A continuous recovery method of ampicillin crystallization mother liquor by enzyme

Abstract

The application discloses a kind of continuous recovery methods of enzyme ampicillin crystallization mother liquor, belong to heterocyclic compound technical field.The application is connected to form ampicillin crystallization mother liquor continuous recovery device by circulating tank, nanofiltration unit, enzyme synthesis tank, crystallization tank, solid-liquid separator;Through the continuous circulation of the steps such as the pH of crystallization mother liquor up-regulation, nanofiltration concentration, enzyme catalysis synthesis, crystallization, ampicillin solid online removal, the effective recovery and utilization of ampicillin, 6-APA, benzoglycine methyl ester in mother liquor are realized.The mild pH operating condition of the whole process reduces the damage of nanofiltration membrane equipment and the degradation of ampicillin to the maximum, and the product yield is high, and the quality is good;6-APA and benzoglycine methyl ester in mother liquor are effectively recovered and utilized, and compared with the content of ampicillin in mother liquor, the total recovery rate of ampicillin is more than 100%.

Description

Technical Field

[0001] This invention relates to the field of heterocyclic compound technology, and specifically to a method for the continuous recovery of mother liquor from enzymatic ampicillin crystallization. Background Technology

[0002] Ampicillin is a broad-spectrum semi-synthetic penicillin-type β-lactam antibiotic, widely used clinically for the treatment of bacterial infections of the respiratory, urinary, and digestive tracts. It is one of the most widely used antibiotics globally and is also a raw material for the synthesis of products such as piperacillin. The current mainstream industrial synthesis process uses immobilized penicillin acylase-catalyzed synthesis: using 6-aminopenicillanic acid (6-APA) and D-phenylglycine methyl ester hydrochloride as substrates, ampicillin is produced under the catalysis of an immobilized enzyme. The product is then obtained after pH adjustment, isoelectric crystallization, and solid-liquid separation. The mother liquor from enzymatic ampicillin crystallization contains uncrystallized ampicillin residue (4-6 mg / mL), unreacted substrate 6-APA (0.8-1.4 mg / mL), unreacted acyl donor D-phenylglycine methyl ester (2-6 mg / mL), inorganic salts, and trace amounts of organic impurities. Direct discharge of the mother liquor presents three major problems: 1. Severe resource waste, with 4%-6% of the effective ingredient being lost annually, resulting in losses of tens of millions of yuan; 2. Immense environmental pressure: high COD (8000-15000 mg / L) and high treatment costs; 3. Incompatible with green manufacturing: contradicting the pharmaceutical industry's energy conservation, emission reduction, and circular economy policies.

[0003] Existing recycling technologies mainly include macroporous resin adsorption, enzymatic hydrolysis, solvent extraction, and membrane concentration and crystallization. Resin recovery methods have long single-batch recovery cycles, require organic solvents for desorption and resin regeneration, and generate a large amount of waste liquid and wastewater. Enzymatic recovery methods destroy ampicillin, resulting in the loss of the value of previous synthesis, and the separation of 6-APA and D-phenylglycine is difficult and results in low purity. Traditional solvent extraction methods have low recovery efficiency (≤65%), high energy consumption, and high safety and environmental risks. Patent application number: 201810136768.X discloses a method for recovering ampicillin from the mother liquor of enzymatic ampicillin synthesis: the mother liquor of enzymatic ampicillin crystallization is acidified with hydrochloric acid to pH 1.3-1.8, concentrated by 150-200Dal organic membrane nanofiltration about 10 times, and the concentrated solution is directly adjusted to pH 5.0 for crystallization. This method has problems: the low pH of the nanofiltration solution causes significant damage to the membrane; ampicillin is unstable under strong acid conditions, impurities increase, 6-APA is not effectively utilized, the product has high impurities, and the yield is low. Therefore, developing a comprehensive recovery technology for ampicillin crystallization mother liquor that can simultaneously recover ampicillin, 6-APA, and D-phenylglycine methyl ester hydrochloride with a simple process and high recovery rate has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a continuous recovery method for the mother liquor of enzymatic ampicillin crystallization.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for the continuous recovery of mother liquor from enzymatic ampicillin crystallization, comprising the following steps: The mother liquor from the enzymatic ampicillin crystallization process is continuously injected into the circulation tank of the continuous recovery unit. After pH adjustment, it enters the nanofiltration concentrator for concentration. The obtained concentrate enters the enzyme synthesis tank. 6-aminopenicillanic acid and phenylglycine methyl ester contained in the mother liquor are used to synthesize ampicillin under the catalysis of immobilized enzymes. The obtained synthesis solution is continuously fed into the crystallization tank for crystallization. The crystallized solution in the crystallization tank continuously enters the separator, where the solid is removed online, which is the ampicillin product. Part of the filtrate is discharged, and the rest is returned to the circulation tank. In the circulating tank, the pH of the adjusted enzymatic ampicillin crystallization mother liquor is 6.5-8.0; The continuous recovery device includes a circulation tank, a nanofiltration unit, an enzyme synthesis tank, a crystallization tank, and a separator; the outlet of the circulation tank is connected to the inlet of the nanofiltration unit, the outlet of the nanofiltration unit is connected to the inlet of the enzyme synthesis tank, the outlet of the enzyme synthesis tank is connected to the inlet of the crystallization tank, the outlet of the crystallization tank is connected to the inlet of the separator, and the outlet of the separator is connected to the inlet of the circulation tank; the circulation tank is also provided with a mother liquor inlet.

[0006] Furthermore, a filter plate with a pore size of 100-150 mesh is installed near the bottom of the enzyme synthesis tank.

[0007] Furthermore, the separator can be a plate and frame filter, a centrifuge, or a filter tank.

[0008] Furthermore, the volume ratio of the circulation tank, enzyme synthesis tank, and crystallization tank is (0.5-1):(1-2):(0.5-1).

[0009] Furthermore, the membrane pore size of the nanofiltration unit is 100-200 dahl.

[0010] Furthermore, the pH range of the feed solution in the enzyme synthesis tank is 5.8-7.0; the pH range of the feed solution in the crystallization tank is 4.9-5.1. The temperature of the circulation tank, enzyme synthesis tank, and crystallization tank is 14℃-20℃.

[0011] Furthermore, the discharge flow rate of the filtrate accounts for 5%-20% of the flow rate of the ampicillin crystallization mother liquor entering the circulation tank; The nanofiltration unit operates in series, and the feed liquid is concentrated 2-3.5 times upon entering the nanofiltration unit; the volume of the concentrated liquid after nanofiltration entering the enzyme synthesis tank per hour is 1-3 times the operating volume of the enzyme synthesis tank.

[0012] Furthermore, during the initial startup of the device, 0.01-0.1% of ampicillin seed crystals are pre-added to the enzyme synthesis tank relative to its operating volume.

[0013] Furthermore, the immobilized enzyme used is a synthetic immobilized penicillin acylase, and the addition amount is 4-10 U / ml relative to the operating volume of the enzyme synthesis tank.

[0014] Furthermore, the acid solution used to adjust the pH is hydrochloric acid solution, and the alkaline solution used is sodium carbonate or ammonia solution.

[0015] The inventors evaluated the stability of ampicillin under different pH conditions and the acceptable range of technical parameters for the industrial recovery of ampicillin crystallization mother liquor. They studied and calculated the product content of the ampicillin crystallization mother liquor under pH 5.0-8.0 conditions. The solubility of ampicillin solution increased with increasing pH after deviating from the isoelectric point, but the solubility was generally low near neutral. Even so, the saturated solubility of ampicillin at pH 7.5 (15.88, 15℃) was still about three times that at pH 5.0 (5.21, 15℃), which provides sufficient space for the implementation of this invention.

[0016] During operation, the mother liquor from the enzymatic ampicillin crystallization process continuously enters the circulation tank, where the pH is adjusted from 5.0 to 7.2-7.5. It then continuously enters a nanofiltration unit operating in series, where it is concentrated 2-3.5 times in one pass. The concentrated solution continuously enters the enzyme synthesis tank, where the pH is adjusted to approximately 6.1 and maintained. The ampicillin in the enzyme synthesis solution becomes supersaturated and precipitates out under seed crystal guidance. Simultaneously, the concentrations of residual 6-APA and phenylglycine methyl ester in the mother liquor increase 2-3.5 times due to nanofiltration concentration, and under the catalysis of synthetic immobilized penicillin acylase, they proceed towards the synthesis of ampicillin. The enzyme synthesis solution continuously enters the crystallization tank, where the pH is adjusted to approximately 5.0 for crystallization, further reducing the concentration of soluble ampicillin. The crystallized solution continuously enters the solid-liquid separator, where the ampicillin solid is retained, part of the filtrate is discharged, and the remainder is returned to the circulation tank for further concentration and recycling.

[0017] This invention can achieve any concentration ratio of more than 2.5 times by adjusting the mother liquor reflux ratio. The higher the concentration ratio, the higher the yield, but the higher the quality and impurities of the recovered product. Considering the product recovery rate and purity of the recovered product in the mother liquor, the total concentration ratio is generally controlled between 8 and 20 times, preferably between 9 and 14 times.

[0018] The beneficial effects of this invention are: This invention, while ensuring the stability of ampicillin, appropriately increases the pH of the mother liquor to control the single nanofiltration concentration ratio within the range of 2.5-3 times, so that the concentration of ampicillin in the concentrate is near the saturation concentration. Under the condition of no seed crystals, a supersaturated solution of ampicillin at 2 times the saturation concentration will not precipitate or crystallize within 0.5 hours, thus avoiding the impact on the working efficiency of the nanofiltration unit due to ampicillin precipitation during the nanofiltration concentration process. After continuously removing part of the ampicillin online, through continuous cyclic concentration, the total concentration ratio reaches 9-14 times, effectively ensuring both the recovery rate and purity of ampicillin.

[0019] This invention utilizes nanofiltration concentration and precise pH control at different stages to continuously remove ampicillin solids online from the recovery equipment system. The concentrations of residual 6-APA and phenylglycine methyl ester are increased after nanofiltration concentration, thereby enabling the catalytic reaction to proceed towards the synthesis of ampicillin. This allows 6-APA and phenylglycine methyl ester in the crystallization mother liquor to be effectively recovered and utilized in the form of ampicillin product.

[0020] This invention operates under mild pH conditions throughout the process, minimizing damage to nanofiltration membrane equipment and degradation of ampicillin, resulting in high product yield and quality. 6-APA and methyl phenylglycine in the mother liquor are effectively recovered and utilized, with a total ampicillin recovery rate exceeding 100% relative to the ampicillin content in the mother liquor. Attached Figure Description

[0021] Figure 1 This invention relates to a continuous recovery device for mother liquor from enzymatic ampicillin crystallization. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0024] The experimental materials used in the embodiments of this invention, unless otherwise specified, are all conventional experimental materials in the art and can be purchased through commercial channels. The penicillin acylase used in this invention is an immobilized penicillin acylase (synthetic type) produced by Hunan Fulaige Company, with an enzyme activity of 100 U / g.

[0025] Example 1 Experimental apparatus and raw materials according to Figure 1The model constructs a continuous recovery device for mother liquor from enzymatic ampicillin crystallization, including two 25L reaction vessels, one 50L enzyme synthesis tank, two nanofiltration units, and two plate and frame filters; the nanofiltration units and plate and frame filters are connected in parallel, with one unit in operation and the other on standby.

[0026] The parameters were set as follows during the experiment: the temperature of the circulation tank, enzyme synthesis tank, and crystallization tank was automatically controlled at 14℃-16℃; the pH range of the circulation tank was automatically controlled at 7.4-7.6, the pH range of the enzyme synthesis tank was automatically controlled at 6.0-6.2, and the pH range of the crystallization tank was automatically controlled at 4.9-5.1.

[0027] The raw material used was the mother liquor of enzymatic ampicillin crystallization. The test results showed that the content of ampicillin was 4.96 mg / ml, the content of 6-APA was 1.34 mg / ml, the content of phenylglycine methyl ester was 2.13 mg / ml, and the pH of the mother liquor was 5.01.

[0028] The experimental steps are as follows: 1. The ampicillin crystallization mother liquor is first continuously fed into the circulation tank, with the operating liquid level controlled at 14-16L. After reaching the liquid level, continuous nanofiltration concentration mode is started, with a concentration ratio set to 3:1. The concentrated liquid is then fed into the enzyme synthesis tank, with the operating liquid level controlled at 29-31L. For the first run, 20g of ampicillin seed crystals and 1500g of penicillin acylase are added. After the enzyme synthesis tank reaches the control liquid level, the liquid is fed into the crystallization tank. The liquid level in the crystallization tank is controlled at 14-16L. 8% hydrochloric acid or 8% sodium carbonate solution is used for pH control.

[0029] 2. Once the crystallization tank reaches the control level, the crystallizing liquid continuously enters the plate and frame filter. A small amount of the filtrate is discharged into the wastewater system, while the remainder is returned to the circulation tank.

[0030] In this operating mode, the nanofiltration unit has an inlet flow rate of 180L / h, an outlet flow rate of 60L / h, and a concentration ratio of 3:1. Most of the filtrate is returned to the circulation tank for further concentration, and the cycle repeats multiple times. The amount of new mother liquor flowing into the circulation tank per hour is 135L, and the amount of mother liquor discharged is 15L. The total concentration factor of the mother liquor (excluding the returned mother liquor) = the amount of new mother liquor flowing into the circulation tank per hour / the amount of mother liquor discharged = 135 / 15 = 9 (times).

[0031] 3. The ampicillin filter cake is dissolved in deionized water, acidified to pH 2.0 until it is clear, filtered, and the pH is adjusted to 5.0 with ammonia water for crystallization for 2 hours. After filtration, the filter cake is dried to obtain the ampicillin product. The filtrate is the ampicillin crystallization mother liquor, which is added to the production mother liquor for recovery.

[0032] Experimental results calculation: The mass of each substance flowing into the mother liquor per hour is shown in Table 1, and the mass of each substance discharging from the mother liquor per hour is shown in Table 2.

[0033] Table 1. Mass of each substance flowing into the mother liquor per hour Table 2. Mass of each substance in the mother liquor discharged per hour According to the results in Tables 1 and 2, the mass difference of 6-APA in the inflow and outflow mother liquor is 163.5g = 180.9g - 17.4g; this portion of 6-APA was synthesized into ampicillin by enzyme catalysis; based on the molecular weights of 6-APA (216.26) and ampicillin (349), it was calculated (m=nM) that the mass of ampicillin newly synthesized from 6-APA per hour is 263.9g. Combining this with Tables 1 and 2, the calculation formula (1) for the hourly ampicillin recovery (g) can be obtained: Ampicillin recovery = Mass of ampicillin in the inflow mother liquor - Mass of ampicillin in the outflow mother liquor + Mass of newly synthesized ampicillin (1) =669.6 - 80.6 + 263.9 = 852.9 Under the above continuous production mode, 135L of ampicillin mother liquor was processed per hour, and the amount of ampicillin recovered per hour was 852.9g. Compared with the amount of ampicillin flowing into the mother liquor per hour, combined with formula (1), the theoretical total recovery rate per hour is calculated by formula (2) as follows: Total recovery rate = Ampicillin recovery amount / Ampicillin mass in the mother liquor flowing in per hour =(mass of ampicillin in the inflow mother liquor - mass of ampicillin in the outflow mother liquor + mass of newly synthesized ampicillin) / mass of ampicillin in the inflow mother liquor (2) =852.9 / 669.6 100% = 127.4% After the actual production was completed, the purity of the recovered product in the separator was measured, and the content of the active ingredient ampicillin in the ampicillin product was 99.34%. Due to the presence of trace amounts of ampicillin and 6-APA in the dialysate (120L) discharged during nanofiltration concentration, and the presence of trace degradation during the recycling process, the actual weight of ampicillin recovered was slightly lower, and the actual total recovery rate was 124.2%.

[0034] Example 2 Experimental apparatus and raw materials according to Figure 1 The model constructs a continuous recovery device for mother liquor from enzymatic ampicillin crystallization, including two 25L reaction vessels, one 50L enzyme synthesis tank, two nanofiltration units, and two plate and frame filters; the nanofiltration units and plate and frame filters are connected in parallel, with one unit in operation and the other on standby.

[0035] The parameters were set as follows during the experiment: the temperature of the circulation tank, enzyme synthesis tank, and crystallization tank was automatically controlled at 14℃-16℃; the pH range of the circulation tank was automatically controlled at 7.2-7.4, the pH range of the enzyme synthesis tank was automatically controlled at 6.0-6.2, and the pH range of the crystallization tank was automatically controlled at 4.9-5.1.

[0036] The raw material used was the mother liquor of enzymatic ampicillin crystallization. The test results showed that the content of ampicillin was 5.08 mg / ml, the content of 6-APA was 1.37 mg / ml, the content of phenylglycine methyl ester was 2.41 mg / ml, and the pH of the mother liquor was 5.12.

[0037] The experimental steps are as follows: 1. The ampicillin crystallization mother liquor is first continuously fed into the circulation tank, with the operating liquid level controlled at 14-16L. After reaching the liquid level, the continuous nanofiltration concentration mode is started. The concentrated liquid is then fed into the enzyme synthesis tank, with the operating liquid level controlled at 29-31L. For the first run, 15g of ampicillin seed crystals and 1800g of penicillin acylase are added. After the enzyme synthesis tank reaches the control liquid level, the liquid is fed into the crystallization tank. The liquid level in the crystallization tank is controlled at 14-16L. 8% hydrochloric acid or 8% sodium carbonate solution is used for pH control.

[0038] 2. Once the crystallization tank reaches the control level, the crystallizing liquid continuously enters the plate and frame filter. A small amount of the filtrate is discharged into the wastewater system, while the remainder is returned to the circulation tank.

[0039] The flow rate of the mother liquor to be recovered into the circulation tank in this device is set to 120 L / h, the nanofiltration concentration ratio is set to 2.8:1, and the total concentration factor of the mother liquor is 14 times.

[0040] 3. The ampicillin filter cake is dissolved in deionized water, acidified to pH 2.0 until it is clear, filtered, and the pH is adjusted to 5.0 with sodium carbonate solution for crystallization for 2 hours. After filtration, the filter cake is dried to obtain the ampicillin product. The filtrate is the ampicillin crystallization mother liquor, which is added to the production mother liquor for recovery.

[0041] Experimental results calculation: The mass of each substance flowing into the mother liquor per hour is shown in Table 3, and the mass of each substance discharging from the mother liquor per hour is shown in Table 4.

[0042] Table 3. Mass of each substance flowing into the mother liquor per hour Table 4. Mass of each substance in the mother liquor discharged per hour According to the results in Tables 3 and 4, the mass difference of 6-APA in the inflow and outflow mother liquor is 153.9 g = 164.4 g - 10.5 g; this portion of 6-APA was catalyzed by enzyme to synthesize ampicillin; based on the molecular weights of 6-APA (216.26) and ampicillin (349), it was calculated (m = nM) that the mass of ampicillin newly synthesized from 6-APA per hour is 248.4 g. Combining this with Tables 1 and 2, the calculation formula (1) for the hourly ampicillin recovery (g) can be obtained: Ampicillin recovery = Mass of ampicillin in the inflow mother liquor - Mass of ampicillin in the outflow mother liquor + Mass of newly synthesized ampicillin (1) =609.6 - 46.0 + 248.4 = 812.0 Under the above continuous production mode, 120L of ampicillin mother liquor is processed per hour, and the amount of ampicillin recovered per hour is 812.0g. Compared with the amount of ampicillin flowing into the mother liquor per hour, combined with formula (1), the theoretical total recovery rate per hour is calculated by formula (2) as follows: Total recovery rate = Ampicillin recovery amount / Ampicillin mass in the mother liquor flowing in per hour =(mass of ampicillin in the inflow mother liquor - mass of ampicillin in the outflow mother liquor + mass of newly synthesized ampicillin) / mass of ampicillin in the inflow mother liquor (2) =812.0 / 609.6 100% = 133.2% After the actual production was completed, the purity of the recovered product in the separator was measured, and the content of the active ingredient ampicillin in the ampicillin product was 99.21%. Due to the presence of trace amounts of ampicillin and 6-APA in the dialysate (120L) discharged during nanofiltration concentration, and the presence of trace degradation during the recycling process, the actual weight of ampicillin recovered was slightly lower, and the actual total recovery rate was 130.2%.

[0043] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for continuous recovery of mother liquor from enzymatic ampicillin crystallization, characterized in that, Includes the following steps: The mother liquor from the enzymatic ampicillin crystallization process is continuously injected into the circulation tank of the continuous recovery unit. After pH adjustment, it enters the nanofiltration concentrator for concentration. The obtained concentrate enters the enzyme synthesis tank. 6-aminopenicillanic acid and phenylglycine methyl ester contained in the mother liquor are used to synthesize ampicillin under the catalysis of immobilized enzymes. The obtained synthesis solution is continuously fed into the crystallization tank for crystallization. The crystallized solution in the crystallization tank continuously enters the separator, where the solid is removed online, which is the ampicillin product. Part of the filtrate is discharged, and the rest is returned to the circulation tank. In the circulating tank, the pH of the adjusted enzymatic ampicillin crystallization mother liquor is 6.5-8.0; The continuous recovery device includes a circulation tank, a nanofiltration unit, an enzyme synthesis tank, a crystallization tank, and a separator; the outlet of the circulation tank is connected to the inlet of the nanofiltration unit, the outlet of the nanofiltration unit is connected to the inlet of the enzyme synthesis tank, the outlet of the enzyme synthesis tank is connected to the inlet of the crystallization tank, the outlet of the crystallization tank is connected to the inlet of the separator, and the outlet of the separator is connected to the inlet of the circulation tank; the circulation tank is also provided with a mother liquor inlet.

2. The continuous recycling method according to claim 1, characterized in that, A filter plate with a pore size of 100-150 mesh is installed near the bottom of the enzyme synthesis tank.

3. The continuous recycling method according to claim 1, characterized in that, The separator can be a plate and frame filter, a centrifuge, or a filter tank.

4. The continuous recycling method according to claim 1, characterized in that, The volume ratio of the circulation tank, enzyme synthesis tank, and crystallization tank is (0.5-1):(1-2):(0.5-1).

5. The continuous recycling method according to claim 1, characterized in that, The membrane pore size of the nanofiltration unit is 100-200 Dal.

6. The continuous recycling method according to claim 1, characterized in that, The pH range of the feed solution in the enzyme synthesis tank is 5.8-7.0; the pH range of the feed solution in the crystallization tank is 4.9-5.

1. The temperature of the circulation tank, enzyme synthesis tank, and crystallization tank is 14℃-20℃.

7. The continuous recycling method according to claim 1, characterized in that, The discharge flow rate of the filtrate accounts for 5%-20% of the flow rate of the ampicillin crystallization mother liquor entering the circulation tank; The nanofiltration unit operates in series, and the feed liquid is concentrated 2-3.5 times upon entering the nanofiltration unit; the volume of the concentrated liquid after nanofiltration entering the enzyme synthesis tank per hour is 1-3 times the operating volume of the enzyme synthesis tank.

8. The continuous recycling method according to claim 1, characterized in that, When the device is first started up, 0.01-0.1% of ampicillin seed crystals are added to the enzyme synthesis tank relative to its operating volume.

9. The continuous recycling method according to claim 1, characterized in that, The immobilized enzyme used was a synthetic immobilized penicillin acylase, and the addition amount was 4-10 U / ml relative to the operating volume of the enzyme synthesis tank.

10. The continuous recycling method according to claim 1, characterized in that, The acid solution used to adjust the pH is hydrochloric acid solution, and the alkaline solution used is sodium carbonate or ammonia solution.

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