JPH05236975A - Production of amides - Google Patents

Abstract

PURPOSE:To obtain industrially useful high-purity amides from nitriles by reacting a microorganism, belonging to the genus Enterobacter and having the ability to decompose the nitriles with the nitriles. CONSTITUTION:A microorganism [e.g. Enterobacter.sp. MCI2707 (FERM P-12801) which is a new microorganism], belonging to the genus Enterobacter and having the ability to decompose nitriles (preferably acrylonitrile) is made to react with the nitriles. The conversion of the nitriles into amides is preferably carried out by a method for culturing the microorganism, suspending a microbial cell collected from the resultant culture solution in a buffer solution at pH4-11 and then adding the nitriles thereto.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing water-soluble amides by adding water to nitriles by the action of microorganisms. More specifically, the present invention relates to amides characterized by the microorganisms used. Related to biological manufacturing methods.

[0002]

2. Description of the Related Art Conventionally, chemical methods such as a sulfuric acid method and a copper catalyst method have been widely industrialized as a method for producing corresponding amides from nitriles as a raw material. A method using an enzyme derived from is also reported. Advantages of the method using a microorganism are that the reaction conditions are mild, so that the polymerization reaction of the raw material and the product is difficult to occur, there are few by-products, and the reaction apparatus is small. Examples of microorganisms having an enzymatic activity of adding water to nitriles to form amides include bacteria (Japanese Patent Publication No. 62-21519), particularly coryneform bacteria (Arthrobacter, Rhodococcus, Corynebacterium, etc.): JP-A-56-17918, JP-A-5
9-2693, JP-A-61-162193, JP-A-62-911189) and fungi (Fusarium:
JP-A 64-86889) and the like, and Pseudomonas has been reported as the only Gram-staining bacterium (JP-B-59-37951).

In terms of taxonomy, microorganisms are classified into eukaryotic cells (slime mold, flagella, zygomycetes, ascomycetes, basidiomycetes, imperfect bacteria, etc.) and prokaryotic cells (bacteria). , Blue algae,
Actinomycetes, etc.) Of the prokaryotic microorganisms,
Bacteria are classified into Gram-staining positive ones (Bacillus subtilis, coryneform bacteria, lactic acid bacteria, Staphylococcus, etc.) and Gram-staining negative ones (enteric bacteria, Pseudomonas aeruginosa, Vibrio bacteria, etc.) according to the difference in cell wall structure. Therefore, the two are taxonomically different.

[0004]

As a result of extensive screening of microorganisms from soil, the inventors have shown that microorganisms belonging to the genus Enterobacter belonging to the family Enterobacteriaceae, which has never been known, as shown below. Has been found to have the ability to convert nitriles to amides, and established a new method for producing amides. That is, the gist of the present invention is a method for producing amides from nitriles by the action of a microorganism, wherein the microorganism belongs to the genus Enterobacter and is a microorganism having the ability to decompose nitriles. Exists in the manufacturing method. The present invention will be described in detail below.

The nitriles used as reaction raw materials in the present invention include: simple nitriles such as acetonitrile, propiononitrile, n-butyronitrile and isobutyronitrile; α-aminopropionitrile, α-aminomethylthio. Α-Aminonitriles such as butyronitrile, α-aminobutyronitrile, aminoacetonitrile; o-lactonitrile, hydroxyacetonitrile, α-hydroxy-γ-methylthiobutyronitrile
Hydroxyl nitriles; -β-amino nitriles such as amino-3-propionitrile; -Dinitriles such as malonnitrile, succinonitrile, adiponitrile; -α-such as acrylonitrile and methacrylonitrile
Unsaturated nitriles; ○ α such as homoveratrine nitrile and benzonitrile
-Benzenenitriles; ○ Heterocyclic nitriles such as nicotinonitrile and isonicotinonitrile, and the like, among which acetonitrile, propiononitrile, acrylonitrile, methacrylonitrile, n-butyronitrile, isobutyronitrile, etc. 2 to 4 carbon atoms
Nitriles are preferred. Particularly preferred is acrylonitrile.

The amides produced from the above nitriles are amides corresponding to the above nitriles, for example, acetamide from acetonitrile, propionamide from propiononitrile, and acrylamide from acrylonitol. It Next, the microorganism used in the present invention will be described. The microorganism used in the present invention is not particularly limited as long as it belongs to the genus Enterobacter and has the ability to decompose nitriles and convert them into amides. Examples of such microorganisms Enterobacter sp (Enterobacte
r sp. ) MCI No. 2707 hereafter may be abbreviated as "the present strain" or "MCI No. 2707 bacterium"), and the present strain may be referred to the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology, Micro Engineering Research Institute No. 12801 (FERM P- Deposited as 12801).

[0007] The MCI 2707 bacterium was
It is a bacterium isolated from natural soil, and its mycological properties are as follows. a) Morphological properties

[Table 1] Culturing on normal agar medium at 30 ° C for 24 hours 1) Cell size and shape: Short rods 2) Presence or absence of cell polymorphism: None 3) Motility: Yes, periflagellates 4) Sporulation : None 5) Gram stain: Negative 6) Anti-acidic: Negative b) Growth state 1) Characteristics of colonies cultured on ordinary agar culture medium at 30 ° C for 2 days 1. Outline: Circular 2. Surface ridge: Semi-lenticular shape 3. Directional shape: Smooth 4. Gloss: Yes 5. Color tone: Yellowish gray 6. Transparency: Opaque 7. Edge: All edges c) Physiological properties 1) Growth in air: + 2) Growth under anaerobic conditions: + 3) Catalase: + 4) Oxidase: -5) OF test: F 6) Gelatin Hydrolysis: -7) Litmus milk: No acid coagulation 8) Nitrate reduction: +9) Denitrification reaction: +10) Methyl red test: -11) VP test: +12) Indole formation: -13) Sulfidation production of hydrogen: - 14) starch hydrolysis: - 15) use of citric acid: + (on Christensen medium) 16) use of inorganic nitrogen source NH _4: + NO _3: + 17) urease: - 18) lysine Decarboxylation: + 19) Decarboxylation of ornithine: + 20) Hydrolysis of arginine: -21) Deamination of tryptophan: -22) DNase: -d) Biophysical properties 23 ) Β-Galactosidase: + 24) Degradation of Tween 80: -25) Pigment production: -26) Growth temperature range: 9 to 45 ° C 27) Growth pH: pH 4 to 10 28) Generation of acid and gas from only carbon source

[0008]

[Table 2]

[0009]

[Table 3] e) Chemotaxonomic properties Isoprenoid quinone Q-8, MK-8 GC content in DNA 57 mole% f) Taxonomic considerations ○ Family level identification This strain MCI2707 is 1) Gram-negative bacillus. ,
2) show facultative anaerobic, 3) ferment glucose,
4) Negative oxidase, 5) Periflagellate and other properties suggest that Bergey's Manual of System
Electronic Bacterology, Volume 1. Factory-teverlyAnaer
It has been revealed that the bacterium belongs to the family Enterobacteriaceae of the group of obic Gram-Negative Rods. -Identification of genus level This strain MCI2707 was Enterobacter
Each genera belonging to the iacae family (Bergey's Man
null of Systematic Bacteri
1) Motility, 2) VP test positive, 3) urease negative, 4) ornithine decarboxylation positive, 5) DNase
It was found to belong to the genus Enterobacter because it has characteristics such as negative, 6) acid production from sorbitol, and positive. The GC content of the DNA of this strain is 5
7%, 52-60 of the genus Enterobacter
It also matches the% range. ○ Identification of species level Currently Bergey's Manual of Sys
The first volume of the Tactical Bacteriology
Seven species are described as the genus Enterobacter . For this strain MCI2707 No. bacteria and these species was subjected to a comparison, Enterobac as shown in the table below
It was suggested to be closely related to ter aerogenes . However, the GC content of this strain is 57%,
E. There was a difference of about 3% from 53-54% of the aerogenes . As a bacterial strain having a GC content of 57%, E. coli was used as shown in the table below . sakazakii and E. agglo
Although merans is known, the physiological properties of this strain were clearly different from those of these species. Therefore, this strain MCI2707 was designated as Enterobacter . s
p. Was identified.

The following are MCI 2707 and Entero
The comparison with various types of Bacter is shown. In the table, + indicates all positive, (+) indicates 89% or more positive, d indicates 10 to 80% positive, and-indicates all negative.

[0011]

[Table 4]

Cultivation of the microorganism used in the present invention can be carried out by a conventional method. The medium to be used includes carbon sources such as glucose, glycerol, starch syrup and starch, inorganic nitrogen sources such as ammonium sulfate and ammonium nitrate, organic nitrogen sources such as soybean flour, yeast extract, peptone and urea, and phosphate, sodium, potassium and magnesium. Ordinary medium containing an inorganic salt such as a suitable ratio is used. It is also desirable to add nitriles such as acetonitrile, amides such as acrylamide, and inorganic salts necessary for enzyme activity such as iron ion and cobalt ion to the medium in order to induce the target enzyme. The pH of these media is 5 to 10, the temperature is 20 to 37 ° C., and shaking or static culture is performed for 1 to 7 days.

The conversion of nitriles to amides can be carried out by culturing the microorganism in the presence of nitrile for the purpose of hydration, but preferably by the following method. Select one of the above-mentioned microorganisms, cultivate it by the method described above, collect the bacterial cells from the culture solution by centrifugation, etc.,
Add this to water, saline, phosphoric acid, Tris, etc.
11 suspended in the buffer solution, to which the desired nitriles such as acrylonitrile were added, and under appropriate temperature conditions,
For example, they may coexist at temperatures above the freezing point and below 40 ° C. In that case, it is also possible to successively add the desired nitriles as the reaction progresses.

Crushing, ammonium sulfate precipitation, ion exchange, gel filtration, microbial cells or culture supernatant cultured as described above,
It is also possible to purify the enzyme by means of column chromatography using a hydrophobic carrier and to carry out the above reaction using the obtained enzyme. Further, the microbial cells or enzyme obtained by the above method is entrapped and immobilized on a gel such as polyacrylamide, photocrosslinkable resin, agar, and carrageenan, and the reaction is agitated under the appropriate pH and temperature conditions as described above. It is also possible to react with the nitriles in the tank or by filling a column and circulating a liquid containing the nitriles.

The amides obtained after the reaction can be used as an aqueous solution as it is or as a powder after being concentrated by a method such as membrane concentration or spray dry concentration. Depending on the case, it is possible to further increase the purity by a method such as activated carbon, ion exchange resin, or ion exchange membrane. The microbiological hydration reaction of nitriles according to the present invention is essentially the same as known methods except that the microorganism used is specific. Therefore, in the present invention, "to convert nitriles into corresponding amides by hydration of microorganisms by the action of microorganisms" means that the microorganisms are cultured in the presence of nitriles, as well as the culture solution and the bacterial cells after the culture of the microorganisms. Also, it includes any of the cases where these treated products are brought into contact with nitrile. Further, it also includes the case of immobilizing a microbial cell or an enzyme produced by the microorganism inside or outside the microbial cell and utilizing it for the reaction.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto unless it exceeds the gist. The quantification of nitriles and amides was performed by high performance liquid chromatography. <Example 1> Enterobacter sp. MCI
No. 2707 is 0.4% glycerol, 0.
2%, polypeptone _{0.05%, FeSO 4 · 7H 2} O
0.001%, CoCl ₂ .6H ₂ O 0.001%, Na
_{Cl0.2%, MgSO 4 · 7H 2} O0.04%, K 2 H
The medium was aerobically cultured at 30 ° C. for 3 days in a medium containing 0.25% PO ₄ and 0.025% acrylamide. After the culture was completed, the cells were separated by centrifugation, and the obtained cells were washed with physiological saline to obtain a phosphate buffer solution (pH 7.0, 0.1).
M). 0.8 ml of this was sampled, mixed with 0.2 ml of 7% acrylonitrile aqueous solution, and mixed at 20 ° C.
And reacted for 1 hour. After completion of the reaction, acrylonitrile was converted to acrylamide, and 0.01% was produced.

[0017]

Industrial Applicability As described above, according to the method of the present invention, industrially useful high-purity amides can be obtained from nitriles by using a novel microorganism belonging to the genus Enterobacter.

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[Procedure amendment]

[Document name] Consignment number change notification

[Submission date] August 5, 1992

[Former name of depositary institution] Institute of Microbiology, Institute of Industrial Technology

[Old consignment number] Micro Engineering Research Institute, Microbiology No. 12801 (FERM
P-12801)

[Name of new depositary institution] Institute of Microbial Technology, Ministry of International Trade and Industry, Institute of Industrial Technology

[New contract number] Mikokenjoyori No. 3955 (FERMB
P-3955)

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yutaka Teranishi 1000 Kamoshida-cho, Midori-ku, Yokohama-shi, Kanagawa Sanryo Kasei Co., Ltd.

Claims (5)

[Claims] 1. A method for producing amides from nitriles by the action of a microorganism, wherein the microorganism is a microorganism belonging to the genus Enterobacter and capable of degrading nitriles. 2. The method for producing amides according to claim 1, wherein nitriles are added to the culture solution of the microorganism to convert the nitriles into corresponding amides. 3. The method for producing amides according to claim 1, wherein the addition of the nitriles is carried out continuously or intermittently. 4. The method for producing amides according to claim 1, wherein the nitriles are acrylonitrile. 5. The microorganism according to claim 1, wherein the microorganism is Enterobacter sp. MCI No. 2707.
4. The method for producing an amide according to any one of 3 above.