A method for synthesizing amoxicillin from penicillin or its salt by one-step enzyme catalysis

Abstract

The present invention belongs to the technical field of semi-synthetic antibiotic preparation, and more particularly to a method for synthesizing amoxicillin from penicillin or its salt by an enzyme-catalyzed one-step method. The current synthesis of amoxicillin mainly utilizes hydrolysis penicillin acylase and synthesis penicillin acylase to catalyze respectively, and the intermediate product 6-APA needs to be separated, and the production economic cost is high, and the production efficiency is low. The present invention utilizes a mutant of Kluyvera citrophila penicillin acylase as the only enzyme in the reaction system, with penicillin potassium salt and D-p-hydroxyphenylglycine methyl ester as substrates, to achieve amoxicillin one-step synthesis, and the product yield can reach 99%. The method has the advantages of fast catalytic rate, high product yield, green environmental protection of pure water phase reaction system, low cost and good economic benefit.

Description

Technical Field

[0001] The invention belongs to the technical field of semi-synthetic antibiotic preparation, and particularly relates to a method for synthesizing amoxicillin from penicillin or its salt in a one-step enzyme-catalyzed method. Background Art

[0002] Amoxicillin, also known as amoxicillin, is a major type of second-generation penicillin. It can inhibit 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 main methods for synthesizing amoxicillin are chemical synthesis and enzyme-catalyzed synthesis. The chemical process is relatively mature, but it has obvious disadvantages. First, the long process route leads to low overall efficiency; second, a large amount of organic solvents are used in the production process, which will produce more toxic and harmful wastes and cause great pollution to the environment; third, the reaction is carried out at low temperature, and the energy consumption and cost are high. Compared with chemical synthesis, in the case of enzyme-catalyzed preparation, due to the mild process conditions and green environmental protection, the quality of the prepared amoxicillin is better than that of chemical synthesis, so the enzyme-catalyzed synthesis of amoxicillin has been increasingly studied and applied. The enzyme-catalyzed synthesis of amoxicillin currently consists of a two-step process, that is, firstly, penicillin (or penicillin salt) is catalyzed by hydrolysis penicillin acylase (PA) to prepare 6-APA (6-aminopenicillanic acid), and then the synthetic penicillin acylase is catalyzed by 6-APA and D-p-hydroxyphenylglycine (or D-p-hydroxyphenylglycine methyl ester) to synthesize amoxicillin (such as application number 201711221286.6, a method for directly synthesizing amoxicillin; application number 201811490254.0, a method for enzymatic synthesis of amoxicillin). However, the two-step process requires separation of the intermediate product, which greatly increases the economic cost of production, and the production efficiency is low. At present, it has been reported that amoxicillin is synthesized in one pot by two enzymes, penicillin acylase for hydrolysis and penicillin acylase for synthesis (WO 2022 / 195603), but this method requires the synergistic action of the two enzymes to obtain a better effect. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention has created a method for synthesizing amoxicillin from penicillin potassium in one step using a single enzyme. The method has the advantages of fast catalytic rate, high product yield, green and environmentally friendly pure water phase reaction system, low cost and good economic benefits.

[0005] The present invention is achieved by a one-step enzyme-catalyzed method for synthesizing amoxicillin from penicillin potassium, using only a penicillin acylase mutant as the only enzyme in the reaction system, taking penicillin or its salt (preferably penicillin potassium) and D-p-hydroxyphenylglycine methyl ester as substrates, and reacting in a buffer system of pH 4 to 8 (preferably pH 4 to 7); the amino acid sequence of the penicillin acylase mutant, compared with the amino acid sequence shown in SEQ ID NO.1, comprises at least one of the following mutations: F146αK, F24βR, F71βY, N241βK, G385βY or G385βR.

[0006] The specific steps include:

[0007] S1: Add a pH 4-8 buffer into the reaction bottle as a reaction buffer system;

[0008] 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;

[0009] 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.

[0010] 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.

[0011] 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.

[0012] Furthermore, in step S3, the amount of enzyme is 3000 to 30000 U / L, and the reaction time is 1 to 6 hours.

[0013] Furthermore, step S3 further includes a step of recovering the immobilized penicillin acylase mutant.

[0014] Furthermore, the step of recovering the immobilized penicillin acylase mutant comprises:

[0015] S4: filtering out the immobilized penicillin acylase using a mesh;

[0016] S5: Add 20-30 mL of deionized water for every 10 g of immobilized enzyme, stir for 5-10 min, drain and rinse repeatedly until the released water is clear and transparent and the enzyme sample has no odor.

[0017] Furthermore, step S5 further includes S6: adding 0.5‰ (mass to volume ratio) of p-hydroxybenzoate solution to the buffer solution, covering the immobilized enzyme, and sealing and storing at 2-8°C.

[0018] Furthermore, when long-term storage is required, the cleaned immobilized penicillin acylase mutant is disinfected with 5‰ glutaraldehyde solution and the enzyme is filtered out; 0.5‰ (mass volume ratio) of p-hydroxybenzoate solution is added to the buffer, the immobilized enzyme is covered, and the solution is sealed and stored at 2-8°C.

[0019] In summary, the advantages and positive effects of the present invention are as follows: the present invention uses a mutant of penicillin acylase from Kluyvera citrophila as the only enzyme in the reaction system, uses penicillin potassium salt and D-p-hydroxyphenylglycine methyl ester as substrates, realizes one-step synthesis of amoxicillin, and the product yield can reach up to 100%. The method has the advantages of fast catalytic rate, high product yield, green and environmentally friendly pure water phase reaction system, low cost and good economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the wild-type penicillin acylase amino acid sequence;

[0021] Figure 2 is a schematic diagram of the construction of the recombinant plasmid pET28a-kcPA;

[0022] Figure 3 This is the agarose electrophoresis detection diagram of the recombinant plasmid PCR;

[0023] Figure 4 This is the SDS-PAGE electrophoresis of protein expressed by E.coli BL21(DE3) / pET28a-kcPA bacteria;

[0024] Figure 5 This is the HPLC chromatogram of the one-step synthesis of amoxicillin by KcPA-catalyzed penicillin potassium salt in Example 3. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] Based on the information contained in this application, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined process, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention. In fact, various changes that the technicians in this field or related fields can obviously make to the embodiments of the present invention are all covered within the scope of the appended claims.

[0027] 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%.

[0028] 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.

[0029] 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.

[0030] The present invention discloses a method for synthesizing amoxicillin from penicillin or its salt by an enzyme-catalyzed one-step method. The enzyme used in the present invention is a mutant of penicillin acylase (PA) of Kluyvera citrophila, and the penicillin acylase mutant is used to catalyze the one-step synthesis of amoxicillin from penicillin potassium. The technical solution provided by the present invention can avoid the separation of intermediates, greatly simplify the production process, and at the same time improve the yield of the reaction product, significantly save production costs, and bring about a revolutionary change in the amoxicillin synthesis industry. The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0031] Example 1

[0032] Construction, prokaryotic expression and functional characterization of a mutant of penicillin acylase from Kluyvera citrophila

[0033] 1. Construction of wild-type PA expression vector pET28a-kcPA

[0034] 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 1 , 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.

[0035] The schematic diagram of the construction of the recombinant plasmid pET28a-kcPA is as follows Figure 2 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 / AGCTTTTAGCGCTGCACCTGCAGC-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.

[0036] PCR reaction system:

[0037]

[0038] The PCR temperature program was designed as follows:

[0039]

[0040]

[0041] Select EcoRI and HindIII restriction endonucleases to double-digest the blank plasmid pET28a vector and the target fragment. Double-digestion system:

[0042]

[0043] 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.

[0044] 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.

[0045] Target fragment and linearized vector connection system:

[0046]

[0047] 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 3 ). The verified expression vector pET28a-kcPA was transformed into BL21 (DE3) to obtain the wild-type PA expression recombinant bacteria E. coli BL21 (DE3) / pET28a-kcPA.

[0048] 2. Obtaining mutant expression vector

[0049] 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.

[0050] Table 1 Mutants and corresponding mutation sites

[0051]

[0052]

[0053] 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):

[0054] F146αK, F: 5'-GGCGAACCGTaaaTCTGACAGCACCAG-3', SEQ ID NO.5;

[0055] R: 5'-ATGGTGCCGACAAAAATCATCGCCA-3', SEQ ID NO.6;

[0056] F24βR, F: 5'-TGGGCCGCAGcgcGGTTGGTATGCG-3', SEQ ID NO.7,

[0057] R: 5'-TTGACCATAATGGCCTTCGCATCCT-3', SEQ ID NO.8;

[0058] F71βY, F: 5'-CACCGCCGGTtatGGTGATGATG-3', SEQ ID NO.9,

[0059] R: 5'-GATCCCCCATGAAATGGTGCCGTTGT-3', SEQ ID NO.10;

[0060] N241βK, F: 5'-CGCCAACTGGaaaAACTCGCCGC-3', SEQ ID NO.11,

[0061] R: 5'-ATATAGCCCGACTGCGGGTTATACAC-3', SEQ ID NO. 12;

[0062] G385βY: F: 5'-CGGGCCAACCtatTCGCTGAACATCAGCGTG-3', SEQ ID NO.13,

[0063] R: 5'-TCCTGGGTGGTTTCATAGCCACTGG-3', SEQ ID NO.14;

[0064] G385βR, F: 5'-CGGGCCAACCcgcTCGCTGAACATC-3', SEQ ID NO.15,

[0065] R: 5'-TCCTGGGTGGTTTCATAGCCACTGG-3', SEQ ID NO.16;

[0066] The primers were synthesized by a nucleic acid synthesis company, then dissolved in sterile water, and then operated according to the kit. As follows:

[0067] ①Use PCR to mutate the corresponding sites

[0068] PCR reaction system:

[0069]

[0070] Cycle program temperature:

[0071]

[0072] 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.

[0073] ② Kinase, Ligase & DpnI (KLD) (a special mixture of kinase, ligase and DpnI) reaction treatment

[0074] The reaction system is as follows:

[0075] volume Final concentration PCR products 1μL 2XKLDReactionBuffer 5μL 1X 10XKLDEnzymeMix 1μL 1X Nuclease-free water 3μL

[0076] React at room temperature for 5 min.

[0077] ③ Heat shock transformation

[0078] 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

[0079] ④ Mutant identification

[0080] 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.

[0081] 3. Expression of wild-type and mutant KcPA

[0082] 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.

[0083] Figure 4 The SDS-PAGE image of the protein expressed by the mutant E.coli BL21(DE3) / pET28a-kcPA18 is given in the figure; wherein, lane M is the protein marker; Lane 1: E.coli BL21(DE3) / pET28a expression supernatant; Lane 2: E.coli BL21(DE3) / pET28a-kcPA uninduced supernatant; Lane 3: E.coli BL21(DE3) / pET28a-kcPA18 uninduced supernatant; Lane 4: IPTG-induced E.coli BL21(DE3) / pET28a-kcPA18 supernatant.

[0084] Example 2

[0085] 1. Determination of KcPGA hydrolysis activity

[0086] 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.

[0087] 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.

[0088] 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.

[0089] Calculation formula: Penicillin acylase hydrolase activity per mL

[0090] 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.

[0091] 2. Determination of KcPA synthesis activity

[0092] 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.

[0093] 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.

[0094] 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.

[0095] Table 2 Comparison of activity between mutants and wild type:

[0096]

[0097]

[0098] 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.

[0099] 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 significantly 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.

[0100] Example 3

[0101] One-step synthesis of amoxicillin by PGK catalyzed by various mutants and wild-type penicillin acylase

[0102] 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.

[0103] 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:

[0104]

[0105] The HPLC detection spectrum of mutant KcPA 18 is as follows Figure 5As 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.

[0106] Table 3 The yield of amoxicillin synthesized by each mutant

[0107]

[0108] 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.

[0109] Example 4 Preparation of immobilized penicillin acylase

[0110] Penicillin acylase enzyme solution was prepared according to Example 1. The enzyme solution was added with epoxy resin (ER) activated by glutaraldehyde and cross-linked for 1.5 hours at 15°C. The cross-linking reaction system was as follows: 1200U of enzyme solution, 0.25% of glutaraldehyde content, 5g of ER, and 50mL of 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 of 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 was 75%.

[0111] Example 5-17 Optimization of process conditions for one-step synthesis of amoxicillin using PGK catalyzed by KcPGA18

[0112] The synthesis process includes the following steps:

[0113] (1) Add a certain amount of PBS or other buffer with a pH of 4 to 8 into the reaction bottle as the reaction buffer system.

[0114] (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 50-200mmol / L, and the concentration of DHPGM is 50-400mmol / L. Then stir thoroughly to make the reactant PGK and the DHPGM substrate evenly dispersed in the solvent.

[0115] (3) Accurately weigh a certain amount of immobilized penicillin acylase with synthetic catalytic performance, and put it into the reaction bottle with an enzyme amount of 3000-30000 U / L (calculated based on the activity of the synthase), and control the reaction temperature to 12-30°C and the reaction time to 1-6 h.

[0116] (4) After the reaction is completed, 2 to 5 volumes of deionized water are added to the reaction system, and then 15% (mass volume ratio) phosphoric acid solution is added dropwise. The addition is stopped when the entire system is observed to be clear and transparent.

[0117] (5) Filter out the immobilized penicillin acylase and rinse it with deionized water for 2-3 times, and mix the collected washing liquid with the filtrate thoroughly.

[0118] (6) The filtered immobilized penicillin acylase is soaked in deionized water for 15-20 min (3-4 drops of phosphoric acid can be added during the soaking period, and the weak acidity can maintain the enzyme activity), then rinsed with deionized water 2-3 times, and the surface moisture is absorbed with absorbent paper (or blown dry with cold air), and stored in a refrigerator at 2-6°C for recycling.

[0119] The HPLC detection conditions are the same as those in Example 3.

[0120] The experimental results are shown in the following table:

[0121]

[0122] Example 18 Enzyme Recovery and Recycling

[0123] The filtered immobilized penicillin acylase was soaked in deionized water for 20 minutes, and 20-30 mL of deionized water was added for every 10 g of immobilized enzyme, and stirred for 5-10 minutes. After being drained, it was washed repeatedly until the released water was clear and transparent and the enzyme sample had no odor. 0.5‰ of p-hydroxybenzoate solution was added to the buffer used, covering the immobilized enzyme, and stored in a refrigerator at 4°C for recycling.

[0124] The recovered enzyme was added into a pH 7 PBS reaction system containing PGK 200mmol / L, DHPGM 300mmol / L, and an enzyme amount of 25000U / L, and reacted at 26°C for 3h. The yield of amoxicillin reached 98%.

[0125] Example 19 Enzyme Recovery and Recycling

[0126] According to "The filtered immobilized penicillin acylase was soaked in deionized water for 20 minutes, 20-30 mL of deionized water was added for every 10 g of immobilized enzyme, stirred for 5-10 minutes, and washed repeatedly after being drained until the released water was clear and transparent and the enzyme sample had no odor." The recovery cycle was repeated 20 times and the following reaction was carried out.

[0127] The recovered enzyme was added into a pH 7 PBS reaction system containing PGK 200mmol / L, DHPGM 300mmol / L, and an enzyme amount of 25000U / L and reacted at 26°C for 3h. The yield of amoxicillin reached 92%.

[0128] 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 synthesizing amoxicillin from penicillin or its salt by an enzyme-catalyzed one-step process, characterized in that: Only one penicillin acylase mutant is used as the only enzyme in the reaction system, penicillin or its salt and D-p-hydroxyphenylglycine methyl ester are used as substrates, and the reaction is carried out in a buffer system of pH 4-8; compared with the amino acid sequence shown in SEQ ID NO.1, the amino acid sequence of the penicillin acylase mutant is mutated in any of the following ways: F146αK; or F24βR; or F71βY; or N241βK; or G385βY; or G385βR; or F146αK & F24βR; or F146αK & F71βY; or F146αK & N241βK; or F146αK & G385βY; or F146αK & G385βR; or F146αK & F24βR & F71βY; or F146αK & N241βK & G385βY; or F146αK & N241βK & G385βR; or F146αK & F71βY & N241βK; or F146αK& F71βY & N241βK & G385βY; or F146αK & F24βR & F71βY & N241βK & G385βY; or F146αK& F24βR & F71βY & N241βK & G385βR.

2. The method for synthesizing amoxicillin from penicillin or its salt by an enzyme-catalyzed one-step method according to claim 1, characterized in that: The following steps are involved: S1: Add pH 4-8 buffer into the reaction bottle as the reaction buffer system; 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 1:1-1:2, and stirring thoroughly; S3: adding the immobilized penicillin acylase mutant to the reaction buffer system, controlling the reaction temperature at 12-30°C, and carrying out the reaction.

3. The method for synthesizing amoxicillin from penicillin or its salt by an enzyme-catalyzed one-step method according to claim 2, characterized in that: 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.

4. The method for synthesizing amoxicillin from penicillin or its salt by an enzyme-catalyzed one-step method according to claim 2, characterized in that: 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.

5. The method for synthesizing amoxicillin from penicillin or its salt by an enzyme-catalyzed one-step method according to claim 2, characterized in that: In step S3, the enzyme amount is 3000-30000 U / L, and the reaction time is 1-6 h.

6. The method for synthesizing amoxicillin from penicillin or its salt by an enzyme-catalyzed one-step method according to claim 2, characterized in that: Step S3 further includes a step of recovering the immobilized penicillin acylase mutant.

7. The method for synthesizing amoxicillin from penicillin or its salt by an enzyme-catalyzed one-step method according to claim 6, characterized in that: The step of recovering the immobilized penicillin acylase mutant comprises: S4: filtering out the immobilized penicillin acylase using a mesh; S5: Add 20-30 mL of deionized water for every 10 g of immobilized enzyme, stir for 5-10 min, drain and rinse repeatedly until the released water is clear and transparent and the enzyme sample has no odor.

8. The method for synthesizing amoxicillin from penicillin or its salt by an enzyme-catalyzed one-step method according to claim 7, characterized in that: Step S5 further includes S6: covering the immobilized enzyme with a buffer solution containing 0.5‰ of a p-hydroxybenzoate solution, and sealing and storing at 2-8°C.

9. The method for synthesizing amoxicillin from penicillin or its salt by an enzyme-catalyzed one-step method according to claim 7, characterized in that: Step S5 also includes S6: using 5‰ glutaraldehyde solution to disinfect the cleaned immobilized penicillin acylase mutant and filter out the enzyme; covering the immobilized enzyme with a buffer solution containing 0.5‰ p-hydroxybenzoate solution, and sealing and storing at 2-8°C.

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