Product separation and recovery process for one-step synthesis of amoxicillin

Abstract

The invention belongs to the technical field of product separation of antibiotics synthesized by an enzyme method, and particularly relates to a product separation and recovery process of amoxicillin synthesized by a one-step method. 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 a Chinese invention patent with the application number 202411522210.7, the application date of October 29, 2024, and the invention title of "A Method for Separating Amoxicillin and Phenylacetic Acid from the Reaction Solution of Enzymatic Catalytic One-step Preparation of Amoxicillin". Technical Field

[0002] The present invention belongs to the technical field of product separation in enzymatic synthesis of antibiotics, and particularly relates to a product separation and recovery process for one-step synthesis of amoxicillin. Background Art

[0003] Amoxicillin, also known as amoxicillin, is a major variety of the second-generation penicillins. Amoxicillin inhibits the synthesis of bacterial cell walls and is a broad-spectrum semi-synthetic antibiotic.

[0004] The preparation methods of amoxicillin include chemical synthesis methods and enzymatic catalytic synthesis methods. Chemical synthesis methods have the disadvantages of long reaction steps, high generation of three wastes, and extensive use 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 enzymatic synthesis process level of amoxicillin, enzymatic catalytic synthesis of amoxicillin has become the main method for preparing amoxicillin. Its main process route is: 6-APA reacts with D-p-hydroxyphenylglycine (or D-p-hydroxyphenylglycine methyl ester) catalyzed by penicillin acylase for synthesis of amoxicillin (such as application number 201711221286.6), and then after separation and purification, crystallization, and drying, the amoxicillin finished product is obtained.

[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 the final product yield of this method is relatively low and the fluidity is poor. Using penicillin acylase mutants, amoxicillin can be prepared from penicillin and its salts in one step. This technology avoids the disadvantages of the existing amoxicillin production technology that requires multiple steps of reaction, and at the same time avoids the separation process of intermediate products such as 6-APA. The two-step reaction and two-time separation and purification and other technologies can be achieved by one-step reaction, which has the advantages of simplified production process, high production efficiency, and greatly reduced production costs. However, in the one-step synthesis, in addition to a large amount of amoxicillin (AMOX) in the reaction solution from the original multiple-step reaction, it also contains an equimolar amount of phenylacetic acid (PAA). In addition to efficiently and highly yielding the separation and purification of amoxicillin products that meet the quality requirements of raw materials, it is also necessary to recover phenylacetic acid and realize the recycling of phenylacetic acid. Therefore, the separation technology for the original multi-step method of producing amoxicillin cannot meet the separation and refining requirements of the one-step method. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention has developed a production technology for the efficient and rapid separation and purification of amoxicillin and the recovery of phenylacetic acid, realizing the rapid separation and purification of amoxicillin and the large-scale recycling of phenylacetic acid. The produced amoxicillin meets the requirements of pharmaceutical production and reaches the standards of bulk drugs.

[0007] The present invention is realized as follows. A process for the separation and recovery of the product of one-step synthesis of amoxicillin is characterized in that: an immobilized penicillin acylase mutant is used to catalyze the one-step synthesis of amoxicillin from potassium penicillin to obtain a reaction suspension, and the reaction suspension is separated, including the following steps:

[0008] (1) Deionized water is added to the reaction suspension, and suction filtration is carried out to obtain an amoxicillin filtrate and a retentate of the immobilized penicillin acylase mutant.

[0009] (2) The retained immobilized penicillin acylase mutant is washed with deionized water, and the washing solution and the amoxicillin filtrate in step (1) are combined.

[0010] (3) The pH of the mixed solution obtained in step (2) is adjusted to 2 with hydrochloric acid to obtain a mixed solution to be separated.

[0011] (4) An NaOH solution is added dropwise to the mixed solution to be separated until the pH is 3.5 - 5.5, and crystallization is carried out at 4 °C.

[0012] (5) After crystallization is completed, filtration is carried out to separate and obtain amoxicillin crystals and a liquid phase containing phenylacetic acid.

[0013] Furthermore, the following steps are further included after step (5):

[0014] (6) The pH of the liquid phase containing phenylacetic acid is adjusted to between 2.0 and 2.5, and extraction is carried out with toluene to obtain an organic phase containing phenylacetic acid.

[0015] (7) Back-extraction is carried out on the organic phase containing phenylacetic acid with an NaOH solution, and phenylacetic acid is converted into sodium phenylacetate and enters the aqueous phase.

[0016] (8) The aqueous phase and the organic phase are separated, and the organic phase toluene is recycled for the extraction in step (6).

[0017] (9) Hydrochloric acid is added to the aqueous phase containing sodium phenylacetate to adjust the pH to 2 - 2.5, and sodium phenylacetate is converted into phenylacetic acid, and crystallization is carried out at 4 °C.

[0018] (10) After crystallization is completed, filtration is carried out to separate and recover phenylacetic acid crystals.

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

[0020] Further, in step (4), the pH for amoxicillin crystallization is 5 and the crystallization time is 9 h.

[0021] Further, in step (5), filtration and separation are carried out to obtain amoxicillin crystals, and the amoxicillin crystals are dried in a vacuum drying oven at 50 °C for 2 hours.

[0022] Further, in step (6), the pH is adjusted with hydrochloric acid with a volume ratio of 15%; extraction is carried out with toluene more than 2 times, and the organic phases are combined.

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

[0024] Specifically, it includes the following steps:

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

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

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

[0028] Further, 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] Further, in step S2, the concentration of potassium penicillin salt is 50 - 200 mmol / L, and the concentration of D-p-hydroxyphenylglycine methyl ester is 50 - 400 mmol / L.

[0030] Further, in step S3, the enzyme amount is 3000 - 30000 U / L, and the reaction time is 1 - 6 h.

[0031] In summary, the advantages and positive effects of the present invention are as follows: In this application, immobilized penicillin acylase mutants are used to catalyze the one-step synthesis of amoxicillin from potassium penicillin, and a method for separating amoxicillin and phenylacetic acid from the obtained reaction mixture is developed. The technical solution mainly includes: First, the immobilized penicillin acylase is separated from the reaction solution by filtration, and then amoxicillin is separated by crystallization; then, phenylacetic acid is separated and recovered by toluene extraction and back-extraction, etc. This separation method can quickly and efficiently separate amoxicillin, and the amoxicillin yield is relatively high, with an average crystallization rate of 93.22%; at the same time, the separation and recovery effect of phenylacetic acid is good; the extraction agent toluene can be recycled. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 FIG. is a process flow diagram of a method for separating amoxicillin and phenylacetic acid from the reaction solution prepared by the one-step enzymatic method for preparing amoxicillin.

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

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

[0035] Figure 4 FIG. is the detection chart of recombinant plasmid PCR agarose electrophoresis;

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

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

[0038] Figure 7 FIG. is the liquid chromatogram of amoxicillin crystal;

[0039] Figure 8 FIG. is the liquid chromatogram of phenylacetic acid crystal. DETAILED DESCRIPTION OF THE INVENTION

[0040] For a better understanding of the present invention rather than limiting the scope thereof, all numbers representing amounts, percentages, and other numerical values used in this application should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may vary according to the different desired properties to be obtained. Each numerical parameter should be regarded as being obtained at least according to the significant figures reported and by the conventional rounding method. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

[0041] In the following examples of the present invention, when the temperature is not specifically defined, it is under normal temperature conditions. Normal temperature refers to the natural room temperature conditions in the four seasons without additional cooling or heating treatment. Generally, the normal 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 the medium contains 50 μg / mL kanamycin.

[0042] In the examples, the experimental methods without specific conditions are usually carried out under conventional conditions, such as those described in "Molecular Cloning: A Laboratory Manual" (Chinese version) (edited by J. Sambrook, M.R. Green, translated by He Fuchu. Fourth Edition, Beijing: Science Press, 2017) and the methods described in the New England Biolabs (NEB) kits.

[0043] The present invention discloses a product separation and recovery process for the one-step synthesis of amoxicillin. The main contents include: First, use an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from potassium penicillin to obtain a reaction suspension. Then, separate and process the reaction suspension. The main steps include: (1) At room temperature, add an equal volume of deionized water to the reaction suspension and perform vacuum filtration at atmospheric pressure to obtain a filtrate containing amoxicillin and a retentate containing the immobilized penicillin acylase. (2) Wash the retained immobilized penicillin acylase with deionized water, and combine the washing solution and the amoxicillin filtrate in step (1). (3) Adjust the pH of the mixed solution obtained in step (2) to 2 with hydrochloric acid and filter to obtain an amoxicillin reaction solution. (4) Adjust the pH of the amoxicillin reaction solution to 5.0 with a 0.25 mol / L NaOH solution for crystallization, crystallize at 4°C for 9 h, and perform solid-liquid separation after crystallization 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 transfer phenylacetic acid from the aqueous phase to the organic phase. (5) Add a 0.25 mol / L NaOH solution to the above organic phase to convert phenylacetic acid into sodium phenylacetate and enter the aqueous phase. (6) Under heating conditions, add 15% hydrochloric acid to the aqueous phase containing sodium phenylacetate to adjust the pH to 2 - 2.5, convert sodium phenylacetate into phenylacetic acid, and crystallize at 4°C. The flow chart is as Figure 1 shown.

[0044] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention.

[0045] Example 1

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

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

[0048] In this example, the wild-type penicillin acylase used 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, they are: positions 1 - 26 are the signal peptide, positions 27 - 235 are the α subunit composed of 209 amino acids, positions 236 - 289 are the intermediate linker peptide composed of 54 amino acids, and positions 290 - 846 are the β subunit composed of 557 amino acids (also see 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), and the nucleotide sequence is shown in SEQ ID NO.2.

[0049] The construction schematic diagram of the recombinant plasmid pET28a-kcPA is as Figure 3 shown. Using the K. citrophila ATCC21285 genome 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; the reverse primer was 5'-CC A / AGCTT TTAGCGCTGCACCTGCAGC-3', SEQ ID NO.4. The EcoR I and HindIII restriction enzyme sites were introduced respectively (the underlined bases are the recognition sites of the restriction endonucleases), and the wild-type target fragment of PA was amplified by PCR.

[0050] PCR reaction system:

[0051]

[0052] The PCR temperature program was designed as:

[0053]

[0054] Two restriction endonucleases, EcoRI and HindIII, were selected to perform double digestion on the plasmid pET28a empty vector and the target fragment. Double digestion system:

[0055]

[0056] The double digestion reaction was carried out at 37°C for 1 h and then inactivated at 80°C for 20 min. The double-digested products were purified and recovered, and their concentrations were estimated according to their gel electrophoresis patterns. The concentration of the plasmid pET28a was about 50 ng / μL, and the concentration of the target gene kcPA was about 140 ng / μL.

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

[0058] Ligation system of the target fragment and the linearized vector:

[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'-GATCCCCATGAAATGGTGCCGTTGT-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, then dissolved in sterile water, and then operated according to the kit. As follows:

[0080] ① Mutate the corresponding site by PCR

[0081] PCR reaction system:

[0082]

[0083]

[0084] Cycling program temperature:

[0085]

[0086] For mutants with more than 2 mutation sites, use the PCR product of the previous mutation site obtained as a template to perform site-directed mutagenesis of the corresponding site successively.

[0087] ② React and process with Kinase, Ligase & DpnI (KLD) (a special mixture of kinase, ligase and DpnI)

[0088] The reaction system is as follows:

[0089] Volume Final concentration PCR product 1 μL 2X KLD Reaction Buffer 5 μL 1X 10X KLD Enzyme Mix 1 μL 1X Nuclease-free water 3 μL

[0090] React at room temperature for 5 min.

[0091] ③ Transformation by heat shock

[0092] Add 5 μL of the KLD reaction mixture to 50 μL of the chemically competent cell suspension of E. coli BL21(DE3). Incubate on ice for 30 min, perform heat shock at 42 °C for 30 s, incubate on ice for 5 min, add 950 μL of sterile SOC liquid medium, and gently shake at 37 °C for 1 h. Spread 40 - 100 μL of the bacterial suspension on an LB plate with Kan50 and incubate overnight at 37 °C. The single colonies grown are the corresponding mutant expression strains, which are respectively named E. coli BL21(DE3) / pET28a-kcPA01 - 18

[0093] ④ Identification of mutants

[0094] Inoculate the obtained mutant expression strains into 25 mL of LB liquid medium containing Kan50 and culture overnight at 37 °C. Extract the plasmid using a plasmid extraction kit. Send it to a third-party biological company for sequencing to determine that the corresponding product is the target product of site-directed mutagenesis.

[0095] 3. Expression of wild-type and mutant KcPA

[0096] Inoculate the constructed recombinant E. coli E. coli BL21(DE3) / pET28a-kcPA and E. coli BL21(DE3) / pET28a-kcPA01 - 18 on an LB agar plate with Kan50 and culture in an incubator at 37 °C for 12 - 16 h. Respectively pick single colonies and inoculate them into 25 mL of LB liquid medium supplemented with Kan50 and culture overnight at 37 °C on a shaker at 300 rpm. Pipette 500 μL of the bacterial liquid and transfer it to 50 mL of LB liquid medium with Kan50 and culture on a shaker at 37 °C and 280 rpm, and monitor OD 600When the change reaches 0.6 - 0.8, add IPTG solution to make the IPTG induction concentration 0.3 mM, and induce expression in a shaker at 25 °C and 220 rpm for 10 h. Centrifuge the fermentation broth to collect the bacteria. Suspend the collected bacterial cells in PBS buffer at pH 7.5 and pre-cool on ice for 10 min, then centrifuge at 4 °C and 12,000 rpm for 6 min to collect the bacteria. Add the bacteria to PBS buffer at 50 mM pH 7.5 to resuspend the bacteria in the centrifuge tube, and then centrifuge at 4 °C and 12,000 rpm for 6 min. Discard the supernatant, collect the final bacteria and resuspend them at a concentration of 0.01 g / mL, and use an ultrasonic crusher to break the cells. The cell disruption conditions are: ice-water bath, at a power of 400 W, each cycle works for 3 s, with an intermittent time of 5 s, for a total of 80 cycles. After disruption, centrifuge the mixture at 4 °C and 12,000 rpm for 15 min to obtain the supernatant, which is the crude enzyme solution. Collect the crude enzyme solution and analyze the expressed protein by SDS-PAGE.

[0097] Figure 5 It is the SDS-PAGE diagram of the protein expressed by the bacteria; among them, lane M is the protein Marker; Lane 1: the expression supernatant of E. coli BL21(DE3) / pET28a; Lane 2: the non-induced supernatant of E. coli BL21(DE3) / pET28a-kcPA; Lane 3: the non-induced supernatant of E. coli BL21(DE3) / pET28a-kcPA18; Lane 4: the IPTG-induced supernatant of E. coli BL21(DE3) / pET28a-kcPA18.

[0098] Example 2

[0099] 1. Determination of the hydrolysis activity of KcPA

[0100] The principle of the determination is as follows: Potassium penicillin (PGK) is hydrolyzed by KcPA to generate 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. Definition of enzyme activity: In PBS buffer at 28 °C and 0.1 M, the amount of enzyme required for penicillin acylase to catalyze 20 mg / mL PGK to generate 1 μmol 6-APA per minute is 1 unit of KcPA enzyme activity, and the unit is U.

[0101] Weigh 0.5 g of PGK and dissolve it in the above buffer solution, and make the volume up to 25 mL. Pipette 2 mL of the PGK solution into a centrifuge tube and add 0.1 mL of the KcPA enzyme solution. Set the control group without adding KcPA, and keep other conditions the same.

[0102] Place the above reaction system in a water bath shaker at 28°C and 200 rpm for 10 min. After the reaction, inactivate the enzyme in a 90°C water bath for 2 min. Take 200 μL of the reaction solution, add 3 mL of 0.1 M sodium citrate buffer with a pH of 3.0, add 1 mL of the color reagent (0.5% PDAB), let it stand at room temperature for 3 min, and then measure the absorbance at 415 nm. According to the 6-APA standard curve, obtain the 6-APA concentration in the sample after the reaction, and calculate the enzyme activity, that is, the hydrolysis activity, according to the formula.

[0103] Calculation formula: Penicillin acylase hydrolysis activity per mL

[0104] In the formula, C 6-APA : 6-APA concentration in the sample, μmol / L; V: volume of the reaction system, mL; V E : volume of penicillin acylase added, mL; t: reaction time, 10 min.

[0105] 2. Determination of KcPA synthesis activity

[0106] Amoxicillin is synthesized from 6-aminopenicillanic acid (6-APA) and methyl D-p-hydroxyphenylglycinate (D-HPGM) under the action of KcPA. The amoxicillin content can be determined by high performance liquid chromatography (HPLC), and then the PA synthesis activity can be calculated. The enzyme activity is defined as: under certain conditions, when 1 μmol of amoxicillin is catalyzed to be generated per minute by 1 unit of penicillin acylase, it is defined as 1 synthesis enzyme activity unit, denoted by U.

[0107] Weigh 1 g of 6-APA and 1.25 g of D-HPGM, dissolve them in 50 mL of 0.1 M PBS buffer with a pH of 6.3, adjust the pH to 6.3, and then make up the volume to 100 mL with the above buffer solution. Take 0.1 mL of KcPA and add it to the above solution. Start the reaction at 25°C and 200 rpm for 30 min, then place it in a 90°C water bath for 2 min to inactivate the enzyme and end the reaction. Take 0.5 mL of the reaction solution and filter it through a 0.22 μm water-based filter membrane, and then make up the volume to 100 mL with phosphate buffer for HPLC detection to obtain the amoxicillin content. Enzyme activity calculation formula: Penicillin acylase synthesis activity per mL In the formula: V: volume of the reaction solution, mL; 200: dilution factor; C 样 : molar concentration of amoxicillin, μmol / L; V E : volume of enzyme added, mL; t: reaction time, min.

[0108] The HPLC detection conditions were as follows: an Agilent ZORBAX SB-C18 4.6x250 mm chromatographic column, column temperature 25 °C, injection volume 10 μL. Mobile phase A (0.02 M NaH2PO4-Na2HPO4 buffer solution with pH 4.7), mobile phase B (methanol). Initially, 90% of mobile phase A and 10% of mobile phase B were maintained for 5 min. From 5 min to 7 min, the proportion of mobile phase B increased from 10% to 50% and was then maintained for 10 min. From 17 to 19 min, the proportion of mobile phase B decreased from 50% to 10%. Finally, 90% of mobile phase A and 10% of mobile phase B were used for equilibration for 5 min, and the total flow rate was 1 mL / min.

[0109] Table 2 Comparison of activities between mutants and wild type

[0110]

[0111]

[0112] Note: The hydrolysis activity of the wild type of KcPA expressed by the recombinant bacterium was 15 U / mL (fermentation broth), and the synthesis activity was 80 U / mL. For the convenience of comparison, the enzyme activity of the wild type of KcPA in Table 2 was defined as 100, and each mutant was compared with it.

[0113] As can be seen from the above table, for the mutants with single mutation sites, the hydrolysis activity and synthesis activity of each mutant were significantly improved compared with the wild type, especially the F146αK mutant on the α subunit and the G385βR mutant on the β subunit. The hydrolysis activity and synthesis activity of the single-point F146αK mutant were 5.8 times and 15.3 times that of the wild type, respectively; the hydrolysis activity and synthesis activity of the G385βR mutant were 4.6 times and about 11.2 times that of the wild type, respectively. Compared with the G385βR mutant, the G385βY mutant had higher hydrolysis activity, but its synthesis activity was not prominent. When multiple mutation sites were superimposed, the enzyme activity of the mutant increased compared with single-point mutation, especially the five-point mutant F146αK&F24βR&F71βY&N241βK&G385βR, which had relatively high hydrolysis activity and synthesis activity.

[0114] Example 3

[0115] One-step synthesis of amoxicillin by penicillin acylase of each mutant and wild type catalyzing PGK

[0116] PGK was added to PBS buffer solution with pH 7.0 to make its concentration reach 200 mM. At the same time, methyl p-hydroxyphenylglycinate (D-HPGM) was added to make its final concentration 300 mM. The enzyme dosage was 30 U / mL (calculated based on the synthesis enzyme activity). The reaction was carried out with constant stirring at 28 °C for 3 h. After the reaction, HPLC detection was carried out to calculate the amoxicillin yield.

[0117] The HPLC detection conditions were as follows: an Agilent ZORBAX SB-C18 4.6 x 250 mm chromatographic column, column temperature 25°C, injection volume 10 μL. Mobile phase A (0.02 M NaH2PO4-Na2HPO4 buffer solution with pH 4.7), mobile phase B (methanol). Initially, 90% of mobile phase A and 10% of mobile phase B were maintained for 5 min. From 5 min to 7 min, the proportion of mobile phase B was increased from 10% to 50% and then maintained for 10 min. From 17 to 19 min, the proportion of mobile phase B was decreased from 50% to 10%. Finally, 90% of mobile phase A and 10% of mobile phase B were used for equilibration for 5 min. The total flow rate was 1 mL / min. The reaction formula is as follows:

[0118]

[0119] The HPLC detection chromatogram of mutant KcPA 18 is as Figure 6 shown, where D-HPG is D-p-hydroxyphenylglycine, AMOX is amoxicillin, D-HPGM is methyl D-p-hydroxyphenylglycinate, PAA is phenylacetic acid, and PGK is potassium penicillin. It can be seen from the figure that the content of the intermediate 6-APA is extremely low, almost none.

[0120] Table 3 Yields of Amoxicillin Catalyzed by Each Mutant

[0121]

[0122]

[0123] From the results in the above table, it can be seen that each mutant can catalyze the reaction of potassium penicillin with methyl p-hydroxyphenylglycinate in a reaction system to synthesize amoxicillin in one step, and the product yield is significantly higher than that of the wild type.

[0124] Example 4 Preparation of Immobilized Penicillin Acylase

[0125] The penicillin acylase enzyme solution was prepared according to Example 1. The enzyme solution was cross-linked with epoxy resin (ER) activated by glutaraldehyde at 15°C for 1.5 h. The cross-linking reaction system was as follows: the enzyme amount in the enzyme solution was 1200 U (calculated based on synthetic enzyme activity), the glutaraldehyde content was 0.25%, 5 g of ER, and 50 mL of potassium dihydrogen phosphate-potassium hydrogen phosphate buffer solution with pH 7.5. After the cross-linking reaction, the immobilized enzyme was collected by filtration through a sieve. Then, the immobilized enzyme was washed with 100 mL of potassium dihydrogen phosphate-potassium hydrogen phosphate buffer solution with pH 7.5. The activity of the immobilized enzyme was 180 U / g, and the recovery rate of the immobilized enzyme activity reached 75%.

[0126] Example 5 One-step synthesis of amoxicillin catalyzed by KcPGA18

[0127] (1) Add 50 mL of pH 7 PBS buffer solution to the reaction flask as the reaction buffer system.

[0128] (2) Weigh a certain amount of potassium penicillin G (PGK) and methyl D-p-hydroxyphenylglycinate (D-HPGM) respectively and put them into the reaction buffer system. The molar ratio of PGK to D-HPGM is 1:1 - 1:2, the concentration of PGK is 50 - 200 mmol / L, and the concentration of D-HPGM is 50 - 400 mmol / L. Then stir well to make the reactant substrates PGK and D-HPGM evenly dispersed in the reaction system. In this example, the final concentrations of PGK and D-HPGM are 200 mmol / L and 300 mmol / L respectively.

[0129] (3) Immobilize the enzyme solution of the penicillin acylase mutant KcPGA18 according to the method in Example 4. Accurately weigh the immobilized penicillin acylase, with an enzyme amount of 20 U / mL (calculated based on the synthetic enzyme activity), put it into the reaction flask, control the reaction temperature at 24 °C, and the reaction time at 4 h to obtain a milky white reaction suspension.

[0130] Example 6 Separation and recovery of amoxicillin and phenylacetic acid

[0131] 1. Separation and crystallization of amoxicillin

[0132] (1) Add an equal volume of deionized water to the 50 mL reaction suspension obtained in Example 5.

[0133] (2) Filter out the immobilized penicillin acylase by suction filtration, and then wash the immobilized penicillin acylase with 50 mL of deionized water. Collect and combine the above filtrate and washing solution, a total of 150 mL.

[0134] (3) Slowly add 15% hydrochloric acid dropwise to the above filtrate to clarify the filtrate to about pH 2 to obtain the mixed solution to be separated.

[0135] (4) Slowly add 0.25 mol / L NaOH solution dropwise to the mixed solution to be separated until pH 5, and place it in a refrigerator at 4 °C to stand and crystallize for 9 h.

[0136] (5) After crystallization is completed, filter by suction to separate, obtaining amoxicillin crystals and a liquid phase containing phenylacetic acid. Finally, put the amoxicillin crystals into an oven and dry them at 85 °C for 30 min.

[0137] To verify whether the crystalline crystals are amoxicillin, in this example, 0.1 g of the crystalline crystals was dissolved in deionized water and made up to 50 mL to obtain a 2 mg / mL solution, and then high-performance liquid chromatography detection was carried out. The detection conditions were the same as in Example 3. The results are asFigure 7 As can be seen from the figure, except for the AMOX peak, there are no other obvious impurity peaks, and it can be basically determined that this crystal is amoxicillin.

[0138] 2. Separation and crystallization of phenylacetic acid

[0139] (1) Take the above-mentioned liquid phase containing phenylacetic acid (150 mL), and add 15% hydrochloric acid dropwise to adjust the pH to between 2.0 and 2.5.

[0140] (2) Add 40 mL of toluene as an extraction agent, extract at 25 °C, stir for 15 min, and then let it stand for phase separation, so that phenylacetic acid is extracted from the aqueous phase into the organic phase of toluene. Extract twice and combine the organic phases.

[0141] (3) Add 30 mL of 0.25 mol / L NaOH solution to the collected organic phase and mix well, then let it stand for phase separation (or centrifuge for phase separation), and phenylacetic acid becomes sodium phenylacetate and is back-extracted from toluene into the aqueous phase. Separate the aqueous phase and the organic phase, and the organic phase of toluene is recycled for the extraction in step (2).

[0142] (4) Collect the aqueous phase (lower phase) from the previous step, add 15% hydrochloric acid dropwise at 60 °C to adjust the pH to between 2.0 and 2.5. Then transfer it to 4 °C for crystallization for 12 h to convert sodium phenylacetate into phenylacetic acid and crystallize.

[0143] (5) After crystallization is completed, perform suction filtration, and the filter residue is the phenylacetic acid crystal. Finally, place the phenylacetic acid crystal in a vacuum drying oven and dry it at 40 °C for 30 min.

[0144] In order to verify whether the crystallized crystal is phenylacetic acid, in this example, 0.1 g of the crystallized crystal was dissolved in deionized water and fixed volume to 50 mL to obtain a 2 mg / mL solution, and then high-performance liquid chromatography detection was carried out. The detection conditions were the same as those in Example 3. The results are as Figure 8 As can be seen from the figure, except for the PAA peak, there are no other obvious impurity peaks, and it can be basically determined that the main component of this crystal is phenylacetic acid.

[0145] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A process for the separation and recovery of the product in the one-step synthesis of amoxicillin, characterized in that: Using an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from potassium penicillin to obtain a reaction suspension, and separating the reaction suspension, including the following steps: (1) Adding deionized water to the reaction suspension, and performing suction filtration to obtain 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 and the amoxicillin filtrate in 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 a NaOH solution dropwise to the mixed solution to be separated until the pH is 3.5 - 5.5, and standing for crystallization at 4°C; (5) After crystallization is completed, filtering and separating to obtain amoxicillin crystals and a liquid phase containing phenylacetic acid; The method for using an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from potassium penicillin includes: only using one immobilized penicillin acylase mutant as the only enzyme in the reaction system, using penicillin or its salt and D-p-hydroxyphenylglycine methyl ester as substrates, and reacting in a buffer system with a pH of 4 - 8; compared with the amino acid sequence shown in SEQ ID NO.1, the mutation mode of the penicillin acylase mutant is: N241βK or F146αK&N241βK&G385βR or F146αK&F71βY&N241βK&G385βY.

2. The product separation and recovery process for the one-step synthesis of amoxicillin according to claim 1, characterized in that, The following steps are further included after step (5): (6) Adjusting the pH of the liquid phase containing phenylacetic acid to between 2.0 - 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, and converting phenylacetic acid into sodium phenylacetate and entering the aqueous phase; (8) Separating the aqueous phase and the organic phase, and recycling the organic phase toluene for the 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 crystallization is completed, filtering and separating to recover phenylacetic acid crystals.

3. The product separation and recovery process for one-step synthesis of amoxicillin according to claim 1, characterized in that: In step (3), the concentration of hydrochloric acid is 15% by volume ratio.

4. The product separation and recovery process for one-step synthesis of amoxicillin according to claim 1, characterized in that: In step (4), the pH for amoxicillin crystallization is 5, and the crystallization time is 9 h.

5. The product separation and recovery process for the one-step synthesis of amoxicillin according to claim 1, characterized in that: In step (5), filtering and separating to obtain amoxicillin crystals, and drying the amoxicillin crystals in a vacuum drying oven at 50°C for 2 hours.

6. The product separation and recovery process for one-step synthesis of amoxicillin according to claim 2, characterized in that: In step (6), adjusting the pH with hydrochloric acid with a volume ratio of 15%.

7. The product separation and recovery process for the one-step synthesis of amoxicillin according to claim 2, characterized in that: In step (6), extracting with toluene more than 2 times, and combining the organic phases.

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