Penicillin G acylase for synthesizing penicillin V

Abstract

The invention discloses penicillin G acylase for synthesizing penicillin V and a mutant of the penicillin G acylase, which can catalyze a reaction of a substrate 6-APA and phenoxyacetate to generate the penicillin V, and provides a new way for enzymatic synthesis of the penicillin V.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology and relates to a penicillin G acylase (PGA) for the enzymatic synthesis of penicillin V and its mutant. Background Technology

[0002] β-lactam antibiotics inhibit bacterial cell wall material peptidoglycan synthesis by acting on transpeptidase and D-alanine carboxypeptidase, thereby inhibiting microbial growth. They possess advantages such as strong bactericidal activity, broad indications, and low toxicity, and are widely used clinically. These include penicillins and their derivatives, cephalosporins, carbapenems, monoamide ring antibiotics, and penicillin enzyme inhibitors. As one of the most important antibiotics, penicillin was the first to be used clinically. Based on its source, it can be divided into natural penicillin antibiotics and semi-synthetic penicillins. Common natural penicillins include penicillin V potassium and penicillin G potassium. Through cleavage or ring expansion, intermediates 6-APA (6-aminopenicillanic acid) and 7-ADCA (7-aminodeacetoxycephalosporanic acid) are produced. Further modification with different side chain groups can prepare various types of semi-synthetic penicillins.

[0003] Penicillin V is chemically named 6-phenoxyacetamidopenicillin, with the chemical formula C60. 16 H 18 N2O5S appears as white crystals or crystalline powder, and its structural formula is shown below.

[0004]

[0005] The molecular structure of penicillin V potassium mainly includes a β-lactam ring, a tetrahydrothiazole ring, and side chains. The β-lactam ring possesses antibacterial activity, while the phenoxymethyl side chain affects the physicochemical properties of penicillin V potassium. Clinically, penicillin is often used as a salt. Compared to penicillin G salt, penicillin V potassium is more acid-stable and less prone to degradation by gastric acid, making it more effective in treating infections caused by drug-resistant Staphylococcus aureus. Furthermore, when penicillin V potassium is used as an injectable preparation, a skin test is not required, resulting in high safety.

[0006] In China, penicillin V is mainly obtained through chemical methods or microbial fermentation. Due to its higher pollution levels, chemical methods are currently the most prevalent form of penicillin V on the market, obtained through fermentation. The fermentation process includes spore and seed preparation, microbial fermentation and filtration of the fermentation broth, acidification extraction, decolorization filtration, carbonate solution extraction, acid crystallization, alkali dissolution, azeotropic crystallization, washing, and drying. However, this process is energy-intensive and produces numerous byproducts. 4-Hydroxypenicillin V, as one of the main impurities, is difficult to completely remove due to its similar physicochemical properties to the product penicillin V, resulting in differences in product morphology compared to the original product. Therefore, it is necessary to develop new penicillin V production processes.

[0007] The enzymatic synthesis of penicillin V from 6-APA and phenoxyacetic acid derivatives has attracted attention in recent years due to its simple process, mild conditions, short production cycle, high conversion rate, and absence of the impurity 4-hydroxypenicillin V. For example, patent document CN109628541A ​​discloses an enzymatic method for synthesizing penicillin V salt, which uses a mutant of penicillin G acylase from *Achromobacter xylosoxidans* or a mutant of penicillin G acylase from *Escherichia coli* ATCC 11105 to catalyze the reaction of 6-APA and phenoxyacetic acid derivatives to produce penicillin V.

[0008] The substrate phenoxyacetic acid derivative is selected from phenoxyacetic acid ethylene glycol ester, phenoxyacetic acid glycerol ester, phenoxyacetic acid butyltetraol ester, etc., that is, the R group is a polyol with a hydroxyl group. However, the patent document does not report the reaction when the phenoxyacetic acid derivative is phenoxyacetic acid ester, that is, when R is an alkoxy group.

[0009] The commercial prices of phenoxyacetic esters, such as methyl phenoxyacetate and ethyl phenoxyacetate, are significantly lower than those of phenoxyacetic acid polyol esters. Using phenoxyacetic esters as substrates would greatly reduce the production cost of penicillin V and improve the economic efficiency of enzymatic penicillin V production. However, our experiments revealed that the penicillin G acylase mutants or wild-type enzymes reported in CN109628541A ​​did not perform well in catalyzing the phenoxyacetic ester reaction, exhibiting low enzyme activity, substrate or product inhibition, and poor tolerance. Summary of the Invention

[0010] Considering that using phenoxyacetic esters, such as methyl or ethyl ester, as substrates for the synthesis of penicillin V is more economical, we conducted experimental comparisons of numerous reported penicillin G acylases (PGA) and penicillin V acylases from various microbial sources. We screened for a penicillin G acylase that can smoothly catalyze the reaction of 6-APA and phenoxyacetic methyl / ethyl ester to produce penicillin V without substrate or product inhibition. This acylase is derived from *Achromobacter sp.* CCM 4824 (Genbank accession number AAY25991.1), and its amino acid sequence is shown in SEQ ID NO: 1. Since natural enzymes generally have low activity, we further mutated this penicillin G acylase to obtain mutants with increased enzyme activity. Specifically, this invention includes the following technical solutions.

[0011] The first aspect of the present invention provides a method for the enzyme-catalyzed synthesis of penicillin V, comprising the following steps: using 6-APA and phenoxyacetic acid ester as substrates, and using penicillin G acylase (Genbank accession number AAY25991.1) or its conserved variant polypeptide as shown in SEQ ID NO: 1 as a catalytic reaction to obtain penicillin V.

[0012] In one embodiment, the conserved variant polypeptide of the above-mentioned penicillin G acylase is a mutant that has more than 90% homology with the amino acid sequence SEQ ID NO: 1 of the wild-type enzyme, preferably more than 92%, more preferably more than 95%, more preferably more than 97%, more preferably more than 98%, and more preferably more than 99% homology, and whose enzyme activity is increased compared to the wild-type enzyme.

[0013] Preferably, the above-mentioned penicillin G acylase mutant refers to the A124T, F330G, T482S, Y486S, Y554F, V763L mutant of the wild-type enzyme SEQ ID NO: 1, with the amino acid sequence shown in SEQ ID NO: 3, and named PGA-V8 in this document.

[0014] MKQQWLSAALLAASSCLPAMAAQPVAPAAGQTSEAVAARPQTADGKVTIRRDAYGMPHVYADTVYGIFYGYGYAVAQDRLFQMEMARRSTQGRVAEVLGASMVGFDKSIRANFSPERIQRQLATLPAADRQVLDGYAAGMNAWLARVRAQPGQLMPKEFNDLGFAPADWTAYDVAMIFVGTMANRFSDANSEIDNLALLTALKDRHGAADAMRIFNQLRWLTDSRAPTTVPAEAGSYQPPVFQPDGADPLAYALPRYDGTPPMLERVVRDPATRGVVDGAPATLRAQLAAQYAQSGQPGIAGFPTTSNMWIVGRDHAKDARSILLNGPQGGWWNPAYTYGIGLHGAGFDVVGNTPFAYPSILFGHNAHVTWGSTAGFGDDVDIFAEKLDPADRTRYFHDGQWKTLEKRTDLILVKDAAPVTLDVYRSVHGLIVKFDDAQHVAYAKARAWEGYELQSLMAWTRKTQSANWEQWKAQAARHALSINWSYADDRGNIGYAHTGFYPRRRPGHDPRLPVPGTGEMDWLGLLPFSTNPQVYNPRQGFIANWNNQPMRGFPSTDLFAIVWGQADRYAEIETRLKAMTANGGKVSAQQMWDLIRTTSYADVNRRHFLPFLQRAVQGLPADDPRVRLVAGLAAWDGMMTSERQPGYFDNAGPAVMDAWLRAMLRRTLADEMPADFFKWYSATGYPTPQAPATGSLNLTTGVKVLFNALAGPEAGVPQRYDFFNGARADDVILAALDDALAALRQAYGQDPAAWKIPAPPMLFAPKNFLGVPQADAKAVLCYRATQNRGTENNMTVFDGKSVRAVDVVAPGQSGFVAPDGTPSPHTRDQFDLYNTFGSKRVWFTADEVRRNATSEETLRYPR (SEQ ID NO: 3). Or

[0015] The aforementioned penicillin G acylase mutant refers to the A124T, R185E, F330G, T482S, Y486S, N547R, Y554F, L559E, I562D, and V763L mutants of the wild-type enzyme SEQ ID NO: 1, with the amino acid sequence shown in SEQ ID NO: 5. In this paper, it is named PGA-V23.

[0016] (SEQ ID NO: 5).

[0017] In one embodiment, the phenoxyacetic ester is selected from alkyl esters such as methyl phenoxyacetate, ethyl phenoxyacetate, n-propyl phenoxyacetate, isopropyl phenoxyacetate, n-butyl phenoxyacetate, and tert-butyl phenoxyacetate. Preferably, the phenoxyacetic ester is methyl phenoxyacetate.

[0018]

[0019] Optionally, in the above method, the penicillin G acylase or its conserved variant polypeptide is in enzyme form or expressed in microbial cell form.

[0020] Furthermore, in the above method, the pH value of the reaction system is 6.0-7.2, preferably 6.2-7.0, preferably 6.5-6.7, and more preferably around 6.6; the reaction system is a buffer solution, and ammonia is used to adjust the pH of the reaction solution.

[0021] Furthermore, the reaction temperature is 20℃~40℃, preferably 25℃~35℃, more preferably 28℃~32℃, and even more preferably around 30℃.

[0022] It should be understood that in this article, when describing numerical characteristics, the terms "approximately" or "around" refer to the fact that the represented number may have an error range or fluctuation range of ±10%, ±9%, ±8%, ±7%, ±6%, or ±5%. The range of numerals appearing in this article includes the numeral itself and any number within that range.

[0023] A second aspect of this invention provides a penicillin G acylase mutant, the amino acid sequence of which is shown in SEQ ID NO: 3, and is named PGA-V8 herein; or

[0024] The amino acid sequence of the penicillin G acylase mutant is shown in SEQ ID NO: 5, and is named PGA-V23 in this paper.

[0025] A third aspect of the invention provides a gene encoding the penicillin G acylase mutant SEQ ID NO: 3 or SEQ ID NO: 5 as described above.

[0026] In one embodiment, the nucleotide sequence of the gene encoding the penicillin G acylase mutant SEQ ID NO: 3 is shown in SEQ ID NO: 4; and the nucleotide sequence of the gene encoding the penicillin G acylase mutant SEQ ID NO: 5 is shown in SEQ ID NO: 6.

[0027] A fourth aspect of the present invention provides a DNA molecule comprising the gene encoding as described above.

[0028] For example, the DNA molecule contains a coding gene with a nucleotide sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 6, and an upstream promoter such as the T7 promoter and / or a downstream terminator such as the T7 terminator. This DNA molecule may be referred to as an expression cassette / expression box of a penicillin G acylase mutant SEQ ID NO: 3 or SEQ ID NO: 5.

[0029] A fifth aspect of the present invention provides a recombinant plasmid, which is an expression plasmid formed by cloning the coding gene as described above on a plasmid vector.

[0030] The plasmid vectors mentioned above can be selected from the pET series (e.g., pET22b, pET24a, pET28a), pMAL series, pGEX series, pQE series, pBAD series, pCAI series, pSH series, pRSFDuet series, or other vectors.

[0031] The sixth aspect of the present invention provides a microbial engineered bacterium that expresses the coding gene as described above.

[0032] In one embodiment, the aforementioned engineered microbial bacteria are transformants that have been transformed with the recombinant plasmids described above; or positive recombinant bacteria that have cloned the gene encoding the above-described gene into the host genome using gene editing technology.

[0033] Preferably, the host bacteria are microorganisms with rapid proliferation rates and suitable for expressing exogenous recombinant proteins, such as those selected from Bacillus subtilis, Lactobacillus brevis, Escherichia coli, Vibrio natriureticis, Candida magnolius, Pichia pastoris, and Saccharomyces cerevisiae. Escherichia coli is a preferred microorganism, and more preferably Escherichia coli BL21(DE3).

[0034] Furthermore, when constructing the aforementioned engineered microorganisms, the transformation of the recombinant plasmids can be performed using conventional chemical transformation or electroporation methods to transfer them into competent cells; the aforementioned gene editing technologies are selected from, for example, the following groups: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), etc.

[0035] This invention screened a penicillin G acylase capable of smoothly catalyzing the reaction of 6-APA and phenoxyacetic acid esters (including alkyl esters such as methyl phenoxyacetate and ethyl phenoxyacetate) to produce penicillin V. Furthermore, a penicillin G acylase mutant with significantly enhanced enzyme activity was obtained through mutation. This penicillin G acylase and its mutant exhibit no substrate or product inhibition and demonstrate high substrate and product tolerance. This provides a more economical new route for the enzymatic synthesis of penicillin V. Attached Figure Description

[0036] Figure 1 The plasmid map of wild-type penicillin G acylase gene expression plasmid pET-PGA-V1 is shown.

[0037] Figure 2 The HPLC chromatograms of the reaction between the penicillin G acylase mutant PGA-V23 and the substrate 6-APA and methyl phenoxyacetate after 90 min are shown in the examples. Detailed Implementation

[0038] In the process development of the synthesis of penicillin V from the enzyme-catalyzed substrate 6-APA and phenoxyacetic acid ester, we screened a penicillin G acylase that meets the expected requirements for substrate and product tolerance. It is derived from Achromobacter sp. CCM 4824 (Genbank accession number AAY25991.1), and its amino acid sequence is shown in SEQ ID NO: 1.

[0039] Wild-type enzymes typically exhibit low activity. To enhance their activity and improve the feasibility of this process, we modified their amino acid sequences using mutagenesis. To this end, we constructed a random mutation library of penicillin G acylase using the classic error-prone PCR method, and combined this with site-directed mutagenesis based on the pocket structure of the enzyme model. Through multiple rounds of experiments, we screened and obtained several mutants with improved enzyme activity. Statistical analysis shows that specific mutations at certain sites can enhance enzyme activity, including mutations at positions such as alanine at position 124 to threonine (A124T), arginine at position 185 to glutamic acid (R185E), phenylalanine at position 330 to glycine (F330G), threonine at position 482 to serine (T482S), tyrosine at position 486 to serine (Y486S), asparagine at position 547 to arginine (N547R), tyrosine at position 554 to phenylalanine (Y554F), leucine at position 559 to glutamic acid (L559E), isoleucine at position 562 to aspartic acid (I562D), and valine at position 763 to leucine (V763L). The two mutants obtained, PGA-V8 (six-site mutations A124T, F330G, T482S, Y486S, Y554F, V763L) and PGA-V23 (ten-site mutations A124T, R185E, F330G, T482S, Y486S, N547R, Y554F, L559E, I562D, V763L), are particularly outstanding.

[0040] As used herein, the terms “(enzyme activity) increase,” “enhancement,” or “increase” mean an increase of at least 50% or more compared to a reference level, such as wild-type, or an increase of at least 80%, at least about 1, at least about 2, at least about 3, at least about 4, or at least about 5 times compared to a reference level.

[0041] In this document, the terms “wild-type penicillin G acylase”, “wild-type enzyme”, “initial enzyme”, and “original enzyme” have the same meaning, for example, referring to the penicillin G acylase with Genbank accession number AAY25991.1 and amino acid sequence SEQ ID NO: 1 (named PGA-V1 in the examples).

[0042] Correspondingly, the terms "penicillin G acylase mutant," "mutant penicillin G acylase," "mutant," and "mutant enzyme" have the same meaning, all referring to mutants of penicillin G acylase with increased enzyme activity, such as SEQ ID NO: 3 or SEQ ID NO: 5. For brevity and convenience, wild-type penicillin G acylase and its mutants may be collectively referred to as "penicillin G acylase (PGA)" in this article, as long as it is not confused with the wild-type enzyme SEQ ID NO: 1.

[0043] The term "mutation" includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably a positive mutation, i.e., a mutation that increases enzyme activity. The substitution can be a non-conservative substitution, a conserved substitution, or a combination of both. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in a polypeptide with amino acids from the same or similar amino acid definition class. However, as used herein, if a conserved mutation can alternatively be an aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restriction residue to restriction residue substitution, then a conserved mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue substitution. As is known in this technical field, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated into another aliphatic residue or another nonpolar residue.

[0044] The penicillin G acylase and its mutants of the present invention contain 863 amino acids with a well-defined sequence. Therefore, those skilled in the art can easily obtain its encoding gene, expression cassettes (DNA molecules) containing these genes, plasmids, and transformants containing the plasmids.

[0045] These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering construction methods well known to those skilled in the art.

[0046] In order to optimally express penicillin G acylase SEQ ID NO: 1 and its mutants SEQ ID NO: 3 and SEQ ID NO: 5 in microbial hosts, such as Escherichia coli, which is most commonly used in genetic engineering, the present invention has optimized the codons of its expression gene.

[0047] Codon optimization is a technique used to maximize protein expression in an organism by increasing the translation efficiency of genes of interest. Different organisms often exhibit a particular preference for one of a set of codons encoding the same amino acid due to mutational predisposition and natural selection. For example, in fast-growing microorganisms such as *E. coli*, optimized codons reflect the composition of their respective genomic tRNA repertoires. Thus, in fast-growing microorganisms, low-frequency codons for amino acids can be used for high-frequency codon substitutions of the same amino acid. Consequently, the expression of optimized DNA sequences is improved in fast-growing microorganisms.

[0048] After codon optimization, the coding gene for wild-type penicillin G acylase SEQ ID NO: 1 can be the nucleotide sequence SEQ ID NO: 2, while the coding gene for the penicillin G acylase mutant SEQ ID NO: 3 can be the nucleotide sequence SEQ ID NO: 4; and the coding gene for the mutant SEQ ID NO: 5 can be the nucleotide sequence SEQ ID NO: 6.

[0049] As used herein, “DNA molecule” and “expression cassette” refer to a gene expression system containing all the necessary elements required to express a target penicillin G acylase, such as SEQ ID NO: 5. Typically, this system includes the following elements: a promoter, a gene sequence encoding a penicillin G acylase, such as SEQ ID NO: 5, SEQ ID NO: 6, and a terminator; additionally, it may optionally include a signal peptide coding sequence, an enhancer sequence, etc.; these biological elements are operatively linked.

[0050] As used herein, “operable linking” refers to the functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region is placed at a specific position relative to the target gene nucleic acid sequence SEQ ID NO: 4, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby “operable linking” the promoter region to the nucleic acid sequence.

[0051] Preferably, the recombinant plasmid (or nucleic acid construct) includes one or more copies, two or more copies, preferably four or more copies, six or more copies, or eight or more copies of the penicillin G acylase, for example, the gene encoding SEQ ID NO: 3 or SEQ ID NO: 5.

[0052] When used as a biocatalyst in the preparation of penicillin V, the penicillin G acylate of the present invention can be in the form of an enzyme or in the form of a bacterial cell. The enzyme form includes free enzymes and immobilized enzymes, including purified enzymes, crude enzymes, fermentation broth, and enzymes immobilized on a carrier; the bacterial cell form includes live cells and dead cells.

[0053] In the field of biocatalysis, it is well known that compared with free enzyme methods, the application of immobilized enzyme technology has advantages such as simplified production processes and improved production efficiency. Furthermore, because the enzyme can be used multiple times and its stability is improved, the productivity per unit enzyme is effectively increased. Secondly, immobilized enzymes are easily separated from substrates and products, simplifying purification processes, resulting in higher yields and better product quality.

[0054] Those skilled in the art will readily understand that bacterial cells themselves are a natural form of enzyme immobilization, and can be used as an enzyme preparation for catalytic reactions without the need for disruption or even extraction and purification. Since the reaction substrates and products can easily cross the bacterial cell membrane—the biological barrier—disruption of the cells is unnecessary, which is economically advantageous.

[0055] On the other hand, compared with the catalysis of isolated enzymes, the present invention can provide a continuous and inexhaustible supply of enzymes or other substances through simple microbial fermentation, without the need for further extraction, purification, or enzyme separation. The economic benefits are obvious, creating conditions for industrial application.

[0056] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0057] Example

[0058] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0059] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0060] Materials and methods

[0061] In the embodiments, the whole gene synthesis, primer synthesis and sequencing were all completed by Suzhou Genewiz Biotechnology Co., Ltd.

[0062] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual" (4th Edition), edited by M.R. Green and J. Sambrook (USA), translated by He Fuchu, Science Press, Beijing, 2017. Specific experimental conditions can be determined through simple experiments if necessary.

[0063] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0064] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2 (LB solid medium with an additional 20 g / L agar powder).

[0065] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4·3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5.

[0066] Fermentation medium for a 5L fermenter: 24g / L yeast extract, 12g / L tryptone, 16.43g / L K2HPO4·3H2O, 2.31g / L KH2PO4, 5g / L glycerol, 0.5g / L defoamer, pH 7.0-7.5, with a fermentation broth volume of 2L per fermenter.

[0067] Feeding medium for a 5L fermenter: 60% glycerol.

[0068] Kanamycin (Kan, 50 μg / mL) should be used as directed based on the antibiotic gene carried by the plasmid.

[0069] Protein purification reagents:

[0070] 1M PB solution: Take 174g of dipotassium hydrogen phosphate and dilute it to 1L of ultrapure water (adjust the pH to 7.1 with potassium dihydrogen phosphate).

[0071] 4M sodium chloride solution: Take 117g of sodium chloride and dilute it to 0.5L of ultrapure water.

[0072] 4M imidazole solution: Dissolve 136g of imidazole in 0.5L of ultrapure water.

[0073] Equilibrium solution: Dissolve 50g glycerol, 13mL sodium chloride stock solution, and 10mL 1M PB solution in 100mL water, then add ultrapure water to bring the volume to 500mL.

[0074] Impurity removal solution: Dissolve 20g glycerol, 5mL 4M sodium chloride solution, 4mL 1M PB solution, and 1.25mL 4M imidazole solution in 100mL ultrapure water, then add ultrapure water to bring the volume to 200mL (adjust the pH to 7.1 with hydrochloric acid).

[0075] Coomassie Brilliant Blue Developing Solution: Weigh 100 mg of Coomassie Brilliant Blue G-250, dissolve it in 50 mL of 90% ethanol, add 100 mL of 85% phosphoric acid, and then add ultrapure water to bring the volume to 1000 mL.

[0076] Eluent: Dissolve 20g glycerol, 5mL 4M sodium chloride solution, 4mL 1M PB solution, and 12.5mL 4M imidazole solution in 100mL ultrapure water, then add ultrapure water to bring the volume to 200mL (adjust pH to 7.1 with hydrochloric acid).

[0077] Desalination equilibrium solution: Take 10 mL of 1 M PB solution and dilute to 500 mL with ultrapure water.

[0078] Dipotassium hydrogen phosphate buffer: Weigh 4.56g K2HPO4·3H2O and dissolve it in 900ml purified water. Adjust the pH to 7.8 with KH2PO4 and bring the volume to 1L with purified water.

[0079] The HPLC detection conditions for substrate 6-APA, methyl phenoxyacetate, and product penicillin V are as follows:

[0080] Agilent 1260 high performance liquid chromatograph; Waters Symmetry ® C18 (4.6×250mm 5μm) column; mobile phase A: 0.05M potassium dihydrogen phosphate (pH adjusted to 4.0 with phosphoric acid); mobile phase B: acetonitrile; column temperature: 30℃; flow rate: 1.0mL / min; detection wavelength: 215nm; injection volume: 10μL.

[0081] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 3 75 25 6 50 50 10 50 50 11 75 25 15 75 25

[0082] For ease of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number can share the same number, that is, the same number can refer to different biological forms in different descriptive scenarios. For example, PGA-V1 can represent the wild-type enzyme SEQ ID NO: 1 number, the wild-type enzyme-encoding gene SEQ ID NO: 2 number, and the wild-type enzyme SEQ ID NO: 1 expressing strain (EcPGA-V1 or WT).

[0083] Example 1: Construction of recombinant Escherichia coli with wild-type penicillin G acylase gene

[0084] Based on the amino acid sequence SEQ ID NO: 1 of wild-type penicillin G acylase derived from *Achromobacterium coli* CCM 4824 (Genbank accession number AAY25991.1), codon optimization was performed according to the codon preference of *E. coli* to obtain the coding gene sequence SEQ ID NO: 2 suitable for expression in *E. coli*. Suzhou Genewise Biotech Co., Ltd. was commissioned to synthesize the whole genome of SEQ ID NO: 2 and clone it into the NdeI and XhoI sites of the plasmid vector pET24a to obtain the recombinant plasmid pET-PGA-V1, the structure of which is shown below. Figure 1 As shown.

[0085] The recombinant plasmid pET-PGA-V1 was transformed into competent Escherichia coli BL21(DE3) cells by electroporation. Positive clones were screened to obtain recombinant E. coli EcPGA-V1 expressing wild-type penicillin G acylase. The expressed penicillin G acylase was designated PGA-V1. Simultaneously, a negative control group (transformed with the empty pET24a vector) was constructed to obtain E. coli EcPGA-V0.

[0086] Example 2: Error-prone PCR method for establishing random mutant libraries and high-throughput screening using PGA

[0087] 2.1 Error-prone PCR method for constructing a random mutation library

[0088] Using plasmid pET-PGA-V1 as a template, a random mutant library was constructed using error-prone PCR technology.

[0089] Design the following primer pair: PGA-UP / PGA-DN:

[0090] Forward primer PGA-UP: 5'-CTTTAAGAAGGAGATATACATATG-3',

[0091] Reverse primer PGA-DN: 5'-GGATAGCGCAGGGTTTCTTC-3'.

[0092] Using plasmid pET-PGA-V1 as a template, PCR amplification was performed to obtain a PGA mutant DNA sequence of approximately 2.6 kb.

[0093] The 50 μL error-prone PCR reaction system includes: 10 ng plasmid (pET-PGA-V1) template, 50 pmol primer pair PGA-UP and PGA-DN, 1×Taq buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl2, (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM) MnCl2, and 2.5 units of Taq enzyme (Takara).

[0094] The PCR reaction conditions were: 95℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min / kbp, 30 cycles; 72℃ for 10 min.

[0095] PCR products were electrophoresed and recovered using an Axygen DNA Gel Recovery Kit AP-GX-50. Using plasmid pET-PGA-V1 as a template and a recovered product (random mutant fragment) of approximately 2.6 kb as a large primer, MegaPrimer PCR was performed using KOD-plus DNA polymerase: 94℃ for 5 min; 98℃ for 10 s, 60℃ for 30 s, 68℃ for 2 min / kb, 25 cycles; 68℃ for 10 min. The plasmid template was digested with DpnI restriction endonuclease (Thermo Fisher Scientific), and electrotransformed into *E. coli* BL21(DE3) to obtain more than 10... 4 A random mutation library of clones.

[0096] 2.2 96-well plate culture of random mutant library clones

[0097] Single colonies were picked and transferred to 96-well plates (each well containing 100 μL of liquid LB-Kan medium), with EcPGA-V1 as the control strain. After incubation at 37°C and 400 rpm for 12 h, 30 μL of the bacterial culture was transferred from each well to a 96-well deep-well plate (each well containing 120 μL of liquid TB-Kan-0.2 mM IPTG), and incubated at 25°C and 400 rpm for 12–16 h. The bacterial cells were collected by centrifugation at 4°C and 4000 rpm for 10 min, and the supernatant was removed. Subsequently, the bacterial cells were washed with pre-cooled physiological saline, centrifuged at 4°C and 4000 rpm for 10 min, and the supernatant was removed.

[0098] 2.3 High-throughput reaction screening

[0099] Add 200 μl of preheated phosphate buffer (50 mM, pH 7.5) containing 0.4 g / L NIPAB (6-nitro-3-phenylacetylcarbamate) to each well. Incubate at 37°C, and measure the absorbance at 405 nm after 2 min and 4 min. Calculate the difference between the absorbance values ​​at 4 min and 2 min; a larger difference indicates higher enzyme activity. Select strains with significantly improved enzyme activity, extract plasmids, and perform nucleic acid sequencing at Suzhou Genewiz Biotechnology Co., Ltd. The penicillin G acylase-related fragments in the genome are compared with SEQ ID NO: 2 to determine the amino acid sequence changes of penicillin G acylase. Select the optimal strain with the highest improvement in enzyme activity as the starting strain for the next round of random mutant library construction.

[0100] Table 1 shows the activity comparison between mutants PGA-V2 to PGA-V8 and wild-type PGA-V1. The coding gene sequence of mutant PGA-V8 is SEQ ID NO: 4, and the encoded protein amino acid sequence is SEQ ID NO: 3.

[0101] Table 1. Results of the first and second rounds of high-throughput screening of random mutant libraries

[0102] Starting strain strain number Penicillin G acylase mutant Mutation sites relative to their respective originating bacteria Absorbance difference increase factor EcPGA-V1 / / / EcPGA-V2 PGA-V2 E96V, L202P, V276M ++ EcPGA-V1 EcPGA-V3 PGA-V3 A28G, I177L +++ EcPGA-V4 PGA-V4 V59A, V780E +++ EcPGA-V5 PGA-V5 T482S, Y554F, V763L +++++ EcPGA-V6 PGA-V6 D410N + EcPGA-V5 EcPGA-V7 PGA-V7 F113C, E406A, G553S, L737I ++ EcPGA-V8 PGA-V8 A124T, F330G, Y486S ++++

[0103] Notes: "+" indicates that the viability percentage relative to the original strain is greater than 0% and less than or equal to 50%; "++" indicates that the viability percentage relative to the original strain is greater than 50% and less than or equal to 100%; "+++" indicates that the viability percentage relative to the original strain is greater than 100% and less than or equal to 150%; "++++" indicates that the viability percentage relative to the original strain is greater than 150% and less than or equal to 200%; "+++++" indicates that the viability percentage relative to the original strain is greater than 200%.

[0104] Example 3: Site-directed mutagenesis further enhances PGA enzyme activity and penicillin V tolerance.

[0105] 3.1 Computer-aided design identifies key sites for PGA to enhance penicillin V tolerance.

[0106] This method uses PGA-V8 as the starting sequence and employs GROMACS (version 2025.2) molecular dynamics simulations and gmx_MMPBSA (version 1.6.4) energy decomposition analysis to systematically identify key residues affecting enzyme-substrate binding and predict mutation effects through computer simulations. First, the structures of the protein and penicillin V were constructed using Alphafold and PubChem. Then, the enzyme-substrate complex system was constructed using Autodock Vina (version 1.1.2). Solvation, ion neutralization, and energy minimization were performed under a Charmm36 force field, followed by a 200 ps NVT and a 50 ns NPT. A Leap-Frog integrator was used during the simulation, with a time step of 2 fs, and LINCS constraints were applied to fix the hydrogen bond length. The system temperature was controlled at 303 K using a V-rescale temperature coupler, and the pressure was maintained using a Parrinello-Rahman pressure coupler. Long-range electrostatic interactions were performed using the PME method with a cutoff distance of 1.2 nm, and the van der Waals force cutoff distance was also set to 1.2 nm. After obtaining a stable dynamic trajectory, the trajectory was aligned and framed, and MM-PBSA calculations were performed using gmx_MMPBSA to decompose the binding free energy into residues, thereby identifying key residues resistant to penicillin V. The study found that mutations at R185E, N547R, L559E, and I562D reduced the binding energy of penicillin V to PGA. Therefore, these site mutations may reduce the affinity of penicillin V for PGA, which on the one hand helps to reduce the contact time between penicillin V and PGA enzymes, thus reducing enzyme activity loss; on the other hand, it facilitates the release of penicillin V, thereby increasing substrate conversion.

[0107] Table 2. Computer Simulation Results

[0108] mutation point Binding energy (kcal / mol) PGA-V8 -28.27 PGA-V8 R185E -19.03 PGA-V8 N547R -24.21 PGA-V8 L559E -27.23 PGA-V8 I562D -21.47

[0109] 3.2 Construction of mutants

[0110] Design R185E-5 / R185E-3 primer pairs to perform R185E mutation:

[0111] R185E-5: 5'-TTGTGGGCACGATGGCGAACGAATTTAGCGATGCGAACAGCGA-3',

[0112] R185E-3: 5'-TCGCTGTTCGCATCGCTAAATTCGTTCGCCATCGTGCCCACAA-3'.

[0113] Design N547R-5 / N547R-3 primer pairs to perform N547R mutation:

[0114] N547R-5: 5'-AAGGCTTTATTGCGAACTGGAGAAATCAGCCGATGCGCGGCTT-3',

[0115] N547R-3: 5'-AAGCCGCGCATCGGCTGATTTCTCCAGTTCGCAATAAAGCCTT-3'.

[0116] Design L559E-5 / L559E-3 primer pairs to perform L559E mutation:

[0117] L559E-5: 5'-GCGGCTTTCCGAGCACCGATGAATTTGCGATTGTGTGGGGCCA-3',

[0118] L559E-3: 5'-TGGCCCCACACAATCGCAAATTCATCGGTGCTCGGAAAGCCGC-3'.

[0119] Design the I562D-5 / I562D-3 primer pair to perform I562D mutation:

[0120] I562D-5: 5'-CGAGCACCGATCTGTTTGCGGATGTGTGGGGCCAAGCGGATCG-3',

[0121] I562D-3: 5'-CGATCCGCTTGGCCCCACACATCCGCAAACAGATCGGTGCTCG-3'.

[0122] Design L559E-I562D-5 / L559E-I562D-3 to perform L559E-I562D mutation:

[0123] L559E-I562D-5:

[0124] 5'-GCGGCTTTCCGAGCACCGATGAATTTGCGGATGTGTGGGGCCAAGCGGATCG-3',

[0125] L559E-I562D-3:

[0126] 5'-CGATCCCGCTTGGCCCCACACATCCGCAAATTCATCGGTGCTCGGAAAGCCGC-3'.

[0127] Using the sequence SEQ ID NO: 4 as a template, plasmid PCR was performed using primer pairs R185E-5 / R185E-3, N547R-5 / N547R-3, L559E-5 / L559E-3, and I562D-5 / I562D-3 to construct site-directed mutagenesis and obtain mutants PGA-V9 (PGA-V8-R185E), PGA-V10 (PGA-V8-N547R), PGA-V11 (PGA-V8-L559E), and PGA-V12 (PGA-V8-I562D).

[0128] Using the PGA-V9 plasmid nucleic acid sequence as a template, site-directed mutagenesis was constructed by plasmid PCR using primer pairs N547R-5 / N547R-3, L559E-5 / L559E-3, and I562D-5 / I562D-3, respectively, to obtain mutants PGA-V13 (PGA-V8-R185E-N547R), PGA-V14 (PGA-V8-R185E-L559E), and PGA-V15 (PGA-V8-R185E-I562D). Using the PGA-V10 plasmid nucleic acid sequence as a template, site-directed mutagenesis was performed by plasmid PCR using primer pairs L559E-5 / L559E-3 and I562D-5 / I562D-3 to obtain the mutants PGA-V16 (PGA-V8-N547R-L559E) and PGA-V17 (PGA-V8-N547R-I562D). Using the sequence SEQ ID NO: 4 as a template, site-directed mutagenesis was performed by plasmid PCR using primer pairs L559E-I562D-5 / L559E-I562D-3 to obtain the mutant PGA-V18 (PGA-V8-L559E-I562D).

[0129] Using the PGA-V13 plasmid nucleic acid sequence as a template, site-directed mutagenesis was constructed by plasmid PCR using primer pairs L559E-5 / L559E-3 and I562D-5 / I562D-3, respectively, to obtain mutants PGA-V19 (PGA-V8-R185E-N547R-L559E) and EcPGA-V20 (PGA-V8-R185E-N547R-I562D). Using the PGA-V18 plasmid nucleic acid sequence as a template, site-directed mutagenesis was constructed by plasmid PCR using primer pairs R185E-5 / R185E-3 and N547R-5 / N547R-3, respectively, to obtain mutants PGA-V21 (PGA-V8-R185E-L559E-I562D) and PGA-V22 (PGA-V8-N547R-L559E-I562D).

[0130] Using the PGA-V12 plasmid nucleic acid sequence as a template, site-directed mutagenesis was constructed by plasmid PCR using primer pair R185E-5 / R185E-3 to obtain the mutant PGA-V23 (PGA-V8-R185E-N547R-L559E-I562D).

[0131] The PCR product was digested with DpnI and then transformed into the host Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli EcPGA-V9~EcPGA-V23 expressing penicillin G acylase mutants.

[0132] 3.3 Expression of penicillin G acylase mutant

[0133] Single colonies of EcPGA-V0 and EcPGA-V8~EcPGA-V23 were picked and cultured overnight at 37°C and 220 rpm in 5 mL of liquid LB medium containing Kans. The next day, the colonies were transferred to shake flasks containing 100 mL of liquid TB medium at a volume concentration of 1%, and cultured at 37°C and 220 rpm until the OD600 nm reached 1.3~1.5. Then, IPTG was added to a final concentration of 0.2 mM for 24 h induction. The bacterial cells were collected by centrifugation at 4°C and 4000 rpm, and the supernatant was discarded.

[0134] 3.4 Protein purification of penicillin G acylase mutant

[0135] EcPGA-V0, EcPGA-V8~EcPGA-V23 bacterial cells were resuspended in 50 mL of equilibration buffer (50 mM potassium phosphate buffer, 200 mM NaCl, pH 8.0), then sonicated. The disrupted cells were centrifuged at 4 °C and 12,000 rpm for 20 min, and the supernatant was collected. The supernatant was added at a rate of 1 mL / min to an affinity chromatography column containing 10 mL of Ni-NAT matrix. The column was then washed with equilibration buffer containing 30 mM imidazole to elute impurities. Finally, the column was washed with equilibration buffer containing 500 mM imidazole to remove the target protein, and the peak eluent was collected. The eluent was desalted by ultrafiltration to obtain pure enzyme.

[0136] 3.5 Penicillin V tolerance assay for penicillin G acylase mutants

[0137] Enzyme activity assay in the experimental group: 10 mL of 50 mM phosphate buffer (pH 6.6) containing 5% sonicated cells (w / v) and 100 g / L penicillin V was incubated at 30 °C and 150 rpm for 20 h, and the hydrolytic activity of penicillin G was then detected.

[0138] Control group viability assay: 10 mL of 50 mM phosphate buffer (pH 6.6) containing 5% of sonicated cells (w / v) was incubated at 30 °C and 150 rpm for 20 h before the hydrolytic activity of penicillin G was measured.

[0139] Method for determining the hydrolytic activity of penicillin G: Weigh 1 g of potassium penicillin G and dissolve it in 20 mL of dipotassium hydrogen phosphate buffer. Adjust the pH to 8.00 with 0.1 mol / L sodium hydroxide solution. After centrifuging the fermentation broth, resuspend the bacterial cells in the buffer solution for enzyme activity determination and dilute accordingly (the input enzyme activity range is 13-16 U / ml, i.e., 0.5 g of bacterial cells). Completely disrupt the cell walls using an ultrasonic cell disruptor. After disruption, pipette an appropriate amount of the disrupted solution and add it to the solution prepared in section 2.1, which has been preheated to 27.8-28.2℃. Maintain the temperature and stir rapidly. Titrate with 0.1 mol / L NaOH solution to maintain the pH at 8.00 ± 0.05 for 3-5 min. Record the amount of alkali added and the reaction time. One unit is defined as 1 μmol of NaOH consumed per unit of enzyme.

[0140] Formula for calculating enzyme activity:

[0141] ,

[0142] in

[0143] V2: Titrator graduations after titration, ml; V1: Titrator graduations before titration, ml;

[0144] min: the number of whole minutes taken for the reaction; S: the number of seconds taken for the reaction after removing the whole minutes;

[0145] V sample: Volume of enzyme solution sample, m; W sample: Weight of immobilized enzyme sample, g;

[0146] C: The molar concentration of the NaOH titrant is 0.1 mol / L.

[0147] As shown in Table 3, the difference in activity between the control group and the experimental group represents the rate of loss of hydrolytic activity of penicillin G. After introducing single-point or combined mutations of R185E, N547R, L559E, and I562D, the rate of loss of hydrolytic activity of penicillin G was reduced to as low as 2.7%.

[0148] Table 3. Loss rate of penicillin G hydrolytic activity of PGA

[0149] strain number Penicillin G acylase mutant Relative PGA-V8 variant sites Loss rate of hydrolytic activity of penicillin G EcPGA-V0 PGA-V0 / 64.6% EcPGA-V8 PGA-V8 / 66.3% EcPGA-V9 PGA-V9 R185E 16.1% EcPGA-V10 PGA-V10 N547R 40.5% EcPGA-V11 PGA-V11 L559E 32.8% EcPGA-V12 PGA-V12 I562D 20.3% EcPGA-V13 PGA-V13 R185E, N547R 18.9% EcPGA-V14 PGA-V14 R185E, L559E 16.7% EcPGA-V15 PGA-V15 R185E, I562D 10.6% EcPGA-V16 PGA-V16 N547R, L559E 41.2% EcPGA-V17 PGA-V17 N547R, I562D 19.5% EcPGA-V18 PGA-V18 L559E, I562D 18.1% EcPGA-V19 PGA-V19 R185E, N547R, L559E 8.3% EcPGA-V20 PGA-V20 R185E, N547R, I562D 7.6% EcPGA-V21 PGA-V21 R185E, L559E, I562D 3.3% EcPGA-V22 PGA-V22 N547R, L559E, I562D 13.3% EcPGA-V23 PGA-V23 R185E, N547R, L559E, I562D 2.7%

[0150] The amino acid sequence of the mutant PGA-V23 is SEQ ID NO: 5, and the nucleic acid sequence of the corresponding encoding gene is SEQ ID NO: 6. Table 3 shows that the mutant PGA-V23 exhibits high tolerance to the product penicillin V.

[0151] Example 4: Fermentation of PGA-producing Escherichia coli strains

[0152] Fermentation of strains EcPGA-V0, EcPGA-V8, and EcPGA-V23 was carried out in a 5L bioreactor. Single colonies were picked and cultured overnight at 37°C and 220 rpm in 5 mL of liquid LB medium containing Kans. The next day, the inoculum was transferred to shake flasks containing 100 mL of liquid TB medium at a concentration of 5% v / v, and cultured at 37°C and 220 rpm until the OD600nm reached 6. This seed culture was then transferred to a 5L fermenter. After inoculation, the culture was carried out at 37°C and 400–800 rpm, with dissolved oxygen controlled within the range of 20–30%. When the bacterial OD600nm reached 20, IPTG was added to induce PGA expression. The final IPTG concentration was 0.2 mM, and the culture was continued at 25°C for 22–24 hours. Throughout the fermentation process, ammonia was used to control the pH at 6.8–7.2, and the aeration rate was controlled within the range of 2.5–3.5 L / min. After fermentation, the cells were collected by centrifugation at 10,000 rpm for 10 min at 4°C, resuspended in 0.1 M dipotassium hydrogen phosphate buffer (pH 8.0), and then subjected to high-pressure disruption.

[0153] Example 5: Enzymatic Synthesis of Penicillin V

[0154] The total reaction system of 100 mL included: 36 mmol 6-APA, 39.6 mmol methyl phenoxyacetate, and 25 mL of 20% (w / v) bacterial cell lysis supernatant. The specific operation procedure was as follows: 10 mL of pure water was added to the enzyme synthesis reactor, and the temperature was controlled at 30℃. 39.6 mmol methyl phenoxyacetate was added, followed by 25 mL of 20% (w / v) bacterial cell lysis supernatant. 36 mmol 6-APA was dissolved in 35 mL of pure water, and approximately 13 mL of 3M ammonia was added between 15-20℃ to adjust the pH to 7.2-7.3. This solution was added to the reactor, and the reactor was brought to a final volume of 100 mL with pure water. The pH of the reaction system was controlled at 6.5-6.7 using 3M ammonia, and the temperature was controlled at 30℃ during the reaction. As shown in Table 4, the substrate conversion rate of the mutant PGA-V23 was 98.6%, and the liquid chromatography analysis results are as follows: Figure 2 As shown.

[0155] Table 4. Substrate conversion efficiency of PGA mutants

[0156] strain number Penicillin G acylase mutant Relative PGA-V8 variant sites Reaction termination time Substrate conversion rate EcPGA-V0 PGA-V0 / 4h30min 23.2% EcPGA-V8 PGA-V8 / 110min 96.4% EcPGA-V23 PGA-V23 R185E, N547R, L559E, I562D 90min 98.6%

[0157] In summary, the penicillin G acylases screened in this invention exhibit high tolerance to substrates and products; the constructed penicillin G acylase mutants SEQ ID NO: 3 and SEQ ID NO: 5 have higher enzyme activity compared to the wild-type enzyme, and mutant SEQ ID NO: 5 exhibits high tolerance to the product penicillin V, showing promising industrialization potential.

[0158] It should be understood that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A method for the enzyme-catalyzed synthesis of penicillin V, characterized in that, The process includes the following steps: using 6-APA and phenoxyacetic acid ester as substrates, penicillin G acylase (Genbank accession number AAY25991.1) or its conserved variant polypeptide as shown in SEQ ID NO: 1 as a catalytic reaction to obtain penicillin V.

2. The method as described in claim 1, characterized in that, The conserved variant polypeptide of the penicillin G acylase is a mutant that has more than 90% homology with the amino acid sequence of the wild-type enzyme (SEQ ID NO: 1) and exhibits increased enzyme activity compared to the wild-type enzyme.

3. The method as described in claim 2, characterized in that, The mutant refers to the A124T, F330G, T482S, Y486S, Y554F, and V763L mutants of the wild-type enzyme SEQ ID NO: 1, with the amino acid sequence shown in SEQ ID NO: 3; or The mutants refer to the A124T, R185E, F330G, T482S, Y486S, N547R, Y554F, L559E, I562D, and V763L mutants of the wild-type enzyme SEQ ID NO: 1, with the amino acid sequences shown in SEQ ID NO:

5.

4. The method as described in claim 1, characterized in that, The phenoxyacetic ester is selected from methyl phenoxyacetate, ethyl phenoxyacetate, n-propyl phenoxyacetate, isopropyl phenoxyacetate, n-butyl phenoxyacetate, and tert-butyl phenoxyacetate. Preferably, the phenoxyacetic ester is methyl phenoxyacetate.

5. The method as described in claim 1, characterized in that, The penicillin G acylase or its conserved variant polypeptide is in enzyme form or expressed in microbial cell form.

6. A penicillin G acylase mutant, characterized in that, The amino acid sequence of the penicillin G acylase mutant is shown in SEQ ID NO: 3; or The amino acid sequence of the penicillin G acylase mutant is shown in SEQ ID NO:

5.

7. The gene encoding the penicillin G acylase mutant SEQ ID NO: 3 or SEQ ID NO: 5 as described in claim 6.

8. The gene as described in claim 7, characterized in that, The nucleotide sequence of the gene encoding the penicillin G acylase mutant SEQ ID NO: 3 is shown in SEQ ID NO: 4; the nucleotide sequence of the gene encoding the penicillin G acylase mutant SEQ ID NO: 5 is shown in SEQ ID NO:

6.

9. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression plasmid formed by cloning the gene as described in claim 7 on a plasmid vector.

10. A type of engineered microbial bacterium, characterized in that, It expresses the gene as described in claim 8.

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