WO2012117999A1 - Mutant hyperactif (s)-sélectif d'arylmalonate décarboxylase - Google Patents

Mutant hyperactif (s)-sélectif d'arylmalonate décarboxylase Download PDF

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WO2012117999A1
WO2012117999A1 PCT/JP2012/054731 JP2012054731W WO2012117999A1 WO 2012117999 A1 WO2012117999 A1 WO 2012117999A1 JP 2012054731 W JP2012054731 W JP 2012054731W WO 2012117999 A1 WO2012117999 A1 WO 2012117999A1
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mutant
acid
cysteine
decarboxylase
activity
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憲二 宮本
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学校法人慶應義塾
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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01076—Arylmalonate decarboxylase (4.1.1.76)

Definitions

  • the present invention relates to a highly active mutant arylmalonate decarboxylase that is selective for S-form, which produces S-form ⁇ -arylpropionic acid from arylmalonate.
  • Arylmalonate decarboxylase (AMDase, EC 4.1.1.76) is an enzyme that catalyzes the decarboxylation of arylmalonic acid to produce R-carboxylic acid derivatives ( ⁇ -arylpropionic acid derivatives: profenes) It is a monomeric enzyme of 240 amino acids (estimated molecular weight 24,734 Da) isolated from Alcaligenes bronchisepticus KU1201 strain (Non-patent Document 1).
  • Wild type arylmalonic acid produces only R-form ⁇ -arylpropionic acid, but S-form ⁇ -arylpropionic acid and its derivatives are non-steroidal anti-inflammatory drugs, liquid crystal materials, insecticides, herbicides And an important group of compounds as intermediates thereof.
  • the triple mutant (S36N / G74C / C188S) obtained by random mutagenesis to the double mutant (G74C / C188S) has about 10 times higher activity than the double mutant. Had. However, it still has an activity of 1% or less as compared with the wild-type enzyme, and is still not practical (Non-Patent Document 4).
  • the present invention is a mutant arylmalonic acid decarboxylase, which produces an S-form ⁇ -arylpropionic acid having an optical purity of almost 100% ee from arylmalonic acid with high activity, and the enzyme. It is an object of the present invention to provide a method for producing S-form ⁇ -arylpropionic acid.
  • the present inventors have already succeeded in reversing enantioselectivity by introducing a mutation of G74C / C188S into wild-type arylmalonate decarboxylase (AMDase), and the S-form of the arylmalonate derivative, which is a substrate, has been successfully reversed. It has succeeded in producing ⁇ -arylpropionic acid. However, the activity is drastically reduced compared to the wild type, and the activity was not sufficient for industrial use.
  • AMDase wild-type arylmalonate decarboxylase
  • Directed evolution here refers to (1) creating a library in which mutations are introduced into the gene of the target protein, (2) selecting mutants having the desired properties from this library, and (3) selecting By repeating the cycle of sequencing the mutant gene and (4) creating the library again from the selected mutant gene, it is possible to repeat the mutation in the target protein as if it were an “evolution” of an organism. It is a protein engineering technique to finally obtain a protein with desirable properties.
  • the present inventors first analyzed the three-dimensional structure of wild-type arylmalonate decarboxylase and S-selective G74C / C188S mutant arylmalonate decarboxylase by X-ray crystal structure analysis, and participated in catalytic activity. The amino acid residues to be identified were identified. A library of mutant arylmalonate decarboxylase was constructed by substituting these amino acids with other amino acids, and Saturation Mutagenesis was performed to measure the activity of the mutant enzyme.
  • G74C / M159L / C188G mutant arylmalonate decarboxylase and Y48F / G74C / M159L / C188G mutant decarboxylase have an S-selective enantioselectivity, while G74C / C188S mutant arylcarbonate. It was found that the enzyme activity was improved as compared with malonate decarboxylase, and the mutant arylmalonate decarboxylase of the present invention was obtained.
  • the present invention is as follows.
  • a method for producing S-form ⁇ -arylpropionic acid which comprises contacting the mutant arylmalonic acid decarboxylase according to any one of [1] to [5] with an arylmalonic acid derivative.
  • a process for producing S-form ⁇ -arylpropionic acid which comprises contacting the mutant arylmalonic acid decarboxylase of [9] with an arylmalonic acid derivative.
  • the S-form selective highly active mutant arylmalonic acid decarboxylase of the present invention can obtain S-form ⁇ -arylpropionic acid with high optical purity of almost 100% ee by decarboxylation of arylmalonic acid derivatives. it can.
  • S-form ⁇ -arylpropionic acid S-type profen
  • NSAID non-steroidal anti-inflammatory agent
  • the S-form selective highly active mutant arylmalonate decarboxylase of the present invention has the following mutation (i) in the amino acid sequence of the wild-type arylmalonate decarboxylase shown in SEQ ID NO: 2, ) And (ii) mutations, (i) and (iii) mutations, or (i), (ii) and (iii) mutations: (i) substitution of the 74th glycine with cysteine and the substitution of the 188th cysteine with glycine; (ii) substitution of the 48th tyrosine with another amino acid; (iii) Replacement of the 159th methionine with another amino acid.
  • amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, lysine and valine. It is selected from 19 types of amino acids excluding amino acids.
  • the 48th tyrosine is preferably substituted with phenylalanine, and the 159th methionine is preferably substituted with leucine.
  • the 74th glycine in the amino acid sequence of the wild-type arylmalonate decarboxylase shown in SEQ ID NO: 2 is substituted with cysteine, and the 159th methionine Is a mutant in which is substituted with leucine and the 188th cysteine is substituted with glycine.
  • the 48th tyrosine of the amino acid sequence of the wild-type arylmalonate shown in SEQ ID NO: 2 is substituted with phenylalanine, and the 74th glycine is substituted.
  • G74C 74th glycine to cysteine mutation
  • G74C mutant enzyme an enzyme mutant
  • G74C mutant enzyme an enzyme mutant
  • the above-mentioned mutant arylmalonate decarboxylase is referred to as G74C / M159L / C188G mutant enzyme and Y48F / G74C / M159L / C188G mutant enzyme, respectively.
  • the 74th glycine in the amino acid sequence of the wild-type arylmalonate decarboxylase shown in SEQ ID NO: 2 is preferably replaced with cysteine, In which methionine is replaced with leucine, 188th cysteine is replaced with glycine, and 156th valine is replaced with isoleucine (G74C / M159L / C188G / V156I).
  • the 74th glycine in the amino acid sequence of the wild-type arylmalonate decarboxylase shown in SEQ ID NO: 2 is preferably replaced with cysteine, In which methionine is substituted with leucine, 188th cysteine is substituted with glycine, and 156th valine is substituted with leucine (G74C / M159L / C188G / V156L).
  • the mutant arylmalonic acid decarboxylase of the present invention acts on arylmalonic acid to produce S-form ⁇ -arylpropionic acid (profen) having S configuration at the ⁇ -position. That is, it has a stereoselectivity opposite to that of the wild type arylmalonate decarboxylase, and the enantioselectivity is S-form.
  • a mutant arylmalonic acid decarboxylase that acts on arylmalonic acid to produce S-form ⁇ -arylpropionic acid (profen) the 74th glycine has been replaced with cysteine, and the 188th cysteine has been substituted.
  • the mutant arylmalonate decarboxylase of the present invention has an enzyme activity improved over that of the G74C / C188S mutant arylmalonate decarboxylase.
  • the mutant arylmalonate decarboxylase activity of the invention as measured by the enzyme activity measurement method described below is 5 times or more, preferably 10 times or more, more preferably that of G74C / C188S mutant arylmalonate decarboxylase. Is 50 times or more, more preferably 100 times or more, and particularly preferably 150 times or more.
  • the enzyme activity of the G74C / C188G mutant arylmalonate decarboxylase of the present invention is about 5 times higher than that of the G74C / C188G mutant arylmalonate decarboxylase, and the G74C / M159L / C188G mutant aryl of the present invention
  • the enzyme activity of malonate decarboxylase is about 200 times higher than that of G74C / C188G mutant arylmalonate decarboxylase
  • the enzyme activity of Y48F / G74C / M159L / C188G mutant arylmalonate decarboxylase of the present invention is G74C.
  • the enzyme activity is G74C / M159L / C188G mutant arylmalonic acid
  • the enzyme activity when using ⁇ -methyl- ⁇ -phenylmalonic acid as a substrate of the G74C / M159L / C188G / V156L mutant arylmalonate decarboxylase of the present invention is about 2 times higher than that of decarboxylase. About three times higher than M159L / C188G mutant arylmalonate decarboxylase.
  • the specific activity of arylmalonate decarboxylase can be measured, for example, by the following method. That is, 50 ⁇ L of an aqueous phenylmalonic acid derivative solution of pH 7 to 8, several tens to several hundreds of mM, 50 ⁇ L of 1 M Tris-HCl pH buffer, and 350 ⁇ L of ultrapure water are mixed and incubated at 30 ° C. for 10 minutes or more. To this, add 50 ⁇ L of arylmalonate decarboxylase solution and mix. The reaction time is adjusted so that the conversion rate of the substrate is within 10%. This is because the enzyme reaction rate varies depending on the concentration of the substrate. After the reaction is completed, reverse phase HPLC analysis is performed. Similarly, under the same conditions, reverse phase HPLC analysis was performed in a system where 10 mM Tris buffer was added instead of the enzyme solution, and the specific activity was calculated from the value subtracted from the enzyme solution data using this measurement result as a blank. Good.
  • K cat ⁇ K m of arylmalonate decarboxylase can be measured, for example, by the following method. That is, the specific activity is measured in a reaction system in which the final concentration of the substrate is 40, 20, 10, 5, 2.5, 1.25, 0.625 mM. Using the measurement result of specific activity as a plot, “ cat [ K] and K m can be determined by subtracting“ 1 / [S] -1 / v plot ”(Lineweaver-Burk plot).
  • the compound used as a substrate by the mutant enzyme of the present invention is an arylmalonic acid derivative, preferably an ⁇ -aryl- ⁇ -methyl-malonic acid derivative having a methyl group at the ⁇ -position.
  • arylmalonic acid derivatives preferably an ⁇ -aryl- ⁇ -methyl-malonic acid derivative having a methyl group at the ⁇ -position.
  • ⁇ -methyl- ⁇ -phenylmalonic acid can be mentioned.
  • the mutant enzyme acts on these arylmalonic acid derivatives to produce S-form ⁇ -arylpropionic acid.
  • the arylmalonic acid is represented by the following general formula (I), and ⁇ -arylpropionic acid represented by the following general formula (II) is produced by the mutant arylmalonic acid decarboxylase of the present invention.
  • R is selected from the group consisting of H, CH 3 , F, OH, and NH 2
  • Ar represents an aryl group that is unsubstituted or substituted with any substituent.
  • Substituents include C1-C6 alkyl groups, C1-C6 alkoxy groups, halogen atoms, nitro groups, cyano groups, amino groups, C1-C6 alkoxycarbonyl groups, phenyl groups, hydroxy groups, etc., aryl groups And phenyl group, ⁇ -naphthyl group, ⁇ -naphthyl group, 2-furyl group, 3-furyl group, 2-thienyl group, 3-thienyl group and the like.
  • optical purity (enantiomeric excess) of S-form ⁇ -arylpropionic acid produced using the mutant arylmalonate decarboxylase of the present invention is 99% ee or higher, preferably 99.5% ee or higher, Preferably it is 99.7% ee or more.
  • the enantiomeric excess can be measured, for example, by the following method. That is, about 50 ⁇ mol of the substrate was transferred to a 1.5 mL tube, dissolved in 500 ⁇ L of ultrapure water, and adjusted to pH 8 using 2M NaOH ultrapure water and 2M HCl ultrapure water. This was made up to 1 mL and vortexed by adding 250 ⁇ L of 1 M Tris-HCl pH buffer. Dispense 125 ⁇ L each of this solution, add 0 to 100 ⁇ L of ultrapure water and 25 to 125 ⁇ L of enzyme solution, and react at 30 ° C. for about one week to react all of the substrate.
  • the reaction was stopped by adding 125 ⁇ L of 2M HCl to this reaction solution, and 300 ⁇ L of diethyl ether was added thereto, vortexed, centrifuged (13,000 rpm, 2 min), and the supernatant was collected.
  • the product is extracted by repeating this operation three times.
  • the enantiomeric excess may be measured from the peak area of the enantiomer of the product.
  • the mutant arylmalonate decarboxylase of the present invention has the mutation described above, and one or several more (for example, at positions other than the 48th, 74th, 159th and 188th amino acids) (1-10, preferably 1-5, more preferably 1-3, particularly preferably 1) amino acid sequence having a deleted, substituted or added amino acid, and decarboxylation of the arylmalonic acid derivative. It includes a mutant arylmalonate decarboxylase that catalyzes to produce S-form ⁇ -arylpropionic acid and has an enzyme activity that is higher than that of G74C / C188G mutant arylmalonate decarboxylase.
  • the amino acid substitutions at the 48th, 74th, 159th and 188th positions are maintained, 90% or more, more preferably 95% or more, particularly preferably 97% or more amino acid sequence having amino acid identity, catalyzing the decarboxylation of arylmalonic acid derivatives to produce S- ⁇ -arylpropionic acid, G74C / C188G mutant aryl It includes a mutant arylmalonate decarboxylase having an enzyme activity that is higher than that of malonate decarboxylase.
  • the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 2 is an arylmalonate decarboxylase isolated from Alcaligenes® bronchisepticus® KU1201 strain (Mikukenken no. 11670).
  • SEQ ID NO: 1 shows the base sequence of the DNA encoding the enzyme.
  • DNA encoding arylmalonate decarboxylase of the present invention is isolated by isolating DNA encoding arylmalonate decarboxylase from the microorganism and introducing the above mutation by site-directed mutagenesis or the like Can be obtained.
  • a DNA encoding the mutant arylmalonate decarboxylase of the present invention is inserted into an appropriate expression vector, the vector is introduced into a host, a transformant containing the DNA is produced, and the transformant is cultured.
  • the mutant arylmalonate decarboxylase of the present invention may be purified from a culture such as a host cell or a culture solution by a known method.
  • examples of the vector include plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, etc.) YEp system, YCp system such as YCp50), etc., and phage DNA such as ⁇ phage can also be used.
  • animal viruses such as retrovirus or vaccinia virus and insect virus vectors such as baculovirus can also be used.
  • bacteria such as Escherichia coli and Bacillus subtilis
  • fungi such as yeast
  • animal cells such as monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells)
  • insect cells such as Sf9 cells
  • Introduction of the vector into which the DNA has been inserted into the host cell can be carried out by electroporation, calcium phosphate, lipofection, spheroplast, lithium acetate, or the like.
  • Purification of the mutant arylmalonate decarboxylase of the present invention from a culture such as a host cell or a culture solution is performed by appropriately combining known protein purification methods such as gel filtration chromatography, ion exchange chromatography, affinity chromatography and the like. It can be carried out.
  • the present invention includes a method for producing S-form ⁇ -arylpropionic acid from arylmalonic acid using the above-mentioned mutant arylmalonic acid decarboxylase.
  • the starting arylmalonic acid is an ⁇ -aryl- ⁇ -methylmalonic acid derivative, which can be obtained from malonic acid by adding a methyl group and an aryl group by a known method. It can also be obtained by carbonating a racemic ⁇ -arylpropionic acid.
  • the S-form ⁇ -arylpropionic acid can be obtained by mixing the arylmalonic acid with the mutant arylmalonic acid decarboxylase of the present invention and bringing it into contact.
  • the reaction conditions in this case are pH 7 to 9, 4 to 50 ° C., and several minutes to several tens of hours depending on the amounts of arylmalonic acid and mutant arylmalonic acid decarboxylase as raw materials.
  • the amount of the enzyme to be mixed with the raw material arylmalonic acid is not limited. For example, 1 to 1000 U of the mutant arylmalonic acid decarboxylase may be mixed with 10 mM of arylmalonic acid.
  • the S-form ⁇ -arylpropionic acid obtained by the mutant arylmalonic acid decarboxylase of the present invention is useful as a pharmaceutical, a liquid crystal raw material, an agrochemical, etc., or an intermediate thereof. In particular, it is useful as a nonsteroidal anti-inflammatory drug (NSAID). These ⁇ -arylpropionic acids are called profenes.
  • the following ⁇ -arylpropionic acid is produced from the following arylmalonic acid.
  • (S) - ⁇ - (p-isobutylphenyl) propionic acid is formed from ⁇ - (p-isobutylphenyl) - ⁇ -methylmalonic acid, and ⁇ - (6-methoxy-2-naphthyl) - ⁇ (S) - ⁇ - (6-Methoxy-2-naphthyl) propionic acid (naproxen) is formed from -methylmalonic acid, and (S) - ⁇ - (6-fluorobiphenyl-4-yl) - ⁇ -methylmalonic acid produces (S ) - ⁇ - (2-Fluorobiphenyl-4-yl) propionic acid (flurbiprofen) is produced from ⁇ - (3-phenoxyphenyl) - ⁇ -methylmalonic acid to (S) - ⁇ - (3- Phenoxyphenyl) propionic acid (phenoprofen) is produced, and (S) - ⁇ - (3-benzoylphen
  • the mutated arylmalonate decarboxylase of the present invention allows oxaprofen, zaltoprofen, aluminoprofen, beoxaprofen, vermoprofen, carprofen, cycloprofen, flunoxaprofen, microprofen, and pyrprofen.
  • Pranoprofen, suprofen, ximoprofen and the like can be produced.
  • the produced S-form ⁇ -arylpropionic acid can be purified by a known method such as chromatography and used as a pharmaceutical, an agricultural chemical, a liquid crystal material, or a raw material thereof.
  • Example 1 Production of mutant arylmalonate decarboxylase and measurement of activity
  • direct evolution evolutionary molecular engineering technique
  • Ser188 and Cys74 side chains are positioned to face each other across the ⁇ -carbon of the product. From this structure, the enantioselectivity is expressed that the 188th and 74th residues are located so as to sandwich the prochiral plane of the product intermediate.
  • the pro-S carboxylate is stabilized by hydrogen bonding with the side chain hydroxyl group or main chain amide, while the pro-R carboxylate is destabilized in a hydrophobic environment. Is done. As a result, the carboxylate of pro-R is eliminated and an enolate intermediate is formed. Finally, the 188th or 74th cysteine with respect to the intermediate undergoes enantioselective protonation to produce a product.
  • Saturation mutagenesis refers to the construction of a mutant library in which all 19 kinds of natural amino acid residues are substituted for a certain amino acid residue.
  • mutation of G74C is indispensable for the expression of enantioselectivity, so that mutation cannot be introduced. Therefore, in 2nd screening, we tried to improve the activity by introducing mutations into other residues.
  • PDB-CODE Molecular modeling based on the four crystal structures of 3DG9 (wild type), 3IP8 (wild type), 3IXL (G74C / C188S mutant type), and 3IXM (G74C mutant type) to form a hydrophobic pocket
  • 3DG9 wild type
  • 3IP8 wild type
  • 3IXL wild type
  • 3IXM G74C mutant type
  • phenylmalonic acid was selected as a substrate. The reason is that it is a fast-reacting substrate and can detect weak AMDase activity, and in previous studies, the activity against phenylmalonic acid was correlated with the activity against other substrates.
  • NNK codon (N is A, G, C or T; K is G or T) or NHK codon (N is A, G, C or T; H is T, C or A: Mutagenesis primer using ⁇ ⁇ K G or T) was introduced, and mutation was introduced according to the protocol of QuickChange IIQuickSite-Directed Mutagenesis Kit (STRATAGENE) (hereinafter referred to as "QuickChange ⁇ protocol "). Escherichia coli was transformed with the prepared mutant plasmids and spread on plates. The resulting colonies were cultured in a 96-well microplate. This culture solution was inoculated into another microplate using Kenzan, and glycerol was added to preserve it as a master plate.
  • STRATAGENE QuickChange IIQuickSite-Directed Mutagenesis Kit
  • the inoculated plate was cultured at 37 ° C. for about 3 hours, and then L-(+)-arabinose was added to induce enzyme expression, followed by further incubation for about 14 hours.
  • the plate was centrifuged, the supernatant was removed, E. coli was dissolved in a solution containing Lysozyme / DNase / PMSF, frozen, and then disrupted by incubation at 37 ° C. for 3 hours.
  • the cell-free extract was added to a phenylmalonic acid solution containing BTB, and the activity was evaluated by confirming the change in pH due to decarboxylation of the substrate with BTB.
  • E. coli of wells judged to have high activity were picked up from the above master plate, and plasmid sequencing was performed to identify the mutant type.
  • the mutant enzyme is purified from the cell-free extract prepared by culturing the mutant expression host, collecting the cells, sonicating and centrifuging, and then purifying the mutant enzyme by affinity chromatography using a Ni column. Evaluated.
  • Table 1 shows the results of measuring G74C / C188S mutant type and wild type specific activity, k cat, and K m using the above QuickChange protocol.
  • the G74C / C188G mutant is purified from about 2 g of cultured cells (collected from 500 mL of liquid LB medium) by column chromatography using 50 mL of Ni Sepharose High Performance (GE Healthcare). did. The G74C mutant was purified from about 1 g of cultured cells by column chromatography using 2 mL of COSMOGEL His-Accept gel.
  • the G74C / C188G mutant improved the specific activity by 2.5 times and the relative activity by 5.6 times compared to the G74C / C188S mutant.
  • the product obtained by the G74C mutant is a racemate, it is not the target mutant. Therefore, the G74C / C188G mutant was used as a 2nd screening template.
  • Table 4 shows the results of purification and analysis of specific activity, k cat, and K m .
  • the G74C / M159L / C188G mutant improved the specific activity by 150 times and the relative activity by 210 times compared to the G74C / C188S mutant. (The specific activity is 60 times and the relative activity is 37 times that of the G74C / C188G mutant.)
  • (2) G74C / M159X / C188S mutant library Next, the G74C / C188S mutant and the G74C / C188G mutant were compared by actually performing directed evolution using the G74C / C188S mutant as a template.
  • a G74C / M159X / C188S mutant library was constructed using NNK codons, and 160 colonies were screened. As a result, one strain of M159S mutation, which was estimated to be more active than the G74C / C188S mutant, was obtained. In addition, 7 strains (with a breakdown of 2 strains of M159L, 1 strain of M159G, and 4 strains of [template]) were obtained that were estimated to have the same activity as the G74C / C188S mutant.
  • Table 5 shows the results obtained by purifying these mutants and analyzing the specific activity, k cat, and K m .
  • the Y48F / G74C / C188G mutant improved the specific activity by 20 times and the relative activity by 23 times compared to the G74C / C188S mutant. (Compared with G74C / C188G mutant, specific activity is 8 times and relative activity is 4 times.) Among the mutants obtained by 2nd screening, the G74C / M159L / C188G mutant had the highest activity. Therefore, we decided to perform 3rd screening using this mutant as a template.
  • Table 7 shows the specific activity, k cat, and K m of the mutants obtained by 3rd screening.
  • the activity of the obtained mutant type was equivalent to that of the G74C / M159L / C188G mutant type, but improved k cat and K m were obtained.
  • the Y48F / G74C / M159L / C188G mutant showed the highest activity, 920-fold relative activity compared to the G74C / C188S mutant.
  • Substrates include ⁇ -methyl- ⁇ -phenylmalonic acid ( ⁇ -methyl- ⁇ -phenylmalonic acid), ⁇ -methyl- ⁇ - (2-naphthyl) malonic acid ( ⁇ -methyl- ⁇ -naphthylmalonic acid) and ⁇ - (6-methoxy-2-naphthyl) - ⁇ -methylmalonic acid ( ⁇ -6- (methoxy-2-naphthyl) - ⁇ -methylmalonic acid) was selected.
  • the enzyme solution used in this experiment took about 1 to 6 months after purification, and there was concern about enzyme deactivation. Therefore, the specific activity was measured using phenylmalonic acid as a substrate, and the value obtained by dividing this measured value by the specific activity measured immediately after purification was defined as the residual activity. As a result, the residual activity was about 50 to 90%.
  • the optical purity of the product was measured as follows. First, the substrate was recrystallized to remove products and impurities that were slightly present in the substrate. Using this substrate, an enzyme reaction was carried out in a system for measuring the specific activity described later. The reaction was stopped by adding 2M HCl to the reaction, and the product was extracted with diethyl ether and methyl esterified with TMS diazomethane. The product was analyzed by normal phase HPLC using a column with a chiral support.
  • the G74C / M159L / C188G mutant not the G74C / Y48F / M159L / C188G mutant, was the most active against a substrate having a methyl group at the ⁇ -position.
  • This mutant type showed a good activity of about 10 to 13 times that of the wild type.
  • the product configuration was all (S) except for the wild type, and the enantiomeric excess was 99% e.e. or more except for the G74C / C188S mutant.
  • Mutant Activity Table 9 summarizes the activity of the mutant type obtained in the 1st to 3rd screenings on phenylmalonic acid.
  • the G74C / M159L / C188G mutant was 210 times more active than the G74C / C188S mutant.
  • the Y48F / G74C / M159L / C188G mutant improved activity 920 times that of the G74C / C188S mutant.
  • G74C / C188S mutant is more active than G74C / C188S mutant because “glycine is less sterically hindered” or “serine hydroxyl group” The hydrogen bond between the substrate and the substrate disappeared.
  • K m is the is improved to less than half, was speculated to be due to the substitution of a serine to a small glycine hindered.
  • Cys74 has larger steric hindrance than Gly and Ala” or “hydrogen bond between Cys74 and substrate”. Therefore, the influence of the 74th Cys was analyzed by molecular modeling.
  • PDB-CODE: 3IXM (G74C / C188S mutant) was docked with phenylmalonic acid as a substrate, and minimization was performed. As a result, a model was obtained in which the 74th cysteine hydrogen bonds with the carboxylate of pro-S.
  • the collected E. coli cells were suspended in pH 8.0, 0.5 mM EDTA, 100 mM Tris-HCl buffer (hereinafter referred to as 100 mM Tris buffer) (35 mL). In order to remove (wash) the medium components, the mixture was centrifuged again (12,000 g, 20 min), and the supernatant was removed. The obtained cultured cells (2 pieces of about 4.5 g) were stored frozen.
  • a cell-free extract was prepared from the cells. Suspend two cells in pH 8.0, 0.5 mM EDTA, 10 mM Tris-HCl buffer (hereinafter referred to as 10 mM Tris buffer) (30 mL), and ultrasonically disrupt while keeping the temperature low in an ice bath. (OUTPUT 8, DUTY 20, 5min, twice, 2 pieces, standard chip). The obtained crushed liquid was centrifuged (12,000 g, 20 min), and a cell-free extract was prepared by removing the precipitate.
  • ammonium sulfate fractionation was performed.
  • the amount of the cell-free extract was measured, ammonium sulfate (ammonium sulfate) was slowly added to 30% saturation, and the mixture was stirred for about 1 hour.
  • ammonium sulfate was slowly added so as to be 60% saturated, and stirred for 2 hours or more. This solution was further centrifuged (12,000 g, 50 min) to obtain a protein precipitate containing AMDase.
  • This precipitate was dissolved in 20 mL of 10 mM Tris buffer, placed in a cellulose tube, and dialyzed against 2 L of 10 mM Tris buffer (dialysis buffer) to remove ammonium sulfate and low molecular weight compounds.
  • the dialysis buffer was changed once after 2 hours, and dialysis was further performed for 6 hours or more.
  • anion exchange column chromatography was performed.
  • the column was filled with 100 mL of DEAE-Toyopearl gel stored in 20% ethanol, and equilibrated by flowing 300 mL of distilled water and 300 mL of 10 mM Tris buffer.
  • the crude enzyme solution after dialysis was poured into this at 1.0 mL / min, and AMDase was adsorbed on the gel.
  • 300 mL of 10 mM Tris buffer was flowed, and then eluted with 600 mL of a linear NaCl concentration gradient from 0 mM to 200 mM at an elution rate of 1.0 mL / min.
  • hydrophobic column chromatography was performed.
  • the active fraction was collected and ammonium sulfate was slowly added to 20%.
  • the column was filled with 60 mL of Butyl-Toyopearl gel stored in 20% ethanol, and equilibrated by flowing 180 mL of distilled water and 180 mL of 10 mM Tris buffer containing 20% ammonium sulfate.
  • an active fraction containing 20% ammonium sulfate was flowed at 1.0 mL / min to adsorb AMDase onto the gel.
  • Each active fraction was analyzed by SDS-PAGE. If there is a fraction in which only AMDase is dissolved (single band), collect the active fraction and dialyze with 2 L of dialysis buffer for 3 hours or more to remove ammonium sulfate and NaCl from the crude enzyme solution. Removed to complete purification. If there is no fraction with a single band, collect active fractions with a strong AMDase band, and then anion exchange column chromatography using SuperQ-Toyopearl gel and hydrophobic column using Phenyl-Toyopearl gel. Chromatography was performed and purification was performed by SDS-PAGE until a single band was obtained.
  • the SuperQ-Toyopearl gel used 60 mL and was eluted with 360 mL linear NaCl concentration gradient from 0 mM to 200 mM. Phenyl-Toyopearl gel was eluted with 300 mL of 25% to 0% linear ammonium sulfate gradient using 50 mL.
  • the reaction time was adjusted so that the substrate conversion rate in a 20 mM reaction system was about 5%. This is because, with this reaction time, the substrate conversion rate is often less than 20% at all substrate concentrations.
  • ⁇ -methyl- ⁇ - Enzymatic reaction was carried out using phenylmalonic acid as a substrate.
  • quenching was performed using 20 mM 2-phenylacetic acid acetonitrile solution, and reverse phase HPLC analysis was performed. Analysis of K cat ⁇ K m was performed in the same manner as in the case of using phenylmalonic acid as a substrate.
  • 2-Phenylpropionic acid was quantified using a calibration curve prepared with phenylacetic acid and 2-phenylpropionic acid.
  • the product was quantified using a calibration curve prepared with the product and 2-naphthylacetic acid.
  • L-(+)-arabinose induces enzyme expression. Therefore, it is necessary to select E. coli that can take in L-(+)-arabinose and does not undergo metabolic degradation as a host. Therefore, E. coli top10 having genotypes of araBADC- and araEFGH + and recommended as a pBAD vector host was used as the host.
  • the precipitated cells were frozen by adding 100 ⁇ L of 10 mM Tris, 300 mM NaCl, 1 ⁇ g / mL DNase, 10 mg / mL Lysozyme, 10 ⁇ g / mL PMSF, and left at -80 ° C. for 30 min or longer. This was incubated at 37 ° C. for 3 hours and then centrifuged (860 g, 2000 rpm, 45 min). This supernatant was used as a cell-free extract. Since PMSF was decomposed in an aqueous solution, it was stored as a methanol solution and adjusted immediately before adding it to the cells.
  • E. coli in wells that were judged to have a fast BTB discoloration compared to the template mutant were inoculated from the above master plate and cultured in 3.5 mL of liquid LB (amp) medium (Round Bottom tube). Of these, 0.5 mL of the culture broth was mixed with an equal amount of 80% glycerol and stored as a glycerol stock at ⁇ 80 ° C. Plasmid extraction was performed from the remaining 3 mL. Subsequently, plasmid sequencing was performed to identify the mutant type.
  • the mutant expression host was pre-cultured overnight from the above glycerol stock, and 1 mL was inoculated into 100 mL of liquid LB (amp) medium. When this is cultured at 30 ° C. for about 2 to 4 hours and when the absorbance at 660 nm (OD660) reaches 0.5, 250 ⁇ L of 10% L-(+)-arabinose is added (final concentration: 0.025%), and further 14 Incubate for hours. The resulting culture was collected by centrifugation (12,000 g, 10 min) and stored frozen. AMDase was purified from the cells by the method described above. In consideration of enzyme inactivation, the activity of the mutant was evaluated within 2 weeks at the longest after purification.
  • AMDase was eluted by flowing 2.5 mL of pH 8.0, 25 mM Tris-HCl, 0.5 M NaCl, 60 mM imidazole buffer (hereinafter referred to as 60 mM imidazole buffer) 4 times.
  • the enzyme solution was concentrated and desalted using Amicon® Ultra-15® Centrifugal® Filter® Units. This is because imidazole may catalyze the decarboxylation or racemization of arylmalonic acid (as pointed out by co-researcher Robert), and imidazole has an absorbance of 280 nm, which adversely affects the determination of enzyme concentration. It is.
  • the column was filled with 45 mL of Ni Sepharose (trademark) High Performance (GE Healthcare) gel stored in 20% ethanol, and equilibrated by flowing 150 mL of distilled water and 150 mL of 10 mM imidazole buffer. To this, 35 mL of cell-free extract was poured, and AMDase was adsorbed on the gel. Subsequently, 450 mL of 10 mM imidazole buffer was flowed, and then eluted with 900 mL of a linear imidazole concentration gradient from 10 mM to 250 mM at an elution rate of 1.0 mL / min.
  • Ni Sepharose trademark
  • GE Healthcare High Performance
  • AMDase activity was identified by BTB assay.
  • AMDase generally eluted in a fraction of 95 to 120 mM imidazole.
  • the active fractions were collected and the enzyme solution was concentrated and desalted using Amicon® Ultra-15® Centrifugal® Filter® Units.
  • mutant type A mutant expression plasmid was prepared by the above-mentioned QuickChange protocol, and recombinant E. coli top10 was transformed. In addition, the heat shock method was used for transformation.
  • the grown colonies were singulated by streaking to another LB (amp) medium plate with a toothpick. Colonies on the toothpick were inoculated into 3.5 mL of liquid LB (amp) medium (Round Bottom tube) and cultured. A glycerol stock was prepared from 0.5 mL of the culture solution, and plasmid extraction was performed from the remaining 3 mL. Subsequently, the plasmid was sequenced to confirm that it was the intended mutant.
  • Table 11 shows the primers used for introducing these mutations.
  • the overlapping primer is only the S36N pair.
  • the reaction was stopped by adding 125 ⁇ L of 2M HCl to the reaction solution. To this was added 300 ⁇ L of diethyl ether, vortexed, centrifuged (13,000 rpm, 2 min), and the supernatant was collected. By repeating this operation three times, the product was extracted.
  • Example 2 Activity measurement when ⁇ -methyl- ⁇ -phenylmalonic acid was used as a substrate for mutant arylmalonate decarboxylase As shown in Example 1, G74C was tested against a substrate having a methyl group at the ⁇ -position. The / M159L / C188G mutant had the highest activity, but an attempt was made to create a more active mutant.
  • the mutant arylmalonate decarboxylase having amino acid mutations G74C / M159L / C188G, G74C / M159L / C188G / V156I and G74C / M159L / C188G / V156L was prepared by the method described in Example 1. Using ⁇ -methyl- ⁇ -phenylmalonic acid as a substrate, the enzyme activity was measured by the same method as 4 in Example 1.
  • Table 12 shows the results of activity measurement.
  • Km values indicating affinity with the enzyme were improved in the order of G74C / M159L / C188G mutant, G74C / M159L / C188G / V156I mutant, and G74C / M159L / C188G / V156L mutant. Also, the activity was improved. As a result, a mutant was obtained in which the relative activity was improved 2- to 3-fold over the G74C / M159L / C188G mutant.

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Abstract

L'objectif de la présente invention est de fournir une arylmalonate décarboxylase mutante de forme hyperactive (S)-sélective qui est apte à produire un acide (S)-α-arylpropionique avec une pureté optique de presque 100 % e.e. à partir de l'acide arylmalonique. L'arylmalonate décarboxylase mutante de forme hyperactive (S)-sélective qui est apte à produire de l'acide (S)-α-arylpropionique à partir de l'acide arylmalonique, présente une mutation (i), des mutations (i) et (ii), des mutations (i) et (iii) ou des mutations (i), (ii) et (iii) dans la séquence d'acides aminés de l'arylmalonate décarboxylase de type sauvage qui est représenté par SEQ ID NO :2 : (i) substitution de la glycine à la position 74 par une cystéine et substitution de la cystéine à la position 188 par une glycine ; (ii) substitution de la tyrosine à la position 48 par un autre acide aminé ; et (iii) substitution de la méthionine à la position 159 par un autre acide aminé.
PCT/JP2012/054731 2011-02-28 2012-02-27 Mutant hyperactif (s)-sélectif d'arylmalonate décarboxylase WO2012117999A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003169685A (ja) * 2001-12-10 2003-06-17 Tosoh Corp 改変アリールマロン酸脱炭酸酵素およびそれを用いた製造法
WO2009068308A2 (fr) * 2007-11-28 2009-06-04 Basf Se Nouvelles malonate décarboxylases pour des applications industrielles

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003169685A (ja) * 2001-12-10 2003-06-17 Tosoh Corp 改変アリールマロン酸脱炭酸酵素およびそれを用いた製造法
WO2009068308A2 (fr) * 2007-11-28 2009-06-04 Basf Se Nouvelles malonate décarboxylases pour des applications industrielles

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
KENJI MIYAMOTO ET AL.: "Hen'i 1 ko de Kotonaru Kino o Fuyo, Hen'i 2 ko de Koso no Enantio Sentakusei Gyakuten", BIOSCIENCE & INDUSTRY, vol. 63, no. 9, 2005, pages 583 - 586 *
KENJI MIYAMOTO: "Arylmalon San Datsu Tansan Koso no Kino Kaihen", KOSO KOGAKU NEWS, vol. 10, no. 64, 2010, pages 5 - 10 *
MIYAUCHI, Y. ET AL.: "Dramatically improved catalytic activity of an artificial (S)- selective arylmalonate decarboxylase by structure-guided directed evolution", CHEMICAL COMMUNICATIONS, vol. 47, no. 26, 14 July 2011 (2011-07-14), pages 7503 - 7505 *
OBATA, R. ET AL.: "Structural Basis for Inverting the Enantioselectivity of Arylmalonate Decarboxylase Revealed by the Structural Analysis of the Gly74Cys/Cysl88Ser Mutant in the Liganded Form", BIOCHEMISTRY, vol. 49, 2010, pages 1963 - 1969 *
TERAO Y. ET AL.: "Improvement of the activity of arylmalonate decarboxylase by random mutagenesis", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 73, 2006, pages 647 - 653 *
YUSUKE MIYAUCHI ET AL.: "Arylmalon San Datsu Tansan Koso no Kino Kaihen", THE 14TH SYMPOSIUM ON BIOCATALYST CHEMISTRY JAPAN IN SHIZUOKA, KOEN YOSHISHU, 23 September 2010 (2010-09-23), pages 28 - 29 *

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