Penicillin G acylase AxPGA mutant, expression plasmid, genetically engineered bacterium and application

Abstract

The invention provides a penicillin G acylase AxPGA mutant, an expression plasmid, a genetically engineered bacterium and application, and belongs to the technical field of biocatalysts. The mutant is obtained by mutation of any one or more of the following sites of an amino acid sequence as shown in SEQ ID NO: 1: (1) methionine at the 103rd site is substituted by cysteine, (2) tyrosine at the 424th site is substituted by phenylalanine, (3) tyrosine at the 442nd site is substituted by phenylalanine, and (4) tyrosine at the 451st site is substituted by alanine. The obtained penicillin G acylase AxPGA mutant is relatively high in enzyme activity and can be applied to high-conversion-rate and high-yield synthesis of beta-lactam antibiotics such as benicillin and amoxicillin.

Description

Technical Field

[0001] The present application relates to a penicillin G acylase Ax PGA mutants and expression plasmids, genetic engineering bacteria and applications belong to the technical field of biocatalysts. Background Art

[0002] β-lactam antibiotics are very important antibacterial drugs. Since β-lactamase can hydrolyze the β-lactam ring into an inactive form, their use is affected by drug resistance. The production of β-lactam antibiotics mainly adopts chemical methods, but chemical methods have strict requirements on chemical substances, there are extreme temperatures in the reaction, and there are complex processes such as selective protection and deprotection of functional groups, which leads to defects in the industrial application of chemical production of β-lactam antibiotics.

[0003] The synthetic pathway of penicillin G acylase has attracted great attention. Compared with chemical catalysts, enzymes, as green natural biocatalysts, have advantages such as superior stereoselectivity and regioselectivity in catalyzing chemical reactions, and the reaction conditions are mild, environmentally friendly, and do not require harsh reaction equipment. Therefore, research in this field is relatively active, but the enzyme activities of penicillin G acylase and its mutants are not ideal, resulting in low catalytic activity and poor reaction yield 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 with high enzyme activity. Ax PGA mutants can be applied to the synthesis of β-lactam antibiotics, such as the high-yield synthesis of ampicillin and amoxicillin.

[0005] Specifically, the present application is implemented through the following scheme: A penicillin G acylase Ax The PGA mutant is obtained by mutation of any one or more of the following sites of the amino acid sequence shown in SEQ ID NO: 1: (1) methionine at position 103 is substituted with cysteine, (2) tyrosine at position 424 is substituted with phenylalanine, (3) tyrosine at position 442 is substituted with phenylalanine, and (4) tyrosine at position 451 is substituted with alanine.

[0006] A penicillin G acylase Ax A PGA mutant is derived from penicillin G acylase from Achromobacterxylosoxidans, wherein the amino acid sequence of the penicillin G acylase is recorded as SEQ ID NO: 2, and the following four sites of SEQ ID NO: 2 are mutated simultaneously to obtain the penicillin G acylaseAx PGA mutants: (1) methionine at position 103 is substituted with cysteine, (2) tyrosine at position 424 is substituted with phenylalanine, (3) tyrosine at position 442 is substituted with phenylalanine, and (4) tyrosine at position 451 is substituted with alanine.

[0007] The second object of the applicant is to provide the above-mentioned penicillin G acylase Ax The expression plasmid of the PGA mutant is composed of pET28a.

[0008] The third object of the applicant is to provide the above-mentioned penicillin G acylase Ax The genetically engineered bacteria of the PGA mutant, wherein the host bacteria of the genetically engineered bacteria is E.coli BL21(DE3).

[0009] The fourth object of the applicant is to provide the above-mentioned penicillin G acylase Ax Application of PGA mutants in the catalytic synthesis of β-lactam antibiotics.

[0010] Specific: Penicillin G acylase Ax The PGA 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-phenylglycine methyl ester is used as a second reaction substrate. Ampicillin or amoxicillin is prepared by reacting at 15 to 40°C.

[0011] More preferred: 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.

[0012] Glycerol is added into the conversion system as a co-solvent.

[0013] The catalyst is added in an amount of 2 to 10 g DCW / L (DCW: dry cell weight, L refers to the volume of the conversion system).

[0014] Penicillin G acylase Ax PGA mutants can be used to express penicillin G acylase Ax The PGA mutant is added in the form of wet bacterial cells of genetically engineered bacteria, or cell lysate of genetically engineered bacteria, or pure enzyme solution separated after protein purification of cell lysate. Penicillin G acylase AxThe expression plasmid of PGA mutant was transferred into E.coli BL21 (DE3) competent cells are used to obtain genetically engineered bacteria. The obtained strains are inoculated, transferred, induced, and recovered. The resuspended bacterial solution is the wet bacterial body of the genetically engineered bacteria, wherein the culture medium can be any culture medium for growing Escherichia coli cells in the art and expressing the target gene of the present invention. The preferred LB culture 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 special restrictions on the culture method and culture conditions, and the culture method and conditions can be appropriately selected according to factors such as the host type and the culture method. As a specific preparation process: contain penicillin G acylase Ax The genetically engineered bacteria of the PGA mutant were inoculated into LB liquid culture medium with a final concentration of 50 μg / mL kanamycin, cultured at 37°C for 10 h, inoculated into LB liquid culture medium with a final concentration of 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, cultured at 37°C and 180 rpm for 2 h, and then 0.1 mM isopropyl β-D-thiogalactoside (IPTG) was added to the culture medium. After culture at 28°C for 12 h, the culture medium was centrifuged at 4°C and 9000 xg (xg 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 min to obtain the wet cells of the genetically engineered bacteria containing the penicillin G acylase AxPGA mutant.

[0015] The preparation method of the cell disruption solution is as follows: resuspend the cells in a pH 5.9, 50 mM potassium phosphate buffer at a dosage of 10 g DCW / L, and disrupt them by ultrasonication on an ice-water mixture for 6 min. The ultrasonication conditions are as follows: power of 400 W, disruption for 1 s, and pause for 2 s.

[0016] The present application directly synthesizes β-lactam antibiotics (such as ampicillin and amoxicillin) through a one-step reaction, in which penicillin G acylase is used as a catalyzer: first, enzymes from four different sources are screened, and the one with the highest enzyme activity is penicillin G acylase from Achromobacter xylosoxidans PX02 Ax PGA, the enzyme was molecularly modified by directed evolution, the amino acid residues near the enzyme-substrate pocket were site-directed mutated, and the enzyme was modified according to the principle of reducing the substrate pocket. The enzyme activity of the mutant strain was increased by 55.12~124.26% compared with the original strain. Ax PGA. AxPGA mutants are applied to the enzymatic synthesis of β-lactam antibiotics. For example, the key enzyme penicillin G acylase catalyzes the synthesis of ampicillin (ampicillin) from the substrates 6-aminopenicillanic acid and D-phenylglycine methyl ester, and the synthesis of hydroxyampicillin (amoxicillin) from the substrates 6-aminopenicillanic acid and D-hydroxyphenylglycine methyl ester. The catalytic activity of the corresponding β-lactam antibiotics is increased by 1.01 to 1.41 times, the conversion rate is increased to more than 99%, and the yield is close to 98%. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 paying any creative work.

[0018] Figure 1 To screen penicillin G acylase from different sources Ax Yield diagram of the PGA-catalyzed preparation of ampicillin and amoxicillin.

[0019] Figure 2 Penicillin G acylase Ax SDS-PAGE electrophoresis of the supernatant of PGA mutants, Lane M: standard protein molecule Marker; Lane 1: control penicillin G acylase Ax PGA pure enzyme; Lane 2: Penicillin G acylase Ax PGA-M103C pure enzyme; Lane 3: Penicillin G acylase Ax PGA-Y424F pure enzyme; Lane 5: Penicillin G acylase Ax PGA-Y442F pure enzyme; Lane 6: Penicillin G acylase Ax PGA-Y451A pure enzyme; Lane 7: Penicillin G acylase Ax PGA-M103C / Y424F / Y442F / Y451A pure enzyme.

[0020] Figure 3 Penicillin G acylase under different pH conditions Ax The yields of ampicillin and amoxicillin synthesized by the supernatant of PGA-M103C / Y424F / Y442F / Y451A.

[0021] Figure 4 Penicillin G acylase at different reaction temperatures Ax The yields of ampicillin and amoxicillin synthesized by the supernatant of PGA-M103C / Y424F / Y442F / Y451A.

[0022] Figure 5 Penicillin G acylase Ax Effect of PGA-M103C / Y424F / Y442F / Y451A on the yield of ampicillin and amoxicillin.

[0023] Figure 6 Penicillin G acylase Ax Effect of PGA-M103C / Y424F / Y442F / Y451A bacterial concentration on the yield of catalytic preparation of ampicillin and amoxicillin.

[0024] Figure 7 Penicillin G acylase Ax Process diagram of the production of ampicillin catalyzed by PGA-M103C / Y424F / Y442F / Y451A.

[0025] Figure 8 Penicillin G acylase Ax Process diagram of the production of ampicillin catalyzed by PGA-M103C / Y424F / Y442F / Y451A. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application clearer, 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 work are within the scope of protection of this application.

[0027] Example 1

[0028] In this example, penicillin G acylase Ax Mining and screening of PGA genetic engineering bacteria. Penicillin G acylase was mined through NCBI.

[0029] Its GenBank: AVC04856.1, after codon optimization using Escherichia coli as the host (the optimized nucleotide sequence is shown in SEQ ID NO:1) and synthesized by Qingke Biotechnology Co., Ltd. Ax The cDNA fragment of PGA was linked to the site after TATACCAT (before the NcoI restriction site) and before CTCGAG (XhoI restriction site) in pET-28a (+). AxPGA Transfer-In E. coli BL21(DE3) was used to obtain the strain E. coli BL21(DE3) / pET28a(+)- Ax PGA.

[0030] SEQ ID NO.1:

[0031] Its GenBank: EJA4313497.1, nucleotide sequence as shown in SEQ ID NO:3, and was synthesized by Qingke Biotechnology Co., Ltd. Ec The cDNA fragment of PGA was linked to the site after TATACCAT (before the NcoI restriction site) and before CTCGAG (XhoI restriction site) in pET-28a (+). Ec PGA Transfer-In E. coli BL21(DE3) was used to obtain the strain E. coli BL21(DE3) / pET28a(+)- Ec PGA.

[0032] Its GenBank: AVC04856.1, after codon optimization using Escherichia coli as the host (the optimized nucleotide sequence is shown in SEQ ID NO:5) and synthesized by Qingke Biotechnology Co., Ltd. As The cDNA fragment of PGA was linked to the site after TATACCAT (before the NcoI restriction site) and before CTCGAG (XhoI restriction site) in pET-28a (+). As PGA Transfer-In E. coli BL21(DE3) was used to obtain the strain E. coli BL21(DE3) / pET28a(+)- As PGA.

[0033] Its GenBank: AVC04856.1, after codon optimization using Escherichia coli as the host (the optimized nucleotide sequence is shown in SEQ ID NO: 7) and synthesized by Qingke Biotechnology Co., Ltd. Ad The cDNA fragment of PGA was linked to the site after TATACCAT (before the NcoI restriction site) and before CTCGAG (XhoI restriction site) in pET-28a (+). Ad PGA Transfer-In E. coli BL21(DE3) was used to obtain the strain E. coli BL21(DE3) / pET28a(+)- Ad PGA.

[0034] 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, inoculated into fresh LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin at an inoculum size of 1% (v / v), cultured at 37°C and 180 rpm for 2 h, and then IPTG with a final concentration of 0.1 mM was added to the culture medium. After culture at 28°C for 12 h, the culture medium was centrifuged at 4°C and 9000 x g for 10 min to obtain the corresponding wet bacterial cells.

[0035] Screening of bacterial catalytic ability: The catalytic ability of the obtained cells was tested, and after adding 40% glycerol to aid solubilization, the following processes were used to synthesize different β-lactam antibiotics.

[0036] (1) Add 200 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester as substrates. The amount of catalyst used is 4 g DCW / L (DCW cell dry weight) based on the total dry weight of the mixed bacteria. The conversion system is composed of 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 liquid is subjected to liquid phase detection after the reaction is terminated. The amount of product ampicillin produced is detected to calculate the product yield.

[0037] (2) Add 200 mM 6-aminopenicillanic acid and 200 mM D-hydroxyphenylglycine methyl ester as substrates. The amount of catalyst used is 4 g DCW / L (DCW cell dry weight) based on the total dry weight of the mixed bacteria. The conversion system is composed of 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 phase detection after the reaction is terminated. The amount of amoxicillin produced is detected to calculate the yield of the product.

[0038] Penicillin G acylase Ax The reaction equation for the preparation of ampicillin and amoxicillin catalyzed by the engineered bacteria of the PGA gene is as follows: .

[0039] The production of ampicillin and amoxicillin is as follows Figure 1 As shown, the source Ax PGA Correspondence Achromobacter xylosoxidans The mutant Ec PGA Correspondence Escherichia coli The mutant As PGA Correspondence Achromobacter sp. Mutants of UMC46, Ad PGA CorrespondenceAchromobacter denitrificans The results showed that: Achromobacter xylosoxidans The yield of penicillin G acylase in producing β-lactam antibiotics (ampicillin and amoxicillin) was the highest among the four penicillin G acylases from different sources. Ax The PGA-based cells were further modified to improve the efficiency of synthesis of β-lactam antibiotics.

[0040] Liquid phase detection conditions: The samples were analyzed by high performance liquid chromatography - using Kromasil liquid chromatography (Dionex P680 HPLC) 100-5 C18 column (4.6 mm × 250 mm, Kromasil, Sweden) and UVD170U detector at 254 nm, mobile phase: 50 mM, pH 7.0 potassium phosphate buffer; column temperature: 35 ° C; flow rate: 0.8 mL / min; injection volume: 10 μL. The retention time of ampicillin is 5.2 min, and the standard curve is: y = 2438.91x-240.14. The retention time of ampicillin is 7.3 min, and the standard curve is: y = 2189.21x-217.31.

[0041] Example 2

[0042] This example constructs and screens a penicillin G acylase mutant library.

[0043] According to the substrate pocket reduction strategy of the bi-substrate molecule, the preparation of the penicillin G acylase mutant library was achieved by site-directed mutagenesis technology. The primers were designed as shown in Table 1. E. coli BL21(DE3) / pET28a(+)- Ax PGA vector pET28a(+)- Ax PGA was used as the template, and M103C-F and M103C-R in Table 1 were used as the mutation primers for amino acid 103. Ax The 103rd methionine in the amino acid sequence of PGA was mutated to cysteine, and the monoclonal mutant was obtained by transformation. Ax PGA-M103C. Using Y383A-F and Y383A-R in Table 1 as the mutation primers for amino acid 383, penicillin G acylase was mutated by site-directed mutagenesis PCR. Ax The tyrosine at position 383 of the amino acid sequence of PGA was mutated to alanine, and the monoclonal mutant was obtained by transformation. Ax PGA-Y383A. Using the mutation primers W401A-F and W401A-R in Table 1 as the 401 amino acid position, site-directed mutagenesis PCR was performed to transform penicillin G acylase Ax The tryptophan at position 401 of the PGA amino acid sequence was mutated to alanine, and the monoclonal mutant was obtained by transformation.Ax PGA-W401A. Using Y424F-F and Y424F-R in Table 1 as the mutation primers for amino acid 424, site-directed mutagenesis PCR was performed to transform penicillin G acylase Ax The 424th tyrosine in the amino acid sequence of PGA was mutated to phenylalanine, and the monoclonal mutant was obtained by transformation. Ax PGA-Y424F. Using Y442F-F and Y442F-R in Table 1 as the mutant primers for amino acid 442, site-directed mutagenesis PCR was performed to transform penicillin G acylase Ax The 442nd tyrosine in the amino acid sequence of PGA was mutated to phenylalanine, and the monoclonal mutant was obtained by transformation. Ax PGA-Y442F. Using W448A-F and W448A-R in Table 1 as the mutation primers for amino acid 448, site-directed mutagenesis PCR was performed to transform penicillin G acylase Ax The tryptophan at position 448 of the PGA amino acid sequence was mutated to alanine, and the monoclonal mutant was obtained by transformation. Ax PGA-W448A. Using Y451A-F and Y451A-R in Table 1 as the mutant primers for amino acid 451, site-directed mutagenesis PCR was performed to transform penicillin G acylase Ax The 451st tyrosine in the amino acid sequence of PGA was mutated to alanine, and the monoclonal mutant was obtained by transformation. Ax PGA-Y451A.

[0044] PCR reaction system (50 µL): 2 µL forward primer (10 μM), 2 µL reverse primer (10 μM), 25 µL 2× Phanta buffer, 1 µL dNTP mixture (10 mM each), 1 µL plasmid template, 1 µL DNA polymerase and 18 µL ultrapure water. The PCR program set according to the Phanta Super-Fidelity DNA polymerase manual is as follows: 95°C pre-denaturation for 5 min, then 29 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 5 min), 72°C final extension for 10 min, and 16°C incubation. The obtained recombinant plasmid was transformed into E. coliBL21 (DE3) competent cells, and evenly spread on a plate containing 50 µg / uL kanamycin resistance LB medium, and cultured at 37℃ for 14 h. Pick out a single clone and transfer it to 1 mL LB liquid medium containing 50 µg / mL kanamycin, culture it in a 100 mL shake flask at 37℃, 180 rpm for 10 h, induce it, culture it at 28℃ for 12 h, and then collect the bacteria by centrifugation. The crude enzyme was prepared as follows: resuspend the total amount of wet bacteria in pH 7.5, 50 mM phosphate buffer at a dosage of 100 g / L, and ultrasonically disrupt it on an ice-water mixture for 6 min. The ultrasonic disruption conditions were: power of 400 W, disruption for 1 s, pause for 2 s, and take the disruption mixture to obtain the crude enzyme solution. The 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 the ammonium sulfate was completely precipitated, it was placed in an ice bath for 1 h, and centrifuged at 12000 rpm at 4°C for 10 min to obtain a crude penicillin G acylase enzyme with the supernatant as the primary precipitate. To the obtained supernatant, ammonium sulfate solid with a final saturation of 80% was added while stirring under ice bath conditions. After the ammonium sulfate was completely dissolved, it was placed in an ice bath for 2 h, and centrifuged at 12000 rpm at 4°C for 10 min. The obtained precipitate was dissolved in phosphate buffer (pH 7.0, 20 mM) to obtain a pure penicillin G acylase enzyme. The collected pure enzyme was dialyzed overnight with 20 mM phosphate buffer (pH 7.0). All purification steps were carried out at 4°C. The protein size was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis results are shown in Figure 2 .

[0045] The yield of catalytic synthesis of ampicillin and amoxicillin by each mutant was determined using the catalytic system in Example 1. Detection of the specific enzyme activity of the mutant of penicillin G acylase AxPGA: 40% glycerol was added to dissolve, and 200 mM of 6-aminopenicillanic acid and 200 mM of D-phenylglycine methyl ester were added as substrates. The mutant enzyme catalyst was catalyzed by pure enzyme with a final concentration of 1 mg / mL, and a conversion system was formed with a 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 completed, the reaction solution was subjected to liquid phase detection after the reaction was terminated, and the amount of product ampicillin generated was detected to calculate the specific enzyme activity of the mutant. 200 mM of 6-aminopenicillanic acid and 200 mM of D-hydroxyphenylglycine methyl ester were added as substrates. The mutant enzyme catalyst was catalyzed by pure enzyme at a final concentration of 1 mg / mL, and the conversion system was composed of 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 liquid phase of the reaction solution was detected, and the amount of amoxicillin produced was detected to calculate the specific enzyme activity of the mutant (Table 2).

[0046] Table 1: Primer design for site-directed mutagenesis of penicillin G acylase AxPGA .

[0047] 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, U. The specific enzyme activity is defined as the number of activity units per milligram of enzyme protein, U / mg.

[0048] The protein concentration was determined using a bicinchoninic acid protein assay kit (Nanjing KeyGen Biotechnology Development Co., Ltd., Nanjing).

[0049] After screening, four single mutants with improved enzyme activity were obtained, and the results are shown in Table 2. The strain with improved enzyme activity is E. coli BL21(DE3) / pET28a(+)- Ax PGA-M103C, E. coli BL21(DE3) / pET28a(+)- Ax PGA-Y424F, E. coli BL21(DE3) / pET28a(+)- Ax PGA-Y442F and E. coli BL21(DE3) / pET28a(+) Ax PGA-Y451A, catalyzes the synthesis of ampicillin. AxThe specific enzyme activity of PGA-M103C was 392.25 U / mg; Ax The specific enzyme activity of PGA-Y424F was 442.77 U / mg; Ax The specific enzyme activity of PGA-Y442F was 563.41 U / mg; Ax The specific enzyme activity of PGA-Y451A is 533.41 U / mg. When catalyzing the synthesis of amoxicillin, Ax The specific enzyme activity of PGA-M103C was 337.62 U / mg; Ax The specific enzyme activity of PGA-Y424F was 335.61 U / mg; Ax The specific enzyme activity of PGA-Y442F was 429.16 U / mg; Ax The specific enzyme activity of PGA-Y451A was 407.42 U / mg.

[0050] Table 2: Catalytic performance and specific enzyme activity of AxPGA and its single mutants .

[0051] The above results show that the penicillin G acylase constructed in this case Ax PGA mutant Ax PGA-M103C, Ax PGA-Y424F, Ax PGA-Y442F and Ax The enzyme activity of PGAY451A was greatly improved. When the mutant of penicillin G acylase catalyzes the synthesis of ampicillin, its Ax PGA-M103C, Ax PGA-Y424F, Ax PGA-Y442F and Ax PGAY451A compared with the control group Ax PGA increased by 56.13%, 76.24%, 124.26% and 112.32% respectively. When the mutant of penicillin G acylase catalyzed the synthesis of amoxicillin, its Ax PGA-M103C, Ax PGA-Y424F, Ax PGA-Y442F and Ax Compared with the control group, PGAY451A increased ribokinase by 56.13%, 76.24%, 124.26% and 112.32%, respectively.

[0052] Example 3

[0053] In this example, penicillin G acylase Ax Construction and screening of a combinatorial mutant library of PGA mutants.

[0054] Penicillin G acylase Ax The preparation of the PGA four-mutant combinatorial mutation library was achieved by batch site-directed saturation mutagenesis. The primers were designed as shown in Table 3. E. coli BL21(DE3) / pET28a(+)- Ax PGA vector pET28a(+)- Ax PGA-M103C was used as a template, and the primers at the three sites 424, 442 and 451 in Table 1 were used for combined mutation. The PCR system and procedure were the same as in Example 2.

[0055] Screening for penicillin G acylase combination mutants: Ax PGA-M103C / Y424F, Ax PGA-M103C / Y442F, Ax PGA-M103C / Y451A, Ax PGA-Y424F / Y442F 、Ax PGA-Y442F / Y451A, Ax PGA-M103C / Y424F / Y442F, Ax PGA-M103C / Y424F / Y442F / Y451A were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin, respectively, and cultured at 37°C for 10 h. Then, they were inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a 1% (v / v) inoculum and cultured at 37°C and 180 rpm for 2 h. Then, IPTG with a final concentration of 0.1 mM was added to the culture medium. After culture at 28°C for 12 h, the culture medium was centrifuged at 4°C and 8000 x g for 10 min to obtain the corresponding wet bacterial cells.

[0056] The yield of each mutant catalyzing the synthesis of ampicillin and amoxicillin was determined using the catalytic system in Example 1. Penicillin G acylase AxDetection of the specific enzyme activity of the PGA mutant: Add 40% glycerol, add 200 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester as substrates. The mutant enzyme catalyst was catalyzed by pure enzyme with a final concentration of 1 mg / mL, and the conversion system was formed with 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 completed, the reaction solution was subjected to liquid phase detection after the reaction was terminated. The amount of product ampicillin generated was detected to calculate the specific enzyme activity of the mutant. Add 40% glycerol, add 200 mM 6-aminopenicillanic acid and 200 mM D-hydroxyphenylglycine methyl ester as substrates. The mutant enzyme catalyst was catalyzed by pure enzyme at a final concentration of 1 mg / mL, and the conversion system was composed of 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 liquid phase of the reaction solution was detected, and the amount of product amoxicillin produced was detected to calculate the specific enzyme activity of the mutant. The results are shown in Table 3.

[0057] Table 3: .

[0058] The results showed that when the mutant catalyzed the synthesis of ampicillin, Ax PGA-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 yield of 78.51% for the catalytic synthesis of ampicillin. Ax PGA-M103C / Y424F / Y442F / Y451A achieved the highest specific enzyme activity and catalytic performance, with a specific enzyme activity of 504.97 U / mg and an amoxicillin yield of 77.81%.

[0059] Example 4

[0060] This embodiment is carried out Ax The best mutant of PGA Ax The optimal pH experiment of PGA-M103C / Y424F / Y442F / Y451A for catalytic synthesis of β-lactam antibiotics (ampicillin and amoxicillin).

[0061] Penicillin G acylase prepared in Example 3 E. coli BL21(DE3) / pET28a(+)- AxThe cells of the PGA-M103C / Y424F / Y442F / Y451A mutant were used as catalysts, and the calculation method of the catalytic performance of the mutants at different pH was the same as in Example 1. Four buffers of different pH were prepared: the preparation method of 50 mM phosphate buffer was as follows: 50 mM (0.087 g) of potassium dihydrogen phosphate was weighed and dissolved in 8 mL dd H2O, and 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, and 40% glycerol was added and the volume was fixed to 10 mL with dd H2O. Screening of the catalytic ability of the mutant cells under the above pH conditions: The catalytic ability of the obtained cells was tested, and the substrates 200 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine 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 h. After the reaction was completed, the reaction solution was subjected to liquid phase detection after the reaction was terminated. The amount of product ampicillin produced was detected to calculate the yield of the product. 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 h. After the reaction was completed, the reaction solution was subjected to liquid phase detection after the reaction was terminated. The amount of product amoxicillin produced was detected to calculate the yield of the product to compare the results under different pH conditions. Ax The best mutant of PGA Ax Catalytic performance of PGA-M103C / Y424F / Y442F / Y451A.

[0062] Under different pH conditions, Ax The yields of ampicillin and amoxicillin synthesized by PGA-M103C / Y424F / Y442F / Y451A are as follows: Figure 3 The results showed that in 50 mM phosphate buffer at pH 5.9, Ax The yield of ampicillin and amoxicillin catalyzed by PGA-M103C / Y424F / Y442F / Y451A was the highest.

[0063] Example 5

[0064] This example performs mutant Ax Reaction temperature experiment of the synthesis of ampicillin and amoxicillin catalyzed by PGA-M103C / Y424F / Y442F / Y451A.

[0065] The penicillin G acylase mutant was obtained by the same method as in Example 2. AxPGA-M103C / Y424F / Y442F / Y451A engineered bacteria. The yield of ampicillin and amoxicillin synthesized by the mutant at different reaction temperatures was investigated. Six temperature gradients (15℃, 20℃, 25℃, 30℃, 35℃, 40℃) were mainly considered. 50 mM phosphate buffer pH 5.9 was used as the buffer system, and 40% glycerol was added. 200 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester were added as substrates. The amount of catalyst was 4 g DCW / L (DCW cell dry weight) based on the total dry weight of the mixed bacteria. The reaction was carried out under different reaction temperature gradients and 800 rpm. After the reaction was completed, the reaction solution was subjected to liquid phase detection after the reaction was terminated. The amount of product ampicillin produced was detected to calculate the yield of the product. 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 different temperature gradients and 800 rpm for 3 h. After the reaction was terminated, the reaction solution was subjected to liquid phase detection. The amount of 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.

[0066] The influence of temperature Figure 4 As shown: When the reaction temperature is 20℃, the mutant Ax The yield of ampicillin and amoxicillin catalyzed by PGA-M103C / Y424F / Y442F / Y451A was the highest.

[0067] Example 6

[0068] This example performs mutant Ax The reaction substrate ratio of PGA-M103C / Y424F / Y442F / Y451A catalyzed synthesis of ampicillin and amoxicillin was optimized.

[0069] The penicillin G acylase mutant was obtained by the same method as in Example 2. AxPGA-M103C / Y424F / Y442F / Y451A engineered bacteria. The yield of ampicillin and amoxicillin synthesized by the mutant at different reaction temperatures was investigated. This patent considers 5 different substrate gradients of 6-aminopenicillanic acid to D-hydroxyphenylglycine methyl ester / D-hydroxyphenylglycine methyl ester molar ratio (1.0:1, 1.05:1, 1.1:1, 1.15:1, 1.2:1). 50 mM phosphate buffer pH 5.9 was used as the buffer system, and 40% glycerol was added. Substrates of 200 mM; 210 mM; 220 mM; 230 mM; 240 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester were added. The amount of catalyst 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 20℃ and 800 rpm. After the reaction was completed, the reaction solution was subjected to liquid phase detection after the reaction was terminated. The amount of product ampicillin produced was detected to calculate the yield of the product. 200 mM; 210 mM; 220 mM; 230 mM; 240 mM of 6-aminopenicillanic acid and 200 mM of D-hydroxyphenylglycine methyl ester were added. The amount of catalyst 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 different temperature gradients and 800 rpm for 3 h. After the reaction was completed, the reaction solution was subjected to liquid phase detection after the reaction was terminated. The amount of 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 mutant strains under different temperature conditions.

[0070] The influence of substrate ratio Figure 5 As shown: When the molar ratio of D-hydroxyphenylglycine methyl ester / D-hydroxyphenylglycine methyl ester to 6-aminopenicillanic acid is 1.2:1, the mutant Ax PGA-M103C / Y424F / Y442F / Y451A catalyzed the synthesis of ampicillin and amoxicillin to achieve the highest yield.

[0071] Example 7

[0072] This example performs mutant Ax Optimization of bacterial dosage for the synthesis of ampicillin and amoxicillin catalyzed by PGA-M103C / Y424F / Y442F / Y451A.

[0073] The penicillin G acylase mutant was obtained by the same method as in Example 2. AxPGA-M103C / Y424F / Y442F / Y451A engineered bacteria. The yield of ampicillin and amoxicillin synthesized by the mutant under different bacterial dosage conditions was investigated. Five different bacterial dosage gradients (2 g DCW / L, 4 g DCW / L, 6 g DCW / L, 8 g DCW / L, 10 g DCW / L) were mainly considered, 50 mM phosphate buffer pH 5.9 was used as the buffer system, and 40% glycerol was added. The substrates were added with 240 mM 6-aminopenicillanic acid and 200 mM D-phenylglycine methyl ester. 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. After the reaction was completed, the reaction solution was subjected to liquid phase detection after the reaction was terminated. The amount of product ampicillin generated was detected to calculate the yield of the product. 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 h. 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 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.

[0074] result Figure 6 As shown: When the cell dosage is 8 g DCW / L, the mutant Ax The yield of ampicillin and amoxicillin catalyzed by PGA-M103C / Y424F / Y442F / Y451A was the highest.

[0075] Example 8

[0076] This example performs mutant Ax Reaction process of the synthesis of ampicillin and amoxicillin catalyzed by PGA-M103C / Y424F / Y442F / Y451A.

[0077] The penicillin G acylase mutant was obtained by the same method as in Example 2. Ax PGA-M103C / Y424F / Y442F / Y451A engineered bacteria. The reaction process of synthesizing ampicillin and amoxicillin by adding substrate was investigated.

[0078] 50 mM phosphate buffer pH 5.9 was used as the buffer system, and 40% glycerol was added. 240 mM of 6-aminopenicillanic acid and 200 mM of D-phenylglycine methyl ester were added as substrates. The amount of catalyst was 8 g DCW / L based on the total dry weight of the mixed bacteria. The reaction was carried out at 20°C and 800 rpm. After every 1 hour of reaction, 120 mM of 6-aminopenicillanic acid and 100 mM of D-phenylglycine methyl ester were added as substrates. The amount of ampicillin generated in the reaction solution was monitored at any time during the reaction. The results are shown in FIG. Figure 7 As shown, after 6 h of reaction, ampicillin could reach 588.90 mM, with a conversion rate of 99% and a yield of 98.15%.

[0079] 50 mM phosphate buffer pH 5.9 was used as the buffer system, and 40% glycerol was added. 240 mM of 6-aminopenicillanic acid and 200 mM of D-hydroxyphenylglycine methyl ester were added as substrates. The amount of catalyst was 8 gDCW / L based on the total dry weight of the mixed bacteria. The reaction was carried out at 20°C and 800 rpm. After every 1 hour of reaction, 120 mM of 6-aminopenicillanic acid and 100 mM of 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, after 6 h of reaction, amoxicillin could reach 587.52 mM, with a conversion rate of 99% and a yield of 97.92%.

Claims

1. A penicillin G acylase Ax A PGA mutant, characterized in that Obtained by mutation of any one or more of the following sites of the amino acid sequence shown in SEQ ID NO: 1: (1) The methionine at position 103 was substituted with cysteine, (2) Tyrosine at position 424 was replaced by phenylalanine, (3) Tyrosine at position 442 was replaced by phenylalanine, (4) The tyrosine at position 451 was substituted with alanine.

2. A penicillin G acylase Ax A PGA mutant, characterized in that The penicillin G acylase is derived from the penicillin G acylase of Achromobacter xylosus. The amino acid sequence of the penicillin G acylase is recorded as SEQ ID NO:

2. The following four sites of SEQ ID NO: 2 are mutated simultaneously to obtain the penicillin G acylase. Ax PGA mutants: (1) The methionine at position 103 was substituted with cysteine, (2) Tyrosine at position 424 was replaced by phenylalanine, (3) Tyrosine at position 442 was replaced by phenylalanine, (4) The tyrosine at position 451 was substituted with alanine.

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

4. A penicillin G acylase according to claim 1 or 2 Ax The genetically engineered bacteria of the PGA mutant are characterized by: The host bacteria of the genetically engineered bacteria is E. coli BL21.

5. A penicillin G acylase according to claim 1 or 2 Ax The application of the PGA mutant is characterized by: Penicillin G acylase Ax PGA mutants are used to catalyze the synthesis of β-lactam antibiotics.

6. A penicillin G acylase according to claim 5 Ax The application of the PGA mutant is characterized by: Penicillin G acylase Ax A PGA 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, D-phenylglycine methyl ester or D-phenylglycine methyl ester is used as a second reaction substrate, and ampicillin or amoxicillin is prepared by reaction 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.

7. A penicillin G acylase according to claim 6. Ax The application of the PGA mutant is characterized by: Glycerol is added into the transformation system.

8. A penicillin G acylase according to claim 6. Ax The application of the PGA mutant is characterized by: The catalyst is added in an amount of 2 to 10 g DCW / L in the conversion system.

9. A penicillin G acylase according to claim 6 Ax The application of the PGA mutant is 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.

10. A penicillin G acylase according to claim 9. Ax The application of the PGA mutant is characterized by: Penicillin G acylase Ax The genetically engineered bacteria of the PGA mutant were inoculated into LB liquid culture medium with a final concentration of 50 μg / mL kanamycin, cultured at 37°C for 10 h, inoculated into LB liquid culture medium with a final concentration of 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, cultured at 37°C and 180 rpm for 2 h, and then 0.1 mM isopropyl thiogalactoside was added to the culture medium. After culture at 28°C for 12 h, the culture medium was centrifuged at 4°C and 9000 xg for 10 min to obtain the wet cells of the genetically engineered bacteria containing the penicillin G acylase AxPGA mutant.

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