WO2017143944A1 - 一种青霉素g酰化酶突变体 - Google Patents

一种青霉素g酰化酶突变体 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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French (fr)
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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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    • 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

通过基因工程构建了一种青霉素G酰化酶突变体，与来源于无色杆菌(Achromobacter sp.CCM 4824)的野生型青霉素G酰化酶相比，该青霉素G酰化酶突变体的合成性能得到大幅度提高，合成产物/水解产物值S/H最高达到22.3，是野生型酶的3.9倍，可高效地催化合成多种β-内酰胺类抗生素。在侧链与母核比为1.05∶1时，母核6-APA、7-ADCA、7-ACCA和7-APRA的转化率达到99.0％以上。

Description

一种青霉素G酰化酶突变体 技术领域
本发明属于基因工程领域，具体地说，涉及一种通过基因定点突变方法获得的合成性能提高的青霉素G酰化酶突变体、及其在生产β-内酰胺类抗生素中的用途。
背景技术
青霉素G酰化酶(Penicillin G Acylase，E.C.3.5.1.11，简称PGA)是制备半合成β-内酰胺类抗生素中的重要用酶，该酶主要用于水解青霉素G和头孢菌素G，生成相应的化合物母核比如6-氨基青霉烷酸(6-AminoPenicillinic acid，6-APA)和7-氨基-3-去乙酰氧基头孢烷酸(7-Amino-deacetoxy-cephalosporanic-acid，7-ADCA)(Abian et al.，Biotechnol Prog，2003，19(6)，1639-42，2003)；还可用于催化母核6-APA、7-ADCA或其他母核与各种D-氨基酸侧链反应生成新的半合成β-内酰胺抗生素(半合成青霉素和头孢霉素)(Bruggink et al.1998；Yang and Wei 2003；Youshko et al.2004；Gabor et al.2005)。
酶法合成β-内酰胺抗生素的研究开始于上世纪60年代。相对于化学法(Wegman et al.，Advanced Synthesis&Catalysis，343(6-7)，559-576，2001)，由于其具有不使用有机溶剂、反应条件温和、以及环保等优点，酶法合成β-内酰胺抗生素逐渐成为β-内酰胺抗生素工业研究中的一个热点(Giordano，Ribeiro，and Giordano，Biotechnology Advances，24(1)，27-41，2006)。理论上，β-内酰胺类抗生素的酶法合成可通过下述两种途径实现(Kasche.Enzyme and Microbial Technology，8(1)，4-16，1986)，即1)热力学控制，即逆转水解反应；和2)动力学控制，即酰基转移。动力学控制的催化机制涉及酶和酰基供体反应，形成酰基酶中间体，当中间体遇到β-内酰胺母核即可发生耦合，形成半合成β-内酰胺抗生素；而水作为竞争性亲核性试剂，引起反应物和产物的水解，当产物合成速率与产物水解速率相当时，该合成产物量达到最大值。
目前，通过动力学控制途径，利用青霉素酰化酶催化供体酰基转移到β-内酰胺类抗生素母核来生产半合成β-内酰胺类抗生素的酶法工艺已日益成为化学合成法的有效替代(Bruggink et al.1998；Wegman et al.2001)。
与传统的化学合成法相比，β-内酰胺抗生素的酶法合成面临的主要问题是：在合成抗生素的同时又发生两个副反应，即1)水解活化的酰基供体和2)水解生成的抗生素，从而导致酰基供体和生成的抗生素的减少，进而造成了母核转化率偏低，即合成产物/水解产物(S/H)的比值偏低，而且酶自身的特性对S/H值的影响依然是最重要的(Alkema et al.，Eur.J.Biochem，270(18)，3675-83，2003)。利用蛋白质工程来改善青霉素G酰化酶的合成性能已有报道(Alkema et al，Protein Eng.，13(12)，857-63，2000)。在大肠杆菌来源的青霉素G酰化酶中，αR 145、αF146和βF24这三种氨基酸位于酰化酶与青霉素G结合的口袋位置，对酶的合成性能具有关键的作用(Alkema et al.，Protein Engineering Design&Selection，17(5)，473-480，2004)。
利用有理设计和定向进化结合的方法，青霉素G酰化酶PAS2把αR160、αF161和βF24这三个氨基酸筛选到在氨苄青霉素和头孢氨苄合成时性能大大提高的工程菌中(Gabor，E.M.and D.B.Janssen，Protein Eng Des Sel，2004，17(7)，571-9)，上述报道都提高了青霉素G酰化酶的合成性能。
发明内容
为了得到合成性能、尤其是S/H值进一步提高的青霉素G酰化酶，本发明利用基因工程技术来对微生物来源的野生型青霉素G酰化酶进行改造，构建高合成性能的青霉素G酰化酶突变体，从而进行β-内酰胺类抗生素的工业化生产。
为此，本发明通过定点突变的方法对野生型青霉素G酰化酶进行改造和筛选，获得了高合成性能的突变体。
因此，本发明的第一个目的在于提供一种青霉素G酰化酶。
本发明的第二个目的在于提供编码上述青霉素G酰化酶的基因。
本发明的第三个目的在于提供包含上述基因的质粒。
本发明的第四个目的在于提供转化了上述质粒的微生物。
本发明的第五个目的在于提供上述酶或微生物在生产β-内酰胺类抗生素中的用途。
为了达到上述目的，本发明提供如下技术方案：
一种青霉素G酰化酶，其氨基酸序列为：
SEQ ID NO：3，其为SEQ ID NO：1第44位的D替换为L的突变体，其氨基酸序列为：
Figure PCTCN2017074028-appb-000001
SEQ ID NO：4，其为SEQ ID NO：1第130位的R替换为M的突变体，其氨基酸序列为：
Figure PCTCN2017074028-appb-000002
Figure PCTCN2017074028-appb-000003
SEQ ID NO：5，其为SEQ ID NO：1第186位的F替换为A的突变体，其氨基酸序列为：
Figure PCTCN2017074028-appb-000005
SEQ ID NO：6，其为SEQ ID NO：1第232位的A替换为E的突变体，其氨基酸序列为：
Figure PCTCN2017074028-appb-000006
SEQ ID NO：7，其为SEQ ID NO：1第330位的F替换为A的突变体，其氨基酸序列为：
Figure PCTCN2017074028-appb-000007
Figure PCTCN2017074028-appb-000008
SEQ ID NO：8，其为SEQ ID NO：1第415位的K替换为E的突变体，其氨基酸序列为：
Figure PCTCN2017074028-appb-000009
SEQ ID NO：9，其为SEQ ID NO：1第798位的F替换为L的突变体，其氨基酸序列为：
Figure PCTCN2017074028-appb-000010
Figure PCTCN2017074028-appb-000011
SEQ ID NO：10，其为SEQ ID NO：1第44位的D替换为L、第130位的R替换为M、第186位的F替换为A、第232位的A替换为E、第330位的F替换为A、第415位的K替换为E、第798位的F替换为L的突变体，其氨基酸序列为：
Figure PCTCN2017074028-appb-000012
优选上述青霉素G酰化酶的氨基酸序列为SEQ ID NO：10。
编码上述青霉素G酰化酶SEQ ID NO：3-10的基因。
优选编码上述青霉素G酰化酶SEQ ID NO：10的基因的碱基序列为：
Figure PCTCN2017074028-appb-000013
Figure PCTCN2017074028-appb-000014
一种包含上述基因比如SEQ ID NO：11的重组表达质粒。
一种转化了上述质粒的微生物。
优选地，上述微生物是大肠杆菌或者酵母菌，更优选上述微生物是大肠杆菌，尤其优选大肠杆菌BL21(DE3)。
本发明的上述青霉素G酰化酶或者上述微生物可用于催化以6-APA、7-ADCA、7-ACCA、7-APRA为母核的各种β-内酰胺类抗生素的合成，所述β-内酰胺类抗生素包括但不仅限于阿莫西林、氨苄西林、头孢氨苄、头孢羟氨苄、头孢克洛、头孢丙烯和头孢拉定等。
本发明的青霉素G酰化酶不仅可以直接以酶的形式用于催化反应，而且可以以表达该青霉素G酰化酶的微生物的形式用于催化反应。
为操作方便、易于控制、以及后处理容易起见，优选使用青霉素G酰化酶作为催化剂来生产β-内酰胺类抗生素。
在上述的β-内酰胺类抗生素生产中，可以以6-氨基青霉烷酸(6-APA)为原料生产阿莫西林或者氨苄西林；可以以7-氨基-3-去乙酰氧基头孢烷酸(7-ADCA)为原料生产头孢氨苄、头孢羟氨苄或者头孢拉定；也可以以7-氨基-3-氯-头孢烯酸(7-ACCA)为原料生产头孢克洛；还可以以7-氨基-3-丙烯基头孢烷酸(7-APRA)为原料生产头孢丙烯。
本发明的青霉素G酰化酶突变体不仅合成活力(或称合成比活)高于野生酶，而且其合成产物/水解产物值S/H也明显高于野生酶，最高可达野生酶S/H值的3.9倍，催化母核6-APA、7-ADCA、7-CCA和7-APRA的转化率达到99.0％以上。因此相对于现有技术而言，能够以更高的催化效率来催化生成半合成抗生素，极具工业化应用前景。
具体实施方式
以下结合具体实施例对本发明做进一步详细说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。
为简要起见，本文中的氨基酸缩写既可以使用英文三字母、也可以采用英文单字母，这是本领域技术人员熟知的，这些缩写列于下表中：
氨基酸中英文对照及缩写
丙氨酸 Alanine A或Ala 脂肪族类
精氨酸 Arginine R或Arg 碱性氨基酸类
天冬酰胺 Asparagine N或Asn 酰胺类
天冬氨酸 Aspartic acid D或Asp 酸性氨基酸类
半胱氨酸 Cysteine C或Cys 含硫类
谷氨酰胺 Glutamine Q或Gln 酰胺类
谷氨酸 Glutamic acid E或Glu 酸性氨基酸类
甘氨酸 Glycine G或Gly 脂肪族类
组氨酸 Histidine H或His 碱性氨基酸类
异亮氨酸 Isoleucine I或Ile 脂肪族类
亮氨酸 Leucine L或Leu 脂肪族类
赖氨酸 Lysine K或Lys 碱性氨基酸类
甲硫氨酸 Methionine M或Met 含硫类
苯丙氨酸 Phenylalanine F或Phe 芳香族类
脯氨酸 Proline P或Pro 亚氨基酸
丝氨酸 Serine S或Ser 羟基类
苏氨酸 Threonine T或Thr 羟基类
色氨酸 Tryptophan W或Trp 芳香族类
酪氨酸 Tyrosine Y或Tyr 芳香族类
缬氨酸 Valine V或Val 脂肪族类
在本发明中，术语“青霉素G酰化酶突变体”、“突变体青霉素G酰化酶”、“突变青霉素G酰化酶”和“突变酶”表示相同的意义，都是指青霉素G酰化酶的突变体。
在本发明中，术语“野生(型)”、“野生酶”、“野生型酶”表示相同的意义，都是指野生型的青霉素G酰化酶AspPGA。
在本文中，术语“合成性能”是指青霉素G酰化酶催化合成产物的能力(表示为合成活力、或者合成比活)和合成产物/水解产物(S/H)比值的综合性能，尤其是指S/H值。为简要起见，本文中有时将“合成产物/水解产物”表示为“合成/水解”或“S/H”。
作为构建青霉素G酰化酶突变体的基础模板，来源于无色杆菌(Achromobacter sp.CCM 4824)的野生型青霉素G酰化酶(AspPGA)的氨基酸序列是序列表中的SEQ ID NO：1。其编码基因是序列表中的SEQ ID NO：2。
为了得到适用于工业化生产半合成β-内酰胺类抗生素应用的合成性能尤其是S/H值更高的青霉素G酰化酶突变体，有必要对野生型青霉素G酰化酶(AspPGA)进行改造。
以前的文献研究发现，青霉素G酰化酶的有些氨基酸对底物结合和催化活性有重 要影响，并通过定点突变提高其合成性能(WO2010/072765A2，LAAN，Van der，Jan Metske，et al.；Alkema et al.，Protein Engineering Design&Selection，17(5)，473-480，2004)，本发明人根据氨基酸序列对比，确定Achromobacter sp.CCM 4824来源的青霉素G酰化酶(AspPGA)中相对应的氨基酸残基，最后选择到合适的突变位点。
根据无色杆菌来源的野生型青霉素G酰化酶基因序列SEQ ID NO：2，设计出了一系列合成突变引物；再以包含该基因的重组质粒为模板质粒，以上述的合成突变引物为引物，利用TaKaRa公司的TaKaRa MuTanBEST Kit对AspPGA进行定点突变，从而获得一系列合成性能提高了的青霉素G酰化酶的突变质粒，同时对这些突变质粒的DNA序列进行测定，经验证，确定该突变质粒的DNA序列为设计预期的选择性突变的青霉素G酰化酶的DNA序列。
发明人再将这些突变后的质粒转化大肠杆菌BL21(DE3)感受态细胞、或者酵母感受态细胞，获得合成性能提高了的青霉素G酰化酶的工程菌。挑选该工程菌的单菌落进行培养，放大发酵，纯化，从而获得了一系列青霉素G酰化酶突变体。
对上述获得的突变酶的性能进行测定，包括水解酶活力、合成活力、S/H值和以6-APA、7-ADCA、7-ACCA和7-APRA为母核合成各种β-内酰胺抗生素的能力。经过筛选，最终得到了数个较为理想的青霉素G酰化酶突变体，其合成性能包括合成活力和S/H值都高于野生酶。其中一种青霉素G酰化酶突变体SEQ ID NO：10在合成各种抗生素中催化母核6-APA、7-ADCA、7-CCA和7-APRA的转化率达到99.0％以上。
野生型青霉素G酰化酶(AspPGA)包括α亚基和β亚基，其中α亚基是从第41个谷氨酰胺到第259位的甘氨酸；β亚基是从第307位点的丝氨酸到最后的第863位精氨酸。与野生型青霉素G酰化酶(AspPGA)相比，本发明的青霉素G酰化酶突变体AspPGAm的氨基酸突变点包括：在位点α4(即SEQ ID NO：1中第44位)上的天冬氨酸被亮氨酸(Dα4L)替代，在位点α90(即SEQ ID NO：1中第130位)上的精氨酸被甲硫氨酸替代(Rα90M)，位点α146(即SEQ ID NO：1中第186位)上的苯丙氨酸被丙氨酸替代(Fα146A)，位点α192(即SEQ ID NO：1中第232位)上的丙氨酸被谷氨酸替代(Aα192E)，在位点β24(即SEQ ID NO：1中第330位)上的苯丙氨酸被丙氨酸替代(Fβ24A)，在位点β109(即SEQ ID NO：1中第415位)上的赖氨酸被谷氨酸替代(Kβ109E)，在位点β488(即SEQ ID NO：1中第798位)上的苯丙氨酸被亮氨酸替代(Fβ488L)。
本发明的青霉素G酰化酶突变体SEQ ID NO：3-10都是在野生型青霉素G酰化酶 SEQ ID NO：1的基础上进行1个、或7个氨基酸的替换而获得的突变体，并且这些位点具有高度的重合性。由于这些突变体包含863个氨基酸，替换的氨基酸数量极少，因此这些突变体保持了99.2％以上的同源性。
本发明的青霉素G酰化酶突变体的氨基酸数量皆为863个，且结构明确，因此本领域技术人员很容易获得其编码基因、包含这些基因的表达盒和质粒、以及包含该质粒的转化体。
这些基因、表达盒、质粒、转化体可以通过本领域技术人员所熟知的基因工程构建方式获得。
上述转化体宿主可以使任何适合表达青霉素G酰化酶的微生物，包括细菌和真菌。优选微生物是大肠杆菌和酵母菌，尤其优选大肠杆菌。
当作为生物催化剂用于生产时，本发明的青霉素G酰化酶可以呈现酶的形式或者菌体的形式。所述酶的形式包括游离酶、固定化酶，包括纯化酶、粗酶、发酵液、载体固定的酶等；所述菌体的形式包括存活菌体和死亡菌体。
本发明的酶分离纯化、包括固定化酶制备技术也是本领域技术人员所熟知的。
实施例
试剂
限制性内切酶，Pyrobest DNA聚合酶，T4连接酶，TaKaRa基因定点突变试剂盒，5-溴-4-氯-3-吲哚-β-D-半乳糖苷(X-gal)购自TaKaRa公司；PCR纯化试剂盒和DNA胶回收试剂盒购自华舜生物制品有限公司；6-APA和青霉素G钾盐由国药威奇达药业有限公司赠送；7-ADCA、D-苯甘氨酸甲酯盐酸盐(DPGM.HCL)由华北制药集团赠送；D-对羟基苯甘氨酸甲酯(DHPGM)、D型双氢苯甘氨酸甲酯(D-DHPGM)，7-ACCA和7-APRA由山西新宝源制药有限公司赠送；双氢苯甘氨酸甲酯盐酸盐，D-对羟基苯甘氨酸甲酯(DHPGM)、7-ADCA标准品、6-APA标准品、头孢氨苄(CEX)标准品、DPGM.HCL、DHPGM、阿莫西林(Amoxicillin，AMXL)、头孢克洛标准品(Cefaclor)、头孢丙烯标准品(Cefprozi)、头孢拉定标准品(Cefradine)标准品均购自Sigma-Aldrich(St.Louis，USA)；琼脂购自西巴斯生物技术有限公司；其它常规试剂均为国产或进口分装。
测定方法
1)酶水解活力的测定方法：
1.1)发酵液水解活力
精密量取5mL发酵液，离心去上清，加入同体积0.02mol/L、pH7.8磷酸缓冲液重悬，超声或者压榨破壁，破壁率大于90％，精密量取1mL，加入预热至28℃的40mL浓度为8wt％的青霉素G钾盐溶液中，保持温度28℃，快速搅拌，用0.1mol/L NaOH滴定液调pH至8.0，计时，保持pH恒定，反应3-5min，记录加入的NaOH量及反应时间(分钟)。
1.2)纯酶水解活力
精密量取1mL酶液，加入预热至28℃的40mL浓度为8wt％的青霉素G钾盐溶液中，保持温度28℃，快速搅拌，用0.1mol/L NaOH滴定液调pH至8.0，计时，保持pH恒定，反应3-5min，记录加入的NaOH量及反应时间(分钟)。
酶水解活力的计算(U/mL)：
Figure PCTCN2017074028-appb-000015
上式中，各符号及单位表示如下：
V2：滴定完后，滴定仪读数，mL；
V1：滴定前，滴定仪读数，mL；
Min：反应用时，分钟；
S：反应用时，秒；
V：酶液体积，mL；
C：NaOH滴定液的摩尔浓度为0.1mol/L。
酶水解活力单位(U)定义为：28℃，pH8.0条件下，每分钟水解1μmol的青霉素G钾盐所需的青霉素G酰化酶(PGA)量为1U。
2)酶合成活力测定方法：
取50mL含有50mM的7-APA和60mM的DHPGM的底物反应液(即pH7.0的0.05M的磷酸钾盐缓冲溶液)，用盐酸将该底物反应液的pH值调至7.0±0.02，然后再加入1.0mg纯化酶，于28℃反应；分别在开始反应后的第10、15和20分钟取样，每次取样量为30μL，并将样品用50mM的磷酸二氢钾溶液稀释100倍，用HPLC测定产物生成量。
酶合成活力单位(SU)定义为：28℃，pH7.0条件下，每分钟生成1μmol阿莫 西林所需的青霉素G酰化酶量为1SU。
3)合成/水解(S/H)值测定方法：
在100mL烧杯中加入纯化水50mL，加入0.5g的DHPGM和0.5g的6-APA，均匀搅拌，控制反应温度25℃；以加入100SU纯酶液体酶为零点计时，分别于2min、4min、6min、8min、10min、12min取样；通过高效液相HPLC检测各样品中DHPG和AMXL的微摩尔浓度(μmol/mL)；分别做AMXL和D-HPG的一次线性回归曲线，取得各斜率K1和K2；
合成/水解(S/H)＝K1/K2。
S/H值为：反应产物中阿莫西林(AMXL)的摩尔数与副产品对羟基苯甘氨酸(DHPG)的摩尔数的比值。
实施例1突变质粒的构建
1.1、引物设计
根据无色杆菌来源的PGA的基因序列SEQ ID NO：2，以及选定7个突变位点α4、α90、α146、α192、β24、β109、β488，设计下列16个突变引物：
表1、构建不同突变AspPGA酶基因的突变引物
突变位点 突变引物名称 引物序列(5’-3’)
Dα4 Dα4S F1 ACGGCCCCAAACCGCCTCGGGCAAGGTCACGAT
Dα4 Dα4S F2 ATCGTGACCTTGCCCGAGGCGGTTTGGGGCCGT
Dα4 Dα4L F1 ACGGCCCCAAACCGCCCTGGGCAAGGTCACGAT
Dα4 Dα4L F2 ATCGTGACCTTGCCCAGGGCGGTTTGGGGCCGT
Rα90 Rα90M F1 TGCCGGCCGCCGACATGCAGGTGCTGGA
Rα90 Rα90M F2 TCCAGCACCTGCATGTCGGCGGCCGGCA
Fα146 Fα146A F1 ACCATGGCCAACCGCGCTTCGGACGCCAACAGCGA
Fα146 Fα146A F2 TCGCTGTTGGCGTCCGAAGCGCGGTTGGCCATGGT
Aα192 Aα192E F1 CGCCGACCACGGTGCCGGAGGAAGCGGGCAGCTA
Aα192 Aα192E F2 TAGCTGCCCGCTTCCTCCGGCACCGTGGTCGGCG
Fβ24 Fβ24A F1 TGAACGGCCCGCAGGCCGGCTGGTGGAATCCGGCCT
Fβ24 Fβ24A F2 AGGCCGGATTCCACCAGCCGGCCTGCGGGCCGTTCA
Rβ109 Kβ109E F1 ACCTGATCCTGGTGGAAGACGCGGCGCCAGT
Rβ109 Kβ109E F2 ACTGGCGCCGCGTCTTCCACCAGGATCAGGT
Fβ488 Fβ488L F1 AACAACATGACGGTGCTGGACGGTAAATCGGTGCG
Fβ488 Fβ488L F2 CGCACCGATTTACCGTCCAGCACCGTCATGTTGTT
1.2、AspPGA的定点突变
以含有AspPGA野生型基因的重组质粒为模板质粒，参照TaKaRa生物产品及操作手册，利用TaKaRa MuTanBEST突变试剂盒，利用设计的相应突变引物对，PCR扩增出BmPGA的定点突变序列，具体操作步骤如下：
(1)反应体系(10μL)：
Figure PCTCN2017074028-appb-000016
(2)PCR条件：94℃3min，94℃30s，55℃30s，72℃300s，30个循环，72℃10min。
以Dα4S F1、Dα4S F2作为定点突变的上/下游引物，PCR获得AspPGADα3S(α3上的天冬氨酸被丝氨酸替代)；
以Dα4L F1、Dα4L F2作为定点突变的上/下游引物，PCR获得AspPGADα3L(α3上的天冬氨酸被亮氨酸替代)；
以Rα90M F1、Rα90M F2作为定点突变的上/下游引物，PCR获得AspPGARα90M(α90的精氨酸被甲硫氨酸替代)；
以Fα146A F1、Fα146A F2作为定点突变的上/下游引物，PCR获得AspPGAFα146A(α146的苯丙氨酸被丙氨酸替代)；
以Aα192E F1、Aα192E F2作为定点突变的上/下游引物，PCR获得AspPGAAα192E(α192的丙氨酸被谷氨酸替代)；
以Fβ24A F1、Fβ24A F2作为定点突变的上/下游引物，PCR获得AspPGAFβ24A(β24上的苯丙氨酸被丙氨酸替代)；
以Kβ109E F1、Kβ109E F2作为定点突变的上/下游引物，PCR获得AspPGAKβ109E(β109上的赖氨酸被谷氨酸替代)；
以Fβ488L F1、Fβ488L F2作为定点突变的上/下游引物，PCR获得AspPGAFβ488L(β488上的苯丙氨酸被亮氨酸替代)；
参考TAKARA公司操作手册，对PCR获得的DNA片断的5’末端进行磷酸化，然后将磷酸化的突变引物进行自身环化，接着转化DH5α感受态细胞，并涂布于加入卡 纳硫酸霉素抗生素的LB选择性平板上，37℃倒置培养约20h。能在加入卡纳硫酸霉素抗生素的LB平板上长出来的就是转化有重组质粒的转化子。最后对转化子进一步培养以对重组质粒进行扩增，并抽提扩增的重组质粒。
在获得定点突变的AspPGADα4L的基础上，进一步以AspPGADα4L质粒作为定点突变的模板，并分别以Rα90M F1和Rα90M F2、Fα146A F1和Fα146A F2、Aα192E F1和Aα192E F2、Fβ24A F1和Fβ24A F2、Kβ109E F1和Kβ109E F2、Fβ488L F1和Fβ488L F2作为上/下游引物，进行6轮连续重叠PCR，获得组合突变酶基因AspPGAm(Dα4L/Rα90M/Fα146A/Aα192E/Fβ24A/Kβ109E/Fβ488L)，PCR方法同上，并按照上述同样的方法获得扩增的重组质粒。
1.3、突变序列鉴定：
分别对筛选到的突变质粒进行测序，确定所获得的质粒在目的突变位点发生了突变而在非目的突变位点没有发生突变。
虽然本申请为在确定的位点α4，α90，α146，α192，β24，β109，β488进行的定点突变，但是该7个位点的定点突变与其他位点的无义突变或同义突变的组合，获得的产物具有与本申请所要求保护的产物具有同样的功能，这对本领域的人员来说是显而易见的。
实施例2工程菌的获得
参照《分子克隆实验指南》(第三版)，J.萨姆布鲁克，D.W.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002，第1章96页，分别将突变后的9种质粒(AspPGADα4S、AspPGADα4L、AspPGARα90M、AspPGAFα146A、AspPGAAα192E、AspPGAFβ24A、AspPGAKβ109E、AspPGAFβ488L和AspPGAm)转化大肠杆菌感受态细胞或酵母菌感受态细胞，获得表达突变酶的基因工程菌株，最后将工程菌株涂布于加入硫酸卡那霉素抗生素的LB选择性平板上，37℃倒置培养约12-18h，能在加入硫酸卡那霉素抗生素的LB平板上长出来的就是转化有重组质粒的转化子。
实施例3工程菌发酵培养
从LB选择性培养平板上挑单菌落，接种至3mL的LB液体培养基中，加入卡那霉素，至其终浓度为100μg/mL，于37℃下250r/min培养过夜；取2mL培养过夜的培养液，接种至200mL的LB液体培养基中，于37℃下250r/min培养4-6h，OD600达到 1.0-1.6之间，获得种子菌液。按1∶15的接种量接入到含有3升TB培养基的5升发酵罐中，在37℃，400rpm，1.3vvm的发酵条件下，发酵培养至OD600达到1.0时(约1小时)，调节温度为30℃，用终浓度为1％的乳糖诱导，继续培养2小时后，再加入终浓度为0.5％乳糖诱导，(乳糖最终浓度达到1.5％)，再继续培养约14-16小时，发酵结束。发酵液8000rpm离心10min，收集菌体，-20℃保存。待发酵至酶水解活力达到最大，即大约在第一次诱导后16-18小时后，即可停止发酵。
细菌生长量测定：取1mL发酵液，根据菌浓度不同，用双蒸水稀释相应不同倍数(1-100倍)，以使测定的OD600的数值在0.2到0.6之间。发酵结束时，OD600数值在15到25之间，可认为发酵正常。
酶水解活力测定：参见上文提到的“酶水解活力的测定方法1.1)发酵液水解活力”，其中，用发酵液替换“酶液”。
实施例4粗酶液提取
取冻融的菌体，按照1∶2(g∶mL)比例用裂解缓冲液(pH 8.0，100mM磷酸钠，5％甘油)重悬菌体，超声波脉冲破壁(每个循环为工作15秒，间歇30秒，功率5W)，工作30个循环；也可利用French Pressure细胞压榨机破碎细胞，细胞破碎液10000rpm离心1h，回收上清液，获得粗酶液，-20℃保存。在4℃条件下进行亲和纯化操作。
实施例5突变酶的纯化
量取5mL预处理的亲和载体FP-IDA-Ni2+，装入纯化柱(Φ10×200)中。样品按照1.0BV/h速度过柱。用3-5BV的Washing缓冲液以1.0BV/h流速去除杂蛋白，同时利用蛋白核酸检测仪在280nm条件下检测流出液的蛋白含量，直到Washing缓冲液澄清以及蛋白核酸检测仪的数值不再变化为止，最后在蛋白核酸检测仪280nm的监控下，用约1-2BV的Elution缓冲液以1-2BV/h的速度洗脱目的酶AspPGAs，酶液4℃保存。
实施例6纯化后酶的测定
参照前述方法，测定酶水解活力、合成活力和S/H值。
6.1、酶水解活力测定
参见上文提到的“酶水解活力的测定方法1.2)纯酶水解活力”，测定各酶的水解活力，结果见表2。如表2所示，与野生型酶AspPGA相比，各个突变酶的水解活力都有不同程度下降，其中AspPGAm的比活下降最大，AspPGADα3S水解活力下降最小。
6.2、酶合成活力测定
酶合成活力的测定的方法，见上文提到的“酶合成活力测定方法”。通过测定各种酶的纯酶催化阿莫西林合成，计算出了它们的合成活力，结果见表2。
与野生酶相比，八种突变青霉素G酰化酶的合成活力(或称合成比活)除了AspPGADα3S降低外，其他突变酶均有不同程度提高，而且突变酶AspPGAKβ109E的合成活力最大，为74.03SU/mg，是野生酶的近2.5倍。
6.3、S/H值测定
S/H值测定的方法，见上文提到的“合成/水解(S/H)值测定方法”，测定结果见表2。如表2数据所示，野生型AspPGA的S/H值为5.72；与野生酶相比，八种突变青霉素G酰化酶的S/H值除了AspPGADα3S降低外，其他突变酶均有不同程度提高，而且组合突变酶AspPGAm的S/H值最大，为22.3，是野生酶的3.9倍。
表2：九种AspPGAs的水解活力、合成活力及S/H值
Figure PCTCN2017074028-appb-000017
实施例7野生酶AspPGA催化6-APA生成阿莫西林
100mL纯酶水解反应体系中含有250mM的6-APA和262.5mM的D-HPGM的底物(D-HPGM与6-APA的摩尔浓度比为1.05∶1)，将其pH调至6.5±0.02，向该反应体系中加入1200SU的野生酶AspPGA的纯酶液体，使反应体系中的酶的合成活力 控制为12SU/mL。于20℃，pH6.5条件下，反应90分钟。6-APA转化率最大可达到74.7％。
实施例8突变酶AspPGAm催化6-APA生成阿莫西林
100mL纯酶水解反应体系中含有250mM的6-APA和262.5mM的D-HPGM的底物(D-HPGM与6-APA的摩尔浓度比为1.05∶1)，将其pH调至6.50±0.02，向该反应体系中加入1200SU的突变酶AspPGAm的纯酶液体，使反应体系中的酶的合成活力控制为12SU/mL。于20℃，pH6.5条件下，反应90分钟。6-APA转化率大于99.0％。
实施例9突变酶AspPGAm催化6-APA生成氨苄西林
100mL纯酶水解反应体系中含有250mM的6-APA和262.5mM的D-PGM.HCL的底物(D-PGM与6-APA的摩尔浓度比为1.05∶1)，将其pH调至6.50±0.02，向该反应体系中加入2000SU的突变酶AspPGAm的纯酶液体，使反应体系中的酶的合成活力控制为20SU/mL。于20℃，pH6.50条件下，反应120分钟。6-APA转化率大于99.0％。
实施例10突变酶AspPGAm催化7-ADCA生成头孢氨苄
100mL纯酶水解反应体系中含有250mM的7-ADCA和262.5mM的D-PGM.HCL的底物(D-PGM与6-APA的摩尔浓度比为1.05∶1)，将其pH调至6.50±0.02，向该反应体系中加入2000SU的突变酶AspPGAm的纯酶液体，使反应体系中的酶的合成活力控制为20SU/mL。于20℃，pH6.5条件下，反应120分钟。7-ADCA转化率大于99.0％。。
实施例11突变酶AspPGAm催化7-ADCA生成头孢羟氨苄
100mL纯酶水解反应体系中含有250mM的7-ADCA和262.5mM的D-HPGM的底物(D-HPGM与6-APA的摩尔浓度比为1.05∶1)，将其pH调至6.50±0.02，向该反应体系中加入1200SU的突变酶AspPGAm的纯酶液体，使反应体系中的酶的合成活力控制为12SU/mL。于20℃，pH6.5条件下，反应90分钟。7-ADCA转化率大于99.0％。。
实施例12突变酶AspPGAm催化7-ADCA生成头孢拉定
100mL纯酶水解反应体系中含有250mM的7-ADCA和262.5mM的D-DHPGM 的底物(D-DHPGM与7-ADCA的摩尔浓度比为1.05∶1)，将其pH调至6.50±0.02，向该反应体系中加入2200SU的突变酶AspPGAm的液体纯酶，使反应体系中的酶的合成活力控制为22SU/mL。于20℃，pH6.5条件下，反应120分钟，整个反应过程中，反应体系通氮气保护。7-ADCA转化率大于99.0％。。
实施例13突变酶AspPGAm催化7-ACCA生成头孢克洛
100mL纯酶水解反应体系中含有250mM的7-ACCA和262.5mM的D-PGM.HCL的底物(D-PGM与6-APA的摩尔浓度比为1.05∶1)，将其pH调至6.50±0.02，向该反应体系中加入1500SU的突变酶AspPGAm的液体纯酶，使反应体系中的酶的合成活力控制为15SU/mL。于20℃，pH6.5条件下，反应120分钟。7-ACCA转化率大于99.0％。
实施例14突变酶AspPGAm催化7-APRA生成头孢丙烯
100mL纯酶水解反应体系中含有250mM的7-APRA和262.5mM的D-HPGM的底物(D-HPGM与6-APA的摩尔浓度比为1.05∶1)，将其pH调至6.50±0.02，向该反应体系中加入1500SU的突变酶AspPGAm的纯酶液体，使反应体系中的酶的合成活力控制为15SU/mL。于20℃，pH6.5条件下，反应90分钟，整个反应过程中，反应体系通氮气保护。7-ACCA转化率大于99.0％。。
综上所述，相比野生型青霉素G酰化酶，本发明所构建的突变酶的合成性能、合成产物/水解产物值(S/H值)均得到了提高，可高效催化多种β-内酰胺母核包括6-APA、7-ADCA、7-ACCA和7-APRA生成阿莫西林、氨苄西林、头孢氨苄、头孢羟氨苄、头孢拉定、头孢克洛和头孢丙烯等β-内酰胺抗生素，具有广阔的工业应用前景。

Claims (10)

  1. 一种青霉素G酰化酶，其氨基酸序列为SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9或者SEQ ID NO：10。
  2. 如权利要求1所述青霉素G酰化酶，其特征在于，其氨基酸序列为SEQ ID NO：10。
  3. 编码如权利要求1所述青霉素G酰化酶的基因。
  4. 编码如权利要求2所述青霉素G酰化酶的基因，其序列为SEQ ID NO：11。
  5. 包含如权利要求3或4所述基因的质粒。
  6. 转化了如权利要求5所述质粒的微生物。
  7. 如权利要求6所述的微生物，其特征在于，所述微生物是大肠杆菌或者酵母菌。
  8. 如权利要求1所述青霉素G酰化酶或者如权利要求6所述微生物在生产β-内酰胺类抗生素中的用途。
  9. 如权利要求1所述青霉素G酰化酶在生产β-内酰胺类抗生素中的用途。
  10. 如权利要求8或9所述的用途，其特征在于，以6-氨基青霉烷酸(6-APA)为原料生产阿莫西林或者氨苄西林；以7-氨基-3-去乙酰氧基头孢烷酸(7-ADCA)为原料生产头孢氨苄、头孢羟氨苄或者头孢拉定；以7-氨基-3-氯-头孢烯酸(7-ACCA)为原料生产头孢克洛；或者以7-氨基-3-丙烯基头孢烷酸(7-APRA)为原料生产头孢丙烯。
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WO2021096298A1 (ko) * 2019-11-15 2021-05-20 아미코젠주식회사 세파졸린 생산성이 증가된 페니실린 g 아실라제 변이체 및 이의 이용
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WO2021140526A1 (en) * 2020-01-08 2021-07-15 Fermenta Biotech Limited Mutant penicillin g acylases of 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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