JPS6140795A - Method for microbiological preparation of 2-4c organic acid and salt thereof - Google Patents

Abstract

PURPOSE:To carry out industrially and advantageously the hydrolysis of a nitrile compound in preparing the corresponding organic acid and salt thereof from the nitrile compound using a microorganism, by using the microorganism of the genus Corynebacterium. CONSTITUTION:A microorganism, belonging to the genus Corynebacterium, and having the ability to hydrolyze a nitrile compound, e.g. Corynebacterium nitrilophilus or Corynebacterium species B-96 strain (FERM-P No. 7733) or Corynebacterium species C-99 strain (FERM-P No.7734), is cultivated in a culture medium containing a saturated nitrile added thereto. The resultant microbial cells and an enzymic extract thereof are suspended in water, etc., and a 2-4C nitrile compound is present therein to advance the hydrolytic reaction and form the corresponding organic acid and ammonia.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing corresponding organic acids and salts thereof from nitrile compounds having 2 to 4 carbon atoms by the action of microorganisms. The generated organic acids and ammonia usually exist in the form of organic acid ammonium salts, but organic acid ammonium salts can be recovered as organic acids by treatment with a roughly stoichiometric amount of strong acid or thermal decomposition treatment. It is possible. Among organic acids having 2 to 4 carbon atoms, acrylic acid and methacrylic acid in particular are used not only as raw materials for synthesizing methyl acrylate and methyl methacrylate, but also as raw materials for various higher esters.

(Prior art) Conventionally, if a nitrile compound is hydrolyzed using a strong acid,
It is well known that organic acids are produced, but the treatment of the waste acids that are produced at the same time has been a major problem.

Regarding the production method of acrylic acid or methacrylic acid, in order to solve these problems when acrylonitrile or methacrylonitrile is hydrolyzed with a strong acid, a two-step gas phase oxidation reaction of propylene or isobutylene is used.
Processes for production via acrolein and methacrolein have already been developed. However, even in this method, a high temperature reaction is required, and deterioration of the catalyst and polymerization of the reaction product remain as major problems, and the development of a new and industrially advantageous production method is desired.

On the other hand, nitrile compounds are assimilated or decomposed by microorganisms, such as acetonitrile [Journal Op.
Fermentation Technology (J, Ferme)
nt, Technol, ) 47@+ 631-pages,
19.69, same magazine volume 49, page 1011, 1971)
, α-Hydroxyisovaleronitrile (Journal Op Fermentation Technology Vol. 51, 39
3, 1973), benzonitrile [Biochemical Journal
al) 165@+309 pages.

1977) etc. are known. In addition, as a method for producing organic acids by microbiological hydrolysis of nitrile compounds,
A method using microorganisms such as Bacillus, Batatteridium, Micrococcus and Brevibacterium (Japanese Patent Publication No. 58-15120), conversion of acrylonitrile to acrylic acid and ammonia, Arthrobacter species I
Reaction using -9 (Journal Op Fermente)
John Technology Vol. 57, p. 8, 1979).

(Problem to be Solved by the Invention) However, the method using microorganisms such as Bacillus, Batatteridium, Micrococcus and Brevibacterium does not sufficiently hydrolyze α-hydroxynitriles and aminonitriles. However, for nitriles having 2 to 4 carbon atoms, i.e. acetonitrile, propionitrile, n-butyronitrile, isobutyronitrile, acrylonitrile and methacrylonitrile,
No industrial reaction activity was observed. In addition, when microorganisms of the genus Arthrobacter are used, when hydrolysis is performed with the same nitrile compound used during culture, the organic acids produced are further decomposed and
It was thought that the yield of the reaction product would decrease because it may be assimilated and disappear.

(Means for solving the problem) - With the aim of solving such industrial problems, the present inventors hydrolyzed a nitrile compound having 2 to 4 carbon atoms, and the organic acid produced was As a result of intensive research into microorganisms that have nitrile compound hydrolyzing activity that does not disappear through decomposition and assimilation, culturing, and reaction conditions, we have developed a method for hydrolyzing nitrile compounds selected from among microorganisms belonging to the genus Corynebacterium. They discovered an active microorganism and completed the present invention. That is, these microorganisms exhibit strong hydrolytic activity toward nitrile compounds having 2 to 4 carbon atoms, and have the property that the organic acids that are reaction products are not destroyed and assimilated.

The microorganism used in the present invention is Corynebacterium nitrilophyllus (C'orynebacterium nitrilophilus).
itrilophylus ATCC214L 9)
, Corynebacterium species B-96 strain and Corynebacterium species C-99 strain,
American Type Culture
Co11ection ATCC) and Microtechnical Research Institute No. 7733 and No. 7734, and its mycological properties are as shown below. In addition, Corynebacterium nitrilophilus ATCC2141
'9 was isolated as a microorganism that assimilates and decomposes nitrile compounds such as acetonitrile, and its properties are described in detail in Journal of Fermentation Technology, Vol. 47, p. 631, 1969.

Corynebacterium species B-96 strain a Morphology■Cell shape and size Zenbacterium 2.1-2.4 x 3.6-5.5μm■Presence or absence of cell pleomorphism Branched and spherical with remarkable polymorphism Shows form.

■Presence or absence of motility No ■Presence or absence of spores Wind ■Durham staining Positive ■Ancid-fast No b Growth status in each medium ■Grass agar plate culture Round, surface rough ζ gold edge, central protrusion, pink color, surface smooth, butter-like ,Opacity.

■Juice agar slant culture Medium growth, filamentous, surface wrinkled, pink, raised, wavy.

■Liquid culture of meat juice Thick but fragile bacterial film formed, transparent or slightly turbid, with sediment.

■Meat juice and gelatin puncture culture: Does not liquefy and grows well at the top.

■ Lidmus Milk No change.

C Physiological properties ■ Nitrate reduction Negative ■ MR test Negative - vP test Negative ■ Indole formation ゛ Negative ■ Hydrogen sulfide formation Negative ■ Starch hydrolysis Negative ■ Utilization of inorganic nitrogen source Positive ■ Soluble pigment formation Negative However, the fungus turns pink.

■Urease Positive.

[Phase] Catalase Positive ■ Hydrolysis of cellulose Negative ■ Range of growth pH 5-9, preferably 6-8° Temperature 18-39°C, preferably 23-36°C 0 Attitude towards oxygen Aerobic ■ <) H to acid and Production of gas Production of acid Production of gas Glucose 10 - Maltose + - Sucrose - Lactose - Corynebacterium species B-99 strain a Morphology ■Cell shape and size Rods 0.7-1.2 Xl, 2 ~1.7 μm ■Presence or absence of pleomorphism in cells Branched and pleomorphic.

■Presence or absence of motility No ■Presence or absence of spores No ■Durham staining positive ■Ancid-fast No b Growth status in each medium ■Juice agar plate culture Round, surface rough, golden rim, central protrusion, pale pink, surface smooth, butter opaque.

■Meat juice agar slant culture Growth, filamentous, with many wrinkles on the surface.
Pink, ridged, wavy.

■ Broth liquid culture Thick but fragile bacterial film formation, transparent or slightly turbid, sediment.

■Meat juice gelatin puncture culture Does not liquefy and grows well at the top.

■ Lidmus Milk No change.

C Physiological properties ■ Nitrate reduction Negative ■ MR test Negative ■ VP test Negative ■ Indole production Negative ■ Hydrogen sulfide production Negative ■ Starch hydrolysis Negative ■ Utilization of inorganic nitrogen source Positive ■ Soluble pigment production Negative However, bacteria turns pink.

■ Urease positive [strain] Catalase positive ■ Hydrolysis of cellulose Negative @Growth range pH 5-10, preferably 6-8 Temperature 10-40°C, preferably 25-35°C 0f11 Attitude towards elements Aerobic ■ From sugars to acids and Gas production Acid production Gas production Glucose 10 - Maltose
+ - Sea monosaccharide - Lactose - The above mycological properties are based on Bergy's bacterial taxonomy.
's Manual of Determ, 1na
tive Bacteriology) 8th edition (19
74), the B-96 strain and C-9 strain
The nine bacterial strains were determined to belong to the genus Corynebacterium because they were Durham-positive, incapable of sporulation, non-acid-fast, aerobic, and pleomorphic bacilli. Corynebacterium nitrilophilus ATCC21419, B-
The 96 strain and the C-99 strain differ in the appearance of the slant, growth conditions, and ability to assimilate nitrile.

These microorganisms have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the following numbers.

Strain Deposit number Deposit date-B-96
Microtechnology Research Institute No. 7733 July 20, 1980 C-
99 No. 7734 Same as above and equity @56-
Corynebacterium N-7 described in 17918
71 and N-774 strains and the microorganisms used in the present invention are clearly different in their ability to reduce nitrate.
They are considered to be different species of microorganisms.゛For culturing the microorganisms used in the present invention, a saturated nitrile compound such as acetonitrile or isobutyronitrile is used as the sole carbon source and nitrogen source, or a carbon source such as glucose or aldose, ammonium sulfate, ammonium nitrate, etc. A medium containing saturated nitrile as a nitrogen source, a carbon source, and a nitrogen source, as well as appropriate inorganic nutrients such as phosphate, chloride, iron, magnesium, manganese, and zinc, is used. In addition, by culturing using a medium to which no saturated nitrile compound is added, and continuing the culture by adding the nitrile compound as appropriate during the culture, it is possible to obtain bacterial cells having nitrile compound hydrolysis activity. The pH of the medium is usually 5 to 9, preferably 6 to 8, and the temperature is usually 20 to 32°C, preferably 25 to 32°C.
Culture is carried out aerobically for 5 days.

The bacterial cell culture thus obtained, the bacterial cells isolated therefrom, and the enzyme extract thereof are suspended in water or a buffer such as a phosphate buffer (for example, pH 7 to 8), and added to a If a nitrile compound is present, the hydrolysis reaction will proceed rapidly, producing the corresponding organic acid and ammoloa.

That is, usually, the microbial cells are 1 to 10% by weight and 2% to 10% by weight.・An aqueous suspension containing 0.2 to 10% by weight of a dill compound was prepared at a temperature of 5 to 35°C and a pH of 10 to 1.
The reaction may be allowed to proceed for 0 minutes to 24 hours.

Furthermore, since the nitrile compound used as a substrate in the reaction is generally highly biotoxic, it can be added sequentially while controlling the substrate concentration in the reaction system to a level that does not inhibit the reaction. Thus, nitrile compounds having 2 to 4 carbon atoms can be converted into the corresponding organic acids and ammonia in nearly 100% molar yield without the formation of by-products, amide compounds, and can be converted into highly concentrated aqueous solutions of organic acid ammonium salts. It can be generated and accumulated as .

In addition, in the above reaction, the bacterial cells or enzymes can also be immobilized using a conventional immobilization method using, for example, acrylamide gel or calcium alginate.

Furthermore, regarding the type of reactor, it is possible to carry out the reaction using any of the following types: batch type, continuous type, or reusable type.

(Effects of the Invention) The present invention provides the following effects by using a microorganism belonging to the genus Corynebacterium that has nitrile compound hydrolysis activity.
Almost 100% yield of nitrile compounds with 2 to 4 carbon atoms.
It was discovered that the reaction can be converted into the corresponding organic acid and ammonia, and because the reaction proceeds under mild conditions of room temperature and pressure, it can be applied to the industrially advantageous hydrolysis reaction of nitrile compounds without polymerization loss. It is possible.

(Example) Next, the present invention will be explained by examples.

Example 1 Corynebacterium nitrilophilus ATCC21
419 was cultured with shaking at 30°C for 24 hours using medium A described below, and then cultured with shaking at 30°C for 24 hours using medium B.

Δ Medium Glucose 1.0% Meat Extract
°1.0% peptone 0.3% salt, 0.1% p)l 7. Q B medium Isobutyronitrile 0.5% Potassium phosphate 0.1% Magnesium sulfate 0.05% Ferrous sulfate o,oos% Manganese sulfate 0.005% Ammonium sulfate 0.1% Potassium nitrate 0.1% pH 7.0 The bacteria grown under the above culture conditions were collected by centrifugation,
A reaction solution containing 2% by weight of dry bacterial cells, 2% by weight of methacrylonitrile, and 96% by weight of a pH 7.0 phosphate buffer solution was prepared, and the reaction was started at 30°C. Three hours after the start of the reaction, methacrylonitrile was hydrolyzed, and the molar yield was approximately 1.
Ammonium methacrylate salt was produced at 00%.

In addition, almost no methacrylamide was observed.

For analysis of the product, after the reaction was completed, the face piece was removed by centrifugation, and methacrylic acid and methacrylamide were determined by gas chromatography, and ammonia was determined by Nessler's method. Ammonium methacrylate salt was detected as methacrylic acid by gas chromatography.

Example 2 Corynebacterium° Nitrilophyllus ATCC2
1419 was cultured under the same culture conditions as in Example 1. When the reaction was started under the same reaction conditions as in Example 1 except that the acrylonitrile was used at 2% by weight, the acrylonitrile was hydrolyzed 30 minutes after the start of the reaction, and the molar yield was approximately 100%.
acrylic acid ammonium salt was produced. In addition, almost no acrylamide was observed. In addition,
The analysis of the product was carried out in the same manner as in Example 1.

Example 3 Corynebacterium nitrilophilus ATCC21
419 was cultured under the same culture conditions as in Example 1. The reaction was carried out under the same reaction conditions as in Examples 1 and 2, except that acetonitrile, propionitrile, n-butyronitrile, and isobutyronitrile were each used in an amount of 2% by weight. The reaction activities were compared and the results are shown in Table 1. Incidentally, the specific activity is expressed in millimole of product/gram of dry bacterial mass/time.

Table 1 Example 4 Corynebacterium species B-96 strain was cultured with shaking at 30° C. for 24 hours using the following C medium.

C medium Glucose 1.0% Meat extract 1.0% Pep-ton 0.3% Salt
0.1% Acetonitrile 0.5% Potassium phosphate 0.1% Magnesium sulfate 0.05% Ferrous sulfate
0.005% Manganese sulfate o, oos% Ammonium sulfate 0.1% Potassium nitrate 0.1% pH 6.0% The bacterial cells grown under the above culture conditions were collected by centrifugation,
The reaction was carried out under the same reaction conditions as in Example 1, except that methacrylonitrile, acrylonitrile, and acetonitrile were each used at 2% by weight, and the reaction activities were compared. The results are shown in Table 2.

Example 5 Corynebacterium species C-991 strain was cultured under the same culture conditions as in Example 4 to produce bacterial cells 3).

A reaction was carried out using methacrylonitrile, acrylonitrile and acetonitrile under the same conditions as in Example 4, and the reaction activities were compared. The results are shown in Table 3.

Example 6 Corynebacterium nitrilophilus ATCC21419 cells prepared in the same manner as in Example 1 were suspended in distilled water at pH 6.0 to a concentration of 2% by dry weight. Methacrylonitrile was continuously added dropwise to this at a ratio of 2% over 3 hours, and the mixture was reacted at 30°C. 12
After reacting for a period of time, the bacterial cells were removed by centrifugation to obtain a clear solution. When the methacrylic acid concentration of this product was determined, it was found to be 9.7% by weight.

Example 7 Bacterial cells were collected from the reaction solution obtained in Example 6 by centrifugation, and suspended again in distilled water with a pH of 6.0 to a dry cell mass of 2% by weight. Methacrylonitrile was continuously added dropwise in the same manner as in 6, and the mixture was reacted for 12 hours. When this operation was repeated a total of 5 times, the results shown in Table 4 were obtained.

(The added substrate is 8% by weight for one reaction) Procedure amendment September 19, 1980

Claims (1)

[Claims] Due to the action of microorganisms that belong to the genus Corynebacterium and have the ability to hydrolyze nitrile compounds, carbon atoms with a carbon number of 2 to 4
1. A microbiological method for producing organic acids having 2 to 4 carbon atoms and salts thereof, which comprises producing a corresponding organic acid and ammonia from a nitrile compound.