CN109762860A - A kind of alkaline degradation liquid - Google Patents
A kind of alkaline degradation liquid Download PDFInfo
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- CN109762860A CN109762860A CN201910259119.3A CN201910259119A CN109762860A CN 109762860 A CN109762860 A CN 109762860A CN 201910259119 A CN201910259119 A CN 201910259119A CN 109762860 A CN109762860 A CN 109762860A
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Abstract
The invention discloses the alkaline degradation liquid that a kind of fermentation prepares oritavancin intermediate A 82846B, the preparation method of the degradation solution is after the fermentation culture for obtain after fermented and cultured by Amycolatopsis orientalis extracts, it extracts obtained impurity waste liquid and carries out alkaline degradation, obtain alkaline degradation liquid.It after alkaline degradation liquid is added to fermentation medium, ferments again, by adding the impurity alkaline degradation liquid of various concentration, A82846B fermentation unit, which has, to be greatly improved, while component has and significantly improves.
Description
Technical field
The invention belongs to microbial fermentation production technical fields, are related to the fermentation of glycopeptide antibiotics, and in particular to a kind of
The method of oritavancin intermediate A 82846B is prepared by fermentation and a kind of alkaline degradation liquid of precursor is synthesized as A82846B.
Background technique
Glycopeptide antibiotic is the big substance for being generated by microorganism, or being generated by microorganism and carried out part modification.Ten thousand
Ancient mycin and teicoplanin are commercially available antimicrobial product.In the glycopeptide of last century the nineties discovery, including it is referred to as A82846A
(also referred to as ereomomycin), A82846B (also referred to as chlorine orientomycin chloroorienticinA), A82846C are (also referred to as
Orientomycin orienticinC) and orientomycin A.A variety of modifications are carried out to naturally occurring glycopeptide, one of which is
Modification to the reductive alkylation of the reactive amine in glycopeptide.
The FDA on the 7th of August in 2014 ratifies antibiotic oritavancin (oritavancin) and is used for by sensitive gram-positive bacteria
The treatment of caused acute bacterial skin and skin structure infection adult patient.Oritavancin is a kind of single-dose regimen
Antibiotic, have good market prospects.
A82846B is the key intermediate for synthesizing oritavancin, can be by Amycolatopsis orientalis (Amycolatopsis
Orientalis) fermentation generates.In addition to A82846B, Amycolatopsis orientalis can also generate two kinds of main components when fermenting
A82846A and A82846C, structural formula are as follows:
A82846A molecular formula is C73H89N10O26Cl, A82846B molecular formula are C73H88N10O26Cl2, A82846C molecular formula
For C73H90N10O26。
From the foregoing, it will be observed that three kinds of structures of matter are very close, isolate and purify that work is extremely difficult, therefore production cost mentions
Height, the height of the component ratio (A82846B proportion in three kinds of substances) of A82846B extracts extreme influence in fermentation liquid
Yield.China is less about the research of A82846B at present, and fermentation unit and component ratio are all lower, causes production cost to occupy high
Under not.
Summary of the invention
The invention discloses a kind of ferment to prepare the new method of A82846B, by the technique of optimization A82846B fermentation, no
Fermentation unit is improved only, and also improves the component ratio of A82846B, yield is improved, reduces production cost.
During the entire process of fermentation prepares A82846B, a large amount of impurity waste liquids can be generated in the extraction process after fermentation, this
Most of A82846A and A82846C and part A82846B in a little impurity waste liquids comprising being generated in fermentation process.The present invention
Technical staff passes through these impurity waste liquids of research and utilization, it has unexpectedly been found that certain ingredients in the catabolite of these impurity waste liquids
The precursor that can be used as A82846B synthesis, helps to improve the fermentation unit and component ratio of A82846B, to reduce production
Cost.
The method that fermentation of the present invention prepares oritavancin intermediate A 82846B are as follows:
(1) Amycolatopsis orientalis is inoculated into seed culture medium and is cultivated, obtain seed liquor；
(2) seed liquor is inoculated in fermentation medium and carries out fermented and cultured；
(3) fermentation liquid obtained to fermentation extracts, and the A82846B purity extracted is used in 90% above section
Product is extracted, purity is below 90%, is impurity waste liquid；
(4) above-mentioned impurity waste liquid is subjected to alkaline degradation, obtains alkaline degradation liquid；
(5) after the alkaline degradation liquid that step (4) obtains being added to fermentation medium, the seed liquor that step (1) obtains is connect
Kind is fermented in the fermentation medium, and fermented and cultured obtains A82846B.
Wherein, in step (1), the Amycolatopsis orientalis can generate in oritavancin to be commonly used in the art
The Amycolatopsis orientalis bacterial strain of mesosome A82846B, preferably Amycolatopsis orientalis NRRL18099.
In step (1), the seed culture medium can be seed culture medium commonly used in the art.Preferred seed training
Supporting base includes maltodextrin, glucose, soybean powder, yeast extract and calcium carbonate.The pH value of the seed culture medium is 6.5
~7.5, preferably 7.0.
In step (2) and step (5), the method and condition of the fermented and cultured can be the method and item of this field routine
Part.The fermentation medium can be normal fermentation liquid used when being fermented using Nocardia orientalis.Preferably comprise wheat
Bud dextrin, molasses, soybean powder, yeast powder, calcium carbonate culture medium.Further preferably comprising maltodextrin, molasses, soybean powder,
The culture medium of yeast powder, calcium carbonate and chlorate.The chlorate can be any inorganic salts that can provide chloride ion, preferably chlorine
Change one of sodium, potassium chloride, calcium chloride and ammonium chloride or a variety of.The pH value of the fermentation medium is 6.5~7.5, excellent
It is selected as 7.0.
In step (3), the method for the extraction can extract Amycolatopsis orientalis for state of the art is disclosed
The method that fermentation prepares the fermentation liquid of A82846B.For example, can according to WO2006061166, CN101440127, CN87106483,
Method disclosed in CN107434823, CN106928323 extracts.When being extracted with above method, obtained impurity is useless
Liquid includes the most of A82846A and A82846C and part A82846B generated in fermentation process.Typical method is China
Method disclosed in patent application CN106928323, specifically:
The fermentation culture pH that step (2) obtains is adjusted to 10~11, separation of solid and liquid obtains filtrate；Filtrate pH is adjusted
Macroporous absorbent resin is imported after to 9.0-9.5 to be enriched with；By resin purification, is then desorbed with stripping liquid and collect group
Point, obtain A82846B desorption mixed liquor；After desorption mixed liquor is concentrated, reverse phase filler chromatography is carried out, it is molten with polarity
The desorption mixed liquor of agent and salt water is desorbed, and the desorbed solution that chromatography is obtained, A82846B purity is in 90% above section
For extracting product, desorbed solution A82846B purity is then impurity waste liquid below 90%.
Solid-liquid separating method described above is preferably centrifuged, plate compression or decompression filter.
Macroporous absorbent resin described above is independent to be selected from low pole or non-polar resin；Preferably LX18,
XAD1600, HP20 or HZ816, adsorbance 5-10g/L, preferably 6-8g/L.
Resin purification described above is the purified water cleansed resin column for using pH as 7-9, preferable amount 2-4BV.
Stripping liquid described above is 0.5%-1.5% (v/v) aqueous acetic acid, and preferably desorbed solution is that 1% acetic acid is water-soluble
Liquid, dosage 2-5BV.
Condensing mode described above is nanofiltration, and being concentrated into concentration is 20-50mg/ml.
Reverse phase filler described above is micro- using the polymer of polystyrene or the preparation of polyacrylate and its derivative
Ball, ODS C18 or Fraclite800；In the desorption mixed liquor of polar solvent and the salt water, with desorbed solution total volume meter
It calculates, salt water is 0.5%-1% (w/v) NH4H2PO4；Polar solvent is the methanol or acetonitrile of 2-10% (v/v), preferably 2-5% (v/
V) methanol or acetonitrile, stripping liquid dosage are 3-5BV.
In step (4), the alkaline degradation is hydrolyzed in the environment of pH is not less than 11.5, and preferred pH is
11.5~12.5.
In step (5), 1.5%~6% alkaline degradation liquid is added (in terms of fermentation volume).
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
And not meaning that has any restrictions to the present invention
The preparation of 1 seed liquor of embodiment
Seed culture medium: maltodextrin 20g/L, glucose 10g/L, soybean powder 15g/L, yeast extract 3g/L, carbonic acid
Calcium 1g/L, pH7.0.
The bottled 150ml seed culture medium of 750ml triangle, 120 DEG C of sterilizing 30min are stand-by.
The A82846B production strain Nocardia orientalis NRRL 18098 of cryo-conservation is inoculated into ready seed bottle
In, mature shake-flask seed can be obtained in the shaking flask culture 40-50h at 30 DEG C of temperature, revolving speed 250rpm.
Embodiment 2
Fermentation medium: maltodextrin 40g/L, molasses 20g/L, soybean powder 20g/L, yeast powder 10g/L, calcium carbonate 3g/
L, calcium chloride 10g/L, pH7.0.
The canned 35L fermentation medium of 50L fermentation, 120 DEG C of sterilizing 30min are stand-by.
The shake-flask seed of 3.5L maturation is inoculated into the fermentor equipped with 35L feed liquid, condition of culture: 30 DEG C of temperature, being led to
Tolerance 1VVM, tank press 0.05Mpa, initial speed 200rpm, control 30% or more dissolved oxygen by adjusting revolving speed.Culture obtains for 5 days
Fermentation liquid, fermentation unit 515mg/L.
Embodiment 3
The fermentation liquid that embodiment 2 is obtained adjusts pH=10.3 with NaOH solution, carries out plate compression after stirring 2h, obtains
Pressing filtering liquid is adjusted pH=9.2 by 35L pressing filtering liquid, is then introduced into LX18 absorption resin.The Water warfare 9L that resin is 8 with pH
Afterwards, then with 1.0% aqueous acetic acid of 8L it is desorbed, and the component by concentration greater than 500mg/L mixes, forms primary desorption
Then desorption mixed liquor is carried out nanofiltration by mixed liquor, being concentrated into unit is 35000mg/L.
Concentrate is imported to the C18 column balanced, with eluent (5% acetonitrile: 0.5%NH4H2PO4=3:97 (v/v))
It is eluted, collects the component that purity is 90% or more and be used to extract product.A82846B purity 90% it is below then be impurity
Waste liquid.
Impurity waste liquid is collected, nanofiltration concentration, being concentrated into A82846A, A82846B, A82846C three's content summation is about
3%~5%, typical batch impurity concentrate component is as follows:
| Component | A82846A | A82846B | A82846C | It amounts to |
| Content | 3.20% | 1.45% | 0.35% | 5.0% |
Embodiment 4
200ml impurity waste liquid is taken, sodium hydroxide adjusts pH to 11.5,50-60 DEG C water-bath.Start to sample liquid after water-bath 10h
Facies analysis stops water-bath when A82846A, A82846B and A82846C can't detect completely.Impurity degradation liquid cooling is but
Afterwards, sulphur acid for adjusting pH is to neutrality, refrigerates spare after aseptic filtration, obtains being alkaline degradation liquid.
Embodiment 5
Fermentation flask, the bottled 50ml fermentation medium of 250ml triangle, 120 DEG C of sterilizings are prepared according to formula shown in embodiment 2
30min is stand-by.
The alkaline degradation liquid that ready fermentation flask addition embodiment 4 is obtained, additional amount is respectively 1ml, 2ml and 3ml, is
One, two, three groups.The fermentation flask for being added without alkaline degradation liquid is the 4th group, is control group.
The shake-flask seed of 5ml maturation is inoculated into above-mentioned fermentation flask, the shaking flask culture at 30 DEG C of temperature, revolving speed 250rpm
5 days, liquid phase analysis.
Test result see the table below:
It can be seen from the results that the impurity alkaline degradation liquid of addition various concentration, A82846B fermentation unit, which has, substantially to be mentioned
Height, while component has and significantly improves.
Claims (8)
1. a kind of alkaline degradation liquid for preparing oritavancin intermediate A 82846B for fermentation, it is characterised in that the alkaline degradation liquid by
Following method preparation:
(1) Amycolatopsis orientalis is inoculated into seed culture medium and is cultivated, obtain seed liquor；
(2) seed liquor is inoculated in fermentation medium and carries out fermented and cultured；
(3) fermentation liquid obtained to fermentation extracts, and the A82846B purity extracted is in 90% above section for extracting
Product, purity is below 90%, is impurity waste liquid；
(4) above-mentioned impurity waste liquid is subjected to alkaline degradation.
2. alkaline degradation liquid as described in claim 1, it is characterised in that east described in the step of preparing its method (1) is quasi- without branch
Sour bacterium is Amycolatopsis orientalis NRRL 18099.
3. alkaline degradation liquid as described in claim 1, it is characterised in that seed culture medium described in the step of preparing its method (1)
For maltodextrin 20g/L, glucose 10g/L, soybean powder 15g/L, yeast extract 3g/L, calcium carbonate 1g/L, pH7.0.
4. alkaline degradation liquid as described in claim 1, it is characterised in that fermentation medium described in the step of preparing its method (2)
For maltodextrin 40g/L, molasses 20g/L, soybean powder 20g/L, yeast powder 10g/L, calcium carbonate 3g/L, calcium chloride 10g/L,
pH7.0。
5. alkaline degradation liquid as described in claim 1, it is characterised in that the method for extraction described in the step of preparing its method (3)
Are as follows: the fermentation culture pH that step (2) obtains is adjusted to 10.3, plate compression is carried out after stirring, obtains pressing filtering liquid, by filters pressing
Liquid adjusts pH=9.2, is conducted into LX18 absorption resin, after resin Water warfare, then is solved with 1.0% aqueous acetic acid
It inhales, and the component by concentration greater than 500mg/L mixes, forms primary desorption mixed liquor, desorption mixed liquor is then subjected to nanofiltration,
Being concentrated into unit is 35000mg/L, and concentrate is imported to the C18 column balanced, is eluted with eluent, which is
5% acetonitrile by volume: 0.5%NH4H2PO4=3:97 collects the component that purity is 90% or more and is used to extract product.
6. alkaline degradation liquid as described in claim 1, it is characterised in that alkaline degradation described in the step of preparing its method (4) is step
What the impurity waste liquid that (3) obtain suddenly was hydrolyzed in the environment of pH is not less than 11.5.
7. alkaline degradation liquid as claimed in claim 6, it is characterised in that alkaline degradation described in the step of preparing its method (4) is step
What the impurity waste liquid that (3) obtain suddenly was hydrolyzed in the environment of pH is 11.5~12.5.
8. alkaline degradation liquid as claimed in claim 7, it is characterised in that alkaline degradation described in the step of preparing its method (4) are as follows:
The impurity waste liquid for taking step (3) to obtain adjusts pH to 11.5,50-60 DEG C water-bath, starts to sample liquid phase analysis after water-bath 10h, when
When A82846A, A82846B and A82846C can't detect completely, stop water-bath, impurity degradation liquid cooling but after, adjust pH to
Neutrality obtains being alkaline degradation liquid.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910259119.3A CN109762860B (en) | 2019-04-02 | 2019-04-02 | Alkali degradation liquid |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910259119.3A CN109762860B (en) | 2019-04-02 | 2019-04-02 | Alkali degradation liquid |
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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 CN201910259119.3A patent/CN109762860B/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 |
|---|
| WEI-YAN WANG: "《Enhancement of A82846B yield and proportion by overexpressing the halogenase gene in Amycolatopsis orientalis SIPI18099》", 《APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY》 * |
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