JPH02167090A - Production of amide compound by microorganism - Google Patents

Abstract

PURPOSE:To obtain an amide of high concentration without markedly lowered activity of the microorganism by combining an amphoteric surfactant with a microorganism of nitrile hydrase activity and allowing them to react with a nitrile compound. CONSTITUTION:A microorganism in Pseudomonas with nitrile hydrase activity is combined with an amphoteric surfactant, preferably phosphatid-yl choline and they are allowed to react with a nitrile such as acetonitrile or acrylonitrile to give the subject amide such as acetamide or methacrylamide. The amphoteric surfactant is more preferably 1,2-dimyristylamide-1,2-deoxyphosphatidylcholine.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention provides a method for producing an amide compound from a nitrile compound using a microorganism (prior art).
Special Publication 1983-37 using microorganisms belonging to the genus Onas)
No. 951 (Yamada et al.), Corynebacterium (Coryn
JP-A-56-17918 (Watanabe et al.) using microorganisms belonging to the genus Rhodococcus and JP-A No. 62-91189 (Kawakami et al.) using microorganisms belonging to the genus Rhodococcus have been reported. This is an industrially useful invention for producing acrylamide and/or methacrylamide using microorganisms.

However, nitrile hydratase active enzymes that have the ability to convert nitrile compounds into amide compounds have a problem in that they easily lose their enzyme activity due to the nitrile compound as a raw material and the amide compound as a product.

Therefore, when the concentration of a nitrile compound is increased for the purpose of increasing the amidation reaction rate, the nitrile hydratase active enzyme is deactivated in a short period of time, making it difficult to obtain the product amide compound in a predetermined time. be. Furthermore, the nitrile hydratase active enzyme easily loses its activity due to the product amide compound, making it difficult to obtain a high concentration of the amide compound.

On the other hand, in a method for producing acrylamide or methacrylamide from acrylonitrile or methacrylonitrile using microorganisms, by setting the reaction temperature in an aqueous medium to a temperature range of freezing point to 5°C,
An excellent production method is known in which the activity of the nitrile hydratase enzyme is maintained and the product acrylamide or methacrylamide is concentrated to a high concentration (Japanese Patent Publication No. 56-38118). According to this method, it is possible to suppress the decrease in the activity of the nitrile hydratase enzyme to some extent, and according to the records of the examples, the acrylamide concentration reaches a maximum of 31.0°C at the reaction temperature of -3 to 0°C.
It becomes 8%.

(Problems to be Solved by the Invention) However, even when the reaction temperature is lowered, the effect of suppressing the decrease in the activity of the nitrile hydratase enzyme is not sufficient, and according to Example 2 of Japanese Patent Publication No. 56-38118,
The dry weight percentage of the microorganism used to obtain an acrylamide concentration of 31.8% was 2.25% at the end of the reaction.
The weight of acrylamide produced per unit weight (g) of dry microorganisms is as small as 14.1 (g). Therefore, the amount of microorganisms used is large, and it is thought that there is room for further improvement in industrial production. Furthermore, the reaction temperature range is low, from the freezing point to 15°C, which is different from the temperature range of 20 to 35°C for enzyme reactions using microorganisms, and the reaction rate is slow.

(Means for Solving the Problems) As a result of intensive research to improve these problems, the present inventors surprisingly discovered that an amphoteric surfactant can coexist with a microorganism having nitrile hydratase activity. The present invention has been achieved based on the discovery that the decrease in the activity of the nitrile hydratase enzyme can be significantly suppressed by That is, the present invention is a method for producing an amide compound from a nitrile compound by the action of a microorganism having nitrile hydratase activity, which is characterized in that an amphoteric surfactant is allowed to coexist.

(Function) The present invention will be explained in detail below. A microorganism having nitrile hydratase activity is a microorganism containing a nitrile hydratase active enzyme having the ability to convert a nitrile compound into an amide compound (hereinafter referred to as the microorganism), and is used, for example, in Japanese Patent Publication No. 59-37951. Microorganisms belonging to the genus Pseudomonas that are used in
microorganisms belonging to the genus Nocardia
Microorganisms belonging to the genus Rdia), Special Publication No. 62-21519
Bacterium (jeridi), a microorganism belonging to the genus Bacillus used in this issue.
Microorganisms belonging to the genus Micrococcus um)
microorganisms belonging to the genus Brevibacterium, microorganisms belonging to the genus Brevibacterium;
Microorganisms belonging to the genus Rhodococcus used in JP-A No. 62-91189 and microorganisms belonging to the genus Δcinetobacter used in the invention previously filed by the present applicant may be mentioned. Yes, but not limited to these.

Examples of the amphoteric surfactants used in the present invention include phosphoglycerides such as phosphatidylethanolamine, phosphatidylcholine, egg yolk lecithin, soybean lecithin, phosphatidylserine, phosphatidylinositol, 3'-o-lysylphosphatidylglycerol, 1.
2-dimyristoylamido-1,2-deoxyphosphatidylcholine (hereinafter abbreviated as DDPC), or the following general formula R7-"→CH, -h coo-R:I (wherein, R1 is a carbon number of 12 to 18 saturated hydrocarbon or unsaturated hydrocarbon, R2, R: l represents hydrogen, methyl group, ethyl group or propyl group, n represents 1 or 2), or a carboxybetaine compound represented by the following general formula (in the formula , R1 is a saturated hydrocarbon or unsaturated hydrocarbon having 12 to 18 carbon atoms, RZ is hydrogen, methyl group, ethyl group or propyl group, X is a methylene group, ethylene group,
Represents a propylene group or a phenyl group.

A sulfobetaine compound represented by the formula %, or an aminocarbon represented by the following general formula % (wherein, R is a saturated hydrocarbon or unsaturated hydrocarbon having 12 to 18 carbon atoms, and n represents 1 or 2). or an imidazolinium betaine compound represented by the following general formula % (wherein R represents a saturated hydrocarbon or unsaturated hydrocarbon having 12 to 1 carbon atoms),
It is not limited to these.

In the present invention, the nitrile compounds hydrolyzed by the microorganisms include mononitrile compounds such as acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, isobutyronitrile, n-butyronitrile, adiponitrile, isophthalonitrile, ortho Mention may be made of dinitrile compounds such as, but not limited to, 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, 1-amide substituted product and 2-amide substituted product are used. For example, acetamide, propionamide, acrylamide, methacrylamide,
Examples include isobutylamide, n-butylamide, etc., but are not limited thereto.

Methods for the coexistence of amphoteric surfactants include, for example, adding an amphoteric surfactant to the culture solution of the microorganism and then reacting with a nitrile compound to obtain the corresponding amide compound; After adding and mixing the activator,
A method for obtaining an isolated microorganism with an amphoteric surfactant adsorbed through filtration, and further dispersing the isolated microorganism in an aqueous medium, and then reacting with a nitrile compound to obtain a corresponding amide compound. After filtering the culture solution to obtain an isolated microorganism, and dispersing the isolated microorganism and an amphoteric surfactant in an aqueous medium, a reaction with a nitrile compound is performed,
A method for obtaining the corresponding amide compound is to obtain an isolated microorganism by filtering the culture solution of the microorganism, and then immobilize it on a carrier such as polyacrylamide, carrageenan, alginates, etc. to obtain an immobilized microorganism, and then use an amphoteric surfactant. A method for obtaining a corresponding amide compound by carrying out a reaction with a nitrile compound in a fixed bed, fluidized bed, etc. format while adding a nitrile compound.After filtering a culture solution of the microorganism and obtaining an isolated microorganism, The isolated microorganism is immobilized on a carrier such as carrageenan or alginates together with an amphoteric surfactant to obtain an immobilized microorganism. Next, the immobilized microorganism is reacted with a nitrile compound in a fixed bed, fluidized bed, etc. format,
There are methods for obtaining the corresponding amide compound, but the method is not limited to these.

The amphoteric surfactant contains O,0 per gram of dry weight of the microorganism.
0.01 g or more, more preferably 0.01 g or more may coexist. By coexisting 0.001 g or more of an amphoteric surfactant per 1 g of the dry weight of the microorganism, the amount of amide compound that can be produced per 1 g of the dry weight of the microorganism in the reaction with the nitrile compound is greatly increased. is advantageous for industrial production. That is, the amount of amphoteric surfactant per 1 g of dried microorganism
．． 001 g or more, more preferably 0.01 g or more, in order to significantly suppress the decrease in the activity 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 the microorganism can be greatly increased. increase This is due to the nitrile hydrolase activity enzyme, such as the change in ion concentration caused by the extraction and removal of lipids in the cell membrane of microorganisms by the raw material nitrile compound and/or product amide compound in the reaction with the nitrile compound. This is thought to be due to the ability of the amphoteric surfactant to suppress the decrease in nitrile hydratase activity caused by changes in the surrounding environment. In other words, the adsorption of amphoteric surfactants to the surface of microorganisms inhibits the extraction and removal of lipids in the cell membranes of microorganisms, or the presence of amphoteric surfactants around microorganisms inhibits the reaction with nitrile compounds. At this time, the amphoteric surfactant dissolves in the dil-IJ compound as the raw material and/or the amide compound as the product, and as a result, the amount of lipid dissolved in the nitrile compound and/or amide compound in the cell membrane of the microorganism This is thought to be due to a decrease in

The amphoteric surfactant is 0°001 per gram of microorganism dry weight.
If the amount is less than 1 g, it becomes difficult to sufficiently suppress the action of extracting and removing lipids in the cell membrane of microorganisms by the raw material nitrile compound and/or product amide compound, and as a result, the amount of amide that can be produced per 1 g of microorganism dry weight becomes difficult. This results in no improvement in the amount of the compound.

In addition, if the amount of amphoteric surfactant is 50 g or more per 11 g of microbial dry type ff, the viscosity of the solvent during reaction with the nitrile compound will increase, and it will be difficult to remove the amphoteric surfactant from the solvent after the reaction, which may cause practical problems. be.

Regarding the reaction conditions for the reaction of microorganisms with nitrile compounds, the 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. It should be added so that 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 using examples.
This example is merely an example specifically describing the method used in the present invention, and it goes without saying that the method of this example does not limit the content of the present invention in any way.

Examples 1 to 4 Rhodococcus AK-32
Bacterial strain (Feikoken Joyori No. 1046) 0.3 parts (moisture content 90
%) and soybean lecithin listed in Table 1, 0°05
moj! /f sodium sulfate aqueous solution and adjust the pH to 7.0. Next, while maintaining the temperature at the temperature listed in Table 1, acrylonitrile is added continuously at the rate listed in Table 1 per hour with stirring for the time listed in Table 1. After the addition of acrylonitrile was completed, stirring was continued for 1 hour, and then the reaction was terminated. The product after the reaction was quantified by gas chromatography. The concentration of the product amide compound and the dry microbial unit weight (
The amount of amide compound produced per g) is listed in Table 1.

Comparative Examples 1 to 4 Reactions with acrylonitrile were 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.

The concentration of the amide compound as a product and the amount of amide compound produced per unit weight (g) of dry microorganism are listed in Table 1.

In Examples 1 to 4, the concentration of acrylamide, which is an 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 the amount of unreacted acrylonitrile at the end of the reaction was 4.55 to 1.3%.
7% remained. Comparative Examples 1-4. In order to achieve an industrially useful acrylamide concentration of 20%, further addition of the microorganism is required. therefore,
This is a practical problem because the amount of amide compound produced further decreases.

Examples 5-8 Acinetobacter genus 5
AIE5 strain (Feikoken Bacteria No. 10432) (water content 9
0%) and the weight listed in Table 2 and the type and weight of the amphoteric surfactant listed in Table 2. 05moffi//!
Disperse in 100 parts of sodium sulfate aqueous solution and adjust the pH to 7.
．． Adjust to 0. Next, while maintaining the temperature at the temperature listed in Table 2, the nitrile compound listed in Table 2 is added continuously at the rate listed in Table 2 per hour with stirring for the time listed in Table 2. After the addition of the nitrile compound was completed, stirring was continued for 1 hour, and then the reaction was terminated. The product after the reaction was quantified in the same manner as in Examples 1 to 4. The results obtained are listed in Table 2.

Comparative Examples 5 to 8 Reactions with nitrile compounds were carried out in the same manner as in Examples 5 to 8, except that no amphoteric surfactant was added. The results obtained are listed in Table 2.

Comparing Examples 5 and 6 and Comparative Example 5, Example 5,
The amide production amount in Comparative Example 5 is 797.781, and the amide production amount in Comparative Example 5 is 635, which is a low value. Furthermore, the concentration of acrylamide, which is an amide compound, was as low as 16.1% in Comparative Example 5, and in order to achieve an industrially useful acrylamide concentration of 20%, it was necessary to add the microorganism.

When Example 7 and Comparative Example 6 are compared, the amide production amount is 793.572, which is a difference of 221.

Comparing Example 8 and Comparative Example 5, the amide production amount was 797.635, which is a difference of 163.

Comparing Example 9 and Comparative Example 5, the amide production amount was 793.635, which is a difference of 158.

When Example 10 and Comparative Example 7 are compared, the production amount of methacrylamide, which is an amide compound, is 467.350, which is a difference of 117.

Comparing Example 11 and Comparative Example 8, the production amount of acetamide, which is an amide compound, is 828.678, which is a difference of 150.

In all comparisons, there was a large difference in the amount of amide compound produced between the Examples and Comparative Examples (the effect of the coexistence of the amphoteric surfactant was clearly observed).

Reference Example 1 Method for producing immobilized microorganisms Rhodococcus genus AK-32
Bacterial strain (vlI Koken Joyori No. 1046) 50.0 parts (moisture content 90%), soybean lecithin 0.1 part, sodium alginate 2 parts at 0.05 mol! ,/i! was mixed with 47.9 parts of an aqueous sodium sulfate solution to form a uniform suspension. The suspension was dropped into a 12.0% by weight aqueous calcium chloride solution at 5° C. to obtain granular immobilized microorganisms with a diameter IM. The microorganism content in the obtained immobilized microorganisms was 5.0% by weight as dry microorganisms. In addition, the content of soybean lecithin in the obtained immobilized microorganism was 0.1% by weight.
Met.

Reference Example 2 Production method of immobilized microorganism Acinetobacter genus 5
50.0 parts of AIE5 strain (Feikoken Bibori No. 10432), (water content 90%), 1 part of DDPCo, 2 parts of sodium alginate at 0.05 IloI! The mixture was mixed with 47.9 parts of an aqueous sodium sulfate solution of 1/l to form a uniform suspension.

The suspension was dropped into a 12.0% by weight aqueous calcium chloride solution at 5°C to obtain granular immobilized microorganisms with a diameter of 1ffl11. The microorganism content in the obtained immobilized microorganisms was 5.0% by weight as dry microorganisms. Furthermore, the content of DDPC in the obtained immobilized microorganism was 0.11% by weight.

Reference Example 3 Method for producing immobilized microorganisms Immobilized microorganisms in the form of granules with a diameter of 1 mm were obtained in the same manner as in Example 9, except that soybean lecithin was not added.

The microorganism content in the obtained immobilized microorganisms was 5.0% by weight as dry microorganisms.

Reference Example 4 Manufacturing method of immobilized microorganism A granular immobilized microorganism having a diameter of ``law'' was obtained in the same manner as in Example 10, except that DDPC was not added. The microorganism content in the obtained immobilized microorganisms was 5.0% by weight as dry microorganisms.

Examples 12-19. Comparative Examples 9 to 16 The immobilized microorganisms prepared in Reference Examples 1 to 4 were added in the amounts listed in Table 3 to 100 parts of the solvent listed in Table 3, and then, while maintaining the solvent at the temperature listed in Table 3, The nitrile compounds listed in Table 3 are added continuously at the rate listed in Table 3 per hour with stirring for the time listed in Table 3. After the addition of the nitrile compound was completed, stirring was continued for 2 hours, and then the reaction was terminated. The unreacted substances and products after the reaction were quantified in the same manner as in Examples 1 to 4. Further, if the concentration of unreacted nitrile compound is less than 1%, the immobilized microorganism used is collected and 2) reaction with the IJ compound is performed again under the same conditions.

When the concentration of the unreacted 2I-IJ compound after the completion of 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 using the following formula.

(Amide compound production amount) = (total amide compound production amount including repeated production)/(dry cell weight of all immobilized microorganisms used) The obtained results are listed in Table 3.

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

Similarly, the amide compound production amounts in Example 13 and Comparative Example 10 have a difference of 449, and in Example 14
And the amide compound production amount in Comparative Example 11 was 449
There is a difference between

There is a difference of 408 between the amide compound production amounts in Example 15 and Comparative Example 12.

In Example 16 and Comparative Example 13, water was used as a solvent, but the amide compound production amount in Example 16 was 453, and the amide compound production amount in Comparative Example 13 was only 60 at most, which was a large difference. ing.

In Example 17 and Comparative Example 14, the amide compound production amount of acetamide, which is an amide compound, was 4.
70.136, with a difference of 334.

In Example 18 and Comparative Example 15, the amide compound production amount of methacrylamide, which is an amide compound, was 375.140, which is a large difference.

Similarly, in Example 19 and Comparative Example 16, the amide compound production amount was 421.192, respectively, which is a large difference.

In both comparisons, there was a large difference in the amount of amide compound produced between the Examples and Comparative Examples, and the coexistence effect of the amphoteric surfactant in the immobilized microorganisms was observed.

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

Claims (4)

[Claims] (1) A method for producing an amide compound from a nitrile compound by the action of a microorganism having nitrile hydratase activity, the method comprising coexisting an amphoteric surfactant. (2) The production method according to claim 1, wherein the amphoteric surfactant is phosphatidylcholine. (3) The production method according to claim 1, wherein the amphoteric surfactant is 1,2-dimyristoylamide-1,2-deoxyphosphatidylcholine. (4) Add 0.0% amphoteric surfactant per 1 g of dry weight of microorganism.
The manufacturing method according to claim 1, characterized in that 0.01 g or more coexist.