阅读说明：本技术 一种7-adca回收方法 (7-ADCA recovery method ) 是由 杜二洪 宋迎权 郑国荣 方金凤 徐成苗 求满金 何丽 胡敏 于 2021-08-30 设计创作，主要内容包括：本申请提供一种7-ADCA回收方法,属于杂环化合物技术领域。取头孢羟氨苄结晶后的母液,升温至10～30℃,将母液pH调节至7.0～8.0,加入EDTA,搅拌；加入固定化酶,维持pH在7.0～8.0,温度10～30℃,搅拌至头孢羟氨苄浓度不超过0.5mg/ml；分离去除固定化酶,分离所得溶液脱色、过滤；滤液pH调节至5.0～6.0,控制温度为10～30℃,养晶；过滤分离,采用漂洗湿品,即得7-ACDA,纯度98.5%以上。该技术路线绿色环保,操作简单,收率高,稳定可靠,优势明显。(The application provides a 7-ADCA recovery method, and belongs to the technical field of heterocyclic compounds. Taking mother liquor after cefadroxil crystallization, heating to 10-30 ℃, adjusting the pH of the mother liquor to 7.0-8.0, adding EDTA, and stirring; adding an immobilized enzyme, maintaining the pH value at 7.0-8.0 and the temperature at 10-30 ℃, and stirring until the concentration of cefadroxil is not more than 0.5 mg/ml; separating to remove immobilized enzyme, decolorizing the separated solution, and filtering; adjusting the pH value of the filtrate to 5.0-6.0, controlling the temperature to be 10-30 ℃, and growing the crystals; filtering and separating, and rinsing the wet product to obtain 7-ACDA with purity of over 98.5%. The technical route is green and environment-friendly, the operation is simple, the yield is high, the stability and the reliability are realized, and the advantages are obvious.)

1. A method for recovering 7-ADCA, comprising the steps of:

(1) taking mother liquor after cefadroxil crystallization, heating to 10-30 ℃, adjusting the pH of the mother liquor to 7.0-8.0, adding EDTA, and stirring for later use;

(2) adding an immobilized enzyme, maintaining the pH value at 7.0-8.0 and the temperature at 10-30 ℃, and reacting until the concentration of cefadroxil is not more than 0.5 mg/ml;

(3) separating to remove immobilized enzyme, decolorizing the separated solution, and filtering;

(4) adjusting the pH value of the filtrate to 5.0-6.0, controlling the temperature to be 10-30 ℃, and growing the crystals;

(5) filtering and separating, and rinsing the wet product to obtain 7-ACDA.

2. A 7-ADCA recovery process according to claim 1, wherein: in the step (1), the concentration of cefadroxil is 15-20 mg/ml.

3. A 7-ADCA recovery process according to claim 1, wherein: in the step (1), the EDTA is 0.01-0.10 times of the total amount of cefadroxil.

4. A 7-ADCA recovery process according to claim 1, wherein: sodium sulfite is also added in the step (1), and the sodium bisulfite is 0.01-0.10 times of cefadroxil.

5. A 7-ADCA recovery process according to claim 1, wherein: in the step (1), the pH is adjusted by ammonia water or sodium bicarbonate.

6. A 7-ADCA recovery process according to claim 1, wherein: in the step (2), the immobilized enzyme is 0.9-3.0 times of the total amount of cefadroxil.

7. A 7-ADCA recovery process according to claim 1, wherein: in the step (3), medicinal carbon is used for decoloring, and the adding amount of the medicinal carbon is 0.02-0.05 time of the total amount of cefadroxil.

8. The 7-ADCA recovery method according to claim 1, wherein in step (4), the seeding process is as follows: firstly, adjusting the pH =6.0, and growing the crystal for 5 min; continuously adjusting the pH to be 5.9 by using hydrochloric acid, growing the crystals for 5min, and repeating the operation until the crystals are grown; or pH =6.0, 0.5% seed-mediated seeding is added.

9. The method for recovering 7-ADCA according to claim 1, wherein in step (4), after the completion of the crystal growth, the pH is adjusted to 3.0-4.0, and the stirring is continued for 30-60 min.

10. A 7-ADCA recovery process according to claim 1, wherein: in the step (5), methanol or ethanol is used for rinsing, and the addition amount of the methanol or ethanol is 0-3.0 times of the total amount of cefadroxil.

Technical Field

The application relates to a 7-ADCA recovery method, belonging to the technical field of heterocyclic compounds.

Background

Cefadroxil (cefadroxil) is the first generation oral cephalosporin, has wide antibacterial spectrum and good oral drug effect, and is used for treating urinary system, respiratory tract, skin, five sense organs, gastrointestinal tract infection and the like caused by staphylococcus, streptococcus, pneumococcus, escherichia coli and the like. The traditional synthesis method mainly comprises the steps of carrying out acylation condensation on a mixed anhydride of 7-ADCA (7-aminodesacetoxycephalosporanic acid) mother nucleus, D-p-hydroxyphenylglycine dane salt and pivaloyl chloride under the condition of liquid nitrogen deep cooling, preparing a cefadroxil DMF compound by acid hydrolysis and a large amount of DMF solvent, and carrying out water phase crystal transformation to obtain cefadroxil, wherein a large amount of solvents such as dichloromethane, tetramethylguanidine and DMF are used in the whole process, and the reaction conditions are severe and have large pollution. Because of adopting twice crystallization, the yield is lower. With the rapid development of biotechnology, especially enzyme catalysis technology in recent years, it has become possible to use enzyme method to perform reactions in aqueous phase, hydrophilic phase, and even organic phase systems, wherein some reactions are harsh in chemical synthesis conditions.

Penicillin G acylase is an applied enzyme which is successful, 7-ADCA parent nucleus and D-p-hydroxyphenylglycine methyl ester are used as side chains in a water phase, cefadroxil can be catalytically synthesized under the normal temperature condition, and the obtained product has high quality, and the research reports already exist. However, research reports that cefadroxil compound is prepared and recovered by adopting 2-naphthol or naphthol analogue mode aiming at cefadroxil mother liquor after crystallization. On one hand, the further purification of the cefadroxil naphthol compound still needs to use an organic solvent, the pollution still exists, and on the other hand, the recovery rate of the recovered cefadroxil naphthol compound of naphthol or naphthol analogues is not high and the yield is low.

Disclosure of Invention

In view of the above, the application provides a 7-ADCA recovery method, which adopts a biological enzyme method to catalyze cefadroxil in a mother liquor back to a mother nucleus of a starting material 7-ADCA, and carries out a recycling technical route. Because the residual concentration of the 7-ADCA crystallization liquid is lower, the yield is greatly improved, the product quality is better, and the pollution is less.

Specifically, the method is realized through the following scheme:

a 7-ADCA recovery process comprising the steps of:

(1) putting a mother solution obtained after cefadroxil crystallization and prepared by an enzymatic cefadroxil process route into a reactor with stirring, adjusting the pH of the mother solution to 7.0-8.0, heating to 10-30 ℃, adding EDTA (ethylene diamine tetraacetic acid), stirring at the speed of 50-70 rpm, and stirring for 30min for later use;

(2) adding immobilized enzyme into a bioreactor, maintaining the pH at 7.0-8.0 and the temperature at 10-30 ℃, sampling every 1 hour, detecting the residual concentration of cefadroxil by adopting high performance liquid chromatography, and when the reaction is carried out until the concentration of cefadroxil is not more than 0.5mg/ml, prolonging for 30min, and separating;

(3) separating and removing the immobilized enzyme, intercepting immobilized enzyme particles on a filter screen, directly recycling to the step (2), decoloring the feed liquid for more than 30 minutes after the feed liquid passes through the screen, and filtering;

(4) adding hydrochloric acid into the filtrate to adjust the pH value to 5.0-6.0, controlling the temperature to be 10-30 ℃, and growing crystals;

(5) filtering and separating, and rinsing the wet product to obtain 7-ACDA.

The method comprises the steps of firstly adjusting the pH value of cefadroxil mother liquor to 7.0-8.0, heating to 10-30 ℃, performing simple pretreatment, hydrolyzing residual cefadroxil in the mother liquor by immobilized (biological) enzyme in a bioreactor with a filter, a stirrer and the like to obtain 7-ADCA mother nuclei, performing crystal growing treatment, and simply rinsing by methanol or ethanol to obtain a mother nucleus 7-ADCA wet product with the purity of more than 98.5%. The technical route is green and environment-friendly, and the operation is simple; compared with the problems of poor yield, large using amount of organic solvent and the like existing in the traditional recovery route (recovery of cefadroxil naphthol compound in mother liquor through 2-naphthol or naphthol analogues and purification through the organic solvent), the recovery route is stable and reliable and has obvious advantages; the recovered 7-ADCA wet product can be directly applied to enzymatic acylation synthesis reaction, so that a path for circularly preparing cefadroxil by a holoenzyme method is realized.

Further, as preferable:

in the step (1), the concentration of cefadroxil is about 15-20 mg/ml, the cefadroxil is in a saturated concentration in a mother liquor after crystallization, the pH of the mother liquor is 4.0-5.0, and t is 0-20 ℃.

In the step (1), EDTA is 0.01-0.10 times (g/g) of the total amount (volume concentration) of cefadroxil, and the EDTA is used as a compounding agent to remove the influence of possible heavy metals of raw and auxiliary materials and equipment on the biological enzyme.

Sodium sulfite is also added in the step (1), and the sodium bisulfite is 0.01-0.10 times (g/g) of cefadroxil.

In the step (1), the pH is adjusted by ammonia water or sodium bicarbonate.

In the step (2), the immobilized enzyme is 0.9-3.0 times (g/g) of the total amount of cefadroxil.

In the step (3), medicinal carbon is used for decoloring, and the carbon amount is 0.02-0.05 time (g/g) of the total amount of cefadroxil.

In the step (4), the crystal growing process is as follows: firstly adjusting the pH value to 6.0, and growing the crystal for 5 min; continuously adjusting pH to 5.9 with hydrochloric acid, growing crystal for 5min, and repeating the above steps until crystal is grown; alternatively, when the pH is 6.0, an appropriate amount of seed crystals are directly added. After the crystal growth is finished, adjusting the pH value to 3.0-4.0, and continuing stirring for 30-60 min.

In the step (5), methanol or ethanol is used for rinsing, wherein the addition amount of the methanol is 0-3.0 times (g/g) of the total amount of cefadroxil, and the addition amount of the ethanol is 0-3.0 times (g/g) of the total amount of cefadroxil.

Compared with the prior art, the invention has the following advantages:

recovering the cefadroxil mother liquor through naphthol or naphthol analogues, and further purifying and reusing the cefadroxil mother liquor, wherein the molar yield is 83.8%; according to the process, immobilized enzyme 1 (hydrolase) is used for catalyzing cefadroxil to generate 7-ADCA mother nucleus, and immobilized enzyme 2 (acylase) is continuously adopted to synthesize cefadroxil after crystallization and solid extraction to obtain 93.3% of molar yield; the yield of the route of the invention is improved by 9.5 percent, and the product quality is obviously improved because naphthol and organic solvent are not used for purification. In addition, the organic solvent is not used, and only common water phase is needed, so that the operation is convenient, and the safety and environmental protection benefits are more obvious.

The recovery process describes the catalytic synthesis of 7-ADCA parent nucleus by the immobilized enzyme 1 (hydrolase) route, and the 7-ADCA parent nucleus is not described in detail in the catalytic synthesis of cefadroxil by the immobilized enzyme 2 (acylase).

The reaction scheme of the above process can be described as follows:

in the reaction formula (1), 7-ADCA is catalytically recovered from the cefadroxil mother liquor by immobilized enzyme 1 (hydrolase), and then the cefadroxil is synthesized by immobilized enzyme 2 (acylase).

The mother liquor is then recovered by chelation as described in equation (2) above for the synthesis of cefadroxil.

Detailed Description

The invention is further described below with reference to specific embodiments.

In the embodiment, the immobilized biological enzyme is purchased from Guangzhou Hengzheng medicine science and technology limited company, model IPA-SPIII; the rest raw materials and auxiliary materials can be obtained commercially.

Example 1: influence of addition amount of auxiliary agent on reaction effect in pretreatment

In the pretreatment process, the auxiliary agents mainly comprise two components: EDTA and sodium bisulfite.

The effect of the amount of EDTA added was first tested in conjunction with the specific recovery procedure.

(1) Adding 1000ml of mother liquor after cefadroxil crystallization prepared by an enzymatic cefadroxil process route into a reactor with stirring, wherein the concentration of cefadroxil is about 15mg/ml, heating to 15 ℃, controlling the addition amount of EDTA and 0.20g of sodium bisulfite, adjusting the pH of the mother liquor to 7.0 by ammonia water, and stirring for later use.

(2) The method comprises the steps of putting 15g of immobilized biological enzyme with a proper amount into a self-designed bioreactor, putting the feed liquid prepared in the step (1), maintaining the pH value of the enzyme reaction at 7.0 by ammonia water and the temperature at 15 ℃, sampling every 1 hour, detecting the residual concentration of cefadroxil by adopting high performance liquid chromatography, and separating after the residual concentration is not more than 0.5mg/ml and the time is prolonged for 30 min.

(3) Immobilized enzyme particles are intercepted on a filter screen, and can be directly put into the next batch (1) for reaction, and the feed liquid passes through the screen to carry out the next working procedure.

(4) Adding appropriate amount of medicinal charcoal, decolorizing for more than 30min, and filtering.

(5) Adding the filtrate, and growing the crystal for 30 minutes. The process is as follows: adjusting pH to 6.0, growing crystal for 5min, adjusting pH to 5.9 with hydrochloric acid, growing crystal for 5min, and optionally adding appropriate amount of seed crystal at pH 6.0. After the crystal growth is finished, the pH value is continuously adjusted to 3.0 by hydrochloric acid, and stirring is continuously carried out for 60min after the adjustment is finished.

(6) Filtering and separating, and rinsing the wet product with 15g of methanol to obtain a 7-ADCA wet product;

(7) the 7-ADCA obtained can be used as a starting material for further use.

The effect of the amount of EDTA added is summarized in Table 1.

TABLE 1 Effect of different EDTA additions on the reaction

In the reaction process, EDTA is used as a pretreating agent, the action effect of the EDTA is mainly reflected in the influence on the enzyme reaction time, and the used raw and auxiliary materials are conventional chemicals and can bring certain metal ions and influence the catalytic activity of the enzyme. Taking table 1 as an example, under the same conditions, with the increase of the addition amount of EDTA (the times of EDTA relative to the mass of cefadroxil in the mother liquor), the enzyme reaction time is gradually shortened, and as 7-ADCA is a nitrogen heterocyclic substance and is easy to degrade, the shorter the reaction time is, the better the appearance of the 7-ADCA wet product is obtained, and the appearance is changed from a light yellow wet product to a white-like wet product; the weight of the wet product is reduced because the content is increased, and the viscosity degree of the 7-ADCA wet product is gradually reduced. The 7-ADCA content (expressed as pure content/total wet weight) presents an increasing trend. When the addition amount of EDTA is more than 0.15g (namely the addition amount of EDTA is 0.01 time of the mass of cefadroxil in the mother solution), the improvement effect is more obvious; this improvement tendency tends to be smooth when the EDTA addition amount is increased to 0.09 times the mass of cefadroxil (in the vicinity of nos. 13 to 14 in table 1), and is optimal when the EDTA addition amount is increased to 0.1 times the mass of cefadroxil (in the case of No. 13 in table 1); the recovery effect of 7-ADCA wet products is not obviously improved by continuously increasing the addition amount of EDTA.

The concentration of cefadroxil in the mother liquor is from 15 to 20 (represented by 15, 16, 17, 18, 19 and 20) mg/ml, and the trend still exists, so that the following can be seen: in the 7-ADCA recovery process, the addition amount of EDTA is preferably controlled to be 0.01 to 0.10 times, more preferably 0.08 to 0.10(1.2 to 1.5g) times, the mass of cefadroxil.

In the pretreatment process, sodium bisulfite is used as an antioxidant, the addition amount of the sodium bisulfite mainly influences the color of the feed liquid and the appearance of the recovered 7-ADCA, and the influence trend is shown as follows: when the adding amount of the sodium bisulfite is less than 0.01 time of the mass of the cefadroxil in the mother liquor, the color of the feed liquid is darker, the obtained 7-ADCA is yellow to faint yellow, and the appearance character improvement effect is not obvious; when the adding amount of the sodium bisulfite is increased to be more than 0.01 time of the mass of the cefadroxil in the mother liquor, particularly when the adding amount of the sodium bisulfite is 0.013-0.05 time of the mass of the cefadroxil in the mother liquor, the color of the feed liquid gradually becomes lighter, the appearance of the recovered 7-ADCA gradually tends to be white, the optimal effect is achieved when the adding amount of the sodium bisulfite is 0.05-0.10 time of the mass of the cefadroxil in the mother liquor, and the appearance color tends to be stable and does not obviously change when the adding amount of the sodium bisulfite is continuously more than 0.10.

Besides the above effects, the addition ratio of the two auxiliary agents is also properly controlled between the addition amount of EDTA: the best effect is achieved when the adding amount of the sodium bisulfite (g/g) is 1: 1-1.7, which is shown in table 2.

TABLE 2 influence of different addition agent addition ratios on the reaction effect

The influence of the addition amount of EDTA on the enzyme activity depends on the degree of EDTA to remove metal ions, and the metal ions influence the enzyme configuration and indirectly influence the enzyme reaction time. The cephalosporin substances are easy to degrade, the longer the enzyme reaction time is, the darker the color of the feed liquid is, the appearance character of the obtained 7-ADCA is shown as the darker the color is, the lower the content and the purity are, and therefore, the shorter the enzyme reaction time is, the better the enzyme reaction time is expected to be; on the other hand, sodium bisulfite is used as an antioxidant, and the addition of a proper amount is beneficial to the protection of the feed liquid, the oxidation degree is reduced, and the color of the feed liquid and the color of 7-ADCA tend to be improved. The 7-ADCA is white-like in appearance, and has the best content and purity according to the optimal proportion (EDTA/NaHSO4, g/g) of 1.0-1.7.

Example 2: effect of parameters on reaction Effect in Pre-treatment

The enzyme-catalyzed reaction needs to be at an appropriate temperature to exhibit optimal catalytic activity. The temperature is too low, the enzyme activity is not high, the main temperature influences the contact probability of the substrate molecules and the enzyme activity catalytic center, the higher the temperature is, the more violent the molecular movement is, the higher the probability of contacting the catalytic center is, on the other hand, the temperature is too high, the spatial conformation of the enzyme molecules is changed, and the enzyme molecules are denatured and inactivated. Meanwhile, due to the fact that the degradation of cephalons is accelerated and the color of cephalons is darkened due to the fact that the excessively high temperature is high, the temperature of feed liquid needs to be pretreated within a proper range. The pretreatment in this step is to provide suitable conditions for the next enzymatic reaction, so the temperature is tested for the enzymatic activity, see table 3.

TABLE 3 Effect of different temperatures on the Activity of immobilized enzymes

Serial number	Temperature of	Enzyme activity U/g
			1	10	355
2	15	393
			3	20	415
4	25	440
			5	30	450

The activity of the immobilized hydrolase is calculated according to the consumption of NaOH, wherein the activity of the immobilized hydrolase is the amount of 1g of immobilized hydrolase (wet product) in a buffer salt system with the pH value of 8.0 to catalyze and hydrolyze the potassium salt of penicillin into 6-APA and phenylacetic acid per minute, and the phenylacetic acid is titrated by a sodium hydroxide standard solution.

Calculating the formula:

in the above formula:

x: the enzyme activity (28 deg.C, wet basis) of the product is U/g;

c: actual concentration of sodium hydroxide titration solution, mol/L;

v: the product consumes the volume of sodium hydroxide titration solution, ml;

m: mass of sample, g;

t: titration time, min.

According to an enzyme activity determination experiment, the enzyme activity of the immobilized enzyme in a test range is increased along with the increase of the temperature and tends to be gentle after 25 ℃, and the feed liquid is unstable at high temperature, so that the temperature is selected to be within 10-30 ℃, and the optimal temperature is 20 ℃.

pH is another important factor affecting enzyme activity, and too high or too low a pH can cause enzyme activity loss. The enzyme activity was measured at 20 ℃ at different pH values.

TABLE 4 Effect of different pH values on enzyme Activity

Serial number	pH	Enzyme activity U/g
			1	6.0	182
2	6.5	330
			3	7.0	423
4	7.5	480
			5	8.0	450
6	8.5	427

Hydrolyzing the amount of the penicillin potassium salt converted into 6-APA and phenylacetic acid by 1g of immobilized hydrolase (wet product) per minute in a salt system with different pH values at 20 ℃, titrating the phenylacetic acid by using a sodium hydroxide standard solution, and calculating the activity of the immobilized hydrolase according to the consumption of NaOH.

Calculating the formula:

in the above formula:

x: the enzyme activity (20 ℃, measured by wet product) of the product is U/g;

c: actual concentration of sodium hydroxide titration solution, mol/L;

v: the product consumes the volume of sodium hydroxide titration solution, ml;

m: mass of sample, g;

t: titration time, min.

The enzyme activity increases with the increase of pH at pH 6.0-7.5, tends to decrease at pH 7.5-8.0, and decreases obviously at pH above 8.0, and the enzyme activity and the degradation behavior of the feed liquid under alkaline condition are considered, and the pH 7.0-8.0 and the optimal pH 7.0 are selected.

There are many pH adjusting reagents, such as ammonia water, sodium carbonate, sodium bicarbonate, triethylamine, diethylamine, etc., and the best of ammonia water and sodium bicarbonate is selected by comprehensively considering the raw material cost.

Example 3: influence of the amount of immobilized enzyme on the reaction effect

The immobilized enzyme is used as a catalyst, an active catalytic center is provided in the reaction process, the input amount influences the enzyme reaction time, and the immobilized enzyme is a key factor of product quality and cost.

In the following, the effect of the amount of immobilized enzyme added was first tested in connection with the specific recovery process.

(1) Adding 1000ml of mother liquor after cefadroxil crystallization prepared by an enzymatic cefadroxil process route into a reactor with stirring, wherein the concentration of cefadroxil is about 15mg/ml, heating to 20 ℃, controlling the addition amount of EDTA and 0.80g of sodium bisulfite, adjusting the pH of the mother liquor to 7.0 by ammonia water, and stirring for later use.

(2) The method comprises the steps of putting 12.75-45 g of proper amount of immobilized biological enzyme into a self-designed bioreactor, putting the feed liquid prepared in the step (1), maintaining the pH value of the enzyme reaction at 7.0 by ammonia water and the temperature at 20 ℃, sampling every 1 hour, detecting the residual concentration of cefadroxil by adopting high performance liquid chromatography, and separating after the residual concentration is not more than 0.5mg/ml and prolonging for 30 min.

(3) Immobilized enzyme particles are intercepted on a filter screen, and can be directly put into the next batch (1) for reaction, and the feed liquid passes through the screen to carry out the next working procedure.

(4) Adding appropriate amount of medicinal charcoal, decolorizing for more than 30min, and filtering.

(5) Adding the filtrate, and growing the crystal for 30 minutes. The process is as follows: adjusting pH to 6.0, growing crystal for 5min, adjusting pH to 5.9 with hydrochloric acid, growing crystal for 5min, and optionally adding appropriate amount of seed crystal at pH 6.0. After the crystal growth is finished, the pH value is continuously adjusted to 3.0 by hydrochloric acid, and stirring is continuously carried out for 60min after the adjustment is finished.

(6) Filtering and separating, and rinsing the wet product with 15g of methanol to obtain a 7-ADCA wet product;

(7) the 7-ADCA obtained can be used as a starting material for further use.

TABLE 5 influence of different immobilized enzyme addition ratios on reaction effect

The feeding proportion of the immobilized enzyme is increased from 0.75 to 2.0 times, the reaction time is shortened, the color of the obtained feed liquid and the color appearance of 7-ADCA are improved due to the shortened reaction time, and the content and the purity are also improved. When the charge ratio of the immobilized enzyme is continuously increased to 2.0-3.0 times, the reaction time does not tend to be continuously shortened any more, the content and the purity tend to be stable, and the reaction speed is not accelerated any more due to the excessive catalytic center increased by the immobilized enzyme in the system. Therefore, the range of the charge ratio of the immobilized enzyme is 0.85-3.0 times, and the optimal charge ratio is 2.0 times.

Example 4: influence of crystal growing mode on wet product

The 7-ADCA belongs to reaction crystallization and is divided into two steps of reaction process and crystallization precipitation, so that the crystallization process is more important and directly influences the quality of the 7-ADCA. The crystal growth can be carried out by adopting the way of gradually reducing the pH value to find the optimal metastable zone, but the way is easy to explode and nucleate, so that 7-ADCA is more viscous, and the crystal growth can also be carried out at constant pH by adopting the way of adding crystal seeds for guidance.

The following experiments were conducted in conjunction with the specific recovery procedure for the grain growth mode.

(1) Adding 1000ml of mother liquor after cefadroxil crystallization prepared by an enzymatic cefadroxil process route into a reactor with stirring, wherein the concentration of cefadroxil is about 15mg/ml, heating to 20 ℃, controlling the addition amount of EDTA and 0.80g of sodium bisulfite, adjusting the pH of the mother liquor to 7.0 by ammonia water, and stirring for later use.

(2) And (2) adding 30g of immobilized biological enzyme into a self-designed bioreactor, adding the feed liquid prepared in the step (1), maintaining the pH value of the enzyme reaction at 20 ℃ by using ammonia water, sampling every 1 hour, detecting the residual concentration of cefadroxil by using high performance liquid chromatography, and separating after prolonging the residual concentration by 30min until the residual concentration is not more than 0.5 mg/ml.

(3) Immobilized enzyme particles are intercepted on a filter screen, and can be directly put into the next batch (1) for reaction, and the feed liquid passes through the screen to carry out the next working procedure.

(4) Adding appropriate amount of medicinal charcoal, decolorizing for more than 30min, and filtering.

(5) Adding the filtrate, and growing the crystal for 30 minutes. The process is as follows: firstly, adjusting the pH value to 6.0, growing crystals for 5min, continuously adjusting the pH value to 5.9 by hydrochloric acid, growing crystals for 5min if no solid appears, and the like until crystals are grown; ② adding proper amount of seed crystal at pH 6.0. After the crystal growth is finished, the pH value is continuously adjusted to 3.0 by hydrochloric acid, and stirring is continuously carried out for 60min after the adjustment is finished.

(6) Filtering and separating, and rinsing the wet product with 15g of methanol to obtain a 7-ADCA wet product;

(7) the 7-ADCA obtained can be used as a starting material for further use.

TABLE 6 influence of different modes of crystallization on the quality of 7-ADCA

By adopting a crystallization mode, a proper metastable zone needs to be found for crystal growth, but explosive crystallization is easy to occur in the process of adjusting pH, and the experiment in Table 6 shows that the explosive crystallization occurs in a narrow range of 0.1 of delta PH. Selecting crystallization mode of ②, adding 0.5 percent of 7-ADCA seed crystal at pH of 6.0 for mediation, stabilizing the precipitation process, improving the appearance viscosity of the obtained 7-ADCA, loosening the obtained 7-ADCA, and having highest content and purity. Therefore, seeding was conducted by seeding with 0.5% 7-ADCA seed at pH 6.0.

Example 5: effect of rinsing on Wet goods

And (3) obtaining a 7-ADCA wet product, rinsing by adopting a proper solvent to further remove impurities and color, and selecting 1-3 times of methanol or ethanol for rinsing in consideration of adopting methyl ester or ethyl ester as a side chain in the 7-ADCA cefadroxil synthesis enzymatic process and not introducing a new solvent.

The rinse mode was tested in conjunction with the specific recovery procedure as follows.

(1) Adding 1000ml of mother liquor after cefadroxil crystallization prepared by an enzymatic cefadroxil process route into a reactor with stirring, wherein the concentration of cefadroxil is about 16mg/ml, heating to 30 ℃, adding 0.20g of EDTA and 0.20g of sodium bisulfite, adjusting the pH of the mother liquor to 8.0 by ammonia water, and stirring for later use.

(2) The method comprises the following steps of putting 15g of immobilized biological enzyme into a self-designed bioreactor (the bioreactor comprises specially designed speed-variable mechanical stirring, a separation net, an air pipe and the like), putting the prepared feed liquid in the step (1), maintaining the pH value of the enzyme reaction to be 8.0 by using ammonia water, carrying out sampling inspection every 1 hour, detecting the residual concentration of cefadroxil by using high performance liquid chromatography, and separating after prolonging for 30min when the residual concentration is not more than 0.5 mg/ml.

(3) Immobilized enzyme particles are intercepted on a filter screen, and can be directly put into the next batch (1) for reaction, and the feed liquid passes through the screen to carry out the next working procedure.

(4) Adding appropriate amount of medicinal charcoal, decolorizing for more than 30min, and filtering.

(5) Hydrochloric acid is added into the filtrate to adjust the pH value, the temperature is 30 ℃, and the crystal growth is carried out for 30 minutes. The process is as follows: adjusting pH to 5.0, growing crystal for 5min, adjusting pH to 4.9 with hydrochloric acid, growing crystal for 5min, and optionally adding appropriate amount of seed crystal at pH 5.0. After the crystal growth is finished, the pH value is continuously adjusted to 3.0 by hydrochloric acid, and stirring is continuously carried out for 60min after the adjustment is finished.

(6) Filtering and separating, rinsing the wet product with 1-3 times of methanol or ethanol to obtain 11.9g (the content of the wet product is 72.8%) of 7-ADCA white wet product with the purity of 98.8%;

(7) the 7-ADCA obtained can be used as a starting material for further use.

TABLE 7 Effect of different rinsing regimes on the quality of 7-ADCA

Methanol and ethanol can be used as rinsing solvents for rinsing, the content and the purity can be further improved by 1.0-3.0 times of rinsing amount, the appearance, the character and the color are improved, and the optimal feeding ratio of the cost is 1.0.

Example 6: exemplary Condition examples

(1) Adding 1000ml of mother liquor after cefadroxil crystallization prepared by an enzymatic cefadroxil process route into a reactor with stirring, wherein the concentration of cefadroxil is about 15mg/ml, heating to 20 ℃, adding 0.80g of EDTA and 0.80g of sodium bisulfite, adjusting the pH of the mother liquor to 7.0 by ammonia water, and stirring for later use.

(2) And (2) adding 30g of immobilized biological enzyme into a self-designed bioreactor, adding the feed liquid prepared in the step (1), maintaining the pH value of the enzyme reaction at 20 ℃ by using ammonia water, sampling every 1 hour, detecting the residual concentration of cefadroxil by using high performance liquid chromatography, and separating after prolonging the residual concentration by 30min until the residual concentration is not more than 0.5 mg/ml.

(3) Immobilized enzyme particles are intercepted on a filter screen, and can be directly put into the next batch (1) for reaction, and the feed liquid passes through the screen to carry out the next working procedure.

(4) Adding appropriate amount of medicinal charcoal, decolorizing for more than 30min, and filtering.

(5) Hydrochloric acid was added to the filtrate to adjust the pH, the temperature was 30 ℃ and 0.5% seed crystals were added at pH 6.0. After the crystal growth is finished, the pH value is continuously adjusted to 3.0 by hydrochloric acid, and stirring is continuously carried out for 60min after the adjustment is finished.

(6) Filtering, separating, rinsing wet product with 15g methanol to obtain 11.9g (wet product content 73%) of 7-ADCA white wet product with purity of 98.9%;

(7) the 7-ADCA obtained can be used as a starting material for further use.

From the above examples 1-7 it can be seen that: through the recovery route, the 7-ADCA parent nucleus with better quality can be obtained and can be used for continuously synthesizing cefadroxil.

Comparative example

Cefadroxil mother liquor was compared with naphthol or naphthol analogs.

The recovery routes and parameters are described below:

(1) adding 1.5 times of 2-naphthol into cefadroxil mother liquor with the pH value of 4.5-5.0, stirring for 1 hour at normal temperature, cooling to below 10 ℃, continuing stirring for 2 hours, and performing suction filtration to obtain a cefadroxil naphthol compound;

(2) adding 2 times of water, 2 times of dichloromethane and 0.5 time of DMF into the cefadroxil naphthol compound, adjusting the pH value to be 1.00-1.20, stirring and dissolving the mixture clearly, standing the mixture for 30min, layering to obtain a water layer which is cefadroxil dissolving solution, and sleeving the water layer into the cefadroxil naphthol compound.

Comparison of recovery modes of two cefadroxil mother solutions

TABLE 8 comparison of two cefadroxil mother liquor recovery modes

As shown in the above reaction formula (2), the enzymatic recovery route of the mother liquor converts cefadroxil in the mother liquor into 7-ADCA to recover the cefadroxil, and the recovery route of the mother liquor, namely the recovery route of the mother liquor (1), recovers cefadroxil in the mother liquor. The routes of the two are not consistent, and the target products are not consistent. The chelating route for recovery of cefadroxil was therefore converted to 7-ADCA for comparison.

The recovery route of the mother liquor by the enzyme method is shown in the reaction formula (1), the total recovery rate of the 7-ADCA crystals is 85.1 percent, and the recovery rate of the 7-ADCA crystals converted from the recovery of the cefadroxil by the mother liquor chelation route, namely the reaction formula (2), is only 47 percent; the enzymatic recovery route has obvious advantages. The method mainly comprises the steps of recovering 7-ADCA by an enzyme method, converting material loss into 7-ADCA to the maximum extent, and crystallizing the 7-ADCA, wherein the saturated concentration is lower than 0.5mg/ml, and the residual quantity is low; and the saturated concentration of the cefadroxil naphthol chelate in the chelating route is more than 5mg/ml, so that the cefadroxil naphthol chelate cannot be taken out, and the loss is large (33% of cefadroxil loss). In addition, in the subsequent organic solvent impurity removal process, the organic layer carries away partial products and the like. Thus, the enzymatic recovery route of 7-ADCA is more advantageous from the yield point of view.

The above-mentioned recovered materials are respectively used in the following routes.

In the reaction formula (2), the cefadroxil mother liquor is recovered through naphthol or naphthol analogues, and is further purified and reused, so that the molar yield is 83.8% (actual product/theoretical yield); the 7-ADCA wet product obtained by the recovery method reflected by the reaction formula (1) continues to adopt the immobilized enzyme 2 (acylase) to synthesize cefadroxil with the process route molar yield of 93.3% (actual product/theoretical yield); compared with the traditional recovery route, the yield is improved by 9.5 percent, and the product quality is obviously improved because naphthol and organic solvent are not used for purification. In addition, because the organic solvent is not used, only common water phase is needed, the ring operation is convenient, and the safety and environmental protection benefits are more obvious.

TABLE 9 comparison of the two cefadroxil routes

Route	7-ADCAg	Cefadroxil g	Mass yield	Molar yield
					1	60	99	1.65	93.3％
2	60	89.7	1.52	83.8％

Scheme 1, equation (1):

scheme 2, equation (2):

mass yield (cefadroxil weight + (produced cefadroxil naphthol complex content-input cefadroxil naphthol complex content))/input 7-ADCA weight

The present invention has been described in detail in order to enable those skilled in the art to practice the invention, and it is not limited to the embodiments shown, and all equivalent changes and modifications made according to the spirit of the present invention are intended to be covered by the scope of the present invention.