JP2006204285A - New aldolase and method for producing optically active ihog[4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid] and monatin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an optically active IHOG useful for producing monatin, to provide a method for producing an optically active monatin, and to provide aldolase to be used for the above methods. <P>SOLUTION: The IHOG of high optical purity useful as an intermediate for synthesizing an optically active monatin is synthesized from indolepyruvic acid and pyruvic acid(or oxaroacetic acid). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel aldolase that produces 4- (indol-3-ylmethyl) -4-hydroxy-2-oxoglutarate (IHOG), a precursor of monatin, a method for producing 4R-IHOG using the same, and The present invention relates to a method for producing 4R-monatin.

  4- (Indol-3-ylmethyl) -4-hydroxy-glutamic acid (3- (1-amino-1,3-dicarboxy-3-hydroxy-butan-4-yl) -indole) represented by the following structural formula ( (Hereinafter referred to as “monatin”) is a compound expected to be a particularly low calorie sweetener since it is contained in the roots of the plant Schlerochiton ilicifolia and has an extremely high sweetness intensity (Japanese Patent Laid-Open No. 64- No. 25757).

  The above monatin has two asymmetry (2nd and 4th positions), and the stereoisomer has been reported as (2S, 4S). In addition, the presence of three types of stereoisomers has been confirmed, and it has been confirmed that all have a sweetness intensity several tens to several thousand times that of sucrose (Table 1).

  As shown in Table 1, not only (2S, 4S) -monatin but all other stereoisomers each have a high degree of sweetness, and in particular, (2R, 4R) -monatin is It has a remarkably high sweetness of 2700 times that of sucrose, and is the most expected isomer as a sweetener or sweetener component (sweetener). Therefore, development of a method for efficiently generating (2R, 4R) -monatin is desired.

The present inventors have developed a new monatin synthesis method comprising the following reactions (a) and (b) using indolepyruvic acid and pyruvic acid, which can be purchased as reagents (Patent Document 1).
(A) Reaction step of synthesizing precursor keto acid (IHOG) by aldol condensation of indolepyruvic acid and pyruvic acid (or oxaloacetic acid) (b) Reaction step of amination of position 2 of IHOG

  Patent Document 2 discloses Pseudomonas taetrolens as an enzyme capable of producing a precursor keto acid (IHOG) from indolepyruvic acid and pyruvic acid (or oxaloacetic acid) in the aldol condensation reaction (a) in the monatin synthesis route. (Pseudomonas taetrolens) and Pseudomonas coronafaciens-derived aldolases are disclosed. These aldolases have been shown to catalyze reactions that produce keto acids such as 4-phenylmethyl-4-hydroxy-2-oxoglutarate (PHOG) in addition to IHOG.

  IHOG has two isomers, 4R and 4S. In order to efficiently produce (2R, 4R) -monatin, which is the most sweet isomer, in the above monatin synthesis route. In the aldol condensation reaction of (a), it is desirable to preferentially generate 4R IHOG (hereinafter, “4R-IHOG”, 4S isomer is referred to as “4S-IHOG”) to obtain 4R-rich IHOG. In addition, chiral molecules often exhibit different physiological activities for different isomers, but IHOG may also have different properties for different isomers. By separately creating 4R and 4S isomers, Other than the precursor of monatin, it can be used for other purposes. Therefore, development of a method for preferentially producing one isomer of 4R-IHOG and 4S-IHOG is extremely useful industrially.

International Publication No. 03/056026 Pamphlet International Publication No. 04/018672 Pamphlet

  However, in the conventional chemical synthesis system, the generated IHOG was a mixture of 4R and 4S (racemic). In addition, the present inventors have obtained an aldolase derived from Pseudomonas taetrolens as an aldolase suitable for the synthesis of IHOG, but the IHOG produced by the aldolase is not rich in 4R form and depends on the reaction conditions. Rather, it has been clarified that IHOG rich in 4S body is generated. (Patent Document 1, Patent Document 2). In addition, no aldolase that preferentially produces 4R-IHOG has been reported so far. Therefore, at present, a method for efficiently generating 4R-IHOG, in particular, 4R-body-rich IHOG has not been established.

  The present invention has been made in view of the above, and an object thereof is to provide a novel aldolase that produces PHOG and IHOG, particularly 4R-IHOG, a method for producing IHOG using the same, and a method for producing monatin. And

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that aldolase that can be suitably used for the synthesis of the target 4R-IHOG exists in certain microorganisms, and by using these, 4R The present invention relating to a process for producing -IHOG and 4R-monatin has been conceived.

That is, the present invention includes the following contents.
[1]
(A) or (b) below
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, or (b) a protein having at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 2 and having 4R-aldolase activity,
Or a microorganism containing the protein according to any one of
Act on indole-3-pyruvic acid and pyruvic acid or oxaloacetic acid,
(4R) -4- (Indol-3-ylmethyl) -4-hydroxy-2-oxoglutaric acid (4R-IHOG) or a salt thereof represented by the following formula (1) having an optical purity of 70% or more is produced. A method for producing 4R-IHOG or a salt thereof.

（式中、波線の結合はＲ−配置及びＳ−配置の両方を含むことを表す。）
［３］
前記第２の工程において、カルボニル基のアミノ基への変換が、４Ｒ−ＩＨＯＧに酵素を作用せしめてアミノ化することにより行なわれるものである、［２］記載の４Ｒ−モナティン又はその塩の製造方法。
［４］
前記第２の工程において、カルボニル基のアミノ基への変換が、反応液に含まれる４−（インドール−３−イルメチル）−４−ヒドロキシ−２−オキソグルタル酸を、中性又はアルカリ性条件下において下記一般式（３）

（上記一般式（３）において、Ｒは水素原子、アルキル基、アリール基又はアラルキル基を表す。）
で表されるアミン化合物又はその塩と反応せしめ、下記式（４）に示される４−ヒドロキシ−４−（３−インドリルメチル）−２−ヒドロキシイミノグルタル酸（ＩＨＯＧ−ｏｘｉｍｅ）を生成させ、生成したＩＨＯＧ−ｏｘｉｍｅ又はその塩の４Ｒ体を晶析し、
得られた４Ｒ体のＩＨＯＧ−ｏｘｉｍｅ又はその塩を還元し、生成した光学純度９０％以上の４Ｒ−モナティン又はその塩を採取するものである、［２］記載の４Ｒ−モナティン又はその塩の製造方法。

［５］
前記一般式（３）で表されるアミン化合物が、ヒドロキシルアミン、メトキシアミン、ベンジルオキシアミンからなる群より選ばれる少なくとも一種のアミン化合物である、［４］記載の４Ｒ−モナティン又はその塩の製造方法。
［６］
４Ｒ体のＩＨＯＧ−ｏｘｉｍｅ又はその塩の還元が、水素及び水素添加触媒の存在下で実施されることを特徴とする［４］又は［５］に記載の４Ｒ−モナティン又はその塩の製造方法。
［７］
前記第２の工程において、晶析により（２Ｒ，４Ｒ）−モナティンを採取することを特徴とする［４］〜［６］のいずれか一項に記載の４Ｒ−モナティン又はその塩の製造方法。
［８］
前記第２の工程において、晶析溶媒として水、アルコール溶媒又は含水アルコール溶媒を用いることを特徴とする［４］〜［７］のいずれか１項に記載の４Ｒ−モナティン又はその塩の製造方法。
［９］
前記方法に用いられるタンパク質が、スフィンゴモナス属又はバークホルデリア属細菌から選ばれる微生物に由来するタンパク質である、［１］〜［８］のいずれか一項に記載の製造方法。
［１０］
前記微生物は、スフィンゴモナス エスピー（Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｐ．）ＡＪ１１０３２９株又はＡＪ１１０３７２株、バークホルデリア エスピー（Ｂｕｒｋｈｏｌｄｅｒｉａ ｓｐ．）ＡＪ１１０３７１株であることを特徴とする［９］記載の製造方法。 [2]
(A) or (b) below
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, or (b) a protein having at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 2 and having 4R-aldolase activity,
Or a microorganism containing the protein according to any one of
Act on indole-3-pyruvic acid and pyruvic acid or oxaloacetic acid,
A first step of preferentially producing 4R-IHOG or a salt thereof; and
A second carbonyl group of 4R-IHOG or a salt thereof obtained in the first step is converted to an amino group to obtain 4R-monatin or a salt thereof represented by the following formula (2) having an optical purity of 90% or more. Process,
A process for producing 4R-monatin or a salt thereof.

(In the formula, the combination of wavy lines indicates that both R-configuration and S-configuration are included.)
[3]
Production of 4R-monatin or a salt thereof according to [2], wherein in the second step, conversion of a carbonyl group to an amino group is carried out by aminating 4R-IHOG with an enzyme. Method.
[4]
In the second step, the conversion of the carbonyl group into an amino group is performed by converting 4- (indol-3-ylmethyl) -4-hydroxy-2-oxoglutaric acid contained in the reaction solution under neutral or alkaline conditions as follows. General formula (3)

(In the general formula (3), R represents a hydrogen atom, an alkyl group, an aryl group, or an aralkyl group.)
To produce 4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid (IHOG-oxime) represented by the following formula (4): Crystallize the produced 4HO form of IHOG-oxime or a salt thereof,
Production of 4R-monatin or a salt thereof according to [2], wherein the obtained 4R-form IHOG-oxime or a salt thereof is reduced, and the produced 4R-monatin or a salt thereof with an optical purity of 90% or more is collected. Method.

[5]
Production of 4R-monatin or a salt thereof according to [4], wherein the amine compound represented by the general formula (3) is at least one amine compound selected from the group consisting of hydroxylamine, methoxyamine, and benzyloxyamine. Method.
[6]
The method for producing 4R-monatin or a salt thereof according to [4] or [5], wherein the reduction of 4R IHOG-oxime or a salt thereof is carried out in the presence of hydrogen and a hydrogenation catalyst.
[7]
The method for producing 4R-monatin or a salt thereof according to any one of [4] to [6], wherein (2R, 4R) -monatin is collected by crystallization in the second step.
[8]
The method for producing 4R-monatin or a salt thereof according to any one of [4] to [7], wherein in the second step, water, an alcohol solvent, or a hydrous alcohol solvent is used as a crystallization solvent. .
[9]
The production method according to any one of [1] to [8], wherein the protein used in the method is a protein derived from a microorganism selected from the genus Sphingomonas or Burkholderia.
[10]
[9] The method according to [9], wherein the microorganism is Sphingomonas sp. AJ110329 strain or AJ110372 strain, Burkholderia sp. AJ110371 strain.

[11]
The protein in any one of the following (a)-(c).
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 (b) a protein having at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 2 and having 4R-aldolase activity (c) a sequence in the sequence listing A protein having an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues in the amino acid sequence of No. 2 and having aldolase activity [12]
The protein according to [11], wherein the protein having at least 70% homology with the amino acid sequence described in SEQ ID NO: 2 and having 4R-aldolase activity is the protein described in either SEQ ID NO: 13 or 15.
[13]
DNA encoding the protein according to [11] or [12].
[14]
DNA of the following (d) or (e).
(D) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or the nucleotide sequence of nucleotide numbers 210 to 1004 in the same sequence
(E) a protein that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequences 210 to 1004 of the same sequence and has an aldolase activity DNA to do
[15]
A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence set forth in SEQ ID NO: 1 or a base sequence complementary to the base sequences 210 to 1004 in the same sequence and that encodes a protein having aldolase activity (F) DNA consisting of the base sequence of SEQ ID NO: 12 or the base sequence of base numbers 399 to 1253 in the same sequence, or (g) the base sequence of SEQ ID NO: 14 or the base sequence of base numbers 531 to 1385 in the same sequence The DNA according to [14], which is any one of DNAs consisting of
[16]
A recombinant DNA obtained by connecting the DNA according to [14] or [15] and a vector DNA.
[17]
[16] A cell transformed with the recombinant DNA according to [16].
[18]
[17] A method for producing a protein having aldolase activity, comprising culturing the cell according to [17] in a medium and accumulating a protein having aldolase activity in the medium and / or the cell.

  By using the aldolase of the present invention, 4R-IHOG can be preferentially produced from indolepyruvic acid and pyruvic acid (or oxaloacetic acid). In addition, since 4R-IHOG can be aminated to produce 4R-monatin, it can be used very advantageously in the production of high-sweetness monatin.

  Conventionally, when 4R isomers are to be isolated from racemic IHOG (4R, 4S-IHOG), 4R, 4S-IHOG is oximed, and the resulting 4-hydroxy-4- (3-indolyl) is obtained. Methyl) -2-hydroxyiminoglutaric acid (hereinafter referred to as “IHOG-oxime”) must be allowed to act on a chiral amine to crystallize 4R IHOG-oxime (hereinafter referred to as “4R-IHOG-oxime”). It was. In contrast, according to the present invention, IHOG rich in 4R isomer can be generated at the stage of the aldol condensation reaction, so that it is not necessary to perform optical resolution using a chiral amine during crystallization. 4R-IHOG-oxime can be crystallized. Therefore, it becomes possible to reduce the purification process of 4R-IHOG.

  The present inventors confirmed that a strain that produces an aldolase having an activity of preferentially synthesizing 4R-IHOG exists in certain microorganisms, and a method for producing 4R-IHOG and 4R-monatin has been confirmed. It was found.

Hereinafter, regarding the present invention,
[I] Production Method of Optically Active IHOG [II] The production method of optically active monatin will be described in detail with reference to the accompanying drawings.

で示されるインドールピルビン酸と、
下記一般式（６）

（式中、Ｒは水素原子又はカルボキシル基）
で示されるピルビン酸又はオキサロ酢酸とを反応せしめることにより、
下記式（１）

で示される４Ｒ−ＩＨＯＧを優先的に製造する方法であって、
当該反応を触媒するタンパク質存在下で反応を実施する点に特徴を有する。 [I] Method for Producing Optically Active IHOG (1) Reaction The method for producing 4R-IHOG of the present invention will be described. The method for producing 4R-IHOG of the present invention has the following formula (5).

Indole pyruvic acid represented by
The following general formula (6)

(Wherein R is a hydrogen atom or a carboxyl group)
By reacting with pyruvic acid or oxaloacetic acid represented by
Following formula (1)

A process for preferentially producing 4R-IHOG represented by:
The reaction is carried out in the presence of a protein that catalyzes the reaction.

  The “protein that catalyzes the reaction” is preferably a protein having 4R-aldolase activity, and may be derived from a microorganism or may be a chemically synthesized protein. That is, 4R-aldolase activity is defined by the above general formula (1) by aldol condensation between indole pyruvic acid represented by the general formula (5) and pyruvic acid or oxaloacetic acid represented by the general formula (6). ) And / or a reaction that preferentially produces 4R-IHOG and / or an activity that can catalyze a reaction that preferentially produces 4R-PHOG from phenylpyruvic acid and pyruvic acid. Any such protein can be used in the present invention without any particular limitation. In the present specification, “to preferentially produce 4R-IHOG” means that the optical purity at the 4-position of the produced IHOG is higher than the ratio in which the R form is converted to the S form, preferably optical It means that the reaction efficiency of converting to R-form can be achieved with a purity of 70% or more, particularly preferably 90% or more. Depending on the reaction conditions, the ratio of optical purity varies, but those skilled in the art can easily set the optimal conditions for the reaction. Even if the ratio is not achieved when the conditions are changed, it is included in the method of the present invention. In addition, for the purpose of appropriately adjusting the mixing ratio of the 4R isomer and the 4S isomer, a protein that can be used in the present invention is used, and the reaction below the above ratio is performed by adjusting the reaction conditions. Included in the method. The optical purity of 4R-IHOG is ([4R-IHOG]-[4S-IHOG]) / ([4R-IHOG] + [4S-IHOG]) * as the enantiomeric excess (% ee). 100.

The protein that catalyzes the reaction is preferably an aldolase described later in the section of (2) a protein having aldolase activity. In the reaction, an aldolase obtained by culturing a cell that produces a protein (aldolase) that catalyzes the reaction may be used, or a transformant that produces a protein that catalyzes the reaction is prepared by recombinant DNA technology. Alternatively, an aldolase obtained by culturing the transformant may be used.
The protein that catalyzes the reaction may be added to the reaction system in any form as long as the reaction that preferentially synthesizes 4R-IHOG can be catalyzed. That is, a protein that catalyzes the reaction may be added alone to the reaction system, or may be added to the reaction system in the form of a composition having an aldolase activity including a protein (aldolase) that catalyzes the reaction.

  Here, the “composition having aldolase activity” only needs to contain a protein (aldolase) that catalyzes the reaction, and specifically, a culture, a medium (one obtained by removing cells from the culture), Bacteria (including both cultured cells and washed cells), treated cells obtained by crushing or lysing the cells, a composition having aldolase activity obtained by purifying the medium and / or cells ( Crude enzyme solution, purified enzyme). For example, in the case of producing optically active IHOG using cells transformed with aldolase-producing bacteria or recombinant DNA, a substrate may be added directly to the culture solution while culturing, or the optically active IHOG may be separated from the culture solution. Both bacterial cells and washed bacterial cells can be used. In addition, a cell-treated product obtained by crushing or lysing cells may be used as it is, or aldolase may be recovered from the cell-treated product and used as a crude enzyme solution, or the enzyme may be purified. May be used. In other words, any form having aldolase activity can be used in the method for producing 4R-IHOG of the present invention.

In order to advance the aldol reaction using an aldolase or a composition having aldolase activity, a reaction solution containing indolepyruvic acid and pyruvic acid or oxaloacetic acid, a protein that catalyzes the reaction or an aldolase-containing material is 20 to 50 ° C. The temperature may be adjusted to an appropriate temperature and kept at pH 6 to 12, while standing for 30 minutes to 5 days, shaking or stirring.
Here, when IHOG is generated more stereoselectively, the desired 4R-IHOG can be generated with higher stereoselectivity by suppressing spontaneous aldol condensation. Here, a case where IHOG is generated by subjecting indole-3-pyruvic acid and pyruvic acid to aldol condensation will be described as an example. This reaction spontaneously undergoes aldol condensation by reacting the pH on the alkali side, for example, around pH 9-12. The IHOG produced by this spontaneous aldol condensation has low selectivity with respect to the 4-position stereo and gives a mixture of 4R and 4S (racemate). Therefore, in one embodiment of the present invention, when the protein that catalyzes the reaction is allowed to act, the reaction pH is controlled to be between pH 9 and pH 7, more preferably around pH 8.7 to pH 8, thereby suppressing spontaneous IHOG production. On the other hand, 4R-IHOG is selectively subjected to aldol condensation by the protein, so that the 4R selectivity of the resulting IHOG can be increased. Regarding such reaction conditions, those skilled in the art can determine the conditions by simple preliminary examination.

  Moreover, it is considered that the aldolase disclosed in the present invention belongs to a so-called class II aldolase whose enzymatic activity is increased by adding a divalent cation. Here, the concentration of the divalent cation added to the reaction system also affects the progress of spontaneous aldol condensation, and thus may affect the stereoselectivity at the 4-position of the resulting IHOG. With regard to the type and concentration of the divalent cation added to the reaction system, those skilled in the art can determine suitable conditions by simple preliminary studies.

In addition, the reaction rate can be improved by adding divalent cations such as Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , and Co ²⁺ to the reaction solution. From the viewpoint of cost and the like, Mg ²⁺ is preferably used. Here, when the divalent cation is added to the reaction solution, any salt may be used as long as the reaction is not inhibited, but MgCl ₂ , MgSO ₄ , MnSO _{4 and the} like may be preferably used. The addition concentration of these divalent cations can be determined by a simple preliminary study by those skilled in the art, but as an example when Mg ²⁺ is added as a divalent cation, the added Mg ²⁺ is 1 mM. Below, preferably 0.5 mM or less, more preferably 0.1 mM or less, the spontaneous IHOG condensation rate is suppressed, and as a result, the 4R selectivity of IHOG produced when aldolase is allowed to act is increased. I can do it.

Examples of preferable reaction conditions for carrying out the method for producing IHOG of the present invention are as follows: 100 mM buffer, 300 mM indole-3-pyruvic acid, 600 mM pyruvic acid, 0.1 mM MgCl ₂ , 1% (v / v ) Expression of aldolase as an enzyme source in a reaction solution comprising toluene. 4- (indol-3-ylmethyl) -4-hydroxy-2-oxoglutaric acid was obtained by adding the washed bacterial cells of E. coli to 10% (w / v) and shaking at 37 ° C. for 4 hours. (IHOG) is obtained.

  The generated IHOG can be separated and purified by a known method. Examples thereof include a method in which a basic amino acid is adsorbed by contacting with an ion exchange resin and crystallized after elution, or a method in which crystallization is performed by decolorizing filtration using activated carbon or the like after elution. Moreover, it is also possible to use the produced | generated reaction solution containing IHOG as it is for the next reaction process.

For example, the production ratio of 4S-IHOG to 4R-IHOG in the case of producing IHOG according to the method of the present invention is, for example, 0. 300 mM indole-3-pyruvic acid and 600 mM pyruvic acid as a substrate. It has been confirmed that SpALD produces S-IHOG and R-IHOG at a ratio of about 4:96 under the reaction conditions of pH 8.7 to pH 8.0 in the presence of 1 mM MgCl ₂ (see Example 12). Furthermore, when the reaction solution containing 4R-IHOG thus obtained is crystallized through an oximation reaction described later, the optical purity at the 4-position of 4R-IHOG-oxime can achieve 90% or more. It becomes possible. In addition, since the reaction which converts IHOG to IHOG-oxime using hydroxylamine does not have optical selectivity at the 4-position, the measurement of optical purity is the same in any form. The 4R-IHOG thus obtained is extremely useful as an intermediate for the production of 4R-monatin.

(2) Protein having aldolase activity (aldolase)
A protein having 4R-aldolase activity (hereinafter sometimes simply referred to as “4R-aldolase”) used in the production method of the present invention has a property of catalyzing the above reaction. It can also be obtained from a microorganism having 4R-aldolase activity, and the microorganism can be obtained by the following screening method.

(I) Microorganism having 4R-aldolase activity (a) Screening method for microorganism having 4R-aldolase activity A microorganism producing 4R-aldolase can be obtained from the natural world such as soil and water. That is, as a carbon source or a nitrogen source, preferably a single carbon source or a single nitrogen source, a compound of which the target aldolase is a substrate such as monatin, IHOG, IHOG-oxime, PHG, PHOG, PHOG-oxime, It is desirable to add it to the medium and inoculate a sample that is a source of microorganisms and incubate. In addition, a racemic mixture may be used as these additives, but it is preferable to use 4R-form, more preferably (2R, 4R) -monatin. As an organic nutrient source other than the carbon source, a normal medium component can be appropriately selected. As the nitrogen source, organic acid ammonium salts, nitrates, organic nitrogen compounds such as peptone, yeast extract and meat extract, or mixtures thereof can be used. In addition, commonly used nutrient sources such as inorganic salts, trace metal salts, and vitamins can be mixed as appropriate. Many microorganisms capable of growing in such enrichment culture contain aldolase-active bacteria.

  Next, a single colony is obtained from the accumulated bacteria in the medium, and the obtained colony is again checked for growth on a culture plate using the target substrate as a single carbon source, and the aldolase activity is evaluated. As the culture conditions for the screening, normal culture conditions can be used except for the carbon source. As an example, the conditions described in the section of the method for culturing a microorganism having aldolase activity (c) described later can be given. It is done.

  As a method for evaluating the aldolase activity produced by the microorganism, it is desirable to purify the enzyme from the microorganism and evaluate the enzyme reaction using the enzyme preparation. Specifically, i) a method of detecting free pyruvic acid using IHOG or PHOG as a substrate (detection of degradation activity), ii) aldol condensation using indole pyruvic acid or phenylpyruvic acid and pyruvic acid (or oxaloacetic acid) as substrates And a method for detecting PHOG produced by HPLC (high-performance liquid chromatography (HPLC) measurement) (synthetic activity detection). Furthermore, it is desirable to evaluate 4R selectivity by confirming asymmetry at the 4-position of IHOG or PHOG produced by aldol condensation in ii) by HPLC.

Specifically, aldolase was added to a reaction solution consisting of 100 mM buffer, 300 mM indole-3-pyruvic acid, 600 mM pyruvic acid, 0.1 mM MgCl ₂ , 1% (v / v) toluene, and shaken at 37 ° C. for 4 hours. The aldolase activity can be estimated by reacting and quantifying the amount of IHOG produced by HPLC.

  IHOG can be quantified by, for example, HPLC analysis using “Inertsil ODS-2” (5 μm, 4.6 × 250 mm) manufactured by GL Sciences. An example of analysis conditions is shown below.

Mobile phase: 40% (v / v) acetonitrile / 5 mM tetrabutylammonium dihydrogen phosphate flow rate: 1 ml / min
Column temperature: 40 ° C
Detection: UV210nm

(B) Microorganisms obtained by screening As a result of the screening, the present inventors selected microorganisms belonging to the genus Sphingomonas and microorganisms belonging to the genus Burkholderia from the accumulated bacteria, and microorganisms belonging to both genera or close to these microorganisms. It has been found that aldolase that can be used in the present invention is produced from related microorganisms. Therefore, microorganisms having aldolase activity that can be used in the present invention include microorganisms belonging to the genus Sphingomonas, Burkholderia, or related genera. Examples of closely related genera of the genus Sphingomonas include the genus Rhizomonas, the genus Blastmonas, the genus erythromicrobium, the genus Porphylobacter, the genus Agrobacterium, and the genus Erytrobacter. In addition, about the genus Sphingomonas, the proposal of the reclassification has been made in recent years, and there are cases where it is called the genus Sphingobiium, Novosphingobium genus or Sphingopixis (International Journal of Evolutionary Microbiology (2001)). , 51, 1405-1417), and in the present specification, the term of the genus Sphingomonas is used to include these.

Examples of microorganisms belonging to the genus Sphingomonas include the following.
Sphingomonas sp. , Sphingomonas trueperi, Sphingomonas parapaucimobilis, Sphingomonas sanguinis, Sphingomonas paucimobilis, Sphingomonas adhaesiva, Sphingomonas pruni, Sphingomonas mali, Sphingomonas asaccharolytica, Sphingomonas echinoids, Sphingomonas yanoikuyae, Sphingomonas herbicidovorans, Sphingomonas chlorophenolica, Sphingomonas agrestis, Sphingomonas rosa, S hingomonas subarctica, Sphingomonas stygia, Sphingomonas subterranean, Sphingomonas aromaticivorans, Sphingomonas capsulate, Sphingomonas macrogoltabidus, Sphingomonas terrae, Rhizomonas suberifaciens, Blastomonas natatoria, Blastomonas ursincola, Agrobacterium sanguineum, Erythrobacter longus, Erythrobacter litoralis, and the like.

Examples of microorganisms belonging to the genus Burkholderia include the following.
Burkholderia sp. , Burkholderia phenazinium, Burkholderia caribensis, Burkholderia graminis, Burkholderia kururiensis, Burkholderia brasilensis, Burkholderia caryophylli, Burkholderia glathei, Burkholderia plantarii, Burkholderia vandii, Burkholderia glumae, Burkholderia cocovenenans, Burkholderia gladioli, Burkholderia vietnamiensis, Burkholderia multivorans, Burkho deria cepacia, Burkholderia pyrrocinia, Burkholderia thailandensis, Burkholderia pseudomallei, Burkholderia mallei, Burkholderia andropogonis the like (Current Microbiology Vol. 42 (2001), pp. 269-275).

Particularly preferred are the following microorganisms. The depository of these microorganisms is shown below.
・ Sphingomonas sp. AJ110329 strain (C77 strain)
(I) Accession number FERM BP-10027
(Ii) Date of application for original deposit application May 21, 2004 (iii) Depositee National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan)
・ Sphingomonas sp. AJ110372 strain (C43 strain)
(I) Accession number FERM BP-10156
(Ii) Date of application for the original deposit application October 28, 2004 (iii) Depositee National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1st, 1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan)
・ Burkholderia sp. AJ110371 strain (C24 strain)
(I) Accession number FERM BP-10155
(Ii) Date of application for the original deposit application October 28, 2004 (iii) Depositee National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1st, 1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan)

In addition, AJ110329 strain (C77 strain, FERM BP-10027) was obtained from the above-mentioned Sphingomonas sp. Was identified.
A region of about 500 bp on the 5 ′ end side of 16S ribosomal RNA gene (16S rDNA) was amplified from the genomic DNA of AJ110329 strain by PCR, and the base sequence was determined (SEQ ID NO: 16). Homology analysis of the resulting sequences was performed using MicroSeq Bacterial 500 Library v. 0023 (Applied Biosystems, CA, USA) as a database, MicroSeq Microbial Identification System Software V. 1.4.1. Was used for analysis. As a result, there was no known sequence corresponding to the AJ110329 strain 16S rDNA partial nucleotide sequence, and the homology was 96.6%, which showed the highest homology to 16S rDNA of Sphingomonas capsula. Even in the molecular phylogenetic tree, 16S rDNA of AJ110329 strain was included in the cluster formed by 16S rDNA of Sphingomonas. In addition, the bacteriological properties shown in Table 2 are not inconsistent with the results of the 16S rDNA nucleotide sequence analysis described above, and the strain AJ110329 was designated as Sphingomonas sp. Estimated.

As a result of the following classification experiment, the AJ110372 strain (C43 strain, FERM BP-10156) Was identified.
From the genomic DNA of the AJ110372 strain, a region of about 500 bp on the 5 ′ end side of the 16S ribosomal RNA gene (16S rDNA) was amplified by PCR, and the base sequence was determined (SEQ ID NO: 17). Homology analysis of the resulting sequences was performed using MicroSeq Bacterial 500 Library v. 0023 (Applied Biosystems, CA, USA) as a database, MicroSeq Microbial Identification System Software V. 1.4.1. Was used for analysis. As a result, there was no known sequence corresponding to the AJ110372 strain 16S rDNA partial base sequence, and the homology was 98.94%, which showed the highest homology to 16S rDNA of Sphingomonas yanokuyae. Also in the molecular phylogenetic tree, the 16S rDNA of the AJ110372 strain is contained in the cluster formed by the 16S rDNA of Sphingomonas yanokuyae, and the AJ110372 strain is included in the Sphingomonas sp. Estimated.

As a result of the following classification experiment, AJ110371 strain (C24 strain, FERM BP-10155) was found to have the above-mentioned Burkholderia sp. Was identified.
From the genomic DNA of the AJ110371 strain, a region of about 500 bp on the 5 ′ end side of the 16S ribosomal RNA gene (16S rDNA) was amplified by PCR, and the base sequence was determined (SEQ ID NO: 18). Homology analysis of the resulting sequences was performed using MicroSeq Bacterial 500 Library v. 0023 (Applied Biosystems, CA, USA) as a database, MicroSeq Microbial Identification System Software V. 1.4.1. Was used for analysis. As a result, there was no known sequence corresponding to the AJ110371 strain 16S rDNA partial base sequence, and the homology was 95.21%, which showed the highest homology to Burkholderia phenazinium 16S rDNA. Even in the molecular phylogenetic tree, 16S rDNA of AJ110371 strain was included in the cluster formed by Burkholderia 16S rDNA. In addition, the bacteriological properties shown in Table 3 are not inconsistent with the results of the 16S rDNA nucleotide sequence analysis, and the AJ110371 strain was identified as Burkholderia sp. Estimated.

Ｂｕｒｋｈｏｌｄｅｒｉａ ｓｐ． ＡＪ１１０３７１株（ＦＥＲＭ ＢＰ−１０１５５）の菌学的性質は、以下の表３に記載の通りである。

参考文献および使用キット
１）ＢＡＲＲＯＷ，（Ｇ．Ｉ．）ａｎｄ ＦＥＬＴＨＡＭ，（Ｒ．Ｋ．Ａ）：Ｃｏｗａｎ ａｎｄ Ｓｔｅｅｌ’ｓ Ｍａｎｕａｌ ｆｏｒ ｔｈｅ Ｉｄｅｎｔｉｆｉｃａａｔｉｏｎ ｏｆ Ｍｅｄｉｃａｌ Ｂａｃｔｅｒｉａ． ３^rd ｅｄｉｔｉｏｎ．１９９３，Ｃａｍｂｒｉｄｇｅ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ．
２）坂崎利一・吉崎悦郎・三木寛二：新細菌培地学講座・下〈第二版〉．１９８８，近大出版，東京．
３）細菌同定キットＡＰＩ２０，ＮＥ，
（ｂｉｏＭｅｒｉｅｕｘ，Ｆｒａｎｃｅ：ｈｔｔｐ：／／ｗｗｗ．ｂｉｏｍｅｒｉｅｕｘ．ｆｒ／ｈｏｍｅ＿ｅｎ．ｈｔｍ）． Sphingomonas sp. The mycological properties of AJ110329 strain (FERM BP-10027) are as shown in Table 2 below.

Burkholderia sp. The mycological properties of AJ110371 strain (FERM BP-10155) are as shown in Table 3 below.

References and kits used 1) BARROW, (GI) and FELTHAM, (RKA): Cowan and Steel's Manual for the Identification of Medical Bacteria. 3 ^rd edition. 1993, Cambridge University Press.
2) Toshikazu Sakazaki, Goro Yoshizaki, Kanji Miki: New Bacteriological Culture Course, 2nd edition. 1988, Jindai Publishing, Tokyo.
3) Bacteria identification kit API20, NE,
(BioMerieux, France: http://www.biomerieux.fr/home_en.htm).

(C) Method for culturing microorganisms having aldolase activity The culture form of microorganisms as an acquisition source of aldolase can be either liquid culture or solid culture, but an industrially advantageous method is the deep aeration stirring culture method. As a nutrient source of the nutrient medium, a carbon source, a nitrogen source, an inorganic salt, and other trace nutrient sources that are usually used for microbial culture can be used. Any nutrient source for which the strain used is available can be used.

  The aerobic condition is adopted as the aeration condition. The culture temperature may be within a range in which bacteria grow and aldolase is produced. Therefore, although there is no strict condition, it is usually 10 to 50 ° C, preferably 30 to 40 ° C. The culture time varies depending on other culture conditions. For example, it may be cultured until the time when the aldolase is most produced, and is usually 5 hours to 7 days, preferably about 10 hours to 3 days.

(D) Method for obtaining aldolase from microorganism After culturing a microorganism having aldolase activity, the cells are collected by centrifugation (for example, 10,000 × g, 10 minutes). Since most of the aldolase is present in the cells, the aldolase is solubilized by crushing or lysing the cells. For crushing the cells, methods such as ultrasonic crushing, French press crushing, and glass bead crushing can be used. In the case of lysis, egg white lysozyme, peptidase treatment, or a combination of these methods is used.

  When purifying aldolase derived from an aldolase-producing bacterium, the enzyme lysate is purified as a starting material. If undisrupted or undissolved bacteria residue is present, the lysate is again subjected to centrifugation. It is advantageous for purification to remove the precipitated residue.

  For the purification of aldolase, all conventional methods usually used for purifying enzymes such as ammonium sulfate salting out method, gel filtration chromatography method, ion exchange chromatography method, hydrophobic chromatography method, hydroxyapatite chromatography, etc. Can be adopted. As a result, an aldolase-containing fraction with higher specific activity can be obtained.

  After preparing a purified aldolase preparation, the N-terminal amino acid sequence can be known by subjecting it to a protein sequencer by Edman degradation method (Edman, P., Acta Chem. Scand. 4, 227 (1950)). Furthermore, after the peptidase treatment, the peptide preparation is separated and purified by reverse phase HPLC, and then subjected to a protein sequencer by Edman degradation, whereby the internal amino acid sequence can be known.

  Based on the revealed amino acid sequence, the base sequence of the DNA encoding it can be deduced. To deduct the base sequence of DNA, universal codons can be employed.

  Based on the deduced base sequence, a DNA molecule of about 30 base pairs is synthesized. A method for synthesizing the DNA molecule is disclosed in Tetrahedron Letters, 22, 1859 (1981). Further, the DNA molecule can be synthesized using a synthesizer manufactured by Applied Biosystems. The DNA molecule can be used as a probe when isolating a full-length DNA encoding an aldolase from an aldolase-producing bacterium chromosomal gene library. Alternatively, it can be used as a primer when the DNA encoding the aldolase of the present invention is amplified by the PCR method. However, since the DNA amplified using the PCR method may not contain the entire DNA encoding the aldolase, the DNA amplified using the PCR method is used as a probe, and the entire DNA encoding the aldolase is converted into the aldolase. Isolate from the production chromosome gene library.

  For the operation of the PCR method, see White, T .; J. et al. et al. , Trends Genet. 5, 185 (1989). A method for preparing chromosomal DNA and a method for isolating a target DNA molecule from a gene library using the DNA molecule as a probe are described in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989) and the like. ing.

  A method for determining the base sequence of the DNA encoding the isolated aldolase is described in A Practical Guide to Molecular Cloning, John Wiley & Sons, Inc. (1985). Further, the base sequence can be determined using a DNA sequencer manufactured by Applied Biosystems.

  Examples of a method for obtaining DNA encoding an aldolase from an aldolase-producing bacterium include a method for obtaining DNA that hybridizes using the full-length DNA of the present invention or a partial sequence as a probe.

As a method for obtaining DNA encoding an aldolase from an aldolase-producing bacterium, a DNA sequence deduced from a highly conserved amino acid portion can be used as a probe DNA by aligning the amino acid sequences of the aldolase of the present invention. . Or it can utilize as a primer of PCR method. Since the DNA amplified using the PCR method may not contain the full length DNA encoding the aldolase, using the DNA amplified using the PCR method as a probe, the full length DNA encoding the aldolase is converted to the aldolase producing bacterial chromosome. It can be isolated from a gene library.
In this manner, a microorganism having aldolase activity can be obtained, and aldolase and DNA encoding the aldolase can be obtained from the obtained microorganism.

(Ii) Aldolase The present inventors obtained aldolase from C77 strain (Sphingomonas sp. AJ110329 strain) selected by the above screening method and named it SpALD. The DNA encoding the SpALD of the present invention identified by the aforementioned method is shown in SEQ ID NO: 1 (CDS: base numbers 210 to 1004) in the sequence listing, and the amino acid sequence of SpALD is shown in SEQ ID NO: 2.

  The present inventors further carried out microorganism screening (detailed in the Examples), and by Southern analysis and colony hybridization using a part of the nucleotide sequence described in SEQ ID NO: 1 as a probe, C43 strain (Sphingomonas sp. AJ110372 strain) ) And C24 strain (Burkholderia sp. AJ110371 strain) (SEQ ID NOs: 12 and 14), a protein having the amino acid sequence of SEQ ID NO: 13 (SpALD2) and a protein having the amino acid sequence of SEQ ID NO: 15 ( BuALD) was obtained. The result of comparing the sequences of SpALD, SpALD2, and BuALD is shown in FIG. The aldolase core sequence (SEQ ID NO: 23) common to the three aldolases is shown in the upper part of the figure. There are 200 amino acid residues common to the three aldolases, accounting for 70.2% of the total, and 76 amino acid residues common to two of the three aldolases are present. The combined total really accounts for 96.8% of the total. Moreover, the homology between SpALD / SpALD2 is 88.4%, and the homology between SpALD / BuALD is 74.7%.

  Furthermore, as a result of investigating homology with other known sequences, the amino acid sequence of SEQ ID NO: 2 in the sequence listing was 2,4-dihydroxyhept-2-ene-1, 7-dioic acid alcoholase (derived from Escherichia coli C strain). HpcH) (Stringfall JM et al., Gene. 1995 166 (1): 73-6) and 20% homology. In addition, IHOG aldolase (gene name PtALD) derived from known Pseudomonas taetrolens bacteria, IHOG aldolase (gene name PcALD) derived from Pseudomonas coronafaciens bacteria (International Publication No. 03/056026 pamphlet) 4-methyl-2-oxoglutarate alcohol (gene name proA) or 4-Hydroxy-4-methyl-2-oxoglutarate alcohol (gene name proA) derived from Pseudomonas straminea is almost less than 10% and almost homologous. There is no present invention etc. Aldolase is found to be extremely novel protein. The three aldolases found by the present inventors also constitute a part of the present invention, and are sometimes collectively referred to as “the aldolase of the present invention”.

  The homology analysis here is a value calculated using the gene analysis software “Geneticx ver. 6” (Genetics) with the parameters as initial setting values.

  Therefore, any protein having 70% or more, preferably 74% or more, more preferably 80% or more, particularly preferably 85% or more, and most preferably 95% or more homology with the amino acid sequence shown in SEQ ID NO: 2 Have the same enzyme activity and can be used in the present invention. Furthermore, any protein having 70% or more homology with the amino acid sequence described in SEQ ID NO: 2 and having the aldolase core sequence described in SEQ ID NO: 23 can be used very suitably.

Therefore, the protein having 4R-aldolase activity used in the production method of the present invention includes the aldolase of the present invention, and preferably includes the following protein (a) or (b).
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 (b) a protein having at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 2 and having 4R-aldolase activity; The protein includes the following protein (c).
(C) a protein comprising the amino acid sequence of any one of SEQ ID NOs: 13 and 15

The aldolase of the present invention includes the following proteins.
(D) a protein comprising the amino acid sequence of SEQ ID NO: 2, 13, or 15 (e) substitution of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 2, 13, or 15 , A protein having an amino acid sequence containing a deletion, insertion, addition or inversion, and having aldolase activity

  Here, “one or several” is a range that does not significantly impair the three-dimensional structure of the protein of the amino acid residue and the aldolase activity, specifically 1 to 90, preferably 1 to 75. More preferably, it is 1 to 57, particularly preferably 1 to 43, and most preferably 1 to 15. However, in the case of an amino acid sequence including substitution, deletion, insertion, addition or inversion of one or several amino acid residues in the amino acid sequence described in any of SEQ ID NO: 2, 11 or 13 in the sequence listing, 10% or more, preferably 30% or more, more preferably 50% or more, still more preferably 10% or more, preferably 30% or more, of the protein having the amino acid sequence of SEQ ID NO: 2, 11 or 13 in the sequence listing under conditions of 33 ° C. and pH 9 It is desirable to retain at least 70% aldolase activity, preferably 4R-aldolase activity.

  Substitutions, deletions, insertions or additions as described above include conservative mutations such that aldolase activity is maintained. Conservative mutations are typically conservative substitutions. Amino acids that replace the original amino acid of the aldolase and are considered conservative substitutions include Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His Or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Asn, Gln, Lys Or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu, Met, Val or Phe, Leu to Ile, Met, Val or Phe Replacement from Lys to Asn, Glu, Gln, His or Arg Substitution, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, substitution from Tyr, Met, Ile or Leu, substitution from Ser to Thr or Ala, substitution from Thr to Ser or Ala, from Trp Substitution to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu.

  Here, the aldolase activity means the aforementioned 4R-aldolase activity when used in the production method of the present invention, and the property of the aldolase (protein) of the present invention does not strictly require optical selectivity. However, 4R-aldolase activity is more preferable. Hereinafter, when the term “aldolase activity” is used, it refers to 4R-aldolase activity when used in the production method of the present invention, and as described in the above (1) reaction section, indole pyruvic acid and pyruvic acid or This activity is capable of catalyzing a reaction that preferentially produces 4R-IHOG by aldol condensation with oxaloacetic acid. Here, “preferentially producing 4R-IHOG” means that the optical purity at the 4-position of the produced IHOG is higher than the ratio of R-form, preferably 70% optical purity. % Or more, particularly preferably 90% or more means that the reaction efficiency to be an R form can be achieved. On the other hand, when the “aldolase activity” means the activity of the protein of the present invention, it means an aldolase activity that does not require optical selectivity, and an aldolase activity that does not require optical selectivity means indole pyruvic acid and pyruvic acid ( Or oxaloacetic acid) means activity to synthesize IHOG, regardless of optical selectivity. This is because the aldolase of the present invention is useful regardless of the degree of optical selectivity.

Next, the enzyme chemical properties examined for the purified SpALD and BuALD are described below.
SpALD and BuALD catalyze the reaction of preferentially producing 4R-IHOG by reacting indolepyruvic acid with pyruvic acid or oxaloacetic acid.

  The optimum pH of SpALD is around 7.1-8.0 at 37 ° C. Moreover, it has pH stability at pH 8 or less. Moreover, it has temperature stability at 50 degrees C or less, and has high temperature stability especially in the range of 30 degrees C or less.

When the molecular weight of SpALD was measured by gel filtration, it was about 155 kDa, and when measured by SDS-PAGE, it was about 30 kDa. Therefore, it was presumed that SpALD had a subunit hexamer structure with a molecular weight of about 30 kDa. The
Thus, another aspect of the protein of the invention is:
(A) an activity of catalyzing a reaction in which indole-3-pyruvic acid and pyruvic acid are subjected to aldol condensation to form 4- (indol-3-ylmethyl) -4-hydroxy-2-oxoglutaric acid, and / or phenyl Having an activity of catalyzing a reaction in which pyruvic acid and pyruvic acid are subjected to aldol condensation to form 4-phenylmethyl-4-hydroxy-2-oxoglutarate,
(B) The optimum pH of the activity described in (A) is about 7.5-8.0 at 37 ° C,
(C) pH stability at pH 8 or lower,
(D) having temperature stability at 50 ° C. or lower, and
(E) A protein characterized in that the molecular weight measured by gel filtration is about 155 kDa, and the molecular weight per subunit measured by SDS-PAGE is about 30 kDa.

  The optimum pH of BuALD is around 6.5-7.5 at 37 ° C. Moreover, it has pH stability at pH 7.5 or less. Moreover, it has temperature stability at 37 degrees C or less, and has high temperature stability especially in the range of 30 degrees C or less.

The molecular weight of BuALD was about 160 kDa as measured by gel filtration, and about 30 kDa as measured by SDS-PAGE, so it was assumed that BuALD had a subunit hexamer structure with a molecular weight of about 30 kDa. The
Accordingly, another aspect of the protein of the present invention is:
(A) an activity of catalyzing a reaction in which indole-3-pyruvic acid and pyruvic acid are subjected to aldol condensation to form 4- (indol-3-ylmethyl) -4-hydroxy-2-oxoglutaric acid, and / or phenyl Having an activity of catalyzing a reaction in which pyruvic acid and pyruvic acid are subjected to aldol condensation to form 4-phenylmethyl-4-hydroxy-2-oxoglutarate,
(B) the optimum pH of the activity described in (A) is about 6.5-7.5 at 37 ° C;
(C) having pH stability at pH 8.5 or lower,
(D) having temperature stability at 37 ° C. or lower, and
(E) A protein having a molecular weight of about 160 kDa measured by gel filtration and a molecular weight of about 30 kDa measured by SDS-PAGE.

  Here, the term “about” attached to a numerical value indicates that a deviation range of 20% of the numerical value can be included, and the allowable deviation is more preferably 10%, further preferably 5%, Preferably it shows 2.5%.

(Iii) DNA encoding aldolase
As described in the above (i) and (ii), the present inventors have identified the sphingomonas sp., Which is an aldolase-producing bacterium isolated by the present inventors. From the AJ110329 strain (C77 strain), the aldolase gene of the present invention having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing, Sphingomonas sp. From the AJ110372 strain (C43 strain), the aldolase gene of the present invention having the nucleotide sequence of SEQ ID NO: 12, and Burkholderia sp. The aldolase gene of the present invention having the nucleotide sequence of SEQ ID NO: 14 was obtained from AJ110371 strain (C24 strain). These genes encode aldolase that catalyzes the reaction of synthesizing IHOG from indolepyruvic acid and pyruvic acid (or oxaloacetic acid) of the present invention, and are included in the present invention.

  The DNA encoding aldolase is not limited to the DNAs shown in SEQ ID NOs: 1, 12, and 14 in the sequence listing. That is, among the species of Sphingomonas and Burkholderia that generate aldolase that catalyzes the reaction of synthesizing IHOG from indolepyruvic acid and pyruvic acid (or oxaloacetic acid), a difference in base sequence should be observed for each species and strain. It is.

  That is, a protein having a homology of 70% or more, preferably 74% or more, more preferably 80% or more, particularly preferably 85% or more, very preferably 95% or more with the amino acid sequence described in SEQ ID NO: 2, A protein having aldolase activity, preferably 4R-aldolase activity, or a protein having 70% or more homology with the amino acid sequence described in SEQ ID NO: 2 and having the aldolase core sequence described in SEQ ID NO: 23 Any DNA encoding a protein having aldolase activity, preferably 4R-aldolase activity, is included in the DNA of the present invention.

  Further, the DNA of the present invention is not limited to DNA encoding isolated aldolase, but of course, DNA obtained by artificially mutating DNA encoding aldolase isolated from chromosomal DNA of aldolase-producing bacteria. However, when encoding an aldolase, it is the DNA of the present invention. As a method frequently used for artificially adding mutation, Method. in Enzymol. 154 (1987), a site-directed mutagenesis method.

  Further, a protein that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1, 12, or 14 in the sequence listing, and has aldolase activity, preferably 4R-aldolase activity The DNA encoding is also the DNA of the present invention. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to clearly quantify this condition, for example, highly homologous DNAs, for example, 50% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95%. % Of DNA that has a homology of at least%, and DNA that has a lower homology than that does not hybridize, or 37 ° C., 0.1 × SSC, 0 The conditions for hybridization at a salt concentration corresponding to 1% SDS, preferably 60 ° C., 0.1 × SSC, 0.1% SDS, more preferably 65 ° C., 0.1 × SSC, 0.1% SDS can give. Here, “aldolase activity” or “4R-aldolase activity” has the same meaning as described in the section (ii) Aldolase. However, in the case of a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence described in SEQ ID NO: 1, 12 or 14 in the sequence listing, under conditions of 33 ° C. and pH 9 Aldolase activity of 10% or more, preferably 30% or more, more preferably 50% or more, more preferably 70% or more, preferably 10% or more of the protein having the amino acid sequence shown in any one of SEQ ID NO: 2, 13 or 15 in the sequence listing Each preferably retains 4R-aldolase activity.

Furthermore, a DNA encoding a protein substantially the same as the aldolase encoded by the DNA described in any one of SEQ ID NOs: 1, 12, or 14 in the sequence listing is also the DNA of the present invention. That is,
(A) DNA consisting of base sequences 210 to 1004 of the base sequence described in SEQ ID NO: 1
(B) DNA comprising the nucleotide sequence of nucleotide numbers 399 to 1253 among the nucleotide sequences described in SEQ ID NO: 12
(C) DNA consisting of the nucleotide sequences of base numbers 531 to 1385 among the base sequences described in SEQ ID NO: 14
(D) the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of nucleotide numbers 210 to 1004 in the same sequence, the nucleotide sequence of SEQ ID NO: 12 or the nucleotide sequence of nucleotide numbers 399 to 1253 of the same sequence, or the nucleotide sequence of SEQ ID NO: 14 A DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence of base numbers 531 to 1385 in the same sequence or that encodes a protein having aldolase activity
(E) DNA encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 2, 13, or 15
(F) having an amino acid sequence including substitution, deletion, insertion, addition, or inversion of one or several amino acid residues in the amino acid sequence of any one of SEQ ID NOs: 2, 13, or 15; DNA encoding a protein having aldolase activity
(G) a DNA comprising an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 2, 13, or 15 and encoding a protein having aldolase activity
Is also included in the DNA of the present invention. Here, the meaning of “one or several” is the same as that described in the section of (ii) aldolase.

(3) Method for Producing Aldolase Next, the method for producing the aldolase of the present invention will be described. The method for producing the aldolase of the present invention includes (i) a method for producing and accumulating aldolase by culturing an aldolase-producing bacterium, and (ii) producing a transformant that produces aldolase by recombinant DNA technology. There are two methods for producing and accumulating aldolase by culturing the transformant. Regarding (i), the method for obtaining an aldolase-producing bacterium and the method for culturing the aldolase-producing bacterium as described in the above section (2) (i). Hereinafter, (ii) will be described.

(Ii) Production method using recombinant DNA technology There are many known examples of producing useful proteins such as enzymes and physiologically active substances using recombinant DNA technology. Protein can be mass-produced.

FIG. 2 is a flowchart of the production process of the aldolase of the present invention.
First, DNA encoding the aldolase of the present invention is prepared (step S1).
Next, the prepared DNA is connected to the vector DNA to produce a recombinant DNA (step S2), and cells are transformed with the recombinant DNA to produce a transformant (step S3). Subsequently, the transformant is cultured in a medium, and aldolase is produced and accumulated in the medium and / or cells (step S4).
Then, it progresses to step S5 and refine | purified aldolase is manufactured by collect | recovering and refine | purifying this enzyme.
Moreover, the target optically active IHOG can be produced in large quantities by using the purified aldolase produced in step S5 or the medium and / or cells in which the aldolase from step S4 is accumulated in the aldol reaction (step S6).

  The DNA connected to the vector DNA only needs to be capable of expressing the aldolase of the present invention.

  Here, as the aldolase gene connected to the vector DNA, any of the DNAs described in the above section (2) Aldolase (iii) DNA can be used.

  When a protein is mass-produced using recombinant DNA technology, it is preferable that the protein associates in a transformant producing the protein to form an inclusion body of the protein. Advantages of this expression production method include that the target protein is protected from digestion by proteases present in the microbial cells, and that the target protein can be easily purified by a centrifugation operation following cell disruption.

  The protein inclusion body obtained in this way is solubilized by a protein denaturing agent, and after being subjected to an activity regeneration operation mainly by removing the denaturing agent, it is converted into a correctly folded physiologically active protein. . For example, there are many examples such as activity regeneration of human interleukin-2 (Japanese Patent Laid-Open No. 61-257931).

  In order to obtain an active protein from a protein inclusion body, a series of operations such as solubilization and activity regeneration are required, and the operation becomes more complicated than when directly producing an active protein. However, when a large amount of a protein that affects the growth of the bacterial cell is produced in the bacterial cell, the effect can be suppressed by accumulating it in the bacterial cell as an inactive protein inclusion body.

  As a method for mass production of the target protein as inclusion bodies, in addition to a method of expressing the target protein alone under the control of a strong promoter, a method of expressing it as a fusion protein with a protein known to be expressed in large quantities is there.

  Furthermore, it is also effective to place a restriction protease recognition sequence at an appropriate position in order to cut out the target protein after expression as a fusion protein.

  When a protein is mass-produced using recombinant DNA technology, bacterial cells, actinomycetes cells, yeast cells, mold cells, plant cells, animal cells, etc. can be used as host cells to be transformed. Bacterial cells for which host-vector systems have been developed include Escherichia bacteria, Pseudomonas bacteria, Corynebacterium bacteria, Bacillus bacteria, etc., preferably Escherichia coli is used. This is because there is a lot of knowledge about the technology for mass production of proteins using Escherichia coli. Hereinafter, a method for producing aldolase using transformed E. coli will be described.

  As a promoter for expressing DNA encoding aldolase, a promoter usually used for heterologous protein production in Escherichia coli can be used. For example, a strong promoter such as T7 promoter, trp promoter, lac promoter, tac promoter, PL promoter, etc. Is mentioned.

  In order to produce aldolase as a fusion protein inclusion body, a gene encoding another protein, preferably a hydrophilic peptide, is linked upstream or downstream of the aldolase gene to form a fusion protein gene. Any gene encoding such other proteins may be used as long as it increases the accumulation amount of the fusion protein and enhances the solubility of the fusion protein after the denaturation / regeneration process. For example, T7gene10, β-galactosidase gene, dehydro Candidates include folate reductase gene, interferon γ gene, interleukin-2 gene, prochymosin gene and the like.

When these genes and the gene encoding aldolase are linked, the codon reading frames are made to coincide. Ligation may be performed at an appropriate restriction enzyme site, or synthetic DNA having an appropriate sequence may be used.
In order to increase the production amount, it is preferable to link a terminator, which is a transcription termination sequence, downstream of the fusion protein gene. Examples of the terminator include T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, E. coli trpA gene terminator, and the like.

  As a vector for introducing a gene encoding an aldolase or a fusion protein of an aldolase and another protein into Escherichia coli, a so-called multi-copy type is preferable, and a plasmid having a replication origin derived from Col E1, such as pUC type Examples include plasmids, pBR322-based plasmids, and derivatives thereof. Here, the “derivative” means one obtained by modifying a plasmid by base substitution, deletion, insertion, addition or inversion. In addition, the modification here includes modification by mutation treatment with a mutation agent, UV irradiation, or natural mutation.

  In order to select transformants, the vector preferably has a marker such as an ampicillin resistance gene. As such a plasmid, expression vectors having a strong promoter are commercially available (pUC system (Takara Shuzo Co., Ltd.), pPROK system (Clontech), pKK233-2 (Clontech), etc.).

  Recombinant DNA is obtained by ligating a promoter, aldolase or a gene encoding a fusion protein of aldolase and another protein, a DNA fragment ligated in the order of terminator, and vector DNA.

  When E. coli is transformed with the recombinant DNA and the E. coli is cultured, an aldolase or a fusion protein of aldolase and another protein is expressed and produced. As a host to be transformed, a strain usually used for expression of a heterologous gene can be used, and Escherichia coli JM109 (DE3) strain and JM109 strain are particularly preferable. Methods for performing transformation and methods for selecting transformants are described in Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989) and the like.

  When expressed as a fusion protein, the aldolase may be excised using a restriction protease such as blood coagulation factor Xa, kallikrein, etc., which has a sequence not present in the aldolase as a recognition sequence.

  As the production medium, a medium usually used for culturing Escherichia coli such as M9-casamino acid medium and LB medium may be used. The culture conditions and production induction conditions are appropriately selected according to the type of the marker, promoter, host fungus and the like used.

  In order to recover aldolase or a fusion protein of aldolase and another protein, there are the following methods. If aldolase or a fusion protein thereof is solubilized in the microbial cells, the microbial cells can be recovered and then disrupted or lysed to be used as a crude enzyme solution. Furthermore, if necessary, aldolase or a fusion protein thereof can be purified and used by a conventional method such as precipitation, filtration, column chromatography and the like. In this case, a purification method using an antibody of aldolase or fusion protein can also be used.

  When a protein inclusion body is formed, it is solubilized with a denaturing agent. Although it may be solubilized together with the bacterial protein, it is preferable to take out the inclusion body and solubilize it in consideration of the subsequent purification operation. In order to recover the inclusion body from the microbial cell, a conventionally known method may be used. For example, the microbial cells are destroyed, and the inclusion bodies are collected by centrifugation operation or the like. Examples of denaturing agents that solubilize protein inclusion bodies include guanidine hydrochloride (eg, 6M, pH 5-8), urea (eg, 8M), and the like.

  When these denaturing agents are removed by dialysis or the like, they are regenerated as active proteins. As a dialysis solution used for dialysis, a Tris-HCl buffer or a phosphate buffer may be used, and the concentration may be 20 mM to 0.5 M, and the pH may be 5 to 8.

  The protein concentration during the regeneration step is preferably suppressed to about 500 μg / ml or less. In order to suppress the regenerated aldolase from self-crosslinking, the dialysis temperature is preferably 5 ° C. or lower. Further, as a method for removing the denaturant, there are a dialysis method, a dilution method, an ultrafiltration method, and the like, and regeneration of activity can be expected by using any of them.

  When the DNA shown in SEQ ID NO: 1, 12 or 14 is used as the DNA encoding aldolase, an aldolase having the amino acid sequence shown in SEQ ID NO: 2, 13 or 15 is produced.

[II] Method for Producing Optically Active Monatin The method for producing the optically active monatin of the present invention is as follows. [I] Optically active IHOG is produced by the method described in the method for producing optically active IHOG, and then converted to monatin It is a method to do. Since IHOG produced according to the method of the present invention is predominantly produced by 4R-IHOG, the 4H-isomer optically active monatin, ie, (2R, 4R) -monatin and ( 2S, 4R) -monatin is produced predominantly (hereinafter, (2R, 4R) -monatin and (2S, 4R) -monatin are collectively referred to as 4R-monatin).

  In order to efficiently produce (2R, 4R) -monatin, which is an isomer having the highest sweetness among the four isomers of monatin, it is preferable to use 4HO-rich IHOG. In this case, the ratio of 4R-IHOG to the total amount of IHOG is preferably more than 55%, more preferably more than 60%, still more preferably more than 70%, and particularly preferably more than 80%.

  The method for converting IHOG to monatin is not particularly limited, and a known method such as a chemical reaction method or an enzymatic method can be used.

(1) Chemical reaction method As a method for producing optically active monatin from optically active IHOG by a chemical reaction method, optically active IHOG is oximed and the corresponding IHOG-oxime or its salt described in the following formula (4) is chemically treated. A method for producing optically active monatin by reduction can be mentioned.

  Preferably, 4R-IHOG-oxime or a salt thereof is isolated by crystallization from a solution containing the 4R-isomer-rich IHOG and crystallizing from the solution containing the 4R-isomer-rich IHOG-oxime, and the 4R-IHOG-oxime or a salt thereof is isolated. Chemical reduction to produce 4R-monatin.

（上記一般式（３）において、Ｒは水素原子、アルキル基、アリール基又はアラルキル基を表す。）
に表されるアミン化合物又はその塩とを反応せしめることによって行う。ここに、Ｒがアルキル基、アリール基又はアラルキル基である場合、Ｒは炭素原子を１〜３有するアルキル基、又は、側鎖に置換基を有していてもよいアリール基又はアラルキル基が好ましく、結晶化の観点からより好ましくは、Ｒはメチル基、エチル基、ベンジル基から選択される。 Oxidation of IHOG is carried out under the neutral or alkaline conditions with IHOG and the following general formula (3)

(In the general formula (3), R represents a hydrogen atom, an alkyl group, an aryl group, or an aralkyl group.)
The reaction is carried out by reacting with the amine compound represented by Here, when R is an alkyl group, an aryl group or an aralkyl group, R is preferably an alkyl group having 1 to 3 carbon atoms, or an aryl group or aralkyl group which may have a substituent in the side chain. From the viewpoint of crystallization, R is more preferably selected from a methyl group, an ethyl group, and a benzyl group.

  This oximation reaction can be performed by directly adding the amine of the above general formula (3) to an aldolase reaction solution containing IHOG. The 4R isomer can be isolated by crystallizing 4R-IHOG-oxime or a salt thereof from the solution containing the 4R isomer-rich IHOG-oxime. As the crystallization solvent, it is preferable to use water, an alcohol solvent or a hydrous alcohol solvent.

  4R-monatin can be obtained by reducing 4R-IHOG-oxime or a salt thereof obtained by crystallization. The reduction of 4R-IHOG-oxime or a salt thereof is carried out in the presence of hydrogen and a hydrogenation catalyst. As the hydrogenation catalyst, it is preferable to use a metal-supported catalyst in which a metal catalyst such as platinum, rhodium, palladium, nickel, or cobalt is supported on a carrier such as silica, alumina, titania, magnesia, zirconia, or activated carbon.

  Conventionally, since optically active IHOG could not be efficiently produced, 4R-IHOG was isolated from racemic IHOG (4R, 4S-IHOG) after oximation of 4R, 4S-IHOG. It was necessary to cause 4R-IHOG-oxime to crystallize by acting a chiral amine. In contrast, according to the present invention, 4R-rich IHOG can be generated at the stage of the aldol condensation reaction, so that it is not necessary to perform optical resolution using a chiral amine during crystallization, and the 4R-rich IHOG is rich. After oximation, the 4R-IHOG-oxime can be crystallized as it is. Therefore, it is possible to reduce the cost required for the purification process of 4R-IHOG.

  The 4R-monatin obtained by the chemical reduction method becomes a racemic mixture of (2R, 4R) -monatin and (2S, 4R) -monatin. When (2R, 4R) -monatin is isolated, (2R, 4R) -monatin may be precipitated by crystallization. Specifically, the method described in International Publication No. 03/059865 pamphlet can be used.

(2) Enzymatic method When 4R-monatin is produced from 4R-IHOG by the enzymatic method, 4R-IHOG may be aminated in the presence of an enzyme that catalyzes the reaction of aminating the 2-position of 4R-IHOG. Examples of the enzyme that catalyzes the reaction include an aminotransferase that catalyzes a transamination reaction to 4R-IHOG, and a dehydrogenase that catalyzes a reductive amination reaction of 4R-IHOG. More preferably, aminotransferase is used.

  When an aminotransferase is used, 4R-monatin can be produced by reacting 4R-IHOG in the presence of an aminotransferase and an amino group donor. Specifically, the method described in International Publication No. 03/056026 pamphlet can be used.

In this case, both L-aminotransferase and D-aminotransferase can be used as the aminotransferase. When L-aminotransferase is used, 2S-monatin can be selectively produced by transferring the amino group of the L-amino acid to the 2-position of IHOG. When D-aminotransferase is used, 2R-monatin can be selectively produced by transferring the amino group of the D-amino acid to the 2-position of IHOG. Therefore, in order to selectively produce (2R, 4R) -monatin having a high sweetness level, it is preferable to react 4R-IHOG using D-aminotransferase.
The reaction for producing monatin using aminotransferase may be performed after isolating and purifying 4R-IHOG produced after the above-mentioned aldol condensation, or in the same system by coexisting aldolase and aminotransferase. The reaction may be carried out at When the reaction is carried out in the same system, a microorganism co-expressing the DNA encoding the aldolase and the DNA encoding the aminotransferase may be used, or a separate enzyme may be prepared and added to the reaction system. . A microorganism (host cell) co-expressing an aldolase-encoding DNA and an aminotransferase-encoding DNA comprises an expression vector functionally containing the aldolase-encoding DNA as described above and an aminotransferase-encoding DNA. Co-transduction with functionally containing expression vectors, or expression vectors containing aldolase-encoding DNA and aminotransferase-encoding DNA in a form that can be expressed in a host cell with activity. It can be prepared by transformation or the like.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to only these examples. In the Examples, IHOG, PHOG and (2R, 4R) -monatin used as substrates were synthesized by the methods described in Reference Examples 1, 2, and 3.

In this example, IHOG and PHOG were quantified by HPLC analysis using “Inertsil ODS-2” (5 μm, 4.6 × 250 mm) manufactured by GL Sciences. The analysis conditions are as shown below.
Mobile phase: 40% (v / v) acetonitrile / 5 mM tetrabutylammonium dihydrogen phosphate solution Flow rate: 1 ml / min
Column temperature: 40 ° C
Detection: UV210nm
In addition, the 4th-position asymmetric analysis of the generated IHOG or PHOG is performed by “Inertsil ODS-3” (5 μm, 4.6 × 160 mm) manufactured by GL Sciences and “SUMICIRAL OA-7100” (5 μm) manufactured by Sumika Chemical Analysis Center. , 4.6 × 250 mm) was analyzed by HPLC analysis directly connected in order. The analysis conditions are as shown below.
Mobile phase A: 5% (v / v) acetonitrile 20 mM potassium phosphate buffer (pH 6.8)
Mobile phase B: 50% (v / v) acetonitrile 20 mM potassium phosphate buffer (pH 6.8)
Elution with mobile phase A from 0 to 90 minutes, elution and washing with mobile phase B from 90 to 120 minutes Flow rate: 0.4 ml / min
Column temperature: 17 ° C
Detection: UV210nm

Example 1 Collection of IHOG aldolase-active bacteria from nature (2R, 4R)-4R-IHOG optically selective aldolase activity by performing enrichment culture using a medium using monatin (hereinafter RR monatin) as a single carbon source The strain was isolated and obtained from nature.

  A test tube containing 1 ml of RR monatin single C source medium (RR-monatin monopotassium salt 0.4 g / l, Yeast Nitrogen base without amino acid (Difco) 0.67 g / l) was inoculated with a soil sample, and 30 ° C. And cultured with shaking for 7 days. 0.1 ml of the obtained culture solution was again inoculated into a test tube containing 1 ml of RR monatin single C source medium, and again cultured with shaking at 30 ° C. for 7 days. The obtained culture solution was appropriately diluted with sterilized physiological saline, and then CM2G plate medium (glucose 5 g / l, yeast extract 10 g / l, peptone 10 g / l, NaCl 5 g / l, agar powder 20 g / l (pH 7.0). )) And cultured at 30 ° C. for 24 hours to isolate colonies.

Aldolase active strains were selected from the strains obtained by accumulation. The test microorganisms were inoculated on a broth flat plate medium (Eiken Chemical) and cultured at 30 ° C. for 24 hours. Enzyme production medium (glycerol 5 g / l, fumaric acid 5 g / l, yeast extract 3 g / l, peptone 2 g / l, ammonium sulfate 3 g / l, K ₂ HPO ₄ 3 g / l, KH ₂ PO ₄ 1 g / l, MgSO _The plate containing 0.5 g / l of 4.7H ₂ O, 2.5 g / l of sodium phthalate, and 20 g / l of agar powder (pH 6.5) was inoculated and cultured at 30 ° C. for 24 hours. 100 mM Tris-HCl (pH 8.0), 50 mM PHOG, 1 mM MgCl ₂ , 5 mM potassium phosphate solution (KPi), so that the obtained bacterial body is about 1% (w / v) by the wet bacterial body weight, A reaction solution composed of 1% (v / v) toluene was inoculated and reacted at 30 ° C. for 24 hours. The concentration of free pyruvate in the reaction solution was quantified by an enzymatic method using lactate dehydrogenae (LDH). 10 μl of a sample was added to 200 μl of a reaction solution consisting of 100 mM Tris-HCl (pH 8.0), 1.5 mM NADH, 5 mM MgCl ₂ , 25 U / ml LDH, and incubated at 30 ° C. for 10 minutes. Absorbance at 340 nm after the reaction was measured, and the amount of pyruvic acid in the sample was quantified from the decreased amount of NADH.

The amount of generated phenylpyruvic acid was quantified by HPLC analysis using “Inertsil ODS-2” (5 μm, 4.6 × 250 mm) manufactured by GL Sciences. The analysis conditions are as shown below.
Mobile phase: 20% (v / v) acetonitrile / 0.05% (v / v) aqueous trifluoroacetic acid Flow rate: 1 ml / min
Column temperature: 40 ° C
Detection: UV 210nm
Under these conditions, PHOG was eluted at about 9.8 minutes and phenylpyruvic acid was eluted at a retention time of about 12 minutes.

  A value obtained by subtracting the production amount of the control group (cell-free group) from the amount of pyruvic acid or phenylpyruvic acid produced from PHOG in the test cell-added group was defined as the production amount by aldolase. As a result, an aldolase active strain using PHOG as a substrate was found.

Next, IHOG synthesis reaction was carried out using the collected aldolase activity strain. The test strains were enzyme production medium (glycerol 5 g / l, fumaric acid 5 g / l, yeast extract 3 g / l, peptone 2 g / l, ammonium sulfate 3 g / l, K ₂ HPO ₄ 3 g / l, KH ₂ PO ₄ 1 g / l. , MgSO ₄ .7H ₂ O 0.5 g / l, sodium phthalate 2.5 g / l (pH 6.5)) was inoculated into a test tube containing 3 ml, and cultured at 30 ° C. for 16 hours. The obtained cells were suspended in the following IHOG synthesis reaction solution and reacted so that the wet cell weight was about 1% (w / v).

IHOG synthesis reaction solution: 100 mM Hepes-KOH (pH 8.5), 300 mM indole pyruvate, 750 mM sodium pyruvate, 1 mM MgCl ₂ , 5 mM potassium phosphate buffer (pH 8.5)

  After incubating the reaction solution at 37 ° C. for 16 hours, the produced IHOG was quantified, and the asymmetry at the 4-position of the reaction product was also analyzed. As a result, 4R-IHOG preferential aldol condensation activity was found as shown in Table 4 in the integrated strain C77.

Example 2 Purification of IHOG aldolase (SpALD) derived from C77 strain (Sphingomonas sp. AJ110329 strain) IHOG aldolase was purified from the soluble fraction of C77 strain (Sphingomonas sp. AJ110329 strain) as follows. In the aldolase activity measurement, the aldol decomposition activity using PHOG as a substrate was measured under the following conditions.
Reaction conditions: 50 mM Hepes-KOH (pH 8.5), 2 mM PHOG, 0.25 mM NADH, 0.2 mM KPi, 1 mM MgCl ₂ , 16 U / ml lactate dehydrogenase, 3 μl enzyme / 600 μl reaction solution, 30 ° C., absorbance at 340 nm Measurement.

(1) Preparation of soluble fraction C77 strain (Sphingomonas sp. AJ110329 strain) cultured in a broth flat plate medium at 30 ° C. for 24 hours was scraped with one platinum ear, and 50 ml of enzyme production medium (5 g / l glycerol, 5 g / l) Fumaric acid, 5 g / l ammonium sulfate, 3 g / l K ₂ HPO ₄ , 1 g / l KH ₂ PO ₄ , 0.5 g / l MgSO ₄ .7H ₂ O, 3 g / l, yeast extract, 2 g / l peptone, 2 Inoculated into a 500 ml flask containing 0.5 g / l sodium phthalate (manufactured by Sigma), adjusted to pH 6.5 with KOH), and cultured with shaking at 30 ° C. for 24 hours. 0.5 ml of the culture solution was inoculated into 20 500 ml flasks containing 50 ml of enzyme production medium and cultured with shaking at 30 ° C. for 24 hours. The cells were collected from the obtained culture broth by centrifugation, suspended in buffer A (20 mM Hepes-KOH (pH 7.6)), washed, and then collected again by centrifugation. The obtained washed cells were suspended in 80 ml of buffer A and sonicated at 4 ° C. for 30 minutes. The disrupted solution was centrifuged (x8000 rpm, 10 minutes × twice) to remove the cell residue, and the resulting supernatant was used as a soluble fraction.

(2) Anion exchange chromatography: Q-Sepharose FF
The soluble fraction (40 ml) was subjected to an anion exchange chromatography column Q-Sepharose FF 26/10 (Pharmacia, CV = 20 ml) equilibrated with buffer A, and adsorbed onto a carrier. After washing away the protein not adsorbed on the carrier (non-adsorbed protein) with buffer A, the KCl concentration was linearly changed from 0 M to 0.7 M (total 140 ml), and the adsorbed protein was eluted. When PHOG aldolase activity was detected for each eluted fraction, a peak of PHOG aldolase activity was detected in a fraction corresponding to about 0.4 M. The same chromatographic operation was repeated twice.

(3) Hydrophobic chromatography: Phenyl Sepharose HP HR 16/10
The solution in which the aldolase activity was detected was dialyzed overnight at 4 ° C. against buffer B (20 mM Hepes-KOH, 1M ammonium sulfate, pH 7.6) and filtered through a 0.45 μm filter. The obtained filtrate was subjected to a hydrophobic chromatography column Phenyl Sepharose HP HR 16/10 (manufactured by Pharmacia) equilibrated with buffer B. By this operation, aldolase was adsorbed on the carrier.
After washing away non-adsorbed protein that was not adsorbed on the carrier with buffer B, the aldolase was eluted by linearly changing the ammonium sulfate concentration from 1M to 0M. The aldolase activity was measured for each of the obtained elution fractions, and the aldolase activity was observed at the elution position where the ammonium sulfate concentration was approximately 0.4 to 0.5M.

(4) Gel filtration chromatography: Sephadex 200 HP 16/60
Each fraction containing aldolase was collected, dialyzed against buffer A, and filtered through a 0.45 μm filter. The obtained filtrate was concentrated using an ultrafiltration membrane centriprep 10. The obtained concentrated solution was subjected to gel filtration Sephadex 200 HP 16/60 (Pharmacia) equilibrated with buffer C (20 mM Hepes-KOH, 0.1 M KCl, pH 7.6) at a flow rate of 1 ml / min. Eluted. By this operation, aldolase was eluted in a fraction of around 66 ml. From the elution position of the activity peak, the molecular weight of the aldolase was estimated to be about 155 kDa.

(5) Anion exchange chromatography: Mono Q HR 5/5
The obtained fraction was filtered through a 0.45 μm filter. The filtrate obtained here was applied to an anion exchange chromatography column Mono-Q HR 5/5 (Pharmacia) equilibrated with buffer A. By this operation, aldolase was adsorbed on the carrier. After washing away non-adsorbed protein with buffer A, protein elution was performed by linearly changing the KCl concentration from 0 mM to 700 mM (Total 24 ml). The aldolase activity was measured for each elution fraction, and the aldolase activity was observed at the elution position where the KCl concentration was about 0.4M.
When the obtained fraction was subjected to SDS-PAGE, about 10 bands were observed in the active fraction. Among them, there was a band of about 30 kDa in which the activity profile and the SDS-PAGE band intensity profile matched, and the band was cut out from SDS-PAGE as a candidate for aldolase and subjected to amino acid sequence analysis.

Example 3 Determination of internal amino acid sequence of IHOG aldolase After the purified aldolase fraction was subjected to SDS-PAGE, a band corresponding to 30 kDa was excised, and the sample in the SDS-PAGE gel was trypsinized (pH 8.5, 35). C., 20 hours), and subjected to reverse phase HPLC to separate the fragment peptides. Among the fractions collected, the amino acid sequences of 17 residues and 12 residues (SEQ ID NOs: 3 and 4) of the two fractions were determined as shown in Table 6 below.

Example 4 Cloning of IHOG aldolase gene derived from C77 strain (Sphingomonas sp. AJ110329) (1) Preparation of chromosomal DNA C77 strain (Sphingomonas sp. AJ110329 strain) was cultured overnight at 30 ° C. using 50 ml of CM2G medium ( Pre-culture). Using 5 ml of this culture as an inoculum, main culture was performed using 50 ml of bouillon medium. After culturing until the late stage of logarithmic growth, 50 ml of the culture solution was subjected to centrifugation (12000 × g, 4 ° C., 15 minutes) to collect the cells. Using these cells, chromosomal DNA was prepared according to a conventional method.

(2) Acquisition of internal sequence by PCR Based on the determined internal amino acid sequence of IHOG aldolase, mix primers (SEQ ID NOs: 5 and 6) described in Table 7 were synthesized.

Using the prepared mix primer, amplification by PCR was performed using the chromosomal DNA of the C77 strain (Sphingomonas sp. AJ110329 strain) as a template. The PCR reaction was performed using PCR Thermal PERSONEL (TaKaRa), and 30 cycles were performed under the following conditions.
94 ° C. 30 seconds 55 ° C. 30 seconds 72 ° C. 1 minute When the PCR product was subjected to agarose gel electrophoresis, amplification of a fragment of about 200 bp was observed. When the DNA fragment was cloned into pT7Blue (manufactured by Novagen) and the nucleotide sequence was determined, the amino acid sequence deduced from the obtained DNA fragment was consistent with the internal amino acid sequence of IHOG aldolase, and the target aldolase gene was It was confirmed that it was acquired.

(3) Acquisition of full-length gene by colony hybridization Using a DNA fragment amplified by PCR, a full-length gene was acquired by Southern analysis and colony hybridization. The DNA probe was prepared by using DIGH High Prime (Roche Diagnostics) and labeling the probe by incubating at 37 ° C. according to the instruction manual. Southern analysis was performed by digesting 1 μg of chromosomal DNA completely with various restriction enzymes, electrophoresis on 0.8% agarose gel, blotting on nylon membrane, and following the manual below. Hybridization was performed using DIG Easy Hyb (Roche Diagnostics), prehybridization was performed at 50 ° C. for 1 hour, a probe was added, and hybridization was performed with O / N. Band detection was performed using the DIG Nucleotide Detection Kit. As a result, an about 3.2 kbp PstI / BamHI fragment that strongly hybridizes using the PCR fragment as a probe was detected. Next, this PstI / BamHI fragment was obtained by colony hybridization. After treating 20 μg of chromosomal DNA with PstI / BamHI, it was subjected to agarose gel electrophoresis, and a fragment having a size of about 3.2 kbp was recovered. This is linked to pUC19. A library was prepared using E. coli JM109. The colony was transferred to a nylon membrane filter (Hybond-N, Amersham) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using DIG Easy Hyb. The filter was immersed in a buffer and prehybridization was performed at 42 ° C. for 1 hour. Thereafter, the prepared labeled probe was added, and hybridization was performed at 42 ° C. for 16 hours. After washing with SSC, colonies that hybridized with the probe were detected using DIG Nucleotide Detection Kit (Roche Diagnostics). As a result, a clone that strongly hybridized with the probe was obtained.

  When the base sequence of plasmid DNA recovered from the obtained clone was determined, it was revealed that it had the base sequence described in SEQ ID NO: 1. An 855 bp orf containing the nucleotide sequence corresponding to the determined internal amino acid sequence (519th to 569th and 666 to 701th in SEQ ID NO: 1) was found, and the full length of the target aldolase was obtained.

Example 5 Mass expression of IHOG aldolase (SpALD) in E. coli (1) Construction of trp promoter and rrnB terminator loaded plasmid pTrp4 The target gene region was amplified by PCR (combination of SEQ ID NO: 7 and SEQ ID NO: 8) using the trp operon promoter region on E. coli W3110 chromosomal DNA as a primer in Table 8, and the resulting DNA fragment was pGEM-Teasy Ligated to a vector (Promega). With this ligation solution E. coli JM109 was transformed, and a strain having the desired plasmid in which the direction of the trp promoter was inserted in the opposite direction to the lac promoter was selected from ampicillin resistant strains. Next, this plasmid was ligated with a DNA fragment containing the trp promoter obtained by treating with EcoO109i / EcoRI and an EcoO109i / EcoRI-treated product of pUC19 (manufactured by Takara). With this ligation solution E. coli JM109 was transformed, a strain having the target plasmid was selected from ampicillin resistant strains, and the plasmid was named pTrp1. Next, pKK223-3 (manufactured by Amersham Pharmacia) was treated with HindIII / HincII, and ligated with the obtained DNA fragment containing the rrnB terminator and the HindIII / PvuII-treated product of pTrp1. With this ligation solution E. coli JM109 was transformed, a strain having the target plasmid was selected from ampicillin resistant strains, and the plasmid was named pTrp2. Next, the trp promoter region was amplified by PCR (combination of SEQ ID NO: 7 and SEQ ID NO: 9) using pTrp2 as a template and oligonucleotides shown in Table 8 as primers. This DNA fragment was treated with EcoO109i / NdeI and ligated with the pTrp2 treated product of EcoO109i / NdeI. With this ligation solution E. coli JM109 was transformed, a strain having the desired plasmid was selected from ampicillin resistant strains, and this plasmid was named pTrp4.

(2) Construction of an aldolase gene expression plasmid ptrpSpALD and E. coli. Expression in E. coli Using the primers shown in Table 9 (SEQ ID NOs: 10 and 11), a fragment amplified from the chromosomal DNA of C77 strain (Sphingomonas sp. AJ110329) was digested with NdeI / PstI and inserted into the NdeI / PstI site of pTrp4 The constructed plasmid ptrpSpALD was constructed. This plasmid expresses an aldolase gene consisting of the amino acid sequence described in SEQ ID NO: 2, which is obtained by translating the 210th ATG of the nucleotide sequence described in SEQ ID NO: 1 to the 1004th as a translation initiation codon.

  The constructed expression plasmid was transformed into E. coli. After introduction into E. coli JM109, one platinum loop was inoculated into 50 ml of LB medium containing 100 μg / ml ampicillin and shaken at 37 ° C. for 16 hours. After completion of the culture, 1 ml of the obtained culture solution was collected, washed, suspended in 1 ml of 20 mM Hepes-KOH (pH 7.6), and the cells were disrupted by ultrasonic disruption. The supernatant obtained by centrifuging the disrupted solution at 15000 rpm for 10 minutes was used as a crude enzyme solution.

Aldolase activity was measured using the crude enzyme solution. In the aldolase activity measurement, the aldol decomposition activity using PHOG as a substrate was measured under the following conditions.
Reaction conditions: 50 mM Hepes-KOH (pH 8.5), 2 mM PHOG, 0.25 mM NADH, 0.2 mM KPi, 1 mM MgCl ₂ , 16 U / ml lactate dehydrogenase, 3 μl enzyme / 600 μl reaction solution, 30 ° C., absorbance at 340 nm Measurement As a result of the measurement, E. coli introduced pTrp4. No aldolase activity was detected in E. coli (control), whereas an aldolase activity of 33.6 U / mg protein was detected in the ptrpSpALD-introduced strain. Thereby, it was confirmed that the target aldolase was certainly cloned together with the construction of the SpALD high expression plasmid.

Example 6 Purification of Aldolase Recombinant Enzyme Derived from C77 Strain (Sphingomonas sp. AJ110329) Aldolase (SpALD) derived from C77 strain (Sphingomonas sp. AJ110329) was highly expressed. Purification of recombinant SpALD from the soluble fraction of E. coli was performed as follows. In the aldolase activity measurement, the aldol decomposition activity using PHOG as a substrate was measured under the following conditions.
Reaction conditions: 50 mM Hepes-KOH (pH 8.5), 2 mM PHOG, 0.25 mM NAD, 1 mM MgCl ₂ , 16 U / ml lactate dehydrogenase, 3 μl enzyme / 600 μl reaction solution, 30 ° C., absorbance at 340 nm is measured

(1) Preparation of soluble fraction:
E. coli cultured in LB-amp plate medium at 37 ° C. for 16 hours. E. coli JM109 / ptrpSpALD cells were scraped off from one platinum ear, inoculated into 10 500 ml flasks containing 50 ml of LB-amp medium, and cultured with shaking at 37 ° C. for 16 hours. Bacteria were collected from the obtained culture broth by centrifugation, suspended in buffer A (20 mM Hepes-KOH, pH 7.6), washed, and collected again by centrifugation. The obtained washed cells were suspended in 20 ml of buffer A and sonicated at 4 ° C. for 30 minutes. The disrupted liquid was centrifuged (x8000 rpm, 10 minutes × twice) to remove cell residue, and the resulting supernatant was used as a crude extract fraction.

(2) Anion exchange chromatography: Q-Sepharose FF
The soluble fraction (20 ml) was subjected to an anion exchange chromatography column Q-Sepharose FF 26/10 (Pharmacia, CV = 20 ml) equilibrated with buffer A, and adsorbed onto a carrier. After washing away the protein not adsorbed on the carrier (non-adsorbed protein) with buffer A, the KCl concentration was linearly changed from 0 M to 0.7 M (total 140 ml), and the adsorbed protein was eluted. When PHOG aldolase activity was detected for each eluted fraction, a peak of PHOG aldolase activity was detected in a fraction corresponding to about 0.4 M.

(3) Hydrophobic chromatography: Phenyl Sepharose HP HR 16/10
The solution in which the aldolase activity was detected was dialyzed overnight at 4 ° C. against buffer B (20 mM Hepes-KOH, 1M ammonium sulfate, pH 7.6) and filtered through a 0.45 μm filter. The obtained filtrate was subjected to a hydrophobic chromatography column Phenyl Sepharose HP HR 16/10 (manufactured by Pharmacia) equilibrated with buffer B. By this operation, aldolase was adsorbed on the carrier.
After washing away non-adsorbed protein that was not adsorbed on the carrier with buffer B, the aldolase was eluted by linearly changing the ammonium sulfate concentration from 1M to 0M. The aldolase activity was measured for each of the obtained elution fractions, and the aldolase activity was observed at the elution position where the ammonium sulfate concentration was approximately 0.4 to 0.5M.

(4) Gel filtration chromatography: Sephadex 200 HP 16/60
Each fraction containing aldolase was collected, dialyzed against buffer A, and filtered through a 0.45 μm filter. The obtained filtrate was concentrated using an ultrafiltration membrane centriprep 10. The obtained concentrated solution is subjected to gel filtration Sephadex 200 HP 16/60 (Pharmacia) equilibrated with buffer C (20 mM Hepes-KOH, 0.1 M KCl, pH 7.6), and a flow rate of 1 ml / min. Eluted with. By this operation, aldolase was eluted in a fraction of around 66 ml.

(5) Anion exchange chromatography: Mono Q HR 5/5
The obtained fraction was filtered through a 0.45 μm filter. The filtrate obtained here was applied to an anion exchange chromatography column Mono-Q HR 5/5 (manufactured by Pharmacia) equilibrated with buffer A. By this operation, aldolase was adsorbed on the carrier. After washing away non-adsorbed protein with buffer A, protein elution was performed by linearly changing the KCl concentration from 0 mM to 700 mM (Total 24 ml). The aldolase activity was measured for each elution fraction, and the aldolase activity was observed at the elution position where the KCl concentration was about 0.4M.

  When the fraction purified by the above column chromatographic operation was subjected to SDS-PAGE, it was detected as a single band by CBB staining at a position corresponding to about 30 kDa. The obtained recombinant SpALD solution was dialyzed against buffer A at 4 ° C. overnight. By the above operation, 1.5 ml of 452 U / ml SpALD solution was obtained.

Example 7 Synthesis of 4- (indol-3-ylmethyl) -4-hydroxy-2-oxoglutarate (IHOG) from indole-3-pyruvic acid and pyruvic acid using SpALD SpALD produced in Example 6 was prepared. Using as an enzyme source, synthesis of 4- (indol-3-ylmethyl) -4-hydroxy-2-oxoglutarate (IHOG) from indole-3-pyruvic acid and pyruvic acid was performed. SpALD was added to a reaction solution consisting of 100 mM Hepes-KOH (pH 8.5), 300 mM indole-3-pyruvic acid, 750 mM pyruvic acid, and 1 mM MgCl ₂ to a concentration of 7.9 mg / ml, and reacted at 37 ° C. for 3 minutes. I let you. The enzyme reaction solution was appropriately diluted and subjected to HPLC analysis, and the generated IHOG was quantified. As a result, IHOG production by the aldolase was confirmed. The initial rate of IHOG synthesis reaction under these conditions was estimated to be 451 U / mg.

Example 8 Various enzymatic properties of SpALD Using SpALD prepared in Example 6, the following items were examined.
Basic measurement conditions: 50 mM Hepes-KOH (pH 8.5), 2 mM PHOG, 5 mM MgCl ₂ , 16 U / ml lactate dehydrogenase, 340 nm absorbance decrease measured at 30 ° C.

(1) Kinetic constant using PHOG as a substrate From the results shown in FIG. 3, Vmax (for PHOG) = 979 μmol / min / mg, Km (for PHOG) = 0.60 mM, Km (for MgCl ₂ ) = 0.034 mM Each was decided. Further, no increase in aldolase activity due to the addition of KPB was observed.

(2) pH stability The pH stability in the range of pH 3-10 was measured. The buffer solution used for the measurement is as follows. Sodium citrate buffer (pH 3, 4, 4.5), sodium acetate buffer (pH 4, 4.5, 5, 5.5, 6, 6.5), potassium phosphate buffer (pH 6, 6.5, 7, 7.5, 8, 8.5), Hepes-KOH buffer (pH 7.5, 8, 8.5, 9, 9.5), CAPS-NAOH buffer (pH 9, 9.5, 10). Residual activity of SpALD incubated in a buffer solution of 100 mM at 37 ° C. for 30 minutes was measured under basic measurement conditions. The results are shown in FIG.

(3) Temperature stability After incubation of SpALD for 30 minutes in 100 mM potassium phosphate buffer (pH 7.0) and 100 mM Hepes-KOH buffer (pH 8.5) at 25 ° C. to 70 ° C., the residual activity was measured under basic measurement conditions. Measured with The results are shown in FIG.
(4) Optimum pH
The PHOG decomposition activity at 30 ° C. was measured by a colorimetric method (FIG. 6). As a result, it was revealed that the optimum pH for the PHOG aldol decomposition reaction was around pH 7.5.
Further, IHOG synthesis activity using 300 mM IPA and 750 mM pyruvate as substrates was measured at each pH (reaction conditions: 100 mM Hepes-KOH (pH 8.5), 300 mM IPA, 750 mM PA, 1 mM MgCl ₂ , 37 ° C., 16 h). ). FIG. 7 shows a value obtained by subtracting the IHOG production amount in the enzyme-free group from the IHOG production amount in the enzyme-added group. As a result, it was revealed that the optimum pH for the IHOG aldol condensation by SpALD is around pH 8.0.

Example 9 IHOG synthesis by SpALD Cultured at 37 ° C. for 16 hours in LB-amp plate medium. E. coli JM109 / ptrpSpALD cells were scraped from one platinum loop, inoculated into 12 500 ml flasks containing 50 ml of LB-amp medium, and cultured with shaking at 37 ° C. for 16 hours. Bacteria were collected from the obtained culture broth by centrifugation, suspended in buffer A (20 mM Hepes-KOH, pH 7.6), washed, and collected again by centrifugation. Bacterial cells prepared by centrifugation (wet cell weight of about 3 g) were suspended in 300 ml of a reaction solution having the following composition.
IHOG synthesis reaction solution: 50 mM Hepes-KOH (pH 8.5), 300 mM indole pyruvate, 750 mM sodium pyruvate, 1 mM MgCl ₂ , 5 mM potassium phosphate buffer (pH 8.5), (pH adjusted to 8.5 with 6N KOH) Adjustment)
Argon gas was bubbled through the reaction solution in which the cells were suspended, and the reaction was carried out in an argon gas atmosphere thereafter. The reaction was carried out for 20 hours while stirring at 37 ° C. After completion of the reaction, the cells were sterilized by centrifugation to obtain 300 ml of an aldol reaction solution.

Example 10 Oxidation of Aldol Reaction Solution and Isolation of 4R-IHOG-Oxime Hydroxylamine hydrochloride was added to the aldol reaction solution obtained in Example 9 while maintaining the pH value at 9 with 8N aqueous sodium hydroxide solution. 8 g (270 mmol) was added, and the mixture was stirred at 25 ° C. for 3 hours and 10 ° C. overnight. The amount of IHOG-oxime in the obtained reaction solution was quantified by HPLC analysis. As a result, 25 mmol of 4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid (IHOG-oxime) was produced in the reaction solution. When the 4th-position asymmetry was analyzed, 4R-IHOG-oxime produced 21.4 mmol, 4S-IHOG-oxime produced 3.6 mmol, and optical purity 71.3% e.e. e. Thus, it was confirmed that the 4R body was preferentially generated.

  The pH value of the reaction solution obtained using concentrated hydrochloric acid was adjusted to 2, and the organic matter was extracted with ethyl acetate. The organic layer was concentrated to give a residue. 12 ml of 28% aqueous ammonia and 25 ml of water are added to the residue, 138 ml of 2-propanol is added for crystallization, and the diammonium salt of 4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid 9.76 g (wet weight) was obtained as crystals. After dissolving the obtained crystals in 60 ml of water, 50 ml of 2-propanol was added at 50 ° C., and 150 ml of 2-propanol was further added dropwise at 50 ° C. over 3 hours. The obtained crystals were filtered and dried under reduced pressure to obtain 5.38 g (15.8 mmol) of IHOG-oxime · 2 ammonium salt. When the asymmetry at the 4-position of the obtained crystal was analyzed, the optical purity as a 4R form was 99.0% e.e. e. The high purity 4R-IHOG-oxime ammonium salt was isolated and obtained by crystallization from 2-propanol.

Example 11 Formation of 4R-monatin by chemical reduction of 4R-IHOG-oxime Ammonium of (4R) -4-hydroxy-4- (3 -indolylmethyl ) -2-hydroxyiminoglutaric acid obtained in Example 10 5.38 g (15.8 mmol) of the salt was dissolved in 60 ml of 28% aqueous ammonia, 2.84 g of 5% rhodium carbon (50% water-containing product) was added, and the reaction was performed at room temperature with a hydrogen pressure of 1 MPa. After 17 hours, the catalyst was filtered (0.2 micron filter), and 1.04 g (7.5 mmol) of potassium carbonate was dissolved in the filtrate. The solution was concentrated, 20 ml of ethanol was added to 15.4 g of the resulting concentrate, and the mixture was stirred at 25 ° C., 30 ml of ethanol was added dropwise over 3 hours, and the mixture was stirred at 25 ° C. for 17 hours.

2.93 g of the obtained wet crystals were dissolved in 4 ml of water, 8 ml of ethanol was added at 35 ° C., and 17 ml of ethanol was further added dropwise at 35 ° C. over 3 hours. The ethanol solution was cooled to 10 ° C. over 4 hours and then stirred at 10 ° C. for 1 hour. 2.30 g of the obtained wet crystals were dried under reduced pressure to obtain 1.99 g of the desired (2R, 4R) -monatin K salt. The resulting monatin has an optical purity of 99.6% d. e. Met.
¹ HNMR (400 MHz, D ₂ O) δ: 2.06 (dd, J = 11.8, 15.3 Hz, 1H), 2.67 (dd, J = 2.0, 15.2 Hz, 1H), 3 .08 (d, J = 14.4 Hz, 1H), 3.28 (d, J = 14.4 Hz, 1H), 3.63 (dd, J = 2.2, 12.2 Hz, 1H), 7. 12-7.16 (m, 1H), 7.20-7.24 (m, 2H), 7.48-7.49 (m, 1H), 7.71-7.73 (m, 1H).
ESI-MS calculated value C ₁₄ H ₁₆ N ₂ O ₅ = 292.29, analysis value 291.28 [M−H] ⁻

Example 12 Improved IHOG synthesis by SpALD E. coli cultured for 16 hours at 37 ° C. in LB-amp plate medium. E. coli JM109 / ptrpSpALD cells were scraped from one platinum loop, inoculated into 12 500 ml flasks containing 50 ml of LB-amp medium, and cultured with shaking at 37 ° C. for 16 hours. Bacteria were collected from the obtained culture broth by centrifugation, suspended in buffer A (20 mM Hepes-KOH, pH 7.6), washed, and collected again by centrifugation. Bacterial cells prepared by centrifugation (wet cell weight of about 3 g) were suspended in 300 ml of a reaction solution having the following composition.
IHOG synthesis reaction solution: 50 mM phosphate buffer (pH 8.7), 300 mM indole pyruvate, 600 mM sodium pyruvate, 0.1 mM MgCl ₂ (adjusted to pH 8.5 with 6N KOH)
Argon gas was bubbled through the reaction solution in which the cells were suspended, and the reaction was carried out in an argon gas atmosphere thereafter. The reaction was carried out for 20 hours while stirring at 37 ° C. After completion of the reaction, the cells were sterilized by centrifugation to obtain 300 ml of an aldol reaction solution. The resulting reaction solution was oximed in the same manner as in Example 10, and the generated IHOG was quantified by HPLC. As a result, 63.6 mM 4R-IHOG had an optical purity of 92.2% e.e. e. The 4R selectivity could be improved by improving the reaction conditions of SpALD.

Example 13 Cloning of aldolase gene (buald) from C24 strain (Burkholderia sp. AJ110371 strain) Mass gene expression in E. coli Collected in Example 1, among the accumulated bacteria that found 4R-IHOG aldolase activity, a gene encoding 4R-IHOG aldolase (hereinafter BuALD) was obtained from the C24 strain. In addition, the bald gene is ligated to ptrp4. The gene was expressed in large quantities in E. coli JM109.

(1) Preparation of Chromosomal DNA C24 strain (Burkholderia sp. AJ110371 strain) was cultured overnight at 30 ° C. using 50 ml of CM2G medium (preculture). Using 5 ml of this culture as an inoculum, main culture was performed using 50 ml of bouillon medium. After culturing until the late stage of logarithmic growth, 50 ml of the culture solution was subjected to centrifugation (12,000 × g, 4 ° C., 15 minutes) to collect the cells. Using these cells, chromosomal DNA was prepared according to a conventional method.

(2) Acquisition of bald gene by Southern analysis / colony hybridization Using the DNA fragment encoding the full length of SpALD gene as a probe, the bald gene was acquired by Southern analysis and colony hybridization. Using the primers shown in Table 9 (SEQ ID NOs: 10 and 11), the full length of the spald gene was amplified by PCR from the chromosomal DNA of the C77 strain (Sphingomonas sp. AJ110329 strain). A DNA probe using the amplified fragment was labeled with a DIG High Prime (Roche Diagnostics) by incubating at 37 ° C. according to the instructions at O / N. Southern analysis was performed by digesting 1 μg of chromosomal DNA prepared from C24 strain completely with various restriction enzymes, electrophoresis on 0.8% agarose gel, blotting on nylon membrane, and following the manual below. Hybridization was performed using DIG Easy Hyb (Roche Diagnostics), prehybridization was performed at 50 ° C. for 1 hour, a probe was added, and hybridization was performed with O / N. Band detection was performed using the DIG Nucleotide Detection Kit. As a result, a PstI / SmaI fragment of about 2.3 kbp that was strongly hybridized using the PCR fragment as a probe was detected. Next, this PstI / SmaI fragment was obtained by colony hybridization. 20 μg of chromosomal DNA was treated with PstI / SmaI and then subjected to agarose gel electrophoresis, and a fragment having a size of about 2.3 kbp was recovered. This is linked to pUC18. A library was prepared using E. coli JM109. The colony was transferred to a nylon membrane filter (Hybond-N, Amersham) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using DIG Easy Hyb. The filter was immersed in a buffer and prehybridization was performed at 42 ° C. for 1 hour. Thereafter, the prepared labeled probe was added, and hybridization was performed at 42 ° C. for 16 hours. After washing with SSC, colonies that hybridized with the probe were detected using DIG Nucleotide Detection Kit (Roche Diagnostics). As a result, a clone that strongly hybridized with the probe was obtained.
When the base sequence of plasmid DNA recovered from the obtained clone was determined, it was revealed that it had the base sequence described in SEQ ID NO: 14, and the full length of the target bald gene was obtained.

(3) Construction of aldolase gene expression plasmid ptrpBuALD and E. coli. Expression in E. coli Using the primers shown in Table 10 (SEQ ID NOs: 19 and 20), the fragment amplified from the chromosomal DNA of C24 strain (Burkholderia sp. AJ110371) was digested with NdeI / PstI and inserted into the NdeI / PstI site of pTrp4 The constructed plasmid ptrpBuALD was constructed. This plasmid expresses an aldolase gene consisting of the amino acid sequence described in SEQ ID NO: 15, which is obtained by translating the 531st ATG of the nucleotide sequence described in SEQ ID NO: 14 to the 1385th position as a translation initiation codon.

The constructed expression plasmid was transformed into E. coli. After introduction into E. coli JM109, one platinum loop was inoculated into 50 ml of LB medium containing 100 μg / ml ampicillin and shaken at 37 ° C. for 16 hours. After completion of the culture, 1 ml of the obtained culture solution was collected, washed, suspended in 1 ml of 20 mM Hepes-KOH (pH 7.6), and the cells were disrupted by ultrasonic disruption. The supernatant obtained by centrifuging the disrupted solution at 15000 rpm for 10 minutes was used as a crude enzyme solution.
Aldolase activity was measured using the crude enzyme solution. In the aldolase activity measurement, the aldol decomposition activity using PHOG as a substrate was measured under the following conditions.
Reaction conditions: 50 mM Hepes-KOH (pH 8.5), 2 mM PHOG, 0.25 mM NADH, 0.2 mM KPi, 1 mM MgCl ₂ , 16 U / ml lactate dehydrogenase, 3 μl enzyme / 600 μl reaction solution, 30 ° C., absorbance at 340 nm Measurement As a result of the measurement, E. coli introduced pTrp4. While no aldolase activity was detected in E. coli (control), 231 U / mg protein aldolase activity was detected in the ptrpBuALD-introduced strain. Thus, it was confirmed that the target aldolase was cloned together with the construction of the BuALD high expression plasmid.

Example 14 Purification of recombinant enzyme of aldolase derived from C24 strain (Burkholderia sp. AJ110371) Aldolase (BuALD) derived from C24 strain (Burkholderia sp. AJ110371) was highly expressed. Recombinant BuALD was purified from the soluble fraction of E. coli as follows. In the aldolase activity measurement, the aldol decomposition activity using PHOG as a substrate was measured under the following conditions.
Reaction conditions: 50 mM Hepes-KOH (pH 8.5), 2 mM PHOG, 0.25 mM NAD, 1 mM MgCl ₂ , 16 U / ml lactate dehydrogenase, 3 μl enzyme / 600 μl reaction solution, 30 ° C., absorbance at 340 nm is measured

(1) Preparation of soluble fraction:
E. coli cultured in LB-amp plate medium at 37 ° C. for 16 hours. E. coli JM109 / ptrpBuALD cells were scraped from one platinum loop, inoculated into 10 500 ml flasks containing 50 ml of LB-amp medium, and cultured with shaking at 37 ° C. for 16 hours. Bacteria were collected from the obtained culture broth by centrifugation, suspended in buffer A (20 mM Hepes-KOH, pH 7.6), washed, and collected again by centrifugation. The obtained washed cells were suspended in 20 ml of buffer A and sonicated at 4 ° C. for 30 minutes. The disrupted liquid was centrifuged (x8000 rpm, 10 minutes × twice) to remove cell residue, and the resulting supernatant was used as a crude extract fraction.

(2) Anion exchange chromatography: Q-Sepharose FF
The above soluble fraction (20 ml) was subjected to an anion exchange chromatography column Q-Sepharose FF 26/10 (Pharmacia, CV = 20 ml) equilibrated with buffer A and adsorbed onto a carrier. After washing away the protein not adsorbed on the carrier (non-adsorbed protein) with buffer A, the KCl concentration was linearly changed from 0 M to 0.7 M (total 140 ml), and the adsorbed protein was eluted. . When PHOG aldolase activity was detected for each eluted fraction, a peak of PHOG aldolase activity was detected in a fraction corresponding to about 0.35M.

(3) Hydrophobic chromatography: Phenyl Sepharose HP HR 16/10
The solution in which the aldolase activity was detected was dialyzed overnight at 4 ° C. against buffer B (20 mM Hepes-KOH, 1M ammonium sulfate, pH 7.6) and filtered through a 0.45 μm filter. The obtained filtrate was subjected to a hydrophobic chromatography column Phenyl Sepharose HP HR 16/10 (manufactured by Pharmacia) equilibrated with buffer B. By this operation, aldolase was adsorbed on the carrier.
After washing away non-adsorbed protein that was not adsorbed on the carrier with buffer B, the aldolase was eluted by linearly changing the ammonium sulfate concentration from 1M to 0M. The aldolase activity was measured for each of the obtained elution fractions, and the aldolase activity was observed at the elution position where the ammonium sulfate concentration was approximately 0.4 to 0.5M.

(4) Gel filtration chromatography: Sephadex 200 HP 16/60
Each fraction containing aldolase was collected, dialyzed against buffer A, and filtered through a 0.45 μm filter. The obtained filtrate was concentrated using an ultrafiltration membrane centriprep 10. The obtained concentrated solution is subjected to gel filtration Sephadex 200 HP 16/60 (Pharmacia) equilibrated with buffer C (20 mM Hepes-KOH, 0.1 M KCl, pH 7.6), and a flow rate of 1 ml / min. Eluted with. By this operation, aldolase was eluted in a fraction of around 66 ml.

(5) Anion exchange chromatography: Mono Q HR 5/5
The obtained fraction was filtered through a 0.45 μm filter. The filtrate obtained here was applied to an anion exchange chromatography column Mono-Q HR 5/5 (Pharmacia) equilibrated with buffer A. By this operation, aldolase was adsorbed on the carrier. After washing away non-adsorbed protein with buffer A, protein elution was performed by linearly changing the KCl concentration from 0 mM to 700 mM (Total 24 ml). The aldolase activity was measured for each elution fraction, and the aldolase activity was observed at the elution position where the KCl concentration was about 0.4M.

  When the fraction purified by the above column chromatographic operation was subjected to SDS-PAGE, it was detected as a single band by CBB staining at a position corresponding to about 30 kDa. The obtained recombinant BuALD solution was dialyzed against buffer A at 4 ° C. overnight. By the above operation, 1.5 ml of 1241 U / ml BuALD solution was obtained.

Example 15 Various enzymatic properties of BuALD Using BuALD prepared in Example 14, the following items were examined.
Basic measurement conditions: 50 mM Hepes-KOH (pH 8.5), 2 mM PHOG, 5 mM MgCl ₂ , 16 U / ml lactate dehydrogenase, 340 nm absorbance decrease measured at 30 ° C.

(1) Kinetic constants using PHOG as a substrate From the results shown in FIGS. 8-1 and 8-2, Vmax (for PHOG) = 483 μmol / min / mg, Km (for PHOG) = 0.66 mM, Km (for MgCl ₂ ) = 0.021 mM, respectively. Further, no increase in aldolase activity due to the addition of KPB was observed.

(2) pH stability The pH stability in the range of pH 3-10 was measured. The buffer solution used for the measurement is as follows. Sodium citrate buffer (pH 3, 4, 4.5), sodium acetate buffer (pH 4, 4.5, 5, 5.5, 6, 6.5), potassium phosphate buffer (pH 6, 6.5, 7, 7.5, 8, 8.5), Hepes-KOH buffer (pH 7.5, 8, 8.5, 9, 9.5), CAPS-NaOH buffer (pH 9, 9.5, 10). Residual activity of BuALD incubated in each buffer solution of 100 mM at 37 ° C. for 30 minutes was measured under basic measurement conditions. The results are shown in FIG.

(3) Temperature stability After incubation of BuALD for 30 minutes in 100 mM potassium phosphate buffer (pH 7.0) and 100 mM Hepes-KOH buffer (pH 8.5) at 25 ° C. to 70 ° C., the residual activity was measured under basic measurement conditions. Measured with The results are shown in FIG.

Example 16 IHOG synthesis by BuALD and SpALD and cultured in oximed LB-amp plate medium at 37 ° C. for 16 hours. E. coli JM109 / ptrpBuALD or E. coli. E. coli JM109 / ptrpSpALD cells were scraped off from one platinum loop, inoculated into 4 500 ml flasks containing 50 ml of LB-amp medium, and cultured with shaking at 37 ° C. for 16 hours. Bacteria were collected from the obtained culture broth by centrifugation, suspended in buffer A (20 mM Hepes-KOH, pH 7.6), washed, and collected again by centrifugation. The cells prepared by centrifugation (about 1 g in wet cell weight) were suspended in 100 ml of a reaction solution having the following composition.
IHOG synthesis reaction solution: 50 mM KPB (pH 8.0), 300 mM indole pyruvate, 600 mM sodium pyruvate, 0.1 mM MgCl ₂ (adjusted to pH 8.0 with 6N KOH)
After argon gas was bubbled through the reaction solution in which the cells were suspended, the reaction was performed at 37 ° C. with stirring for 18 hours. After completion of the reaction, the cells were sterilized by centrifugation to obtain about 100 ml of an aldol reaction solution.
To approximately 96 ml of the obtained aldol reaction solution, 6.25 g (90 mmol) of hydroxylamine hydrochloride was added while maintaining the pH value at 9 with a 6N aqueous sodium hydroxide solution, and at 25 ° C. for 6 hours and at 10 ° C. overnight. Stir. The amount of IHOG-oxime in the obtained reaction solution was quantified by HPLC analysis. As a result, (Table 11) 4R-IHOG-oxime was preferentially generated in both the BuALD and SpALD sections.

Example 17 Cloning of aldolase gene (SpALD2) from C43 strain (Sphingomonas sp. AJ110372 strain) Overexpression was collected in Example 1 in coli, among integrated bacteria found 4R-IHOG aldolase activity, Sphingomonas sp. A gene encoding 4R-IHOG aldolase (hereinafter referred to as SpALD2) was obtained from the C43 strain. In addition, the spad2 gene is linked to ptrp4, The gene was expressed in large quantities in E. coli JM109.

(1) Preparation of Chromosomal DNA C43 strain (Sphingomonas sp. AJ110372 strain) was cultured overnight at 30 ° C. using 50 ml of CM2G medium (preculture). Using 5 ml of this culture as an inoculum, main culture was performed using 50 ml of bouillon medium. After culturing until the late stage of logarithmic growth, 50 ml of the culture solution was subjected to centrifugation (12,000 × g, 4 ° C., 15 minutes) to collect the cells. Using these cells, chromosomal DNA was prepared according to a conventional method.

(2) Acquisition of the spald2 gene by Southern analysis / colony hybridization Using the DNA fragment encoding the full length of the SpALD gene as a probe, the spald2 gene was acquired by Southern analysis and colony hybridization. Using the primers shown in Table 9 (SEQ ID NOs: 10 and 11), the full length of the spald gene was amplified by PCR from the chromosomal DNA of the C77 strain (Sphingomonas sp. AJ110329 strain). A DNA probe using the amplified fragment was labeled with a DIG High Prime (Roche Diagnostics) by incubating at 37 ° C. according to the instructions at O / N. Southern analysis was performed by digesting 1 μg of chromosomal DNA prepared from C43 strain completely with various restriction enzymes, electrophoresis on 0.8% agarose gel, blotting on nylon membrane, and following the manual below. Hybridization was performed using DIG Easy Hyb (Roche Diagnostics), prehybridization was performed at 50 ° C. for 1 hour, a probe was added, and hybridization was performed with O / N. Band detection was performed using the DIG Nucleotide Detection Kit. As a result, an approximately 3 kbp PstI fragment that strongly hybridizes using the PCR fragment as a probe was detected. Next, this PstI fragment was obtained by colony hybridization. After treating 20 μg of chromosomal DNA with PstI, it was subjected to agarose gel electrophoresis, and a fragment having a size of about 3 kbp was recovered. This is linked to pUC118. A library was prepared using E. coli JM109. The colony was transferred to a nylon membrane filter (Hybond-N, Amersham) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using DIG Easy Hyb. The filter was immersed in a buffer and prehybridization was performed at 42 ° C. for 1 hour. Thereafter, the prepared labeled probe was added, and hybridization was performed at 42 ° C. for 16 hours. After washing with SSC, colonies that hybridized with the probe were detected using DIG Nucleotide Detection Kit (Roche Diagnostics). As a result, a clone that strongly hybridized with the probe was obtained.
When the base sequence of the plasmid DNA recovered from the obtained clone was determined, it was revealed that it had the base sequence described in SEQ ID NO: 12, and the full length of the desired spald2 gene was obtained.

(3) Construction of aldolase gene expression plasmid pUCSpALD2 and E. coli. Expression in E. coli Using the primers shown in Table 12 (SEQ ID NOs: 21 and 22), Sphingomonas sp. A fragment amplified from the chromosomal DNA of the C43 strain was digested with EcoRI / PstI to construct plasmid pUCSpALD2 inserted into the EcoRI / PstI site of pUC18. This plasmid expresses an aldolase gene consisting of the amino acid sequence described in SEQ ID NO: 13, which is obtained by translating the 399th ATG from the nucleotide sequence described in SEQ ID NO: 12 to the 1253rd using the translation start codon.

  The constructed expression plasmid was transformed into E. coli. E. coli JM109 was introduced, and one transformant was inoculated into 50 ml of LB medium containing 100 μg / ml ampicillin and 0.1 mM Isopropyl-b-D-thiogalactopyroside (IPTG) and shaken at 30 ° C. for 16 hours. After completion of the culture, 1 ml of the obtained culture solution was collected, washed, suspended in 1 ml of 20 mM Hepes-KOH (pH 7.6), and the cells were disrupted by ultrasonic disruption. The supernatant obtained by centrifuging the disrupted solution at 15000 rpm for 10 minutes was used as a crude enzyme solution.

Aldolase activity was measured using the crude enzyme solution. In the aldolase activity measurement, the aldol decomposition activity using PHOG as a substrate was measured under the following conditions.
Reaction conditions: Hepes-KOH (pH 8.5), 2 mM PHOG, 0.25 mM NADH, 1 mM MgCl ₂ , 16 U / ml lactate dehydrogenase, 3 μl enzyme / 600 μl reaction solution, 30 ° C., absorbance at 340 nm was measured. As a result, pUC18 E. was introduced. While no aldolase activity was detected from E. coli (control), an aldolase activity of 0.68 U / mg protein was detected in the pUCSpALD2-introduced strain. Thereby, it was confirmed that the target aldolase was certainly cloned together with the construction of the SpALD2 high expression plasmid.

Example 18 Synthesis of IHOG and IHOG-oxime by SpALD2 Cultured at 30 ° C. for 16 hours in an LB-amp plate medium. E. coli JM109 / pUCSpALD2 cells were scraped off from one platinum ear and inoculated into 4 500 ml flasks each containing 50 ml of LB medium containing 100 μg / ml ampicillin and 0.1 mM IPTG, and cultured with shaking at 34 ° C. for 16 hours. Bacteria were collected from the obtained culture broth by centrifugation, suspended in buffer A (20 mM Hepes-KOH, pH 7.6), washed, and collected again by centrifugation. The cells prepared by centrifugation (about 1 g in wet cell weight) were suspended in 100 ml of a reaction solution having the following composition.
IHOG synthesis reaction solution: 50 mM KPB (pH 8.0), 300 mM indole pyruvate, 600 mM sodium pyruvate, 0.1 mM MgCl ₂ (adjusted to pH 8.0 with 6N KOH)
After argon gas was bubbled through the reaction solution in which the cells were suspended, the reaction was performed at 37 ° C. with stirring for 18 hours. After completion of the reaction, the cells were sterilized by centrifugation to obtain about 100 ml of an aldol reaction solution.
To about 96 ml of the obtained aldol reaction solution, 5.95 ml (90 mmol) of 50% aqueous hydroxylamine solution was added and stirred at 25 ° C. for 6 hours and at 10 ° C. overnight. The amount of IHOG-oxime in the obtained reaction solution was quantified by HPLC analysis. As a result, 4R-IHOG-oxime produced 3.84 mmol, and 4S-IHOG-oxime produced 0.15 mmol, with an optical purity of 92.4% e.e. e. Thus, it was confirmed that the 4R body was preferentially generated.

Reference Example 1 Synthesis of 4-hydroxy-4- (3-indolylmethyl) -2-ketoglutaric acid (IHOG) To 64.45 ml of water in which 18.91 g (286.5 mmol, 85% by weight) of potassium hydroxide was dissolved. Indole-3-pyruvic acid 7.50 g (35.8 mmol, content 97.0 wt%) and oxaloacetic acid 14.18 g (107.4 mmol) were added and dissolved. This mixed solution was stirred at 35 ° C. for 24 hours.
Further, 40.0 ml of 3N-hydrochloric acid was added for neutralization (pH = 7.0) to obtain 153.5 g of a reaction neutralized solution. This reaction neutralized solution contained 5.55 g of 4-hydroxy-4- (3-indolylmethyl) -2-ketoglutaric acid, and the yield was 53.3% (vs. indolepyruvic acid). .
Water was added to the reaction neutralized solution to make 168 ml, and the solution was passed through a resin tower (diameter 4.8 cm) filled with 840 ml of a synthetic adsorbent (Diaion-SP207 manufactured by Mitsubishi Chemical). Further, pure water was passed at a flow rate of 23.5 ml / min, and 1.73 to 2.55 (L / LR) was collected, whereby high purity 4-hydroxy-4- (3-yne An aqueous solution containing 3.04 g of (dolylmethyl) -2-ketoglutaric acid was obtained in a yield of 54.7% (relative to the amount charged into the resin).
(NMR measurement)
¹ H-NMR (400 MHz, D ₂ O): 3.03 (d, 1H, J = 14.6 Hz), 3.11 (d, 1H, J = 14.6 Hz), 3.21 (d, 1H, J = 18.1 Hz), 3.40 (d, 1H, J = 18.1 Hz), 7.06-7.15 (m, 3H), 7.39 (d, 1H, J = 7.8 Hz), 7.66 (d, 1H, J = 7.8 Hz).
¹³ C-NMR (100 MHz, D ₂ O): 35.43, 47.91, 77.28, 109.49, 112.05, 119.44, 119.67, 121.91, 125.42, 128. 41,136.21,169.78,181.43,203.58

Reference Example 2 Synthesis of 4-phenylmethyl-4-hydroxy-2-ketoglutaric acid (PHOG) To 25 ml of water in which 13.8 g of potassium hydroxide (purity 85%) was dissolved, 5.0 g (30.5 mmol) of phenylpyruvic acid ), 12.1 g (91.4 mmol) of oxalacetic acid was added and reacted at room temperature for 72 hours. The pH value of the reaction solution was adjusted to 2.2 using concentrated hydrochloric acid, and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated to give a residue. The residue was recrystallized from ethyl acetate and toluene to obtain 2.8 g (11.3 mmol) of 4-phenylmethyl-4-hydroxy-2-ketoglutaric acid as crystals.
(NMR measurement)
¹ H NMR (D ₂ O) δ: 2.48 (d, J = 14.4 Hz, 0.18H), 2.60 (d, J = 14.4 Hz, 0.18H), 2.85-3. 30 (m, 3.64H), 7.17-7.36 (m, 5H)
(Molecular weight measurement)
ESI-MS calculated value C ₁₂ H ₁₂ O ₆ = 252.23, analyzed value 251.22 (MH ⁻ )

Reference Example 3 Production of 4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid To 917 g of 1.6 wt% sodium hydroxide aqueous solution, 73.8 g (352 mmol) of indole-3-pyruvic acid was added. In addition, it was dissolved. The reaction solution was adjusted to 35 ° C., and 310.2 g (1761 mmol) of 50% pyruvic acid aqueous solution was added dropwise over 2 hours while maintaining the pH value at 11.1 using 30% sodium hydroxide aqueous solution. The reaction was further continued for 4.5 hours to obtain a reaction solution containing 4-hydroxy-4- (3-indolylmethyl) -2-ketoglutaric acid. To this was added 367.2 g (2114 mmol) of 40% hydroxylamine hydrochloride aqueous solution while maintaining the pH value at 7 with 30% aqueous sodium hydroxide solution, and the mixture was stirred at 5 ° C. for 17.5 hours. The pH of the reaction solution was adjusted to 2 using concentrated hydrochloric acid, and the organic matter was extracted with ethyl acetate. The organic layer was washed with saturated brine and concentrated to give a residue. The residue was recrystallized from 60 ml of 28% aqueous ammonia and 1350 ml of 2-propanol to give 43.4 g (142 mmol: yield of 2-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid diammonium salt. 40% ratio vs. indole-3-pyruvate) was obtained as crystals.

Reference Example 4 Production of (R)-(+)-1-phenylethylamine salt of (4S) -4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid 4-hydroxy-4- ( After dissolving 44.7 g (0.131 mol) of ammonium salt of 3-indolylmethyl) -2-hydroxyiminoglutaric acid in 500 ml of water at 25 ° C., the pH of the aqueous solution was adjusted to 2 with 25.5 g of 36% hydrochloric acid. did. The acidic solution was extracted with 1300 ml of ethyl acetate, and the ethyl acetate solution was washed with 200 ml of saturated brine. To the obtained ethyl acetate solution, 500 ml of an aqueous sodium carbonate solution (13.9 g of sodium carbonate 0.131 mol) was added and stirred to separate the aqueous alkali solution and ethyl acetate. The pH of the solution was adjusted to 2 by adding 23.1 g of 36% hydrochloric acid to the obtained aqueous alkaline solution. To this acidic aqueous solution, 6.99 g (57.6 mmol) of (R)-(+)-1-phenylethylamine is added dropwise and stirred at 25 ° C. for 1 hour. The obtained crystals were filtered and dried under reduced pressure to give (R)-(+)-1-phenylethylamine salt of (4S) -4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid 21.8 g (47.8 mmol) was obtained (yield 72.7%, optical purity 87.4%).
(NMR measurement)
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 1.48 (d, 3H, J = 6.8 Hz), 2.63 (d, 1H, J = 14.0 Hz), 2.70 (d, 1H, J = 14.0 Hz), 2.90 (d, 1H, J = 14.1 Hz), 3.06 (d, 1H, J = 14.1 Hz), 4.40 (q, 1H, J = 6) .8 Hz), 6.91-7.54 (m, 10H).

Reference Example 5 Production of (S)-(−)-1-phenylethylamine salt of (4R) -4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid Obtained in Reference Example 4 To the crystal filtrate, (S)-(−)-1-phenylethylamine (7.12 g, 58.7 mmol) was further added dropwise and stirred at 25 ° C. for 1 hour. The obtained crystals were filtered and dried under reduced pressure to give (S)-(−)-1-phenylethylamine salt of (4R) -4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid 23.8 g (53.3 mol) was obtained (yield 81.1%, optical purity 92.1%).

Reference Example 6
(1) Preparation of ammonium salt of (4S) -4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid at 25 ° C., (4S) -4-hydroxy-4- (3- 200 ml of water and 18.5 g of 28% aqueous ammonia are dissolved in 21.8 g (51.0 mmol) of (R)-(+)-1-phenylethylamine salt of indolylmethyl) -2-hydroxyiminoglutaric acid. After that, 200 ml of toluene was further added and stirred. The aqueous layer obtained by layering was heated to 60 ° C., and 900 ml of 2-propanol was added dropwise to the aqueous solution over 2 hours. After cooling this 2-propanol aqueous solution to 10 degreeC over 5 hours, it stirred at 10 degreeC for 10 hours. The obtained crystals were filtered and dried under reduced pressure to obtain 14.75 g of ammonium salt of (4S) -4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid (yield 85. 1%, optical purity 99.0%).
Melting point: 205 ° C (decomposition)
Specific rotation [α] ²⁰ _D +13.4 (c = 1.00, H ₂ O)
(2) Production of ammonium salt of (4R) -4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid In the same manner as in the above Reference Example, (4R) -4-hydroxy-4- ( From 33.8 g (53.3 mmol) of (R)-(+)-1-phenylethylamine salt of 3-indolylmethyl) -2-hydroxyiminoglutaric acid to (4R) -4-hydroxy-4- (3- 16.2 g of ammonium salt of indolylmethyl) -2-hydroxyiminoglutaric acid was obtained (yield 89.3%, optical purity 99.9%).
Specific rotation [α] ²⁰ _D -13.6 (c = 1.00, H ₂ O)

Reference Example 7 Production of (2R, 4R) monatin 13.2 g of ammonium salt of (4R) -4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid obtained in Reference Example 6 (38. 7 mmol) was dissolved in 135 ml of 28% aqueous ammonia, 6.93 g of 5% rhodium carbon (50% water-containing product) was added, and the reaction was carried out at 25 ° C. under a hydrogen pressure of 1 MPa. After 24 hours, the catalyst was filtered (0.2 micron filter), and 2.54 g (18.4 mmol) of potassium carbonate was dissolved in the filtrate. The solution was concentrated, 20 ml of water and 45 ml of ethanol were added to 32.7 g of the resulting concentrate, and the mixture was stirred at 25 ° C. Further, 60 ml of ethanol was added dropwise over 3 hours, and the mixture was stirred at 25 ° C for 20 hours for crystallization. Went. 9.78 g of the obtained wet crystal was dissolved in 12 ml of water, 24 ml of ethanol was added, and 51 ml of ethanol was further added dropwise over 3 hours. The ethanol solution was cooled to 15 ° C. over 4 hours and then stirred at 15 ° C. for 10 hours. 7.08 g of the obtained wet crystals were dried under reduced pressure to obtain 5.7 g of the target (2R, 4R) monatin potassium salt.

  As described above, by using the aldolase of the present invention, IHOG and PHOG can be optically selected. The aldolase of the present invention enables efficient asymmetric introduction at the stage of the aldol condensation reaction in the monatin synthesis route, and can be suitably used for the production of optically active IHOG and optically active monatin. it can.

It is a figure which shows the homology comparison of the aldolase of this invention. It is a flowchart which shows the manufacturing process of the aldolase of this invention. It is a graph which shows the reaction rate which used PHOG of SpALD as a substrate. It is a graph showing the rate of reaction MgCl ₂ concentration of SpALD. It is a graph which shows the result of having measured the pH stability of SpALD. It is a graph which shows the result of having measured the temperature stability of SpALD. It is a graph which shows reaction optimal pH in the aldol decomposition | disassembly activity of SpALD. It is a graph which shows reaction optimal pH in the aldol condensation activity of SpALD. It is a graph which shows the reaction rate which used the PHOG of BuALD as a substrate. It is a graph showing the rate of reaction MgCl ₂ concentration of BuALD. It is a graph which shows the result of having measured the pH stability of BuALD. It is a graph which shows the result of having measured the temperature stability of BuALD.

Claims (18)

(A) or (b) below
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, or (b) a protein having at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 2 and having 4R-aldolase activity,
Or a microorganism containing the protein according to any one of
Act on indole-3-pyruvic acid and pyruvic acid or oxaloacetic acid,
(4R) -4- (Indol-3-ylmethyl) -4-hydroxy-2-oxoglutaric acid (4R-IHOG) or a salt thereof represented by the following formula (1) having an optical purity of 70% or more is produced. A method for producing 4R-IHOG or a salt thereof.

(A) or (b) below
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, or (b) a protein having at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 2 and having 4R-aldolase activity,
Or a microorganism containing the protein according to any one of
Act on indole-3-pyruvic acid and pyruvic acid or oxaloacetic acid,
A first step of preferentially producing 4R-IHOG or a salt thereof; and
A second carbonyl group of 4R-IHOG or a salt thereof obtained in the first step is converted to an amino group to obtain 4R-monatin or a salt thereof represented by the following formula (2) having an optical purity of 90% or more. Process,
A process for producing 4R-monatin or a salt thereof.

(In the formula, the combination of wavy lines indicates that both R-configuration and S-configuration are included.)   The production of 4R-monatin or a salt thereof according to claim 2, wherein in the second step, the conversion of the carbonyl group into an amino group is carried out by aminating 4R-IHOG with an enzyme. Method. In the second step, the conversion of the carbonyl group into an amino group is performed by converting 4- (indol-3-ylmethyl) -4-hydroxy-2-oxoglutaric acid contained in the reaction solution under neutral or alkaline conditions as follows. General formula (3)

(In the general formula (3), R represents a hydrogen atom, an alkyl group, an aryl group, or an aralkyl group.)
To produce 4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutaric acid (IHOG-oxime) represented by the following formula (4): Crystallize the produced 4HO form of IHOG-oxime or a salt thereof,
The 4R-monatin or salt thereof according to claim 2, wherein the 4R-form IHOG-oxime or salt thereof obtained is reduced, and the produced 4R-monatin or salt thereof having an optical purity of 90% or more is collected. Production method.

  The 4R-monatin or a salt thereof according to claim 4, wherein the amine compound represented by the general formula (3) is at least one amine compound selected from the group consisting of hydroxylamine, methoxyamine, and benzyloxyamine. Production method.   The method for producing 4R-monatin or a salt thereof according to claim 4 or 5, wherein the reduction of 4R IHOG-oxime or a salt thereof is carried out in the presence of hydrogen and a hydrogenation catalyst.   The method for producing 4R-monatin or a salt thereof according to any one of claims 4 to 6, wherein (2R, 4R) -monatin is collected by crystallization in the second step.   The method for producing 4R-monatin or a salt thereof according to any one of claims 4 to 7, wherein in the second step, water, an alcohol solvent or a hydrous alcohol solvent is used as a crystallization solvent.   The production method according to any one of claims 1 to 8, wherein the protein used in the method is a protein derived from a microorganism selected from the genus Sphingomonas or Burkholderia.   The method according to claim 9, wherein the microorganism is Sphingomonas sp. Strain AJ110329 or AJ110372 strain, Burkholderia sp. Strain AJ110371. The protein in any one of the following (a)-(c).
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 (b) a protein having at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 2 and having 4R-aldolase activity (c) a sequence in the sequence listing A protein having an amino acid sequence including substitution, deletion, insertion, addition, or inversion of one or several amino acid residues in the amino acid sequence of No. 2 and having aldolase activity   The protein according to claim 11, wherein the protein having at least 70% homology with the amino acid sequence described in SEQ ID NO: 2 and having 4R-aldolase activity is the protein according to SEQ ID NO: 13 or 15. .   A DNA encoding the protein according to claim 11 or 12. DNA of the following (d) or (e).
(D) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or the nucleotide sequence of nucleotide numbers 210 to 1004 in the same sequence
(E) a protein that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequences 210 to 1004 of the same sequence and has an aldolase activity DNA to do   A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence set forth in SEQ ID NO: 1 or a base sequence complementary to the base sequences 210 to 1004 in the same sequence and that encodes a protein having aldolase activity (F) DNA consisting of the base sequence of SEQ ID NO: 12 or the base sequence of base numbers 399 to 1253 in the same sequence, or (g) the base sequence of SEQ ID NO: 14 or the base sequence of base numbers 531 to 1385 in the same sequence The DNA according to claim 14, which is any one of DNAs consisting of   A recombinant DNA obtained by connecting the DNA according to claim 14 or 15 and a vector DNA.   A cell transformed with the recombinant DNA according to claim 16. A method for producing a protein having aldolase activity, comprising culturing the cell according to claim 17 in a medium and accumulating the protein having aldolase activity in the medium and / or the cell.

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