CN117106844A - Method for synthesizing phenoxyacetamido penicillin potassium salt by fermentation method - Google Patents

Method for synthesizing phenoxyacetamido penicillin potassium salt by fermentation method Download PDF

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CN117106844A
CN117106844A CN202310991180.3A CN202310991180A CN117106844A CN 117106844 A CN117106844 A CN 117106844A CN 202310991180 A CN202310991180 A CN 202310991180A CN 117106844 A CN117106844 A CN 117106844A
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fermentation
catalyst
phenoxyacetamido
solution
potassium salt
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陈英新
王路路
韩贺东
白雪峰
安志强
彭涛
郑兵
邢明亮
王艳枫
解荣慧
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Inner Mongolia Changsheng Pharmaceutical Co ltd
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    • C12R2001/19—Escherichia coli

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Abstract

The invention provides a method for preparing phenoxyacetamido penicillin potassium salt by a fermentation method, which comprises the following steps: (1) fermenting the catalyst; (2) separating the catalyst; (3) purifying the catalyst; (4) synthesizing phenoxyacetamido penicillin potassium salt; (5) separating the product and recycling the catalyst; (6) azeotropic crystallization. Compared with the prior art, the method adopts the fermentation catalyst to catalyze and synthesize and adopts the one-step crystallization method to prepare the phenoxyacetamido penicillin potassium salt, has the advantages of short process flow, short production period, simple operation, environmental protection, no pollution, low production cost, high Performance Liquid Chromatography (HPLC) purity of the product being more than or equal to 99.80 percent, undetected p-hydroxyphenoxyacetamido penicillin, polymer impurity content being less than or equal to 0.013 percent, clarity being less than or equal to 0.5#, product quality being superior to the phenoxyacetamido penicillin potassium salt produced by the fermentation method and the two-step crystallization method, not only having good production and application value, but also having important significance for guaranteeing medication safety and effectiveness.

Description

Method for synthesizing phenoxyacetamido penicillin potassium salt by fermentation method
Technical Field
The invention belongs to the technical field of biological medicine and chemical industry, and particularly relates to a method for synthesizing phenoxyacetamido penicillin potassium salt by a fermentation method.
Background
The phenoxyacetamido penicillin potassium is a clinically common antibiotic drug, is the first choice drug for treating gram positive bacteria and partial negative bacteria infection, and has been widely applied to the clinic for treating abscess, suppurative meningitis, pneumonia, gonorrhea and other diseases. Because the oral preparation is stable to acid, is not easy to be damaged by gastric acid, is good in oral absorption, and is especially suitable for children. At present, phenoxyacetamido penicillin potassium is mainly produced by a microbial fermentation method and a two-step crystallization method.
The microbial fermentation method is that fermentation liquor is subjected to the steps of acidification extraction, decoloration filtration, carbonate solution extraction, azeotropic crystallization, washing, drying and the like to obtain phenoxyacetamido penicillin potassium, the production period of the process is as long as half a month, and the product possibly contains synthesis precursors, process byproducts and various degradation products; in addition, potassium phenoxyacetamido penicillin may also polymerize itself, producing a high molecular weight polymer. This is mainly due to the fact that the microbial fermentation method has complex raw material components, complex microbial metabolites and metabolic pathways, and the longer the production process is, the greater the probability of impurity generation.
The two-step crystallization method is to synthesize intermediate crystalline acid by using 6-APA and phenoxyacetic acid derivatives under the catalysis of immobilized penicillin acylase, the crystalline acid is salinized into potassium salt solution again, and then the potassium salt product is obtained through azeotropic distillation. In addition, as a part of immobilized enzyme, the structure and performance of the carrier material can cause large enzyme activity loss, low enzyme loading rate and low enzyme activity in the immobilization process, and the carrier of the immobilized enzyme has low affinity with the enzyme and poor mechanical stability, so that the enzyme is easy to fall off from the carrier in the catalytic reaction or cyclic catalytic reaction process, and the product quality and the recycling times of the enzyme are affected; secondly, the enzyme activity is seriously reduced due to the harsh condition of the enzyme immobilization process, and the use of an organic solvent is not beneficial to the enzyme activity; meanwhile, the immobilized enzyme has complex production process and high production cost.
The preparation of phenoxyacetamido penicillin potassium salt by adopting the catalytic synthesis of the fermentation catalyst and the one-step crystallization method has the advantages of short production period, simple process, mild reaction condition, high conversion rate, few byproducts, few impurities such as polymers, high product quality and the like. However, the quality of the domestic produced phenoxyacetamido penicillin potassium bulk drug is quite far from that of foreign bulk drugs. Therefore, how to produce the phenoxyacetamido penicillin potassium with low impurity content, especially low polymer impurity content, reaches or even exceeds the quality level of foreign raw materials, and has important significance for guaranteeing the medication safety and effectiveness.
Disclosure of Invention
The invention aims to provide a method for preparing phenoxyacetamido penicillin potassium salt by a fermentation method.
In order to achieve the aim of the invention, the invention provides a method for synthesizing phenoxyacetamido penicillin potassium salt by a fermentation method, which comprises the following steps:
(1) preparing a fermentation catalyst by fermentation;
(2) separating a fermentation catalyst;
(3) purifying a fermentation catalyst;
(4) under the catalysis of a fermentation catalyst, controlling the temperature to be 20-28 ℃, and controlling the pH to be 6.5-8.0 by using 10-30% potassium carbonate solution to enable 6-APA and phenoxyacetic acid derivatives to react to generate a product phenoxyacetamido penicillin potassium salt;
(5) separating products and recycling a fermentation catalyst;
(6) azeotropic crystallization to obtain final product phenoxyacetamido penicillin potassium salt.
In the present invention, the fermentation catalyst is penicillin acylase.
Further, step (1) includes: culturing strains with fermentation catalyst production capacity in a primary seed culture medium and a secondary seed culture medium in sequence under the conditions of natural pH and 33-36 ℃ to obtain a secondary seed solution; inoculating the secondary seed solution to a fermentation medium, and culturing for 20-30 hours at the pH of 6.5-6.8 and the temperature of 33-35 ℃ to obtain fermentation liquor; the bacterial content of the fermentation liquor is 1.0X10 9 -1.0×10 11 CFU/mL；
Wherein, the components of the seed culture medium comprise: peptone, yeast powder, sodium chloride, etc.; the components of the fermentation medium comprise: peptone, yeast powder, glycerol, potassium dihydrogen phosphate, disodium hydrogen phosphate, etc.
Preferably, the inoculation amount of the secondary seed liquid is 3-6v/v%.
Further, step (2) includes: centrifuging the fermentation liquor at 5-10deg.C and 8000-12000g, collecting thallus, and crushing thallus by high-pressure homogenizer to release fermentation catalyst; adding flocculant into the obtained homogeneous solution to flocculate cell debris, centrifuging, and collecting supernatant.
Wherein the operation pressure of the high-pressure homogenizer is 800-1000bar, and homogenizing is carried out for 2-4 times.
The flocculant can be at least one selected from benzalkonium chloride, benzalkonium bromide, polyethylene oxide, polystyrene sulfonate, polyacrylamide derivative, polyethyleneimine and the like.
Further, step (3) includes: concentrating the supernatant by ultrafiltration to remove small molecular compounds, polypeptides and amino acids; adding ammonium sulfate to the obtained concentrated solution to precipitate a fermentation catalyst, and removing the impurity protein; then phosphate buffer solution is used for dissolving the fermentation catalyst, and ammonium sulfate and small molecular compounds are removed through ultrafiltration dialysis; and (3) obtaining fermentation catalyst concentrated solution, and performing anion exchange resin column chromatography to obtain the purified fermentation catalyst.
The enzyme purification method comprises the following steps: ultrafiltering the supernatant with ultrafiltration membrane with molecular weight of 10000-50000MW at 5-10deg.C, concentrating until concentration of concentrated solution fermentation catalyst is 60-100mg/mL, stopping ultrafiltering, and removing small molecular compounds, polypeptides and amino acids; adding ammonium sulfate into the concentrated solution to precipitate a fermentation catalyst at 5-10 ℃ to remove the impurity protein; dissolving the fermentation catalyst in phosphate buffer solution with pH of 7.8-8.2 and 0.02-0.025M at 5-10deg.C, ultrafiltering the solution with ultrafiltration membrane with molecular weight of 10000-50000MW at 5-10deg.C until the conductivity of the fermentation catalyst solution is less than or equal to 4MS, concentrating to fermentation catalyst concentration of 80-100mg/mL, and removing ammonium sulfate and small molecular compounds; and (3) subjecting the fermentation catalyst concentrated solution to anion exchange resin column chromatography at the temperature of 5-10 ℃ and the rate of 0.5BV/h, and finally, performing top washing with 2BV (2 column volumes) and 0.02-0.025M phosphate buffer solution with the pH of 7.8-8.0 to obtain the purified fermentation catalyst.
Further, step (4) includes: under the condition of 20-28 ℃,6-APA and phenoxyacetic acid derivatives react for 1-1.5 hours under the action of a fermentation catalyst by using 10-30% potassium carbonate solution to control pH to 6.5-8.0, and the obtained reaction liquid contains phenoxyacetamido penicillin potassium salt.
Wherein the molar ratio of the 6-APA to the phenoxyacetic acid derivative is 1 (1-1.3).
The total activity of the fermentation catalyst is 120-200U/g calculated by 6-APA.
The phenoxyacetic acid derivative may be at least one selected from methyl phenoxyacetate, ethyl phenoxyacetate, glyceryl phenoxyacetate, alcohol phenoxyacetate, etc.
In the present invention, the fermentation catalyst is added in liquid form.
Further, step (5) includes: ultrafiltering the reaction solution with ultrafilter membrane with molecular weight of 10000-50000MW at 5-10deg.C to concentrate and recover fermentation catalyst, and the concentrated fermentation catalyst solution is used for synthesizing phenoxyacetamido penicillin potassium salt; nanofiltration concentration of the dialyzate with nanofiltration membrane with molecular weight of 200-300MW at 10-25deg.C to obtain concentrated solution of phenoxyacetamido penicillin potassium salt.
Further, step (6) includes: adding a water carrying agent into the concentrated solution of phenoxyacetamido penicillin potassium salt, and concentrating by reduced pressure distillation until crystals are separated out.
The water-carrying agent can be at least one selected from n-butanol, isobutanol, benzene, toluene and the like.
Preferably, the strain used for the fermentative production of the catalyst according to the invention is E.coli.
The synthetic route of the phenoxyacetamido penicillin potassium salt is shown in figure 1.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the invention solves the problems of complex production process, long production period, high production cost, and high probability of producing impurities such as process byproducts, degradation products, high molecular polymers and the like in the fermentation method.
The invention effectively avoids the complex production process flow and long production procedure of the two-step crystallization method, and increases the possibility of producing degradation products, polymers and other impurities. Meanwhile, the problems that the carrier of the immobilized enzyme has low affinity with the enzyme and poor mechanical stability, so that the enzyme is easy to fall off from the carrier in the catalytic reaction or cyclic catalytic reaction process, the product quality and the recycling frequency of the enzyme are further affected are solved.
The invention adopts the fermentation catalyst to catalyze and synthesize and adopts a one-step crystallization method to prepare the phenoxyacetamido penicillin potassium salt, and simultaneously adopts an ultrafiltration membrane concentration process to recycle the catalyst, thereby ensuring the repeatability of the catalyst and being applicable in large scale; the process for preparing the phenoxyacetamido penicillin potassium salt by the method has the advantages of short production period, simple operation, environmental protection, no pollution, low production cost, high Performance Liquid Chromatography (HPLC) purity of the product being more than or equal to 99.80 percent, undetected p-hydroxyphenoxyacetamido penicillin, polymer impurity content being less than or equal to 0.013 percent, clarity being less than or equal to 0.5#, product quality being superior to that of phenoxyacetamido penicillin potassium salt produced by a fermentation method and a two-step crystallization method, and the method not only has good production and application values, but also has important significance for guaranteeing medication safety and effectiveness.
Drawings
FIG. 1 shows the synthetic route of the potassium salt of phenoxyacetamido penicillin according to the present invention.
Detailed Description
The invention provides a method for preparing phenoxyacetamido penicillin potassium salt by a fermentation method, which comprises the following steps:
(1) and (3) fermenting a catalyst: e.coli is inoculated to a fermentation medium by a primary seed culture medium and a secondary seed culture medium under the conditions of natural pH and 33-36 ℃ and is cultured for 20-30 hours under the conditions of pH6.5-6.8 and 33-35 ℃ to obtain fermentation liquor.
(2) Separation of the catalyst: centrifuging fermentation liquor, collecting thalli, crushing thalli by a high-pressure homogenizer to release a catalyst; adding flocculant into the obtained homogeneous solution to flocculate cell debris, centrifuging and collecting clear liquid.
(3) Purification of the catalyst: concentrating the clear solution by ultrafiltration to remove small molecular compounds, polypeptides and amino acids; precipitating the catalyst and removing the impurity protein from the concentrated solution; dissolving the catalyst, ultrafiltering the solution, and dialyzing to remove ammonium sulfate and small molecular compounds; and (3) subjecting the concentrated solution to anion exchange resin column chromatography to obtain the purified catalyst.
(4) Synthesis of phenoxyacetamido penicillin potassium salt: under the condition of 20-28 ℃,6-APA and phenoxyacetic acid derivatives react for 1-1.5 hours under the action of a catalyst by using 10-30% potassium carbonate solution to control pH to 6.5-8.0, and the obtained reaction liquid contains phenoxyacetamido penicillin potassium salt.
(5) Product separation and catalyst recycling: the reaction solution is ultrafiltered, concentrated and used for recovering the catalyst, the concentrated catalyst is used for synthesizing phenoxyacetamido penicillin potassium salt, and the dialyzate is subjected to nanofiltration concentration to obtain the phenoxyacetamido penicillin potassium salt concentrated solution.
(6) Azeotropic crystallization: adding water-carrying agent into the concentrated solution of phenoxyacetamido penicillin potassium salt, and concentrating by reduced pressure distillation until crystals are separated out.
Preferably, the seed culture medium component in the step (1) comprises peptone, yeast powder and sodium chloride; the fermentation medium comprises peptone, yeast powder, glycerol, potassium dihydrogen phosphate and disodium hydrogen phosphate; the inoculation amount of the fermentation medium is 3-6%.
Preferably, the centrifugal force in the step (2) is 8000-12000g, and the centrifugal temperature is 5-10 ℃; the running pressure of the high-pressure homogenizer is 800-1000bar, and the homogenizing operation is carried out for 2-4 times; the flocculant is benzalkonium chloride, benzalkonium bromide, polyoxyethylene, polystyrene sulfonate, polyacrylamide derivatives, polyethyleneimine and the like.
Preferably, the supernatant ultrafiltration concentration conditions in step (3) include: molecular weight 10000-50000MW and temperature 5-10deg.C; the catalyst precipitant is ammonium sulfate, and the precipitation temperature is 5-10 ℃; the catalyst dissolution solvent is phosphate buffer solution with pH of 7.8-8.2 and concentration of 0.02-0.025M, and the dissolution temperature is 5-10 ℃; the condition of ultrafiltration dialysis of the dissolved solution comprises: molecular weight 10000-50000MW and temperature 5-10deg.C; the column chromatography conditions are as follows: the temperature is 5-10 ℃ and the speed is 0.5BV/h, and 2BV and 0.02-0.025M phosphate buffer solution are used for top washing.
More preferably, the ultrafiltration is stopped when the supernatant fluid in the step (3) is concentrated to the concentration of 60-100mg/mL of concentrated solution catalyst; the dissolved solution is ultrafiltered and dialyzed until the catalyst conductivity is less than or equal to 4MS, and then further concentrated until the catalyst concentration is 80-100mg/mL.
Preferably, the molar ratio of the 6-APA synthesized by the phenoxyacetamido penicillin potassium salt and the phenoxyacetic acid derivative in the step (4) is 1 (1-1.3); the phenoxyacetic acid derivatives include, but are not limited to, methyl phenoxyacetate, ethyl phenoxyacetate, glycerol phenoxyacetate and alcohol phenoxyacetate; the catalyst is added in a liquid form, and the total activity of the fermentation catalyst is 120-200U/g calculated by 6-APA.
Preferably, the ultrafiltration concentration conditions in step (5) include: molecular weight 10000-50000MW and temperature 5-10deg.C; the dialysate nanofiltration concentration conditions include: the molecular weight is 200-300MW and the temperature is 10-25 ℃.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
The strain used for the catalyst fermentation in the following examples was E.coli, specifically penicillin acylase (E.Coli254) strain, purchased from North pharmaceutical Co., ltd.
The formula of the seed culture medium comprises: 1% of peptone, 0.5% of yeast powder, 1% of sodium chloride, 0.05% of defoaming agent and natural pH.
The formula of the fermentation medium comprises: 1% of peptone, 0.5% of yeast powder, 10ml/L of glycerin, 0.15% of potassium dihydrogen phosphate, 0.23% of disodium hydrogen phosphate, 0.05% of defoaming agent and natural pH.
The anion exchange resin column was purchased from GE company as DEAE fast flow, agarose gel material.
Example 1 fermentation process for preparing potassium salt of phenoxyacetamido penicillin
1. And (3) fermenting a catalyst: mixing peptone, yeast powder, sodium chloride, etc. with deionized water according to the formula of the seed culture medium, sterilizing at 121deg.C for 30min, and cooling to room temperature for use. Uniformly mixing peptone, yeast powder, glycerol, potassium dihydrogen phosphate, disodium hydrogen phosphate and the like with deionized water according to a fermentation medium formula, naturally adjusting the pH, sterilizing at 121 ℃ for 30min, and cooling to room temperature for later use. E.coli is inoculated into the seed culture, primary and secondary seed expansion culture is carried out based on the condition of 33+/-0.5 ℃, when the OD value of a seed tank is between 1.8 and 2.3 and the pH value is 7.4 to 7.8, the secondary seed liquid is inoculated into the fermentation culture medium according to the inoculation amount of 6 percent of the volume of the fermentation culture medium, and the fermentation liquid is obtained after the secondary seed liquid is cultured for 20 hours under the condition of pH value of 6.5+/-0.05 and temperature of 33 ℃. The bacterial content of the fermentation broth was about 1.0X10 9 CFU/mL。
2. Separation of the catalyst: centrifuging the fermentation broth at 5-6deg.C under centrifugal force 8000g, and collecting lower layer thallus to obtain crude catalyst with activity of 46000u/L. The cells were homogenized 4 times by a high-pressure homogenizer under a high-pressure condition of 800bar, and the catalyst was released after the cells were disrupted. Adding flocculating agent benzalkonium chloride into the obtained homogeneous solution, adjusting pH7.54, controlling temperature to 10deg.C, flocculating for 30min, centrifuging the flocculated solution by a centrifuge to remove cell debris, and collecting clear liquid.
3. Purification of the catalyst: controlling the temperature of the centrifugated clear liquid to be 5-6 ℃, adopting an ultrafiltration membrane of 10000MW to carry out ultrafiltration concentration, and stopping ultrafiltration when the concentration of the catalyst is 60 mg/mL. Adding ammonium sulfate into the concentrated solution until the concentration is 30.28%, controlling the temperature to be 5-6 ℃ to precipitate the catalyst, filtering and collecting the catalyst precipitate. The catalyst precipitate is dissolved in 0.02M phosphate buffer solution with pH of 8.0 at 5-6deg.C, filtered, and insoluble substances are removed. The temperature of the solution is controlled to be 5-6 ℃, and the solution is dialyzed by an ultrafiltration membrane with 10000MW until the catalyst conductivity is 3.7MS, and then the solution is further concentrated until the catalyst concentration is 80mg/mL. The concentrated solution is subjected to anion exchange resin column chromatography at the temperature of 5-6 ℃ and the speed of 0.5BV/h, the initially discharged 0.5BV chromatographic solution is discarded, the 2BV chromatographic solution is collected, finally, the 2BV chromatographic solution is subjected to top washing by using phosphate buffer solution with the pH of 2BV and 0.02M, and the catalyst is obtained after uniform mixing and is preserved at the temperature of 5 ℃ for standby.
4. Synthesis of phenoxyacetamido penicillin potassium salt: 30.0Kg of 6-APA is suspended by 200L of purified water, 10 percent of potassium carbonate solution is dripped to fully dissolve the 6-APA, 23.05Kg of methyl phenoxyacetate is added to be stirred and mixed uniformly, a catalyst is added to ensure that the total activity of the catalyst system is 120U/g of 6-APA, the pH value is controlled to 6.5 by 10 percent of potassium carbonate solution, the temperature is 25 ℃, the reaction is carried out for 1 hour, sampling and detecting that the 6-APA residue is 0.45g/L, and the synthesis reaction is finished, wherein the obtained reaction solution contains phenoxyacetamido penicillin potassium salt.
5. Product separation and catalyst recycling: controlling the temperature of the reaction solution to be 5-6 ℃, performing ultrafiltration concentration by adopting an 10000MW ultrafiltration membrane until the concentration of the catalyst in the dialysate is 0.001mg/mL, after the ultrafiltration is finished, collecting the catalyst concentrate and preserving at 5 ℃ for later use; the dialyzate is concentrated by 2 times by adopting a 200MW nanofiltration membrane at the temperature of 10 ℃, and the phenoxyacetamido penicillin potassium salt concentrated solution is collected.
6. Azeotropic crystallization: the concentrated solution of phenoxyacetamido penicillin potassium salt is diluted by n-butanol, distilled under reduced pressure and azeotropy crystallized until crystals are separated out, and n-butanol is added in the crystallization process until the water content of the crystallization solution is 0.58%, and 48.29Kg of phenoxyacetamido penicillin potassium is obtained through crystal filtration, washing and drying, wherein the molar yield is 89.62%. The HPLC purity of the phenoxyacetamido penicillin potassium is 99.87%, the p-hydroxyphenoxyacetamido penicillin is not detected, the polymer impurity is 0.011%, and the clarity is 0.5#.
Example 2 fermentation process for preparing potassium salt of phenoxyacetamido penicillin
1. And (3) fermenting a catalyst: uniformly mixing peptone, yeast powder, sodium chloride and the like with deionized water according to a seed culture medium formula,Natural pH, sterilizing at 121deg.C for 30min, cooling to room temperature, and standing. Uniformly mixing peptone, yeast powder, glycerol, potassium dihydrogen phosphate, disodium hydrogen phosphate and the like with deionized water according to a fermentation medium formula, naturally adjusting the pH, sterilizing at 121 ℃ for 30min, and cooling to room temperature for later use. E.coli is inoculated into the seed culture, primary and secondary seed expansion culture is carried out under the condition of 35+/-0.5 ℃, when the OD value of a seed tank is between 1.8 and 2.3 and the pH value is 7.4 to 7.8, the secondary seed liquid is inoculated into the fermentation culture medium according to the inoculation amount of 4 percent of the volume of the fermentation culture medium, and the fermentation liquid is obtained after the culture is carried out for 25 hours under the condition of pH value 6.7+/-0.05 and temperature 34 ℃. The bacterial content of the fermentation broth was about 1.0X10 11 CFU/mL。
2. Separation of the catalyst: centrifuging the fermentation broth with a centrifuge at a centrifugal force of 10000g and a temperature of 7-8deg.C, and collecting lower layer thallus to obtain crude catalyst with an activity of 43500u/L. The cells were homogenized 3 times by a high-pressure homogenizer under a high-pressure condition of 900bar, and the catalyst was released after the cells were disrupted. Adding flocculating agent polyethylene oxide into the obtained homogeneous solution, regulating pH to 7.72, controlling temperature to 10deg.C, flocculating for 30min, centrifuging the flocculated solution by a centrifuge to remove cell debris, and collecting clear liquid.
3. Purification of the catalyst: controlling the temperature of the centrifugate to 7-8 ℃, carrying out ultrafiltration concentration by adopting an ultrafiltration membrane of 30000MW, and stopping ultrafiltration when the catalyst concentration is 80mg/mL. Adding ammonium sulfate into the concentrated solution until the concentration is 29.97%, controlling the temperature to 7-8 ℃ to precipitate the catalyst, filtering and collecting the catalyst precipitate. The catalyst precipitate is dissolved in 0.02M phosphate buffer solution with pH of 8.0 at 7-8deg.C, filtered, and insoluble substances are removed. The temperature of the solution is controlled to 7-8 ℃, and the solution is dialyzed by an ultrafiltration membrane of 30000MW until the catalyst conductivity is 2.8MS, and then the solution is further concentrated to the catalyst concentration of 90mg/mL. The concentrated solution is subjected to anion exchange resin column chromatography at the temperature of 7-8 ℃ and the speed of 0.5BV/h, the initially discharged 0.5BV chromatographic solution is discarded, the 2BV chromatographic solution is collected, finally, the 2BV chromatographic solution is subjected to top washing by using phosphate buffer solution with the pH of 2BV and 0.02M, and the catalyst is obtained after uniform mixing and is preserved at the temperature of 8 ℃ for standby.
4. Synthesis of phenoxyacetamido penicillin potassium salt: 30.0Kg of 6-APA is suspended by 200L of purified water, 20 percent of potassium carbonate solution is dripped to fully dissolve the 6-APA, 27.50Kg of phenoxyacetic acid ethyl ester is added to be stirred and mixed uniformly, a catalyst is added to ensure that the total activity of the catalyst system is 150U/g of 6-APA, the pH7.0 is controlled by 20 percent of potassium carbonate solution, the temperature is 26 ℃, the reaction is carried out for 1.5 hours, the sampling detection is carried out, the 6-APA residue is 0.31g/L, and the synthesis reaction is finished, wherein the obtained reaction solution contains phenoxyacetamido penicillin potassium salt.
5. Product separation and catalyst recycling: controlling the temperature of the reaction liquid to be 7-8 ℃, carrying out ultrafiltration concentration by adopting an ultrafiltration membrane of 30000MW until the concentration of the catalyst in the dialyzate is 0.0008mg/mL, after the ultrafiltration is finished, collecting the catalyst concentrated solution and preserving at 7 ℃ for later use; the dialyzate is concentrated by adopting a 250MW nanofiltration membrane for 3 times at the temperature of 18 ℃, and the phenoxyacetamido penicillin potassium salt concentrated solution is collected.
6. Azeotropic crystallization: the concentrated solution of phenoxyacetamido penicillin potassium salt is diluted by n-butanol, distilled under reduced pressure and azeotropy crystallized until crystals are separated out, and n-butanol is added in the crystallization process until the water content of the crystallization solution is 0.46%, and 48.53Kg of phenoxyacetamido penicillin potassium is obtained through crystal filtration, washing and drying, and the molar yield is 90.06%. The HPLC purity of the phenoxyacetamido penicillin potassium is 99.92%, the p-hydroxyphenoxyacetamido penicillin is not detected, the polymer impurity is 0.009%, and the clarity is less than 0.5#.
Example 3 fermentation process for preparing potassium salt of phenoxyacetamido penicillin
1. And (3) fermenting a catalyst: mixing peptone, yeast powder, sodium chloride, etc. with deionized water according to the formula of the seed culture medium, sterilizing at 121deg.C for 30min, and cooling to room temperature for use. Uniformly mixing peptone, yeast powder, glycerol, potassium dihydrogen phosphate, disodium hydrogen phosphate and the like with deionized water according to a fermentation medium formula, naturally adjusting the pH, sterilizing at 121 ℃ for 30min, and cooling to room temperature for later use. E.coli is inoculated into the seed culture, primary and secondary seed expansion culture is carried out under the condition of 36+/-0.5 ℃, when the OD value of a seed tank is between 1.8 and 2.3 and the pH value is 7.4 to 7.8, the secondary seed liquid is inoculated into the fermentation culture medium according to the inoculation amount of 3 percent of the volume of the fermentation culture medium, and the fermentation liquid is obtained after the culture is carried out for 30 hours under the condition of pH value of 6.8+/-0.05 and the temperature of 35 ℃. The bacterial content of the fermentation broth was about 1.0X10 10 CFU/mL。
2. Separation of the catalyst: and (3) centrifuging the fermentation broth by using a centrifuge at the centrifugal force of 12000g and the temperature of 9-10 ℃ and collecting the lower layer thalli to obtain a crude catalyst product, wherein the activity of the crude catalyst product is 47800u/L. The cells were homogenized by a high-pressure homogenizer under a high pressure of 1000bar for 2 times to break the cells and release the catalyst. Adding polyacrylamide as flocculant into the obtained homogeneous solution, regulating pH to 7.95, controlling temperature to 10deg.C, flocculating for 30min, centrifuging the flocculated solution by a centrifuge to remove cell debris, and collecting clear liquid.
3. Purification of the catalyst: controlling the temperature of the centrifugated clear liquid to 9-10 ℃, carrying out ultrafiltration concentration by adopting an ultrafiltration membrane with 50000MW, and stopping ultrafiltration when the concentration of the catalyst is 100mg/mL. Adding ammonium sulfate into the concentrated solution until the concentration is 31.08%, controlling the temperature to 9-10 ℃ to precipitate the catalyst, filtering and collecting the catalyst precipitate. The catalyst precipitate is dissolved in 0.02M phosphate buffer solution with pH of 8.0 at 9-10deg.C, filtered, and insoluble substances are removed. The temperature of the dissolved solution is controlled to be 9-10 ℃, and an ultrafiltration membrane with 50000MW is adopted for dialysis until the electric conductivity of the catalyst is 1.4MS, and then the dissolved solution is further concentrated to the concentration of the catalyst of 100mg/mL. The concentrated solution is subjected to anion exchange resin column chromatography at the temperature of 9-10 ℃ and the speed of 0.5BV/h, the initially discharged 0.5BV chromatographic solution is discarded, the 2BV chromatographic solution is collected, finally, the 2BV chromatographic solution is subjected to top washing by using phosphate buffer solution with the pH of 2BV and 0.02M, and the catalyst is obtained after uniform mixing and is preserved at the temperature of 10 ℃ for standby.
4. Synthesis of phenoxyacetamido penicillin potassium salt: suspending 30.0Kg of 6-APA with 200L of purified water, dropwise adding 30% potassium carbonate solution to fully dissolve the 6-APA, then adding 37.62Kg of phenoxyacetic glyceride, stirring and mixing uniformly, adding a catalyst to ensure that the total activity of the catalyst of the system is 200U/g of 6-APA, controlling the pH value to 8.0 by using 30% potassium carbonate solution at 28 ℃, sampling and detecting that the 6-APA residue is 0.16g/L after 1.5h of reaction, and finishing the synthesis reaction, wherein the obtained reaction solution contains phenoxyacetamido penicillin potassium salt.
5. Product separation and catalyst recycling: controlling the temperature of the reaction solution to be 9-10 ℃, carrying out ultrafiltration concentration by adopting an ultrafiltration membrane with the concentration of 50000MW until the concentration of the catalyst in the dialyzate is 0.0013mg/mL, and collecting the catalyst concentrated solution after ultrafiltration is finished and preserving the catalyst concentrated solution at the temperature of 9 ℃ for later use; the dialyzate is concentrated by adopting a 300MW nanofiltration membrane for 4 times at the temperature of 25 ℃, and the phenoxyacetamido penicillin potassium salt concentrated solution is collected.
6. Azeotropic crystallization: the phenoxyacetamido penicillin potassium salt concentrated solution is diluted by n-butanol, distilled under reduced pressure and azeotropy crystallized until crystals are separated out, and n-butanol is added in the crystallization process until the water content of the crystallized solution is 0.64%, and 49.39Kg phenoxyacetamido penicillin potassium is obtained through crystal filtration, washing and drying, and the molar yield is 91.66%. The HPLC purity of the phenoxyacetamido penicillin potassium is 99.80%, the p-hydroxyphenoxyacetamido penicillin is not detected, the polymer impurity is 0.013%, and the clarity is 0.5#.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (9)

1. The method for preparing phenoxyacetamido penicillin potassium salt by a fermentation method is characterized by comprising the following steps:
(1) preparing a fermentation catalyst by fermentation;
(2) separating a fermentation catalyst;
(3) purifying a fermentation catalyst;
(4) under the catalysis of a fermentation catalyst, controlling the temperature to be 20-28 ℃, and controlling the pH to be 6.5-8.0 by using 10-30% potassium carbonate solution to enable 6-APA and phenoxyacetic acid derivatives to react to generate a product phenoxyacetamido penicillin potassium salt;
(5) separating products and recycling a fermentation catalyst;
(6) azeotropic crystallization;
wherein the fermentation catalyst is penicillin acylase.
2. The method of claim 1, wherein step (1) comprises: culturing strains with fermentation catalyst production capacity in a primary seed culture medium and a secondary seed culture medium in sequence under the conditions of natural pH and 33-36 ℃ to obtain a secondary seed solution;inoculating the secondary seed solution to a fermentation medium, and culturing for 20-30 hours at the pH of 6.5-6.8 and the temperature of 33-35 ℃ to obtain fermentation liquor; the bacterial content of the fermentation liquor is 1.0X10 9 -1.0×10 11 CFU/mL；
Wherein, the components of the seed culture medium comprise: peptone, yeast powder and sodium chloride; the components of the fermentation medium comprise: peptone, yeast powder, glycerol, potassium dihydrogen phosphate and disodium hydrogen phosphate;
preferably, the inoculation amount of the secondary seed liquid is 3-6v/v%.
3. The method of claim 2, wherein step (2) comprises: centrifuging the fermentation liquor at 5-10deg.C and 8000-12000g, collecting thallus, and crushing thallus by high-pressure homogenizer to release fermentation catalyst; adding a flocculating agent into the obtained homogeneous liquid to flocculate cell fragments, centrifuging and collecting supernatant;
wherein, the operation pressure of the high-pressure homogenizer is 800-1000bar, and the homogenization is carried out for 2-4 times;
the flocculant is at least one selected from benzalkonium chloride, benzalkonium bromide, polyoxyethylene, polystyrene sulfonate, polyacrylamide derivative and polyethyleneimine.
4. A method according to claim 3, wherein step (3) comprises: concentrating the supernatant by ultrafiltration to remove small molecular compounds, polypeptides and amino acids; adding ammonium sulfate to the obtained concentrated solution to precipitate a fermentation catalyst, dissolving the fermentation catalyst with phosphate buffer solution, ultrafiltering and dialyzing to obtain a fermentation catalyst concentrated solution, and passing through an anion exchange resin column to obtain a purified fermentation catalyst.
5. The method according to claim 4, wherein the fermentation catalyst is purified by the following steps: ultrafiltering the supernatant with ultrafiltration membrane with molecular weight of 10000-50000MW at 5-10deg.C, concentrating until concentration of concentrated solution fermentation catalyst is 60-100mg/mL, stopping ultrafiltering, and removing small molecular compounds, polypeptides and amino acids; adding ammonium sulfate into the concentrated solution to precipitate a fermentation catalyst at 5-10 ℃ to remove the impurity protein; dissolving the fermentation catalyst in phosphate buffer solution with pH of 7.8-8.2 and 0.02-0.025M at 5-10deg.C, ultrafiltering the solution with ultrafiltration membrane with molecular weight of 10000-50000MW at 5-10deg.C until the conductivity of the fermentation catalyst solution is less than or equal to 4MS, concentrating to fermentation catalyst concentration of 80-100mg/mL, and removing ammonium sulfate and small molecular compounds; and (3) subjecting the fermentation catalyst concentrated solution to anion exchange resin column chromatography at the temperature of 5-10 ℃ and the rate of 0.5BV/h, and finally, performing top washing with 0.02-0.025M phosphate buffer solution with the pH of 7.8-8.2 to obtain the purified fermentation catalyst.
6. The method of claim 4, wherein step (4) comprises: under the condition of 20-28 ℃,6-APA and phenoxyacetic acid derivatives react for 1-1.5 hours under the action of a fermentation catalyst by using 10-30% potassium carbonate solution to control pH to 6.5-8.0, and the obtained reaction liquid contains phenoxyacetamido penicillin potassium salt;
wherein, the mol ratio of the 6-APA to the phenoxyacetic acid derivative is 1 (1-1.3); the total activity of the fermentation catalyst is 120-200U/g based on 6-APA;
the phenoxyacetic acid derivative is at least one selected from methyl phenoxyacetate, ethyl phenoxyacetate, glyceryl phenoxyacetate and alcohol phenoxyacetate.
7. The method of claim 6, wherein step (5) comprises: ultrafiltering the reaction solution with ultrafilter membrane with molecular weight of 10000-50000MW at 5-10deg.C to concentrate and recover fermentation catalyst, and the concentrated fermentation catalyst solution is used for synthesizing phenoxyacetamido penicillin potassium salt; nanofiltration concentration of the dialyzate with nanofiltration membrane with molecular weight of 200-300MW at 10-25deg.C to obtain concentrated solution of phenoxyacetamido penicillin potassium salt.
8. The method of claim 7, wherein step (6) comprises: adding a water carrying agent into the phenoxyacetamido penicillin potassium salt concentrated solution, and concentrating by reduced pressure distillation until crystals are separated out;
the water carrying agent is at least one selected from n-butanol, isobutanol, benzene and toluene.
9. The method according to any one of claims 1 to 8, wherein the strain used for the fermentative production of the fermentation catalyst is escherichia coli.
CN202310991180.3A 2023-08-08 2023-08-08 Method for synthesizing phenoxyacetamido penicillin potassium salt by fermentation method Pending CN117106844A (en)

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