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Method for cumulatively producing Sirolimus by using streptomyces hygroscopicus

A technology of Streptomyces hygroscopicus and sirolimus, which is applied in the field of sirolimus production, can solve the problems of long fermentation period, many intermediate products, high-concentration products and substrate inhibition, and achieve the effect of increasing production

Inactive Publication Date: 2011-11-02
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the levels of RPM produced by fermentation of Streptomyces hygroscopicus reported in literature at home and abroad are all low, mostly at 100-300mg / L
Research progress at home and abroad has found that the fermentation production of sirolimus has problems such as low sirolimus-producing ability of the original strain, long fermentation cycle, many intermediate products, high concentration of products and substrate inhibition, and complicated separation and extraction of fermentation broth. Realize industrialization

Method used

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  • Method for cumulatively producing Sirolimus by using streptomyces hygroscopicus
  • Method for cumulatively producing Sirolimus by using streptomyces hygroscopicus

Examples

Experimental program
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Effect test

Embodiment 1

[0024] The slant was cultured in ISP3 medium for 9 days and placed in a 4°C refrigerator to store the slant. Get the slant seeds and use 20% glycerol to elute the spores to form a spore suspension, and 2wt% is inoculated into the seed shake flask medium (g / L): glucose (C 6 h 12 o 6 ·H 2 O) 10.0, peptone (Peptone) 6.0, yeast extract (Yeast Extract) 6.0, casein hydrolyzate (casamino acid) 1.5, magnesium sulfate (MgSO 4 ·7H 2 O) 0.25, dipotassium hydrogen phosphate (K 2 HPO 4 ) 1.0, pH 7.0. Activation culture at 30°C for 60h, inoculated into 7L fermenter according to 6wt% inoculation amount. The optimized fermentation medium (g / L) was used in the initial stage of fermentation: glucose (Glucose) 15.0, glycerol (Glycerol) 10.0, soybean cake powder (Soybean) 10.0, yeast extract (Yeast Extract) 6.0, peptone (Peptone) 2.0, phosphoric acid Potassium dihydrogen (KH 2 PO 4 )5.0, dipotassium hydrogen phosphate (K 2 HPO 4 ) 5.0, sodium chloride (NaCL) 5.0, L-lysine (L-Lysine) 1....

Embodiment 2

[0028] The slant was cultured in ISP3 medium for 9 days and placed in a 4°C refrigerator to store the slant. Get the slant seeds and use 20% glycerol to elute the spores to form a spore suspension, and 2wt% is inoculated into the seed shake flask medium (g / L): glucose (C 6 h 12 o 6 ·H 2 O) 10.0, peptone (Peptone) 6.0, yeast extract (Yeast Extract) 6.0, casein hydrolyzate (casamino acid) 1.5, magnesium sulfate (MgSO 4 ·7H 2 O) 0.25, dipotassium hydrogen phosphate (K 2 HPO 4 ) 1.0, pH 7.5. Activation culture at 30°C for 60 hours, inoculate 6 wt% of the inoculum into a 7L fermenter based on the volume of the medium. The optimized fermentation medium (g / L) was used in the initial stage of fermentation: glucose (Glucose) 15.0, glycerol (Glycerol) 10.0, soybean cake powder (Soybean) 10.0, yeast extract (Yeast Extract) 6.0, peptone (Peptone) 2.0, phosphoric acid Potassium dihydrogen (KH 2 PO 4 )5.0, dipotassium hydrogen phosphate (K 2 HPO 4 ) 5.0, sodium chloride (NaCL) 5....

Embodiment 3

[0031] The slant was cultured in ISP3 medium for 9 days and placed in a 4°C refrigerator to store the slant. Get the slant seeds and use 20% glycerol to elute the spores to form a spore suspension, and 2wt% is inoculated into the seed shake flask medium (g / L): glucose (C 6 h 12 o 6 ·H 2 O) 10.0, peptone (Peptone) 6.0, yeast extract (Yeast Extract) 6.0, casein hydrolyzate (casamino acid) 1.5, magnesium sulfate (MgSO 4 ·7H 2 O) 0.25, dipotassium hydrogen phosphate (K 2 HPO 4 ) 1.0, pH 7.0. Activation culture was carried out at 30°C for 60 hours, and the inoculum was inoculated into a 7L fermenter according to the inoculum size of 6wt%. The optimized fermentation medium (g / L) was used in the initial stage of fermentation: glucose (Glucose) 15.0, glycerol (Glycerol) 10.0, soybean cake powder (Soybean) 10.0, yeast extract (Yeast Extract) 6.0, peptone (Peptone) 2.0, phosphoric acid Potassium dihydrogen (KH2PO4) 5.0, dipotassium hydrogen phosphate (K 2 HPO 4 ) 5.0, sodium ch...

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Abstract

The invention relates to a method for producing Sirolimus by using streptomyces hygroscopicus, which comprises: inoculating streptomyces hygroscopicus into a seed culture medium, and performing activated culture for 60 hours under a condition that the temperature is 30 DEG C; inoculating the streptomyces hygroscopicus into a fermentation tank according to an inoculation amount of 6 weight percent; at the early stage of fermentation, namely at the growth stage of bacteria, fermenting for 8 hours, performing exponential feeding of glucose, controlling a specific growth rate to be between 0.02 to 0.06 hours, performing high-cell density culture, and obtaining high bacterium concentration; and after fermentation lasts 32 hours, namely in the rear middle stage of the fermentation, and providing a precursor for synthesizing Sirolimus by constant-speed feeding of shikimic acid and L-lysine, which are precursors to improve fermentation level. Compared with the prior art, the peak titer of the Sirolimus synthesized by the method reaches 700mg / L, which is 30 percent higher than that synthesized by batch fermentation. According to the fermentation policy, the control is simple and the yield of Sirolimus is high.

Description

technical field [0001] The invention relates to a method for producing sirolimus, in particular to a method for accumulatively producing sirolimus by using Streptomyces hygroscopicus. Background technique [0002] Sirolimus (SRL), formerly known as Rapamycin (RPM). It is a nitrogen-containing triene macrolide antibiotic composed of a thirty-one-membered ring. It is a new type of potent immunosuppressant with anti-fungal, anti-proliferation and anti-tumor effects. In 1975, Ayerster laboratory isolated sirolimus produced by Streptomyces hygroscopicus from the soil of Ester Island (Raps Nui) in the Pacific Ocean. Sirolimus was initially identified as an antifungal antibiotic. It was discovered in 1977 that it had an important immunosuppressive effect. In 1989, it was studied as a new type of high-efficiency immunosuppressant for anti-organ transplant rejection. At present (2010 ) Phase I and Phase II clinical trials of RAPA have ended, and Phase III clinical trials are under...

Claims

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Application Information

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IPC IPC(8): C12P17/18C12R1/55
Inventor 孙爱友魏东芝王丽华董玉国徐瑞俞鹏飞
Owner EAST CHINA UNIV OF SCI & TECH
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