Method for preparing ansamitocin P-3 from precious orange actinosynnema pretiosum

A technology of ansesomycin and actinomycetes, which is applied in the field of biological fermentation, can solve the problems of low proportion of anseomycin P-3, difficulty in large-scale production, inability to synthesize maytansinol and the like, and achieves low cost, The effect of increasing output and saving production costs

Active Publication Date: 2015-09-09
QILU PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In other prior art US4162940, US4450234, US4228239, US4331598 and US4356265, the output of ansamectin is generally 12-100 mg / L, the proportion of ansamectin P-3 is less than 70%, and there are many different Desired production of ansamitocin components, such as N-demethylation, 20-O-demethylation and 19-dechlorination modified products, these products cannot synthesize maytansinol under reductive deacylation
[0008] At present, the yield of ansamitocin fermented by actinomycetes is low, the production cost is high, the proportion of ansamitocin P-3 is low, and the purification process is cumbersome, involving the treatment of a variety of highly toxic materials, and large-scale production is very difficult. difficulty

Method used

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  • Method for preparing ansamitocin P-3 from precious orange actinosynnema pretiosum
  • Method for preparing ansamitocin P-3 from precious orange actinosynnema pretiosum

Examples

Experimental program
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Effect test

Embodiment 1

[0058] Embodiment 1: 50L tank fed-batch fermentation (during fermentation and cultivation, no hot-pressed bean cake powder was added, and the addition method of isobutanol was fed-batch)

[0059] (1) Spore culture: the spores of Actinomyces fasciculatus orientalis spores frozen in 20% glycerol tubes were cultured by streaking on a solid medium culture dish, and cultured upside down at 28°C for 6 days;

[0060] Solid medium formula: yeast extract 0.3%, soybean peptone 0.5%, malt extract 0.3%, glycerin 1%, agar powder 2%, natural pH;

[0061] (2) Seed culture: Add 6L liquid seed medium to a 10L fermenter, and after sterilizing at 121°C for 30 minutes, scrape the spores from the culture dish and inoculate them into the liquid seed medium; Stir at a speed of 80rpm; during the cultivation process, the dissolved oxygen is kept above 30% by adjusting the ventilation and strengthening the stirring, the maximum ventilation is 1vvm, the maximum rotation speed is 350rpm, and the seed liq...

Embodiment 2

[0067] Embodiment 2: 50L tank fed-batch fermentation (add hot-pressed bean cake powder during fermentation and cultivation, and the supplementary mode of isobutanol is fed-batch)

[0068] (1) Spore culture: as in Example 1;

[0069] (2) seed culture: as embodiment 1;

[0070] (3) Fermentation culture: as in Example 1, the difference is that the liquid fermentation medium is: cornstarch 5%, corn steep liquor dry powder 2%, hot-pressed bean cake powder 2%, glucose 0.5%, K 2 HPO 4 ·3H 2 O 0.025%, MgSO 4 ·7H 2 O 0.3%, CaCO 3 0.4%, CaCl 2 0.1%, isobutanol 0.3%, CoCl 2 ·6H 2 O 0.0005%, FeSO 4 ·7H 2 O 0.0002%, initial pH 7.2;

[0071] For 0-60 hours of fermentation culture, 22.5% glucose solution was added at a flow rate of 0.5mL / L / h. After 60 hours of fermentation, 25% glucose, 10% hot-pressed bean cake powder and 10% glucose solution were added at a rate of 1mL / L / h. % isobutanol feed solution, fermented and cultivated for 412 hours, until the expression of ansamitocin P-3...

Embodiment 3

[0073] Example 3: 50L tank fed-batch fermentation

[0074] (1) Spore culture: as in Example 1;

[0075] (2) seed culture: as embodiment 1;

[0076] (3) Fermentation culture: as in Example 2, the difference is the feeding process, fermentation culture 0-60h, add 22.5% glucose solution with a flow rate of 0.5mL / L / h, after fermentation 60h, add 22.5% glucose solution with 1mL / L Feed solution containing 25% glucose and 10% hot-pressed bean cake powder was added at a rate of / h, and 55mL of isobutanol was added once every 24h; The expression level of is no longer increasing, the fermentation has ended, and the expression level reaches 1211mg / L (such as figure 1 shown);

[0077] (4) Sample processing and detection methods are as in Example 1.

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Abstract

The invention relates to a method for preparing ansamitocin P-3 from precious orange actinosynnema pretiosum. The method includes steps of (1), cultivating pores, namely subjecting actinosynnema pretiosum spores cryopreserved in a 20%-glycerinum tube to streak cultivation in a solid-medium-contained culture dish at the temperature of 25-30 DEG C for 3-9 days until rich spores grow; (2), cultivating seeds, namely scraping the spores from the cultivation dish, and inoculating the spores into a liquid seed cultivating medium at the temperature of 25-30 DEG C for 30-50h to obtain seed liquid; (3) fermenting and cultivating the seed liquid, namely inoculating cultivated seed liquid into a liquid fermentation cultivation medium, fermenting and cultivating the seed liquid at the temperature of 25-30 DEG C for 350-450h, and controlling dissolved oxygen to be above 20%. The method has the advantages that ansamitocin in fermentation liquid is high in yield, the ansamitocin P-3 accounts for a high proportion, production cost is saved, a subsequent purge process is benefited, and suitability for industrial production is achieved.

Description

technical field [0001] The invention relates to a method for preparing ansamitocin P-3 from actinomycetes precious A. orange, belonging to the technical field of biological fermentation. Background technique [0002] Ansamitocin is a highly cytotoxic compound produced from actinomycetes such as Actinosynnema pretiosum. The mechanism of action of ansamitocin is to inhibit the assembly of tubulin, thereby inhibiting cell division. The cytotoxicity of ansamitocin is very strong, which is 100-1000 times that of traditional chemotherapy drugs vinblastine, methotrexate and daunorubicin. [0003] Ansamitocin belongs to macrolide compounds, and its structure is similar to that of maytansinol. The ansamitocin expressed by A. precious orange mainly includes 6 homologues, including P-1, P-2, P-3, P-3', P-4 and P-4', among which P- 3 is its main product, and the structure of ansamitocin homologs is shown in the following formula. [0004] [0005] Ansamitocin has highly effective...

Claims

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Application Information

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IPC IPC(8): C12P17/18C12R1/01
Inventor 刘升波孙丽霞张超江波王克波王德明肖娜樊乐艾丽静
Owner QILU PHARMA
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