JPS63222696A - Production of alpha-hydroxycarboxylic acid - Google Patents

Abstract

PURPOSE:To efficiently obtain an alpha-hydroxycarboxylic acid useful as a food, raw material for agricultural chemicals medicines, organic synthesis, etc., by bringing a specific microorganism into contact with an alpha-hydroxynitrile. CONSTITUTION:One or more microorganisms (e.g. Pseudomonas sp. MY-1), belonging to any of the genuses Pseudomonas, Arthrobacter, Aspergillus, Penicillium, Cochliobolus and Fusarium and capable of hydrolyzing an alpha-hydroxynitrile (alpha-H) are aerobically or anaerobically cultivated in a culture medium containing a carbon source, such as glucose, a nitrogen source, such as yeast extract, and alpha-H, such as lactonitrile, at 5-60 deg.C and pH4-11 to form an alpha- hydroxycarboxylic acid (e.g. lactic acid) which is a hydrolyzate of the alpha-H in the culture. The resultant alpha-hydroxycarboxylic acid is then separated and recovered.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing α-hydroxycarboxylic acid, and more specifically, a method for producing α-hydroxycarboxylic acid from α-hydroxynitrile using specific microorganisms. Regarding.

[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. For example, the microorganism used is Bacillus.

Method using bacteria belonging to the genus Batatteridium, Micrococcus, and Brevibacterium (Special Publication No. 5B-151
No. 20); Method using bacteria belonging to the genus Corynebacterium (Japanese Unexamined Patent Publication No. 61-56086); genus Corynebacterium, genus Nocardia.

There is a method using bacteria belonging to the genus Bacillus, Bacteridium, Micrococcus, and Brevibacterium (Japanese Patent Application Laid-Open 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 problems]

Therefore, the inventor of the present invention aimed to establish an efficient production method for α-hydroxycarboxylic acid (after studying the microorganisms to be used, the inventors found that by selecting and using specific microorganisms other than the microorganisms described in the above publication). The inventors have discovered that the object can be achieved and have completed the present invention.

That is, the present invention is directed to Pseudomonas
nas) genus, Arthrobacter
The present invention relates to a method for producing α-hydroxycarboxylic acid belonging to the genus Er), Aspergillus, Cochliobolus, and Fusarium, characterized by contacting with α-lyl.

Microorganisms that can be used in the present invention belong to the various genera mentioned above, and α
- Any substance that has the ability to hydrolyze hydroxynitrile to produce α-hydroxycarboxylic acid, specifically Pseudomonas sp.
p. ) MY-1 (FERM P-9174) + Arthrobacter ararecens
aurescens) (IAM 12340), Aspergillus niger (J
CM 1925 and JCM 2261).

Penicillium chrysogenum
cr so enum (IFO 5473), Cochliobolus myabeanus
(OUT 2074) + Fusarium sp. (Fusar force + a + sp.) MY-2
(FBRM P-9187).

Fusarium sp.
MY-3 (FBRMP-9188), etc., and these can be used alone or in combination of two or more. 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 O. Presence or absence of pleomorphism of 8-1.4μm cells None Presence or absence of spores None Durham staining Negative motility Yes Flagella Scratching flagella (1) (2) Growth status in medium Broth agar plate culture Size 4-4.5 Cranes Round Gold rim. Smooth, opaque and glossy meat juice agar 1 & culture Good growth, smooth surface.

Translucent and glossy meat juice Liquid culture Strong turbidity and sweetened lidmus milk Alkalinization (3) Physiological properties Growth conditions Temperature (optimal temperature) 5-40℃ (20-37) pH (
Optimal pH) 4.5-9.5 (4.5-9.0)
Attitude towards oxygen Availability of obligate aerobic inorganic sources Ammonium salts, nitrate catalase + oxidase + 0-F test Hydrolysis of gelatin without reaction - Hydrolysis of starch - Hydrolysis of casein Hydrolysis of decacellulose - Formation of indole - VR test - MR test - Generation of Hzs - Acid and gas production from sugar Production yellow (King A medium, ) (in
g R culture ■ya) As mentioned above, country 1 is a bacillus with polar flagella, and country 0 was identified as a bacterium belonging to the genus Pseudomonas because it is Durham negative, absolutely aerobic, and positive for catalase and oxidase. It has been deposited with the Institute of Microbial Technology, Shima Institute of Industrial Science and Technology, and its accession number is FE.
It is RM P-9174.

(2) Microscopic characteristics Size of vegetative hyphae Width 1 to 3 μm Separation with interlocking connections None Sclerotia None Conidia Type of conidia formation Phialo-type macroconidial shape New moon Type conidia size 3-5 μ×20-35 Number of large conidial septa: 3-5 septated microconidial shape Long round microconidial size: 2-3μ x 5-7μ Number of microconidial septa: None Chlamydospore shape Spherical Shape size: 3-4μ CA medium 23℃7 Day culture slide culture method (3) Growth environment Growth temperature range 15°C to 39°C Unsuitable growth temperature 10°C or lower and 42°C or higher Optimal growth temperature 23°C to 25°C Growth pH temperature pH 2 to pH 10 Raw MPG medium on HH5p 7 days Culture (4) Phenoloxidase reaction laccase fraction tJ6ゝ〉 positive tyrosinase ′
C) a) MPG medium + 0.1% tannic acid b) Potato agar + 0.0005. M naphthol C) Potato agar + 0.1% cresol IQI
/i-01mm (2) Microscopic characteristics Size of vegetative hyphae Width 1 to 3μ Separation walls With sintering connections None Sclerotia None Conidia Type of conidia formation Phialo-type macroconidia Rugby ball-shaped conidia size 3 ~4μ x 10-15μ large conidial septa number
2-septate microconidial shape Rugby ball-shaped microconidial size 2-4μ x 8-10μ Microconidial septum number
None chlamydospore form Spheroidal form (prolific) size
μ CA medium 23°C 7-day culture Slide culture method Growth temperature range 15°C to 39°C Unsuitable growth temperature Below 10°C and above 42°C Optimum growth temperature 23°C to 25°C Growth pH temperature pH 4 to p) 110 Maximum growth HH
Culture on MPG medium on 5p for 7 days (4) Secretion of phenol oxidase-reactive laccase ゛) Positive tyrosinase ゛go) a) MPG medium + 0.1% tannic acid b) Potato agar + 0.0005M naphthol C
) Potato agar + 0.1% cresol Since they showed a t-like pattern, both MY-2 and MY-3 strains were identified as fungi belonging to the genus Fusarium. Strains MY-2 and MY-3 have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the former is P
ERMP-9187, the latter being FERM P-9188.

In addition to the above new strain isolated by the present inventor,
Even mutant strains obtained by natural or artificial mutation treatments can be used in the present invention as long as they have the ability to hydrolyze α-hydroxynitrile to produce α-hydroxycarboxylic acid. Of course it can be done.

Microorganisms can be used in various forms, such as cells in the growth phase, cells in the resting phase, immobilized cells, and even as extracts from the cells. It may be. The immobilization of microorganisms can be carried out by applying conventional immobilization techniques such as carrier binding method, crosslinking method, and entrapping method. Further, as an extraction method, a method may be employed in which a suspension of microbial cells is disrupted using ultrasound, a French press, a high-pressure homogenizer, etc., and then a soluble extract is obtained by centrifugation or the like.

Next, there are various types of α-hydroxynitrile used as a raw material in the present invention, such as lactonitrile (acetaldehyde cyanohydrin), acetone cyanohydrin, and manzolonitrile (benzaldehyde cyanohydrin).

Examples include α-hydroxybutyronitrile, α-hydroxypentanenitrile, α-hydroxy-α-phenylpropionitrile, α-hydroxy-α-(p-isobutylphenyl)-propionitrile, and the like. In addition, when using microorganisms other than bacterial cells in the growth phase, part or all of the above-mentioned nitriles in the raw material should be replaced with nitriles such as acetonitrile and propionitrile, or amides such as acetamide and propionamide, and then added to the medium. Can be done.

The medium for culturing the microorganism to produce α-hydroxycarboxylic acid from the raw material nitrile includes a carbon source,
It is necessary to use a substance that contains a substance that serves as an energy source, such as a nitrogen source. As the carbon source, any carbon source can be used as long as it can be assimilated by the test surface, such as sugars such as glucose and shakerose, and alcohols such as ethanol, propylene glycol, and glycerin. Further, as the nitrogen source, those that do not adversely affect the induction of the α-hydroxycarboxylic acid producing enzyme, such as nitrates, ammonium salts, yeast extract, and corn staple liquor, are used.

In addition, magnesium salts, calcium salts,
Inorganic salts such as phosphates, iron salts, copper salts, and nutritional substances necessary for the growth of microorganisms can be added to the medium as appropriate.

During culturing, the raw material nitrile compound may be added to the medium from the beginning or at an appropriate time after the start of culturing, and may be added all at once or divided into several portions. You may do so.

The reaction between the above-mentioned microorganisms and the raw material nitrile may be carried out under either aerobic or anaerobic conditions, and the reaction may be determined appropriately taking into consideration the properties of the microorganisms used. When reacting at a temperature of 5 to 60°C, preferably 10 to 40°C, and a pH of 4 to 11, preferably 6.
What is necessary is just to make it react in the range of -9. In addition, when reacting with something other than bacterial cells in the growth phase, the temperature is 0 to 50°C, preferably 0 to 30°C, and the pH is 4 to 11. Preferably 6~
It is sufficient to react within the range of 9. In the present invention, various reaction modes such as a culture method, a resting bacterial cell reaction method, and an immobilized bacterial cell reaction method can be carried out alone or in combination. In either case, the raw material nitrile compound is hydrolyzed by an enzyme derived from the microorganism, and the resulting α-hydroxycarboxylic acid can be separated and purified by a conventional method.Products include, for example, lactic acid, Examples include α-hydroxybutyric acid, α-hydroxyisobutyric acid, α-hydroxypentanoic acid, α-hydroxy-α-phenylpropionic acid, mandelic acid, α-hydroxy-α-(p-isobutylphenyl)-propionic acid, etc. can.

〔Example〕

Next, the present invention will be explained in detail with reference to examples.

Example 1 Shademonas sp. MY-1 (FERM P
-9174) with 5g/j of glycerin! , acetonitrile 5 g/l.

KHzPO* 0.5g/L Na2HPO4・12H
z05 g/1. Mg 304・7H! OO,2
g/l.

caclg・2HgOO,01g/L Fe5Oa・
7H, 00,001g/I1. Yeast extract 0.05g/
l.

100aj of medium containing 0.05g/J of corn steep liquor! and cultured for 72 hours.

After the culture was completed, bacteria were collected and washed. The obtained bacterial cells were adjusted to pH 8.
The suspension was suspended in 3 ml of 1/15M phosphate buffer, and 300 ml of lactonitrile was added thereto. After reacting at 22°C for 2 hours, gas chromatography (column; Th
ereon 30005his+alite TP, column temperature 160°C). As a result, the production amount of lactic acid was 100 g/J.

Example 2 Culture 2 reaction was carried out in the same manner as in Example 1 except that 150 μl of acetone cyanohydrin was used instead of lactonitrile and 15 μl of the same buffer was used instead of 3 mz of phosphoric acid solution. . Quantification is done by gas chromatography (
Column; Thermon 3000 celite5
45, column temperature 150°C). The production amount of α-hydroxyisobutyric acid was 8 g/J.

Example 3 In Example 1, α-hydroxy-α-phenylpropionitrile was used instead of lactonitrile (however, the raw material was used because it was unstable. Namely, acetophenone 4 g/j
! Culture 2 reaction was carried out in the same manner as in Example 1, except that an aqueous solution of acetophenone and potassium cyanide at a concentration of 2.2 g/j1) was added. Quantification is done by gas chromatography (column; Thern+on 3
000 celtte 545+ram temperature 200℃)
I went there. The production amount of α-hydroxy-α-phenylpropionic acid was 2 g/It.

Example 4 Example 1 except that 5 g/l of mandelamide Ig/L was used instead of acetonitrile and 5 g/l of manzolonitrile was used instead of lactonitrile, and 15 ml of the same buffer was used instead of phosphate buffer 3II11. Culture 1 was reacted in the same manner as above. Quantification is done by gas chromatography (
Column; Thermon 3000 celite5
45, column temperature 245°C). The production amount of mandelic acid is 0.3g/l! Met.

Example 5 In Example 1, Pseudomonas sp. MY-1 (
The cells were cultured in the same manner as in Example 1 using Fusarium sp. MY-2 (FBRM P-9187) instead of FERM P-9174). After culturing, collect bacteria and 1/1
Washed with 5M phosphate buffer (pl+8). The obtained bacterial cells were added to 151111 1/15M phosphate buffer (pH
8), 1500 g of lactonitrile was added, and the reaction was carried out for 2 hours. 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. MY-1 (
Fill! The cells were cultured in the same manner as in Example 1 using Fusarium sp. MY-2 (FERM P-9187) instead of Fusarium sp. MY-2 (FERM P-9187). After culturing, collect the bacteria,
Washed with 1/15M phosphate buffer (pH 8). The obtained bacterial cells were added to 15 ml of 1/15M phosphate buffer (pH 8").
) and added 75 μ of acetone cyanohydrin and reacted for 2 hours. After the reaction was completed, gas chromatography (column; Therw+on 3000
Quantification was performed using Celite 545 (column temperature: 150°C). The production amount of α-hydroxyisobutyric acid is 4.2g/l
Met.

Example 7 In Example 1, Pseudomonas sp. MY-1 (
PI! The cells were cultured in the same manner as in Example 1 using Fusarium sp. MY-3 (PI!RM P-9188) instead of Fusarium sp. MY-3 (PI!RM P-9188). After culturing, collect the bacteria,
Washed with 1/15M phosphate buffer (pH 8). The obtained bacterial cells were added to 15Ill of 1/15M phosphate buffer (pH 8).
150 μl of lactonitrile was added and the reaction was carried out for 2 hours. After the reaction was completed, the amount of lactic acid produced was determined in the same manner as in Example 1, and the amount of lactic acid produced was 2 g/l.

Example 8 Arthrobacter ararecens (TAM 12340
) for glucose 5 g/L and acetonitrile 3 g/L.

KHzPOn 1.5 g/l, NazHPOn・12
HzO1, 5g/1, MgSO4・7
HzOO, 2g/l.

CaClt-2HzOO, 01g/l, Fe5Oa・7
HzOo, o O1g/1. Yeast extract 0.03g/
The cells were inoculated into 10 μl of a medium containing 0.03 g/l of 6° corn steep liquor and cultured for 24 hours. After culturing, collect bacteria, wash, and reduce to 1/15 of 2a+1(7) pH8.
After suspending in M phosphate buffer, 10 mg of lactonitrile was added and reaction was carried out for 2 hours. After the reaction was completed, the amount of lactic acid produced was determined to be 1.5 g/l in the same manner as in Example 1.

Example 9 Aspergillus niger (JCK 1925) was grown in 10 m7 of Czapek medium containing 3 g/l of acetonitrile. The cells were inoculated and cultured for 6 days. After culturing, collect bacteria by centrifugation, wash, and add p118 1/15M phosphate buffer 2 rm
10 ml 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 In Example 4, Aspergillus niger (30M19
25) instead of Aspergillus niger (JCM 22)
Culture 1 reaction was carried out in the same manner as in Example 4 except that 61) was used. When the product was quantified in the same manner as in Example 1, the production amount of lactic acid was 0.4 g/l. Example 11 In Example 9, Aspergillus niger (30M19
25) instead of Penicillium chrysogenum (IFO5).
Culture was carried out in the same manner as in Example 9 except that 473) was used.

After the reaction was completed, the product was quantified in the same manner as in Example 1, and the production amount of lactic acid was 0.1 g/l.

Example 12 In Example 9, Aspergillus niger (30M19
25) instead of Cochliobolus myabeanus (OUT
Culture and reaction were carried out in the same manner as in Example 9 except that 20'r4) was used. After the reaction was completed, the amount of lactic acid produced was quantified in the same manner as in Example 1, and the amount of lactic acid produced was 0.8 g/l.

〔Effect of the invention〕

According to the present invention, α-hydroxycarboxylic acid can be efficiently produced by hydrolyzing α-hydroxynitrile using a specific microorganism. This α-hydroxycarboxylic acid is a food.

It is useful as a raw material for agricultural chemicals, medicines, organic synthesis, etc.

Claims (2)

[Claims] (1) Pseudomonas genus,
Genus Arthrobacter, Genus Aspergillus, Genus Penicillium, Genus Cochliobolus (
Cochliobolus and Fusarium
Production of α-hydroxycarboxylic acid, which comprises contacting one or more microorganisms belonging to the genus Salium and having the ability to hydrolyze α-hydroxynitrile with α-hydroxynitrile. Law. (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.