JPWO2006109632A1 - Novel α-keto acid reductase, gene thereof, and use thereof - Google Patents

Abstract

The present invention provides an α-keto acid reductase having the following physicochemical properties (a) and (b), a DNA encoding the enzyme, a transformant producing the enzyme, and the enzyme or the transformation The present invention relates to a method for producing an S-form α-hydroxy acid by reducing α-keto acid using the body. (A) α-keto acid is asymmetrically reduced to produce S-form α-hydroxy acid, (b) α-keto acid reduction activity in the presence of 150 mM of S-form α-hydroxy acid, It is 10% or more of the value of α-keto acid reduction activity in the absence of α-hydroxy acid of S form. ADVANTAGE OF THE INVENTION According to this invention, the S-form alpha-hydroxy acid useful as intermediates, such as a pharmaceutical and an agrochemical, can be efficiently manufactured with a high accumulation concentration.

Description

  The present invention relates to an α-keto acid reductase, a DNA encoding the enzyme, a vector containing the DNA, and a transformant transformed with the vector. The present invention also relates to a method for producing an S-form α-hydroxy acid, particularly an S-form α-hydroxy acid having an aryl group, using the enzyme or the transformant. S-form α-hydroxy acids, particularly S-form α-hydroxy acids having an aryl group, are very useful compounds as intermediates for various pharmaceuticals and agricultural chemicals.

  Several methods for producing S-form α-hydroxy acids by asymmetric reduction of α-keto acids using biocatalysts such as microbial cells and enzymes have been reported so far.

  As a method for producing (S) -phenyllactic acid, which is one of S-form α-hydroxy acids, for example, L-lactic acid derived from Bacillus stearothermophilus having an artificially modified amino acid sequence Examples include a method using a dehydrogenase (Non-patent Document 1). Examples of the method for producing optically active 2-hydroxy-4-phenylbutyric acid include a method using various microorganisms (Patent Document 1) and a method using L-lactic acid dehydrogenase derived from bovine heart (Patent Document 2). ) And the like. Furthermore, examples of the method for producing optically active 2-hydroxy-4-phenyl-3-butenoic acid include methods using various microorganisms (Patent Documents 3 and 4). As described above, although there are many reported examples, the accumulated amount (concentration) of the product is low with any of the methods and is not satisfactory.

Moreover, the manufacturing method of the optically active alpha-hydroxy acid using the continuous membrane reactor which incorporated the film | membrane in the apparatus has also been reported (patent document 5, nonpatent literature 2, 3). However, since the equipment is complicated and the equipment is expensive, it is difficult to say that it is an advantageous method for industrial use.
Japanese Patent No. 2752754 Japanese Patent No. 2873236 Japanese Patent No. 2519980 Japanese Patent No. 3067901 Shoko 63-35 J. Am. Chem. Soc., 114, 4551 (1992) Organic Process Research & Development, 6, 520 (2002) J. Biotechnol., 24, 315 (1992)

  In view of the above, the present invention provides an α-keto acid reductase that is extremely useful in the production of S-form α-hydroxy acids, particularly S-form α-hydroxy acids having an aryl group, DNA encoding the enzyme, and the DNA And a transformant transformed with the vector.

  Another object of the present invention is to provide an efficient process for producing various S-form α-hydroxy acids including S-form phenyllactic acid using the enzyme or the transformant.

  In the conventional reduction method using a biocatalyst, the S-form α-hydroxy acid, which is the product, inhibits the activity of the reductase. As a result of intensive studies, the present inventors have found that α-keto acid reductase having high α-keto acid reductase activity even in the presence of S-form α-hydroxy acid, particularly S-form α-hydroxy acid having an aryl group. Newly found. In addition, in connection with the enzyme, DNA encoding the enzyme was isolated, and a recombinant vector and a transformant that expresses the enzyme in large quantities were newly created. Furthermore, by asymmetric reduction of α-keto acid using the above α-keto acid reductase or transformant, S-form α-hydroxy acid, particularly S-form α-hydroxy acid having an aryl group, is increased in the reaction solution. The present invention has been completed by successfully accumulating at a concentration.

That is, the present invention relates to an α-keto acid reductase having the following physicochemical properties (a) and (b):
(A) a-keto acid is asymmetrically reduced to produce S-form α-hydroxy acid,
(B) The value of the α-keto acid reduction activity in the presence of 150 mM of the α-hydroxy acid of the S form is 10% or more of the value of the α-keto acid reduction activity in the absence of the α-hydroxy acid of the S form. .

  The present invention also relates to DNA encoding the enzyme, a vector containing the DNA, and a transformant transformed with the vector.

  The present invention also provides a method for producing S-form α-hydroxy acid using the enzyme or the transformant, particularly the following general formula (1):

(Wherein n represents an integer of 1 to 3, X represents a hydrogen atom, an alkali metal or an alkaline earth metal, and Ar represents an optionally substituted aryl group). The following general formula (2), wherein the enzyme or the transformant is reacted with an α-keto acid having the following formula:

(Wherein n, X and Ar are the same as above) and a method for producing an S-form α-hydroxy acid having an aryl group.

  Provided is an efficient process for producing S-form α-hydroxy acids, particularly S-form α-hydroxy acids having an aryl group, which are very useful compounds as intermediates for various pharmaceuticals and agricultural chemicals.

The production method and structure of the recombinant vector pNTPA are shown. The production method and structure of the recombinant vector pNTPAG are shown.

Hereinafter, the present invention will be described in detail.
Unless otherwise specified, genetic manipulations such as DNA isolation, vector preparation, and transformation described in this specification are described in documents such as Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989). It can be carried out by the method described. Further,% used in the description of the present specification means% (w / v) unless otherwise specified.

The α-keto acid reductase of the present invention has the following physicochemical properties (a) and (b).
(A) a-keto acid is asymmetrically reduced to produce S-form α-hydroxy acid,
(B) The value of the α-keto acid reduction activity in the presence of 150 mM of the α-hydroxy acid of the S form is 10% or more of the value of the α-keto acid reduction activity in the absence of the α-hydroxy acid of the S form. .

Any α-keto acid reductase having these physicochemical properties (a) and (b) is included in the present invention. Here, the α-hydroxy acid (α-hydroxy acid as a reduction product) in (a) and the α-hydroxy acid (α-hydroxy acid as an inhibitory factor of the enzyme) in (b) are not necessarily the same. Not necessarily. Preferably, the α-hydroxy acid of the S form in (b) is (S) -phenyllactic acid, and the α-keto acid reductase has an α-keto acid reductive activity that is phenylpyruvate reductive activity. That is, the α-keto acid reductase of the present invention preferably has the following physicochemical property (b ′) as the physicochemical property of (b):
(B ′) The value of phenylpyruvate reducing activity in the presence of 150 mM (S) -phenyllactic acid is 10% or more of the value of phenylpyruvic acid reducing activity in the absence of (S) -phenyllactic acid.

  In addition, in the physicochemical properties of (b), the value of α-keto acid reducing activity in the presence of 150 mM of S-form α-hydroxy acid is the same as that of S-form in the absence of α-hydroxy acid. However, it is preferably 20% or more, more preferably 30% or more.

  Regarding the method for determining whether or not the α-keto acid reductase has the physicochemical property (b) of the present invention, (S) -phenyllactic acid as the α-hydroxy acid of the S form in (b), The case where phenylpyruvic acid is used as the α-keto acid serving as a substrate will be described below as an example.

  The value of the phenylpyruvate reducing activity in the absence of (S) -phenyllactic acid is 3.0 ml of reaction containing 20 mM phenylpyruvic acid, 0.25 mM NADH and 0.05 ml enzyme solution in a 100 mM phosphate buffer (pH 6). It can be calculated from the rate of decrease in absorbance at a wavelength of 340 nm when reacted in a liquid at 30 ° C. for 3 minutes. The value of phenylpyruvate reducing activity in the presence of 150 mM (S) -phenyllactic acid can be measured by adding (S) -phenyllactic acid 150 mM to the above activity measurement system and carrying out the same. That is, the concentration of the α-hydroxy acid of the S form in the above (b) is the concentration at the initial stage of the reaction. By measuring the enzyme activity value under both of these conditions, it can be easily determined whether or not it has the physicochemical properties of (b) of the present invention.

  In the above determination method, when the phenylpyruvic acid is replaced with another α-keto acid, such as pyruvic acid, α-keto acid reducing activity (for example, pyruvic acid reducing activity) other than phenylpyruvic acid reducing activity is determined. The Further, the determination can be made by replacing (S) -phenyllactic acid with another α-hydroxy acid. In these cases, other reaction conditions are adjusted separately as necessary.

In addition, the α-keto reductase of the present invention has the following physicochemical properties (c) and / or (d) simultaneously with the physicochemical properties (a) and (b). preferable.
(C) The value of pyruvate reducing activity in the presence of 300 mM (S) -phenyllactic acid is 20% or more of the value of reducing activity in the absence of (S) -phenyllactic acid.
(D) The value of pyruvate reducing activity in the presence of 420 mM (S) -phenyllactic acid is 10% or more of the value of reducing activity in the absence of (S) -phenyllactic acid.

  Whether or not the α-keto acid reductase has the physicochemical properties (c) and (d) of the present invention is determined by the reducing activity in the method for determining whether or not it has the physicochemical properties (b) described above. The measurement substrate may be changed in the same manner by changing 20 mM phenylpyruvic acid to 30 mM pyruvic acid and changing the concentration of (S) -phenyllactic acid to be added from 150 mM to 300 mM or 420 mM.

  The α-keto acid reductase of the present invention can be isolated from a microorganism having a reducing activity for α-keto acid. The microorganism can be found, for example, by the following method. Microorganisms are cultured in an appropriate medium, collected, and then reacted with α-keto acid in a buffer solution in the presence of nutrients such as glucose. After the reaction, the reaction liquid may be analyzed by a known method such as high performance liquid chromatography or gas chromatography to confirm the production of α-hydroxy acid. As a medium for culturing a microorganism, a normal liquid nutrient medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients and the like can be used as long as the microorganism grows.

  The microorganism that is the source of the α-keto acid reductase of the present invention is not particularly limited. And microorganisms belonging to Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus brevis, Pediococcus acidilactici, Pedococcus (Pediococcus pentosaceus), Enterococcus faecalis, Achromobacter xylosoxidans subsp. Xylosoxidans, etc. That.

  More preferred is Pediococcus acidilactici, and particularly preferred is Pediococcus acidilactici JCM8797. The microorganism can be obtained from the Institute for Microbial Materials Development, RIKEN BioResource Center (JCM: 2-1 Hirosawa, Wako, Saitama 351-0198).

  The α-keto acid reductase of the present invention is isolated from a microorganism having α-keto acid reductive activity as described above. Isolation of the enzyme can be carried out by appropriately combining known protein purification methods. For example, it can be implemented as follows.

  First, the microorganism is cultured in an appropriate medium, and the cells are collected from the culture solution by centrifugation or filtration. The obtained bacterial cells are crushed by an ultrasonic crusher or a physical method using glass beads or the like, and then the microbial cell residue is removed by centrifugation to obtain a cell-free extract. And salting out (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fraction precipitation with acetone or ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, ultrafiltration The α-keto acid reductase of the present invention is isolated from the cell-free extract by using such a technique alone or in combination.

  The α-keto acid reductase of the present invention includes any α-keto acid reductase as long as it has the physicochemical properties (a) and (b) described above, preferably lactate dehydrogenase, more preferably Is an L-lactate dehydrogenase.

  The DNA of the present invention is a DNA encoding the above-described α-keto acid reductase of the present invention, and any DNA can be used as long as it can express the enzyme in a host cell introduced according to the method described below. Any untranslated region may be included. If the enzyme can be obtained, such a DNA can be obtained by a person skilled in the art from a microorganism that is the source of the enzyme by a known method. For example, it can be acquired by the method shown below.

  First, the isolated α-keto acid reductase of the present invention is digested with an appropriate endopeptidase, and the resulting peptide fragment is fractionated by reverse phase HPLC. Then, for example, part or all of the amino acid sequences of these peptide fragments are determined by an ABI492 type protein sequencer (Applied Biosystems).

  Based on the amino acid sequence information thus obtained, a PCR (Polymerase Chain Reaction) primer for amplifying a part of the DNA encoding the polypeptide is synthesized. Next, chromosomal DNA of the microorganism that is the origin of the polypeptide is prepared by a conventional DNA isolation method, for example, the method of Visser et al. (Appl. Microbiol. Biotechnol., 53, 415 (2000)). Using this chromosomal DNA as a template, PCR is carried out using the PCR primers described above, a part of the DNA encoding the polypeptide is amplified, and the base sequence is determined. The base sequence can be determined using, for example, ABI373A DNA Sequencer (Applied Biosystems).

  If the partial base sequence of DNA encoding the polypeptide is clarified, the entire sequence can be determined by, for example, the inverse PCR method (Nucl. Acids Res., 16, 8186 (1988)).

  Examples of the DNA of the present invention thus obtained include DNA containing the base sequence shown in SEQ ID NO: 2 in the sequence listing.

  Examples of the α-keto acid reductase of the present invention include a reductase consisting of the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing encoded by the base sequence shown in SEQ ID NO: 2 of the sequence listing.

  The vector used for introducing the DNA of the present invention into a host microorganism and expressing it in the introduced host microorganism is not particularly limited as long as it can express the gene encoded by the DNA in an appropriate host microorganism. Not. Examples of such vectors include plasmid vectors, phage vectors, cosmid vectors, and shuttle vectors that can exchange genes with other host strains can also be used.

  Such vectors usually contain regulatory elements such as lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter, pL promoter and the like, and are operably linked to the DNA of the present invention. It can be suitably used as an expression vector containing a unit. For example, pUCNT (WO94 / 03613) can be suitably used.

  As used herein, the term “regulatory element” refers to a base sequence having a functional promoter and any associated transcription element (eg, enhancer, CCAAT box, TATA box, SPI site, etc.).

  As used herein, the term “operably linked” means that a gene is operably linked to various regulatory elements such as promoters and enhancers that regulate the expression of the gene. It is well known to those skilled in the art that the type and kind of the control factor can vary depending on the host.

  As an example of the recombinant vector of the present invention, a plasmid pNTPA (FIG. 1) in which the DNA shown in SEQ ID NO: 2 is introduced into the vector pUCNT can be mentioned. This recombinant vector has the deposit number of FERM P-20434. The National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (March 1-1-Higashi 1-11-1, Tsukuba, Ibaraki, Japan) 6).

  Examples of the host cell into which the vector containing the DNA of the present invention is introduced include bacteria, yeast, filamentous fungi, plant cells, animal cells, and the like. Bacteria are preferred from the introduction and expression efficiency, and Escherichia coli is particularly preferred. A vector containing the DNA of the present invention can be introduced into a host cell by a known method. When E. coli is used as the host cell, for example, commercially available E. coli. By using an E. coli HB101 competent cell (manufactured by Takara Bio Inc.), the vector can be introduced into a host cell.

  Examples of the transformant of the present invention include the above-mentioned E.I. E. coli obtained by introducing the plasmid pNTPA into HB101. coli HB101 (pNTPA).

  The α-keto acid reductase of the present invention is contacted with a coenzyme such as α-keto acid and NADH as a substrate and reacted to asymmetrically reduce α-keto acid, thereby producing α-form of S-form. Hydroxy acids can be produced.

  In particular, as the α-keto acid, the following general formula (1)

(Wherein n represents an integer of 1 to 3, X represents a hydrogen atom, an alkali metal or an alkaline earth metal, and Ar represents an optionally substituted aryl group). By using an α-keto acid having the following general formula (2)

It is possible to efficiently produce an S-form α-hydroxy acid having an aryl group represented by (n, X and Ar are the same as above).

  In this case, being able to produce efficiently means accumulating the product α-hydroxy acid at a concentration of 100 mM, preferably 150 mM, more preferably 200 mM or more in the reaction solution as the final production amount.

  In the above formulas (1) and (2), n represents an integer of 1 to 3, but is preferably 1. X represents a hydrogen atom, an alkali metal, or an alkaline earth metal, and is not particularly limited. Examples thereof include a hydrogen atom, lithium, sodium, potassium, magnesium, and calcium. Ar represents an optionally substituted aryl group, preferably a halogen atom, a hydroxyl group, an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, or a thioalkyl group having 1 to 5 carbon atoms. A phenyl group or a naphthyl group which may be substituted with one or more substituents selected from the group consisting of an amino group, a nitro group and a mercapto group, more preferably an unsubstituted phenyl group or a para-position It is a phenyl group substituted with a tert-butyloxy group.

  Further, the α-keto acid reductase used in the reduction reaction may be a purified enzyme or a crude enzyme. Furthermore, the microorganisms having the α-keto acid reduction activity as described above and the processed products thereof are also included in the enzyme of the present invention. The “processed product” as used herein means, for example, a cell treated with a surfactant or an organic solvent, a dried cell, a disrupted cell, a crude cell extract, or the like. These enzymes, bacterial cells, processed products thereof, and the like may be immobilized by known methods.

  In these reduction reactions, as the reaction proceeds, a coenzyme such as NADH is converted to an oxidized form. However, a polypeptide having the ability to convert this oxidized coenzyme into a reduced form (hereinafter referred to as coenzyme regeneration ability) and a compound serving as a substrate for the polypeptide coexist with the polypeptide of the present invention. By performing this reaction, the amount of expensive coenzyme used can be greatly reduced. Examples of the polypeptide having coenzyme regeneration ability include hydrogenase, formate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, glucose-6-phosphate dehydrogenase, and glucose dehydrogenase. Preferably, glucose dehydrogenase is used.

  Even if the transformant of the present invention containing DNA encoding the enzyme is used instead of the α-keto acid reductase of the present invention, S-form α-hydroxy acid can be produced in the same manner. In addition, even when a transformant containing both the DNA encoding the enzyme of the present invention and the DNA encoding a polypeptide having coenzyme regeneration ability is used, the S-form α-hydroxy acid can be produced in the same manner. Can do. In particular, when a transformant containing both the DNA encoding the enzyme of the present invention and the DNA encoding a polypeptide having coenzyme regeneration ability is used, an enzyme for regenerating the coenzyme is separately prepared. -It is not necessary to add and an alpha-hydroxy acid can be manufactured more efficiently.

  A transformant containing a DNA encoding the α-keto acid reductase of the present invention, or a DNA encoding a polypeptide encoding the α-keto acid reductase of the present invention and a polypeptide capable of coenzyme regeneration. The transformant containing both can be used for the production of S-α-hydroxy acid as a processed product, not to mention cultured cells. The term “transformed product” as used herein refers to, for example, cells treated with a surfactant or an organic solvent, dried cells, disrupted cells, crude cell extracts, and the like by known means. It means something fixed.

  A transformant containing both the DNA encoding the α-keto acid reductase of the present invention and the DNA encoding a polypeptide having a coenzyme regenerating ability is a DNA encoding the enzyme of the present invention and a coenzyme regenerating ability. In addition to incorporating both of the DNAs encoding the polypeptides having a DNA into one vector and introducing it into a host cell, these two types of DNA are incorporated into two different vectors of different incompatibility groups, respectively. It can also be obtained by introducing these two types of vectors into the same host cell.

  Examples of vectors in which both the DNA encoding the α-keto acid reductase of the present invention and the DNA encoding a polypeptide having a coenzyme regenerating ability are incorporated include Pediococcus acidilactici in the expression vector pUCNT. PNTPAG1E (FIG. 2) into which both an α-keto acid reductase gene derived from Pediococcus acidilactici JCM8797 strain and a glucose dehydrogenase gene derived from Bacillus megaterium have been introduced.

  Examples of transformants containing both the DNA encoding the α-keto acid reductase of the present invention and the DNA encoding a polypeptide having a coenzyme regeneration ability include E. obtained by transforming E. coli HB101. coli HB101 (pNTPAG1E).

  Cultivation of transformant containing DNA encoding α-keto acid reductase of the present invention, and transformation containing both DNA encoding polypeptide of the present invention and DNA encoding polypeptide having coenzyme regeneration ability As long as they grow, the body can be cultured using a normal liquid nutrient medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients and the like.

  The activity of α-keto acid reductase in the transformant can be measured in the same manner as in the method for measuring the activity of the isolated enzyme described above. In this case, a culture solution or other solution containing the transformant or a processed product thereof may be used as the enzyme solution. The activity of the polypeptide having the ability to regenerate the coenzyme in the transformant can be measured by a conventional method. For example, the activity of glucose dehydrogenase was obtained by adding 100 mM glucose, 2 mM coenzyme NADP or NAD, and an enzyme solution to 1 M Tris-HCl buffer (pH 8.0) and allowing them to react at 25 ° C. for 1 minute. It can be calculated from the rate of increase in absorbance at a wavelength of 340 nm.

  Production of S-form α-hydroxy acid using either the α-keto acid reductase of the present invention or a transformant containing DNA encoding the enzyme is carried out in the presence of α- serving as a substrate in an appropriate solvent. It can be carried out by adding a coenzyme such as keto acid, NADH, NADPH and the like, and the α-keto acid reductase of the present invention or a transformant containing DNA encoding the enzyme, and stirring under pH adjustment.

  On the other hand, when the reaction is carried out by combining the α-keto acid reductase of the present invention and a polypeptide having a coenzyme regeneration ability, the above reaction composition contains a polypeptide having a coenzyme regeneration ability (for example, glucose dehydrogenase). Then, a compound (for example, glucose) serving as the substrate is further added. Even in the latter case, a transformant comprising both the DNA encoding the α-keto acid reductase of the present invention and the DNA encoding a polypeptide having a coenzyme regeneration ability (for example, glucose dehydrogenase), Or when using the processed material, it is not necessary to add separately the polypeptide (for example, glucose dehydrogenase) which has coenzyme reproduction | regeneration ability.

  For the reaction, an aqueous solvent may be used, or an aqueous and organic solvent may be mixed and used. Examples of the organic solvent include toluene, ethyl acetate, n-butyl acetate, hexane, isopropanol, diisopropyl ether, methanol, acetone, dimethyl sulfoxide and the like. The reaction is carried out at a temperature of 10 ° C. to 70 ° C., and the pH of the reaction solution is maintained at 3-10. In the case of a batch system, the reaction substrate is added at a preparation concentration of 0.1% to 70% (w / v), and may be added all at once or dividedly.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by these. Detailed operation methods relating to the recombinant DNA technology used in the following examples are described in the following documents:
Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989),
Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience).

(Comparative example 1) Reduction reaction of phenylpyruvic acid using L-lactate dehydrogenase (BSLDH) derived from commercially available Bacillus stearothermophilus in 40 ml of 100 mM phosphate buffer (pH 6.5) , Glucose 5.3 g, L-lactate dehydrogenase derived from Bacillus stearothermophilus (Sigma) (hereinafter abbreviated as BSLDH) 2,900 U, glucose dehydrogenase (Amano Enzyme) After adding 2,270 U, 5 mg of NAD ⁺ and 0.4 g of sodium salt of phenylpyruvic acid, the reaction mixture was stirred at 30 ° C. while adjusting the pH of the reaction solution to 6.5 by adding an aqueous 5N sodium hydroxide solution. . 0.4 g of sodium salt of phenylpyruvic acid was added at 0.6 hours of reaction, and sodium salt of phenylpyruvic acid was added at 3.5 hours, 5 hours, 7 hours, 9 hours and 24 hours, respectively. An additional 0.2 g was added. During the reaction, the reaction solution was sampled in a timely manner, and the sampled solution was acidified with hydrochloric acid, extracted with ethyl acetate, and analyzed under the above conditions to measure the amount of phenyllactic acid produced. The increase in the amount of phenyl lactic acid almost stopped 30 hours after the start of the reaction, and the accumulated concentration of phenyl lactic acid at this time was 32.1 g / l (193 mM). The absolute configuration of the generated phenyllactic acid was S-form, and its optical purity was 99% ee or higher.

In addition, the quantitative determination of phenyllactic acid was performed by analyzing under the following HPLC conditions.
[Quantitative analysis (HPLC analysis conditions)]
Column: YMC-Pack ODS-A (A303) (4.6 mm ID × 250 mm) (manufactured by YMC), column temperature: room temperature, mobile phase: 0.1% potassium dihydrogen phosphate (pH 3) / acetonitrile = 7/3, flow rate: 1 ml / min, detection wavelength: 254 nm, retention time: 5.9 minutes.

The optical purity of phenyl lactic acid was determined by analyzing it under the following conditions after derivatized with phenacyl chloride.
[Optical purity analysis (HPLC conditions)]
Column: CHIRALPAK AD-H (4.6 mm ID × 250 mm) (manufactured by Daicel Chemical Industries), column temperature: room temperature, mobile phase: n-hexane / ethanol = 1/1, flow rate: 1 ml / min, detection wavelength: 243 nm, retention time: S-form-18.5 minutes, R-form-20.7 minutes.

(Comparative Example 2) on the BSLDH of reducing activity (S) - Effect of phenyl lactic acid (S) - was measured reducing activity against pyruvate BSLDH in phenyl acid presence. The reduction activity for pyruvic acid was dissolved in 100 mM phosphate buffer (pH 6.5) so that pyruvic acid had a final concentration of 30 mM and coenzyme NADH had a final concentration of 0.25 mM. This was calculated from the rate of decrease in absorbance at a wavelength of 340 nm of the reaction solution when the reaction was performed for 1 minute. Under this reaction condition, the activity of oxidizing 1 μmol of NADH to NAD per minute was defined as 1U. The reduction activity of BSLDH was measured under the condition that (S) -phenyllactic acid was added to this activity measurement system so that the final concentration was 0 to 210 mM. The results are summarized in Table 1 as the enzyme activity when (S) -phenyllactic acid was not added was 100%, and the enzyme activity when (S) -phenyllactic acid was added as relative activity. In the examination, an about 2 U / ml BSDDH solution was prepared and used as an enzyme solution.

  As the (S) -phenyllactic acid concentration increased, the reduction activity of BSLDH decreased, and the activity almost disappeared in the presence of 180 mM (S) -phenyllactic acid. From this result, it was speculated that the reason why the product phenyllactic acid could not be accumulated at a high concentration in Comparative Example 1 was that the inhibition of BSLDH activity was caused by phenyllactic acid, which is an α-hydroxy acid.

(Example 1) Search for α-keto acid reductase resistant to inhibition by α-hydroxy acid ((S) -phenyl lactic acid) Various microorganisms shown in Table 2 were cultured in a large test tube, and 5 ml of each culture solution was centrifuged. The cells were collected through separation. Among the microorganisms shown in Table 2, microorganisms belonging to any of Lactobacillus, Lactococcus, Pediococcus, Enterococcus or Streptococcus are under the conditions (a) below, and microorganisms of other genera are under the conditions (b) below. Cultured.
(A) 15 ml of a commercially available Difco Lactobacilli MRS Broth (Becton, Dickinson) liquid medium (pH 7) was dispensed into a large test tube and steam sterilized at 120 ° C. for 20 minutes. These liquid media were aseptically inoculated with one microbe of microorganisms and cultured at 30 ° C. for about 72 to 96 hours.
(B) Dispense 7 ml of liquid medium (pH 7) consisting of 10 g of meat extract, 10 g of peptone, 5 g of yeast extract and 3 g of sodium chloride (each per 1 L) into a large test tube, and steam sterilize at 120 ° C. for 20 minutes went. These liquid media were aseptically inoculated with one platinum loop of microorganisms and cultured with shaking at 30 ° C. for 36 hours.

  Each collected microbial cell was suspended in 1 ml (pH 7.0) of 100 mM phosphate buffer containing 8% glucose. This cell suspension was added to a test tube containing 10 mg of sodium salt of phenylpyruvic acid in advance and reacted at 30 ° C. for 20 hours. The reaction solution was acidified with hydrochloric acid, extracted with ethyl acetate, and analyzed by the method described in Comparative Example 1 to determine the amount of phenyllactic acid produced and its optical purity. Table 2 summarizes the molar yield of phenyllactic acid, its optical purity, and absolute configuration in a column for cell reaction. All of these microorganisms showed high reduction activity and high stereoselectivity for phenylpyruvic acid.

  Further, 10 ml of the culture solution cultured as described above was centrifuged to collect the cells, and each cell was suspended in 2 ml of 100 mM phosphate buffer (pH 7.0) containing 5 mM β-mercaptoethanol. The cells were crushed using a UH-50 type ultrasonic homogenizer (manufactured by SMT), and then the cell residue was removed by centrifugation to obtain each cell-free extract. The reduction activity of these cell-free extracts against phenylpyruvic acid was measured. The measurement of the reduction activity for phenylpyruvic acid was carried out in the same manner by adding 20 mM phenylpyruvic acid instead of 30 mM pyruvic acid in the measurement system for the reduction activity against pyruvic acid described in Comparative Example 2. For those in which reduction activity was observed, activity measurement was similarly performed under the condition that (S) -phenyllactic acid was added to the activity measurement system so that the final concentration was 150 mM (2.5% (wt / vol)). Went.

  Table 2 shows the relative ratios of enzyme activity when 150 mM (S) -phenyllactic acid was added to enzyme activity when (S) -phenyllactic acid was not added. A relatively high reduction activity even in the presence of 150 mM (S) -phenyllactic acid, specifically, 10% or more of the reduction activity in the absence of (S) -phenyllactic acid even in the presence of 150 mM (S) -phenyllactic acid We were able to find seven new microorganisms with active enzymes.

(Example 2) In examination of the cloning destination of the L-lactate dehydrogenase gene from Pediococcus acidilactici JCM8797, it has a relatively high reducing activity even in the presence of (S) -phenyllactic acid. The gene of L-lactate dehydrogenase (hereinafter abbreviated as PALDH), which is one of α-keto acid reductases, was cloned from the obtained Pediococcus acidilactici JCM8797 strain by the following method.

(Create PCR primers)
Primer 1 (5'-ACNTAYGCNACNTGGAARYT-3) for amplifying a part of the gene encoding PALDH by PCR based on the deduced amino acid sequence information of known L-lactate dehydrogenase registered in the gene data bank ': SEQ ID NO: 3 in the sequence listing) and primer 2 (5'-CCRTARAANGTNGCNCCYTT-3': SEQ ID NO: 4 in the sequence listing) were synthesized.

(Amplification of genes by PCR)
Preparation of chromosomal DNA from cells of Pediococcus acidilactici JCM8797 cultured in the same manner as in Example 1 was performed using an UltraClean Microbiol DNA Isolation kit (manufactured by MO BIO). It was carried out according to the instruction manual. Next, PCR was performed using the DNA primers 1 and 2 prepared above and the obtained chromosomal DNA as a template. As a result, a DNA fragment of about 0.3 kbp considered to be a part of the target gene was amplified. PCR was performed using TaKaRa Ex Taq (manufactured by Takara Bio Inc.) as a DNA polymerase, and the reaction conditions were in accordance with the instruction manual.

  This DNA fragment was cloned into the vector pT7Blue T-Vector (Novagen), and its base sequence was analyzed using BigDye Terminator Sequencing Standard Kit (Applied Biosystem) and ABI PRISM 3100 Genetic Analyzer (Applied Biosystem) did.

(Determining the full-length sequence of the gene of interest by the inverse PCR method)
The chromosomal DNA of Pediococcus acidilactici JCM8797 strain prepared above was completely digested with the restriction enzyme FbaI, and the resulting mixture of DNA fragments was intramolecularly cyclized with T4 ligase. Using this as a template, the entire nucleotide sequence of the enzyme gene including the region corresponding to the above-mentioned DNA fragment of about 0.3 kbp was determined by the Inverse PCR method (Nucl. Acids Res., 16, 8186 (1988)). The results are shown in SEQ ID NO: 2 in the sequence listing. Inverse PCR was performed using TaKaRa Ex Taq (manufactured by Takara Bio Inc.) as a DNA polymerase, and the reaction conditions were in accordance with the instruction manual. The amino acid sequence encoded by the base sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1.

(Example 3) Expression vector construction Primer 3 (5'-GGGAATGGACATATGTCTAATATTCAAAATCATC-3 ': SEQ ID NO: 5 in the sequence listing) and primer 4 (5'-GATAAGAATTCTTATTATTTGTCTTGTTTTTCAGCAAGAG-3': SEQ ID NO: 6 in the sequence listing) PCR was performed using the chromosomal DNA of Pediococcus acidilactici JCM8797 obtained in Example 2 as a template. As a result, double-stranded DNA in which an NdeI recognition site was added to the start codon portion of the gene consisting of the base sequence shown in SEQ ID NO: 2 in the sequence listing and the stop codon TAA and EcoRI recognition site were added immediately after the stop codon was obtained. Obtained. PCR was performed using TaKaRa Ex Taq (manufactured by Takara Bio Inc.) as a DNA polymerase, and the reaction conditions were in accordance with the instruction manual. This DNA was partially digested with NdeI and EcoRI and inserted between the NdeI recognition site and the EcoRI recognition site downstream of the lac promoter of the vector pUCNT (WO94 / 03613) to construct a recombinant vector pNTPA. This recombinant vector is the deposit number of FERM P-20434, and is the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (March 1-1-Higashi, Tsukuba City, Ibaraki Prefecture 305-28566) 6). A method for preparing pNTPA and its structure are shown in FIG.

Example 4 Construction of Expression Vector Further Containing Glucose Dehydrogenase Gene Primer 5 (5′-GCCGAATTCTAAGGAGGTTAACAATGTATAAA-3 ′: SEQ ID NO: 7 in Sequence Listing) and Primer 6 (5′-GCGGTCGACTTATCCGCGTCCTGCTTGG-3 ′: Sequence Listing PCR No. 8) and a vector pGDK1 (Eur. J. Biochem., 186, 389 (1989)) as a template, and a glucose dehydrogenase (hereinafter referred to as “Bacillus megaterium” IAM1030 strain). A double-stranded DNA in which an Escherichia coli ribosome binding sequence is added 5 bases upstream from the start codon of the gene, an EcoRI breakpoint is added immediately before it, and a SalI breakpoint is added immediately after the stop codon. Acquired. The obtained DNA fragment was digested with EcoRI and SalI, and inserted between the EcoRI recognition site and SalI recognition site downstream of the lac promoter of the vector pUCNT (WO94 / 03613) to construct a recombinant vector pNTG.

  Next, double-stranded DNA in which an NdeI recognition site was added to the start codon portion of the gene consisting of the base sequence shown in SEQ ID NO: 2 in the sequence listing and an EcoRI recognition site was added immediately after the termination codon was prepared in Example 3. This was prepared in the same manner as described above and inserted between the NdeI recognition site and EcoRI recognition site of the above-described recombinant vector pNTG to construct a recombinant vector pNTPAG. A method for producing pNTPAG and its structure are shown in FIG.

(Example 5) Production of transformant Using the recombinant vector pNTPA constructed in Example 3, E. coli HB101 competent cells (Takara Bio Inc.) were transformed. E. coli HB101 (pNTPA) was obtained. Similarly, using the recombinant vector pNTPAG constructed in Example 4, E. E. coli HB101 competent cells (Takara Bio Inc.) were transformed. coli HB101 (pNTPAG) was obtained.

(Example 6) Expression of gene in transformant Two types of transformants obtained in Example 5 and E. a transformant containing the vector pUCNT. E. coli HB101 (pUCNT) is inoculated into 50 ml of 2 × YT medium (1.6% tryptone, 1.0% yeast extract, 0.5% NaCl, pH 7.0) containing 200 μg / ml ampicillin and incubated at 37 ° C. for 24 hours. Cultured with shaking for hours. The cells were collected by centrifugation and suspended in 50 ml of 100 mM phosphate buffer (pH 6.5). This was crushed using a UH-50 type ultrasonic homogenizer (manufactured by SMT), and then the cell residue was removed by centrifugation to obtain a cell-free extract. Table 3 shows the specific activity of pyruvate reducing activity (hereinafter referred to as PALDH activity) and GDH activity of this cell-free extract. In any of the two types of transformants obtained in Example 5, expression of PALDH was observed. In addition, E. coli containing the GDH gene. In E. coli HB101 (pNTPAG), expression of GDH activity was also observed. The PALDH activity was measured in the same manner as the method for measuring the reducing activity against pyruvic acid described in Comparative Example 2. GDH activity was determined by adding 0.1 M glucose, 2 mM coenzyme NADP, and crude enzyme solution to 1 M Tris-HCl buffer (pH 8.0), reacting at 25 ° C. for 1 minute, and increasing absorbance at a wavelength of 340 nm. Calculated. Under these reaction conditions, the enzyme activity for reducing 1 μmol of NADP to NADPH per minute was defined as 1 U. The protein concentration in the cell-free extract was measured using a protein assay kit (manufactured by BIO-RAD).

(Example 7) Effect of (S) -phenyl lactic acid on the reduction activity of PALDH By the same method as described in Comparative Example 2, the reduction activity of PALDH on pyruvate in the presence of 0-600 mM (S) -phenyl lactic acid was measured. did. In the examination, as in the case of BSLDH, an about 2 U / ml PALDH solution was prepared and used as an enzyme solution. The results are summarized in Table 4 with the enzyme activity when (S) -phenyllactic acid was not added as 100% and the enzyme activity when (S) -phenyllactic acid was added as relative activity.

  Although the reduction activity of PALDH decreased with increasing (S) -phenyllactic acid concentration, the activity of BSDDH almost disappeared even in the presence of 180 mM (S) -phenyllactic acid, and had a relative activity of 49%. In addition, enzyme activity could be detected even in the presence of 600 mM (S) -phenyllactic acid. From these results, it was found that PALDH retains activity even in the presence of a higher concentration of phenyllactic acid than BSLDH.

(Example 8) E. cultured in the same manner as the reduction reaction in Example 6 of the transformants phenylpyruvic acid used Glucose 8 g, NAD 5 mg, and phenylpyruvic acid 0.8 g were added to 40 ml of a culture solution of E. coli HB101 (pNTPAG), and the mixture was stirred at 30 ° C. while adjusting the pH to 6.5 by dropwise addition of 5 M sodium hydroxide. Add 0.8 g each of sodium salt of phenylpyruvic acid at 0.4, 0.8, 1.2, 1.6, 2.3, and 2.9 hours of reaction. Added. Further, an additional 0.4 g of phenylpyruvic acid sodium salt was added at 3.6 hours, 4.2 hours, and 5 hours, respectively. During the reaction, the reaction solution was sampled in a timely manner, acidified with hydrochloric acid, extracted with ethyl acetate, and analyzed under the conditions described in Comparative Example 1 to determine the amount of phenyllactic acid produced. The accumulated concentration of phenyllactic acid at 7.3 hours after the start of the reaction was 104.1 g / l (626 mM). The produced phenyllactic acid was S-form, and its optical purity was 99% ee or higher.

Reference Example 1 Preparation of sodium 2-oxo-3- (4-tert-butoxyphenyl) propionate 4-tert-butoxybenzaldehyde (30.0 g, 168 mmol), hydantoin (33.73 g, 337 mmol, 2 eq.), A suspension of 1-amino-2-propanol (12.73 g, 168 mmol, 1 eq.) In distilled water (300 mL) was stirred under reflux conditions for 4 hours. After cooling to room temperature, the precipitate was filtered under reduced pressure to obtain 5- (4-tert-butoxybenzylidene) hydantoin (43.0 g, yield 98%).

Subsequently, a suspension of 5- (4-tert-butoxybenzylidene) hydantoin (30.0 g, 115 mmol) synthesized above and NaOH (24.0 g, 576 mmol, 5 eq.) In distilled water (300 mL) was refluxed. Stir for 4.5 hours. After cooling to room temperature, the pH was adjusted to 7.94 with hydrochloric acid. After adding NaCl (35.3 g, 600 mmol), the mixture was further stirred at room temperature for 15 hours. The precipitated target product was collected by filtration under reduced pressure, washed with methanol, and then vacuum dried to obtain the target sodium 2-oxo-3- (4-tert-butoxyphenyl) propionate (25.9 g, yield 88). %)was gotten.
¹ H NMR (400 MHz, D ₂ O / ppm): δ 1.19 (s, 9H), 3.92 (s, 2H), 6.91-6.94 (m, 2H), 7.04-7. 06 (m, 2H).

(Example 9) Reduction reaction of 2-oxo-3- (4-tert-butoxyphenyl) propionic acid using a transformant E. coli cultured in the same manner as in Example 6. The cells were collected by centrifuging 60 l of a culture solution of E. coli HB101 (pNTPAG), and suspended in 20 ml of the culture supernatant. To this was added 2.5 g of glucose, 3 mg of NAD, and 0.25 g of sodium 2-oxo-3- (4-tert-butoxyphenyl) propionate synthesized in Reference Example 1, and the pH was adjusted to 6 by adding 5 M sodium hydroxide dropwise. It stirred at 30 degreeC, adjusting to .5. An additional 0.4 g of sodium 2-oxo-3- (4-tert-butoxyphenyl) propionate was added at 0.6 hour and 1.0 hour respectively. Furthermore, 1.5 hours, 2.0 hours, 2.5 hours, 3.1 hours, 3.8 hours, 4.7 hours, 5.6 hours, 6.3 hours and 7 hours. In the third hour, 0.188 g of sodium 2-oxo-3- (4-tert-butoxyphenyl) propionate was added in increments of 0.188 g. During the reaction, the reaction solution was sampled in a timely manner, acidified with hydrochloric acid, extracted with ethyl acetate, and analyzed under the following HPLC conditions to analyze 2-hydroxy-3- (4-tert-butoxyphenyl) propionic acid. The production amount and optical purity were measured.
[HPLC analysis conditions]
Column: CHIRALPAK AD-H (4.6 mm ID × 250 mm) (manufactured by Daicel Chemical Industries), column temperature: room temperature, mobile phase: n-hexane / ethanol / trifluoroacetic acid = 950/50/1, flow rate: 1 ml / Min, detection wavelength: 230 nm, retention time: R-form-15.6 minutes, S-form-21.3 minutes.

The accumulated concentration of 2-hydroxy-3- (4-tert-butoxyphenyl) propionic acid 27 hours after the start of the reaction was 81.3 g / l. The absolute configuration of 2-hydroxy-3- (4-tert-butoxyphenyl) propionic acid produced was S-form, and its optical purity was 99% ee or higher.

Claims (22)

Α-keto acid reductase having the following physicochemical properties (a) and (b):
(A) a-keto acid is asymmetrically reduced to produce S-form α-hydroxy acid,
(B) The value of the α-keto acid reduction activity in the presence of 150 mM of the α-hydroxy acid of the S form is 10% or more of the value of the α-keto acid reduction activity in the absence of the α-hydroxy acid of the S form. . The α-keto acid reductase according to claim 1, wherein the physicochemical property of (b) has the following physicochemical property (b ′):
(B ′) The value of phenylpyruvate reducing activity in the presence of 150 mM (S) -phenyllactic acid is 10% or more of the value of phenylpyruvic acid reducing activity in the absence of (S) -phenyllactic acid. Furthermore, the α-keto acid reductase according to claim 1 or 2, which has the following physicochemical properties (c):
(C) The value of pyruvate reducing activity in the presence of 300 mM (S) -phenyllactic acid is 20% or more of the value of pyruvate reducing activity in the absence of (S) -phenyllactic acid. Furthermore, (alpha) -keto acid reductase of any one of Claims 1-3 which has the following physicochemical property:
(D) The value of pyruvate reducing activity in the presence of 420 mM (S) -phenyllactic acid is 10% or more of the value of pyruvate reducing activity in the absence of (S) -phenyllactic acid. It is derived from a microorganism selected from the group consisting of Lactobacillus genus, Pediococcus genus, Enterococcus genus and Achromobacter genus. Α-keto acid reductase. Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus brevis, Pediococcus acidilactici, Pediococcus pentocus edo (Α) according to any one of claims 1 to 4, which is derived from a microorganism selected from the group consisting of (Enterococcus faecalis) and Achromobacter xylosoxidans subsp. Xylosoxidans. A keto acid reductase. The α-keto acid reductase according to any one of claims 1 to 4, wherein the α-keto acid reductase is derived from a microorganism belonging to Pediococcus acidilactici. The α-keto acid reductase according to claim 1, which is derived from Pediococcus acidilactici JCM8797 strain. The α-keto acid reductase according to any one of claims 1 to 8, which is L-lactate dehydrogenase. The α-keto acid reductase according to any one of claims 1 to 9, comprising an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. DNA which codes the alpha-keto acid reductase of any one of Claims 1-10. A DNA comprising the base sequence represented by SEQ ID NO: 2 in the sequence listing. A recombinant vector containing the DNA according to claim 11 or 12. A recombinant vector which is the plasmid pNTPA (FERM P-20434) shown in FIG. The recombinant vector according to claim 13, further comprising DNA encoding a polypeptide having glucose dehydrogenase activity. The vector according to claim 15, wherein the polypeptide having glucose dehydrogenase activity is a glucose dehydrogenase derived from Bacillus megaterium. A transformant obtained by transforming a host cell with the recombinant vector according to any one of claims 13 to 16. The transformant according to claim 17, wherein the host cell is Escherichia coli. An α-keto acid reductase according to any one of claims 1 to 10 or a transformant according to claim 17 or 18 is reacted with α-keto acid. A method for producing an α-hydroxy acid. The α-keto acid is represented by the general formula (1)

(Wherein n represents an integer of 1 to 3, X represents a hydrogen atom, an alkali metal or an alkaline earth metal, and Ar represents an optionally substituted aryl group). An α-keto acid having the general formula (2)

20. The production method according to claim 19, wherein an S-form [alpha] -hydroxy acid having an aryl group represented by (n, X and R are the same as above) is produced. Ar is selected from the group consisting of a halogen atom, a hydroxyl group, an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, a thioalkyl group having 1 to 5 carbon atoms, an amino group, a nitro group, and a mercapto group. The production method according to claim 20, which is a phenyl group or a naphthyl group, which may be substituted with one or more substituents. The production method according to any one of claims 19 to 21, wherein the S-form α-hydroxy acid is accumulated in a concentration of 200 mM or more in the reaction solution.

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