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Method for preparing daptomycin by fermentation

A technology of daptomycin and fermentation method, which is applied in the field of producing daptomycin and daptomycin artificially mutagenizing high-yield strains

Active Publication Date: 2013-03-20
SHANDONG LUBEI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The technical problem to be solved in the present invention is to disclose a high-yield strain of daptomycin and a method for preparing daptomycin by fermentation of the strain, so as to overcome The existing daptomycin fermentation technology has the defects of long fermentation cycle, low unit yield and low conversion rate

Method used

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  • Method for preparing daptomycin by fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] (1) Seed cultivation

[0033] Primary seed medium: 2.0% trypsin soybean broth, 2.0% dextrin, supplemented with deionized water, pH 6.0.

[0034] Secondary seed medium: soybean powder 0.4%, yeast extract 0.4%, calcium gluconate 0.8%, KCl 0.01%, MgSO 4 ·7H 2 O 0.01%, FeSO 4 ·7H 2 O 0.0003%, Sag 471 (organic silicon defoamer) 0.02%, supplemented with deionized water, the pH value is 6.0.

[0035] Culture conditions: After recovery, the strains were directly inoculated into a 250mL Erlenmeyer flask containing 50mL of primary seed medium, and cultured on a shaker at 30°C at 250rpm for 48h. Take 10 mL of primary seed medium, inoculate it into a 500 mL Erlenmeyer flask with 250 mL of secondary seed medium, and incubate at 250 rpm at 30°C for 24 hours.

[0036] (2) Fermentation culture

[0037] Fermentation medium: glucose 0.85%, CaCO 3 0.3%, soybean flour 3.0%, yeast extract 0.15%, Fe(NH 4 ) 2 SO 4 ·6H 2 O 0.068%, KCl 0.025%, MgSO 4 ·7H 2 O 0.025%, FeSO 4 ·7H 2...

Embodiment 2

[0043] (1) Seed cultivation

[0044] Primary seed medium: 3.0% trypsin soybean broth, 2.5% dextrin, supplemented with deionized water, pH 7.0.

[0045] Secondary seed medium: soybean powder 0.45%, yeast extract 0.5%, calcium gluconate 0.9%, KCl 0.02%, MgSO 4 ·7H 2 O 0.02%, FeSO 4 ·7H 2 O 0.0004%, Sag 471 (organic silicon defoamer) 0.03%, supplemented with deionized water, the pH value is 7.0.

[0046] Culture conditions: After recovery, the strains were directly inoculated into a 250mL Erlenmeyer flask containing 50mL of primary seed medium, and cultured on a shaker at 30°C at 250rpm for 48h. Take 10 mL of primary seed medium, inoculate it into a 500 mL Erlenmeyer flask with 250 mL of secondary seed medium, and incubate at 250 rpm at 30°C for 24 hours.

[0047] (2) Fermentation culture

[0048] Fermentation medium: glucose 0.83%, CaCO 3 0.2%, soybean flour 2.0%, yeast extract 0.1%, Fe(NH 4 ) 2 SO 4 ·6H 2 O 0.066%, KCl 0.02%, MgSO 4 ·7H 2 O 0.02%, FeSO 4 ·7H 2 O...

Embodiment 3

[0054] (1) Seed cultivation

[0055] Primary seed medium: 4.0% trypsin soybean broth, 3.0% dextrin, supplemented with deionized water, pH 8.0.

[0056] Secondary seed medium: soybean powder 0.5%, yeast extract 0.6%, calcium gluconate 1.0%, KCl 0.03%, MgSO 4 ·7H 2 O 0.03%, FeSO 4 ·7H 2 O 0.0005%, Sag 471 (organic silicon defoamer) 0.04%, supplemented with deionized water, the pH value is 8.0.

[0057] Culture conditions: After recovery, the strains were directly inoculated into a 250mL Erlenmeyer flask containing 50mL of primary seed medium, and cultured on a shaker at 30°C at 250rpm for 48h. Take 10 mL of primary seed medium, inoculate it into a 500 mL Erlenmeyer flask with 250 mL of secondary seed medium, and incubate at 250 rpm at 30°C for 24 hours.

[0058] (2) Fermentation culture

[0059] Fermentation medium: glucose 0.80%, CaCO 3 0.1%, soybean flour 1.0%, yeast extract 0.05%, Fe(NH 4 ) 2 SO 4 ·6H 2 O 0.065%, KCl 0.015%, MgSO 4 ·7H 2 O 0.015%, FeSO 4 ·7H 2...

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Abstract

The invention discloses a high-yield daptomycin strain and a method for preparing daptomycin by its fermentation method, belonging to the technical field of biochemical industry. The present invention uses Streptomyces roseosporus as the starting bacterium, and obtains a high-yielding mutagenic strain WKL-126 through protoplast fusion and gene recombination technology, which has been registered in the General Microorganism Center of China Microbiological Culture Collection Management Committee ( CGMCC), the preservation number is: 3731; the present invention also discloses a method for preparing daptomycin by using the strain, and compared with the existing method, the fermentation unit of the method reaches more than 2000 mg / L.

Description

technical field [0001] The invention relates to a daptomycin artificial mutagenesis high-yield strain and a method for preparing daptomycin by fermentation of the strain, belonging to the technical field of biochemical industry. Background technique [0002] Daptomycin (trade name cubecin) is a cyclic ester peptide extracted from the fermentation broth of Streptomyces roseosporus. The molecular formula of daptomycin is C 72 h 101 N 17 o 26 , with a molecular weight of 1619.71, in which the 4th threonine and the 13th kynuric acid in the 13 amino acid molecules are closed to form a ring with peptide bonds, and the amino acid tryptophan at the end of the side chain is connected with capric acid with an amide bond to form an acidic cyclic lipopeptide antibiotic. Its structural formula is shown in the figure below: [0003] [0004] In general, daptomycin is a yellow or pale yellow crystalline substance that is soluble in water, methanol, ethanol, propanol, n-butanol, d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P21/04C12R1/465
Inventor 王克柳王美海
Owner SHANDONG LUBEI PHARMA
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