JPH1189575A - Production of amide compound by using microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing an amide corresponding to a nitrile compound from the nitrile compound by utilizing cell bodies of microorganisms producing a nitrile hydratase, or a treated material of the cell bodies, by which a reaction liquid having a higher amide concentration is obtained by using smaller amount of the cell bodies. SOLUTION: This method for producing acrylamide from acrylonitrile by utilizing cell bodies of microorganisms producing a nitrile hydratase, or a treated material of the cell bodies comprises addition of the acrylonitrile to a reaction liquid so that the concentration of the acrylonitrile may be not less than the solubility of the acrylonitrile at the time of the start or on the way of the reaction for forming the acrylamide by bringing the cell bodies of the microorganisms capable of keeping the nitrile hydratase activities after treating the microorganisms in 30 wt.% acrylamide aqueous solution at 10 deg.C for 60 min, or the treated material thereof, into contact with the acrylonitrile in an aqueous medium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a reaction method for producing a corresponding amide compound from a nitrile compound in an aqueous medium by using a microbial cell having nitrile hydratase activity or a processed product thereof. More specifically,
The present invention relates to a method for efficiently producing a corresponding amide compound from a nitrile compound by setting the concentration of the nitrile compound at the start of or during the reaction to be equal to or higher than the saturation concentration of the nitrile compound in an aqueous medium.

[0002]

2. Description of the Related Art In recent years, nitrile hydratase, an enzyme having nitrile hydration activity for hydrating a nitrile group and converting it to an amide group, has been discovered. There is known a method for producing a corresponding amide compound from a nitrile compound in an aqueous medium using the enzyme, cells of a microorganism containing the enzyme, or a processed product of the cells (Japanese Patent Publication No. 62-21519). The method for producing an amide compound using nitrile hydratase has an advantage that the conversion and selectivity of the nitrile compound are high as compared with the conventional chemical methods. However, on the other hand, nitrile compounds serving as substrates for enzymatic reactions and amide compounds as products are generally organic compounds having strong biotoxicity, and as the concentration in the reaction system increases, the enzyme rapidly reacts. It has been conventionally pointed out that it is deactivated. Therefore, when industrially producing an amide compound from a nitrile compound, by keeping the concentrations of the nitrile compound and the amide compound in the reaction system low, the reaction proceeds smoothly by maintaining the nitrile hydratase activity stably. Has been considered important.

As an example, as a method for industrially producing an amide compound from a nitrile compound, a report by Nagasawa and Yamada (Genetics, separate volume 1, pages 36 to 45, March 1988) is known. I have. In the report (p. 38, right column, 8th line from the bottom), the method for producing acrylamide from acrylonitrile is described as “Static cells at 20 mg / ml.
With acrylonitrile at a concentration of 5 to 15 ° C and a pH of around 7.0. At this time, since acrylonitrile at a high concentration inhibits nitrile hydratase activity, the acrylonitrile is added in portions over time as the reaction proceeds. "

Japanese Patent Publication No. 56-38118 discloses a method in which the hydration reaction using acrylonitrile or methacrylonitrile as a substrate is carried out at a low temperature from the freezing point to 15 ° C. Regarding the concentration, it is described that "in the present enzyme reaction, it is appropriate to add the substrate so that it is always in a dissolved state in the reaction system". Further, "in the case of the batch method, usually a feeding method is employed, but acrylonitrile or methacrylonitrile is added dropwise to the aqueous suspension containing the immobilized cells while stirring, while keeping the temperature at the freezing point to 15 ° C. do it."
And the necessity of divided addition of the nitrile compound.

In Japanese Patent Publication No. 57-1234, when acrylamide or methacrylamide is continuously produced from acrylonitrile or methacrylonitrile, the concentration of the nitrile compound is continuously supplied in an amount within a range that dissolves in the reaction mixture. In this publication, the importance of controlling the concentration of the nitrile compound low is also shown.

When an amide compound is produced by hydrating a nitrile compound in an aqueous medium using nitrile hydratase, the amide compound produced continues to accumulate in the aqueous medium as the reaction proceeds. The maximum concentration of the amide compound produced is determined by the nature of the nitrile hydratase from the individual microorganism and the reaction temperature. That is, when the enzyme and the amide compound are brought into contact at the reaction temperature, the concentration of the amide compound in the reaction solution may be higher than the lower limit of the amide compound concentration at which the enzyme is immediately deactivated. Absent. In the following, this concentration of the amide compound will be referred to as the limit amide compound concentration.

According to the above-mentioned Nagasawa-Yamada report, "Acrylamide production as high as 40% is considered impossible by current chemical methods, and the effectiveness of the enzymatic method as an industrial production method of acrylamide is considered here. It was strongly suggested. "(Page 39, left column, second line from the top). Thus, industrially, it is important that the concentration of the amide compound at the end of the reaction is high, and nitrile hydratase having a high amide compound concentration at this limit is useful.

When the reaction is carried out while controlling the concentration of the nitrile compound in the reaction solution to be low, the contact time of the nitrile hydratase with the nitrile compound and the amide compound becomes relatively long, and the amide compound concentration reaches the limit amide compound concentration. To accumulate the concentration, it is necessary to relatively increase the nitrile hydratase activity in the reaction system. An increase in the amount of the enzyme used is disadvantageous in terms of cost for industrial production of amide compounds. Therefore, a reaction method for controlling the concentration of the nitrile compound in the reaction solution to be low is not necessarily an efficient reaction method.

[0009] Microbial strains having nitrile hydratase activity almost always have amidase, an enzyme that hydrates amide compounds into the corresponding carboxylic acid compounds. Therefore, when a corresponding amide compound is produced from a nitrile compound using the microorganism strain, it is known that the corresponding carboxylic acid compound is by-produced by the amidase. In order to avoid this, it was necessary to obtain a mutant microbial strain having no amidase activity or to devise a method for preparing a microbial cell having a relatively low amidase activity.

[0010]

It is an object of the present invention to provide a more efficient method for producing the corresponding amide compound from a nitrile compound using a microorganism.

An object of the present invention is to provide a method for producing a corresponding amide from a nitrile compound by using a cell of a nitrile hydratase-producing microorganism or a treated cell thereof, and a higher amide concentration with a smaller amount of cells. To obtain a reaction solution of

[0012]

Means for Solving the Problems The present inventors have found that nitrile hydratase having the above-mentioned limit amide compound concentration higher than a certain level has high resistance to the corresponding nitrile compound. When a reaction is performed to produce a corresponding amide from a nitrile compound using a cell of a microorganism having a nitrile hydratase having a resistance to such an amide compound that is higher than a certain level or a treated cell thereof, In this case, the deactivation of the enzyme by a highly biotoxic nitrile compound such as acrylonitrile or methacrylonitrile does not proceed rapidly, and the reaction rate of the enzyme becomes relatively higher as the concentration of the nitrile compound in the aqueous medium increases. It was found to increase. That is, the reaction method of adding acrylonitrile to the reaction solution so that the concentration of the nitrile compound in the reaction system does not need to be constantly controlled to be lower than the conventional method, so that the concentration becomes equal to or higher than the saturation concentration of the nitrile compound in the aqueous medium is known. Newly found.

As a result of the study by the present inventors, according to the newly discovered reaction method, when the same amount of enzyme (active amount) is used, the nitrile can be obtained in a shorter time than the conventional method. It was confirmed that the compound can be converted to the corresponding amide compound, and that a higher concentration of the amide compound aqueous solution can be obtained with a smaller amount of enzyme (active amount). The present inventors have completed the present invention based on the above findings.

That is, the present invention relates to a method for producing an amide compound from a nitrile compound by using the cells of a nitrile hydratase-producing microorganism or a treated product thereof, wherein the amide compound is prepared in a 30% by weight aqueous solution of acrylamide. After treating the cells of the microorganism at 10 ° C. for 60 minutes, the cells of the microorganism or the treated product of the microorganism that retain the activity of nitrile hydratase are contacted with a nitrile compound in an aqueous medium. Production of an amide compound characterized by adding a nitrile compound to a reaction solution such that the concentration of the nitrile compound at the start of or during the reaction for producing the amide compound is equal to or higher than the saturation concentration of the nitrile compound in the aqueous medium. It provides a method.

According to the present invention, when a corresponding amide compound is produced from a nitrile compound in an aqueous medium using a cell or a treated product of a microorganism producing nitrile hydratase, the corresponding amide compound is produced from the nitrile compound. At the beginning of or during the reaction to produce the compound,
By adding the nitrile compound to the reaction system at or above the saturation concentration of the nitrile compound in an aqueous medium, it becomes possible to efficiently and quickly produce a corresponding amide compound from the nitrile compound at a high concentration. .

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The details of the present invention will be outlined below. The nitrile compound in the present invention is a nitrile compound having 2 to 8 carbon atoms. Among them, acrylonitrile, methacrylonitrile, and crotonnitrile can be mentioned as preferred examples.

The nitrile hydratase in the present invention is a microorganism of a microorganism having the ability to hydrolyze a nitrile compound to produce a corresponding amide compound and produce nitrile hydratase in a 30% by weight aqueous solution of acrylamide. There is no particular limitation as long as the activity of the nitrile hydratase is maintained even after the body is treated at 10 ° C. for 60 minutes. Preferably, a nitrile hydratase having the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing in the α subunit can be mentioned.

The resistance of nitrile hydratases of various microorganisms to amide compounds varies. Taking acrylamide as an example of the amide compound, when nitrile hydratase is treated in an aqueous acrylamide solution at 10 ° C., there is nitrile hydratase which is completely inactivated after 60 minutes in a 10% by weight aqueous acrylamide solution. And nitrile hydratase which is not inactivated even when treated in a 50% by weight aqueous solution of acrylamide for 60 minutes. Thus, the above-mentioned limit acrylamide concentration of nitrile hydratase differs depending on the individual nitrile hydratase.

Industrially, it is important that the concentration of the amide compound at the end of the reaction is high, and the concentration of the acrylamide that does not deactivate (retains the activity) after treatment in an aqueous acrylamide solution at 10 ° C. for 60 minutes. It is particularly effective to use nitrile hydratase having a content of at least 30% by weight. Also,
When an amide compound is produced industrially, it is advantageous from the viewpoint of cost to use the cells of a microorganism that produces nitrile hydratase or a processed product of the cells.

That is, in the present invention, when the cells of a nitrile hydratase-producing microorganism are treated in an aqueous acrylamide solution at 10 ° C. for 60 minutes, the upper limit of the acrylamide concentration at which the activity of nitrile hydratase is maintained is limited. It is preferable to use cells of a microorganism that produces 30% by weight or more of nitrile hydratase, or a treated product thereof.

The microorganism of the present invention which retains nitrile hydratase activity even after being treated in a 30% by weight aqueous solution of acrylamide at 10 ° C. for 60 minutes is defined as a microorganism which hydrolyzes a nitrile compound and converts the corresponding amide compound. Produce nitrile hydratase capable of producing
Even if the cells of the microorganism are treated in a 0% by weight aqueous solution of acrylamide at 10 ° C. for 60 minutes, the microorganism is not particularly limited as long as it retains nitrile hydratase activity. Preferably, the microorganism may be a microorganism producing nitrile hydratase having the amino acid sequence described in SEQ ID NO: 1 or the amino acid sequence described in SEQ ID NO: 2 in its α subunit. Specifically, Nocardia genus, Corynebacterium ( Corynebacteriu)
m) the genus Bacillus (Bacillus), Bacillus thermophilic, Pseudomonas (Pseudomonas) genus Micrococcus (MicroC
occus) genus, Rodokokkkasu (Rhodococcus) genus is represented by rhodochrous (rhodochrous) species, Acinetobacter (Ac
inetobacter) genus, key Santo Enterobacter (Xanthobacter) genus,
Streptomyces (Streptomyces) genus Rhizobium (Rhi
zobium ), Klebsiella ( Klebsiella ), Enterobacter ( Enterobacter ), Erwinia ( Erwinia ),
Earomonasu (Aeromonas) genus, Citrobacter (Citrobac
ter) genus Achromobacter (Achromobacter) genus, Agrobacterium (Agrobacterium) genus or thermophila (th
ermophila) shoe de Nocardia represented by species (Pseud
Microorganisms belonging to the genus onocardia ) can be mentioned as preferred examples.

Further, a transformant expressing the nitrile hydratase gene cloned from the microorganism in any host is also included in the microorganism producing nitrile hydratase referred to in the present invention. In addition, as an arbitrary host referred to herein, Escherichia coli ( Escherichia coli ) is mentioned as a typical example as described in Examples below, but it is not particularly limited to Escherichia coli, and Bacillus subtilis ( Bacillus subtilis ) and the like. Other microbial strains such as genera, yeasts and actinomycetes are also included. As an example of such, MT-10822 (this strain is 1
International approval of the deposit of microorganisms in patent procedures under the accession number FERM BP-5785 to the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry of 1-3-1 Higashi, Tsukuba, Ibaraki Prefecture on February 7, 996 Has been deposited under the Budapest Treaty. ). In addition, recombinant D
One or two of the constituent amino acids of the enzyme using NA technology
A transformant expressing a mutant nitrile hydratase which further improves amide compound resistance, nitrile compound resistance and temperature resistance by substituting, deleting, deleting or inserting at least one of the amino acids with another amino acid is also the present invention. Included in microorganisms that produce nitrile hydratase.

When the microorganism of the present invention is used in the production method of the present invention, cells of the microorganism or a processed product of the microorganism are used. The cells may be prepared using a general method known in the fields of molecular biology, biotechnology, and genetic engineering. For example, after inoculating the microorganism in a normal liquid medium such as LB medium or M9 medium, an appropriate culture temperature (generally 20 ° C. to 50 ° C .;
C or higher. ), Followed by centrifugation to separate and collect the microorganism from the culture solution.

The treated product of the microorganism in the present invention may be an extract or a crushed product of the above-mentioned microbial cell, or a nitrile hydratase active fraction of the extract or the crushed product. Products, immobilized products obtained by immobilizing the microbial cells and extracts, crushed products, and post-separated products of the cells using a suitable carrier, etc., as long as they have nitrile hydratase activity. This corresponds to the treated product of the present invention.

The microbial cells of the present invention or processed microbial cells can be subjected to a batch reaction or a continuous reaction. Further, it may be used as a suspension bed or as a fixed bed. At this time, the concentration of the cells of the microorganism or the treated product of the microorganism in the reaction solution is not particularly limited as long as the mixture of the aqueous medium and the nitrile compound is not hindered.

The aqueous medium in the present invention is water or a buffer such as phosphate, an inorganic salt such as sulfate or carbonate,
This shows an aqueous solution in which an alkali metal hydroxide / amide compound or the like is dissolved at an appropriate concentration.

In the present invention, the nitrile compound in the aqueous medium is consumed as the reaction proceeds and is supplied from the nitrile phase at the same time, so that the concentration is maintained for a relatively long time before or after the saturation concentration. This is very important. Therefore, in either the batch reaction or the continuous reaction, the entire amount of the necessary nitrile compound is added all at once at the start of the reaction, or the nitrile compound concentration in the aqueous medium during the reaction is maintained at a saturation concentration or higher. It is preferable to sequentially add a nitrile compound to the mixture. It is also important to sufficiently mix the aqueous medium phase and the nitrile phase, which separate into two phases under standing, using a suitable mixing device such as a rotary blade or a line mixer.

When the nitrile compound is added all at once at the start of the reaction, the lower limit of the concentration of the nitrile compound may be at least the saturation concentration of the nitrile compound at the start of the reaction.
On the other hand, the upper limit of the concentration is not particularly limited,
The concentration may be arbitrarily determined depending on the concentration of the amide compound and the concentration of the nitrile compound at the end of the expected reaction. Unreacted nitrile compounds can also be removed from the reaction solution after the reaction by means such as distillation. Therefore, the nitrile compound can be added so that the nitrile compound becomes excessive even when the concentration of the amide compound at the end of the expected reaction is reached.

Specifically, when acrylonitrile is a nitrile compound, the saturation concentration of the present compound with respect to water is about 7% by weight, so that the range of about 7% by weight to 50% by weight is preferable. When methacrylonitrile or crotonnitrile is a nitrile compound, the saturation concentration of these compounds with respect to water is about 2% by weight, so the range of about 2% by weight to 50% by weight is preferable.

The reaction in the present invention is generally carried out under normal pressure, but may be carried out under pressure in order to increase the solubility of the nitrile compound in the aqueous medium. The reaction temperature is not particularly limited as long as it is equal to or higher than the freezing point of the aqueous medium, but is preferably in the range of 0 ° C to 50 ° C, more preferably 10 ° C to 30 ° C. On the other hand, pH
Is not particularly limited as long as the nitrile hydratase activity is maintained, but is preferably pH 6 to pH 10,
It is more preferably in the range of pH 7 to pH 9.

In the reaction for producing an amide compound in the method of the present invention, the nitrile hydratase does not immediately deactivate, but rather the nitrile hydration proceeds well. In addition, since the nitrile compound in the aqueous medium phase is consumed as the reaction proceeds and supplied from the nitrile phase, its concentration is always maintained near the saturation concentration. Therefore, the nitrile hydration reaction proceeds at or near the maximum reaction rate at the reaction temperature, so that the corresponding amide compound is efficiently produced from the nitrile compound in a relatively short time.

After the nitrile compound is added to a concentration higher than the saturation concentration in the aqueous medium, the concentration of the nitrile compound in the reaction system gradually decreases with the progress of the reaction, and becomes lower than the saturation concentration in the aqueous medium. As a result, the reaction rate also gradually decreases, but up to that point, the nitrile hydration reaction proceeds at or near the maximum reaction rate, so that the corresponding amide compound can be efficiently converted from the nitrile compound in a substantially short time. Will be generated.

Further, according to the production method of the present invention, by increasing the concentration of the nitrile compound in the aqueous medium, the production of the corresponding carboxylic acid compound by the amidase in the microbial cells is relatively suppressed. The secondary effect described above can also be obtained.

The obtained amide compound may be used as it is as an aqueous solution after separating cells or processed cells from the reaction solution by a usual known method such as centrifugation, or may be used for membrane concentration or spraying. Crystals may be obtained by concentration by a method such as dry concentration. In addition, after the cells or the treated cells are separated, the purity of the amide compound can be further increased by removing impurities such as coloring substances using activated carbon, an ion exchange resin, an ion exchange membrane or the like.

[0035]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. In addition, the HPL in each of the examples and the comparative examples
The C analysis was performed using Finepak manufactured by JASCO as a column.
Using SIL C18-5 (250 × 4.6φmm)
A 10 mM phosphoric acid aqueous solution containing 4% by volume of acetonitrile was used as a developing solution. Acrylamide, methacrylamide and crotonamide were detected by absorbance at 220 nm, and acrylonitrile, acrylic acid, methacrylonitrile and crotonnitrile were detected by absorbance at 210 nm.

Example 1 Measurement of Acrylamide Concentration Resistance (1) 100 ml of a medium having the following composition was prepared in a 500 ml baffled Erlenmeyer flask, and sterilized in an autoclave at 121 ° C. for 20 minutes. A final concentration of 50 μg /
ml of ampicillin, and then MT-1
The 0822 strain (FERM BP-5785) was inoculated with a monobacterial ear and cultured at 37 ° C. and 130 rpm for 20 hours. Only the cells were separated from the culture solution by centrifugation (15000 G × 15 minutes). Subsequently, after resuspending the cells in 50 ml of physiological saline, the cells were centrifuged again to obtain wet cells. .

Medium Composition Yeast Extract 5.0 g / L Polypeptone 10.0 g / L NaCl 5.0 g / L Cobalt chloride hexahydrate 10.0 mg / L Ferric sulfate heptahydrate 40.0 mg / L L pH 7.5

10 mg of the wet cells were suspended in 10 g of a 50 mM aqueous solution of tris hydrochloride (pH 8.0), and added to the suspension so that the final concentration of acrylonitrile was 14% by weight. The reaction was performed for 5 minutes while stirring. After adding 90 g of a 10 mM phosphoric acid aqueous solution to terminate the reaction, the acrylamide concentration in the reaction-terminated liquid was measured by HPLC analysis. Subsequently, the production rate of acrylamide per unit wet cell and unit reaction time (= reaction rate) was calculated.

Subsequently, 50 mg of the same wet cells were suspended in 40 g of a 30% by weight aqueous solution of acrylamide (pH 8.0) containing 50 mM of tris hydrochloride, and contacted at 10 ° C. for 60 minutes with gentle stirring. Was. After this,
Only the cells were separated from the suspension by centrifugation (15000 G × 15 minutes). Subsequently, the cells were resuspended in 50 g of a 50 mM aqueous solution of Tris hydrochloride (pH 8.0), and the operation of centrifuging again was repeated twice to wash the cells. 10 g of the washed wet cells 10 mg
Was suspended in a 50 mM aqueous solution of tris hydrochloride (pH 8.0), and the suspension was added so that the final concentration of acrylonitrile was 14% by weight. The mixture was stirred at 10 ° C. for 5 minutes while stirring. . 90 mM phosphoric acid aqueous solution
After completion of the reaction by adding g, the acrylamide concentration in the reaction-completed solution was measured by HPLC analysis. continue,
The production rate of acrylamide per unit wet cell and unit reaction time (= reaction rate) was calculated.

The reaction rate before contact with a 30% by weight aqueous solution of acrylamide was taken as 100%, and the reaction rate after contacting at 10 ° C. for 60 minutes was compared as a relative value. As a result, the relative value was 70%. That is, the residual activity ratio was 70%.

Example 2 Comparison of Nitrile Compound Concentration and Reaction Rate In the same manner as in Example 1, wet cells of strain MT-10822 were prepared. 10 mg of the wet cells was added to 10 g of 50 mM
Acrylonitrile was added to this suspension to a final concentration as shown in Table 1 (Table 1), and the mixture was stirred at 10 ° C. for 5 minutes while stirring. The reaction was performed. 90 g of 10 mM phosphoric acid aqueous solution
After the addition, the reaction was terminated, and the acrylamide concentration in the reaction-terminated liquid was measured by HPLC analysis. The production rate of acrylamide per unit wet cell and unit reaction time (= reaction rate) was calculated, and the reaction rate when the acrylonitrile concentration at the start of the reaction was 1% by weight was taken as 100%.
The relative value at each acrylonitrile concentration was compared as a relative reaction rate (Table 1 (Table 1) and FIG. 1 (FIG. 1)).

[0042]

[Table 1]

Example 3 Conversion of Nitrile Compound to Amide Compound (1) In the same manner as in Example 1, wet cells of strain MT-10822 were prepared. 1.5 g of the wet cells was added to 98.5 g of 50
The suspension was suspended in an aqueous solution of mM tris hydrochloride (pH 8.0), and 36 g of acrylonitrile was added to the suspension at a time.
The reaction was carried out at 0 ° C. while stirring. Eight hours after the start of the reaction, the reaction solution was analyzed by HPLC analysis.
As a result, only acrylamide was present (concentration = 35.0% by weight) in the reaction solution, and acrylonitrile was not observed. That is, the conversion was 100%.

Example 4 Conversion of Nitrile Compound to Amide Compound (2) In the same manner as in Example 1, wet cells of strain MT-10822 were prepared. 1.5 g of the wet cells was added to 98.5 g of 50
The suspension was suspended in an aqueous solution of tris hydrochloride (mM) (pH 8.0).
The reaction was started while stirring at 0 ° C. Further, acrylonitrile was added at the same time as the reaction started,
g of acrylonitrile in a total amount of 36 g. Eight hours after the start of the reaction, the reaction solution was analyzed by the same HPLC analysis as in Example 1. As a result, only acrylamide was present in the reaction solution (concentration =
35.0% by weight), and no acrylonitrile was observed. That is, the conversion was 100%.

Comparative Example 1 Conversion of Nitrile Compound to Amide Compound (1) In the same manner as in Example 1, wet cells of strain MT-10822 were prepared. 1.5 g of the wet cells was added to 98.5 g of 50
The suspension was suspended in a mM aqueous tris hydrochloride solution (pH 8.0), and acrylonitrile was added to the suspension at a rate of 2 g per hour for 18 hours. Example 1 8 hours after the start of the reaction
The reaction solution was analyzed by the same HPLC analysis as described above. As a result, the acrylamide concentration in the reaction solution was 17.4% by weight, and the acrylonitrile concentration was 0.8% by weight. That is, the conversion was 94%. Further, 26 hours after the start of the reaction, the reaction solution was analyzed by the same HPLC analysis as in Example 1. As a result, the acrylamide concentration in the reaction solution was 33.0% by weight, and the acrylonitrile concentration was 1.9% by weight. That is, the conversion was 93%.

Comparative Example 2 Conversion of Nitrile Compound to Amide Compound (2) In the same manner as in Example 1, wet cells of MT-10822 strain were prepared. 1.5 g of the wet cells was added to 98.5 g of 50
It was suspended in an aqueous solution of mM tris hydrochloride (pH 8.0), and acrylonitrile was added to the suspension at a rate of 1.2 g per hour for 30 hours. Eight hours after the start of the reaction, the reaction solution was analyzed by the same HPLC analysis as in Example 1. As a result, the acrylamide concentration in the reaction solution was 10.3% by weight, and the acrylonitrile concentration was 1.0% by weight. . That is, the conversion was 88%. Further, 38 hours after the start of the reaction, the reaction solution was analyzed by the same HPLC analysis as in Example 1. As a result, the acrylamide concentration in the reaction solution was 32.6% by weight, and the acrylonitrile concentration was 2.1% by weight. That is, the conversion was 92%.

Example 5 Conversion of Nitrile Compound to Amide Compound (3) In the same manner as in Example 1, wet cells of strain MT-10822 were prepared. 3.0 g of the wet cells were added to 97.0 g of 50
The suspension was suspended in an aqueous solution of mM tris hydrochloride (pH 8.0), and 8.6 g of methacrylonitrile was added to the suspension at a time, and the reaction was started while stirring at 10 ° C.
24 hours after the start of the reaction, the same HPLC as in Example 1 was performed.
The reaction solution was analyzed by analysis. As a result, only methacrylamide was present (concentration = 10.0% by weight) in the reaction solution, and methacrylonitrile was not observed. That is, the conversion was 100%.

Example 6 Conversion of Nitrile Compound to Amide Compound (4) Wet cells of strain MT-10822 were prepared in the same manner as in Example 1. 1.5 g of the wet cells was added to 98.5 g of 50
The suspension was suspended in an aqueous solution of tris hydrochloride (mM) (pH 8.0), and crotonnitrile (provided that cis isomer and tra
(a mixture of ns forms) was added all at once, and the reaction was started while stirring at 10 ° C. The crystals of crotonamide precipitated 24 hours after the start of the reaction were completely dissolved at 30 ° C., and the reaction solution was analyzed by the same HPLC analysis as in Example 1. As a result, only crotonamide (a mixture of cis- and trans-forms) was present (concentration = 20.0% by weight) in the reaction solution, and crotonnitrile was not recognized. That is, the conversion was 100%.

Example 7 Conversion of Nitrile Compound to Amide Compound (5) 100 ml of a medium having the following composition was prepared in a 500 ml baffled Erlenmeyer flask, and sterilized in an autoclave at 121 ° C. for 20 minutes. This medium is described in JP-A-08-566.
Pseudonocardia thermophila described in No. 84 ( Ps
Eudonocardia thermophila ) (JCM3095) was inoculated with monobacterial ears and cultured at 50 ° C. and 130 rpm for 72 hours. Only the cells were separated from the culture solution by centrifugation (15000 G × 15 minutes). Subsequently, after resuspending the cells in 50 ml of physiological saline, the cells were centrifuged again to obtain wet cells. .

Medium Composition Yeast Extract 5.0 g / L Polypeptone 10.0 g / L trans-crotonamide 2.0 g / L Cobalt chloride hexahydrate 10.0 mg / L Ferric sulfate heptahydrate 40 0.0 mg / L pH 7.0

10.0 g of the wet cells was added to 100 g of 50 mM
Was suspended in an aqueous solution of tris hydrochloride (pH 8.0), and 36 g of acrylonitrile was added to the suspension at a time, and 10 ° C.
The reaction was started while stirring. Eight hours after the start of the reaction, the reaction solution was analyzed by the same HPLC analysis as in Example 1. As a result, only acrylamide was present (concentration = 35.0% by weight) in the reaction solution, and acrylonitrile was not observed. That is, the conversion was 100%.

Example 8 Measurement of Resistance to Acrylamide Concentration (2) 100 ml of a medium having the following composition was prepared in a 500 ml Erlenmeyer flask with a baffle, and sterilized in an autoclave at 121 ° C. for 20 minutes. In this medium, Japanese Patent Publication No. 06-551
No. 48, Rhodococcus rhodochrous J-1 strain (F
ERM BP-1478 has been deposited with the depositary institution under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedures, and will be distributed to all persons upon request). And 30 ℃ ・ 130rpm
m for 72 hours. Centrifugation (15000G x 1
5 minutes) to separate only the cells from the culture solution.
After resuspending the cells in 0 ml of physiological saline, the cells were again centrifuged to obtain wet cells.

Medium Composition Glucose 10.0 g / L Monopotassium dihydrogen phosphate 0.5 g / L Dipotassium monohydrogen phosphate 0.5 g / L Magnesium sulfate / heptahydrate 0.5 g / L Yeast extract 0 g / L Peptone 7.5 g / L Urea 7.5 g / L Cobalt chloride hexahydrate 10.0 mg / L pH 7.2

10 mg of the wet cells were suspended in 10 g of a 50 mM aqueous solution of tris hydrochloride (pH 8.0), and added to the suspension so that the final concentration of acrylonitrile was 14% by weight. The reaction was performed for 5 minutes while stirring. After adding 90 g of a 10 mM phosphoric acid aqueous solution to terminate the reaction, the acrylamide concentration in the reaction-terminated liquid was measured by HPLC analysis. Subsequently, the production rate of acrylamide per unit wet cell and unit reaction time (= reaction rate) was calculated.

Subsequently, 50 mg of the same wet cells were suspended in 40 g of a 30% by weight aqueous solution of acrylamide (pH 8.0) containing 50 mM of tris hydrochloride, and contacted at 10 ° C. for 60 minutes with gentle stirring. Was. After this,
Only the cells were separated from the suspension by centrifugation (15000 G × 15 minutes). Subsequently, the cells were resuspended in 50 g of a 50 mM aqueous solution of Tris hydrochloride (pH 8.0), and the operation of centrifuging again was repeated twice to wash the cells. 10 g of the washed wet cells 10 mg
Was suspended in a 50 mM aqueous solution of tris hydrochloride (pH 8.0), and the suspension was added so that the final concentration of acrylonitrile was 14% by weight. The mixture was stirred at 10 ° C. for 5 minutes while stirring. . 90 mM phosphoric acid aqueous solution
After completion of the reaction by adding g, the acrylamide concentration in the reaction-completed solution was measured by HPLC analysis. continue,
The production rate of acrylamide per unit wet cell and unit reaction time (= reaction rate) was calculated.

The reaction rate before contacting with a 30% by weight aqueous solution of acrylamide was taken as 100%, and the reaction rate after contacting at 10 ° C. for 60 minutes was compared as a relative value. As a result, the relative value was 95%. That is, the residual activity ratio was 95%.

[Example 9] Conversion of nitrile compound to amide compound (6) In the same manner as in Example 8, wet cells of Rhodococcus rhodochrous J-1 strain were prepared. 0.15 g of the wet cells
With 100 g of a 50 mM Tris hydrochloride aqueous solution (pH 8.
0), and 60 g of acrylonitrile was added to this suspension.
The reaction was added at once and the reaction was started while stirring at 20 ° C. 8 hours after the start of the reaction, the same H
The reaction solution was analyzed by PLC analysis. As a result, only acrylamide was present in the reaction solution (concentration = 50.0
% By weight), and neither acrylonitrile nor acrylic acid was found. That is, the conversion was 100%.

Example 10 Conversion of Nitrile Compound to Amide Compound (7) In the same manner as in Example 8, wet cells of Rhodococcus rhodochrous J-1 strain were prepared. 0.15 g of the wet cells
With 100 g of a 50 mM Tris hydrochloride aqueous solution (pH 8.
0), and 60 g of acrylonitrile was added to this suspension.
The reaction was started at a time while stirring at 10 ° C. 8 hours after the start of the reaction, the same H
The reaction solution was analyzed by PLC analysis. As a result, the acrylamide concentration in the reaction solution was 47.7% by weight,
The acrylonitrile concentration was 1.9% by weight. That is, the conversion was 95%. In addition, 1
Two hours later, the reaction solution was analyzed by the same HPLC analysis as in Example 1. As a result, the concentration of acrylamide in the reaction solution was 50.0% by weight, the concentration of acrylic acid was 0.05% by weight, and acrylonitrile was not observed. That is, the conversion was 100%.

[Example 11] Conversion of nitrile compound to amide compound (8) In the same manner as in Example 8, wet cells of Rhodococcus rhodochrous J-1 strain were prepared. 0.15 g of the wet cells
With 100 g of a 50 mM Tris hydrochloride aqueous solution (pH 8.
0), and 20 g of acrylonitrile was added to this suspension.
The reaction was started at a time while stirring at 10 ° C. Further, acrylonitrile was added at a rate of 20 g per hour for 2 hours simultaneously with the start of the reaction, and a total of 60 g of acrylonitrile was added. Eight hours after the start of the reaction, the reaction solution was analyzed by the same HPLC analysis as in Example 1. As a result, the acrylamide concentration in the reaction solution was 46.7% by weight, and the acrylonitrile concentration was 2.6.
% By weight. That is, the conversion was 93%.
Further, 12 hours after the start of the reaction, the same H as in Example 1 was used.
The reaction solution was analyzed by PLC analysis. As a result, the concentration of acrylamide in the reaction solution was 50.0% by weight, the concentration of acrylic acid was 0.05% by weight, and acrylonitrile was not observed. That is, the conversion was 100%.

Comparative Example 3 Conversion of Nitrile Compound to Amide Compound (3) Wet cells of Rhodococcus rhodochrous J-1 strain were prepared in the same manner as in Example 8, and 0.15 g of the wet cells was prepared. With 100 g of a 50 mM Tris hydrochloride aqueous solution (pH 8.
0), and acrylonitrile was added to the suspension at a rate of 3 g per hour for 20 hours. 8 from the start of the reaction
After a lapse of time, the reaction solution was analyzed by the same HPLC analysis as in Example 1. The acrylamide concentration in the reaction solution was 23.6% by weight, and the acrylonitrile concentration was 1.7.
% By weight. That is, the conversion was 91%.
Further, 32 hours after the start of the reaction, the same H
The reaction solution was analyzed by PLC analysis. As a result, the acrylamide concentration in the reaction solution was 46.1% by weight, the acrylic acid concentration was 0.2% by weight, and the acrylonitrile concentration was 2.9% by weight. That is, the conversion was 92%.

[Comparative Example 4] Conversion of nitrile compound to amide compound (4) A wet cell of Rhodococcus rhodochrous J-1 strain was prepared in the same manner as in Example 8, and 0.15 g of the wet cell was prepared. With 100 g of a 50 mM Tris hydrochloride aqueous solution (pH 8.
0), and acrylonitrile was added to the suspension at a rate of 2 g per hour for 30 hours. 8 from the start of the reaction
After a lapse of time, the reaction solution was analyzed by the same HPLC analysis as in Example 1. As a result, the acrylamide concentration in the reaction solution was 16.4% by weight, and the acrylonitrile concentration was 1.5% by weight. That is, the conversion was 89%. Further, 42 hours after the start of the reaction, the reaction solution was analyzed by the same HPLC analysis as in Example 1. As a result, the acrylamide concentration in the reaction solution was 43.5% by weight,
The acrylic acid concentration was 0.3% by weight, and the acrylonitrile concentration was 4.8% by weight. That is, the conversion is 8
7%.

[0062]

According to the method of the present invention, it is possible to obtain a reaction solution having a high nitrile concentration with a small amount of enzyme within a shorter period of time than in the conventional method, and to efficiently convert a corresponding amide compound from a nitrile compound. Can be manufactured. Moreover, according to the method of the present invention, the by-product of the carboxylic acid compound corresponding to the nitrile compound is also suppressed. Therefore, the method of the present invention is extremely useful for industrial amide compound production using a microorganism that produces nitrile hydratase.

[0063]

[Sequence list]

SEQ ID NO: 1 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence characteristics Other information: Partial sequence of nitrile hydratase α subunit Sequence Val Cys Xaa Leu Cys Ser Cys Tyr Pro Trp Pro Xaa Leu Gly Leu Pro 5 10 15 Pro Xaa Trp Xaa Lys 20 21

SEQ ID NO: 2 Sequence length: 20 Sequence type: amino acid Topology: linear Sequence characteristics Other information: Partial sequence of nitrile hydratase α subunit Sequence Val Cys Xaa Leu Cys Ser Cys Xaa Trp Pro Xaa Leu Gly Leu Pro Pro 5 10 15 Xaa Trp Tyr Lys 20

[Brief description of the drawings]

FIG. 1 is a diagram comparing the reaction rates with respect to the acrylonitrile concentration at the start of the reaction. The horizontal axis is the acrylonitrile concentration at the start of the reaction (% by weight), and the vertical axis is the relative reaction rate (%).
Is represented.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:01) (72) Inventor Tadashi Suzuki 1144 Togo, Mobara-shi, Chiba Mitsui Toatsu Chemicals Co., Ltd. (72) Inventor Takeshi Nakamura Chiba 1144 Togo, Mobara City Mitsui Toatsu Chemicals Co., Ltd.

Claims (7)

[Claims] 1. A method for producing acrylamide from acrylonitrile using the cells of a microorganism that produces nitrile hydratase or a treated product thereof, comprising the steps of: 10
At the beginning of the reaction for producing acrylamide by contacting the cells of the microorganism or the treated cells with acrylonitrile in an aqueous medium after maintaining the nitrile hydratase activity even after treatment at 60 ° C. for 60 minutes. Alternatively, a method for producing acrylamide, comprising adding acrylonitrile to a reaction solution such that the acrylonitrile concentration during the reaction is equal to or higher than the acrylonitrile saturation concentration in an aqueous medium. 2. The method according to claim 1, wherein acrylonitrile is added so that the acrylamide concentration in the reaction solution after the reaction is 30% by weight or more. 3. A method for producing methacrylamide from methacrylonitrile using the cells of a microorganism producing nitrile hydratase or a treated product thereof, comprising the steps of:
After treating the cells of the microorganism in a 0% by weight aqueous solution of acrylamide at 10 ° C. for 60 minutes, the cells of the microorganism or the treated product of the microorganism which retain the nitrile hydratase activity in an aqueous medium The method is characterized in that methacrylonitrile is added to the reaction solution such that the methacrylonitrile concentration at the start of the reaction for producing methacrylamide by contact with methacrylonitrile or during the reaction is equal to or higher than the saturated concentration of acrylonitrile in the aqueous medium. A method for producing methacrylamide. 4. A method for producing crotonamide from crotonnitrile using the cells of a microorganism that produces nitrile hydratase or a treated product thereof, comprising the steps of producing the crotonamide in a 30% by weight aqueous solution of acrylamide. 10 body
Reaction of the reaction for producing crotonamide by contacting the cells of the microorganism or the treated product thereof with crotonnitrile in an aqueous medium after retaining the nitrile hydratase activity even after treatment at 60 ° C for 60 minutes. A method for producing crotonamide, comprising adding crotonnitrile to a reaction solution such that the crotonnitrile concentration at the start or during the reaction is equal to or higher than the saturated concentration of crotonnitrile in an aqueous medium. 5. A method of producing a nitrile hydratase-producing microorganism in a 30% by weight aqueous solution of acrylamide at 10 ° C.
The nitrile hydratase expressed by the microorganism which retains the nitrile hydratase activity even after the treatment for 60 minutes in the α subunit has an amino acid sequence described in SEQ ID NO: 1 in the sequence listing or a sequence listing in the α subunit. Having the amino acid sequence of SEQ ID NO: 2.
The production method according to claim 4. 6. The cells of a nitrile hydratase-producing microorganism are placed in a 30% by weight aqueous solution of acrylamide at 10 ° C.
The microorganism which retains nitrile hydratase activity even after treatment for 60 minutes with Nocardia ( Nocdia)
ardia) genus Corynebacterium (Corynebacterium) genus,
Bacillus (Bacillus), Bacillus thermophilic, Pseudomonas (Pseudomonas) genus, Micrococcus (Micrococcus)
Genus, Rhodococcus ( Rhodococcus ), Acinetobacter ( Acinetobacter ), Xanthobacter ( Xanthobacte
r ) genus, Streptomyces ( Streptomyces ) genus, Rhizobium ( Rhizobium ) genus, Klebsiella ( Klebsiella ) genus, Enterobacter ( Enterobacter ) genus, Erwinia ( Erwini
a) the genus, Earomonasu (Aeromonas) genus, Citrobacter (Ci
trobacter ) genus, Achromobacter ( Achromobacter ) genus,
The method according to any one of claims 1 to 4, wherein the microorganism is a microorganism belonging to the genus Agrobacterium or the genus Pseudonocardia . 7. The cells of a nitrile hydratase-producing microorganism are placed in a 30% by weight aqueous solution of acrylamide at 10 ° C.
Microorganisms that retain nitrile hydratase activity even after treatment for 60 minutes with Pseudonocardia thermophila JCM
30. The method according to any one of claims 1 to 4, wherein the microorganism is a genetically modified microorganism expressing a nitrile hydratase gene derived therefrom.