Method for synthesizing key enzyme cofactor by adding daptomycin to improve daptomycin yield

A technology of daptomycin and enzyme cofactor, which is applied in the field of microbial fermentation to achieve the effects of simple operation, improved yield, novel and effective technology

Inactive Publication Date: 2010-06-09
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no relevant literature and reports on the use of cofactors in the optimization of daptomycin fermentation medium.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Take Streptomyces reseosporus slant spores at room temperature and make spore suspension with sterile water. Then use 0.5 milliliters of spore suspension to inoculate in the shaking flask of 250 milliliters that 30 milliliters of seed culture medium are housed, 28 ℃, cultivate 48 hours in the shaker of 180~220rpm, wherein seed liquid culture medium (composition and quality content are: paste Essence 1.5g, glucose 0.5g, peptone 0.5g, yeast extract powder 0.5g, K 2 HPO 4 ·3H 2 O 0.05g, MgSO 4 ·7H 2 O 0.05, CaCO 3 0.02g, dissolved in 1L distilled water, initial pH 7.5. ). Then with 1% inoculum size, automatic 7.5LDE fermenter. Wherein the fermentation medium (composed of: glucose 0.2g / L, dextrin 2g / L, casein 0.5g / L, peanut cake powder 0.4g / L, L-asparagine 0.8g / L, K SO 0.4g / L , initial pH 7.5.) Add 0.5 mg / L of pyridoxal phosphate (VB6). Then, ferment for 120 hours at 28°C, under the conditions of controlling the ventilation rate to 0.8v / v / m and pH6.5. The daptomyc...

Embodiment 2

[0017] Take Streptomyces reseosporus slant spores at room temperature and make spore suspension with sterile water. Then remove 0.5 milliliters of spore suspension and inoculate in 250 milliliters shake flasks that 30 milliliters of seed culture medium are housed, 30 ℃, cultivate 48 hours in the shaker of 180~220rpm. Wherein seed liquid culture medium (composition and mass content are: dextrin 1g, glucose 0.2g, peptone 0.3g, yeast extract powder 0.2g, K 2 HPO 4 ·3H 2 O 0.01g, MgSO 4 ·7H 2 O 0.01g, CaCO 3 0.01g, dissolved in 1L distilled water, initial pH6.5. ) and then in an automatic 7.5LDE fermenter with a 1% inoculation amount. Wherein the fermentation medium (composed of: glucose 1g / L, dextrin 4g / L, casein 1g / L, peanut cake powder 0.6g / L, L-asparagine 1.2g / L, K SO 0.6g / L, initial pH 7.5.) Add 30mg / L heme (heme). Then, ferment for 120 hours at 32° C., under the conditions of controlling the ventilation rate to 1.2 v / v / m and pH 7.5. The daptomycin concentration in t...

Embodiment 3

[0019] Take Streptomyces reseosporus slant spores at room temperature and make spore suspension with sterile water. Then remove 0.5 milliliters of spore suspension and inoculate in 250 milliliters shake flasks that 30 milliliters of seed culture medium are housed, 30 ℃, cultivate 48 hours in the shaker of 180~220rpm. Wherein seed liquid culture medium (composition and mass content are: dextrin 1.5g, glucose 0.3g, peptone 0.4g, yeast extract powder 0.35g, K 2 HPO 4 ·3H 2 O 0.03g, MgSO 4 ·7H 2 O0.03g, CaCO 3 0.015g, dissolved in 1L distilled water, initial pH 7.5. ) and then in an automatic 7.5LDE fermenter with a 1% inoculation amount. Wherein the fermentation medium (composed of: glucose 1.0g / L, dextrin 4g / L, casein 1.0g / L, peanut cake powder 0.6g / L, L-asparagine 1.2g / L, K 2 SO 4 0.6g / L, initial pH 7.5. ) Add 15 mg / L leucovorin (MTHF). Then, ferment for 90 hours at 30° C., under the conditions of controlling the ventilation rate to 0.8 v / v / m and pH 7.0. The daptomyc...

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Abstract

The invention relates to a method for synthesizing a key enzyme cofactor by adding daptomycin to improve the daptomycin yield, which comprises the following steps of: putting streptomyces reseosporus slant spores into sterile water to prepare a spore suspension at room temperature; then inoculating the 0.5-ml spore suspension into a 250ml shake flask filled with a 30ml seed culture medium at the temperature of 28-32 DEG C, and cultivating in a 180-220rpm shaking bed for 48 hours; then adding the mixture with a 1-percent inoculated dose into an automatic 7.5LDE fermentation tank filled with a fermentation culture medium; adding one or the composition of 0.5-30mg / l cofactor heme or calcium leucovorin or pyridoxal phosphate VB6; controlling the ventilation volume to be 0.8-1.2v / v / m at the temperature of 28-32 DEG C, and fermenting for 90-144 hours with a pH value of 6.5-7.5. The method is utilized for regulating biological enzyme activity, so that an intracellular metabolic flux leading to daptomycin synthesis is increased, and the yield of the daptomycin is improved; the method has simple operation and low cost, and the fermentation unit of the daptomycin in the fermented cultivated streptomyces reseosporus can be improved by 0.24-4.6 times than a reference substance.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for adding a key enzyme cofactor for daptomycin synthesis to increase the yield of daptomycin. Background technique [0002] The widespread resistance of pathogenic bacteria to antibiotics is a severe challenge facing the world, and hundreds of drug-resistant pathogenic strains appear every year. Vancomycin was once recognized as the last line of defense against Gram-positive bacteria, and now many resistant strains of the drug have been found at home and abroad. Therefore, it is imminent to develop a new type of antibiotic with a new mechanism of action that can effectively inhibit drug-resistant pathogens. [0003] "Streptomyces roseosporus" (Latin name: "Streptomyces roseosporus") is an actinomycete of the genus Streptomyces, and its synthetic daptomycin (English name "daptomycin") is N -Decanoyl-Tryptophanyl-Asparagyl-Aspartyl-Threonylgly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12R1/465
Inventor 闻建平宇光海贾晓强
Owner TIANJIN UNIV
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