JP3277930B2 - Method for producing D-malic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an industrial method for producing optically active D-malic acid using microorganisms. Optically active D-malic acid is an intermediate useful for synthesizing various optically active natural products or physiologically active substances such as pharmaceuticals.

[0002]

2. Description of the Related Art An industrially useful method for producing D-malic acid is a method of optically resolving a racemic malic acid by a diastereomer salt method (Japanese Patent Application Laid-Open No. 57-19757). No. 56439, JP-A-60-204
No. 741). However, optical resolution by the diastereomer salt method is not an economical method because a resolving agent such as quinine is expensive.

[0003] As a method of using a microorganism for the production of D-malic acid, L-malic acid is contained in a medium containing racemic malic acid.
A method of culturing a bacterium that selectively assimilates only malic acid and recovering the remaining D-malic acid (US Pat. No. 4,912,204)
No. 2, JP-A-2-242699), but it cannot be said to be an efficient method such that the optical purity of D-malic acid obtained is low and the yield cannot exceed 50%. .

[0004] Enzymatic reactions that act on maleic acid to produce D-malic acid have been described in the kidneys of plants and animals (S).
acks, W.C. Jensen, C.A. O. (1951)
J. Biol. Chem. , 192 231-236,
England, S.M. Britten, J .; S. (19
67) J. Amer. Biol. Chem. , 242 2255-
2259). Among microorganisms, production of D-malic acid from maleic acid by a cell extract of a bacterium belonging to Pseudomonas has been reported (HI Rahateka).
r (1968) Indian J. et al. Biochem. ,
3, 143-144). However, these studies
Although the possibility of producing D-malic acid from maleic acid by a biological method was shown, it is a basic study,
It did not indicate a method for industrial mass production of malic acid.

A method for producing D-malic acid by reacting maleic acid with a certain microorganism (Japanese Patent Laid-Open No. 3-53)
888)), but in addition to using expensive citraconic acid for culture, L-malic acid is produced as a by-product and D-malic acid is reduced in the late stage of the reaction, so that the product yield and optical purity are low. However, it cannot be said that it is an industrially inexpensive production method due to the low enzyme activity of the cells.

[0006]

Means for Solving the Problems In producing D-malic acid from maleic acid by using a microorganism, the present inventors improve the cell membrane permeability represented by an organic solvent such as toluene during the reaction. By coexisting the compound, it was found that D-malic acid was efficiently produced,
The present invention has been completed. That is, the gist of the present invention is to produce D-malic acid from maleic acid using a microorganism or a processed product thereof, wherein the D-malic acid is reacted in the presence of a cell membrane permeability improving agent for the microorganism.
The present invention relates to a method for producing malic acid. Hereinafter, the present invention will be described in detail.

[0007] Arthrobacter sp. MCI
2612 (hereinafter sometimes abbreviated as “MCI 2612 bacterium”) and Arthrobacter globifo
rmis MCI 2613 (hereinafter sometimes abbreviated as “MCI 2613 bacterium”) is a bacterium isolated from natural soil by the present inventors, and has been sent to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology. 12562 (FER
MP-12562) and No. 12561 (FERM)
P-12561). The bacteriological properties are as follows. 1. Morphological characteristics ○ Characteristics of colonies on a heart infusion agar medium at 30 ° C for 1 week

[0008]

[Table 1]

○ On a heart infusion agar medium, 3
Morphological properties during culturing at 0 ° C. for 3 to 48 hours 1) Cell morphology: In both strains, about 6 to 12 hours after culturing, cells elongate unevenly and become long rod-shaped. Thereafter, a septum is formed at the center, and the cell bends in a curved manner, and gradually repeats segmentation. After 18 hours, almost all cells change into a short rod-like uniform form.

2) Cell division mode: Bending type Bending type 3) Motility: None None 4) Sporulation: None None 5) Gram stain: Indefinite 6) Antiacidity: Negative negative Physiological properties

[0010]

[Table 3]

25) Generation of acid from only carbon source (observed after 1-2 weeks after culture)

[0012]

[Table 4]

26) Utilization of organic acids

[0014]

[Table 5]

3. Chemical taxonomic properties

[0016]

[Table 6]

4. Taxonomic considerations ○ Identification of the genus level The strains MCI2612 and 2613 are 1)
Has a rod-coccus polymorphism in the cell cycle, 2)
It is an absolute aerobic bacterium. 3) It does not produce acid from most saccharides such as glucose. 3) The GC content in DNA is 6
It shows a high GC of 4.4%. 4) Diamino-amino acids in the cell wall have lysine. 5) Major menaquinone is MK-.
9 (H ₂ ).

[0018] From these characteristics, this bacterium is Bergey's
s Manual of Systematic Ba
Ir, which is described in Volume 2 of cteriology.
regular, Nonsporing, Gram-P
It has been found that it belongs to the genus Arthrobacter of the positive Rods group.

同 定 Species level identification About 18 species of Arthrobacter spp. Although these species are distinguished by various physiological and chemotaxonomic properties, differences in menaquinone composition, cross-linked peptide structures that make up the cell wall, and differences in sugar composition are important classification criteria at the species level. Is considered. (KH Schleifer & O. Kandl
er, 1972, Bacteriol. Rev .. Vo
l. 36: 407-377, Bergey's Man
ual of Systematic Bacteri
ology vol.2)

About MCI2612 This strain is 1) the main menaquinone is MK-9 (H ₂ ), 2)
The crosslinked peptide structure of the cell wall is Lys-Ser-Thr-
Having Ala, 3) the sugar composition of the cell wall is galactose,
And rhamnose. Bergey's Manual of Sy
Among the species described in S.Bacteriology, Arthrobacter ox is a species having the same cell wall cross-linked peptide structure as the present strain.
ydans Only one species is known. However,
This strain contains galactose and rhamnose in the sugar composition of the cell wall . oxydans (galactose and glucose).

Therefore, based on the cross-linked peptide structure of the cell wall and other chemical taxonomic properties, this strain (MCI 2612) was used.
Although was estimated to closely related species Arthrobacter oxydans, Arthroba from differences in sugar composition
cter sp. Was identified.

About MCI 2613 strain This strain is 1) the main menaquinone is MK-9 (H ₂ ), 2)
Crosslinked peptide structure of the cell wall has an Lys-Ala _{3, 3)} sugar composition of the cell wall has a chemical taxonomic feature of having galactose, and glucose, 4) Bergey's Manual of since there is no motility
Arthrobacter globi described in Systematic Bacteriology
well matched to the characteristics of formis . Therefore, this strain (MCI 2613) was transformed into Arthrobacter.
globiformis .

In the present invention, the above-mentioned bacteria can be easily grown by culturing them in a medium containing nutrients which can be generally used by microorganisms. As a nutrient source,
Glucose, starch syrup, dextrin, sucrose, starch, sugar alcohol, molasses, animal and vegetable oils and the like can be used. As a nitrogen source, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, ammonium nitrate, urea and the like can be used. In addition, if necessary, it is effective to add inorganic salts capable of generating sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions.

In addition, a medium in which nutrients used for ordinary culturing, such as vitamins, are appropriately mixed can be used. As a culture method, the nutrient medium is cultured aerobically at a pH of 4.0-9.5 and at a temperature of 20-40 ° C for 10-96 hours.

As a method of converting maleic acid into D-malic acid by the action of a microorganism, the microorganism is cultured in a nutrient medium, and the obtained cell suspension, cell lysate, or acrylamide resin is obtained. Water-insoluble polymers such as carrageenan, carrageenan, etc., adsorbing, binding, entrapping and suspending in a suitable buffer the enzyme or cells by a known method using a synthetic adsorbent, or immobilized microorganism or immobilized A substrate is added to a column filled with immobilized enzyme,
-A method of obtaining malic acid, a method of adding a substrate to a medium and performing culturing and reaction at the same time, or a method of adding a substrate and further reacting after completion of the culturing can be used. Therefore, the “processed material” in the present invention means these. The temperature during the reaction is preferably 20-50 ° C, and the pH is 5.0-
Perform in the range of 10.0. The substrate concentration is preferably in the range of 0.1% to 10%. During the reaction, the reaction rate can be increased by adding an organic solvent such as toluene.

As a method for collecting the optically active D-malic acid obtained by the culture and the reaction, a commonly known extraction and purification method can be used, but the following method can also be used. For example,
The pH of the resulting D-malic acid-containing solution is lowered to around 1.0 with sulfuric acid or the like, and ammonium sulfate is further added until saturation is reached. Thereafter, extraction is performed several times with an equal volume of ethyl acetate. By removing the solvent under reduced pressure, a D-malic acid-containing substance is obtained. Further, this substance is dissolved in a small amount of hexane and subjected to silica gel chromatography eluting with a mixed solvent of hexane and ethyl acetate to easily separate it from other impurities.

The product thus obtained was confirmed to be high-purity malic acid by elemental analysis, NMR and liquid chromatography. Determination of optical purity is S-α
Synthesizing an ester with -methoxy-α-trifluoromethyl-phenylacetyl chloride (MTPA-Cl),
It was performed by analyzing the ratio of the purified diastereomer by gas chromatography. The amount of D-malic acid in the examples was determined by liquid chromatography.

[0028]

EXAMPLES The method of the present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the scope of the invention. Example 1 Sorbitol 10 g, NH ₄ NO ₃ 6 g, KH ₂ PO ₄
3 g, K ₂ HPO ₄ 10.5 g, MgSO ₄ . 7H ₂ O
Arthrobacter was added to a liquid medium consisting of 250 mg, 5.5 g of yeast extract, 2 g of L-proline, 2 g of maleic acid, 1 l of water, and pH 6.0.
globiformis MCI-2613 and 2
The cells were cultured at 8 ° C for 20 hours.

The obtained culture solution is centrifuged to collect the bacterial cells.
The plate was washed with a mM phosphate buffer (pH 7.0). 25 mM containing 23.7 g of maleic acid and 10 ml of toluene
Phosphate buffer (pH 7.0) was added to bring the total volume to 1 l.
Table 1 shows the time-dependent changes at 7 ° C. The reaction proceeded quickly, and after 45 hours, D-malic acid having an optical purity of 100% was obtained in a yield of 88%.

[0030]

[Table 7]

Example 2 A 25 mM phosphate buffer (pH 7.0) containing 3% maleic acid and 1% toluene was added to the cells obtained by using the microorganism strain, the growth medium and the culture method described in Example 1. Add 1l
The reaction was carried out at 37 ° C., and the reaction was carried out by successively adding maleic acid during the reaction. After 30 hours of reaction,
The generated malic acid was 81 g / l and the optical purity of D-malic acid was 100%.

Example 3 As a strain to be used, Arthrobacter sp. M
The culture and reaction method described in Example 1 were carried out using CI-2612, and the results shown in Table 2 were obtained.

[0033]

[Table 8]

Example 4 The strain used was Arthrobacter sp. M
As a result of performing the same method as in Example 2 using CI-2612, malic acid produced by the reaction for 30 hours was 81 g /
At 1 the optical purity of D-malic acid was 100%.

[0035]

According to the present invention, D-malic acid can be efficiently produced from maleic acid at a high reaction rate in a microbial reaction.

────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI C12R 1:06) C12R 1:06) (72) Inventor Reiko Sashida 1000 Kamoshida-cho, Aoba-ku, Aoba-ku, Yokohama-shi, Kanagawa-ken Mitsubishi Chemical Corporation Yokohama (56) References JP-A-3-53888 (JP, A) JP-A-60-102191 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 7/46 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A method for producing D-malic acid, comprising producing a D-malic acid from maleic acid using a microorganism belonging to the genus Arthrobacter or a treated product thereof, in the presence of toluene. Method.