An artificially designed penicillin g acylase, encoding nucleotide and application thereof

Abstract

This invention belongs to the field of enzyme catalysis technology, and particularly relates to an artificially designed penicillin G acylase, its encoding nucleotide, and its applications. Compared with the amino acid sequence shown in SEQ ID NO.1, the mutant exhibits the following mutations: G385βY; or G385βR; or F146αK&N241βK&G385βY. This invention, through mutation of penicillin acylase derived from Kluyvera citrophila, yielded a penicillin acylase mutant with strong hydrolytic and synthetic activities, exhibiting coordinated activities of both enzymes. It can be used in the synthetic production of β-lactam antibiotics, particularly catalyzing the one-step preparation of amoxicillin from potassium penicillin, avoiding the separation of the intermediate 6-APA. This invention provides a key enzyme for the efficient preparation of β-lactam antibiotics, which will greatly advance the technological innovation of β-lactam antibiotic preparation.

Description

[0001] This application is a divisional application of Chinese invention patent application number 202411396069.0, filed on October 8, 2024, entitled "A penicillin acylase mutant and its application". Technical Field

[0002] This invention belongs to the field of enzyme catalysis technology, and particularly relates to an artificially designed penicillin G acylase, its encoded nucleotide, and its applications. Background Technology

[0003] β-lactam antibiotics are a large class of antibiotics with a β-lactam ring in their chemical structure. These antibiotics have advantages such as strong bactericidal activity, low toxicity, broad indications, and good clinical efficacy. However, naturally occurring β-lactam antibiotics (such as penicillin and cephalosporins) have disadvantages such as a narrow antibacterial spectrum, insensitivity to acid, easy development of drug resistance, and a tendency to cause allergic reactions. Semi-synthetic β-lactam antibiotics, such as semi-synthetic penicillin and semi-synthetic cephalosporins, have overcome these disadvantages and have become the main antibiotics used in the current medical field.

[0004] Utilizing enzyme catalysis to prepare semi-synthetic β-lactam antibiotics offers a more efficient, green, and sustainable production method, yielding products of superior quality compared to traditional chemical synthesis methods. Penicillin acylase is the key enzyme catalyzing the synthesis of these products. The current enzyme-catalyzed synthesis of semi-synthetic penicillins, such as ampicillin and amoxicillin, mainly consists of a two-step process: first, hydrolysis using penicillin acylase catalyzes the reaction of penicillin and its salt (to prepare 6-APA (6-aminopenicillanic acid)); then, synthesis using penicillin acylase catalyzes the reaction of 6-APA with an acyl donor side chain (such as D-p-hydroxyphenylglycine methyl ester) to prepare the semi-synthetic β-lactam antibiotic. Semi-synthetic cephalosporins primarily involve the reaction of cephalosporin intermediates, such as 7-ACA and 7-ADCA, with an acyl donor, catalyzed by penicillin acylase. Penicillin acylase (Penicillin Acylase...) PA (EC 3.5.1.11) is the key enzyme in this preparation technology. Current technologies all use two enzymes—a penicillin acylase for hydrolysis and a penicillin acylase for synthesis—to catalyze a two-step reaction: the hydrolysis of 6-APA (or 7-ACA, 7-ADCA) and the synthesis of the intermediate core and side chain. This two-step reaction is not only lengthy but also requires the separation of intermediates, making the production process complex. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an artificially designed penicillin G acylase, its encoding nucleotide, and its applications, aiming to solve the problems in the prior art. The mutant provided by this invention can catalyze the reaction of β-lactam raw materials, including penicillin or cephalosporins, and activated acyl donors to synthesize semi-synthetic β-lactam antibiotics, including semi-synthetic penicillin and semi-synthetic cephalosporins, realizing a new technology for rapid and efficient synthesis of semi-synthetic β-lactam antibiotics.

[0006] This invention is achieved by providing a penicillin G acylase mutant, which, compared to the amino acid sequence shown in SEQ ID NO. 1, contains at least one of the following mutation sites: F146αK, F24βR, F71βY, N241βK, G385βY, and G385βR. Explanation of amino acid abbreviations: F: phenylalanine; K: lysine; R: arginine; Y: tyrosine; N: asparagine; G: glycine.

[0007] This invention also protects nucleotides encoding any of the above-mentioned penicillin acylase mutants.

[0008] The present invention also provides the application of the penicillin acylase mutant as described above in the preparation of β-lactam antibiotics.

[0009] Furthermore, β-lactam antibiotics include semi-synthetic penicillins and semi-synthetic cephalosporins. Semi-synthetic penicillins include amoxicillin, ampicillin, or pimecrolimus; semi-synthetic cephalosporins include cephalexin, cefprozil, cefaclor, cefadroxil, cefamandole, cefazolin, cefnicotinic acid, cefotaxime, or cefotaxime.

[0010] Furthermore, by using only one of the penicillin acylase mutants as the sole penicillin acylase in the reaction system, the reaction of penicillin potassium salt with the acyl donor is catalyzed to synthesize a semi-synthetic penicillin β-lactam antibiotic in one step.

[0011] Furthermore, the acyl donor includes methyl phenylglycine or methyl p-hydroxyphenylglycine.

[0012] Furthermore, using any of the penicillin acylase mutants as penicillin acylases, catalyzing the reaction of 7-ACCA or 7-ADCA with methyl phenylglycine, a semi-synthetic cephalosporin β-lactam antibiotic is synthesized.

[0013] This invention mutates the amino acids in the substrate-binding region of wild-type penicillin acylase to improve its binding performance to the substrate and reduce substrate binding steric hindrance. At the same time, it lowers the free energy of the substrate-active site transition state in the active center to promote the conversion of the intermediate state to the product, thereby promoting the accumulation of the final product and ultimately significantly improving the ability of penicillin acylase to synthesize semi-synthetic β-lactam antibiotics.

[0014] In summary, the advantages and positive effects of this invention are as follows: By mutating penicillin acylase derived from Kluyvera citrophila, this invention yields a penicillin acylase mutant with strong hydrolytic and synthetic activities, coordinated activity between the two, and better stability. This mutant can be used for the synthesis of β-lactam antibiotics such as semi-synthetic penicillins (e.g., amoxicillin, ampicillin, or pimecrolimus), semi-synthetic cephalosporins (e.g., cephalexin, cefprozil, cefaclor, cefadroxil), and for the one-step preparation (synthesis) of amoxicillin and ampicillin from potassium penicillin, avoiding the separation of intermediates such as 6-APA. This invention provides a key enzyme for the efficient preparation of β-lactam antibiotics, which will greatly advance the technological innovation of β-lactam antibiotic preparation. Attached Figure Description

[0015] Figure 1 It is the amino acid sequence of wild-type penicillin acylase;

[0016] Figure 2 This is a schematic diagram of the construction of the recombinant plasmid pET28a-kcPA;

[0017] Figure 3 This is a graph from recombinant plasmid PCR agarose gel electrophoresis detection.

[0018] Figure 4 This is an SDS-PAGE electrophoresis image of the protein expressed in E. coli BL21(DE3) / pET28a-kcPA cells;

[0019] Figure 5 This is the HPLC chromatogram of the one-step synthesis of amoxicillin from potassium penicillin catalyzed by KcPA in Example 3;

[0020] Figure 6 This refers to the changes in the content of each substance during the reaction process in Example 4;

[0021] Figure 7 This refers to the changes in the content of each substance during the reaction process in Example 5;

[0022] Figure 8 This refers to the changes in the content of each substance during the reaction process in Example 6;

[0023] Figure 9 This refers to the changes in the content of each substance during the reaction process in Example 7. Detailed Implementation

[0024] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "about". Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. In this invention, "about" means within 10%, preferably within 5%, of a given value or range.

[0025] Unless otherwise specified, all embodiments of the present invention are based on ambient temperature conditions. Ambient temperature refers to the natural room temperature during the four seasons, without additional cooling or heating treatment. Generally, ambient temperature is controlled between 10 and 30°C, preferably between 15 and 25°C. The abbreviations are as follows: "min" represents minutes, "s" represents seconds, "U" represents enzyme activity units, "mM" represents millimoles per liter, "M" represents moles per liter, "rpm" represents revolutions per minute, "mol" represents moles, "μg" represents micrograms, "mg" represents milligrams, "g" represents grams, "μL" represents microliters, "mL" represents milliliters, "bp" represents base pairs, LB medium represents Luria-Bertani medium, and Kan50 indicates that the medium contains 50 μg / mL kanamycin.

[0026] In the examples, experimental methods without specific conditions are generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (Chinese version) (edited by J. Sambrook and MR. Green, translated by He Fuchu, 4th edition, Beijing: Science Press, 2017) and the methods described in the New England Biolabs (NEB) kit.

[0027] This invention discloses a penicillin acylase mutant and its applications. The technical solution provided by this invention avoids the separation of intermediates, greatly simplifies the production process, improves the yield of reaction products, and significantly saves production costs, thus revolutionizing the semi-synthetic antibiotic industry. The technical solution of this invention will be clearly and completely described below with reference to embodiments.

[0028] Example 1

[0029] Construction, prokaryotic expression and functional identification of penicillin acylase mutants from Kluyvera citrophila

[0030] 1. Construction of wild-type PA expression vector pET28a-kcPA

[0031] The wild-type penicillin acylase used in this embodiment is derived from Kluyveracitrophila ATCC21285, and its amino acid sequence is shown in SEQ ID NO. 1. This amino acid sequence consists of four parts, from the N-terminus to the C-terminus of the protein: positions 1-26 are the signal peptide; positions 27-235 are the α subunit consisting of 209 amino acids; positions 236-289 are the intermediate linker peptide consisting of 54 amino acids; and positions 290-846 are the β subunit consisting of 557 amino acids (see also...). Figure 1 (The single underlined portion represents the α subunit, the wavy underline represents the linker peptide, and the double underlined portion represents the β subunit), the nucleotide sequence is shown in SEQ ID NO. 2.

[0032] A schematic diagram of the construction of recombinant plasmid pET28a-kcPA is shown below. Figure 2 As shown. Using the K. citrophila ATCC21285 genome as a template, primers were designed based on the PA nucleotide sequence (SEQ ID NO. 2). The forward primer was: 5'-CG G / AATTC ATGAAAAACCGCAATCGCAT-3', SEQ ID NO. 3; Reverse primer is 5'-CC. A / AGCTT TTAGCGCTGCACCTGCAGC-3', SEQ ID NO. 4. EcoRI and HindIII restriction enzyme sites were introduced respectively (underlined bases are restriction endonuclease recognition sites), and the PA wild-type target fragment was amplified by PCR.

[0033] PCR reaction system:

[0034]

[0035] The PCR temperature program was designed as follows:

[0036]

[0037] Two restriction endonucleases, EcoRI and HindIII, were used to double-digest the plasmid pET28a blank vector and the target fragment. Double digestion system:

[0038]

[0039] Double digestion was performed at 37°C for 1 h, followed by inactivation at 80°C for 20 min. The double digestion products were purified and recovered, and the concentrations were estimated based on their gel electrophoresis patterns. The concentrations of plasmid pET28a were approximately 50 ng / μL, and the concentrations of the target gene kcPA were approximately 140 ng / μL.

[0040] 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 thermally transfected into competent E. coli DH5α cells.

[0041] Target fragment and linearized vector linkage system:

[0042]

[0043] To verify the successful transformation of the recombinant plasmid, a single colony was picked from an LB agar plate containing Kan50 and transferred to LB liquid medium containing Kan50. The plasmid was extracted the next day using a plasmid extraction kit and subjected to PCR identification. A 2500 bp target band was obtained by agarose gel electrophoresis (e.g., ...). Figure 3 The validated expression vector pET28a-kcPA was transformed into E. coli BL21(DE3) to obtain the wild-type PA expression recombinant strain E. coli BL21(DE3) / pET28a-kcPA.

[0044] 2. Obtaining mutant expression vectors

[0045] In this embodiment, a total of 18 mutants were obtained through site-directed mutagenesis, as shown in the table below. Among them, "F146αK" means that the 146th amino acid on the α subunit is mutated from F to K. The interpretation of other mutation sites is the same.

[0046] Table 1. Mutants and their corresponding mutation sites

[0047]

[0048] First, primers corresponding to each mutation site were designed. Then, using the PA wild-type target fragment as the initial template, site-directed mutagenesis was performed using the NEB Q5® Site-Directed Mutagenesis Kit (Q5 SDM Kit). The primers for each mutation site are as follows (lowercase letters represent the bases at the mutation sites):

[0049]

[0050] Primers were synthesized by a nucleic acid synthesis company, then dissolved in sterile water, and the kit was followed accordingly. See below:

[0051] Mutation of the corresponding sites using PCR

[0052] PCR reaction system:

[0053]

[0054] Cyclic program temperature:

[0055]

[0056] For mutants with more than two mutation sites, the PCR product of the previous mutation site is used as a template to perform site-directed mutagenesis at the corresponding sites one after another.

[0057] Kinase, Ligase & DpnI (KLD) (a special mixture of kinase, ligase and DpnI) reaction treatment

[0058] The reaction system is as follows:

[0059]

[0060] React at room temperature for 5 minutes.

[0061] Thermal shock conversion

[0062] Add 5 μL of KLD reaction mixture to 50 μL of chemicompetent E. coli BL21(DE3) cell suspension, incubate on ice for 30 min, subject to heat shock at 42°C for 30 s, incubate on ice for 5 min, add 950 μL of SOC sterile liquid medium, and gently shake at 37°C for 1 h. Spread 40-100 μL of the bacterial suspension onto Kan50 LB agar plates and incubate overnight at 37°C. Single colonies that grow are the corresponding mutant expression strains, named E. coli BL21(DE3) / pET28a-kcPA01~18.

[0063] Mutant identification

[0064] The obtained mutant expression strain was inoculated into 25 mL of liquid medium containing Kan50 LB and cultured overnight at 37°C. Plasmids were extracted using a plasmid extraction kit. The samples were sent to a third-party biotechnology company for sequencing to confirm that the corresponding product was the target product of the site-directed mutagenesis.

[0065] 3. Expression of wild-type and mutant KcPA

[0066] The recombinant *E. coli* BL21(DE3) / pET28a-kcPA and *E. coli* BL21(DE3) / pET28a-kcPA01~18 were inoculated onto Kan50 LB agar plates and cultured at 37°C for 12–16 h. Single colonies were picked and inoculated into 25 mL of LB liquid medium supplemented with Kan50 and cultured overnight at 37°C and 300 rpm. 500 μL of the bacterial culture was transferred to 50 mL of Kan50 LB liquid medium and cultured at 37°C and 280 rpm. OD was monitored. 600 When the concentration of the cytokine reached 0.6-0.8, IPTG solution was added to induce expression at a concentration of 0.3 mM. Expression was induced for 10 h at 25°C and 220 rpm in a shaker. The fermentation broth was then centrifuged to collect the cells. The collected cells were resuspended in pH 7.5 PBS buffer and pre-cooled on ice for 10 min, followed by centrifugation at 12000 rpm for 6 min at 4°C to collect the cells. The cells were resuspended in 50 mM pH 7.5 PBS buffer and centrifuged again at 12000 rpm for 6 min at 4°C. The supernatant was discarded, and the remaining cells were collected and resuspended at a concentration of 0.01 g / mL. The cells were then disrupted using an ultrasonic cell disruptor. The cell disruption conditions were: ice-water bath, 400 W power, 3 s per cycle, 5 s interval, for a total of 80 cycles. After crushing, the mixture was centrifuged at 4℃ 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.

[0067] Figure 4 SDS-PAGE images of proteins expressed in bacterial cells; lane M represents the protein marker; Lane 1: supernatant of E. coli BL21(DE3) / pET28a expression; Lane 2: supernatant of E. coli BL21(DE3) / pET28a-kcPA without induction; Lane 3: supernatant of E. coli BL21(DE3) / pET28a-kcPA18 without induction; Lane 4: supernatant of E. coli BL21(DE3) / pET28a-kcPA18 induced by IPTG.

[0068] Example 2

[0069] 1. Determination of KcPA hydrolytic activity

[0070] The principle of the assay is as follows: potassium penicillin (PGK) hydrolyzes under the action of KcPA to produce 6-aminopenicillanic acid (6-APA) and phenylacetic acid. 6-APA reacts with p-dimethylaminobenzaldehyde (PDAB) under acidic conditions to form a yellow-green substance with a maximum absorption peak at 415 nm. Enzyme activity is defined as the amount of enzyme required for penicillin acylase to catalyze the generation of 1 μmol of 6-APA per minute from 20 mg / mL PGK at 28℃ in 0.1 M PBS buffer; this amount is defined as 1 unit of KcPA enzyme activity, expressed in units of U.

[0071] Weigh 0.5 g of PGK and dissolve it in the above buffer solution, then bring the volume to 25 mL. Pipette 2 mL of PGK solution into a centrifuge tube and add 0.1 mL of KcPA enzyme solution. A control group was set up without KcPA, while keeping all other conditions the same.

[0072] The above reaction system was placed in a water bath at 28℃ and 200 rpm for 10 min. After the reaction, the enzyme was inactivated by incubating in a water bath at 90℃ for 2 min. 200 μL of the reaction solution was taken and 3 mL of pH 3.0, 0.1 M sodium citrate buffer was added, along with 1 mL of chromogenic solution (0.5% PDAB). After standing at room temperature for 3 min, the absorbance was measured at 415 nm. The concentration of 6-APA in the sample after the reaction was obtained according to the 6-APA standard curve, and the enzyme activity, i.e., the hydrolytic activity, was calculated according to the formula.

[0073] Calculation formula: per mL of penicillin acylase hydrolase activity

[0074] In the formula, C 6-APA V: 6-APA concentration in the sample, μmol / L; V: Reaction volume, mL; E : Amount of penicillin acylase added, mL; t: Reaction time, 10 min.

[0075] 2. Determination of KcPA Synthetic Activity

[0076] Amoxicillin is synthesized from 6-aminopenicillanic acid (6-APA) and methyl p-hydroxyphenylglycine (DHPGM) under the action of KcPA. The amoxicillin content can be determined by high-performance liquid chromatography (HPLC) to calculate the PA synthesis activity. Enzyme activity is defined as: under certain conditions, one unit of penicillin acylase catalyzes the production of 1 μmol of amoxicillin per minute, which is denoted as U.

[0077] Weigh 1 g of 6-APA and 1.25 g of D-HPGM and dissolve them in 50 mL of 0.1 M, pH 6.3 PBS buffer. Adjust the pH to 6.3, then bring the volume to 100 mL with the buffer solution. Add 0.1 mL of KcPA to the solution and react at 25°C and 200 rpm for 30 min. Inactivate the enzyme by placing the solution in a 90°C water bath for 2 min to stop the reaction. Filter 0.5 mL of the reaction solution through a 0.22 μm aqueous filter membrane and bring the volume to 100 mL with phosphate buffer. Perform HPLC analysis to determine the amoxicillin content. Enzyme activity calculation formula: Activity per mL of penicillin acylase synthase. In the formula: V: volume of reaction liquid, mL; 200: dilution factor; C 样 Amoxicillin molar concentration, μmol / L; V E : Volume of enzyme added (mL); t: Reaction time (min).

[0078] The HPLC detection conditions were as follows: Agilent ZORBAX SB-C18 4.6x250 mm column, column temperature 25℃. Injection volume was 10 μL. Mobile phase A (0.02 M pH 4.7 NaH2PO4-Na2HPO4 buffer) and mobile phase B (methanol) were used. Initially, the mobile phase was maintained at 90% mobile phase A and 10% mobile phase B for 5 min. From 5 min to 7 min, the mobile phase B was increased from 10% to 50% and maintained for 10 min. From 17 to 19 min, the mobile phase B was reduced from 50% to 10%. Finally, the mobile phase was equilibrated with 90% mobile phase A and 10% mobile phase B for 5 min. The total flow rate was 1 mL / min.

[0079] Table 2 Comparison of activities between mutants and wild-type

[0080]

[0081] Note: The hydrolytic activity of the wild-type KcPA expressed by the recombinant bacteria was 15 U / mL (fermentation broth), and the synthetic activity was 80 U / mL. For ease of comparison, the enzyme activity of the wild-type KcPA was defined as 100 in Table 2, and each mutant was compared with it.

[0082] As shown in the table above, for mutants with single mutation sites, the hydrolytic and synthetic activities of each mutant are significantly improved compared to the wild type, especially the F146αK mutant on the α subunit and the G385βR mutant on the β subunit. The hydrolytic and synthetic activities of the single-site F146αK mutant are 5.8 times and 15.3 times that of the wild type, respectively; the hydrolytic and synthetic activities of the G385βR mutant are 4.6 times and approximately 11.2 times that of the wild type, respectively. Compared with the G385βR mutant, the G385βY mutant has higher hydrolytic activity, but its synthetic activity is not outstanding. When multiple mutation sites are superimposed, the enzyme activity of the mutants is increased compared to single-site mutations, especially the five-site mutants F146αK & F24βR & F71βY & N241βK & G385βR, which have high hydrolytic and synthetic activities.

[0083] Example 3

[0084] Amoxicillin synthesized in one step via PGK catalysis by various mutant and wild-type penicillin acylases

[0085] PGK was added to PBS buffer (pH 7.0) to achieve a concentration of 200 mM, and p-hydroxyphenylglycine methyl ester (D-HPGM) was added to achieve a final concentration of 300 mM. The enzyme concentration was 30 U / mL (calculated based on synthase activity). The reaction was carried out at 28°C with stirring for 3 h. After the reaction was completed, HPLC analysis was performed to calculate the amoxicillin yield.

[0086] The HPLC detection conditions were as follows: Agilent ZORBAX SB-C18 4.6x250 mm column, column temperature 25℃. Injection volume was 10 μL. Mobile phase A (0.02 M pH 4.7 NaH2PO4-Na2HPO4 buffer) and mobile phase B (methanol) were used. Initially, the mobile phase was maintained at 90% of mobile phase A and 10% of mobile phase B for 5 min. From 5 to 7 min, mobile phase B was increased from 10% to 50% and maintained for 10 min. From 17 to 19 min, mobile phase B was decreased from 50% to 10%. Finally, the system was equilibrated with 90% mobile phase A and 10% mobile phase B for 5 min. The total flow rate was 1 mL / min. The reaction formula is as follows:

[0087]

[0088] The HPLC chromatogram of mutant KcPA 18 is shown below. Figure 5 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 potassium penicillin. It can be seen from the figure that the content of the intermediate product 6-APA is extremely low, almost non-existent.

[0089] Table 3. Yields of amoxicillin synthesized by each mutant

[0090]

[0091] The results in the table above show that each mutant can catalyze the reaction of potassium penicillin with methyl p-hydroxyphenylglycine in a single reaction system to synthesize amoxicillin in one step, and the product yield is significantly higher than that of the wild type.

[0092] Example 4

[0093] One-step synthesis of amoxicillin via KcPA18-catalyzed PGK method

[0094] The reaction system differs from that in Example 3 only in that the pH of the PBS buffer in this example is 7.5. The HPLC detection conditions are the same as in Example 3.

[0095] Changes in the content of each substance during the reaction are as follows: Figure 6 The reaction yield was 98%.

[0096] Example 5

[0097] One-step synthesis of ampicillin via KcPA18-catalyzed PGK method

[0098] PGK was added to PBS buffer at pH 7.5 to achieve a concentration of 240 mM, and methyl phenylglycine (D-PGM) was added to achieve a final concentration of 480 mM. The amount of enzyme added was 30 U / mL (calculated based on synthase activity). The reaction was carried out at 25°C with stirring for 3 hours, and samples were taken at regular intervals during the reaction.

[0099] The HPLC detection conditions are the same as in Example 3, and the reaction formula is as follows:

[0100]

[0101] Changes in the content of each substance during the reaction are as follows: Figure 7 The reaction yield was 98%.

[0102] Example 6

[0103] KcPA18 catalyzes the synthesis of cefaclor using 7-ACCA.

[0104] 7-ACCA was added to PBS buffer at pH 7.5 to achieve a concentration of 200 mM, and methyl phenylglycine (D-PGM) was added to achieve a final concentration of 240 mM. The amount of enzyme added was 20 U / mL (calculated based on synthase activity). The reaction was carried out at 15°C with stirring for 2.25 h, and samples were taken at regular intervals during the reaction.

[0105] The HPLC analysis conditions were as follows: 0.01 M sodium phosphate (pH 6.8) and methanol (95:5) as the mobile phase, flow rate 1.0 mL / min, Agilent ZORBAX SB-C18 4.6 x 250 mm column, injection volume 10 μL. The reaction formula is as follows:

[0106]

[0107] Changes in the content of each substance during the reaction are as follows: Figure 8 The reaction yield was 95%.

[0108] Example 7

[0109] KcPA18 catalyzes the synthesis of cephalosporin from 7-ADCA.

[0110] 7-ADCA was added to PBS buffer at pH 8.0 to achieve a concentration of 180 mM, and methyl phenylglycine (D-PGM) was added to achieve a final concentration of 270 mM. The amount of enzyme added was 25 U / mL (calculated based on synthase activity). The reaction was carried out at 10°C with stirring for 2.25 h, and samples were taken at regular intervals during the reaction.

[0111] The HPLC analysis conditions were as follows: 0.01 M sodium phosphate (pH 5.5) and methanol (93:7) as the mobile phase, flow rate 1.0 mL / min, Agilent ZORBAX SB-C18 4.6 x 250 mm column, injection volume 10 μL. The reaction formula is as follows:

[0112]

[0113] Changes in the content of each substance during the reaction are as follows: Figure 9 The reaction yield was 99%.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An artificially designed penicillin G acylase, characterized in that: Compared with the amino acid sequence shown in SEQ ID NO. 1, the mutation mode is: G385βY; or G385βR; or F146αK & N241βK & G385βY.

2. A nucleotide encoding the penicillin G acylase as described in claim 1.

3. The use of penicillin G acylase as described in claim 1 in the preparation of β-lactam antibiotics, wherein the β-lactam antibiotics include amoxicillin, ampicillin, cefaclor, or cefadroxil.

4. The application according to claim 3, characterized in that: Using only the penicillin G acylase as the sole enzyme in the reaction system, a semi-synthetic penicillin-type β-lactam antibiotic is synthesized in one step by catalyzing the reaction of penicillin potassium salt with an acyl donor; the acyl donor includes methyl phenylglycine or methyl p-hydroxyphenylglycine.

5. The application according to claim 3, characterized in that: Using the penicillin G acylase as a penicillin acylase, 7-ACCA or 7-ADCA is catalyzed to react with methyl phenylglycine to synthesize semi-synthetic cephalosporin β-lactam antibiotics.

Images