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Process of screening phosphonomycin producing strain

A screening method and technology of fosfomycin, applied in the field of biomedicine, can solve problems such as failure of screening work, undetectable antibiotics, etc., and achieve the effects of improving the sensitivity and efficiency of identification, the improvement of sensitivity and identification efficiency, and the simple operation.

Inactive Publication Date: 2010-06-16
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that when the antibiotic content in the fermentation broth is low, the presence of antibiotics (such as fosfomycin) cannot be detected on the bioassay plate, which leads to the failure of the screening work

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0027] Preparation of indicator bacteria suspension: Pour 100mL sterilized normal saline into an eggplant-shaped bottle, scrape and wash the bacterial lawn with an inoculation loop, then pour the scraping solution into a sterilized empty conical flask, and measure the absorbance (O.D. value) 0.54 to 0.57.

[0028] 2. Primary screening and secondary screening of fosfomycin biotransformation strains

[0029] Taking D-fosfomycin as the composition of the selection medium of the sole carbon source: by weight percentage, D-fosfomycin 0.5%, (NH 4 ) 2 SO 4 0.5%, NaCl 0.2%, KCl 0.1%, MgSO4 7H 2 O 0.01%, FeSO 4 ·7H 2 O 0.01%, KH 2 PO 4 0.05%, agar 2%, the balance is tap water, pH 7.5. 121 ℃ damp heat sterilization for 20min.

[0030] Broth slant medium composition: by weight percentage, beef extract 0.5%, peptone 1%, NaCl 0.2%, agar 2%, pH 7.5, and the balance is tap water. Sterilize with damp heat at 121°C for 20 minutes.

[0031] The composition of primary screening cultu...

Embodiment

[0048] Take 1 g of the soil sample collected in this laboratory and add it to 100 mL of selection medium, and culture it on a shaker at 30°C for 2 weeks. After melting the selection medium, pour 25mL into a 90mm diameter petri dish and let it stand to solidify. The above soil suspension was serially diluted to 10 -3 、10 -4 、10 -5 , take 0.1 mL of the soil suspension of each dilution and spread it on the selection medium plate, and cultivate it at 30°C for 3 days. Pick a single colony and plant it on the control plate without carbon source and the selective medium plate respectively, and then culture at 30°C for 3 days, pick the colony that only grows on the selective medium plate, and inoculate it into the broth slant medium.

[0049] Pick two rings of the slant strains and insert them into a 250mL Erlenmeyer flask containing 30mL of the primary screening medium, and after 24 hours of shaking culture at 30°C and 180 rpm shaker, inoculate the bacteria with a volume ratio of ...

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Abstract

The present invention relates to biomedicine technology, and is especially process of screening phosphonomycin producing strain. The screening process includes: culturing strain to be identified withdextro phosphonomycin as substrate and colibacillus as the bioassay indicator; pre-cooling the biotransformed phosphonomycin fermenting liquid to 0-10 deg.c, setting the liquid in alcohol-ice coolingagent at -30 deg.c to -10 deg.c to freeze at vacuum degree of 10-20 Pa and temperature -30 deg.c to -10 deg.c for 15 min to 2 hr to form concentrate; and identifying whether to producing inhibition zone with Oxford cup on bioassay plate to screen biotransformed strain. The present invention has the advantages of no deactivation on antibiotic, simple operation, low cost, etc.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for screening antibiotic-producing bacteria-fosfomycin-producing strains. Background technique [0002] Fosfomycin [(-)-cis-(1R,2S)-1,2-epoxypropyl phosphate, FOM] is a broad-spectrum antibiotic. It has a unique chemical structure and antibacterial mechanism, not only has no cross-resistance with other antibiotics or antibacterial drugs, but most of them show synergistic effects. At the same time, because fosfomycin also has the advantages of stable physical and chemical properties, safety and low toxicity, no antigenicity (no need for allergy testing), etc., it has been widely used in clinical treatment, and is called a new drug in the 21st century by the international medical and pharmaceutical circles. antibiotic. [0003] FOM can be obtained through microbial transformation. The traditional screening method for fosfomycin-producing strains is to use shaking culture to s...

Claims

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Application Information

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IPC IPC(8): C12Q1/04G01N30/90
Inventor 周丽沙徐慧李佰广张颖李慧任瑞霞张忠泽
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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