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Ribosome resistance mutagenesis breeding vancomycin production bacterial strain and application thereof

A technology for vancomycin and strain production, which can be used in microorganisms, mutant preparation, microorganism-based methods, etc., and can solve problems such as time-consuming and labor-intensive

Inactive Publication Date: 2011-08-31
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Researchers have obtained some vancomycin-producing strains by isolating, breeding or mutating the strains by various means, but it is time-consuming and labor-intensive

Method used

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  • Ribosome resistance mutagenesis breeding vancomycin production bacterial strain and application thereof
  • Ribosome resistance mutagenesis breeding vancomycin production bacterial strain and application thereof
  • Ribosome resistance mutagenesis breeding vancomycin production bacterial strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1) Purchased from the American Culture Collection, numbered ATCC No.19795 The starting strain AO-1 of the vancomycin-producing bacteria was inoculated on Gao’s No. 1 agar medium plate and cultured at 28°C for 7 days. After the spores grew out, take an appropriate amount and use sterile water to make 10 4 mL -1 spore suspension. Spread 0.1 mL of the spore suspension on Gaoshi No. 1 agar medium plate containing 500, 1000 mg / L vancomycin and culture it at 28°C for 7 days, and randomly select 50 single colonies that grow out and inoculate it in Gaoshi No. 1 On the agar plate medium, cultivate at 28°C for 7 days, and measure the biological activity of the grown mutagenized strains by the agar block inhibition zone method, and select 12 mutagenized strains whose diameter of the inhibition zone is 2mm larger than that of the starting strain AO-1 (see figure 1 ).

[0023] 2) Inoculate the above 12 mutagenized strains into a 250mL Erlenmeyer flask equipped with 40mL liquid...

Embodiment 2

[0028] Take 0.1 mL of the spores of the starting strain with a concentration of 10,000 / mL and smear it on the mixed resistance Gowell No. 1 agar plate with a concentration of 500 mg / L of vancomycin and a concentration of 400 mg / L of streptomycin. -1 spores were diluted into different concentration gradients and spread on ordinary Goose No. 1 agar plates as a control, and cultured at 28°C for 7 days to obtain several mutant strains. The difference in the growth of the grown strains is as follows Figure 4 and shown in Table 1.

[0029] Table 1 Comparison of colony morphological differences between the vancomycin production strain and the starting strain AO-1

[0030] Strain characteristics

Embodiment 3

[0032] Insert the vancomycin production strain obtained in Example 1 into the liquid seed culture medium (the liquid fermentation seed culture medium consists of (g / L): yeast powder 5, malt extract 4, peptone 13, glucose 17, pH 6.8; Liquid volume 40 / 250mL), at 28°C, 200rpm cultured for 48h, then transferred to shake flask fermentation medium (liquid fermentation medium composition (g / L): dextrin 100-150, potato protein 15-25, soybean powder 15~25, dipotassium hydrogen phosphate 0.1~0.5, antifoaming agent 1~3, sodium chloride 1.2~1.6, pH 6.8~7.2, liquid volume 40 / 250mL), cultivated at 28°C, 200rpm for 120h, passed HPLC Determination of vancomycin content in fermentation broth. According to the chromatographic conditions provided in Example 1, the yield of vancomycin in the fermentation broth was measured to be 8.5 g / L. Its production curve see image 3 .

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Abstract

The invention provides ribosome resistance mutagenesis breeding vancomycin production bacterial strain and an application thereof, specifically the mutagenesis breeding method of mutagenesis bacterial strain of Amycolatopsis orientalis and the application thereof in preparing vancomycin, relating to breeding of microorganism. The method comprises the following steps: inoculating the Amycolatopsisorientalis to Gause-1 agar culture medium panel to be cultured; taking out spore and using sterile water to prepare spore suspension and coating the spore suspension in Gause-1 agar culture medium panel featuring mixing resistance of vancomycin and streptomycin to be cultured; randomly selecting produced single bacterial colony and streak-inoculating the single bacterial colony to the Gause-1 agar culture medium panel to be cultured, carrying out biological activity determination on produced mutagenesis bacterial strain; selecting mutagenesis bacterial strain with inhibition zone diameter thereof being 2mm larger than the inhibition zone diameter of original strain and inoculating the mutagenesis bacterial strain to liquid fermentation medium to be cultured; determining the content of vancomycin in fermentation liquor by HPLC method to verify the obtained vancomycin mutagenesis bacterial strain; subjecting the obtained vancomycin mutagenesis bacterial strain to 5 times of continuous passage stability inspection to obtain the vancomycin production bacterial strain.

Description

technical field [0001] The present invention relates to the breeding of a kind of microorganism, especially relate to the mutagenic strain V500-S400-1 of Amycolatopsis orientalis (Amycolatopsis orientalis) AO-1 (the mutagenic strain V500-S400-1 is called vancomycin production strain) and its application to vancomycin producing strains. Background technique [0002] Vancomycin was obtained from Indonesian soil in 1956 by McCormick et al. (McCormick MH, Stark WM, Plttenger GE, et al. A glycopeptide antibiotic with resistance to Gram-positive bacteria isolated from the fermentation broth of a strain called Amycolatopsis orientalis selected and bred in the study. Twenty years after its introduction, vancomycin was reserved only for the treatment of severe severe disease caused by a small number of drug-resistant Staphylococcus aureus due to the marketed use of penicillins and cephalosporins and their strong oto-nephrotoxicity. Infectious diseases, clinical use is rare. Later,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/01C12R1/01
Inventor 卢英华曾宪海敬科举姚传义凌雪萍
Owner XIAMEN UNIV
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