JP2001340091A - Method of production for amide compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of production for amide compounds by which nitrile compounds are converted with high conversion rate and a high purity and concentration of amide compound solution can readily be obtained without necessitating concentration processes. SOLUTION: On the method for successively producing amide compounds by reacting the microbial cell body of a microorganism containing nitride hydrase or the treated product of the microbial cell body with the nitrile compounds in an aqueous solvent, this method of production for amide compounds features acting the microbial cell body or the treated product of the microbial cell body on the nitrile compounds in the aqueous solvent followed by further reacting the resultant reaction solution under the condition having a plug flow basin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an amide compound, and more particularly, to a method for continuously producing a high-concentration amide compound aqueous solution by hydrating a nitrile compound at a high conversion rate. The present invention also relates to a method for producing an aqueous solution of an amide compound, which has little residual nitrile compound after the reaction and is industrially suitable.

[0002]

2. Description of the Related Art As one of the main methods for producing amide compounds, a hydration method using a nitrile compound as a raw material has been used in many cases. In particular, acrylamide has long been used as a metal copper catalyst such as Raney copper. Alternatively, in recent years, it has been known that acrylonitrile is used as a raw material by hydration catalysts such as microbial cells containing nitrile hydratase and processed cells thereof.

[0003] In the above, the product acrylamide is usually supplied in the form of an aqueous solution or crystals, and when it is used, it is generally diluted with an aqueous solvent or used after being dissolved. For this reason, especially in recent years, the supply in the form of an aqueous solution has been mostly performed.

In the supply of an aqueous acrylamide solution,
From the viewpoint of cost for transportation and storage, the product form is required to have a higher concentration. On the other hand, it is necessary to prevent precipitation of crystals during transportation or storage. For this reason, the acrylamide concentration suitable for transportation and storage at around normal temperature is usually about 40 to 50% by weight.

However, in the conventional industrial production technology, the acrylamide concentration in the reaction step is usually less than 40% by weight, depending on the production process and the type of catalyst used. The reason is that when the concentration of acrylonitrile and / or acrylamide is increased in the reaction step, there is a tendency that the catalytic activity is deactivated, the reaction is incomplete and the raw material acrylonitrile remains, and by-products increase. In addition, there are problems such as limitations due to the ability to remove the heat of reaction, and it is usually difficult to directly obtain a final product concentration of acrylamide at the end of the reaction.

Therefore, in the current industrial production process of acrylamide, a method of supplying acrylonitrile as a raw material in multiple stages (Japanese Patent Publication No. 57-1234) and a method of diluting a raw material by partially circulating a reaction solution (Japanese Patent Publication No. 58-35077). Gazette) to limit the concentration of the raw material acrylonitrile, or keep the conversion of the raw material acrylonitrile low,
By reducing the acrylamide concentration in the reaction solution to less than 40% by weight or the like, deactivation of the catalyst and an increase in by-products are suppressed. For this reason, usually, concentration for the purpose of improving the concentration of the obtained acrylamide aqueous solution and / or removing residual acrylonitrile is often performed.

However, in general, acrylamide-containing liquids are concentrated under reduced pressure. However, since acrylamide is a highly reactive polymerizable monomer, there is a high risk of polymerization during concentration. For this reason, for example, a method of stabilizing by introducing oxygen at the time of concentration, a method of coexisting nitric oxide, and a method of coexisting metal ions have been performed, but it is difficult to completely prevent polymerization. It is.

Japanese Patent Application Laid-Open No. H11-89575 discloses that
A method for producing acrylamide using microbial cells or processed cells is disclosed.In this publication, the raw material acrylonitrile, at the start of the reaction or during the reaction, the acrylonitrile concentration is equal to or higher than the acrylonitrile saturation concentration in the aqueous medium. Thus, it is described that a high concentration of acrylamide concentration can be obtained with a smaller amount of cells by adding the acrylamide as described above. Thus, it is possible to obtain an aqueous solution of acrylamide having a concentration of 40% by weight or more without concentration, but it is inconvenient depending on the relationship between the amount of catalyst used in the reaction, the reaction temperature, and the concentration of acrylonitrile at the start or during the reaction. May occur. For example, when the reaction is completed in a short time, the initial reaction heat is large, and a relatively large heat exchanger is required for the reactor, or conversely,
A long reaction time is required to achieve an acrylonitrile conversion of 99% or more.

[0009] For the above reasons, there is a long-awaited need for an industrial process for producing an amide compound in which the conversion of a nitrile compound is higher, a concentration step is not required, and a high-concentration amide compound can be obtained more efficiently. Was.

[0010]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and an object thereof is to convert a nitrile compound at a high conversion to produce a high-concentration amide compound. It is an object of the present invention to provide an industrially suitable production method capable of continuous operation, having a very small amount of residual nitrile compounds.

[0011]

Means for Solving the Problems The inventors of the present invention have made intensive studies to achieve the above object, and as a result, contacted the cells of a microorganism containing nitrile hydratase or a treated product of the microorganism with a nitrile compound in an aqueous medium. And then reacting the reaction solution under a condition having a plug flowable basin was found to be a very effective means for achieving the object, and the present invention was completed. .

That is, the present invention relates to (1) a method for continuously producing an amide compound by reacting a cell of a microorganism containing nitrile hydratase or a treated product thereof with a nitrile compound in an aqueous medium. After contacting the cells or the treated cells with a nitrile compound in an aqueous medium,
A method for producing an amide compound, characterized by further reacting the obtained reaction solution under conditions having a plug flow basin, and (2) the nitrile compound is acrylonitrile; The ratio of water and acrylonitrile at the time of contact with the treated bacterial cells is 0.4 to 1.5 parts by weight of acrylonitrile per 1 part by weight of water.
The method according to the above (1), wherein the microorganism is a transformant in which the nitrile hydratase gene cloned from the microorganism is expressed in any host. The production method according to (1) or (2).

[0013]

DETAILED DESCRIPTION OF THE INVENTION An important point of the present invention is that a microorganism cell containing nitrile hydratase or a treated product thereof is used as a catalyst for amidating a nitrile group of a nitrile compound, and in particular, the formation of by-products is suppressed. In addition, it is possible to efficiently produce an amide compound aqueous solution having a high conversion rate and a high concentration.

The nitrile hydratase referred to in the present invention is:
An enzyme capable of hydrolyzing a nitrile compound to produce a corresponding amide compound. Here, the microorganisms containing nitrile hydratase include nitrile hydratase having the ability to hydrolyze nitrile compounds to produce the corresponding amide compounds, and produce nitrile hydratase in a 30% by weight aqueous solution of acrylamide. There is no particular limitation on the microorganism as long as it retains its activity. Specifically, Nocardia genus, Corynebacterium genus, Bacillus (Bacill
us), thermophilic Bacillus, Pseudomo
nas), Rhodococcus (Rhodococcus) represented by the genus Micrococcus, rhodochrous species
cus), Acinetobacter (Acinetobacter), Xanthobacter (Xanthobacter), Streptomyces (Str
eptomyces), Rhizobium, Klebsiella, Enterobacter
Genus, Erwinia, Aeromona
s), Citrobacter, Achromobacter, Agrobacterium
Microorganisms belonging to the genus Pseudonocardia represented by the genus terium or thermophila species can be mentioned as preferred examples.

[0015] A transformant in which the nitrile hydratase gene cloned from the microorganism is expressed in any host is also included in the microorganism according to the present invention. In addition, as described in Examples described later, E. coli (Esc
herichia coli) as a typical example, but it is not particularly limited to Escherichia coli, but Bacillus subt.
ilis), and other microbial strains such as yeasts and actinomycetes. An example of such is the MT-10
822 (this strain was obtained on February 7, 1996, at 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan under the accession number FERM BP-5785 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, Japan). Has been deposited under the Budapest Treaty on the International Recognition of Deposits.) Further, by using recombinant DNA technology, one, two or more of the constituent amino acids of the enzyme are substituted, deleted, deleted or inserted with another amino acid, thereby further improving acrylamide resistance, acrylonitrile resistance, and temperature resistance. A transformant expressing the mutant nitrile hydratase is also included in the microorganism according to the present invention.

When an amide compound is produced using the above-mentioned microorganism, cells of the microorganism or a treated product of the microorganism are usually used. Bacteria are molecular biology, biotechnology,
It may be prepared using a general method known in the field of 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 .; Good), followed by separating and recovering the microorganism from the culture solution by centrifugation.

[0017] The treated product of the microorganism in the present invention is an extract or a crushed product of the above-mentioned microbial cell, and a post-isolate obtained by separating and purifying a nitrile hydratase active fraction of the extract or the crushed product. Refers to immobilized products obtained by immobilizing the microbial cells, extracts of the cells, crushed products, and post-separated products using a suitable carrier. These are immobilized as long as they have nitrile hydratase activity. This corresponds to the treated product of the present invention. These may be of a single type, or two or more different types may be used simultaneously or alternately.

In the present invention, when a microbial cell containing nitrile hydratase or a processed product of the microbial cell is used to obtain an amide compound from a nitrile compound, the reaction is carried out using two or more reactors. Then, the microbial cells or processed cells thereof, the nitrile compound and the aqueous medium are supplied to the reactor at the preceding stage. The reaction mode at this time is not particularly limited, and may be, for example, a suspension bed or a fixed bed.However, a stirrer is usually used because of the ease of heat removal of the reaction heat. A suspended bed in a tank reactor provided is more preferably used.

In the present invention, the type of the nitrile compound is not particularly limited. Specifically, the nitrile compound is a nitrile compound having about 2 to 20 carbon atoms, and has a wide range of nitriles such as aliphatic nitrile and aromatic nitrile. And nitrile. Aliphatic nitriles have 2 to 6 carbon atoms
Saturated or unsaturated nitriles such as acetonitrile, propionitrile, butyronitrile, isobutyronitrile, valernitrile, isovaleronitrile, and capronitrile; aliphatic saturated mononitrile; aliphatic such as malononitrile, succinonitrile, adiponitrile; Saturated dinitrile; aliphatic unsaturated nitriles such as acrylonitrile, methacrylonitrile, and crotonnitrile; Aromatic nitriles include benzonitrile, o-, m-, and p-chlorobenzonitrile, o-, m-, and p-fluorobenzonitrile,
o-, m-, and p-nitrobenzonitrile, o-,
m-, and p-tolunitrile, benzyl cyanide and the like. Among them, acrylonitrile, methacrylonitrile, crotonnitrile and the like are mentioned as preferable examples.

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

In the present invention, the concentration of the nitrile compound to be supplied to the reactor at the preceding stage is a concentration which is higher than the saturation concentration of the nitrile compound at the start of the reaction. The upper limit of the concentration is not particularly limited, but the supply of the nitrile compound in an excessively large amount may cause a large amount of catalyst and an excessive volume in a reactor to complete the reaction, and an excessive amount for removing heat. Heat exchangers and the like are required, and the economic burden on the equipment increases. For this reason, the supply concentration of the nitrile compound is more specifically set so that the theoretical concentration of the product solution when it is converted into the corresponding amide compound is 40 to 80% by weight in the case of acrylamide. Specifically, acrylonitrile 0.4 to 1. 1 part by weight of water is used.
Preferably, it is supplied as 5 parts by weight. In the case of methacrylamide, the theoretical product concentration is in the range of 10 to 40% by weight, more specifically, 0.08 to 0.5 part by weight of methacrylonitrile per 1 part by weight of water. It is preferred to supply the nitrile compound and water.

Next, in the present invention, the reaction solution taken out from the reactor at the preceding stage is further reacted under conditions having a plug flow area. More specifically, the reaction solution obtained in the former reactor is sent to the latter reactor having a plug flow area. Here, the reactor having a plug-flow basin is a reactor which is generally called a tubular reactor or a tubular reactor, and a reaction solution in a pipe having a pipe shape. The reaction proceeds while moving the piston flow.
In order to remove the heat of reaction, double tube type and shell &
A tube type or the like can be used.

However, in the present invention, the reactor having the plug-flow basin is not limited to the above-mentioned type, and a reactor having a type in which a short path of the reaction liquid hardly occurs even in other types of reactors. If so, you can use it enough. That is, if the reaction solution can have a plug flow basin depending on conditions such as flow rate, and if the reaction solution has a structure capable of removing heat of reaction,
It can be used as a reactor having a plug flow basin. For example, various types such as a spiral type or a plate type in a heat exchanger or a tower type reactor can be used. Furthermore, baffles (baffles) for bringing the flow state of the reaction solution into these inside evenly
In addition, even if it has a packing, it can be used as a reactor having a plug flow basin if it has a plug flow basin depending on conditions such as flow rate. It is.

It is to be noted that each of the above-described first-stage reactor and the second-stage reactor having a plug-flow-type basin is not limited to one each, and each may be used alone or in series. Alternatively, the data may be arranged in parallel. In addition, the reaction time (residence time) in the above-mentioned first and second-stage reactions is not fixed depending on the conditions such as the amount of catalyst used and the temperature.
5 to 40 hours, preferably 1 to
The range is 20 hours. Also, of the total reaction time, the reaction time of the former reactor is 20 to 99 of the total reaction time.
%, Preferably 40 to 90%, and the reaction time of the latter half of the reactor is 1 to 80% of the total reaction time, preferably 1 to 80%.
0 to 60%.

The amount of the catalyst used varies depending on the reaction conditions, the type of the catalyst, and the form thereof.
00 ppm by weight, preferably 50 to 30,000 ppm by weight.

The amidation reaction is usually carried out at or near normal pressure, but can also be carried out under pressure 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 usually preferably in the range of 0 to 50 ° C, more preferably 10 to 40 ° C. Further, the reaction can be performed even in a slurry state in which the product is crystallized in the reaction solution. The pH of the reaction solution during the amidation reaction is not particularly limited as long as the nitrile hydratase activity is maintained, but is preferably in the range of pH 6 to 10, more preferably in the range of pH 7 to 9. Range.

In this example, amino acid substitutions retaining nitrile hydratase activity are obtained by site-specific mutation. However, even if a recombinant plasmid is constructed by a method other than site-specific mutation based on the types of bases to be replaced with the mutation points disclosed in the examples and then introduced into host cells, Similar results can be obtained.

For example, a DNA fragment having a nucleotide sequence such that the nucleotide sequence of the DNA corresponding to the mutation point disclosed in the Examples is a sequence after amino acid substitution is synthesized by a DNA synthesizer or the like, and the obtained fragment is obtained. By substituting the region corresponding to the fragment of pPT-DB1 which has been separately separated from the above, the desired recombinant plasmid can be obtained.

[0029]

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 the following, the HPLC analysis of the reaction solution was performed using ULTRON 80HG as an HPLC column.
(50 × 8φmm) using 10 mM phosphoric acid aqueous solution as a developing solution, and acrylamide and acrylonitrile were detected by absorbance at 220 nm.
It is a measurement of the concentration.

Example 1 (1) Acquisition of an Amino Acid Substitute Retaining Nitrile Hydratase Activity In order to replace the sixth Leu of the α subunit with Met, the pPT-DB obtained in Japanese Patent Application Laid-Open No. 9-275978 was used.
1 "LA" manufactured by Takara Shuzo Co., Ltd.
PCR in vitro mutagenesis K
site-directed mutagenesis using “it”. Since then
"LA PCR in vitro mutagenesis
s Kit "is simply referred to as a kit. In the following example,
Basically, the principle and operation method of the kit were followed.

10 ml of LB liquid medium was prepared in a 30 ml test tube, and sterilized in an autoclave at 121 ° C. for 20 minutes. After adding ampicillin to this medium to a final concentration of 100 μg / ml, MT-10822
Inoculate one platinum loop of the strain and incubate at 37 ° C and 300 rpm for about 20 minutes.
Cultured for hours. After 1 ml of the culture solution was collected in a suitable centrifuge tube, the cells were separated by centrifugation (15000 rpm × 5 minutes). Subsequently, plasmid DNA of pPT-DB1 was prepared from the cells by alkaline SDS extraction.

1 μg of plasmid DNA of pPT-DB1
Was used as a template to perform two types of PCR reactions. PCR reaction No. 1 is a heat-denaturing system (50 μl in total, containing 50 pmol each of the primer described in SEQ ID NO: 1 of the sequence listing and the M13 primer M4 (sequence described in SEQ ID NO: 2 in the sequence listing)). (98 ° C) 15
Second, annealing (55 ° C.) for 30 seconds, extension reaction (72
C.) for 120 seconds was repeated 25 times. PCR reaction No. No. 2 corresponds to MUT4 primer (sequence described in SEQ ID NO: 3 in the sequence listing) and M13 primer RV (sequence described in SEQ ID NO: 4 in the sequence listing) for 5
In a system with a total volume of 50 μl containing 0 pmol (composition is based on the conditions described in the kit), PCR The same operation as in Example 1 was performed. PCR reaction No. 1 and No. When the DNA amplification product was analyzed by agarose electrophoresis (agarose concentration: 1.0% by weight) using 5 μl of each of the reaction completion liquids of 2, the presence of the amplified DNA product was confirmed. Mi
Using crocon100 (manufactured by Takara Shuzo Co., Ltd.), primers and dNTPs in excess of each PCR reaction end solution
After removal of TE, TE (trishydroxymethylaminomethane / EDTA buffer) was added to prepare 50 μl of each solution. A total volume of 4 containing 0.5 μl each of the TE solution
After preparing 7.5 μl of an annealing solution (composition according to the conditions described in the kit), performing a heat denaturation treatment (98 ° C.) for 10 minutes, cooling to 37 ° C. at a constant rate over 60 minutes, Annealing treatment was performed by holding at 37 ° C. for 15 minutes. T for the annealing solution
Add 0.5 μl of AKARALA Taq and add 3 at 72 ° C.
Heat treatment was performed for a minute to complete a heteroduplex. To this, M13 primer M4 (sequence is described in SEQ ID NO: 2 in the sequence listing) and M13 primer RV (SEQ ID NO: 4 in the sequence listing)
50 pmol), and the total amount is 50 μm.
After that, the conditions of heat denaturation (98 ° C.) for 15 seconds, annealing (55 ° C.) for 30 seconds, and extension reaction (72 ° C.) for 120 seconds are repeated for 25 cycles. Three
Was done. PCR reaction No. The DNA amplification product was analyzed by agarose electrophoresis (using Sigma type VII low-melting point agarose; agarose concentration of 0.8% by weight) using 5 μl of the reaction-finished solution of Example 3. As a result, about 2.0 kb was obtained.
The presence of the amplified DNA product of p was confirmed. Subsequently, only a DNA fragment of about 2.0 Kbp was cut out from the agarose gel, and the agarose piece (about 0.1 g) was pulverized into fine pieces.
After suspending in 55 ml of TE solution, the solution was kept at 55 ° C. for 1 hour to completely melt the agarose. This melt was subjected to phenol / chloroform extraction and ethanol precipitation according to a conventional method to purify the DNA fragment, and finally 10 μl of T
E. After the purified amplified DNA fragment of about 2.0 kbp was digested with restriction enzymes EcoRI and HindIII, this restriction enzyme-treated solution was subjected to phenol / chloroform extraction and ethanol precipitation to purify the DNA fragment. In TE. Similarly, pP
EcoRI, the only restriction enzyme site on T-DB1
Then, pPT-DB1 was digested with HindIII and subjected to agarose gel electrophoresis (using Sigma type VII low melting point agarose; agarose concentration of 0.7%) to cut out only a DNA fragment of about 2.7 Kbp from the agarose gel. The cut agarose pieces (about 0.1 g) were finely pulverized, suspended in 1 ml of a TE solution, and kept at 55 ° C. for 1 hour to completely melt the agarose. The melt was subjected to phenol / chloroform extraction and ethanol precipitation to purify the DNA fragment, and finally 10 μl of T
E. The thus obtained amplified DNA product and the pPT-DB1 fragment were ligated using a DNA ligation kit (Takara Shuzo) and transformed into competent cells of Escherichia coli HB101 (Toyobo Co., Ltd.). A bank was prepared.

In a 30 ml test tube, 40 μg / ml ferric sulfate heptahydrate and 10 μg / ml cobalt chloride
A 10 ml LB liquid medium containing dihydrate (hereinafter referred to as an activity expression medium) was prepared and sterilized by an autoclave at 121 ° C. for 20 minutes. A final concentration of 100μ
After adding ampicillin to a concentration of g / ml, 5 clones arbitrarily selected from the Escherichia coli bank were inoculated with one platinum loop and cultured at 37 ° C. and 300 rpm for about 20 hours. After dispensing 1 ml of the culture termination solution into an appropriate centrifuge tube, centrifugation (15000 rpm × 5 minutes)
Was used to separate the cells. The cells were suspended in 200 μl of potassium phosphate buffer (pH 7.0), and 1% by weight of acrylonitrile was added thereto, followed by a reaction at 10 ° C. for 2 minutes. The reaction was stopped by adding an equal volume of a 1 M phosphoric acid aqueous solution to the reaction solution, and the concentration of the formed acrylamide was measured by the same HPLC analysis as in Example 2. As a result, generation of acrylamide was detected in 4 out of 5 clones, and it was confirmed that the clone retained nitrile hydratase activity.

The cells of each of the four clones were separated from the remaining 1 ml of the culture solution used for the measurement of nitrile hydratase activity, and the plasmid DNA of each clone was prepared by alkaline SDS extraction. Subsequently, a sequencing kit manufactured by ABI and an auto sequencer 373A were used.
Was used to determine the nucleotide sequence of the nitrile hydratase structural gene of each clone. As a result, clone No. 1 shown in Table 1 (Table 1) was obtained.
In 1 the 6 of the α subunit of nitrile hydratase
The th Leu was replaced by Met.

[0035]

[Table 1]

Subsequently, in order to replace the 126th Phe of the α subunit with Tyr, clone no. Using the plasmid DNA of No. 1 as a template, site-specific mutagenesis was performed by the same operation as described above. That is, 30 ml
Prepare 10 ml of LB liquid medium in a test tube at 121 ° C.
-Sterilized by autoclave for 20 minutes. Ampicillin was added to this medium to a final concentration of 100 μg / ml. One strain was inoculated with one platinum loop and cultured at 37 ° C. and 300 rpm for about 20 hours. After 1 ml of the culture solution was collected in an appropriate centrifuge tube, the cells were separated by centrifugation (15000 rpm × 5 minutes). Subsequently, clone No. 1 was obtained from the cells by alkaline SDS extraction. One strain of plasmid DNA was prepared.

This clone No. One strain of plasmid DN
Two types of PCR reactions were performed using A1 μg as a template. P
CR reaction no. No. 4 is a 50 μl total system containing 50 pmol of each of the primer described in SEQ ID NO: 5 in the sequence listing and the M13 primer M4 (sequence described in SEQ ID NO: 2 in the sequence listing) (composition depends on the conditions described in the kit). (98
C.) for 15 seconds, annealing (55 ° C.) for 30 seconds, and extension reaction (72 ° C.) for 120 seconds by repeating 25 cycles. PCR reaction No. 5 is a 50 μl total system containing 50 pmol each of MUT4 primer (sequence described in SEQ ID NO: 3 in the sequence listing) and M13 primer RV (sequence described in SEQ ID NO: 4 in the sequence listing) (composition depends on the conditions described in the kit) ), PCR reaction No. The same operation as in Example 4 was performed. PCR reaction No. 4 and no. Analysis of the DNA amplification product by agarose electrophoresis (agarose concentration: 1.0% by weight) using 5 μl of each of the reaction completion solutions of No. 5 confirmed the presence of the amplified DNA product.
Thereafter, clone No. An E. coli bank was prepared by exactly the same operation as in the case of 1.

Five clones arbitrarily selected from the E. coli bank were clone No. The same activity expression medium 10 as in 1
One ml of each platinum loop was inoculated into each ml and cultured at 37 ° C. and 300 rpm for about 20 hours. After dispensing 1 ml of the culture termination solution into an appropriate centrifuge tube, nitrile hydratase activity was measured. As a result, generation of acrylamide was detected in 4 out of 5 clones, and it was confirmed that the clone retained nitrile hydratase activity.

The cells of each of the four clones were separated from the remaining 1 ml of the culture solution used for the measurement of nitrile hydratase activity, and plasmid DNA of each clone was prepared by alkaline SDS extraction. Subsequently, clone No.
The nucleotide sequence of the nitrile hydratase structural gene of each clone was determined by the same operation as in the case of 1. as a result,
Clone No. shown in Table 2 (Table 2) In Example 2, the sixth Leu of the α subunit of nitrile hydratase is M
et is the 126th Phe of the α subunit as Tyr
Has been replaced respectively.

[0040]

[Table 2]

Subsequently, the 212th S in the β subunit
In order to replace Tyr with Tyr, clone No. Using the plasmid DNA of No. 2 as a template, site-specific mutagenesis was performed by the same operation as described above. That is, 10 ml of LB liquid medium was prepared in a 30 ml test tube,
Sterilized by autoclaving for 20 minutes. Ampicillin was added to this medium to a final concentration of 100 μg / ml. Two strains were inoculated with one platinum loop and cultured at 37 ° C. and 300 rpm for about 20 hours.
After 1 ml of the culture solution was collected in an appropriate centrifuge tube, the cells were separated by centrifugation (15000 rpm × 5 minutes). Subsequently, clone No. 1 was obtained from the cells by alkaline SDS extraction. One strain of plasmid DNA was prepared.

This clone No. Plasmid DNA of 2
Two types of PCR reactions were performed using 1 μg as a template. PC
R reaction no. No. 6 is a 50 μl total system (composition is based on the conditions described in the kit) containing 50 pmol of each of the primer described in SEQ ID NO: 6 in the sequence listing and the M13 primer M4 (sequence described in SEQ ID NO: 2 in the sequence listing). (98
C.) for 15 seconds, annealing (55 ° C.) for 30 seconds, and extension reaction (72 ° C.) for 120 seconds by repeating 25 cycles. PCR reaction No. 7 is a 50 μl total system containing 50 pmol each of the MUT4 primer (sequence described in SEQ ID NO: 3 in the sequence listing) and the M13 primer RV (sequence described in SEQ ID NO: 4 in the sequence listing) (composition depends on the conditions described in the kit) ), PCR reaction No. The same operation as in Example 6 was performed. PCR reaction No. 6 and no. The DNA amplification product was analyzed by agarose electrophoresis (agarose concentration: 1.0% by weight) using 5 μl each of the reaction completed solution of No. 7, and the presence of the amplified DNA product was confirmed.
Thereafter, clone No. An E. coli bank was prepared by exactly the same operation as in the case of 1.

Five clones arbitrarily selected from the E. coli bank were clone No. The same activity expression medium 10 as in 1
One ml of each platinum loop was inoculated into each ml and cultured at 37 ° C. and 300 rpm for about 20 hours. After dispensing 1 ml of the culture termination solution into an appropriate centrifuge tube, nitrile hydratase activity was measured. As a result, generation of acrylamide was detected in 4 out of 5 clones, and it was confirmed that the clone retained nitrile hydratase activity.

The cells of each of the four clones were separated from the remaining 1 ml of the culture solution used for the measurement of nitrile hydratase activity, and plasmid DNA of each clone was prepared by alkaline SDS extraction. Subsequently, clone No.
The nucleotide sequence of the nitrile hydratase structural gene of each clone was determined by the same operation as in the case of 1. as a result,
Clone No. shown in Table 3 (Table 3) 3 Ser at position 212 of the β subunit of nitrile hydratase
Was replaced with Tyr.

[0045]

[Table 3]

This clone NO. 3 cells were cultured to obtain the cells required for the reaction. Hereinafter, typical culture examples will be described. 5
100 ml of a medium having the following composition was prepared in a 00 ml baffled conical flask, and sterilized by an autoclave at 121 ° C. for 20 minutes. This medium has a final concentration of 50 μg / m
1 after adding ampicillin to the above clone NO. Inoculate one platinum loop of the cells of No. 3 at 37 ° C and 130r
The cells were cultured at pm for 20 hours. Centrifugation (15000G ×
15 minutes) to separate only the cells from the culture solution, and then resuspended in 50 ml of physiological saline.
Centrifugation was performed 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

(2) Synthesis reaction of acrylamide by hydration of acrylonitrile 2 parts by weight of wet bacterial cells obtained by the above-mentioned culturing method was added to 0.3 parts by weight.
The suspension was suspended in 98 parts by weight of an aqueous solution of mM-NaOH, and the suspension and acrylonitrile were added at 50 g / h and 30 g / h, respectively.
As a first reactor, a 1 L glass flask charged with 400 g of water in advance is continuously fed while stirring.
The reaction solution was continuously withdrawn at 80 g / h so as to keep the liquid level constant. The solution was used as a second reactor and had an inner diameter of 5
mm of Teflon (registered trademark) tube 20 m. The reaction temperature is about 10 to 20 ° C.
Immersing the first reactor and the second reactor in a water bath of
The internal liquid temperature was controlled to be 15 ° C. The reaction solution was analyzed at the outlet of the second reactor by HPLC analysis 200 hours after the start of the reaction. As a result, only acrylamide was present in the reaction solution (concentration = 50% by weight), and acrylonitrile was detected at the detection limit. (100 ppm by weight).

Comparative Example 1 The reaction time was the same as in the above example, and the reactor was
The test was performed only with a stirring tank serving as a reactor. That is, the operation was performed in the same manner as in Example except that the reactor was changed to a 2 L glass flask, and the internal liquid amount was changed to 800 g. 200 hours after the start of the reaction, by HPLC analysis,
As a result of analyzing the reaction solution at the reactor outlet, acrylamide in the reaction solution was 48% by weight, and acrylonitrile was 1.9.
% By weight was detected. Thus, in this comparative example, unreacted acrylonitrile remained, and it was confirmed that the reaction was not completed.

COMPARATIVE EXAMPLE 2 As a second reactor, the reaction solution at the outlet of the first reactor was continuously fed to a 1-L glass flask previously charged with 400 g of water while stirring as in the first reactor. The operation was performed in the same manner as in Example 1 except that the reaction solution was continuously extracted so as to keep the liquid level constant.
200 hours after the start of the reaction, the second
As a result of analyzing the reaction solution at the outlet of the reactor, acrylamide was found to be 49.5% in the reaction solution, and acrylonitrile was detected at 3000 ppm by weight. Also in this comparative example, unreacted acrylonitrile remained, and it was confirmed that the reaction was not completed.

[0051]

According to the present invention, a nitrile compound can be hydrated at a high conversion to produce an aqueous solution of a high concentration amide compound continuously, and no residual nitrile compound is observed. And the method of the present invention can be suitably used as an industrial method for producing an amide compound.

[0052]

[Sequence List] SEQUENCE LISTING <110> Mitsui Chemicals, Inc <120> Process for Preparing Amide Compound <130> 50010029 <140> <141> <150> JP 2000-091203 <151> 2000-03-29 <160> 6 <170> PatentIn Ver. 2.1 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide for PCR primer <400> 1 aacatcatgc gcaagtcg 18 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide for PCR primer <400> 2 caggaaacag ctatgac 17 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide for PCR primer <400> 3 ggccagtgcc tagcttacat 20 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide for PCR primer <400> 4 gttttcccag tcacgac 17 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide fir PCR primer <400> 5 aactggtacaaggagccg 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide for PCR primer <400> 6 ccgaactaca gcgtctac 18

 ──────────────────────────────────────────────────の Continued on the front page (72) Inventor Kenki Sasaki 1900 Togo, Mobara-shi, Chiba Mitsui Chemicals Co., Ltd. (72) Inventor Seiichi Watanabe 1900 Togo, Togo, Mobara-shi, Chiba Mitsui Chemicals Co., Ltd. (72) Invention Toshihisa Tachibana 1900 Togo, Mobara-shi, Chiba Mitsui Chemicals Co., Ltd. (72) Inventor Tamotsu Asano 1900, Togo, Togo, Mobara-shi, Chiba Mitsui Chemicals Co., Ltd.F-term (reference) 4B024 AA03 BA07 CA04 DA06 EA04 GA11 HA01 4B064 AE02 CA02 CA19 CA21 CC24 DA16

Claims (3)

[Claims] 1. A method for continuously producing an amide compound by reacting a cell of a microorganism containing nitrile hydratase or a treated product of the cell with an nitrile compound in an aqueous medium, the method comprising continuously producing the cell or a cell thereof. A method for producing an amide compound, which comprises contacting a body-treated product with a nitrile compound in an aqueous medium, and further reacting the obtained reaction solution under conditions having a plug-flow basin. 2. The nitrile compound is acrylonitrile, and the ratio of water and acrylonitrile is 0.4 to 1.5 parts by weight of acrylonitrile to 1 part by weight of water when the compound is brought into contact with a microorganism cell or a treated product thereof. The production method according to claim 1, which is a part. 3. The method according to claim 1, wherein the cells of the microorganism are transformants in which the nitrile hydratase gene cloned from the microorganism is expressed in any host.