JP2007300809A - Method for producing shikimic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing shikimic acid capable of becoming a raw material of a medicine effective for bird influenza, etc., and many other medicines, agrochemicals, etc., simply in a good efficiency and without giving a load to the environment. <P>SOLUTION: This method for producing shikimic acid is provided by passing through each of the processes of (1) a first process of producing 3-dehydroshikimic acid from quinic acid in the presence of an enzyme derived from an acetobactor and (2) a process of producing shikimic acid from the 3-dehydroshikimic acid under the joint actions of shikimic acid dehydrogenase (SKDH) and glucose dehydrogenase (GDH). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an efficient method for producing shikimic acid using acetic acid bacteria. Specifically, the present invention relates to a method for synthesizing 3-dehydroshikimic acid by acetic acid bacteria using quinic acid as a raw material, and further producing shikimic acid from 3-dehydroshikimic acid using an enzyme derived from acetic acid bacteria.

  Shikimic acid is an important intermediate of aromatic amino acids used as a useful raw material for many antibiotics, alkaloids, herbicidal active substances and the like. Particularly in recent years, it has become an important raw material compound of Tamiflu (registered trademark), which is expected to be most effective against avian influenza, which is feared to cause a global epidemic. Despite wide warnings from the World Health Organization (WHO), there is not enough reserve available around the world. One of the reasons is that it is difficult to prepare shikimic acid as a raw material. From glucose to shikimic acid, the shikimic acid pathway must go through a multi-step metabolic pathway starting from the condensation reaction of phosphoenolpyruvate and erythrose-4-phosphate obtained from two different metabolic pathways. . Therefore, even if the metabolic intermediate of the shikimic acid pathway is efficiently produced using the latest metabolic control method and gene amplification method, there is a drawback that the barrier is too large to reach shikimic acid. The present inventors have developed a method for producing 3-dehydroshikimic acid, which is an intermediate of the shikimic acid pathway, from quinic acid using cell membrane-bound quinate dehydrogenase (QDH) in acetic acid bacteria. Clarification (Patent Document 1) and a method for producing shikimic acid from quinic acid in a single culture system of acetic acid bacteria are also provided (Non-Patent Document 1).

  At present, in China, shikimic acid is extracted from plant shikimi (Illicium anisatum) and Toshikimi (Illicium verum), but since it is a natural product, it is difficult to always obtain raw materials of the same quality.

  Regarding the method of producing shikimic acid organically, a method of producing a shikimic acid derivative by dehydration from a quinic acid derivative using a Filsmeier reagent (Patent Document 2), a diaminoshikimic acid derivative produced from an isophthalic acid derivative (Patent Document 3) or a method of manufacturing from furan (Patent Document 4) is known. These are shikimic acid derivatives and may require difficult reactions in the production of the target substance.

  In addition, as a method for producing shikimic acid at low cost, quinic acid is not isolated from a quinic acid-containing raw material liquid containing a large amount of an inorganic salt extracted from an extraction source, but reacted with alcohols in the presence of an acid or a base catalyst. Also disclosed is a method for producing a shikimic acid precursor and shikimic acid from the quinic acid derivative by converting it to a quinic acid ester and then reacting with a ketone derivative or an aldehyde derivative in the presence of an acid catalyst to produce an acetal form of the quinic acid ester. (Patent Document 5).

Furthermore, a method for producing shikimic acid by a fermentation method is also known, and a method for efficiently producing shikimic acid using a microorganism belonging to the genus Citrobacter having the property of secreting shikimic acid outside the cell is disclosed. (Patent Document 6). However, in the production method by the fermentation method, the purification process is complicated because many substances other than shikimic acid are present in the culture solution.
JP 2003-70497 A Japanese Patent No. 3641384 JP 2001-354635 A JP 2001-288152 A Japanese Patent Application Laid-Open No. 11-349583 JP 2002-281993 A Adachi O. et al. Biosci. Biotecnol. Biochem. 67: 2124-2131 (2003)

  The present invention provides a method for producing shikimic acid, which is a raw material for a large number of pharmaceuticals, agricultural chemicals, etc., in addition to pharmaceuticals effective against avian influenza etc., in an efficient and simple manner and in a manner that does not burden the environment. The purpose is to do.

  As a result of intensive studies to solve the above problems, a method for easily producing shikimic acid using an acetic acid bacterium from an intermediate metabolite in the shikimic acid pathway starting from glucose was developed. That is, the present invention has the following configuration.

  (1) In the method for producing shikimic acid, 1) the first step of producing 3-dehydroshikimic acid from quinic acid in the presence of an enzyme derived from acetic acid bacteria, 2) shikimic acid dehydrogenase (SKDH) and glucose dehydration A method for producing shikimic acid, wherein each step of the second step of producing shikimic acid from 3-dehydroshikimic acid under the conjugation of elementary enzyme (GDH) is conducted.

  (2) In the method for producing shikimic acid, the reaction in the first step of producing 3-dehydroshikimic acid from quinic acid is performed at pH 7 to pH 9, and shikimic acid dehydrogenase (SKDH) and glucose dehydrogenase (GDH) The method for producing shikimic acid according to (1), wherein the reaction in the second step of producing shikimic acid from 3-dehydroshikimic acid is carried out at pH 6 to pH 7 under conjugation.

  (3) The method for producing shikimic acid according to (1) or (2), wherein the reaction solution in the second step contains NADP and glucose at a molar concentration of 100 to 10,000 times that of NADP.

  (4) The method for producing shikimic acid according to (1) or (2), wherein the enzyme derived from acetic acid bacteria used in the first step is quinic acid dehydrogenase (QDH).

  (5) The shikimic acid according to (1) or (2), wherein the shikimate dehydrogenase (SKDH) and glucose dehydrogenase (GDH) used in the second step are derived from acetic acid bacteria. Manufacturing method.

  (6) Shikimate dehydrogenase (SKDH) and glucose dehydrogenase (GDH) used in the second step include both enzymes obtained from acetic acid bacteria having the ability to produce both of these enzymes together. The method for producing shikimic acid according to (5), wherein the method is used in the state of a crude enzyme solution or is used as two enzymes isolated and purified from acetic acid bacteria.

  (7) The method for producing shikimic acid according to any one of (1) to (6), wherein the acetic acid bacterium is a genus Gluconobacter.

  (8) The method for producing shikimic acid according to (7), wherein the genus Gluconobacter is Gluconobacter oxydans IFO 3244.

  The method for producing shikimic acid according to the present invention facilitates the production of shikimic acid and metabolic intermediates of the shikimic acid pathway. Therefore, it is possible to produce useful substances using these as raw materials.

  The present inventors previously described that cell membrane-bound quinate dehydrogenase (QDH) and 3-dehydroquinate dehydratase (DQD) are widely present on the cytoplasmic membrane coat of acetic acid bacteria. In particular, in the genus Gluconobacter, it was found and reported that quinic acid is oxidized to 3-dehydroshikimic acid via 3-dehydroquinic acid by a normal fermentation production method. (Adachi O. et al. Biosci. Biotechnol. Biochem., 67: 2124-2131 (2003) (Non-Patent Document 1), Adachi O. et al. Biosci. Biotechnol. Biochem., 67: 2111-2123 (2003) Vangnai AS et al. FEMS Microbiol. Lett., 241: 157-162 (2004)).

  Furthermore, in the cytoplasm of acetic acid bacteria, NADP-dependent shikimate dehydration has a reversible action, in which shikimate is produced from 3-dehydroshikimate, and on the contrary, shikimate is oxidized to produce 3-dehydroshikimate. The presence of elementary enzyme (Shikimate dehydrogenase: SKDH) was clarified as a result of carrying out in one cell line. (Adachi O. et al. Biosci. Biotechnol. Biochem., 67: 2124-2131 (2003) (Non-Patent Document 1), Adachi O. et al. Biosci. Biotechnol. Biochem., 67: 2111-2123 (2003) ), Adachi O. et al. Biochem. Biophys. Acta, 1647: 10-17 (2003), Adachi O. et al. Appl. Microbiol. Biotechnol., 60: 643-653 (2003))

  From this, it was found that in order to increase the production amount of shikimic acid, it was necessary to use two independent enzyme reaction systems as shown in FIG. 1, and a method for producing shikimic acid comprising two steps was found. Completed.

<First reaction step>
In the first step, 3-dehydroquinic acid is produced from quinic acid by QDH, and 3-dehydroshikimic acid is produced from 3-dehydroquinic acid by DQD, and quinic acid and dried cells or cell membranes are separated from conventional ones. This is a reaction step in which 3-dehydroshikimic acid is produced by QDH and DQD existing in the culture medium after culturing by the method. In the culture solution, it is necessary that dry cells or QDH and DQD derived from cell membranes exist, and in particular, a strain producing QDH must be selected and used. Any strain may be used as long as it can produce QDH.

<Reaction second step>
In the second step, shikimic acid is produced from 3-dehydroshikimic acid. This is a step of conjugating two kinds of enzymes present in the cytoplasmic gel to carry out an enzyme reaction. That is, under NADP, 3-dehydroshikimic acid is produced by reacting NADP-dependent glucose dehydrogenase (glucose dehydrogenase: GDH) having an activity of converting glucose into glucono-δ-lactone and NADPH simultaneously with SKDH. Shikimic acid can be produced from In this case, before adding 3-dehydroshikimic acid, it is desirable to make excess glucose exist in the reaction system with respect to NADP.

<Selection of acetic acid bacteria>
As the acetic acid bacterium used in the present invention, a bacterium having a large amount of QDH is desirable, and gluconobacter oxydans and gluconobacter melanogenus are preferable, and gluconobacter oxydans IFO 3244 is most preferable.

<Preparation of enzyme>
GDH and SKDH used in the second step can be obtained by purification from acetic acid bacteria. GDH and SKDH are both obtained by culturing an acetic acid bacterium having the ability to produce both enzymes together, accumulating the enzyme in the cytoplasm of the acetic acid bacterium, and extracting it from the disruption solution of the microbial cell. It can also be used in the state of a crude enzyme solution containing any of these enzymes, or it can be used as two enzymes that have been isolated and purified separately from the above-mentioned microbial cell disruption solution. For purification of GDH, the method of Adachi et al. (Adachi O. et al. Agric. Biol. Chem., 44: 301-108 (1980)) can be used.

  SKDH can be obtained by purification by the following method. Acetic acid bacteria are homogenized in a phosphate buffer containing mercaptoethanol, and the supernatant obtained by centrifugation is purified by column chromatography. Column chromatography is performed according to a conventional method. In the purification of SKDH, a DEAE-cellulose column, a DEAE-Sephadex column, a Blue-Dextran Sepharose column or the like is used and eluted with mercaptoethanol containing KCl. The eluate can be purified by an ammonium sulfate precipitation method according to a conventional method.

<Enzyme reaction conditions>
The reaction temperature in the first step and the second step in the present invention is not particularly limited as long as the reaction proceeds. Considering the solubility of the substrate and the stability of the enzyme used, the reaction is desirably carried out at a temperature of 20 ° C. to 50 ° C., preferably 25 ° C. to 30 ° C.

The pH of the enzyme reaction solution in the first step needs to consider the stability of the enzyme used and the stability of the product. The state of conversion from quinic acid to 3-dehydroshikimic acid when an enzyme reaction is carried out in a reaction solution of pH 4 to pH 9 for 20 hours can be confirmed by a paper chromatography method. From the results shown in FIG. 2, no conversion to 3-dehydroshikimic acid was confirmed at pH 4 to pH 5, about 50% conversion was observed at pH 6, and highly efficient conversion was confirmed at pH 7 to pH 9. From this result, it was estimated that the reaction was preferably performed at pH 7 to pH 9. However, it has been confirmed that by-product protocatechinic acid is produced when reacted for a long time, and the reaction is preferably completed in a short time (about 20 hours).

In the second step, SKDH used in the reaction system for producing shikimic acid by the reduction reaction of 3-dehydroshikimic acid is a reduction reaction in which shikimic acid is produced from 3-dehydroshikimic acid, and 3-dehydroshikimic acid from shikimic acid. As described above, the reverse oxidation reaction generated by ## STR4 ## reaches an equilibrium state depending on the pH. The present inventors have further obtained the following knowledge. SKDH purified to high purity shows SKDH oxidation activity at pH 10.0 and enzyme activity against shikimic acid is 60 units / mg, and pH 6 to pH 7 shows reduction activity and enzyme activity against 3-dehydroshikimic acid. 25 units / mg. The Km (Michaelis-Menten constant) was 0.5 mM in the oxidation reaction to shikimic acid at pH 10.0, and 0.2 mM in the reduction reaction to 3-dehydroshikimic acid at pH 5 to pH 7. Therefore, in the second step of the present invention, in order to effectively produce shikimic acid from 3-dehydroshikimic acid, it is desirable to keep the reaction solution at pH 6 to pH 7, preferably pH 7.

<Paper chromatography method>
Confirmation of 3-dehydroquinic acid, 3-dehydroshikimic acid, shikimic acid and quinic acid which is a raw material produced by the enzyme reaction of the present invention can be performed by a paper chromatography method. According to the method of Yoshida and Hasegawa (Yoshida S. and Hasegawa M. Arch. Biochem. Biophys., 70: 377-388 (1957)), the method previously reported by the present inventors (Adachi O. et al. Biosci). Biotechnol.Biochem., 67: 2124-2131 (2003)), 2% formic acid, benzyl alcohol: 2-butanol: 2-propanol: water = 3: 1: 1: 1 Using a developing solvent of (W / V), the existing substances are separated on paper and then dried. A solution of 160 mg of sodium metaperiodate dissolved in 12.5 ml of a mixed solution of 1M acetic acid and 1M sodium acetate is sprayed, and after 20 minutes, an alcohol solution of 3% aniline is sprayed. Under this condition, the raw material quinic acid and the oxidative metabolite 3-dehydroquinic acid show almost the same Rf value, but the spot colors are different, quinic acid has a light pink color, and 3-dehydroquinic acid has a yellow color. . Further, a red spot of shikimic acid can be confirmed above 3-dehydroquinic acid, and a yellow spot of 3-dehydroshikimic acid can be confirmed further above.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

Preparation of 3-dehydroshikimic acid 1 g of quinic acid and 2.5 g of acetic acid bacterium Gluconobacter oxydans IFO 3244 cell membrane were placed in a 500 ml Erlenmeyer flask, 100 ml of McIlvaine buffer (pH 8) was added, and at 30 ° C. The reaction was shaken for 20 hours. To complete the reaction, trichloroacetic acid (TCA) was added to 5.5%, and the cells were removed by centrifugation to obtain a supernatant containing 3-dehydroshikimic acid.

Confirmation of 3-dehydroshikimic acid To the supernatant obtained above, 0.1 M acetate buffer (pH 6.0) was added and diluted 50 times. In order to confirm the formation of 3-dehydroshikimic acid, 1 part of the diluted solution was taken and 0.25 μmol NADPH (manufactured by Oriental Yeast) and SKDH (1 unit as the reducing activity of 3-dehydroshikimic acid) were added. The volume was adjusted to 1 ml with 1M acetate buffer (pH 6.0) and reacted at 25 ° C. When the conversion of NADPH to NADP reached equilibrium, the final concentration of 3-dehydroshikimic acid was about 40 mM. This indicates that 75% of the quinic acid has been converted to 3-dehydroshikimic acid.

Purification of 3-dehydroshikimic acid The above-mentioned fraction containing 3-dehydroshikimic acid diluted 50-fold by adding 0.1 M acetic acid buffer (pH 6.0) was subjected to column chromatography packed with Dowex 50, After neutralization with lyophilization, it was freeze-dried. This lyophilized product was dissolved in a small amount of water, and insoluble substances were removed by centrifugation. As a result, the 3-dehydroshikimic acid concentration used as a raw material for shikimic acid production was 2.1M.

Purification of shikimate dehydrogenase Cells (wet weight of about 300 g) were suspended in 1 liter of 2 mM potassium phosphate buffer (pH 7.2: KPB) containing 5 mM β-mercaptoethanol. This bacterial cell suspension was applied twice to a pressure type homogenizer (Mini-Lab, type 8.30H, manufactured by Rannie, Denmark) to disrupt the bacterial cells. The obtained cell disruption solution was centrifuged at 68,000 × g for 90 minutes to remove insoluble solids, and the supernatant was taken out. 1100 ml of the supernatant obtained here was applied to a DEAE-cellulose column (5 × 30 cm) and equilibrated with KPB. The column was washed away with KPB containing 100 mM KCl, and then SKDH was eluted with KPB containing 200 mM KCl. Next, the eluted SKDH was adjusted to a concentration of 100 mM KCl with KPB and applied to a DEAE-Sephadex A-50 column (2.5 × 20 cm). The column was washed away with KPB containing 150 mM KCl, and then SKDH was eluted stepwise with KPB containing 175 mM, 200 mM, 220 mM KCl. Next, the fractions eluted with KPB containing 175 mM and 200 mM KCl were combined, adjusted to 100 mM KCl with KPB, and applied to a Blue-Dextran Sepharose 4B column (1.5 × 20 cm). After the column was washed with KPB containing 100 mM KCl, SKDH was eluted with KPB containing 300 mM KCl. The SKDH fraction was treated with an ammonium sulfate precipitation method to obtain SKDH precipitate. The precipitate was dissolved in a small amount of KPB and applied to a Sephadex G-75 column (1.5 × 125 cm) equilibrated with KPB. The eluate was fractionated by 35 drops (about 1.2 ml). The 92nd fraction had the highest SKDH activity. Fractions having SKDH activity were collected and subjected to ammonium sulfate precipitation to obtain SKDH having a specific activity increased 2550 times compared to the supernatant obtained by disrupting the cells.

Production of shikimic acid 250 mg (about 1.4 mmol) of 3-dehydroshikimic acid obtained in Example 1, SKDH (20 units as the reducing activity of 3-dehydroshikimic acid) obtained by the above method, NADP (5 μm) Mole: manufactured by Oriental Yeast Co., Ltd., GDH (1000 units), glucose (about 5.0 mmol), 5 mM β-mercaptoethanol and 5 mM EDTA, and 30 mM KPB (pH 7.0) to make a total volume of 20 ml . The reaction was carried out at 25 ° C., 0.1 ml of the reaction solution was taken out, 10 μl of 60% TCA was added to terminate the reaction, and the precipitate was removed by centrifugation. In the middle of the reaction and at the end of the reaction, a part of the reaction solution was taken out over time and analyzed by the paper chromatography method as shown in FIG. 3 (upper). It was revealed that the reduction reaction from 3-dehydroshikimic acid to shikimic acid was completed in 90 minutes when unreacted 3-dehydroshikimic acid disappeared.

  Separately, SKDH (1 unit as oxidation activity) and 50 mM glycine-NaOH buffer (pH 10.0) were added to the sample solution in the presence of 0.25 μmol of NADP and reacted for 1 hour, and the amount of shikimic acid was enzymatically converted 3 -The result of measuring the absorbance at 340 nm as the amount of dehydroshikimic acid is shown in Fig. 3 (bottom). As a result, the enzyme reaction from 3-dehydroshikimic acid to shikimic acid proceeded 100%, which was consistent with paper chromatography.

  According to the present invention, it becomes possible to effectively use a material containing quinic acid such as coffee lees, and it is possible to produce useful substances such as pharmaceuticals using shikimic acid and metabolic intermediates of shikimic acid pathway as raw materials.

It is drawing which showed two processes in the manufacturing method of shikimic acid. The reaction first step represents that 3-dehydroquinic acid is produced from quinic acid by GDH in the presence of acetic acid bacteria cell membrane, and 3-dehydroshikimic acid is produced from 3-dehydroquinic acid by DQD. This shows that 3-dehydroshikimic acid is converted to shikimic acid by cytoplasm-derived SKDH by converting glucose into glucono-δ-lactone and NADPH in the presence of NADP by cytoplasm-derived GDH. It is the photograph replaced with drawing of the paper chromatography performed in order to confirm 3-dehydroshikimic acid production | generation in various pH. It represents spots of quinic acid (QA), 3-dehydroquinic acid (DQA), and 3-dehydroshikimic acid (DSA). (Upper) It is a photograph replacing a drawing of paper chromatography performed by taking out 0.1 ml of the reaction solution over time and stopping the reaction in order to confirm the formation of shikimic acid. It represents spots of shikimic acid (SKA) and 3-dehydroshikimic acid (DSA). (Lower) It is a drawing showing the ratio of 3-dehydroshikimic acid converted to shikimic acid by taking out part of the reaction solution over time and carrying out an enzyme reaction.

Claims (8)

  In the method for producing shikimic acid, 1) the first step of producing 3-dehydroshikimic acid from quinic acid in the presence of an enzyme derived from acetic acid bacteria, 2) shikimate dehydrogenase (SKDH) and glucose dehydrogenase ( A method for producing shikimic acid, wherein each step of the second step of producing shikimic acid from 3-dehydroshikimic acid under conjugation of GDH) is conducted.   In the method for producing shikimic acid, the reaction in the first step of producing 3-dehydroshikimic acid from quinic acid is performed at pH 7 to pH 9, and the conjugate of shikimic acid dehydrogenase (SKDH) and glucose dehydrogenase (GDH) The method for producing shikimic acid according to claim 1, wherein the reaction in the second step of producing shikimic acid from 3-dehydroshikimic acid is performed at pH 6 to pH 7.   The method for producing shikimic acid according to claim 1 or 2, wherein the reaction solution in the second step contains NADP and glucose having a molar concentration of 100 to 10,000 times that of NADP.   The method for producing shikimic acid according to claim 1 or 2, wherein the enzyme derived from acetic acid bacteria used in the first step is quinic acid dehydrogenase (QDH).   The method for producing shikimic acid according to claim 1 or 2, wherein shikimate dehydrogenase (SKDH) and glucose dehydrogenase (GDH) used in the second step are derived from acetic acid bacteria.   Shikimate dehydrogenase (SKDH) and glucose dehydrogenase (GDH) used in the second step are crude enzyme solutions containing both enzymes obtained from acetic acid bacteria having the ability to produce both of these enzymes together. The method for producing shikimic acid according to claim 5, wherein the method is used in the state of (2), or is used as two enzymes isolated and purified from acetic acid bacteria.   The method for producing shikimic acid according to any one of claims 1 to 6, wherein the acetic acid bacterium is a genus Gluconobacter.   The method for producing shikimic acid according to claim 7, wherein the genus Gluconobacter is Gluconobacter oxydans IFO 3244.

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