CN109929895B

CN109929895B - Acid degradation liquid

Info

Publication number: CN109929895B
Application number: CN201910259134.8A
Authority: CN
Inventors: 袁建栋; 刘会明
Original assignee: Brightgene Bio Medical Technology Co Ltd
Current assignee: Brightgene Bio Medical Technology Co Ltd
Priority date: 2019-04-02
Filing date: 2019-04-02
Publication date: 2021-05-18
Anticipated expiration: 2039-04-02
Also published as: CN109929895A

Abstract

The invention discloses an acid degradation liquid for preparing an oritavancin intermediate A82846B through fermentation. 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

Acid degradation liquid

Technical Field

The invention belongs to the technical field of microbial fermentation production, relates to fermentation of glycopeptide antibiotics, and particularly relates to a method for preparing an oritavancin intermediate A82846B through fermentation and an acid degradation liquid serving as a synthetic precursor of A82846B.

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.

In the step (5), 1.5-6% of acid degradation liquid (based on fermentation volume) is added.

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 NRRL18099 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 pressure filtration to obtain 35L of a filtrate, and the filtrate was adjusted to pH 9.2 and 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)₄H₂PO₄97(v/v)) and fractions with a purity of 90% or more were collected for product extraction. 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

1. An acid degradation liquid for preparing an oritavancin intermediate A82846B through fermentation is characterized by being prepared by the following method:

(4) carrying out acid degradation on the impurity waste liquid; wherein the content of the first and second substances,

in the step (1), the amycolatopsis orientalis is amycolatopsis orientalis NRRL18099, the seed culture medium is maltodextrin 20g/L, glucose 10g/L, soybean meal 15g/L, yeast extract 3g/L, calcium carbonate 1g/L, pH7.0;

in the step (2), the fermentation medium is maltodextrin 40g/L, molasses 20g/L, soybean powder 20g/L, yeast powder 10g/L, calcium carbonate 3g/L, calcium chloride 10g/L and pH7.0;

in the step (3), the extraction method 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% by volumeAcetonitrile 0.5% NH₄H₂PO₄97, collecting the components with the purity of more than 90 percent for extracting the product;

in the step (4), the 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 the pH value is adjusted to be neutral after the impurity degradation liquid is cooled, so that the acid degradation liquid is obtained.

2. The acid degradation solution according to claim 1, wherein in the step (4), the acid degradation is performed by hydrolyzing the impurity waste liquid obtained in the step (3) in an environment of pH 1.0-2.0.

3. The acid degradation liquid according to claim 2, wherein in the step (4), the acid degradation is performed by adjusting the pH of the waste impurity liquid obtained in the step (3) to 2.0.