阅读说明：本技术 一种去乙酰氧基头孢菌素c水溶液提纯的方法 (Method for purifying aqueous solution of deacetoxycephalosporin C ) 是由 朱洪生 高旋 阮卫国 苏彦波 杨勇 王树林 王瑞 刘建 杨伟 尼米夏·巴特次仁 于 2021-08-03 设计创作，主要内容包括：本发明提供了一种去乙酰氧基头孢菌素C水溶液提纯的方法,通过大孔树脂吸附DAOC水溶液、水洗、解吸的步骤得到纯度高的DAOC。本发明纯化DAOC水溶液的方法工艺流程短、操作简单、收率高、纯度高、质量稳定、成本低,能够获得高纯度的DAOC,具有很高的工业应用价值。(The invention provides a method for purifying a deacetoxycephalosporin C aqueous solution, which comprises the steps of adsorbing a DAOC aqueous solution by macroporous resin, washing with water and desorbing to obtain high-purity DAOC. The method for purifying the DAOC aqueous solution has the advantages of short process flow, simple operation, high yield, high purity, stable quality and low cost, can obtain high-purity DAOC, and has high industrial application value.)

1. A method for purifying an aqueous DAOC solution, comprising the steps of:

(1) adsorbing the DAOC aqueous solution by using macroporous resin to obtain macroporous resin containing DAOC; the temperature of the DAOC aqueous solution is 15.0-21.0 ℃, the pH value is 2.5-3.5, and the DAOC purity is 80-85%;

(2) washing the DAOC-containing macroporous resin obtained in the step (1) with water;

(3) desorbing the resin obtained in the step (2) by using a desorption agent.

2. The method for purifying DAOC aqueous solution with adsorption resin as claimed in claim 1, wherein the titer of the DAOC aqueous solution in step (1) is 8000-12000 μ g/mL.

3. The method for purifying DAOC aqueous solution by using adsorption resin as claimed in claim 1, wherein the adsorption capacity of the macroporous adsorption resin in step (1) is 35-45 g/L.

4. The method for purifying DAOC aqueous solution with adsorption resin of claim 1, wherein the flow rate of adsorption in step (1) is 0.4BV/h to 0.6 BV/h.

5. The method for purifying DAOC aqueous solution with adsorption resin as claimed in claim 1, wherein in step (2), the water is one or a mixture of deionized water and purified water.

6. The method for purifying DAOC aqueous solution with adsorption resin as claimed in claim 1, wherein in step (2), the water washing flow rate is 0.4BV/h to 0.6BV/h, and the total amount of water washing is 4BV to 6 BV.

7. The method for purifying DAOC aqueous solution with adsorption resin as claimed in claim 1, wherein in step (3), the resolving agent is one or more of sodium bicarbonate, sodium acetate and sodium carbonate.

8. The method for purifying DAOC aqueous solution with adsorption resin as claimed in claim 1, wherein in step (3), the concentration of the resolving agent is 0.40% -0.50%, and/or the amount of the resolving agent is 3.0-4.0 BV.

9. The method for purifying DAOC aqueous solution with adsorption resin as claimed in claim 1, wherein the desorption rate in step (3) is 0.4-0.6 BV/h.

10. DAOC product, characterized in that it is obtained by the purification process according to claims 1 to 9.

Technical Field

The invention belongs to the field of biochemistry, and particularly relates to a method for purifying a deacetoxy cephalosporin C aqueous solution.

Background

7-ADCA (7-aminodesacetoxycephalosporanic acid) is used as a novel intermediate of cephalosporin antibiotics, is used for synthesizing medicines such as cefalexin, cephradine, cefadroxil and the like in the medical industry, and is an antibiotic with larger market consumption.

DAOC (desacetoxycephalosporanic acid C) is used as an important raw material for preparing 7-ADCA (7-aminodesacetoxycephalosporanic acid) by using the current enzyme method, is a metabolite of microbial fermentation, and the filtrate generated after DAOC fermentation liquor is subjected to multi-stage filtration has high impurity content and low DAOC purity, so that if the filtrate is not further treated, the impurities are reduced, the purity is improved, the subsequent conversion yield and the finished product quality are influenced, and the production cost is increased, so that a method for removing and purifying DAOC feed liquid is urgently needed, and the subsequent production yield and the product quality are improved.

However, no literature reports a method for purifying DAOC feed liquid at present.

Disclosure of Invention

The invention aims to provide a method for purifying DAOC aqueous solution, which has the advantages of short process flow, simple operation, high yield, high purity, stable quality and low cost.

The invention provides a method for purifying DAOC aqueous solution, which comprises the following steps:

(1) adsorbing the DAOC aqueous solution by using macroporous resin to obtain macroporous resin containing DAOC; the temperature of the DAOC aqueous solution is 15.0-21.0 ℃, the pH value is 2.5-3.5, and the DAOC purity is 80-85%;

(2) washing the DAOC-containing macroporous resin obtained in the step (1) with process water;

(3) desorbing the resin obtained in the step (2) by using a desorption agent.

Further, in the step (1), the titer of the DAOC aqueous solution is 8000-12000 mu g/mL.

Further, in the step (1), the adsorption capacity of the macroporous adsorption resin is 35-45 g/L.

Further, in the step (1), the flow rate of the adsorption is 0.4BV/h-0.6 BV/h.

Further, in the step (2), the process water is one or a mixture of deionized water and purified water.

Further, in the step (2), the flow rate of water washing is 0.4BV/h-0.6BV/h, and the total amount of water washing is 4BV-6 BV.

In step (3), the resolving agent is one or a mixture of two or more of sodium bicarbonate, sodium acetate, and sodium carbonate.

Further, in the step (3), the concentration of the resolving agent is 0.40% -0.50%, and/or the dosage of the resolving agent is 3.0-4.0 BV.

Further, in the step (3), the flow rate of desorption is 0.4-0.6 BV/h.

Further, in the above method, the macroporous resin is DM-700, LX-3020, LX-18, LX-1180, LX-67, LX-16, DM-825, DM1180S or LKA53 macroporous resin, preferably DM-700, LX-3020, LX-18 or LX-1180 macroporous resin, more preferably DM-700 macroporous resin.

The invention also provides a high purity DAOC product purified by the process of claims 1-9, having a purity of greater than 96%.

Experimental results show that the method for purifying the DAOC aqueous solution has the advantages of short process flow, simple operation, high yield, stable quality and low cost, can obtain high-purity DAOC, and has high industrial application value.

Explanation of the terms of the present invention:

the DAOC aqueous solution of the invention refers to: the filtrate is obtained by multi-stage filtration of a fermentation liquid of desacetoxycephalosporan C (DAOC) obtained by fermenting cephalosporium acremonium.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Detailed Description

The DAOC aqueous solution used in the invention is self-made: the method comprises the following steps of firstly carrying out solid-liquid separation on a deacetoxy cephalosporin C (DAOC) fermentation liquid obtained by fermenting cephalosporium acremonium by membrane or plate-and-frame equipment, and then carrying out nanofiltration and ultrafiltration to obtain a DAOC aqueous solution with low impurity content.

The remaining raw materials and equipment were known products, obtained by purchasing commercially available products, unless otherwise specified.

Example 1

A method for purifying DAOC aqueous solution by using adsorption resin specifically comprises the following steps:

(1) adjusting the pH of DAOC aqueous solution with the titer of 8000 mu g/mL to 2.8 by using sulfuric acid, and controlling the temperature to be 15 ℃;

(2) according to the adsorption capacity of 35g/L, carrying out adsorption treatment on the mixture by using DM700 macroporous adsorption resin at the flow rate of 0.4BV/h to obtain macroporous adsorption resin containing DAOC;

(3) washing 4BV of deionized water with 0.5BV/h of macroporous adsorption resin containing DAOC in the step (1);

(4) desorbing 3.5BV of 0.5 percent sodium bicarbonate solution by using the DAOC-containing macroporous adsorption resin in the step (3) at the flow rate of 0.5BV/h, and collecting desorption solution;

the DAOC purity of the desorption solution in the above example is 96.97%, and the desorption rate is 92.11%.

Example 2

A method for purifying DAOC aqueous solution by using adsorption resin specifically comprises the following steps:

(1) adjusting the pH of a DAOC aqueous solution with the titer of 9000 mu g/mL to 2.7 by using sulfuric acid, and controlling the temperature to be 17 ℃;

(2) according to the adsorption capacity of 36g/L, carrying out adsorption treatment on the mixture by using DM700 macroporous adsorption resin at the flow rate of 0.5BV/h to obtain macroporous adsorption resin containing DAOC;

(3) washing 4.5BV of deionized water with 0.6BV/h of macroporous adsorption resin containing DAOC in the step (1);

(4) desorbing 3.8BV of 0.4 percent sodium bicarbonate solution by using the DAOC-containing macroporous adsorption resin in the step (3) at the flow rate of 0.6BV/h, and collecting desorption solution;

the DAOC purity of the desorption solution in the above example is 97.19%, and the desorption rate is 91.82%.

Example 3

A method for purifying DAOC aqueous solution by using adsorption resin specifically comprises the following steps:

(1) adjusting pH of DAOC aqueous solution with titer of 10000 μ g/mL to 2.9 with sulfuric acid, and controlling temperature at 20 deg.C;

(2) according to the adsorption capacity of 37g/L, carrying out adsorption treatment on the mixture by using DM700 macroporous adsorption resin at the flow rate of 0.55BV/h to obtain macroporous adsorption resin containing DAOC;

(3) washing 4.5BV of deionized water with 0.5BV/h of macroporous adsorption resin containing DAOC in the step (1);

(4) desorbing 3.0BV of 0.45 percent sodium bicarbonate solution by using the DAOC-containing macroporous adsorption resin in the step (3) at the flow rate of 0.4BV/h, and collecting desorption solution;

the DAOC purity of the desorption solution in the above example is 97.13%, and the desorption rate is 91.87%.

Example 4

A method for purifying DAOC aqueous solution by using adsorption resin specifically comprises the following steps:

(1) adjusting the pH of DAOC aqueous solution with the titer of 11000 mu g/mL to 3.0 by using sulfuric acid, and controlling the temperature to be 21 ℃;

(2) according to the adsorption capacity of 42g/L, carrying out adsorption treatment on the mixture by using DM700 macroporous adsorption resin at the flow rate of 0.6BV/h to obtain macroporous adsorption resin containing DAOC;

(3) washing 5.0BV of deionized water with 0.5BV/h of macroporous adsorption resin containing DAOC in the step (1);

(4) desorbing 4.0BV of 0.4 percent sodium bicarbonate solution by using the DAOC-containing macroporous adsorption resin in the step (3) at the flow rate of 0.6BV/h, and collecting desorption solution;

the DAOC purity of the desorption solution in the above example is 96.85%, and the desorption rate is 92.08%.

Example 5

A method for purifying DAOC aqueous solution by using adsorption resin specifically comprises the following steps:

(1) adjusting the pH of DAOC aqueous solution with the titer of 9500 mu g/mL to 2.5 by using sulfuric acid, and controlling the temperature at 18 ℃;

(2) according to the adsorption capacity of 40g/L, carrying out adsorption treatment on the mixture by using DM700 macroporous adsorption resin at the flow rate of 0.55BV/h to obtain macroporous adsorption resin containing DAOC;

(3) washing 6BV of deionized water with 0.4BV/h of macroporous adsorption resin containing DAOC in the step (1);

(4) desorbing 4.0BV of 0.47% sodium bicarbonate solution with the flow rate of 0.5BV/h by the DAOC-containing macroporous adsorption resin in the step (3), and collecting desorption solution;

the DAOC purity of the desorption solution in the above example is 97.11%, and the desorption rate is 90.21%.

Example 6

A method for purifying DAOC aqueous solution by using adsorption resin specifically comprises the following steps:

(1) adjusting the pH of DAOC aqueous solution with the titer of 12000 mu g/mL to 3.5 by using sulfuric acid, and controlling the temperature to be 16 ℃;

(2) according to the adsorption capacity of 45g/L, carrying out adsorption treatment on the mixture by using DM700 macroporous adsorption resin at the flow rate of 0.5BV/h to obtain macroporous adsorption resin containing DAOC;

(3) washing 5.0BV of deionized water with 0.5BV/h of macroporous adsorption resin containing DAOC in the step (1);

(4) desorbing 4BV of 0.48 percent sodium bicarbonate solution by using the DAOC-containing macroporous adsorption resin in the step (3) at the flow rate of 0.6BV/h, and collecting desorption solution;

the DAOC purity of the desorption solution in the above example is 97.12%, and the desorption rate is 91.52%.

Example 7

A method for purifying DAOC aqueous solution by using adsorption resin specifically comprises the following steps:

(1) adjusting pH of DAOC aqueous solution with titer of 10000 μ g/mL to 3.0 with sulfuric acid, and controlling temperature at 19 deg.C;

(2) according to the adsorption capacity of 39g/L, carrying out adsorption treatment on the mixture by using DM700 macroporous adsorption resin at the flow rate of 0.5BV/h to obtain macroporous adsorption resin containing DAOC;

(3) washing 5.2BV of deionized water with 0.45BV/h of macroporous adsorption resin containing DAOC in the step (1);

(4) desorbing 3.2BV of 0.46 percent sodium bicarbonate solution by using the DAOC-containing macroporous adsorption resin in the step (3) at the flow rate of 0.4BV/h, and collecting desorption solution;

the DAOC purity of the desorption solution in the above example is 97.17%, and the desorption rate is 91.98%.

Example 8

A method for purifying DAOC aqueous solution by using adsorption resin specifically comprises the following steps:

(1) adjusting the pH of DAOC aqueous solution with the titer of 10500 mu g/mL to 2.8 by using sulfuric acid, and controlling the temperature to be 17 ℃;

(2) according to the adsorption capacity of 36g/L, carrying out adsorption treatment on the mixture by using DM700 macroporous adsorption resin at the flow rate of 0.5BV/h to obtain macroporous adsorption resin containing DAOC;

(3) washing 4.3BV of deionized water with 0.47BV/h of macroporous adsorption resin containing DAOC in the step (1);

(4) desorbing 3.3BV of 0.44 percent sodium bicarbonate solution by using the DAOC-containing macroporous adsorption resin in the step (3) at the flow rate of 0.6BV/h, and collecting desorption solution;

the DAOC purity of the desorption solution in the above example is 97.06%, and the desorption rate is 92.3%.

The purity of the DAOC before and after purification in the above examples by the process of the invention is shown in Table 1:

therefore, the purity of the DAOC purified by the method is obviously improved, and the purity of the obtained desorption solution DAOC is up to over 96.5 percent.

The beneficial effects of the method of the invention are demonstrated by the following experimental examples.

Experimental example 1 screening test of Process parameters of the present invention

1. Screening of temperature and pH conditions:

(1) experimental methods

a. Taking the feed liquid with different temperatures to carry out adsorption experiments, and taking the adsorption residual liquid to carry out titer detection in the experiment process under the same conditions as in example 7, and calculating the total adsorption amount of the resin to the target object under the condition of similar leakage titer;

b. the adsorption experiment was performed on the feed liquid with different pH ranges, and the adsorption ratio was calculated under the same conditions as in example 7.

(2) The experimental results are shown in tables 1 and 2:

TABLE 1 feed liquid experimental data at different temperatures

TABLE 2 pH Material liquid Experimental data in different ranges

The results show that only in the temperature and pH range of the invention, the resin has stronger adsorption capacity to DAOC solution, and the feed liquid is not easy to degrade, thus being beneficial to further purification and impurity removal.

2. Titer screening of aqueous DAOC solutions:

(1) experimental methods

In the temperature and pH ranges of the feed liquid screened in the previous step, the feed liquids with different titers are taken to carry out an adsorption experiment, the other conditions are the same as those in example 7, and the yield is calculated.

(2) The results are shown in Table 3:

TABLE 3 adsorption results for different titers of DAOC aqueous solutions

The above results indicate that the titer of the aqueous DAOC solution before purification is too low, the adsorption amount of the resin is decreased, which is disadvantageous for purification and separation, and that when the titer is more than 12000. mu.g/mL, although the adsorption ratio is not so large, the titer leakage is early at the time of washing with water, which results in a lower yield. The titer of the DAOC aqueous solution is preferably in the range of 8000-12000 mu g/mL, and the best purification effect and higher yield can be obtained.

3. Screening the types of macroporous resin:

(1) experimental methods

The adsorption test was performed using different types of resins, and the adsorption amount was counted under the same conditions as in example 7.

(2) The results of the experiment are shown in table 4:

TABLE 4 results of adsorption on different types of macroporous resins

The results show that the preferred macroporous adsorption resin is DM-700, LX-3020, LX-18 or LX-1180, the adsorption capacity is high, which is beneficial to achieving excellent purification effect, and the other types of resin have low adsorption capacity and poor separation effect.

4. Screening of adsorption flow rate:

(1) experimental methods

Adsorption experiments were performed at different flow rates, and the conditions were the same as in example 7, and the feed solution after adsorption was taken for detection.

(2) The results of the experiment are shown in table 5:

TABLE 5 adsorption results for different adsorption flow rates

The above results indicate that too large adsorption flow rate results in leakage of titer, while too small results in too long adsorption period, which is not favorable for industrial production applications, and therefore, the adsorption flow rate is preferably in the range of 0.4BV/h to 0.6BV/h, the adsorption period is suitable and the treatment efficiency is high.

5. Screening the flow rate of water washing:

(1) experimental methods

The resin column after completion of the adsorption was washed with water at different flow rates under the same conditions as in example 7, and the titer was measured at the end of the washing.

(2) The results of the experiment are shown in table 6:

TABLE 6 results for different water wash flow rates

The above results show that the excessive washing flow rate can cause the target DAOC to leak out in advance, and the impurity separation effect is influenced; on the other hand, if the flow rate is too low, the water system time is too long, which is not favorable for industrial production and application, and the washing time is shortened as much as possible while the titer and the separation effect are ensured as much as possible, therefore, the washing flow rate is preferably in the range of 0.4BV/h to 0.6BV/h, the washing cycle is appropriate, and the treatment efficiency is high.

6. Screening of the concentration of the resolving agent:

(1) experimental methods

Desorbing the washed resin column with different concentrations of the desorbing agent under the same conditions as in example 7, detecting the titer, purity and other quality indexes of the final desorbing solution, and calculating the yield.

(2) The results of the experiment are shown in table 7:

TABLE 7 desorption results for different concentrations of desorption agent

The above results show that the concentration of the resolving agent is preferably in the range of 0.4 to 0.5%, and the effects of good separation and purification and high yield can be achieved.

7. Screening the dosage of the resolving agent:

(1) experimental methods

Desorbing the washed resin column with different volumes of the same concentration of the desorbing agent, collecting the desorbing solution and calculating the desorbing rate under the same conditions as in example 7.

(2) The results of the experiment are shown in table 8:

TABLE 8 desorption results for different amounts of desorbent

The results show that the dosage of the resolving agent is too small, the desorption effect is poor, the yield is low, and the change of the desorption rate is not large after the dosage of the resolving agent is higher than 3BV, so that the dosage of the resolving agent is preferably within the range of 3.0-4.0BV, the good desorption effect can be ensured, and the resource cost can be saved.

8. Screening of desorption flow rate:

(1) experimental methods

Desorbing the washed resin column with the desorbing agent at different flow rates, collecting the desorbing solution for detection under the same conditions as in example 7.

(2) The results of the experiment are shown in table 9:

TABLE 9 desorption results for different desorption flow rates

The results show that the flow rate of the desorption agent is preferably in the range of 0.4-0.6BV/h, good separation and purification effects can be ensured, the desorption period is shortened, and the yield is improved.

In conclusion, the method for purifying the DAOC aqueous solution provided by the invention has the advantages of short process flow, simple operation, high yield, stable quality and low cost, can obtain high-purity DAOC, and has high industrial application value.