A method for the separation and recovery of amoxicillin products synthesized in a one-step process catalyzed by Kluyveromycin β-subunit G385 mutant penicillin acyltransferase.

Abstract

This invention belongs to the field of product separation technology in enzymatic antibiotic synthesis, and particularly relates to a method for separating and recovering amoxicillin products from a one-step synthesis catalyzed by a Kluyveromycin β-subunit G385 mutant penicillin acylase. This application uses an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from potassium penicillin. A method for separating amoxicillin and phenylacetic acid is developed for the obtained reaction mixture. The technical solution mainly includes: firstly, separating the immobilized penicillin acylase mutant from the reaction solution by filtration; then separating amoxicillin by crystallization; and finally, separating and recovering phenylacetic acid by toluene extraction, back-extraction, etc. This separation method can rapidly and efficiently separate amoxicillin with a high yield, with an average crystallization rate of 93.22%; simultaneously, the separation and recovery of phenylacetic acid is good; and the extractant toluene can be recycled.

Description

[0001] This application is a divisional application of Chinese invention patent application number 202411522210.7, filed on October 29, 2024, entitled "A method for separating amoxicillin and phenylacetic acid from a reaction solution for one-step preparation of amoxicillin by enzyme catalysis". Technical Field

[0002] This invention belongs to the field of product separation technology in enzymatic synthesis of antibiotics, and particularly relates to a method for separating and recovering amoxicillin products synthesized in a one-step process based on the catalysis of the G385 mutant penicillin acylase of Kluyveromycin β subunit. Background Technology

[0003] Amoxicillin, also known as amoxicillin, is a major type of second-generation penicillin. Amoxicillin inhibits bacterial cell wall synthesis and is a broad-spectrum semi-synthetic antibiotic. Due to its highly effective, broad-spectrum antibacterial activity and minimal side effects, the World Health Organization (WHO) recommends it as the first-line β-lactam oral antibiotic.

[0004] Amoxicillin can be prepared through chemical synthesis and enzymatic synthesis. Chemical synthesis methods suffer from drawbacks such as long reaction times, high levels of waste, and extensive use of chemical solvents. In recent years, with the widespread application of green synthesis concepts in the pharmaceutical industry and the development of amoxicillin enzymatic synthesis technology, enzymatic synthesis of amoxicillin has become the main method for its preparation. The main process route is as follows: amoxicillin is synthesized by reacting 6-APA with D-p-hydroxyphenylglycine (or D-p-hydroxyphenylglycine methyl ester) catalyzed by penicillin acylase (e.g., application number 201711221286.6). The amoxicillin product is then obtained after separation, purification, crystallization, and drying.

[0005] However, some problems still exist in the 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 results in a low final product yield and poor flowability. Using a penicillin acylase mutant, amoxicillin can be prepared from penicillin and its salts in a one-step process. This technology avoids the disadvantages of existing amoxicillin production technologies that require multiple steps, and also avoids the separation process of intermediate products such as 6-APA. A one-step reaction can achieve the original two-step reaction and two separation and purification techniques, offering advantages such as simplified production process, high production efficiency, and significantly reduced production costs. However, in addition to the large amount of amoxicillin (AMOX) from the original multi-step reaction, the one-step synthesis also contains an equimolar amount of phenylacetic acid (PAA) in the reaction solution. Besides efficiently and with high yield separating and purifying amoxicillin to meet the quality requirements of the active pharmaceutical ingredient, it is also necessary to recover phenylacetic acid for reuse. Therefore, the separation technology of the original multi-step amoxicillin production cannot meet the separation and purification requirements of the one-step production. Summary of the Invention

[0006] To address the problems existing in current technologies, this invention develops a highly efficient and rapid production technology for separating and purifying amoxicillin and recovering phenylacetic acid, achieving rapid separation and purification of amoxicillin and large-scale recovery and utilization of phenylacetic acid. The produced amoxicillin meets pharmaceutical production requirements and reaches the standards for active pharmaceutical ingredients.

[0007] This invention is implemented as follows: a method for separating and recovering amoxicillin products synthesized in a one-step process catalyzed by a Kluyveromycin β-subunit G385 mutant penicillin acylase, characterized by: using an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from potassium penicillin, obtaining a reaction suspension, and performing separation treatment on the reaction suspension, including the following steps:

[0008] (1) Add deionized water to the reaction suspension, filter, and obtain amoxicillin filtrate and the retentate of immobilized penicillin acylase mutant;

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

[0010] (3) Adjust the pH of the mixture obtained in step (2) to 2 with hydrochloric acid to obtain the mixture to be separated;

[0011] (4) Add NaOH solution dropwise to the mixture to be separated until the pH reaches 3.5-5.5, and allow it to stand at 4℃ to crystallize;

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

[0013] Furthermore, step (5) is followed by the following steps:

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

[0015] (7) The organic phase containing phenylacetic acid is back-extracted using NaOH solution, and the phenylacetic acid is converted into sodium phenylacetate and enters the aqueous phase;

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

[0017] (9) Add hydrochloric acid to the aqueous phase containing sodium phenylacetate to adjust the pH to 2-2.5. Sodium phenylacetate is converted into phenylacetic acid and crystallizes at 4°C.

[0018] (10) After crystallization, phenylacetic acid crystals are obtained by filtration and separation.

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

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

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

[0022] Further, in step (6), the pH is adjusted using 15% hydrochloric acid by volume; the organic phases are extracted with toluene more than twice and then combined.

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

[0024] Specifically, the following steps are included:

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

[0026] S2: Add potassium penicillin and D-p-hydroxyphenylglycine methyl ester to the reaction buffer system. The molar ratio of potassium penicillin to D-p-hydroxyphenylglycine methyl ester is 1:1 to 1:2. Stir thoroughly.

[0027] S3: Add the immobilized penicillin acylase mutant to the reaction buffer system and carry out the reaction at a controlled temperature of 12–30°C. The preferred reaction temperature is 12–28°C.

[0028] Further, the buffer solution in step S1 includes any one of citrate buffer, acetate buffer, PBS buffer, sodium dihydrogen phosphate-citrate buffer, sodium barbital-hydrochloric acid buffer, and pure water.

[0029] Furthermore, in step S2, the concentration of potassium penicillin 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 concentration is 3000–30000 U / L, and the reaction time is 1–6 h.

[0031] In summary, the advantages and positive effects of this invention are as follows: This application uses an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from potassium penicillin. A method for separating amoxicillin and phenylacetic acid has been developed for the obtained reaction mixture. The main technical solution includes: firstly, separating the immobilized penicillin acylase from the reaction solution by filtration; then separating amoxicillin by crystallization; and finally, recovering phenylacetic acid by toluene extraction, back-extraction, etc. This separation method can rapidly and efficiently separate amoxicillin with a high yield, achieving an average crystallization rate of 93.22%. Simultaneously, the separation and recovery of phenylacetic acid is effective; and the extractant toluene can be recycled. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the process for separating amoxicillin and phenylacetic acid in the reaction solution of a one-step enzyme-catalyzed amoxicillin preparation.

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

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

[0035] Figure 4 This is a graph from recombinant plasmid PCR agarose gel electrophoresis detection.

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

[0037] Figure 6 This is the HPLC chromatogram of the one-step synthesis of amoxicillin from potassium penicillin catalyzed by KcPA 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 Implementation

[0040] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "about". Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. In this invention, "about" means within 10%, preferably within 5%, of a given value or range.

[0041] Unless otherwise specified, all embodiments of the present invention are based on ambient temperature conditions. Ambient temperature refers to the natural room temperature during the four seasons, without additional cooling or heating treatment. Generally, ambient temperature is controlled between 10 and 30°C, preferably between 15 and 25°C. The abbreviations are as follows: "min" represents minutes, "s" represents seconds, "U" represents enzyme activity units, "mM" represents millimoles per liter, "M" represents moles per liter, "rpm" represents revolutions per minute, "mol" represents moles, "μg" represents micrograms, "mg" represents milligrams, "g" represents grams, "μL" represents microliters, "mL" represents milliliters, "bp" represents base pairs, LB medium represents Luria-Bertani medium, and Kan50 indicates that the medium contains 50 μg / mL kanamycin.

[0042] In the examples, experimental methods without specific conditions are generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (Chinese version) (J. Sambrook and M. Green, eds., translated by He Fuchu, 4th edition, Beijing: Science Press, 2017) and the methods described in the New England Biolabs (NEB) kit.

[0043] This invention discloses a method for separating and recovering amoxicillin products synthesized in a one-step process using a mutant penicillin acylase catalyzed by Kluyveromycin β-subunit G385. The main contents include: firstly, using an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from potassium penicillin, obtaining a reaction suspension; then, separating the reaction suspension, the main steps of which include: (1) adding an equal volume of deionized water to the reaction suspension at room temperature, filtering under normal pressure to obtain a filtrate containing amoxicillin and a retentate containing immobilized penicillin acylase. (2) washing the retentate immobilized penicillin acylase with deionized water, combining the washings and the amoxicillin filtrate from step (1). (3) adjusting the pH of the mixture obtained in step (2) to 2 with hydrochloric acid, filtering to obtain the amoxicillin reaction solution. (4) Adjust the pH of the amoxicillin reaction solution to 5.0 with 0.25 mol / L NaOH solution for crystallization. Crystallize at 4°C for 9 h. After crystallization, perform solid-liquid separation to obtain amoxicillin crystals and a liquid phase containing phenylacetic acid. (5) Adjust the pH of the liquid phase containing phenylacetic acid to between 2.0 and 2.5, and extract with toluene to allow phenylacetic acid to enter the organic phase from the aqueous phase. (6) Add 0.25 mol / L NaOH solution to the above organic phase to convert phenylacetic acid into sodium phenylacetate, which then enters the aqueous phase. (7) Under heating conditions, add 15% hydrochloric acid to the above aqueous phase containing sodium phenylacetate to adjust the pH to 2-2.5, converting sodium phenylacetate into phenylacetic acid. Crystallize at 4°C. Flowchart as follows: Figure 1 As shown.

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

[0045] Example 1

[0046] Construction, prokaryotic expression and functional identification of penicillin acylase mutants from Kluyvera citrophila

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

[0048] The wild-type penicillin acylase used in this embodiment is derived from Kluyveracitrophila 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: positions 1-26 are the signal peptide; positions 27-235 are the α subunit consisting of 209 amino acids; positions 236-289 are the intermediate linker peptide consisting of 54 amino acids; and positions 290-846 are the β subunit consisting of 557 amino acids (see also...). Figure 2(where the single underlined part is the α subunit, the wavy part is the linker peptide, and the double underlined part is the β subunit), the nucleotide sequence is shown in SEQ ID NO.2.

[0049] A schematic diagram of the construction of recombinant plasmid pET28a-kcPA is shown below. Figure 3 As shown. Using the K. citrophila ATCC21285 genome as a template, primers were designed based on 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 (underlined bases are restriction endonuclease recognition sites), and the PA wild-type target fragment was amplified by PCR.

[0050] PCR reaction system:

[0051]

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

[0053]

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

[0055]

[0056] Double digestion was performed at 37°C for 1 hour, followed by inactivation at 80°C for 20 minutes. The double digestion products were purified and recovered, and the concentrations were estimated based on their gel electrophoresis patterns. The concentrations of plasmid pET28a were approximately 50 ng / μL, and the concentrations of the target gene kcPA were approximately 140 ng / μL.

[0057] The double-digested 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 E.coli DH5α cells.

[0058] Target fragment and linearized vector linkage system:

[0059]

[0060] To verify the successful transformation of the recombinant plasmid, a single colony was picked from an LB agar plate containing Kan50 and transferred to LB liquid medium containing Kan50. The plasmid was extracted the next day using a plasmid extraction kit and subjected to PCR identification. A 2500 bp target band was obtained by agarose gel electrophoresis (e.g., ...). Figure 4 The validated expression vector pET28a-kcPA was transformed into E. coli BL21(DE3) to obtain the wild-type PA expression recombinant strain E. coli BL21(DE3) / pET28a-kcPA.

[0061] 2. Obtaining mutant expression vectors

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

[0063] Table 1. Mutants and corresponding mutation sites

[0064]

[0065]

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

[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] Primers were synthesized by a nucleic acid synthesis company, then dissolved in sterile water, and the procedure was followed according to the kit instructions. See below:

[0080] ①Mutation of the corresponding site using PCR

[0081] PCR reaction system:

[0082]

[0083]

[0084] Cyclic program temperature:

[0085]

[0086] For mutants with more than two mutation sites, the PCR product of the previous mutation site is used as a template to perform site-directed mutagenesis at the corresponding sites one after another.

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

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

[0089] React at room temperature for 5 minutes.

[0090] ③ Thermal shock conversion

[0091] Add 5 μL of KLD reaction mixture to 50 μL of chemicompetent E. coli BL21(DE3) cell suspension, incubate on ice for 30 min, subject to heat shock at 42°C for 30 s, incubate on ice for 5 min, add 950 μL of SOC sterile liquid medium, and gently shake at 37°C for 1 h. Spread 40-100 μL of the bacterial suspension onto Kan50 LB agar plates and incubate overnight at 37°C. Single colonies that grow are the corresponding mutant expression strains, named E. coli BL21(DE3) / pET28a-kcPA01~18.

[0092] ④ Mutant identification

[0093] The obtained mutant expression strain was inoculated into 25 mL of liquid medium containing Kan50 LB and cultured overnight at 37°C. Plasmids were then extracted using a plasmid extraction kit. The samples were 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 recombinant *E. coli* strains *E. coli* BL21(DE3) / pET28a-kcPA and *E. coli* BL21(DE3) / pET28a-kcPA01–18 were inoculated onto Kan50 LB agar plates and cultured at 37°C for 12–16 h. Single colonies were picked and inoculated into 25 mL of LB liquid medium supplemented with Kan50, and cultured overnight at 37°C with a shaker at 300 rpm. 500 μL of the bacterial culture was then transferred to 50 mL of Kan50 LB liquid medium and cultured at 37°C with a shaker at 280 rpm. OD was monitored. 600When the concentration of saturation reached 0.6-0.8, IPTG solution was added to induce expression at a concentration of 0.3 mM. Expression was induced for 10 h at 25°C and 220 rpm in a shaker. The fermentation broth was then centrifuged to collect the cells. The collected cells were resuspended in pH 7.5 PBS buffer and pre-cooled on ice for 10 min, followed by centrifugation at 4°C and 12000 rpm for 6 min to collect the cells. The cells were resuspended in 50 mM pH 7.5 PBS buffer and centrifuged again at 4°C and 12000 rpm for 6 min. The supernatant was discarded, and the remaining cells were collected and resuspended at a concentration of 0.01 g / mL. The cells were then disrupted using an ultrasonic cell disruptor. The cell disruption conditions were: ice-water bath, 400 W power, 3 s operation per cycle, 5 s interval, for a total of 80 cycles. After crushing, the mixture was centrifuged at 4℃ and 12000rpm for 15min 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 SDS-PAGE images of proteins expressed in bacterial cells; lane M represents the protein marker; Lane 1: supernatant of E. coli BL21(DE3) / pET28a expression; Lane 2: uninduced supernatant of E. coli BL21(DE3) / pET28a-kcPA; Lane 3: supernatant of uninduced E. coli BL21(DE3) / pET28a-kcPA18; Lane 4: supernatant of IPTG-induced E. coli BL21(DE3) / pET28a-kcPA18.

[0097] Example 2

[0098] 1. Determination of KcPA hydrolytic activity

[0099] The principle of the assay is as follows: potassium penicillin (PGK) hydrolyzes under the action of 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, which has a maximum absorption peak at 415 nm. Enzyme activity is defined as the amount of enzyme required for penicillin acylase to catalyze the generation of 1 μmol of 6-APA per minute from 20 mg / mL PGK at 28℃ in 0.1 M PBS buffer. One unit of KcPA enzyme activity is expressed in units of U.

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

[0101] The above reaction system was placed in a water bath shaker at 28℃ and 200 rpm for 10 min. After the reaction, the enzyme was inactivated by incubating in a water bath at 90℃ for 2 min. 200 μL of the reaction solution was taken and 3 mL of 0.1 M sodium citrate buffer (pH 3.0) was added, along with 1 mL of chromogenic solution (0.5% PDAB). After standing at room temperature for 3 min, the absorbance was measured at 415 nm. The concentration of 6-APA in the sample after the reaction was obtained based on the 6-APA standard curve, and the enzyme activity, i.e., hydrolytic activity, was calculated using the formula.

[0102] Calculation formula: per mL of penicillin acylase hydrolase activity

[0103] In the formula, C 6-APA V: 6-APA concentration in the sample, μmol / L; V: Reaction volume, mL; E : Amount of penicillin acylase added, mL; t: Reaction time, 10 min.

[0104] 2. Determination of KcPA Synthetic Activity

[0105] Amoxicillin is synthesized from 6-aminopenicillanic acid (6-APA) and methyl p-hydroxyphenylglycine (D-HPGM) under the action of KcPA. The amoxicillin content can be determined by high-performance liquid chromatography (HPLC) to calculate the PA synthesis activity. Enzyme activity is defined as: under certain conditions, one unit of penicillin acylase catalyzes the production of 1 μmol of amoxicillin per minute, which is denoted as U.

[0106] Weigh 1 g of 6-APA and 1.25 g of D-HPGM and dissolve them in 50 mL of 0.1 M, pH 6.3 PBS buffer. Adjust the pH to 6.3, then bring the volume to 100 mL with the buffer solution. Add 0.1 mL of KcPA to the above solution and react at 25 °C and 200 rpm for 30 min. Inactivate the enzyme by placing the solution in a 90 °C water bath for 2 min to stop the reaction. Filter 0.5 mL of the reaction solution through a 0.22 μm aqueous filter membrane and bring the volume to 100 mL with phosphate buffer. Perform HPLC analysis to determine the amoxicillin content. Enzyme activity calculation formula: [Formula for amoxicillin activity calculation is missing from the original text]. Where: V: volume of reaction liquid, mL; 200: dilution factor; C 样 Amoxicillin molar concentration, μmol / L; V E : Volume of enzyme added (mL); t: Reaction time (min).

[0107] The HPLC detection conditions were as follows: Agilent ZORBAX SB-C18 4.6x250 mm column, column temperature 25℃, injection volume 10 μL. Mobile phase A (0.02 M pH 4.7 NaH2PO4-Na2HPO4 buffer) and mobile phase B (methanol) were used. Initially, the mobile phase was maintained at 90% mobile phase A and 10% mobile phase B for 5 min. From 5 min to 7 min, the mobile phase B was increased from 10% to 50% and maintained for 10 min. From 17 min to 19 min, the mobile phase B was reduced from 50% to 10%. Finally, the mobile phase was equilibrated with 90% mobile phase A and 10% mobile phase B for 5 min. The total flow rate was 1 mL / min.

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

[0109]

[0110]

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

[0112] As shown in the table above, for mutants with single mutation sites, the hydrolytic and synthetic activities of each mutant are significantly improved compared to the wild type, especially the F146αK mutant on the α subunit and the G385βR mutant on the β subunit. The hydrolytic and synthetic activities of the single-site F146αK mutant are 5.8 times and 15.3 times that of the wild type, respectively; the hydrolytic and synthetic activities of the G385βR mutant are 4.6 times and approximately 11.2 times that of the wild type, respectively. Compared with the G385βR mutant, the G385βY mutant has higher hydrolytic activity, but its synthetic activity is not outstanding. When multiple mutation sites are superimposed, the enzyme activity of the mutants is increased compared to single-site mutations, especially the five-site mutant F146αK&F24βR&F71βY&N241βK&G385βR, which has high hydrolytic and synthetic activities.

[0113] Example 3

[0114] Amoxicillin synthesized in one step via PGK catalysis by various mutant and wild-type penicillin acylases

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

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

[0117]

[0118] The HPLC chromatogram of the mutant KcPA18 is shown below. Figure 6 As shown in the figure, D-HPG is D-p-hydroxyphenylglycine, AMOX is amoxicillin, D-HPGM is D-p-hydroxyphenylglycine methyl ester, PAA is phenylacetic acid, and PGK is potassium penicillin. It can be seen from the figure that the content of the intermediate product 6-APA is extremely low, almost non-existent.

[0119] Table 3 Yields of amoxicillin synthesized by each mutant

[0120]

[0121]

[0122] The results in the table above show that each mutant can catalyze the reaction of potassium penicillin with methyl p-hydroxyphenylglycine in a single reaction system to synthesize amoxicillin in one step, and the product yield is significantly higher than that of the wild type.

[0123] Example 4: Preparation of immobilized penicillin acylase

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

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

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

[0127] (2) Weigh out a certain amount of potassium penicillin (PGK) and D-hydroxyphenylglycine methyl ester (D-HPGM) and add them to the reaction buffer system. The molar ratio of PGK to D-HPGM is 1:1 to 1:2, the concentration of PGK is 50 to 200 mmol / L, and the concentration of D-HPGM is 50 to 400 mmol / L. Then stir thoroughly to ensure that the reactants PGK and D-HPGM are uniformly dispersed 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 penicillin acylase mutant KcPGA18 was immobilized according to the method in Example 4. The immobilized penicillin acylase was accurately weighed, and the enzyme amount was 20 U / mL (calculated based on the synthetic enzyme activity). It was added to the reaction flask, and the reaction temperature was controlled at 24°C for 4 hours 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) Add an equal volume of deionized water to 50 mL of the reaction suspension obtained in Example 5.

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

[0133] (3) Add 15% hydrochloric acid dropwise to the above filtrate to dissolve the filtrate until the pH is about 2, and obtain the mixture to be separated.

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

[0135] (5) After crystallization, the amoxicillin crystals and phenylacetic acid-containing liquid phase were obtained by filtration. Finally, the amoxicillin crystals were dried in an oven at 85°C for 30 min.

[0136] To verify whether the crystalline crystals were 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. This solution was then detected by high-performance liquid chromatography (HPLC) under the same conditions as in Example 3. The results are as follows: Figure 7As can be seen from the figure, apart from the AMOX peak, there are no other obvious impurity peaks, which basically confirms that the crystal is amoxicillin.

[0137] 2. Isolation and crystallization of phenylacetic acid

[0138] (1) Take the above liquid phase containing phenylacetic acid (150 mL) and add 15% hydrochloric acid 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. After stirring for 15 min, allow the mixture to stand and separate into layers, allowing phenylacetic acid to be extracted from the aqueous phase into the organic phase toluene. Repeat the extraction 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 and separate into layers (or centrifuge to separate into layers). Phenylacetic acid is converted into sodium phenylacetate and back-extracted from toluene into the aqueous phase. Separate the aqueous phase and the organic phase, wherein the toluene in the organic phase is recycled for extraction in step (2).

[0141] (4) Collect the aqueous phase (lower phase) from the previous step, and adjust the pH to between 2.0 and 2.5 by adding 15% hydrochloric acid at 60°C. Then transfer it to 4°C for crystallization for 12 hours to convert sodium phenylacetate into phenylacetic acid and crystallize it.

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

[0143] To verify whether the crystalline crystals were phenylacetic acid, 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. This solution was then analyzed by high-performance liquid chromatography (HPLC) under the same conditions as in Example 3. The results are as follows: Figure 8 As can be seen from the figure, apart from 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 within the protection scope of the present invention.

Claims

1. A method for separating and recovering amoxicillin products synthesized in a one-step process using Kluyveromycin β-subunit G385 mutant penicillin acyltransferase catalyzed by Kluyveromycin β-subunit, characterized in that: Amoxicillin was synthesized in one step from potassium penicillin using an immobilized penicillin acylase mutant, yielding a reaction suspension. The reaction suspension was then separated and processed, including the following steps: (1) Add deionized water to the reaction suspension, filter, and obtain amoxicillin filtrate and the retentate of immobilized penicillin acylase mutant; (2) Wash the retained immobilized penicillin acylase mutant with deionized water, and combine the washing solution and the amoxicillin filtrate from step (1) to obtain a mixture; (3) Adjust the pH of the mixture obtained in step (2) to 2 with hydrochloric acid to obtain the mixture to be separated; (4) Add NaOH solution dropwise to the mixture to be separated until the pH reaches 3.5-5.5, and allow it to stand at 4℃ to crystallize; (5) After crystallization, the amoxicillin crystals and the liquid phase containing phenylacetic acid were obtained by filtration and separation; A method for one-step synthesis of amoxicillin from potassium penicillin using an immobilized penicillin acylase mutant includes: using only one immobilized penicillin acylase mutant as the sole enzyme in the reaction system, with potassium penicillin and... D - Using p-hydroxyphenylglycine methyl ester as a substrate, the reaction is carried out in a buffer system with pH 4-8; the amino acid sequence of the penicillin acylase mutant is selected from one of the following (a), (b), and (c) compared with the amino acid sequence shown in SEQ ID NO. 1: (a): One of the following mutations occurs: G385βY; (b): One of the following mutations occurs: G385βR; (c): The following five mutations occur simultaneously: F146αK & F24βR & F71βY & N241βK & G385βY; In SEQ ID NO. 1, from the N-terminus to the C-terminus of the protein, the following are the subunits: the α subunit, consisting of 209 amino acids, from position 27 to 235; and the β subunit, consisting of 557 amino acids, from position 290 to 846. F146αK indicates that the 146th amino acid of the α subunit is changed from F to K; F24βR indicates that the 24th amino acid of the β subunit is changed from F to R; F71βY indicates that the 71st amino acid of the β subunit is changed from F to Y; N241βK indicates that the 241st amino acid of the β subunit is changed from N to K; G385βR indicates that the 385th amino acid of the β subunit is changed from G to R; and G385βY indicates that the 385th amino acid of the β subunit is changed from G to Y.

2. The method for separating and recovering amoxicillin products synthesized in a one-step process based on the β-subunit G385 mutant penicillin acylase catalyzed by Kluyveromycin citrate bacteria, as described in claim 1, is characterized in that... Step (5) is followed by the following steps: (6) Adjust the pH of the liquid phase containing phenylacetic acid to between 2.0 and 2.5, and extract with toluene to obtain an organic phase containing phenylacetic acid; (7) The organic phase containing phenylacetic acid is back-extracted using NaOH solution, and the phenylacetic acid is converted into sodium phenylacetate and enters the aqueous phase; (8) Separate the aqueous phase and the organic phase, wherein the toluene phase of the organic phase is recycled for extraction in step (6); (9) Add hydrochloric acid to the aqueous phase containing sodium phenylacetate to adjust the pH to 2-2.

5. Sodium phenylacetate is converted into phenylacetic acid and crystallizes at 4°C. (10) After crystallization, phenylacetic acid crystals are obtained by filtration and separation.

3. The method for separating and recovering amoxicillin products synthesized in a one-step process based on the β-subunit G385 mutant penicillin acylase catalyzed by Kluyveromycin citrate bacteria, as described in claim 1, is characterized in that: In step (3), the concentration of hydrochloric acid is 15% by volume.

4. The method for separating and recovering amoxicillin products synthesized in a one-step process based on the β-subunit G385 mutant penicillin acylase catalyzed by Kluyveromycin citrate bacteria, as described in claim 1, is characterized in that: In step (4), the amoxicillin crystallization pH is 5 and the crystallization time is 9 hours.

5. The method for separating and recovering amoxicillin products synthesized in a one-step process based on the β-subunit G385 mutant penicillin acylase catalyzed by Kluyveromycin citrate bacteria, as described in claim 1, is characterized in that: In step (5), the amoxicillin crystals were obtained by filtration and separation. The amoxicillin crystals were then 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 based on the β-subunit G385 mutant penicillin acylase catalyzed by Kluyveromycin citrate bacteria according to claim 2, characterized in that: In step (6), the pH is adjusted using 15% hydrochloric acid by volume.

7. The method for separating and recovering amoxicillin products synthesized in a one-step process based on the β-subunit G385 mutant penicillin acylase catalyzed by Kluyveromycin citrate bacteria according to claim 2, characterized in that: In step (6), the organic phases are extracted with toluene more than twice and then combined.

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