CN103695510A - Method for producing Daptomycin by fed-batch methyl oleate fermentation - Google Patents
Method for producing Daptomycin by fed-batch methyl oleate fermentation Download PDFInfo
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- CN103695510A CN103695510A CN201310644851.5A CN201310644851A CN103695510A CN 103695510 A CN103695510 A CN 103695510A CN 201310644851 A CN201310644851 A CN 201310644851A CN 103695510 A CN103695510 A CN 103695510A
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Abstract
The invention provides a method for producing Daptomycin by fed-batch methyl oleate fermentation. A methyl oleate solution is supplemented into a fermentation tank in a Streptomyces roseosporus fermentation culture process to produce the Daptomycin. Thus, the method provided by the invention can enhance the yield of the Daptomycin. The method provided by the invention widens the path for producing Daptomycin by a fermentation process; and the methyl oleate solution can exist in the form of liquid at normal temperature, and thus, can not crystallize due to low temperature, thereby ensuring the fed-batch effect. Besides, the method is suitable for laboratory small-tank fermentation of Daptomycin, and also suitable for scale-up production in industrialized big tanks.
Description
Technical field
The invention belongs to the production field of daptomycin, relate in particular to a kind of method that stream adds Witconol 2301 producing daptomycin by fermentation.
Background technology
Over nearly 10 years, the trend of Gram-positive drug-fast bacteria infection is in continuous rising.The difficult point for the treatment of clinically bacterium infection at present mainly concentrates on methicillin-resistant staphylococcus aureus (MRSA), the faecalis of vancomycin resistance (VRE), the golden Portugal bacterium (GISA) of glycopeptide class sensitivity etc., they are also the very high pathogenic agent of spreading rate profit lethality rate in Hospitals at Present, although the glycopeptide antibiotics such as vancomycin are treated this class infection and have been obtained certain curative effect, but in recent years, found clinically the resistant organism of vancomycin, and this trend is increasing gradually.Therefore the research of antimicrobial agent new antibiotic becomes again heat subject.Daptomycin all has good sterilization effect to above resistant organism, and preparation medication is convenient, and toxic side effect is little, becomes the best substitute of " pathogenic bacteria last line of defense---vancomycin "; What have more meaning is, daptomycin is as first product of cyclic lipopeptide microbiotic family, its chemical structure and mechanism of action are different from the microbiotic of existing all categories, be over nearly 40 years after mouthful oxazolidinones microbiotic, be applied to clinical unique new texture classification microbiotic, and daptomycin seldom causes generation resistance strain isolated in clinical trial.
Daptomycin (Daptomycin) is a kind of cyclic lipopeptide microbiotic obtaining from Streptomyces roseosporus (Streptomyces roseosporus) separation of fermentative broth, and its chemical structure is suc as formula shown in (I).From formula (I), can find out, daptomycin is comprised of 13 amino acid and 1 longer chain fatty acid, and wherein 10 amino acid form an amino acid ring, separately have 3 amino acid to be arranged in wire, and L-tryptophane (L-Trp) is endways upper, connects 1 n-capric acid.
Daptomycin not only has novel chemical structure, its binding mode is also different from the microbiotic of any existing listing: daptomycin can be upset cytolemma to amino acid whose transhipment, thereby hinder the biosynthesizing of bacteria cell wall peptidoglycan, change the character of cytoplasmic membrane; In addition, it can also be by destroying the cytolemma of bacterium, its content is leaked and reaches the object of sterilization.Daptomycin, by destroy bacterial cell membrane function from many aspects, can kill rapidly gram-positive microorganism, and make bacterium be difficult to its produce resistance (A.Raja et al., Nature Rev.Drug Discov., 2003,2,943-944).Daptomycin, except acting on most of clinical relevant gram-positive microorganisms, the more important thing is, it also presents powerful activity to the resistance isolated strains of X-1497, vancomycin and linwzolid etc. in testing in vitro.
The synthetic method of daptomycin has complete synthesis, the molecular design of chemistry and three kinds of techniques of biosynthesizing, and wherein the first two is planted due to complex technical process and the low commercial value that there is no of productive rate.At present, in industry, the ordinary method of production daptomycin is mainly to make by Streptomyces roseosporus strain fermentation.
Up to the present, thus also not yet relevantly in feed supplement process, add separately the report that Witconol 2301 improves daptomycin output.
Summary of the invention
For addressing the above problem, the present invention adopts following technical scheme:
Stream adds a method for Witconol 2301 producing daptomycin by fermentation, comprises fermentation culture, it is characterized in that: after fermentation culture 20h, add oleic acid methyl ester solution, until fermentation ends in every liter of fermented liquid with the flow velocity of 1.5～3.0ml/h toward feed supplement stream in fermentor tank.
Preferably, the mass concentration of described Witconol 2301 solution is 2.0%; The solvent of described Witconol 2301 solution is the mixed solution that second alcohol and water is made into according to the volume ratio of 1:20.
Beneficial effect of the present invention is as follows:
Widen the path of fermentative Production daptomycin, Witconol 2301 solution is less to the toxicity of cell, and it can exist with the form of liquid at normal temperatures, can Yin Wendu not low and crystallization has ensured the effect that stream adds.In addition, the present invention is not only applicable to laboratory canister fermenting daptomycin, can on the large tank of industrialization, amplify production yet.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art further to understand the present invention, but not limit in any form the present invention.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make some distortion and improvement.These all belong to protection scope of the present invention.
embodiment 1
Stream adds a method for Witconol 2301 producing daptomycin by fermentation, comprises following content:
Cultivate Streptomyces roseosporus seed liquor; Seed liquor is inoculated in fermention medium; Then cultivate and ferment, fermentation culture temperature is 30～32 ℃; After fermentation culture 20h, to stream in fermented liquid, adding mass concentration is 2.0% Witconol 2301 solution (solvent is the mixed solution that second alcohol and water is made into according to the volume ratio of 1:20), the speed that stream adds is: in every liter of fermented liquid with the flow velocity of 1.5～3.0ml/h toward feed supplement in fermentor tank, until after fermentation culture 48～144h, finish fermentation, the fermented liquid obtaining is extracted to separation, obtain described daptomycin.
embodiment 2
Stream adds a method for Witconol 2301 producing daptomycin by fermentation, comprises following content:
A. cultivate Streptomyces roseosporus seed liquor: the Streptomyces roseosporus bacterial strain of preservation is transferred on slant medium, cultivate 120h for 30 ℃, be then inoculated in liquid nutrient medium, in the shaking table of 30 ℃, cultivate 36h, obtain described Streptomyces roseosporus seed liquor.
Wherein, the compound method of described slant medium is as follows: Zulkovsky starch 20g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate heptahydrate 0.5g, green vitriol 0.01g, saltpetre 1g are added in 1L deionized water, regulate pH=7.2, and then add agar 20g, through steam sterilizing, then contra bevel is dull and stereotyped, obtains slant medium;
B. inoculation: the seed liquor obtaining in step a is inoculated in fermention medium (pH=6.5 ~ 7.0 of fermention medium), and inoculum size is 10%;
C. fermentation culture: the postvaccinal substratum obtaining in step b is cultivated and fermented, and fermentation culture temperature is 30～32 ℃;
D. add Witconol 2301: after fermentation culture 20h, to stream in fermented liquid, adding mass concentration is 2.0% Witconol 2301 solution (solvent is the mixed solution that second alcohol and water is made into according to the volume ratio of 1:20), the speed that stream adds is: in every liter of fermented liquid with the flow velocity of 1.5～3.0ml/h toward feed supplement in fermentor tank, until fermentation ends;
E. fermentation ends separation: after fermentation culture 48～144h, finish fermentation, the fermented liquid obtaining is extracted to separation, obtain described daptomycin.
Claims (2)
1. a stream adds the method for Witconol 2301 producing daptomycin by fermentation, comprise fermentation culture, it is characterized in that: after fermentation culture 20h, in every liter of fermented liquid, with the flow velocity of 1.5～3.0ml/h, toward feed supplement stream in fermentor tank, add oleic acid methyl ester solution, until fermentation ends.
2. according to the above-mentioned stream of claim 1, add the method for Witconol 2301 producing daptomycin by fermentation, it is characterized in that, the mass concentration of described Witconol 2301 solution is 2.0%; The solvent of described Witconol 2301 solution is the mixed solution that second alcohol and water is made into according to the volume ratio of 1:20.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310644851.5A CN103695510A (en) | 2013-12-05 | 2013-12-05 | Method for producing Daptomycin by fed-batch methyl oleate fermentation |
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| CN201310644851.5A CN103695510A (en) | 2013-12-05 | 2013-12-05 | Method for producing Daptomycin by fed-batch methyl oleate fermentation |
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| CN103695510A true CN103695510A (en) | 2014-04-02 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104131053A (en) * | 2014-08-15 | 2014-11-05 | 苏州丰倍生物科技有限公司 | Method for producing erythromycin by adding methyl oleate |
| CN106086104A (en) * | 2016-06-08 | 2016-11-09 | 山东大学 | A kind of additive for improving slimeball rhzomorph fermentation yield and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100047873A1 (en) * | 2008-08-01 | 2010-02-25 | Antibioticos S.P.A | Process for the production of daptomycin |
| CN102796680A (en) * | 2012-07-04 | 2012-11-28 | 鲁南新时代生物技术有限公司 | Streptomyces roseosporus and method for producing daptomycin by utilizing combined precursor |
-
2013
- 2013-12-05 CN CN201310644851.5A patent/CN103695510A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100047873A1 (en) * | 2008-08-01 | 2010-02-25 | Antibioticos S.P.A | Process for the production of daptomycin |
| CN102796680A (en) * | 2012-07-04 | 2012-11-28 | 鲁南新时代生物技术有限公司 | Streptomyces roseosporus and method for producing daptomycin by utilizing combined precursor |
Non-Patent Citations (2)
| Title |
|---|
| 吴旻等: "达托霉素发酵过程中前体癸酸的添加策略研究", 《药物生物技术》, vol. 19, no. 2, 31 December 2012 (2012-12-31), pages 142 - 145 * |
| 韩培等: "达托霉素生物合成研究进展及临床研究", 《中国医药工业杂志》, vol. 42, no. 6, 31 December 2011 (2011-12-31), pages 466 - 471 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104131053A (en) * | 2014-08-15 | 2014-11-05 | 苏州丰倍生物科技有限公司 | Method for producing erythromycin by adding methyl oleate |
| CN106086104A (en) * | 2016-06-08 | 2016-11-09 | 山东大学 | A kind of additive for improving slimeball rhzomorph fermentation yield and application thereof |
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