WO2017143944A1 - Mutant de la pénicilline g acylase - Google Patents

Mutant de la pénicilline g acylase Download PDF

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WO2017143944A1
WO2017143944A1 PCT/CN2017/074028 CN2017074028W WO2017143944A1 WO 2017143944 A1 WO2017143944 A1 WO 2017143944A1 CN 2017074028 W CN2017074028 W CN 2017074028W WO 2017143944 A1 WO2017143944 A1 WO 2017143944A1
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penicillin
acylase
enzyme
seq
mutant
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PCT/CN2017/074028
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Chinese (zh)
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王金刚
梁岩
陈舒明
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上海星维生物技术有限公司
山西新宝源制药有限公司
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Publication of WO2017143944A1 publication Critical patent/WO2017143944A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/84—Penicillin amidase (3.5.1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
    • C12P35/04—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin by acylation of the substituent in the 7 position
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01011—Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/025—Achromobacter

Definitions

  • the present invention belongs to the field of genetic engineering, and in particular to a synthetic performance-enhanced penicillin G acylase mutant obtained by a gene site-directed mutagenesis method, and its use in producing a ⁇ -lactam antibiotic.
  • Penicillin G Acylase (EC3.5.1.11, PGA for short) is an important enzyme for the preparation of semi-synthetic ⁇ -lactam antibiotics. This enzyme is mainly used to hydrolyze penicillin G and cephalosporin G to produce corresponding
  • the parent core of the compound such as 6-AminoPenicillinic acid (6-APA) and 7-Amino-deacetoxy-cephalosporanic-acid (7-ADCA) (Abian et al., Biotechnol Prog, 2003, 19(6), 1639-42, 2003); can also be used to catalyze the side chain reaction of the parental nucleus 6-APA, 7-ADCA or other parent nucleus with various D-amino acids.
  • New semi-synthetic ⁇ -lactam antibiotics (semi-synthetic penicillin and cephalosporin) were generated (Bruggink et al. 1998; Yang and Wei 2003; Youshko et al. 2004; Gabor et al. 2005).
  • thermodynamic control ie reverse hydrolysis Reaction
  • kinetic control ie acyl transfer.
  • the catalytic mechanism of kinetic control involves the reaction of an enzyme with an acyl donor to form an acylase intermediate, which can be coupled when the intermediate encounters the ⁇ -lactam nucleus to form a semi-synthetic ⁇ -lactam antibiotic; water is competitive
  • the nucleophilic reagent causes hydrolysis of the reactants and products, and the amount of the synthesized product reaches a maximum when the product synthesis rate is comparable to the product hydrolysis rate.
  • penicillin G acylase PAS2 screens three amino acids, ⁇ R160, ⁇ F161 and ⁇ F24, into engineered bacteria with greatly improved performance in the synthesis of ampicillin and cephalexin (Gabor, EMand DBJanssen , Protein Eng Des Sel, 2004, 17(7), 571-9), all of which have been reported to enhance the synthetic properties of penicillin G acylase.
  • the present invention utilizes genetic engineering technology to modify microbial-derived wild type penicillin G acylase to construct a highly synthetic penicillin G acylation. Enzyme mutants for industrial production of ⁇ -lactam antibiotics.
  • the present invention modifies and screens the wild-type penicillin G acylase by site-directed mutagenesis, and obtains a mutant having high synthetic properties.
  • a first object of the present invention is to provide a penicillin G acylase.
  • a second object of the present invention is to provide a gene encoding the above penicillin G acylase.
  • a third object of the present invention is to provide a plasmid comprising the above gene.
  • a fourth object of the present invention is to provide a microorganism transformed with the above plasmid.
  • a fifth object of the present invention is to provide the use of the above enzyme or microorganism for producing a ⁇ -lactam antibiotic.
  • the present invention provides the following technical solutions:
  • SEQ ID NO: 3 which is a mutant in which D at position 44 of SEQ ID NO: 1 is replaced by L, the amino acid sequence of which is:
  • SEQ ID NO: 4 which is a mutant in which the R at position 130 of SEQ ID NO: 1 is replaced by M, and the amino acid sequence thereof is:
  • SEQ ID NO: 5 which is a mutant in which the F at position 186 of SEQ ID NO: 1 is replaced by A, and the amino acid sequence thereof is:
  • SEQ ID NO: 6 which is a mutant in which A of position 232 of SEQ ID NO: 1 is replaced by E, and the amino acid sequence thereof is:
  • SEQ ID NO:7 which is a mutant in which the F at position 330 of SEQ ID NO: 1 is replaced by A, and the amino acid sequence thereof is:
  • SEQ ID NO:8 which is a mutant in which K at position 415 of SEQ ID NO: 1 is replaced by E, the amino acid sequence of which is:
  • SEQ ID NO:9 which is a mutant in which the 798th position of SEQ ID NO: 1 is replaced by L, and the amino acid sequence thereof is:
  • SEQ ID NO: 10 which is the D of the 44th position of SEQ ID NO: 1 is replaced by L, the R of the 130th position is replaced by M, the F of the 186th place is replaced by A, and the A of the 232th place is replaced by E, The 330-position F is replaced by A, the 415th K is replaced by E, and the 798th F is replaced by L.
  • the amino acid sequence is:
  • amino acid sequence of the above penicillin G acylase is SEQ ID NO: 10.
  • the base sequence of the gene encoding the above penicillin G acylase SEQ ID NO: 10 is:
  • a recombinant expression plasmid comprising the above gene, such as SEQ ID NO:11.
  • a microorganism transformed with the above plasmid A microorganism transformed with the above plasmid.
  • the microorganism is Escherichia coli or yeast, and more preferably the microorganism is Escherichia coli, and particularly preferably Escherichia coli BL21 (DE3).
  • the above penicillin G acylase of the present invention or the above microorganism can be used for catalyzing the synthesis of various ⁇ -lactam antibiotics having 6-APA, 7-ADCA, 7-ACCA, and 7-APRA as the mother nucleus
  • the ⁇ - Lactam antibiotics include, but are not limited to, amoxicillin, ampicillin, cephalexin, cefadroxil, cefaclor, cefprozil, and cefradine.
  • the penicillin G acylase of the present invention can be used not only for the catalytic reaction in the form of an enzyme but also for the catalytic reaction in the form of a microorganism expressing the penicillin G acylase.
  • a penicillin G acylase as a catalyst to produce a beta-lactam antibiotic.
  • amoxicillin or ampicillin can be produced from 6-aminopenicillanic acid (6-APA); 7-amino-3-desacetoxycephalosporin can be used.
  • Acid (7-ADCA) is used as raw material to produce cephalexin, cefadroxil or cefradine; 7-amino-3-chloro-cephem acid (7-ACCA) can also be used as raw material to produce cefaclor; 7-amino can also be used.
  • 3-propenyl cephalosporanic acid (7-APRA) is used as a raw material to produce cefprozil.
  • the penicillin G acylase mutant of the present invention not only has higher synthesis activity (or synthesis specific activity) than wild enzyme, but also has a synthetic product/hydrolysate value S/H which is significantly higher than wild enzyme, and up to wild enzyme S/ At 3.9 times the H value, the conversion rates of the catalytic cores 6-APA, 7-ADCA, 7-CCA and 7-APRA reached 99.0% or more. Therefore, compared with the prior art, semi-synthetic antibiotics can be catalyzed by higher catalytic efficiency, which has great industrial application prospects.
  • amino acid abbreviations herein may be either English three-letter or English single-letter, which are well known to those skilled in the art and are listed in the following table:
  • penicillin G acylase mutant means the same meaning, and all refer to penicillin G. A mutant of the enzyme.
  • wild (type) wild enzyme
  • wild type enzyme wild type enzyme
  • synthetic performance refers to the combined performance of the ability of penicillin G acylase to catalyze the synthesis of a product (expressed as synthetic activity, or synthesis specific activity) and synthetic product/hydrolysate (S/H) ratio, especially Refers to the S/H value.
  • synthetic product/hydrolysate is sometimes referred to herein as “synthesis/hydrolysis” or "S/H.”
  • the amino acid sequence of wild-type penicillin G acylase (AspPGA) derived from Achromobacter sp. CCM 4824 is SEQ ID NO: 1 in the Sequence Listing. Its coding gene is SEQ ID NO: 2 in the Sequence Listing.
  • a series of synthetic mutation primers were designed based on the wild type penicillin G acylase gene sequence of SEQ ID NO: 2, and the recombinant plasmid containing the gene was used as a template plasmid, and the above synthetic mutant primers were used as primers.
  • the TaKaRa MuTanBEST Kit of TaKaRa was used to carry out site-directed mutagenesis of AspPGA to obtain a series of mutant plasmids with improved synthetic performance of penicillin G acylase, and the DNA sequences of these mutant plasmids were determined, and the mutant plasmid was confirmed.
  • the DNA sequence is designed to visualize the DNA sequence of the selective mutation of penicillin G acylase.
  • the inventors further transformed these mutated plasmids into E. coli BL21 (DE3) competent cells or yeast competent cells to obtain an engineering strain of penicillin G acylase having improved synthesis performance.
  • E. coli BL21 DE3 competent cells or yeast competent cells to obtain an engineering strain of penicillin G acylase having improved synthesis performance.
  • a single colony of the engineered bacteria was selected for cultivation, amplified and purified, thereby obtaining a series of penicillin G acylase mutants.
  • the properties of the mutant enzyme obtained above were determined, including hydrolase activity, synthetic activity, S/H value, and synthesis of various ⁇ -lactams using 6-APA, 7-ADCA, 7-ACCA and 7-APRA as the mother nucleus.
  • the ability of antibiotics After screening, several ideal penicillin G acylase mutants were obtained, and their synthetic properties, including synthetic activity and S/H values, were higher than those of wild enzymes.
  • One of the penicillin G acylase mutants SEQ ID NO: 10 catalyzes the conversion of the mother nucleus 6-APA, 7-ADCA, 7-CCA and 7-APRA to 99.0% or more in the synthesis of various antibiotics.
  • the wild type penicillin G acylase includes an ⁇ subunit and a ⁇ subunit, wherein the ⁇ subunit is glycine from the 41st glutamine to the 259th position; the ⁇ subunit is from the serine at the 307th point to The last 863th arginine.
  • the amino acid mutation point of the penicillin G acylase mutant AspPGAm of the present invention includes: day at position ⁇ 4 (ie, position 44 in SEQ ID NO: 1) compared to wild type penicillin G acylase (AspPGA) Aspartate is replaced by leucine (D ⁇ 4L), and arginine at position ⁇ 90 (ie, position 130 in SEQ ID NO: 1) is replaced by methionine (R ⁇ 90M), site ⁇ 146 (ie SEQ ID)
  • the phenylalanine at position 186 of NO:1 was replaced by alanine (F ⁇ 146A), and the alanine at position ⁇ 192 (ie, position 232 of SEQ ID NO:1) was replaced by glutamic acid (A ⁇ 192E) ), the phenylalanine at position ⁇ 24 (ie, position 330 in SEQ ID NO: 1) is replaced by alanine (F ⁇ 24A) at position ⁇ 109 (ie position 415 in SEQ ID NO: 1)
  • the penicillin G acylase mutants of the present invention SEQ ID NOs: 3-10 are all in the wild type penicillin G acylase A mutant obtained by performing one or seven amino acid substitutions based on SEQ ID NO: 1, and these sites have a high degree of overlap. Since these mutants contain 863 amino acids and the number of amino acids replaced is extremely small, these mutants maintain a homology of more than 99.2%.
  • the penicillin G acylase mutant of the present invention has 863 amino acid numbers and has a well-defined structure, and thus a gene encoding the same, an expression cassette and a plasmid containing the same, and a transformant containing the same are easily obtained by those skilled in the art. .
  • genes, expression cassettes, plasmids, transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.
  • the above transformant host can be any microorganism suitable for expressing penicillin G acylase, including bacteria and fungi.
  • the microorganism is Escherichia coli and yeast, and E. coli is particularly preferred.
  • the penicillin G acylase of the present invention may be in the form of an enzyme or a form of a fungus.
  • the form of the enzyme includes a free enzyme, an immobilized enzyme, and includes a purified enzyme, a crude enzyme, a fermentation broth, a carrier-immobilized enzyme, and the like; the form of the bacterial body includes a living cell and a dead cell.
  • V 1 before titration, the titration readings, mL;
  • the enzymatic hydrolysis activity unit (U) is defined as: the amount of penicillin G acylase (PGA) required to hydrolyze 1 ⁇ mol of penicillin G potassium salt per minute at 28 ° C and pH 8.0 is 1 U.
  • substrate reaction solution containing 50 mM 7-APA and 60 mM DHPGM (ie, 0.05 M potassium phosphate buffer solution at pH 7.0), and adjust the pH of the substrate reaction solution to 7.0 ⁇ 0.02 with hydrochloric acid. Then, 1.0 mg of purified enzyme was further added and reacted at 28 ° C; samples were taken at 10, 15 and 20 minutes after the start of the reaction, respectively, and the sample amount was 30 ⁇ L, and the sample was diluted 100 times with 50 mM potassium dihydrogen phosphate solution. The amount of product produced was determined by HPLC.
  • the enzyme synthesis activity unit is defined as: 1 °mol Amo per minute at 28 ° C, pH 7.0
  • the amount of penicillin G acylase required for Xilin is 1SU.
  • 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 by-product p-hydroxyphenylglycine (DHPG).
  • the following 16 mutant primers were designed based on the gene sequence of SEQ ID NO: 2 of P. aeruginosa-derived PGA, and the selected 7 mutation sites ⁇ 4, ⁇ 90, ⁇ 146, ⁇ 192, ⁇ 24, ⁇ 109, ⁇ 488:
  • the recombinant plasmid containing AspPGA wild-type gene was used as a template plasmid, and the TaKaRa MuTanBEST mutation kit was used to PCR-amplify the site-directed mutant sequence of BmPGA using the TaKaRa MuTanBEST mutation kit.
  • the specific steps are as follows: :
  • the 5' end of the DNA fragment obtained by PCR was phosphorylated by reference to the TAKARA operating manual, and then the phosphorylated mutant primer was subjected to self-cyclization, followed by transformation of DH5 ⁇ competent cells, and application to the addition card.
  • the sulfamycin antibiotic was plated on an LB selective plate at 37 ° C for about 20 h.
  • a transformant that can be transformed with a recombinant plasmid can be grown on an LB plate to which kanamycinmycin antibiotic is added. Finally, the transformant is further cultured to amplify the recombinant plasmid, and the amplified recombinant plasmid is extracted.
  • the AspPGAD ⁇ 4L plasmid was further used as a template for site-directed mutagenesis, and R ⁇ 90M F1 and R ⁇ 90M F2, F ⁇ 146A F1 and F ⁇ 146A F2, A ⁇ 192E F1 and A ⁇ 192E F2, F ⁇ 24A F1 and F ⁇ 24A F2, K ⁇ 109E F1 and K ⁇ 109E F2, F ⁇ 488L F1 and F ⁇ 488L F2 were used as upstream/downstream primers for 6 rounds of continuous overlapping PCR to obtain the combined mutant enzyme gene AspPGAm (D ⁇ 4L/R ⁇ 90M/F ⁇ 146A/A ⁇ 192E/F ⁇ 24A/K ⁇ 109E/F ⁇ 488L), the PCR method was the same as above, and according to the above The same method was used to obtain an amplified recombinant plasmid.
  • the selected mutant plasmids were sequenced, and it was confirmed that the obtained plasmid was mutated at the target mutation site and no mutation occurred at the non-target mutation site.
  • the LB-selective plate to which the kanamycin sulfate antibiotic was added was cultured at 37 ° C for about 12-18 hours, and the transformant transformed with the recombinant plasmid was grown on the LB plate to which the kanamycin sulfate antibiotic was added.
  • Single colonies were picked from LB selective culture plates, inoculated into 3 mL of LB liquid medium, and kanamycin was added to a final concentration of 100 ⁇ g/mL, and cultured at 37 ° C for 250 r/min overnight; 2 mL was cultured overnight.
  • the culture solution was inoculated into 200 mL of LB liquid medium, cultured at 250 ° C for 4-6 h at 37 ° C, and the OD 600 was between 1.0 and 1.6 to obtain a seed broth.
  • Determination of bacterial growth Take 1 mL of fermentation broth, and dilute the corresponding multiples (1-100 times) with double distilled water according to the different bacterial concentrations, so that the measured OD 600 value is between 0.2 and 0.6. At the end of the fermentation, the OD 600 value is between 15 and 25 and the fermentation is considered normal.
  • Enzymatic hydrolysis activity assay Refer to the "Method 1 of the determination of enzymatic hydrolysis activity" mentioned above for the hydrolysis activity of the fermentation broth", wherein the "enzyme solution” is replaced with a fermentation broth.
  • the freeze-thawed cells were resuspended in a 1:2 (g:mL) ratio with a lysis buffer (pH 8.0, 100 mM sodium phosphate, 5% glycerol), and the ultrasonic pulse was broken (15 seconds per cycle) , intermittent 30 seconds, power 5W), work 30 cycles; can also use the French Pressure cell crusher to break the cells, the cell disruption solution was centrifuged at 10000 rpm for 1 h, the supernatant was recovered, and the crude enzyme solution was obtained and stored at -20 °C.
  • the affinity purification operation was carried out at 4 °C.
  • the target enzyme AspPGAs was eluted with an 1-2 BV Elution buffer at a rate of 1-2 BV/h, and the enzyme solution was stored at 4 °C.
  • the enzymatic hydrolysis activity, the synthesis activity, and the S/H value were determined by referring to the aforementioned methods.
  • the synthesis activity (or synthesis specific activity) of the eight mutant penicillin G acylases was increased in addition to AspPGAD ⁇ 3S, and the other mutant enzymes were increased to different extents, and the mutant enzyme AspPGAK ⁇ 109E had the highest synthesis activity, which was 74.03 SU. /mg, which is nearly 2.5 times that of wild enzymes.
  • S/H value measurement method For the method of determining the S/H value, see the "Synthesis/Hydrolysis (S/H) value measurement method" mentioned above, and the measurement results are shown in Table 2. As shown in the data in Table 2, the S/H value of wild-type AspPGA was 5.72. Compared with wild-type enzyme, the S/H values of eight mutant penicillin G acylases were increased in addition to AspPGAD ⁇ 3S, and other mutant enzymes were increased to different extents. And the combined mutant enzyme AspPGAm has the largest S/H value of 22.3, which is 3.9 times that of the wild enzyme.
  • Example 7 Wild-enzyme AspPGA catalyzes the formation of 6-APA amoxicillin
  • the 100 mL pure enzyme hydrolysis reaction system contained 250 mM 6-APA and 262.5 mM D-HPGM substrate (the molar concentration ratio of D-HPGM to 6-APA was 1.05:1), and the pH was adjusted to 6.5 ⁇ 0.02. Adding 1200SU of pure enzyme solution of wild enzyme AspPGA to the reaction system to synthesize the enzyme activity in the reaction system The control is 12SU/mL. The reaction was carried out for 90 minutes at 20 ° C and pH 6.5. The 6-APA conversion rate can reach up to 74.7%.
  • Example 8 Mutant enzyme AspPGAm catalyzes the formation of 6-APA amoxicillin
  • the substrate of 250 mM 6-APA and 262.5 mM D-HPGM (the molar concentration ratio of D-HPGM to 6-APA was 1.05:1) in a 100 mL pure enzymatic hydrolysis reaction system, and the pH was adjusted to 6.50 ⁇ 0.02.
  • a pure enzyme liquid of the 1200SU mutant enzyme AspPGAm was added to control the synthesis activity of the enzyme in the reaction system to 12 SU/mL. The reaction was carried out for 90 minutes at 20 ° C and pH 6.5.
  • the 6-APA conversion rate is greater than 99.0%.
  • Example 9 Mutant enzyme AspPGAm catalyzes the formation of ampicillin by 6-APA
  • the 100 mL pure enzyme hydrolysis reaction system contained 250 mM 6-APA and 262.5 mM D-PGM.HCL substrate (the molar concentration ratio of D-PGM to 6-APA was 1.05:1), and the pH was adjusted to 6.50 ⁇ . 0.02, a pure enzyme liquid of 2000SU mutant enzyme AspPGAm was added to the reaction system to control the synthesis activity of the enzyme in the reaction system to 20 SU/mL. The reaction was carried out for 120 minutes at 20 ° C and pH 6.50. The 6-APA conversion rate is greater than 99.0%.
  • Example 10 Mutant enzyme AspPGAm catalyzes the production of cephalexin by 7-ADCA
  • the 100 mL pure enzyme hydrolysis reaction system contained 250 mM 7-ADCA and 262.5 mM D-PGM.HCL substrate (the molar concentration ratio of D-PGM to 6-APA was 1.05:1), and the pH was adjusted to 6.50 ⁇ . 0.02, a pure enzyme liquid of 2000SU mutant enzyme AspPGAm was added to the reaction system to control the synthesis activity of the enzyme in the reaction system to 20 SU/mL. The reaction was carried out for 120 minutes at 20 ° C and pH 6.5. The 7-ADCA conversion is greater than 99.0%. .
  • Example 11 Mutant enzyme AspPGAm catalyzes the production of cephalosporin by 7-ADCA
  • the 100 mL pure enzyme hydrolysis reaction system contained 250 mM 7-ADCA and 262.5 mM D-HPGM substrate (the molar concentration ratio of D-HPGM to 6-APA was 1.05:1), and the pH was adjusted to 6.50 ⁇ 0.02.
  • a pure enzyme liquid of the 1200SU mutant enzyme AspPGAm was added to control the synthesis activity of the enzyme in the reaction system to 12 SU/mL. The reaction was carried out for 90 minutes at 20 ° C and pH 6.5. The 7-ADCA conversion is greater than 99.0%. .
  • Example 12 Mutant enzyme AspPGAm catalyzes the production of cephalosporin by 7-ADCA
  • 100mL pure enzymatic hydrolysis reaction system contains 250mM 7-ADCA and 262.5mM D-DHPGM
  • the substrate (the molar concentration ratio of D-DHPGM to 7-ADCA is 1.05:1)
  • the pH is adjusted to 6.50 ⁇ 0.02
  • 2200SU of the pure enzyme AspPGAm of the mutant enzyme is added to the reaction system to make the reaction system
  • the synthetic activity of the enzyme was controlled to 22 SU/mL.
  • the reaction was carried out for 120 minutes at 20 ° C and pH 6.5. During the whole reaction, the reaction system was protected with nitrogen.
  • the 7-ADCA conversion is greater than 99.0%. .
  • Example 13 Mutant enzyme AspPGAm catalyzes the production of cefaclor by 7-ACCA
  • the 100 mL pure enzyme hydrolysis reaction system contained 250 mM 7-ACCA and 262.5 mM D-PGM.HCL substrate (the molar concentration ratio of D-PGM to 6-APA was 1.05:1), and the pH was adjusted to 6.50 ⁇ . 0.02, a liquid pure enzyme of the 1500 SU mutant enzyme AspPGAm was added to the reaction system to control the synthesis activity of the enzyme in the reaction system to 15 SU/mL. The reaction was carried out for 120 minutes at 20 ° C and pH 6.5. The 7-ACCA conversion rate is greater than 99.0%.
  • Example 14 Mutant enzyme AspPGAm catalyzes the formation of cefprozil by 7-APRA
  • a pure enzyme liquid of 1500 SU of the mutant enzyme AspPGAm was added to control the synthesis activity of the enzyme in the reaction system to 15 SU/mL.
  • the reaction was carried out for 90 minutes at 20 ° C and pH 6.5. During the entire reaction, the reaction system was purged with nitrogen.
  • the 7-ACCA conversion rate is greater than 99.0%. .
  • the lactam core includes 6-APA, 7-ADCA, 7-ACCA and 7-APRA to form ⁇ -lactam antibiotics such as amoxicillin, ampicillin, cephalexin, cefadroxil, cefradine, cefaclor and cefprozil.
  • ⁇ -lactam antibiotics such as amoxicillin, ampicillin, cephalexin, cefadroxil, cefradine, cefaclor and cefprozil.

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Abstract

La présente invention concerne un mutant de la pénicilline G acylase construit par génie génétique; par comparaison avec une pénicilline G acylase de type sauvage dérivée d'Achromobacter sp. CCM 4824, la performance de synthèse du présent mutant de pénicilline G acylase est considérablement améliorée, la valeur du produit de synthèse/produit d'hydrolyse S/H atteignant un maximum de 22,3, 3,9 fois celle de l'enzyme de type sauvage, étant apte à catalyser efficacement la synthèse de divers antibiotiques de type β-lactame. Lorsque le rapport de la chaîne latérale au noyau mère est de 1,05:1, le taux de conversion de 6-APA, 7-ADCA, 7-ACCA et 7-APRA du noyau-mère est supérieur à 99,0 %.
PCT/CN2017/074028 2016-02-23 2017-02-19 Mutant de la pénicilline g acylase WO2017143944A1 (fr)

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Application Number Priority Date Filing Date Title
CN201610097503.4A CN105483105B (zh) 2016-02-23 2016-02-23 一种青霉素g酰化酶突变体
CN201610097503.4 2016-02-23

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CN109971742A (zh) * 2017-12-28 2019-07-05 艾美科健株式会社 来源于无色杆菌属ccm 4824的青霉素g酰化酶突变体及其应用
CN109971743A (zh) * 2017-12-28 2019-07-05 艾美科健株式会社 来源于无色杆菌属ccm4824的青霉素g酰化酶突变体及其应用
CN109971743B (zh) * 2017-12-28 2022-09-06 艾美科健株式会社 来源于无色杆菌属ccm 4824的青霉素g酰化酶突变体及其应用
WO2021096298A1 (fr) * 2019-11-15 2021-05-20 아미코젠주식회사 Mutant de pénicilline g acylase ayant une productivité accrue de céfazoline et son utilisation
KR20210059533A (ko) * 2019-11-15 2021-05-25 아미코젠주식회사 세파졸린 생산성이 증가된 페니실린 g 아실라제 변이체 및 이의 이용
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CN115397979A (zh) * 2019-11-15 2022-11-25 艾美科健株式会社 具有增加的头孢唑啉生产率的青霉素g酰化酶突变体及其用途
WO2021140526A1 (fr) * 2020-01-08 2021-07-15 Fermenta Biotech Limited Acylases mutantes de pénicilline g d'achromobacter ccm4824
CN113009034A (zh) * 2021-03-04 2021-06-22 广东华南药业集团有限公司 一种头孢拉定的高效液相分析方法
CN116120343A (zh) * 2023-02-06 2023-05-16 艾美科健（中国）生物医药有限公司 一种从酶法合成头孢丙烯原料药废液中提取原料母核7-apra及侧链d-hpg的方法

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