JP2008194037A - Method for optical resolution of 4-halo-3-hydroxybutyric acid ester by biocatalyst - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing the S-isomer of a 4-halo-3-hydroxybutyric acid ester having high optical purity from a mixture of both enantiomers of a 4-halo-3-hydroxybutyric acid ester, a method for producing the R-isomer of 3-hydroxy-γ-butyrolactone from the R-isomer of a 4-halo-3-hydroxybutyric acid ester, an enzyme CHBH (4-chloro-3-hydroxybutyrate hydrolase) derived from microorganisms and usable for the production method, and a gene encoding the enzyme. <P>SOLUTION: The method uses (a) a specific base sequence or (b) a base sequence hybridizable with the specific base sequence under a stringent condition and encoding a protein having RhCHBH enzyme (Rhizobium-derived 4-chloro-3-hydroxybutyrate hydrolase) activity. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to optically active S-form 4-halo-3-hydroxybutyrate and R-form 3-hydroxy-γ that can be useful intermediates in the synthesis of optically active compounds such as pharmaceuticals, agricultural chemicals, physiologically active substances and ferroelectric liquid crystals. -It relates to a method for producing butyrolactone by asymmetric hydrolysis using microorganisms, processed products thereof, microorganism-derived enzymes or genes, microorganism-derived enzymes used in these methods, and genes encoding the enzymes.

  As a method for producing an optically active compound, a chemical synthesis method in which a corresponding optically active starting material is converted into a target product, and a method in which a corresponding racemic body is treated with an optical resolution agent to be resolved into an optically active material are used. In recent years, many methods for separating optically active substances by biocatalytic reaction using asymmetric reduction or asymmetric hydrolysis reaction using microorganisms or enzymes have been reported.

  As a method for producing 4-halo-3-hydroxybutyric acid ester, a method by asymmetric reduction using a ruthenium-optically active phosphine complex as a catalyst (Patent Document 1) is known as a synthesis method. The synthesis of is not easy and has a problem in terms of cost. In addition, a method of reduction using an optically active amino acid derivative and an alcohol (Patent Document 2) is known, but has a disadvantage that the optical purity of the product is low, and the reaction is performed at a low temperature such as −78 ° C. There is a problem of having to.

  Further, as a production method using a biocatalytic reaction of 4-halo-3-hydroxybutyric acid ester, an asymmetric reduction method of a β-ketoester using a microbial cell or an enzyme (Patent Documents 3, 4, and 5) is known. In addition, improvement of the asymmetric reduction method by gene recombination technology has been carried out (Patent Document 6). However, in the production of S-form 4-halo-3-hydroxybutyrate from β-ketoester by an asymmetric reduction method using these microorganisms or enzymes, expensive NADH (nicotinamide adenine dinucleotide) or Since a coenzyme such as NADPH (nicotinamide adenine dinucleotide phosphate) is required and a reaction for converting the oxidized form into a reduced form is required, an enzyme such as glucose oxidase or formate dehydrogenase is required. From the viewpoint that the reaction process becomes a rate-limiting reaction, it is difficult to say that it is an industrial production method.

  In addition, a method for producing S-form 4-chloro-3-hydroxybutyric acid ester by asymmetric decomposition reaction of both enantiomeric mixtures of 4-halo-3-hydroxybutyric acid ester using microorganisms or enzymes is reported (Non-patent Documents 1 and 2). However, these production methods cannot produce products with high optical purity. In addition, in the method using Agrobacterium (Patent Document 7), there is no report of an enzyme or a gene involved in this reaction, so the improvement in productivity is limited to the improvement in wild strains.

The optically active 3-hydroxy-γ-butyrolactone was produced by asymmetric reduction of ethyl-4-t-butoxy-3-oxobutyric acid using baker's yeast, and the resulting R-form ethyl-4-t-butoxy- A method of deriving R-form 3-hydroxy-γ-butyrolactone from 3-hydroxybutyric acid in the presence of fluoroacetic acid (Non-patent Document 3) has been reported. It is difficult to obtain a stable product with high optical purity. Further, a method for producing a corresponding steric 3-hydroxy-γ-butyrolactone from optically active 4-halo-3-hydroxybutyric acid ester using a microorganism (Patent Document 8) has been reported. Stereoselectivity is not disclosed. Furthermore, since there are no reports of enzymes or genes involved in this reaction, the improvement in productivity is limited to the improvement in wild strains.
Japanese Patent Laid-Open No. 1-211551 JP-A-61-155354 JP-A-61-146191 Japanese Patent Application Laid-Open No. 6-209782 Japanese Patent Laid-Open No. 11-187869 JP 2000-236883 A JP-A-9-322786 JP 2002-204699 A Tetrahedron: Asymmetry 12 pp545-556, 2001 Tetrahedron: Asymmetry 7 pp3109-3112, 1996 Seebach, Dieter; Eberle, Martin, Synthesis (1), pp37-40, 1986

  An object of the present invention is to solve the above-mentioned problems, and a 4-halo-3-hydroxybutyric acid ester is obtained by an asymmetric hydrolysis reaction using a biocatalyst with a microorganism, an enzyme derived from a microorganism or a gene encoding the enzyme. A method for producing S-isomer 4-halo-3-hydroxybutyric acid ester with high optical purity from a mixture of both enantiomers of the above, and production of R-isomer 3-hydroxy-γ-butyrolactone from R-isomer 4-halo-3-hydroxybutyric acid ester It is an object of the present invention to provide a microorganism-derived enzyme used in these production methods, and a gene encoding the enzyme.

The present invention also improves the stereo-discriminating ability by using a recombinant DNA technique, can dramatically increase the catalytic ability of microorganisms as compared with the prior art, and can mass-produce optically active substances. An object is to provide a halo-3-hydroxybutyrate asymmetric hydrolase (RhCHBH (Rhizobium sp. 4-Chloro-3-HydroxyButyrate Hydrolase)) and a gene encoding the same.
Another object of the present invention is to provide a vector containing the gene, a transformant, and a method for producing an optically active substance using the enzyme.

  The present inventors have conducted various studies to find a method for industrially producing S-form 4-halo-3-hydroxybutyrate ester and R-form 3-hydroxy-γ-butyrolactone. As a result, certain microorganisms ( Accession No. FERM P-21129) shows a highly selective reactivity with respect to the R form in a mixture of both enantiomers of 4-halo-3-hydroxybutyrate, and converts it to 3-hydroxy-γ-butyrolactone, It was found that S-form 4-chloro-3-hydroxybutyric acid ester remains.

  Further, it has been found that in this reaction by microorganisms, R-form 3-hydroxy-γ-butyrolactone can be obtained without degrading the optical purity by using R-form 4-halo-3-hydroxybutyrate as a starting material.

  Furthermore, since the microorganism (Accession No. FERM P-21129) has low reactivity, a gene encoding an enzyme that catalyzes the reaction (hereinafter also referred to as RhCHBH) (hereinafter also referred to as RhCHBH gene) is isolated and recombined. We succeeded in improving productivity with E. coli.

式[１]
［式中、Ｘは、ハロゲン原子を表し、Ｒは炭素原子数１〜４のアルキル基を表す］。 Therefore, the present invention provides a gene comprising the following base sequence (a) or (b):
(A) a nucleotide sequence consisting of the 44th base to the 1240th base of SEQ ID NO: 1,
(B) a 4-halo-3-hydroxybutyric acid represented by the following formula [1], which is hybridized under stringent conditions with the nucleotide sequence consisting of the 44th to 1240th nucleotides of SEQ ID NO: 1 A nucleotide sequence encoding a protein that acts on an enantiomeric mixture of esters, selectively hydrolyzes the R-form of the mixture, and selectively leaves the S-form (hereinafter also referred to as RhCHBH activity):

Formula [1]
[Wherein X represents a halogen atom and R represents an alkyl group having 1 to 4 carbon atoms].

The present invention also provides
(C) a base sequence consisting of the 44th to 1240th bases of SEQ ID NO: 1, wherein one or several bases are deleted, added or substituted, and encodes a protein having RhCHBH activity Base sequence,
(D) a base sequence encoding the amino acid sequence set forth in SEQ ID NO: 2,
(E) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2, and a base sequence encoding an amino acid sequence having RhCHBH activity; and
(F) an amino acid sequence having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2, and a base sequence encoding an amino acid sequence having RhCHBH activity;
The gene which consists of one of these base sequences is included.

The present invention also provides the following protein (a ′) or (b ′):
(A ′) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2,
(B ′) a protein having an RhCHBH activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2;

The present invention also provides
(C ′) A protein comprising an amino acid sequence having at least 75% homology with the amino acid sequence shown in SEQ ID NO: 2 and having RhCHBH activity is also included.

  The present invention also provides a bacterium having the above gene, preferably a bacterium DS-S-51 strain (accession number FERM P-21129) presumed to belong to the genus Rhizobium.

  Moreover, according to this invention, the vector containing the gene of this invention is provided. Preferably the vector is the plasmid pKK-RhCHBH. Here, the plasmid pKK-RhCHBH refers to a plasmid obtained by introducing the RhCHBH gene of the present invention into a commercially available plasmid pKK223-3 (manufactured by Pharmacia).

  Moreover, according to this invention, the transformant containing said vector is provided. Preferably, the host is E. coli, more preferably E. coli strain JM109.

  Moreover, this invention provides the manufacturing method of RhCHBH protein including expressing a RhCHBH gene.

式[１]
［式中、Ｘは、ハロゲン原子を表し、Ｒは炭素原子数１〜４のアルキル基を表す］。 Furthermore, the present invention allows RhCHBH protein to act on an enantiomeric mixture of 4-halo-3-hydroxybutyrate ester represented by the following formula [1], selectively hydrolyze the R form of the mixture, and S form A method for producing S-form 4-halo-3-hydroxybutyric acid ester, which comprises selectively remaining:

Formula [1]
[Wherein X represents a halogen atom and R represents an alkyl group having 1 to 4 carbon atoms].

式[１]
［式中、Ｘは、ハロゲン原子を表し、Ｒは炭素原子数１〜４のアルキル基を表す］。

式[２] Furthermore, the present invention includes the action of RhCHBH protein on the R form of 4-halo-3-hydroxybutyric acid ester represented by the following formula [1], R-form 3-hydroxy- represented by the following formula [2] A method for producing γ-butyrolactone is provided:

Formula [1]
[Wherein X represents a halogen atom and R represents an alkyl group having 1 to 4 carbon atoms].

Formula [2]

  In the method for producing the S-form 4-halo-3-hydroxybutyrate and the method for producing the R-form 3-hydroxy-γ-butyrolactone, R in the formula [1] is preferably methyl, and preferably in the formula [1]. X is a chlorine atom.

  According to the present invention, a microorganism (DS-S-51 strain (Accession No. FERM P-21129)) presumed to belong to Rhizobium sp. Is utilized to produce 4-halo-3-hydroxybutyric acid ester. The R-form 4-halo-3-hydroxybutyrate can be selectively decomposed from the mixture of both enantiomers to leave the S-form 4-halo-3-hydroxybutyrate in the system. Further, when R-form 4-halo-3-hydroxybutyrate is used as a substrate, R-form 3-hydroxy-γ-butyrolactone can be obtained without degrading the optical purity. Furthermore, productivity can be improved by using recombinant E. coli.

  In this specification, the “biocatalytic reaction” refers to a reaction using a living body including a microorganism, an enzyme, a gene recombinant, and the like.

  In the present specification, the “asymmetric hydrolysis reaction” refers to an optical resolution method using a difference in hydrolysis rate for each optically active (isomer) isomer in both enantiomeric mixtures.

  In the present specification, the “4-halo-3-hydroxybutyric acid ester compound group represented by the formula [1]” means that in the formula [1], X represents a halogen atom and R has 1 to 4 carbon atoms. Includes all compounds representing alkyl groups.

式[１]
とは、式[１]で示される４−ハロ−３−ヒドロキシ酪酸エステル化合物群に属する少なくともいずれか一つの化合物のエナンチオマー混合物としてラセミ体を基質として用いた場合に、出発物質として用いたエナンチオマー混合物のうちＲ体基質の少なくとも９５％を加水分解し、Ｓ体基質の少なくとも２０％を残存させ、その結果、光学純度が９０％ｅｅ（エナンチオ過剰）以上のＳ体エステルを生成物として生じる酵素活性をいう。該酵素は、Ｒ体基質の好ましくは少なくとも９７％以上、さらに好ましくは９９％以上、特に好ましくは９９．５％以上加水分解する。また、該酵素は、Ｓ体基質の好ましくは４０％以上、さらに好ましくは５０％以上、特に好ましくは６０％以上を残存させる。さらに、該酵素による反応の結果得られるＳ体エステルは、好ましくは９５％ｅｅ以上、さらに好ましくは９８％ｅｅ以上、特に好ましくは９９％ｅｅ以上の光学純度である。なお、式[１]で示される化合物としては、Ｘが塩素原子であるものが好ましく、Ｒがメチルのものが好ましい。 In the present specification and claims, “acting on an enantiomeric mixture of 4-halo-3-hydroxybutyric acid ester represented by the following formula [1], selectively hydrolyzing the R form of the mixture; and Enzyme activity to selectively leave S-form "

Formula [1]
Is a mixture of enantiomers used as a starting material when a racemate is used as a substrate as an enantiomer mixture of at least one compound belonging to the 4-halo-3-hydroxybutyric acid ester compound group represented by the formula [1] Enzyme activity that hydrolyzes at least 95% of the R-form substrate and leaves at least 20% of the S-form substrate, resulting in an S-form ester having an optical purity of 90% ee (enantio-excess) or higher as a product Say. The enzyme preferably hydrolyzes at least 97% or more, more preferably 99% or more, particularly preferably 99.5% or more of the R-form substrate. In addition, the enzyme preferably leaves 40% or more, more preferably 50% or more, particularly preferably 60% or more of the S-form substrate. Furthermore, the S-form ester obtained as a result of the reaction with the enzyme preferably has an optical purity of 95% ee or more, more preferably 98% ee or more, and particularly preferably 99% ee or more. As the compound represented by the formula [1], those in which X is a chlorine atom are preferable, and those in which R is methyl are preferable.

  In the present specification and claims, “acting on an enantiomeric mixture of 4-halo-3-hydroxybutyric acid ester represented by the following formula [1], selectively hydrolyzing the R form of the mixture; and “Selectively leaving the S-form” means that the racemate was used as a substrate as an enantiomeric mixture of at least one compound belonging to the 4-halo-3-hydroxybutyric acid ester compound group represented by the formula [1]. In this case, it means that at least 95% of the R-form substrate of the enantiomeric mixture used as the starting material is hydrolyzed to leave at least 20% of the S-form substrate. Preferably, at least 97%, more preferably 99%, particularly preferably 99.5% or more of the R-form substrate is hydrolyzed. Further, preferably 40% or more, more preferably 50% or more, particularly preferably 60% or more of the S-form substrate remains.

式[１]

式[２] In the present specification and claims, “without degrading optical purity” means R of at least one compound belonging to the 4-halo-3-hydroxybutyric acid ester compound group represented by the following formula [1]. In the process for producing R-form 3-hydroxy-γ-butyrolactone represented by the following formula [2], which comprises acting on the body, R-form 4-halo-3-hydroxybutyric acid having an optical purity of 90% ee or more as a substrate When ester is used, it means to obtain R-form 3-hydroxy-γ-butyrolactone having an optical purity of 90% ee or higher as a product. The optical purity of 4-halo-3-hydroxybutyric acid ester of the R form of the substrate is preferably 95% ee, more preferably 98% ee, particularly preferably 99% ee or more. The optical purity of -hydroxy-γ-butyrolactone is preferably 95% ee, more preferably 98% ee, particularly preferably 99% ee or higher.

Formula [1]

Formula [2]

In the present specification and claims, “microorganism, processed product”
1) microorganism culture solution,
2) A microorganism obtained by centrifugation and / or a treated product thereof (a crushed cell or a cell extract) or 3) 1) and 2) immobilized by a conventional method.
In the present specification and claims, “acting a protein on an enantiomeric mixture of 4-halo-3-hydroxybutyrate” does not only cause the protein itself to act on a substrate, but also a microorganism, treatment thereof Or a transformant containing the gene according to the present invention is allowed to act on a substrate.

  In the present specification and claims, “high optical purity” means 90% e.e. e. It means that it is (enantio excess) or more. The high optical purity is preferably 95% ee, more preferably 98% ee, and particularly preferably 99% ee or more.

式[１]
［式中、Ｘは、ハロゲン原子を表し、Ｒは炭素原子数１〜４のアルキル基を表す］。
（ｃ）配列番号１の第４４塩基〜第１２４０塩基からなる塩基配列において、１または数個の塩基が欠失、付加または置換されている塩基配列であって、ＲｈＣＨＢＨ活性を有するタンパク質をコードする塩基配列、
（ｄ）配列番号２に記載のアミノ酸配列をコードする塩基配列、
（ｅ）配列番号２に記載のアミノ酸配列において１または数個のアミノ酸が欠失、置換または付加されているアミノ酸配列であって、ＲｈＣＨＢＨ活性を有するアミノ酸配列をコードする塩基配列、および、
（ｆ）配列番号２に記載のアミノ酸配列と少なくとも７５％以上の相同性を有するアミノ酸配列であって、ＲｈＣＨＢＨ活性を有するアミノ酸配列をコードする塩基配列、
のいずれかの塩基配列からなる遺伝子が提供される。 Hereinafter, embodiments of the present invention will be described in detail.
According to the present invention, the following base sequence:
(A) a nucleotide sequence consisting of the 44th base to the 1240th base of SEQ ID NO: 1,
(B) a 4-halo-3-hydroxybutyric acid represented by the following formula [1], which is hybridized under stringent conditions with the nucleotide sequence consisting of the 44th to 1240th nucleotides of SEQ ID NO: 1 A base sequence encoding a protein that acts on an enantiomeric mixture of esters, selectively hydrolyzes the R form of the mixture, and selectively leaves the S form (hereinafter also referred to as RhCHBH activity);

Formula [1]
[Wherein X represents a halogen atom and R represents an alkyl group having 1 to 4 carbon atoms].
(C) a base sequence consisting of the 44th to 1240th bases of SEQ ID NO: 1, wherein one or several bases are deleted, added or substituted, and encodes a protein having RhCHBH activity Base sequence,
(D) a base sequence encoding the amino acid sequence set forth in SEQ ID NO: 2,
(E) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2, and a base sequence encoding an amino acid sequence having RhCHBH activity; and
(F) an amino acid sequence having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2, and a base sequence encoding an amino acid sequence having RhCHBH activity;
A gene comprising any of the nucleotide sequences is provided.

  In the present invention, “one or several amino acids are deleted, substituted or added” means, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 Means any number of ~ 5 amino acids have been deleted, substituted or added. The phrase “one or several bases are deleted, added or substituted” means, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, and further preferably 1 to 5. It means that any number of bases has been deleted, substituted or added.

  In the present invention, “RhCHBH” is a protein having the amino acid sequence of SEQ ID NO: 2, and its molecular weight is 42 kDa as measured by SDS-PAGE.

  In the present invention, “stringent conditions” refers to conditions under which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions are usually about 0.2xSSC, 0.1% SDS, 65 ° C. The DNA obtained by hybridization preferably has a high homology of 70% or more with the DNA comprising the base sequence (a), and preferably has a homology of 80% or more. Hybridization can be performed according to the method described in Molecular Cloning: A laboratory Mannual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989.).

  In the present invention, “homology” means the degree of sequence similarity between two polypeptides or polynucleotides, and is in an optimum state over the region of the amino acid sequence or base sequence to be compared (maximum sequence match). Determined by comparing two sequences aligned to the (state). The homology number (%) determines the number of matching sites by determining the same amino acid or base present in both (amino acid or base) sequences, and then the number of matching sites within the sequence region to be compared. Divided by the total number of amino acids or bases and multiplied by 100. Examples of algorithms for obtaining optimal alignment and homology include various algorithms (for example, BLAST algorithm, FASTA algorithm, etc.) that are usually available to those skilled in the art. The homology of amino acid sequences is determined using sequence analysis software such as BLASTP and FASTA. The base sequence homology is determined using software such as BLASTN and FASTA.

  The method for obtaining the gene of the present invention is not particularly limited. The gene of the present invention can be obtained by cloning from the chromosome of a microorganism producing a desired enzyme, or by synthesis using a DNA synthesizer, and the form thereof may be either single-stranded or double-stranded.

  As a chromosome donor microorganism when obtained by cloning, a bacterial DS-S-51 strain (accession number FERM P-21129) presumed to belong to the genus Rhizobium can be mentioned as a suitable one.

  The DNA cloning of the present invention is not particularly limited, and various methods can be used. The shotgun cloning method shown in the examples is preferably used.

  In addition, the gene of the present invention may be prepared by preparing appropriate probes and primers based on the nucleotide sequence set forth in SEQ ID NO: 1 or the amino acid sequence set forth in SEQ ID NO: 2, and using them. It can also be isolated by screening a predicted DNA library.

  For example, a DNA fragment having the RhCHBH gene can be obtained from the gene donor based on the partial amino acid sequence of the polypeptide chain of the purified enzyme RhCHBH. That is, the purified enzyme is digested with endopeptidase, a part of the amino acid sequence of each fragment is determined with a protein sequencer, and a primer for polymerase chain reaction (PCR) is synthesized based on this. Next, PCR was performed using the chromosomal DNA of the bacterial DS-S-51 strain (Accession No. FERM P-21129) presumed to belong to the genus Rhizobium as a template, and a part of the RhCHBH gene was amplified to reveal its base sequence. To. Using the obtained partial base sequence of the RhCHBH gene as a probe, a DNA fragment of the target gene can be obtained from a DNA library prepared by a conventional method from the chromosomal DNA of the gene donor using a hybridization method.

  Examples of the method for determining the DNA base sequence of the obtained RhCHBH gene include the dideoxy sequencing method. This method includes various methods commonly used in the field of genetic engineering, such as a method of amplifying a gene by PCR and a method of deleting by a nucleolytic enzyme. By these methods, an open reading frame (ORF) encoding all amino acids in the target DNA base sequence represented by SEQ ID NO: 1 can be confirmed.

The DNA encoding RhCHBH of the present invention is characterized in that the amino acid sequence having RhCHBH activity substantially comprises a base sequence encoding the polypeptide shown in SEQ ID NO: 2. Here, as long as it has RhCHBH activity, the amino acid sequence shown in SEQ ID NO: 2 may have one or several amino acid deletions, insertions, substitutions, and the like. For example, modification of DNA to cause some deletion, insertion, substitution, etc. of amino acids in the amino acid sequence encoded by DNA is performed by a known method such as site-directed mutagenesis using a synthetic oligonucleotide. It can be performed appropriately. In addition, using the DNA shown in SEQ ID NO: 1 or a DNA obtained by appropriately modifying the DNA as a template, in the presence of Mn ²⁺ ions (usually a concentration of 0.5 to 10 mM) or a specific nucleotide concentration is lowered. By carrying out the PCR method, it is possible to obtain DNA into which mutations are randomly introduced. It goes without saying that the DNA encoding the protein having RhCHBH activity among the DNAs thus obtained is included in the present invention.

The present invention also provides the following protein:
(A ′) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2,
(B ′) a protein having an RhCHBH activity consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2;
(C ′) A protein comprising an amino acid sequence having at least 75% homology with the amino acid sequence shown in SEQ ID NO: 2 and having RhCHBH activity is provided.

  Here, the “amino acid sequence having at least 75% homology with the amino acid sequence described in SEQ ID NO: 2” is not particularly limited as long as the homology with the amino acid sequence of SEQ ID NO: 2 is 75% or more, For example, it means 75% or more, preferably 80% or more, more preferably 85% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.

  The method for purifying the enzyme of the present invention is not particularly limited, but it can be purified by appropriately combining ordinary protein purification methods. For example, after disrupting the cells with ultrasound, salting out with ammonium sulfate, and combining the precipitate lysate with hydrophobic chromatography, anion exchange chromatography, and gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis It can be purified to single by electrophoresis (hereinafter abbreviated as SDS-PAGE).

  Furthermore, the present invention provides a vector comprising a nucleotide sequence of a gene encoding an amino acid sequence exhibiting RhCHBH activity.

  A cloning vector such as pUC or pBluescript (both commercially available from Takara Bio Inc.) is not particularly limited, and can be used depending on the type of host. Preferably pUC19 is used.

  Recombinant DNA by incorporating a structural gene encoded by a DNA fragment having the above base sequence (SEQ ID NO: 1) into a vector having a functional region such as a promoter enabling the expression of the enzyme and a replication origin in the host Recombinant DNA for obtaining a transformant having this asymmetric hydrolysis reaction using the technology can be constructed.

  The expression vector is not particularly limited, such as a pUC system, a pBluescript system (Takara Bio Inc., etc.), a pET system (Invitrogen), and can be used depending on the type of host. Preferred examples of the expression vector include those that are capable of autonomous replication in a host cell and that are further provided with an appropriate selection marker that can be used to select only recombinant host cells. The gene of the present invention can be expressed. Further, such a vector may be one that can be easily produced by a trader using a known technique from a known vector or the like, or may be commercially available. Particularly preferred is pKK223-3 (Pharmacia).

  By incorporating a necessary part from a DNA fragment carrying genetic information of the RhCHBH gene into a vector, it can be introduced into a host cell to obtain a transformant.

  Any host cell can be used without particular limitation as long as it is transformed with the obtained recombinant vector and can express the present gene. The expression host can be used without being limited by microorganisms, plants, animals and the like as long as expression of the gene can be achieved in accordance with the object of the present invention. The microorganism is selected from, for example, Escherichia coli, Enterobacter, Saccharomyces, Xanthomonas, Acetobacter, Pseudomonas, Gluconobacter, Azotobacter, Rhizobium, Klebsiella, Salmonella and Serratia Among them, E. coli is preferable, and E. coli JM109 is particularly preferable. E. coli JM109 (pKK-RhCHBH) has been deposited as FERM P-21128 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center.

  Transformants can be obtained in large quantities by culturing in an appropriate nutrient medium. The medium used for the culture may be any normal medium containing a carbon source, a nitrogen source, an inorganic substance, and the like necessary for the growth of microorganisms. For example, carbohydrates such as glucose and fructose as a carbon source, alcohols such as glycerol, mannitol, sorbitol, and propylene glycol, organic acids such as acetic acid, citric acid, malic acid, maleic acid, fumaric acid, gluconic acid and salts thereof, or the like Can be used. Examples of the nitrogen source include inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate, and ammonium phosphate, and organic nitrogen compounds such as urea, peptone, casein, yeast extract, meat extract, corn steep liquor, and mixtures thereof. In addition, vitamins such as phosphates, magnesium salts, potassium salts, manganese salts, iron salts, zinc salts, copper salts and the like may be added as necessary as inorganic salts.

  In addition, as an enzyme-inducing additive for obtaining transformed bacterial cells having high enzyme activity, the target DNA is effectively applied to the above-mentioned medium and nutrient medium such as peptone medium and bouillon medium depending on the strain used. Antibiotics such as ampicillin, kanamycin, and chloramphenicol may be added to the culture solution for expression, and activation of a promoter such as isopropyl β-D (−)-thiogalactopyranoside (IPTG) is induced. An agent can also be used. Cultivation may be performed for 16 to 24 hours under aerobic conditions by controlling the pH within a range of 6.0 to 7.5 and a temperature of 25 to 40 ° C. Under optimum conditions depending on the transformant used. Then it is more effective.

Extraction of the enzyme RhCHBH from the cells of the obtained RhCHBH-producing transformant can be performed by destroying the transformant by the following method.
1) Mechanical (physical) method by French press or ultrasonic crushing,
2) Enzyme treatment method such as lysozyme,
3) Self-dissolution method,
4) Extraction method using osmotic pressure.

  E. coli (including pKK-RhCHBH) such as JM109 can be used as a highly active enzyme in the state of a culture solution without disrupting the cells.

  Enzymes produced by this method are not limited to those that match the polypeptide sequence of RhCHBH in the origin DS-S-51 strain (Accession No. FERM P-21129). It should be understood that a polypeptide obtained by using a recombinant technique exhibits RhCHBH activity. That is, it includes those in which one or several amino acids in the peptide sequence are deleted, or those in which one or several amino acids in the peptide sequence are replaced with other amino acids.

  The obtained transformant can be suitably used for mass production of the enzyme RhCHBH and for production of S-form 4-chloro-3-hydroxybutyrate and R-form 3-hydroxy-γ-butyrolactone using the transformant itself. .

  Next, a method for producing S-form 4-halo-3-hydroxybutyric acid ester and a method for producing R-form 3-hydroxy-γ-butyrolactone according to the present invention will be described.

  A raw material 4-halo-3-hydroxybutyric acid ester, for example, a mixture of both enantiomers of 4-chloro-3-hydroxybutyric acid ester, can be produced at low cost from, for example, epichlorohydrin.

  In addition, R-form 4-halo-3-hydroxybutyrate is produced by stereoselectively hydrolyzing a mixture of both enantiomers of 4-halo-3-hydroxybutyrate using a microorganism or an enzyme to leave R-form ( JP-A-9-47296) can be easily obtained.

  The microorganism that produces the RhCHBH enzyme used in the present invention may be a transformant introduced with the RhCHBH gene as described above, or a natural R of a mixture of both enantiomers of 4-halo-3-hydroxybutyric acid ester. As a microorganism capable of selectively degrading the R form of both enantiomeric mixtures of such 4-halo-3-hydroxybutyric acid esters, there may be mentioned Rhizobium sp. ) Strain DS-S-51 (accession number FERM P-21129) presumed to belong to

[Detailed classification method]
The DS-S-51 strain, which is a specific example of the microorganism used in the present invention, is a bacterium isolated from soil by the present inventors. Results of entrustment and identification based on the morphological and physiological properties of this strain, results of morphology, growth state, physiological test results, gram staining negative, catalase positive, oxidase positive, motilityless spore-forming bacilli Pseudomonas and other similar properties were shown. Subsequent biochemical and assimilation tests showed similar properties to Rhizobium radiobacter. However, the properties have not been estimated in detail because the NAGa and GATa test results are different from the typical properties of Rhizobium radiobacter.

  Thus, as a result of entrusting and presuming the sample taxonomic group using a partial base sequence of about 500 bp of the 16S rRNA gene, it showed high homology with bacteria belonging to the genus Rhizobium. The 16S rRNA partial nucleotide sequence was 98.9% homologous and showed the highest homology to 16S rRNA of Rhizobium daejeonense. Furthermore, phylogenetic branches were formed on the molecular phylogenetic tree.

  Based on these results and various properties of the following strains, the DS-S-51 strain was identified as a bacterium belonging to the genus Rhizobium. The scientific properties of the DS-S-51 strain are as follows.

A. Morphological cell shape Bacilli cell size 0.6-0.7 × 1.5-2.0 μm
Gram staining Presence of positive spores Presence of negative motility Positive

B. Growth state (30 ℃, 48 hours culture)
The shape of the colony The shape of the surface of the circular colony The smooth state of the colony on all edges The gloss of the low convex colony The glossy color of the colony Cream color
Growth condition at 37 ℃
Growth condition at 45 ° C Negative

C. Physiological test Catalase Positive oxidase Positive glucose, Acid production Negative glucose, Gas production Negative glucose, O test Positive glucose, F test Negative

D. Biochemical test nitrate reduction ＋
Indole production −
Glucose acidification −
Arginine dihydrolase −
Urease +
Esculin hydrolysis ＋
Gelatin hydrolysis-
β-galactosidase +

E. Utilization test glucose +
L-arabinose +
D-Mannose +
D-mannitol +
N-acetyl-D-glucosamine −
Maltose +
Potassium gluconate −
n-Capric acid −
Adipic acid −
dl-malic acid +
Sodium citrate −
Phenyl acetate −
Cytochrome oxidase +

Growth test on MacConkey agar medium −
Simmons citric acid utilization test −

  A mixture of both enantiomers of 4-halo-3-hydroxybutyric acid ester or R-form 4-halo-3-hydroxybutyric acid ester is allowed to act on the enzyme RhCHBH contained in a bacterium containing the RhCHBH gene or in a transformant, thereby producing an S-form 4- As a method for obtaining halo-3-hydroxybutyric acid ester or R-form 3-hydroxy-γ-butyrolactone, the enzyme RhCHBH itself may be contacted with a substrate, a culture solution of a bacterium or a transformant containing the RhCHBH gene, or Alternatively, the cells obtained by centrifugation thereof and the treated cells thereof (crushed cells or extracts of the cells) or those immobilized by a conventional method may be brought into contact with the substrate.

  The contact between the substrate and the enzyme or the microorganism containing the enzyme includes a method of adding the substrate to a microbial cell mixture mixed in a buffer solution. The reaction temperature is preferably 15 to 50 ° C., and the reaction pH is preferably 6 to 9. The substrate may be added all at once or in divided portions. The reaction is usually carried out with stirring or shaking, and the reaction time varies depending on the substrate concentration, microbial cells or amount of enzyme, but should be completed in 1 to 120 hours. Preferably, the end point is determined by measuring the concentration and optical purity of the target optically active substance by analysis such as gas chromatography.

  Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto. In the examples,% is expressed as (w / v) unless otherwise specified.

(Example 1) Culture of DS-S-51 strain 2% of inoculum from a cryopreservation vial into a test tube containing 5 ml of a liquid nutrient medium (pH 7.0) each containing 1.0% peptone, yeast extract, and glycerol (V / v) amount was inoculated aseptically and cultured at 30 ° C. for 24 hours.

(Example 2) Cloning of the gene The cells were collected from the culture solution obtained in Example 1 by centrifugation. The cells were suspended in 490 μl of TE solution (10 mM Tris-HCl pH 8.0, 1 mM EDTA), 30 μl of 10% SDS and 50 μl of 20 mg / ml protease K were added and reacted at 50 ° C. for 1 hour. Then, after extraction with an equal amount of phenol: chloroform: isoamyl alcohol (25: 24: 1), 0.1 volume of 3M sodium acetate solution was added, and 0.6 volume of 2-propanol was gently added. Layered. As a result, the DNA generated at the interface was collected by winding it into a thread with a glass rod. The obtained DNA was dissolved in 500 μl of TE solution. About 1.1 mg of chromosomal DNA was obtained.

  Using restriction enzyme Sau3AI, 60 μg of the chromosomal DNA obtained above was partially digested with 0.75 U of enzyme in a 250 μl reaction system to obtain a chromosomal DNA fragment. Further, the plasmid vector pUC19 was digested with the restriction enzyme BamHI by a conventional method and subjected to BAP treatment.

  14 μg of the chromosomal DNA fragment digested as described above and 800 ng of the plasmid vector pUC19 were subjected to a ligation reaction at 16 ° C. in a conventional manner using 1400 U of T4 DNA ligase (available from Takara Bio Inc.), and this was used for heat shock at 42 ° C. E. coli DH5α strain (available from Toyobo Co., Ltd.) was transformed by the method.

  After heat shock, after culturing at 37 ° C. for 1 hour in SOC medium, LB agar medium (100 μg / ml ampicillin, 0.5% methyl 4-chloro-3-hydroxybutyric acid, 0.0008% BTB (bromothyl blue)) About 100 μl of the above bacterial cell solution was applied to each sheet and incubated at 37 ° C. for 16 hours.

  Plates with a neutral pH due to BTB are green, but out of a total of 47600 transformant colonies that appeared on the plate, the pH was reduced by hydrolysis with dechlorination of methyl 4-chloro-3-hydroxybutyrate. As the value decreased, one colony whose periphery turned yellow was obtained.

  The resulting positive colonies were isolated and cultured, plasmids were prepared according to conventional methods, digested with restriction enzymes EcoRI and HindIII, and subjected to 1% agarose electrophoresis. As a result, about 4 kb of DNA was inserted into pUC19. I understood.

(Example 3) Determination of DNA base sequence Among the approximately 4 kb gene fragment obtained in Example 2, the sequence of 1.8 kb around ORF was determined by a DNA sequencer. As a result, an open reading frame (ORF) consisting of 1197 bp shown in the 44th to 1240th bases of SEQ ID NO: 1 was confirmed. From this, it was found that it encoded an amino acid of 398 residues. The amino acid sequence is shown in SEQ ID NO: 2.

(Example 4) Production of highly expressed recombinant Escherichia coli PCR primers shown in SEQ ID NOs: 3 and 4 so as to give an EcoRI site immediately upstream of the ORF and a HindIII site downstream of the ORF:
5'-ttgaattcatgccccataatctg-3 '(SEQ ID NO: 3)
5'-aaaagcttgtggccgtcga-3 '(SEQ ID NO: 4)
Then, the amplified fragment was introduced into the EcoRI and HindIII sites of pKK223-3, and E. coli JM109 strain was transformed in the same manner as in Example 2 to obtain JM109 (pKK-RhCHBH). Further, JM109 (pKK223-3) was obtained by transformation using only the vector pKK223-3 as a control sample.

  The recombinant E. coli prepared in LB medium containing 100 μg / ml ampicillin was cultured for 20 hours.

  The prepared recombinant Escherichia coli culture was collected by centrifugation, suspended in 20 mM potassium phosphate buffer, and then sonicated. 5 μg of the extracted protein sample was subjected to SDS-PAGE (10% separation gel) and stained with Coomassie Brilliant Green. As a result, the molecular weight was found to be 42 kDa. The molecular weight markers and molecular weights used are shown below. Phosphorylase B (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100), α-lactalbumin (14, 400).

(Example 5) Production of S-form 4-chloro-3-hydroxybutyrate
100 ml of a culture solution of DS-S-51 strain obtained by culturing in the same manner as in Example 1 or Example 4 except that the culture was performed at 130 rpm on a rotary shaker using a 500 ml baffled conical flask, and Racemic methyl 4-chloro-3-hydroxybutyric acid was added as a substrate to 100 ml of the culture solution of recombinant E. coli JM109 (pKK-RhCHBH), and the mixture was allowed to react at 30 ° C. with shaking. After completion of the reaction, methyl 4-chloro-3-hydroxybutyric acid remaining in the reaction solution was analyzed by gas chromatography (column carrier: PEG20M, 60-80 mesh). As a result, the residual rate (yield) was DS-S. It was 30.9% for the -51 strain and 30.5% for the recombinant E. coli. The residual ratio (yield) represents the base mass remaining after the reaction when the amount of added racemate is 100%. The reaction solution was centrifuged to remove the cells, and then extracted with the same amount of ethyl acetate as the centrifuged supernatant. The optical purity of methyl 4-chloro-3-hydroxybutyric acid in this extract was measured by gas chromatography using capillary column G-TA (0.25 mm × 30 m) manufactured by Advanced Separation Technologies Inc., NJ, USA. As a result of the analysis, the recovered methyl 4-chloro-3-hydroxybutyric acid was found to be an S form having an optical purity of 99% ee or higher. The DS-S-51 strain required 40 hours to optically resolve 1.0% (v / v) substrate. In contrast, recombinant E. coli optically resolved 2.0% (v / v) substrate in 4 hours. Next, ethyl 4-chloro-3-hydroxybutyric acid and propyl 4-chloro-3-hydroxybutyric acid were used so that the substrate of the recombinant E. coli obtained in Example 4 was 2.0% (v / v). As a result of adding a racemic body of butyl 4-chloro-3-hydroxybutyric acid to each, and similarly reacting and analyzing for 4 hours, the residual ratios (yields) were 30.5%, 30.0%, 31.0, respectively. It was found that various 4-chloro-3-hydroxybutyric acid esters were S-forms with an optical purity of 99% ee or higher. Optical purity analysis conditions are as follows: column temperature: 100 ° C., detector temperature: 200 ° C., carrier gas: nitrogen, flow rate: 0.5 ml / min; detector, FID; split ratio, 100/1, retention time Street. Methyl 4-chloro-3-hydroxybutyric acid: R-form 14.4 minutes, S-form 15.2 minutes, ethyl 4-chloro-3-hydroxybutyric acid: R-form 18.4 minutes, S-form 19.2 minutes, propyl 4 -Chloro-3-hydroxybutyric acid: R form 28.8 minutes, 30.2 minutes, butylmethyl 4-chloro-3-hydroxybutyric acid: R form 49.2 minutes, S form 50.2 minutes.

(Example 6) Production of R-form 3-hydroxy-γ-butyrolactone To 100 ml of a culture solution of DS-S-51 strain prepared in the same manner as in Example 5 and 100 ml of a culture solution of recombinant Escherichia coli JM109 (pKK-RhCHBH) Then, R-form methyl 4-chloro-3-hydroxybutyric acid having an optical purity of 99% ee was added as a substrate, and the reaction was carried out while shaking at 30 ° C. After completion of the reaction, the produced R-form 3-hydroxy-γ-butyrolactone was analyzed by gas chromatography (column carrier: PEG20M, 60-80 mesh). As a result, the molar yield was DS-S-51 strain, recombinant It was 100% for both E. coli. The optical purity analysis of 3-hydroxy-γ-butyrolactone was performed as follows. The reaction solution was centrifuged to remove the cells, and then concentrated using a rotary evaporator. Extraction with an appropriate amount of 1,2-dichloroethane gave a syrup. To 1 ml of dichloromethane, 20 μl of syrup and 200 μl of trifluoroacetic anhydride were added and stirred well. After 30 minutes, the mixture was concentrated with a desiccator, dissolved in 1 ml of ethanol, and then applied to the same capillary column G-TA as in Example 5. As a result, the recovered 3-hydroxy-γ-butyrolactone was found to be an R form having an optical purity of 99.0% ee. Analysis conditions are column temperature: 100 ° C., detector temperature: 200 ° C., carrier gas: nitrogen, flow rate: 0.5 ml / min; detector, FID; split ratio, 100/1. Retention of 3-hydroxy-γ-butyrolactone The time was 29.5 minutes for S-form and 31.2 minutes for R-form. The DS-S-51 strain required 80 hours to convert all 1.0% of the substrate to R form. In contrast, recombinant E. coli converted 2.0% (v / v) substrate in 8 hours.

FIG. 1 is a diagram showing the construction of a RhCHBH gene construct. FIG. 2 is a diagram showing SDS-PAGE of recombinant E. coli.

Claims (14)

A gene comprising any of the following base sequences (a) or (b):
(A) a nucleotide sequence consisting of the 44th base to the 1240th base of SEQ ID NO: 1,
(B) a 4-halo-3-hydroxybutyric acid represented by the following formula [1], which is hybridized under stringent conditions with the nucleotide sequence consisting of the 44th to 1240th nucleotides of SEQ ID NO: 1 A nucleotide sequence encoding a protein having an enzymatic activity that acts on an enantiomeric mixture of esters, selectively hydrolyzes the R-form of the mixture, and selectively leaves the S-form:

Formula [1]
[Wherein X represents a halogen atom and R represents an alkyl group having 1 to 4 carbon atoms]. Any of the following proteins (a ′) or (b ′):
(A ′) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2,
(B ′) 4-halo-3-hydroxy represented by the following formula [1], comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 A protein having an enzymatic activity that acts on an enantiomeric mixture of butyric ester, selectively hydrolyzes the R-form of the mixture, and selectively leaves the S-form:

Formula [1]
[Wherein X represents a halogen atom and R represents an alkyl group having 1 to 4 carbon atoms].   A vector comprising the gene according to claim 1.   The vector according to claim 3, which is a plasmid pKK-RhCHBH.   A bacterium having the gene according to claim 1.   The bacterium according to claim 5, which is a bacterium DS-S-51 strain (Accession No. FERM P-21129).   A transformant comprising the gene according to claim 1 or the vector according to claim 3 or 4.   The transformant according to claim 7, which is Escherichia coli.   The transformant according to claim 8, which is Escherichia coli JM109 (pKK-RhCHBH) (Accession No. FERM P-21128).   The method for producing a protein according to claim 2, comprising expressing the gene according to claim 1. The protein according to claim 2 is allowed to act on an enantiomeric mixture of 4-halo-3-hydroxybutyric acid ester represented by the following formula [1] to selectively hydrolyze the R form of the mixture, and the S form A method for producing S-form 4-halo-3-hydroxybutyrate, which comprises selectively leaving

Formula [1]
[Wherein X represents a halogen atom and R represents an alkyl group having 1 to 4 carbon atoms]. The R-form 3-hydroxy- represented by the following formula [2], comprising allowing the protein of claim 2 to act on the R-form of a 4-halo-3-hydroxybutyrate ester represented by the following formula [1] Method for producing γ-butyrolactone:

Formula [1]
[Wherein X represents a halogen atom and R represents an alkyl group having 1 to 4 carbon atoms].

Formula [2]   The method of Claim 11 or 12 using the compound whose R of Formula [1] is methyl.   The method in any one of Claims 11-13 using the compound whose X is a chlorine atom.

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