JP4344572B2 - Method for producing D-kilo-inositol - Google Patents

Description

  The first aspect of the present invention relates to a method for producing D-kilo-inositol, which comprises reacting D-kilo-inosose with myo-inositol 2-dehydrogenase and NADH to convert to D-kilo-inositol.

In addition, the second aspect of the present invention is that, starting from myo-inositol, myo-inositol is reacted with myo-inositol 2-dehydrogenase and NAD ^+, and the produced scyllo-inosose is reacted with scyllo-inosose isomerase. The present invention relates to a method for producing D-kilo-inositol, which comprises reacting D-kilo-1-inosose with myo-inositol 2-dehydrogenase and NADH to produce D-kilo-inositol.

The third invention relates to an enzymatic reaction of myo-inositol with myo-inositol 2-dehydrogenase and NAD ⁺ , an enzymatic reaction of scyllo-inosose with scyllo-inosose isomerase, D-kilo-1-inosose and myo-inositol. The present invention relates to a method for producing D-kilo-inositol capable of efficiently producing D-kilo-inositol by sequentially proceeding with an enzymatic reaction of 2-dehydrogenase and NADH in one aqueous reaction medium.

  Furthermore, the fourth aspect of the present invention relates to an aqueous solution of a mixture of myo-inositol and D-kilo-inositol as obtained by the second or third method of the present invention, from D-kilo-inositol produced by an enzymatic reaction. The present invention relates to a method for separating myo-inositol from a mixture containing myo-inositol and D-kilo-inositol, which can efficiently separate mixed myo-inositol.

  The fifth aspect of the present invention relates to a method for separating D-kilo-inositol by chromatography from a mixture containing myo-inositol and D-kilo-inositol in a certain ratio.

The sixth aspect of the present invention also relates to a method for producing myo-inositol 2-dehydrogenase and scyllo-inosose isomerase from Bacillus bacteria.
However, the second invention relates to the invention according to claim 1, the third invention relates to the invention according to claim 4, and the sixth invention relates to the invention according to claim 8.

  When administered orally or parenterally, D-kilo-inositol acts on an inositol phospholipid system that activates intracellular signal transduction, and has an improvement effect on type II diabetes (Patent Document 1: (Japanese Patent Publication No. 4-505218) and the effect of improving polycystic ovary syndrome (Patent Document 2: USP5906979, Non-Patent Document 1: "The New England Journal of Medicine", JE Nestler et al., 1999, 340, p. 1314) are proposed therapeutic agents. D-Chiro-inositol is functionally considered to be involved in a wide range of metabolic diseases, and is a substance that is expected to expand its application in the future.

  D-kilo-inositol (sometimes abbreviated as DCI) is one of nine inositol stereoisomers. As a method for producing DCI, a method for producing DCI by hydrolysis of kasugamycin, which is an antibiotic, has been hitherto (see Patent Document 3: USP5091596, Patent Document 4: USP5463142, and Patent Document 5: USP5714643). No. specification, Patent Document 6: USP6342645 specification), a method of producing D-kilo-inositol by hydrolyzing D-vinitol contained in legumes (see Patent Document 7: USP5827896 specification) Patent Document 8: PCT / US01 / 15353, Patent Document 9: Japanese Patent Laid-Open No. 13-261600, and D-kilo-inositol by organic synthesis (Patent Document 10: USP5406005) Reference, Patent Document 11: Refer to pamphlet of WO96 / 25381, Patent Document 12: Refer to Japanese Patent Publication No. 8-502255, Patent Document 13: Refer to Japanese Patent Application Laid-Open No. 11-280068, and make microorganisms act on myo-inositol. By A method for producing D-kilo-inositol by converting myo-inositol into D-kilo-inositol is known (see Patent Document 14: JP-A-9-140388).

  However, when starting from kasugamycin or D-vinitol, these materials are expensive, and when produced by organic synthesis, the yield and optical purity are low, and the final product, D-kilo-inositol, is expensive. It will be something. Furthermore, the reaction in which myo-inositol is allowed to act on microorganisms to convert to D-kilo-inositol is difficult to control, and the microorganisms produce other metabolites at the same time. Purification is difficult.

  In recent years, myo-inositol has been utilized by utilizing a bacterium belonging to the genus Agrobacterium and capable of producing D-myo-inositol 1-epimerase as a constituent enzyme capable of converting myo-inositol into D-kilo-inositol. There is known a method for producing D-kilo-inositol from Pt (see Patent Document 15: Japanese Patent Application Laid-Open No. 2001-8694 and Patent Document 16: WO 02/055715 pamphlet). However, it is difficult to allow bacteria to act at a high substrate concentration by the method of converting to D-kilo-inositol by acting on myo-inositol while culturing the bacteria, and therefore, handling a large amount of medium. Must-have.

  Furthermore, the entire nucleotide sequence of chromosomal DNA of Bacillus subtilis (Bacillus subtilis) has been published (see Non-Patent Document 2: “Microbiology”, 140, p. 2289-2298 (1994)). The nucleotide sequence of myo-inositol 2-dehydrogenase that oxidizes myo-inositol into scyllo-inosose, which is present in 12 open reading frames (ORFs) present in the inositol metabolism operon in the nucleotide sequence of this chromosomal DNA. And the amino acid sequence of the D-myo-inositol 2-dehydrogenase is known (see Patent Document 17: Japanese Patent Laid-Open No. 05-192163, Non-Patent Document 3: “Journal of Biological Chemistry”, Vol. 254, p.7684. ~ 7690 (1979)). In addition, the base sequence of the iolI gene present in the inositol metabolism operon is known (see Non-Patent Document 2 and Non-Patent Document 3).

  Myo-inositol 2-dehydrogenase is a well-known enzyme that has been confirmed to exist in 1956 (EC, 1.1.1.18). One example is myo-inositol 2-dehydrogenase derived from Aerobacter aerogenes. (See Non-Patent Document 4: “Archives of Biochemistry and Biophysics”, J. Larner et al., 60, p.352-363 (1956)). Myo-inositol 2-dehydrogenase exists widely in prokaryotes, yeasts, and animals and plants. For example, the amino acid sequence of myo-inositol 2-dehydrogenase derived from Bacillus subtilis is shown in Patent Document 17 (Japanese Patent Laid-Open No. 05-192163). The amino acid sequence of myo-inositol 2-dehydrogenase derived from sp. No. 3, ie, Bacillus halodurans, is described in Patent Document 18 (see Japanese Patent Application Laid-Open No. 06-007158). In addition, the amino acid sequences of these myo-inositol 2-dehydrogenases are published on the Internet. The optimum pH of this enzyme is 8-9.

It is known that when myo-inositol 2-dehydrogenase is allowed to act on myo-inositol together with NAD ⁺ , myo-inosose is produced and NAD ⁺ becomes NADH (Patent Document 18: JP 06-007158 A and Patent Document 1). 19: See Japanese Patent No. 3251978). Myo-inosose is a substance also called scyllo-inosose.

Further, D-kilo-1-inosose is a known substance (Non-patent Document 5: “Carbohydrate Research”, 79, p. 121-130 (1979)), and D-kilo-1-inosose is chemically reduced. The present inventors have previously found that D-kilo-inositol is produced by reduction with an agent (Japanese Patent Application No. 2003-128065).
Japanese translation of PCT publication No. 4-505218 (the whole sentence: effect of D-kilo-inositol for type II diabetes), USP5906979 specification (full text: effect of D-kilo-inositol on polycystic ovary syndrome) USP5091596 specification (full text: method for obtaining D-kilo-inositol by hydrolysis of kasugamycin) USP5463142 specification (full text: method for obtaining D-kilo-inositol by acetylation degradation of kasugamycin) USP5714643 (full text: method for obtaining D-kilo-inositol by hydrolysis of kasugamycin) USP6342645 specification (full text: method for obtaining D-kilo-inositol by hydrolysis of kasugamycin) USP5827896 specification (full text: method of hydrolyzing vinylitol to obtain D-kilo-inositol) PCT / US01 / 15353 specification (full text: Method for obtaining D-kilo-inositol by hydrolyzing vinylitol obtained from soybean seeds) JP-A No. 13-261600 (full text: method for obtaining D-kilo-inositol by hydrolyzing vinylitol obtained from edible resources) USP5406005 specification (full text: Method of synthesizing D-kilo-inositol from glucuronic acid in 10 steps) WO96 / 25381 pamphlet (full text: method for obtaining D-kilo-inositol from dialdose by organic synthesis) JP-T 8-502255 Publication (Method for obtaining D-kilo-inositol from acetanilide by organic synthesis) JP 11-280068 A (Method for obtaining D-kilo-inositol from myo-inositol by organic synthesis) JP-A-9-140388 (full text: method for producing D-kilo-inositol by allowing myo-inositol to act while culturing Agrobacterium bacteria) JP 2001-8694 (full text: method for obtaining D-kilo-inositol by allowing Agrobacterium sp. AB10121 strain to act on myo-inositol while culturing) WO 02/055715 pamphlet (full text: method for producing D-kilo-inositol by allowing microorganisms (E. coli) in which epimerase is expressed by genetic recombination to act on myo-inositol while culturing) JP 05-192163 A (full text: Myo-inositol 2-dehydrogenase derived from Bacillus subtilis and its amino acid sequence) Japanese Patent Application Laid-Open No. 06-007158 (full text: Myo-inositol 2-dehydrogenase derived from Bacillus halodurans and its amino acid sequence) Patent No. 3251976 (full text: Myo-inositol 2-dehydrogenase derived from Bacillus halodurans) “The New England Journal of Medicine”, JE Nestler et al., 1999, 340, p. 1314 (full text: effects of D-kilo-inositol on polycystic ovarian syndrome) “Microbiology”, 140, pp. 2289-2298 (1994) (full text: paper on genes in the inositol metabolism operon in the total DNA of Bacillus subtilis chromosomes) “Journal of Biological Chemistry”, 254, p.7684-7690 (1979) (full text: paper on myo-inositol 2-dehydrogenase from Bacillus subtilis) "Archives of Biochemistry and Biophysics", J. Larner et al., 60, 352-363 (1956) (full text: paper on inositol 2-dehydrogenase from Aerobacter aerogenes) “Carbohydrate Research” Stephan J. et al., 79, p. 121-130 (1979) Previously, the present inventors used scyllo-inosose, which can be produced by acting bacteria on myo-inositol, as a starting material. In addition, research has been conducted to develop a new method capable of producing D-kilo-inositol from scyllo-inosose through reaction with an enzyme and reaction with a chemical reducing agent.

  In the course of this research, a known iolI gene isolated from the Bacillus subtilis chromosome was incorporated into a plasmid, E. coli was transformed with the plasmid containing the gene, and the iolI gene was then cultured by culturing the transformed E. coli. A series of experiments for expression were performed. In this series of experiments, the enzyme encoded by the iolI gene was successfully isolated from the cultured cells of the transformed E. coli. The properties of this enzyme were examined, and the isolated enzyme was isomerized from scyllo-inosose to D-kilo-1-inosose and from D-kilo-1-inosose to scyllo-inosose. It has been found to have an action of catalyzing an equilibrium reaction of interconversion consisting of This new enzyme was named scyllo-inosose isomerase (see Japanese Patent Application No. 2003-128065 filed on May 6, 2003 by the present applicant).

The enzyme encoded by the iolI gene, scyllo-inosose isomerase, has an optimum pH of pH 5 to pH 9, and has the property of being activated by Mn ²⁺ , Zn ²⁺ or Co ²⁺ as an activator And an enzyme having an amino acid sequence consisting of 278 amino acid residues described in SEQ ID NO: 1 in the sequence listing shown below (molecular weight 31.6 kDalton). 2003-128065 specification).

Furthermore, it was confirmed that the above-mentioned new enzyme, scyllo-inosose isomerase, can produce D-kilo-1-inosose when it is reacted with scyllo-inosose in an aqueous scyllo-inosose solution. It was confirmed that D-kilo-inositol can be produced by chemically reducing inosource with NaBH ₄ (see the aforementioned Japanese Patent Application No. 2003-128065).

  In a method for producing D-kilo-inositol using an inexpensive raw material, D-kilo-inositol can be efficiently produced, and the produced D-kilo-inositol can be easily isolated and purified. A new method of producing kilo-inositol has been desired.

  By using only the reaction that causes the enzyme to act on the raw material, the present inventors can allow several enzymes to act on myo-inositol at a high substrate concentration, thereby efficiently producing D-kilo-inositol as a final product. We thought that it was possible to manufacture. Therefore, research was repeated for the purpose of developing a new method for producing D-kilo-inositol by reaction with various enzymes starting from myo-inositol.

During the course of the study, the inventors paid attention to myo-inositol 2-dehydrogenase encoded by a known iolG gene of Bacillus subtilis. This enzyme can recognize the axial hydroxyl group of myo-inositol and catalyzes the oxidation reaction of myo-inositol in the presence of the coenzyme NAD ⁺ to produce the corresponding ketone (ie, scyllo-inosose) from myo-inositol. It is known to be an enzyme that can be used. Furthermore, the present inventors conducted basic experiments using myo-inositol 2-dehydrogenase derived from Enterobacter aerogenesis (commercially available product from Sigma). As a result, myo-inositol 2-dehydrogenase derived from Enterobacter As is known, it not only catalyzes the oxidation reaction of myo-inositol to scyllo-inosose in the presence of NAD ⁺ , but also reduces D-kilo-1-inosose to D-chiro-inositol in the presence of NADH. The present inventors have now found that the reaction is also catalyzed.

  Furthermore, the present inventors have succeeded in cultivating Bacillus subtilis and separating myo-inositol 2-dehydrogenase, an enzyme encoded by the iolG gene of Bacillus subtilis, directly from the lysate of the cultured cells (described later). Example 1). Thus, the myo-inositol 2-dehydrogenase derived from Bacillus subtilis obtained in Example 1 described later by the present inventors is the same as the myo-inositol 2-dehydrogenase having the amino acid sequence of the formula (I) shown in Patent Document 17. Presumed to be an enzyme.

  When the present inventors acted on the D-kilo-1-inosose together with NADH, the present inventors obtained the Bacillus subtilis-derived myo-inositol 2-dehydrogenase obtained in Example 1 below, from the D-kilo-1-inosose. It has now been found that the reduction reaction to D-kilo-inositol can be catalyzed.

のD−キロ−1−イノソースにミオ−イノシトール 2−デヒドロゲナーゼと補酵素として還元型のニコチンアミドアデニンジヌクレオチド（NADH）とを反応させて次式（IV）

のD−キロ−イノシトールを生成させ、得られた反応液からD−キロ−イノシトールを回収することを特徴とする、D−キロ−イノシトールの製造方法が提供される。 Therefore, in the first present invention, the following formula

D-kilo-1-inosose is reacted with myo-inositol 2-dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH) as a coenzyme to give the following formula (IV)

Of D-kilo-inositol is produced, and D-kilo-inositol is recovered from the resulting reaction solution.

  The reaction of the first method of the present invention can be performed using water as a reaction medium. The myo-inositol 2-dehydrogenase used in this reaction can be myo-inositol 2-dehydrogenase derived from Bacillus subtilis or Enterobacter aerogenesis, preferably a known iolG gene present in the Bacillus subtilis chromosome Myo-inositol 2-dehydrogenase encoded by

  In the first inventive method, the raw material substrate used is D-kilo-1-inosose, which is a ketose or cyclic sugar ketone that can be dissolved in water at 36 ° C. at about 40%. The resulting D-kilo-inositol is a cyclic sugar alcohol that can be dissolved in water at 36 ° C. to a concentration of about 60%. However, the substrate concentration (D-kilo-1-inosose) in water in the enzymatic reaction of this method is 3% as the maximum D-kilo-1-inosose concentration when the reaction temperature is 36 ° C. This is because NADH required as a reactant has a solubility of 10% in water at 36 ° C., and the D-kilo-1-inosose concentration is about 1/3 of that.

  The reaction temperature in this method is not particularly limited as long as the enzyme reaction proceeds. Considering the solubility of the substrate, the stability of NADH, and the heat resistance of the enzyme used, it is desirable to carry out the reaction at a temperature of 20 ° C. to 50 ° C., preferably 36 ° C. Further, since the reaction is a homogeneous aqueous solution, it is not necessary to stir, but it is desirable to stir in order to make the temperature in the reaction mixture uniform.

In this enzyme reaction, NADH is required as a reactant, and NADH is converted to NAD ⁺ after performing a reducing action in the reaction solution. NAD ⁺ and NADH differ in pH stability in aqueous solution, NAD ⁺ is stable at pH 8.0 or lower, and NADH is stable at pH 8.0 or higher. Therefore, it is estimated that the pH of the reaction medium should be kept at about pH 8.0. In addition, since myo-inositol 2-dehydrogenase has an optimum pH of about pH 8.0, it is appropriate to proceed this reaction at pH 8.0, and this was confirmed experimentally.

Myo-inositol 2-dehydrogenase is used in an amount necessary to complete the desired reaction within the desired reaction time. Usually, the amount of activity to convert 1 μmole myo-inositol and NAD ⁺ to 1 μmole scyllo-inosose and NADH per minute at pH 9.0 at 25 ° C. is defined as 1 unit.

Furthermore, since myo-inositol 2-dehydrogenase is activated by Mg ²⁺ ions, it was experimentally confirmed that the reaction rate was increased by adding this metal ion.

Accordingly, in the first method of the present invention, NADH is added to the reaction medium for performing the enzyme reaction in an amount of 0.0001% to 10%, preferably 0.004% to 0.1%, and Mg ²⁺ ions are 0.001 mM to 10 mM, preferably 1 mM. By adding the Mg salt so that the pH of the reaction medium becomes preferably 7.7 to 8.3, D-kilo-inositol is efficiently produced.

  The reactant used in the enzyme reaction of the first method of the present invention is NADH, but NADPH can also be used. In view of stability, NADH is desirable, and the NADH concentration is desirably 0.0001% to 10%, preferably 0.04% to 0.1%.

  The pH of the enzyme reaction solution is adjusted in the range of pH 7.7 to pH 8.3, and the reaction is preferably carried out at pH 8.0. In addition, a buffer may be added if necessary to keep this pH during the reaction during the reaction. The type of the buffer solution to be added is not particularly limited, but a buffer solution having a buffering ability to maintain pH 8.0 is desirable, and more preferable examples include a phosphate buffer solution and a Tris buffer solution.

In the enzyme reaction in the first method of the present invention, D-kilo-inositol once produced from D-kilo-1-inosose is caused by the action of myo-inositol 2-dehydrogenase in the presence of NAD ⁺ produced from NADH. There is also a reverse reaction that is oxidized to D-kilo-1-inosose. Thus, a reduction reaction in which D-kilo-inositol is produced and a reverse reaction in which D-kilo-1-inosose is produced from D-kilo-inositol. It has been experimentally observed that the oxidation reaction reaches an equilibrium state.

  Furthermore, the myo-inositol 2-dehydrogenase used can also be used as an immobilized enzyme, and a general enzyme immobilization method such as a gel embedding method or an ion exchange resin adsorption method can be applied as the enzyme immobilization method. .

  Termination of the enzyme reaction can be achieved by deactivating the enzyme by heating, changing the pH, adding a protein denaturing agent, and the like. However, in consideration of the recovery and purification of D-kilo-inositol in the next step, it is desirable to deactivate the enzyme by heating. For example, the enzyme can be inactivated by heating the enzyme reaction solution at 70 ° C. to 120 ° C., preferably 80 ° C. to 90 ° C. for 10 to 20 minutes.

  In order to stop the enzyme reaction, the reaction can be stopped by separating and recovering the enzyme from the reaction solution. Such enzyme recovery is performed by separating the enzyme by passing the reaction solution through an ion exchange resin column. When an immobilized enzyme is used, the immobilized enzyme can be recovered by centrifuging or filtering the reaction solution.

In the first invention method, the enzyme reaction solution after the reaction stop treatment by heating needs to be centrifuged or filtered in order to remove insoluble matters generated by protein denaturation. Furthermore, the reaction solution from which insoluble matter has been removed is composed of ionic substances other than D-kilo-inositol existing in the liquid, that is, NAD ⁺ , metal ions, etc., and residual D-kilo-1-inosose and hydrophobic substances. Is passed through an ion exchange resin column for the purpose of removing water. The ion exchange resin column used at this time is either a strong basic ion exchange resin or a weak basic ion exchange resin, or a mixture of both, a strong acid ion exchange resin, and a weak acid ion exchange. It can be a column of any one of the resins or a mixture of both. As described above, the method of passing the reaction solution through the ion exchange resin column can also be used, but it is also possible to deionize the reaction liquid from which insoluble matter has been removed with the ion exchange resin particles in a batch-type stirring and filtration. It is.

  The eluate finally discharged from the ion exchange resin column is an aqueous solution containing D-kilo-inositol.

  Furthermore, the obtained filtrate or centrifugation supernatant is an aqueous solution of D-kilo-inositol, and if necessary, it can be decolorized using activated carbon. The activated carbon treatment method can be a method in which a solution is passed through activated carbon packed in a column shape, but it is also possible to decolorize by stirring and mixing in a batch system and filtering. When the decolorized D-kilo-inositol aqueous solution is concentrated and dried, a high-purity D-kilo-inositol solid is obtained.

  In addition, the inventors proceeded with research. That is, as described above, in the invention described in Japanese Patent Application No. 2003-128065 related to the earlier application of the present applicant, E. coli transformed with a plasmid incorporating a known iolI gene of Bacillus subtilis was prepared, The transformed Escherichia coli is cultured, and the enzyme encoded by the iolI gene, scyllo-inosose isomerase (protein having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing below) is obtained from the disrupted solution of the cultured cells. Successful. Based on the experience and knowledge gained at this time, Bacillus subtilis was cultured, the cultured cells of Bacillus subtilis were disrupted, and the experiment was conducted to separate and collect scyllo-inosose isomerase directly from the obtained Bacillus subtilis cell lysate. Tried.

  Based on the results of this experiment, the above-mentioned scyllo-inosose isomerase was successfully produced and separated directly from Bacillus subtilis (see the description of the sixth invention described later and Example 1 described later). .

  The scyllo-inosose isomerase directly produced and isolated from Bacillus subtilis by the method of Example 1 described later also has the amino acid sequence of SEQ ID NO: 1 in the sequence listing below and is obtained in the invention of Japanese Patent Application No. 2003-128065. The inventors have confirmed that they have enzymological properties consistent with those obtained. Then, it was confirmed that when scyllo-inosose isomerase derived from Bacillus subtilis obtained in Example 1 described later is allowed to act on scyllo-inosose, D-kilo-1-inosose can be produced.

のミオ−イノシトールにミオ−イノシトール 2−デヒドロゲナーゼと、補酵素として知られる酸化型のニコチンアミドアデニンジヌクレオチド（NAD⁺）とを水性反応媒質中で反応させて次式（II）

のシロ−イノソースを生成させる第１の反応ステップを行った。続いて、得られた式（II）のシロ−イノソースに、バチルス ズブチリス染色体中に存在する既知のiolI遺伝子のコードする酵素、すなわちシロ−イノソース イソメラーゼを水性反応媒質中で反応させて次式（III）

のD−キロ−1−イノソースを生成させる第２の反応ステップを実験し、さらに得られた式（III）のD−キロ−1−イノソースに、第１の反応ステップで使用したものと同じミオ−イノシトール 2−デヒドロゲナーゼと、を還元型のニコチンアミドアデニンジヌクレオチド（NADH）とを水性反応媒質中で反応させて次式(IV)

のD−キロ−イノシトールを生成させる第３の反応ステップを実験したところ、第３の反応ステップの反応溶液中に式（IV）のD−キロ−イノシトールが生成できることを知見した。 Based on the above-mentioned findings of the present inventors, the present inventors have first described the following formula (I) according to a known enzymatic method for producing scyllo-inosose from myo-inositol.

Myo-inositol was reacted with myo-inositol 2-dehydrogenase and an oxidized form of nicotinamide adenine dinucleotide (NAD ⁺ ), known as a coenzyme, in an aqueous reaction medium to give the following formula (II)

A first reaction step was performed to produce the scyllo-inosose. Subsequently, the obtained scyllo-inosose of the formula (II) is reacted with an enzyme encoded by a known iolI gene present in the Bacillus subtilis chromosome, that is, scyllo-inosose isomerase, in an aqueous reaction medium to give the following formula (III )

A second reaction step to produce a D-kilo-1-inosose of formula (III), and the resulting D-kilo-1-inosose of formula (III) is subjected to the same myo as used in the first reaction step. -Inositol 2-dehydrogenase is reacted with reduced nicotinamide adenine dinucleotide (NADH) in an aqueous reaction medium to give the following formula (IV)

When the third reaction step for producing D-kilo-inositol was tested, it was found that D-kilo-inositol of the formula (IV) can be produced in the reaction solution of the third reaction step.

のミオ−イノシトールにミオ−イノシトール 2−デヒドロゲナーゼと酸化型のニコチンアミドアデニンジヌクレオチド（NAD⁺）とを水性反応媒質中で反応させて次式（II）

のシロ−イノソースを生成させる第１の反応ステップを行い、式（II）のシロ−イノソースに、バチルス ズブチリス染色体中に存在する既知のiolI遺伝子のコードする酵素、すなわち配列表の配列番号１のアミノ酸配列を有する酵素であって、しかもシロ−イノソース イソメラーゼと命名された酵素を水性反応媒質中で反応させて次式（III）

のD−キロ−1−イノソースを生成させる第２の反応ステップを行い、式（III）のD−キロ−1−イノソースに、第１の反応ステップで使用したものと同じミオ−イノシトール 2−デヒドロゲナーゼと、還元型のニコチンアミドアデニンジヌクレオチド（NADH）とを水性反応媒質中で反応させて次式(IV)

のD−キロ−イノシトールを生成させる第３の反応ステップを行い、第３の反応ステップの反応溶液から式（IV）のD−キロ−イノシトールを回収する工程を行うことから成る、D−キロ−イノシトールの製造方法が提供される。 Accordingly, in the second invention (the invention of claim 1) , the following formula (I)

Myo-inositol was reacted with myo-inositol 2-dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) in an aqueous reaction medium to give the following formula (II)

A first reaction step of generating a scyllo-inosose of the enzyme, and the scyllo-inosose of formula (II) is converted into an enzyme encoded by a known iolI gene present in the Bacillus subtilis chromosome, that is, the amino acid of SEQ ID NO: 1 in the sequence listing An enzyme having a sequence, which is also named scyllo-inosose isomerase, is reacted in an aqueous reaction medium to give the following formula (III)

A second reaction step to produce a D-kilo-1-inosose of formula (III) and the same myo-inositol 2-dehydrogenase as used in the first reaction step. Is reacted with reduced nicotinamide adenine dinucleotide (NADH) in an aqueous reaction medium to give the following formula (IV)

A third reaction step of producing D-kilo-inositol, and a step of recovering D-kilo-inositol of formula (IV) from the reaction solution of the third reaction step. A method for producing inositol is provided.

  The myo-inositol 2-dehydrogenase used in the first reaction step and the third reaction step of the second method of the present invention can be myo-inositol 2-dehydrogenase derived from Bacillus subtilis or enterobacter aerogenesis, and preferably Is a myo-inositol 2-dehydrogenase encoded by a known iolG gene present in the Bacillus subtilis chromosome.

In the second method of the present invention, the reaction solution obtained in the first reaction step, that is, the above-mentioned 2-dehydrogenase, NADH converted from NAD ⁺ , generated scyllo-inosose and residual myo-inositol are contained. The reaction solution can be used immediately in the second reaction step as it is, and scyllo-inosose isomerase can be added to the reaction solution to proceed with the second reaction step. The reaction solution obtained in the second reaction step, that is, a reaction solution containing NADH, the produced D-kilo-1-inosose, the remaining scyllo-inosose isomerase, the 2-dehydrogenase, and myo-inositol. Can be used immediately in the third reaction step, and NADH and / or myo-inositol 2-dehydrogenase can be added or supplemented to the reaction solution as necessary to proceed with the third reaction step. In this way, in the reaction solution of the third reaction step, D-kilo-inositol can be formed and some unreacted D-kilo-1-inosose remains, and NAD ⁺ converted from NADH and the residual The scyllo-inosose isomerase and myo-inositol 2-dehydrogenase and myo-inositol are contained.

  In order to recover D-kilo-inositol from the reaction solution obtained in the third reaction step, first, the remaining enzyme is denatured by heating and precipitated, the precipitate is removed, and the ionic substance is removed from the remaining supernatant. Inosose can be removed by treatment with a strongly basic ion exchange resin, and D-kilo-inositol can be collected from an aqueous solution of a mixture of D-kilo-inositol and myo-inositol thus obtained by a concentration method or the like.

  The substrate used in the first reaction step of the second method of the present invention is myo-inositol, which is a cyclic sugar alcohol that can be dissolved up to 21% in water at 36 ° C. D-kilo-inositol produced in the third reaction step is a cyclic sugar alcohol that can be dissolved in water at 36 ° C. to about 60%. When using a reaction temperature of 36 ° C., myo-inositol can be added up to 21% in the first reaction step, but the reaction can also be carried out at a concentration of 21% or more by further raising the temperature.

The reaction temperatures in the first, second and third reaction steps in the second method of the present invention are not particularly limited as long as the reaction proceeds. Considering the solubility of the substrate, the stability of NAD ⁺ and NADH, and the heat resistance of the enzyme used, it is desirable to carry out the reaction at a temperature of 20 ° C. to 50 ° C., preferably 36 ° C. Further, when the enzyme is dissolved in water and used, it is a reaction in a homogeneous solution, so that it is not necessary to stir the reaction solution, but it is desirable to stir to make the temperature uniform.

The pH of the enzyme reaction solution in each reaction step is adjusted to a range of pH 7.0 to pH 9.0 to allow the reaction to proceed, but considering the stability of NAD ⁺ and NADH and the stability of scyllo-inosose, The pH is preferably 7.7 to 8.3, more preferably 8.0. In addition, a buffer solution can be added if necessary to keep an appropriate pH during the reaction. The type of buffer solution to be added is not particularly limited, but a buffer solution having a buffering ability that maintains pH 8.0 is desirable, and more preferably, a phosphate buffer solution, a Tris buffer solution, and the like are exemplified.

In the first reaction step of the second method of the present invention, in the reaction system in which myo-inositol is reacted with myo-inositol 2-dehydrogenase and NAD ⁺ to generate scyllo-inosose (ie, myo-inosose) by oxidation, The presence of NAD ⁺ as a reactant is necessary, and NAD ⁺ is converted to NADH as the reaction proceeds in the reaction solution. NAD ⁺ and NADH have different pH stability in an aqueous solution, NAD ⁺ is stable at pH 8.0 or lower, and NADH is stable at pH 8.0 or higher. Therefore, the pH of the reaction medium should be maintained at about 8.0 in the first reaction step.

Furthermore, in this first reaction step, since myo-inositol 2-dehydrogenase is activated by Mg ²⁺ ions, it was experimentally confirmed that the reaction rate is increased by adding this metal ion. .

Therefore, in the first reaction step of the second method of the present invention, NAD ⁺ is added as a reactant to the aqueous myo-inositol solution at a concentration of 0.0001% to 10%, preferably 0.004%, and Mg ²⁺ ions By adding Mg salt so that the pH of the reaction medium is 0.001 mM to 10 mM, preferably 1 mM, and maintaining the pH of the reaction medium at pH 7.0 to 9.0, preferably pH 7.7 to 8.3. It is done efficiently.

In the first reaction step, the 2-hydroxyl group of myo-inositol is oxidized under the action of NAD ⁺ due to the catalytic action of myo-inositol 2-dehydrogenase, and an oxidation reaction that generates scyllo-inosose occurs at the same time. NADH is generated from NAD ⁺ . In addition, the reductive reaction in which the produced scyllo-inosose once produces myo-inositol by the catalytic action of the 2-dehydrogenase under the action of NADH also occurs. Therefore, it was experimentally observed that the oxidation reaction in which scyllo-inosose is produced from myo-inositol and the reverse reduction reaction in which myo-inositol is produced from scyllo-inosose reach an equilibrium state.

  In the second reaction step of the second method of the present invention, the reaction of scyllo-inosose with scyllo-inosose isomerase to produce D-kilo-1-inosose is obtained in the first reaction step. To the aqueous solution containing scyllo-inosose, scyllo-inosose isomerase is added. In the aqueous solution of scyllo-inosose, the scyllo-inosose concentration at the start of the reaction is desirably 1% to 15%, preferably 10%.

  The scyllo-inosose isomerase acting on the scyllo-inosose aqueous solution may be an isolated scyllo-inosose isomerase or a crude enzyme solution containing the same. Furthermore, the scyllo-inosose isomerase used can also be used as an immobilized enzyme. As an enzyme immobilization method, a general enzyme immobilization method such as a gel embedding method or an ion exchange resin adsorption method can be applied.

The pH of the aqueous solution of scyllo-inosose to be used is desirably adjusted to pH 5-9, preferably pH 5-9, more preferably pH 7.5, which is the optimum pH of scyllo-inosose isomerase. Furthermore, as an activator, Mn ² + and Zn ²⁺ ions to a final concentration 0.01MM～10mM, preferably adding Mn salt and Zn salt to be 0.5 mM, there is to be desirable. The Mn salt and Zn salt used can be used as long as they are water-soluble salts. The reaction temperature is 20 ° C. to 50 ° C., preferably 40 ° C., when using a Bacillus derived scyllo-inosose isomerase.

  The reaction mixture obtained by adding the aqueous scyllo-inosose isomerase solution to the aqueous scyllo-inosose solution does not need to be stirred in order to become a homogeneous solution, but is preferably stirred to make the temperature uniform. The reaction of this second reaction step can proceed until the scyllo-inosose concentration and the D-kilo-1-inosose concentration reach an equilibrium value of 70:30.

  After completion of the reaction in the second reaction step, the enzyme can be recovered from the reaction solution. The enzyme can be separated and recovered by passing the reaction solution obtained when the aqueous isomerase enzyme solution is used through an ion exchange resin column. When an immobilized enzyme is used, the immobilized enzyme can be recovered by centrifuging or filtering the reaction solution.

In this way, the reaction solution, which is the remaining solution after removing the enzyme, contains unreacted scyllo-inosose and the produced D-kilo-1-inosose, and optionally contains metal ions, NADH and NAD ⁺ . Contains. Next, in order to remove metal ions, ionic substances such as NADH and NAD ⁺ from the residual liquid, the residual liquid is passed through an ion exchange resin column. When the obtained column effluent is concentrated and the concentrate is allowed to stand at room temperature, scyllo-inosose is selectively crystallized, so that it is separated and washed with water. By this operation, unreacted scyllo-inosose can be recovered. The D-kilo-1-inosose produced by the reaction is dissolved in the mother liquor after the crystal fractionation, and the D-kilo-1-inosose powder can be recovered by concentrating the mother liquor to dryness.

  In the second reaction step of the second method of the present invention, the ketone group of scyllo-inosose is regiospecifically transferred to the adjacent alcohol group by the catalytic action of scyllo-inosose isomerase, and D-kilo-1- An isomerization reaction that produces inosose occurs. In addition, since the once-generated D-kilo-1-inosose also undergoes a reverse isomerization reaction that returns to the scyllo-inosose by the action of the isomerase, the reaction of producing D-kilo-1-inosose from the scyllo-inosose It has been observed that the formation reaction of scyllo-inosose from D-kilo-1-inosose reaches an equilibrium state.

In the third reaction step of the second method of the present invention, an aqueous solution containing D-kilo-1-inosose obtained in the second reaction step (the reaction solution after completion of the reaction in the second reaction step can be used as it is). D-kilo-1-inosose is added with or supplemented with myo-inositol 2-dehydrogenase and NADH as necessary, and reacted with D-kilo-1-inosose in a reductive manner. Is generated. This third reaction step can be carried out in the same manner as in the first method of the present invention. That is, the reaction can be performed at a reaction temperature of 20 ° C. to 50 ° C., preferably 36 ° C., while maintaining the pH of the reaction medium at pH 7.7 to 8.3. NADH and Mg ²⁺ ions can be replenished prior to the start of the reaction or as needed during the reaction.

In this third reaction step, the 1-position ketone group of D-kilo-1-inosose is reduced by the reaction of NADH by the catalytic action of myo-inositol 2-dehydrogenase which is the same as the enzyme used in the first reaction step, A reduction reaction is performed to produce D-kilo-inositol. At the same time, NAD ⁺ is generated from NADH.

  In this third reaction step, a reduction reaction for producing D-kilo-inositol from D-kilo-1-inosose occurs by the action of the 2-dehydrogenase. It can be seen that the reverse oxidation reaction producing -1-inosose also occurs and both reactions reach equilibrium.

After the completion of this third reaction step, it contains the produced D-kilo-inositol, the remaining D-kilo-1-inosose, NAD ⁺ , NADH, and the remaining enzyme, and possibly residual. An aqueous solution containing scyllo-inosose is obtained as the final reaction solution.

  From this final reaction solution, the enzyme is first recovered, and then the desired D-kilo-inositol is recovered from the reaction solution after the enzyme recovery. The recovery of D-kilo-inositol from the reaction solution can be performed in the same manner as described for the first method of the present invention.

  A series of reactions occurring in the first to third reaction steps of the second method of the present invention is shown as a reaction chart (A) as follows.

第２の本発明方法は、その第１、第２および第３の各々の反応ステップをそれぞれ別々の反応容器中で行うことによりバッチ方式でも実施できるものであるけれども、第２の本発明方法を、単一の反応容器中でワン・ポット（one-pot）方式で実施できるように改変できるのではないかと本発明者らは考えた。

The second method of the present invention can be carried out in a batch mode by carrying out the first, second and third reaction steps in separate reaction vessels. The present inventors thought that it could be modified so that it could be carried out in a one-pot system in a single reaction vessel.

That is, referring to the result of studying the second method of the present invention, the first reaction step of scyllo-inosose formation in a reaction mixture comprising adding myo-inositol 2-dehydrogenase and NAD ⁺ to an aqueous myo-inositol solution is described. Prior to carrying out, the above-mentioned sciro which catalyzes the isomerization reaction from scyllo-inosose to D-kilo-1-inosose in an aqueous solution (ie reaction mixture) containing myo-inositol, the 2-dehydrogenase and NAD ^+. -Inosource When isomerase is also added and coexists, in the above reaction mixture, the conversion from myo-inositol to scyllo-inosose and the conversion from scyllo-inosose to D-kilo-1-inosose D-kilo-1-inosose can be converted to D-kilo-inositol, and D Km - inositol is produced as a final product, it was considered to be Shutoku this.

Therefore, scyllo-inosose isomerase and myo-inositol 2-dehydrogenase are simultaneously added to myo-inositol aqueous solution, and coenzyme NAD ⁺ is also added to coexist and act on myo-inositol to proceed a series of enzyme reactions. As a result, it was found that D-kilo-inositol was generated in the reaction solution at the final stage and could be detected. Therefore, for the second method of the present invention, the reaction obtained by adding both myo-inositol 2-dehydrogenase and scyllo-inosose isomerase and NAD ⁺ as a coenzyme from the beginning to an aqueous solution of myo-inositol. Even when the process is modified to consist of carrying out the first reaction step, the second reaction step and the third reaction step in sequence in the mixture, it is finally possible to add D-kilo in the reaction solution. -The inventors have found that inositol can be produced.

Therefore, as a modification of the second method of the present invention, in the third present invention (the invention of claim 4) , myo-inositol 2-dehydrogenase and oxidized or reduced nicotine with respect to an aqueous myo-inositol solution are used. An enzyme having the amidoadenine dinucleotide (NAD ⁺ or NADH) and the amino acid sequence of SEQ ID NO: 1 in the sequence list, which is also named scyllo-inosose isomerase, was added, and NAD was obtained in the resulting reaction mixture. ^A first reaction for producing scyllo-inosose from myo-inositol by the action of ⁺ and catalysis of myo-inositol 2-dehydrogenase, a conversion reaction from NAD ⁺ to NADH, and the amino acid sequence of SEQ ID NO: 1 The enzyme scyllo-inosose is converted from scyllo-inosose to D-kilo-1-inos by the catalytic action of isomerase. A second reaction for producing a source, a third reaction for producing D-kilo-inositol from D-kilo-1-inosose by the action of the produced NADH and the catalytic action of myo-inositol 2-dehydrogenase, and NADH There is provided a method for producing D-kilo-inositol, which comprises carrying out a conversion reaction to NAD ⁺ and recovering D-kilo-inositol from the obtained reaction liquid after the completion of these reactions.

  In the third method of the present invention, myo-inositol can be added to the aqueous myo-inositol solution used at a temperature of 20 to 50 ° C. at a concentration of 10 to 21% (by weight). By increasing the temperature further, Also, it can be added at a concentration of 21% or more.

  Furthermore, the reaction rate of scyllo-inosose isomerase can be increased experimentally by adding a small amount of scyllo-inosose or D-kilo-1-inosose, which is a reaction intermediate, to the aqueous myo-inositol solution. confirmed. The reaction intermediate scyllo-inosose added to the aqueous myo-inositol solution can also be added as a mixture with D-kilo-1-inosose, but in consideration of stability, it is desirable to add scyllo-inosose alone. It is desirable that the concentration of the scyllo-inosose in the inositol aqueous solution is 0.0001% to 10%, preferably 0.3% (weight).

The reactant or coenzyme added to the aqueous myo-inositol solution is NAD ⁺ , but NADH can also be used instead. In view of stability, NAD ⁺ is desirable, and the concentration of NAD ⁺ added is desirably 0.0001% to 0.2%, preferably 0.004% to 0.1%.

The pH of the reaction medium during the series of enzyme reactions is adjusted to a range of pH 7.0 to pH 9.0. In consideration of the stability of NAD ⁺ and NADH and the stability of scyllo-inosose, it is desirable to adjust the pH to pH 7.7 to pH 8.3, more preferably pH 8.0. In addition, a buffer solution can be added if necessary to maintain this pH during the reaction. The type of buffer solution to be added is not particularly limited, but a buffer solution having a buffering ability that maintains pH 8.0 is desirable, and more preferably, a phosphate buffer solution, a Tris buffer solution, and the like are exemplified.

Furthermore, since myo-inositol 2-dehydrogenase is activated by Mg ²⁺ ions and scyllo-inosose isomerase is activated by Mn ²⁺ ions, the reaction rate is increased by adding these metal ions. It was confirmed experimentally that it increased.

Therefore, in the third method of the present invention, NAD ⁺ is added to the myo-inositol aqueous solution at a concentration of 0.0001% to 0.2%, preferably 0.004% to 0.1%, and scyllo-inosose is added to 0.0001% to 10%. , Preferably at a concentration of 0.3%, and Mg salt is added so that the Mg ²⁺ ion is 0.001 mM to 10 mM, preferably 1 mM, and the Mn ²⁺ ion is 0.0001 mM to 1 mM, Preferably, it is preferable to add Mn salt to 0.1 mM, and to maintain the pH of the reaction mixture at pH 7.0 to 9.0, preferably pH 7.7 to 8.3, thereby producing D-kilo-inositol. Is done efficiently.

  The myo-inositol 2-dehydrogenase and scyllo-inosose isomerase added to the aqueous myo-inositol solution can be an isolated myo-inositol 2-dehydrogenase and an isolated scyllo-inosose isomerase, or these It can also be a crude enzyme solution containing.

  Furthermore, the myo-inositol 2-dehydrogenase and scyllo-inosose isomerase used can be immobilized enzymes, and the enzyme immobilization methods include general methods such as gel embedding and ion exchange resin adsorption. Enzyme immobilization methods can be applied.

The reaction temperature is not particularly limited as long as the reaction proceeds. Considering the solubility of the substrate, the stability of NAD ⁺ and NADH, and the heat resistance of the enzyme, it is desirable to react at a temperature of 20 ° C. to 50 ° C., preferably 36 ° C.

  The enzyme reaction that occurs in this method is a reaction that reaches equilibrium, so that myo-inositol can be added to the extent that myo-inositol does not precipitate during the reaction.

  In the third method of the present invention, the enzymatic reaction can be stopped when the reaction product produced by a series of enzymatic reactions reaches equilibrium. The enzymatic reaction is preferably allowed to proceed until the concentration ratio of myo-inositol and D-kilo-inositol in the resulting reaction solution reaches an equilibrium value of 88:12. The measurement of these concentration values can be carried out by various analytical instruments by a method capable of quantifying one or both of myo-inositol and D-kilo-inositol. Specifically, it can be measured by an optical rotation meter or analysis by HPLC.

  Termination of the enzyme reaction can be achieved by deactivating the enzyme by heating, changing the pH, adding a protein denaturing agent, or the like. However, in consideration of the recovery and purification of D-kilo-inositol in the next step, it is desirable to deactivate the enzyme by heating. For example, the enzyme can be deactivated and precipitated by heating the reaction solution after completion of the enzyme reaction at 70 ° C. to 120 ° C., preferably 80 ° C. to 90 ° C. for 10 to 20 minutes.

  In order to stop the enzyme reaction, the reaction can be stopped by recovering the enzyme from the reaction solution. Such enzyme recovery is performed by separating the enzyme by passing the reaction solution through an ion exchange resin column. When an immobilized enzyme is used, the immobilized enzyme can be recovered by centrifuging or filtering the reaction solution.

In the third invention method, the enzyme reaction solution after the reaction stop treatment needs to be centrifuged or filtered in order to remove protein insoluble matter produced by protein denaturation. Further, the deproteinized solution from which protein insoluble matter has been removed contains D-kilo-inositol and myo-inositol dissolved in the solution, and the remaining D-kilo-1-inosose and scyllo-inosose as well as ionic substances and Contains hydrophobic material. In order to remove D-kilo-1-inosose and scyllo-inosose remaining in the deproteinized solution from which protein insolubles have been removed, as well as ionic substances and hydrophobic substances such as NAD ⁺ , NADH and metal ions, The deproteinized solution is passed through an ion exchange resin column. The ion exchange resin column used at this time is either a strong basic ion exchange resin or a weak basic ion exchange resin, or a mixture of both, a strong acid ion exchange resin, and a weak acid ion exchange. It can consist of any one of the resins, or a column of a mixture of both. As described above, a method of passing a deproteinized solution through an ion exchange resin column can also be used. However, the deproteinized solution from which protein insolubles have been removed is mixed with ion exchange resin particles in a batch manner and filtered, and the inosource compound is filtered. It is also possible to remove and deionize.

  The eluate finally discharged from the ion exchange resin column is an aqueous solution containing D-kilo-inositol and myo-inositol. When this is concentrated, D-kilo-inositol remains dissolved but myo-inositol is not dissolved. It precipitates and can be collected by filtration.

As described above, the eluate finally leaving the ion exchange resin column is usually an aqueous solution containing D-kilo-inositol and myo-inositol, but the remaining scyllo-inosose and / or D In some cases, it still contains significant amounts of kilo-1-inosose. In this case, an aqueous solution containing D-kilo-inositol, myo-inositol, scyllo-inosose, and D-kilo-1-inosose, which has been discharged as the eluate, is used as a strongly basic ion exchange resin such as duo. When passed through a column of Wright A116 (OH ^- type), scyllo-inosose and D-kilo-1-inosose can be adsorbed and removed by the column resin, and the eluate that can finally be collected from the last resin column is: It is an aqueous solution containing only D-kilo-inositol and myo-inositol.

  Furthermore, if necessary, the eluate can be treated with activated carbon for decolorization and for removal of neutral lipids and glycolipids. The activated carbon treatment can be carried out by passing the eluate through activated carbon packed in a column, but it can also be decolorized by stirring and mixing in a batch system and filtering.

  In the third method of the present invention, after stopping or ending the series of enzyme reactions, the enzyme is denatured from the obtained reaction solution by the above-mentioned method by heating or the like and precipitated, and the precipitate is removed by centrifugation or filtration. When the obtained supernatant is processed through a strongly acidic ion exchange resin column and a strongly basic resin column, an aqueous solution containing myo-inositol and D-kilo-inositol in a weight ratio of approximately 9: 1 is obtained. It is normal.

  Therefore, research was conducted to develop a new method capable of recovering myo-inositol and D-kilo-inositol separately from an aqueous solution containing myo-inositol and D-kilo-inositol.

  In that study, a solid mixture of myo-inositol and D-kilo-inositol at a weight ratio of 9: 1 was first dissolved in a mixed solvent of water and various water-soluble organic solvents at a volume ratio of 1: 1. A series of trials were conducted. As a result of these tests, when the solid mixture was dissolved in a 1: 1 mixed solvent of water and isopropanol or n-propanol (water-soluble organic solvent) for dissolution, the mixed solvent It has been found that the solubility of myo-inositol in water is quite low and that of D-kilo-inositol is high. When the solid mixture is mixed and dissolved in a 1: 1 mixed solvent of water and another water-soluble organic solvent, the solubility of myo-inositol and the solubility of D-kilo-inositol in the mixed solvent is determined. There was no significant difference between them. As a result of these tests, an appropriate proportion of isopropanol and / or n-propanol or both are added to and mixed with an aqueous solution containing myo-inositol and D-kilo-inositol in a weight ratio of 80:20 to 90:10. It was then found that myo-inositol can preferentially precipitate and that D-kilo-inositol can remain in solution and a small amount of myo-inositol remains dissolved in the solution.

  Therefore, in the fourth aspect of the present invention, isopropanol or n-propanol or both are mixed in an aqueous solution containing myo-inositol and D-kilo-inositol in a weight ratio of 80:20 to 90:10, and obtained. A method for separating myo-inositol from D-kilo-inositol is provided, comprising preferentially precipitating myo-inositol from the resulting mixture, but leaving D-kilo-inositol dissolved in the aqueous solution.

  The aqueous solution containing myo-inositol and D-kilo-inositol used in the fourth method of the present invention contains myo-inositol and D-kilo-inositol in a weight ratio of 80:20 to 90:10, and myo. It may be an aqueous solution having a total concentration of inositol and D-kilo-inositol of 40-60% by weight and a water content of 60-40% by weight. By mixing 30% to 100% (volume) of isopropanol or n-propanol with stirring into the aqueous solution, myo-inositol can be precipitated and separated from the mixture. However, if the mixture containing the precipitated myo-inositol is left overnight, and then the precipitated myo-inositol crystals are filtered or centrifuged, the myo-inositol crystals can be collected and myo-inositol can be collected. And an aqueous solution containing D-kilo-inositol in a weight ratio of 40:60 to 20:80 was found to be obtained as a mother liquor or supernatant.

  Further, when the aqueous solution containing myo-inositol and D-kilo-inositol in a weight ratio of 40:60 to 20:80 obtained as the filtrate or supernatant is concentrated next, isopropanol or n-propanol is obtained. It is removed from the concentrate by azeotropy. By this concentration operation, a powder or syrup containing myo-inositol and D-kilo-inositol in a weight ratio of 40:60 to 20:80 can be obtained. 30-90 parts by weight of the powder or syrup thus obtained is dissolved in 70-10 parts by weight of water to form an aqueous solution, which is then subjected to myo-chromatography by chromatography through a strongly basic ion exchange resin column. It has been found that the inositol fraction and the D-kilo-inositol fraction can be separated and recovered from each other.

  Here, the aqueous solution of D-kilo-inositol, which is finally obtained as a D-kilo-inositol fraction from the myo-inositol fraction, can be concentrated to dryness and powdered. It is also possible to add and dissolve water-miscible organic solvent to recrystallize D-kilo-inositol. The water-miscible organic solvent added here is not particularly limited, but is preferably ethanol.

  In the study of the fourth method of the present invention, generally, myo-inositol and D-kilo-inositol are contained in a weight ratio of 20:80 to 30:70 and myo-inositol and D-kilo- By chromatography an aqueous solution of myo-inositol and D-kilo-inositol, which has a total concentration of 30-80% (by weight) with inositol, through a strongly basic ion exchange resin column, only myo-inositol is obtained. It was found that the eluate fraction containing and the eluate fraction containing only D-kilo-inositol can be separated and obtained separately.

  Accordingly, in the fifth aspect of the present invention, myo-inositol and D-kilo-inositol are contained in a weight ratio of 20:80 to 30:70, and the total concentration of myo-inositol and D-kilo-inositol is Preparing an aqueous solution of myo-inositol and D-kilo-inositol, which is 30-80% (by weight), and subjecting the aqueous solution to chromatography on a strongly basic ion exchange resin column, an eluate fraction containing only myo-inositol; And the eluate fraction containing only D-kilo-inositol, myo-inositol and D, consisting of concentrating the latter fraction and isolating D-kilo-inositol from the concentrate. A method for separating D-kilo-inositol from an aqueous solution containing kilo-inositol is provided.

  The myo-inositol 2-dehydrogenase used in the first invention, the second invention, and the third invention method is a myo-inositol 2-dehydrogenase encoded by a known iolG gene present in the Bacillus subtilis chromosome. Can do. Myo-inositol 2-dehydrogenase encoded by the iolG gene is an enzyme having a molecular weight of 38.3 k Dalton consisting of 344 amino acid residues shown in Sequence-1 of Sequence Listing of Patent Document 17 (Japanese Patent Laid-Open No. 05-192163). Yes (see the right column on page 7 of Patent Document 17).

  Furthermore, the scyllo-inosose isomerase used in the second and third methods of the present invention can be an enzyme having a molecular weight of about 31.6 k Dalton consisting of 278 amino acid residues shown in SEQ ID NO: 1 described later. As described above, this scyllo-inosose isomerase is an enzyme encoded by a known iolI gene present in the Bacillus subtilis chromosome, and this enzyme is disclosed in Japanese Patent Application No. 2003-128065 according to the earlier application of the present applicant. According to the invention described in 1), it was isolated by genetic engineering techniques from transformed E. coli introduced with the iolI gene.

  This time, the present inventors conducted further research, and as a result, cultured Bacillus subtilis and succeeded in isolating myo-inositol 2-dehydrogenase and scyllo-inosose isomerase separately from the cultured cells. . That is, Bacillus subtilis is cultured in a known bacterial culture medium containing myo-inositol at a culture temperature of 30 ° C. to 55 ° C., and the cells of the cultured bacteria are collected from the obtained culture solution. Bacteria and the collected pellet-like cells are washed with water and then centrifuged to collect the washed cells, and the washed cells are further added to 100 mM Tris buffer (pH 7.5) containing 0.6% Triton X-100. And the cells were crushed by ultrasonic waves. After disrupting the cells, the mixture was centrifuged, and the crude enzyme extract was collected as a supernatant. Thereafter, ammonium sulfate was added to the crude enzyme extract to 50% to salt out the protein.

  A solution in which the protein thus separated was dissolved in water or 100 mM Tris buffer (pH 7.5) was further centrifuged from this to remove insoluble matter, and a supernatant was obtained. The crude enzyme solution obtained as the supernatant was added to a Sephadex G100 column (diameter 12 cm, height 50 cm) for gel filtration at 4 ° C., and eluted with 20 mM Tris buffer (pH 7.0), followed by column chromatography. When the eluate is fractionated into fractions, a fraction containing myo-inositol 2-dehydrogenase is obtained as a fraction containing a substance having a molecular weight of 100 kD or more, and scyllo-inosose isomerase is used as a fraction containing a substance having a molecular weight of 20 to 50 kD. A fraction containing was obtained.

  Next, the fraction containing the above-mentioned substance having a molecular weight of 100 k Dalton or more is collected, added to a DEAE Toyopearl column, passed through, and then eluted with a NaCl concentration gradient in 20 mM Tris buffer (pH 7.0). The solution was subjected to column chromatography using an eluent. When the eluate was fractionated by fraction, myo-inositol 2-dehydrogenase activity was detected in the fraction eluted with about 250 mM NaCl. This fraction was collected to obtain a solution of myo-inositol 2-dehydrogenase. The enzyme can be precipitated from the solution by ammonium sulfate precipitation.

  Further, the fraction containing the substance having a molecular weight of 20 to 50 kDa is collected, added to a DEAE Toyopearl column, passed, and then eluted with a NaCl concentration gradient in 20 mM Tris buffer (pH 7.0). Column chromatography was performed using the solution. When the eluate was fractionated by fraction, scyllo-inosose isomerase activity was detected in the fraction eluted with about 30 mM NaCl. This fraction was collected to obtain a solution of scyllo-inosose isomerase. The enzyme can be precipitated from this solution by the ammonium sulfate precipitation method. The target enzyme obtained as a protein precipitate can be dissolved and stored in 100 mM Tris buffer (pH 7.5).

  In the above description, the protein precipitate obtained by salting out ammonium sulfate from the above crude enzyme solution is dissolved in 20 mM Tris buffer (pH 7.0), and after dialysis, adsorbed on an ion exchange resin and linear concentration gradient due to salt concentration. Although it isolate | separated and refine | purified by column chromatography using, purification by molecular weight fractionation by gel filtration is also possible. Also, since myo-inositol 2-dehydrogenase uses NAD as a coenzyme, it can be purified using an affinity column such as Brute Yopal.

Accordingly, in the present invention of the sixth (the invention of claim 8), myo encoding known iolG gene present in the chromosome of Bacillus subtilis - and inositol 2-dehydrogenase, known to be present in the chromosome iolI gene code for enzymes, an enzyme having the amino acid sequence of SEQ ID NO: 1, yet scyllo - culturing a Bacillus ShokuHoso bacteria capable of producing both the inosose isomerase named enzyme, cultured is obtained by accumulating the two enzymes in the cells, then harvested cultured cells were disrupted were harvested in buffer, centrifuged and the resulting cell lysate, supernatant and recovering separately from the resulting crude enzyme extract the two enzymes Examples, myo - inositol and 2-dehydrogenase, of SEQ ID No. 1 amino An enzyme having the sequence, yet scyllo - inosose isomerase and named the preparation of the enzyme is provided.

  In the sixth method of the present invention, a known strain such as an ATCC deposited strain can be used as the Bacillus subtilis to be cultured. The liquid medium to be used has a known composition, and it is preferable to add 1 to 10% (by weight) of myo-inositol for the induction of gene expression. The culture temperature is preferably in the range of 30 to 55 ° C. In order to disrupt the cells collected after culturing, it is convenient to irradiate the cells with a ultrasonic wave after suspending the cells in a buffer solution such as 100 mM Tris buffer (pH 7.5). The obtained cell lysate is centrifuged, and various enzyme separation means can be used to collect the two target enzymes from the crude enzyme extract obtained as a supernatant.

  For example, 50% strength ammonium sulfate is added to the crude enzyme extract obtained as the above supernatant, the protein is salted out, the precipitated protein is collected, dissolved in water, the aqueous solution is centrifuged, and the precipitate is discarded. The resulting protein-containing supernatant (crude enzyme solution) is subjected to gel fraction chromatography using a Sephadex G100 column. Thereby, the fraction containing myo-inositol 2-dehydrogenase and the fraction containing scyllo-inosose isomerase can be obtained separately. Each fraction was passed through a DEAE Toyopearl column and then subjected to column chromatography eluting with 20 mM Tris buffer (pH 7.0) under a linear NaCl gradient, and myo-inositol 2- A fraction containing only dehydrogenase and a fraction containing only scyllo-inosose isomerase are obtained as separate enzyme solutions. From each of these enzyme solutions, the enzyme protein can be precipitated and separated as a solid by ammonium sulfate precipitation.

  According to the first aspect of the present invention, D-kilo-1-inosose that can be prepared by enzymatic reaction from myo-inositol is used as a substrate, and myo-inositol 2-dehydrogenase and NADH are allowed to act on D-kilo-1-inosose. As a result, D-kilo-inositol can be produced easily and rapidly, and D-kilo-inositol with higher purity can be easily recovered.

  Further, according to the second and third methods of the present invention, starting from inexpensive myo-inositol, a series of enzyme reactions are performed at a high substrate concentration, so that D-kilo can be easily and rapidly performed. -Inositol can be produced, and D-kilo-inositol with high purity can be recovered from the final enzyme reaction solution by a simple method.

  Furthermore, according to the fourth method of the present invention and the fifth method of the present invention, myo-inositol or D-kilo-inositol can be easily and efficiently separated from an aqueous solution containing myo-inositol and D-kilo-inositol. Can be recovered.

  According to the sixth method of the present invention, myo-inositol 2-dehydrogenase, which is an enzyme useful in various fields, can be obtained from cultured cells of Bacillus bacteria, particularly Bacillus subtilis, without using genetic engineering techniques. Scyllo-inosose isomerase can be produced efficiently.

  Hereinafter, the sixth aspect of the present invention will be specifically described with reference to Example 1.

Preparation of myo-inositol 2-dehydrogenase and scyllo-inosose isomerase from Bacillus subtilis cells
(a) Preparation of crude enzyme extract liquid containing tryptone 1.0%, yeast extract 1.5%, KH ₂ PO ₄ 0.23%, K ₂ HPO ₄ 1.25%, MgCl ₂ 0.1% and myo-inositol 2% added Each 100 ml of medium (pH 7.0) was placed in 10 Sakaguchi flasks, autoclaved at 121 ° C. for 15 minutes, and cooled to room temperature. One platinum loop from a slant culture of Bacillus subtilis ATCC6633 was added to each medium in 10 flasks and inoculated. Thereafter, the inoculated medium (1000 ml in total) was cultured with shaking at 36 ° C. for 16 hours on a reciprocating shaker. The resulting culture was then aseptically added to 100 liters of medium having the same composition as above and autoclaved in a jar fermenter. The inoculated medium was aerobically cultured for 7 hours at 36 ° C. with aeration of 1 vvm under stirring at 200 rpm in a jar fermenter. The obtained culture broth was centrifuged to collect the cells, and the collected cells (wet weight of about 400 g) were suspended in 400 ml of water. The bacterial cell suspension was pumped at a temperature of 10 ° C. or less to an ultrasonic crusher (output 600 W, cooling water circulation type) at a flow rate of 50 ml / min to crush the bacterial cells. 100 ml of water was used for washing the crushing apparatus, and the washing solution was added to the cell disruption solution. The obtained bacterial cell disruption solution was centrifuged to discard insoluble solids, and 500 ml of the supernatant was taken out. This supernatant is a crude enzyme extract (500 ml) containing myo-inositol 2-dehydrogenase and scyllo-inosose isomerase.

(b) Preparation of myo-inositol 2-dehydrogenase crude enzyme solution from crude enzyme extract and preparation of scyllo-inosose isomerase crude enzyme solution Next, 450 g of ammonium sulfate was added to the above crude enzyme extract at 4 ° C. The protein was salted out. The resulting mixture was centrifuged to obtain a protein precipitate. The supernatant was discarded. The obtained protein precipitate was dissolved in 300 ml of water, and insoluble matters generated in the obtained aqueous solution were removed by centrifugation. The supernatant obtained here is passed through a Sephadex G100 column (diameter 12 cm, height 50 cm) for gel filtration at 4 ° C., and the column is then eluted with 20 mM Tris buffer (pH 7.0) to obtain column chromatography. Was done. When the column eluate is fractionated into 10 ml fractions, a crude enzyme solution of myo-inositol 2-dehydrogenase is obtained as a fraction containing a substance having a molecular weight of 100 kDa or more, and as a fraction containing a substance having a molecular weight of 20 to 50 kDa. A crude enzyme solution of inosource isomerase was obtained.

(c) Separation of myo-inositol 2-dehydrogenase enzyme The crude enzyme solution obtained by collecting the above fractions having a molecular weight of 100 kDa or more was passed through a DEAE Yovar column (diameter 5 cm, height 25 cm), and then the column was subjected to 20 mM Tris buffer. Column chromatography was performed by elution with a solution (pH 7.0) under a linear NaCl gradient. When the column eluate was fractionated into 10 ml fractions, myo-inositol 2-dehydrogenase activity was detected in the fraction eluted at a concentration of about 250 mM NaCl. These fractions were collected to obtain 100 ml of a purified enzyme solution containing myo-inositol 2-dehydrogenase. When ammonium sulfate was added to the enzyme solution at a concentration of 50%, 100 mg of myo-inositol 2-dehydrogenase was precipitated.

(d) Separation of scyllo-inosose isomerase enzyme The crude enzyme solution obtained by collecting the above fractions having a molecular weight of 20 to 50 kDa was passed through a DEAE Yovar column (diameter 5 cm, height 25 cm), and then the column was subjected to 20 mM Tris buffer ( Column chromatography was performed by eluting under a linear concentration gradient of NaCl at pH 7.0). When the column eluate was fractionated into 10 ml fractions, scyllo-inosose isomerase activity was detected in the fraction eluted at a concentration of about 300 mM NaCl. These fractions were collected to obtain 100 ml of scyllo-inosose isomerase enzyme solution. When ammonium sulfate was added to the enzyme solution at a concentration of 50%, 100 mg of scyllo-inosose isomerase was precipitated.

The following method was used for the assay of the enzyme activities of the two enzymes described above.
The assay for myo-inositol 2-dehydrogenase activity is performed as follows. That is, 100 μl of enzyme-containing fraction solution, 100 μl of 10% myo-inositol aqueous solution, 100 μl of 1M Tris buffer (pH 8.0), 10 μl of 100 mM MgSO ₄ aqueous solution, 10 μl of 1% NAD ⁺ aqueous solution, diaphorase 5U and 2 μl of 1% NTB solution A reaction mixture solution was prepared by adding water to the mixture so that the total volume was 1 ml. This reaction mixture solution was kept at 27 ° C. for 10 minutes for enzyme reaction. After the reaction, myo-inositol 2-dehydrogenase activity can be assayed by measuring absorbance at 565 nm light derived from reduced NTB.

The assay for scyllo-inosose isomerase activity is performed as follows. That is, 100 μl of enzyme-containing fraction solution, 100 μl of 10% scyllo-inosose solution, 100 μl of 1M Tris buffer (pH 7.5), 10 μl of 100 mM MnCl ₂ solution and 10 μl of 100 mM ZnCl ₂ solution were added to make a total volume of 1 ml. A reaction mixture solution was prepared. The basic reaction mixture prepared in this way was kept at 27 ° C. for 16 hours for enzyme reaction. After the reaction, 10 μl of 1M phosphoric acid (pH 2.0) was added to stop the enzyme reaction. Furthermore, the obtained mixture was heated at 70 ° C. for 10 minutes to denature and precipitate the protein, and then centrifuged. The obtained supernatant was subjected to HPLC analysis, and when the amount of D-kilo-1-inosose produced was assayed, scyllo-inosose isomerase activity could be assayed. For the HPLC operation, a WakosilNH ₂ column (inner diameter 4.6 mm × 25 cm) was used, the mobile phase was 80% acetonitrile aqueous solution, the column temperature was 20 ° C., the flow rate was 2 ml / min, the detector was a UV detector, and the OR detector. Used. HPLC under these conditions can detect D-kilo-1-inosose with an elution time of 18 minutes.

  Next, the first embodiment of the present invention will be specifically described with reference to Example 2.

Production of D-kilo-inositol from D-kilo-1-inosose by the action of myo-inositol 2-dehydrogenase and NADH
In a 100 ml Erlenmeyer flask, 20 units of myo-inositol dehydrogenase (manufactured by Sigma, Enterobacter), 2 g of D-kilo-1-inosose (containing 97% purity and 3% scyllo-inosose), NADH (oriental yeast) 9 g), 1 ml of 100 mM magnesium chloride solution and 5 ml of 1M Tris-HCl buffer solution (pH 8.0) were added, and water was added to a volume of 100 ml to prepare a reaction mixture solution. This solution was allowed to undergo an enzyme reaction by standing at 36 ° C. for 16 hours.

After completion of the reaction, 100 ml of the obtained enzyme reaction solution is mixed with a 50 ml strongly acidic ion exchange resin (Duolite C20 H ⁺ type) column, a 50 ml strongly basic ion exchange resin (Duolite A1160OH ^- type) column, and 20 ml. Were sequentially passed through the activated carbon column at a flow rate of 2 ml / min. Finally, each column was washed with 80 ml of ion-exchanged water, and these washing solutions were combined to obtain a passing solution having a total amount of 180 ml. This solution was concentrated to dryness with an evaporator to obtain 0.82 g of a solid.

  When this solid was analyzed by HPLC, it was a mixture of D-kilo-inositol and myo-inositol having a composition of D-kilo-inositol: myo-inositol (reduced form of scyllo-inosose) = 97: 3.

Then, 500 ml of a strong basic ion exchange resins ^- a (Duolite A1160 OH type) column was prepared and the chromatographic separation was passed through the solution and 4ml of the above mentioned solid 0.82g this column was dissolved in water went. Unreacted D-kilo-1-inosose could be adsorbed and removed by the strongly basic ion exchange resin. Fractions were fractionated every 4 ml, and the myo-inositol fraction eluting first and the D-kilo-inositol fraction eluting later were obtained separately. From the HPLC analysis of each fractionated solution, fractions containing only D-kilo-inositol were collected and concentrated with an evaporator to obtain 0.74 g of a white powder of D-kilo-inositol.

  This white powder was D-kilo-inositol having a purity of 99.5% or more from the results of HPLC analysis and NMR analysis. From 2 g of D-kilo-1-inosose, 0.74 g of D-kilo-inositol was obtained, and the yield was 37%.

  Furthermore, the second aspect of the present invention will be specifically described with respect to Example 3 (batch method).

D by a method comprising the steps of myo-inositol to scyllo-inosose, scyllo-inosose to D-kilo-1-inosose, and D-kilo-1-inosose to D-kilo-inositol. -Production of kilo-inositol
(1) As the first reaction step, in a 1 liter Erlenmeyer flask, 200 units of myo-inositol dehydrogenase (manufactured by Sigma, Enterobacter), 20 g of myo-inositol, 90 g of NAD ⁺ (manufactured by Oriental Yeast), 100 ml of 100 mM magnesium chloride solution and 50 ml of 1M Tris-HCl buffer solution (pH 8.0) were added, and water was added to make 1 liter to prepare a reaction mixture solution. This prepared solution was allowed to undergo an enzyme reaction by standing at 36 ° C. for 16 hours.

After completion of the reaction, the enzyme reaction solution 1 liter obtained a strongly acidic ion exchange resin (Duolite C20 H ⁺ type) column of 500 ml, weakly basic ion exchange resin of 500 ml (Duolite 368S OH ^- type) column and The solution was sequentially passed through a 200 ml activated carbon column at a flow rate of 10 ml / min. Finally, each column was washed with 800 ml of ion-exchanged water, and the washing solution was combined to obtain a passing solution having a total amount of 1.8 liters. This solution was concentrated to dryness with an evaporator to obtain 19.3 g of a solid.

  When this solid was analyzed by HPLC, it was a mixture of scyllo-inosose and myo-inositol having a composition of scyllo-inosose: myo-inositol = 56: 44.

  (2) As the second reaction step, in a 500 ml Erlenmeyer flask, 20 ml of the syleuno source isomerase enzyme solution (enzyme solution purified from 1 liter of Bacillus subtilis culture solution) prepared in Example 1 (c) and the above reaction 19.3 g of the solid obtained in the step, 0.2 ml of 100 mM manganese chloride solution and 10 ml of 1M Tris-HCl buffer solution (pH 7.5) were added, and water was added to 200 ml to prepare a reaction mixture solution. This prepared solution was allowed to stand still at 36 ° C. for 16 hours to produce D-kilo-1-inosose from scyllo-inosose.

After completion of the reaction, the enzyme reaction solution 200ml obtained, and strongly acidic ion exchange resin (Duolite C20 H ⁺ type) column of 100 ml, weakly basic ion exchange resin 100 ml ^- and (Duolite 368S OH type) column, The solution was sequentially passed through a 40 ml activated carbon column at a flow rate of 2 Aml / min. Finally, each column was washed with 160 ml of ion-exchanged water, and the washing solutions were combined to obtain a total amount of 360 ml of passing solution. This solution was concentrated to dryness with an evaporator to obtain 17.9 g of a solid.

  When this solid was analyzed by HPLC, it was a mixture having a composition of D-kilo-1-inosose: shiro-inosose: myo-inositol = 15: 36: 49.

  (3) As the third reaction step, in a 1-liter Erlenmeyer flask, 100 units of myo-inositol dehydrogenase (manufactured by Sigma, Enterobacter), 17.9 g of solid obtained in the second reaction step, NADH ( (Oriental Yeast Co., Ltd.) (41 g), 100 mM magnesium chloride solution (5 ml), 1M Tris-HCl buffer solution (pH 8.0) (25 ml) was added, and water was added to a volume of 500 ml to prepare a reaction mixture solution. This prepared solution was allowed to undergo an enzyme reaction by standing at 36 ° C. for 16 hours. D-kilo-inositol was produced from D-kilo-1-inosose.

After completion of the reaction, 500 ml of the resulting enzyme reaction solution is added to a 250 ml strongly acidic ion exchange resin (Duolite C20 H ⁺ type) column, a 250 ml strongly basic ion exchange resin (Duolite A1160 OH ⁻ type) column, and 100 ml. The activated carbon column was sequentially passed at a flow rate of 5 ml / min. Finally, the column was washed with 150 ml of ion-exchanged water, and the water washes were combined to obtain a passing solution having a total amount of 650 ml. This solution was concentrated to dryness with an evaporator to obtain 14.0 g of a solid.

  When this solid was analyzed by HPLC, it was a mixture of D-kilo-inositol and myo-inositol consisting of D-kilo-inositol: myo-inositol = 11: 89.

Next, a 3 liter strongly basic ion exchange resin (Duolite A1160 OH ^- type) column was prepared, and 14.0 g of the solid was dissolved in water in this column. Chromatographic separation was performed by passing 30 ml of the solution. Fractionation was carried out for each 15 ml fraction, and the myo-inositol fraction eluting first and the D-kilo-inositol fraction eluting later were obtained separately. From the HPLC analysis of each fractionated solution, fractions containing only D-kilo-inositol were collected and concentrated with an evaporator to obtain 1.22 g of a white powder.

  This white powder was D-kilo-inositol having a purity of 99.5% or more from the results of HPLC analysis and NMR analysis. From 20 g of myo-inositol, 1.22 g of D-kilo-inositol was obtained through the first, second and third reaction steps, and the yield was 6.1%.

  Next, the third aspect of the present invention will be specifically described with respect to Example 4 (one-pot system) and Example 5.

Production of D-kilo-inositol by conducting a series of enzymatic reactions in a reaction mixture containing myo-inositol and myo-inositol 2-dehydrogenase, NAD ⁺ and scyllo-inosose isomerase
(a) In a jar fermenter, 1.0 kg of myo-inositol, 1.2 g of magnesium sulfate heptahydrate and 0.1 g of manganese chloride tetrahydrate were dissolved in 1.7 liters of water at 70 ° C., and the total amount was 4.5 Water was added to make a liter to make an aqueous solution. The aqueous solution was cooled to 40 ° C. and 0.3 liter of 5% aqueous scyllo-inosose solution was added. The resulting mixture solution was further cooled to 36 ° C., and the pH was adjusted to 8.0 by addition of 1N NaOH aqueous solution. Next, 0.1 liter of each of the two purified enzyme solutions prepared in Examples 1 (c) and (d), that is, 0.1 liter of the purified enzyme solution of myo-inositol 2-dehydrogenase prepared in Example 1 (c) 0.1 liter of purified enzyme solution of scyllo-inosose isomerase prepared in Example 1 (d) was added to the above mixture solution. The container containing the enzyme solution was washed with 0.02 liters of water, and the washings were also added. When the above mixture solution to which the enzyme solution was added showed pH 8.0, 0.2 g of NAD ⁺ (manufactured by Oriental Yeast) powder was added and dissolved. The initial concentration of myo-inositol in 5.0 liters of reaction mixture obtained here corresponded to 20% (weight).

  The reaction mixture obtained above was incubated by keeping it under agitation at 36 ° C., thereby proceeding with the enzyme reaction. While the enzyme reaction was in progress, the pH value of the reaction mixture was adjusted to pH 8.0 by appropriate addition of 0.05N aqueous NaOH and 0.05N aqueous hydrochloric acid. The enzyme reaction was thus continued at 36 ° C. for 10 hours.

  After performing the enzyme reaction for 10 hours, the resulting enzyme reaction solution was heated in a jar fermenter at 85 ° C. for 25 minutes to denature the enzyme and stop the enzyme reaction. Thereafter, the reaction solution was cooled to 60 ° C. to precipitate the denatured protein from the reaction solution, and 5.1 liter of supernatant was obtained by centrifugation. The jar fermenter was washed with 0.1 liter of water and the wash was added to the supernatant.

  The enzyme reaction solution that was stopped after the enzyme reaction for 10 hours in the above experiment was subjected to denaturation of the enzyme protein by heating, and when subjected to centrifugation for removal of the denatured protein, the above supernatant was Obtained. This supernatant was analyzed by HPLC and was found to contain the desired D-kilo-inositol, plus D-kilo-1-inosose, scyllo-inosose and myo-inositol.

  In order to recover D-kilo-inositol from the supernatant and to separate myo-inositol, the supernatant was treated by the method shown in Example 5 below.

(b) Another experiment was performed in which the above reaction mixture was allowed to undergo an enzymatic reaction at 36 ° C. and pH 8.0 for 13 hours. In this separate experiment, during a reaction time of 13 hours, 20 μl of the reaction solution was sampled from the reaction solution at 1 hour intervals, immediately the sample solution was heated at 80 ° C. for 10 minutes, 300 μl of water was added, mixed, Further, the precipitate was discarded by centrifugation, and a supernatant was obtained. The supernatant was analyzed by HPLC.

The HPLC analysis of the above sample supernatant was performed by passing the sample supernatant through a WakosilNH ₂ column (φ4.6 mm × 250 mm), and then flowing 80% acetonitrile aqueous solution (flow rate 2 ml / min) as the mobile phase. An RI detector was used and the column temperature was 40 ° C.

  By this HPLC analysis, the concentration of D-kilo-inositol, myo-inositol, D-kilo-1-inosose and scyllo-inosose in a series of sample supernatants collected at regular intervals was measured. From the D-kilo-inositol concentration and myo-inositol concentration in the sample supernatant collected every reaction time, the D-kilo-inositol conversion rate (%) was calculated by the following formula.

D-kilo-inositol conversion rate (%) = [(D-kilo-inositol concentration) / myo-inositol concentration at the start of reaction]] × 100
The relationship between the measured conversion rate (%) of D-kilo-inositol (DCI) and reaction time is shown in the following table.

算定されたDCI変換率（％）と反応時間（ｈ）との関係曲線を添付図面の図１に示す。図１に示すように、反応時間が８時間目で反応液中のミオ−イノシトールからD−キロ−イノシトールへの変換率(%)が12％になって、ほぼこの点で平衡に達したことが判る。

A relationship curve between the calculated DCI conversion rate (%) and the reaction time (h) is shown in FIG. 1 of the accompanying drawings. As shown in FIG. 1, the conversion time (%) from myo-inositol to D-kilo-inositol in the reaction solution was 12% at the reaction time of 8 hours, and the equilibrium was almost reached at this point. I understand.

  Next, the fourth aspect of the present invention and the fifth aspect of the present invention will be specifically described with reference to Example 5 below.

Recovery and purification of D-kilo-inositol from the enzyme reaction solution and separation of myo-inositol The enzyme was precipitated from the reaction solution obtained by enzymatic reaction in Example 4 (a) for 10 hours and removed by centrifugation. The resulting supernatant 5.1 liter was used in this example.

(a) Separation of aqueous solutions of D-kilo-inositol and myo-inositol from the enzyme reaction supernatant The above supernatant (5.1 L) contains the target product D-kilo-inositol, in addition to this An aqueous solution containing myo-inositol, D-kilo-1-inosose and scyllo-inosose, and further containing metal ions, NAD ⁺ and NADH and a hydrophobic substance. To remove D-kilo-1-inosose and scyllo-inosose as well as ionic and hydrophobic substances from this supernatant, 0.3 liters of strongly acidic ion exchange resin (Duolite C20 , H ⁺ type) column and 0.4 liter of strongly basic ion exchange resin (Duolite A116, OH ⁻ type) column sequentially at a flow rate of 5 ml / min, and sequentially through a 0.1 liter activated carbon column at a flow rate of 5 ml / min. Allowed to pass in minutes. The passing liquid (5.0 liters) from the activated carbon column was stored.

  Subsequently, 1.0 liter of deionized exchange water was sequentially passed through the three columns to wash the column, and the resulting washing solution was combined with the activated carbon column passing solution. As a result, an aqueous solution of D-kilo-inositol and myo-inositol (total amount 6.0 liters) was obtained.

  The above aqueous solution (6.0 liters) of D-kilo-inositol and myo-inositol was placed in an evaporator, and the aqueous solution was concentrated to 2.0 kg (about 1.6 liters). The resulting concentrated solution was in the form of a slurry because part of myo-inositol was crystallized. The slurry concentrate was centrifuged to separate crystallized myo-inositol and to obtain a supernatant. When the myo-inositol and D-kilo-inositol aqueous solution obtained as supernatants were analyzed by HPLC, the concentration ratio of myo-inositol: D-kilo-inositol was 69:31.

(b) Separation of myo-inositol solids from myo-inositol and D-kilo-inositol aqueous solution (according to the fourth invention)
To the myo-inositol and D-kilo-inositol aqueous solutions obtained as the supernatant obtained in (a) above, 0.3 liters of isopropanol was added with stirring. With the mixing of isopropanol, myo-inositol solid precipitated. This solid could be separated by centrifugation. The whole mixture containing the solid was allowed to stand, and the supernatant was analyzed by HPLC. The myo-inositol: D-kilo-inositol concentration ratio in the supernatant was 46:54.

  When the above mixture consisting of the precipitated myo-inositol solid and the supernatant was allowed to stand at room temperature overnight, solid precipitation of myo-inositol progressed, and the myo-inositol: D-kilo-inositol concentration ratio in the supernatant portion was 30:70.

  The above mixture that was allowed to stand overnight at room temperature was stirred to obtain an aqueous solution of myo-inositol and D-kilo-inositol containing crystals and forming a slurry. This slurry aqueous solution was filtered to obtain crystals containing myo-inositol and myo-inositol and D-kilo-inositol aqueous solution (1.34 liters) as filtrate. The crystals were washed with 50% aqueous isopropanol (0.2 liter) to obtain crystals (820 g). The weight ratio of myo-inositol and D-kilo-inositol contained in this crystal was 99: 1. The concentration ratio of myo-inositol: D-kilo-inositol contained in the filtrate was 29:71.

  The myo-inositol and D-kilo-inositol aqueous solution obtained as the above filtrate was then concentrated by an evaporator to obtain 146 g of crystals. This crystal contains 42 g of myo-inositol and 104 g of D-kilo-inositol.

(c) Chromatographic separation of D-kilo-inositol from myo-inositol and D-kilo-inositol aqueous solution (according to the fifth invention)
Crystals (146 g) containing 42 g of myo-inositol and 104 g of D-kilo-inositol obtained in (b) above were dissolved in water to prepare an aqueous solution (0.3 liter).

This prepared myo-inositol and D-chiro-inositol aqueous solution (myo-inositol: D-chiro-inositol concentration ratio of about 12:15) was added to 6.0 liter of strongly basic ion exchange resin, Duolite A116 (OH ^- type) was passed through the column, it was carried out chromatographic separation. The column eluate was fractionated with 200 ml fraction to obtain the myo-inositol fraction eluting first, and the D-kilo-inositol fraction eluting later was obtained and separated. From HPLC analysis of fractionated solutions of each fraction, fractions containing only D-kilo-inositol were selected and collected. The collected aqueous solution was concentrated with an evaporator to obtain 201 g of a white powder.

  From the results of HPLC analysis and NMR analysis, this white powder was found to be D-kilo-inositol with a purity higher than 99.5%. When the results of Example 4 and Example 5 were combined, 101 g of D-kilo-inositol was obtained from 1.0 kg of myo-inositol, and the yield from myo-inositol was 10.1%.

  Moreover, as myo-inositol (including a small amount of D-kilo-inositol) obtained in the recovery stage, a total of 888 g of myo-inositol was recovered in the crystallization stage and the chromatographic fractionation stage. This can again be used for enzymatic reactions.

  As is clear from the above description, any of the first to sixth methods of the present invention can be used for the production of D-kilo-inositol which is useful as a medicine, and thus is industrially useful.

6 is a curve graph showing the relationship between the conversion rate (%) of D-kilo-inositol produced in the reaction solution of the series of enzyme reactions performed in Example 4 and the reaction time.

Claims (8)

Formula (I)

Myo-inositol was reacted with myo-inositol 2-dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) in an aqueous reaction medium to give the following formula (II)

A first reaction step of generating a scyllo-inosose of the enzyme, and the scyllo-inosose of formula (II) is converted into an enzyme encoded by a known iolI gene present in the Bacillus subtilis chromosome, that is, the amino acid of SEQ ID NO: 1 in the sequence listing An enzyme having a sequence, which is also named scyllo-inosose isomerase, is reacted in an aqueous reaction medium to give the following formula (III)

A second reaction step to produce a D-kilo-1-inosose of formula (III) and the same myo-inositol 2-dehydrogenase as used in the first reaction step. Is reacted with reduced nicotinamide adenine dinucleotide (NADH) in an aqueous reaction medium to give the following formula (IV)

A third reaction step of producing D-kilo-inositol, and a step of recovering D-kilo-inositol of formula (IV) from the reaction solution of the third reaction step. A method for producing inositol. 2. The method according to claim 1, wherein the myo-inositol 2-dehydrogenase used is myo-inositol 2-dehydrogenase from Bacillus subtilis or from Enterobacter aerogenesis. The method according to claim 1, wherein the myo-inositol 2-dehydrogenase used is myo-inositol 2-dehydrogenase encoded by a known iolG gene in the Bacillus subtilis chromosome. Myo-inositol 2-dehydrogenase and oxidized or reduced nicotinamide adenine dinucleotide (NAD) against myo-inositol aqueous solution ⁺ Or NADH) and an enzyme having the amino acid sequence of SEQ ID NO: 1 in the Sequence Listing, and an enzyme designated as scyllo-inosose isomerase, and NAD in the resulting reaction mixture. ⁺ And the first reaction of generating scyllo-inosose from myo-inositol by the catalytic action of myo-inositol 2-dehydrogenase, and NAD ⁺ A second reaction of generating D-kilo-1-inosose from scyllo-inosose by the catalytic action of scyllo-inosose isomerase, which is the aforementioned enzyme having the amino acid sequence of SEQ ID NO: 1, A third reaction in which D-kilo-1-inositol is produced from D-kilo-1-inosose by the action of NADH and myo-inositol 2-dehydrogenase, and NADH to NAD ⁺ And a method for producing D-kilo-inositol, which comprises recovering D-kilo-inositol from the obtained reaction solution after completion of the reaction. (1) NAD from the beginning in myo-inositol aqueous solution ⁺ Or add NADH at a concentration of 0.0001% to 0.2%, (2) Mg ²⁺ Adding Mg salt so that the ion concentration is 0.001 mM to 10 mM, (3) Mn ²⁺ The method according to claim 4, characterized in that the Mn salt is added so that the ions have a concentration of 0.0001 mM to 1 mM, and (4) the pH of the reaction mixture is kept at pH 7.7 to 8.3. The method according to claim 4, wherein scyllo-inosose is initially added to the aqueous myo-inositol solution at a concentration of 0.0001% to 10% (by weight). The myo-inositol 2-dehydrogenase initially added to the aqueous solution of myo-inositol and the enzyme having the amino acid sequence of SEQ ID NO: 1 in the sequence listing and also named scyllo-inosose isomerase are these 2 Bacillus subtilis as a bacterium having the ability to produce two enzymes together, accumulate the two enzymes in the cells of the bacterium, disrupt the cultured cells, and extract from the obtained cell lysate Myo-inositol 2 in the form of a crude enzyme solution containing the two enzymes obtained as described above, or isolated and purified from the cell lysate by the known enzyme separation method and enzyme purification method, respectively. -Dehydrogenase and the above-mentioned scyllo-inosose isomerase having the amino acid sequence of SEQ ID NO: 1 The method of claim 4. It has a myo-inositol 2-dehydrogenase encoded by a known iolG gene present in the chromosome of Bacillus subtilis and an enzyme encoded by the known iolI gene present in the chromosome, that is, the amino acid sequence of SEQ ID NO: 1 in the sequence listing. A Bacillus bacterium, which is an enzyme and has the ability to produce both the enzyme named scyllo-inosose isomerase, was cultured, the two enzymes were accumulated in the cultured cells, and then cultured. Cells are collected, the collected cells are crushed in a buffer solution, the obtained cell lysate is centrifuged, and the above two enzymes are collected separately from the crude enzyme extract obtained as a supernatant. A myo-inositol 2-dehydrogenase, an enzyme having the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and scyllo-inosose Preparation of an enzyme, designated isomerase.

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