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A method for preparing ansamitocin p-3 by using actinomycetes precious orange fasciculatus

A technology of ansamectin and actinomycetes, applied in the field of biological fermentation, can solve the problems of low proportion of ansamectin P-3, inability to synthesize maytansinol, low output of ansamectin, etc. Low cost, saving production cost, high purity effect

Active Publication Date: 2018-04-27
QILU PHARMA CO LTD
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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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  • A method for preparing ansamitocin p-3 by using actinomycetes precious orange fasciculatus
  • A method for preparing ansamitocin p-3 by using actinomycetes precious orange fasciculatus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: 50L tank fed-batch fermentation (the hot-squeezed soybean meal powder was not added during the fermentation culture, and the supplementary method of isobutanol was flow-feeding)

[0059] (1) Spore culture: Streak culture of spores of the precious orange fascicle actinomycete frozen in a 20% glycerol tube in a solid medium petri dish, and inverted culture at 28°C for 6 days;

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

[0061] (2) Seed culture: Add 6L of liquid seed culture medium to a 10L fermentor, sterilize at 121°C for 30 minutes, scrape the spores from the petri dish and inoculate the liquid seed culture medium; culture at 28°C, with a ventilation volume of 0.4vvm, Stirring at 80 rpm; during the cultivation process, the dissolved oxygen is maintained above 30% by adjusting the aeration rate and enhanced stirring, the maximum aeration rate is 1 vvm, the maximum rotation speed is ...

Embodiment 2

[0067] Example 2: 50L tank fed-batch fermentation (add hot-squeezed soybean meal powder during fermentation culture, and the supplementary method of isobutanol is fed-batch)

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

[0069] (2) Seed cultivation: as in Example 1;

[0070] (3) Fermentation culture: as in Example 1, the difference is that the liquid fermentation medium is: corn starch 5%, corn syrup dry powder 2%, hot pressed soybean meal 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] Fermentation culture 0-60h, supplemented with 22.5% glucose solution at a flow rate of 0.5mL / L / h, after 60h fermentation, supplemented with 25% glucose, 10% hot-pressed soybean meal and 10% glucose at a rate of 1mL / L / h % Isobutanol feed liquid, fermented for 412 hours, until the expression level of ansamcin P-3 no longer increased, the fermentation has ended, the expressi...

Embodiment 3

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

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

[0075] (2) Seed cultivation: as in Example 1;

[0076] (3) Fermentation culture: as in Example 2, the difference is the feeding process. Fermentation culture is 0-60h, and 22.5% glucose solution is added at a flow rate of 0.5mL / L / h. After 60h of fermentation, 1mL / L Supplement with a feed liquid containing 25% glucose and 10% hot-pressed soybean meal at a rate of 1 / h. Isobutanol supplementation adopts a one-time addition of 55mL every 24h; fermentation culture is to 408h, to ansamicin P-3 The expression level no longer increases, the fermentation has ended, the expression level reached 1211mg / L (such as figure 1 Shown);

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

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Abstract

The present invention relates to a method for preparing ansamitocin P-3 from precious actinomycetes orange, comprising the following steps: (1) spore culture: the spores of actinomycetes cryopreserved in 20% glycerol tubes in Streak culture on a solid medium petri dish, culture at 25-30°C for 3-9 days, until abundant spores grow; (2) Seed culture: scrape spores from the petri dish, inoculate in liquid seed medium at 25-30°C Cultivate for 30-50 hours to obtain seed liquid; (3) Fermentation culture: take the cultivated seed liquid and inoculate it into a liquid fermentation medium, ferment and cultivate at 25-30° C., control dissolved oxygen above 20%, and ferment for 350-450 h. The ansamitocin yield is high in the fermented liquid prepared by the method of the invention, and the proportion of ansamitocin P-3 is large, which saves production costs, is beneficial to the purification process in the later stage, and is suitable for industrial production.

Description

Technical field [0001] The invention relates to a method for preparing ansamicin P-3 from precious orange bundle filament actinomycetes, and belongs to the technical field of biological fermentation. Background technique [0002] Ansamitocin is a highly cytotoxic compound produced from actinomycetes such as Actinosynnemapretiosum. The mechanism of action of ansamicin is to inhibit the assembly of tubulin, thereby inhibiting cell division. The cytotoxicity of Ansamicin is very strong, 100-1000 times that of the traditional chemotherapy drugs vinblastine, methotrexate and daunorubicin. [0003] Ansamicin is a macrolide compound, and its structure is similar to maytansinol. The ansamidin expressed by A. aurantiacus 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 the ansamicin homologue is shown in the following formula. [0004] [0005] Ansamicin has high anti-B-16 melanoma, malignant tumor 180 an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 CO LTD