JP3173176B2 - Method for producing S (+)-citramalic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an industrial method for producing optically active S (+)-citramalic acid using microorganisms. Optically active S (+)-citramalic acid is a useful intermediate for synthesizing various optically active natural products or physiologically active substances such as pharmaceuticals.

[0002]

2. Prior Art and Problems to be Solved by the Invention
Regarding the method for producing (+)-citramalic acid, Hasegawa et al. Discloses a method of preparing from mesaconic acid using Hansenula nonfermentans or Kleetschella africana (Japanese Patent Laid-Open No. 60-196191). However, since mesaconic acid as a raw material is expensive, it cannot be said to be an economical method.

[0003]

[Means for Solving the Problems] Accordingly, the present inventors
As a result of conducting various studies to advantageously produce S (+)-citramalic acid, microorganisms belonging to the genus Alcaligenes from itaconic acid produced at a low cost by microorganisms can efficiently convert S (+)-citramalic acid. It was found to be produced and completed the present invention.

That is, the gist of the present invention is to provide a method for producing S (+)-citramalic acid from itaconic acid by converting itaconic acid into S (+)-citramic acid in the presence of a microorganism or a treated product thereof.
S, characterized in that (+)-citramalic acid is produced.
(+)-Citramalic acid. Hereinafter, the present invention will be described in detail.

The microorganism used in the present invention is not particularly limited as long as it has an ability to produce S (+)-citramalic acid from itaconic acid. Examples of such microorganisms include microorganisms belonging to the genus Alcaligenes, and more specifically, Alcaligenes denitrificans.
cans ) MCI 2775 (hereinafter "MCI 277
No. 5 bacteria), Alcaligenes denitrificans MCI 2776 (hereinafter referred to as “MCI
No. 2776 bacteria). MCI No. 2775 and MCI 2776 are bacteria isolated from the natural soil by the present inventors, and each of them is sent to the Institute of Microbial Industry and Technology by the Ministry of Industrial Science and Technology, No. 13192 (FERM P-13192). And No. 13193 (FERM P-1319)
3). The bacteriological properties of MCI 2775 and MCI 2776 are as follows.

[0006] 1. Morphological characteristics 特 徴 Characteristics of colonies after cultivation on ordinary agar medium at 30 ℃ for 24 hours

[Table 1] MCI 2775 Bacteria MCI 2776 Bacteria 1) Outer shape: Circular Circular 2) Surface protrusion: Semi-lens shape Semi-lens shape 3) Surface shape: Smooth Smooth 4) Gloss: Dull dull 5) Color: Yellow White Yellowish white 6) Transparency: Opaque Opaque 7) Peripheral edge: All edges All edges 8) Viscosity: None None

[0007] Morphological properties after cultivation on ordinary agar medium at 30 ° C for 24 hours

Table 2 MCI 2775 Bacteria MCI 2776 Bacteria 1) Cell morphology: rod-shaped rod-shaped 2) Polymorphism: none None 3) Motility: Yes, with periflagellate Yes, with periflagellate 4) Sporulation: None None 5 6) Gram stain: Negative Negative 6) Acid-fast: Negative Negative

[0008] 2. Physiological properties

[Table 3] MCI 2775 Bacteria MCI 2776 Bacteria 1) Growth under anaerobic conditions: Can grow slightly but can grow weakly 2) Growth in the air: Positive Positive 3) Catalase: Positive Positive 4) Oxidase: Positive Positive 5 6) Hydrolysis of gelatin: Negative Negative 7) Litmus milk: Alkaline Alkaline No peptone conversion No peptone conversion 8) Nitrate reduction: Positive Positive 9) Denitrification reaction : Positive Positive 10) VP test: Negative Negative 11) Indole formation: Negative Negative 12) Hydrogen sulfide formation: Negative Negative 13) Starch hydrolysis: Negative Negative 14) Use of citric acid: Positive Positive (on Christensen medium) 15) Urease: Negative Negative 16) NaCl resistance: Can grow at 5% Can grow at 5% Growth is impossible at 7%. Growth is impossible at 7%. 17) Pigment formation: None None 18) Growth temperature range: 9-45 ° C. 9-45 ° C. Growth pH: pH 4-11 pH 4-11 19) Decomposition of Tween 80: Negative Negative 20 ) Degradation of esculin: negative negative 21) Hydrolysis of arginine: negative negative 22) β-galactosidase: negative negative 23) Use of N-acetylglucosamine: negative negative

24) Formation of acid from carbon source

[Table 4]

25) Use of organic acids

[Table 5]

3. Chemical taxonomic properties

Table 6 MCI No. 2775 MCI No. 2776 1) GC content in DNA (Tm method): 68.7 68.4 mol% 2) Ubiquinone: Q-8 Q-8

4. Taxonomic considerations 同 定 Identification of genus levels MCI 2775 and 2776 have 1) gram negative, 2) aerobic, 3) periflagellate,
4) no acid is produced from glucose; 5) morphological and physiological properties such as negative urease activity; and 6) ubiquinone Q-8 as isoprenoid quinone as chemical taxonomic properties. 7) GC content in DNA. Is 68.4
特 徴 68.7%. Bergey's Manual for genera having these characteristics
of Systematic Bacteriology
y When the genus level was searched based on Volume 1, Gr
am-negative Aerobic Rods
and Cocci group, the characteristics of the genus Alcaligenes were well matched.
Bacteria No. 6 was identified as the genus Alcaligenes .

(3) Species level identification Bergey's Manual of System
According to Volume 1, the genus Alcaligenes currently contains about 10 species, including 5 species isolated from the marine environment. Therefore, a comparison was made between five strains which seemed to be closely related to MCI No. 2775 and No. 2776. As a result, as shown in Table 1 below, this strain was found to be Alcaligene.
s denitrificans or Alcali
Genes eutrophus showed similar properties. But Alcaligenes eutrophu
s means reduction of nitrite, use of D-fructose, Tw
As distinction was made in the hydrolysis of een80, the utilization of P-OH-benzoic acid, MCI 2775 and 2776
Bacteria No. Alcaligenes denitrific
ans .

[0014]

[Table 7]

[0015]

[Table 8]

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, organic acids, 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, urea and the like can be used. In addition, if necessary, sodium, potassium, calcium, magnesium, cobalt, manganese, chlorine,
It is effective to add phosphoric acid, sulfuric acid and other inorganic salts capable of generating ions. In addition, a medium in which nutrient sources used for normal culture such as vitamins are appropriately mixed can be used. As a culture method, the pH of the nutrient medium is aerobically adjusted to 20-40 in the range of 4.0-9.5.
Incubate for 10-96 hours in the range of ° C.

Microbes act on itaconic acid to produce S (+)
-As a method of converting to citramalic acid, a microorganism is cultured in a nutrient medium, and the obtained cell suspension or cell lysate, or a water-soluble polymer such as acrylamide resin or carrageenan, a synthetic adsorbent, etc. The substrate is adsorbed, bound, and entrapped on an immobilized carrier by a known method, and suspended in an appropriate buffer, or the immobilized microorganism or immobilized enzyme is packed into a column, and the substrate is immobilized on the immobilized carrier using a known method. A method of adding and reacting to obtain S (+)-citramalic acid, a method of adding a substrate to a medium to perform culturing and reaction at the same time, or a method of adding a substrate after the culturing and further reacting can be used. The reaction temperature is preferably 20-50 ° C.
H is in the range of 5.0-10.0. Substrate concentration is 0.1
A range of -10% is preferred. During the reaction, the reaction rate can be increased by adding an organic solvent such as toluene or a surfactant.

Optical activity S (+) obtained by culture and reaction
As a method for collecting citramalic acid, a commonly known extraction method can be used, but the following method can also be used. For example, the pH of the obtained S (+)-citramalic 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. This is dehydrated with magnesium sulfate or the like, and then the solvent is removed under reduced pressure to obtain a substance containing S (+)-citramalic acid. Further, this substance is dissolved in a small amount of ethyl acetate and subjected to silica gel chromatography eluted with a mixed solvent of hexane and ethyl acetate, whereby it can be easily separated from other impurities.

The product thus obtained is confirmed to be high-purity citramalic acid by elemental analysis, NMR, liquid chromatography, and IR. The determination of optical purity can be performed by synthesizing an ester with S-α-trifluoromethyl-phenylacetyl chloride (MTPA-Cl) and analyzing the ratio of purified diastereomers by gas chromatography. The determination of S (+)-citramalic acid in the examples was performed by liquid chromatography.

[0020]

The present invention will be described in more detail with reference to the following examples.
, But are limited to these without exceeding the gist.
It is not something to be done. Example 1 Itaconic acid 10 g, sodium succinate 2 g, NH
_FourNO_Three 2g, KH _TwoPO_Four 5g, K_TwoHPO_Four 
5g, MgSO_Four・ 7H_TwoO 250mg, MnCl_Two
・ 4H_TwoO 10mg, calcium pantothenate 20
mg, yeast extract 10g, L-leucine 2g,
Liquid composed of 11 (liter) water and pH 7.5
In a somatic medium, Alcaligenes denitrificans MC
I 2775 was inoculated and cultured at 28 ° C. for 20 hours. Obtained
The culture is centrifuged, and the cells are collected and buffered with 50 mM Tris-HCl.
The buffer was washed with (pH 8.0). Itaconic acid 2
0 g, 50 mM Tris-HCl buffer containing 10 ml of toluene
The solution was added (pH 8.0) and the total amount was adjusted to 11 (liter).
And reacted at 28 ° C. Optical purity 100% after 48 hours
Of S (+)-citramalic acid was obtained in an amount of 20 g.

Example 2 Alcaligenes denitrificans was used as a bacterial strain.
Using MCI 2776, the culture and reaction methods described in Example 1 were performed. 48 hours later, S with 100% optical purity
10 g of (+)-citramalic acid was obtained.

[0022]

Industrial Applicability The method for producing S (+)-citramalic acid using the microorganism of the present invention produces S (+)-citramalic acid favorably from itaconic acid. It is very effective in performing effective production.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Reiko Sashida 1000 Kamoshita-cho, Midori-ku, Yokohama-shi, Yokohama, Japan Mitsubishi Chemical Research Institute (58) Field surveyed (Int.Cl. ⁷ , DB name) C12P 7 / 44 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A method for producing S (+)-citramalic acid from itaconic acid, comprising the steps of:
S, characterized in that (+)-citramalic acid is produced.
A method for producing (+)-citramalic acid.