JPH0681597B2 - Method for producing amide compound - Google Patents

Description

TECHNICAL FIELD The present invention relates to amidation of nitrile compounds. More specifically, it relates to a method for producing an amide compound by hydrolyzing a nitrile compound by the action of a microorganism.

(Prior Art) To date, as a method for producing an amide compound from a nitrile compound using a microorganism, for example, Pseudomonas (Pseu
Japanese Patent Publication No. 59-37951 (Yamada et al.) using microorganisms belonging to the genus Domonas, Corynebacterium
JP-B-56-17918 (Watanabe et al.) Using microorganisms belonging to the genus and JP-A-62-91189 (Kawakami et al.) Using microorganisms belonging to the genus Rhodococcus have been reported. It is an industrially useful invention for producing and / or methacrylamide by using a microorganism.

(Problems to be described by the invention) Microorganisms that produce industrially useful amide compounds are considered to be more likely to exist in nature, and an object of the present invention is to search for such microorganisms, It is to provide a method for producing an amide compound which is industrially useful.

(Means for Solving the Problem) As a result of broadly seeking microorganisms that produce amide compounds in nature, the present inventors have obtained a suspension of microorganisms belonging to the genus Acinetobacter collected from soil and a nitrile compound at room temperature. The present invention has been completed, and it was found that the corresponding amide compound is promptly produced by mixing in.

That is, the present invention, in the method of producing an amide compound by hydrolyzing a nitrile compound microbially, the microorganism to be used belongs to the genus Acinetobacter and the nitrate reduction test is negative, and the nitrile compound is hydrolyzed. The method for producing an amide compound is characterized in that it has the ability to be converted into the corresponding amide compound.

The present invention will be described in detail below.

The microorganism used in the present invention is Acinetobacter (Acin).
etobacter) genus and negative nitrate reduction test,
Hydrolyze the tolyl compound to convert it to the corresponding amide compound.
It is a microorganism that has the ability to convert. The nitrate reduction test is
Pore media for nitrate reduction test of Eiken Chemical Co., Ltd. N
Judgment is performed using the broth set. For more details, see
15 g of peptone, 2 g of disodium phosphate per 1000 ml of ground,
Mix 1 g of potassium nitrate and adjust to pH 7.2 ±, then heat
Dissolve and dispense 3 ml into a small test tube with a cap, and sterilize under high pressure.
Inoculate a fresh culture medium with a platinum wire and
Loosen well and incubate at 30 ℃ for 48 hours. Square filter paper
Aseptically containing the reagent (improved Griess reagent) in (5 x 5 mm)
Let it dry, then stick it on a stick.
Immerse the immer in the culture medium and immediately pull it up. N paper
What turns red immediately is regarded as positive. N paper is unchanged
In case of chemical conversion, add a trace amount of zinc dust to unused N paper
Then, it is immersed in the culture solution and immediately pulled up. 1-2 minutes later N
If the paper turns red, it is negative; if it is unchanged, it is positive.
To do.

Examples of the microorganism include, for example, Acinetobacter calcoaceticus S.
AII25 strain (deposited as Micromachine Research Institute No. 10433), and Acinetobacter spp.
ter calcoaceticus) SAIE5 strain (deposited as Micromachine Research Institute No. 10432). The mycological properties are shown in Table 1.

The above-mentioned mycological properties can be found in the Vergy classification documents (Berg
er's Manual of Systematic Bacteriology) (1984
Years), both SAII25 and SAIE5 strains were aerobic, gram-negative, catalase-positive, oxidase-negative, non-motile, bacilli or cocci, nitrate-reducing negative, acid production from glucose, and OF test oxidizes Therefore, it was identified as a microorganism contained in Calcoaceticus species belonging to the genus Acinetobacter of the family Neisseriaceae.

This microorganism has been deposited with the following number at the Institute of Microbial Technology, National Institute of Industrial Science and Technology.

Culture of microorganisms used in the present invention, glucose, dextrin, carbon sources such as maltose, ammonium sulfate, nitrogen sources such as ammonium nitrate, yeast extract, malt extract, peptone, organic nutrient sources such as meat extract and phosphates, Sodium, potassium, iron, magnesium, manganese, a normal medium containing inorganic nutrient sources such as zinc as appropriate, isobutyronitrile, methacrylonitrile, acrylonitrile, isophthalonitrile, nitrile compounds such as adiponitrile, isobutyramide, A method of adding one or more kinds of compounds selected from amide compounds such as methacrylamide, acrylamide, and isophthalamide and performing the culture can be mentioned, but the method is not limited thereto.

The nitrile compound hydrolyzed by the microorganism in the present invention includes, for example, acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, isobutyronitrile, mononitrile compounds such as n-butyronitrile, adiponitrile, isiphthalonitrile, orthophthalonitrile. Examples thereof include, but are not limited to, dinitrile compounds such as nitrile. The amide compound produced from the nitrile compound by the microorganism in the present invention is an amide compound obtained by hydrolyzing the corresponding nitrile compound, and in the dinitrile compound, 1-
Mention may be made of amide-substituted products and 2-amide-substituted products.

The method of hydrolyzing the nitrile compound using the microorganism includes, for example, a method of obtaining the corresponding amide compound by adding the nitrile compound to a culture solution of the microorganism, an aqueous solution of bacterial cells separated from the culture solution of the microorganism. Method of obtaining the corresponding amide compound by adding the nitrile compound to the dispersion, a method of immobilizing the cells separated from the culture solution of the microorganism on a carrier such as polyacrylamide, k-carrageenan, alginate, etc., as an immobilized microorganism , Or a treated product of a microorganism (for example, a crushed product of the microorganism or an enzyme separated from the microorganism)
Is used in place of the above-mentioned microorganism, but is not limited thereto.

Incidentally, the hydrolysis reaction of the nitrile compound according to the present invention,
It is considered to proceed by the nitrile hydratase produced by the microorganism.

(Example) Next, the method of the present invention will be specifically described with reference to Examples.
It goes without saying that the present embodiment is merely an example in which the method used in the present invention is specifically described, and the method of the present embodiment does not limit the content of the present invention in any way.

Example 1 (1) Culture The SAII25 strain was cultured under the following conditions.

Medium Isobutyronitrile 0.5 wt% Glucose 1.0 〃 Meat extract 1.0 〃 Peptone 1.0 〃 Dietary salt 0.1 〃 Ferrous potassium phosphate 0.1 〃 Magnesium sulfate 0.05 〃 Ferrous sulfate 0.005 〃 Manganese sulfate 0.005 〃 Ammonium sulfate 0.1 〃 Potassium nitrate 0.1 〃 Water 0.1 〃 96.04〃 pH 7.0 Culture conditions Cultured at 30 ℃ for 30 hours under aerobic condition.

(2) Hydrolysis of nitrile compound Acinetobacter calcoaceticus SAII25 strain bacterial cells obtained by culturing are centrifuged, washed with 0.05M sodium sulfate aqueous solution, and the centrifugation is repeated twice to wash SAII25 strain. A body (water content 90%) was prepared.

2.0 parts of the washed cells and 96.0 parts of a 0.05 M sodium sulfate aqueous solution were mixed, 2 parts of acrylonitrile was further added, and the hydrolysis reaction was carried out at 18 ° C. for 1 hour while stirring. Acrylonitrile contained in the reaction solution is 0.05%, acrylamide is
It was 2.61% and the acrylamide yield was 90.8%.
In addition, the by-product acrylate was not detected.

Example 2 Washed bacterial cells of strain SAII25 obtained by culturing under the same conditions as in Example 1 were used and various nitrile compounds were used under the following conditions to react with stirring to obtain the corresponding amide compound. I asked for the rate.

<Reaction conditions> (1) SAII25 strain washed bacterial cells 2.0 parts (2) 0.05M sodium sulfate aqueous solution 96.0 parts (3) Nitrile compound 2.0 parts (4) Temperature 20 ° C (5) Reaction time 1 hour <Reaction result> Example 3 (1) Culture The SAIE5 strain was cultured under the following conditions.

Medium Isobutyramide 0.8 wt% Potassium phosphate 0.1 〃 Magnesium sulfate 0.05 〃 Ferrous sulfate 0.005 〃 Manganese sulphate 0.005 〃 Ammonium sulfate 0.1 〃 Potassium nitrate 0.1 〃 Water 98.84 〃 pH 7.0 Culture conditions 30 ℃, 48 hours aerobically did.

(2) Hydrolysis of nitrile compound Acinetobacter calcoaceticus strain SAIE5 strain bacterial cells obtained by culturing were washed under the same conditions as in Example 1 and centrifuged to wash bacterial cells (water content 90%). Was prepared.

Using the washed cells, under the same conditions as in Example 2,
Reaction with a nitrile compound was performed to determine the yield of the corresponding amide compound.

<Result of reaction> Example 4 (1) Culture The SAIE5 strain was cultured under the same culture conditions as in Example 3.

(2) Hydrolysis of nitrile compound Acinetobacter calcoaceticus strain SAIE5 strain bacterial cells obtained by culturing were washed under the same conditions as in Example 1 and centrifuged to wash bacterial cells (water content 90%). Was prepared.

Using the washed cells, under the same conditions as in Example 2,
Reaction with a nitrile compound was performed to determine the yield of the corresponding amide compound.

<Result of reaction> (Effects of the Invention) The present invention enables a reaction of a nitrile compound to a corresponding amide compound at room temperature by using a microorganism belonging to the genus Acinetobacter, and thus an industrially useful amide compound can be easily obtained. it can.

Claims (1)

[Claims] 1. A method for producing an amide compound by microbially hydrolyzing a nitrile compound, wherein the microorganism used belongs to the genus Acinetobacter and the nitrate reduction test is negative, and the nitrile compound is hydrolyzed. A method for producing an amide compound, which has an ability to be converted into a corresponding amide compound.