A method for separating amoxicillin and phenylacetic acid from a reaction solution for preparing amoxicillin by an enzyme-catalyzed one-step method

Abstract

The present invention belongs to the technical field of product separation of enzymatically synthesized antibiotics, and in particular, relates to a method for separating amoxicillin and phenylacetic acid from a reaction solution of preparing amoxicillin by an enzyme-catalyzed one-step method. In the present application, an immobilized penicillin acylase mutant is used to catalyze the one-step synthesis of amoxicillin by penicillin potassium, and a set of methods for separating amoxicillin and phenylacetic acid is developed for the obtained reaction mixture. The technical scheme mainly includes: firstly, the immobilized penicillin acylase mutant is separated from the reaction solution by filtration, and then amoxicillin is separated by crystallization; then, the phenylacetic acid is crystallized, separated and recovered by toluene extraction, back extraction, etc. The separation method can quickly and efficiently separate amoxicillin, and the yield of amoxicillin is high, with an average crystallization rate of 93.22%; at the same time, the separation and recovery effect of phenylacetic acid is good; the extractant toluene can be recycled.

Description

Technical Field

[0001] The invention belongs to the technical field of product separation of enzymatically synthesized antibiotics, and in particular relates to a method for separating amoxicillin and phenylacetic acid from a reaction solution in which amoxicillin is prepared by an enzyme-catalyzed one-step method. Background Art

[0002] Amoxicillin, also known as amoxicillin, is a major type of second-generation penicillin. Amoxicillin inhibits the synthesis of bacterial cell walls and is a broad-spectrum semi-synthetic antibiotic. The World Health Organization (WHO) recommends it as the preferred β-lactam oral antibiotic because of its high efficiency, broad-spectrum antibacterial effect, and minimal toxic side effects.

[0003] 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 large amounts 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 enzyme synthesis process of amoxicillin, enzyme-catalyzed synthesis of amoxicillin has become the main method for preparing amoxicillin. Its main process route is: synthesizing amoxicillin (such as application number 201711221286.6) by the reaction of 6-APA and D-p-hydroxyphenylglycine (or D-p-hydroxyphenylglycine methyl ester) catalyzed by synthetic penicillin acylase, and then obtaining the finished amoxicillin after separation, purification, crystallization, and drying.

[0004] 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 the method is low and the fluidity is poor. Amoxicillin can be prepared from penicillin and its salt in a one-step method using a penicillin acylase mutant. This technology avoids the disadvantages of the existing amoxicillin production technology requiring multi-step reactions, and at the same time avoids the separation process of the intermediate product 6-APA. The original two-step reaction can be achieved by a one-step reaction. The separation and purification techniques have the advantages of simplified production process, high production efficiency, and greatly reduced production costs. However, the one-step synthesis contains equimolar phenylacetic acid (PAA) in addition to a large amount of amoxicillin (AMOX) in the original multi-step reaction in the reaction solution. How 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 phenylacetic acid and realize the recycling of phenylacetic acid. Therefore, the separation technology of the original multi-step method for producing amoxicillin cannot meet the separation and purification requirements of the one-step method. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention develops a highly efficient and rapid production technology for separating and refining amoxicillin and recovering phenylacetic acid, realizing rapid separation and purification of amoxicillin and large-scale recovery and utilization of phenylacetic acid. The produced amoxicillin meets the requirements of pharmaceutical production and reaches the standards of raw materials.

[0006] The present invention is achieved by a method for separating amoxicillin and phenylacetic acid from a reaction solution of preparing amoxicillin by an enzyme-catalyzed one-step method, characterized in that: an immobilized penicillin acylase mutant is used to catalyze the one-step synthesis of amoxicillin from penicillin potassium to obtain a reaction suspension, and the reaction suspension is subjected to separation treatment, comprising the following steps:

[0007] (1) adding deionized water to the reaction suspension, filtering with suction to obtain an amoxicillin filtrate and a retentate of the immobilized penicillin acylase mutant;

[0008] (2) washing the retained immobilized penicillin acylase mutant with deionized water, and combining the washing solution with the amoxicillin filtrate in step (1);

[0009] (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;

[0010] (4) adding NaOH solution to the mixed solution to be separated until the pH value reaches 3.5-5.5, and standing at 4°C for crystallization;

[0011] (5) After the crystallization is completed, the amoxicillin crystals and the liquid phase containing phenylacetic acid are separated by filtration.

[0012] Furthermore, step (5) further includes the following steps:

[0013] (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;

[0014] (7) using a NaOH solution to back-extract the organic phase containing phenylacetic acid, and converting phenylacetic acid into sodium phenylacetate and entering the aqueous phase;

[0015] (8) separating the aqueous phase and the organic phase, wherein the toluene in the organic phase is recycled for extraction in step (6);

[0016] (9) adding hydrochloric acid to the aqueous phase containing sodium phenylacetate to adjust the pH to 2-2.5, and converting the sodium phenylacetate into phenylacetic acid, which crystallizes at 4° C.;

[0017] (10) After the crystallization is completed, the phenylacetic acid crystals are recovered by filtration and separation.

[0018] Furthermore, in step (3), the concentration of hydrochloric acid is 15% by volume.

[0019] Furthermore, in step (4), the pH of amoxicillin crystallization is 5, and the crystallization time is 9 hours.

[0020] 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.

[0021] 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.

[0022] The method for synthesizing amoxicillin from penicillin potassium in one step by using an immobilized penicillin acylase mutant to catalyze the synthesis comprises: using only one immobilized penicillin acylase mutant as the only enzyme in a reaction system, taking penicillin or its salt and D-p-hydroxyphenylglycine methyl ester as substrates, and reacting in a buffer system of pH 4 to 8; the amino acid sequence of the penicillin acylase mutant comprises at least one of the following mutations compared with the amino acid sequence shown in SEQ ID NO.1: F146αK, F24βR, F71βY, N241βK, G385βY or G385βR.

[0023] The specific steps include:

[0024] S1: Add a pH 4-8 buffer into the reaction bottle as a reaction buffer system;

[0025] 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;

[0026] S3: adding the immobilized penicillin acylase mutant to the reaction buffer system, controlling the reaction temperature to 12-30° C., and performing the reaction, preferably the reaction temperature is 12-28° C.

[0027] 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.

[0028] 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.

[0029] Furthermore, in step S3, the amount of enzyme is 3000 to 30000 U / L, and the reaction time is 1 to 6 hours.

[0030] In summary, the advantages and positive effects of the present invention are as follows: in the present application, an immobilized penicillin acylase mutant is used to catalyze the one-step synthesis of amoxicillin from penicillin potassium, and a method for separating amoxicillin and phenylacetic acid is developed for the obtained reaction mixture. The technical scheme mainly includes: firstly, the immobilized penicillin acylase is separated from the reaction solution by filtration, and then amoxicillin is separated by crystallization; and then the phenylacetic acid is crystallized, separated and recovered by toluene extraction, back extraction, etc. The separation method can separate amoxicillin quickly and efficiently, and the yield of amoxicillin is high, with an average crystallization rate of 93.22%; at the same time, the separation and recovery effect of phenylacetic acid is good; the extractant toluene can be recycled. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The present invention is a schematic flow chart of a method for separating amoxicillin and phenylacetic acid from a reaction solution of preparing amoxicillin by an enzyme-catalyzed one-step method.

[0032] Figure 2 is the wild-type penicillin acylase amino acid sequence;

[0033] Figure 3 is a schematic diagram of the construction of the recombinant plasmid pET28a-kcPA;

[0034] Figure 4 This is the agarose electrophoresis detection diagram of the recombinant plasmid PCR;

[0035] Figure 5 This is the SDS-PAGE electrophoresis of protein expressed by E.coli BL21(DE3) / pET28a-kcPA bacteria;

[0036] Figure 6 This is the HPLC chromatogram of the one-step synthesis of amoxicillin by KcPA-catalyzed penicillin potassium salt in Example 3;

[0037] Figure 7 It is a liquid chromatogram of amoxicillin crystals;

[0038] Figure 8 This is a liquid chromatogram of crystalline phenylacetic acid. DETAILED DESCRIPTION

[0039] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in this application to express the amount, percentage, and other numerical values ​​should be understood as 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%.

[0040] When the temperature is not particularly limited in the following embodiments of the present invention, it is all room temperature conditions. Room temperature refers to the natural room temperature conditions in the four seasons, without additional cooling or heating treatment, and is generally 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 that the medium contains 50μg / mL karanomycin.

[0041] In the examples, the experimental methods without specifying the specific conditions are usually carried out according to conventional conditions, such as the methods described in the Molecular Cloning Laboratory Manual (Chinese version) (J. Sambrook, MR Green, ed., translated by He Fu. Fourth edition, Beijing: Science Press, 2017) and New England Biolabs (NEB) kits.

[0042] The present invention discloses a method for separating amoxicillin and phenylacetic acid from a reaction solution of preparing amoxicillin by an enzyme-catalyzed one-step method. The main contents include: firstly, 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 the 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 toluene is used for extraction 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.

[0043] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0044] Example 1

[0045] Construction, prokaryotic expression and functional characterization of a mutant of penicillin acylase from Kluyvera citrophila

[0046] 1. Construction of wild-type PA expression vector pET28a-kcPA

[0047] 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. The amino acid sequence consists of four parts, which are: a signal peptide at positions 1 to 26, an α subunit consisting of 209 amino acids at positions 27 to 235, an intermediate connecting 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.

[0048] The schematic diagram of the construction of the recombinant plasmid pET28a-kcPA is as follows Figure 3 Using the genome of K. citrophila ATCC21285 as a template, primers were designed based on the PA nucleotide sequence (SEQ ID NO.2), and 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. EcoR I and HindIII restriction enzyme sites were introduced respectively (the underlined bases are restriction endonuclease recognition sites), and the wild-type PA target fragment was amplified by PCR.

[0049] PCR reaction system:

[0050]

[0051] The PCR temperature program was designed as follows:

[0052]

[0053] Select EcoRI and HindIII restriction endonucleases to double-digest the blank plasmid pET28a vector and the target fragment. Double-digestion system:

[0054]

[0055]

[0056] The double enzyme digestion was reacted 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 the concentrations were estimated based on the gel electrophoresis diagram, and 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 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 was performed on it. 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 vector

[0062] In this example, 18 mutants were obtained by site-directed mutagenesis, as shown in the following table, where "F146αK" indicates that the 146th amino acid on the α subunit mutated from F to K, and 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 (Q5SDM 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, then dissolved in sterile water, and then operated according to the kit. As follows:

[0080] ①Use PCR to mutate the corresponding sites

[0081] PCR reaction system:

[0082]

[0083] Total volume 25 μL

[0084] Cycle program temperature:

[0085]

[0086] For mutants with more than two mutation sites, the PCR product of 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

[0088] The reaction system is as follows:

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

[0090] React at room temperature for 5 min.

[0091] ③ Heat shock transformation

[0092] Add 5 μL of KLD reaction mixture to 50 μL of chemical competent cell E. coli BL21 (DE3) suspension, incubate on ice for 30 min, 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 bacterial suspension on Kan50 LB plate, incubate at 37°C overnight, and the single colonies grown are the corresponding mutant expression strains, named E. coli BL21 (DE3) / pET28a-kcPA01~18

[0093] ④ Mutant identification

[0094] The mutant expression strain was inoculated into 25 mL of LB liquid medium containing Kan50, cultured overnight at 37°C, and the plasmid was extracted using a plasmid extraction kit. The plasmid was sent to a third-party biological company for sequencing to confirm that the corresponding product was the target product of the site-directed mutation.

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

[0096] 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 a single colony and inoculate it into 25 mL of LB liquid culture medium with Kan50, and culture it 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 culture medium with Kan50, culture it at 37 ° C and 280 rpm on a shaker, and monitor the OD 600, when it reaches 0.6-0.8, add IPTG solution to make the IPTG induction concentration 0.3mM, induce expression in a shaker at 25℃ and 220rpm for 10h, and collect the bacteria by centrifugation. The collected bacterial cells were suspended in pH7.5 PBS buffer and pre-cooled on ice for 10min, and then centrifuged at 4℃ and 12000rpmin for 6min to collect the bacteria. Add 50mM pH 7.5 PBS buffer to resuspend the bacteria in the centrifuge tube, and then centrifuge at 4℃ and 12000rpm for 6min, discard the supernatant, collect the final bacteria and resuspend them at a concentration of 0.01g / mL, and use an ultrasonic disruptor to disrupt the cells. The cell disruption conditions are: ice water bath, at 400W power, each cycle works for 3s, rests for 5s, and 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.

[0097] Figure 5 It is the SDS-PAGE diagram of the protein expressed by the bacteria; wherein, lane M is the 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.coliBL21(DE3) / pET28a-kcPA18 supernatant.

[0098] Example 2

[0099] 1. Determination of KcPA hydrolysis activity

[0100] The principle of the assay is: penicillin (PGK) potassium salt is hydrolyzed under the action of KcPA to generate 6-aminopenicillanic acid (6-APA) and phenylacetic acid. 6-APA reacts with p-dimethylaminobenzaldehyde (PDAB) under acidic conditions to generate a yellow-green substance with a maximum absorption peak at 415nm. Enzyme activity definition: At 28°C and 0.1M PBS buffer, the amount of enzyme required for penicillin acylase to catalyze 20mg / mL PGK to generate 1μmol 6-APA per minute is 1 unit of KcPA enzyme activity, the unit is U.

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

[0102] The reaction system was placed in a water bath shaker at 28°C and 200 rpm for 10 minutes. After the reaction, the enzyme was inactivated in a water bath at 90°C for 2 minutes. 200 μL of the reaction solution was added to 3 mL of pH 3.0, 0.1 M sodium citrate buffer, and 1 mL of a colorimetric solution (0.5% PDAB). After standing at room temperature for 3 minutes, the absorbance was measured at 415 nm. The 6-APA concentration in the sample after the reaction was obtained according to the 6-APA standard curve, and the enzyme activity, i.e., the hydrolysis activity, was calculated according to the formula.

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

[0104] In the formula, 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.

[0105] 2. Determination of KcPA synthesis activity

[0106] 6-aminopenicillanic acid (6-APA) and 4-hydroxyphenylglycine methyl ester (DHPGM) synthesize amoxicillin under the action of KcPA. The amoxicillin content can be determined by high performance liquid chromatography to calculate the PA synthesis activity. Enzyme activity is defined as: under certain conditions, the unit of penicillin acylase catalyzes the production of 1 μmol of amoxicillin per minute, which is 1 unit of synthetic enzyme activity, represented by U.

[0107] Weigh 1g 6-APA and 1.25g D-HPGM and dissolve them in 50mL 0.1M PBS buffer at 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.

[0108] HPLC detection conditions are: Agilent ZORBAX SB-C18 4.6x250 mm chromatographic column, column temperature 25°C. Injection volume 10μL. Mobile phase A (0.02M pH4.7 NaH2PO4-Na2HPO4 buffer), mobile phase B (methanol), initially 90% mobile phase A and 10% mobile phase B for 5 min, 5min to 7min mobile phase B increased from 10% to 50% and then maintained for 10 min, 17min to 19min mobile phase B decreased from 50% to 10%, and finally 90% mobile phase A and 10% mobile phase B were balanced for 5 min, and the total flow rate was 1mL / min.

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

[0110]

[0111]

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

[0113] As can be seen from the above table, for mutants with single mutation sites, the hydrolysis activity and synthesis activity of each mutant are 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 are 5.8 times and 15.3 times that of the wild type, respectively; the hydrolysis activity and synthesis activity of the G385βR mutant are 4.6 times and about 11.2 times that of the wild type, respectively. Compared with the G385βR mutant, the G385βY mutant has higher hydrolysis activity, but its synthesis activity is not outstanding. When mutations are superimposed at each mutation site, the enzyme activity of the mutant is increased compared with the single-point mutation, especially the five-point mutant F146αK&F24βR&F71βY&N241βK&G385βR, which has higher hydrolysis activity and synthesis activity.

[0114] Example 3

[0115] One-step synthesis of amoxicillin by PGK catalyzed by various mutants and wild-type penicillin acylase

[0116] PGK was added to a pH 7.0 PBS buffer to a concentration of 200 mM, and p-hydroxyphenylglycine methyl ester (D-HPGM) was added to a final concentration of 300 mM. The amount of enzyme added was 30 U / mL (calculated based on the activity of the synthase), and the reaction was stirred at a constant temperature of 28°C for 3 hours. After the reaction was completed, HPLC detection was performed to calculate the yield of amoxicillin.

[0117] HPLC detection conditions are: Agilent ZORBAX SB-C18 4.6x250 mm chromatographic column, column temperature 25°C. Injection volume 10μL. Mobile phase A (0.02M pH4.7 NaH2PO4-Na2HPO4 buffer), mobile phase B (methanol), initially 90% mobile phase A and 10% mobile phase B for 5 minutes, 5min to 7min mobile phase B increased from 10% to 50% and then maintained for 10 minutes, 17 to 19min mobile phase B decreased from 50% to 10%, and finally 90% mobile phase A and 10% mobile phase B were balanced for 5 minutes, and the total flow rate was 1mL / min. The reaction formula is as follows:

[0118]

[0119] The HPLC detection spectrum of mutant KcPA 18 is as follows Figure 6 As shown in the figure, DHPG is D-p-hydroxyphenylglycine, AMOX is amoxicillin, DHPGM is D-p-hydroxyphenylglycine methyl ester, PAA is phenylacetic acid, and PGK is penicillin potassium salt. It can be seen from the figure that the content of the intermediate product 6-APA is extremely low, almost non-existent.

[0120] Table 3 The yield of amoxicillin synthesized 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 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.

[0124] Example 4 Preparation of immobilized penicillin acylase

[0125] 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 hours. The cross-linking reaction system was as follows: the enzyme amount of the enzyme solution was 1200U (calculated as synthetase activity), the glutaraldehyde content was 0.25%, ER 5g, 50mL pH7.5 potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer. After the cross-linking reaction was completed, the immobilized enzyme was collected by sieve filtration. The immobilized enzyme was then washed with 100mL pH7.5 potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer. The activity of the immobilized enzyme was 180U / g, and the recovery rate of the immobilized enzyme activity reached 75%.

[0126] Example 5 KcPGA18 catalyzes PGK to synthesize amoxicillin in one step

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

[0128] (2) Weigh a certain amount of penicillin potassium salt (PGK) and D-p-hydroxyphenylglycine methyl ester (DHPGM) respectively and put them into the reaction buffer system, the molar ratio of PGK to DHPGM is 1:1-1:2, the concentration of PGK is 50-200 mmol / L, and the concentration of DHPGM is 50-400 mmol / L, and then stir well to make the reactant PGK and the DHPGM substrate uniformly dispersed in the reaction system. In this embodiment, the final concentrations of PGK and DHPGM are 200 and 300 mmol / L, respectively.

[0129] (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 the enzyme amount was 20 U / mL (calculated based on the activity of the synthetase), and was added into the reaction bottle. The reaction temperature was controlled at 24° C. and the reaction time was 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 50 mL of the reaction suspension obtained in Example 5.

[0133] (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 form a total of 150 mL.

[0134] (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.

[0135] (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.

[0136] (5) After the crystallization is completed, the amoxicillin crystals and the liquid phase containing phenylacetic acid are separated by suction filtration. Finally, the amoxicillin crystals are placed in an oven at 85° C. and dried for 30 min.

[0137] 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 the volume was adjusted to 50 mL to obtain a 2 mg / mL solution, which was then subjected to high performance liquid chromatography detection under the same detection conditions as in Example 3. The results are shown in FIG. Figure 7,It can be seen from the figure that: except the AMOX peak, there are no other obvious impurity peaks, which basically confirms that the crystal is amoxicillin.

[0138] 2. Separation and crystallization of phenylacetic acid

[0139] (1) Take the above liquid phase containing phenylacetic acid (150 mL), 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 extractant, extract at 25°C, stir for 15 min, and then stand to separate the layers, so that phenylacetic acid is extracted from the aqueous phase into the organic phase 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 thoroughly. Allow to stand for stratification (or centrifuge for stratification). Phenylacetic acid is converted into 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).

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

[0143] (5) After the crystallization is completed, the filtrate is filtered and the filter residue is the phenylacetic acid crystal. Finally, the phenylacetic acid crystal is placed in a vacuum drying oven at 40° C. and dried for 30 min.

[0144] In order to verify whether the crystalline crystals are phenylacetic acid, in this example, 0.1 g of the crystalline crystals were dissolved in deionized water and the volume was adjusted to 50 mL to obtain a 2 mg / mL solution, which was then tested by high performance liquid chromatography under the same testing 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.

[0145] 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 protection scope of the present invention.

Claims

1. A method for separating amoxicillin and phenylacetic acid from a reaction solution of preparing amoxicillin by an enzyme-catalyzed one-step method, characterized in that: The method uses an immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from penicillin potassium to obtain a reaction suspension, and separates and treats the reaction suspension, comprising the following steps: (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 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) Add NaOH solution to the mixed solution to be separated until the pH value reaches 3.5-5.5, and allow to stand at 4°C for crystallization; (5) After the crystallization is completed, filtering and separating to obtain amoxicillin crystals and a liquid phase containing phenylacetic acid; The method for synthesizing amoxicillin from penicillin potassium in one step by using an immobilized penicillin acylase mutant to catalyze the synthesis comprises: using only one immobilized penicillin acylase mutant as the only enzyme in a reaction system, taking penicillin or its salt and D-p-hydroxyphenylglycine methyl ester as substrates, and reacting in a buffer system of pH 4 to 8; the amino acid sequence of the penicillin acylase mutant is compared with the amino acid sequence shown in SEQ ID NO.1, and the mutation pattern is: F146αK & F24βR & F71βY & N241βK & G385βR.

2. The method for separating amoxicillin and phenylacetic acid from the reaction solution of preparing amoxicillin by an enzyme-catalyzed one-step method according to claim 1, characterized in that: Step (5) may further include 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) using a NaOH solution to back-extract the organic phase containing phenylacetic acid, and 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, and converting the sodium phenylacetate into phenylacetic acid, which crystallizes at 4°C; (10) After the crystallization is completed, the phenylacetic acid crystals are recovered by filtration and separation.

3. The method for separating amoxicillin and phenylacetic acid from the reaction solution of preparing amoxicillin by an enzyme-catalyzed one-step method according to claim 1, characterized in that: The concentration of hydrochloric acid in step (3) is 15% by volume.

4. The method for separating amoxicillin and phenylacetic acid from the reaction solution of preparing amoxicillin by an enzyme-catalyzed one-step method according to claim 1, characterized in that: In step (4), the pH of amoxicillin crystallization is 5 and the crystallization time is 9 hours.

5. The method for separating amoxicillin and phenylacetic acid from the reaction solution of preparing amoxicillin by an enzyme-catalyzed one-step method according to claim 1, characterized in that: In step (5), 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 amoxicillin and phenylacetic acid from the reaction solution of preparing amoxicillin by an enzyme-catalyzed one-step method 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 amoxicillin and phenylacetic acid from the reaction solution of preparing amoxicillin by an enzyme-catalyzed one-step method according to claim 2, characterized in that: In step (6), extract with toluene for more than 2 times and combine the organic phases.

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