Method for separating and recycling amoxicillin product synthesized by one-step method based on catalysis of kluyveromyces citrate beta subunit G385 mutant penicillin acylase

Abstract

The invention belongs to the technical field of separation of products for synthesizing antibiotics by an enzymic method, and particularly relates to a method for separating and recycling an amoxicillin product synthesized by a one-step method based on catalysis of kluyveromyces citricacidophilus beta subunit G385 mutant penicillin acylase. An immobilized penicillin acylase mutant is used for catalyzing penicillin potassium to synthesize amoxicillin in one step, a set of method for separating amoxicillin and phenylacetic acid is developed for an obtained reaction mixture, and the technical scheme mainly comprises the following steps: firstly, separating the immobilized penicillin acylase mutant from a reaction solution through filtration; then, amoxicillin is separated out through crystallization; and then crystallizing, separating and recovering phenylacetic acid through toluene extraction, back extraction and the like. According to the separation method, the amoxicillin can be rapidly and efficiently separated, the yield of the amoxicillin is relatively high, and the average crystallization rate reaches 93.22%; meanwhile, the separation and recovery effects of phenylacetic acid are good; and the extraction agent toluene can be recycled.

Description

[0001] This application is a divisional application of the Chinese invention patent with application number 202411522210.7, application date 2024.10.29, and invention name “A method for separating amoxicillin and phenylacetic acid from the reaction solution of amoxicillin prepared by enzyme-catalyzed one-step method”. Technical Field

[0002] The present invention belongs to the technical field of product separation of enzymatically synthesized antibiotics, and in particular relates to a method for separating and recovering amoxicillin products synthesized in a one-step process catalyzed by Kluyveromyces citriodora beta subunit G385 mutant penicillin acylase. Background Art

[0003] Amoxicillin, also known as amoxycillin, is a major second-generation penicillin. Amoxicillin inhibits bacterial cell wall synthesis and is a broad-spectrum semisynthetic antibiotic. Due to its high efficacy, broad-spectrum antibacterial activity, and minimal toxic side effects, the World Health Organization (WHO) recommends it as the preferred oral β-lactam antibiotic.

[0004] The preparation methods of amoxicillin include chemical synthesis and enzyme-catalyzed synthesis. The chemical synthesis method has the disadvantages of long reaction steps, high production of three wastes, and the use of a large amount of chemical solvents in the process. In recent years, with the popularization and application of the concept of green synthesis in the pharmaceutical preparation industry and the development of the level of amoxicillin enzyme synthesis technology, the enzyme-catalyzed synthesis of amoxicillin has become the main method for preparing amoxicillin. Its main process route is: synthetic penicillin acylase catalyzes the reaction of 6-APA and D-p-hydroxyphenylglycine (or D-p-hydroxyphenylglycine methyl ester) to synthesize amoxicillin (such as application number 201711221286.6), and then the amoxicillin product is obtained after separation, purification, crystallization, and drying.

[0005] However, there are still some problems in the practice of enzymatic synthesis of amoxicillin. For example, patent CN102660621A provides a process for synthesizing amoxicillin from 6-APA and D-phenylglycine methyl ester hydrochloride, but this method has a low final product yield and poor fluidity. Amoxicillin can be prepared from penicillin and its salts in a one-step process using a penicillin acylase mutant. This technology avoids the shortcomings of existing amoxicillin production technologies that require multiple reactions and also avoids the separation process of the intermediate product 6-APA. The single-step reaction can achieve the original two-step separation and purification technology, which has the advantages of simplified production process, high production efficiency, and greatly reduced production costs. However, in addition to the large amount of amoxicillin (AMOX) in the original multi-step reaction, the reaction solution also contains an equimolar amount of phenylacetic acid (PAA). In order to efficiently and efficiently separate and purify the amoxicillin product that meets the quality requirements of the raw material drug, it is also necessary to recover the phenylacetic acid and realize its recycling. Therefore, the separation technology of the original multi-step production of amoxicillin cannot meet the separation and purification requirements of the one-step production. Summary of the Invention

[0006] To address the challenges of existing technologies, the present invention develops a highly efficient and rapid production technology for separating and purifying amoxicillin and recovering phenylacetic acid. This technology enables rapid separation and purification of amoxicillin and large-scale recovery of phenylacetic acid. The amoxicillin produced meets pharmaceutical production requirements and meets API standards.

[0007] The present invention is achieved by providing a method for separating and recovering amoxicillin products from a one-step synthesis using a Kluyveromycin β-subunit G385 mutant penicillin acylase catalyzed by Kluyveromycin β-subunit G385. The method is characterized by using an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from penicillin potassium to obtain a reaction suspension, and separating and treating the reaction suspension, comprising the following steps:

[0008] (1) adding deionized water to the reaction suspension, filtering, and obtaining an amoxicillin filtrate and a retentate of the immobilized penicillin acylase mutant;

[0009] (2) washing the retained immobilized penicillin acylase mutant with deionized water, and combining the washing solution with the amoxicillin filtrate from step (1);

[0010] (3) adjusting the pH of the mixed solution obtained in step (2) to 2 with hydrochloric acid to obtain a mixed solution to be separated;

[0011] (4) adding NaOH solution dropwise to the mixed solution to be separated until the pH reaches 3.5-5.5, and standing at 4°C for crystallization;

[0012] (5) After the crystallization is completed, the amoxicillin crystals and the liquid phase containing phenylacetic acid are separated by filtration.

[0013] Furthermore, step (5) further includes the following steps:

[0014] (6) adjusting the pH of the liquid phase containing phenylacetic acid to between 2.0 and 2.5, and extracting with toluene to obtain an organic phase containing phenylacetic acid;

[0015] (7) back-extracting the organic phase containing phenylacetic acid with a NaOH solution, converting phenylacetic acid into sodium phenylacetate and entering the aqueous phase;

[0016] (8) separating the aqueous phase and the organic phase, wherein the toluene in the organic phase is recycled for extraction in step (6);

[0017] (9) adding hydrochloric acid to the aqueous phase containing sodium phenylacetate to adjust the pH to 2-2.5, converting sodium phenylacetate into phenylacetic acid and crystallizing at 4°C;

[0018] (10) After the crystallization is completed, the phenylacetic acid crystals are recovered by filtration and separation.

[0019] Furthermore, the concentration of hydrochloric acid in step (3) is 15% by volume.

[0020] Furthermore, in step (4), the pH of amoxicillin crystallization is 5, and the crystallization time is 9 hours.

[0021] Furthermore, in step (5), the amoxicillin crystals are obtained by filtration and separation, and the amoxicillin crystals are dried in a vacuum drying oven at 50° C. for 2 hours.

[0022] Furthermore, in step (6), 15% by volume hydrochloric acid is used to adjust the pH; extraction is performed with toluene for more than 2 times, and the organic phases are combined.

[0023] The method for catalyzing the one-step synthesis of amoxicillin from penicillin potassium using an immobilized penicillin acylase mutant comprises: using only one immobilized penicillin acylase mutant as the sole enzyme in a reaction system, using penicillin or its salt and D-p-hydroxyphenylglycine methyl ester as substrates, and conducting the reaction in a buffer system with a pH of 4 to 8; the amino acid sequence of the penicillin acylase mutant, compared with the amino acid sequence shown in SEQ ID NO.1, comprises at least one of the following mutations: F146αK, F24βR, F71βY, N241βK, G385βY, or G385βR.

[0024] The specific steps include:

[0025] S1: Add pH 4-8 buffer to the reaction flask as the reaction buffer system;

[0026] S2: adding penicillin potassium salt and D-p-hydroxyphenylglycine methyl ester to the reaction buffer system, wherein the molar ratio of penicillin potassium salt to D-p-hydroxyphenylglycine methyl ester is 1:1 to 1:2, and stirring thoroughly;

[0027] S3: Add the immobilized penicillin acylase mutant to the reaction buffer system and control the reaction temperature to 12-30° C., preferably 12-28° C., to carry out the reaction.

[0028] Furthermore, the buffer in step S1 includes any one of citric acid buffer, acetic acid buffer, PBS buffer, sodium dihydrogen phosphate-citric acid buffer, sodium barbital-hydrochloric acid buffer, and pure water.

[0029] Furthermore, in step S2, the concentration of penicillin potassium salt is 50-200 mmol / L, and the concentration of D-p-hydroxyphenylglycine methyl ester is 50-400 mmol / L.

[0030] Furthermore, in step S3, the enzyme amount is 3000 to 30000 U / L, and the reaction time is 1 to 6 hours.

[0031] In summary, the advantages and positive effects of the present invention are as follows: This application utilizes an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from penicillin potassium. A method for separating amoxicillin and phenylacetic acid from the resulting reaction mixture has been developed. The technical solution primarily comprises: first, separating the immobilized penicillin acylase from the reaction solution by filtration, then isolating the amoxicillin by crystallization; and then, separating and recovering the phenylacetic acid through toluene extraction and back extraction. This separation method enables rapid and efficient separation of amoxicillin, with a high amoxicillin yield, reaching an average crystallization rate of 93.22%. Furthermore, the separation and recovery of phenylacetic acid is effective, and the extractant, toluene, can be recycled. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The present invention is a schematic flow chart of a method for separating amoxicillin and phenylacetic acid from a reaction solution of amoxicillin prepared by an enzyme-catalyzed one-step method.

[0033] Figure 2 is the wild-type penicillin acylase amino acid sequence;

[0034] Figure 3 is a schematic diagram of the construction of the recombinant plasmid pET28a-kcPA;

[0035] Figure 4 This is the agarose electrophoresis detection diagram of the recombinant plasmid PCR;

[0036] Figure 5 This is the SDS-PAGE electrophoresis of the protein expressed in E. coli BL21 (DE3) / pET28a-kcPA bacteria;

[0037] Figure 6 This is the HPLC chromatogram of the one-step synthesis of amoxicillin by KcPA-catalyzed penicillin potassium salt in Example 3;

[0038] Figure 7 This is a liquid chromatogram of amoxicillin crystals;

[0039] Figure 8 This is a liquid chromatogram of phenylacetic acid crystals. DETAILED DESCRIPTION

[0040] In order to better understand the present invention and not to limit the scope of the present invention, all numbers used in this application to express amounts, percentages, and other numerical values should be understood to be modified by the word "approximately" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

[0041] In the following embodiments of the present invention, when the temperature is not particularly limited, the conditions are all room temperature. Room temperature refers to the natural room temperature conditions in all seasons, without additional cooling or heating treatment. Generally, the room temperature is controlled at 10-30°C, preferably 15-25°C. The abbreviations have the following meanings: "min" means minute, "s" means second, "U" means enzyme activity unit, "mM" means millimole per liter, "M" means mole per liter, "rpm" means revolutions per minute, "mol" means mole, "μg" means microgram, "mg" means milligram, "g" means gram, "μL" means microliter, "mL" means milliliter, "bp" means base pair, LB medium means Luria-Bertani medium, and Kan50 means medium containing 50μg / mL karatomycin.

[0042] In the examples, the experimental methods without specific conditions are generally performed under conventional conditions, such as those described in "Molecular Cloning Laboratory Manual" (Chinese version) (J. Sambrook and MR Green, translated by He Fu. Fourth edition, Beijing: Science Press, 2017) and New England Biolabs (NEB) kits.

[0043] The present invention discloses a method for separating and recovering amoxicillin products synthesized in a one-step process based on the catalysis of Kluyveromyces citriodora β-subunit G385 mutant penicillin acylase. The main contents include: first, using an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from penicillin potassium to obtain a reaction suspension, and then separating and treating the reaction suspension. The main steps include: (1) adding an equal volume of deionized water to the reaction suspension at room temperature, filtering under normal pressure, and obtaining a filtrate containing amoxicillin and a retentate containing immobilized penicillin acylase. (2) washing the entrapped immobilized penicillin acylase with deionized water, combining the washing liquid and the amoxicillin filtrate in step (1). (3) adjusting the pH of the mixed solution obtained in step (2) to 2 with hydrochloric acid, filtering, and obtaining an amoxicillin reaction solution. (4) The pH of the amoxicillin reaction solution is adjusted to 5.0 with 0.25 mol / L NaOH solution for crystallization, and crystallization is carried out at 4°C for 9 hours. After the crystallization is completed, solid-liquid separation is performed to obtain amoxicillin crystals and a liquid phase containing phenylacetic acid. (5) The pH of the liquid phase containing phenylacetic acid is adjusted to between 2.0-2.5, and extracted with toluene to allow phenylacetic acid to enter the organic phase from the aqueous phase. (5) 0.25 mol / L NaOH solution is added to the above organic phase to convert phenylacetic acid into sodium phenylacetate and enter the aqueous phase. (6) Under heating conditions, 15% hydrochloric acid is added to the above aqueous phase containing sodium phenylacetate to adjust the pH to 2-2.5, so that sodium phenylacetate is converted into phenylacetic acid and crystallized at 4°C. The flow chart is as follows Figure 1 shown.

[0044] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0045] Example 1

[0046] Construction, prokaryotic expression and functional characterization of a Kluyvera citrophila penicillin acylase mutant

[0047] 1. Construction of wild-type PA expression vector pET28a-kcPA

[0048] The wild-type penicillin acylase used in this example is derived from Kluyvera citrophila K. citrophila ATCC21285, and its amino acid sequence is shown in SEQ ID NO. 1. This amino acid sequence consists of four parts, from the N-terminus to the C-terminus of the protein: a signal peptide at positions 1 to 26, an α subunit consisting of 209 amino acids at positions 27 to 235, an intermediate linker peptide consisting of 54 amino acids at positions 236 to 289, and a β subunit consisting of 557 amino acids at positions 290 to 846 (see also). Figure 2, where the single underlined part is the α subunit, the wavy part is the connecting peptide, and the double underlined part is the β subunit). The nucleotide sequence is shown in SEQ ID NO.2.

[0049] The schematic diagram of the construction of the recombinant plasmid pET28a-kcPA is as follows Figure 3 As shown. Using the genome of K. citrophila ATCC21285 as a template, primers were designed according to the PA nucleotide sequence (SEQ ID NO.2). The forward primer was: 5'-CG G / AATTC ATGAAAAACCGCAATCGCAT-3', SEQ ID NO.3; reverse primer is 5'-CC A / AGCTT TTAGCGCTGCACCTGCAGC-3', SEQ ID NO. 4. EcoRI and HindIII restriction enzyme sites were introduced respectively (the underlined bases are restriction enzyme recognition sites), and the wild-type PA target fragment was amplified by PCR.

[0050] PCR reaction system:

[0051]

[0052] The PCR temperature program was designed as follows:

[0053]

[0054] Select EcoRI and HindIII restriction endonucleases to double-digest the plasmid pET28a blank vector and the target fragment. Double enzyme digestion system:

[0055]

[0056] The double enzyme digestion reaction was carried out at 37°C for 1 hour and then inactivated at 80°C for 20 minutes. The double enzyme digestion products were purified and recovered, and their concentrations were estimated based on the gel electrophoresis pattern. The concentration of the plasmid pET28a was approximately 50 ng / μL, and the concentration of the target gene kcPA was approximately 140 ng / μL.

[0057] The double-enzyme digestion products were ligated overnight using T4 DNA ligase in a 16°C metal bath to obtain the recombinant plasmid pET28a-kcPA, which was then heat-transformed into competent cells E. coli DH5α.

[0058] Target fragment and linearized vector connection system:

[0059]

[0060] To verify whether the recombinant plasmid was successfully transferred, a single colony was picked from the LB plate containing Kan50 and transferred to the LB liquid medium containing Kan50. The next day, the plasmid was extracted using a plasmid extraction kit and PCR identification was performed. The target band of 2500 bp was obtained by agarose electrophoresis (such as Figure 4 The verified expression vector pET28a-kcPA was transformed into E. coli BL21 (DE3) to obtain the wild-type PA expression recombinant bacteria E. coli BL21 (DE3) / pET28a-kcPA.

[0061] 2. Obtaining mutant expression vectors

[0062] In this example, a total of 18 mutants were obtained by site-directed mutagenesis, as shown in the table below. Among them, "F146αK" indicates that the 146th amino acid on the α subunit is mutated from F to K. The interpretation of other mutation sites is similar.

[0063] Table 1 Mutants and corresponding mutation sites

[0064]

[0065]

[0066] First, primers corresponding to each mutation site were designed, and then the wild-type PA target fragment was used as the initial template. Site-Directed Mutagenesis Kit (Q5 SDM Kit) was used for site-directed mutagenesis. The primers for each mutation site are as follows (lowercase letters represent the bases at the mutation site):

[0067] F146αK, F: 5'-GGCGAACCGTaaaTCTGACAGCACCAG-3', SEQ ID NO.5;

[0068] R: 5'-ATGGTGCCGACAAAAATCATCGCCA-3', SEQ ID NO.6;

[0069] F24βR, F: 5'-TGGGCCGCAGcgcGGTTGGTATGCG-3', SEQ ID NO.7,

[0070] R: 5'-TTGACCATAATGGCCTTCGCATCCT-3', SEQ ID NO.8;

[0071] F71βY, F: 5'-CACCGCCGGTtatGGTGATGATG-3', SEQ ID NO.9,

[0072] R: 5'-GATCCCCCATGAAATGGTGCCGTTGT-3', SEQ ID NO.10;

[0073] N241βK, F: 5'-CGCCAACTGGaaaAACTCGCCGC-3', SEQ ID NO.11,

[0074] R: 5'-ATATAGCCCGACTGCGGGTTATACAC-3', SEQ ID NO. 12;

[0075] G385βY: F: 5'-CGGGCCAACCtatTCGCTGAACATCAGCGTG-3', SEQ ID NO.13,

[0076] R: 5'-TCCTGGGTGGTTTCATAGCCACTGG-3', SEQ ID NO.14;

[0077] G385βR, F: 5'-CGGGCCAACCcgcTCGCTGAACATC-3', SEQ ID NO.15,

[0078] R: 5'-TCCTGGGTGGTTTCATAGCCACTGG-3', SEQ ID NO.16;

[0079] The primers were synthesized by a nucleic acid synthesis company, dissolved in sterile water, and then operated according to the kit.

[0080] ①Use PCR to mutate the corresponding sites

[0081] PCR reaction system:

[0082]

[0083]

[0084] Cycle program temperature:

[0085]

[0086] For mutants with more than two mutation sites, the PCR product obtained from the previous mutation site was used as a template to perform site-directed mutagenesis of the corresponding sites one by one.

[0087] ② Kinase, Ligase & DpnI (KLD) (a special mixture of kinase, ligase and DpnI) reaction treatment reaction system is as follows:

[0088] volume Final concentration PCR products 1 μL 2X KLD Reaction Buffer 5μL 1X 10X KLD Enzyme Mix 1 μL 1X Nuclease-free water 3μL

[0089] React at room temperature for 5 minutes.

[0090] ③ Heat shock transformation

[0091] 5 μL of KLD reaction mixture was added to 50 μL of chemically competent E. coli BL21 (DE3) suspension, incubated on ice for 30 minutes, heat-shocked at 42°C for 30 seconds, incubated on ice for 5 minutes, and then 950 μL of SOC sterile liquid medium was added. The suspension was gently shaken at 37°C for 1 hour. 40-100 μL of bacterial suspension was spread on Kan50 LB plates and incubated at 37°C overnight. The single colonies that grew were the corresponding mutant expression strains, named E. coli BL21 (DE3) / pET28a-kcPA01 to 18, respectively.

[0092] ④ Mutant identification

[0093] The mutant expression strain was inoculated into 25 mL of LB liquid medium containing Kan50 and cultured overnight at 37°C. The plasmid was extracted using a plasmid extraction kit and sent to a third-party biotechnology company for sequencing to confirm that the corresponding product was the target product of the site-directed mutagenesis.

[0094] 3. Expression of wild-type and mutant KcPA

[0095] The constructed recombinant E. coli BL21 (DE3) / pET28a-kcPA and E. coli BL21 (DE3) / pET28a-kcPA01 ~ 18 were inoculated on LB agar plates with Kan50 and cultured in a 37 ° C incubator for 12-16 hours. Pick single colonies and inoculate them into 25 mL of LB liquid medium supplemented with Kan50, and culture them overnight at 37 ° C and 300 rpm on a shaker. Pipette 500 μL of bacterial solution and transfer it to 50 mL of LB liquid medium supplemented with Kan50, culture it at 37 ° C and 280 rpm on a shaker, and monitor the OD 600When the protein concentration reaches 0.6-0.8, IPTG solution is added to an induction concentration of 0.3 mM. Expression is induced in a shaker at 25°C and 220 rpm for 10 hours. The fermentation broth is then centrifuged to collect the cells. The collected cells are suspended in PBS buffer at pH 7.5, pre-chilled on ice for 10 minutes, and then centrifuged at 4°C and 12,000 rpm for 6 minutes to collect the cells. 50 mM PBS buffer at pH 7.5 is added to the cells in the centrifuge tube to resuspend them. The cells are then centrifuged at 4°C and 12,000 rpm for 6 minutes. The supernatant is discarded, and the final cells are collected and resuspended at a concentration of 0.01 g / mL. The cells are disrupted using an ultrasonic disruptor. Cell disruption conditions are: ice-water bath, 400 W power, 3 seconds per cycle, 5 seconds rest, for a total of 80 cycles. The mixture after crushing was centrifuged at 4°C and 12000 rpm for 15 min to obtain the supernatant, which was the crude enzyme solution. The crude enzyme solution was collected and the expressed protein was analyzed by SDS-PAGE.

[0096] Figure 5 The figure is an SDS-PAGE diagram of protein expressed in bacteria; wherein, lane M is a protein marker; Lane 1: E.coliBL21(DE3) / pET28a expression supernatant; Lane 2: E.coli BL21(DE3) / pET28a-kcPA uninduced supernatant; Lane 3: uninduced E.coli BL21(DE3) / pET28a-kcPA18 supernatant; Lane 4: IPTG-induced E.coli BL21(DE3) / pET28a-kcPA18 supernatant.

[0097] Example 2

[0098] 1. Determination of KcPA hydrolysis activity

[0099] The assay principle is as follows: Penicillin K (PGK) potassium salt is hydrolyzed by KcPA to produce 6-aminopenicillanic acid (6-APA) and phenylacetic acid. 6-APA reacts with p-dimethylaminobenzaldehyde (PDAB) under acidic conditions to form a yellow-green substance with a maximum absorption peak at 415 nm. Enzyme activity is defined as the amount of penicillin acylase required to produce 1 μmol of 6-APA per minute in 0.1 M PBS buffer at 28°C. KcPA activity (unit: U) is the amount of enzyme required to catalyze the conversion of 20 mg / mL PGK to 1 μmol of 6-APA per minute.

[0100] Weigh 0.5g of PGK and dissolve it in the aforementioned buffer solution, then dilute to 25mL. Pipette 2mL of PGK solution into a centrifuge tube and add 0.1mL of KcPA enzyme solution. Set up a control group without KcPA, keeping all other conditions unchanged.

[0101] The reaction system was shaken in a water bath at 28°C and 200 rpm for 10 minutes. After completion, the enzyme was inactivated in a 90°C water bath for 2 minutes. 200 μL of the reaction solution was added to 3 mL of 0.1 M sodium citrate buffer (pH 3.0), followed by 1 mL of a colorimetric solution (0.5% PDAB). The sample was allowed to stand at room temperature for 3 minutes, and absorbance was measured at 415 nm. The 6-APA concentration in the sample after the reaction was determined using a 6-APA standard curve, and the enzyme activity (i.e., hydrolytic activity) was calculated using the following formula.

[0102] Calculation formula: Penicillin acylase hydrolase activity per mL

[0103] Where C 6-APA : 6-APA concentration in the sample, μmol / L; V: reaction system volume, mL; V E : amount of penicillin acylase added, mL; t: reaction time, 10 min.

[0104] 2. Determination of KcPA synthesis activity

[0105] Amoxicillin is synthesized from 6-aminopenicillanic acid (6-APA) and p-hydroxyphenylglycine methyl ester (D-HPGM) in the presence of KcPA. High-performance liquid chromatography (HPLC) can be used to determine the amoxicillin content and calculate the PA synthesis activity. Enzyme activity is defined as the amount of penicillin acylase that catalyzes the production of 1 μmol of amoxicillin per minute under specific conditions. This activity is expressed as 1 unit (U).

[0106] Weigh 1g 6-APA and 1.25g D-HPGM and dissolve them in 50mL of 0.1M PBS buffer solution with pH 6.3, adjust the pH to 6.3, and then dilute to 100mL with the above buffer solution. Take 0.1mL KcPA and add it to the above solution. Start the reaction at 25℃ and 200rpm for 30min. Put it in a 90℃ water bath for 2min to inactivate the enzyme and end the reaction. Take 0.5mL of the reaction solution and filter it with a 0.22μm water filter membrane. Use phosphate buffer to dilute to 100mL for HPLC detection to obtain the amoxicillin content. Enzyme activity calculation formula: Penicillin acylase synthase activity per mL Where: V: reaction solution volume, mL; 200: dilution factor; C 样 : Amoxicillin molar concentration, μmol / L; V E : Volume of enzyme added, mL; t: reaction time, min.

[0107] HPLC detection conditions were: an Agilent ZORBAX SB-C18 4.6 x 250 mm column, column temperature 25°C. The injection volume was 10 μL. Mobile phase A (0.02 M NaH2PO4-Na2HPO4 buffer, pH 4.7) and mobile phase B (methanol) were initially set at 90% mobile phase A and 10% mobile phase B for 5 minutes. From 5 to 7 minutes, the mobile phase B concentration was increased from 10% to 50% and then maintained for 10 minutes. From 17 to 19 minutes, the mobile phase B concentration was decreased from 50% to 10%. Finally, the concentration was equilibrated with 90% mobile phase A and 10% mobile phase B for 5 minutes. The total flow rate was 1 mL / min.

[0108] Table 2 Comparison of activity between mutants and wild type

[0109]

[0110]

[0111] Note: The hydrolytic activity of the wild-type KcPA expressed by the recombinant strain was 15 U / mL (fermentation broth) and the synthetic activity was 80 U / mL. For ease of comparison, the activity of the wild-type KcPA was defined as 100 in Table 2, and the mutants were compared with this value.

[0112] As shown in the table above, for mutants with single mutations, the hydrolytic and synthetic activities of each mutant were significantly improved compared to the wild type, particularly the F146αK mutant in the α subunit and the G385βR mutant in the β subunit. The hydrolytic and synthetic activities of the single-point F146αK mutant were 5.8-fold and 15.3-fold higher than those of the wild type, respectively; the hydrolytic and synthetic activities of the G385βR mutant were 4.6-fold and approximately 11.2-fold higher than those of the wild type, respectively. The G385βY mutant exhibited higher hydrolytic activity compared to the G385βR mutant, but its synthetic activity was not significant. When mutations were combined at each site, the enzyme activity of the mutants increased compared to the single-point mutations, particularly the five-point mutant F146αK, F24βR, F71βY, N241βK, and G385βR, which exhibited both higher hydrolytic and synthetic activities.

[0113] Example 3

[0114] One-step synthesis of amoxicillin by PGK catalyzed by various mutants and wild-type penicillin acylase

[0115] PGK was added to a pH 7.0 PBS buffer to a concentration of 200 mM. Parahydroxyphenylglycine methyl ester (D-HPGM) was also added to a final concentration of 300 mM. The enzyme was added at a concentration of 30 U / mL (calculated based on synthase activity). The reaction was stirred at 28°C for 3 h. After completion of the reaction, HPLC analysis was performed to calculate the amoxicillin yield.

[0116] HPLC detection conditions were: Agilent ZORBAX SB-C18 4.6x250 mm column, column temperature 25°C. Injection volume 10 μL. Mobile phase A (0.02 M NaH2PO4-Na2HPO4 buffer, pH 4.7) and mobile phase B (methanol). Initially, the concentration was 90% mobile phase A and 10% mobile phase B for 5 minutes. From 5 to 7 minutes, the concentration of mobile phase B was increased from 10% to 50% and then maintained for 10 minutes. From 17 to 19 minutes, the concentration of mobile phase B was decreased from 50% to 10%. Finally, the concentration was equilibrated with 90% mobile phase A and 10% mobile phase B for 5 minutes. The total flow rate was 1 mL / min. The reaction equation is as follows:

[0117]

[0118] The HPLC detection spectrum of mutant KcPA18 is as follows Figure 6 As shown in the figure, D-HPG is D-parahydroxyphenylglycine, AMOX is amoxicillin, D-HPGM is D-parahydroxyphenylglycine methyl ester, PAA is phenylacetic acid, and PGK is penicillin potassium salt. As can be seen from the figure, the content of the intermediate product 6-APA is extremely low, almost non-existent.

[0119] Table 3 Yield of the mutants catalyzing the synthesis of amoxicillin

[0120]

[0121]

[0122] From the results in the above table, it can be seen that each mutant can catalyze the reaction of penicillin potassium and p-hydroxyphenylglycine methyl ester in a reaction system to synthesize amoxicillin in one step, and the product yield is significantly improved compared with the wild type.

[0123] Example 4 Preparation of immobilized penicillin acylase

[0124] Penicillin acylase enzyme solution was prepared according to Example 1. The enzyme solution was cross-linked with epoxy resin (ER) activated with glutaraldehyde at 15°C for 1.5 hours. The cross-linking reaction system was as follows: 1200 U of enzyme solution (calculated as synthase activity), 0.25% glutaraldehyde content, 5 g of ER, and 50 mL of potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer at pH 7.5. After the cross-linking reaction, the immobilized enzyme was collected by filtration using a mesh. The immobilized enzyme was then washed with 100 mL of potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer at pH 7.5. The immobilized enzyme activity was 180 U / g, and the recovery rate of immobilized enzyme activity reached 75%.

[0125] Example 5: One-step synthesis of amoxicillin using PGK catalyzed by KcPGA18

[0126] (1) Add 50 mL of pH 7 PBS buffer into the reaction flask as the reaction buffer system.

[0127] (2) Weigh a certain amount of penicillin potassium salt (PGK) and D-p-hydroxyphenylglycine methyl ester (D-HPGM) into the reaction buffer system, with a PGK to D-HPGM molar ratio of 1:1 to 1:2, a PGK concentration of 50 to 200 mmol / L, and a D-HPGM concentration of 50 to 400 mmol / L. The mixture is then stirred thoroughly to uniformly disperse the PGK and D-HPGM substrates in the reaction system. In this example, the final concentrations of PGK and D-HPGM are 200 and 300 mmol / L, respectively.

[0128] (3) The enzyme solution of the penicillin acylase mutant KcPGA18 was immobilized according to the method in Example 4. The immobilized penicillin acylase was accurately weighed and added to a reaction flask at an enzyme dosage of 20 U / mL (calculated based on synthase activity). The reaction temperature was controlled at 24°C and the reaction time was 4 h to obtain a milky white reaction suspension.

[0129] Example 6 Separation and Recovery of Amoxicillin and Phenylacetic Acid

[0130] 1. Isolation and crystallization of amoxicillin

[0131] (1) To 50 mL of the reaction suspension obtained in Example 5, an equal volume of deionized water was added.

[0132] (2) The immobilized penicillin acylase was separated by filtration, and then washed with 50 mL of deionized water. The filtrate and the washing solution were collected and combined to total 150 mL.

[0133] (3) 15% hydrochloric acid was added dropwise to the filtrate to dissolve the filtrate to a pH of about 2 to obtain a mixed solution to be separated.

[0134] (4) Add 0.25 mol / L NaOH solution dropwise to the mixed solution to be separated until the pH reaches 5, and place it in a refrigerator at 4°C for 9 h to allow crystallization.

[0135] (5) After crystallization is complete, the amoxicillin crystals and the liquid phase containing phenylacetic acid are separated by suction filtration. Finally, the amoxicillin crystals are placed in an oven and dried at 85°C for 30 minutes.

[0136] In order to verify whether the crystalline crystals are amoxicillin, in this example, 0.1 g of the crystalline crystals were dissolved in deionized water and diluted to 50 mL to obtain a 2 mg / mL solution, which was then subjected to high performance liquid chromatography under the same detection conditions as in Example 3. The results are shown in FIG. Figure 7As can be seen from the figure: except for the AMOX peak, there are no other obvious peaks, which basically confirms that the crystal is amoxicillin.

[0137] 2. Separation and crystallization of phenylacetic acid

[0138] (1) Take the above liquid phase containing phenylacetic acid (150 mL) and add 15% hydrochloric acid dropwise to adjust the pH to between 2.0 and 2.5.

[0139] (2) Add 40 mL of toluene as the extractant and extract at 25°C. Stir for 15 minutes and then allow the mixture to stand for separation, allowing phenylacetic acid to be extracted from the aqueous phase into the organic phase of toluene. Extract twice and combine the organic phases.

[0140] (3) Add 30 mL of 0.25 mol / L NaOH solution to the collected organic phase and mix thoroughly. Allow to stand (or centrifuge) to separate the layers. Phenylacetic acid is converted to sodium phenylacetate and extracted from toluene into the aqueous phase. The aqueous phase and the organic phase are separated, and the toluene in the organic phase is recycled for extraction in step (2).

[0141] (4) The aqueous phase (lower phase) from the previous step was collected and 15% hydrochloric acid was added dropwise at 60°C to adjust the pH to between 2.0 and 2.5. The mixture was then transferred to 4°C for crystallization for 12 hours to convert sodium phenylacetate into phenylacetic acid and crystallize.

[0142] (5) After crystallization is complete, the phenylacetic acid crystals are filtered and the residue is the phenylacetic acid crystals. Finally, the phenylacetic acid crystals are dried in a vacuum drying oven at 40°C for 30 min.

[0143] In order to verify whether the crystals are phenylacetic acid, 0.1 g of the crystals were dissolved in deionized water and the volume was adjusted to 50 mL to obtain a 2 mg / mL solution, which was then subjected to high performance liquid chromatography under the same conditions as in Example 3. The results are shown in FIG. Figure 8 ,It can be seen from the figure that: except for the PAA peak, there are no other obvious impurity peaks, which basically confirms that the main component of the crystal is phenylacetic acid.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for separating and recovering amoxicillin products by a one-step synthesis catalyzed by Kluyveromyces citriodora β-subunit G385 mutant penicillin acylase, characterized by: The method comprises the following steps: using an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from penicillin potassium to obtain a reaction suspension, and separating and treating the reaction suspension: (1) adding deionized water to the reaction suspension, filtering, and obtaining an amoxicillin filtrate and a retentate of the immobilized penicillin acylase mutant; (2) washing the retained immobilized penicillin acylase mutant with deionized water, and combining the washing solution with the amoxicillin filtrate from step (1); (3) adjusting the pH of the mixed solution obtained in step (2) to 2 with hydrochloric acid to obtain a mixed solution to be separated; (4) adding NaOH solution dropwise to the mixed solution to be separated until the pH reaches 3.5-5.5, and standing at 4°C for crystallization; (5) After the crystallization is completed, the amoxicillin crystals and the liquid phase containing phenylacetic acid are separated by filtration; The method for catalyzing the one-step synthesis of amoxicillin from penicillin potassium using an immobilized penicillin acylase mutant comprises: using only one immobilized penicillin acylase mutant as the sole enzyme in a reaction system, using penicillin or its salt and D-p-hydroxyphenylglycine methyl ester as substrates, and conducting the reaction in a buffer system with a pH of 4 to 8; the amino acid sequence of the penicillin acylase mutant, compared with the amino acid sequence shown in SEQ ID NO.1, has the following mutation pattern: G385βY or G385βR or F146αK&F24βR&F71βY&N241βK&G385βY.

2. The method for separating and recovering amoxicillin products by one-step synthesis catalyzed by Kluyveromyces citriodora β-subunit G385 mutant penicillin acylase according to claim 1, characterized in that: Step (5) may be followed by the following steps: (6) adjusting the pH of the liquid phase containing phenylacetic acid to between 2.0 and 2.5, and extracting with toluene to obtain an organic phase containing phenylacetic acid; (7) back-extracting the organic phase containing phenylacetic acid with a NaOH solution, converting phenylacetic acid into sodium phenylacetate and entering the aqueous phase; (8) separating the aqueous phase and the organic phase, wherein the toluene in the organic phase is recycled for extraction in step (6); (9) adding hydrochloric acid to the aqueous phase containing sodium phenylacetate to adjust the pH to 2-2.5, converting sodium phenylacetate into phenylacetic acid and crystallizing at 4°C; (10) After the crystallization is completed, the phenylacetic acid crystals are recovered by filtration and separation.

3. The method for separating and recovering amoxicillin products synthesized in a one-step process catalyzed by Kluyveromyces citriodora β-subunit G385 mutant penicillin acylase according to claim 1, characterized in that: The concentration of hydrochloric acid in step (3) is 15% by volume.

4. The method for separating and recovering amoxicillin products synthesized in a one-step process catalyzed by Kluyveromyces citriodora β-subunit G385 mutant penicillin acylase according to claim 1, characterized in that: In step (4), the pH value of amoxicillin crystallization is 5, and the crystallization time is 9 hours.

5. The method for separating and recovering amoxicillin products synthesized in a one-step process catalyzed by Kluyveromyces citriodora β-subunit G385 mutant penicillin acylase according to claim 1, characterized in that: In step (5), the amoxicillin crystals are obtained by filtration and separation, and the amoxicillin crystals are dried in a vacuum drying oven at 50° C. for 2 hours.

6. The method for separating and recovering amoxicillin products synthesized in a one-step process catalyzed by Kluyveromyces citriodora β-subunit G385 mutant penicillin acylase according to claim 2, characterized in that: In step (6), 15% by volume hydrochloric acid is used to adjust the pH.

7. The method for separating and recovering amoxicillin products synthesized in a one-step process catalyzed by Kluyveromyces citriodora β-subunit G385 mutant penicillin acylase according to claim 2, characterized in that: In step (6), the product is extracted with toluene for more than 2 times, and the organic phases are combined.

Images