CN102786586A

CN102786586A - Discovery of peptide toxins in Amanita pallidorosea fermentation mycelium

Info

Publication number: CN102786586A
Application number: CN2012102517812A
Authority: CN
Inventors: 包海鹰; 张楠
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-07-20
Filing date: 2012-07-20
Publication date: 2012-11-21

Abstract

The invention relates to peptide toxins in an Amanita pallidorosea fermentation mycelium, and provides a fermentation preparation method and a fermentation preparation condition. Research results show that the Amanita pallidorosea fermentation mycelium contains alpha-amatoxin and beta-amatoxin. The researches provide a new resource for the obtaining of the toxins.

Description

The anatoxic discovery of peptide in the rose-red goose cream fermentation mycelium

Technical field:

The present invention has studied the peptide toxoid in the rose-red goose cream fermentation mycelium, and fermentation preparation and condition are provided, and research shows that rose-red Amanita fuliginea filament contains α-amanita hemolysin and β-amanita hemolysin.This research obtains to provide new resource for toxin.

Background technology:

Amanita fuliginea is a universal big genus, and in the poisonous mushroom most important one type, that has named at present has kind more than 500 approximately.This genus belongs to Basidiomycotina (Basidiomycotina), Hymenomycetes (Hymenomycetes), Agaricales (Agaricales), amanitaceae (Amanitaceae), Amanita (Amanita Pers.:Gray).Toxin main in the Amanita fuliginea is the peptide toxoid, has 3 big types 22 kinds, is respectively 6 kinds of 9 kinds of amanita hemolysins (amatoxins), 7 kinds of Phallus phallotoxins (phallotoxins) and phalloidins (virotoxins).The peptide toxoid is because the unique biological characteristic; Be widely used in biology, medical science, genetics, biological chemistry, genetically engineered at present; Research fields such as virus; People hope and can make it become the good medicine of curing the disease through its toxic component and pharmaceutical research are found a kind of ideal medicine that this also is when the important research project of forward swing in face of pharmacology and bacteriology worker.Aspect biology, amatoxin is as the molecular biological a kind of instrument of research.

Because most kind of Amanita is the ectomycorrhiza fungi, is difficult to artificial culture, the toxin basic source is in the sporophore of field acquisition.Every milligram of price of toxin source U.S. Sigma company is at 12～140,000 dollars.Because key status and the singularity of its toxin of Amanita in fungi, so the effort of artificial culture Amanita fuliginea never stopped.

Summary of the invention:

The present invention has adopted conventional known method to prepare rose-red goose cream fermentation mycelium, and is specific as follows:

(1) optimum medium (improvement PDM): yam 200g/L, glucose 20g/L, potassium primary phosphate 3g/L; Sal epsom 0.5g/L, phosphoric acid hydrogen ammonia 0.5g/L, calcium chloride 0.05g/L; Saltpetre 0.1g/L, wort (12 degree Beaume) 120ml, vitaminB10 .01g/L.

(2) spawn culture: slant strains inserts an amount of mycelia in the optimum medium after 26 ℃ of constant incubators are cultivated 10d, places on the constant temperature oscillator 150r/min shaking culture 10d then.

Shake flask fermentation: the bottled 100mL nutrient solution of 250mL triangle, inoculum size 5% (v/v) places on 26 ℃ of constant temperature oscillators 150r/min shaking culture 25d.

The present invention has adopted conventional known method to detect the peptide toxoid in the rose-red goose cream fermentation mycelium, and method and result are following:

1. experimental technique

(1) preparation of crude venom: after spawn culture is accomplished,, collect filter residue and filtrating, grind mycelium after the oven dry down at 60 ℃ with filtering fermentation liquor.Get the mycelium powder after 1g grinds, be dissolved in 10ml 50% methyl alcohol, vibration extracting 24h under the room temperature, the centrifugal collection supernatant of 8000r/min.Deposition is with the vibration extracting centrifugal collection of 24h 8000r/min under the 10ml 50% methyl alcohol room temperature and merge supernatant.With sherwood oil degrease twice, lyophilize then, freeze dried sample dissolves with ultrapure water, uses the filtering with microporous membrane of 0.22um again, obtains goose cream crude venom.

(2) separation and purification of toxin: the HPLC location parameter uses Agilent 1100 high performance liquid chromatographs separation gradient mode to be: 0 → 15min, and B 0 → 5%, and A 100% → 95%; 15 → 35min, B 5 → 80%, and A 95% → 20%; 35 → 40min, B 80%, and A 20%; 40-45min, B 80 → 100%, and A 20 → 0%; 45-50min, B 100%; 50 → 55min, B 100 → 0%, and A 0 → 100% also keeps 10min; Purifying gradient mode: with to separate gradient mode identical.Detect wavelength: 295nm; Column temperature: 40 ℃; Flow velocity: analytical column: lmL/min; Semipreparative column: 2mL/min.Sample size: analytical column: 10 μ L, semipreparative column: lmL.

HPLC analyzes separation and the purifying that on semipreparative column, carries out earlier toxin, obtains 5 kinds of compounds, after the lyophilize, identifies with the liquid chromatography mass combined instrument with the HPLC separation and purification again.

2. experimental result

The liquid phase RT, because of different separation conditions and different chromatographic columns, RT has certain difference, but the peak sequence of each compound on liquid phase remains unchanged.Simultaneously confirm compound according to liquid chromatography mass combined instrument and marker method.

Shown in the detected result (like Figure of description 1, the HPLC of Fig. 1 compound 1 schemes spectrogram), detection compound 1 RT is 11.41min to compound 1 on analytical column, and purity is higher.Electrospray ionization mass spectrum [M+Na] peak is 941, and MW is 918 (like Figure of description 2, the EFI thing figure spectrogram of Fig. 2 compound 1).According to chromatographic retention and molecular weight, the judgement component is that (α-AMA), molecular formula is C to α-amanita hemolysin ₃₉H ₅₄N ₁₀O ₁₄S.

Compound 2 is being analyzed shown in the detected result (like Figure of description 3, the HPLC of Fig. 3 compound 2 schemes spectrogram), and detection compound 2 RTs are 7.92min.Electrospray ionization mass spectrum [M-H] peak is 918, and MW is 919 (like Figure of description 4, the EFI thing figure spectrogram of Fig. 4 compound 2).According to chromatographic retention and molecular weight, the judgement component is β-amanita hemolysin (β-AMA), molecular formula C ₃₉H ₅₃N ₉O ₁₅S.

In addition, also separation and purification the peptide toxoid of three kinds of the unknowns.

Claims

1. the peptide toxoid is because the unique biological characteristic is widely used in biology, medical science, genetics, biological chemistry, genetically engineered, research fields such as virus at present.This research obtains to provide new resource for toxin.

2. contain α-amanita hemolysin (α-AMA) and β-amanita hemolysin (β-AMA) and other unknown toxin by the fermentation mycelium of the rose-red goose cream of preparing of claim 1.