A penicillin acylase mutant and its application

Abstract

The present invention belongs to the technical field of enzyme catalysis, and particularly relates to a penicillin acylase mutant and its application. Compared with the amino acid sequence shown in SEQ ID NO.1, this mutant contains at least one of the following mutation sites: F146αK, F24βR, F71βY, N241βK, G385βY or G385βR. By mutating the penicillin acylase derived from Kluyvera citrophila, the present invention obtains a penicillin acylase mutant with strong hydrolysis activity and synthesis activity, and the two enzyme activities are coordinated. It can be used for the synthesis and production of β-lactam antibiotics, especially for the one-step preparation of amoxicillin from potassium penicillin, etc., avoiding the separation of the intermediate 6-APA. The present invention provides a key enzyme for the efficient preparation of β-lactam antibiotics, which will greatly promote the innovation of β-lactam antibiotic preparation technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme catalysis, and particularly relates to a mutant of penicillin acylase (PA) and its application. Background Art

[0002] β-lactam antibiotics are a large class of antibiotics with a β-lactam ring in their chemical structure. This class of antibiotics has the advantages of strong bactericidal activity, low toxicity, wide indications, and good clinical efficacy. However, naturally occurring β-lactam antibiotics (such as penicillin and cephalosporin) have disadvantages such as a narrow antibacterial spectrum, acid intolerance, easy generation of drug resistance, and easy induction of allergic reactions. Semi-synthetic β-lactam antibiotics such as semi-synthetic penicillins and semi-synthetic cephalosporins have overcome these disadvantages and have become the main antibiotics used in the current medical field.

[0003] Using enzyme catalysis technology to prepare semi-synthetic β-lactam antibiotics can produce semi-synthetic β-lactam antibiotics more efficiently, greenly, and sustainably, and the quality of the prepared products is better than that of traditional chemical synthesis methods. Penicillin acylase is the key enzyme for catalyzing the synthesis of this class of products. The enzyme-catalyzed synthesis of semi-synthetic penicillins such as ampicillin and amoxicillin currently mainly consists of two steps, that is, first, penicillin acylase for hydrolysis catalyzes penicillin and its salts to prepare 6-APA (6-aminopenicillanic acid), and then penicillin acylase for synthesis catalyzes the reaction of 6-APA with an acyl donor side chain (such as methyl D-p-hydroxyphenylglycinate) to prepare semi-synthetic β-lactam antibiotics. Semi-synthetic cephalosporins are mainly prepared by penicillin acylase catalyzing the reaction of cephalosporin intermediate nuclei such as 7-ACA and 7-ADCA with an acyl donor. Penicillin acylase (PA, EC3.5.1.11) is the key enzyme for this preparation technology. Currently, all existing technologies use two enzymes, penicillin acylase for hydrolysis and penicillin acylase for synthesis, to catalyze the hydrolysis step by step to prepare 6-APA (or 7-ACA, 7-ADCA) and the two-step reaction of the synthesis of the intermediate nucleus and the side chain. The two-step reaction not only has a long reaction process but also requires the separation of intermediates, and the production process is relatively complex. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the present invention provides a mutant of penicillin acylase and its application, aiming to solve the problems in the prior art. The mutant provided by the present invention can catalyze the reaction of β-lactam raw materials including penicillin or cephalosporin and an activated acyl donor to synthesize semi-synthetic β-lactam antibiotics, including semi-synthetic penicillins and semi-synthetic cephalosporins, and realize a new technology for the rapid and efficient synthesis of semi-synthetic β-lactam antibiotics.

[0005] The present invention is implemented as follows. A penicillin acylase mutant, compared with the amino acid sequence shown in SEQ ID NO.1, contains at least one of the following mutation sites: F146αK, F24βR, F71βY, N241βK, G385βY, G385βR. Explanation of amino acid abbreviations: F: phenylalanine; K: lysine; R: arginine; Y: tyrosine; N: asparagine; G: glycine.

[0006] The present invention also protects the nucleotide encoding any one of the above penicillin acylase mutants.

[0007] The present invention also provides the application of the penicillin acylase mutant as described above in the preparation of β-lactam antibiotics.

[0008] Furthermore, the β-lactam antibiotics include semi-synthetic penicillins and semi-synthetic cephalosporins. The semi-synthetic penicillins include amoxicillin, ampicillin or pivampicillin; the semi-synthetic cephalosporins include cephalexin, cefprozil, cefaclor, cefradine, cefadroxil, cefamandole, cefazolin, cefonicid, cefalotin or cefglycin.

[0009] Furthermore, only any one of the penicillin acylase mutants is used as the only penicillin acylase in the reaction system to catalyze the reaction of potassium penicillin with an acyl donor to synthesize semi-synthetic penicillin β-lactam antibiotics in one step.

[0010] Furthermore, the acyl donor includes phenylglycine methyl ester or p-hydroxyphenylglycine methyl ester.

[0011] Furthermore, any one of the penicillin acylase mutants is used as the penicillin acylase to catalyze 7-ACCA or 7-ADCA to react with phenylglycine methyl ester to synthesize semi-synthetic cephalosporin β-lactam antibiotics.

[0012] The present invention mutates the amino acids in the original substrate-binding region of wild-type penicillin acylase to improve the binding performance with the substrate and reduce the substrate-binding steric effect; at the same time, the free energy of the transition state of the substrate-active site in the active center is reduced to promote the conversion of the intermediate state to the product, thereby promoting the accumulation of the final product, and ultimately greatly improving the ability of penicillin acylase to synthesize semi-synthetic β-lactam antibiotics.

[0013] In summary, the advantages and positive effects of the present invention are as follows: By mutating the penicillin acylase derived from Kluyvera citrophila, a penicillin acylase mutant with strong hydrolysis activity and synthesis activity, coordinated two activities, and better stability is obtained. It can be used for the synthesis of β-lactam antibiotics such as semi-synthetic penicillins (such as amoxicillin, ampicillin or pivampicillin), semi-synthetic cephalosporins (such as cephalexin, cefprozil, cefaclor, cefradine), and the one-step preparation (synthesis) of amoxicillin and ampicillin from potassium penicillin, etc., avoiding the separation of intermediates such as 6-APA. The present invention provides a key enzyme for the efficient preparation of β-lactam antibiotics, which will greatly promote the innovation of β-lactam antibiotic preparation technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the amino acid sequence of wild-type penicillin acylase;

[0015] Figure 2 is the schematic diagram of the construction of recombinant plasmid pET28a-kcPA;

[0016] Figure 3 is the detection map of recombinant plasmid PCR agarose electrophoresis;

[0017] Figure 4 is the SDS-PAGE electrophoresis map of the expressed protein of E. coli BL21(DE3) / pET28a-kcPA;

[0018] Figure 5 is the HPLC chromatogram of the one-step synthesis of amoxicillin from potassium penicillin catalyzed by KcPA in Example 3;

[0019] Figure 6 is the change of the content of each substance during the reaction in Example 4;

[0020] Figure 7 is the change of the content of each substance during the reaction in Example 5;

[0021] Figure 8 is the change of the content of each substance during the reaction in Example 6;

[0022] Figure 9 is the change of the content of each substance during the reaction in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0023] For a better understanding of the present invention rather than limiting the scope thereof, all numbers representing amounts, percentages, and other numerical values used in this application shall be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may vary according to the different desired properties to be obtained. Each numerical parameter should be regarded as being obtained at least according to the significant figures reported and by the conventional rounding method. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

[0024] In the following examples of the present invention, when the temperature is not specifically limited, it is under normal temperature conditions. Normal temperature refers to the natural room temperature conditions in the four seasons without additional cooling or heating treatment. Generally, the normal temperature is controlled at 10 - 30 °C, preferably 15 - 25 °C. The abbreviations have the following meanings: "min" represents minute, "s" represents second, "U" represents enzyme activity unit, "mM" represents millimole per liter, "M" represents mole per liter, "rpm" represents revolutions per minute, "mol" represents mole, "μg" represents microgram, "mg" represents milligram, "g" represents gram, "μL" represents microliter, "mL" represents milliliter, "bp" represents base pair, LB medium represents Luria - Bertani medium, and Kan50 represents the medium containing 50 μg / mL kanamycin.

[0025] In the examples, for the experimental methods without specific conditions noted, they are usually carried out under conventional conditions, such as those described in "Molecular Cloning: A Laboratory Manual" (Chinese version) (edited by J. Sambrook, M.R. Green, translated by He Fuchu. Fourth Edition, Beijing: Science Press, 2017) and the methods described in New England Biolabs (NEB) kits.

[0026] The present invention discloses a penicillin acylase mutant and its application. The technical solution provided by the present invention can avoid the separation of intermediates, greatly simplify the production process, improve the yield of reaction products, significantly save production costs, and bring about a revolutionary change in the semi - synthetic antibiotic industry. The following will clearly and completely describe the technical solution of the present invention in combination with the examples of the present invention.

[0027] Example 1

[0028] Construction, prokaryotic expression and functional identification of a penicillin acylase mutant from Kluyvera citrophila

[0029] 1. Construction of the wild - type PA expression vector pET28a - kcPA

[0030] In this example, the wild-type penicillin acylase used is derived from Kluyveracitrophila K. citrophila ATCC21285, and its amino acid sequence is shown in SEQ ID NO.1. This amino acid sequence consists of four parts. From the N-terminus to the C-terminus of the protein, they are as follows: positions 1-26 are the signal peptide, positions 27-235 are the α subunit composed of 209 amino acids, positions 236-289 are the intermediate linker peptide composed of 54 amino acids, and positions 290-846 are the β subunit composed of 557 amino acids (also see Figure 1 , where the single-underlined part is the α subunit, the wavy-line part is the linker peptide, and the double-underlined part is the β subunit), and the nucleotide sequence is shown in SEQ ID NO.2.

[0031] The schematic diagram of the construction of the recombinant plasmid pET28a-kcPA is as shown in Figure 2 . Using the genome of K. citrophila ATCC21285 as a template, primers were designed according to the PA nucleotide sequence (SEQ ID NO.2). The forward primer was: 5'-CG G / AATTC ATGAAAAACCGCAATCGCAT-3', SEQ ID NO.3; the reverse primer was 5'-CC A / AGCTT TTAGCGCTGCACCTGCAGC-3', SEQ ID NO.4. The EcoR I and HindIII restriction enzyme cleavage sites were introduced respectively (the underlined bases are the recognition sites of the restriction endonucleases), and the wild-type target fragment of PA was amplified by PCR.

[0032] PCR reaction system:

[0033]

[0034] The PCR temperature program was designed as:

[0035]

[0036] Two restriction endonucleases, EcoRI and HindIII, were selected to perform double digestion on the plasmid pET28a blank vector and the target fragment. Double digestion system:

[0037]

[0038] The double digestion reaction was carried out at 37°C for 1 h and then inactivated at 80°C for 20 min. The double digestion products were purified and recovered, and the concentration was estimated according to their gel electrophoresis patterns. The concentration of the plasmid pET28a was about 50 ng / μL, and the concentration of the target gene kcPA was about 140 ng / μL.

[0039] The double-digested product was ligated overnight at 16 °C in a metal bath using T4 DNA ligase to obtain the recombinant plasmid pET28a-kcPA, which was then heat-transformed into the competent cell E. coli DH5α.

[0040] Ligation system of the target fragment and the linearized vector:

[0041]

[0042] To verify whether the recombinant plasmid was successfully transferred, single colonies were picked from the LB plate containing Kan50 into the LB liquid medium containing Kan50. The next day, the plasmid was extracted using a plasmid extraction kit and subjected to PCR identification. The target band of 2500 bp was obtained by agarose gel electrophoresis (as Figure 3 ). The verified expression vector pET28a-kcPA was transformed into E. coli BL21(DE3) to obtain the recombinant strain E. coli BL21(DE3) / pET28a-kcPA expressing wild-type PA.

[0043] 2. Obtaining of the mutant expression vector

[0044] In this example, a total of 18 mutants were obtained by site-directed mutagenesis as shown in the following table. Among them, "F146αK" means that the 146th amino acid on the α subunit was mutated from F to K, and the explanations for other mutation sites are the same.

[0045] Table 1 Mutants and corresponding mutation sites

[0046]

[0047]

[0048] First, primers corresponding to each mutation site were designed, and then using the wild-type PA target fragment as the initial template, site-directed mutagenesis was carried out using a site-directed mutagenesis kit (NEB Site-Directed Mutagenesis Kit (Q5 SDM Kit)). Primers for each mutation site are as follows (lowercase letters are the mutated site bases):

[0049] F146αK, F: 5'-GGCGAACCGTaaaTCTGACAGCACCAG-3', SEQ ID NO.5;

[0050] R: 5'-ATGGTGCCGACAAAAATCATCGCCA-3', SEQ ID NO.6;

[0051] F24βR, F: 5'-TGGGCCGCAGcgcGGTTGGTATGCG-3', SEQ ID NO.7,

[0052] R: 5'-TTGACCATAATGGCCTTCGCATCCT-3', SEQ ID NO.8;

[0053] F71βY, F: 5'-CACCGCCGGTtatGGTGATGATG-3', SEQ ID NO.9,

[0054] R: 5'-GATCCCCATGAAATGGTGCCGTTGT-3', SEQ ID NO.10;

[0055] N241βK, F: 5'-CGCCAACTGGaaaAACTCGCCGC-3', SEQ ID NO.11,

[0056] R: 5'-ATATAGCCCGACTGCGGGTTATACAC-3', SEQ ID NO.12;

[0057] G385βY: F: 5'-CGGGCCAACCtatTCGCTGAACATCAGCGTG-3', SEQ ID NO.13,

[0058] R: 5'-TCCTGGGTGGTTTCATAGCCACTGG-3', SEQ ID NO.14;

[0059] G385βR, F: 5'-CGGGCCAACCcgcTCGCTGAACATC-3', SEQ ID NO.15,

[0060] R: 5'-TCCTGGGTGGTTTCATAGCCACTGG-3', SEQ ID NO.16;

[0061] The primers were synthesized by a nucleic acid synthesis company, then dissolved in sterile water, and then the operations were carried out according to the kit. As follows:

[0062] ① Mutate the corresponding site by PCR

[0063] PCR reaction system:

[0064]

[0065] Cycling program temperature:

[0066]

[0067]

[0068] For mutants with more than 2 mutation sites, using the PCR product of the previous mutation site obtained as a template, site-directed mutagenesis of the corresponding sites is carried out successively.

[0069] ②Reaction treatment with Kinase, Ligase & DpnI (KLD) (a special mixture of kinase, ligase and DpnI)

[0070] The reaction system is as follows:

[0071] Volume Final concentration PCR product 1 μL 2X KLD Reaction Buffer 5 μL 1X 10X KLD Enzyme Mix 1 μL 1X Nuclease-free water 3 μL

[0072] React at room temperature for 5 min.

[0073] ③Transformation by heat shock method

[0074] Add 5 μL of the KLD reaction mixture to 50 μL of the suspension of chemically competent cells E. coli BL21(DE3), incubate on ice for 30 min, perform heat shock at 42 °C for 30 s, incubate on ice for 5 min, add 950 μL of sterile SOC liquid medium, and gently shake at 37 °C for 1 h. Spread 40 - 100 μL of the bacterial suspension on an LB plate with Kan50 and incubate overnight at 37 °C. The single colonies grown are the corresponding mutant expression strains, which are respectively named E. coli BL21(DE3) / pET28a-kcPA01 - 18

[0075] ④Identification of mutants

[0076] Inoculate the obtained mutant expression strains into 25 mL of LB liquid medium containing Kan50 and culture overnight at 37 °C. Use a plasmid extraction kit to extract the plasmids. Send them to a third-party biological company for sequencing to determine that the corresponding products are the target products of site-directed mutagenesis.

[0077] 3. Expression of wild-type and mutant KcPA

[0078] The constructed recombinant E. coli E. coli BL21(DE3) / pET28a-kcPA and E. coli BL21(DE3) / pET28a-kcPA01 - 18 are inoculated on an LB agar plate with Kan50 and cultured in an incubator at 37 °C for 12 - 16 h. Pick single colonies respectively and inoculate them into 25 mL of LB liquid medium supplemented with Kan50 and culture overnight at 37 °C on a shaker at 300 rpm. Pipette 500 μL of the bacterial liquid and transfer it to 50 mL of LB liquid medium with Kan50 and culture on a shaker at 37 °C and 280 rpm, and monitor OD 600When the change reaches 0.6 - 0.8, add IPTG solution to make the IPTG induction concentration 0.3 mM, and induce expression at 25 °C in a shaker at 220 rpm for 10 h. Centrifuge the fermentation broth to collect the bacterial cells. Suspend the collected bacterial cells in PBS buffer at pH 7.5 and pre-cool on ice for 10 min, then centrifuge at 4 °C and 12,000 rpm for 6 min to collect the bacterial cells. Add the bacterial cells to PBS buffer at 50 mM pH 7.5 to resuspend the bacterial cells in the centrifuge tube, and then centrifuge at 4 °C and 12,000 rpm for 6 min. Discard the supernatant, collect the final bacterial cells and resuspend them at a concentration of 0.01 g / mL, and use an ultrasonic disruptor to disrupt the cells. The cell disruption conditions are: ice-water bath, at a power of 400 W, each cycle works for 3 s and pauses for 5 s, for a total of 80 cycles. After disruption, the mixture is centrifuged at 4 °C and 12,000 rpm for 15 min, and the supernatant obtained is the crude enzyme solution. Collect the crude enzyme solution and analyze the expressed protein by SDS-PAGE.

[0079] Figure 4 It is the SDS-PAGE diagram of the protein expressed by the bacterial cells; among them, lane M is the protein Marker; Lane 1: the expression supernatant of E. coli BL21(DE3) / pET28a; Lane 2: the non-induced supernatant of E. coli BL21(DE3) / pET28a-kcPA; Lane 3: the non-induced supernatant of E. coli BL21(DE3) / pET28a-kcPA18; Lane 4: the IPTG-induced supernatant of E. coli BL21(DE3) / pET28a-kcPA18.

[0080] Example 2

[0081] 1. Determination of the hydrolysis activity of KcPA

[0082] The principle of the determination is as follows: Potassium penicillin (PGK) is hydrolyzed by KcPA to generate 6-aminopenicillanic acid (6-APA) and phenylacetic acid. 6-APA reacts with p-dimethylaminobenzaldehyde (PDAB) under acidic conditions to form a yellow-green substance, which has a maximum absorption peak at 415 nm. Enzyme activity definition: In 0.1 M PBS buffer at 28 °C, the amount of enzyme required for penicillin acylase to catalyze 20 mg / mL PGK to generate 1 μmol 6-APA per minute is 1 unit of KcPA enzyme activity, and the unit is U.

[0083] Weigh 0.5 g of PGK and dissolve it in the above buffer solution, and make the volume up to 25 mL. Pipette 2 mL of the PGK solution into a centrifuge tube and add 0.1 mL of the KcPA enzyme solution. Set the control group without adding KcPA, and keep other conditions the same.

[0084] The above reaction system was placed in a water bath shaker at 28 °C and 200 rpm for 10 min. After the reaction, the enzyme was inactivated in a 90 °C water bath for 2 min. 200 μL of the reaction solution was taken and added to 3 mL of 0.1 M sodium citrate buffer with a pH of 3.0. 1 mL of the chromogenic solution (0.5% PDAB) was added. 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, that is, the hydrolysis activity, was calculated according to the formula.

[0085] Calculation formula: Penicillin acylase hydrolysis activity per mL

[0086] In the formula, C 6-APA : Concentration of 6-APA in the sample, μmol / L; V: Volume of the reaction system, mL; V E : Volume of penicillin acylase added, mL; t: Reaction time, 10 min.

[0087] 2. Determination of KcPA synthesis activity

[0088] Amoxicillin is synthesized from 6-aminopenicillanic acid (6-APA) and methyl p-hydroxyphenylglycinate (DHPGM) under the action of KcPA. The content of amoxicillin can be determined by high performance liquid chromatography (HPLC) to calculate the PA synthesis activity. The enzyme activity is defined as: Under certain conditions, when 1 μmol of amoxicillin is catalyzed to be generated per minute by 1 unit of penicillin acylase, it is defined as 1 synthesis enzyme activity unit, denoted by U.

[0089] 1 g of 6-APA and 1.25 g of D-HPGM were weighed and dissolved in 50 mL of 0.1 M PBS buffer with a pH of 6.3. The pH was adjusted to 6.3, and then the solution was made up to 100 mL with the above buffer. 0.1 mL of KcPA was taken and added to the above solution. The reaction was started at 25 °C and 200 rpm for 30 min, and then the enzyme was inactivated in a 90 °C water bath for 2 min to end the reaction. 0.5 mL of the reaction solution was filtered through a 0.22 μm aqueous filter membrane and made up to 100 mL with phosphate buffer for HPLC detection to obtain the amoxicillin content. Enzyme activity calculation formula: Penicillin acylase synthesis activity per mL In the formula: V: Volume of the reaction solution, mL; 200: Dilution factor; C 样 : Molar concentration of amoxicillin, μmol / L; V E : Volume of enzyme added, mL; t: Reaction time, min.

[0090] The HPLC detection conditions were as follows: an Agilent ZORBAX SB-C18 4.6 x 250 mm chromatographic column, column temperature 25 °C, injection volume 10 μL. Mobile phase A (0.02 M NaH2PO4-Na2HPO4 buffer solution with pH 4.7), mobile phase B (methanol). Initially, 90% of mobile phase A and 10% of mobile phase B were maintained for 5 min. From 5 min to 7 min, the proportion of mobile phase B was increased from 10% to 50% and then maintained for 10 min. From 17 to 19 min, the proportion of mobile phase B was decreased from 50% to 10%. Finally, 90% of mobile phase A and 10% of mobile phase B were used for equilibration for 5 min. The total flow rate was 1 mL / min.

[0091] Table 2 Comparison of activities between mutants and wild type

[0092]

[0093] Note: The hydrolysis activity of the wild type of KcPA expressed by the recombinant bacteria was 15 U / mL (fermentation broth), and the synthesis activity was 80 U / mL. For the convenience of comparison, the enzyme activity of the wild type of KcPA in Table 2 was defined as 100, and each mutant was compared with it.

[0094] As can be seen from the above table, for the mutants with single mutation sites, the hydrolysis activity and synthesis activity of each mutant were significantly improved compared with the wild type, especially the F146αK mutant on the α subunit and the G385βR mutant on the β subunit. The hydrolysis activity and synthesis activity of the single-site F146αK mutant were 5.8 times and 15.3 times that of the wild type respectively; the hydrolysis activity and synthesis activity of the G385βR mutant were 4.6 times and about 11.2 times that of the wild type respectively. Compared with the G385βR mutant, the G385βY mutant had higher hydrolysis activity, but its synthesis activity was not prominent. When multiple mutation sites were superimposed, the enzyme activity of the mutant increased compared with single-site mutation, especially the five-site mutant F146αK&F24βR&F71βY&N241βK&G385βR, which had relatively high hydrolysis activity and synthesis activity.

[0095] Example 3

[0096] One-step synthesis of amoxicillin by penicillin acylase of each mutant and wild type catalyzing PGK

[0097] PGK was added to the PBS buffer solution with pH 7.0 to make its concentration reach 200 mM. At the same time, methyl D-p-hydroxyphenylglycinate (D-HPGM) was added to make its final concentration 300 mM. The enzyme dosage was 30 U / mL (calculated based on the synthesis enzyme activity). The reaction was carried out with constant stirring at 28 °C for 3 h. After the reaction, HPLC detection was carried out to calculate the amoxicillin yield.

[0098] The HPLC detection conditions were as follows: an Agilent ZORBAX SB-C18 4.6x250 mm chromatographic column, column temperature 25 °C, injection volume 10 μL. Mobile phase A (0.02 M NaH2PO4-Na2HPO4 buffer solution with pH 4.7), mobile phase B (methanol). Initially, 90% of mobile phase A and 10% of mobile phase B were maintained for 5 min. From 5 min to 7 min, the proportion of mobile phase B was increased from 10% to 50% and then maintained for 10 min. From 17 to 19 min, the proportion of mobile phase B was decreased from 50% to 10%. Finally, 90% of mobile phase A and 10% of mobile phase B were used for equilibration for 5 min, and the total flow rate was 1 mL / min. The reaction formula is as follows:

[0099]

[0100] The HPLC detection chromatogram of mutant KcPA18 is as Figure 5 shown, where DHPG is D-p-hydroxyphenylglycine, AMOX is amoxicillin, DHPGM is methyl D-p-hydroxyphenylglycinate, PAA is phenylacetic acid, and PGK is potassium penicillin. It can be seen from the figure that the content of the intermediate 6-APA is extremely low, almost none.

[0101] Table 3 Yields of amoxicillin catalytically synthesized by each mutant

[0102]

[0103] From the results in the above table, it can be seen that each mutant can catalyze the reaction of potassium penicillin with methyl p-hydroxyphenylglycinate in a reaction system to synthesize amoxicillin in one step, and the product yield is significantly higher than that of the wild type.

[0104] Example 4

[0105] One-step synthesis of amoxicillin by KcPA18 catalyzing PGK

[0106] The difference between the reaction system and that in Example 3 was only that: in this example, the pH of the PBS buffer solution was 7.5. The HPLC detection conditions were the same as those in Example 3.

[0107] The content changes of each substance during the reaction were as Figure 6 , and the reaction yield was 98%.

[0108] Example 5

[0109] One-step synthesis of ampicillin by KcPA18 catalyzing PGK

[0110] PGK was added to PBS buffer at pH 7.5 to a concentration of 240 mM, and at the same time, phenylglycine methyl ester (D-PGM) was added to a final concentration of 480 mM. The enzyme dosage was 30 U / mL (calculated based on synthase activity). The reaction was carried out with constant stirring at 25 °C for 3 h, and samples were taken at regular intervals during the reaction for detection.

[0111] The HPLC detection conditions were the same as those in Example 3. The reaction formula is as follows:

[0112]

[0113] The changes in the contents of various substances during the reaction are as Figure 7 , and the reaction yield was 98%.

[0114] Example 6

[0115] KcPA18 catalyzes the synthesis of cefaclor from 7-ACCA

[0116] 7-ACCA was added to PBS buffer at pH 7.5 to a concentration of 200 mM, and at the same time, phenylglycine methyl ester (D-PGM) was added to a final concentration of 240 mM. The enzyme dosage was 20 U / mL (calculated based on synthase activity). The reaction was carried out with constant stirring at 15 °C for 2.25 h, and samples were taken at regular intervals during the reaction for detection.

[0117] The HPLC analysis conditions were as follows: 0.01 M sodium phosphate (pH 6.8) and methanol (95:5) were used as the mobile phase, the flow rate was 1.0 mL / min, an Agilent ZORBAX SB-C18 4.6x250 mm chromatographic column was used, and the injection volume was 10 μL. The reaction formula is as follows:

[0118]

[0119] The changes in the contents of various substances during the reaction are as Figure 8 , and the reaction yield was 95%.

[0120] Example 7

[0121] Reaction effect of KcPA18 catalyzing the synthesis of cefradine from 7-ADCA

[0122] 7-ADCA was added to PBS buffer at pH 8.0 to a concentration of 180 mM, and at the same time, phenylglycine methyl ester (D-PGM) was added to a final concentration of 270 mM. The enzyme dosage was 25 U / mL (calculated based on synthase activity). The reaction was carried out with constant stirring at 10 °C for 2.25 h, and samples were taken at regular intervals during the reaction for detection.

[0123] The HPLC analysis conditions were as follows: 0.01 M sodium phosphate (pH 5.5) and methanol (93:7) were used as the mobile phase, the flow rate was 1.0 mL / min, an Agilent ZORBAX SB-C18 4.6 x 250 mm chromatographic column was used, and the injection volume was 10 μL. The reaction formula is as follows:

[0124]

[0125] The content changes of each substance during the reaction are as Figure 9 , and the reaction yield is 99%.

[0126] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A penicillin acylase mutant, characterized in that: Compared with the amino acid sequence shown in SEQ ID NO.1, the mutation modes are: F146αK & F24βR & F71βY & N241βK & G385βR.

2. Nucleotides encoding the penicillin acylase mutant as claimed in claim 1.

3. Use of the penicillin acylase mutant as claimed in claim 1 in the preparation of β-lactam antibiotics, wherein the β-lactam antibiotics include amoxicillin, ampicillin, cefaclor or cefradine.

4. The application according to claim 3, wherein: Only using the penicillin acylase mutant as the sole enzyme in the reaction system to catalyze the reaction of potassium penicillin with an acyl donor to synthesize semi-synthetic penicillin β-lactam antibiotics in one step; the acyl donor includes phenylglycine methyl ester or p-hydroxyphenylglycine methyl ester.

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

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