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

Active Publication Date: 2013-11-27
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the isolation of endophyte Lysobacter enzymogenes from Artemisia annua and its metabolism of 2,5-diketopiperazine dipeptides

Method used

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  • 2,5-diketopiperazine dipeptide derived from lysobacter enzymogenes as well as preparation method and application thereof
  • 2,5-diketopiperazine dipeptide derived from lysobacter enzymogenes as well as preparation method and application thereof
  • 2,5-diketopiperazine dipeptide derived from lysobacter enzymogenes as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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;

[0...

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Abstract

The invention provides 2,5-diketopiperazine dipeptide (I) derived from lysobacter enzymogenes as well as a preparation method and an application thereof and an endophytic bacterium producing 2,5-diketopiperazine dipeptide, namely lysobacter enzymogenes R-2-1. 2,5-diketopiperazine dipeptide (I) has the main beneficial effects that (1) lysobacter enzymogenes R-2-1 producing 2,5-diketopiperazine dipeptide (I) is provided and is the first lysobacter enzymogenes produced in the strain; (2) 2,5-diketopiperazine dipeptide (I) has strong inhabiting effects on aspergillus flavus and candida albicans, can serve as a novel bactericide and can be further used for preparing medicament for treating relevant diseases caused by aspergillus flavus or candida albicans; (3) 2,5-diketopiperazine dipeptide (I) is produced by liquid fermentation of lysobacter enzymogenes R-2-1 and has the advantages of simple and convenient operation process, short period, low cost and ensured origin; (4) 2,5-diketopiperazine dipeptide (I) is synthesized by a biological method and does not pollute the environment.

Description

(1) Technical field [0001] The invention relates to a 2,5-diketopiperazine dipeptide derived from Lysobacter enzymogenes, its preparation and application, and a method for producing the 2,5-diketopiperazine dipeptide Endophytic bacteria - Lysobacter enzymogenes R-2-1. (2) Background technology [0002] Pathogenic bacteria not only seriously endanger human health, but also threaten agricultural production and bring great losses to the economy and life of people all over the world. Although there are many types of fungicides in my country, they can no longer effectively inhibit new pathogenic bacteria and strong resistant pathogenic bacteria. Therefore, fungicides must be continuously innovated. Biological drugs have the advantages of high efficiency, low toxicity, safety, and environmental friendliness, which cater to the needs of drug development in my country. The microbial drug industry has great potential for development in the future. [0003] Expanding microbial reso...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D487/04A61K31/4985A61P31/10A61P39/02C12P17/18C12N1/20C12R1/01
Inventor 章华伟应晨杨婷媛何青汤逸飞
Owner ZHEJIANG UNIV OF TECH
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