JPH05308980A - Production of amide - Google Patents

Abstract

(57) [Summary] [Structure] In a method for producing amides from nitriles by the action of a microorganism, the microorganism or a treated product thereof is treated with an organic solvent and then subjected to a reaction. [Effect] The yield of amides can be increased as compared with conventional microbiological methods, which is practically advantageous.

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 corresponding amides from nitriles by a microbiological method, and more specifically, it is characterized by a method for activating a microorganism or a processed product thereof. The present invention relates to a method for producing amides.

[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 methods for producing corresponding amides from nitriles as raw materials. A method using an enzyme (called nitrile hydratase) derived from A. has also been reported. The advantages of the method using a microorganism-derived enzyme 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. Bacteria (Japanese Patent Publication No. 59-37951 and Japanese Patent Publication No. 62-215) are known as microorganisms having an enzymatic activity to produce amides by adding water to nitriles.
19), particularly Coneri type bacteria ( Arthrobacter , Rhodococcus ( Rho ).
dococcus ), Corynebacterium ( Coryn
ebacterium ), etc .: Japanese Patent Publication No. 56-17918
JP-A-59-2693, JP-A-61-16219
No. 3, JP-A No. 62-91189) and fungi ( Fusarium ): JP-A No. 64-86.
889).

Although these microorganisms are separated from soil, waste water, etc., they often have low activity as they are and are not practical. For this reason, attempts have been made to investigate the culture method, enzyme inducer, stabilizer, prosthetic group, etc. and to increase the activity by mutation treatment, but it cannot be said to be sufficient for actual production.

[0004]

[Means for Solving the Problems] As a result of examination on a method of activating nitrile hydratase derived from a microorganism, it was found that treatment of an organic solvent, which has not been known so far, increases the activity of this enzyme. A new manufacturing method was established. That is, the gist of the present invention is a method for producing amides from nitriles by the action of a microorganism having the ability to decompose nitriles, characterized in that the microorganism or a treated product thereof is treated with an organic solvent and then subjected to a reaction. It exists in the method for producing amides.

The present invention will be described in detail below. In the present invention, the nitriles used as a reaction raw material include: simple nitriles such as acetonitrile, propionitrile, n-butyronitrile and isobutyronitrile; α-aminopropionitrile, α-aminomethylthiobutyronitrile Α-Aminonitriles such as α-aminobutyronitrile and aminoacetonitrile; α-such as lactonitrile, hydroxyacetonitrile and 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,
That is, acetonitrile produces acetamide, propionitrile produces propionamide, and acrylonitrile produces acrylamide.

The microorganisms used in the present invention include those obtained by screening from soil, drainage, etc.,
Also, there is no particular limitation as long as it is capable of decomposing nitriles and converting them into amides among those stored as type culture. Examples of such microorganisms include Enterobacter sp .
bacteria sp. ) MCI 2707 bacterium [deposited as Micro Engineering Research Institute No. 12801 (FERM P-12801) at the Institute for Microbial Technology, Institute of Industrial Technology].

Cultivation of the microorganism used in the present invention can be carried out in a conventional manner. 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 set to 5 to 10 and the temperature is 20 to 37 ° C. for 1 to 7 days with shaking culture or static culture.

After selecting one of the above-mentioned microorganisms, culturing the same by the above-mentioned method and collecting the bacterial cells from the culture solution by centrifugation or the like, the following suitable organic solvent such as acrylamide 10-40 % Suspension in aqueous solution, freezing point above 40
Hold at 1 ° C. or lower for 1 hour to 6 hours. Then collect the cells again by centrifugation etc. and wash them to 10 parts with physiological saline, etc., and then add water, physiological saline, phosphoric acid, Tris, etc. to pH 4
It may be suspended in a buffer solution of ~ 11, to which the desired nitriles, for example, acrylonitrile, may be added and coexist at an appropriate temperature condition, for example, above the freezing point and below 40 ° C.
In that case, it is possible to add nitrites successively as the reaction progresses.

Further, the enzyme was purified by crushing, ammonium sulfate precipitation, ion exchange, gel filtration, column chromatography using a hydrophobic carrier, etc. to obtain the enzyme from the microbial cells or culture supernatant cultured as described above. It is also possible to carry out the reaction with the above organic solvent treatment using an enzyme. Further, the microbial cells or enzyme obtained by the above culture and organic solvent treatment method, polyacrylamide, photocrosslinkable resin, agar, entrapping immobilized with a gel such as carrageenan,
Alternatively, the microbial cells or enzyme obtained by the above culture are immobilized, then treated with an organic solvent, and reacted with nitriles in a stirred reaction tank under the appropriate pH and temperature conditions as described above, or in a column. It is also possible to carry out the reaction by filling the above and circulating a liquid containing nitriles.

As the organic solvent used in the organic solvent treatment of the present invention, in addition to the above-mentioned acrylamide, various concentrations of the following organic solvents and mixtures thereof can be used. Hydrocarbons such as n-hexane, cyclohexane, 1-hexene, cyclohexene and butadiene; Esters such as ethyl acetate, γ-butyrolactone and sugar ester; Ethers such as diethyl ether, tetrahydrofuran, dioxane and ethylene glycol dimethyl ether O Ethanol, isopropyl alcohol, 2-methoxyethanol, cyclohexanol, ethylene glycol, glycerin, erythritol and other alcohols, polyols; o Acetone, cyclohexanone, methyl ethyl ketone,
Ketones such as acetophenone; ○ Aldehydes such as acetaldehyde, glutaraldehyde, benzaldehyde, furfural;

Organic acids such as acetic acid, palmitic acid, muconic acid, benzoic acid, trifluoroacetic acid, methanesulfonic acid or salts thereof; Acid anhydrides such as acetic anhydride and maleic anhydride; Benzene, toluene, xylene, Aromatic compounds such as naphthalene, anthracene and indole; ○ Phenols such as phenol, cresol, catechol and hydroquinone; ○ Amides such as acetamide, malonamide, nicotinamide, dimethylformamide; ○ Acetonitrile, malonnitrile, nicotinonitrile, methacryl Nitriles such as ronitrile; ○ Pyridines such as pyridine, picoline, quinoline; ○ Amines such as piperidine, triethylamine, cyclohexylamine, ethylenediamine, morpholine, pyrrole, aniline;

○ Nitrogen-containing compounds such as phthalimide, maleimide, imidazole, urea, nitromethane, hydrazine, n-butyl isocyanate, diazomethane; ○ Silicon-containing compounds such as trimethylsilylimidazole; ○ Chloroform, bromoform, bromobenzene, iodomethane, Halogen-containing compounds such as N-bromosuccinimide; ○ Sulfur-containing compounds such as dimethyl sulfoxide, carbon disulfide, mercaptoethanol, thiophene, dimethyl sulfide, p-toluenesulfonic acid; ○ Methylphosphonate, isopentenyl pyrophosphate, A
Phosphorus-containing compounds such as TP; ○ Water-soluble polymer compounds such as polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone; ○ Surfactants such as Tween 60, Triton X100, NP-40, sodium dodecyl sulfate, cetyltrimethylammonium chloride Kind.

In the present invention, the amides obtained after the reaction can be used as an aqueous solution as they are, or can be used 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 using activated carbon, an ion exchange resin, an ion exchange membrane or the like. The microbiological hydration reaction of nitriles according to the present invention is characterized in that the microorganism to be used is cultured and then treated with an organic solvent before the reaction. Therefore, in the present invention, "to hydrate nitriles by the action of microorganisms to convert them into corresponding amides" means that the nitrites are brought into contact with the culture solution after culturing the microorganisms, bacterial cells or treated products thereof. , And the case where the microorganism or the enzyme produced by the microorganism inside or outside the microorganism is immobilized and used in the reaction.

[0014]

EXAMPLES The present invention will be specifically described below with reference to examples, but the invention is not limited to the following unless the gist thereof is exceeded. 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 completion of the culture, the cells were separated by centrifugation and treated with a 40% acrylamide aqueous solution at 25 ° C for 1 hour. After that, the bacterial cells were washed with physiological saline and suspended in a phosphate buffer (pH 7.0, 0.1M). 0.8 ml of this was sampled, mixed with 0.2 ml of 7% acrylonitrile aqueous solution, and reacted at 30 ° C. for 1 hour. After completion of the reaction, acrylonitrile was converted into acrylamide, and 0.18% was produced.

<Example 2> The cells cultured in the same manner as in <Example 1> and centrifuged were treated with an aqueous solution of 30% acrylamide to give 25 cells.
It was treated at 1 ° C for 1 hour. Then wash the cells with physiological saline,
It was suspended in a phosphate buffer (pH 7.0, 0.1 M). 0.8 ml of this was sampled, mixed with 0.2 ml of 7% acrylonitrile aqueous solution, and reacted at 30 ° C. for 1 hour. After completion of the reaction, acrylonitrile was converted into acrylamide, and 0.19% was produced.

<Example 3> The cells cultured and centrifuged in the same manner as in <Example 1> were diluted with 20% acrylamide aqueous solution to give 25 cells.
It was treated at 1 ° C for 1 hour. Then wash the cells with physiological saline,
It was suspended in a phosphate buffer (pH 7.0, 0.1 M). 0.8 ml of this was sampled, mixed with 0.2 ml of 7% acrylonitrile aqueous solution, and reacted at 20 ° C. for 1 hour. After completion of the reaction, acrylonitrile was converted into acrylamide, and 0.15% was produced.

<Comparative Example 1> The cells cultured and centrifuged in the same manner as in <Example 1> were washed with physiological saline and suspended in a phosphate buffer (pH 7.0, 0.1 M). 0.8 of this
ml was sampled and a 7% acrylonitrile aqueous solution was added.
It was mixed with 2 ml and reacted at 30 ° C. for 1 hour. After completion of the reaction, acrylonitrile was converted to acrylamide, and
It produced 01%. From this, it can be said that the organic solvent treatment of the present invention is effective in increasing the yield of amides.

[0018]

EFFECTS OF THE INVENTION According to the method of the present invention, in producing amides from nitriles by a microbiological method, the yield of amides can be increased, which is practically advantageous.

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

Claims (3)

[Claims] 1. A method for producing amides from nitriles by the action of a microorganism having the ability to decompose nitriles, characterized in that the microorganism or a treated product thereof is treated with an organic solvent and then subjected to a reaction. Manufacturing method. 2. The method according to claim 1, wherein the organic solvent is water-soluble. 3. The method according to claim 1, wherein the organic solvent is an aqueous solution of amide.