WO2012117999A1 - (s)-selective hyperactive mutant arylmalonate decarboxylase - Google Patents

Abstract

The purpose of the present invention is to provide a mutant arylmalonate decarboxylase of (S)-selective hyperactive form which is capable of producing (S)-α-arylpropionic acid with an optical purity of almost 100%e.e. from arylmalonic acid. The mutant arylmalonate decarboxylase of (S)-selective hyperactive form, which is capable of producing (S)-α-arylpropionic acid from arylmalonic acid, has mutation (i), mutations (i) and (ii), mutations (i) and (iii), or mutations (i), (ii) and (iii) in the amino acid sequence of wild-type arylmalonate decarboxylase that is represented by SEQ ID NO:2: (i) substitution of glycine at 74 position by cysteine, and substitution of cysteine at 188 position by glycine; (ii) substitution of tyrosine at 48 position by another amino acid; and (iii) substitution of methionine at 159 position by another amino acid.

Description

S-selective highly active mutant arylmalon decarboxylase

The present invention relates to a highly active mutant arylmalonate decarboxylase that is selective for S-form, which produces S-form α-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 (α-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 α-arylpropionic acid, but S-form α-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.

In the amino acid sequence of arylmalonate decarboxylase, a mutant (G74C / C188S) introduced with a double mutation in which the 74th glycine is replaced with cysteine and the 188th cysteine is replaced with serine is an α-form α -It has been reported to become arylpropionic acid. However, the activity of the mutant was very low, about 1 / 10,000 of that of the wild-type enzyme, and was not practical (Non-patent Documents 2 and 3).

Furthermore, 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 α-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 α-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 α-arylpropionic acid. However, the activity is drastically reduced compared to the wild type, and the activity was not sufficient for industrial use.

The present inventors thought to improve the activity of G74C / C188S mutant arylmalonate decarboxylase by directed evolution (evolutionary molecular engineering technique). 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.

As a result, 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.

That is, the present invention is as follows.

[1] The following mutation (i) in the amino acid sequence of the wild-type arylmalonate decarboxylase shown in SEQ ID NO: 2 capable of generating S-form α-arylpropionic acid from arylmalonic acid, (i) And (ii) mutations, (i) and (iii) mutations, or (i), (ii) and (iii) mutations, and S-selective highly active mutant arylmalonate decarboxylase :
(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.

[2] G74C / C188G mutation in which 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 188th cysteine is substituted with glycine The mutant arylmalonate decarboxylase of [1], which is a type.

[3] The 74th glycine in the amino acid sequence of the wild-type arylmalonate decarboxylase shown in SEQ ID NO: 2 is replaced with cysteine, the 159th methionine is replaced with leucine, and the 188th cysteine The mutant arylmalonate decarboxylase of [1], which is a G74C / M159L / C188G mutant in which is substituted with glycine.

[4] The 48th tyrosine of the amino acid sequence of the wild type arylmalonate decarboxylase shown in SEQ ID NO: 2 is replaced with phenylalanine, the 74th glycine is replaced with cysteine, and the 159th methionine is replaced with The mutant arylmalonate decarboxylase according to [1], which is a Y48F / G74C / M159L / C188G mutant in which cysteine is substituted with leucine and 188th cysteine is substituted with glycine.

[5] G74C / C188S mutation in which 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 188th cysteine is substituted with serine The mutant arylmalonate decarboxylase according to any one of [1] to [3], wherein the enzyme activity is improved as compared with the type enzyme.

[6] A method for producing S-form α-arylpropionic acid, which comprises contacting the mutant arylmalonic acid decarboxylase according to any one of [1] to [5] with an arylmalonic acid derivative.

[7] The process according to [6], wherein the S-form α-arylpropionic acid is a prophene.

[8] The production method of [7], wherein the S-form α-arylpropionic acid is selected from the group consisting of ibuprofen, naproxen, flurbiprofen, fenoprofen, ketoprofen, and indoprofen.

[9] The following mutations (i) and (ii) in the amino acid sequence of the wild-type arylmalonate decarboxylase shown in SEQ ID NO: 2 that can generate S-form α-arylpropionic acid from arylmalonic acid S-selective highly active mutant arylmalonate decarboxylase having:
(i) the replacement of the 74th glycine with cysteine, the replacement of the 159th methionine with cysteine, and the replacement of the 188th cysteine with glycine;
(iii) Replacement of the 156th methionine with isoleucine or leucine.

[10] A process for producing S-form α-arylpropionic acid, which comprises contacting the mutant arylmalonic acid decarboxylase of [9] with an arylmalonic acid derivative.

[11] The process according to [10], wherein the S-form α-arylpropionic acid is a prophene.

[12] The production method of [11], wherein the S-form α-arylpropionic acid is selected from the group consisting of ibuprofen, naproxen, flurbiprofen, fenoprofen, ketoprofen, and indoprofen.

This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2011-043085 which is the basis of the priority of the present application.

The S-form selective highly active mutant arylmalonic acid decarboxylase of the present invention can obtain S-form α-arylpropionic acid with high optical purity of almost 100% ee by decarboxylation of arylmalonic acid derivatives. it can. S-form α-arylpropionic acid (S-type profen) can be used as a non-steroidal anti-inflammatory agent (NSAID) or the like.

It is a figure which shows the structural formula of ibuprofen, naproxen, flurbiprofen, fenoprofen, ketoprofen, and indoprofen. It is a figure which shows the scheme for improving the activity of G74C / C188S mutant type arylmalonate decarboxylase by the evolution molecular engineering method (direct evolution).

Hereinafter, the present invention will be described in detail.

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.

(1) Mutation of (i) reverses the stereoselectivity of wild-type arylmalonic acid decarboxylase, and produces S-form α-arylpropionic acid from arylmalonic acid derivatives. However, due to the mutation (i), the enzyme activity is reduced compared to the wild-type enzyme. The decreased activity is improved by the mutation of (ii) or (iii).

Other 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.

In the above mutation, the 48th tyrosine is preferably substituted with phenylalanine, and the 159th methionine is preferably substituted with leucine.

In the mutant arylmalonate decarboxylase of the present invention, preferably, 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. Furthermore, in the mutant arylmalonate decarboxylase of the present invention, preferably, 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. It is a mutant in which cysteine is substituted, 159th methionine is substituted with leucine, and 188th cysteine is substituted with glycine. In the present invention, for example, the 74th glycine to cysteine mutation is referred to as G74C, and an enzyme mutant is referred to as a G74C mutant enzyme. According to this expression, the above-mentioned mutant arylmalonate decarboxylase is referred to as G74C / M159L / C188G mutant enzyme and Y48F / G74C / M159L / C188G mutant enzyme, respectively.

Furthermore, in the mutant arylmalonate decarboxylase of the present invention, 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). Furthermore, in the mutant arylmalonate decarboxylase of the present invention, 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 α-arylpropionic acid (profen) having S configuration at the α-position. That is, it has a stereoselectivity opposite to that of the wild type arylmalonate decarboxylase, and the enantioselectivity is S-form. As a mutant arylmalonic acid decarboxylase that acts on arylmalonic acid to produce S-form α-arylpropionic acid (profen), the 74th glycine has been replaced with cysteine, and the 188th cysteine has been substituted. G74C / C188S mutant arylmalonate decarboxylase in which is substituted with serine was known (Terao et al., J Mol Catat B: Enzym, 2007, 15-20). 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. Preferably, 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, and the enzyme activity of Y48F / G74C / M159L / C188G mutant arylmalonate decarboxylase of the present invention is G74C. About 920 times higher than / C188G mutant arylmalonate decarboxylase.

In addition, when α-methyl-α-phenylmalonic acid is used as a substrate of the G74C / M159L / C188G / V156I mutant arylmalonate decarboxylase of the present invention, the enzyme activity is G74C / M159L / C188G mutant arylmalonic acid The enzyme activity when using α-methyl-α-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 μL of an aqueous phenylmalonic acid derivative solution of pH 7 to 8, several tens to several hundreds of mM, 50 μL of 1 M Tris-HCl pH buffer, and 350 μL of ultrapure water are mixed and incubated at 30 ° C. for 10 minutes or more. To this, add 50 μL 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.

In addition, 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 α-aryl-α-methyl-malonic acid derivative having a methyl group at the α-position. For example, α-methyl-α-phenylmalonic acid can be mentioned. The mutant enzyme acts on these arylmalonic acid derivatives to produce S-form α-arylpropionic acid.

The arylmalonic acid is represented by the following general formula (I), and α-arylpropionic acid represented by the following general formula (II) is produced by the mutant arylmalonic acid decarboxylase of the present invention.

In the formula, R is selected from the group consisting of H, CH ₃ , F, OH, and NH ₂ , and 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, α-naphthyl group, β-naphthyl group, 2-furyl group, 3-furyl group, 2-thienyl group, 3-thienyl group and the like.

The optical purity (enantiomeric excess) of S-form α-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 μmol of the substrate was transferred to a 1.5 mL tube, dissolved in 500 μL 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 μL of 1 M Tris-HCl pH buffer. Dispense 125 μL each of this solution, add 0 to 100 μL of ultrapure water and 25 to 125 μL 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 μL of 2M HCl to this reaction solution, and 300 μL 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 resulting product is methylated by adding a small amount of TMS diazomethane, the diethyl ether is volatilized, dissolved in 500 μL of diethyl ether: isopropanol = 1: 1 solution and subjected to normal layer HPLC analysis. 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 α-arylpropionic acid and has an enzyme activity that is higher than that of G74C / C188G mutant arylmalonate decarboxylase. Further, with respect to the amino acid sequence of the above-mentioned 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-α-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. Thus, 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.

At this time, 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 λ phage can also be used. Furthermore, animal viruses such as retrovirus or vaccinia virus and insect virus vectors such as baculovirus can also be used.

As the host, 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), and insect cells such as Sf9 cells can be used. .

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 α-arylpropionic acid from arylmalonic acid using the above-mentioned mutant arylmalonic acid decarboxylase. The starting arylmalonic acid is an α-aryl-α-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 α-arylpropionic acid.

The S-form α-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 α-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 α-arylpropionic acids are called profenes.

For example, the following α-arylpropionic acid is produced from the following arylmalonic acid.

(S) -α- (p-isobutylphenyl) propionic acid (ibuprofen) is formed from α- (p-isobutylphenyl) -α-methylmalonic acid, and α- (6-methoxy-2-naphthyl) -α (S) -α- (6-Methoxy-2-naphthyl) propionic acid (naproxen) is formed from -methylmalonic acid, and (S) -α- (6-fluorobiphenyl-4-yl) -α-methylmalonic acid produces (S ) -α- (2-Fluorobiphenyl-4-yl) propionic acid (flurbiprofen) is produced from α- (3-phenoxyphenyl) -α-methylmalonic acid to (S) -α- (3- Phenoxyphenyl) propionic acid (phenoprofen) is produced, and (S) -α- (3-benzoylphenyl) propionic acid (ketoprofen) is produced from α- (3-benzoylphenyl) -α-methylmalonic acid, α- {4-[(2-oxocyclopentyl) methyl] phenyl} -α-methylmalonic acid to (S) -α- {4-[(2-oxocyclopentyl) methyl ] Phenyl} propionic acid (loxoprofen) is produced from α- [4- (1-oxo-2-isoindolinyl) phenyl] -α-methylmalonic acid ((S) -α- [4- (1-oxo- 2-isoindolinyl) phenyl] propionic acid (indoprofen) is produced, as described above for ibuprofen, naproxen, flurbiprofen, fenoprofen, ketoprofen, indopro The structural formula of fen (indoprofen) is shown in FIG.

Furthermore, 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 α-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.

The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Production of mutant arylmalonate decarboxylase and measurement of activity By direct evolution (evolutionary molecular engineering technique), the activity of an arylmalonate mutant (G74C / C188S) already reported has been improved. Tried.

First, when the catalytic reaction of the enzyme was analyzed using the structural information obtained from the X-ray crystal structure analysis, the following was found about the catalytic reaction of the arylmalonate decarboxylase.

(1) Ser188 and Cys74 side chains are positioned to face each other across the α-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.

(2) Cys74 and Ser188 adversely affect decarboxylation activity by directly interacting with the substrate.

(3) When the substrate binds to the active site, 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.

(4) Both the wild type and G74C / C188S mutants are rate-limiting at the first stage.

From the above results, it was suggested that the G74C / C188S mutation had an adverse effect on the first-stage pro-R carboxylate elimination reaction and the activity decreased.

Therefore, first, as the 1st screening, we decided to perform the saturation mutagenesis for the 188th. 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.

On the other hand, 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.

For mutations other than the 74th and 188th mutations, we try to promote “destabilization of pro-R carboxylate” by introducing mutations in the residues constituting the hydrophobic pocket (hydrophobic pocket) and its vicinity. It was decided.

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 We confirmed that Pro14, 、 Leu40, Val43, Tyr48, Leu77, Val156, and Met159 are located near pro-R carboxylate.

From the above, we have planned the Directed Evolution scheme shown in Figure 2. That is, using G74C / C188S as a template, saturation mutagenesis is performed on the 188th residue, and a highly active mutant is selected (1st screening). Using the obtained mutant type as a template, selection is performed by performing saturation mutagenesis on the residues of Leu40, Val43, Tyr48, Met73, Leu77, Val156, and Met159 (2nd screening). Furthermore, it is a scheme in which a mutant type having the highest activity is used as a template to select other six hydrophobic residues (3rd screening).

Next, a system was established for screening mutants with high decarboxylation activity from the saturation mutagenesis library. When performing molecular evolution, it is necessary to rapidly carry out enzyme purification and activity evaluation. For this purpose, it is effective to add a His-tag to the enzyme. Therefore, for expression of the enzyme, an expression vector provided by BioSpring of Germany and incorporating the ORF gene of AMDase derived from KU1201 strain into a pBAD vector was used (WO / 2005/078111). The N-terminus of AMDase expressed by this vector is extended by 3 residues of “MGQ”, and the C-terminal side is extended by 9 residues of “GGSHHHHHH”.

Also, 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 用 い 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. 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.

1. Evaluation of activity of G74C / C188S mutant type 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 specific activity of the G74C / C188S mutant was almost consistent with previous studies.

2. 1st screening-saturation mutagenesis for 188th
(1) Screening using decarboxylation activity as an index First, a mutant library was prepared using the NNN codon, and 68 colonies were screened using decarboxylation activity as an index. As a result, the G74C mutant type had the highest activity, and then the G74C / C188G mutant type had the highest activity. Furthermore, a G74C / C188X (X ≠ C, W, R, G) mutant library was constructed using NHK codons, and 76 colonies were screened. As a result, a mutant having a higher activity than the G74C / C188G mutant could not be obtained. Therefore, in the screening of the 188th residue, it was judged that the G74C mutant type had the strongest activity, and then the G74C / C188G mutant type had the strongest activity.

(2) Purification and activity measurement 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.

As a result of analysis of specific activity, k _cat, and K _m , it was found that the activity of the mutant was higher in the order of G74C mutant> G74C / C188G mutant> G74C / C188S mutant, as predicted at the screening stage. (Table 2).

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.

Since 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.

3. 2nd & 3rd screening-Directed evolution targeting hydrophobic pockets
(1) G74C / M159X / C188G mutant library Next, it was decided to screen the G74C / M159X / C188G mutant library. The 159th residue is a saturation mutagenesis by the group of Micklefield et al. As a result, a residue proven with improved 50-fold relative activity with K _m basis. Using the NNK codon, a G74C / M159X / C188G mutant library was constructed and 138 colonies were screened. As a result, 11 strains estimated to be more active than the G74C / C188G mutant and 6 strains estimated to be equivalent in activity to the G74C / C188G mutant were obtained. These strains were classified into groups 1 to 4 in descending order of activity.

As a result of sequencing, most of the strains having higher activity than the G74C / C188G mutant were M159L mutations, and other M159V mutations were obtained (Table 3).

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 .

Furthermore, we decided to continue the screening of hydrophobic pockets using the G74C / C188G mutant as a template.

(3) Summary of 2nd screening Table 6 shows the specific activities, k _cat, and K _m of the mutants obtained by 2nd screening.

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.

(4) Summary of 3rd screening 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.

4). Analysis of Substrate Specificity and Optical Purity (1) Method Next, G74C / Y48F / M159L / C188G mutant, G74C / M159L / C188G mutant, Y48F / G74C / C188G were particularly active in the 1st to 3rd screening. We decided to analyze the substrate specificity and optical purity of the mutant type, G74C / C188G mutant type, G74C / C188S mutant type, and wild type.

Substrates include α-methyl-α-phenylmalonic acid (α-methyl-α-phenylmalonic acid), α-methyl-α- (2-naphthyl) malonic acid (α-methyl-α-naphthylmalonic acid) and α- (6-methoxy-2-naphthyl) -α-methylmalonic acid (α-6- (methoxy-2-naphthyl) -α-methylmalonic acid) was selected. By comparing phenylmalonic acid and α-methyl-α-phenylmalonic acid, the effect of the methyl group at the α-position, and also comparing α-methyl-α-phenylmalonic acid and α-methyl-α-naphthylmalonic acid Therefore, it was thought that the influence of the aryl group could be analyzed. In addition, α- (6-methoxy-2-naphthyl) -α-methylmalonic acid corresponding to naproxen, which is important as a pharmaceutical, was selected.

As the activity index, only specific activity was measured. Similar to phenylmalonic acid, 20 mM substrate was converted with a mutant enzyme and the reaction was stopped by adding acetonitrile. This reaction solution was analyzed by reverse phase HPLC using a Cosmosil 5C ₁₈ -AR-II (Nacalai Tesque) column, and the reaction product was quantified.

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%.

In addition, a correction was made by dividing the measured value of the specific activity for this substrate by the residual activity. (The subsequent analysis results are the values after correction.)
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.

(2) Analysis results Table 8 shows the analysis results.

First, 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 α-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.

In addition, the activity of α- (6-methoxy-2-naphthyl) -α-methylmalonic acid was about half that of α-methyl-α-naphthylmalonic acid regardless of the mutant type. From this result, it can be presumed that the electronic effect of the methoxy group at the 6-position of the naphthyl group affects the activity.

5. Summary of Mutant Activity (1) 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.

(2) Comparison between G74C / C188S mutant and G74C / C188G mutant G74C / C188G 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.

Also, 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.

Also, as a result of screening, since the activity of G74C / C188G was higher than that of G74C / C188A, it was found that the 188th steric hindrance also affected the activity.

(3) Consideration of G74C mutation The activity of G74C mutant is lower than that of wild type or G74A mutant, and the activity of G74C / C188S mutant is lower than that of C188S mutant. In the G74C / C188S mutant decarboxylation reaction, no kinetic isotope effect of D ₂ O is observed. From this, it can be concluded that the mutation of G74C significantly reduced the activity of the first-stage pro-R carbosylate elimination reaction.

The reason can be presumed that “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.

From this result, it was speculated that Cys74 hydrogen-bonded with pro-S carboxylate and disturbed the binding mode of the substrate, thereby destabilizing pro-R carboxylate and decreasing decarboxylation activity.

In this example, purification of arylmalonate decarboxylase, measurement of enzyme activity, and molecular evolution studies by creating mutants were carried out by the following methods.

(1) Purification of arylmalonate decarboxylase E. coli XL1-Blue transformed with a mutant expression plasmid was cultured overnight at 30 ° C in two test tubes containing liquid LB (amp) medium (10 mL). . This is inoculated into two 5L Erlenmeyer flasks containing liquid LB (amp) medium (1L), shaken at 30 ° C for about 2 to 5 hours, and then measured for OD660 to be within the range of 0.3 to 0.4. It was confirmed. The expression of the enzyme was induced by adding 1 mL of 0.1 M IPTG to a final concentration of 0.1 mM, and further cultured with shaking at 30 ° C. for 18 hours. The obtained culture broth was collected by centrifugation (12,000 g, 10 min).

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.

Next, 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.

The subsequent operations were performed in an ice bath or a refrigerator at 4 ° C.

Next, 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. To the supernatant obtained by centrifuging this solution (12,000 mm, 25 min), 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.

Next, 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. Subsequently, 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. After collecting 30 bottles of 20 mL per fraction, a fraction having AMDase activity was identified by the BTB assay method described below.

Next, 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. To this, an active fraction containing 20% ammonium sulfate was flowed at 1.0 mL / min to adsorb AMDase onto the gel. Subsequently, 180 mL of dialysis buffer containing 20% ammonium sulfate was flowed, and then eluted with a linear ammonium sulfate concentration gradient of 360 mL from 20% to 0% at an elution rate of 1.0 mL / min. 30 bottles of 12 mL were collected per fraction. A fraction having AMDase activity was identified by BTB assay.

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.

(2) mutant activity measurement wild type arylmalonate decarboxylase and mutant arylmalonate decarboxylase specific activity, K _cat and K _m were measured by the following methods.

(A) When phenylmalonic acid is used as a substrate
(i) Measurement of specific activity 50 μL of a pH 7.0, 200 mM phenylmalonic acid aqueous solution, 50 μL of 1 M Tris-HCl pH buffer solution, and 350 μL of ultrapure water were mixed and incubated on a heat block at 30 ° C. for 10 minutes or more. To this, 50 μL of AMDase solution was added and mixed by pipetting about 3 times. The reaction time was adjusted so that the conversion rate of the substrate was within 10%. This is because the enzyme reaction rate varies depending on the concentration of the substrate. After completion of the reaction, 500 μL of acetonitrile was added to quench the reaction. In acetonitrile used for quenching, 20 mM 2-phenylpropionic acid was dissolved in advance as an internal standard substance. This reaction solution was directly subjected to reverse phase HPLC analysis.

Under exactly the same conditions, 10 mM Tris buffer was added instead of AMDase solution, quenching was performed in the same manner, and reverse phase HPLC analysis was performed. Using this measurement result as a blank, the specific activity was calculated from the value subtracted from the data of the AMDase solution.

(ii) Analysis of K _cat · K _m Specific activity was measured in a reaction system in which the final concentration of the substrate was 40, 20, 10, 5, 2.5, 1.25, 0.625 mM. As the substrate solution, 50 μL of a serially diluted 200 mM phenylmalonic acid solution was used. Blanks were measured for each substrate concentration.

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.

Using the measurement results of specific activity as a plot, “1 / [S] -1 / v plot” (Lineweaver-Burk plot) was drawn to determine k _cat and K _m .

(iii) Reversed phase HPLC analysis The solution which quenched the reaction liquid with acetonitrile was analyzed as it was. Phenylacetic acid was quantified using a calibration curve prepared with phenylacetic acid and 2-phenylpropionic acid.

The analysis conditions were as follows.

Column Cosmosil 5C ₁₈ -AR-II (Nacalai Tesque)
Developing solvent Methanol / Ultra pure water / TFA = 60: 40: 0.05
Flow rate 0.8mL / min
Detection 254nm
The retention time of each substance was as follows.

Phenylmalonic acid (substrate) About 4.6-4.8min
Phenylacetic acid (product) Approx. 6.6-6.8min
2-Phenylpropionic acid (internal standard) Approx. 8.9-9.5min
(B) Measurement of specific activity when α-methyl-α-phenylmalonic acid is used as a substrate In a system in which specific activity is measured using phenylmalonic acid as a substrate, α-methyl-α- Enzymatic reaction was carried out using phenylmalonic acid as a substrate. Then, 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.

The column used was Cosmosil 5C ₁₈ -AR-II (Nacalai Tesque), the developing solvent was methanol / ultra pure water / TFA = 60: 40: 0.05, the flow rate was 0.8 mL / min, and the detection was performed at 254 nm. 2-Phenylpropionic acid was quantified using a calibration curve prepared with phenylacetic acid and 2-phenylpropionic acid.

(C) Measurement of specific activity when α-methyl-α-naphthylmalonic acid or α- (6-methoxy-2-naphthyl) -α-methylmalonic acid is used as a substrate Ratio using phenylmalonic acid as a substrate In the system for measuring the activity, an enzyme reaction was carried out using α-methyl-α-naphthylmalonic acid or α- (6-methoxy-2-naphthyl) -α-methylmalonic acid at about pH 8 and 20 mM as a substrate. Then, quenching was performed using 20 mM 2-naphthyl acetate 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.

The column used was Cosmosil 5C ₁₈ -AR-II (Nacalai Tesque), the developing solvent was methanol / ultra pure water / TFA = 65: 35: 0.05, the flow rate was 0.8 mL / min, and the detection was performed at 254 nm. The product was quantified using a calibration curve prepared with the product and 2-naphthylacetic acid.

(3) Molecular evolution of arylmalonate decarboxylase
(i) Design of Mutagenesis primer The primer pairs used for mutagenesis were those shown in Table 10.

(ii) Creation of mutant library Mutation was introduced by the “QuickChange protocol”. PfuUltra was used as the PCR enzyme. PCR conditions were determined with reference to “Manual for QuickChange II Site-Directed Mutagenesis Kit”, “Manual for PfuUltra ^™ HF DNA polymerase” and Lei Zheng, Jean-Louis Reymond, Nucleic Acids Research, 2004, 32, No. 14.

When using a pBAD vector, 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.

(iii) Screening of decarboxylation activity 150 μL of liquid LB (amp) medium was added to all wells of a 96-well microplate. These wells were inoculated with a mutant library and a template mutant expression host as a control with a toothpick and cultured overnight at 37 ° C. This culture solution was inoculated at Kenzan into 100 μL (96-well microplate) of a liquid LB (amp) medium for enzyme expression. To the original cultured cells, 50 μL of 80% glycerol was added and stored at −80 ° C. This was used as a master plate.

Incubate 100 μL of liquid LB (amp) medium for enzyme expression at 37 ° C for about 3 hours, and then add 50 μL of liquid LB (amp) medium containing 0.075% L-(+)-arabinose to induce enzyme expression. (The final concentration of L-(+)-arabinose was 0.025%), and further cultured at 37 ° C. for 14 hours. After completion of the culture, the absorbance at 660 nm was measured with a plate reader to confirm the growth of E. coli in each well. Next, after centrifugation (860 g, 2000 rpm, 20 min), the supernatant was removed with a Pipetman.

The precipitated cells were frozen by adding 100 μL of 10 mM Tris, 300 mM NaCl, 1 μg / mL DNase, 10 mg / mL Lysozyme, 10 μg / 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.

20 μL of cell-free extract was transferred to a new 96-well microplate. To this was added 180 μL of 10 mM MOPS, 25 mM phenylmalonic acid, and 11 g / L BTB (reaction solution). Immediately after that, the absorbance at 660 nm was continuously measured with a plate reader, and it was visually confirmed that the reaction solution was changed from green to blue. When the crushed microbial cells that had precipitated entered the reaction solution, it was difficult to see the absorbance and BTB discoloration activity.

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 μL 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.

(4) Purification of AMDase containing His-tag
(i) Purification of small amount system About 0.5 g of cultured cells were suspended in 10 mL of pH 8.0, 25 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole buffer (hereinafter referred to as 10 mM imidazole buffer). While keeping the temperature low in an ice bath, it was crushed by ultrasonic crushing (OUTPUT 2-3, DUTY 20, 5min, 2 times, elongated chip). Subsequent operations were performed in an ice bath or a refrigerator at 4 ° C. The obtained crushed liquid was centrifuged (8000 g, 40 min), and a cell-free extract was prepared by removing the precipitate.

2 mL of COSMOGEL His-Accept (Nacalai Tesque) gel stored in 20% ethanol was packed into a small column and equilibrated by flowing 10 mL of distilled water and 10 mL of 10 mM imidazole buffer. To this, 10 mL of cell-free extract was poured, and AMDase was adsorbed on the gel. Subsequently, excess protein was removed by flowing 2.5 mL of 10 mM imidazole buffer four times. Subsequently, 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.

Finally, 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.

(ii) Purification of large-scale system Since G74C / C188S mutant and G74C / C188G mutant had weak AMDase activity, they were purified by the following method to obtain an enzyme solution of about 1 mg / mL.

Approximately 2 g of cultured cells were suspended in 35 mL of 10 mM imidazole buffer. While keeping the temperature low in an ice bath, it was crushed by ultrasonic crushing (OUTPUT 8, DUTY 20, 20, 5 min, 2 times, standard chip). Subsequent operations were performed in an ice bath or a refrigerator at 4 ° C. The obtained crushed liquid was centrifuged (12,000 g, 20 min), and a cell-free extract was prepared by removing the precipitate.

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. After collecting 41 bottles of 22 mL per fraction, a fraction having 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.

(5) Production of mutant arylmalonate decarboxylase
(i) Preparation of 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.

(ii) Mutagenesis primer design By using the above-mentioned QuickChange protocol, from the G74A mutant expression plasmid, wild type, G74C mutant, C188S mutant, G74C / C188S mutant, S36N / G74C / C188S mutant, S36N / G74C / An expression plasmid of C188G mutant / S36N / G74C / M159L / C188G mutant was prepared.

Table 11 shows the primers used for introducing these mutations. The overlapping primer is only the S36N pair.

(6) Analysis of substrate specificity
(i) Recrystallization of substrate Substrates α-methyl-α-phenylmalonic acid, α-methyl-α-naphthylmalonic acid, α- (6-methoxy-2-naphthyl) -α-methylmalonic acid The sample was dissolved in diethyl ether and saturated, a small amount of hexane was added, and the mixture was sealed and allowed to stand at room temperature. The produced crystals were collected by filtration with Kiriyama filter paper, transferred to a desiccator, and dried by drawing with a vacuum pump.

(ii) Measurement of enantiomeric excess of product Transfer about 50 μmol of substrate to a 1.5 mL tube, dissolve in 500 μL of ultrapure water, and adjust to pH 8 using 2M NaOH ultrapure water and 2M HCl ultrapure water. It was. After making up this to 1 mL, 250 μL of 1 M Tris-HCl pH buffer solution was added and vortexed. 125 μL of this solution was dispensed, and 0 to 100 μL of ultrapure water and 25 to 125 μL of enzyme solution were added and reacted at 30 ° C. for about 1 week to react all of the substrate.

The reaction was stopped by adding 125 μL of 2M HCl to the reaction solution. To this was added 300 μL 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.

The product was methylated by adding a small amount of TMS diazomethane. Diethyl ether was volatilized and dissolved in 500 μL of diethyl ether: isopropanol = 1: 1 solution and subjected to normal layer HPLC analysis. The enantiomeric excess was determined from the peak area of the enantiomer of the product.

Example 2 Activity measurement when α-methyl-α-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 α-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 α-methyl-α-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.

All publications, patents and patent applications cited in this specification shall be incorporated into this specification as they are.

Production of S-form α-arylpropionic acid, which is industrially useful as medical pesticides, liquid crystal materials and intermediates thereof, by using the S-form selective highly active mutant arylmalonate decarboxylase of the present invention can do.

SEQ ID NO: 3 to 32 primer

Claims (12)

1. The following mutations (i) and (ii) in the amino acid sequence of the wild-type arylmalonate decarboxylase shown in SEQ ID NO: 2 capable of generating S-form α-arylpropionic acid from arylmalonic acid ) Mutation, (i) and (iii) mutation, or (i), (ii) and (iii) mutations, S-isomer selective highly active mutant arylmalonate decarboxylase:
   (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.
2. A G74C / C188G mutant in which 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 188th cysteine is substituted with glycine The mutant arylmalonate decarboxylase according to claim 1.
3. The 74th glycine in the amino acid sequence of the wild type arylmalonate decarboxylase shown in SEQ ID NO: 2 is replaced with cysteine, the 159th methionine is replaced with leucine, and the 188th cysteine is replaced with glycine. The mutant arylmalonate decarboxylase according to claim 1, which is a substituted G74C / M159L / C188G mutant.
4. The 48th tyrosine of the amino acid sequence of the wild type arylmalonate decarboxylase shown in SEQ ID NO: 2 is replaced with phenylalanine, the 74th glycine is replaced with cysteine, and the 159th methionine is replaced with leucine. The mutant arylmalonate decarboxylase according to claim 1, which is a mutant of Y48F / G74C / M159L / C188G in which the 188th cysteine is substituted with glycine.
5. The G74C / C188S mutant enzyme in which 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 188th cysteine is substituted with serine The mutant arylmalonate decarboxylase according to any one of claims 1 to 3, wherein the enzyme activity is improved as compared with that.
6. A process for producing S-form α-arylpropionic acid, which comprises contacting the mutant arylmalonic acid decarboxylase according to any one of claims 1 to 5 with an arylmalonic acid derivative.
7. The production method according to claim 6, wherein the S-form α-arylpropionic acid is a profene.
8. The production method according to claim 7, wherein the S-form α-arylpropionic acid is selected from the group consisting of ibuprofen, naproxen, flurbiprofen, fenoprofen, ketoprofen and indoprofen.
9. Having the following mutations (i) and (ii) in the amino acid sequence of the wild-type arylmalonate decarboxylase shown in SEQ ID NO: 2 capable of generating S-form α-arylpropionic acid from arylmalonic acid, S-selective highly active mutant arylmalonate decarboxylase:
   (i) the replacement of the 74th glycine with cysteine, the replacement of the 159th methionine with cysteine, and the replacement of the 188th cysteine with glycine;
   (iii) Replacement of the 156th methionine with isoleucine or leucine.
10. A process for producing S-form α-arylpropionic acid, which comprises contacting the mutant arylmalonic acid decarboxylase according to claim 9 with an arylmalonic acid derivative.
11. The production method according to claim 10, wherein the S-form α-arylpropionic acid is a profene.
12. The production method according to claim 11, wherein the S-form α-arylpropionic acid is selected from the group consisting of ibuprofen, naproxen, flurbiprofen, fenoprofen, ketoprofen and indoprofen.

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