Penicillin g acylase axpga mutant, expression plasmid, genetically engineered bacteria and application

Abstract

The application provides a penicillin G acylase Ax The PGA mutant and the expression plasmid, the genetically engineered bacteria and the application belong to the technical field of biological catalysts. The PGA mutant is obtained by mutation of any one or more of the following positions of the amino acid sequence shown in SEQ ID NO: 1: (1) methionine at the 103th position is replaced by cysteine, (2) tyrosine at the 424th position is replaced by phenylalanine, (3) tyrosine at the 442th position is replaced by phenylalanine, and (4) tyrosine at the 451th position is replaced by alanine. The obtained penicillin G acylase Ax The PGA mutant has high enzyme activity and can be applied to high-conversion-rate and high-yield synthesis of beta-lactam antibiotics such as benzylpenicillin and amoxicillin.

Description

Technical Field

[0001] The present application relates to a penicillin G acylase AxPGA mutant, an expression plasmid, a genetically engineered bacterium and applications, and belongs to the technical field of biocatalysts. Background Art

[0002] β-lactam antibiotics are very important antimicrobial drugs. However, their use is affected by the development of drug resistance, as β-lactamases hydrolyze the β-lactam ring into an inactive form. β-lactam antibiotics are primarily produced by chemical methods, but these methods are demanding on chemical substances, involve extreme temperatures, and involve complex processes such as the selective protection and deprotection of functional groups. This results in limitations in the industrial application of chemical production of β-lactam antibiotics.

[0003] The synthetic pathway catalyzed by penicillin G acylase has garnered significant attention. Compared to chemical catalysts, enzymes, as green, natural biocatalysts, offer superior stereoselectivity and regioselectivity in catalyzing chemical reactions. Furthermore, they operate under mild, environmentally friendly conditions, and do not require demanding reaction equipment. Consequently, research in this area remains active. However, the reported enzymatic activities of penicillin G acylase and its mutants have been suboptimal, resulting in low catalytic activity and poor yields when used in the synthesis of related drugs. Summary of the Invention

[0004] In view of this, the first object of the present application is to provide a penicillin G acylase AxPGA mutant with higher enzyme activity, which can be used for the synthesis of β-lactam antibiotics, such as ampicillin and amoxicillin, with high yield.

[0005] Specifically, this application is implemented through the following solutions:

[0006] A penicillin G acylase AxPGA mutant, wherein the amino acid sequence of SEQ ID NO: 2 is mutated to one of the following:

[0007] (1) methionine at position 103 is substituted with cysteine,

[0008] (2) Based on (1), the tyrosine at position 424 was replaced by phenylalanine.

[0009] (3) Based on (1), the tyrosine at position 442 was replaced by phenylalanine.

[0010] (4) Based on (1), the tyrosine at position 451 was replaced by alanine.

[0011] (5) Based on (1), the tyrosine at position 424 was replaced by phenylalanine, and the tyrosine at position 442 was replaced by phenylalanine.

[0012] (6) Based on (1), the tyrosine at position 424 was substituted with phenylalanine, the tyrosine at position 442 was substituted with phenylalanine, and the tyrosine at position 451 was substituted with alanine.

[0013] The second purpose of the applicant is to provide an expression plasmid for the above-mentioned penicillin G acylase AxPGA mutant, wherein the vector of the plasmid is pET28a.

[0014] The third purpose of the applicant is to provide a genetically engineered bacterium producing the aforementioned penicillin G acylase AxPGA mutant, wherein the host bacterium of the genetically engineered bacterium is E. coli BL21 (DE3).

[0015] The fourth purpose of the applicant is to provide the use of the above-mentioned penicillin G acylase AxPGA mutant in catalyzing the synthesis of β-lactam antibiotics.

[0016] Specifically:

[0017] The penicillin G acylase AxPGA mutant is used as a catalyst, a potassium phosphate buffer with a pH of 5.3 to 7.4 is used as a reaction medium to form a conversion system, 6-aminopenicillanic acid is used as a first reaction substrate, and D-phenylglycine methyl ester or D-hydroxyphenylglycine methyl ester is used as a second reaction substrate. Ampicillin or amoxicillin is prepared by reacting at 15 to 40°C.

[0018] More preferred:

[0019] The molar ratio of the first reaction substrate to the second reaction substrate is 1:1 to 1.2. More preferably, the final concentration of the first reaction substrate in the conversion system is 200 to 600 mM, and the final concentration of the second reaction substrate in the conversion system is 200 to 720 mM.

[0020] Glycerol is added into the conversion system as a cosolvent.

[0021] The catalyst is added to the conversion system in an amount of 2 to 10 g DCW / L (DCW: dry cell weight, L refers to the volume of the conversion system).

[0022] The penicillin G acylase AxPGA mutant can be added in the form of wet cells of genetically engineered bacteria expressing the penicillin G acylase AxPGA mutant, or cell lysate of genetically engineered bacteria, or pure enzyme solution separated after protein purification of the cell lysate.

[0023] The expression plasmid of the penicillin G acylase AxPGA mutant is transferred into E. coli BL21 (DE3) competent cells by heat shock to obtain genetically engineered bacteria. The resulting strain is subjected to methods such as inoculation, transfer, induction, and bacterial cell recovery. The resuspended bacterial liquid is the genetically engineered bacterial wet cell. The culture medium can be any culture medium for growing E. coli cells and expressing the target gene of the present invention in the art. Preferably, LB medium is composed of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in distilled water, and adjusted to pH 7.0. There are no particular limitations on the culture method and culture conditions, and the culture method and conditions can be appropriately selected based on factors such as the host type and culture method. As a specific preparation process, a genetically engineered bacterium containing a penicillin G acylase AxPGA mutant was inoculated into a LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin, cultured at 37° C. for 10 hours, inoculated into a LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, and cultured at 37° C. and 180 rpm for 2 hours. Isopropyl β-D-thiogalactoside (IPTG) was then added to the culture medium at a final concentration of 0.1 mM. After cultured at 28° C. for 12 hours, the culture was centrifuged at 4° C. and 9000×g (×g refers to the ratio of the centripetal acceleration to the gravitational acceleration of an object in rotational motion, also known as the relative central acceleration) for 10 minutes to obtain wet cells of the genetically engineered bacterium containing the penicillin G acylase AxPGA mutant.

[0024] The cell disruption solution was prepared by resuspending the cells in 50 mM potassium phosphate buffer at pH 5.9 at a dosage of 10 g DCW / L, and ultrasonically disrupting the cells on an ice-water mixture for 6 minutes. The ultrasonic disruption conditions were: power 400 W, disruption for 1 second, and pause for 2 seconds.

[0025] The present application directly synthesizes β-lactam antibiotics (such as ampicillin and amoxicillin) through a one-step reaction catalyzed by penicillin G acylase. First, enzymes from four different sources were screened, and the one with the highest enzyme activity, penicillin G acylase AxPGA, from Achromobacter xylosoxidans PX02, was obtained. This enzyme was molecularly modified through directed evolution, with site-directed mutagenesis of amino acid residues near the enzyme-substrate pocket. The enzyme was modified based on the principle of reducing the substrate pocket, resulting in a mutant strain AxPGA with an enzyme activity that was 55.12 to 124.26% higher than that of the original strain. The AxPGA mutant was applied to the enzymatic synthesis of β-lactam antibiotics. For example, the key enzyme penicillin G acylase catalyzed the synthesis of ampicillin (ampicillin) from the substrates 6-aminopenicillanic acid and D-phenylglycine methyl ester, and the synthesis of amoxicillin (amoxicillin) from the substrates 6-aminopenicillanic acid and D-hydroxyhydroxyphenylglycine methyl ester. The catalytic activity of the corresponding β-lactam antibiotics increased by 1.01 to 1.41 times, the conversion rate increased to more than 99%, and the yield reached around 98%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0027] Figure 1 This figure shows the yield of ampicillin and amoxicillin catalyzed by penicillin G acylase AxPGA from different sources.

[0028] Figure 2 This is the SDS-PAGE electrophoresis diagram of the supernatant of the penicillin G acylase AxPGA mutant.

[0029] Lane M: standard protein molecule marker; Lane 1: control penicillin G acylase AxPGA pure enzyme; Lane 2: penicillin G acylase AxPGA-M103C pure enzyme; Lane 3: penicillin G acylase AxPGA-Y424F pure enzyme; Lane 4: penicillin G acylase AxPGA-Y442F pure enzyme; Lane 5: penicillin G acylase AxPGA-Y451A pure enzyme; Lane 6: penicillin G acylase AxPGA-M103C / Y424F / Y442F / Y451A pure enzyme.

[0030] Figure 3 The yields of ampicillin and amoxicillin synthesized by the supernatant of penicillin G acylase AxPGA-M103C / Y424F / Y442F / Y451A at different pH conditions.

[0031] Figure 4 The yields of ampicillin and amoxicillin synthesized by the supernatant of penicillin G acylase AxPGA-M103C / Y424F / Y442F / Y451A at different reaction temperatures.

[0032] Figure 5 The effect of different substrate ratios on the yield of ampicillin and amoxicillin catalyzed by penicillin G acylase AxPGA-M103C / Y424F / Y442F / Y451A was investigated.

[0033] Figure 6 The effect of different penicillin G acylase AxPGA-M103C / Y424F / Y442F / Y451A bacterial concentrations on the catalytic preparation of ampicillin and amoxicillin yields.

[0034] Figure 7 This is a process diagram of the production of ampicillin catalyzed by penicillin G acylase AxPGA-M103C / Y424F / Y442F / Y451A using substrate D-phenylglycine methyl ester.

[0035] Figure 8 This is a process diagram of the production of amoxicillin catalyzed by penicillin G acylase AxPGA-M103C / Y424F / Y442F / Y451A using substrate D-hydroxyphenylglycine methyl ester. DETAILED DESCRIPTION

[0036] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the technical solutions in the embodiments of this application will be further described in detail below in conjunction with the drawings in the embodiments of this application. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit the technical solutions of this application. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of this application.

[0037] Example 1

[0038] This example is about mining and screening of genetically engineered bacteria for penicillin G acylase AxPGA. Penicillin G acylase was mined through NCBI.

[0039] Its GenBank: AVC04856.1 was codon-optimized using Escherichia coli as a host (the optimized nucleotide sequence is shown in SEQ ID NO: 1) and synthesized by Qingke Biotechnology Co., Ltd., and the AxPGA cDNA fragment was linked to the site after TATACCAT (before the NcoI restriction site) and before CTCGAG (XhoI restriction site) in pET-28a(+). The resulting plasmid pET28a(+)-AxPGA was transformed into E. coli BL21(DE3) to obtain the strain E. coli BL21(DE3) / pET28a(+)-AxPGA.

[0040] SEQ ID NO.1:

[0041]

[0042] Its GenBank: EJA4313497.1, nucleotide sequence is shown in SEQ ID NO: 3, and was synthesized by Qingke Biotechnology Co., Ltd., and the EcPGA cDNA fragment was linked to the site after TATACCAT (before the NcoI restriction site) and before CTCGAG (XhoI restriction site) in pET-28a(+), and the resulting plasmid pET28a(+)-EcPGA was transformed into E. coli BL21(DE3) to obtain the strain E. coli BL21(DE3) / pET28a(+)-EcPGA.

[0043] After codon optimization using Escherichia coli as the host (the optimized nucleotide sequence is shown in SEQ ID NO: 5) and synthesised by Qingke Biotechnology Co., Ltd., the AsPGA cDNA fragment was linked to the site after TATACCAT (before the NcoI restriction site) and before CTCGAG (XhoI restriction site) in pET-28a(+), and the resulting plasmid pET28a(+)-AsPGA was transformed into E. coli BL21(DE3) to obtain the strain E. coli BL21(DE3) / pET28a(+)-AsPGA.

[0044] After codon optimization using Escherichia coli as the host (the optimized nucleotide sequence is shown in SEQ ID NO: 7) and synthesis by Qingke Biotechnology Co., Ltd., the AdPGA cDNA fragment was linked to the sites after TATACCAT (before the NcoI restriction site) and before CTCGAG (XhoI restriction site) in pET-28a(+), and the resulting plasmid pET28a(+)-AdPGA was transformed into E. coli BL21(DE3) to obtain the strain E. coli BL21(DE3) / pET28a(+)-AdPGA.

[0045] The above-mentioned strains containing penicillin G acylase and its mutants were inoculated into LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin, cultured at 37°C for 10 h, and then inoculated into fresh LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin at a 1% (v / v) inoculum size. The culture was cultured at 37°C and 180 rpm for 2 h. Then, a final concentration of 0.1 mM IPTG was added to the culture medium. After culture at 28°C for 12 h, the culture was centrifuged at 4°C and 9000×g for 10 min to obtain the corresponding wet bacterial cells.

[0046] Screening of the catalytic ability of the bacteria: The catalytic ability of the obtained cells was tested, and after adding 40% glycerol to assist in solubilization, different β-lactam antibiotics were synthesized using the following process.

[0047] (1) Add 200 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester as substrates. The catalyst dosage is 4 g DCW / L (DCW cell dry weight) based on the total dry weight of the mixed cells. The conversion system is constructed using a pH 7.0, 50 mM potassium phosphate buffer as the reaction medium. The reaction is carried out at 30°C and 800 rpm. After the reaction is completed, the reaction solution is subjected to liquid chromatography detection after the reaction is terminated. The amount of the product ampicillin produced is detected to calculate the product yield.

[0048] (2) Add 200 mM 6-aminopenicillanic acid and 200 mM D-hydroxyphenylglycine methyl ester as substrates. The catalyst dosage is 4 g DCW / L (DCW cell dry weight) based on the total dry weight of the mixed cells. The conversion system is formed using a pH 7.0, 50 mM potassium phosphate buffer as the reaction medium. The reaction is carried out at 30°C and 800 rpm. After the reaction is terminated, the reaction solution is subjected to liquid chromatography detection to detect the amount of amoxicillin produced to calculate the product yield.

[0049] The reaction equation for preparing ampicillin and amoxicillin catalyzed by the engineered bacteria containing the penicillin G acylase AxPGA gene is as follows:

[0050]

[0051] The production of ampicillin and amoxicillin is as follows Figure 1 As shown in the figure, AxPGA corresponds to Achromobacter xylosoxidans, EcPGA to Escherichia coli, AsPGA to Achromobacter sp.UMC46, and AdPGA to Achromobacter denitrificans. The results showed that the penicillin G acylase from Achromobacter xylosoxidans produced the highest yield of β-lactam antibiotics (ampicillin and amoxicillin) among the four penicillin G acylases from different sources. AxPGA was selected as a chassis cell for further modification to improve the efficiency of β-lactam antibiotic synthesis.

[0052] Liquid chromatography conditions: Samples were analyzed using a Kromasil HPLC (Dionex P680 HPLC) with a 100-5C18 column (4.6 mm × 250 mm, Kromasil, Sweden) and a UVD170U detector at 254 nm. Mobile phase: 50 mM potassium phosphate buffer, pH 7.0; column temperature: 35°C; flow rate: 0.8 mL / min; injection volume: 10 μL. The retention time of ampicillin was 5.2 min, and the calibration curve was: y = 2438.91x - 240.14. The retention time of ampicillin was 7.3 min, and the calibration curve was: y = 2189.21x - 217.31.

[0053] Example 2

[0054] This example constructs and screens a penicillin G acylase mutant library.

[0055] Based on the substrate pocket reduction strategy for bisubstrate molecules, a penicillin G acylase mutant library was prepared using site-directed mutagenesis. Primer designs are shown in Table 1. Using the E. coli BL21(DE3) / pET28a(+)-AxPGA vector pET28a(+)-AxPGA as the template, the primers M103C-F and M103C-R (see Table 1) were used as mutagenesis primers for amino acid position 103. By site-directed mutagenesis PCR, the methionine at position 103 of the penicillin G acylase AxPGA amino acid sequence was mutated to cysteine, resulting in the monoclonal mutant AxPGA-M103C. By site-directed mutagenesis PCR, the tyrosine at position 383 of the penicillin G acylase AxPGA amino acid sequence was mutated to alanine, resulting in the monoclonal mutant AxPGA-Y383A. Using the mutagenesis primers W401A-F and W401A-R in Table 1 for amino acid position 401, the tryptophan at position 401 in the penicillin G acylase AxPGA amino acid sequence was mutated to alanine by site-directed mutagenesis PCR, resulting in the monoclonal mutant AxPGA-W401A. Using the mutagenesis primers Y424F-F and Y424F-R in Table 1 for amino acid position 424, the tyrosine at position 424 in the penicillin G acylase AxPGA amino acid sequence was mutated to phenylalanine by site-directed mutagenesis PCR, resulting in the monoclonal mutant AxPGA-Y424F. Using the mutagenesis primers Y442F-F and Y442F-R in Table 1 for amino acid position 442, the tyrosine at position 442 in the penicillin G acylase AxPGA amino acid sequence was mutated to phenylalanine by site-directed mutagenesis PCR, resulting in the monoclonal mutant AxPGA-Y442F. Using the mutagenesis primers W448A-F and W448A-R in Table 1 for the tryptophan at position 448, site-directed mutagenesis PCR was used to mutate the tryptophan at position 448 of the penicillin G acylase AxPGA to alanine, resulting in the monoclonal mutant AxPGA-W448A. Using the mutagenesis primers Y451A-F and Y451A-R in Table 1 for the tryptophan at position 451, site-directed mutagenesis PCR was used to mutate the tyrosine at position 451 of the penicillin G acylase AxPGA to alanine, resulting in the monoclonal mutant AxPGA-Y451A.

[0056] The PCR reaction system (50 μL) consisted of 2 μL of forward primer (10 μM), 2 μL of reverse primer (10 μM), 25 μL of 2× Phanta buffer, 1 μL of dNTP mix (10 mM each), 1 μL of plasmid template, 1 μL of DNA polymerase, and 18 μL of ultrapure water. The PCR protocol, according to the Phanta Super-Fidelity DNA Polymerase manual, was as follows: initial denaturation at 95°C for 5 min, followed by 29 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 5 min, followed by a final extension at 72°C for 10 min, followed by a 16°C incubation period. The resulting recombinant plasmid was transformed into E. coli BL21(DE3) competent cells via heat shock at 42°C, plated evenly on plates containing 50 μg / μL kanamycin, and incubated at 37°C for 14 h. Single colonies were picked and transferred to 1 mL of LB liquid medium containing 50 μg / mL kanamycin. Cultured in a 100 mL shake flask at 37°C, 180 rpm for 10 h, induced, and cultured at 28°C for 12 h before centrifugation to collect the cells. Crude enzyme was prepared as follows: 100 g / L of wet cells were resuspended in 50 mM phosphate buffer, pH 7.5, and ultrasonically disrupted over ice-water for 6 min. Ultrasonication conditions: 400 W power, 1 second disruption, 2 second pause. The disrupted mixture was collected to obtain a crude enzyme solution. This crude enzyme solution was purified by ammonium sulfate fractional precipitation: 100 mL of culture supernatant was placed in an ice bath, and 50% saturation ammonium sulfate solid powder was added while stirring. After complete precipitation of the ammonium sulfate, the supernatant was placed in an ice bath for 1 h and centrifuged at 12,000 rpm at 4°C for 10 min to obtain a crude penicillin G acylase enzyme as the primary precipitate. To the obtained supernatant, add ammonium sulfate solid with a final saturation of 80% while stirring in an ice bath. After the ammonium sulfate is completely dissolved, continue to stand in an ice bath for 2 hours, centrifuge at 12000 rpm and 4°C for 10 minutes, and dissolve the resulting precipitate in phosphate buffer (pH 7.0, 20mM) to obtain pure penicillin G acylase. The collected pure enzyme is dialyzed overnight with 20mM phosphate buffer (pH 7.0). All purification steps are carried out at 4°C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is used to identify the protein size. The electrophoresis results are shown in Figure 2 .

[0057] The yields of ampicillin and amoxicillin catalyzed by each mutant were determined using the catalytic system described in Example 1. The specific enzymatic activity of the penicillin G acylase AxPGA mutant was determined by adding 40% glycerol for solubilization and adding the substrates 200 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester. The mutant enzyme catalyst was catalyzed by pure enzyme at a final concentration of 1 mg / mL in a 50 mM potassium phosphate buffer (pH 7.0) as the reaction medium. The reaction was carried out at 30°C and 800 rpm for 3 hours. After the reaction was terminated, the reaction solution was analyzed by liquid chromatography to determine the amount of ampicillin produced, which was used to calculate the specific enzymatic activity of the mutant. The substrates 200 mM 6-aminopenicillanic acid and 200 mM D-hydroxyphenylglycine methyl ester were added. The mutant enzyme catalyst was catalyzed by pure enzyme at a final concentration of 1 mg / mL. The conversion system was constructed using pH 7.0, 50 mM potassium phosphate buffer as the reaction medium. The reaction was carried out at 30°C and 800 rpm for 3 h. After the reaction was terminated, the reaction solution was subjected to liquid phase detection. The amount of amoxicillin produced was detected to calculate the specific enzyme activity of the mutant (Table 2).

[0058] Table 1: Primer design for site-directed mutagenesis of penicillin G acylase AxPGA

[0059]

[0060] The enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 micromole of ampicillin / amoxicillin per minute at 30°C and pH 7.0. Specific enzyme activity is defined as the number of activity units per milligram of enzyme protein, U / mg.

[0061] The protein concentration was determined using a bicinchoninic acid protein assay kit (Nanjing KeyGen Biotechnology Development Co., Ltd., Nanjing).

[0062] After screening, four single mutants with improved enzyme activity were obtained. The results are shown in Table 2. The strains with improved enzyme activity were E. coli BL21(DE3) / pET28a(+)-AxPGA-M103C, E. coli BL21(DE3) / pET28a(+)-AxPGA-Y424F, E. coli BL21(DE3) / pET28a(+)-AxPGA-Y442F and E. coli BL21(DE3) / pET28a(+)AxPGA-Y451A. When catalyzing the synthesis of ampicillin, the specific enzyme activity of AxPGA-M103C was 392.25 U / mg; the specific enzyme activity of AxPGA-Y424F was 442.77 U / mg; the specific enzyme activity of AxPGA-Y442F was 563.41 U / mg; and the specific enzyme activity of AxPGA-Y451A was 533.41 U / mg. When catalyzing the synthesis of amoxicillin, the specific enzymatic activity of AxPGA-M103C was 337.62 U / mg; the specific enzymatic activity of AxPGA-Y424F was 335.61 U / mg; the specific enzymatic activity of AxPGA-Y442F was 429.16 U / mg; and the specific enzymatic activity of AxPGA-Y451A was 407.42 U / mg.

[0063] Table 2: Catalytic performance and specific enzyme activity of AxPGA and its single mutants

[0064]

[0065]

[0066] These results demonstrate that the enzymatic activity of the penicillin G acylase mutants AxPGA-M103C, AxPGA-Y424F, AxPGA-Y442F, and AxPGAY451A constructed in this study was significantly enhanced. When catalyzing the synthesis of ampicillin, the penicillin G acylase mutants AxPGA-M103C, AxPGA-Y424F, AxPGA-Y442F, and AxPGAY451A increased their activity by 56.13%, 76.24%, 124.26%, and 112.32%, respectively, compared to the control AxPGA. When penicillin G acylase mutants catalyzed the synthesis of amoxicillin, the mutants AxPGA-M103C, AxPGA-Y424F, AxPGA-Y442F and AxPGAY451A increased by 55.12%, 63.18%, 97.18% and 87.20% respectively compared with the control group AxPGA.

[0067] Example 3

[0068] This example constructs and screens a combinatorial mutant library of penicillin G acylase AxPGA mutants.

[0069] Preparation of a combinatorial mutation library of four penicillin G acylase AxPGA mutants was achieved by batch site-directed saturation mutagenesis. The primer designs are shown in Table 3. The vector pET28a(+)-AxPGA-M103C in E. coli BL21(DE3) / pET28a(+)-AxPGA was used as a template. Combinatorial mutagenesis was performed using primers targeting sites 424, 442, and 451 in Table 1. The PCR system and procedure were the same as in Example 2.

[0070] Penicillin G acylase combination mutants obtained by screening: AxPGA-M103C / Y424F, AxPGA-M103C / Y442F, AxPGA-M103C / Y451A, AxPGA-Y424F / Y442F, AxPGA-Y442F / Y451A, AxPGA-M103C / Y424F / Y442F, AxPGA-M103C / Y424F / Y442F, AxPGA-M103C / Y424F / Y442F / Y451A were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin, cultured at 37°C for 10 h, and inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a 1% (v / v) inoculum size, cultured at 37°C and 180 rpm for 2 h, and then a final concentration of 0.1 mM daptomycin was added to the culture medium. IPTG was added, cultured at 28°C for 12 h, and then centrifuged at 8000×g for 10 min at 4°C to obtain the corresponding wet bacterial cells.

[0071] The yields of ampicillin and amoxicillin catalyzed by each mutant were determined using the catalytic system described in Example 1. The specific enzymatic activity of the penicillin G acylase AxPGA mutant was determined by adding 40% glycerol and the substrates 200 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester. The mutant enzyme catalyst was catalyzed by pure enzyme at a final concentration of 1 mg / mL in a 50 mM potassium phosphate buffer (pH 7.0) at 30°C and 800 rpm for 3 hours. After the reaction was terminated, the reaction solution was analyzed by liquid chromatography to determine the amount of ampicillin produced, which was used to calculate the specific enzymatic activity of the mutant. 40% glycerol was added and the substrates 200 mM 6-aminopenicillanic acid and 200 mM D-hydroxyphenylglycine methyl ester were added. The mutant enzyme catalyst was catalyzed by pure enzyme at a final concentration of 1 mg / mL. A conversion system was formed using pH 7.0, 50 mM potassium phosphate buffer as the reaction medium. The reaction was carried out at 30°C and 800 rpm for 3 h. After the reaction was terminated, liquid phase detection of the reaction solution was performed to detect the amount of product amoxicillin produced to calculate the specific enzyme activity of the mutant. The results are shown in Table 3.

[0072] Table 3

[0073]

[0074] The results showed that when the mutant catalyzed the synthesis of ampicillin, AxPGA-M103C / Y424F / Y442F / Y451A achieved the highest specific enzyme activity and catalytic performance, with a specific enzyme activity of 605.26 U / mg and a catalytic yield of ampicillin of 78.51%. When the mutant catalyzed the synthesis of amoxicillin, AxPGA-M103C / Y424F / Y442F / Y451A achieved the highest specific enzyme activity and catalytic performance, with a specific enzyme activity of 504.97 U / mg and a catalytic yield of amoxicillin of 77.81%.

[0075] Example 4

[0076] In this example, an optimal pH experiment was conducted to determine the optimal pH for the catalytic synthesis of β-lactam antibiotics (ampicillin and amoxicillin) using the optimal mutant of AxPGA, AxPGA-M103C / Y424F / Y442F / Y451A.

[0077] The E. coli BL21(DE3) / pET28a(+)-AxPGA-M103C / Y424F / Y442F / Y451A mutant of penicillin G acylase prepared in Example 3 was used as a catalyst. The catalytic performance of the mutant at different pH values ​​was calculated using the same method as in Example 1. Four buffer solutions of different pH values ​​were prepared: 50 mM phosphate buffer was prepared by weighing 0.087 g of 50 mM potassium dihydrogen phosphate and dissolving it in 8 mL of dd H2O. The pH was adjusted to 5.3, 5.6, 5.9, 6.2, 6.5, 6.8, 7.1, and 7.4 with potassium dihydrogen phosphate, respectively. 40% glycerol was added and the volume was adjusted to 10 mL with dd H2O. Under the aforementioned pH conditions, the catalytic activity of the mutant cells was screened: The catalytic activity of the obtained cells was assessed by adding 200 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester as substrates. The catalyst dosage was 4 g DCW / L (DCW cell dry weight) based on the total dry weight of the mixed cells. The reaction was carried out at 30°C and 800 rpm for 3 hours. After the reaction was terminated, the reaction solution was analyzed by liquid chromatography to determine the amount of ampicillin produced and calculate the product yield. 200 mM 6-aminopenicillanic acid and 200 mM D-hydroxyphenylglycine methyl ester were added. The amount of catalyst used was 4 g DCW / L (DCW cell dry weight) based on the total dry weight of the mixed bacteria. The reaction was carried out at 30°C and 800 rpm for 3 hours. After the reaction was completed, the reaction liquid was subjected to liquid phase detection. The amount of product amoxicillin produced was detected to calculate the product yield, so as to compare the catalytic performance of the optimal mutant AxPGA-M103C / Y424F / Y442F / Y451A of AxPGA under different pH conditions.

[0078] The yields of ampicillin and amoxicillin catalyzed by AxPGA-M103C / Y424F / Y442F / Y451A under different pH conditions are as follows: Figure 3 The results showed that the yield of ampicillin and amoxicillin catalyzed by AxPGA-M103C / Y424F / Y442F / Y451A was the highest in 50 mM phosphate buffer at pH 5.9.

[0079] Example 5

[0080] In this example, a reaction temperature experiment was conducted on the synthesis of ampicillin and amoxicillin catalyzed by the mutant AxPGA-M103C / Y424F / Y442F / Y451A.

[0081] Using the same method as in Example 3, engineered strains containing the penicillin G acylase mutant AxPGA-M103C / Y424F / Y442F / Y451A were obtained. The yields of ampicillin and amoxicillin synthesized by the mutant at different reaction temperatures were investigated. Six temperature gradients (15°C, 20°C, 25°C, 30°C, 35°C, and 40°C) were considered. A 50 mM phosphate buffer solution, pH 5.9, supplemented with 40% glycerol was used as the buffer system. Substrates included 200 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester. The catalyst dosage was 4 g DCW / L (DCW cell dry weight) based on the total dry weight of the mixed strains. Reactions were performed at 800 rpm under the various reaction temperature gradients. After termination, the reaction solution was subjected to liquid chromatography to determine the amount of ampicillin produced and calculate the product yield. 200 mM 6-aminopenicillanic acid and 200 mM D-hydroxyphenylglycine methyl ester were added. The catalyst dosage was 4 g DCW / L (DCW cell dry weight) based on the total dry weight of the mixed cells. The reaction was carried out at 800 rpm under different temperature gradients for 3 hours. After the reaction was terminated, the reaction solution was subjected to liquid chromatography to detect the amount of amoxicillin produced to calculate the product yield. The samples were processed and analyzed by HPLC to compare the yields of ampicillin and amoxicillin produced by the mutant strains under different temperature conditions.

[0082] The influence of temperature Figure 4 As shown: When the reaction temperature is 20℃, the mutant AxPGA-M103C / Y424F / Y442F / Y451A has the highest yield in the synthesis of ampicillin and amoxicillin.

[0083] Example 6

[0084] This example optimizes the substrate ratio of the reaction in which the mutant AxPGA-M103C / Y424F / Y442F / Y451A catalyzes the synthesis of ampicillin and amoxicillin.

[0085] Using the same method as in Example 3, engineered bacteria containing the penicillin G acylase mutant AxPGA-M103C / Y424F / Y442F / Y451A were obtained. The yields of ampicillin and amoxicillin synthesized by the mutant under different reaction substrate ratios were investigated. This patent considered five different substrate gradients: 6-aminopenicillanic acid to D-hydroxyphenylglycine methyl ester / D-hydroxyphenylglycine methyl ester molar ratios of 1.0:1, 1.05:1, 1.1:1, 1.15:1, and 1.2:1. A 50 mM phosphate buffer solution at pH 5.9 was used as the buffer system, supplemented with 40% glycerol. Substrates of 200 mM, 210 mM, 220 mM, 230 mM, and 240 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester were added. The catalyst dosage was 4 g DCW / L (DCW cell dry weight) based on the total dry weight of the mixed cells. Reactions were performed at 20°C and 800 rpm. After the reaction was terminated, the reaction solution was analyzed by liquid chromatography to determine the amount of ampicillin produced and to calculate the product yield. 200 mM, 210 mM, 220 mM, 230 mM, and 240 mM of 6-aminopenicillanic acid and 200 mM of D-hydroxyphenylglycine methyl ester were added. The catalyst dosage was 4 g DCW / L (DCW cell dry weight) based on the total dry weight of the mixed cells. Reactions were performed at different temperature gradients and 800 rpm for 3 hours. After the reaction was terminated, the reaction solution was analyzed by liquid chromatography to determine the amount of amoxicillin produced and to calculate the product yield. Samples were processed and analyzed by HPLC to compare the ampicillin and amoxicillin yields of the mutant strains under different temperature conditions.

[0086] The effect of substrate ratio Figure 5 As shown: When the molar ratio of D-phenylglycine methyl ester / D-hydroxyphenylglycine methyl ester to 6-aminopenicillanic acid is 1.2:1, the mutant AxPGA-M103C / Y424F / Y442F / Y451A can achieve the highest yield in the synthesis of ampicillin and amoxicillin.

[0087] Example 7

[0088] This example optimizes the amount of bacteria used to catalyze the synthesis of ampicillin and amoxicillin using the mutant AxPGA-M103C / Y424F / Y442F / Y451A.

[0089] Using the same method as in Example 3, engineered bacteria containing the penicillin G acylase mutant AxPGA-M103C / Y424F / Y442F / Y451A were obtained. The yield of ampicillin and amoxicillin synthesized by the mutant under varying bacterial cell dosages was investigated. Five different bacterial cell dosage gradients (2 g DCW / L, 4 g DCW / L, 6 g DCW / L, 8 g DCW / L, and 10 g DCW / L) were considered. The buffer system was 50 mM phosphate buffer, pH 5.9, supplemented with 40% glycerol. Substrates of 240 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester were added. Catalyst dosages were 2 g DCW / L, 4 g DCW / L, 6 g DCW / L, 8 g DCW / L, and 10 g DCW / L, based on the total dry weight of the mixed cells. Reactions were conducted at 20°C and 800 rpm. After termination, the reaction solution was analyzed by liquid chromatography to determine the amount of ampicillin produced and calculate the yield. 240 mM 6-aminopenicillanic acid and 200 mM D-hydroxyphenylglycine methyl ester were added. The amount of catalyst used was 2 g DCW / L, 4 g DCW / L, 6 g DCW / L, 8 g DCW / L, and 10 g DCW / L based on the total dry weight of the mixed bacteria. The reaction was carried out at 20°C and 800 rpm for 3 hours. After the reaction was completed, the reaction liquid was subjected to liquid phase detection after the reaction was terminated. The amount of the product amoxicillin produced was detected to calculate the yield of the product. The samples were processed and analyzed by HPLC to compare the yields of ampicillin and amoxicillin produced by the mutant strains under different temperature conditions.

[0090] result Figure 6 As shown in the figure: when the bacterial dosage was 8 g DCW / L, the mutant AxPGA-M103C / Y424F / Y442F / Y451A had the highest yield in the synthesis of ampicillin and amoxicillin.

[0091] Example 8

[0092] This example describes the reaction process of synthesizing ampicillin and amoxicillin catalyzed by the mutant AxPGA-M103C / Y424F / Y442F / Y451A.

[0093] The engineered bacteria containing the penicillin G acylase mutant AxPGA-M103C / Y424F / Y442F / Y451A were obtained by the same method as in Example 3. The reaction process of synthesizing ampicillin and amoxicillin by the mutant was investigated by feeding substrate.

[0094] 50mM phosphate buffer, pH 5.9, was used as the buffer system, with 40% glycerol added. The substrates were 240mM 6-aminopenicillanic acid and 200mM D-phenylglycine methyl ester. The catalyst dosage was 8g DCW / L based on the total dry weight of the mixed cells. The reaction was carried out at 20°C and 800rpm. After every 1 hour of reaction, 120mM 6-aminopenicillanic acid and 100mM D-phenylglycine methyl ester were added. The amount of ampicillin produced in the reaction solution was monitored at all times during the reaction. The results are shown in Figure 2. Figure 7 As shown, after 6 h of reaction, the concentration of ampicillin reached 588.90 mM, the conversion rate was 99%, and the yield was as high as 98.15%.

[0095] 50mM phosphate buffer pH 5.9 was used as the buffer system, and 40% glycerol was added. 240mM 6-aminopenicillanic acid and 200mM D-hydroxyphenylglycine methyl ester were added as substrates. The catalyst dosage was 8gDCW / L based on the total dry weight of the mixed bacteria. The reaction was carried out at 20°C and 800rpm. After every 1 hour of reaction, 120mM 6-aminopenicillanic acid and 100mM D-hydroxyphenylglycine methyl ester were added as substrates. The amount of amoxicillin in the reaction solution was monitored at any time during the reaction. The results are shown in FIG. Figure 8 As shown in the figure, after 6 h of reaction, the concentration of amoxicillin reached 587.52 mM, the conversion rate was 99%, and the yield was as high as 97.92%.

Claims

1. A penicillin G acylase Ax A PGA mutant, characterized in that The mutant is a mutant in which the amino acid sequence shown in SEQ ID NO: 2 is mutated into one of the following: (1) Substituting methionine at position 103 with cysteine, (2) Based on (1), the tyrosine at position 424 was replaced by phenylalanine. (3) Based on (1), the tyrosine at position 442 was replaced by phenylalanine. (4) Based on (1), the tyrosine at position 451 was replaced with alanine. (5) Based on (1), the tyrosine at position 424 was replaced by phenylalanine, and the tyrosine at position 442 was replaced by phenylalanine. (6) Based on (1), the tyrosine at position 424 was replaced by phenylalanine, the tyrosine at position 442 was replaced by phenylalanine, and the tyrosine at position 451 was replaced by alanine.

2. A penicillin G acylase according to claim 1 Ax The expression plasmid of the PGA mutant is characterized by: The plasmid vector is pET28a.

3. The penicillin G acylase according to claim 1 Ax The genetically engineered bacteria of the PGA mutant are characterized by: The host bacteria of the genetically engineered bacteria is E. coli BL21.

4. The penicillin G acylase according to claim 1 Ax Application of PGA mutants in catalytic synthesis of ampicillin or amoxicillin.

5. The use according to claim 4, characterized in that: Penicillin G acylase Ax A PGA mutant is used as a catalyst, a potassium phosphate buffer solution with a pH of 5.3 to 7.4 is used as a reaction medium to form a conversion system, 6-aminopenicillanic acid is used as a first reaction substrate, and D-phenylglycine methyl ester or D-hydroxyphenylglycine methyl ester is used as a second reaction substrate. Ampicillin or amoxicillin is prepared by reacting at 15 to 40° C., and the molar ratio of the first reaction substrate to the second reaction substrate is 1:1 to 1.

2.

6. The use according to claim 5, characterized in that: Glycerol is added into the transformation system.

7. The use according to claim 5, characterized in that: The catalyst is added in an amount of 2 to 10 g DCW / L in the conversion system.

8. The use according to claim 5, characterized in that Penicillin G acylase Ax Penicillin G acylase Ax The PGA mutant is added in the form of wet bacterial cells of genetically engineered bacteria, cell lysate of genetically engineered bacteria, or pure enzyme solution separated after protein purification of cell lysate.

9. The use according to claim 8, characterized in that: Penicillin G acylase Ax The genetically engineered bacteria containing the PGA mutant were inoculated into LB liquid medium with a final concentration of 50 μg / mL kanamycin and cultured at 37°C for 10 h. The inoculum size was then inoculated into LB liquid medium with a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 2 h. Isopropylthiogalactoside was then added to the culture medium at a final concentration of 0.1 mM. The culture was cultured at 28°C for 12 h and then centrifuged at 4°C and 9000 x g for 10 min to obtain wet cells of the genetically engineered bacteria containing the penicillin G acylase AxPGA mutant.

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