Method for preparing ampicillin by enzyme catalysis method

Abstract

The invention belongs to the technical field of biological medicines, and relates to a method for preparing ampicillin by an enzyme catalysis method. The method comprises the following steps: 6-APA and D-phenylglycine methyl ester hydrochloride are respectively dissolved and then mixed, the mixed solution passes through an immobilized enzyme column bed filled with immobilized penicillin acylase for synthesis from top to bottom, and 6-APA and D-phenylglycine methyl ester are subjected to condensation reaction under the action of enzyme when flowing through the immobilized enzyme column bed to generate ampicillin. According to the method, the condensation reaction of 6-APA and D-phenylglycine methyl ester is successfully realized by innovatively using the immobilized enzyme column bed, and compared with the traditional stirring type enzyme reaction, the feeding amount of D-phenylglycine methyl ester is reduced, degraded impurities in the product are reduced, and the yield of the product is improved. In addition, ampicillin meeting CP quality standards can be prepared by adopting an immobilized enzyme column bed reaction mode without a recrystallization process, the acid and alkali consumption is also greatly reduced, and the process safety and the product quality are also greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a method for preparing ampicillin by an enzyme catalysis method, and more specifically, to a method for preparing ampicillin by an enzyme catalysis method using an immobilized enzyme column bed. Background Art

[0002] Ampicillin, also known as ampicillin or ampicillin, is a β-lactam antibiotic belonging to the penicillin class. It is primarily used to treat various bacterial infections caused by susceptible bacteria, such as respiratory and urinary tract infections, and to prevent group B streptococcal infections in neonates. Ampicillin can be administered orally, intramuscularly, or intravenously, with common dosage forms including injections and capsules. It exerts its bactericidal effect by inhibiting bacterial cell wall synthesis and has strong antibacterial activity against Gram-positive cocci and bacilli, as well as Gram-negative bacteria.

[0003] Currently, ampicillin is primarily synthesized through the enzymatic reaction of 6-APA (6-aminopenicillanic acid) and D-phenylglycine methyl ester hydrochloride. This process relies primarily on an enzymatic synthesis process, with immobilized penicillin acylase serving as the key catalyst. During the synthesis process, 6-APA and D-phenylglycine methyl ester hydrochloride are dissolved and mixed with an enzyme. Under appropriate temperature and pH conditions, the enzyme catalyzes the condensation reaction between 6-APA and D-phenylglycine methyl ester to produce ampicillin. Compared to traditional chemical methods, enzymatic synthesis significantly reduces residual solvent in the product, improving the purity of the finished product and production safety. This helps reduce potential toxic side effects for patients and enhances drug safety and efficacy. However, due to the non-specificity of the immobilized penicillin acylase used in the synthesis, a certain amount of hydrolysis still occurs during the catalytic synthesis reaction, namely, hydrolysis of ampicillin to 6-APA and D-phenylglycine, and hydrolysis of D-phenylglycine methyl ester to D-phenylglycine. Although this hydrolysis is relatively weak, prolonged contact between ampicillin and D-phenylglycine methyl ester with the enzyme causes the hydrolysis products to accumulate to high concentrations. This not only reduces the yield of the ampicillin product, but also transfers the hydrolysis products to the crystallization process, thereby affecting the quality of the ampicillin product. Given these limitations of existing ampicillin preparation processes, achieving enzymatic synthesis of ampicillin with high yield, high quality, and low cost is of great practical significance, necessitating continued research and improvement. Summary of the Invention

[0004] Technical issues

[0005] The present invention is designed to solve the above-mentioned problems of the prior art. The purpose of the present invention is to provide a method for preparing ampicillin by enzyme catalysis. The preparation method innovatively uses an immobilized enzyme column bed to prepare high-quality ampicillin without the need for a recrystallization process. The process design is innovative, the operation is simple, and the energy consumption is low.

[0006] Technical Solution

[0007] In order to achieve the above-mentioned object of the present invention, the technical solution adopted by the present invention is as follows: after 6-APA and D-phenylglycine methyl ester hydrochloride are dissolved separately, they are mixed and the pH is controlled to 5.5-6.5. The mixed solution passes from top to bottom through an immobilized enzyme column bed containing immobilized penicillin acylase for synthesis. When flowing through the immobilized enzyme column bed, under the action of the enzyme, 6-APA and D-phenylglycine methyl ester undergo a condensation reaction to produce ampicillin, and the reaction solution is received at the outlet below the immobilized enzyme column bed. The reaction solution is crystallized by adjusting the pH, growing the crystals, filtering, and drying to obtain ampicillin crystals with product quality meeting the CP standard.

[0008] According to the present invention, the method for preparing ampicillin by enzyme catalysis provided by the present invention comprises the following steps:

[0009] (1) Dissolution of 6-APA and D-phenylglycine methyl ester hydrochloride

[0010] Mixing 6-APA with water and adding a base to dissolve it to obtain a 6-APA solution; dissolving D-phenylglycine methyl ester hydrochloride in water to obtain a D-phenylglycine methyl ester hydrochloride solution; then, mixing the 6-APA solution and the D-phenylglycine methyl ester hydrochloride solution, and controlling the pH of the mixture to be within the range of 5.5 to 6.5;

[0011] (2) Synthesis of Ampicillin

[0012] The mixed solution obtained in the above step (1) is passed from top to bottom through an immobilized enzyme column bed containing immobilized penicillin acylase for synthesis. When the mixed solution flows through the immobilized enzyme column bed, 6-APA and D-phenylglycine methyl ester undergo a condensation reaction under the action of the enzyme to produce ampicillin. The reaction solution is received at the outlet below the immobilized enzyme column bed. The reaction solution is subjected to pH adjustment for crystallization, crystal growth, filtration, and drying to obtain ampicillin crystals.

[0013] Beneficial effects

[0014] In the present invention, the innovative use of an immobilized enzyme column bed successfully achieves the preparation of ampicillin. Compared with traditional stirred enzyme reactions, a mixture of the raw materials 6-APA and D-phenylglycine methyl ester hydrochloride passes through the immobilized enzyme column bed. Under the action of the enzyme, 6-APA and D-phenylglycine methyl ester undergo a condensation reaction to produce ampicillin. The generated ampicillin remains in the column for a short time before flowing out of the immobilized enzyme column bed with the reaction liquid. This reaction method reduces the contact time between 6-APA, D-phenylglycine methyl ester, and ampicillin and the immobilized enzyme bed, thereby significantly reducing the degradation of D-phenylglycine methyl ester and ampicillin. As a result, the amount of D-phenylglycine methyl ester added is reduced, the ampicillin yield is increased, and impurities are correspondingly reduced. Moreover, the immobilized enzyme column bed reaction method does not require a recrystallization step to produce ampicillin that meets CP quality standards, significantly reduces the amount of acid and base used, and significantly improves process safety and product quality.

[0015] Taking the above advantages into consideration, compared with the existing conventional technology, the ampicillin prepared by the enzymatic method of the present invention has better quality, higher yield and lower cost, and the present invention is more suitable for promotion and application in industrial production. DETAILED DESCRIPTION

[0016] The enzymatic method for preparing ampicillin according to the present invention will be described in detail below to facilitate understanding of the present invention. However, various modifications may be made to the embodiments of the present invention, and the scope of the present invention is not limited to the embodiments described below. The embodiments of the present invention are provided to make this disclosure clear and complete, and to fully illustrate the present invention to those skilled in the art.

[0017] According to one embodiment of the present invention, in the method for preparing ampicillin by the enzymatic method of the present invention, in the dissolution of 6-APA and D-phenylglycine methyl ester hydrochloride in the step (1), 6-APA is mixed with water and a base is added to dissolve it to obtain a 6-APA solution; D-phenylglycine methyl ester hydrochloride is dissolved in water to obtain a D-phenylglycine methyl ester hydrochloride solution; then, the 6-APA solution is mixed with the D-phenylglycine methyl ester hydrochloride solution, and the pH of the mixed solution is controlled within the range of 5.5 to 6.5.

[0018] In the present invention, since the immobilized penicillin acylase for synthesis is used in the form of an immobilized enzyme column bed, the reaction solution flows through the immobilized enzyme column bed, which reduces the contact time between D-phenylglycine methyl ester and the immobilized enzyme column bed, thereby significantly reducing the degradation of D-phenylglycine methyl ester. Therefore, the amount of D-phenylglycine methyl ester fed can be reduced. Specifically, the amount of 6-APA and D-phenylglycine methyl ester hydrochloride used can be such that the molar ratio of 6-APA to D-phenylglycine methyl ester hydrochloride is 1:1.00 to 1.10, preferably 1:1.00 to 1.05, which is converted to a mass ratio of 1:0.93 to 1.03, preferably 1:0.93 to 0.98. At this feed ratio, a 6-APA conversion rate exceeding 99% can still be ensured.

[0019] 6-APA is mixed with water and a base is added to dissolve the mixture to obtain a 6-APA solution. Specifically, the ratio of 6-APA to water can be 1:3 to 7, preferably 1:3 to 6, by weight. After the mixture, a base is added to dissolve the mixture, such as 3 to 6 mol / L ammonia water, and stirred to dissolve the mixture to obtain a 6-APA solution. The pH of the 6-APA solution is generally 7.5 to 8.5.

[0020] Dissolve D-phenylglycine methyl ester hydrochloride in water to obtain a D-phenylglycine methyl ester hydrochloride solution. Specifically, the ratio of D-phenylglycine methyl ester hydrochloride to water can be 1:3 to 7, preferably 1:3 to 5, by weight. Mix the two and stir until dissolved to obtain a D-phenylglycine methyl ester hydrochloride solution. The pH of the solution is generally 1.5 to 2.5.

[0021] Then, the D-phenylglycine methyl ester hydrochloride solution is slowly added to the 6-APA solution. At this time, the pH of the mixed solution is within the range of 5.5 to 6.5. If it is not within this range, 3 to 6 mol / L ammonia water or 3 to 6 mol / L hydrochloric acid can be used to adjust the pH of the mixed solution to this range. Within the above pH range, the immobilized enzyme can play a good catalytic role and ensure a high conversion rate of the product. In addition, in order to avoid degradation of 6-APA and ampicillin, the dissolution process and the enzyme reaction process in step (2) should be within the range of 5°C to 20°C. Therefore, the water temperature used in the dissolution process of 6-APA and D-phenylglycine methyl ester hydrochloride should be within the above temperature range.

[0022] In the present invention, purified water can be used as the water.

[0023] According to one embodiment of the present invention, in the method for preparing ampicillin by the enzyme-catalyzed method of the present invention, in the synthesis of ampicillin in step (2), the mixed solution obtained in the above step (1) is passed from top to bottom through an immobilized enzyme column bed containing immobilized penicillin acylase for synthesis. When the mixed solution flows through the immobilized enzyme column bed, under the action of the enzyme, 6-APA and D-phenylglycine methyl ester undergo a condensation reaction to produce ampicillin, and the reaction solution is received at the lower outlet of the immobilized enzyme column bed. The reaction solution is subjected to pH adjustment for crystallization, crystal growth, filtration, and drying to obtain ampicillin crystals.

[0024] The immobilized enzyme column bed is used in the form of a circular column bed, with an 80-100 mesh stainless steel metal screen embedded at the bottom of the column bed to retain the immobilized enzyme in the column bed. The immobilized penicillin acylase for synthesis can be yellow or brown spherical particles, with a synthetic activity of 100-300 U / g, preferably 200-300 U / g, and more than 90% of the particles have a particle size within the range of 250-500 μm. The immobilized penicillin acylase for synthesis can be commercially available, for example, from Shanxi Shuangyan Biotechnology Co., Ltd. and Hunan Fulaige Biotechnology Co., Ltd. The immobilized penicillin acylase for synthesis is packed using a wet method, i.e., the immobilized penicillin acylase for synthesis is mixed with water and then poured into the immobilized enzyme column bed. During the packing process, a 2-3 cm excess liquid is left above the immobilized enzyme column bed to prevent the column from drying out. At the same time, the height-to-diameter ratio (i.e., aspect ratio) of the immobilized enzyme bed is 4 or greater, preferably greater than or equal to 6, and more preferably greater than or equal to 8. After the column is loaded, the immobilized enzyme column bed is rinsed with water 3 to 5 times the volume of the immobilized enzyme column bed, and 2 to 3 cm of liquid is left above the immobilized enzyme column bed.

[0025] The mixed solution obtained in the above step (1) is passed from top to bottom through an immobilized enzyme column bed containing immobilized penicillin acylase for synthesis. The mixed solution contacts the immobilized penicillin acylase for synthesis, i.e., 6-APA and D-phenylglycine methyl ester undergo a condensation reaction under the action of the enzyme. The mixed solution passes through the column bed at a flow rate of 1.0 to 2.0 times the volume of the immobilized enzyme column bed per hour, preferably at a flow rate of 1.5 to 2.0 times the volume of the immobilized enzyme column bed per hour. Within this flow rate range, the residence time of the mixed solution in the immobilized enzyme column bed is 30 to 60 minutes. When leaving the lower outlet of the column bed, 6-APA and D-phenylglycine methyl ester have been converted into ampicillin. The generated ampicillin immediately leaves the immobilized enzyme column bed and will not be enriched in the enzyme column bed. Therefore, the contact time between ampicillin and the immobilized enzyme column bed is also short, and the degradation impurities in the product will be very small.

[0026] In the synthesis of ampicillin in step (2), the immobilized penicillin acylase for synthesis is used in the form of a column bed, and the mixed solution is controlled to flow through the column bed at a flow rate of 1.0 to 2.0 times the volume of the immobilized enzyme column bed per hour, that is, the residence time of the mixed solution in the immobilized enzyme column bed is 30 to 60 minutes, so that 6-APA and D-phenylglycine methyl ester react completely and then flow out of the column bed. Therefore, there is no limit on the amount of immobilized penicillin acylase used for synthesis, as long as the mixed solution is ensured to flow through the column bed at a flow rate of 1.0 to 2.0 times the volume of the immobilized enzyme column bed per hour, that is, the residence time of the mixed solution in the immobilized enzyme column bed is 30 to 60 minutes. After the immobilized enzyme column bed completes the catalytic reaction, the mixed solution can be re-injected for reaction. The immobilized enzyme in the immobilized enzyme column bed exhibits excellent stability and durability, and the attenuation of enzyme activity is extremely weak during long-term, high-frequency cyclic use. During continuous operation, if the enzyme activity decreases to a certain extent due to various factors, an appropriate amount of new immobilized penicillin acylase for synthesis can be accurately added to the immobilized enzyme column bed to effectively maintain the reaction rate during the substrate conversion process, thereby ensuring the substrate conversion efficiency and the continuous and efficient operation of the immobilized enzyme column bed.

[0027] The reaction solution is received at the outlet below the immobilized enzyme column bed, and its pH is generally in the range of 6.0 to 6.5. It is continuously fed into the crystallization container, into which 3 to 6 mol / L hydrochloric acid is continuously added to ensure that the pH of the entire crystallization system is maintained at 4.8 to 5.0, until the mixed solution has completely passed through the immobilized enzyme column bed and is collected in the crystallization container. At this time, 0.5 times the volume of the immobilized enzyme column bed is used to rinse the immobilized enzyme bed at the same flow rate, and this part of the rinse liquid is also collected in the crystallization container. After the above operation steps are completed, the crystallization liquid in the crystallization container is cooled to 0°C to 5°C, and the crystals are grown for 30 to 60 minutes. Then, after filtration and drying, ampicillin crystals containing three crystal waters are obtained.

[0028] The following examples further illustrate the method for preparing ampicillin by enzyme catalysis of the present invention. The scope of protection of the present invention is not limited to the following examples. These examples are listed for illustrative purposes only and do not limit the present invention in any way.

[0029] Preparation of immobilized enzyme column bed:

[0030] Synthetic immobilized penicillin acylase (activity 259 U / g, more than 90% of the particles have a particle size range of 250-500 μm, obtained from Shanxi Shuangyan Biotechnology Co., Ltd.) was rinsed with a large amount of purified water. The immobilized penicillin acylase was then packed into a column using the wet column packing method described above, resulting in an immobilized enzyme column bed volume of 100 ml and an aspect ratio of 4. The immobilized enzyme column bed was then rinsed with 500 ml of purified water until ready for use (ensuring that 2-3 cm of liquid remained above the immobilized enzyme column bed).

[0031] Example 1

[0032] Weigh 100 g of 6-APA, add 400 g of 10°C purified water, adjust the pH to 7.8 with 6 mol / L ammonia water, dissolve 6-APA to obtain a 6-APA solution; weigh 98 g of D-phenylglycine methyl ester hydrochloride, add 392 g of 10°C purified water, stir and dissolve D-phenylglycine methyl ester hydrochloride to obtain a D-phenylglycine methyl ester hydrochloride solution with a pH of 1.9; slowly add the D-phenylglycine methyl ester hydrochloride solution to the 6-APA solution. At this time, the pH of the mixed solution is 6.1 and the temperature is 10°C.

[0033] The mixed solution was passed from top to bottom through the immobilized enzyme column bed prepared above at a flow rate of 150 ml / h. The reaction solution was received at the outlet below the immobilized enzyme column bed and continuously input into the crystallization container. After the mixed solution was completely processed, 50 ml of 10 ° C purified water was used to rinse the immobilized enzyme bed and flowed into the crystallization container. During this process, 3-6 mol / L hydrochloric acid was continuously used to maintain the liquid pH in the crystallization container to 5.0, and ampicillin crystals were precipitated. Then the temperature was lowered to 0 ° C to 5 ° C, the crystals were grown for 30 minutes, and 182 g of ampicillin (containing three crystal waters) was obtained after filtration and drying, with a molar yield of 97.3%. The product was tested according to the method recorded in the 2020 edition of the Chinese Pharmacopoeia. The results showed that it met the CP quality standards. The specific test results are shown in Table 1 below.

[0034] Example 2

[0035] Weigh 100 g of 6-APA, add 600 g of 10°C purified water, adjust the pH to 7.6 with 6 mol / L ammonia water, dissolve 6-APA to obtain a 6-APA solution; weigh 93 g of D-phenylglycine methyl ester hydrochloride, add 465 g of 10°C purified water, stir and dissolve D-phenylglycine methyl ester hydrochloride to obtain a D-phenylglycine methyl ester hydrochloride solution with a pH of 2.1; slowly add the D-phenylglycine methyl ester hydrochloride solution to the 6-APA solution. At this time, the pH of the mixed solution is 6.2 and the temperature is 10°C.

[0036] The mixed solution was passed from top to bottom through the immobilized enzyme column bed prepared above at a flow rate of 200 ml / h. The reaction solution was received at the outlet below the immobilized enzyme column bed and continuously input into the crystallization container. After the mixed solution was completely processed, 50 ml of 10 ° C purified water was used to rinse the immobilized enzyme bed and flowed into the crystallization container. During this process, 3-6 mol / L hydrochloric acid was continuously used to maintain the liquid pH in the crystallization container to 5.0, and ampicillin crystals were precipitated. Then the temperature was lowered to 0 ° C to 5 ° C, the crystals were grown for 30 minutes, and 179 g of ampicillin (containing three crystal waters) was obtained after filtration and drying, with a molar yield of 95.5%. The product was tested according to the method recorded in the 2020 edition of the Chinese Pharmacopoeia. The results showed that it met the CP quality standards. The specific test results are shown in Table 1 below.

[0037] Example 3

[0038] Weigh 100 g of 6-APA, add 400 g of 20°C purified water, adjust the pH to 7.7 with 6 mol / L ammonia water, and dissolve 6-APA to obtain a 6-APA solution; weigh 95 g of D-phenylglycine methyl ester hydrochloride, add 285 g of 20°C purified water, stir and dissolve D-phenylglycine methyl ester hydrochloride to obtain a D-phenylglycine methyl ester hydrochloride solution with a pH of 1.7; slowly add the D-phenylglycine methyl ester hydrochloride solution to the 6-APA solution. At this time, the pH of the mixed solution is 5.7 and the temperature is 20°C.

[0039] The mixed solution was passed from top to bottom through the immobilized enzyme column bed prepared above at a flow rate of 200 ml / h. The reaction solution was received at the outlet below the immobilized enzyme column bed and continuously input into the crystallization container. After the mixed solution was completely processed, 50 ml of 20 ° C purified water was used to rinse the immobilized enzyme bed and flowed into the crystallization container. During this process, 3-6 mol / L hydrochloric acid was continuously used to maintain the liquid pH in the crystallization container to 5.0, and ampicillin crystals were precipitated. Then the temperature was lowered to 0 ° C to 5 ° C, the crystals were grown for 60 minutes, and 175 g of ampicillin (containing three crystal waters) was obtained after filtration and drying, with a molar yield of 93.1%. The product was tested according to the method recorded in the 2020 edition of the Chinese Pharmacopoeia. The results showed that it met the CP quality standards. The specific test results are shown in Table 1 below.

[0040] Table 1: Summary of test results of ampicillin products prepared in Examples 1-3:

[0041]

Claims

1. A method for preparing ampicillin by enzyme catalysis, comprising the following steps: (1) Dissolution of 6-APA and D-phenylglycine methyl ester hydrochloride Mixing 6-APA with water and adding a base to dissolve it to obtain a 6-APA solution; dissolving D-phenylglycine methyl ester hydrochloride in water to obtain a D-phenylglycine methyl ester hydrochloride solution; then, mixing the 6-APA solution and the D-phenylglycine methyl ester hydrochloride solution, and controlling the pH of the mixture to be within the range of 5.5 to 6.5; (2) Synthesis of Ampicillin The mixed solution obtained in the above step (1) is passed from top to bottom through an immobilized enzyme column bed containing immobilized penicillin acylase for synthesis. When the mixed solution flows through the immobilized enzyme column bed, 6-APA and D-phenylglycine methyl ester undergo a condensation reaction under the action of the enzyme to produce ampicillin. The reaction solution is received at the outlet below the immobilized enzyme column bed. The reaction solution is subjected to pH adjustment for crystallization, crystal growth, filtration, and drying to obtain ampicillin crystals.

2. The method for preparing ampicillin by enzyme catalysis according to claim 1, characterized in that: The amount of 6-APA and D-phenylglycine methyl ester hydrochloride used is such that the molar ratio of 6-APA to D-phenylglycine methyl ester hydrochloride is 1:1.00 to 1.10, preferably 1:1.00 to 1.

05.

3. The method for preparing ampicillin by enzyme catalysis according to claim 1, characterized in that In the dissolution of 6-APA and D-phenylglycine methyl ester hydrochloride in step (1), 6-APA is mixed with water in a ratio of 6-APA to water of 1:3 to 7, preferably 1:3 to 6, by weight. After the two are mixed, 3 to 6 mol / L ammonia water is added and stirred to dissolve the mixture to obtain a 6-APA solution having a pH of 7.5 to 8.

5.

4. The method for preparing ampicillin by enzyme catalysis according to claim 1, characterized in that: D-phenylglycine methyl ester hydrochloride is dissolved in water, and the ratio of D-phenylglycine methyl ester hydrochloride to water is 1:3-7, preferably 1:3-5, by weight. The two are mixed and stirred to dissolve clearly to obtain a D-phenylglycine methyl ester hydrochloride solution, and the pH of the solution is 1.5-2.

5.

5. The method for preparing ampicillin by enzyme catalysis according to claim 1, characterized in that In the synthesis of ampicillin in step (2), the immobilized penicillin acylase used for synthesis is in the form of yellow or brown spherical particles with a synthetic activity of 100 to 300 U / g, preferably 200 to 300 U / g, and more than 90% of the particles have a particle size within the range of 250 to 500 μm.

6. The method for preparing ampicillin by enzyme catalysis according to claim 1 or 5, characterized in that In the synthesis of ampicillin in step (2), the immobilized enzyme column bed containing immobilized penicillin acylase for synthesis is obtained by the following method: a circular column bed is selected, and a stainless steel metal screen of 80-100 mesh is embedded in the bottom of the column bed. The immobilized penicillin acylase for synthesis is packed into the column using a wet method, that is, the immobilized penicillin acylase for synthesis is mixed with water and poured into the circular column bed. During the packing process, it is ensured that 2-3 cm of liquid is left above the immobilized enzyme column bed, and the height to diameter ratio of the immobilized enzyme bed is 4 or greater. After the packing is completed, the immobilized enzyme column bed is rinsed with water 3-5 times the volume of the immobilized enzyme column bed, and it is ensured that 2-3 cm of liquid is left above the immobilized enzyme column bed.

7. The method for preparing ampicillin by enzyme catalysis according to claim 1 or 5, characterized in that In the synthesis of ampicillin in step (2), the mixed solution obtained in step (1) is passed through the column bed at a flow rate of 1.0 to 2.0 times the volume of the immobilized enzyme column bed per hour. Within this flow rate range, the residence time of the mixed solution in the immobilized enzyme column bed is 30 to 60 minutes.

8. The method for preparing ampicillin by enzyme catalysis according to claim 7, characterized in that In the synthesis of ampicillin in step (2), the mixed solution obtained in step (1) is passed through the column bed at a flow rate of 1.5 to 2.0 times the volume of the immobilized enzyme column bed per hour.

9. The method for preparing ampicillin by enzyme catalysis according to claim 7, wherein In the synthesis of ampicillin in step (2), the reaction solution is received at the outlet below the immobilized enzyme column bed, and its pH is in the range of 6.0 to 6.

5. The reaction solution is continuously fed into the crystallization container, and 3 to 6 mol / L hydrochloric acid is continuously added to the crystallization container to ensure that the pH of the entire crystallization system is maintained at 4.8 to 5.0, until the mixed solution has all passed through the immobilized enzyme column bed and is collected in the crystallization container. At this time, 0.5 times the volume of the immobilized enzyme column bed is used to rinse the immobilized enzyme bed at the same flow rate, and this part of the rinsing liquid is also collected in the crystallization container. After the above operation steps are completed, the crystallization solution in the crystallization container is cooled to 0°C to 5°C, and the crystals are grown for 30 to 60 minutes. Then, the crystals are filtered and dried to obtain ampicillin crystals containing three waters of crystallization.

10. The method for preparing ampicillin by enzyme catalysis according to claim 1, wherein In the step (1) dissolving 6-APA and D-phenylglycine methyl ester hydrochloride and the step (2) synthesis of ampicillin, the dissolution process and the step (2) enzyme reaction process should be within the range of 5°C to 20°C, and the water used is purified water.

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