CN109811023B - Fermentation preparation method of A82846B - Google Patents
Fermentation preparation method of A82846B Download PDFInfo
- Publication number
- CN109811023B CN109811023B CN201910259121.0A CN201910259121A CN109811023B CN 109811023 B CN109811023 B CN 109811023B CN 201910259121 A CN201910259121 A CN 201910259121A CN 109811023 B CN109811023 B CN 109811023B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- liquid
- acid degradation
- solution
- impurity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing A82846B by fermentation. The method comprises the steps of extracting a fermentation culture solution obtained after fermentation culture of Amycolatopsis orientalis, and carrying out acid degradation on impurity waste liquid obtained by extraction to obtain an acid degradation solution. The acid degradation liquid is added into the fermentation medium and then fermented again, and the A82846B fermentation unit is greatly improved and the components are obviously improved by adding the impurity acid degradation liquid with different concentrations.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation production, relates to a fermentation method of glycopeptide antibiotics, and particularly relates to a method for preparing an oritavancin intermediate A82846B through fermentation.
Background
Glycopeptide antibiotics are a broad class of substances produced by, or partially modified by, microorganisms. Vancomycin and teicoplanin are commercially available antibacterial products. Among the glycopeptides found in the nineties of the last century are those known as A82846A (also known as ereomomycin), A82846B (also known as Chloroorganicin A), A82846C (also known as Orienticin C) and Orienticin A. Various modifications have been made to naturally occurring glycopeptides, one of which is the modification of reductive alkylation of reactive amines in glycopeptides.
The FDA approved the antibiotic oritavancin (oritavancin) for treatment of acute bacterial skin and skin structure infections caused by sensitive gram-positive bacteria in adult patients on 8/7/2014. Oritavancin is an antibiotic with a single dose treatment scheme, and has good market prospect.
A82846B is a key intermediate for synthesizing oritavancin, and can be generated by fermenting Amycolatopsis orientalis (Amycolatopsis orientalis). In addition to A82846B, Amycolatopsis orientalis produces two main components A82846A and A82846C when fermented, and the structural formula is as follows:
A82846A molecular formula is C73H89N10O26Cl, A82846B molecular formula is C73H88N10O26Cl2And the molecular formula of A82846C is C73H90N10O26。
From the above, the three substances have very similar structures, the separation and purification work is quite difficult, the production cost is increased, and the extraction yield is greatly influenced by the height of the component ratio of A82846B in the fermentation liquor (the ratio of A82846B in the three substances). At present, China has less research on A82846B, and fermentation units and component proportion are lower, so that the production cost is high.
Disclosure of Invention
The invention discloses a novel method for preparing A82846B by fermentation, which improves the fermentation unit, improves the component proportion of A82846B, improves the yield and reduces the production cost by optimizing the A82846B fermentation process.
In the whole process of preparing A82846B by fermentation, a large amount of impurity waste liquid is generated in the extraction process after fermentation, and the impurity waste liquid contains most of A82846A and A82846C and part of A82846B generated in the fermentation process. Through research and utilization of the impurity waste liquid, the technical personnel of the invention unexpectedly find that certain components in the degradation products of the impurity waste liquid can be used as precursors for synthesizing A82846B, which is beneficial to improving the fermentation unit and the component proportion of A82846B, thereby reducing the production cost.
The method for preparing the oritavancin intermediate A82846B by fermentation comprises the following steps:
(1) inoculating the amycolatopsis orientalis into a seed culture medium for culture to obtain a seed solution;
(2) inoculating the seed liquid into a fermentation culture medium for fermentation culture;
(3) extracting fermentation liquor obtained by fermentation, wherein the part with the purity of more than 90 percent of A82846B obtained by extraction is used for extracting products, and the part with the purity of less than 90 percent is impurity waste liquor;
(4) carrying out acid degradation on the impurity waste liquid to obtain an acid degradation liquid;
(5) and (3) adding the acid degradation liquid obtained in the step (4) into a fermentation culture medium, inoculating the seed liquid obtained in the step (1) into the fermentation culture medium for fermentation, and performing fermentation culture to obtain A82846B.
Wherein, in the step (1), the amycolatopsis orientalis can be an amycolatopsis orientalis strain which is used conventionally in the field and can produce an oritavancin intermediate A82846B, and preferably the amycolatopsis orientalis NRRL 18099.
In step (1), the seed culture medium may be a seed culture medium conventionally used in the art. Preferred seed media comprise maltodextrin, glucose, soy flour, yeast extract and calcium carbonate. The pH value of the seed culture medium is 6.5-7.5, and preferably 7.0.
In step (2) and step (5), the method and conditions for the fermentation culture may be those conventional in the art. The fermentation medium can be conventional fermentation liquid used in fermentation by utilizing Nocardia orientalis. Preferably a medium comprising maltodextrin, molasses, soy flour, yeast powder, calcium carbonate. Further preferred are media comprising maltodextrin, molasses, soybean flour, yeast powder, calcium carbonate and chloride salts. The chloride salt may be any inorganic salt that can provide chloride ions, preferably one or more of sodium chloride, potassium chloride, calcium chloride and ammonium chloride. The pH value of the fermentation medium is 6.5-7.5, and preferably 7.0.
In the step (3), the extraction method may be a method for extracting a fermentation broth for preparing a82846B by fermenting amycolatopsis orientalis disclosed in the prior art in the field. For example, extraction can be performed according to the methods disclosed in WO2006061166, CN101440127, CN87106483, CN107434823, and CN 106928323. When the extraction is carried out by the method, the obtained impurity waste liquid contains most of A82846A and A82846C and part of A82846B generated in the fermentation process. A typical method is disclosed in chinese patent application CN106928323, which specifically comprises:
adjusting the pH value of the fermentation culture solution obtained in the step (2) to 10-11, and performing solid-liquid separation to obtain a filtrate; adjusting the pH of the filtrate to 9.0-9.5, and introducing into macroporous adsorbent resin for enrichment; purifying the resin, desorbing with desorption solution, and collecting the components to obtain A82846B desorption mixed solution; concentrating the desorption mixed solution, performing reversed phase filler chromatography separation, desorbing with the desorption mixed solution of polar solvent and saline water, and extracting the part of A82846B with purity of more than 90% from the desorption solution obtained by chromatography separation. When the purity of the analysis liquid A82846B is less than 90%, the analysis liquid is an impurity waste liquid.
The solid-liquid separation method is preferably centrifugation, plate-and-frame filter pressing or vacuum filtration.
The macroporous adsorption resin is independently selected from weak polar or non-polar resin; preferably LX18, XAD1600, HP20, or HZ816, in an adsorbed amount of 5-10g/L, preferably 6-8 g/L.
The resin purification adopts a purified water resin column with the pH value of 7-9, and the optimal dosage is 2-4 BV.
The desorption solution is 0.5-1.5% (v/v) acetic acid aqueous solution, preferably the desorption solution is 1% acetic acid aqueous solution, and the dosage of the desorption solution is 2-5 BV.
The concentration mode is nanofiltration, and the concentration is carried out until the concentration is 20-50 mg/ml.
The reversed-phase filler is polymer microspheres prepared from polystyrene or polyacrylate and derivatives thereof, ODS C18 or Fraclite 800; in the desorption mixed solution of the polar solvent and the brine, the brine is 0.5 to 1 percent (w/v) NH calculated by the total volume of the desorption solution4H2PO4(ii) a The polar solvent is 2-10% (v/v) methanol or acetonitrile, preferably 2-5% (v/v) methanol or acetonitrile, and the desorption solution is 3-5 BV.
In step (4), the acid degradation is carried out at a pH of not more than 2.0, preferably 1.0 to 2.0.
And (5) adding 1.5-6% of acid degradation liquid (based on the fermentation volume).
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not meant to limit the invention in any way
Example 1 preparation of seed liquid
Seed culture medium: 20g/L of maltodextrin, 10g/L of glucose, 15g/L of soybean meal, 3g/L of yeast extract, 1g/L of calcium carbonate and pH 7.0.
150ml of seed culture medium is bottled in 750ml triangular bottles and sterilized for 30min at 120 ℃ for standby.
Inoculating A82846B strain NRRL 18099 to the seed bottle, and shake-culturing at 30 deg.C and 250rpm for 40-50 hr to obtain mature shake-flask seed.
Example 2
Fermentation medium: 40g/L of maltodextrin, 20g/L of molasses, 20g/L of soybean meal, 10g/L of yeast powder, 3g/L of calcium carbonate, 10g/L of calcium chloride and pH 7.0.
50L of fermentation tank is filled with 35L of fermentation medium, and sterilized for 30min at 120 ℃ for standby.
3.5L of mature shake flask seeds are inoculated into a fermentation tank filled with 35L of feed liquid, and the culture conditions are as follows: the temperature is 30 ℃, the ventilation volume is 1VVM, the tank pressure is 0.05Mpa, the initial rotating speed is 200rpm, and the dissolved oxygen is controlled to be more than 30 percent by adjusting the rotating speed. Culturing for 5 days to obtain fermentation liquor with a fermentation unit of 515 mg/L.
Example 3
The fermentation liquid obtained in example 2 was adjusted to pH =10.3 with NaOH solution, stirred for 2 hours and subjected to frame filter pressing to obtain 35L of a press filtrate, and the press filtrate was adjusted to pH =9.2 and then introduced into LX18 adsorbent resin. Purifying the resin with water of pH 8 for 9L, desorbing with 8L 1.0% acetic acid water solution, mixing the components with concentration higher than 500mg/L to obtain a first desorption mixed solution, nano-filtering the desorption mixed solution, and concentrating to 35000 mg/L.
The concentrate was introduced into a well-equilibrated C18 column, and eluted with an eluent (5% acetonitrile: 0.5% NH)4H2PO4=3:97 (v/v)), and the fraction having a purity of 90% or more is collected for extraction of the product. The A82846B is impurity waste liquid with purity of less than 90%.
Collecting impurity waste liquid, carrying out nanofiltration concentration, and concentrating until the total content of A82846A, A82846B and A82846C is about 3% -5%, wherein the typical batch impurity concentrated solution comprises the following components:
| components | A82846A | A82846B | A82846C | Total of |
| Content (wt.) | 3.20% | 1.45% | 0.35% | 5.0% |
Example 4
200ml of impurity waste liquid is taken, the pH value is adjusted to 2.0 by sulfuric acid, and water bath is carried out at 50-60 ℃. Sampling of the liquid phase was started after 5h of water bath and stopped when A82846A, A82846B and A82846C were completely undetectable. And (4) cooling the impurity degradation liquid, adjusting the pH value to be neutral by using sodium hydroxide, performing aseptic filtration, and refrigerating for later use to obtain the acid degradation liquid.
Example 5
A fermentation bottle is prepared according to the formula shown in the example 2, 50ml of fermentation medium is bottled in a 250ml triangular bottle, and the fermentation medium is sterilized for 30min at 120 ℃ for standby.
The prepared fermentation bottle was added to the acid degradation solution obtained in example 4 in an amount of 1ml, 2ml and 3ml, one, two and three groups, respectively. The fermentation bottles without acid degradation liquid are the fourth group and are the control group.
5ml of mature shake flask seeds were inoculated into the above-mentioned fermentation flask, shake-cultured at 30 ℃ and 250rpm for 5 days, and subjected to liquid phase analysis.
The results of the tests are given in the following table:
the results show that the addition of the impurity acid degradation liquid with different concentrations greatly improves the fermentation unit of A82846B, and simultaneously, the components are obviously improved.
Claims (4)
1. A method for preparing oritavancin intermediate a82846B by fermentation, comprising:
(1) inoculating the amycolatopsis orientalis into a seed culture medium for culture to obtain a seed solution;
(2) inoculating the seed liquid into a fermentation culture medium for fermentation culture;
(3) extracting fermentation liquor obtained by fermentation, wherein the part with the purity of more than 90 percent of A82846B obtained by extraction is used for extracting products, and the part with the purity of less than 90 percent is impurity waste liquor;
(4) carrying out acid degradation on the impurity waste liquid to obtain an acid degradation liquid;
(5) adding the acid degradation liquid obtained in the step (4) into a fermentation culture medium, inoculating the seed liquid obtained in the step (1) into the fermentation culture medium for fermentation, and performing fermentation culture to obtain A82846B; wherein, the amycolatopsis orientalis of the step (1) is amycolatopsis orientalis NRRL 18099;
the seed culture medium of the step (1) is 20g/L of maltodextrin, 10g/L of glucose, 15g/L of soybean meal, 3g/L of yeast extract, 1g/L of calcium carbonate and pH 7.0;
the fermentation culture medium in the step (2) and the step (5) is 40g/L of maltodextrin, 20g/L of molasses, 20g/L of soybean meal, 10g/L of yeast powder, 3g/L of calcium carbonate, 10g/L of calcium chloride and pH7.0;
the extraction method in the step (3) comprises the following steps: adjusting the pH value of the fermentation culture solution obtained in the step (2) to 10-11, and performing solid-liquid separation to obtain a filtrate; adjusting the pH of the filtrate to 9.0-9.5, and introducing into macroporous adsorbent resin for enrichment; purifying the resin, desorbing with desorption solution, and collecting the components to obtain A82846B desorption mixed solution; concentrating the desorption mixed solution, performing reversed phase filler chromatographic separation, desorbing the desorption mixed solution of a polar solvent and saline water, and using the part of the desorption solution obtained by chromatographic separation, the A82846B with the purity of more than 90 percent, for extracting a product;
the step (4) of acid degradation is that the impurity waste liquid obtained in the step (3) is hydrolyzed under the environment that the pH value is not more than 2.0, the impurity waste liquid obtained in the step (3) is taken, water bath is carried out at 50-60 ℃, sampling liquid phase analysis is started after the water bath is carried out for 5h, when the A82846A, the A82846B and the A82846C cannot be completely detected, the water bath is stopped, and after the impurity degradation liquid is cooled, the pH value is adjusted to be neutral, so that the acid degradation liquid is obtained;
in the step (5), 1.5-6% of acid degradation liquid is added according to the fermentation volume.
2. The method of claim 1, wherein the step (3) comprises the following steps: adjusting the pH value of the fermentation culture solution obtained in the step (2) to 10.3, stirring, performing plate-frame pressure filtration to obtain a pressure filtrate, adjusting the pH value of the pressure filtrate to 9.2, introducing the pressure filtrate into LX18 adsorption resin, purifying the resin with water, desorbing with 1.0% acetic acid aqueous solution, mixing the components with the concentration of more than 500mg/L to form a primary desorption mixed solution, performing nanofiltration on the desorption mixed solution, concentrating to 35000mg/L, introducing the concentrated solution into a balanced C18 column, and eluting with an eluent, wherein the eluent is 5% acetonitrile/0.5% NH by volume4H2PO4The fractions with purity above 90% were collected for product extraction at 97: 3.
3. The method of claim 1, wherein the acid degradation in step (4) is carried out by hydrolyzing the waste impurity solution obtained in step (3) in an environment of pH 1.0-2.0.
4. The method of claim 3, wherein the acid degradation of step (4) is: and (4) taking the impurity waste liquid obtained in the step (3), and adjusting the pH value to 2.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910259121.0A CN109811023B (en) | 2019-04-02 | 2019-04-02 | Fermentation preparation method of A82846B |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910259121.0A CN109811023B (en) | 2019-04-02 | 2019-04-02 | Fermentation preparation method of A82846B |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109811023A CN109811023A (en) | 2019-05-28 |
| CN109811023B true CN109811023B (en) | 2021-08-06 |
Family
ID=66611217
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910259121.0A Active CN109811023B (en) | 2019-04-02 | 2019-04-02 | Fermentation preparation method of A82846B |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109811023B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112430608B (en) * | 2020-12-04 | 2022-03-25 | 浙江大学 | Method for constructing high-yield engineering bacteria of oritavancin precursor and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170157206A1 (en) * | 2014-07-17 | 2017-06-08 | The Medicines Company | High purity oritavancin and method of producing same |
| CN106928323A (en) * | 2017-03-02 | 2017-07-07 | 重庆乾泰生物医药有限公司 | A kind of preparation method of high-purity oritavancin key intermediate A82846B |
| CN107434823A (en) * | 2016-05-26 | 2017-12-05 | 江苏恒瑞医药股份有限公司 | A kind of oritavancin intermediate A 82846B purification process |
-
2019
- 2019-04-02 CN CN201910259121.0A patent/CN109811023B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170157206A1 (en) * | 2014-07-17 | 2017-06-08 | The Medicines Company | High purity oritavancin and method of producing same |
| CN107434823A (en) * | 2016-05-26 | 2017-12-05 | 江苏恒瑞医药股份有限公司 | A kind of oritavancin intermediate A 82846B purification process |
| CN106928323A (en) * | 2017-03-02 | 2017-07-07 | 重庆乾泰生物医药有限公司 | A kind of preparation method of high-purity oritavancin key intermediate A82846B |
Non-Patent Citations (1)
| Title |
|---|
| 《Enhancement of A82846B yield and proportion by overexpressing the halogenase gene in Amycolatopsis orientalis SIPI18099》;Wei-Yan Wang;《Applied Genetics and Molecular Biotechnology》;20180505;第102卷(第13期);第5635-5643页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109811023A (en) | 2019-05-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2003251902B2 (en) | Tiacumicin production | |
| CN106928323B (en) | Preparation method of high-purity oritavancin key intermediate A82846B | |
| CN113354877A (en) | Epsilon-polylysine compound and preparation method and application thereof | |
| CN109811023B (en) | Fermentation preparation method of A82846B | |
| CN107557415B (en) | Fermentation medium and production process for producing oritavancin precursor A82846B | |
| CN104402976B (en) | A kind of method preparing enramycin fine powder | |
| CN109811024B (en) | Method for preparing oritavancin intermediate by fermentation | |
| CN109929895B (en) | Acid degradation liquid | |
| CN109762860B (en) | Alkali degradation liquid | |
| CN113321580A (en) | Method for producing malic acid | |
| CN109971809B (en) | Fermentation preparation method of glycopeptide antibiotic intermediate | |
| CN110055294B (en) | Fermentation method of orlistat intermediate | |
| CN106434443A (en) | Production process of sodium hyaluronate | |
| CN112409426B (en) | Preparation method of sisomicin sulfate | |
| CN105671112A (en) | Method of preparing A82846B through fermentation | |
| CN111100823B (en) | Polymyxin B sulfate production strain, preparation method and application of polymyxin B sulfate | |
| CN111718863B (en) | Method for producing leap-year-old mycin by fermentation | |
| CN109705174B (en) | Method for extracting tobramycin | |
| CN110105435B (en) | Fermentation medium and fermentation method for producing A40926 | |
| CN102690333A (en) | Preparation method of high-purity teicoplanin | |
| CN112143759B (en) | Method for improving yield of orange pigment in monascus mycelium and application | |
| CN103130874B (en) | A kind of preparation method of high purity ramoplanin | |
| CN114437963B (en) | Streptomyces olive and application thereof in biosynthesis of vanillin | |
| CN118307605A (en) | Method for separating lactose-N-neotetraose from microbial fermentation liquid | |
| CN106632551A (en) | Method for preparing fidaxomicin by flash chromatography |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |