CN101078022A - Method for preparing taxanes by epimerization biological transformation 7-epimer taxanes - Google Patents
Method for preparing taxanes by epimerization biological transformation 7-epimer taxanes Download PDFInfo
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- CN101078022A CN101078022A CN 200610010914 CN200610010914A CN101078022A CN 101078022 A CN101078022 A CN 101078022A CN 200610010914 CN200610010914 CN 200610010914 CN 200610010914 A CN200610010914 A CN 200610010914A CN 101078022 A CN101078022 A CN 101078022A
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Abstract
The present invention relates to a method of forming taxane diterpene by difference transformation 7- taxane using microorganisms. The present invention comprised the following steps: 1. Completely dissolve the 7- taxane compound which has a content of over 1% into the organic solvent that can be mixed with water at any ratio; 2. Add the solution of step 1 into the microbes biological culture, or abstraction enzyme from themicro-organism. An alternative is that after the extractenzyme immobilization, the solution of step 1 is added and the conversion time is 3-100 hours; 3. Extract taxane diterpene using conventional methods from the convert product of step 2. The technical problem solved in this invention is that the no valueble 7- taxane is converted into a valuble enantiomer, so as to save the resources of Yew.
Description
Technical field
The present invention relates to the microorganism difference to changing conversion 7-difference forms method from Taxan to Taxan.
Technical background
Taxol is the cancer therapy drug of the value costliness extracted from Chinese yew, and the raw materials for production wretched insufficiency causes the taxol price high.To the 7-that will not also have pharmaceutical use at present, is present in a large number in the Ramulus et folium taxi cuspidatae by bio-transformation and differ from, can increase the yield of taxol, have great economic worth to changing the precursor that the taxanes material changes into taxol and can be used for semi-synthetic taxol.Difference has multiple compound to changing Taxan, content is more have the 7-difference to taxol, 10-take off acetyl-7-difference take off to Cephalomannine, 10-to taxol, 7-difference that acetyl-7-difference is peaceful to Cephalotaxus fortunei, 10-takes off acetyl-7-difference is to baccatin III etc.Their common features structurally all are that 7 hydroxyls on the Taxan parent nucleus are compared with 7 hydroxyls on the Taxan parent nucleus with pharmaceutical use, difference has taken place to changing variation, can not be used for synthetic is the cancer therapy drug of architecture basics with the Taxan parent nucleus, as taxol or Docetaxel.
Bio-transformation is to utilize enzyme or organism (cell, organoid) to realize the process of chemical conversion as catalyzer, and the most frequently used organism is a microorganism.Bio-transformation has multiple advantages such as high-level efficiency, highly selective, environmental friendliness.Produce the hot subject that drug component has become current biotechnology research with biocatalysis technology.Resemble taxol and be worth costliness, many chiral centres, complex structure, chance heat easy again destructive medicine, the ample scope for abilities of bio-transformation just like this.
Shi Guibao (BMS) company is the enterprise that develops taxol the earliest; also doing deep research aspect the microbial transformation of Taxan; two strain class Nocardia bacterias (Nocardioides) have been obtained; have the Taxan enzyme that to slough taxone C-13 side chain and the deacetylase that removes taxone C-10 ethanoyl; and reached the scale of 5000L; the deacetylase of C-10 position can also will take off acetyl taxol reversible taxol biosynthesis, does not need C-7 position hydroxyl is protected (US Patent 5523219).Successively using Taxan enzyme and Taxan deacetylase many Taxans can be changed into 10-removes the acetyl baccatin III, is used for the semi-synthetic of taxol then.Shi Guibao company has found that also a strain catarrhalis (Morexella sp) can produce 7-β-xyloside enzyme, the xylosyl enzymolysis that this enzyme can take off 10-in the acetyl-7 wood sugar Taxan falls, thereby obtain not contain the Taxan of xylosyl, conversion through 8 hours, what 10-took off that acetyl-7 wood sugar Taxan almost can 100% is transformed (US Patent5700669; Ramesh N.Patel, Annu.Rev.Microbiol, 1998,98:361-394; Aleksey Zaks, current opinion in Chemical Biology, 2001,5:130-136).The Mr. Zhang increases etc. and to have found the isolating microorganism that in the taxusyunnanensis bark 3 bacterial strains have the ability that transforms the natural Japanese yew alkyl compound, wherein Microsphaeropsis onychiun and Mucor (Mucor sp) all hydrolyzable 10-go acetyl-7-difference to 13 side chains of taxol and can make 7 hydroxyl epimerizations (Zhang JZ et al, J Nat Prod, 1998,61 (4): 497-500), this conversion reaction has obtained three products, difference has taken place to changing conversion in one of them product, but this is reflected at difference when changing conversion, sloughed 10-position side chain, 10-takes off acetyl-7 difference and has become 10-to taxol and take off acetyl crust Ka Ting, rather than its enantiomer 10-takes off the acetyl taxol.Owing to formed 3 reaction product simultaneously, target product yield is low, and incubation time reaches 8 days, and this conversion reaction economic worth is little.
Chemical process is not found can effectively finish this difference to changing conversion reaction as yet.Though the chemical conversion 7-difference report to the Taxan method is arranged, the conversion reaction in these reports formed a plurality of products or the result indeterminate, agents useful for same is worth expensive (Kuang Yunyan etc., Chinese pharmaceutical chemistry magazine, 2005,64 (2): 94-96; Zhang Zhiqiang etc., the biotechnology journal, 2000,16 (3): 378-382), economic worth is little.
Summary of the invention
The object of the present invention is to provide a kind of microbial technique that utilizes effectively 7-to be differed to changing narrow spectrum its enantiomer that is transformed into of Taxan, the difference that other structural variation can't take place is to changing the method that bio-transformation forms Taxan.
Difference of the present invention is made up of following steps to the method for changing bio-transformation formation Taxan:
One, with content greater than 1% 7-difference to bearing taxanes be dissolved in fully can with the unlimited miscible organic solvent of water in;
Two, step 1 gained solution is joined cultured microorganisms cultures or the enzyme that from these microorganisms, extracts in, maybe, add step 1 gained solution again with behind the enzyme immobilization that extracts, transform 3-100 hour;
Three, in the converted product of step 2, extract Taxan with ordinary method.
The above-mentioned 7-difference that is used for conversion reaction is to taxane compounds, and purity can be between 1% to 99.9%.Described microorganism such as Mucor (Mucor sp), Rhizopus (Rhizopus.sp), Bacillaceae (Bacillus.Sp), Cunninghammella (Cunninghamella sp), streptomyces (Streptomyces.sp) etc., concrete as: Bacillus subtilus (Bacillus subtilis), mucor spinosus (Mucor spinosus).These microorganisms are known bacterial classification, and its cultural method also is an ordinary method.The concentration of the substrate that is added in microbial culture medium is envelope-bulk to weight ratio (W/V) ten thousand/to one of percentage.When the enzyme that extracts with Institute of Micro-biology transforms, the concentration of the substrate that is added in saccharase is ten thousand/to 10.The organic solvent that is added can be infinitely miscible with water, and as methyl alcohol, ethanol, acetone, pyridine, dimethyl formamide, N,N-DIMETHYLACETAMIDE, dimethyl sulfoxide (DMSO) (DMSO) etc., the amount of its adding can be the 10-10000 times of weight with degree of being dissolved as fully.Invert point is in the microbial growth temperature range that is adopted; When adopting saccharase, invert point is between 30-100 ℃.
The technical problem to be solved in the present invention is: will not have the 7-difference of pharmaceutical use to change into the enantiomer that it has pharmaceutical use to Taxan, thereby reach the purpose of saving taxus resource.The generation Taxan difference that adopts the present invention's acquisition can be effectively poor to changing narrow spectrum its enantiomer that is transformed into of Taxan with 7-to the microorganism strains of changing enzyme, and other structural variation can't take place.For example, be substrate if take off acetyl-7-difference to taxol with 10-, only 10-is taken off acetyl-7-difference and be transformed into enantiomer 10-to taxol and take off the acetyl taxol, rather than become other compound.
Specific embodiments
Embodiment 1: soybean powder medium 1000ml average mark is contained in the triangular flask of 10 500ml, after 28 ℃ of 160rpm cultivate 3 days, the 7-difference to taxol 0.1 gram, is dissolved among the dimethyl formamide DMF of 100ml.Add the N that contains substrate in each triangular flask, dinethylformamide (DMF) solvent 10ml, after 28 ℃ of 160rpm transform 3 days on the shaking table, merge the conversion culture, the fermented liquid centrifugation obtains slag and fermentation clear liquid, fermentation clear liquid is restrained Amberlite XAD-16 macroporous resin adsorption with 200, methyl alcohol with the 10-30% of 4 times of volumes: water flushing macroporous resin, the 30%-100% methanol solution with 5 times of volumes washes macroporous resin again, and this section washing fluid concentrating under reduced pressure is got the brown mixture.About 20 grams of wet slag divide the acetone extraction three times that adds 100ml for three times, and concentrating under reduced pressure must the brown mixture.Merge two kinds of enriched materials, about 10 grams of weight contain the taxol 75mg that transforms formation, and transformation efficiency is 75%.
Embodiment 2: the cultured Bacillus subtilus beef-protein medium of 1000ml is centrifugal, obtain wet thallus 5 grams, with thalline with sodium alginate to embed after, dress up the immobilized cell post.Take off acetyl-7-difference to taxol with dimethyl sulfoxide (DMSO) (DMSO) 100ml dissolving 1 gram 10-, add entry 900ml again, glucose 10 grams add sample solution from the pillar top, the control flow velocity is 4 of per minutes, and preceding 10 hours conversion fluid joins the top of pillar again.Collect conversion fluid, concentrating under reduced pressure vapors away solvent, obtains converted product 5 grams, contains 10-and takes off acetyl taxol 0.41 gram, and transformation efficiency is 41%.
Embodiment 3: with cultured 3000ml mucor spinosus, behind ultrasonic cell-break, the ammonium sulfate precipitation of 20%-500% times of weight obtains saccharase 25 grams, with the saccharase that obtains with macroporous resin adsorption after, the dress post.1 gram 10-is taken off acetyl-7 difference be dissolved in the 100ml methyl alcohol to taxol, add 900ml water again, the solvent that will contain substrate adds from immobilization pillar top, flows out from the lower end, and flow rate control is 2 of per minutes.After effusive solution decompression concentrated mixture 2 grams of converted product and unconverted substrate, contain 10-and take off acetyl taxol 0.35 gram, after acetylize, promptly become taxol.The substrate of unconverted obtain solution is again proceeded to transform.
Embodiment 4: cultivate streptomycete fermentation liquid 5000ml, to contain 10% 10-takes off acetyl-7-difference and is dissolved in the 200ml methanol solvate to Ramulus et folium taxi cuspidatae medicinal extract 2 grams of taxol, medicinal extract solution after the dissolving directly joins in the substratum, continuing to cultivate streptomycete transforms, after 5 days, separate solid composition and fermentation clear liquid, fermentation clear liquid with macroporous resin adsorption after with the methyl alcohol of the 10-30% of 4 times of volumes: water flushing macroporous resin, with the 30%-100% first solution flushing macroporous resin of 5 times of volumes, this section washing fluid concentrating under reduced pressure is got the brown mixture again.The solids component acetone extract.Concentrating under reduced pressure meoh eluate and acetone extract liquid, the extractives 38 that merges the two restrains, and contains 10-and takes off acetyl taxol 1.4 grams.Transformation efficiency is 70%.
Claims (5)
1, a kind of difference is characterized in that being made up of following steps to changing bio-transformation 7-difference to changing the method that Taxan forms Taxan:
One, with content greater than 1% 7-difference to bearing taxanes be dissolved in fully can with the unlimited miscible organic solvent of water in;
Two, step 1 gained solution is joined cultured microorganisms cultures or the enzyme that from these microorganisms, extracts in, maybe, add step 1 gained solution again with behind the enzyme immobilization that extracts, transform 3-100 hour;
Three, in the converted product of step 2, extract Taxan with ordinary method.
2, difference as claimed in claim 1 forms the method for Taxan to changing bio-transformation 7-difference to Taxan, it is characterized in that described can be a kind of in methyl alcohol, ethanol, acetone, pyridine, dimethyl formamide, N,N-DIMETHYLACETAMIDE, the dimethyl sulfoxide (DMSO) with the unlimited miscible organic solvent of water.
3, difference as claimed in claim 1 forms the method for Taxan to changing bio-transformation 7-difference to Taxan, it is characterized in that described microorganism is a Mucor, a kind of in Rhizopus, Bacillaceae, Cunninghammella, the streptomyces.
4, difference as claimed in claim 1 is to changing the method that bio-transformation forms Taxan, it is characterized in that described 7-difference is an envelope-bulk to weight ratio ten thousand/to one of percentage to the concentration of taxane compounds in microbial culture medium.
5, difference as claimed in claim 1 is to changing the method that bio-transformation forms Taxan, it is characterized in that described 7-difference to the concentration of taxane compounds in saccharase be ten thousand/to 10.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988127A (en) * | 2019-04-15 | 2019-07-09 | 云南汉德生物技术有限公司 | It is poor to the method for -10- deacetylate pacilitaxel taxol from 7 |
| CN110105312A (en) * | 2019-06-05 | 2019-08-09 | 云南大学 | A kind of method that 7- difference forms taxane to change conversion to taxane difference |
| CN112126596A (en) * | 2020-08-20 | 2020-12-25 | 云南大学 | Streptomyces spectabilis with disease-resistant and growth-promoting effects and separation method and application thereof |
-
2006
- 2006-05-23 CN CN 200610010914 patent/CN101078022A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988127A (en) * | 2019-04-15 | 2019-07-09 | 云南汉德生物技术有限公司 | It is poor to the method for -10- deacetylate pacilitaxel taxol from 7 |
| CN109988127B (en) * | 2019-04-15 | 2023-05-23 | 云南汉德生物技术有限公司 | Method for synthesizing taxol from 7-differential-10-deacetyltaxol |
| CN110105312A (en) * | 2019-06-05 | 2019-08-09 | 云南大学 | A kind of method that 7- difference forms taxane to change conversion to taxane difference |
| CN110105312B (en) * | 2019-06-05 | 2023-05-09 | 云南大学 | Method for forming taxane through differential conversion of 7-differential taxane |
| CN112126596A (en) * | 2020-08-20 | 2020-12-25 | 云南大学 | Streptomyces spectabilis with disease-resistant and growth-promoting effects and separation method and application thereof |
| CN112126596B (en) * | 2020-08-20 | 2022-06-24 | 云南大学 | Streptomyces spectabilis with disease-resistant and growth-promoting effects and separation method and application thereof |
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