinomycetes comprise corn flour 15 g/L (boiling for 20min, filtering with four layers of gauze to obtain juice), peptone 5 g/L, and glucose 5 g/L31 g/L40 g/L, adjusting pH to 7.2-7.4, inoculating 6% of the inoculum size into a fermentation medium, and performing shake culture at 28 deg.C and 180rpm for 7 days.
The method for separating and purifying actinomycin D from fermentation liquor specifically comprises the following steps: after the fermentation liquor is filtered, loading the fermentation liquor to D101 or AB-8 macroporous resin chromatographic column macroporous resin by a wet method; then eluting with clear water, 10% ethanol solution, 30% ethanol solution, 50% ethanol solution, 70% ethanol solution and 95% ethanol solution with 2 times of column volume respectively; mixing 70-95% ethanol eluates, concentrating under reduced pressure to remove solvent to obtain 70-95% ethanol extract of fermentation broth rich in actinomycin D.
The method for separating and purifying actinomycin D from fermentation liquor further comprises the following steps: drying 70-95% ethanol extract of fermentation liquid, ultrasonic dissolving with methanol at normal temperature, dissolving methanol soluble part with acetone, recovering acetone solvent from acetone soluble part to obtain orange red powder with actinomycin D content of above 90%.
The method for separating and purifying actinomycin D from the fermentation liquor further comprises the following steps of dissolving orange red powder obtained by acetone extraction with methanol, then passing the orange red powder through a Sephadex L H-20 chromatographic column, collecting and combining brown yellow to orange red parts, and recovering a methanol solvent to obtain an orange red actinomycin D pure product with the purity of more than 98%.
The invention has the following beneficial effects: streptomyces mutabilis TRM45540 and actinomycin D, a main metabolite of the Streptomyces mutabilis TRM45540 have broad-spectrum and high-efficiency antagonistic activity on pathogenic bacteria such as cotton fusarium verticillium, walnut pythium, staphylococcus aureus, staphylococcus epidermidis, candida albicans and the like, have important application values on safe production of crops such as cotton, walnuts and the like and prevention and control of diseases such as cow mastitis, female vaginitis and the like, and can be used for developing high-efficiency biological pesticides and biological medicines.
Drawings
FIG. 1 taxonomic status and phylogenetic tree of Streptomyces mutabilis TRM 45540;
FIG. 2 Electron micrograph of Streptomyces mutabilis TRM45540 (showing spiral sporotriches and oval spores);
FIG. 3 chemical structure of actinomycin D;
FIG. 4 of actinomycin D1HNMR spectrogram;
FIG. 5 of actinomycin D13CNMR spectrogram;
FIG. 6 HSQC spectrum of actinomycin D;
FIG. 7 HMBC spectrum of actinomycin D.
Detailed Description
The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.
Example A Source and isolation culture of Streptomyces mutabilis TRM45540
A coating plate method is adopted to culture 8 culture mediums containing 5%, 10%, 15%, 20% and 25% of sodium chloride at a constant temperature of 28 ℃ from 0-30cm soil samples of ancient city of Loulan (with an altitude of 1400m, an east diameter of 89 degrees 22 '22 degrees, and a north latitude of 40 degrees 29' 55 degrees) in Xinjiang, 35 actinomycetes of 7 genera are separated out altogether, and the biocontrol bacterium TRM45540 with the best antibacterial effect is finally separated and screened through primary screening by a plate confronting method and secondary screening by a tube-disc method.
Example identification of Streptomyces changensis TRM45540
In order to further determine the taxonomic status of the biocontrol bacterium TRM45540, the strain is classified and identified by adopting the traditional classification method and the molecular classification method.
1 Medium for morphological examination
Inorganic salt starch agar (ISP4) culture medium, starch 10 g. L-1,(NH4)2SO44g·L-1,MgSO42g·L-1,K2HPO42g·L-1,CaCO31g·L-1,NaCl 40g·L-1Agar 18g L-1,pH 7.2-7.4。
2 primer
The primer was synthesized by Olympic Biotechnology (Beijing) Ltd. The sequences are respectively as follows: 27F (5'-AGAGTTTGATCCTGGCTC-3') and 1492R (5'-CGGCTACCTTGTTACGACTT-3')
3 test method
3.1 morphological Observation
And (3) observation by a common microscope: the biocontrol strain TRM45540 was cultured at 28 ℃ by the plug-in method. Observing and recording the hypha form, the growth conditions of aerial hyphae and substrate hyphae, and whether the hyphae produce spore filaments and the arrangement mode and the shape of the spore filaments by using an optical microscope; spore shape and size; the presence or absence, shape, size and mode of formation of spores.
3.2 molecular biological identification of biocontrol bacterium TRM45540
(A) Extraction of biocontrol bacterium TRM45540 genome DNA
The TRM45540 cells on the culture plate were collected and placed in a 1.5m L sterile centrifuge tube, and 480. mu. L was added
1 × TE buffer 20. mu. L lysozyme (50 mg. m L) was added-1) Put into a shaker at 37 ℃ and shaken at 200rpm overnight, 50 mu L20% SDS was added to each tube, and 5 mu L20 mg · m L was added-1The protease K is put into a shaking table at 60 ℃, shaken at 200r/min for 1h, added with phenol of 550 mu L, chloroform and isoamylol (25:24:1), centrifuged at 12000rpm for 10min, taken supernatant is transferred into another centrifuge tube, repeatedly extracted for 2-3 times, taken supernatant is added with absolute ethyl alcohol with the same volume, and added with 0.1 time of volume of sodium acetate (3mol L)-1) The DNA is put into a refrigerator at 4 ℃ for precipitating the DNA for about 0.5 h.12000rpm and centrifuging for 10min, the supernatant is discarded, the centrifuged product is washed for 2 times by 70 percent ethanol with 200 mu L, the centrifuged product is centrifuged for 5min at 12000rpm, the supernatant is discarded, the ethanol is completely volatilized, the DNA at the bottom is fully dissolved by sterile ultrapure water with 50 mu L, the DNA extraction quality is detected by 1 percent agarose gel electrophoresis, and the extracted DNA is put into the refrigerator at-20 ℃ for storage and standby.
(B) Amplification of biocontrol bacterium TRM4554016S rDNA gene
A16S rDNA gene fragment in the genomic DNA of actinomycetes was amplified using the actinomycetes 16S rDNA gene universal primers 27F (5'-AGAGTTTGATCCTGGCTC-3') and 1492R (5'-CGGCTACCTTGTTACGACTT-3').
25 mu L PCR reaction system of ddH2O20.4. mu. L, 10 × Buffer (Buffer containing Mg)2+) 2.5. mu. L. mu. L, primer 27F (10. mu. mol. L)-1) 0.5. mu. L, primer 1492R (10. mu. mol. L)-1)0.5 mu L, Taq DNA polymerase 0.1 mu L, template DNA 0.5 mu L.
The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 deg.C for 1min, annealing at 56 deg.C for 1min, extension at 72 deg.C for 2min, and 30 cycles; total extension 72 ℃ for 8 min. After the reaction was completed, the detection was carried out by 1% agarose gel electrophoresis. And (4) carrying out sequence determination on qualified PCR products.
(C) Alignment analysis of sequencing results
The sequencing result is spliced by DNAMAN5.2 software, the sequence is compared with the sequence of the strain which has been effectively published in a GenBank database through B L AST, the 16S rDNA gene sequence of the strain which has been effectively published with higher similarity is downloaded, the MEGA5.0 software is used for constructing a phylogenetic tree for the sequence, and the taxonomic position of actinomycetes is determined.
4 results of the test
4.1 morphological observation results of biocontrol bacterium TRM45540
The biocontrol bacterium TRM45540 grows well on an ISP4 culture medium, a colony is round and flat, the surface is dry, the edge of the colony is neat, aerial hyphae are rich, the spore pile is white, the intrabasal hyphae are yellow, and yellow soluble pigment is generated.
The biocontrol bacterium TRM45540 is gram-positive in staining, has the optimal growth temperature of 37 ℃, the optimal growth pH of 7.2 and the optimal NaCl (W/V) of 5 percent, is cultured on an ISP4 culture medium at 37 ℃ for 7d in an inserting sheet mode, and is observed through an optical microscope to find that: the intrabasal hyphae of the biocontrol bacterium TRM45540 have a plurality of branches and no transverse septa; the aerial mycelium is rich, the aerial mycelium forms spore chains with different lengths, the spores grow in strings, and spore filaments are straight. According to the determination of colony, thallus morphology and physiological characteristics of the biocontrol bacterium TRM45540, the biocontrol bacterium TRM45540 belongs to Streptomyces (Streptomyces).
4.2 molecular biology identification results of biocontrol bacterium TRM45540
4.2.1 determination of 16S rDNA sequence of biocontrol bacterium TRM45540
The sequencing result is spliced by DNAMAN5.2 software, the fragment is determined to be composed of 1404 bases, and the 16S rDNA sequence of the biocontrol bacterium TRM45540 is obtained as follows:
AATCGCCAGTCCCACCTTCGACAGCTCCCTCCCACAAGGGGTTGGGCCACCGGCTTCGGGTGTTACCGACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCGACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACCGGCTTTTTGAGATTCGCTCCACCTCGCGGTATCGCAGCTCATTGTACCGGCCATTGTAGCACGTGTGCAGCCCAAGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCTCCCGTGAGTCCCCAGCACCACAAGGGCCTGCTGGCAACACGGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTACACCGACCACAAGGGGGACCCTGTCTCCAGGGTTTTCCGGTGTATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAGCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCACTTAATGCGTTAGCTGCGGCACGGACAACGTGGAATGTTGCCCACACCTAGTGCCCACCGTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTATCGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCGATCTCCCCTACCGAACTCTAGCCTGCCCGTATCGACTGCAGACCCGGGGTTAAGCCCCGGGCTTTCACAACCGACGTGACAAGCCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACGCTTGCGCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGCGCTTCTTCTGCAGGTACCGTCACTTTCGCTTCTTCCCTGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTGATAGGCCGCGGGCTCATCCTGCACCGCCGGAGCTTTCGAACCTCGCAGATGCCTGCGAGGGTCAGTATCCGGTATTAGACCCCGTTTCCAGGGCTTGTCCCAGAGTGCAGGGCAGATTGCCCACGTGTTACTCACCCGTTCGCCACTAATCCCCACCCGAAGGTGGTTCATCG
4.2.2 homologous evolutionary Tree construction
Through B L AST comparison, downloading a sequence which is relatively similar to the 16S rDNA gene sequence of the actinomycetes to be detected in a GenBank database, performing multiple sequence comparison on the 16S rDNA gene sequence of the actinomycetes by MEGA5.0 software to construct a phylogenetic tree, wherein the biocontrol bacterium TRM45540 evolutionary tree is shown in a figure 1-1, and the biocontrol bacterium TRM45540 and the 16S rDNA gene sequence (with the consistency of 99.87%) of Streptomyces mutabilis are gathered on the same phylogenetic branch, so that the biocontrol bacterium is identified as the Streptomyces mutabilis TRM 45540.
EXAMPLE III fermentation and preparation of actinomycin D Using Streptomyces mutabilis TRM45540
Inoculating a small amount of mycelia in an ISP4 culture medium, culturing at 28 ℃ for 4 days to obtain a seed solution of streptomyces mutabilis TRM45540, inoculating 2m L (8% of inoculum size) seed solution into a corn flour fermentation culture medium for fermentation and culture under the conditions that the rotation speed is 180rpm, the initial pH is 7.2-7.4, the liquid loading amount is 250m L, the fermentation temperature is 28 ℃, fermenting for 7 days, collecting fermentation liquor, filtering the fermentation liquor by using four layers of gauze, loading the fermentation liquor on a macroporous resin chromatographic column by a wet method (every 100L of fermentation filtrate is filled with D101 or AB-8 macroporous resin 2.0kg), then respectively using clear water, 10% ethanol solution, 30% ethanol solution, 50% ethanol solution, 70% ethanol solution and 95% ethanol solution for elution, combining 70-95% ethanol, removing the solvent by decompression concentration to obtain 70-95% ethanol extract rich in actinomycin D, drying the 70-95% ethanol extract, firstly using methanol for ultrasonic dissolution at normal temperature, then using acetone for dissolution, recovering acetone soluble acetone part of acetone, obtaining orange powder, and then using Sephadex to obtain orange red acetone, and extracting orange red.
Example quantitative analysis and structural characterization of Tetraactinomycin D
(1) Quantitative analysis method of actinomycin D
Quantitative analysis of actinomycin D by high performance liquid chromatography under the conditions of 4.6mm × 150mm C18 chromatographic column (0.5 μm) and 80% methanol-water as mobile phase at flow rate of 1.0m L. min-1The detection wavelengths are 254nm,280nm,330nm and 445 nm.
(2) Structural characterization of actinomycin D
TABLE 1 of actinomycin D1H NMR and13c NMR spectrum data
Example antibacterial Activity assay of Streptomyces pentalabile TRM45540 fermentation broth and actinomycin D
(1) The cultured pathogenic fungi spores are eluted from a culture plate by sterile water to prepare a spore suspension with 106 and 107 spores/m L, the spore suspension with 100 mu L is absorbed on a PDA (personal digital assistant) plate, the bacterial suspension is uniformly coated by a sterilized coating rod and is slightly dried on a super clean bench, Oxford cups are arranged at equal distance on the bacteria-carrying plate, 200 mu L of purified actinomycin D is added into each Oxford cup, the Oxford cups in the same plate are added according to the concentration gradient of the purified actinomycin D, the solvent acetone of the actinomycin D (which has no inhibitory effect on pathogenic bacteria) is used as a contrast, the bacteria inhibition ring diameter is measured by a cross method at 28 ℃ for 4-5D, the bacteria inhibition ring diameter is measured by the cross method, the treatment is repeated for 3 times, the plant pathogenic fungi (including fusarium wilt of cotton, penicillium botrytis, fusarium oxysporum and pythium Juglandis) are used for carrying out bacteriostatic experiments on a PDA (PDA) culture medium, and the pathogenic bacteria (Staphylococcus aureus and Staphylococcus epidermidis) of cow mastitis are used for carrying out bacteriostatic experiments on a L B culture medium.
(2) Antagonism of actinomycin D against Staphylococcus aureus and Staphylococcus epidermidis
Selecting single pathogenic bacteria colony in L B culture medium, activating overnight, transferring the bacteria colony into fresh L B culture medium at 1/100 concentration, culturing at 37 deg.C until OD value is 0.4-0.6 to obtain bacterial suspension of pathogenic bacteria, sucking 100 μ L bacterial suspension onto L B plate, coating the bacterial suspension with sterilized coating rod, slightly drying on ultra-clean bench, placing Oxford cups at equal distance on the bacteria-carrying plate, sequentially adding 200 μ L purified actinomycin D into each Oxford cup at concentration of 10 mg.m L-1,1mg·mL-1,0.1mg·mL-1,0.01mg·mL-1Culturing at 37 deg.C overnight with fermentation medium as control, and crossing methodThe zone of inhibition diameter was measured and repeated 3 times per treatment.
(3) Antagonism of actinomycin D against Candida albicans
Eluting cultured pathogenic fungus spores from a culture plate by using sterile water to prepare 106-107 spores/m L spore suspension, sucking 100 mu L spore suspension onto an SDA plate, uniformly coating the bacterial suspension by using a sterilized coating rod, slightly drying the bacterial suspension on an ultraclean workbench, placing oxford cups at equal distance on the bacteria-carrying plate, sequentially adding 200 mu L purified actinomycin D with different concentration gradients into each oxford cup, wherein the concentration is respectively 10 mg. L-1,1mg·mL-1,0.1mg·mL-1,0.01mg·mL-1The diameter of the zone of inhibition was measured by a cross method using fermentation medium as a control and cultured overnight at 28 ℃ and repeated 3 times per treatment.
The results of the above experiments can be seen in Table 2.
TABLE 2 antibacterial Effect of actinomycin D
Claims (4)
1. Application of actinomycin D producing actinomycin D in producing actinomycin D, wherein the strain is streptomyces mutabilis (Streptomyces shibatae)Streptomyces mutabilis) The TRM45540 which is deposited in U.S. Department of Agricultural Research Service (ARS) Culture Collection on 14 th month 4 in 2014 and has the Collection number of NRR L B-59117;
the method comprises the following steps: culturing the actinomycetes under a proper condition to obtain a fermentation liquid; separating and purifying actinomycin D from the fermentation liquor;
wherein the culture medium formula and culture conditions for culturing the actinomycetes comprise corn flour 15 g/L, peptone 5 g/L, and glucose 5 g/L31 g/L40 g/L, adjusting pH to 7.2-7.4, boiling corn flour for 20min, filtering with four layers of gauze to obtain juice, inoculating 6% of the strain into fermentation medium, and shake culturing at 28 deg.C and 180rpm for 7 days.
2. The use of claim 1, wherein: the method for separating and purifying actinomycin D from fermentation liquor specifically comprises the following steps:
after the fermentation liquor is filtered, loading the fermentation liquor to D101 or AB-8 macroporous resin chromatographic column macroporous resin by a wet method; then eluting with clear water, 10% ethanol solution, 30% ethanol solution, 50% ethanol solution, 70% ethanol solution and 95% ethanol solution with 2 times of column volume respectively; mixing 70-95% ethanol eluates, concentrating under reduced pressure to remove solvent to obtain 70-95% ethanol extract of fermentation broth rich in actinomycin D.
3. Use according to claim 2, characterized in that: the method for separating and purifying actinomycin D from fermentation liquor further comprises the following steps: drying 70-95% ethanol extract of fermentation liquid, ultrasonic dissolving with methanol at normal temperature, dissolving methanol soluble part with acetone, recovering acetone solvent from acetone soluble part to obtain orange red powder with actinomycin D content of above 90%.
4. The use according to claim 3, wherein the method for separating and purifying actinomycin D from a fermentation broth further comprises the steps of dissolving orange-red powder obtained by acetone extraction with methanol, passing the solution through Sephadex L H-20 chromatographic column, collecting and combining the brown-yellow to orange-red parts, and recovering methanol solvent to obtain an orange-red actinomycin D pure product with a purity of 98% or more.
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