JP2006115726A - METHOD FOR PRODUCING OPTICALLY ACTIVE beta-HYDROXYAMINO ACID BY SOLUBILIZATION - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing optically active β-hydroxyamino acids capable of retrieving the D- or L-β-hydroxyamino acid deposited in the reaction liquid. <P>SOLUTION: The method for producing the D- or L-β-hydroxyamino acid using an enzyme-active material is provided. That is, the method for retrieving the above D- or L-β-hydroxyamino acid by solubilizing the product deposited in the reaction liquid is established. By refining the optically active β-hydroxyamino acid thus retrieved, the objective compound can be obtained efficiently. Thus, the optically active β-hydroxyamino acids useful as intermediates for pharmaceuticals or agrochemicals can be efficiently produced on an industrial scale. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing an optically active β-hydroxyamino acid.

  D- or L-β-hydroxy amino acids, especially D-β-hydroxy amino acids such as D-erythro-β-hydroxy amino acids and D-threo-β-hydroxy amino acids are useful substances as synthetic intermediates for pharmaceuticals and agricultural chemicals. There is a need for an inexpensive manufacturing method. Hereinafter, the term “optically active β-hydroxyamino acid” means “D-β-hydroxyamino acid or L-β-hydroxyamino acid” unless otherwise specified.

  As a method for producing an optically active β-hydroxyamino acid, a chemical synthesis method by organic synthesis (Patent Document 1) and a biological method using an enzyme active substance (Patent Document 2, Patent Document 3) are known. As an industrial production method, a method using an enzyme active substance that can be synthesized at a lower cost is preferable. Biological techniques generally consist of a reaction step using an enzyme active substance and a step of separating and purifying an optically active β-hydroxyamino acid that is a target product from the reaction solution. Separation of the optically active β-hydroxyamino acid, which is an enzyme active substance and a product, is often difficult in the case of industrial reactions.

  In the industrial production of substances, it is economically advantageous to improve the production amount per reaction tank, that is, to accumulate the product at a high concentration. On the other hand, the optically active β-hydroxyamino acid may have very low solubility in water depending on its structure. When the accumulated amount of optically active β-hydroxyamino acid having low solubility increases, a part or most of the optically active β-hydroxyamino acid is precipitated in the reaction solution. In many cases, the precipitated product makes it difficult to separate it from an enzyme active substance or the like present as a solid.

  Insoluble compounds are often recovered by dissolving the compound of interest by some means. That is, a method of once dissolving the optically active β-hydroxyamino acid, separating the enzyme active substance, and further treating with an ion exchange resin is conceivable. Specifically, water can be added to the reaction solution to dissolve the optically active β-hydroxyamino acid.

  The dissolved optically active β-hydroxyamino acid can be converted into a crude crystal through crystallization operation such as concentration after ion exchange treatment. Thereafter, in order to obtain a product of higher purity, crystals of optically active β-hydroxyamino acid can be obtained by decolorization operation and recrystallization operation. However, the optically active β-hydroxyamino acid has extremely low solubility in water depending on its structure, and the above-described method requires a large amount of water to be dissolved once. As a result, there is a problem that the concentration of the target optically active β-hydroxyamino acid is lowered and the subsequent separation and purification process becomes complicated.

Thus, no effective technique that can be endured at the industrial level has been reported as a method for separating and purifying the raw material precipitated in the reaction solution and the optically active β-hydroxyamino acid.
JP-A-1-317391 JP-A-2-207793 JP-A-6-165893

  An object of the present invention is to provide a method for producing an optically active β-hydroxyamino acid capable of recovering an optically active β-hydroxyamino acid precipitated in a reaction solution in the presence of an enzyme active substance.

  It is often difficult to recover a poorly soluble product in the presence of enzyme actives. As a result of intensive studies, the present inventors have established a method for recovering a product precipitated in a reaction solution by dissolving it in the production of an optically active β-hydroxyamino acid using an enzyme active substance. By purifying the optically active β-hydroxyamino acid recovered in this way, it was clarified that the target compound can be efficiently obtained, and the present invention was completed. That is, the present invention provides the following method for producing an optically active β-hydroxyamino acid.

で表されるβ−ヒドロキシアミノ酸である、上記［１］乃至上記［４］記載の製造方法。
［６］ Ｒが置換されていてもよいシクロヘキシル基である上記［５］記載の製造方法。
［７］ 酵素活性物質がL-フェニルセリンアルドラーゼ活性を有している、上記［１］乃至上記［６］記載の製造方法。
［８］ 酵素活性物質が下記(a)から(e)のいずれかに記載のポリヌクレオチドによりコードされる蛋白質、該蛋白質を発現する微生物およびそれらの処理物からなる群から選択される少なくとも１つである、上記［７］記載の製造方法。
(a)配列番号：１に記載された塩基配列を含むポリヌクレオチド；
(b)配列番号：２に記載のアミノ酸配列からなる蛋白質をコードするポリヌクレオチド；
(c)配列番号：２に記載のアミノ酸配列において、１若しくは複数のアミノ酸が置換、欠失、挿入、および／または付加したアミノ酸配列からなる蛋白質をコードするポリヌクレオチド；
(d)配列番号：１に記載された塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするポリヌクレオチド；および
(e)配列番号：２に記載のアミノ酸配列と７０%以上の相同性を有するアミノ酸配列をコードするポリヌクレオチド [1] A step of causing an enzyme active substance to act on DL-β-hydroxyamino acid in the reaction solution to accumulate D- or L-β-hydroxyamino acid in the reaction solution to a concentration higher than the precipitation concentration;
A step of dissolving the accumulated D or L-β-hydroxyamino acid by maintaining at 30 ° C. to 80 ° C. and recovering the dissolved D or L-β-hydroxyamino acid. Production method.
[2] The enzyme active substance coexists with D or L-β-hydroxyamino acid, and after removing the enzyme active substance from the reaction solution in which D or L-β-hydroxyamino acid is dissolved, D- or L-β -The manufacturing method of said [1] description further including the process of crystallizing a hydroxyamino acid.
[3] The method according to [1] or [2] above, wherein the D- or L-β-hydroxyamino acid is dissolved by adjusting the reaction solution to pH = 0.1 to 2.5.
[4] The reaction solution according to the above [3], wherein the D- or L-β-hydroxyamino acid is dissolved by adjusting the reaction solution to pH = 0.2 to 2.0 and holding at 40 to 70 ° C. for 4 hours or more. Production method.
[5] β-hydroxyamino acid is represented by the following formula 1 (wherein R represents an optionally substituted aliphatic group, alicyclic group, aromatic group or heterocyclic group)

The production method according to [1] to [4] above, which is a β-hydroxyamino acid represented by the formula:
[6] The production method of the above-mentioned [5], wherein R is an optionally substituted cyclohexyl group.
[7] The production method according to [1] to [6] above, wherein the enzyme active substance has L-phenylserine aldolase activity.
[8] At least one selected from the group consisting of a protein encoded by the polynucleotide according to any one of (a) to (e) below, a microorganism expressing the protein, and a processed product thereof The method according to [7] above, wherein
(a) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1;
(b) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2;
(c) a polynucleotide encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2;
(d) a polynucleotide that hybridizes with the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions; and
(e) a polynucleotide encoding an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 2

  According to the present invention, there has been provided a production method capable of recovering an optically active β-hydroxyamino acid having low solubility in the presence of an enzyme active substance. In the present invention, particularly when the step of solubilizing the optically active β-hydroxyamino acid is adopted by adjusting the pH, the target optically active β-hydroxyamino acid can be sufficiently dissolved. For example, under acidic conditions, the optically active β-hydroxyamino acid can be easily dissolved and recovered by adding a small amount of acid. Under acidic conditions, an enzyme active substance such as a microbial cell and an optically active β-hydroxyamino acid can be easily separated. The present invention provides a method for producing an optically active β-hydroxyamino acid that can be applied to an industrial level.

  The present invention comprises a step of causing an enzyme active substance to act on DL-β-hydroxyamino acid to accumulate optically active β-hydroxyamino acid in a reaction solution at a deposition concentration or higher, dissolving the accumulated optically active β-hydroxyamino acid, And a method for producing an optically active β-hydroxyamino acid comprising a step of removing an enzyme active substance.

  In the production of the optically active β-hydroxyamino acid of the present invention, enzymatic active materials (enzyme active materials) that act optically specifically on either the D-form or L-form of DL-β-hydroxyamino acid. ) Is used. In the present invention, the enzyme active substance is an optically specific action on a D-form or L-form compound of DL-β-hydroxyamino acid to catalyze a reaction for conversion to another substance. Is defined as a substance capable of The optically specific action refers to having a higher catalytic action on one optical isomer than on the other isomer. For example, the following enzymes, microorganisms having the enzyme activity, or processed microorganism products can be used as the enzyme active substance.

(1) Aldolases: L-amino acid aldolases having an activity to specifically decompose L-β-hydroxyamino acids into aldehydes corresponding to glycine, or activities to synthesize D-β-hydroxyamino acids from aldehydes corresponding to glycine (2) Decarboxylase: L-amino acid decarboxylase having an activity to specifically decarboxylate and decompose L-β-hydroxyamino acid (3) Acylase: N-acyl- Amino acid acylases that have the activity of hydrolyzing D-form or L-form using DL-β-hydroxyamino acid as a raw material

Specific examples of preferable enzymes include the following enzymes.
L-phenylserine aldolase,
L-threonine aldolase,
L-allo-threonine aldolase,
D-low substrate specificity threonine aldolase,
L-low substrate specificity threonine aldolase,
D-threonine aldolase,
D-Allo-threonine aldolase, etc.

Among these, L-phenylserine aldolase is a preferred enzyme in the present invention. These enzymes are known and can be obtained by, for example, the methods described below.
JP-A-9-238680
Vitamins, 75, 2, 51-61, 2001
Bioscience and industry, Vol. 56, No. 11, 23-26, 1998
Nature, 181, 1533-1534, 1958
Biochim. Biophys. Acta, 258, 779-790 (1972)
FEMS Microbiology Letters, 151, 245-248, 1997
Appl. Microbiol. Biotechnol., 49, 702-708, 1998
Appl. Microbiol. Biotechnol., 54, 44-51, 2000
Eur. J. Biochem., 248, 385-393, 1997
The amount of the enzyme active substance to be used is usually 0.01 to 10000 U / mL, preferably 0.1 to 1000 U / mL, more preferably about 1 to 500 U / mL.

For example, the decomposition activity of L-phenylserine aldolase for L-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid can be measured as follows. 20 mM DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid, 200 mM TAPS-NaOH buffer (pH 8.5), 50 μM pyridoxal-5′-phosphate (hereinafter abbreviated as PLP) and enzyme The reaction is carried out at 30 ° C. for 10 minutes in 0.5 mL of the reaction solution, and the reaction is stopped by adding 0.5 mL of 1 N HCl. The produced cyclohexyl aldehyde is quantified by the following method.

Add 0.5 mL of a 1.2 N hydrochloric acid solution containing 2.5 mM 2,4-dinitrophenylhydrazine to 1.0 mL of the reaction-terminated solution, stir rapidly, and let stand at 30 ° C. for 20 minutes. Furthermore,
Add 3 mL of ethanol and stir rapidly, then add 0.85 mL of 3N NaOH and add 10
Let stand for a minute. The absorbance of the solution at 475 nm is measured. Enzyme activity is 30 ° C
The amount of enzyme that catalyzes the production of 1 μmol of cyclohexyl aldehyde per minute was 1 unit (U).

  The reaction temperature can be selected at a temperature at which the enzyme active substance can maintain its enzyme action. Specifically, 5-60 degreeC normally, Preferably it is 10-50 degreeC, More preferably, it can show 20-40 degreeC. The reaction pH can also be selected within the range in which the enzyme active substance can maintain its enzymatic action. Usually, pH 6-11, Preferably, pH 7-10, More preferably, pH 8-9.5 can be shown. The reaction can be carried out with stirring.

L-phenylserine aldolase can be prepared, for example, from a microorganism belonging to the genus Pseudomonas putida and having the ability to produce L-phenylserine aldolase characterized by the following properties (1)-(3). A preferred example of the microorganism belonging to the genus Pseudomonas putida is Pseudomonas putida biobar A 24-1 strain. That is, L-phenylserine aldolase isolated from Pseudomonas putida and characterized by the following properties can be used as an enzyme active substance in the present invention. Alternatively, Pseudomonas putida microorganisms themselves that produce the enzyme, or processed products thereof can be used as the enzyme active substance of the present invention.
(1) Action
Acts on L-phenylserine to produce benzaldehyde and glycine.
(2) Substrate specificity
(a) It acts on both L-threo-phenylserine and L-erythro-phenylserine, but does not substantially act on D-threo-phenylserine and D-erythro-phenylserine.
(b) Among DL-erythro 2-amino-3-cyclohexyl-3-hydroxypropionic acids, it acts on the L-erythro form, but does not substantially act on the D-erythro form.
(3) Molecular weight The molecular weight in gel filtration is 190,000-210,000, and the molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 35,000.

The L-phenylserine aldolase activity in the present invention can be confirmed, for example, as follows.
Activity measurement method:
20 mM DL-threo-phenylserine, 200 mM TAPS-NaOH buffer (pH 8.5), 5
The reaction is performed at 30 ° C. for 10 minutes in 0.5 mL of a reaction solution containing 0 μM PLP and the enzyme, and the reaction is stopped by adding 0.5 mL of 1 N HCl. The produced benzaldehyde is quantified by the following method. Add 0.5 mL of 2.
A 1.2N hydrochloric acid solution containing 5 mM 2,4-dinitrophenylhydrazine is added, stirred rapidly, and allowed to stand at 30 ° C. for 20 minutes. Add 3 mL of ethanol and stir quickly, then add 0.85 mL of 3N NaOH and let stand for 10 minutes. The absorbance of the solution at 475 nm is measured. The amount of enzyme that catalyzes the production of 1 μmol of benzaldehyde per minute at 30 ° C. was defined as 1 unit (U).

Further, in the present invention, the fact that each compound is given as a substrate means that the activity with respect to the compound is, for example, 10% when the activity with respect to both substrates measured under the same conditions is 100. Hereinafter, it is said that it is not affected when it is preferably 5% or less. Specifically, the activity of L-phenylserine aldolase derived from Pseudomonas putida biobar A 24-1 against the following compounds is as follows for both corresponding substrates.
(a) When the activity for L-threo-phenylserine or L-erythro-phenylserine is defined as 100, the activity against D-threo-phenylserine and D-erythro-phenylserine cannot be confirmed.
(b) In DL-erythro 2-amino-3-cyclohexyl-3-hydroxypropionic acid, when the activity on the L-erythro form is defined as 100, the action on the D-erythro form cannot be confirmed.

  Pseudomonas putida biobar A 24-1 strain can be cultured in a common medium used for bacterial culture. Since this enzyme is induced by DL-threo-phenylserine or the like, it is preferable to add an inducer to the medium. For example, 1.0% peptone, 0.2% potassium dihydrogen phosphate, 0.2% potassium dihydrogen phosphate, 0.2% sodium chloride, 0.01% magnesium sulfate heptahydrate, 0 A medium containing 0.2% DL-threo-phenylserine in a peptone medium having a pH of 7.2 containing 0.01% yeast extract is preferably used.

L-phenylserine aldolase can be purified from a culture of microorganisms, for example, as follows. The Pseudomonas putida biobar A 24-1 strain was sufficiently grown in the peptone medium containing 0.2% DL-threo-phenylserine, and then the cells were collected, disrupted in a buffer solution and cell-free. Use as extract. At this time, it is desirable to protect the enzyme by adding the following components to the buffer.
Reducing agent: 2-mercaptoethanol, etc. Protease inhibitor: Phenylmethanesulfonyl fluoride, pepstatin A, leupeptin, metal chelator, etc. PLP
The target enzyme can be purified from the cell-free extract thus obtained by appropriately combining, for example, the following various protein fractionation methods and chromatography.
Fractionation based on protein solubility (precipitation with organic solvents, salting out with ammonium sulfate, etc.)
Cation exchange, anion exchange, gel filtration, hydrophobic chromatography Affinity chromatography using chelates, dyes, antibodies, etc. More specifically, for example, according to the following steps, the target enzyme is obtained from a cell-free extract. Can be electrophoretically purified to a single band. The detailed conditions of each process can be made as described in the examples, for example.
40-60% ammonium sulfate split;
DEAE-cellulose ion exchange chromatography;
Hydroxyapatite chromatography;
DEAE-cellulose anion exchange chromatography (2nd)
Hydroxyapatite chromatography; (second time)
MonoQ anion exchange chromatography;

L-phenylserine aldolase derived from Pseudomonas putida biobar A 24-1 strain consists of the amino acid sequence set forth in SEQ ID NO: 2. The enzyme active substance used in the present invention also includes a protein homolog having the amino acid sequence set forth in SEQ ID NO: 2. That is, the enzyme active substance having L-phenylserine aldolase activity in the present invention is a protein encoded by the polynucleotide according to any one of (a) to (e) below, a microorganism expressing the protein, or a transformant And at least one enzyme active substance selected from the group consisting of those processed products.
(a) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1;
(b) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2;
(c) a polynucleotide encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2;
(d) a polynucleotide that hybridizes with the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions; and
(e) a polynucleotide encoding an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 2

  Alternatively, as long as the protein having an additional amino acid sequence in addition to the amino acid sequence shown in SEQ ID NO: 2 has the same activity as the protein consisting of the amino acid sequence shown in SEQ ID NO: 2, the enzyme active substance in the present invention Can be used. For example, a protein obtained by adding a His tag or the like to the amino acid sequence shown in SEQ ID NO: 2 is included in the enzyme active substance in the present invention. Furthermore, transformants expressing these proteins and recombinants produced by them are included in the enzyme active substances in the present invention.

  The homologue of L-phenylserine aldolase used in the present invention consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 2. It means a protein functionally equivalent to the protein consisting of the amino acid sequence described in No. 2. In the present invention, functionally equivalent to the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 means that the protein has the physicochemical and enzymatic chemical properties shown in the above (1)-(3). means.

  Those skilled in the art will be able to introduce site-directed mutagenesis into the DNA described in SEQ ID NO: 1 (Nucleic Acid Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning 2ndEdt. , Cold Spring Harbor Laboratory Press (1989), PCR A Practical Approach IRL Press pp.200 (1991)) etc. to introduce L-phenyl as appropriate by introducing substitutions, deletions, insertions, and / or addition mutations. A polynucleotide encoding a serine aldolase homolog can be obtained. By introducing a polynucleotide encoding the homologue of L-phenylserine aldolase into a host and expressing it, the homologue of L-phenylserine aldolase described in SEQ ID NO: 2 can be obtained.

  In the amino acid sequence shown in SEQ ID NO: 2, for example, mutation of amino acid residues of 100 or less, usually 50 or less, preferably 30 or less, more preferably 15 or less, still more preferably 10 or less, or 5 or less is allowed. In general, in order to maintain the function of a protein, the amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution. Such substitution of amino acid residues is called conservative substitution. For example, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp are all classified as nonpolar amino acids, and thus have similar properties to each other. Further, examples of the non-chargeability include Gly, Ser, Thr, Cys, Tyr, Asn, and Gln. Moreover, Asp and Glu are mentioned as an acidic amino acid. Examples of basic amino acids include Lys, Arg, and His. Amino acid substitutions within each of these groups are allowed.

  The polynucleotide homologue in the present invention is a polynucleotide that can hybridize under stringent conditions with the polynucleotide comprising the base sequence shown in SEQ ID NO: 1, and the physicochemical properties (1) and ( Also included is a polynucleotide encoding a protein having 2). The polynucleotide capable of hybridizing under stringent conditions is one or more selected from any at least 20, preferably at least 30, for example, 40, 60 or 100 consecutive sequences in SEQ ID NO: 1. The probe DNA is used as a probe DNA, for example, using ECL direct nucleic acid labeling and detection system (Amersham Pharmaica Biotech) under the conditions described in the manual (for example, wash: 42 ° C, primary wash buffer containing 0.5x SSC) , Refers to a hybridizing polynucleotide. More specific “stringent conditions” are, for example, usually 42 ° C., 2 × SSC, 0.1% SDS conditions, preferably 50 ° C., 2 × SSC, 0.1% SDS conditions, The conditions are preferably 65 ° C., 0.1 × SSC and 0.1% SDS, but are not particularly limited to these conditions. A plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.

  Furthermore, the homologue of the L-phenylserine aldolase of the present invention refers to a protein having at least 70%, preferably at least 80%, more preferably 90% or more homology with the amino acid sequence shown in SEQ ID NO: 2. Protein homology searches include, for example, databases related to amino acid sequences of proteins such as SWISS-PROT, PIR, and DAD, databases related to DNA sequences such as DDBJ, EMBL, and Gene-Bank, databases related to predicted amino acid sequences based on DNA sequences, etc. For example, it can be performed through the Internet using programs such as BLAST and FASTA.

  As a result of homology search using Blast based on the amino acid sequence shown in SEQ ID NO: 2, the highest homology was found to be the expected low substrate-specific aldolase derived from Ralstonia solanacearum (Ralstonia solanacearum). 68%). Among the proteins whose functions have been identified, the amino acid sequence of the low substrate specificity L-threonine aldolase derived from Pseudomonas aeruginosa PAO1 strain was 41% homologous to SEQ ID NO: 2.

  The target DNA can also be obtained by screening from DNA directly obtained from an environmental sample such as soil using a polynucleotide encoding L-phenylserine aldolase as a probe. As the library, a library obtained by introducing a physical digest or a digest of DNA obtained from an environmental sample into a phage or plasmid and transforming Escherichia coli can be used. For screening, colony hybridization or plaque hybridization can be used. As a result of analyzing the obtained DNA base sequence after obtaining the hybridizing DNA by the above method, the encoded amino acid sequence having the homology of 70% or more with L-phenylserine aldolase has the same function. You can expect to have. E. coli transformed with a plasmid having the DNA thus obtained can also be used in the present invention.

A polynucleotide encoding L-phenylserine aldolase can be isolated by, for example, the following method. For example, a target DNA can be obtained by designing a primer for PCR based on the base sequence described in SEQ ID NO: 1 and performing PCR using a chromosomal DNA of an enzyme production strain or a cDNA library as a template.
Alternatively, the target DNA can be obtained by screening a library of enzyme producing strains using a DNA fragment obtained by PCR as a probe. As the library, a library or cDNA library obtained by introducing a restriction enzyme digest of chromosomal DNA into a phage or plasmid and transforming Escherichia coli can be used. For screening, colony hybridization or plaque hybridization can be used.

Further, based on the nucleotide sequence information of the DNA fragment obtained by PCR, the 5′-side or 3′-side nucleotide sequence can also be obtained. As such a method, the RACE method (Rapid Amplification of cDNA End, “PCR Experiment Manual” p25-33, HBJ Publishing Bureau) can be used. Alternatively, as a method for obtaining an unknown region based on known fragment sequence information, inverse PCR (Inverse PCR; Genetics 120, 621-623, 1988) is also known. In inverse PCR, a DNA library that has been circularized by self-cyclization is used. Such a library can be obtained by digesting the chromosomal DNA of an enzyme-producing strain with an appropriate restriction enzyme, followed by self-cyclization. On the other hand, a primer for inverse PCR is designed so that a complementary strand synthesis reaction proceeds toward the outside (unknown region) of a known cDNA base sequence. Since the template DNA is circularized, an unknown region can be obtained as an amplification product by inverse PCR.
The polynucleotide of the present invention includes genomic DNA cloned by the above method, or cDNA obtained by synthesis in addition to cDNA.

  By inserting the isolated polynucleotide encoding the homologue into a known expression vector, an L-phenylserine aldolase homologous expression vector can be obtained. Further, by culturing a transformant transformed with this expression vector, a homologue of L-phenylserine aldolase can be obtained as a recombinant. The transformant thus obtained and the homologous recombinant produced by the transformant are included in the enzyme active substance in the present invention.

  In order to express L-phenylserine aldolase or a homologue thereof in the present invention, a microorganism to be transformed is transformed with a recombinant vector containing a polynucleotide encoding a polypeptide having L-phenylserine aldolase activity. There is no particular limitation as long as it is an organism capable of expressing L-phenylserine aldolase activity. Examples of the microorganisms that can be used include the following microorganisms.

Escherichia genus Bacillus genus Pseudomonas genus Serratia genus Brevibacterium genus Corynebacterium genus Streptococcus genus Lactobacillus genus Lactobacillus genus Developed bacteria Rhodococcus genus Streptomyces genus Streptomyces genus Actinomycetes developed Saccharomyces genus Kluyveromyces genus Schizosaccharomyces genus Saccharomyces (Zygosaccharomyces) genus Yarrowia genus Trichosporon genus Rhodosporidium genus Pichia genus Candida genus Molds for which host vector systems such as the genus Sporasp (Neurospora), the genus Aspergillus (Cephalosporium), and the genus Trichoderma have been developed.

  The procedure for producing transformants and the construction of a recombinant vector suitable for the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Sambrook et al., Molecular cloning, Cold Spring Harbor Laboratories).

  In order to express the L-phenylserine aldolase gene of the present invention in microbial cells, the DNA of the present invention is first introduced into a plasmid vector or a phage vector that is stably present in the microorganism, and the genetic information is transcribed and transferred. Let them translate. For that purpose, a promoter corresponding to a unit controlling transcription / translation may be incorporated upstream of the 5′-side of the DNA strand of the present invention, more preferably a terminator downstream of the 3′-side. As the promoter and terminator, a promoter and terminator known to function in a microorganism used as a host is used. Regarding vectors, promoters, terminators and the like that can be used in these various microorganisms, for example, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan”, especially regarding yeast, Adv. Biochem. Eng. 43, 75-102 (1990), Yeast 8, 423-488 (1992).

  In the genus Escherichia, especially Escherichia coli, for example, pBR and pUC plasmids can be used as plasmid vectors, and lac (β-galactosidase), trp (tryptophan operon), tac, trc (lac, trp) Fusion), promoters derived from λ phage PL, PR, etc. can be used. Moreover, as the terminator, a terminator derived from trpA, phage, or rrnB ribosomal RNA can be used.

  In the genus Bacillus, pUB110 series plasmids, pC194 series plasmids and the like can be used as vectors, and can be integrated into chromosomes. Moreover, apr (alkaline protease), npr (neutral protease), amy (α-amylase), or the like can be used as a promoter or terminator.

  In the genus Pseudomonas, host vector systems such as Pseudomonas putida and Pseudomonas cepacia have been developed. A broad host range vector (including genes necessary for autonomous replication derived from RSF1010) pKT240 based on the plasmid TOL plasmid involved in the degradation of toluene compounds can be used. As the promoter or terminator, a lipase (Japanese Patent Laid-Open No. 5-284973) gene or the like can be used.

    In the genus Brevibacterium, especially Brevibacterium lactofermentum, plasmid vectors such as pAJ43 (Gene 39, 281 (1985)) can be used. As the promoter or terminator, promoters and terminators used in E. coli can be used as they are.

  In the genus Corynebacterium, especially Corynebacterium glutamicum, plasmid vectors such as pCS11 (JP 57-183799) and pCB101 (Mol. Gen. Genet. 196, 175, 1984) are available. is there.

  In the genus Streptococcus, pHV1301 (FEMS Microbiol. Lett. 26, 239 (1985), pGK1 (Appl. Environ. Microbiol. 50, 94 (1985)), etc. can be used as a plasmid vector.

  In the genus Lactobacillus, pAMβ1 (J. Bacteriol. 137, 614, 1979) developed for Streptococcus can be used, and those used in Escherichia coli as a promoter can be used.

  In the genus Rhodococcus, plasmid vectors isolated from Rhodococcus rhodochrous can be used (J. Gen. Microbiol. 138, 1003, 1992).

  In the genus Streptomyces, plasmids can be constructed according to the method described in Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory Manual Cold Spring Harbor Laboratories (1985). In particular, in Streptomyces lividans, pIJ486 (Mol. Gen. Genet. 203, 468-478, 1986), pKC1064 (Gene 103,97-99,1991), pUWL-KS (Gene 165,149- 150, 1995) can be used. The same plasmid can also be used in Streptomyces virginiae (Actinomycetol. 11, 46-53, 1997).

  In the genus Saccharomyces (especially Saccharomyces cerevisiae), YRp, YEp, YCp, and YIp plasmids can be used, and homologous recombination with multiple copies of ribosomal DNA in the chromosome can be performed. The integration vector used (EP 537456, etc.) is extremely useful because it can introduce a gene in multiple copies and can stably hold the gene. ADH (alcohol dehydrogenase), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PHO (acid phosphatase), GAL (β-galactosidase), PGK (phosphoglycerate kinase), ENO (enolase) And other promoters and terminators are available.

  In the genus Klyberomyces, especially Kluyveromyces lactis, 2 μm-type plasmid derived from Saccharomyces cerevisiae, pKD1-type plasmid (J. Bacteriol. 145, 382-390, 1981), involved in killer activity A plasmid derived from pGKl1, an autonomously proliferating gene KARS system plasmid in the genus Kleberomyces, a vector plasmid (such as EP 537456) that can be integrated into the chromosome by homologous recombination with ribosomal DNA and the like can be used. In addition, promoters and terminators derived from ADH, PGK and the like can be used.

  In the genus Schizosaccharomyces, plasmid vectors containing ARS (genes involved in autonomous replication) derived from Schizosaccharomyces pombe and selection markers that complement auxotrophy derived from Saccharomyces cerevisiae are available. (Mol. Cell. Biol. 6, 80, 1986). Further, an ADH promoter derived from Schizosaccharomyces pombe can be used (EMBO J. 6, 729, 1987). In particular, pAUR224 is commercially available from Takara Shuzo and can be easily used.

In the genus Zygosaccharomyces, plasmid vectors derived from pSB3 (Nucleic Acids Res. 13, 4267, 1985) derived from Zygosaccharomyces rouxii are available, and the PHO5 promoter derived from Saccharomyces cerevisiae GAP-Zr (Glyceraldehyde-3-phosphate dehydrogenase) promoter (Agri. Biol. Chem. 54, 2521, 1990) derived from Tigosaccharomyces rouxi can be used.

  In the genus Pichia, a host vector system has been developed in Pichia angusta (former name: Hansenula polymorpha). As vectors, genes involved in autonomous replication derived from Pichia Angusta (HARS1, HARS2) can be used, but because they are relatively unstable, multicopy integration into the chromosome is effective (Yeast 7, 431- 443, 1991). In addition, AOX (alcohol oxidase), FDH (formic acid dehydrogenase) promoters induced by methanol, etc. can be used. In addition, a host vector system using Pichia pastoris and other genes (PARS1, PARS2) etc. involved in autonomous replication from Pichia has been developed (Mol. Cell. Biol. 5, 3376, 1985). Strong promoters such as high concentration culture and methanol-inducible AOX can be used (Nucleic Acids Res. 15, 3859, 1987).

  In the genus Candida, host vector systems in Candida maltosa, Candida albicans, Candida tropicalis, Candida utilis, etc. Has been developed. In Candida maltosa, ARS derived from Candida maltosa has been cloned (Agri. Biol. Chem. 51, 51, 1587, 1987), and vectors using this have been developed. In Candida utilis, a strong promoter for a chromosome integration type vector has been developed (Japanese Patent Laid-Open No. 08-173170).

  In the genus Aspergillus, Aspergillus niger, Aspergillus oryzae, etc. are the most studied among molds, and integration into plasmids and chromosomes is possible, Promoters derived from extracellular proteases or amylases are available (Trends in Biotechnology 7, 283-287, 1989).

  In the genus Trichoderma, a host vector system using Trichoderma reesei has been developed, and an extracellular cellulase gene-derived promoter or the like can be used (Biotechnology 7, 596-603, 1989).

  In addition to microorganisms, various host / vector systems have been developed in plants and animals. Specifically, systems have been developed for expressing heterologous proteins in large quantities in insects such as moths (Nature 315, 592-594, 1985) or plants such as rapeseed, corn, and potato. These host / vector systems can also be used in the present invention.

In the present invention, the contact form of the reaction solution containing the enzyme active substance and the raw material is arbitrary. For example, both can be brought into contact by mixing an enzyme active substance with a solvent containing raw materials. When the enzyme active substance is insoluble in the solvent, the enzyme active substance is dispersed in the solvent, and the two can be separated if necessary. Alternatively, the solvent containing the raw material and the enzyme active substance can be separated and contacted with a substrate-permeable membrane. Such a contact configuration facilitates recovery and reuse of the enzyme active substance. In the present invention, the contact form between the two is not limited to these specific examples.
In the present invention, as the enzyme active substance, a transformant that functionally expresses the protein described in SEQ ID NO: 2 or a homologue thereof and a processed product thereof can be used. For example, E. coli transformed with pKK-PSA1 is preferred as the transformant in the present invention.

Furthermore, the enzyme active substance in the present invention specifically includes the enzyme active substances shown below.
Microorganisms that have changed the permeability of cell membranes by treatment with organic solvents such as surfactants and toluene;
Dry cells prepared by freeze drying or spray drying;
Cell-free extract obtained by crushing bacterial cells with glass beads or enzyme treatment;
Partially purified cell-free extract;
Purified enzyme;
A transformant or an immobilized enzyme on which an enzyme is immobilized, an immobilized microorganism;

  In the present invention, any additive can be added to the reaction solution containing the enzyme active substance and the raw material. The additive is added for the purpose of enhancing the enzyme activity, stabilizing the enzyme activity, or increasing the fluidity of the reaction solution. The enzyme active substance that can be suitably used in the present invention utilizes PLP as a coenzyme. Therefore, by adding PLP to the reaction solution, its enzyme activity can be enhanced and stabilized.

  The concentration of PLP in the reaction solution is usually 0.0001 to 10 mM, preferably 0.001 to 1 mM, more preferably 0.005 to 0.1 mM. Moreover, when an aldehyde derivative is produced in the reaction, the enzyme activity can be stabilized by adding sodium bisulfite. The concentration of sodium bisulfite in the reaction solution is usually 0.05 to 5 equivalents, preferably 0.1 to 2 equivalents, and more preferably 0.5 to 1 equivalents of the aldehyde derivative produced.

  In the present invention, the optically active β-hydroxyamino acid that can be isolated is not particularly limited as long as it precipitates under high concentration conditions. For example, the following formula 1 (wherein R represents an optionally substituted aliphatic group, alicyclic group, aromatic group or heterocyclic group)

で表される構造を有するβ-ヒドロキシアミノ酸は原料として有用である。
本発明によるDL-β-ヒドロキシアミノ酸は、DL-エリスロβ−ヒドロキシアミノ酸およびDＬ−スレオ-β-ヒドロキシアミノ酸のいずれか、または両方であることを意味する。

A β-hydroxyamino acid having a structure represented by is useful as a raw material.
A DL-β-hydroxy amino acid according to the present invention means either or both of DL-erythro β-hydroxy amino acid and DL-threo-β-hydroxy amino acid.

  In the formula, R represents an optionally substituted aliphatic group, alicyclic group, aromatic group or heterocyclic group. Specifically, a lower alkyl group having 10 or less carbon atoms, a halogen atom, a nitro group, an alkoxy group having 10 or less carbon atoms, an alkyl group which may be substituted with a hydroxyl group, an alkenyl group, an alkynyl group, an alicyclic hydrocarbon A compound which is a substituent selected from a group, an aromatic hydrocarbon group or a heterocyclic hydrocarbon group. More specific examples include a cyclohexyl group, a phenyl group, a naphthyl group, an alkyl group having 20 or less carbon atoms, preferably 10 or less, an allyl group, and a thienyl group.

  In particular, compounds in which R is a cyclohexyl group are preferred compounds in the present invention. That is, for example, the present invention is useful in a method for producing D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid using DL-erythro 2-amino-3-cyclohexyl-3-hydroxypropionic acid as a raw material. is there.

  The concentration of DL-β-hydroxyamino acid as a reaction raw material in the reaction solution is not limited. Usually, a raw material having a concentration of 1 to 60%, preferably 2 to 30%, more preferably 5 to 15%, more preferably 2.5 to 7.5% can be used. The concentration of the raw material exemplified here is the weight percent of the raw material with respect to the weight of the solvent constituting the reaction solution. In the present invention, the concentration of the raw material relative to the reaction solution is independent of the dissolved state of the raw material. Therefore, the concentration is constant when the added raw materials are the same regardless of the dissolved state of the raw materials. That is, the raw material may be in a completely dissolved state, or may be in a suspended state that is not completely dissolved as long as it can be suitably handled in the present invention.

  In the present invention, DL-erythro-β-hydroxyamino acid that is preferable as a raw material is a mixture of D-erythro-β-hydroxyamino acid and L-erythro-β-hydroxyamino acid. The ratio of D form to L form in the mixture is D form: L form = 10: 90 to 90:10, preferably D form: L form = 25: 75 to 75:25, more preferably D form. : L form = 50: 50 (racemic form).

  Similarly, a preferable DL-threo-β-hydroxyamino acid as a raw material is a mixture of D-threo-β-hydroxyamino acid and L-threo-β-hydroxyamino acid, and the ratio of D-form to L-form in the mixture Is D form: L form = 10: 90 to 90:10, preferably D form: L form = 25: 75 to 75:25, more preferably D form: L form = 50: 50 (racemic form). ).

  According to the present invention, the target optically active β-hydroxyamino acid is recovered from the reaction solution in which the optically active β-hydroxyamino acid is deposited in the presence of the enzyme active substance having the above activity through a dissolution step. The recovered optically active β-hydroxyamino acid can be isolated from high-purity optically active β-hydroxyamino acid without requiring operations such as decolorization and recrystallization. In the present invention, the method for dissolving the optically active β-hydroxyamino acid is not limited.

  For example, the optically active β-hydroxyamino acid partially precipitated in the reaction solution can be dissolved under acidic conditions. The pH for dissolution can be selected from the range of usually pH 0.1 to 2.5, preferably pH 0.2 to pH 2.0, more preferably pH 0.4 to 1.5. The pH can be adjusted by adding an acid. Any acid can be used to adjust the pH. For example, mineral acids such as sulfuric acid, hydrochloric acid, and phosphoric acid are useful for adjusting the pH in the present invention. In the present invention, dissolution of the optically active β-hydroxyamino acid under acidic conditions is a preferred embodiment.

  The pH can be appropriately selected according to conditions such as the type and amount of the target optically active β-hydroxyamino acid, the temperature of the solvent, or the type of solvent. If the dissolution of the optically active β-hydroxyamino acid is insufficient, the recovery rate may decrease. On the other hand, the addition of an excessive acid causes unnecessary inorganic salts to be generated in the process of neutralization crystallization. Large amounts of inorganic salts can hinder the purification of optically active β-hydroxy amino acids. Therefore, in the present invention, when the optically active β-hydroxyamino acid is dissolved under acidic conditions, it is desirable to select an appropriate pH according to the type and amount of the target compound.

  In addition, the optically active β-hydroxyamino acid can be dissolved by alkalinization. However, when a microbial cell is used as the enzyme active substance, lysis may be induced by setting the pH to the alkaline side. It is often difficult to remove the contents of microbial cells eluted in the reaction solution by lysis from the optically active β-hydroxyamino acid. Therefore, it is preferable to avoid making the reaction solution alkaline in the state where microbial cells are present in the reaction solution.

  On the other hand, the temperature condition for dissolving the optically active β-hydroxyamino acid is usually 30 ° C. to 80 ° C., preferably 40 ° C. to 70 ° C., more preferably 40 ° C. to 60 ° C. In general, the higher the temperature, the higher the solubility. However, when treated at a high temperature, the optically active β-hydroxyamino acid may be decomposed or racemized.

  Furthermore, when the optically active β-hydroxyamino acid precipitated by heating is dissolved while maintaining the pH, the target optically active β-hydroxyamino acid can also be dissolved by heating operation.

Therefore, for example, specific conditions for sufficiently dissolving the optically active β-hydroxyamino acid precipitated in a general buffer and recovering it with high purity include the following conditions: it can.
Temperature: 40 ° C-70 ° C
pH: 0.2-2.0

  In the present invention, the time for dissolution is not limited. Under the given conditions, the dissolution step can be terminated when the target optically active β-hydroxyamino acid is sufficiently dissolved. For example, when the conditions as described above are given, the optically active β-hydroxyamino acid can be sufficiently dissolved in 2 to 72 hours, preferably 4 to 48 hours, more preferably 6 to 24 hours. . In order to promote dissolution of the optically active β-hydroxyamino acid, it is more preferable to carry out the stirring.

In the present invention, the reaction solution containing the optically active β-hydroxyamino acid to be dissolved may contain an enzyme active substance in addition to the target optically active β-hydroxyamino acid. Furthermore, coexistence of various additives useful for promoting or maintaining the reaction by the enzyme active substance is allowed. Examples of such additives include the following.
Additives to enhance enzyme activity,
Additives for stabilizing enzyme activity,
Additives for maintaining or enhancing the fluidity of the reaction solution

  For example, the addition of a coenzyme is effective for enhancing or maintaining the enzyme activity. The L-phenylserine aldolase exemplified above has its enzyme activity enhanced by adding PLP which is a coenzyme.

  Therefore, the reaction solution after the reaction with the enzyme active substance can be used in the production method according to the present invention while containing the enzyme active substance. Alternatively, the dissolution process of the present invention can be carried out on a solid that once separated in the reaction solution and containing the optically active β-hydroxyamino acid and then redispersed in water. The solid content in the reaction solution can be separated by centrifugation or membrane filtration.

In the present invention, the dissolved optically active β-hydroxyamino acid is converted into an aqueous solution of optically active β-hydroxyamino acid through a step of removing the enzyme active substance. The method for removing the enzyme active substance is not limited. For example, an enzyme active substance composed of microbial cells can be removed by centrifugation or membrane filtration.
The target optically active β-hydroxyamino acid can be purified from the aqueous solution of the optically active β-hydroxyamino acid obtained by the present invention by a method such as crystallization. For example, when the optically active β-hydroxyamino acid is dissolved in an acidic state, the target optically active β-hydroxyamino acid crystal can be obtained by neutralization crystallization by adding an alkali. The alkali salt used for neutralization crystallization is not limited.

  In general, in neutralization crystallization using acidic conditions, it is recommended to use an alkali salt that does not generate an insoluble salt by reaction with an acid. For example, sodium hydroxide, ammonia, potassium hydroxide, sodium carbonate, potassium carbonate, calcium hydroxide and the like can be used. The pH at which neutralization crystallization is selected from the range of pH at which the target optically active β-hydroxyamino acid is obtained as crystals. Specifically, for example, the pH is 2.5 to 9.0, preferably 4.0 to 8.0, and more preferably 5.0 to 7.0.

In the present invention, when the optically active β-hydroxyamino acid is dissolved by heating, an optically active β-hydroxyamino acid crystal is obtained by lowering the temperature of the recovered aqueous solution after removing the enzyme active substance. Can do.
Crystals of optically active β-hydroxyamino acid obtained by crystallization can be recovered by centrifugation or membrane filtration.

  Impurities generated by the reaction, such as glycine in the case of the reaction with aldolase, can be effectively removed by washing the crystal with water or a mixture of water and a hydrophilic organic solvent such as methanol. The aldehyde produced by the reaction can be removed by washing the crystal with an organic solvent having high solubility of aldehyde and low solubility of optically active β-hydroxyamino acid. Examples of the organic solvent include isopropyl alcohol, methanol, alcohol and the like.

  Alternatively, extraction can be performed before the sterilization step or the crystallization step using an organic solvent having low solubility in water. Examples of the organic solvent for extraction include toluene, hexane, ethyl acetate, t-methylbutyl ether and the like.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this. “%” Shown below means “weight of raw material or product / weight of reaction liquid (w / w)”.
In the case of D-erythro-β-hydroxyamino acid, ee means a numerical value represented by the following formula.
(([D-erythro concentration]-[L-erythro concentration]))
/ ([D-erythro concentration] + [L-erythro concentration])) x 100

Similarly, in the case of D-threo-β-hydroxyamino acid, it means a numerical value represented by the following formula.
(([D-threo concentration]-[L-threo concentration])
/ ([D-threo concentration] + [L-threo concentration])) x 100

In the case of D-erythro-β-hydroxyamino acid, de means a numerical value represented by the following formula.
(([D-erythro concentration] + [L-erythro concentration]-[D-threo concentration]-[L-threo concentration]) / ([D-erythro concentration] + [ L-erythro concentration] + [D-threo concentration] + [L-threo concentration])) x 100

Similarly, in the case of D-threo-β-hydroxyamino acid, it means a numerical value represented by the following formula.
(([D-threo body concentration] + [L-threo body concentration] − [D-erythro body concentration] − [L-erythro body concentration]) / ([D-threo body concentration] + [ L-threo concentration] + [D-erythro concentration] + [L-erythro concentration])) x 100

  DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid and D- or L-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid can be obtained by the method of “Analysis Method-1” below. Were analyzed quantitatively. The optical purity of D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was analyzed by the method of “Analysis Method-2”.

Analysis Method-1 (Quantitative analysis method of DL or D or L-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid)
Column: Wakosil II 5C18 HG (φ4.6 mm × 250 mm)
Temperature: 40 ° C
Detection: UV210 nm
Flow rate: 1.0 mL / min
Solvent: 50 mM potassium phosphate buffer (pH 2.5) / acetonitrile = 95/5

Analytical method-2 (Optical purity analyzing method of D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid)
Column: CHIRALPAK AD-H (φ4.6 mm × 250 mm)
Temperature: 25 ° C
Detection: UV210 nm
Flow rate: 1.0 mL / min
Solvent: hexane / ethanol / trifluoroacetic acid = 90/10 / 0.1

(Example 1) Preparation of cell suspension expressing L-phenylserine aldolase derived from Pseudomonas putida biovar A 24-1 Pseudomonas putida biovar A 24-1 Pseudomonas putida biobar A 24- E. coli strain HB101 (HB101 (pKK-PSA1)) carrying plasmid pKK-PSA1 (FERM P-20054) expressing L-phenylserine aldolase derived from one strain (Pseudomonas putida biovar A 24-1) was ampicillin (50 μg / LB medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 1% sodium chloride, pH 7.2), and the expression of the target gene was induced with 0.1 mM IPTG. The obtained cells were collected to obtain cells of E. coli strain HB101 (HB101 (pKK-PSA1)).

(Example 2) Optical purity analysis of erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid 0.05 g of the reaction solution was sampled, and 0.5 mL of 50 mM sodium borate buffer (pH 10.8) was added. The supernatant obtained by suspending and centrifuging was added with 0.5 mL of methanol and 0.01 g of anhydrous N-tertiary butoxycarbonyl and reacted at 30 ° C. for 1 hour. Thereafter, 0.4 mL of 5% aqueous potassium hydrogen sulfate solution was added to the reaction solution, and the mixture was extracted with 2.0 mL of ethyl acetate. The organic layer was dried and dissolved in a solvent of hexane / ethanol = 90/10, and the measurement was performed by Analytical Method-2.

(Comparative Example 1)
D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was synthesized using the cells of E. coli strain HB101 (HB101 (pKK-PSA1)) obtained in Example 1.

  457 g of a reaction solution containing 50 μM PLP, 7% DL-erythro 2-amino-3-cyclohexyl-3-hydroxypropionate sodium (D-ACHP · Na) and bacterial cells was adjusted to pH 8.5 and stirred. , Reacted at 30 ° C. overnight. After completion of the reaction, the optical purity analysis of the remaining D-ACHP was analyzed by the method described in Example 2. As a result of analyzing the optical purity, they were 97.1% ee (D-erythro isomer) and 93.7% dee (erythro isomer). D-ACHP was refine | purified from the reaction liquid which complete | finished reaction.

After the reaction was completed, sulfuric acid was added to adjust the pH to 0.5, and the mixture was allowed to stand at 25 ° C. for about 10 minutes. Next, the solution was sterilized by centrifugation, and the supernatant was collected. D-ACHP could be dissolved in this supernatant at a recovery rate of 21.9%. The recovery rate here represents how much the product in the reaction solution is dissolved, and was calculated as follows.
Recovery (%) = [ACHP amount dissolved in the supernatant obtained by centrifugation] / [ACHP amount present in the solution at the end of the reaction]
The ACHP present in the solution at the end of the reaction was quantified by the method of Analysis Method-1 using the sampled reaction solution suspended and dissolved in an equal amount of 1N NaOH and centrifuging the supernatant as a sample.

  No decrease in optical purity was observed before and after acid treatment at room temperature. The obtained supernatant was adjusted to pH 6.0 with 25% NaOH solution and neutralized crystallization was performed. Thereafter, the crystallization liquid was filtered through 5C filter paper. The obtained filter cake was rinsed with cold water and cold isopropyl alcohol to obtain 4.92 g of wet crystals of D-ACHP. The wet crystals were dried under reduced pressure, and 3.25 g of white D-ACHP crystals (purity 90.8%, 99.4% ee (D-erythro form), 97.3% de (erythro form), consistent isolation The yield was 10.3%.

(Comparative Example 2)
D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was synthesized using the cells of E. coli strain HB101 (HB101 (pKK-PSA1)) obtained in Example 1.
40 g containing 50 mM potassium phosphate buffer (pH 8.5), 50 μM PLP, 6% sodium DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionate (D-ACHP · Na) and cells The reaction solution was reacted at 30 ° C. overnight with stirring. After completion of the reaction, the optical purity analysis of the remaining D-ACHP was analyzed by the method described in Example 2. As a result of analyzing optical purity, they were 94.2% ee (D-erythro isomer) and 94.2% de (erythro isomer). D-ACHP was purified from this reaction solution.

  After completion of the reaction, sulfuric acid was added to the reaction solution to adjust to pH 3.0, and then the temperature was raised to 95 ° C. and heat treatment was performed for 6 hours with stirring. Next, the acid-treated and heat-treated solution was sterilized by centrifugation. As a result of optical purity analysis, 93.9% ee (D-erythro form) and 95.5% de (erythro form) before heating were 91.7% ee (D-erythro form) after heating. , 83.7% de (erythro), and a significant decrease in optical purity was observed by heating at 95 ° C.

(Example 3)
Using E. coli HB101 strain (HB101 (pKK-PSA1)) obtained in Example 1, D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was synthesized.
10 g containing 50 mM potassium phosphate buffer (pH 8.5), 50 μM PLP, 6% sodium DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionate (D-ACHP · Na) and cells The reaction solution was reacted at 30 ° C. overnight with stirring. After completion of the reaction, the optical purity analysis of the remaining D-ACHP was analyzed by the method described in Example 2. As a result of analyzing the optical purity, they were 98.8% ee (D-erythro isomer) and 92.0% de (erythro isomer).

  After completion of the reaction, the reaction solution was equally divided into 5 g portions, each was adjusted to pH 0.5 by adding sulfuric acid, and then stirred at 25 ° C. and 50 ° C. The degree of dissolution of the product by stirring at each temperature is shown in FIG.

  What was stirred at 50 ° C. was able to dissolve D-ACHP with a recovery rate of almost 100% in 5 hours, but what was stirred at 25 ° C. was only about 80% recovery after 24 hours. -ACHP could not be dissolved. Here, the recovery rate represents how much the product in the reaction solution is dissolved, and was calculated as follows.

Recovery (%) = [ACHP amount dissolved in the supernatant obtained by centrifugation] / [ACHP amount present in the solution at the end of the reaction]
The ACHP present in the solution at the end of the reaction was quantified by the method of Analysis Method-1 using the sampled reaction solution suspended and dissolved in an equal amount of 1N NaOH and centrifuging the supernatant as a sample.

Example 4
Using E. coli HB101 strain (HB101 (pKK-PSA1)) obtained in Example 1, D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was synthesized.
10 g containing 50 mM potassium phosphate buffer (pH 8.5), 50 μM PLP, 6% sodium DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionate (D-ACHP · Na) and cells The reaction solution was reacted at 30 ° C. overnight with stirring. After completion of the reaction, the optical purity analysis of the remaining D-ACHP was analyzed by the method described in Example 2. As a result of analyzing the optical purity, they were 93.4% ee (D-erythro isomer) and 92.5% de (erythro isomer). D-ACHP was purified from this reaction solution.

  After the reaction was completed, sulfuric acid was added to adjust the pH to 0.5, and then the temperature was raised to 50 ° C. and heated for 5 hours with stirring. Next, the solution was sterilized by centrifugation, and the supernatant was collected. D-ACHP could be dissolved in this supernatant at a recovery rate of 93.9%. Here, the recovery rate represents how much the product in the reaction solution is dissolved, and was calculated as follows.

Recovery (%) = [ACHP amount dissolved in the supernatant obtained by centrifugation] / [ACHP amount present in the solution at the end of the reaction]
The ACHP present in the solution at the end of the reaction was quantified by the method of Analysis Method-1 using the sampled reaction solution suspended and dissolved in an equal amount of 1N NaOH and centrifuging the supernatant as a sample.

  No decrease in optical purity was observed before and after acid treatment and heat treatment. The obtained supernatant was adjusted to pH 6.0 with 25% NaOH solution and neutralized crystallization was performed. Thereafter, the crystallization liquid was filtered through 5C filter paper. The obtained filter cake was rinsed with cold water and cold isopropyl alcohol to obtain 0.44 g of wet crystals of D-ACHP. Wet crystals were dried under reduced pressure, 0.27 g of white D-ACHP crystals (purity 90.6%, 97.9% ee (D-erythro isomer), 96.0% de (erythro isomer), consistent isolation) Yield 45.5%).

(Example 5)
Using E. coli HB101 strain (HB101 (pKK-PSA1)) obtained in Example 1, D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was synthesized.
70 g of a reaction solution containing 50 μM PLP, 6% DL-erythro 2-amino-3-cyclohexyl-3-hydroxypropionate sodium (D-ACHP • Na) and cells was adjusted to pH 8.0, and stirred. , Reacted at 30 ° C. overnight. After completion of the reaction, the optical purity analysis of the remaining D-ACHP was analyzed by the method described in Example 2. As a result of analyzing optical purity, they were 98.1% ee (D-erythro form) and 94.2% de (erythro form). D-ACHP was purified from this reaction solution.

  After completion of the reaction, concentrated sulfuric acid was added to adjust the pH to 0.9, and then the temperature was raised to 50 ° C. and heated for 24 hours while stirring. Next, the acid-treated and heat-treated solution was sterilized by centrifugation, and the supernatant was collected. D-ACHP could be dissolved in this supernatant at a recovery rate of 98.8%. The recovery rate here represents how much the product in the reaction solution is dissolved, and was calculated as follows.

Recovery (%) = [ACHP amount dissolved in the supernatant obtained by centrifugation] / [ACHP amount present in the solution at the end of the reaction]
The ACHP present in the solution at the end of the reaction was quantified by the method of Analysis Method-1 using the sampled reaction solution suspended and dissolved in an equal amount of 1N NaOH and centrifuging the supernatant as a sample.

  No decrease in optical purity was observed before and after acid treatment and heat treatment. The obtained supernatant was adjusted to pH 6.0 with a 25% NaOH solution and subjected to neutralization crystallization. Thereafter, the crystallization liquid was filtered through 5C filter paper. The obtained filter cake was rinsed with cold water and cold isopropyl alcohol to obtain 31.77 g of D-ACHP wet crystals. The wet crystals were dried under reduced pressure, and 1.47 g of white D-ACHP crystals (purity 99.9%, 99.8% ee (D-erythro form), 98.2% de (erythro form), consistent isolation Yield 39.2%).

The degree of dissolution of the product by stirring at each temperature is shown. In the figure, the vertical axis represents the recovery rate (%), and the horizontal axis represents the stirring time (hour / hr).

Claims (8)

A step of causing an enzyme active substance to act on DL-β-hydroxyamino acid in the reaction solution to accumulate D- or L-β-hydroxyamino acid in the reaction solution to a concentration higher than the precipitation concentration;
A step of dissolving the accumulated D or L-β-hydroxyamino acid by maintaining at 30 ° C. to 80 ° C. and recovering the dissolved D or L-β-hydroxyamino acid. Production method. The enzyme active substance coexists with D or L-β-hydroxyamino acid, and after removing the enzyme active substance from the reaction solution in which D or L-β-hydroxyamino acid is dissolved, D- or L-β-hydroxyamino acid The manufacturing method of Claim 1 which further includes the process of crystallizing. The production method according to claim 1 or 2, wherein D- or L-β-hydroxyamino acid is dissolved by adjusting the reaction solution to pH = 0.1 to 2.5. The production method according to claim 3, wherein the D- or L-β-hydroxyamino acid is dissolved by adjusting the reaction solution to pH = 0.2 to 2.0 and holding at 40 to 70 ° C for 4 hours or more. β-hydroxyamino acid is represented by the following formula 1 (wherein R represents an optionally substituted aliphatic group, alicyclic group, aromatic group or heterocyclic group)

The manufacturing method of Claim 1 thru | or 4 which is (beta) -hydroxyamino acid represented by these. 6. The production method according to claim 5, wherein R is an optionally substituted cyclohexyl group. 7. The production method according to claim 1, wherein the enzyme active substance has L-phenylserine aldolase activity. The enzyme active substance is at least one selected from the group consisting of a protein encoded by the polynucleotide according to any of the following (a) to (e), a microorganism expressing the protein, and a processed product thereof: The manufacturing method of Claim 7.
(a) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1;
(b) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2;
(c) a polynucleotide encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2;
(d) a polynucleotide that hybridizes with the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions; and
(e) a polynucleotide encoding an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 2

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