JPH0463675B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing α-hydroxycarboxylic acid, and more particularly to a method for producing α-hydroxycarboxylic acid from α-hydroxynitrile using a specific microorganism. [Prior art and problems to be solved by the invention] A method for producing α-hydroxycarboxylic acid from α-hydroxynitrile using microorganisms is already known. A method using bacteria belonging to the genus Brevibacterium (Japanese Patent Publication No. 58-15120); A method using bacteria belonging to the genus Corynebacterium (Japanese Patent Publication No. 56086/1986); There are methods using bacteria belonging to the genus Bacteridium, Micrococcus, and Previbacterium (Japanese Unexamined Patent Publication No. 162191/1983). However, these methods are not necessarily satisfactory in stably producing the target α-hydroxycarboxylic acid, and also have problems such as there is room for improvement in terms of production efficiency. There is. [Means for solving the problem] Therefore, in order to establish an efficient method for producing α-hydroxycarboxylic acid, the present inventor investigated microorganisms to be used, and found that microorganisms other than those described in the above publication were used. The inventors discovered that the objective can be achieved by selecting and using specific microorganisms, and have completed the present invention. That is, the present invention is directed to the genus Pseudomonas, the genus Arthrobacter, the genus Aspergillus, and the genus Penicillium.
contacting one or more microorganisms belonging to the genus Cochliobolus, genus Cochliobolus, and genus Fusarium, and having the ability to hydrolyze α-hydroxynitrile, with α-hydroxynitrile. Featured α
-Regarding a method for producing hydroxycarboxylic acid. The microorganisms that can be used in the present invention belong to the above-mentioned various genera and can hydrolyze α-hydroxynitrile to produce α-
- Any substance that has the ability to produce hydroxycarboxylic acid is sufficient, specifically Pseudomonas
Pseusomonas sp. MY-1 (FERM
P-9174), Arthrobacter aurescens (IAM 12340), Aspergillus niger (JCM
1925 and JCM 2261), Penicillium chrysogenum (IFO
5473), Cochlioboius miyabeanus (OUT 2074), Fusarium sp. MY-2
(FERM P-9187), Fusarium. Examples include Fusarium sp. MY-3 (FERM P-9188), and these can be used alone or in combination of two or more. In addition,
Among the above microorganisms, those belonging to the genus Pseudomonas and Fusarium are new strains isolated by the inventor. The mycological properties of these microorganisms are shown below. Pseudomonas sp. MY-1 (1) Morphology Cell shape and size Short Bacillus 0.7 - 0.9 x 0.8 - 1.4 μm Presence or absence of cell pleomorphism None Presence or absence of spores None Gram staining Negative Motility Yes Flagella Polar flagella (1 ) (2) Growth status in culture medium Juice agar plate culture Size 4 to 4.5 mm Circular all edges, smooth, opaque and glossy Juice agar slant culture Good growth, surface smooth, translucent and glossy Meat juice liquid culture Strong turbidity; Sweet odor Litmus milk Alkalinization (3) Physiological properties Growth conditions Temperature (optimal temperature) 5-40℃ (20-37) PH (optimum PH) 4.5-9.5 (4.5-9.0) Attitude towards oxygen Absolute aerobic Inorganic nitrogen Source availability Ammonium salts, nitrates Catalase + Oxidase O-F test No reaction Gelatin hydrolysis − Starch hydrolysis − Casein hydrolysis + Cellulose hydrolysis − Indole formation − VR test − MR test − H ₂ − Production of acids and gases from sugars Xylose − Arabinose − Glucose Acid Fructose − Mannose − Galactose − Sucrose − Lactose − Maltose − Trehalose − Mannitol − Saltobis − Inositol − Availability of citric acid − Nitrate reduction − Denitrification reaction − Pigment As described above, this bacterium is a bacillus with polar flagella, Gram-negative, obligate aerobic, and positive for catalase and oxidase, so it is a bacterium belonging to the genus Pseudomonas. It was identified that there is. This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its accession number is FERM P-9174.

[Table] (2) Microscopic characteristics Size of vegetative hyphae Width 1 to 3μ Septa present Grasping connections None Sclerotia None Conidia present Type of conidia formation Fiarot type Macroconidial shape New moon type Macroconidial size 3 to 5μ×20 ~35μ Number of macroconidial septa 3-5 septa Microconidial shape Oblong circular Microconidial size 2-3μ x 5-7μ Number of microconidial septa None Chlamydospore shape Globular chlamydospore size 3-4μ CA medium 23 Culture for 7 days at °C Slide culture method (3) Growth environment Growth temperature range 15°C to 39°C Unsuitable growth temperature Below 10°C and above 42°C Optimal growth temperature 23°C to 25°C Growth PH temperature PH2 to PH10 Optimum growth PH PH5 or more MPG Medium 7-day culture (4) Phenol oxidase reaction strain name SI-8 Phenol oxidase reaction ^a) Negative Lactucase secretion ^b) Positive tyrosinase secretion ^c) Negative a MPG medium + 0.1% tannic acid b Gyam agar + 0.0005M naphthol c Potato agar + 0.1% cresol

〔Example〕

Next, the present invention will be explained in detail with reference to examples. Example 1 Pseudomonas sp. MY-1 (FERM P
-9174) in glycerin 5g/l, acetonitrile
5g/l, KH ₂ PO ₄ 0.5g/l, Na ₂ HPO ₄・12H ₂
O5g/l, MgSO ₄・7H ₂ O0.2g/l, CaCl ₂・2H ₂
O0.01g/l, FeSO ₄ , 7H ₂ O0.001g/l, yeast extract 0.05g/l, cornsteep liquor
The cells were inoculated into 100 ml of a medium containing 0.05 g/l and cultured for 72 hours. After the culture was completed, bacteria were collected and washed. The obtained bacterial cells were suspended in 3 ml of 1/15M phosphate buffer at pH 8, and 300 mg of lactonitrile was added thereto. At 22℃, 2
After reacting for an hour, gas chromatography (column: Thermon3000Shimalite TP, column temperature
160°C). As a result, the amount of lactic acid produced is
It was 100g/l. Example 2 Culture and reaction were carried out in the same manner as in Example 1, except that 150 mg of ascencyanohydrin was used instead of lactonitrile, and 15 ml of the same buffer was used instead of 3 ml of phosphate buffer. Quantification was performed using gas chromatography (column: Thermon 3000celite545,
The column temperature was 150°C). The production amount of α-hydroxyisobutyric acid was 8 g/l. Example 3 In Example 1, α-
Addition of hydroxy-α-phenylpropionitrile (however, raw materials were used because they were unstable; i.e., an aqueous solution of acetophenone and potassium cyanide in a concentration of 4 g/l of acetophenone and 2.2 g/l of potassium cyanide). Cultivation and reaction were carried out in the same manner as in Example 1 except for this. Quantification was performed by gas chromatography (column: Thermon 3000celite 545, column temperature 200°C). The production amount of α-hydroxy-α-phenylpropionic acid was 2 g/l. Example 4 Example 1 except that 1 g/l of mandelamide was used instead of acetonitrile, 5 g/l of mandelonitrile was used instead of lactonitrile, and 15 ml of the same buffer was used instead of 3 ml of phosphate buffer. Cultured and reacted in the same manner. Quantification was performed using gas chromatography (column: Thermon 3000 Celite545,
The column temperature was 245°C). The production amount of mandelic acid was 0.3 g/l. Example 5 In Example 1, Pseudomonas sp.
Culture was carried out in the same manner as in Example 1, using Fusarium sp. MY-2 (FERM P-9187) instead of MY-1 (FERM P-9174). After the culture was completed, the bacteria were collected and washed with 1/15M phosphate buffer (PH8). The obtained bacterial cells were added to 15 ml of 1/15M phosphate buffer (PH8).
2 by adding 150 mg of lactonitrile.
A time reaction was performed. After the reaction was completed, lactic acid was quantified in the same manner as in Example 1, and 10.5 g/l of lactic acid was obtained. Example 6 In Example 1, Pseudomonas sp.
Culture was carried out in the same manner as in Example 1, using Fusarium sp. MY-2 (FERM P-9187) instead of MY-1 (FERM P-9174). After the culture was completed, the bacteria were collected and washed with 1/15M phosphate buffer (PH8). The obtained bacterial cells were added to 15 ml of 1/15M phosphate buffer (PH8).
75 mg of acetone cyanohydrin was added and the reaction was carried out for 2 hours. After the reaction is complete, gas chromatography (column; Thermon
3000celite545, column temperature 150°C). The production amount of α-hydroxyisobutyric acid is 4.2g/l
It was hot. Example 7 In Example 1, Pseudomonas sp.
Culture was carried out in the same manner as in Example 1, using Fusarium sp. MY-3 (FERM P-9188) instead of MY-1 (FERMP-9174). After the culture was completed, the bacteria were collected and washed with 1/15M phosphate buffer (PH8). The obtained bacterial cells were added to 15 ml of 1/15M phosphate buffer (PH8).
2 by adding 150 mg of lactonitrile.
A time reaction was performed. After the reaction was completed, the amount of lactic acid produced was 1.2 g/l as a result of quantitative determination in the same manner as in Example 1. Example 8 Arthrobacter aurescens (IAM
12340), glucose 5g/l, acetonitrile
3g/l, KH ₂ PO ₄ 1.5g/l, Na ₂ HPO ₄・12H ₂
O1.5g/l, MgSO ₄・7H ₂ O0.2g/l, CaCl ₂・
The cells were inoculated into 10 ml of a medium containing 0.01 g/l of 2H ₂ O, 0.001 g/l of FeSO _{4.7H 2} O, 0.03 g/l of yeast extract, and 0.03 g/l of corn steep liquor, and cultured for 24 hours. After culturing, collect bacteria, wash, and add 2 ml of pH8
After suspending in 1/15M phosphate buffer, lactonitrile
10 mg was added and the reaction was carried out for 2 hours. After the reaction is complete,
As a result of quantitative determination in the same manner as in Example 1, the production amount of lactic acid was 1.5 g/l. Example 9 Aspergillus niger (JCK 1925) was inoculated into 10 ml of Czapek medium containing 3 g/l of acetonitrile and cultured for 6 days. After culturing, collect bacteria by centrifugation, wash, and add 1/15M phosphate buffer 2, pH 8.
ml, and 10 mg of lactonitrile was added to carry out the reaction. After the reaction was completed, the amount of lactic acid produced was 2.5 g/l as determined in the same manner as in Example 1. Example 10 Cultivation and reaction were carried out in the same manner as in Example 4, except that Aspergillus niger (JCM2261) was used instead of Aspergillus niger (JCM1925). Example 1: Determination of the product
In the same manner as above, the amount of lactic acid produced was
It was 0.4g/l. Example 11 Culture and reaction were carried out in the same manner as in Example 9 except that Penicillium chrysogenum (IFO5473) was used instead of Aspergillus niger (JCM1925). After the reaction was completed, the product was quantified in the same manner as in Example 1, and the amount of lactic acid produced was 0.1 g/l. Example 12 Culture and reaction were carried out in the same manner as in Example 9, except that Cochliobolus miyabeanus (OUT2074) was used instead of Aspergillus niger (JCM1925) and Cochliobolus miyabeanus (OUT1925). After the reaction was completed, the amount of lactic acid produced was 0.8 g/l as determined in the same manner as in Example 1. [Effects of the Invention] According to the present invention, α-
By hydrolyzing hydroxynitrile, α
-Hydroxycarboxylic acid can be efficiently produced. This α-hydroxycarboxylic acid is useful as a food, an agrochemical/medical raw material, a raw material for organic synthesis, etc.

Claims (1)

[Scope of Claims] 1. Any of the genus Pseudomonas, Arthrobacter, Aspergillus, Penicillium, Cochliobolus and Fusarium. A method for producing α-hydroxycarboxylic acid, which comprises contacting one or more types of microorganisms having the ability to hydrolyze α-hydroxynitrile with α-hydroxynitrile. 2. The method according to claim 1, wherein the microorganism is any one of cells in a growth phase, cells in a resting phase, immobilized cells, and a processed cell extract.