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Toxoflavin high-producing strain and application thereof

A high-yield technology of toxoflavin, applied in the field of microorganisms, can solve the problem of low toxoflavin production and achieve the effect of stabilizing toxoflavin

Active Publication Date: 2020-05-19
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the production of toxin in microorganisms is low, and there is still a lot of room for improving its production.

Method used

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  • Toxoflavin high-producing strain and application thereof
  • Toxoflavin high-producing strain and application thereof
  • Toxoflavin high-producing strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Extraction of toxigenic flavin from solid culture of Burkholderia sp.HDXY-02

[0030] 1. Activation of strains

[0031] Pick the preserved Burkholderia sp.HDXY-02 strain, streak on the seed solid medium (0.5% beef extract; 1% peptone; 0.5% NaCl; 2% agar; distilled water; pH7.0), 30-35 ° C, Cultivate at constant temperature for 48-72 hours.

[0032] 2. Solid plate culture

[0033] Pick a single colony of Burkholderia sp.HDXY-02 and inoculate it on the seed medium (0.5% beef extract; 1% peptone; 0.5% NaCl; 2% agar; distilled water; pH7.0) and shake it for 24 hours. KMB medium (peptone 20.0g, dipotassium hydrogen phosphate 1.5g, magnesium sulfate 0.75g, glycerol 15mL, pH 5.5-8.5, constant volume to 1000mL, 1.5% agar), 30-35℃, static culture at constant temperature for 48-72h .

[0034] 3. Extraction of Toflavin

[0035] Scrape off the bacterial lawn on the surface of the solid medium, chop the solid medium rich in toxin flavin, add 1 / 2 volume of chloroform ...

Embodiment 2

[0036] Example two. Burkholderia sp.HDXY-02 liquid fermentation toxaflavin production

[0037] 1. Activation of strains

[0038]Pick the preserved Burkholderia sp.HDXY-02 strain, streak on the seed solid medium (0.5% beef extract; 1% peptone; 0.5% NaCl; 2% agar; distilled water; pH7.0), 30-35 ° C, Cultivate at constant temperature for 48-72 hours.

[0039] 2. Liquid fermentation process

[0040] Pick the Burkholderia sp.HDXY-02 strain on the seed medium and dilute it with normal saline and mix it evenly. Draw 1-10mL overnight culture and inoculate it into 100mL KMB liquid medium (peptone 20.0g, dipotassium hydrogen phosphate 1.5g, sulfuric acid Magnesium 0.75g, glycerin 15mL, pH 5.5-8.5, constant volume to 1000mL) in a 250mL Erlenmeyer flask, 30-35°C, 150-200rpm, continuous shaking culture for 48-72h.

[0041] 3. Extraction of Toflavin

[0042] After shaking culture, centrifuge the fermentation broth at 4-20°C, 4000-6000g for 4-5min, absorb the supernatant, add 1 / 2 volume ...

Embodiment 3

[0043] Example three. Toxoflavin detection

[0044] Secondary purification of toxin: the above crude extract is separated and purified by silica gel column chromatography, and the gradient elution process is: (dichloromethane: methanol) 90:10→80:20→60:40→50:50 →30:70→0:100 (v / v). And further recrystallized twice through n-butanol to obtain the purified toxin.

[0045] The toxin extracted from the strain was compared with the toxin standard.

[0046] Thin-layer chromatography: respectively dissolve the fermentation broth extract and standard toxin of the bacterial strain in the methanol of the same volume, spot on one end of the thin-layer silica gel G plate, develop solvent (chloroform: methanol: water) (70:25 :5, V / V) (see figure 1 ).

[0047] Mass spectrometry detection: Burkholderia sp.HDXY-02 liquid fermentation toxoflavin fermentation liquid extract has the same ion peak of m / z=194.0714 as the standard product toxoflavin. The result shows that the fermentation liquid...

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Abstract

The invention discloses a Burkholderia sp.HDXY-02 capable of being stably produce toxoflavin with a high yield, and belongs to the technical field of applied microorganisms. The strain is preserved onApril 20, 2017 in China General Microbiological Culture Collection Center (abbreviation CGMCC), and the preservation number is CGMCC No.14054. After the strain is subjected to fermentation in a liquid culture medium or spread plate culture in a solid plate for 48-72 h, extraction is performed by dichloromethane or chloroform, separation purification is performed to obtain the toxoflavin, and thetoxoflavin has the activity of inhibiting pathogenic bacteria aspergillus fumigatus and resisting tumor Lovo cells. The strain provided by the invention can synthesize the toxoflavin by fermentation,and the toxoflavin has a high yield; and compared with a current chemical synthetic method, the strain provided by the invention avoids the pollution to the environment by chemical substances, and haspotential application prospects.

Description

technical field [0001] The invention relates to a strain of Burkholderia sp. HDXY-02 capable of efficiently synthesizing toxins, and belongs to the technical field of microorganisms. Background technique [0002] Natural products are substances produced by microorganisms, plants or other organisms, wherein microbial natural products provide a rich source of chemical diversity. Toflavin is a small molecular fatty acid exotoxin synthesized by microorganisms, which has been found to be used as antibacterial and antitumor agents. The molecular formula of toxin is C 7 h 7 N 5 o 2 , with a molar mass of 193g / mol, can produce resistance to a variety of pathogenic bacteria, and its strong antibacterial properties may be due to its inhibition of the respiratory chain and production of peroxides. Toxoflavin also has strong anti-tumor activity and can kill cancer cells at lower concentrations. Studies have proved that toxin has good inhibitory activity on various cancer cells suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P17/18C12R1/01
CPCC12N1/20C12P17/182C12R2001/01C12N1/205Y02A50/30
Inventor 李晓丹汪仁陆玲周佳宇李宜奎王蓉王松风
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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