WO2009107821A1

WO2009107821A1 - Process for production of optically active carboxylic acid using esterase derived from thermophilic archaebacterium

Info

Publication number: WO2009107821A1
Application number: PCT/JP2009/053783
Authority: WO
Inventors: 宮本憲二; 太田博道
Original assignee: 学校法人慶應義塾
Priority date: 2008-02-26
Filing date: 2009-02-23
Publication date: 2009-09-03
Also published as: JP5506658B2; JPWO2009107821A1

Abstract

The object is to achieve a novel domino-type reaction of a hydrolysis reaction and a decarboxylation reaction using an esterase ST0071 as a catalyst to thereby produce an optically active carboxylic acid such as an optically active α-arylalkanoic acid. Disclosed is a process for producing an optically active carboxylic acid by using a dicarboxylic acid ester as a starting material, which can produce the optically active carboxylic acid through an one-step domino reaction comprising reacting the dicarboxylic acid ester with an esterase derived from Sulfolobus tokodaii strain 7.

Description

Method for producing optically active carboxylic acid using esterase derived from thermophilic archaea

The present invention relates to a method for producing an optically active carboxylic acid used as an intermediate raw material for pharmaceuticals, agricultural chemicals, and the like, and more specifically, optically in one step using a domino reaction catalyzed by a thermophilic archaeal esterase. The present invention relates to a method for producing an active carboxylic acid.

Akita

Background art

There are many types of enzymes that catalyze hydrolysis. Among them, lipases and esterases that act on ester bonds, and proteases that act on amide bonds, such as amidases, are the most studied. Research on substance conversion using hydrolases began about 40 years ago, and studies on esterases that catalyze the hydrolysis of steroid compounds have been made. At present, hydrolytic enzymes are widely used industrially, such as detergents and food additives. Among them, enzymes with high thermal stability are highly industrially useful because they exhibit high stability under high temperature conditions and in organic solvents.

In recent years, archaea has attracted attention as a suitable screening target for such thermostable enzymes. Archaea, Eubacteria (Eubacteria), a third group of organisms alongside eukaryotes (eukaryote), high temperature, high pressure, can grow in good UNA under extreme conditions of low _P H and a high salt concentration known It has been. In this way, archaea are becoming increasingly important from the perspective of biotechnology applications that take advantage of the feature of thermal stability.

In research using biocatalysts, synthesis of optically active substances has become the mainstream. This is because the enzyme itself has an asymmetric environment composed of optically active amino acids. In the synthesis of optically active compounds, prochiral compounds are often used as substrates. A prochiral compound is a compound that can be converted to a chiral compound with an asymmetric carbon by a one-step reaction (Figure 1). In other words, an optically pure compound can be obtained with a yield of 100%, which is an efficient reaction. For example, many studies of reactions using compounds such as Compound 1 shown in Fig. 1 have been reported, for example, asymmetric hydrolysis of dicarboxylic esters using porcine liver esterase (PLE). (See Non-Patent Document 1).

When the substrate is a prochiral dicarboxylic acid ester, one of the equivalent esters is selectively hydrolyzed to obtain an optically active substance. Furthermore, unlike the optical resolution method, a reaction using a protic substrate is theoretically capable of obtaining an optically active form with a 100% enantiomeric excess in a yield of 100%, which is a very efficient reaction. Yes.

For example, stereoselective hydrolysis of disubstituted malonic esters is a useful reaction that can theoretically yield optically pure malonic half esters in 100% yield. There have been many reports on the use of PLE (pig liver esterase), but there have been no reports on other enzymes, and the resulting configuration was limited to one. In addition, PLE was not thermally stable and could not be used at high temperatures where the reaction rate could be improved.

Depending on the compound, it was found that different enantiomers showed different physiological activities, so optically active substances became important. The importance of technology for producing single enantiomers has increased, and the proportion of only one enantiomer as a pharmaceutical has increased dramatically. In addition to pharmaceuticals, optically active substances are used in a wide range of fields such as agricultural chemicals, foods and fragrances, and recently liquid crystals not related to physiological activity. As pharmaceuticals, for example, flurbiprofen, ibuprofen, ketoprofen, naproxen and the like shown in FIG. 2 are known as optically active substances. These optically active α-arylpropionic acids are known as non-steroidal anti-inflammatory drugs and have useful physiological activities for S-enantiomers. Therefore, it is required to efficiently synthesize only one enantiomer of optically active α-arylpropionic acid.

A domino-type reaction is a reaction in which multiple reactions occur in a single reaction solution. The characteristic of the domino-type reaction is that when the yield of the elementary reaction is high, the product can be obtained efficiently because it eliminates the trouble of extracting and purifying the intermediate of the reaction. This method is also used for the synthesis of complex compounds from simple compounds such as organic synthesis. (See Non-Patent Document 2).

From the above, performing such a domino-type reaction with a biocatalyst has the advantage that the synthesis method becomes simpler and more efficient and can be performed with a low load on the environment. However, there has been no report on a domino-type reaction using one enzyme.

Non-Patent Document 1 C. J. Sih, J. Am. Chera. Soc., 1975, 97, 4144

Non-Patent Document 2 D. Enders, C. Grondal, M.R.M.Huttl, Angew.Chem. Int.

Ed., 2007, 46, 1570-1581 Disclosure of the Invention

The purpose of this study is to produce a novel domino-type reaction of hydrolysis and decarboxylation using esterase ST0071 as a catalyst, and to produce optically active carboxylic acids such as α-arylalkanoic acid.

The present inventors cloned the esterase ST0071 having high homology with a known esterase from the genome sequence of the thermoacidophilic archaeon Sulfolobus tokodai i strain 7, and revealed that the expressed enzyme has esterase activity as intended. (Y. Suzuki, K. Miyamoto, H. Ohta, FEMS Microbiol. Lett., 2004, 236, 97-102). Esterase ST0071 was found to have high thermal stability with an optimum reaction temperature of 70 ° C. and an optimum pH of 7.5 to 8.0. Furthermore, homology search confirmed that esterase ST0071 is closely related to thermostable esterase whose X-ray crystal structure has been solved. However, the X-ray structural analysis and detailed features of this enzyme are not yet known. Therefore, the present inventors have clarified the detailed characteristics of this enzyme by using esterase ST0071 having high thermostability, and aimed to construct a substance production process that takes advantage of the thermophilic property. We conducted an intensive study.

The present inventors designed a novel domino-type reaction represented by Chemical Formula 1 using the isolated and purified thermophilic archaeal esterase ST0071 as a catalyst.

1

Decarboxylation reaction

New domino reaction for the purpose of this study

In other words, allylmalonic acid ethyl ester having prochiral and large steric hindrance was selected as the substrate. From this substrate, half ester was first obtained by hydrolysis reaction using esterase ST0071 as a catalyst. This half ester was found to be stable at room temperature. Thus, it was found that optically active aryl propionic acid can be obtained by decarboxylation within the active center of the enzyme by using a substrate that is easy to decarboxylate under high temperature conditions.

By the method of the present invention, the stereoselective hydrolysis of the disubstituted malonic acid ester can be carried out with high optical purity, and the absolute configuration of the resulting compound is opposite to that obtained with PLE. Furthermore, by carrying out the reaction at a high temperature, the present inventors have succeeded in obtaining a product in which a decarboxylation reaction has proceeded subsequent to hydrolysis, thereby completing the present invention.

That is, the present invention is as follows.

[1] A method for producing an optically active carboxylic acid using a dicarboxylic acid ester as a raw material, wherein an esterase derived from a microorganism belonging to the genus Sulfolobus is allowed to act on the dicarboxylic acid ester, and an optically active carboxylic acid is produced by a one-step domino reaction Method.

[2] The method for producing an optically active carboxylic acid according to [1], wherein the esterase derived from a microorganism belonging to the genus Sulfolobus is an esterase derived from Sulfolobus tokodai i.

[4] A method for producing an optically active carboxylic acid from a dicarboxylic acid ester as a raw material, wherein the following (a) or (b) esterase is allowed to act on the dicarboxylic acid ester and optically active by a one-step domino reaction. Method for producing carboxylic acid: (a) an esterase consisting of the amino acid sequence represented by SEQ ID NO: 2

(b) An esterase consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2.

[4] The starting dicarboxylic acid ester is a compound represented by the following chemical formula I,

Formula I

[Wherein R ¹ and R ² are different from each other, and are substituted or unsubstituted alkyl groups; substituted or unsubstituted aryl groups or aralkyl groups; substituted or unsubstituted cycloalkyl groups; A substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted sentence represents an unsubstituted alkoxy group, and R ³ and R ⁴ independently represent H or an alkyl group. ]

The method for producing the optically active carboxylic acid according to any one of [1] to [3], wherein the obtained optically active carboxylic acid is a compound represented by the following chemical formula III.

2

R ² , COOH chemical formula in

[Wherein R ¹ and R ² are the same as the compound represented by Formula I above. ]

[5] The starting dicarboxylic acid ester is a compound represented by the following chemical formula I:

Chemical formula _Σ

[Wherein R ¹ and R ² are different from each other, substituted or unsubstituted with a phenyl group An alkyl group substituted or unsubstituted by an alkyl group having 1 to 5 carbon atoms, R ³ and R ⁴ independently represent H or an alkyl group having 1 to 5 carbon atoms. The method for producing an optically active carboxylic acid according to [5], wherein the obtained optically active carboxylic acid is a compound represented by the following chemical formula III:

4

R ² COOH chemical formula in

[6] The method for producing an optically active carboxylic acid according to [4], wherein the starting dicarboxylic acid ester is an arylcarboxylic acid ester, and the obtained optically active carboxylic acid is α-arylalkanoic acid.

[7] The method for producing an optically active carboxylic acid according to [4], wherein the dicarboxylic acid ester used as a raw material is an allylic malonic acid ester, and the obtained optically active carboxylic acid is α-aryl propionic acid.

[8] The starting dicarboxylic acid ester is 2-methyl-2-phenylmalonic acid diester or 2-methyl-2-benzylmalonic acid diester, and the resulting optically active rubonic acid is 2-phenylpropionic acid. Or the method for producing an optically active carboxylic acid according to [4], which is 2-benzylpropionic acid.

[9] The method for producing an optically active carboxylic acid according to any one of [1] to [8], wherein the reaction between the dicarboxylic acid ester and the esterase derived from the genus Sulfolobus is performed at room temperature to 80 ° C.

[1 0] An optically active carboxylic acid is produced in one step from a dicarboxylic acid ester which is a raw material substrate by catalyzing a domino-type reaction with enantioselectivity, which contains esterase derived from Sulfolobus genus microorganisms as an active ingredient. Catalyst composition for carboxylic acid production.

[1 1] The composition for producing an optically active carboxylic acid according to [10], wherein the esterase derived from a microorganism belonging to the genus Sulfolobus is an esterase derived from Sulfolobus tokodaii. [1 2] Catalyze a domino-type reaction having the following esterase (a) or (b) as an active ingredient and having an enantioselectivity, and in one step from dicarboxylic acid ester as a raw material substrate Catalyst composition for producing an optically active carboxylic acid that generates an acid:

(a) an esterase consisting of the amino acid sequence represented by SEQ ID NO: 2

[1 3] A method for producing an optically active malonic acid half ester by stereoselective hydrolysis using a dicarboxylic acid ester as a raw material, which comprises reacting a dicarboxylic acid ester with an esterase derived from a microorganism belonging to the genus Sulfolobus. A method for producing an optically active malonic acid half ester, wherein the absolute configuration of the resulting malonic acid half ester is opposite to that of a malonic acid half ester obtained using porcine liver esterase (PLE).

[1 4] The method for producing an optically active malonic acid half ester according to [1 3], wherein the esterase derived from a microorganism belonging to the genus Sulfolobus is an esterase derived from Sulfolobus tokodai i.

[15] Manufacture photoactive malonic acid half ester by stereoselective hydrolysis using dicarboxylic acid ester as a raw material, including the action of esterase (a) or (b) below on dicarboxylic acid ester A method for producing an optically active malonic acid half ester in which the absolute configuration of the resulting malonic acid half ester is opposite to that obtained using porcine liver esterase (PLE):

[16] The dicarboxylic acid ester as a raw material is a compound represented by the following chemical formula I,

5 R ¹ NO COOR ³

\.ヽ

R ² COOR ⁴ Chemical formula I

[Wherein R ¹ and R ² are different from each other, and are substituted or unsubstituted alkyl groups; substituted or unsubstituted aryl groups or aralkyl groups; substituted or unsubstituted cycloalkyl groups; A substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted alkoxy group is represented, and R ³ and R ⁴ independently represent H or an alkyl group. ]

The method for producing an optically active malonic acid half ester according to any one of [13 :! to [15], wherein the obtained optically pure malonic acid half ester is a compound represented by the following chemical formula II:

Chemical 6

R ¹ COOR ³ R ¹ \, ヽ COOR ⁴

C ゝ; ^cゝ

R ² COOH or COOH Formula Π

[Wherein, RR ² , R ³ and R ⁴ are the same as the compound represented by the above chemical formula I. [1 7] A method for producing an optically active malonic acid half ester according to any one of [1 3] to [16], wherein the reaction between a dicarboxylic acid ester and a Sulfolobus microorganism-derived esterase is carried out at room temperature.

This specification includes the contents described in the specification and Z or drawings of Japanese Patent Application No. 2008-045055, which is the basis of the priority of the present application. Brief Description of Drawings

FIG. 1 shows an example of a prochiral compound.

FIG. 2 shows an example of an optically active pharmaceutical product.

FIG. 3 is a diagram showing an example of a substrate of Sulfolobus tokodaiii strain 7-derived estebuse ST0071. FIG. 4 is a diagram showing a half ester produced from the substrate shown in FIG. 3 by hydrolysis with Sulfolobus tokodai i strain 7-derived esterase ST0071.

FIG. 5 is a diagram showing the procedure of the enzyme reaction.

FIG. 6 shows the absolute configuration of the product produced by porcine liver esterase (PLE).

FIG. 7 shows the results of HPLC analysis of 2-methyl-2-phenylmalonic acid ethyl ester.

FIG. 8 is a graph showing the yield and enantiomeric excess of half ester produced from the substrate shown in FIG. 3 by hydrolysis with Sulfolobus tokodai i strain 7-derived esterase ST0071.

FIG. 9 is a diagram showing a TLC analysis result of a reaction using 2-methyl-2- (p-diphenyl) maltyl ester malonate as a substrate.

FIG. 10 is a view showing a basic skeleton of a substrate of Sulfolobus tokodai i strain 7-derived esterase ST0071.

Figure 11 shows the electronic effects of substituents that affect the hydrolysis reaction.

FIG. 12 is a diagram showing the structure of 2-methylol-2-phenylmalonic acid jetyl ester.

FIG. 13 shows the structure of 2-methyl-2-benzylmalonic acid jetty / resester. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

The esterase used in the present invention is an esterase derived from a microorganism belonging to the genus Sulfolobus, and includes esterase derived from Sulfolobus' tokodai i strain and esterase power S derived from Sulfolobus tokodai i strain 7. Esterase from Sulfolobus tokodai i strain 7

ST0071 is a thermoacidophilic archaeon Sulfolobus tokodai i strain 7 from Y Suzuki et al.

FEMS Microbiology Letters 236 (2004) 97-102. The esterase ST0071 can be purified from a culture by culturing Sulfolobus tokodai i strain 7. In addition, Sulfolobus tokodai i strain 7 The gene to be transferred can be isolated and manufactured by genetic engineering techniques.

SUI IO IODUS tokodan strain is available from JCM u'apan collection of Microorgani sms (registration number JCM10545). The base sequence and amino acid sequence of the esterase ST0071 are registered under the registration number BAB65028.1 in DDBJ (DNA Data Bank of Japan). The base sequence and amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively.

The esterase of the present invention has a deletion or substitution of at least one, preferably one or several amino acids in the amino acid sequence as long as the protein comprising the amino acid sequence represented by SEQ ID NO: 2 has esterase activity. Even if a mutation such as addition occurs. Here, 1 or several means 1 to 10, more preferably 1 to 5.

The amino acid sequence of SEQ ID NO: 2 and the basic amino acid search tool at the National Center are as amino acid sequences in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2. for Biological Information (e.g., US National Biological Information Center Basic Local Alignment Search)) etc. (e.g., default or default parameters) at least 85%, preferably 90 % Or more, more preferably 95% or more, particularly preferably 97% or more.

A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 is substantially the same as a protein having the amino acid sequence of SEQ ID NO: 2.

In addition, a DNA that can hybridize with a DNA that is complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 under the following stringent conditions and that encodes a protein having esterase activity: The esterase encoded by is also included in the esterase of the present invention. That is, using a filter with DNA immobilized, hybridization was performed at 68 ° C in the presence of 0 · 7 to 1.0M NaCl, and then a 0 · 1 to 2 fold concentration of SSC solution ( A 1-fold concentration of SSC is a condition that can be identified by washing at 68 ° C using 150raM NaCl and 15raM sodium citrate). Alternatively, on the nitrocellulose membrane by Southern blotting After transcription and fixation of DNA, hybridization buffer (50% formamide, 4 X SSC, 50 mM HEPES (pH 7.0), 10 X Denhardf s solution, 100 / zg / ml salmon sperm DNA ] DNA that can form a hybrid by reaction at 42 ° C. Sulfolobus-derived microorganism esterases such as Sulfolobus tokodai i strain 7 derived esterase ST0071, etc., use prochiral dicarboxylic acid ester (disubstituted malonic acid ester) as a substrate and preferentially hydrolyze one of the esters. And malonic acid half ester (monoester form). The half ester is optically pure and can be obtained in high yield. By this reaction, an optically active substance can be obtained.

When the resulting optically active half ester is further reacted with an esterase derived from a microorganism belonging to the genus Sulfolobus such as esterase ST0071 from Sulfolobus tokodaii strain 7, a hydrolysis reaction and a decarboxylation reaction occur, which is optically active and has an asymmetric carbon atom. To produce a chiral monocarboxylic acid.

That is, Sulfolobus microorganism-derived esterases such as Sulfolobus tokodai i strain 7-derived esterase ST0071 catalyze a domino-type reaction with enantioselectivity.

The substrate used as a raw material for the above reaction is a dicarboxylic acid ester represented by the following chemical formula I.

8

Formula I

In the formula, R ¹ and R ² are different from each other, and are substituted or unsubstituted alkyl groups; substituted or unsubstituted aryl groups or aralkyl groups; substituted or unsubstituted cyclohexanes; It represents an alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, or a substituted or unsubstituted alkoxy group.

Examples of the alkyl group include linear or molecular chain alkyl groups such as carbon number. ; ~ 20, preferably 1 to 10 carbon atoms, more preferably a linear or molecular alkyl group having 1 to 5 carbon atoms, such as methyl, ethyl, propyl, methylethyl, butyl, methylpropyl, Examples include pentyl, methylbutyl, ethylpropyl and the like. Examples of the alkenyl group include a straight chain or branched chain alkenyl group. For example, the straight chain or branched chain having 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms, and more preferably 2 to 5 carbon atoms. Alkenyl groups such as bulle, propenyl and the like. Examples of the alkynyl group include a straight chain or branched chain alkynyl group. For example, the straight chain or branched chain having 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms, and more preferably 2 to 5 carbon atoms. Alkynyl groups such as echel and probule. Examples of the alkoxy group include straight-chain or branched alkoxy groups, for example, carbon number:! To 20, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Examples include branched chain alkoxy groups, including methoxy, ethoxy, propoxy and the like. Examples of the aryl group include aryl groups having 6 to 20 carbon atoms, preferably 6 to 10 carbon atoms, and include phenyl, 1-naphthyl, 2-naphthyl and the like. Examples of the substituted alkyl group, cycloalkyl group, alkenyl group, alkynyl group, alkoxyl group, and aryl group include, for example, an aryl group, a cycloalkyl group, an alkyl group, an alkenyl group, an alkynyl group, and an analkoxy group. Amino group, nitro group, hydroxyl group, octalogen atom and the like. These substituents may be further substituted.

Preferably, R ¹ and R ² are different from each other, and a phenyl group, 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 5 alkyl groups, 1 to 20 carbon atoms, preferably 1 to 10, more preferably 1 to 5 alkoxy or carboxy substituted or unsubstituted alkyl groups; 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms An alkyl group, an alkoxy group having 1 to 5 carbon atoms, a hydroxyl group, or a halogen-substituted or unsubstituted aryl group or aralkyl group; Preferably, R ¹ and R ² are different from each other and each represents a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group. Particularly preferably, R ¹ and R ² are different from each other, and are substituted or unsubstituted alkyl groups substituted with a phenyl group, having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms. A It represents an aryl group substituted or unsubstituted with an alkyl group. One of R ¹ and R ² is preferably an alkyl group having 1 to 2 carbon atoms, more preferably a methyl group, and the other is an alkyl group substituted with a phenyl group or an alkyl group having 1 to 5 carbon atoms. A substituted or unsubstituted aryl group. R ³ and R ⁴ independently represent H or an alkyl group having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms. Examples of the compound I include aryl carboxylic acid esters such as allyl malonic acid ester, and specifically, 2-methyl-2-phenylmalonic acid diester, particularly 2-methyl-2-phenylmalonic acid ester. Mention may be made of oleic acid ethyl ester or 2-methyl-2-benzylmalonic acid diester, in particular 2-methyl-2-benzylmalonic acid diethyl ester. The first stereoselective hydrolysis reaction produces a half ester represented by the following chemical formula II.

9 R ¹ , COOFT

No

Or R ² COOH Chemical formula n In the formula, R ² , R ³ and R ⁴ are the same as the compound represented by the chemical formula I.

The present invention also includes a method for producing a half ester represented by Formula II. The half ester can be used as an intermediate for producing pharmaceuticals and agricultural chemicals.

By the above reaction, an optically active carboxylic acid represented by the following chemical formula III is generated.

1 0

/ ^Cゝ

R ² COOH chemical formula in formula wherein R ¹ and R ² are the same as the compound represented by the chemical formula I above. The raw material compound I force S, aryl carboxylic acid ester such as aryl malonate, The resulting compound is α-arylpropyl. When it is an α-arylalkanoic acid such as on-acid, and is a compound I force, 2-methyl-2-phenolmalonic acid diester or 2-methyl_2-benzylmalonic acid diester, -Phenylpropionic acid or 2-benzylpropionic acid. Examples of other α-arylpropionic acid obtained by the method of the present invention include optically active flurbiprofen, ibuprofen, ketoprofen, naproxen and the like.

The enantiomeric excess of the half ester produced in the first hydrolysis reaction is 20ee (%) or more, preferably 80ee (%) or more, and the yield is 50% or more, preferably 90% or more.

'In the present invention, the optically active carboxylic acid means that one optical isomer is contained more than the other optical isomer, and the enantiomeric excess of the carboxylic acid finally obtained is 20ee ( %) Or more, preferably 80ee (%) or more, and the yield is 20% or more, preferably 50% or more.

The reaction is performed at 0 to 100 ° C, preferably at room temperature to 80 ° C, such as 25 ° C to 80 ° C, more preferably 50 to 80 ° C. Depending on the type of R ¹ or R ^{2 in} Formula I above, domino-type reactions are unlikely to occur at low temperatures below room temperature, and only reactions that produce half esters proceed. In this case, it is preferably carried out at 50 to 80 ° C.

The esterase concentration and substrate concentration during the reaction can be determined as appropriate according to the type of substrate. For example, the substrate concentration may be 10 mM, the enzyme is 500 U, and the reaction volume is 20 mL. Here, the unit of esterase U (Unit) is 1 / ¾1 _nitrophenylbutanoate hydrolyzed at pH 8.0 and 25 ° C, 1 // \ 1 _nitrophenol for 1 minute Refers to the enzyme activity produced. The pH during the reaction is 5 to 10 and preferably 6 to 9.5. The reaction may be performed in a buffer solution. .

The reaction time is:! ~ 300 hours.

'Sulfolobus tokodai i strain 7-derived esterase ST0071 etc. of the present invention

The absolute configuration of optically active ruponic acid obtained by reaction using esterase derived from Sulfolobus genus microorganism is the porcine liver esterase (PLE) described in CJ Sih, J. Am. Chera. Soc., 1975, 97, 4144. The opposite is true. Conventionally, no enzyme having an absolute configuration opposite to that of porcine liver esterase (PLE) has been obtained by enzymatic reaction. Further, Sulfolobus tokodaii strain 7-derived esterase ST0071 may be used by immobilizing it on an insoluble carrier. Sulfolobus tokodai i strain 7-derived esterase ST0071 is thermoacidophilic and stable to heat and acid. Therefore, it can be used repeatedly.

By using esterase derived from Sulfolobus genus microorganisms such as Sulfolobus tokodai i strain 7 derived esterase ST0071, the compound represented by the above chemical formula 1 is used as a raw material, and the chemical formula 2 is used as an intermediate material for drugs such as anti-inflammatory agents and agricultural chemicals. The optically active carboxylic acid represented can be produced in one step in a short time.

The present invention is a one-step optical process from a dicarboxylic acid ester as a raw material substrate that catalyzes a domino-type reaction with enantioselectivity, which contains an esterase derived from a genus Sulfolobus such as esterase ST0071 derived from Sulfolobus tokodai i strain 7 as an active ingredient. The catalyst composition for optically active carboxylic acid production which produces | generates active carboxylic acid is included.

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

Example 1 Production of Optically Active Carboxylic Acid Using Esterase ST0071 Crude Enzyme Solution Esterase ST0071 used in this example was simply purified by the following method and used as a crude enzyme solution.

(a) E. coli BL21 Star (DE3) was transformed with plasmid pETstEst and cultured overnight at 37 ° C in 10 ml of LB medium (containing 50 ig / ral ampicillin). It was transferred to 1000 ml of LB medium and further cultured at 30 ° C for 5 hours. To induce enzyme expression, IPTG was added to a concentration of 0.1 mM, and the cells were further cultured for 3 hours.

(b) The culture solution was centrifuged (8500 rpm, 10 rain, 4 ° C).

(c) The cells were suspended in 50 ml of 100 mM Glycine-NaOH buffer (pH 9.2).

(d) The cells were sonicated (15 min, 0 ° C) and centrifuged (12000 rpm, 20 rain, 4 ° C).

(e) The supernatant was incubated at 60 ° C for 25 minutes and centrifuged (12000 rpm, 20 min, 4 ° C).

(f) The supernatant was collected to obtain a crude enzyme solution.

In this example, JE0L JNM was used to measure the nuclear magnetic resonance spectrum.

EX-270 (270 MHz) Fourier transform nuclear magnetic resonance spectrum measuring instrument and JEOL A JNM AL-300 (300 MHz) Fourier transform nuclear magnetic resonance spectrum measuring device was used. JASCO JASCO FT / IR-410 was used to measure the infrared absorption spectrum. Sensyu Science SSC-3461 was used for high-speed liquid chromatography. MERCK 1.005715 was used for analytical silica gel thin layer chromatography (TLC). MERCK1.05744. Was used for preparative Siri-Force gel thin layer chromatography (pTLC).

In this example, enzyme reaction and product analysis were performed by the following method. The substrate and the crude enzyme solution were added to a 50 mL eggplant flask so that the substrate concentration was 10 mM, and the reaction was carried out by stirring at room temperature or 60 ° C for 1 to 10 days. After 2M hydrochloric acid was added to complete the reaction, the mixture was filtered through celite and extracted with ethyl acetate according to a conventional method. The solvent was distilled off under reduced pressure, and the residue was purified by preparative thin-layer column chromatography (hexane / ethyl acetate I acetic acid = 2/1 / 0.1) to obtain the product.

The product was determined by nuclear magnetic resonance spectrum and infrared absorption spectrum. The nuclear magnetic resonance spectrum and infrared absorption spectrum of the product were as follows.

(S) -Monoethyl 2-methyl_2-phenyl-malonate (o)

NMR (300 MHz, CDC1 ₃ ) δ (ppm): 1.26 (3H, t, J = 6.0 Hz), 1.90 (3H, s), 4.26 (2H, q, J = 6. 0 Hz), 7. 24-7. 38 (5H, m)

IR (NaCl) v _max : 3063, 2989, 2641, 1955, 1711, 1600, 1585, 1498, 1447, 1379, 1251, 1117, 1074, 1029, 1017, 930, 857, 727, 698 cnf ¹

(S) -2-Phenyl-propionate (7)

'H-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 1.52 (3H, d, J = 6.0 Hz), 3.75 (1H, q, J = 6.0 Hz), 7. 24-7. 38 (5H, ra)

IR (NaCl) v _max : 3030, 2982, 2938, 2728, 2634, 1949, 1877, 1706, 1601, 1585, 1497, 1454, 1413, 1378, 1262, 1232, 1185, 1064, 1030, 937, 861, 727 , 698 cm— ¹ (S) -Monoethyl 2-ethyl-2-phenyl-mlonate (12)

'H-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 0.98 (3H, t, J = 6.0 Hz), 1.25 (3H, t, J = 6.0

Hz), 2.45 (2H, qq, J = 6.0 Hz), 4.28 (2H, qq, J = 6.0 Hz), 7.24—7.38 (5H, ra)

IR (NaCl) v _max : 3063, 3030, 2981, 2939, 2645, 1953, 1710, 1601, 1498, 1447,

1408, 1234, 1128, 1095, 1024, 935, 857, 723, 698 cm— ¹ (R) -Monoethyl 2-methyl 2_benzyl-malonate (13)

'H-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 1. 28 (3H, t, J = 6.0 Hz), 1. 47 (3H, s), 3. 25 (2H, s), 4. 23 (2H, q, J = 6.0 Hz), 7. 12-7. 27 (5H, ra)

IR (KBr) v _oax : 3031, 2986, 2939, 1712, 1605, 1496, 1455, 1379, 1215, 1113, 1020,

936, 861, 738, 701, 666 cm— ¹

2- (p-Nitro-phenyl) -propionate (14)

^: H-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 1. 58 (3H, d, J = 6.0 Hz), 3. 89 (1H, q, J = 6.0 Hz), 7. 49-8. 22 (4H, m)

Monoethyl 2-(p-methyl-phenyl) -malonate (15)

'H-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 1.28 (3H, t, J = 6.0 Hz), 1.89 (3H, s), 2.34 (3H, s), 4. 28 (2H, q, J = 6.0 Hz), 7. 15-7. 27 (4H, m)

IR (NaCl) v _max : 2987, 2639, 1712, 1557, 1514, 1459, 1377, 1249, 1098, 1069, 1020, 857, 744, 722, 666 cm— ¹

Furthermore, the enantiomeric excess was determined by high performance liquid chromatography. Here, the retention times and the conditions of each compound for the R-form and S-form are shown. Detection was carried out at 214 nm only for monoethyl 2-ethyto 2-phenyto mlonate (12) and 254 nra for all others.

Separation data in the high performance liquid chromatography was as follows. Monoethyl 2-methyl- 2-phenyl- malonate (6)

Column: CHIRALCEL 0D-H (Daicel Chemical)

Development system: hexane / isopropanol / trifluoroacetic acid = 98/2 / 0.5% flow rate: 0.5 ml / min R form: 34.7 min, S form: 30.8 min

2-Phenyl-propionate w)

Column: CHIRALCEL 0D-H (Daicel Chemical)

Development system: hexane / isopropanol / trifluoroacetic acid = 93 I 7 I 1.5% Flow rate: 0.3 ml / rain R-form: 19.1 rain, S-form: 20. 6 min

Monoethyl 2-ethyl-2_phenyl-mlonate (12)

Column: CHIRALCEL 0D (Daicel Chemical)

Development system: hexane / isopropanol / trifluoroacetic acid = 99/1 / 0.1% Flow rate: 0.4 ml / min R body: 109 min, S body: 92.6 min

Monoethyl 2-methyl_2- benzyl-malonate (13)

Column: CHIRALCEL OJ-H (Daicel Chemical)

Development system: hexane / isopropanol / trifluoroacetic acid = 98/2 / 1.0% Flow rate: 1.0 ml / min R-form: 18.2 min, S-form: 13. 0 rain

2- -Nitro-phenyl) -propionate (14)

Column: CHIRALCEL AD-RH (Daicel Chemical)

Deployment system: water / acetonitrinole / phosphoric acid = 5/1 / 0.1%

Flow rate: 0.5 ml / min 70. 6 min, 83.5 min

2-Methyl as a prochiral and highly sterically hindered compound that is difficult to hydrolyze

-2-Phenenolemalonic acid jetyl ester (5 compounds in the following chemical formula) was selected to examine whether this enzyme catalyzes a domino-type reaction. Then, we examined whether hydrolysis products (6 compounds in the following chemical formula) and products by domino-type reaction (7 compounds in the following chemical formula) can be obtained at various temperatures.

1 1

The compounds used to examine the substrate specificity of this enzyme are shown in Figure 3. 1. Synthesis of compounds

(1) Substrate synthesis

2-Methyl-2-phenylmalonic acid jetyl ester (5), 2-ethyl-2-phenylmalonic acid jetyl ester (8), 2-methyl-2-benzylmalonic acid jetyl ester (8) 9), 2-Methyl-2- (ρ-Nittofenenole) malonic acid ethyl ester (10) and 2-methyl-2- (ρ-methylphenyleno) malonic acid jetyl ester (11) It was synthesized by the method shown in 2-6. 1 2

Chemical formula 2 Synthesis of methyl-2-phenylmalonic acid jetyl ester

1 3

Chemical formula 3 Synthesis of ethyl-2-phenylmalonic acid jetyl ester 1 4

Chemical formula 4 Synthesis of 2-methyl-2 · benzylmalonic acid jetyl ester 1 5

Chemical formula 5 Synthesis of 2-methyl-2- (ρ-nitrophenyl) malonic acid jetyl ester 1 6

Chemical formula 6 Synthesis of 2-methyl-2- (p-methylfur) maciatic acid ethyl ester

2-Methyl_2_phenylmalonic acid jetyl ester (5) was synthesized by methylation of 2-phenyl-2-remalonic acid jetyl ester with methyl fluoride using sodium methoxide as the base (Formula 2) . 2-Ethyl-2-phenylmalonic acid jetyl ester (8) and 2-methyl-2-benzenolemalonic acid jetyl ester (9) use sodium hydride as a base and use 2-substituted malonic acid jetyl ester as a base. Synthesized by alkylation with alkyl (chemical formulas 3 and 4). 2-Methyleno-2- (p-diphenyl) malonic acid jetyl ester (10) is obtained by arylating methylmalonic acid ethyl ester with sodium hydride as base and P-nitrochlorobenzene. (Chemical formula 5). 2-Methyl-2- (P-methylphenyl) malonic acid ethyl ester (11) is obtained by esterifying 2- (p-methylphenyl) propionic acid, and using lithium diisopropylamine (LDA) as the base, And was synthesized by ethoxycarbonylation (Chemical Formula 6).

The specific synthesis method was as follows.

(i) Synthesis of Diethyl 2-methy-to 2-pheny-to malonate (5)

Sodium methoxide (0.817 g, 12 讓 ol) I Phenylmalonic acid jetyl ester (2.36 g, 10 mraol) / absolute ethanol (10 ml) was added dropwise to absolute ethanol (8 ml). After completion of dropping, the mixture was stirred at 0 ° C for 30 minutes. Next, methyl iodide (0.934 mL, 15 ol) was added, the temperature was allowed to rise naturally to room temperature (about 22 ° C), and the mixture was further stirred for 3 hours. 2 M Hydrochloric acid was added to make it acidic, followed by extraction using a conventional method, and purification by silica gel column chromatography (hexane I ethyl acetate = 7 I 1) to obtain the desired product (1.89 g, 76%). Obtained as a colorless liquid. ^] H-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 1.25 (6H, t, J = 6.0 Hz), 1.86 (3H, s), 4.22 (4H, q, J = 6.0 Hz), 7. 25-7. 38 (5H, m)

(i i) Synthesis of Diethyl 2_ethy and 2-pheny malonate (8)

Sodium hydride (0.365 g, 15.2 mmol) was washed 3 times with 10 mL of anhydrous hexane. Hexane was removed and 10 mL of absolute ethanol was added. While maintaining the temperature at 0 ° C, a solution of phenylmalonic acid jetyl ester (3.02 g, 12.7 mmol) in absolute ethanol (5 mL) was added. . After stirring for 30 minutes, an aqueous ethanol solution (5 mL) of Yohka chill (3.10 g, 19.1 olol) was added and stirred overnight at room temperature. Add 2 M hydrochloric acid to acidify, extract by a conventional method, and purify by silica gel column chromatography (hexane / ethyl acetate = 30 I 1) to obtain the desired product (1.22 g, 19%) as a colorless liquid. Got as.

Ή-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 0.88 (3H, t, J = 6.0 Hz), 1. 24 (6H, t, J = 6.0 Hz), 2. 35 (2H, q, J = 6.0 Hz), 4. 17 (4H, q, J = 6.0 Hz), 7. 25-7. 41 (5H, ra) IR (NaCl) v _max : 3062, 3030, 2981, 2939, 1732, 1602, 1497, 1447, 1239, 1027, 859, 744, 727, 698, 666 cm— ¹

(i i i) Synthesis of Diethyl 2_methyl_2_benzyl_malonate (9)

Sodium hydride (0.575 g, 14.4 mraol) was washed 3 times with 10 mL of anhydrous hexane. Hexane was removed, 15 mL of THF was added, and a THF solution (10 mL) of benzylmalonic acid jetyl ester (3.00 g, 12.0 olol) was added while maintaining the temperature at 0 ° C. After stirring for 30 minutes, a THF solution (10 mL) of methyl iodide (2.04 g, 14.4 ol) was added, and the mixture was heated to reflux at 80 ° C. for 6 hours. Add 2 M hydrochloric acid to acidify, extract by a conventional method, and purify by silica gel column chromatography (hexane I ethyl acetate = 5/1) to obtain the desired product (2.54 g, 80%) as a colorless liquid. Obtained.

^! H-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 1.25 (6H, t, J = 6.0 Hz), 1.33 (3H, s), 3.22 (2H, s), 4. 19 (4H, q, J = 6.0 Hz), 7.11—7.26 (5H, m)

IR (NaCl) v _max : 3086, 3063, 3030, 2983, 2938, 1956, 1882, 1733, 1605, 1496, 1454, 1241, 1108, 1023, 862, 787, 743, 702, 666 cm— ¹

(iv) Synthesis of Diethyl 2-methyl_2- (p-nitro-phenyl)-malonate (lO)

The solution was washed three times with sodium hydride (1.12 g, 28.1 ol) in 10 mL of anhydrous hexane. Remove hexane, add DMFlO mL, and maintain the temperature at 0 ° C. A DMF solution (15 mL) of acid jetyl ester (3.00 g, 18.7 thigh ol) was added. After stirring for 30 minutes, a DMF solution (15 raL) of p-nitrochlorobenzene (4.43 g, 28.1 mraol) was added and heated to reflux at 100 ° C for 4 hours. Add 2 M hydrochloric acid to acidify, extract by a conventional method, and purify by silica gel column chromatography (hexane I ethyl acetate = 5/1) to obtain the desired product (2.42 g, 44%) as a colorless liquid. Got as.

'H-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 1.26 (6H, t, J = 6.0 Hz), 1.91 (3H, s), 4.25 (4H, q, J = 6.0 Hz), 7.55—8.21 (4H, m)

IR (NaCl) v _max : 3084, 2985, 2941, 2907, 2874, 1930, 1736, 1606, 1524, 1497, 1350, 1258, 1107, 925, 857, 752, 736, 665 cm— ¹

(v) Synthesis of Ethyl 2-(p-methyl-phenyl) propionate

Add 2- (p-methylphenyl) propionic acid (5.00 g, 30.5 mmol) to p-toluenesulfonic acid monohydrate (90 mg) and ethanol (50 mL) and heat at 90 ° C overnight. Refluxed. Ethanol was distilled off under reduced pressure, saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted by a conventional method. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 2I1) to obtain the desired product (5.60 g, 96%) as a colorless liquid.

-Thigh (300 MHz, CDC1 ₃ ) δ (ppm): 1. 20 (3H, t, J = 6.0 Hz), 1. 47 (3H, d, J = 6.0 Hz), 2. 33 ( 3H, s), 3.67 (1H, q, J = 6.0 Hz), 4.19 (2H, q, J = 6.0 Hz), 7.11-7.26 (5H, m)

IR (NaCl) v _max : 3094, 2980, 2936, 2875, 1901, 1732, 1617, 1514, 1453, 1374, 1330 1202, 1173, 1071, 1025, 860, 821, 783, 726, 665 cm " ¹

(vi) Synthesis of Diethyl 2-methyl_2_ (p-methyl-phenyl) malonate (l l)

To a solution of diisopropylamine (0.795 g, 7.80 mmol) in THF (5 ml) was added 1.58M n-BuLi hexane solution (4.61 ml, 7.28 mmol) at 0 ° C for 30 minutes. Stir for minutes. While maintaining the temperature at 0 ° C, a THF solution (10 mL) of 2- (p-methylphenyl) propionic acid ethyl ester (1.0 g, 5.20 mmol) was added and stirred for 30 minutes. Thereafter, the temperature of the reaction system was cooled from 0 ° C. to −78 ° C., and a THF solution (5 mL) of ethyl chloroformate (0.677 g, 6.24 mmol) was added. After stirring for 30 minutes, the temperature of the reaction system was raised to room temperature and further stirred overnight. After adding 2 M hydrochloric acid to acidify, extract by a conventional method, purify by silica gel chromatography (hexane I ethyl acetate = 9/1) and purify the desired product (0.391). g, 28%) as a colorless liquid.

NMR-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 1. 25 (6H, t, J = 6.0 Hz), 1. 85 (3H, s), 2. 33 (3H, s), 4 22 (4H, q, J = 6.0 Hz), 7. 13—7.26 (5H, m)

IR (NaCl) v _max : 2983, 2939, 1730, 1617, 1515, 1463, 1375, 1253, 1105, 1070, 1021, 921, 861, 763, 742, 688, 666 cm " ¹

(2) Product synthesis

In order to know the retention time of high-performance liquid chromatography, a half ester, which is a product of the enzyme reaction, was synthesized (Fig. 4 and chemical formula 7).

1 7

Chemical formula 7 Synthesis of 2- ( _P -nitrophenyl) propionic acid From Fig. 4, 2-methyl-2-phenylmalonic acid monoethyl ester (6), 2-ethyl-2-phenylmalonic acid monoethyl ester (12), 2- Methyl-2-benzylmalonic acid monoethyl ester (13) and 2-methyl_2- (p-methylphenyl) malonic acid monoethyl ester (15) were synthesized by hydrolyzing only one of the esters using potassium hydroxide. . When the nitro compound (10) was hydrolyzed in the same manner, 2- (P-nitrophenyl) propionic acid (14) was obtained in which half ester was not obtained and decarboxylation proceeded.

In addition, a commercially available product was used for analysis of the product 2-phenylpropionic acid (7) of the reaction of substrate 5 at a high temperature.

The specific method for synthesizing the half ester was as follows.

u) Synthesis of Monoethyl 2 -methyl-2-phenyl-malonate (6)

Ethanol solution of 2-methinole-2-phenenolemalonic acid jetinolester (1.81 g, 10.9 mmol) (6 mU in aqueous potassium hydroxide (0.609 g, 7.23 mmol) ( After 6 hours, the mixture was neutralized with hydrochloric acid and aqueous sodium hydroxide solution, and the solvent was evaporated under reduced pressure, and the residue was added with an equal amount of water. After that The aqueous layer was extracted using tellurium and basified with hydrochloric acid. Furthermore, extraction was performed using jetyl ether, and the organic layer was washed with saturated brine. After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 5 I 1) to give the desired product (0.250 g, 11%) as a colorless liquid. Obtained.

Awakening (300 MHz, CDC1 ₃ ) δ (ppra): 1.25 (3H, t, J = 6.0 Hz), 1.90 (3H, s), 4.25 (2H, q, J = 6. 0 Hz), 7. 24—7. 38 (5H, m)

IR (NaCl) v _max : 3063, 2989, 2647, 1954, 1710, 1601, 1585, 1498, 1447, 1379, 1251, 1074, 1029, 1017, 930, 857, 751, 698 era— ¹

(i i) Synthesis of Monoethyl 2-ethyl-2-phenyto mlonate (12)

2-Ethyl-2-phenylmalonic acid jetyl ester (0. 500 g, 1. 89 mmol) in ethanol (3 mL) was added to potassium hydroxide (0.159 g, 2.83 mmol) in water (3 mL). And stirred at room temperature. After 6 hours, the mixture was neutralized with hydrochloric acid and aqueous sodium hydroxide solution, the solvent was distilled off under reduced pressure, and water equivalent to the residue was added. After that, the aqueous layer was extracted using JETLE TERTEL and made basic with hydrochloric acid. Furthermore, extraction was performed using jetyl ether, and the organic layer was washed with saturated brine. After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure and the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 5 I 1) to give the desired product (0.102 g, 23%) as colorless. Obtained as a liquid.

¾—NMR (300 MHz, CDC1 ₃ ) δ (ppm): 0.98 (3H, t, J = 6.0 Hz), 1.25 (3H, t, J = 6.0

Hz), 2.45 (2H, qq, J = 6.0 Hz), 4.28 (2H, qq, J = 6.0 Hz), 7. 24-7.38 (5H, m)

IR (NaCl) v _max : 2981, 2636, 1955, 1877, 1708, 1601, 1584, 1498, 1447, 1388,

1232, 1127, 1095, 1024, 857, 760, 727, 698 cm " ¹

Synthesis of (1 1 1) Monoethyl 2-methyl-2-benzyl-malonate (13)

Ethanol solution of 2_methyl-2-benzylmalonic acid jetyl ester (1.00 g, 3.78 匪 ol) (3 mU in aqueous solution of hydroxylated lithium (0.323 g, 5.76 mmol) ( After 3 hours, the mixture was neutralized using hydrochloric acid and aqueous sodium hydroxide solution, and the solvent was evaporated under reduced pressure, and the residue was added with an equal amount of water. Thereafter, the aqueous layer was extracted with jetyl ether, made basic with hydrochloric acid, extracted with jetyl ether, and the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and then decompressed. The bottom solvent was distilled off and silica gel column chromatography (hexane / acetic acid Ethyl = 1 I 1) to obtain the desired product (0.409 g, 46%) as a colorless liquid

'H-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 1. 28 (3H, t, J = 6.0 Hz), 1. 47 (3H, s), 3. 25

(2H, s), 4.23 (2H, q, J = 6.0 Hz), 7.12-7.27 (5H, m)

IR (NaCl) v _max : 3076, 2991, 1936, 1707, 1598, 1518, 1458, 1382, 1346, 1220, 1092, 925, 855, 754, 741, 698, 666 cm " ¹

(iv) Synthesis of 2- (p-Nitro-phenyl) -propionate (14)

2-Methyl-2- (p-diphenyl) malonic acid ethyl ester (1.00 g, 3.39 ramol) in ethanol (3 mL) was added to potassium hydroxide (0.399 g, 5.33). (Ol) Aqueous solution (3 mL) was added, and the mixture was stirred at room temperature. After 8 hours, the mixture was neutralized with hydrochloric acid and aqueous sodium hydroxide solution, the solvent was distilled off under reduced pressure, and water equivalent to the residue was added. Thereafter, the aqueous layer was extracted with diethyl ether and made basic with hydrochloric acid. Further, extraction was performed using Jetillertel, and the organic layer was washed with saturated saline. After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (hexane I ethyl acetate = 1 I 1) to give the desired product (0.146 g, 22%) as a colorless liquid. Obtained.

Thigh (300 MHz, CDCI3) δ (ppm): 1. 58 (3H, d, J = 6.0 Hz), 3. 89 (1H, q, J = 6.0 Hz), 7. 49-8. 22 (4H, m)

(v) Synthesis of Monoethyl 2_ (p_methyl-phenyl)-malonate (15)

2-Methyl-2- (p-methylphenyl) malonic acid jetinolester (320 mg, l. 21 ramol) in ethanol (2 mL) was added to a hydroxylated aqueous solution (102 mg, 1.82 mmol) in water (3 mL) was added and stirred at room temperature. After 6 hours, the mixture was neutralized using hydrochloric acid and aqueous sodium hydroxide solution, the solvent was distilled off under reduced pressure, and water equivalent to the residue was added. Thereafter, the aqueous layer was extracted with diethyl ether and made basic with hydrochloric acid. Further, extraction was performed using jetyl ether, and the organic layer was washed with saturated saline. After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by preparative thin-layer column chromatography (hexane I ethyl acetate I acetic acid = 2 I 1 I 0.1) to obtain the desired product (88 9 mg, 31%) was obtained as a colorless liquid.

• H-NMR (300 MHz, CDC1 ₃ ) δ (ppm): 1. 27 (3H, t, J = 6.0 Hz), 1. 89 (3H, s), 2. 34 (3H, s), 4. 27 (2H, q, J = 6.0 Hz), 7. 15-7. 27 (4H, m)

(3) Enzyme reaction procedure First, as an enzyme solution, E. coli transformed with a plasmid containing the gene encoding ST0071 was mass-cultured, and the cells were suspended in 100 mM Glycine-NaOH buffer pH 9.2, followed by ultrasonic disruption and heat treatment. The supernatant was used. Then, the substrate was added to 20 mL of the enzyme solution so as to have a concentration of 10 raM and stirred. The progress of the reaction was confirmed by thin layer chromatography. When it was confirmed that all the substrate had disappeared, the reaction solution was acidified by adding 2M HC1, and the product was extracted by a conventional method. The product was purified by preparative thin-layer chromatography and the resulting product was identified by ¹ H-NMR and IR (FIG. 5).

(4) Method for determining the absolute configuration of the product

The optically active form of the half ester product is not commercially available, and the literature values for retention time and optical rotation of high performance liquid chromatography are not known. However, the reaction with porcine liver esterase (PLE) shown in Fig. 6 has been reported (CJ Sih, J. Am. Chem. Soc., 1975, 97, 4144; J. Boutelje, S. Gatenbeck, P. Szmul). ik, Tetrahedron, 1985, 41, 1347-1352; J. Boutel je, S. Gatenbeck, P. Szmul ik, Bioorganic Chem., 1986, 14, 176-186; EJ Toone, JB Jone, Tetrahedron Asymmetry, 1991, 2 , 1041-1052; B. Klotz-Berendes, S. Koti la, Tetrahedron Asymmetry, 1997, 8, 1821-1823; B. Kossmann, 0. May, H. Groger, SYNLETT, 2005, 11, 1746-1748).

Therefore, the absolute configuration was determined by comparing the product of the reaction with PLE and the product of the reaction with this enzyme using high-speed liquid chromatography.

2. Results and discussion

(1) Study of domino-type reaction

In order to investigate whether esterase ST0071 catalyzes a domino-type reaction, an enzymatic reaction was first carried out using 2-methyl-2-phenylmalonic acid jetyl ester (5) as a substrate. At this time, the reaction temperature was examined at room temperature and 60 ° C.

As a result of thin layer chromatography analysis, spots with Rf values different from those of Substrate 5 were confirmed in the reaction solution at both temperatures, and it was confirmed that the hydrolysis reaction proceeded at both temperatures. In the reaction at 60 ° C, the number of spots estimated to be hydrolysis products increased from one to two over time. In other words, it was found that some kind of multi-step reaction progressed and a new product was generated. Therefore, after confirming that all of the substrate 5 had reacted by thin layer chromatography, the product was extracted and purified, and the resulting product was identified by ¾-NMR and IR. As a result of analysis, it was found that the product of the reaction at room temperature was 2_methyl-2-phenylmalonic acid monoethyl ester (6). In other words, it was found that only one of the diester moieties of 2-methyl-2-phenylmalonic acid jetyl ester (5) was hydrolyzed (see Formula 8).

1 8

Yield: 96% Formula 8 Results at room temperature On the other hand, the products of the reaction at 60 ° C were found to be 2-methyl-2-phenylmalonic acid monoethyl ester (6) and 2-phenylpropionic acid (7) . In other words, in the reaction at 60 ° C, it is considered that the hydrolysis reaction first occurred to produce compound 6, followed by the decarboxylation reaction to produce compound 7 (Chemical formula 9).

1 9

Yield; 27% Yield; 56% Chemical formula 9 Reaction results at 60 ΐ From chemical formulas 9 and 10, formation of compound 7 was not detected at room temperature conditions, even though the reaction time was longer than at 60 ° C . This requires activation energy that cannot be exceeded at room temperature for the decarboxylation reaction from compound 6 to compound 7. I understand.

In addition, since the yield of compound 7 is higher than that of compound 6 in the reaction at 60 ° C, it can be inferred that if the reaction time is lengthened, it is all converted to compound 7.

From the above, it was found that this enzyme can hydrolyze 2-methyl-2-phenylmalonic acid jetyl ester (5) and catalyze a domino-type reaction under high temperature conditions.

(2) Detailed analysis of products in domino-type reactions

The 2-methyl-2-phenylmalonic acid jetyl ester (5) used in the enzymatic reaction is prochiral and the resulting 2-methyl-2-phenylmalonic acid monoethyl ester (6) and 2-phenylpropionic acid (7) has an asymmetric carbon. Therefore, if these reactions occur within the active site of the enzyme, the product can be expected to be an optically active form. The absolute configuration of product 6 cannot be determined because the retention time of high performance liquid chromatography (HPLC) is not known and there is no literature value of optical rotation. Therefore, it was determined by comparing the absolute configuration of the product with porcine liver esterase (PLE).

As a result of analysis by HPLC, the peaks of the product 6 of ST0071 and the product 6 of PLE did not overlap (Fig. 7), and it was found that the product 6 of both enzymes had different absolute configurations. Therefore, it is known that the product 6 of R form can be obtained by PLE (B. Klotz-Berendes, S. Kotila, Tetrahedron Asymmetry, 1997, 8, 1821-1823). I was able to decide. The absolute configuration of 2-phenylpropionic acid (7) was determined by HPLC since the retention time of HPLC was known.

From the above, the absolute configuration and enantiomeric excess of each product by ST0071 were analyzed using HPLC (Table 1).

table 1 Results of product analysis by HPLC

Product temp ratljre ^{Yield (%) ee} ( ^{%) Confi} 9 ^uration

Table 1 shows that the product 6 at both temperatures is S-isomer with a high enantiomeric excess of 96-99% ee. Furthermore, the other product 7 in the reaction at 60 ° C was also found to be an optically active form of S form. Since two optically active products were obtained in this way, it was confirmed that enzymes were involved in this reaction.

Based on the above results and discussion, the reaction route from 2-methyl-2-phenylmalonic acid monoethyl ester (6) to 2-phenylpropionic acid (7) was predicted (Chemical Formula 10).

2 0

Prediction of reaction pathway in formula 1 0 60 The first route predicted from Chemical Formula 10 is that decarboxylation occurs, 2-phenylpropionic acid ethyl ester (16) is formed, and further ester hydrolysis occurs. The second is that ester hydrolysis takes place first, 2-methyl-2-phenylmalonic acid (17) is produced, and decarboxylation takes place. The intermediates (16, 17) of these two predicted pathways have not been detected. However, since this enzyme does not have AMDase activity, it is presumed that it is a pathway via intermediate 16. These reaction pathways can be determined by conducting an enzymatic reaction using each of the intermediates 16 and 17 and examining the progress of the reaction.

In either route, the decarboxylation reaction leads to an enol intermediate, so if proton is donated enantioselectively at the active site at this time, the optically active 2-phenylpropionic acid (7) is added. (Formula 1 1)

2 1

Chemical formula 11 1 Surface-selective proton donation during decarboxylation reaction Based on the above results, we succeeded in the construction of a new domino-type reaction with enantioselectivity at 60 ° C. Furthermore, S-form half-ester (6) was produced with a high enantiomeric excess even at room temperature.

Therefore, the substrate specificity of this enzyme was examined, and it was decided whether the domino-type reaction would proceed with other substrates.

(3) Examination of substrate specificity

By the previous section, it was found that this enzyme strongly recognizes 2-methyl-2-ferromalonic acid jetyl ester (5), which is prochiral and has large steric hindrance.

8-11 were used for the enzyme reaction. At this time, the reaction temperature was examined at room temperature. While confirming the progress of the reaction using thin layer chromatography, the reaction was stopped when all the substrates had reacted, and the product was extracted and purified. The resulting product was analyzed using -NMR, IR and HPLC (Table 2). The absolute configuration was determined by comparison with the product by PLE as in the previous section.

Table 2 Analysis results of each product

Reaction time Yield ee

Substrate Product (hour) (%) (%)

(Substrate concentration: 10 mM, reaction temperature: room temperature, number of enzyme units = 500 U)

* Unconfirmed From Table 2, the progress of the reaction was confirmed for all substrates, and the product was obtained in high yield.

(4) Estimation of reaction pathways for each substrate Table 2 shows that each product is a half ester except for the reaction to substrate 10. In other words, when substrates 5, 8, 9, and 11 were used, a reaction in which only one of the substrate diesters was hydrolyzed proceeded (Fig. 8).

On the other hand, the product of the reaction to substrate 10 was carboxylic acid 14 instead of half ester. The following results were obtained by following the reaction with TLC (Fig. 9). From Fig. 9, when all of the substrate 10 has been reacted (144 hours later), the product was only carboxylic acid 14. However, 12 hours after the start of the reaction, TLC analysis confirmed 2 Hafster 18 I was able to do it. Therefore, the reaction route of Chemical Formula 12 could be estimated.

2 2

Chemical reaction 1 2 Reaction pathway in substrate 10 In other words, when substrate 10 was used, the domino-type reaction of hydrolysis and decarboxylation was successful at room temperature.

However, when substrate 5 was used, the decarboxylation reaction did not proceed unless heat was applied. The fact that domino-type reactions occurred only at substrate 10 at room temperature can be explained by the electronic effect of the ditro group. The ditro group attached to the p-position of the phenyl group has electron withdrawing properties. Due to this electron withdrawing property, the electron density between the carbon of the substrate 10 and the carbon of the ester decreases. Therefore, it is considered that half ester 18 is very easy to decarboxylate, and the CC bond at the _α- position is quickly broken to produce carboxylic acid 14.

From the above, we succeeded in realizing the domino-type reaction not only by applying heat but also by designing the substrate in a room temperature condition.

(5) Estimation of reactivity on each substrate

From the above results, it was found that the reaction time varies greatly depending on the substrate. The biggest cause of this is thought to be the electron density of the carbonyl carbon of the ester. The electron density of the carbonyl carbon varies depending on the electronic effects of the alkyl and aryl groups. The basic skeleton of the substrate is shown in FIG. The electronic effects of the substituents are shown in Table 3.

Table 3 Electronic effects of substituents

These electronic effects are thought to affect the hydrolysis reaction as shown in Fig. 11. From Fig. 11 (a), the electron-donating substituent increases the electron density of the carbonyl carbon. This makes the carbonyl carbon less susceptible to nucleophilic attack of the enzyme's catalytic residues, reducing the reaction rate. On the other hand, in the case of electron-withdrawing substituents (Fig. 11 (b)), strong sulfonyl carbon has a reduced electron density and is susceptible to nucleophilic attack. Therefore, the reaction speed increases.

2-Ethyl-2-phenylmalonic acid jetyl ester (8) and 2-methyl-2-benzylmalonic acid jetylesterol (9) are compared to 2-methyl-2-phenylmalonate jetyl ester (5). The reaction time became longer.

Substrate 8 whose structure is shown in Fig. 12 has the methyl group of Substrate 5 substituted with an ethyl group. This suggests that the reason for the longer reaction time is that the electron donating property of the alkyl group is higher than that of the methyl group. Another possible factor is the steric hindrance of the ethyl group. The ethyl group is more sterically hindered than the methyl group, so it is presumed that it was less susceptible to hydrolysis.

Therefore, it is suggested that the influence of the reaction time is due to the high electron donating property and steric hindrance of the ethyl group.

Substrate 9 whose structure is shown in Fig. 13 has the phenyl group of Substrate 5 substituted with a benzyl group. By becoming a benzyl group, a methylene group was inserted between the phenyl group and the α carbon. This has an effect on the carbonyl carbon inserted more than the phenyl group. The electron donating property of the methylene group is larger (Fig. 13). Therefore, it is suggested that this reaction time is also caused by the electron donating property of the methylene group inserted next to the phenyl group.

Next, let us consider 2-methyl-2- (p-diphenyl) malonic acid jetyl ester (10). Substrate 10 is a substrate in which a nitro group is added to the p-position of the phenyl group of substrate 5. Since the nitro and phenyl groups are electron withdrawing, the electron density of the carbonyl carbon of this substrate is significantly reduced. Therefore, the reaction time should be shortened. However, the reaction time was longer than that of substrate 5. As described above, since the substrate 10 is a domino type reaction, the reactivity of hydrolysis cannot be compared based on the reaction time. Therefore, we compare it with the reaction of substrate 5 at 60 ° C.

Note that the reaction time is the time until all of the substrate has reacted. Then, half-ester 6 was obtained in Chemical Formula 1, while half ester 18 could not be obtained in Chemical Formula 1-4. From this, it can be inferred that in the decarboxylation reaction, the reactivity of 18 was higher than that of Ifest 6 due to the electron withdrawing property of the nitro group.

2 3

Yield; 27% Yield; 56% Chemical formula 1 3 Reaction results at 60 using substrate 5 2 4

Yield: 0% Chemical formula 1 4 Reaction results using substrate 10 Finally, let us consider 2-methyl-2- (p-methylphenyl) malonic acid jetyl ester (11). Substrate 11 is a substrate in which a methyl group is added to the p-position of the phenyl group of substrate 5. This p-position methyl group is electron donating, so the electron density of carbonyl carbon of this substrate is higher than that of substrate 5. Therefore, it was predicted that the reaction time would be longer as with substrates 8 and 9, but the reaction time was shorter (Chemical Formula 15).

2 5

Yield; 77%

From the above, the electronic effects of the substituents greatly affected the progress of the hydrolysis reaction, and it was speculated that the electron-withdrawing group could promote the decarboxylation reaction. .

(6) Enantiomeric excess and absolute configuration of each product

Table 4 Enantiomeric excess and absolute configuration of each product

(Substrate concentration; 10 mM reaction temperature; room temperature, number of enzyme units = 500 U)

* Unconsidered First, 2_ethyl-2-phenylmalonic acid monoethyl ester is the product of substrate 8

Consider (12). From Table 4, the enantiomeric excess is 25% ee, which is a low value although it is an optically active substance. On the other hand, since the enantiomeric excess of product 6 is as high as 96% ee, it is speculated that the steric hindrance of the alkyl group affects the enantioselectivity of the enzyme as described in the previous section. In addition, since the absolute configuration of product 12 was the same S-form as product 6, the size of the steric hindrance of the alkyl group is not related to the stereoselectivity of the enzyme.

Next, 2-methyl-2-benzylmalonic acid jetyl ester, the product of substrate 9 Consider (13). The reaction time using this substrate 9 was as long as that using substrate 8. However, unlike the product 12, the enantiomeric excess of the product 13 is as high as 83% ee. From this, it can be inferred that the steric hindrance of the aryl group is not related to the enantioselectivity, and that the product 13 with a high enantiomeric excess was obtained as the reaction proceeded slowly. In addition, this absolute configuration was R, which was the opposite of the absolute configuration of other products. This is presumed that the stereoselectivity of the enzyme was reversed only for substrate 9.

Next, let us consider the product of substrate 10, 2- (p-nitrotropenyl) propionic acid (14). From Table 4, the enantiomeric excess of product 14 was 2.2% ee, almost a racemate. In contrast, the enantiomeric excess of the product 2-phenylpropionic acid (7) in the reaction of substrate 5 at 60 ° C was as low as 16% ee, but it was an optically active substance. Therefore, the decarboxylation reaction that produces product 14 is not due to the enzyme, but is strongly influenced by the electronic effect.

In the above examination, the reaction conditions for the substrate 10 are alkaline, which is a condition for promoting the decarboxylation reaction. By allowing the reaction to proceed slowly under acidic conditions, the electron effect of the nitro group <the enzyme's ability to recognize the substrate, and an increase in the enantiomeric excess can be expected.

Here, the absolute configuration of the product of this enzyme and the product of PLE (CJ Sih, J. Am. Chem. Soc., 1975, 97, 4144; J. Boutelje, S. Gatenbeck, P. Szmul ik, Tetrahedron , 1985, 41, 1347-1352; J. Boutelje, S. Gatenbeck, P. Szmul ik, Bioorganic Chem., 1986, 14, 176-186; EJ Toone, JB Jone, Tetrahedron Asymmetry, 1991, 2, 1041-1052 B. Klotz-Berendes, S. Koti la, Tetrahedron Asymmetry, 1997, 8, 1821-1823; B. Kossraann, 0. May, H. Groger, SYNLETT, 2005, 11, 1746-1748) (Table) Five ).

Table 5 Absolute configuration of product by ST0071 and PLE

Configuration

* Not yet examined Table 5 shows that the absolute configuration of the products of both enzymes is different. This suggests that the stereoselectivity of esterase ST0071 and PE is opposite. Therefore, the absolute configuration of 2-methyl-2_ (p-methylphenyl) malonic acid monoethyl ester (15) can be assumed to be S.

From the above, it was found that esterase ST0071 showed the same stereoselectivity with respect to all the substrates used in this example, and that stereoselectivity was reversed with PLE. The size of the steric hindrance of the alkyl group was found to affect the enantiomeric excess. Esterase ST0071 was found to hydrolyze α-arylmalic acid ketyl ester, which is highly sterically hindered and hardly hydrolyzed, with high stereoselectivity to produce a half ester. Furthermore, by carrying out the enzymatic reaction at a high temperature, the production of carboxylic acid, which was thought to have progressed through decarboxylation due to hydrolysis, was confirmed. In other words, a domino-type reaction was successfully constructed under high temperature conditions (Chemical Formula 16).

2 6 Esterase ST0071

_Ar , C0 ₂ Et Room temperature or _{6 0 ¾} 'Ar-;'"" C ^C 02 ₂ 2EEtt _{6 0.} _{C Ar} / "C0 ₂ H C0 ₂ Et C0 ₂ HH

Results of enzyme reaction at room temperature and high temperature It was also found that the domino-type reaction can be realized by designing a substrate having an electron-attracting substituent such as a nitro group.

It was suggested that the steric hindrance of the alkyl group affects the reaction progress and stereoselectivity at room temperature. Similarly, it was suggested that the electron density of carbonyl carbon affects the progress of the reaction.

It was found that the stereoselectivity of esterase ST0071 was reversed from that of PLE. Example 2 Production of optically active carboxylic acid using purified esterase ST0071

E. coli BL21 Star a (DE3) and Development $ quality transformed with plasmid PETstEst, in 10 ml of LB medium (containing 50 μ _§ / ηι1 ampicillin), and cultured overnight at 37 ° C. It was transferred to 1000 ml of LB medium and further cultured at 30 ° C for 5 hours. For induction of enzyme expression

0. IPTG was added so that the concentration was 1 mM, and the cells were further cultured for 5 hours. The culture solution was centrifuged (8500 rpra, 10 min, 4 ° C) to collect the cells. The obtained cells were suspended in 40 ml of 20 raM, pH 7.4 phosphate buffer solution (containing 0.5 NaCl and 20 mM Imidazole). Cell-free extracts were obtained by sonicating the cells (15 min, 0 ° C) and centrifuging (12000 rpm, 20 min, 4 ° C). Discard the supernatant at 60 ° C for 25 minutes and centrifuge (12000 rpm, 20 min,

Proteins denatured by 4 ° C) were removed. The obtained crude enzyme solution was HiTrap Chelating.

After adsorbing on HP (made by Araersham Biosciences) Ni ²⁺ Chelating affinity column, it was washed with a phosphate buffer solution containing 0.5 M NaCl and 20 mM Imidazole. Then

Elution was performed with a 20 mM phosphate buffer solution containing 0.5 M NaCl and 0.5 M Imidazole. Fractions containing esterase activity were collected and dialyzed to obtain a uniform enzyme.

A substrate (phenylmethylmalonic acid ester) and enzyme (18 U) were added to a 50 mL eggplant flask so that the substrate concentration was 2 raM, and the mixture was stirred at 70 ° C. for 2 days to carry out the reaction. After 2M hydrochloric acid was added to complete the reaction, extraction was performed with ethyl acetate according to a conventional method. Solvent under reduced pressure The residue was purified by preparative thin-layer column chromatography (hexane / ethyl acetate I acetic acid = 2 I 1 I 0.1), and the product (hyphenylpropionic acid) Got.

Yield 95%, Optical purity 48% e.e., Absolute configuration S body Industrial applicability

By using esterase derived from Sulfolobus tokodai i strain 7, a half-ester with the opposite configuration to that obtained with PLE was obtained. Furthermore, a domino-type reaction was successfully performed by using a thermophilic enzyme. A continuous reaction that takes place in one reaction vessel is called a domino-type reaction. This is the first time such an example.

By using the method or catalyst composition of the present invention, an optically active carboxylic acid serving as an intermediate for pharmaceuticals and agricultural chemicals can be produced from a dicarboxylic acid ester.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims

1. A method for producing an optically active carboxylic acid using a dicarboxylic acid ester as a raw material, wherein a sulfolobus microorganism-derived esterase is allowed to act on the dicarboxylic acid ester to produce an optically active carboxylic acid by a one-step domino reaction.

2. The method for producing an optically active carboxylic acid according to claim 1, wherein the esterase derived from a microorganism belonging to the genus Sulfolobus is an esterase derived from Sulfolobus tokodai i.

3. A process for producing optically active rubonic acid from a dicarboxylic acid ester as a raw material, wherein the following (a) or (b) esterase is allowed to act on the dicarboxylic acid ester and optical activity is achieved by a one-step domino reaction. Method for producing carboxylic acid:

4. The starting dicarboxylic acid ester is a compound represented by the following chemical formula I:

1

R -COOR ³

, C

, \

R ² COOFT Chemical Formula I

[Wherein R ¹ and R ² are different from each other, and are substituted or unsubstituted alkyl groups; substituted or unsubstituted aryl groups or aralkyl groups; substituted or unsubstituted cycloalkyl groups; Represents a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted alkoxy group, and R ⁴ independently represents H or an alkyl group. ]

The method for producing optically active power / reponic acid according to any one of claims 1 to 3, wherein the obtained optically active carboxylic acid is a compound represented by the following chemical formula III.

2

5. The starting dicarboxylic acid ester is a compound represented by the following chemical formula I:

Chemical 3

Formula I

[Wherein, R ¹ and R ² are different from each other, and each represents a phenyl group-substituted or unsubstituted alkyl group, a C 1-5 alkyl group-substituted or unsubstituted aryl group, R ³ and R ⁴ independently represent H or an alkyl group having 1 to 5 carbon atoms. 5. The method for producing an optically active carboxylic acid according to claim 4, wherein the obtained optically active carboxylic acid is a compound represented by the following chemical formula III.

4

/

R ² COOH chemical formula in

6. The method for producing an optically active carboxylic acid according to claim 4, wherein the dicarboxylic acid ester as a raw material is an allylic carboxylic acid ester, and the obtained optically active carboxylic acid is an α-aryl alkanoic acid.

7. The method for producing an optically active carboxylic acid according to claim 4, wherein the dicarboxylic acid ester used as a raw material is an allylic malonic acid ester, and the obtained optically active carboxylic acid is α-aryl propionic acid.

8. The starting dicarboxylic acid ester is 2-methyl-2-phenylmalonic acid ester or 2-methyl-2-benzylmalonic acid diester, and the resulting optically active carboxylic acid is 2_phenylpropionic acid or 2 A method for producing an optically active carboxylic acid according to claim 4, which is benzylpropionic acid.

9. The method for producing an optically active carboxylic acid according to any one of claims 1 to 8, wherein the reaction between the dicarboxylic acid ester and the esterase derived from the genus Sulfolobus is performed at room temperature to 80 ° C.

1 0. An optically active carboxylic acid is produced in one step from a dicarboxylic acid ester, which is a raw material substrate, by catalyzing a domino-type reaction having enantioselectivity, which contains an esterase derived from a microorganism of the genus Sulfolobus Catalyst composition for carboxylic acid production.

11. The composition for producing an optically active carboxylic acid according to claim 10, wherein the sterase derived from a microorganism belonging to the genus Sulfolobus is a sterase derived from Sulfolobus tokodai i.

1 2. Catalyze an enantioselective domino-type reaction containing the following esterase (a) or (b) as an active ingredient, and form an optically active carboxylic acid in one step from a dicarboxylic acid ester as a raw material substrate. The resulting catalyst composition for producing optically active carboxylic acid:

1 3. A method for producing an optically active malonic acid half ester by stereoselective hydrolysis using a dicarboxylic acid ester as a raw material, which comprises reacting a dicarboxylic acid ester with an esterase derived from a microorganism belonging to the genus Sulfolobus, A method for producing an optically active malonic acid half ester, wherein the absolute configuration of the resulting malonic acid half ester is opposite to that of a malonic acid half ester obtained using porcine liver esterase (PLE).

14. The method for producing an optically active malonic acid half ester according to claim 13, wherein the esterase derived from a microorganism belonging to the genus Sulfolobus is a esterase derived from Sulfolobus tokodaii.

15 5. Production of optically active malonic acid half ester by stereoselective hydrolysis using dicarboxylic acid ester as a raw material, which comprises reacting dicarboxylic acid ester with esterase (a) or (b) below A method for producing an optically active malonic acid half ester, wherein the absolute configuration of the resulting malonic acid half ester is opposite to that of malic acid half ester obtained using porcine liver esterase (PLE) :

1 6. The dicarboxylic acid ester used as a raw material is a compound represented by the following chemical formula I,

5-

Formula I

The optically pure malonic acid half ester obtained is a compound represented by the following chemical formula II, The method for producing an optically active malonic acid half ester according to any one of claims 13 to 15 .

Formula II [Where I? ¹ , R ² , R ³ and R ⁴ are the same as the compound represented by the above chemical formula I. ]

17. The method for producing an optically active malonic acid half ester according to any one of claims 13 to 16, wherein the reaction between the dicarboxylic acid ester and the esterase derived from Sulfolobus genus microorganism is performed at room temperature.