A penicillin G acylase mutant

Abstract

This invention discloses a penicillin G acylase mutant SEQ ID NO:3, which can efficiently catalyze the reaction of substrate 6-APA with DHPGM to produce amoxicillin. The synthesis / hydrolysis ratio S / H reaches a maximum of 18.1. When the side chain to parent nucleus ratio is 1.05:1, the conversion rate of parent nucleus 6-APA reaches more than 99.0%. This mutant has high thermal stability, which helps to improve the efficiency of industrial production of amoxicillin.

Description

Technical Field

[0001] This invention belongs to the field of enzyme catalysis technology and relates to a penicillin G acylase mutant, specifically a penicillin G acylase mutant for the synthesis of amoxicillin. Background Technology

[0002] Penicillin G acylase (EC3.5.1.11, abbreviated as PGA) is an important enzyme used in the preparation of semi-synthetic β-lactam antibiotics. This enzyme is a reversible reaction enzyme involved in the hydrolysis and synthesis of β-lactam antibiotics. In the antibiotic industry, its hydrolytic function is used to produce 6-aminopenicillic acid (6-APA, also known as 6-aminopenicillic acid) and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA), important parent nuclei for β-lactam antibiotics. Its synthetic function catalyzes the reaction of 6-APA, 7-ADCA, or other parent nuclei with various D-amino acid side chains to generate new semi-synthetic β-lactam antibiotics (semi-synthetic penicillins and cephalosporins). In addition, penicillin G acylase can be used in the synthesis of some chiral compounds to protect hydroxyl and amino groups and to resolve chiral compounds.

[0003] Penicillin G acylases from different microbial sources have their own reaction characteristics. Among them, the hydrolytic performance of PGA from *Escherichia coli* and *Bacillus megaterium* has been reported relatively frequently, with most research focusing on enhancing their enzyme activity. The inventors previously developed some penicillin G acylase mutants with relatively ideal hydrolytic activity, as described in studies such as CN201610097503.4 and CN202210270135.4.

[0004] In the structural model of penicillin G acylase derived from *E. coli*, the amino acid βF24 plays a crucial role in the enzyme's synthetic performance because it is located in the pocket position where the acylase binds to penicillin G. Similarly, αR145 and αF146 also play key roles (Alkema et al., *Protein Engineering Design & SelAvtion*, 17(5), 473-480, 2004). The construction of these enzyme models has a certain promoting effect on the exploration of improvements in enzyme performance.

[0005] Enzyme-catalyzed reactions involve multiple aspects in actual processes, including specific activity, selectivity, substrate / product inhibition, pH, temperature, and stability. To cope with fierce competition in the antibiotic raw material industry, comprehensively improving the various properties of enzymes and meeting the diverse needs of actual industrial production processes is one of the most effective means of reducing production costs.

[0006] Although the inventors reported some penicillin G acylase mutants with relatively ideal hydrolytic activity in patent documents CN201610097503.4 and CN202210270135.4, it was found in actual production that they are relatively delicate, sensitive to higher temperatures, have poor stability above 35℃, and have a rapid rate of enzyme activity decay. This is an adverse factor affecting the production of amoxicillin by catalyzing the 6-APA reaction and the production of cephalexin by catalyzing the 7-ADCA reaction. Summary of the Invention

[0007] In order to find penicillin G acylase with high enzyme activity, strong synthetic performance and good thermostability, the inventors have always focused on continuously modifying the penicillin G acylase reported in the existing technology, in order to obtain an ideal enzyme or its mutant that meets as many requirements as possible. Our research found that, compared with wild-type penicillin G acylases from *Escherichia coli*, *Bacillus megaterium*, and *Achromobacter* (p. CCM 4824), the penicillin G acylase derived from *Arthrobacter viscosus* (amino acid sequence shown in SEQ ID NO:1, corresponding to the 95bp-2548bp nucleic acid sequence of GenBank L04471.1) exhibited better pro-synthetic performance in catalyzing the reaction of substrate 6-APA with DHPGM (D-hydroxyphenylglycine methyl ester, D-HPGM) to produce amoxicillin (AMXL). It had a relatively high synthesis / hydrolysis ratio (S / H, the molar ratio of the target product amoxicillin to the byproduct p-hydroxyphenylglycine (D-HPG)) and weak hydrolytic performance. However, its enzyme activity was limited, with a conversion rate of only about 45%, which, according to analysis, was also related to its low thermostability.

[0008] Enhanced enzyme thermostability is beneficial for extending the reaction time catalyzed by the enzyme and reducing the production cost of amoxicillin. Therefore, based on bioinformatics, an enzyme structure model was constructed, and strategies such as rational design, site-directed saturation mutagenesis of amino acids, and random mutation of gene sequences were used to attempt to modify penicillin G acylase SEQ ID NO:1. After multiple rounds of high-throughput screening, several mutants with significantly improved enzyme activity and thermostability were obtained. Specifically, the technical solution of this invention is as follows.

[0009] A penicillin G acylase mutant, which is selected from the following polypeptides:

[0010] (a) A polypeptide with the amino acid sequence SEQ ID NO:3;

[0011] (b) A polypeptide having 85% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, more preferably 99% or more homology with SEQ ID NO:3 in an amino acid sequence, and having increased enzyme activity compared with SEQ ID NO:3 in a reaction environment at 35°C or above.

[0012] (SEQ ID NO:3).

[0013] In this article, the penicillin G acylase mutant with the amino acid sequence SEQ ID NO:3 is designated AvPGA25, which is the wild-type penicillin G acylase SEQ ID NO:3. The following are mutants of NO:1, with the following mutations: proline at position 58 mutated to alanine (P58A), phenylalanine at position 138 mutated to leucine (F138L), asparagine at position 147 mutated to aspartic acid (N147D), serine at position 226 mutated to glycine (S226G), valine at position 305 mutated to alanine (V305A), phenylalanine at position 307 mutated to alanine (F307A), tyrosine at position 352 mutated to aspartic acid (Y352D), alanine at position 445 mutated to threonine (A445T), isoleucine at position 566 mutated to threonine (I566T), tyrosine at position 601 mutated to phenylalanine (Y601F), proline at position 738 mutated to glutamine (P738Q), and aspartic acid at position 783 mutated to asparagine (D783N).

[0014] The enzyme activity mentioned above refers to the enzyme activity that catalyzes the reaction of the substrate 6-aminopenicillanic acid (6-APA) with D-type p-hydroxyphenylglycine methyl ester (DHPGM) to produce amoxicillin (AMXL).

[0015] Another aspect of the present invention provides a gene encoding the above-mentioned penicillin G acylase mutant.

[0016] For example, the gene encoding the penicillin G acylase mutant SEQ ID NO:3 can be a polynucleotide with a nucleotide sequence as shown in SEQ ID NO:4, or a polynucleotide with a nucleotide sequence that is 90% or more, preferably 92% or more, preferably 95% or more, preferably 97% or more, preferably 98% or more, more preferably 99% or more homologous to SEQ ID NO:4.

[0017] The present invention also provides plasmids containing the above-mentioned coding genes. For example, the above-mentioned plasmids can be pET vectors such as pET22b, pET24a, and pET28a, or other commonly used vectors such as pSH plasmids.

[0018] Another aspect of the present invention provides a microorganism for expressing the above-mentioned penicillin G acylase mutant, such as SEQ ID NO:3, whose genome integrates the above-mentioned coding gene, such as SEQ ID NO:4, or a microorganism transformed with the above-mentioned plasmid.

[0019] The plasmids described above can be transformed into competent cells using conventional chemical or electroporation methods. The gene editing technologies mentioned above are selected from 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.

[0020] Preferably, the microorganisms mentioned above are microorganisms with rapid proliferation rates and suitable for expressing exogenous recombinant proteins, such as those selected from Bacillus subtilis, Lactobacillus brevis, Escherichia coli, Candida magnoliae, Pichia pastoris, and Saccharomyces cerevisiae. Escherichia coli is a preferred microorganism, and more preferably Escherichia coli BL21(DE3).

[0021] The aforementioned penicillin G acylase mutant or the aforementioned microorganism can be used to produce amoxicillin. For example, amoxicillin can be produced by catalyzing the reaction of substrate 6-APA with DHPGM using the aforementioned penicillin G acylase mutant or the aforementioned microorganism.

[0022] The above reaction system is preferably pH 7.0±0.5, more preferably pH 7.0±0.2, and even more preferably pH 7.0±0.1.

[0023] The reaction temperature is preferably 25℃-35℃, more preferably 26℃-32℃, and even more preferably 28℃-30℃.

[0024] Optionally, the above reaction system is a phosphate buffer system.

[0025] Compared to wild-type penicillin G acylase SEQ ID NO:1, the penicillin G acylase mutant selected by mutation screening in this invention exhibits significantly improved synthetic performance in catalyzing the reaction of 6-APA with DHPGM to produce amoxicillin. The S / H ratio of the synthesized product to the hydrolysate reaches a maximum of 18.1, an increase of 2.6 times. At a side chain to core ratio of 1.05:1, the conversion rate of 6-APA in the core reaches over 99.0%. The enzyme's thermostability index, half-life, is increased by 10 times, making it suitable for industrial application. Detailed Implementation

[0026] When amoxicillin is synthesized from the substrate 6-APA via enzyme catalysis, two side reactions generally occur: 1) hydrolysis of the activated acyl donor DHPGM and 2) hydrolysis of the generated amoxicillin. This leads to a reduction in both the acyl donor DHPGM and the generated amoxicillin, resulting in a lower conversion rate of the parent nucleus, i.e., a lower ratio of synthesized product to hydrolyzed product (S / H). Moreover, the enzyme's own characteristics still have the most significant impact on the S / H value. Protein engineering has been reported to improve the synthetic performance of penicillin G acylase (Alkema, et al., Protein Eng., 13(12), 857-63, 2000).

[0027] Experiments revealed that wild-type penicillin G acylases derived from *Escherichia coli*, *Bacillus megaterium*, and *Achromobacterium* exhibited strong hydrolytic activity in catalyzing the reaction of 6-APA with DHPGM to synthesize amoxicillin, resulting in a lower S / H ratio. In contrast, wild-type penicillin G acylases derived from *Arthrobacter* showed lower hydrolytic activity, meaning their synthetic activity was significantly greater than their hydrolytic activity, which facilitated forward control of the reaction. This invention improves the synthetic activity, thereby increasing the S / H ratio, thus improving the amoxicillin production rate and reducing the amoxicillin production cost.

[0028] In this paper, the terms "wild-type," "wild-type enzyme," and "wild-type enzyme" have the same meaning, referring to wild-type penicillin G acylase (GenBank No. L04471.1, amino acid sequence SEQ ID NO:1). Similarly, the terms "penicillin G acylase mutant," "mutant penicillin G acylase," "mutant penicillin G acylase," and "mutant enzyme" have the same meaning, referring to mutants formed by mutations in individual amino acid residues in the amino acid sequence of penicillin G acylase, such as SEQ ID NO:3. Sometimes, for the sake of convenience, the wild-type enzyme and its mutants, such as SEQ ID NO:3, may be collectively referred to as "penicillin G acylase" in this paper.

[0029] In this document, the terms “enzyme activity”, “enzyme activity”, or “enzyme synthetic activity” specifically refer to the synthetic performance of the enzyme in catalyzing the synthesis of amoxicillin from the substrates 6-APA and DHPGM, and obviously do not include the hydrolytic activity of DHPGM and / or amoxicillin, because the present invention does not expect the penicillin G acylase to hydrolyze DHPGM and / or amoxicillin.

[0030] In this article, the terms “(enzyme activity) increase” or “enhancement” as used above mean an increase of at least 100% compared to the reference level, such as at least about 1, at least about 2, or at least about 3, or at least about 5, or at least about 10, or at least about 20 times compared to the reference level.

[0031] 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 the art, 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 to another aliphatic residue or another nonpolar residue. Exemplary conservative substitutions can be performed according to the table below, wherein amino acids belonging to the same partition in the second column can be substituted for each other, and preferably, amino acids in the same row in the third column can be substituted for each other:

[0032]

[0033] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can be performed between, rather than within, the amino acids defined above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0034] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletion can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, while preserving enzyme activity and / or the modified properties of the engineered aldolase. Deletion can target the interior and / or ends of the peptide. In several embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0035] "Insertion" refers to a modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, modified engineered aldolases include inserting one or more amino acids into a naturally occurring aldolase and inserting one or more amino acids into other modified aldolase polypeptides. The insertion can be internal to the polypeptide, or at the carboxyl terminus or amino terminus. Insertions as used herein include fusion proteins as known in the art. The insertion can be a continuous amino acid segment or separated by one or more amino acids in a naturally occurring polypeptide.

[0036] To perform site-directed mutagenesis, a three-dimensional model of penicillin G acylase can be constructed using bioinformatics techniques. This model can be used to infer the enzyme's catalytic active site, substrate / product entry / exit channels, rigid / flexible structural regions, etc., and potential structurally influential sites can be selected and saturation mutagenesis can be attempted.

[0037] This invention employs a site-directed mutagenesis combined with error-prone PCR random mutagenesis method to perform multiple rounds of mutagenesis and high-throughput screening on wild-type penicillin G acylase SEQ ID NO:1, obtaining several mutants with significantly enhanced enzyme activity, including mutant SEQ ID NO:3 and other mutant enzymes with amino acid sequences highly homologous to SEQ ID NO:3. These enzymes all fall within the scope of protection of this invention. Mutant SEQ ID NO:3 exhibits 12 site mutations relative to the wild-type enzyme: P58A, F138L, N147D, S226G, V305A, F307A, Y352D, A445T, I566T, Y601F, P738Q, and D783N.

[0038] For the sake of brevity, the abbreviations for amino acids in this article may use either three letters or single letters, as is well known to those skilled in the art. These abbreviations are listed in Table 1 below:

[0039] Table 1. Comparison of Amino Acids in Chinese and English and Their Abbreviations

[0040]

[0041]

[0042] The penicillin G acylase mutant of the present invention, SEQ ID NO:3, has 818 amino acids and a well-defined structure. Therefore, those skilled in the art can easily obtain its encoding gene, expression cassettes and plasmids containing these genes, and transformants containing the plasmids. These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering construction methods well known to those skilled in the art.

[0043] To optimally express penicillin G acylase mutants in *E. coli*, one of the most commonly used organisms in genetic engineering, codon optimization can be performed on the expression genes of these enzymes. Codon optimization is a technique that can be 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. Therefore, in fast-growing microorganisms, low-frequency codons for amino acids can be used for high-frequency codon substitutions of the same amino acid. Thus, the expression of optimized DNA sequences is improved in fast-growing microorganisms.

[0044] The host of the aforementioned transformant can be any microorganism suitable for expressing penicillin G acylase, including bacteria and fungi. Preferred microorganisms are Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, with Escherichia coli being preferred, and Escherichia coli BL21(DE3) being more preferred.

[0045] When used as a biocatalyst in the production of amoxicillin, the penicillin G acylase mutant of the present invention can be in the form of an enzyme or in the form of bacterial cells. 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, dead cells, and immobilized cells.

[0046] 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.

[0047] Example

[0048] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage.

[0049] Materials and methods

[0050] In the embodiments, the whole gene synthesis, primer synthesis and sequencing were all completed by Suzhou Genewiz Biotechnology Co., Ltd.

[0051] 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" (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. Specific experimental conditions can be determined through simple experiments if necessary.

[0052] 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.

[0053] 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.)

[0054] 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. (TB solid medium with an additional 20 g / L agar powder.)

[0055] 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 tank.

[0056] Feeding medium for a 5L fermenter: 60% glycerol.

[0057] Enzyme synthesis activity assay method:

[0058] Take 50 mL of substrate reaction solution containing 50 mM 6-APA and 60 mM DHPGM (i.e., 0.05 M potassium phosphate buffer solution at pH 7.0), adjust the pH of the substrate reaction solution to 7.0 ± 0.2 with hydrochloric acid, then add penicillin G acylase and react at 28 °C. Take a 30 μL sample at 20 minutes after the start of the reaction, and dilute the sample 100 times with 50 mM potassium dihydrogen phosphate solution. Determine the amount of amoxicillin produced by HPLC.

[0059] HPLC detection conditions for amoxicillin: Agilent 1260, column C18 (4.6 × 250 mm, 5 μm); mobile phase A: mobile phase B = 97.5: 2.5 (mobile phase A: 50 mM KH2PO4, 0.1% triethylamine, pH 5.0; mobile phase B: acetonitrile); flow rate 1.0 mL / min; detection wavelength 210 nm.

[0060] The enzyme activity unit (SU) is defined as the amount of penicillin G acylase required to produce 1 μmol of amoxicillin per minute at 28°C and pH 7.0.

[0061] Methods for determining the synthesis / hydrolysis (S / H) value:

[0062] Add 50 mL of purified water to a 100 mL beaker, then add 0.5 g of D-HPGM (D-type p-hydroxyphenylglycine methyl ester) and 0.5 g of 6-APA. Stir well and control the reaction temperature at 25 °C. Taking the addition of 100 SU of purified enzyme as the zero point, samples were taken at 2 min, 4 min, 6 min, 8 min, 10 min, and 12 min. The micromolar concentration (μmol / mL) of D-HPG (p-hydroxyphenylglycine) and AMXL in each sample was detected by high performance liquid chromatography (HPLC). Linear regression curves of AMXL and D-HPG were plotted to obtain the slopes K1 and K2.

[0063] Synthesis / hydrolysis (S / H) = K1 / K2.

[0064] The S / H value is the ratio of the number of moles of amoxicillin (AMXL) in the reaction product to the number of moles of the byproduct p-hydroxyphenylglycine (D-HPG).

[0065] Kanamycin (Kan, 50 μg / mL) should be used as directed based on the antibiotic gene carried by the plasmid.

[0066] It should be noted that, for the sake of convenience, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number may share the same number. This is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0067] Example 1: Construction of wild-type penicillin G acylase expression strain

[0068] Based on the amino acid sequence SEQ ID NO:1 (the amino acid sequence corresponding to the 95bp-2548bp nucleic acid sequence of GenBank No. L04471.1) of wild-type penicillin G acylase from Arthrobacter viscosus, Suzhou Genewise Biotechnology Co., Ltd. was commissioned to synthesize the nucleotide sequence of its encoding gene as SEQ ID NO:2. Restriction endonuclease sites Nde I and Xho I were designed at both ends of the gene, and subcloned into the corresponding sites of the vector pET24a (Novagen) to obtain plasmid pET-PGA1.

[0069] The recombinant plasmid pET-PGA1 was transformed into the host Escherichia coli BL21(DE3) by electroporation to obtain recombinant Escherichia coli AvPGA1 expressing wild-type penicillin G acylase.

[0070] Example 2: Site-directed saturation mutagenesis and reaction screening

[0071] 2.1 Site-directed saturation mutation library

[0072] Using plasmid pET-PGA1 as a template, a site-directed saturation mutant library targeting the V305 site (position 24 of the β subunit in the enzyme structure model) was constructed using PCR technology.

[0073] Design the following primer pair: PGA1-5 / PGA1-3:

[0074] Forward primer PGA1-5:

[0075] 5'-TTCAGTGGACCACAANNKGGTTTTGTTGCTCCTGGATT-3';

[0076] Reverse primer PGA1-3:

[0077] 5'-AATCCAGGAGCAACAAAACCMNNTTGTGGTCCACTGAA-3'.

[0078] Using plasmid pET-PGA1 as a template, PCR amplification was performed to obtain a penicillin G acylase mutant DNA sequence of approximately 2.4 kb.

[0079] The 50 μl PCR reaction system includes: 10 ng plasmid (pET-PGA1) template, 50 pmol primer pair PGA1-5 and PGA1-3, 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).

[0080] 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.

[0081] The PCR products were electrophoresed and gel-extracted (Axygen DNA Gel Extraction Kit AP-GX-50). The plasmid template was digested with DpnI restriction endonuclease (Thermo). The plasmid was then electrotransformed into *E. coli* BL21(DE3) to obtain more than 10... 4 A site-directed saturation mutant library of clones.

[0082] 2.2 High-throughput screening of mutant libraries

[0083] Single colonies were picked and transferred to 96-well plates (each well containing 110 μL of liquid LB-Kan medium). After incubation at 37°C and 400 rpm for 5 h, 60 μL of the bacterial culture from each well was transferred to 96-well deep-well plates (each well containing 240 μL of liquid TB-Kan-0.2 mM IPTG). The plates were incubated at 25°C and 400 rpm for 12–16 h. The cells were collected by centrifugation at 4°C and 4000 rpm for 10 min, and the supernatant was discarded. The cells were then washed with pre-cooled physiological saline and collected by centrifugation at 4°C and 4000 rpm for 10 min, and the supernatant was discarded. 200 μL of enzyme reaction solution (50 mM 6-APA and 60 mM DHPGM, adjusted to pH 7.0) was added to each well to resuspend the cells. The reaction was carried out at 28°C and 250 rpm for 10–30 min, and the amoxicillin concentration was determined by HPLC.

[0084] 2.3 Strains with significantly enhanced activity were selected, plasmids were extracted, and nucleic acid sequencing was performed by Suzhou Genewiz Biotechnology Co., Ltd. The penicillin G acylase-related fragments in the genome were compared with SEQ ID NO:2 to determine the amino acid sequence changes of penicillin G acylase. The strain with the highest improvement in enzyme activity was selected as the starting strain for the next round of random mutant library construction. The screening results are shown in Table 2.

[0085] Table 2. Results of high-throughput screening of site-specific saturated mutant libraries

[0086]

[0087] Note: "+" 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 200%; "++++" indicates that the viability percentage relative to the original strain is greater than 200%.

[0088] Among them, the mutant PGA5 (corresponding to mutant strain AvPGA5) formed after the wild enzyme undergoes the V305A mutation showed a significant increase in enzyme synthesis activity, and it was used for the next round of error-prone PCR random mutation screening.

[0089] Example 3: Establishment of random mutation point libraries and high-throughput reaction screening in rounds 1 to 2

[0090] 3.1 Error-prone PCR method for constructing a random mutation library

[0091] Based on the amino acid sequence of the penicillin G acylase mutant PGA5, the expression plasmid pET-PGA5 and recombinant Escherichia coli AvPGA5 expressing the penicillin G acylase mutant PGA5 were constructed according to the method in Example 1.

[0092] Using plasmid pET-PGA5 as a template, a random mutant library was constructed using error-prone PCR technology.

[0093] Design the following primer pair: PGA5-5 / PGA5-3:

[0094] Forward primer PGA5-5: 5'-ATGATTCGTGATATAAGTGTTATAA-3';

[0095] Reverse primer PGA5-3: 5'-CTTACTCATATTTAATGCGCTT-3'.

[0096] Using plasmid pET-PGA5 as a template, PCR amplification was performed to obtain a penicillin G acylase mutant DNA sequence of approximately 2.4 kb.

[0097] The 50 μL error-prone PCR reaction system includes: 10 ng plasmid (pET-ATA64) template, 50 pmol primer pair PGA5-5 and PGA5-3, 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).

[0098] 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.

[0099] PCR products were electrophoresed and gel-recovered (Axygen DNA Gel Recovery Kit AP-GX-50). Using plasmid pET-PGA5 as a template and approximately 2.0 kb of the recovered product (random mutant fragment) 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.

[0100] 3.2 High-throughput screening of mutant libraries

[0101] Single colonies were picked and transferred to 96-well plates (each well containing 110 μL of liquid LB-Kan medium). After incubation at 37°C and 400 rpm for 5 h, 60 μL of the bacterial culture from each well was transferred to 96-well deep-well plates (each well containing 240 μL of liquid TB-Kan-0.2 mM IPTG). The plates were incubated at 25°C and 400 rpm for 12–16 h. The cells were collected by centrifugation at 4°C and 4000 rpm for 10 min, and the supernatant was discarded. The cells were then washed with pre-cooled physiological saline and collected by centrifugation at 4°C and 4000 rpm for 10 min, and the supernatant was discarded. 200 μL of enzyme reaction solution (50 mM 6-APA and 60 mM DHPGM, adjusted to pH 7.0) was added to each well to resuspend the cells. The reaction was carried out at 28°C and 250 rpm for 10–30 min, and the amoxicillin concentration was determined by HPLC.

[0102] 3.2 Strains with significantly enhanced enzyme activity were selected for nucleic acid sequencing to determine amino acid mutation sites. The strain with the highest improvement in enzyme activity was selected as the starting strain for the next round of random mutant library construction. Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to perform genome sequencing and alignment on the strain with the highest enzyme activity to determine its amino acid sequence changes. The screening results are shown in Table 3.

[0103] Table 3. Results of high-throughput screening of random mutant libraries in rounds 1 and 2.

[0104]

[0105] Note: "+" 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 200%.

[0106] Among them, the mutant PGA13 (corresponding to mutant strain AvPGA13) formed by the F138L, Y352D, P58A, N147D and A445T mutations of enzyme mutant PGA5 showed a significant increase in enzyme synthesis activity, and was used for the next round of error-prone PCR random mutation screening.

[0107] Example 4: Establishment of random mutation point libraries and high-throughput reaction screening in rounds 3 to 5

[0108] 4.1 Error-prone PCR method for constructing a random mutation library

[0109] Based on the amino acid sequence of the penicillin G acylase mutant PGA13, the expression plasmid pET-PGA13 and recombinant Escherichia coli AvPGA13 expressing the penicillin G acylase mutant PGA13 were constructed according to the method in Example 1.

[0110] Following the method in Example 3.1, a library of random mutation points was constructed using plasmid pET-PGA13 as a template and error-prone PCR technology.

[0111] 4.2 High-throughput screening of mutant libraries

[0112] Single colonies were picked and transferred to 96-well plates (each well containing 110 μL of liquid LB-Kan medium). After incubation at 37°C and 400 rpm for 5 h, 60 μL of the bacterial culture from each well was transferred to 96-well deep-well plates (each well containing 240 μL of liquid TB-Kan-0.2 mM IPTG). The plates were incubated at 25°C and 400 rpm for 12–16 h. The cells were collected by centrifugation at 4°C and 4000 rpm for 10 min, and the supernatant was discarded. The cells were then washed with pre-cooled physiological saline and collected by centrifugation at 4°C and 4000 rpm for 10 min, and the supernatant was discarded. 200 μL of enzyme reaction solution (50 mM 6-APA and 60 mM DHPGM, adjusted to pH 7.0) was added to each well to resuspend the cells. The reaction was carried out at 28°C and 250 rpm for 10–30 min, and the amoxicillin concentration was determined by HPLC.

[0113] 4.3 Strains with significantly enhanced enzyme activity were selected for nucleic acid sequencing to determine amino acid mutation sites. The strain with the highest improvement in enzyme activity was selected as the starting strain for the next round of random mutant library construction. Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to perform genome sequencing and alignment on the strain with the highest enzyme activity to determine its amino acid sequence changes. The screening results are shown in Table 4.

[0114] Table 4. Results of high-throughput screening of random mutant libraries from rounds 3 to 5.

[0115]

[0116] Note: "+" 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 200%.

[0117] Among them, the enzyme mutant PGA22 (corresponding to the mutant strain AvPGA22) showed a significant increase in enzyme synthesis activity. It was used in the next round of error-prone PCR random mutation screening in order to obtain mutants with further enhanced enzyme activity.

[0118] Example 5: Sixth round of high-throughput reaction screening for thermal stability

[0119] 5.1 Error-prone PCR method for constructing a random mutation library

[0120] Based on the amino acid sequence of the penicillin G acylase mutant PGA22, the expression plasmid pET-PGA22 and recombinant Escherichia coli AvPGA22 expressing the penicillin G acylase mutant PGA22 were constructed according to the method in Example 1.

[0121] Following the method in Example 3.1, a library of random mutation points was constructed using plasmid pET-PGA22 as a template and error-prone PCR technology.

[0122] 5.2 High-throughput screening of mutant libraries

[0123] Single colonies were picked and transferred to 96-well plates (each well containing 110 μL of liquid LB-Kan medium). After incubation at 37°C and 400 rpm for 5 h, 60 μL of the bacterial culture from each well was transferred to 96-well deep-well plates (each well containing 240 μL of liquid TB-Kan-0.2 mMIPTG). The plates were then incubated at 25°C and 400 rpm for 12–16 h. The cells were collected by centrifugation at 4°C and 4000 rpm for 10 min, and the supernatant was discarded. Subsequently, the bacterial cells were washed with pre-cooled physiological saline, centrifuged at 4°C and 4000 rpm for 10 min to collect the cells, the supernatant was removed, and the cells were resuspended in 100 μl of pH 7.0 PBS. The cells were then heat-treated at 55°C for 30 min, and 100 μL of enzyme reaction solution (100 mM 6-APA and 120 mM DHPGM, adjusted to pH 7.0) was added to each well to resuspend the cells. The cells were then reacted at 28°C and 250 rpm for 10–30 min, and the amoxicillin concentration was determined by HPLC.

[0124] 5.3 Strains with significantly enhanced enzyme activity were selected for nucleic acid sequencing to determine amino acid mutation sites. Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to perform genome sequencing and alignment on the strains with the highest enzyme activity to determine their amino acid sequence changes. The screening results are shown in Table 5.

[0125] Table 5. Results of the 6th round of high-throughput screening of thermally stable random mutation libraries

[0126]

[0127] Note: "+" 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 200%.

[0128] After multiple rounds of mutation and screening, an enzyme mutant with higher enzyme synthesis activity, PGA25 (corresponding to mutant strain AvPGA25), was obtained. It has undergone 12 site mutations relative to the wild enzyme, and its amino acid sequence is SEQ ID NO:3.

[0129] Recombinant Escherichia coli AvPGA25 expressing the penicillin G acylase mutant PGA25 was constructed according to the method in Example 1. The feasibility of industrial application of PGA25 catalyzing the reaction of 6-APA and DHPGM to synthesize amoxicillin was further investigated.

[0130] Example 6: Fermentation culture of penicillin G acylase expression strain

[0131] Using a 5L fermenter, strains AvPGA1, AvPGA22, and AvPGA25 were cultured separately. Single colonies were picked and transferred to 5 mL of liquid LB medium containing Kans, and cultured overnight at 37°C and 220 rpm. The next day, at an inoculum concentration of 5 v / v%, they were transferred to shake flasks containing 100 mL of liquid TB medium and cultured at 37°C and 220 rpm until OD. 600nm Once the culture reaches level 6, it is transferred to a 5L fermenter as a seed culture. After inoculation, it is cultured at 37°C and 400–800 rpm / min, with dissolved oxygen controlled within the range of 20–30%. When the cell OD... 600nm After reaching a concentration of 20, IPTG was added to induce penicillin G acylase expression. The final IPTG concentration was 0.2 mM. The cells were then cultured at 25-28℃ for 16-24 hours, and collected by centrifugation. 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.

[0132] After fermentation, the cells were collected by centrifugation at 4°C and 10,000 rpm for 10 minutes and then frozen for later use.

[0133] Example 7: Enzyme Extraction and Purification

[0134] Frozen-thawed cells of AvPGA1, AvPGA22, and AvPGA25 were taken separately and resuspended in lysis buffer (pH 8.0, 100mM sodium phosphate, 5% glycerol) at a ratio of 1:2 (g:mL). The cells were then subjected to ultrasonic pulse disruption (15 seconds of operation followed by a 30-second interval, 5W power) for 30 cycles. Alternatively, a French Pressure cell disruptor could be used.

[0135] The cell lysate was centrifuged at 10,000 rpm for 1 hour, and the supernatant was collected to obtain the crude enzyme solution, which was stored at -20°C. Affinity purification was then performed at 4°C.

[0136] Measure 5 mL of the pretreated affinity vector FP-IDA-Ni2+ and pack it into a purification column (Φ10×200). Pass the sample through the column at a rate of 1.0 BV / h. Remove contaminating proteins with 3-5 BV of Washing buffer at a flow rate of 1.0 BV / h, while simultaneously monitoring the protein content of the eluent using a protein and nucleic acid analyzer at 280 nm. Continue until the Washing buffer becomes clear and the protein and nucleic acid analyzer readings no longer change. Finally, under the monitoring of the protein and nucleic acid analyzer at 280 nm, elute the target enzyme with approximately 1-2 BV of Elution buffer at a rate of 1-2 BV / h to obtain pure enzymes of PGA1, PGA22, or PGA25. Store the pure enzyme solution at 4°C for later use.

[0137] Example 8: Determination of the thermal stability of enzymes

[0138] The stability of penicillin G acylase was evaluated using the enzyme activity half-life at a set temperature as an indicator.

[0139] half-life of an enzyme t 1 / 2 The half-life (t) refers to the time required for the initial activity of an enzyme to decrease by 50% at a given temperature. It is one of the commonly used parameters characterizing the thermostability of enzymes. 1 / 2 The higher the value, the better the enzyme's thermal stability.

[0140] 100 μl of pure PGA1, PGA22, and PGA25 enzyme solutions were incubated at 65℃ for different time intervals (0, 10, 20, 30, 40, 50, 60 min up to 120 min) at 10 min intervals. The enzyme synthesis activities were then measured using the heat-treated pure PGA1, PGA22, and PGA25 enzymes, with the initial activity at 0 min incubation considered as 100%. The results are shown in Table 6.

[0141] Table 6: Results of Enzyme Half-Life Test

[0142]

[0143] As shown in Table 6, under thermal fatigue test conditions, the thermostability of mutant enzymes PGA22 and PGA25 was more than double that of the wild-type enzymes. The specific reasons for this require further investigation. This improved thermostability could further promote the practical application of mutant enzymes PGA22 and PGA25 in amoxicillin synthesis.

[0144] Example 9: Application Experiment of Amoxicillin Synthesis Using Purified Mutant Enzyme PGA25

[0145] In a 100 mL reaction system, purified enzymes of wild-type enzyme PGA1 and mutant enzyme PGA25 were used to catalyze the synthesis of amoxicillin from 6-APA and D-HPGM at 30 °C. The total reaction system consisted of 250 mM of 6-APA and 262.5 mM of D-HPGM substrate (molar ratio of DHPGM to 6-APA was 1.05:1), with the pH adjusted to 7.0 ± 0.20, and 1200 SU of purified enzyme.

[0146] HPLC analysis showed that after 90 min of reaction, the 6-APA conversion rate catalyzed by PGA25 exceeded 99%. Under the same conversion conditions, the 6-APA conversion rate catalyzed by the wild-type enzyme PGA1 was only 45%. The calculated S / H ratio of the wild-type enzyme PGA1 was 5.03; compared to the wild-type enzyme, the S / H ratio of the mutant PGA25 was 18.1, which is 3.60 times that of the wild-type enzyme PGA1.

[0147] The above experiments demonstrate that the penicillin G acylase mutant PGA25 exhibits significantly higher enzyme synthesis activity and thermostability than the wild-type enzyme PGA1, showing its potential for industrial application.

Claims

1. A mutant of penicillin G acylase, which is a polypeptide having an amino acid sequence shown in SEQ ID NO:

3.

2. A gene encoding the mutant of penicillin G acylase according to claim 1.

3. The gene of claim 2, wherein The nucleotide sequence of the gene is shown in SEQ ID NO:

4.

4. A plasmid, characterized in that, The plasmid comprises the gene according to claim 3.

5. A microorganism for expressing the mutant penicillin G acylase according to claim 1, characterized in that, The gene according to claim 3 is integrated into the genome of the microorganism, or the microorganism is a transformant transformed with the plasmid according to claim 4.

6. The microorganism of claim 5, wherein, The microorganism is Escherichia coli.

7. Use of the mutant of penicillin G acylase according to claim 1 or the microorganism according to claim 6 in the production of amoxicillin.

8. Use according to claim 7, characterized in that, The mutant of penicillin G acylase according to claim 1 or the microorganism according to claim 6 is used to catalyze the reaction of the substrate 6-aminopenicillanic acid with D-p-hydroxyphenylglycine methyl ester to generate amoxicillin.

9. Use according to claim 8, characterized in that, The pH of the reaction system is 7.0±0.5, and the reaction temperature is 25-35°C.

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