JP2006121983A - METHOD FOR PRODUCING OPTICALLY ACTIVE beta-HYDROXYAMINO ACID IN PRESENCE OF ADDITIVE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an optically active β-hydroxyamino acid. <P>SOLUTION: This method for producing the optically active β-hydroxyamino acid, includes a step for incubating a raw material and an enzyme activating substance in the presence of an alcohol, an amino alcohol, an amine, glycine, phosphoric acid or a phosphate. The fluidity of a reaction liquid containing the β-hydroxyamino acid having low water-solubility is improved by the presence of additives such as the alcohol, the amino alcohol, the amine, glycine, phosphoric acid or the phosphate. The fluidity-improving effect is kept even in reaction. The reaction efficiency of the enzyme activating substance is improved thereby. <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.

Conventionally, the following method has been known as a method for synthesizing optically active β-hydroxyamino acids.
(1) A chemical synthesis method in which an aldehyde derivative and glycine are condensed in the presence of a strong alkali, then the threo / erythro isomers are mutually separated, and optical resolution is performed using an optical resolution agent. (2) Glycine or glycine metal complex Synthesis method in which aldehyde derivative is reacted with aldehyde derivative in the presence of D-threonine aldolase (Patent Documents 1 and 2)
(3) Enzymatic synthesis method in which DL-erythro-β-hydroxyamino acid is allowed to act on L-alurothreonine aldolase that specifically decomposes L-erythro-β-hydroxyamino acid into an aldehyde derivative corresponding to glycine (Patent Document 3) )

However, the above process has the following problems, for example. Therefore, it is difficult to say that it is a practical manufacturing method at an industrial level.
-Complicated process,
-Yield is poor,
-High production costs
-Low diastereomeric selectivity,
-The reaction is hindered by high concentrations of raw materials,

  Generally, when producing a substance industrially, in order to reduce the manufacturing cost, it is extremely effective to improve the production amount per reaction tank, that is, increase the accumulated amount of the product. A method using an enzyme active substance such as a microorganism or an enzyme is one of the effective methods for satisfying the condition.

  When an optically active β-hydroxyamino acid is produced by the action of a microorganism or an enzyme, DL-β-hydroxyamino acid is used as a raw material. DL-β-hydroxyamino acid may have very low solubility in water depending on its structure. If the solubility of the raw material is low, increasing the amount of DL-β-hydroxyamino acid added to the reaction tank significantly reduces the fluidity of the reaction solution at the start of the reaction. Or, in an extreme case, the reaction solution is solidified.

As a result, the mixing efficiency of the reaction solution is reduced, leading to a reduction in the contact frequency between the enzyme active substance and the raw material. When the reaction solution is solidified, the contact between the two is significantly inhibited. In such a state, the reaction efficiency is significantly reduced. That is, in the production of an optically active β-hydroxyamino acid, a reduction in reaction efficiency due to its low solubility has been a major obstacle in developing an industrial production method.
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 using a DL-β-hydroxyamino acid having low water solubility as a raw material. More specifically, an object of the present invention is to provide a method for producing an optically active β-hydroxyamino acid with improved fluidity of the reaction solution.

  In the method for producing an optically active β-hydroxyamino acid, DL-β-hydroxyamino acid having low water solubility is used as a raw material. And by making an enzyme active substance act on a raw material, the target optically active beta-hydroxy amino acid accumulates in a reaction liquid. The optically active β-hydroxyamino acid itself that accumulates is also a compound with low water solubility. Any of these compounds can be dissolved using acid or alkali. However, when utilizing the action of the enzyme active substance, the reaction solution is adjusted to a neutral pH that is the optimum pH for the enzyme reaction. That is, the reaction by the enzyme active substance proceeds with the raw material deposited. Therefore, the precipitated β-hydroxyamino acid is present in both the reaction solution containing the raw material and the reaction solution in which the optically active β-hydroxyamino acid is accumulated.

  The enzymatic reaction itself proceeds with the raw material partially dissolved. The fact that a part of the raw material is deposited does not disturb the enzyme reaction. However, when insoluble β-hydroxyamino acid is present in the reaction solution, the fluidity of the reaction solution is lowered. A decrease in the fluidity of the reaction solution may limit the contact between the enzyme active substance and the raw material, leading to a decrease in reaction efficiency or an inhibition of the reaction itself.

  The present inventors have repeated research on a method that can suppress a decrease in fluidity of a reaction solution even if the raw material or the target optically active β-hydroxyamino acid is present in the reaction solution in an insoluble state. . As a result, by adding one or a combination of compounds selected from alcohols, amino acids, amino alcohols, amines and phosphoric acid to the reaction solution, most of D- or It has been found that even when L-β-hydroxyamino acid is accumulated to the extent that it precipitates, the fluidity of the reaction solution can be improved or solidification can be suppressed, and the present invention has been completed. That is, the present invention provides the following method for producing an optically active β-hydroxyamino acid.

で表される、上記〔１〕乃至〔４〕記載の製造方法。
〔６〕Ｒが置換され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〕酵素活性物質は、L-フェニルセリンアルドラーゼ、L-フェニルセリンアルドラーゼを発現する微生物、または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〔１〕乃至上記〔６〕に記載の方法。
〔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] Contact an enzyme active substance with DL-β-hydroxyamino acid in a reaction solution containing at least one compound selected from alcohols, amino alcohols, amines, glycine or phosphoric acid or phosphate And a step of recovering D or L-β-hydroxyamino acid accumulated in the reaction solution.
[2] The production method of the above-mentioned [1], wherein the concentration of alcohol, amino alcohol, amine, glycine, phosphoric acid or phosphate contained in the reaction solution is 0.05% to 25%.
[3] The production method of the above-mentioned [1], wherein the concentration of alcohol, amino alcohol, amine, glycine, phosphoric acid or phosphate contained in the reaction solution is 0.2% to 5%.
[4] The alcohol is methanol, ethanol, glycerol, 1,3-butanediol, ethylene glycol, the amino alcohol is 1-amino-2,3-propanediol, tris (hydroxymethyl) aminomethane, and an amine The production method according to the above [1] to [3], wherein the kind is ammonia.
[5] DL-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 said [1] thru | or [4] represented by these.
[6] The production method of the above-mentioned [5], wherein R is an optionally substituted cyclohexyl group.
[7] The enzyme active substance is at least one selected from the group consisting of L-phenylserine aldolase, a microorganism expressing L-phenylserine aldolase, or a processed product thereof, [1] to [6] ] The method of description.
[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, the fluidity of the reaction solution in the method for producing an optically active β-hydroxyamino acid utilizing the action of an enzyme active substance has been improved. In a reaction solution containing a β-hydroxyamino acid having low water solubility for both the raw material and the product, the fluidity often decreases under normal conditions. However, according to the present invention, the fluidity of the reaction solution is improved, and the effect continues until the reaction is completed. The decrease in the fluidity of the reaction solution restricts the contact between the enzyme active substance and the raw material, and thus significantly reduces the reaction efficiency. According to the present invention, inhibition of the catalytic action of an enzyme active substance due to a decrease in fluidity can be effectively suppressed. Thus, according to the present invention, the reaction efficiency of the method for producing an optically active β-hydroxyamino acid using an enzyme active substance is improved.

  The present invention provides an enzyme active substance as a DL-β-hydroxyamino acid in a reaction solution containing at least one compound selected from alcohols, amino alcohols, amines, glycine, or phosphoric acid or phosphate. There is provided a method for producing β-hydroxyamino acid, which comprises a step of contacting and recovering D or L-β-hydroxyamino acid accumulated in a reaction solution.

  In the present invention, the reaction liquid is a liquid capable of maintaining a reaction in which an enzyme active substance, which will be described in detail later, acts on either D- or L-β-hydroxyamino acid as a raw material to convert it into another substance. Say. Specifically, the reaction liquid is composed of components necessary for the reaction and provides a suitable environment for the reaction. For example, a coenzyme necessary for the enzyme reaction, a stabilizer for enzyme activity, or an accelerator can be included.

  The reaction solution can be composed of an aqueous solvent, an organic solvent that is difficult to dissolve in water, or a two-phase system of an organic solvent that is difficult to dissolve in water and an aqueous solvent. Examples of the organic solvent that is hardly soluble in water that can be used in the present invention include ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, methyl isobutyl ketone, methyl tertiary butyl ether, and diisopropyl ether. . On the other hand, examples of the aqueous solvent in the present invention include water or a buffer solution that maintains the enzyme activity of the enzyme active substance.

  Furthermore, the raw material and the enzyme active substance can be contacted in an organic solvent that dissolves in water or in a mixed system of an aqueous solvent and an organic solvent that dissolves in water. Examples of the organic solvent that dissolves in water include isopropyl alcohol, acetonitrile, acetone, dimethyl sulfoxide, and the like.

  In the present invention, DL-β-hydroxyamino acid is an enzyme active substance in a reaction solution containing at least one compound selected from alcohols, amino alcohols, amines, glycine, or phosphoric acid or phosphate. To act on. By adding the above-mentioned compound, it is possible to suppress a significant deterioration in the fluidity and mixing properties of the reaction liquid when these compounds are not added, and to maintain the fluidity of the reaction liquid. As a result, the contact efficiency between the suitable bioactive catalyst and the raw material is improved, and the reaction can proceed efficiently. Therefore, alcohols, amino acids, amino alcohols, amines, glycine or phosphoric acid or phosphates that can be used in the present invention can improve the fluidity of the reaction solution under the reaction conditions using the bioactive catalyst described later. There is no particular limitation as long as the maintenance or solidification can be suppressed.

For example, in the present invention, the following can be preferably used.
(1) Alcohols: methanol or ethanol or glycerol or 1,3-butanediol or ethylene glycol (2) Amino acids: Glycine (3) Amino alcohols: 1-amino-2, 3-propanediol, Tris (hydroxymethyl) ) Aminomethane (4) Amines: Ammonia (5) Phosphoric acid or phosphates such as sodium phosphate or potassium phosphate

  As the compound, one selected compound may be added to the reaction solution, or a plurality of compounds may be added. When a plurality of compounds are used in combination, a plurality of compounds can be arbitrarily selected from the above compounds.

  The concentration of the compound in the reaction solution is within a range where the fluidity of the reaction solution can be improved and solidification can be suppressed, and can be arbitrarily adjusted as long as the bioactive catalyst can maintain its enzymatic action. it can. Such a concentration can be, for example, 0.05% to 25%, preferably 0.2% to 5%, and more preferably 0.5% to 3%. The concentration of the compound may be constant during the reaction or may be changed as long as the fluidity of the reaction solution can be maintained.

  The above compound can be supplied to the reaction solution by any method. The timing of addition to the reaction solution is also arbitrary. When a plurality of compounds are added, the plurality of compounds may be added simultaneously or at different timings.

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。 The DL-β-hydroxyamino acid used as a raw material in the present invention means either DL-erythro-β-hydroxyamino acid or DL-threo-β-hydroxyamino acid. It will not specifically limit if 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)

A β-hydroxyamino acid having a structure represented by is useful as a raw material.

  DL-β-hydroxyamino acid according to the present invention means either DL-erythro-β-hydroxyamino acid or DL-threo-β-hydroxyamino 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 specifically, 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, a thienyl group, and the like can be given.

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

  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). ).

In the present invention, “%” means “weight of raw material or product / weight of reaction solution (w / w)”. In the case of D-erythro-β-hydroxyamino acid, ee means a numerical value represented by the following formula.
(([D-erythro body concentration] − [L-erythro body concentration]) / ([D-erythro body concentration] + [L-erythro body concentration])) x 100

Similarly, in D-threo-β-hydroxyamino acid, ee 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 present invention, DL-β-hydroxyamino acid as a raw material often has low solubility in the vicinity of neutrality. In particular, DL-β-hydroxyamino acid having low solubility near neutrality can be usually prepared as an acidic or alkaline solution. The pH is not limited as long as the required amount of DL-β-hydroxyamino acid can be dissolved in a required volume of solvent. In order to construct the reaction solution having the raw material concentration as described above, the raw material can be dissolved usually at pH 10-15, preferably pH 11-14, more preferably pH 12-14. The target pH can be adjusted with NaOH, KOH, or the like.

  Moreover, in order to melt | dissolve a raw material on acidic conditions, pH 0-2.0 normally, Preferably pH 0-1.0 or less, More preferably, pH 0-0.5 conditions can be shown. It can be adjusted to the required pH with hydrochloric acid, sulfuric acid, or nitric acid.

  The raw material solution containing the necessary amount of raw material is incubated with components necessary for the reaction with the enzyme active substance described later in a reaction vessel. The pH suitable for the reaction of the enzyme active substance is around neutral. Accordingly, during the reaction, the pH of the raw material solution is adjusted to near neutrality suitable for the reaction, and thus a part thereof is precipitated. However, in the present invention, the above-described alcohols, amino alcohols, amines, glycine, or phosphoric acid or phosphate coexist in the reaction solution, so that the reaction solution flows despite the precipitation of the raw materials. It becomes possible to maintain sex.

  In the production of the optically active β-hydroxyamino acid of the present invention, enzyme active materials that act stereospecifically on either the D-form or L-form of DL-β-hydroxyamino acid. ) Is used. In the present invention, the enzyme active substance is a compound that acts stereospecifically on a D-form or L-form compound of DL-β-hydroxyamino acid and catalyzes a reaction for conversion to another substance. Is defined as a substance capable of Stereospecific action refers to having a higher catalytic action on either 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, for example, Japanese Patent Application Laid-Open No. 9-238680 obtained by the method described below.
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-finished solution, stir rapidly, and let 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 cyclohexyl aldehyde per minute at 30 ° C. was defined as 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:
The reaction was performed at 30 ° C. for 10 minutes in 0.5 mL of a reaction solution containing 20 mM DL-threo-phenylserine, 200 mM TAPS-NaOH buffer (pH 8.5), 50 μM PLP, and enzyme. The reaction is stopped by adding 5 mL. The produced benzaldehyde 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. 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: Phenylmethane sulfonyl fluoride, pepstatin A, leupeptin, metal chelator PLP, etc.

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, the target enzyme can be electrophoretically purified to a single band from the cell-free extract according to the following steps.
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 to the amino acid sequence shown in SEQ ID NO: 2. : Means a protein functionally equivalent to the protein consisting of the amino acid sequence described in 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.
Host vector systems such as Escherichia genus Bacillus genus Pseudomonas genus Serratia genus Brevibacterium genus Corynebacterium genus Streptococcus genus Lactobacillus genus Lactobacillus genus Bacteria Rhodococcus spp. Streptomyces spp. Host vector systems such as Streptomyces spp. Saccharomyces spp. Kluyveromyces spp. Schizosaccharomyces spp. Yeast Neurospora that has been developed as a host vector system such as Saccharomyces genus Yarrowia genus Trichosporon genus Rhodosporidium genus Pichia genus Candida genus ora) Aspergillus genus Cephalosporium genus Trichoderma genus has developed host vector systems

  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), etc.

  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 whose cell membrane permeability has been altered 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 other than the alcohols, amino alcohols, amines, glycine or phosphoric acid or phosphate 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, maintaining the fluidity of the reaction solution, and the like. The enzyme active substance that can be suitably used in the present invention utilizes PLP as a coenzyme. Therefore, by adding PLP in the reaction solution, the 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.

  The production conditions of the optically active β-hydroxyamino acid using the enzyme active substance can be appropriately set. For example, the raw material concentration during the reaction is usually 1 to 60%, preferably 2 to 30%, more preferably 5 to 15%, and still more preferably 2.5 to 7.5%. The raw materials can be added all at once at the start of the reaction, or may be added continuously or intermittently into the reaction solution.

  The reaction temperature of the reaction solution can be selected at a temperature at which the enzyme active substance can maintain its enzymatic 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.

  Auxiliary components such as raw material solution, enzyme active substance, and coenzyme are mixed with one or more compounds selected from the above alcohols, amino alcohols, amines, glycine or phosphoric acid or phosphate, Finally, the mixing amount and pH of each component are adjusted so that the pH of the reaction solution becomes such a suitable pH. Furthermore, an appropriate buffer solution that gives a pH suitable for the reaction can be added to the reaction solution. The reaction of the present invention can be carried out under stirring or standing.

  In the present invention, the completion of the reaction means a state in which there is almost no L form (or D form) in the reaction solution. Usually, it refers to a state in which 90% e.e. or more, preferably 95% e.e. or more of D-form (or L-form) is accumulated in the reaction solution.

  D-β-Hydroxyamino acid (or L form) accumulated by the reaction is solubilized, separated by centrifugation or filtration, extracted with organic solvent, ion-exchange chromatography, etc., adsorption by adsorbent, flocculant Further, it can be purified by appropriately combining dehydration or aggregation with a dehydrating agent, crystallization, distillation, and the like. D-β-hydroxyamino acid can be solubilized by alkalinization or acidification.

  After dissolving the product, if necessary, a flocculant is added, and then sterilization and protein removal can be performed by centrifugation, membrane filtration, or the like. The aldehyde produced by the reaction can be removed by extraction with an organic solvent having high solubility of aldehyde and low solubility of D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid. . Examples of such an organic solvent include hexane, toluene, xylene, ethyl acetate, butyl acetate, methyl tertiary butyl ether, methyl isobutyl ketone, and the like. Aldehydes extracted with organic solvents can be recovered and reused. From the aqueous phase after extraction with an organic solvent, D-erythro-2-amino-3-cyclohexyl-3-hydroxy is obtained by known methods such as crystallization by concentration, isoelectric precipitation, ion exchange resin treatment, membrane separation, etc. Propionic acid can be recovered.

  In the present invention, by causing an enzyme active substance to act on the raw material, L-β-hydroxyamino acid in the raw material is consumed, and the target compound, D-β-hydroxyamino acid, may remain in the reaction system. . In the present invention, the formation of a target compound stereospecifically is sometimes referred to as generation. In the present invention, the L-form of the raw material containing D-form and L-form is enzymatically removed, and the remaining D-form is recovered as the target product. The D form itself is a substance originally contained in the raw material. However, making the D-form mixed with the L-form have a high optical purity is nothing other than “production of D-form” or “accumulation of D-form”.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this. In the examples shown below, the optical purity analysis of D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was carried out by the following “Analysis Method-1” method.

Analysis Method-1 (Optical purity analysis method for D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid)
Column: CHIRALPAK AD-H (φ4.6 mm × 250 mm)
Temperature: 25 ° C
Detection: UV 210 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 biobar A 24-1 1 strain (Pseudomonas putida biovar A 24-1) -derived L-phenylserine aldolase expressing plasmid pKK-PSA1 E. coli strain HB101 (HB101 (pKK-PSA1)) containing ampicillin (50 μg / mL) in LB medium ( The cells were cultured in 1% bacto-tryptone, 0.5% bacto-yeast extract, 1% sodium chloride, pH 7.2), and expression of the target gene was induced with 0.1 mM IPTG. The obtained cells were collected to obtain cells of Escherichia coli HB101 strain (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. Then, 0.5 mL of methanol and 0.01 g of anhydrous N-tertiary butoxycarbonyl were added to the supernatant obtained by suspending and centrifuging, 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 extraction was performed 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 the analysis method-1 described above.

[Example 3] Synthesis of D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid under conditions with additives added Escherichia coli HB101 obtained in Example 1 (HB101 (pKK-PSA1)) And using a DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid solution in the form of a strong alkaline solution as a raw material, a D-erythro-2-amino-3-cyclohexyl-3-hydroxypropion Acid synthesis was performed.

  After charging the following additive-1 and additive-2 and DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid as a raw material so that the final concentration becomes 6%, Further, sulfuric acid was added to adjust the pH of the solution to about 8.5. Furthermore, 50 μM PLP and bacterial cells were added to give a total weight of 20 g, or 70 g of the reaction solution was reacted overnight at 30 ° C. with stirring. After completion of the reaction, the optical purity of the remaining D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was analyzed by the method described in Example 2.

  Table 1 shows the optical purity of additive-1, additive-2, liquid properties after the overnight reaction, and D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid remaining in the reaction solution. Even when any additive was added, decomposition of unnecessary isomers proceeded, and it was confirmed that D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was obtained with high optical purity. . With respect to the solidification inhibiting effect of the reaction solution, all of the investigated additives had the solidification inhibiting effect of the reaction solution.

(Comparative Example 1) Synthesis of D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid solution Using the cells of Escherichia coli HB101 strain (HB101 (pKK-PSA1)) obtained in Example 1, D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was synthesized using a DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid solution in the form of a strong alkaline solution as a raw material.

  The raw material DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was added to the reaction vessel so that the concentration was 6%, and then the pH was adjusted to about 8.5 using sulfuric acid. Further, a reaction solution having a total weight of 20 g to which 50 μM PLP and bacterial cells were added was reacted at 30 ° C. overnight with stirring. After completion of the reaction, the optical purity analysis of the remaining D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was analyzed by the method described in Example 2. The results are shown in Table 1. Although the decomposition of unnecessary isomers proceeded, it was confirmed that D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was obtained with high optical purity, but the reaction solution was not fluid. It was in a solidified state.

Claims (8)

The reaction is carried out by contacting the enzyme active substance with DL-β-hydroxyamino acid in a reaction solution containing at least one compound selected from alcohols, amino alcohols, amines, glycine or phosphoric acid or phosphate. A method for producing β-hydroxyamino acid, comprising a step of recovering D or L-β-hydroxyamino acid accumulated in a liquid. The production method according to claim 1, wherein the concentration of alcohol, amino alcohol, amine, glycine, phosphoric acid or phosphate contained in the reaction solution is 0.05% to 25%. The production method according to claim 1, wherein the concentration of alcohol, amino alcohol, amine, glycine, phosphoric acid or phosphate contained in the reaction solution is 0.2% to 5%. Alcohols are methanol, ethanol, glycerol, 1,3-butanediol, ethylene glycol,
The amino alcohol is 1-amino-2,3-propanediol, tris (hydroxymethyl) aminomethane,
The production method according to claim 1, wherein the amine is ammonia. DL-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 Claims 1 thru | or 4 represented by these. 6. The production method according to claim 5, wherein R is a cyclohexyl group which may be substituted. The method according to any one of claims 1 to 6, wherein the enzyme active substance is at least one selected from the group consisting of L-phenylserine aldolase, a microorganism expressing L-phenylserine aldolase, or a processed product thereof. 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