JP2006129843A - METHOD FOR PRODUCING OPTICALLY ACTIVE beta-HYDROXYAMINO ACID UNDER PH REGULATION - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an optically active β-hydroxyamino acid by which the fluidity of a reaction liquid is improved. <P>SOLUTION: The method for producing the D- or L-β-hydroxyamino acid involves reacting DL-β-hydroxyamino acid with an enzyme-active substance in a reaction liquid having the pH regulated to 5.5-10. The pH is regulated in an alkaline region at the start of the reaction, and regulated again from weak alkaline to acidic on the way of the reaction according to the rheology change of the reaction liquid, e.g. the change by solidification of the liquid. The fluidity of the reaction liquid containing the β-hydroxyamino acid having low water solubility is improved by such regulation of the pH at the reaction time. The invention contributes to the improvement of the reaction efficiency of an enzyme-active substance. <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, and 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.

  As a result of diligent research, the present inventors have found that the pH from the start of the reaction to the end of the reaction has an important effect on the rheological change of the reaction solution, and is optimal for maintaining the fluidity of the reaction solution. The present inventors have found a pH condition and completed the present invention. That is, the present invention provides the following method for producing an optically active β-hydroxyamino acid.

である、上記〔１〕乃至上記〔１２〕記載の方法。
〔１４〕前記式１中、Rが置換されてもよいシクロヘキシル基である、上記〔１３〕記載の方法。 [1] A method for producing D or L-β-hydroxyamino acid by reacting an enzyme active substance with DL-β-hydroxyamino acid in a reaction solution having a pH adjusted to 5.5 to 10.
[2] The method according to [1] above, wherein the pH is adjusted to a pH capable of maintaining the fluidity of the reaction solution after the reaction starts.
[3] The method according to [2] above, wherein the pH adjustment after the start of the reaction is performed according to a change in the rheology of the solution.
[4] The method according to [2] or [3] above, wherein the pH adjustment is an adjustment in a direction of decreasing the pH.
[5] The method described in [1] to [4] above, wherein the pH at the start of the reaction is in an alkaline region.
[6] The method according to [5] above, wherein the pH in the alkaline region is 8.0 to 10.0.
[7] The method described in [1] to [6] above, wherein the pH is adjusted from weakly alkaline to acidic pH after the reaction starts.
[8] The method according to [7] above, wherein the pH range from weakly alkaline to acidic is 7.5 to 5.5.
[9] The method described in [1] to [8] above, wherein the pH adjustment is performed using phosphoric acid.
[10] The method described in [3] to [9] above, wherein the rheological change of the solution is a change from a liquid to a solid.
[11] The method according to [1] to [10] above, wherein the enzyme active substance is L-phenylserine aldolase, a microorganism expressing L-phenylserine aldolase, or a processed product thereof.
[12] 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 that expresses the protein, and a processed product thereof The method described in [1] to [11] above.
(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. [13] DL-β-hydroxyamino acid is represented by the following formula 1 (wherein R is substituted) Represents a good aliphatic, alicyclic, aromatic or heterocyclic group)

The method described in [1] to [12] above.
[14] The method according to [13] above, wherein in formula 1, R is a cyclohexyl group which may be substituted.

  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 a method for producing D or L-β-hydroxyamino acid by reacting an enzyme active substance with DL-β-hydroxyamino acid in a reaction solution adjusted to a pH of 5.5 to 10.

  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 a promoter 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 methanol, ethanol, isopropyl alcohol, acetonitrile, acetone, dimethyl sulfoxide, and the like.

  In the present invention, the pH of the reaction solution is adjusted to 5.5 to 10, and the enzyme active substance, which will be described in detail later, is reacted with the raw material DL-β-hydroxyamino acid. In general, the pH of a reaction solution in a reaction using an enzyme active substance is adjusted at the end of the reaction in order to maintain the stability of the enzyme active substance to be used and to maintain a pH condition in which an undesirable side reaction does not occur. It is preferable. However, β-hydroxyamino acids have extremely low solubility in water depending on their structures. Therefore, when increasing the amount of β-hydroxyamino acids having a specific configuration, the amount of liquid is significantly increased between the start of the reaction and the end of the reaction. The fluidity of the liquid drops or solidifies. Once the fluidity is lowered and solidification occurs, it becomes difficult to control the pH by adding an acid or an alkali.

  Therefore, in the present invention, the pH of the reaction solution is adjusted to 5.5 to 10.0 as described above in order to maintain the fluidity of the reaction solution. The pH of the reaction solution is preferably readjusted in accordance with the rheological change of the reaction solution after the start of the reaction in order to maintain the fluidity of the reaction solution. The rheological change of the reaction solution requiring pH readjustment is a change in which the liquid is solidified, more preferably a change before the liquid is changed to a solid. Such a rheological change may be detected based on, for example, the viscosity of the reaction solution, the load (pressure) when stirring the reaction solution, and the like. Based on these detection means, the pH is readjusted with the change in the rheology of the detected reaction solution. Alternatively, the elapsed time after the start of the reaction may be experimentally analyzed and the pH may be readjusted before the elapsed time. For example, the elapsed time for pH readjustment is usually in the range of 0 to 24 hours, preferably 20 minutes to 10 hours from the start of the reaction. Thus, in the present invention, it is preferable to readjust the pH from the pH adjusted at the start of the reaction to an appropriate time zone after the start of the reaction. The readjustment of pH after the start of the reaction is not limited to one time, and may be adjusted in a plurality of times.

  The pH at the start of the reaction is within the range of 5.5 to 10.0 described above, preferably neutral to alkaline, for example, pH 7.0 to 9.5, more preferably pH 8.0 to 9.5. is there. In addition, when the reaction is started at a pH exceeding pH 10, the reaction with the enzyme active substance may be stopped in the middle, and a high optical purity target product cannot be obtained.

  In order to maintain the fluidity of the reaction solution from the start of the reaction to the end of the reaction or to suppress solidification, the pH is adjusted to be lower than the pH at the start of the reaction according to the change in the rheology of the reaction solution. The pH after the start of the reaction is adjusted to a weak alkaline region to an acidic region, for example, pH 5.5 to 7.5, preferably pH 6.0 to 7.0.

  The pH at the start can be adjusted by adding a mineral acid to a basic solution of DL-β-hydroxyamino acid as a raw material. Specifically, after adding a mineral acid to the raw material solution to adjust the pH to a suitable pH at the start, an enzyme active substance and auxiliary components such as a coenzyme may be mixed. Alternatively, an enzyme active substance and auxiliary components such as a coenzyme are mixed in the raw material solution, and the pH of the reaction solution is finally adjusted to a suitable pH at the start by adjusting the pH with mineral acid. Good.

  Moreover, pH readjustment during the reaction can be performed by adding a mineral acid to the reaction solution. Mineral acids that can be used for pH adjustment include sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, and the like, with phosphoric acid being particularly preferred. These mineral acids can also be added to the reaction solution in the form of an appropriate buffer that gives a pH suitable for the reaction. Of course, the reaction can be carried out under stirring or standing. In the present invention, it is possible to maintain the fluidity of the reaction solution by adjusting the pH, improve the contact between the enzyme active substance and the raw material, and increase the accumulation amount of the optically active β-hydroxyamino acid.

  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)

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。

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 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, DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid is useful in producing D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid by the method of the present invention. is there.

  The concentration of DL-β-hydroxyamino acid as a reaction raw material in the reaction solution is not limited. Usually, use a raw material having a concentration of 1 to 60%, preferably 2 to 30%, more preferably 2 to 15%, further preferably 2.5 to 7.5%, most preferably 5 to 7.5%. Can do. 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 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. When the raw material is dissolved in an alkaline solution, the pH of the solution can be usually pH 10 to 14, preferably pH 11 to 14, and more preferably pH 12 to 14. The target pH can be adjusted with NaOH, KOH, or the like.

  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 compound of DL-β-hydroxyamino acid are used. Used. In the present invention, the enzyme active substance can act on a D- or L-form compound of DL-β-hydroxyamino acid stereospecifically and catalyze a reaction for conversion to another substance. Defined as a substance. 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 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. 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 and the like-Protease inhibitor: Phenylmethane fluoride 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, 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 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, for example, usually 1 to 60%, preferably 2 to 30%, more preferably 2 to 15%, still more preferably 2.5 to 7.5%, and most preferably 5 to 7. It can be 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.

  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 generated by the reaction can be removed by extraction using an organic solvent having high solubility of aldehyde and low solubility of D-β-hydroxyamino acid (or L-form). 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-β-hydroxyamino acid (or L form) should be recovered by a known method such as crystallization by concentration, isoelectric point precipitation, ion exchange resin treatment, membrane separation, etc. Can do.

  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. “%” 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

[Example 1] Preparation of cell suspension expressing L-phenylserine aldolase derived from Pseudomonas putida biovar A 24-1 (Pseudomonas putida biovar A 24-1)

  Plasmid pKK-PSA1 expressing L-phenylserine aldolase derived from Pseudomonas putida biovar A 24-1 (incorporated administrative agency National Institute of Advanced Industrial Science and Technology, Patent Biology Center) E. coli strain HB101 (HB101 (pKK-PSA1)), which was retained on April 3, 2012), was added to LB medium (1% bacto-tryptone, 0.5% bacto) containing ampicillin (50 μg / mL). -Cultured in yeast extract, 1% sodium chloride, pH 7.2). IPTG was added to the medium to a final concentration of 0.1 mM to induce expression of the L-phenylserine aldolase gene. After induction of IPTG expression, cells of HB101 (pKK-PSA1) were recovered.

The plasmid pKK-PSA1 expressing the L-phenylserine aldolase is deposited as follows.
Accession number: FERM P-20054
Depositary institution: National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center
Chuo 6th, 1-1 1-1 East 1-chome, Tsukuba, Ibaraki, Japan (zip code 305-8566)
Deposit date: April 13, 2004

[Example 2] Optical purity analysis of erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid

0.05 g of the reaction solution was sampled, 0.5 mL of 50 mM sodium borate buffer (pH 10.8) was added and suspended, and then centrifuged to obtain a supernatant. To this supernatant, 0.5 mL of methanol and 0.01 g of anhydrous N-tertiary butoxycarbonyl were added 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 measurement was performed by the following “Analysis method-1”.
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 3 Synthesis of D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid using Escherichia coli HB101 strain (pKK-PSA1)

  Using the cells of Escherichia coli HB101 strain (HB101 (pKK-PSA1)) obtained in Example 1, DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid in the form of a solution (strong alkalinity) As a raw material, D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was synthesized. The raw material, DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid, was added to the reaction vessel to a final concentration of 6%, and phosphoric acid was added at pH 5.7, 6.5, 7.0, 8.5, 9.0, 9.5. , Adjusted to 10.1. Thereafter, 50 μM PLP and the above cells were added to give a total weight of 70 g, and this reaction solution was allowed to react at 30 ° C. overnight without re-adjusting the pH while 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. Decomposition of unnecessary isomers proceeds at any reaction start pH other than the reaction start pH 10.1, and D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid can be obtained with high optical purity. Was confirmed. On the other hand, a decrease in the optical purity of the product caused by an undesirable reverse reaction (condensation reaction between glycine and aldehyde) by L-phenylserine aldolase was not observed at any pH.

［実施例４］大腸菌ＨＢ１０１株（ｐＫＫ−ＰＳＡ１）を用いたＤ−エリスロ−２−アミノ−３−シクロヘキシル−３−ヒドロキシプロピオン酸の合成 Further, the fluidity of the reaction solution was good at pH 8.5 or more at the start of the reaction. On the other hand, at the end of the reaction, contrary to the start of the reaction, the pH was in the range of 5.7 to 7.0.

[Example 4] Synthesis of D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid using Escherichia coli HB101 strain (pKK-PSA1)

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

  DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was added to the reaction vessel to a final concentration of 6%, and the pH was adjusted to 9.0 with phosphoric acid. Thereafter, 50 μM PLP and bacterial cells were added to give a total weight of 70 g. The reaction was started at 30 ° C. with stirring. After 30 minutes from the start of the reaction (before the reaction solution solidified), the pH was adjusted again to 6.5 with phosphoric acid, and thereafter the reaction was allowed to proceed overnight without controlling the pH. 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.

［比較例１］大腸菌ＨＢ１０１株（ｐＫＫ−ＰＳＡ１）を用いたＤ−エリスロ−２−アミノ−３−シクロヘキシル−３−ヒドロキシプロピオン酸の合成 The results are shown in Table 2. The 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. Regarding the mixing property of the reaction solution, solidification of the reaction solution was not observed at the start of the reaction and at the end of the reaction.

[Comparative Example 1] Synthesis of D-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid using Escherichia coli HB101 strain (pKK-PSA1)

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

  Raw material DL-erythro-2-amino-3-cyclohexyl-3-hydroxypropionic acid was added to the reaction vessel so that the final concentration was 6%, and the reaction solution was adjusted to pH 9.0 with phosphoric acid. Thereafter, 50 μM PLP and bacterial cells were added to give a total weight of 70 g. The reaction solution prepared here was allowed to react at 30 ° C. overnight under stirring without controlling the pH. 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 3. The 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. The mixing property of the reaction solution was good at the start of the reaction, but solidification of the reaction solution was confirmed at the end of the reaction.

Claims (14)

A method for producing D or L-β-hydroxyamino acid by reacting an enzyme active substance with DL-β-hydroxyamino acid in a reaction solution having a pH adjusted to 5.5 to 10. The method according to claim 1, wherein the pH is adjusted to a pH capable of maintaining the fluidity of the reaction solution after the start of the reaction. The method according to claim 2, wherein the pH adjustment after the start of the reaction is performed according to a change in the rheology of the solution. The method according to claim 2 or 3, wherein the pH adjustment is an adjustment in a direction of decreasing the pH. The method according to claims 1 to 4, wherein the pH at the start of the reaction is in an alkaline region. 6. The method according to claim 5, wherein the pH in the alkaline region is 8.0 to 10.0. The method according to claim 1, wherein the pH is adjusted from weakly alkaline to acidic pH after the start of the reaction. The method according to claim 7, wherein the pH range from weakly alkaline to acidic range is 7.5 to 5.5. The method according to claim 1, wherein the pH adjustment is performed using phosphoric acid. 10. A method according to claims 3 to 9, wherein the rheological change of the solution is a change from liquid to solid. The method according to claim 1, wherein the enzyme active substance is 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: 12. A method according to claims 1-11.
(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 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 method according to claim 1, wherein The method according to claim 13, wherein R in the formula 1 is an optionally substituted cyclohexyl group.