Method for preparing oxacillin sodium by enzyme method

Abstract

The invention belongs to the technical field of medicine synthesis, and particularly relates to a method for preparing oxacillin sodium through an enzyme method. The method comprises the following steps: taking 6-APA and 5-methyl-3-phenylisoxazole-4-carboxylic acid methyl ester as raw materials, and adding immobilized penicillin G acylase for reaction to obtain an oxacillin sodium solution; and filtering, dialyzing, concentrating and crystallizing the oxacillin sodium solution to obtain the oxacillin sodium. The method has the advantages of mild reaction conditions, reduction of the generation of wastes, low production cost, accordance with a green production process, and very good industrialization value; and a nanofiltration membrane dialysis process is introduced into the post-treatment of the reaction liquid, so that the product quality control is better guaranteed.

Description

Enzymatic preparation method of oxacillin sodium Technical Field

[0001] This invention belongs to the field of drug synthesis technology, specifically relating to an enzymatic method for preparing oxacillin sodium. Background Technology

[0002] Oxacillin sodium is a semi-synthetic, penicillinase-resistant, and acid-resistant penicillin that can be administered orally or by injection. It is a bactericidal agent used during the reproductive phase, exerting its bactericidal effect by inhibiting bacterial cell wall synthesis. Oxacillin sodium is not destroyed by penicillinase produced by Staphylococcus aureus and is effective against enzyme-producing Staphylococcus aureus strains. However, its antibacterial activity against non-resistant fungi is less than that of penicillin G. Oxacillin sodium can be used to treat various infections caused by penicillin-resistant Staphylococcus aureus, such as sepsis, respiratory tract infections, and soft tissue infections, as well as mixed infections caused by Streptococcus pyogenes and penicillin-resistant cocci, playing an important role in clinical practice.

[0003] Oxacillin sodium is a white or crystalline powder, odorless or slightly odorous, readily soluble in water, very slightly soluble in acetone or butanol, and practically insoluble in ethyl acetate or petroleum ether. Its chemical name is (2S,5R,6R)-3,3-dimethyl-6-(5-methyl-3-phenyl-4-isoxazolamide)-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate sodium salt monohydrate, with a molecular weight of 441.44, and its structural formula is shown in Formula I.

[0004]

[0005] I.

[0006] Currently, the main method for synthesizing oxacillin sodium in China is the acyl chloride method, which uses 6-aminopenicillanic acid (6-APA) and 5-methyl-3-phenyl-4-isozolecarboxylic acid chloride as raw materials, which are then condensed to form the sodium salt. The synthetic route is as follows:

[0007]

[0008] However, the existing acyl chloride method for preparing oxacillin sodium has problems such as complex process routes, high costs, high emissions of waste, and significant pollution.

[0009] To overcome the aforementioned problems, some existing technologies have explored the synthesis process of oxacillin sodium. For example, patent CN102161668B discloses a method for preparing oxacillin sodium and oxacillin sodium for injection, using 6-APA and benzoisoxazoline chloride as raw materials, and preparing oxacillin sodium and oxacillin sodium for injection through steps such as condensation, acidification, crystallization, washing, pulverization, and packaging. Another example is patent CN116514839B, which discloses a method for synthesizing oxacillin sodium, using 6-APA and MPCC as raw materials, and synthesizing oxacillin sodium through steps such as condensation, extraction to form salt, and purification crystallization. This invention employs a low-temperature phase separation process during condensation, introduces a phase transfer catalyst, shortens the reaction time, inhibits raw material degradation, and improves product quality.

[0010] Therefore, it is necessary to develop a completely new method for synthesizing oxacillin sodium to overcome the problems of complex process routes, high production costs, high emissions of waste, and significant pollution associated with existing processes. Summary of the Invention

[0011] In view of this, the present invention proposes an enzymatic method for preparing oxacillin sodium. The present invention uses 6-APA and methyl 5-methyl-3-phenylisoxazole-4-carboxylate in the presence of immobilized penicillin G acylase to generate oxacillin, effectively overcoming the problems of complex process routes, high costs, high emissions of waste, and significant pollution associated with existing technologies. This method has the advantages of a simple synthetic route, mild reaction conditions, low production costs, and environmental friendliness.

[0012] One of the objectives of this invention is to provide an enzymatic method for preparing oxacillin sodium.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] The enzymatic method for preparing oxacillin sodium includes the following steps:

[0015] (1) Using 6-APA and 5-methyl-3-phenylisoxazole-4-carboxylic acid methyl ester as raw materials, immobilized penicillin G acylase was added to react and oxacillin sodium solution was obtained.

[0016] (2) The oxacillin sodium solution obtained in step (1) is filtered, dialyzed, concentrated and crystallized to obtain oxacillin sodium.

[0017] Preferably, in step (1), the molar ratio of 6-APA to methyl 5-methyl-3-phenylisoxazole-4-carboxylate is 1:1~2; and the mass ratio of 6-APA to immobilized penicillin G acylase is 1:0.5~2.

[0018] Preferably, in step (1), the molar ratio of 6-APA and methyl 5-methyl-3-phenylisoxazole-4-carboxylic acid is 1:1.1~1.5, more preferably 1:1.4.

[0019] Preferably, in step (1), the mass ratio of the 6-APA to the immobilized penicillin G acylase is 1:1.1~1.5, more preferably 1:1.3.

[0020] Preferably, in step (1), the pH is controlled at 6-8 before immobilized penicillin G acylase is added for reaction; the pH adjuster is sodium carbonate.

[0021] Preferably, the pH is controlled at 6.7~7.2.

[0022] Preferably, the pH adjuster is an aqueous solution of sodium carbonate.

[0023] Preferably, in step (1), the reaction temperature is 5~25℃ and the reaction time is 2~5 hours.

[0024] Preferably, in step (1), the reaction temperature is 15°C and the reaction time is 3 hours.

[0025] Preferably, in step (1), the reaction solvent is water.

[0026] Preferably, in step (2), the dialysis includes: performing dialysis using a 500-800 Dalton nanofiltration membrane ①, performing dialysis on the obtained dialysate using a 300 Dalton nanofiltration membrane ②, and collecting the concentrate.

[0027] Preferably, the nanofiltration membrane ① has a pore size of 500 Daltons.

[0028] As a preferred option, the concentrate obtained from dialysis using nanofiltration membrane ① is recycled and reused in the preparation of the next batch of oxacillin sodium.

[0029] As a preferred option, the dialysate obtained from dialysis using nanofiltration membrane ② is discharged as waste.

[0030] Preferably, in step (2), the enzyme obtained from filtration is recycled and reused in the preparation of the next batch of oxacillin sodium.

[0031] Anhydrous oxacillin sodium has a molecular weight of 423.41 Daltons. After enzyme recovery by filtration, dialysis is performed using a 500 Dalton nanofiltration membrane. Since the product's molecular weight is smaller than the nanofiltration membrane's pore size, the product enters the dialysate during dialysis. Large molecules larger than 500 Daltons (polymers generated during the reaction, enzyme-related proteins, DNA, RNA, total sugars, etc.) are retained in the concentrate, which is used in subsequent processes. After dialysis, the dialysate is further dialyzed using a 300 Dalton filter membrane. 6-APA (molecular weight 216.25 Daltons), methyl ester of the side chain acid (molecular weight 217.22 Daltons), byproduct side chain acid (molecular weight 203.19 Daltons), and other byproducts with molecular weights smaller than 300 Daltons are discharged with the dialysate during dialysis, and the concentrate is used in subsequent processes.

[0032] As a preferred embodiment, the filtration and dialysis specifically include the following steps:

[0033] The oxacillin sodium solution obtained in step (1) is filtered to separate the enzyme and the supernatant; the enzyme is washed with purified water, the washing liquid and the supernatant are combined, and dialyzed using nanofiltration membrane ① to obtain dialysate 1 and concentrate 1; the concentrate 1 is diluted with purified water and dialyzed again, and combined with dialysate 1 to obtain dialysate 2 and concentrate 2; the dialysate 2 is dialyzed using nanofiltration membrane 2, and concentrate 3 is collected.

[0034] Preferably, in step (2), the crystallization solvent is acetone.

[0035] Preferably, in step (2), the crystallization step specifically includes: adjusting the pH of the concentrated solution obtained from dialysis to 6-8, decolorizing with activated carbon, crystallizing, filtering, and drying to obtain sodium oxacillin.

[0036] Preferably, the pH is adjusted to 6.7-7.2 during the crystallization step.

[0037] Preferably, in the crystallization step, the pH adjuster is an aqueous solution of sodium hydroxide, more preferably a 10% aqueous solution of sodium hydroxide.

[0038] As a preferred option, the crystallization conditions include: maintaining the temperature at -5~5℃ for 3~6 hours, and more preferably maintaining the temperature at 0℃ for 4 hours.

[0039] Preferably, the method includes the following steps:

[0040] S1: Mix 6-APA, methyl 5-methyl-3-phenylisoxazole-4-carboxylate and water, adjust the pH to 6-8, add immobilized penicillin G acylase to react, and obtain oxacillin sodium solution.

[0041] S2: Filter the oxacillin sodium solution obtained in S1 to separate the enzyme and the supernatant; wash the enzyme with purified water, combine the washing solution and the supernatant, and dialyze using a nanofiltration membrane ① to obtain dialysate 1 and concentrate 1; dilute the concentrate 1 and dialyze it again, and combine it with dialysate 1 to obtain dialysate 2 and concentrate 2.

[0042] S3: Dialysate 2 obtained from S2 is dialyzed using nanofiltration membrane 2, and concentrate 3 is collected;

[0043] S4: Adjust the pH of the concentrated solution 3 obtained from S3 to 6-8, and then decolorize it with activated carbon, crystallize it, filter it, and dry it to obtain oxacillin sodium.

[0044] The second objective of this invention is to provide an application of immobilized penicillin G acylase in the synthesis of oxacillin sodium.

[0045] The beneficial effects of this invention are as follows:

[0046] 1. This invention uses 6-APA and methyl 5-methyl-3-phenylisoxazole-4-carboxylic acid ester as raw materials to synthesize oxacillin sodium using enzymatic and nanofiltration dialysis processes. It has the advantages of inexpensive materials, fewer process steps, and good atom economy.

[0047] 2. Currently, there is no existing technology for preparing oxacillin sodium using immobilized penicillin G acylase. This invention uses an enzymatic method to replace the acyl chloride method for preparing oxacillin sodium, shortening the reaction by two steps compared to the three steps of the chemical method: hydrolysis of side-chain methyl esters, preparation of acyl chloride, and condensation. Simultaneously, it effectively avoids the use of toxic and harmful substances such as thionyl chloride, oxalyl chloride, and N,N-dimethylformamide (DMF) in the preparation of 5-methyl-3-phenyl-4-isozolecarboxyl chloride, reducing production costs and environmental pressure, resulting in higher economic value for industrial production.

[0048] 3. The inventors' previous research found that the oxacillin sodium prepared using conventional methods with immobilized penicillin G acylase resulted in poor clarity of the oxacillin sodium solid. This invention combines enzymatic methods with nanofiltration dialysis. First, the oxacillin sodium reaction solution is prepared enzymatically, and then the reaction solution is dialyzed using a nanofiltration membrane to remove residual proteins, DNA, RNA, total sugars, and other substances from the reaction system, thereby improving product quality. Attached Figure Description

[0049] Figure 1 shows the HPLC chromatogram of oxacillin sodium prepared in Example 1;

[0050] Figure 2 shows the HPLC chromatogram of oxacillin sodium prepared in Example 2;

[0051] Figure 3 shows the HPLC chromatogram of oxacillin sodium prepared in Example 4;

[0052] Figure 4 shows the HPLC chromatogram of oxacillin sodium prepared in Example 5;

[0053] Figure 5 shows the HPLC chromatogram of oxacillin sodium prepared in Example 6;

[0054] Figure 6 shows the oxacillin sodium prepared in Example 6. 1 H-NMR spectrum;

[0055] Figure 7 shows the oxacillin sodium prepared in Example 6. 13 C-NMR spectrum;

[0056] Figure 8 is a synthetic route diagram of oxacillin sodium according to the present invention. Detailed Implementation

[0057] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0058] In this embodiment of the invention, the chromatographic conditions for HPLC detection are shown in Table 1.

[0059] Table 1. Chromatographic conditions

[0060]

[0061] In this embodiment of the invention, when using nanofiltration membrane dialysis, due to equipment design issues, the liquid level of the concentrate is too low each time the volume is concentrated to less than 1.0L, resulting in a large number of bubbles and severe pressure fluctuations during the concentration process, which causes significant damage to the equipment. Therefore, the concentration volume is controlled at 1.0~1.2L each time.

[0062] Example 1. Enzyme catalysis investigation at 25℃

[0063] (1) Preparation of reaction solution

[0064] Weigh 40 g of 6-APA (0.185 mol, 1.0 eq), 5-methyl-3-phenylisoxazole-4-carboxylic acid methyl ester (56.2 g, 0.259 mol, 1.4 eq), and 120 ml of purified water into a 500 ml three-necked flask. Stir and mix well. Adjust the pH to 6.7-7.2 with saturated sodium carbonate. Add 52 g of immobilized penicillin G acylase and incubate at 25 °C for 3 hours.

[0065] (2) Product crystallization

[0066] After the reaction was complete, the enzyme and filtrate were separated by filtration. 4.0 g of activated carbon was added to the filtrate for decolorization. Acetone (960 ml) was then added dropwise to the decolorized filtrate. After the addition was complete, the solution was incubated at 0°C for 4 hours to allow crystal growth. The solution was then filtered and dried to obtain 75.1 g of oxacillin sodium. The HPLC purity of the product was 99.213%, and the molar yield was 91.98%. The HPLC results are shown in Figure 1 and Table 2.

[0067] Table 2. Integration results of Figure 1

[0068]

[0069] Example 2. Enzyme catalysis investigation at 15℃

[0070] (1) Preparation of reaction solution

[0071] Weigh 40 g of 6-APA (0.185 mol, 1.0 eq), 56.2 g of methyl 5-methyl-3-phenylisoxazole-4-carboxylic acid ester (0.259 mol, 1.4 eq), and 120 ml of purified water into a 500 ml three-necked flask. Stir and mix well. Adjust the pH to 6.7-7.2 with saturated sodium carbonate. Add 52 g of immobilized penicillin G acylase and incubate at 15 °C for 3 hours.

[0072] (2) Product crystallization

[0073] After the reaction was complete, the enzyme and filtrate were separated by filtration. 4.0 g of activated carbon was added to the filtrate for decolorization. Acetone (960 ml) was then added dropwise to the decolorized filtrate. After the addition was complete, the solution was incubated at 0°C for 4 hours to allow crystal growth. The solution was then filtered and dried to obtain 76.1 g of oxacillin sodium. The HPLC purity of the product was 99.297%, and the molar yield was 93.20%. The HPLC results are shown in Figure 2 and Table 3.

[0074] Table 3. Integral Results of Figure 2

[0075]

[0076] Example 3. Enzyme catalysis investigation at 5℃

[0077] (1) Preparation of reaction solution

[0078] Weigh 40 g of 6-APA (0.185 mol, 1.0 eq), 56.2 g of methyl 5-methyl-3-phenylisoxazole-4-carboxylic acid ester (0.259 mol, 1.4 eq), and 120 ml of purified water into a 500 ml three-necked flask. Stir and mix well. Adjust the pH to 6.7-7.2 with saturated sodium carbonate. Add 52 g of immobilized penicillin G acylase and incubate at 5 °C for 3 hours.

[0079] (2) Product crystallization

[0080] After the reaction was completed, the enzyme and the supernatant were separated by filtration. The filtration was very difficult, and it was suspected that a small amount of product precipitated at low temperature and adhered to the surface of the immobilized enzyme, which made filtration difficult. Therefore, we abandoned further processing.

[0081] The experimental results of Examples 1-3 are analyzed and discussed:

[0082] 1. Comparing Examples 1, 2, and 3, it can be seen that the yield at a reaction temperature of 15°C is 93.9%, which is better than the yield of 92.1% at a reaction temperature of 25°C. Reducing the temperature to 5°C has a significant impact on the post-processing.

[0083] 2. The solids obtained in Examples 1 and 2 had poor clarity. This is because during enzyme use, substances such as proteins, DNA, RNA, and total sugars were introduced into the reaction system and crystallized onto the solid. The post-processing steps of the enzyme catalysis need to be optimized.

[0084] Example 4. Post-processing technology investigation

[0085] (1) Reaction: Weigh 400g of 6-APA (1.85mol, 1.0eq), 5-methyl-3-phenylisoxazole-4-carboxylic acid methyl ester (562g, 2.59mol, 1.4eq), and 1.2L of purified water into a 3L three-necked flask, stir and mix well, adjust the pH value to 6.7~7.2 with saturated sodium carbonate, add 520g of immobilized penicillin G acylase, and keep the reaction at 15℃ for 3 hours.

[0086] (2) Dialysis: After the reaction is complete, filter to separate the enzyme and the supernatant. Wash the enzyme with 2.0L of purified water. Combine the washing solution with the supernatant and dialyze using a 500 Dalton nanofiltration membrane ①. Collect the dialysate. Dilute the concentrate with 2.0L of purified water and dialyze again. Combine the concentrate with the first dialysate to obtain dialysate ② and concentrate ①.

[0087] (3) Concentration: Dialyze the dialysate ② using a 300 Dalton nanofiltration membrane ②, collect the concentrated solution ② (volume 1.0~1.2L), and discharge the dialysate as waste.

[0088] (4) Crystallization: The concentrate ② was brought to a final volume of 1.2 L, and 10% sodium hydroxide aqueous solution was added dropwise to adjust the pH to 6.7-7.2. After pH adjustment, 40.0 g of activated carbon was added for decolorization. 9.6 L of acetone was added dropwise to the decolorized filtrate. After the addition was complete, the solution was kept at 0 °C for 4 hours to allow crystal growth. The product was filtered and dried to obtain 728.1 g of oxacillin sodium. The HPLC purity of the product was 99.755%, and the molar yield was 89.17%. The HPLC detection results are shown in Figure 3 and Table 4. The synthetic route of oxacillin sodium is shown in Figure 8.

[0089] Table 4. Integration results from Figure 3

[0090]

[0091] Example 5. Investigation of enzyme application

[0092] (1) Reaction: Weigh 6-APA (400g, 1.85mol, 1.0eq), methyl 5-methyl-3-phenylisoxazole-4-carboxylic acid (562g, 2.59mol, 1.4eq), and 1.2L of purified water into a 3L three-necked flask, stir and mix well, adjust the pH value to 6.7~7.2 with saturated sodium carbonate, and then add the immobilized penicillin G acylase recovered in Example 4 (equivalent to 520g), and keep the reaction at 15℃ for 3 hours.

[0093] (2) Dialysis: After the reaction is complete, filter to separate the enzyme and the supernatant. Wash the enzyme with 2.0L of purified water. Combine the washing solution with the supernatant and dialyze using a 500 Dalton nanofiltration membrane ①. Collect the dialysate. Dilute the concentrate with 2.0L of purified water and dialyze again. Combine the concentrate with the first dialysate to obtain dialysate ② and concentrate ①.

[0094] (3) Concentration: Dialyze the dialysate ② using a 300 Dalton nanofiltration membrane ②, collect the concentrated solution ② (volume 1.0~1.2L), and discharge the dialysate as waste.

[0095] (4) Crystallization: The concentrate ② was brought to a final volume of 1.2L, and 10% sodium hydroxide aqueous solution was added dropwise to adjust the pH to 6.7~7.2. After pH adjustment, 40.0g of activated carbon was added for decolorization. 9.6L of acetone was added dropwise to the decolorized filtrate. After the addition was complete, the solution was kept at 0℃ for crystal growth for 4 hours. The solution was filtered and dried to obtain 733.2g of oxacillin sodium. The HPLC purity of the product was 99.777%, and the molar yield was 89.80%. The HPLC detection results are shown in Figure 4 and Table 5.

[0096] Table 5. Integral Results Table of Figure 4

[0097]

[0098] Example 6. Investigation on the reuse of enzymes and concentrates

[0099] (1) Reaction: Weigh 6-APA (400g, 1.85mol, 1.0eq) and methyl 5-methyl-3-phenylisoxazole-4-carboxylic acid (562g, 2.59mol, 1.4eq), add 1.2L of concentrated solution ① from Example 5 to a 3L three-necked flask (if the volume is less than 1.2L, add purified water to the total volume of 1.2L), stir and mix well, adjust the pH value to 6.7~7.2 with saturated sodium carbonate, then add the immobilized penicillin G acylase recovered in Example 5 (equivalent to 520g), and keep the reaction at 15℃ for 3 hours.

[0100] (2) Dialysis: After the reaction is complete, filter to separate the enzyme and the supernatant. Wash the enzyme with 2.0L of purified water. Combine the washing solution with the supernatant and dialyze using a 500 Dalton nanofiltration membrane ①. Collect the dialysate. Dilute the concentrate with 2.0L of purified water and dialyze again. Combine the concentrate with the first dialysate to obtain dialysate ② and concentrate ①.

[0101] (3) Concentration: Dialyze the dialysate ② using a 300 Dalton nanofiltration membrane ②, collect the concentrated solution ② (volume 1.0~1.2L), and discharge the dialysate as waste.

[0102] (3) Crystallization: The concentrate ② was brought to a final volume of 1.2L, and 10% sodium hydroxide aqueous solution was added dropwise to adjust the pH to 6.7~7.2. After pH adjustment, 40.0g of activated carbon was added for decolorization. 9.6L of acetone was added dropwise to the decolorized filtrate. After the addition was complete, the solution was kept at 0℃ for crystal growth for 4 hours. After filtration and drying, 767.2g of oxacillin sodium was obtained. The HPLC purity of the product was 99.756%, and the molar yield was 93.96%. The HPLC detection results are shown in Figure 5 and Table 6.

[0103] Table 6. Integral Results of Figure 5

[0104]

[0105] Example 7

[0106] The experimental data of Examples 1, 2, and 4-6 are compared and summarized in Table 7 below.

[0107] Table 7. Summary of Experimental Data Comparison for Examples 1, 2, and 4-6

[0108]

[0109] Samples from Examples 4, 5, and 6 were tested for RNA, DNA, protein, and total sugar residues. The test data are summarized in Table 8.

[0110] Table 8. Summary of RNA, DNA, protein, and total sugar residue detection data for samples from Examples 4, 5, and 6

[0111]

[0112] The experimental results of Examples 1-6 are analyzed and discussed:

[0113] 1. Compared with Examples 2 and 4, the addition of dialysis resulted in a small amount of product remaining in the concentrate ① that was not completely dialyzed, reducing the yield from 93.9% to 89.8%.

[0114] 2. Compared with Examples 4 and 5, the immobilized penicillin G acylase still exhibits high catalytic activity.

[0115] 3. Compared with Examples 2, 5, and 6, the yield was restored to the level of Example 2 by adding the concentrate reuse.

[0116] 4. Compared with Examples 1-6, the product quality was further improved by using nanofiltration membrane dialysis process.

[0117] In summary, the production scheme of the present invention (especially Example 6) has the characteristics of high yield, low cost, short route, simple process, less waste, and high capacity, and has good commercial value.

Claims

1. A method for preparing oxacillin sodium using an enzymatic method, characterized in that, The steps include: (1) using 6-APA and 5-methyl-3-phenylisoxazole-4-carboxylic acid methyl ester as raw materials, adding immobilized penicillin G acylase to react and obtain oxacillin sodium solution; (2) the oxacillin sodium solution obtained in step (1) is filtered, dialyzed, concentrated and crystallized to obtain oxacillin sodium.

2. The method according to claim 1, characterized in that, In step (1), the molar ratio of 6-APA to methyl 5-methyl-3-phenylisoxazole-4-carboxylate is 1:1~2; the mass ratio of 6-APA to immobilized penicillin G acylase is 1:0.5~2.

3. The method according to claim 1, characterized in that, In step (1), the pH is controlled at 6-8 before immobilized penicillin G acylase is added for reaction; the pH adjuster is sodium carbonate.

4. The method according to claim 1, characterized in that, In step (1), the reaction temperature is 5~25℃ and the reaction time is 2~5 hours.

5. The method according to claim 1, characterized in that, In step (1), the reaction solvent is water.

6. The method according to claim 1, characterized in that, In step (2), the dialysis includes: dialysis using a 500-800 Dalton nanofiltration membrane ①, dialysis of the obtained dialysate using a 300 Dalton nanofiltration membrane ②, and collection of the concentrate.

7. The method according to claim 6, characterized in that, The concentrated solution obtained from nanofiltration membrane ① is recovered and reused in the preparation of the next batch of oxacillin sodium.

8. The method according to claim 1, characterized in that, In step (2), the enzyme obtained from filtration is recycled and reused in the preparation of the next batch of oxacillin sodium.

9. The method according to claim 1, characterized in that, The method includes the following steps: S1: Mix 6-APA, methyl 5-methyl-3-phenylisoxazole-4-carboxylate, and water, adjust the pH to 6-8, add immobilized penicillin G acylase to react, and obtain oxacillin sodium solution; S2: Filter the oxacillin sodium solution obtained in S1 to separate the enzyme and the supernatant; wash the enzyme with purified water, combine the washing liquid and the supernatant, and dialyze using a nanofiltration membrane ① to obtain dialysate 1 and concentrate 1; dilute the concentrate 1 and dialyze it again, and combine it with dialysate 1 to obtain dialysate 2 and concentrate 2; S3: Dialyze the dialysate 2 obtained in S2 using a nanofiltration membrane 2, and collect concentrate 3; S4: Adjust the pH of the concentrate 3 obtained in S3 to 6-8, decolorize with activated carbon, crystallize, filter, and dry to obtain oxacillin sodium.

10. Application of immobilized penicillin G acylase in the synthesis of oxacillin sodium.

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