2,5-diketopiperazine dipeptide derived from lysobacter enzymogenes as well as preparation method and application thereof
A technology of diketopiperazines and enzyme-producing lysobacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, and medical preparations containing active ingredients
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Embodiment 1
[0055] Example 1: Isolation and purification of endophytic bacteria Lysobacter enzymogenes R-2-1
[0056] The composition of the final concentration of double-antibody WA medium: 200IU / mL amphotericin B, 150IU / mL streptomycin, 20g agar, 1L distilled water, natural pH.
[0057] LB medium final concentration composition: yeast extract 5g / L, tryptone 10g / L, NaCl 10g / L, agar 20g / L, solvent is water, pH7.4.
[0058] Take fresh and healthy Artemisia annua roots (Artemisia annua Linn., Asteraceae, Artemisia, Artemisia annua species), put them into fresh-keeping bags and seal them, and isolate endophytes within 24 hours. The roots of Artemisia annua were washed with clean water, cut into small pieces of about 1 cm, and then soaked in 75% ethanol aqueous solution for 1 min, 1% sodium hypochlorite aqueous solution for 10 min, and 75% ethanol aqueous solution for 1 min for pretreatment. Take the pretreated roots of Artemisia annua and culture them on a double-antibody WA medium plate at...
Embodiment 2
[0061] Embodiment 2: the preparation of endophytic bacterium producing enzyme Lysobacterium R-2-1 fermentation medium
[0062] (1) Slant culture: Inoculate the endophytic fungus Lysobacterium lysobacter R-2-1 into the slant medium and culture at 37°C for 2 days to obtain the slant of the bacteria; the final concentration of the slant medium consists of: 5g of yeast extract / L, tryptone 10g / L, NaCl10g / L, agar 20g / L, solvent is water, pH7.4;
[0063] (2) Seed culture: pick an inoculation ring from the slant of the bacterium and inoculate it into the LB seed medium, and culture it on a shaker at 100-200 rpm and 37°C for 2 days to obtain the seed liquid; the LB seed medium is finally Concentration composition: yeast extract 5g / L, tryptone 10g / L, NaCl 10g / L, solvent is water, pH7.4;
[0064] (3) Fermentation culture: Inoculate the seed solution obtained in step (2) into the fermentation medium with an inoculum volume ratio of 1:20, and culture it on a shaking table at 100-200 rpm ...
Embodiment 3
[0065] Embodiment 3: Extraction, separation and identification of alkaloids
[0066] 1. Extraction and separation of alkaloids
[0067] (1) Filter the fermentation culture solution obtained in Example 2 with gauze, extract the filtrate three times with ethyl acetate (volume ratio 1:1), combine the organic layers, concentrate and dry in vacuum at low temperature to obtain brown crude extract F25g;
[0068] (2) The extract F was subjected to silica gel column chromatography, eluted with a mixed organic solvent (chloroform:methanol, volume ratio = 100:4) 10 times the column retention volume, combined the eluents, and concentrated in vacuum at low temperature to obtain the extract Paste F1;
[0069] (3) The extract F1 was subjected to silica gel column chromatography, and 5 retention volumes were eluted with a mixed organic solvent (chloroform:methanol, volume ratio=100:2), and the eluents were combined and concentrated in vacuum at low temperature to obtain the extract F2;
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