JPH0746997B2 - Method for producing amide compound by microorganism - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing an amide compound from a nitrile compound using a microorganism.

(Prior Art) A method for producing an amide compound from a nitrile compound using a microorganism is, for example, Pseudomonas.
Japanese Patent Publication No. 59-37951 (Yamada et al.) Using microorganisms belonging to the genus, Japanese Patent Publication No. 56-17918 (Watanabe et al.) Using microorganisms belonging to the genus Corynebacterium and microorganisms belonging to the genus Rhodococcus Japanese Unexamined Patent Publication No. 62-91189 (Kawakami et al.) Has been reported.
Both are industrially useful inventions for producing acrylamide and / or methacrylamide using a microorganism.

However, the nitrile hydratase active enzyme having the ability to convert a nitrile compound into an amide compound has a problem that the enzyme activity is easily lost by the nitrile compound as a raw material and the amide compound as a product.
Therefore, when the concentration of the nitrile compound is increased for the purpose of increasing the amidation reaction rate, the nitrile hydratase active enzyme is deactivated within a short time, and it is difficult to obtain the product amide compound in a predetermined time. is there. Further, the amide compound as a product easily loses the activity of the nitrile hydrolase active enzyme, so that it is difficult to obtain a high concentration of the amide compound.

On the other hand, in the method of producing acrylamide or methacrylamide from acrylonitrile or methacrylonitrile using a microorganism, by setting the reaction temperature in the aqueous medium to a temperature range of freezing point to 15 ° C, the activity of the nitrile hydratase active enzyme It is known that an excellent manufacturing method is known in which the product acrylamide or methacrylamide is concentrated to a high concentration while maintaining
56-38118). According to this method, it is possible to suppress the decrease in the activity of the nitrile hydratase active enzyme to some extent, and according to the record of the example, the acrylamide concentration becomes 31.8% at the maximum at the reaction temperature of -3 to 0 ° C. .

(Problems to be Solved by the Invention) However, even when the reaction temperature is lowered, the activity reduction inhibitory effect of the nitrile hydratase active enzyme is not sufficient, and according to Example 2 of JP-B-56-38118, The dry weight% of the microorganisms used to obtain the acrylamide concentration of 31.8% was 2.25% at the end of the reaction, and the acrylamide weight produced per unit weight (g) of dry microorganisms was as small as 14.1 (g). Therefore, the amount of microorganisms used is large, and there is room for further improvement in industrial production. In addition, the reaction temperature range is as low as a freezing point to 15 ° C, which is different from the enzyme reaction temperature range of 20 to 35 ° C which generally uses microorganisms, and there is a problem that the reaction rate becomes slow.

(Means for Solving the Problems) As a result of intensive studies to improve these various problems, the present inventors have surprisingly found that an amphoteric surfactant coexists with a microorganism having a nitrile hydratase active enzyme. It was discovered that the decrease in the activity of the nitrile hydratase active enzyme was remarkably suppressed by this, and the present invention was reached. That is, the present invention is a method for producing an amide compound from a nitrile compound in the method of producing an amide compound from a nitrile compound by the action of a microorganism having a nitrile hydratase activity, which is characterized by allowing an amphoteric surfactant to coexist.

(Operation) The present invention will be described in detail below. A microorganism having a nitrile hydratase activity is a microorganism containing a nitrile hydratase active enzyme having an ability to convert a nitrile compound into an amide compound (hereinafter, abbreviated as the microorganism), and is used, for example, in JP-B-59-37951. Microorganisms belonging to the known genus Pseudomonas,
A microorganism belonging to the genus Corynebacterium and a microorganism belonging to the genus Nocardia used in Japanese Patent Publication No. 56-17918, Japanese Patent Publication No. 62-21519
Used in No. 6 of the genus Bacillus, microorganisms belonging to the genus Bacteridium, microorganisms belonging to the genus Micrococcus, microorganisms belonging to the genus Brevibacterium. -91189 can be mentioned the microorganisms belonging to the genus Rhodococcus (Rhodococcus) and the microorganism belonging to the genus Acinetobacter (Acinetobacter) used in the invention previously filed by the present applicant, and the like. It is not limited.

The amphoteric surfactant used in the present invention is, for example, phosphatidylethanolamine which is a phosphoglyceride, phosphatidylcholine, egg yolk lecithin, soybean lecithin, phosphatidylserine, phosphatidylinositol, 3'-o-lysylphosphatidylglycerol, 1,
2-dimyristoylamide-1,2-deoxyphosphatidylcholine (hereinafter abbreviated as DDPC) or the following general formula (In the formula, R ₁ is a saturated or unsaturated hydrocarbon having 12 to 18 carbon atoms, R ₂ and R ₃ are hydrogen, a methyl group, an ethyl group or a propyl group, and n is 1 or 2.) Carboxybetaine compound, or the following general formula (In the formula, R ₁ is a saturated or unsaturated hydrocarbon having 12 to 18 carbon atoms, R ₂ and R ₃ are hydrogen, a methyl group, an ethyl group or a propyl group, X is a methylene group, an ethylene group, a propylene group or Or a sulfobetaine compound represented by the following general formula: R-NHCH _{2 n} COOH (wherein R is a saturated or unsaturated hydrocarbon having 12 to 18 carbon atoms, and n is 1 or 2). Represents an aminocarboxylic acid salt represented by or a general formula (In the formula, R represents a saturated hydrocarbon or an unsaturated hydrocarbon having 12 to 18 carbon atoms.) An imidazolinium betaine compound represented by
It is not limited to these.

In the present invention, the nitrile compound hydrolyzed by the microorganism is, for example, a mononitrile compound such as acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, isobutyronitrile, n-butyronitrile, adiponitrile, isophthalonitrile, ortho. Examples thereof include, but are not limited to, dinitrile compounds such as phthalonitrile. Also,
In the present invention, the amide compound produced from the nitrile compound by the microorganism is an amide compound obtained by hydrolyzing the corresponding nitrile compound, and in the dinitrile compound, a 1-amide-substituted compound and a 2-amide-substituted compound are used. Mention may be made of, for example, acetamide, propionamide, acrylamide, methacrylamide,
Examples thereof include isobutyramide and n-butyramide, but are not limited thereto.

The method of coexisting with an amphoteric surfactant is, for example, a method of obtaining a corresponding amide compound by reacting with a nitrile compound after adding an amphoteric surfactant to a culture solution of the microorganism, and an amphoteric interface in the culture solution of the microorganism. After adding and mixing the activator,
Filtering to obtain a separated microorganism adsorbed with an amphoteric surfactant, further, after redispersing the separated microorganism in an aqueous medium, a reaction with a nitrile compound to obtain a corresponding amide compound, the method of the microorganism A method of obtaining a corresponding amide compound by filtering the culture solution to obtain a separated microorganism, further dispersing the separated microorganism and the amphoteric surfactant in an aqueous medium, and then reacting with a nitrile compound, culturing the microorganism The liquid is filtered to obtain separated microorganisms, which are then immobilized on a carrier such as polyacrylamide, κ-carrageenan, alginates, etc. to form immobilized microorganisms, and then fixed bed, fluidized bed, etc. form while adding an amphoteric surfactant. In a reaction with a nitrile compound to obtain a corresponding amide compound, and after filtering the culture solution of the microorganism to obtain a separated microorganism, polyacrylamide, κ-color The isolated microorganisms are immobilized together with an amphoteric surfactant on a carrier such as genin or alginates to obtain immobilized microorganisms. Then, the immobilized microorganism is reacted with a nitrile compound in the form of a fixed bed, a fluidized bed or the like to obtain the corresponding amide compound, but the method is not limited thereto.

The amphoteric surfactant may be present together with 0.001 g or more, and more preferably 0.01 g or more per 1 g of the dry weight of the microorganism. By coexisting 0.001 g or more of the amphoteric surfactant per 1 g of the dry weight of the microorganism, in the reaction with the nitrile compound, the amount of the amide compound that can be produced per 1 g of the dry weight of the microorganism is significantly increased, so that the amount of the microorganism used is Less advantageous for industrial production. That is, the amphoteric surfactant 0.001 g or more per 1 g of the microorganism dry weight, more preferably by coexisting with 0.01 g or more, in order to significantly suppress the activity decrease of the nitrile hydratase active enzyme in the reaction with the nitrile compound, The amount of amide compound that can be produced per 1 g of dry weight of microorganisms is significantly increased. This is a nitrile hydrolase active enzyme such as a change in ion concentration caused by extraction and removal of a lipid in a cell membrane of a microorganism by a nitrile compound as a raw material and / or an amide compound as a product in a reaction with a nitrile compound. It is considered that this is because the amphoteric surfactant can suppress the decrease in the activity of the nitrile hydratase activity due to the environmental change in the vicinity. That is, the adsorption of the amphoteric surfactant on the surface of the microorganism suppresses the extraction and removal of lipids in the cell membrane of the microorganism, or the presence of the amphoteric surfactant around the microorganism causes the reaction with the nitrile compound. At this time, the amphoteric surfactant is dissolved in the raw material nitrile compound and / or the product amide compound, and as a result, the amount of the lipid in the cell membrane of the microorganism dissolved in the nitrile compound and / or the amide compound is reduced. Is thought to be the cause.

If the amount of the amphoteric surfactant is less than 0.001 g per 1 g of the dry weight of the microorganism, it is difficult to sufficiently suppress the extraction and removal action of the lipid in the cell membrane of the microorganism by the raw material nitrile compound and / or the product amide compound. As a result, there is no improvement in the amount of amide compound that can be produced per 1 g of dry weight of the microorganism.

Further, if the amphoteric surfactant is 50 g or more per 1 g of dry weight of the microorganism, the solvent viscosity during the reaction with the nitrile compound increases, or it becomes difficult to remove the amphoteric surfactant from the solvent after the reaction, which is a practical problem. is there.

As the reaction conditions in the reaction with the nitrile compound of the microorganism, the addition amount of the nitrile compound used is preferably within the solubility of the nitrile compound, and more preferably, the concentration of the nitrile compound in the solvent is always 1% by weight or less. Is to be added. The reaction temperature of the microorganism with the nitrile compound is preferably 0 to 30 ° C.

(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.

Examples 1 to 4 Rhodococcus AK-32 strain (Microtechnology Research Institute No. 1046) 0.3 part (water content 90%) and soybean lecithin The amount shown in Table 1 was adjusted to 0.05 mol / l sulfuric acid. Disperse in 100 parts of sodium aqueous solution and adjust pH to 7.0. Next, while maintaining the temperature at the temperature shown in Table 1, 1 acrylonitrile was added.
It is continuously added at a rate shown in Table 1 per hour with stirring for the time shown in Table 1. After the addition of acrylonitrile is completed, stirring is continued for 1 hour, and then the reaction is completed. The product after the reaction was quantified by gas chromatography. The concentration of the amide compound as the product and the amount of the amide compound produced per unit weight (g) of the dry microorganism were determined as follows:
Listed in the table.

Comparative Examples 1 to 4 Reaction with acronitrile was carried out in the same manner as in Examples 1 to 4 except that soybean lecithin was not added. The product after the reaction was quantified by gas chromatography. Table 1 shows the concentration of the product amide compound and the amount of the amide compound produced per unit weight (g) of dry microorganisms.

In Examples 1 to 4, the acrylamide concentration of the amide compound was 19.7 to 20.3%, and acrylamide was obtained almost quantitatively from the added acrylonitrile. On the other hand, in Comparative Examples 1 to 4, the acrylamide concentration was as low as 13.8 to 18.0%, and unreacted acrylonitrile remained at 4.55 to 1.37% at the end of the reaction. Comparative Example 1
4 is an industrially beneficial acrylamide concentration
To achieve 20%, further addition of the microorganism is necessary. Therefore, the production amount of the amide compound is further reduced, which is a practical problem.

Examples 5 to 8 Acinetobacter genus SAIE5 strain (Microtechnology Research Institute No. 10432) (water content 90%) in Table 2 weight and amphoteric surfactant type and weight described in Table 2,
Disperse in 100 parts of 0.05 mol / l sodium sulfate aqueous solution and adjust the pH.
To 7.0. Next, while maintaining the temperature shown in Table 2, the nitrile compound shown in Table 2 is continuously added at a rate shown in Table 2 per hour continuously with stirring for the time shown in Table 2. After the addition of the nitrile compound is completed, stirring is continued for 1 hour, and then the reaction is completed. The product after the reaction was quantified by the same method as in Examples 1 to 4. The results obtained are shown in Table 2.

Comparative Examples 5-8 The reaction with the nitrile compound was carried out in the same manner as in Examples 5-8 except that the amphoteric surfactant was not added. The results obtained are shown in Table 2.

Comparing Examples 5 and 6 with Comparative Example 5, the amide production amounts in Examples 5 and 6 were 797 and 781, respectively.
The production amount of amide in 635 is low. In addition, the acrylamide concentration of the amide compound was 1 in Comparative Example 5.
In order to achieve an industrially beneficial acrylamide concentration of 20%, which is as low as 6.1%, it is necessary to further add the microorganism.

Comparing Example 7 and Comparative Example 6, the amide production amounts are 793 and 572, respectively, with a difference of 221.

Comparing Example 8 and Comparative Example 5, the amide production amounts are 797 and 635, respectively, with a difference of 163.

Comparing Example 9 and Comparative Example 5, the amide production amounts are 793 and 635, respectively, with a difference of 158.

Comparing Example 10 and Comparative Example 7, the production amounts of methacrylamide, which is an amide compound, are 467 and 350, respectively, as the amide compound production amount, which is a difference of 117.

Comparing Example 11 and Comparative Example 8, the production amounts of acetamide, which is an amide compound, are 828 and 678, respectively, as the production amount of the amide compound, which is a difference of 150.

In all comparisons, the difference in the amide compound production amount between the examples and the comparative examples was large, and the effect of coexistence of the amphoteric surfactant was clearly recognized.

Reference Example 1 Production Method of Immobilized Microorganisms Rhodococcus sp. AK-32 Strain (Microtechnology Research Institute No. 1046) 50.0 parts (water content 90%), soybean lecithin 0.
1 part and 2 parts of sodium alginate were mixed with 47.9 parts of a 0.05 mol / l sodium sulfate aqueous solution to form a uniform suspension.
The suspension was added dropwise to a 2.0 wt% calcium chloride aqueous solution at 5 ° C to obtain a granular immobilized microorganism having a diameter of 1 mm. The content of microorganisms in the obtained immobilized microorganisms is as dry microorganisms.
It was 5.0% by weight. The content of soybean lecithin in the obtained immobilized microorganism was 0.1% by weight.

Reference Example 2 Production Method of Immobilized Microorganisms Acinetobacter genus SAIE5 strain (Microtechnology Research Institute No. 10432) 50.0 parts, (water content 90%), DDPC0.1
And 27.9 parts of sodium alginate were mixed with 47.9 parts of a 0.05 mol / l sodium sulfate aqueous solution to form a uniform suspension.
The suspension was added dropwise to a 2.0 wt% calcium chloride aqueous solution at 5 ° C to obtain a granular immobilized microorganism having a diameter of 1 mm. The content of microorganisms in the obtained immobilized microorganisms is as dry microorganisms.
It was 5.0% by weight. In addition, D in the obtained immobilized microorganisms
The DPC content was 0.1% by weight.

Reference Example 3 Production Method of Immobilized Microorganism A granular immobilized microorganism having a diameter of 1 mm was obtained in the same manner as in Example 9 except that soybean lecithin was not added. The content of microorganisms in the obtained immobilized microorganisms was 5.0% by weight as dry microorganisms.

Reference Example 4 Preparation of immobilized microorganisms The same method as in Example 10 except that DDPC was not added, and the diameter was 1 mm.
The granular immobilized microorganisms were obtained. The content of microorganisms in the obtained immobilized microorganisms was 5.0% by weight as dry microorganisms.

Examples 12 to 19 and Comparative Examples 9 to 16 The immobilized microorganisms prepared in Reference Examples 1 to 4 were added in an amount described in Table 3 to 100 parts of the solvent described in Table 3, and then the solvent was described in Table 3. The nitrile compound shown in Table 3 was continuously added at a rate shown in Table 3 per hour to the third
Add while stirring for the time indicated in the table. After the addition of the nitrile compound is completed, the reaction is completed after stirring for 2 hours. Unreacted substances and products after the reaction were quantified by the same method as in Examples 1 to 4. Further, when the concentration of the unreacted nitrile compound is less than 1%, the immobilized microorganism used is recovered and again reacted with the nitrile compound under the same conditions.

When the concentration of the unreacted nitrile compound after the reaction was 1% or more, the reaction with the nitrile compound was not performed again, and the number of reactions with the nitrile compound up to that point was taken as the number of repetitions.

The amide compound production amount was calculated by the following formula.

(Production amount of amide compound) = (Production amount of total amide compound including repeated production) / (Weight of dry cells in all immobilized microorganisms used) The results obtained are shown in Table 3.

Comparing Example 12 and Comparative Example 9, the production amount of acrylamide, which is an amide compound, is 591 and 195 as the production amount of amide compound, which is a difference of 396.

Similarly, the amide compound production amount in Example 13 and Comparative Example 10 has a difference of 449, and the amide compound production amount in Example 14 and Comparative Example 11 has a difference of 449.

The amide compound production in Example 15 and Comparative Example 12 is
It has a difference of 408.

In Example 16 and Comparative Example 13, water was used as a solvent, but the amide compound production amount of Example 16 was 453, and in Comparative Example 13, the amide compound production amount was no more than 60, which had a large difference. ing.

In Example 17 and Comparative Example 14, amide compound production of acetamide, which is an amide compound, was 470 and 1, respectively.
36, with a difference of 334.

In Example 18 and Comparative Example 15, the amide compound production of methacrylamide, which is an amide compound, was 37%, respectively.
5, 140, which is a big difference.

Similarly, in Example 19 and Comparative Example 16, the amide compound production amounts are 421 and 192, respectively, which are large differences.

In all the comparisons, the difference in the amide compound production amount between the Examples and Comparative Examples was large, and the coexistence effect of the amphoteric surfactant in the immobilized microorganism was recognized.

(Effect of the invention) The present invention remarkably suppresses the activity decrease of nitrile hydratase active enzyme in the reaction with the microorganism and the nitrile compound by allowing an amphoteric surfactant to coexist with a microorganism having a nitrile hydratase active enzyme. Therefore, it is possible to significantly reduce the amount of the microorganism used in the reaction.

Claims (4)

[Claims] 1. A process for producing an amide compound from a nitrile compound by the action of a microorganism having a nitrile hydratase activity, wherein an amphoteric surfactant is allowed to coexist with the amide compound. 2. The process according to claim 1, wherein the amphoteric surfactant is phosphatidylcholine. 3. The method according to claim 1, wherein the amphoteric surfactant is 1,2-dimyristoylamide-1,2-deoxyphosphatidylcholine. 4. An amphoteric surfactant per 1 g of dry weight of microorganisms.
The manufacturing method according to claim 1, wherein 0.01 g or more is allowed to coexist.