JPH08154691A - Production of amide compound - Google Patents

Abstract

PURPOSE: To provide a method for producing an amide compound by which the stabler amide compound can be produced even in accumulating the amide compound in a reactional solution at a high concentration. CONSTITUTION: This method for producing an amide compound comprises a step for converting an immobilized microorganism prepared by immobilizing a microorganism having hydrating activities in converting a nitrile compound into the amide compound into particles having 250-1000μm diameter before reacting the nitrile compound with the immobilized microorganism and treating the resultant fine particles with a water-soluble dialdehyde at 0.2-1.0wt.% final concentration in the method for reacting the nitrile compound with the immobilized microorganism and converting the nitrile compound into the amide compound. Furthermore, this method for improving the stability of the hydrating activities in converting the nitrile compound into the amide compound is provided. The immobilized microorganism is improved in the stability of the hydrating activities in converting the nitrile compound into the amide compound.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a nitrile compound, which is allowed to act on an immobilized microorganism obtained by immobilizing a microorganism having a hydration activity for converting the nitrile compound into an amide compound, thereby converting the nitrile compound into an amide compound. Regarding how to make.

[0002]

2. Description of the Related Art Conventionally, as a method for producing an amide compound from a nitrile compound, a chemical method such as a sulfuric acid method and a copper catalyst method has been industrially carried out. Microorganisms having a sum activity, for example, the genus Bacillus, the genus Brevibacterium (US Pat. No. 4,010,081), Pseudomonas (P).
genus seudomonas (US Pat. No. 4,555,487)
No.), Agrobacterium (Agrobacterium)
Methods using genus m) (Japanese Patent Laid-Open No. 6-14786) and the like have also been investigated, and among these microorganisms, some microorganisms are also used for industrial production of amide compounds from nitrile compounds. In the method using such a microorganism, the bacterial cells of the microorganism can be directly used in the reaction, but industrially, by using an immobilized microorganism immobilized on a polyacrylamide gel or the like in the reaction, the above-mentioned hydration It stabilizes the activity.

[0003]

However, when the amide compound is accumulated at a high concentration in the reaction solution, the hydration activity of the immobilized microorganisms may be inactivated or decreased. It was not always easy to stably produce an amide compound. Therefore, a method for further stabilizing the hydration activity in the reaction solution has been desired.

[0004]

Under the circumstances, the inventors of the present invention have conducted diligent studies, and as a result, after immobilizing the microorganisms, the immobilized microorganisms were obtained by pulverizing the immobilized microorganisms into small particles in advance. It was found that the stability of the hydration activity for converting a nitrile compound into an amide compound possessed by an immobilized microorganism can be improved by treating the fine particles with a specific concentration of a water-soluble dialdehyde, and thus the present invention was completed. did. That is, the present invention, the nitrile compound, in the method of converting the nitrile compound to an amide compound by acting an immobilized microorganism obtained by immobilizing a microorganism having a hydration activity for converting the nitrile compound to an amide compound, Before allowing the immobilized microorganisms to act on the nitrile compound, the immobilized microorganisms are treated with a diameter of 250 μm to 1000 μm.
The final concentration of the fine particles obtained is 0.2% by weight.
To 1.0% by weight of a water-soluble dialdehyde, the method for producing an amide compound (hereinafter referred to as the production method of the present invention), the nitrile compound being converted into the amide compound. In the method of reacting an immobilized microorganism obtained by immobilizing a microorganism having a hydration activity to convert the nitrile compound into an amide compound, before the immobilized microorganism acts on the nitrile compound, the immobilized microorganism is Diameter from 250 μm to 1000
The final concentration of the fine particles obtained is 0.2% by weight.
To 1.0% by weight of a water-soluble dialdehyde, the method for improving the stability of hydration activity of an immobilized microorganism having a nitrile compound converted to an amide compound (hereinafter, referred to as the activity improving method of the present invention and And an immobilized microorganism having improved stability of hydration activity for converting a nitrile compound into an amide compound by the activity improving method.

The present invention will be described in detail below.

As the nitrile compound in the present invention,
For example, aliphatic nitrile compounds such as n-butyronitrile, n-valeronitrile, isobutyronitrile, acetonitrile and pivalonitrile, nitrile compounds containing a halogen atom such as 2-chloropropionitrile, acrylonitrile, crotononitrile and methacrylonitrile. Aliphatic nitrile compounds containing unsaturated bonds such as, lactonitrile,
Hydroxynitrile compounds such as mandelonitrile, 2-
Amino nitrile compounds such as phenylglycinonitrile,
Examples thereof include aromatic nitrile compounds such as benzonitrile and cyanopyridine, and dinitrile compounds such as malononitrile, succinonitrile and adiponitrile. Preferably, n-butyronitrile, n-valeronitrile, isobutyronitrile, acetonitrile, pivalonitrile, 2-
Chloropropionitrile, acrylonitrile, crotononitrile, methacrylonitrile, benzonitrile, 2-
Mention may be made of cyanopyridine, 3-cyanopyridine, 4-cyanopyridine, malononitrile, succinonitrile or adiponitrile. The amide compound produced from the above nitrile compound is an amide compound corresponding to each of the above nitrile compounds, that is, n-butylamide from n-butyronitrile, n-valeramide from n-valeronitrile, and isobutyronitrile. Yields isobutyramide and acrylonitrile yields acrylamide.

The microorganism used in the present invention is a microorganism having a hydration activity for converting a nitrile compound into an amide compound, and is, for example, Bacillus.
s) genus, Brevibacterium
ium, Pseudomona
s) genus, Agrobacterium
um). A preferable example thereof is a microorganism belonging to Agrobacterium. For culturing the microorganism used in the present invention, various media which are appropriately used for ordinary culturing in general microorganisms, such as a carbon source, a nitrogen source, an organic or inorganic salt and the like can be used. Glucose as a carbon source,
Glycerin, dextrin, sucrose, animal and vegetable oils, molasses and the like can be mentioned. As a nitrogen source, meat extract,
Peptone, yeast extract, malt extract, soybean flour, corn
Steep liquor
or), cottonseed flour, dried yeast, casamino acid, ammonium chloride, ammonium sulfate, ammonium acetate, organic or inorganic nitrogen sources such as urea, and the like. As the organic or inorganic salt, potassium, sodium, magnesium,
Chlorides such as iron, manganese, cobalt, zinc, sulfates,
Acetates, carbonates and phosphates, specifically potassium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium acetate, calcium carbonate, calcium carbonate, phosphorus Examples thereof include monopotassium hydrogenphosphate, dipotassium hydrogenphosphate, monosodium hydrogenphosphate, and disodium hydrogenphosphate. The pH of these media can be appropriately changed depending on the microorganism used, but is, for example, about 5 to 10. The culture temperature can be appropriately changed within the range where the microorganism grows. The culture time varies depending on various conditions, but is usually preferably about 1 to 7 days. The culturing method is carried out according to the usual culturing method for general microorganisms, and it is preferable to carry out shaking culture in a liquid medium under aerobic conditions. In addition, it is preferable to add an inducer for inducing the target enzyme, although it depends on the kind of the microorganism, because it is effective. For example, Agrobacterium radiobacter (A
(grobobacterium radiobacterium)
In the case of, the inducer may be a nitrile compound such as isovaleronitrile or crotononitrile, or an amide compound such as crotonamide, and the amide compound may be added in an amount of about 0.01 to 1% by weight relative to the medium. .

After culturing the microorganism as described above, for example, cells are collected from the culture solution by centrifugation or the like, washed with water, physiological saline, phosphate buffer, etc., and then the obtained microorganism is immobilized. By doing so, an immobilized microorganism is obtained. Examples of the method for immobilizing microorganisms include entrapping immobilization method using an acrylamide polymer. Here, the entrapping immobilization method using an acrylamide polymer means an entrapping type immobilization method using an acrylamide polymer in which acrylamide or methacrylamide is used as a main component, and other ethylenically unsaturated monomers are copolymerized as necessary. The immobilization method. In the entrapping immobilization method using an acrylamide polymer, for example, the microorganism or a suspension thereof is treated with a water-soluble unsaturated monomer such as acrylamide and N,
It is suspended in an aqueous solution containing a water-soluble polyfunctional unsaturated monomer such as N′-methylenebisacrylamide, a polymerization initiator is added, and polymerization is carried out at a temperature of about 0 to 40 ° C., preferably about 0 to 20 ° C. The solution polymerization method or the suspension is dispersed in a hydrophobic liquid, a polymerization initiator is added, and the temperature is adjusted to about 0-40.
It can be immobilized by a suspension polymerization method or the like in which the polymerization is carried out at a temperature of preferably 0 to 20 ° C. The content of the microorganism in the polymer gel depends on the kind of the microorganism, the method of use, etc., but is usually about 0.1 to 50% by weight, preferably about 5 to 40% by weight. The amount of the water-soluble unsaturated monomer used for the polymerization is about 5 to 35% by weight, preferably about 10 to 20% by weight in the suspension.
Is. The amount of the water-soluble polyfunctional unsaturated monomer is about 1 to 50% by weight, preferably about 3 to 20% by weight, based on the amount of the water-soluble unsaturated monomer. As the polymerization initiator, potassium persulfate, ammonium persulfate or the like can be used. Further, it is preferable to use 3- (dimethylamino) propionitrile, N, N, N ′, N′-tetramethylethylenediamine, or the like as the polymerization accelerator. As the hydrophobic liquid for carrying out suspension polymerization, any organic solvent can be used as long as it is a hydrophobic organic solvent, but an organic solvent having little influence on microorganisms, for example, n-hexane, n-heptane, n-
It is preferable to use octane or the like. These organic solvents may be used alone or in combination of two or more. Furthermore, in suspension polymerization, it is preferable to use a suitable dispersant. If necessary, in addition to the water-soluble dialdehyde treatment after the immobilization-microparticulation step described below, the microorganism obtained by centrifuging and washing from the culture solution before the immobilization of the microorganism is treated with the water-soluble dialdehyde. The method described above may be used in combination (two-step treatment with a water-soluble dialdehyde). In this case, as the treatment with a water-soluble dialdehyde before immobilization, for example, microbial cells are adjusted to a pH of about 5 to about 10, preferably about 6 to about 8, about 0.01.
~ 0.5 M phosphate buffer, etc., and then suspended in water-soluble dialdehyde final concentration of the treatment solution about 0.1 ~ 5.0 wt%,
Preferably, about 0.2 to 2.0% by weight is added, and the temperature is about 0.
A method of reacting at 40 ° C., preferably about 0 to 20 ° C. for about 0.5 to 6 hours can be mentioned. The microorganisms treated with the water-soluble dialdehyde before being immobilized in this manner are usually washed with water, physiological saline, phosphate buffer, etc. by centrifugation or the like, and then immobilized as described above.

Before the immobilized microorganisms thus obtained by immobilization are allowed to act on the nitrile compound, the immobilized microorganisms are granulated to have a diameter of 250 μm to 1000 μm, and the resulting fine particles have a final concentration of 0.2% by weight to 1. It is essential in the present invention to carry out the step of treating with 0% by weight of water-soluble dialdehyde. Immobilized microorganisms with a diameter of 250 μm to 100
A pulverization method using a device such as a blender can be used for forming particles to 0 μm. Next, the resulting fine particles are treated with a water-soluble dialdehyde having a final concentration of 0.2% by weight to 1.0% by weight. Specifically, about 0.0
After immersing in 1 to 0.5M phosphate buffer (pH about 5 to about 10, preferably about 6 to about 8), the final concentration of the solution is changed from 0.2% by weight to 1.0% by weight A water-soluble dialdehyde is added so that it is incubated at a temperature of about 0 to 40 ° C, preferably about 0 to 20 ° C for about 0.5 to 30 hours, preferably about 1 to 24 hours. And after finishing the water-soluble dialdehyde treatment, the obtained immobilized microorganisms,
Wash with water, saline, phosphate buffer, etc. Examples of the water-soluble dialdehyde used in the present invention include glutaraldehyde and glyoxal, and glutaraldehyde is particularly preferable.

The thus-prepared water-soluble dialdehyde-treated particle-immobilized microorganisms (hereinafter, also referred to as water-soluble dialdehyde-treated immobilized microorganism fine particles) is allowed to act to convert the nitrile compound into an amide compound. For example, the immobilized microorganisms are packed in a column or placed in a reaction vessel, an aqueous solution of a nitrile compound as a raw material is supplied, and the reaction is performed at about 0 to 20 ° C., for example. Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

[0011]

【Example】

Example 1 Glycerol 1.0% by weight, polypeptone 0.5% by weight, yeast extract 0.3% by weight, malto extract 0.3% by weight, isovaleronitrile 0.1% by weight and ferrous sulfate, chloride Sterilized medium (pH of 0.001% by weight each of cobalt, zinc sulfate and manganese sulfate)
7.2), aerobically cultivated at 30 ° C. for 2 days to obtain Agrobacterium radiobacterium (Agroba)
cterium radiobacterium) SC-C1
The cells of strain 5-1 [FERM BP-3843] were collected by centrifugation (12,000xg, 20 min, 4 ° C). Collected bacterial cells in 50 mM potassium phosphate buffer (pH 7.7)
After washing with, the cells were collected by centrifugation (12,000 xg, 20 min, 4 ° C) to obtain washed cells (water content 77%). After suspending 228 g of the obtained washed cells in 60 g of 50 mM potassium phosphate buffer (pH 7.7), a 2.5% (w / w) acrylamide polymer aqueous solution (Nacalai Tesque reagent) was added as a dispersant to the microorganism suspension. Degree of polymerization 10,0
00g, diluted with 10% aqueous solution) and added well. Furthermore, an aqueous solution of acrylamide monomer (acrylamide 81
g, N, N'-methylenebisacrylamide 9 g, water 1
26 g) and 30 g of a 5% (w / w) aqueous solution of 3- (dimethylamino) propionitrile, which is a polymerization accelerator, were added and then sufficiently mixed to obtain an aqueous microbial suspension monomer solution. Next, 616 g (900 ml) of n-heptane was charged into an apparatus in which one Faudler type stirring rod and one baffle rod were installed in a 2 L separable round bottom flask, and the apparatus was kept at 5 ° C. in a water bath. The inside of the separable round-bottomed flask was replaced with nitrogen, and thereafter, the amount of nitrogen gas required for maintaining the nitrogen atmosphere was blown into the separable round-bottomed flask. Next, the microbial suspension monomer aqueous solution (600 ml) was charged into the separable round bottom flask while stirring at 400 rpm. Stirring was continued for 15 minutes, and when the temperature reached 5 ° C., 60 g of a 2.5% (w / w) potassium persulfate aqueous solution as a polymerization initiator was added to the separable round bottom flask. The polymerization started a few minutes after the addition of the polymerization initiator. In this state, the stirring was continued for 1 hour to continue the polymerization. The temperature in the separable round-bottomed flask system rose up to 10 ° C. due to the heat of polymerization, but was then cooled. After the polymerization was completed, the obtained immobilized microorganism was crushed using a blender. The crushed immobilized microorganisms are washed with 50 mM potassium phosphate buffer (pH 7.7), sieved with a sieve (16 to 60 mesh), and spherical particles having a diameter of 250 μm to 1000 μm (hereinafter referred to as immobilized microbial particles). ) Was recovered. Next, 20 g of the recovered immobilized microbial microparticles (water content 88%) and 20 g of a 0.6 wt% glutaraldehyde-50 mM potassium phosphate buffer (pH 7.7) aqueous solution were mixed, and the mixture was kept at 2 ° C. for 4 hours. The reaction was carried out (the final concentration of glutaraldehyde is 0.3% by weight). After the reaction is completed, the reaction product is 50 m
It was thoroughly washed with M potassium phosphate buffer (pH 7.7). The following enzyme stability test was carried out using immobilized microbial particles treated with glutaraldehyde. First, 30% (w / v) acrylamide-5 of immobilized microbial microparticles was used.
After immersing in a 0 mM potassium phosphate buffer (pH 7.7) aqueous solution at 2 ° C. for 17 hours, the immobilized microbial microparticles were collected, and 50 mM potassium phosphate buffer (pH
After thorough washing with 7.7), the activity of converting acrylonitrile to acrylamide was measured. The activity is measured by
0.2 g of immobilized microbial microparticles and 0.8 g of 50 mM potassium phosphate buffer (pH 7.7) were mixed, and 9 ml of 2.78 wt% acrylonitrile was added to the mixture.
0 mM potassium phosphate buffer (pH 7.7) was added to start the reaction at a reaction temperature of 10 ° C. 10 minutes later,
The reaction was stopped by adding 1 ml of 2N hydrochloric acid. A part of the reaction solution was analyzed by gas chromatography, and the hydration activity was calculated from the amount of acrylamide produced. (Gas chromatography analysis conditions) Column: Packed column Carrier: Porapak type Q (mesh 80-100) Length: 1.1 m Column temperature: 210 ° C Carrier gas flow rate: 50 ml / min Sample injection amount: 2 μl As a result, glutaraldehyde The residual activity (relative value to that before the enzyme stability test) after the enzyme stability test in the treated immobilized microbial microparticles (water content 88%) was 47.0%.
Met. That is, in the present invention, as is apparent from comparison with Comparative Examples 1 and 2 described later, about 2.7 to about 3.7.
A double improvement in stability to acrylamide was observed.

[0012]

Example 2 Immobilized microbial microparticles were obtained in the same manner as in Example 1. The obtained immobilized microbial microparticles (water content 88
%) 20 g and 0.4% by weight of glutaraldehyde-50
mM potassium phosphate buffer (pH 7.7) aqueous solution 2
0 g were mixed and the mixture was treated at 2 ° C. for 4 hours (final glutaraldehyde concentration is 0.2% by weight). After the reaction was completed, the reaction product was thoroughly washed with 50 mM potassium phosphate buffer (pH 7.7). The following enzyme stability test was carried out using immobilized microbial particles treated with glutaraldehyde. First, 30% (w
/ V) After being immersed in an aqueous solution of acrylamide-50 mM potassium phosphate buffer (pH 7.7) at 2 ° C. for 17 hours, the immobilized microbial microparticles were recovered, and 50 mM potassium phosphate buffer (pH 7.7) was sufficient. After washing, the activity of converting acrylonitrile to acrylamide was measured by the same method as in Example 1. As a result, glutaraldehyde-treated immobilized microbial microparticles (water content 88
%), The residual activity after the enzyme stability test (relative value to that before the enzyme stability test) was 34.7%. That is, in the present invention, as is clear from comparison with Comparative Examples 1 and 2 described later, about 2.0 to about 2.7 times improvement in stability against acrylamide was observed.

[0013]

Comparative Example 1 Immobilized microbial microparticles were obtained in the same manner as in Example 1. The obtained immobilized microbial microparticles (water content 88
%) 20 g and 0.2% by weight of glutaraldehyde-50
mM potassium phosphate buffer (pH 7.7) aqueous solution 2
0 g was mixed and the mixture was treated at 2 ° C. for 4 hours (final glutaraldehyde concentration is 0.1% by weight). After the reaction was completed, the reaction product was thoroughly washed with 50 mM potassium phosphate buffer (pH 7.7). The following enzyme stability test was carried out using immobilized microbial particles treated with glutaraldehyde. First, 30% (w
/ V) After being immersed in an aqueous solution of acrylamide-50 mM potassium phosphate buffer (pH 7.7) at 2 ° C. for 17 hours, the immobilized microbial microparticles were recovered, and 50 mM potassium phosphate buffer (pH 7.7) was sufficient. After washing, the activity of converting acrylonitrile to acrylamide was measured by the same method as in Example 1. As a result, glutaraldehyde-treated immobilized microbial microparticles (water content 88
%), The residual activity after the enzyme stability test (relative value to that before the enzyme stability test) was 17.7%.

[0014]

Comparative Example 2 Immobilized microbial microparticles were obtained in the same manner as in Example 1. The obtained immobilized microbial microparticles (water content 88
%) Was used to perform the following enzyme stability test. First, 30% (w / v) acrylamide-
After immersing in a 50 mM potassium phosphate buffer (pH 7.7) aqueous solution at 2 ° C. for 17 hours, the immobilized microbial microparticles were collected, and 50 mM potassium phosphate buffer (p
After thorough washing with H7.7), the activity of converting acrylonitrile to acrylamide was measured by the same method as in Example 1. As a result, the residual activity after the enzyme stability test (relative value to that before the enzyme stability test) of the immobilized microbial fine particles not treated with glutaraldehyde (water content 88%) was 12.7%.

[0015]

Example 3 Glycerol 1.0% by weight, polypeptone 0.5% by weight, yeast extract 0.3% by weight, malto extract 0.3% by weight, isovaleronitrile 0.1% by weight and ferrous sulfate, chloride Sterilized medium (pH of 0.001% by weight each of cobalt, zinc sulfate and manganese sulfate)
7.2), aerobically cultivated at 30 ° C. for 2 days to obtain Agrobacterium radiobacterium (Agroba)
cterium radiobacterium) SC-C1
The cells of the 5-1 strain [FERM BP-3843] were collected by centrifugation (12,000 xg, 20 min, 4 ° C). The collected cells were treated with 50 mM potassium phosphate buffer (pH 7.
After washing in 7), the cells were collected by centrifugation (12,000 xg, 20 min, 4 ° C) to obtain washed cells (water content 77%). After thoroughly mixing 230 g of the obtained washed cells, 8.28 g of 25% (w / w) glutaraldehyde monomer aqueous solution, 55.5 g of 50 mM potassium phosphate buffer (pH 7.7) and 51.22 g of water, The mixture was left under ice cooling for 1 hour (final glutaraldehyde concentration was 0.6
% By weight). After the treatment, it was washed with 50 mM potassium phosphate buffer (pH 7.7) and centrifuged (12,000 xg,
The bacterial cells were collected by 20 min at 4 ° C. The collected bacterial cells were suspended in 50 mM potassium phosphate buffer (pH 7.7), and the total amount of the microorganism suspension was adjusted to 300 g. Next, 2.88 g of the above-mentioned microbial suspension was used as a dispersant.
12 g of a 5% (w / w) acrylamide polymer aqueous solution (Nacalai Tesque reagent, degree of polymerization 10,000, diluted 10% aqueous solution) was added and mixed well. Further, the mixture was previously degassed under reduced pressure to obtain an aqueous solution of acrylamide monomer (81 g of acrylamide, 9 g of N, N'-methylenebisacrylamide, 126 g of water) and 3% of 5% (w / w) which was a polymerization accelerator. After adding 30 g of a-(dimethylamino) propionitrile aqueous solution, they were thoroughly mixed to obtain a microbial suspension monomer aqueous solution. Next, in a 2 L separable round-bottomed flask equipped with one Faudler-type stirring rod and one baffle rod, n-heptane (616 g)
(900 ml) was charged and the device was put on a water bath for 5
It was kept at ℃. Replace the inside of the separable round bottom flask with nitrogen,
After that, the amount of nitrogen gas necessary for maintaining the nitrogen atmosphere was blown into the separable round bottom flask. Next, add 400 rp of the microbial suspension monomer aqueous solution (600 ml).
It was charged into the above separable round bottom flask while stirring at m. Stirring is continued for 15 minutes, and when the temperature reaches 5 ° C., a polymerization initiator of 2.5 is added in a separable round bottom flask.
60 g of a% (w / w) aqueous potassium persulfate solution was added. The polymerization started a few minutes after the addition of the polymerization initiator. In this state, the stirring was continued for 1 hour to continue the polymerization. The temperature in the separable round-bottomed flask system rose up to 10 ° C. due to the heat of polymerization, but was then cooled. After the polymerization was completed, the obtained immobilized microorganism was crushed using a blender. The pulverized immobilized microorganism was treated with 50 mM potassium phosphate buffer (p
H7.7) washed and sieved (16 to 60 mesh)
Sieve with a diameter of 250μm to 1000μm
The spherical particles (hereinafter referred to as immobilized microbial particles) were collected. Next, the immobilized microbial microparticles (water content 8
8%) 20 g and 0.6% by weight of glutaraldehyde-5
20 g of 0 mM potassium phosphate buffer (pH 7.7) aqueous solution was mixed, and the mixture was reacted at 2 ° C. for 4 hours (final glutaraldehyde concentration was 0.3% by weight).
After the reaction was completed, the reaction product was thoroughly washed with 50 mM potassium phosphate buffer (pH 7.7). The following enzyme stability test was carried out using immobilized microbial particles treated with glutaraldehyde. First, 30% of immobilized microbial particles
(W / v) Acrylamide-50 mM potassium phosphate buffer (pH 7.7) After being immersed in an aqueous solution at 2 ° C. for 17 hours, the immobilized microbial microparticles were recovered and treated with 50 mM potassium phosphate buffer (pH 7.7). After thorough washing, the activity of converting acrylonitrile to acrylamide was measured by the same method as in Example 1. As a result, the immobilized microbial microparticles treated with glutaraldehyde (water content 8
8%), the residual activity after the enzyme stability test (relative value to that before the enzyme stability test) was 48.1%. The residual activity after the enzyme stability test is in the case of the glutaraldehyde-treated immobilized microbial microparticles obtained in this Example as compared with the glutaraldehyde-treated immobilized microbial microparticles obtained in Example 1. Was a better value.

[0016]

Example 4 Glycerol 1.0% by weight, polypeptone 0.5% by weight, yeast extract 0.3% by weight, malto extract 0.3% by weight, isovaleronitrile 0.1% by weight and ferrous sulfate, chloride Sterilized medium (pH of 0.001% by weight each of cobalt, zinc sulfate and manganese sulfate)
7.2), aerobically cultivated at 30 ° C. for 2 days to obtain Agrobacterium radiobacterium (Agroba)
cterium radiobacterium) SC-C1
The cells of the 5-1 strain [FERM BP-3843] were collected by centrifugation (12,000 xg, 20 min, 4 ° C). The collected cells were treated with 50 mM potassium phosphate buffer (pH 7.
After washing in 7), the cells were collected by centrifugation (12,000 xg, 20 min, 4 ° C) to obtain washed cells (water content 77%). 8 g of the obtained washed cells and 2 g of 50 mM potassium phosphate buffer (pH 7.7) were placed in a 50 ml beaker and mixed thoroughly. Further, an aqueous solution of acrylamide monomer (1.9 g of acrylamide, 0.1 g of N, N'-methylenebisacrylamide, 5 g of water) that had been degassed in advance under reduced pressure was added to the mixture, and the mixture was sufficiently stirred and mixed using a stirrer. , 5% which is a polymerization accelerator while stirring
An aqueous microbial suspension monomer solution was obtained by adding 1 g of a (w / w) aqueous solution of 3- (dimethylamino) propionitrile. 2.0 g of 2.5% (w / w) potassium persulfate aqueous solution as a polymerization initiator in the microbial suspension monomer aqueous solution
Was added. The polymerization started a few minutes after the addition of the polymerization initiator, but was left standing for 1 hour in this state. All of these polymerization operations were performed under a nitrogen atmosphere. After the polymerization was completed, the obtained immobilized microorganism was crushed using a blender. The crushed immobilized microorganisms were washed with 50 mM potassium phosphate buffer (pH 7.7) and sieved with a sieve (16 to 60 mesh) to obtain a diameter of 250 μm to 10
The spherical particles of 00 μm (hereinafter referred to as immobilized microbial particles) were collected. 20 g of recovered immobilized microbial microparticles (water content 88%) and 0.6% by weight of glutaraldehyde-50 mM potassium phosphate buffer (pH 7.7)
20 g of the aqueous solution was mixed, and the mixture was reacted at 2 ° C. for 4 hours (final concentration of glutaraldehyde is 0.3% by weight). After the reaction was completed, the reaction product was thoroughly washed with 50 mM potassium phosphate buffer (pH 7.7). The following enzyme stability test was carried out using immobilized microbial particles treated with glutaraldehyde. First, 3
After soaking in a 0% (w / v) acrylamide-50 mM potassium phosphate buffer (pH 7.7) aqueous solution at 2 ° C. for 17 hours, the immobilized microbial microparticles were collected,
After thoroughly washing with mM potassium phosphate buffer (pH 7.7), the activity of converting acrylonitrile into acrylamide was measured by the same method as in Example 1. As a result, the residual activity after the enzyme stability test (relative value to that before the enzyme stability test) of the immobilized microbial particles treated with glutaraldehyde (water content 88%) was 29.9%. That is, in the present invention, as is clear from a comparison with Comparative Example 3 described later, about 6.0-fold improvement in stability against acrylamide was recognized.

[0017]

Comparative Example 3 Immobilized microbial microparticles were obtained in the same manner as in Example 4. The following enzyme stability test was carried out using the obtained immobilized microbial microparticles. First, after immersing the immobilized microbial microparticles in a 30% (w / v) acrylamide-50 mM potassium phosphate buffer (pH 7.7) aqueous solution at 2 ° C. for 17 hours, the immobilized microbial microparticles were collected, and 50
After thoroughly washing with mM potassium phosphate buffer (pH 7.7), the activity of converting acrylonitrile into acrylamide was measured by the same method as in Example 1. As a result, the residual activity after the enzyme stability test (relative value to that before the enzyme stability test) in the immobilized microbial fine particles not treated with glutaraldehyde (water content 88%) was 5.0%.

[0018]

Example 5 1.0% by weight of glycerol, 0.5% by weight of polypeptone, 0.3% by weight of yeast extract, 0.3% by weight of malto extract, 0.1% by weight of isovaleronitrile and ferrous sulfate, chloride Sterilized medium (pH of 0.001% by weight each of cobalt, zinc sulfate and manganese sulfate)
7.2), aerobically cultivated at 30 ° C. for 2 days to obtain Agrobacterium radiobacterium (Agroba)
cterium radiobacterium) SC-C1
5-1 strain FERM BP-3843 was collected by centrifugation (12,000 xg, 20 min, 4 ° C). The collected cells were treated with 50 mM potassium phosphate buffer (pH 7.
After washing in 7), the cells were collected by centrifugation (12,000 xg, 20 min, 4 ° C) to obtain washed cells (water content 77%). 8 g of the obtained washed cells, 0.12 g of 25% (w / w) glutaraldehyde aqueous solution and 1.88 g of 50 mM potassium phosphate buffer were placed in a 50 ml beaker and mixed thoroughly, and then the mixture was cooled with ice. It was left for 1 hour (final concentration of glutaraldehyde is 0.3% by weight). Further, the mixture was degassed under reduced pressure in advance to obtain an aqueous solution of acrylamide monomer (acrylamide: 1.9 g,
N, N'-methylenebisacrylamide 0.1 g, water 5
g) was added, and the mixture was sufficiently stirred and mixed by using a stirrer, and then 5% (w / w) of a polymerization accelerator of 3% was added with stirring.
An aqueous microbial suspension monomer solution was obtained by adding 1 g of a-(dimethylamino) propionitrile aqueous solution. 2.5% (w
/ W) aqueous potassium persulfate solution (2.0 g) was added. The polymerization started a few minutes after the addition of the polymerization initiator, but was left standing for 1 hour in this state. All of these polymerization operations were performed under a nitrogen atmosphere. After the polymerization was completed, the obtained immobilized microorganism was crushed using a blender. The crushed immobilized microorganism was treated with 50 mM potassium phosphate buffer (pH 7.
After washing with 7) and sieving with a sieve (16 to 60 mesh), spherical particles having a diameter of 250 μm to 1000 μm (hereinafter referred to as immobilized microbial particles) were collected. Recovered immobilized microbial microparticles (water content 88%) 2
0 g and 20 g of a 0.6 wt% glutaraldehyde-50 mM potassium phosphate buffer (pH 7.7) aqueous solution were mixed, and the mixture was reacted at 2 ° C. for 4 hours (final glutaraldehyde concentration was 0.3 wt%. is there). After the reaction is completed, the reaction product is treated with 50 mM potassium phosphate buffer (pH
It was thoroughly washed with 7.7). The following enzyme stability test was carried out using immobilized microbial particles treated with glutaraldehyde. First, 30% (w / v) of immobilized microbial particles
After being immersed in an acrylamide-50 mM potassium phosphate buffer (pH 7.7) aqueous solution at 2 ° C. for 17 hours, the immobilized microbial microparticles were recovered and thoroughly washed with 50 mM potassium phosphate buffer (pH 7.7). ,
The activity of converting acrylonitrile to acrylamide was measured by the same method as in Example 1. As a result, immobilized microbial microparticles treated with glutaraldehyde (water content 88%)
The residual activity after the enzyme stability test (relative value to that before the enzyme stability test) was 32.5%.

[0019]

Example 6 1.0% by weight of sucrose, 0.5% by weight of polypeptone, 0.3% by weight of yeast extract, 0.3% by weight of malto extract, and ferrous sulfate, cobalt chloride, zinc sulfate, manganese sulfate. Of Bacillus smithie (Bac
illus smthii) SC-J05-1 [FE
RMP-14037] cells were centrifuged (12,000xg, 20
min, 4 ° C). 50mM of collected cells
After washing with a potassium phosphate buffer (pH 7.0), cells were collected by centrifugation (12,000 xg, 20 min, 4 ° C) to obtain washed cells (water content 77%). 8 g of the obtained washed cells and 50 mM potassium phosphate buffer (pH
7.0) 2g was put into a 50 ml beaker and mixed well. Further, the mixture was degassed under reduced pressure in advance to obtain an aqueous solution of acrylamide monomer (acrylamide: 1.9 g,
N, N'-methylenebisacrylamide 0.1 g, water 5
g) was added, and the mixture was sufficiently stirred and mixed by using a stirrer, and then 5% (w / w) of a polymerization accelerator of 3% was added with stirring.
An aqueous microbial suspension monomer solution was obtained by adding 1 g of a-(dimethylamino) propionitrile aqueous solution. 2.5% (w
/ W) aqueous potassium persulfate solution (2.0 g) was added. The polymerization started a few minutes after the addition of the polymerization initiator, but was left standing for 1 hour in this state. All of these polymerization operations were performed under a nitrogen atmosphere. After the polymerization was completed, the obtained immobilized microorganism was crushed using a blender. The crushed immobilized microorganism was treated with 50 mM potassium phosphate buffer (pH 7.
After washing with 0) and sieving with a sieve (16 to 60 mesh), spherical particles having a diameter of 250 μm to 1000 μm (hereinafter, referred to as immobilized microbial particles) were collected. Recovered immobilized microbial microparticles (water content 88%) 2
0 g and 20 g of a 0.6 wt% glutaraldehyde-50 mM potassium phosphate buffer (pH 7.0) aqueous solution were mixed, and the mixture was reacted at 2 ° C. for 4 hours (final glutaraldehyde concentration was 0.3 wt%. is there). After the reaction is completed, the reaction product is treated with 50 mM potassium phosphate buffer (pH
It was thoroughly washed with 7.0). The following enzyme stability test was carried out using immobilized microbial particles treated with glutaraldehyde. First, 30% (w / v) of immobilized microbial particles
After being immersed in an acrylamide-50 mM potassium phosphate buffer (pH 7.0) aqueous solution at 2 ° C. for 17 hours, the immobilized microbial microparticles were collected and thoroughly washed with 50 mM potassium phosphate buffer (pH 7.0). ,
The activity of converting acrylonitrile to acrylamide was measured by the same method as in Example 1. As a result, immobilized microbial microparticles treated with glutaraldehyde (water content 88%)
The residual activity after the enzyme stability test (relative value to that before the enzyme stability test) was 83.7%. That is,
As is clear from the comparison with Comparative Example 4 described later, the present invention was found to have about 6.7-fold improvement in stability against acrylamide.

[0020]

Comparative Example 4 Immobilized microbial microparticles were obtained in the same manner as in Example 6. The following enzyme stability test was carried out using the obtained immobilized microbial microparticles. First, after immersing the immobilized microbial microparticles in a 30% (w / v) acrylamide-50 mM potassium phosphate buffer (pH 7.0) aqueous solution at 2 ° C. for 17 hours, the immobilized microbial microparticles were collected and
After sufficiently washing with mM potassium phosphate buffer (pH 7.0), the activity of converting acrylonitrile into acrylamide was measured by the same method as in Example 1. As a result, the residual activity after the enzyme stability test (relative value to that before the enzyme stability test) of the immobilized microbial fine particles not treated with glutaraldehyde (water content 88%) was 12.5%.

[0021]

Example 7 1.0% by weight sucrose, 0.5% by weight polypeptone, 0.3% by weight yeast extract, 0.3% by weight malt extract, and ferrous sulfate, cobalt chloride, zinc sulfate, manganese sulfate. Of Bacillus smithie (Bac
illus smthii) SC-J05-1 [FE
RMP-14037] cells were centrifuged (12,000xg, 20
min, 4 ° C). 50mM of collected cells
After washing with a potassium phosphate buffer (pH 7.0), cells were collected by centrifugation (12,000 xg, 20 min, 4 ° C) to obtain washed cells (water content 77%). The resulting washed cells (8 g), 25% (w / w) glutaraldehyde aqueous solution (0.12 g) and 50 mM potassium phosphate buffer (pH 7.0) (1.88 g) were placed in a 50 ml beaker and thoroughly mixed, and then the mixture was mixed. Was left under ice-cooling for 1 hour (final concentration of glutaraldehyde is 0.3% by weight). Further, the mixture was degassed under reduced pressure in advance to obtain an aqueous solution of acrylamide monomer (acrylamide: 1.9 g,
N, N'-methylenebisacrylamide 0.1 g, water 5
g) was added, and the mixture was sufficiently stirred and mixed by using a stirrer, and then 5% (w / w) of a polymerization accelerator of 3% was added with stirring.
An aqueous microbial suspension monomer solution was obtained by adding 1 g of a-(dimethylamino) propionitrile aqueous solution. 2.5% (w
/ W) aqueous potassium persulfate solution (2.0 g) was added. The polymerization started a few minutes after the addition of the polymerization initiator, but was left standing for 1 hour in this state. All of these polymerization operations were performed under a nitrogen atmosphere. After the polymerization was completed, the obtained immobilized microorganism was crushed with a blender. The crushed immobilized microorganisms are washed with 50 mM potassium phosphate buffer (pH 7.0), sieved with a sieve (16 to 60 mesh), and spherical particles having a diameter of 250 μm to 1000 μm (hereinafter referred to as immobilized microbial particles). ) Was recovered. 20 g of the recovered immobilized microbial microparticles (water content 88%) was mixed with 20 g of a 0.6 wt% glutaraldehyde-50 mM potassium phosphate buffer (pH 7.0) aqueous solution, and the mixture was reacted at 2 ° C. for 4 hours. (The final concentration of glutaraldehyde is 0.3% by weight). After completion of the reaction, the reaction product was treated with 50 mM potassium phosphate buffer (pH 7.
It was thoroughly washed with 0). The following enzyme stability test was carried out using immobilized microbial particles treated with glutaraldehyde. First, 30% (w / v) acrylamide-50 mM potassium phosphate buffer (pH
7.0) After being immersed in an aqueous solution at 2 ° C. for 17 hours, the immobilized microbial microparticles are collected and thoroughly washed with 50 mM potassium phosphate buffer (pH 7.0), and then acrylonitrile is converted into acrylamide. Was measured in the same manner as in Example 1. As a result, the residual activity after the enzyme stability test (relative value to that before the enzyme stability test) of the immobilized microbial particles treated with glutaraldehyde (water content 88%) was 87.1%.

[0022]

According to the present invention, the immobilized microorganism has
It was possible to suppress the deactivation or decrease of the hydration activity for converting the nitrile compound into the amide compound, and improve the stability of the hydration activity. Therefore, even when the amide compound is accumulated in the reaction solution at a high concentration, it is possible to more stably manufacture the amide compound.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ken Mitsuda 4-2-1 Takashi, Takarazuka-shi, Hyogo Sumitomo Kagaku Kogyo Co., Ltd.

Claims (3)

[Claims] 1. A method for converting a nitrile compound into an amide compound by allowing an immobilized microorganism obtained by immobilizing a microorganism having a hydration activity for converting the nitrile compound into an amide compound into the nitrile compound, the method comprising: A step of granulating the immobilized microorganisms to a diameter of 250 μm to 1000 μm and treating the resulting fine particles with a water-soluble dialdehyde having a final concentration of 0.2% by weight to 1.0% by weight, before the immobilized microorganisms act on the nitrile compound. A method for producing an amide compound, which comprises: 2. A method for converting a nitrile compound into an amide compound by allowing an immobilized microorganism, which is obtained by immobilizing a microorganism having a hydration activity for converting the nitrile compound into an amide compound, to the nitrile compound and converting the nitrile compound into the amide compound. Before the immobilized microorganism is allowed to act on the nitrile compound, the immobilized microorganism is granulated to a diameter of 250 μm to 1000 μm, and the resulting fine particles are treated with a water-soluble dialdehyde having a final concentration of 0.2% by weight to 1.0% by weight. A method for improving the stability of hydration activity of an immobilized microorganism, which comprises converting a nitrile compound into an amide compound. 3. An immobilized microorganism having improved stability of hydration activity for converting a nitrile compound into an amide compound by the method according to claim 2.