Method for screening daptomycin high-producing strain by adopting sodium glutamate tolerance model

A technology of sodium glutamate and daptomycin, applied in the field of biomedicine, to achieve the effect of wide source, high potency and low cost

Inactive Publication Date: 2015-07-15
SICHUAN INDAL INST OF ANTIBIOTICS CHINA NAT PHARMA GROUP CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, due to the lack of 3-methylglutamic acid raw materials, it is very difficult to modify the daptomycin core by chemical synthesis or chemical and enzyme-catalyzed methods

Method used

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  • Method for screening daptomycin high-producing strain by adopting sodium glutamate tolerance model
  • Method for screening daptomycin high-producing strain by adopting sodium glutamate tolerance model

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Experimental program
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Effect test

Embodiment 1

[0027] Mutagenesis: A single spore suspension of Streptomyces roseosporus was subjected to ultraviolet (UV) mutagenesis.

[0028] Primary screening: Prepare 2L of separation medium according to the following formula: soluble starch 20.0 g / L, magnesium sulfate 1.0 g / L, potassium nitrate 1.0 g / L, ferrous sulfate 0.01 g / L, sodium chloride 0.5 g / L, phosphoric acid Dipotassium hydrogen 0.5 g / L, sodium glutamate 150 g / L, agar 15.0 g / L, pH7.3. Prepare 50 sterile petri dishes, and pour the medium into the petri dishes after sterilization to form a flat plate. Make a monospore suspension from the mature slant of the starting strain with physiological saline, oscillate, disperse with glass beads for 15 minutes, and collect about 5ml of the monospore suspension by sterile filtration in a sterile petri dish (to make the final concentration of spores reach 10 6 -10 7 perml), UV (power 30W, lamp distance 35cm) irradiated for 60s. Appropriately dilute the single spore suspension after th...

Embodiment 2

[0039] Mutagenesis: The monospore suspension of Streptomyces roseosporus was mutagenized by ethyl methanesulfonate (EMS).

[0040] Primary screening: Prepare 2L of separation medium according to the following formula: soluble starch 20.0 g / L, magnesium sulfate 1.0 g / L, potassium nitrate 1.0 g / L, ferrous sulfate 0.01 g / L, sodium chloride 0.5 g / L, phosphoric acid Dipotassium hydrogen 0.5 g / L, sodium glutamate 200 g / L, agar 15.0 g / L, pH7.4. Prepare 60 sterile petri dishes, and pour the culture medium into the petri dishes after sterilization to form a flat plate. Use potassium phosphate buffer solution of pH 7.0 to make a single spore suspension from the mature slant of the starting strain, oscillate, disperse the glass beads for 15 minutes, and collect about 5 ml of the single spore suspension by sterile filtration (to make the final concentration of spores reach 10 6 —10 7 each / ml), add the alkylating agent EMS, the final concentration is 5%, shake for 45 minutes, add sodium...

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Abstract

The invention discloses a method for screening daptomycin high-producing strain by adopting a sodium glutamate tolerance model, and aims to solve the problems that the raw materials of the culture medium in the existing method are less in source and high in cost, and the probability of improved daptomycin strains is small. The method comprises the following steps: (1) performing preliminary screening: diluting and coating a mutagenized monospore suspension liquid on an isolation culture medium with sodium glutamate tolerance amount of 150-200g / L to culture, picking and inoculating a single colony resistant to the sodium glutamate on an inclined surface culture medium to culture, then inoculating the single colony in a seed culture medium to culture after spores are produced on the inclined surface, inoculating the cultured single colony to a fermentation culture medium with different sodium glutamate contents to culture for 168 hours to pick out the bacterial strain with high titer; (2) re-screening, namely re-screening the preliminarily-screened bacterial strain, and picking out the bacterial strain with high titer; and (3) performing a fermentation culture medium test, namely performing the fermentation culture medium test on the bacterial strain with high tier to determine the optimal fermentation culture medium formula of the mutant strain.

Description

Technical field: [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for screening high-daptomycin-producing strains by an amino acid tolerance model. Background technique: [0002] Methicillin-resistant Staphylococcus aureus, known as a "superbug", [0003] (Methicillin-resistant Staphylococcus aureus, MRSA) is spreading more and more rapidly in various countries, which has seriously threatened human health and life. Clinically, only vancomycin (Vancomycin) has inhibitory effect on MRSA. With the increase of the application of vancomycin in MRSA infection, clinically infected Staphylococcus aureus has been reduced to moderately sensitive to vancomycin. The phenomenon. [0004] Daptomycin (Daptomycin) is recognized as the best backup antibiotic after vancomycin. It is a new lipopeptide antibiotic discovered by Eli Lilly and Company in the 1980s. It has obvious inhibitory effect on MRSA. In addition, daptomycin also inhibits th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N13/00C12R1/465
Inventor 俞岩青王昆蓉田敏曾志刚雷叶明陈林
Owner SICHUAN INDAL INST OF ANTIBIOTICS CHINA NAT PHARMA GROUP CORP
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