JP2001270857A - Method for purifying amide compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently removing impurities included in an amide compound-containing liquid, especially the amide compound- containing liquid produced by a hydration reaction of a nitrile compound using microorganismic cells containing nitrile hydratase or a treated product of the microorgansmic cells. SOLUTION: An amide compound is purified by bringing a liquid containing the amide compound into contact with active carbon in an acidic condition.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying an amide compound. More specifically, the present invention relates to a method for efficiently obtaining a high-purity amide compound by treating a liquid containing the amide compound with activated carbon.

[0002]

2. Description of the Related Art Amide compounds, especially amide compound production liquids obtained by hydrating nitrile compounds, although the type varies depending on the production method, are usually high molecular substances, surfactants, coloring matters, eluates, or There are other impurities and the like.
In order to remove these, for example, see JP-A-61-1154.
No. 95 and JP-A-61-122253 disclose a purification treatment method using activated carbon.
No. 8 discloses a purification treatment method using an ion exchange membrane, and Japanese Patent Application Laid-Open No. 61-122227 describes a purification treatment method using a porous hollow fiber membrane.

[0003] However, in the purification method using the ion exchange membrane or the porous hollow fiber membrane, a special purification device is required. In addition, in the purification method using activated carbon, although special equipment is not usually required, impurities are still present in the obtained product, and the effect is still insufficient. In particular, when an amide compound is produced by directly hydrating a nitrile compound using nitrile hydratase, which is an enzyme having nitrile hydration ability, the purification method using activated carbon of the prior art described above requires microorganisms contaminated in the reaction solution. Insufficient removal of proteins derived from the like, as a result, if the amount is very small, the reaction solution tends to foam, and if it is too large, the reaction solution becomes cloudy, adversely affecting product quality .

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for efficiently removing impurities contained in an amide compound-containing solution. More specifically,
An object of the present invention is to provide a simple and efficient purification method when an amide compound-containing liquid obtained when a corresponding amide compound is produced from a nitrile compound is treated with activated carbon.

[0005]

Means for Solving the Problems The present inventors have intensively studied a method for purifying the above-mentioned amide compound-containing liquid using activated carbon. As a result, the amide compound-containing liquid is brought into contact with activated carbon under acidic conditions. Thereby, furthermore, by contacting with activated carbon in a specific pH region, impurities contained in the amide compound-containing liquid, particularly proteins,
It has been found that it can be removed very efficiently.

In particular, according to conventional knowledge, compounds having an unsaturated bond in an amide compound (for example, acrylamide, methacrylamide, etc., which are relatively important compounds in industry) easily undergo a polymerization reaction in an acidic region, and the compound is unstable. In order to avoid this, it has been important to keep the solution containing the amide compound neutral. From such a viewpoint, the purification treatment technique under the above-mentioned conditions has never been expected from the prior art.

That is, the present invention provides a method for purifying an amide compound, which comprises contacting (1) an amide compound-containing liquid with activated carbon under acidic conditions, and (2) an amide compound-containing liquid. The purification method according to (1), which is a product liquid obtained by a hydration reaction of a nitrile compound, and (3) the purification method according to (1) or (2), wherein the amide compound has 2 to 20 carbon atoms. And (4) the amide compound has an unsaturated bond. (5) The amide compound is acrylamide or methacrylic. The purification method according to (4), wherein the amide compound is an amide, and the (6) amide compound is synthesized using a microorganism cell containing nitrile hydratase or a processed product of the microorganism cell. That, (1) a purification method according to any one of - (5), and (7) microbial cells is,
It is a transformant according to (6), which is a transformant expressing a nitrile hydratase gene cloned from a microorganism in an arbitrary host, and (8) containing an amide compound upon contact with activated carbon. PH of the liquid is 3.5 to
The purification method according to any one of (1) to (7), which is 6.5, and (9) using an organic acid having an acid dissociation index of 3.5 to 5.5, or using the organic acid and a base. (1) to (8), wherein the amide compound-containing liquid is adjusted to be acidic.
And (10) the purification method according to (9), wherein the organic acid is acrylic acid or methacrylic acid. Or (1) the purification method according to any one of (1) to (10), wherein the activated carbon is a raw material made from coconut shell, and (12) the temperature at the time of contact with activated carbon is 10 to 50 ° C. The method according to any one of (1) to (11), wherein (13) after contacting the amide compound-containing liquid with activated carbon, the activated carbon is separated, and the remaining liquid is reduced to a saturation temperature or lower. Characterized by the precipitation of
A purification method according to any one of (1) to (12).

[0008]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The amide compound-containing liquid in the present invention is not particularly limited. Specifically, the amide compound-containing product liquid obtained by hydrating a nitrile compound having about 2 to 20 carbon atoms is obtained. There are some. The nitrile compound subjected to the hydration reaction includes a wide range of nitriles, such as aliphatic nitriles and aromatic nitriles. Examples of the aliphatic nitrile include saturated or unsaturated nitriles having 2 to 6 carbon atoms, for example, acetonitrile, propionitrile, butyronitrile, isobutyronitrile, valernitrile, aliphatic saturated mononitrile such as isovaleronitrile, capronitrile; Examples include aliphatic saturated dinitrile such as malononitrile, succinonitrile and adiponitrile; and aliphatic unsaturated nitrile such as acrylonitrile, methacrylonitrile and crotononitrile. Aromatic nitriles include benzonitrile, o-, m-, and p-chlorobenzonitrile,
o-, m-, and p-fluorobenzonitrile, o
-, M-, and p-nitrobenzonitrile, o-, m
-, And an aqueous solution containing acrylamide or methacrylamide obtained by hydrating a nitrile compound having an unsaturated bond, such as acrylonitrile or methacrylonitrile, such as p-tolunitrile or benzyl cyanide. On the other hand, the purification method of the present invention is preferable.

In the present invention, the amide compound-containing liquid to be treated may be obtained by any known hydration method. That is, a hydration method using sulfuric acid, a catalytic hydration method using a catalyst containing metallic copper such as a Raney copper catalyst, an enzyme capable of hydrating a nitrile compound (nitrile hydratase), and a microbial cell containing the same, And any of those hydrated using the above-mentioned enzyme and processed microorganisms.

Among them, nitrile hydratase, an enzyme having the ability to hydrate a nitrile compound, a treated product of nitrile hydratase, a microbial cell containing nitrile hydratase, or a processed product of the microbial cell is used. The amide compound-containing liquid obtained by the above method is suitable for the purification method of the present invention. The above-mentioned nitrile hydratase refers to an enzyme capable of hydrolyzing a nitrile compound to produce a corresponding amide compound.

The microorganisms containing nitrile hydratase include nitrile hydratase having the ability to hydrolyze nitrile compounds to produce the corresponding amide compounds, and are prepared in a 30% by weight aqueous solution of acrylamide. The microorganism is not particularly limited as long as it is a microorganism that retains the activity of hydratase. Specifically, Nocardia genus, Corynebacterium (C
orynebacterium), Bacillus, thermophilic Bacillus, Pseudomonas, Micrococcus, rhodochrous
Rhodococcus (Rhodococcus) genus, Acinetobacter (Acinetobacter) genus, Xanthobacter (Xa
nthobacter), Streptomyces
Genus, Rhizobium, Klebsiel
la), Enterobacter, Erwinia, Aeromonas, Citrobacter, Achromob
Suitable examples include microorganisms belonging to the genus Pseudonocardia represented by the genus Acter), the genus Agrobacterium or the genus Thermophila.

Further, 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 not particularly limited to Escherichia coli, and 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 amide compound resistance, nitrile compound resistance, and temperature resistance. A transformant expressing the improved 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.

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. Product, an extract of the microbial cell or the microbial cell, a ground product, an immobilized product obtained by immobilizing the separated product using a suitable carrier, and the like, as long as they have nitrile hydratase activity. This corresponds to the treated product of the present invention.

In the amide compound to be purified according to the present invention, a microorganism cell containing nitrile hydratase,
Alternatively, when a nitrile compound is hydrated to obtain an amide compound by using a treated product of the microbial cell, a reaction method in which the corresponding nitrile compound is subjected to a batch reaction or a continuous reaction may be used. The type of the reaction is not particularly limited, and may be, for example, a suspension bed or a fixed bed. At this time, the concentration of the catalyst, for example, the cells of the microorganisms or the treated cells of the microorganisms in the reaction solution is not particularly limited as long as the mixture of the aqueous medium and the nitrile compound is not hindered.

In the above, when the nitrile compound is added at the start of the reaction, the concentration of the nitrile compound may be at least the saturation concentration of the nitrile compound. On the other hand, the upper limit of the concentration is not particularly limited, and may be arbitrarily determined according to the assumed amide compound concentration and nitrile compound concentration at the end of the reaction.

Unreacted nitrile compounds can 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, for example, when the nitrile compound is acrylonitrile, the saturation concentration of the present compound in water is about 7% by weight at 20 ° C., and therefore, about 7% by weight or more is preferable. When methacrylonitrile or crotonnitrile is a nitrile compound, the saturation concentration of these compounds in water is 20 ° C.
Is about 2% by weight, so about 2% by weight or more is preferable.

The above amidation reaction is usually 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. When the catalyst contains nitrile hydratase, the reaction is preferably performed at 0 to 50 ° C, more preferably at 10 to 30 ° C. .

The pH of the reaction solution during the above amidation reaction is not particularly limited as long as the nitrile hydratase activity is maintained.
It is in the range of H6-10, more preferably in the range of pH7-9.

In the purification of the amide compound in the present invention, the solution containing the amide compound is prepared under acidic conditions, preferably at pH 2
As mentioned above, more preferably, it is carried out by contacting with activated carbon in the range of pH 3.5 to 6.5.

In the present invention, it is usually necessary to add an acid to the amide compound-containing solution in order to make the amide compound-containing solution acidic, and the type of the acid may affect the stability of the amide compound. Otherwise, a wide range can be used, for example, a mineral acid such as sulfuric acid or nitric acid, or an organic acid such as acetic acid or acrylic acid, and two or more kinds may be used. Among them, in the present invention, the pH adjustment is easier and more stable, and the use of a weak acid is effective in obtaining a higher purification effect and the stability of the amide compound. From the viewpoint of imparting, it is very preferable to adjust the pH to the above range using both a weak acid and a base.

The weak acid preferably used has an acid dissociation index (pKa; 25 ° C., in water) of 2.0 to 6.0, preferably 3.5, in consideration of the pH control range.
It is more preferable that it is 5.5. Representative of these acids are acetic acid, propionic acid, octanoic acid, aliphatic saturated monocarboxylic acids such as valeric acid, acrylic acid, crotonic acid, aliphatic unsaturated monocarboxylic acids such as methacrylic acid,
Examples thereof include aliphatic polycarboxylic acids such as oxalic acid, adipic acid, succinic acid, and maleic acid, and aromatic carboxylic acids such as benzoic acid. The base is preferably a strong base, such as sodium hydroxide or potassium hydroxide.

In the present invention, when the amide compound to be treated particularly has an unsaturated bond, when the amide compound is subjected to a polymerization reaction after purification, the amide compound in the obtained polymer is However, there is a disadvantage that the acid remains or is released. Therefore, when an amide compound having an unsaturated bond as described above is to be purified, an acid having an unsaturated bond and capable of copolymerizing with the amide compound, specifically Specifically, it is preferable to use acrylic acid, methacrylic acid, crotonic acid, or the like.

In the present invention, the concentration of the above-mentioned acid or the above-mentioned acid and base depends on the properties of the amide compound-containing solution and the pKa of the acid used. It is in the range of 5% by weight.

In the present invention, the amide compound-containing liquid is brought into contact with activated carbon under the above-mentioned acidic conditions. When the amide compound-containing liquid is in contact with activated carbon, the amide compound-containing liquid is made acidic or acidic. If there is no particular limitation,
A method in which the amide compound-containing liquid is prepared under acidic conditions simultaneously with the addition of the activated carbon may be used.

The activated carbon used in the present invention is not particularly limited, and any of powdered and granular activated carbon can be used. Further, in an apparatus for performing a purification treatment,
What is necessary is just to use the thing suitable for the particle size of the activated carbon to be used. For example, when powdered activated carbon is used, it can be carried out in either a batch type or a continuous type in a tank capable of stirring the liquid. In the case where granular activated carbon is used, a continuous treatment in a packed column format is also possible in addition to the above-mentioned format.

Activated carbon generally includes those using coal, wood, coconut shell and the like as a raw material, but there is no particular limitation as long as it has an adsorbing ability. It can be used even if there is. However, if the amide compound to be treated has a particularly unsaturated bond, the activated carbon having a low metal content should be used in consideration of the storage stability and easiness of polymerization of the amide compound. Preferably, the raw material is wood or coconut shell.

In the present invention, if the amount of activated carbon used for purifying the amide compound is too small, it is difficult to obtain a sufficient purification effect, and if it is used too much, it becomes uneconomical. The amount used is usually 0.01 to 20% by weight based on the amide compound-containing liquid.
, More preferably in the range of 0.05 to 10% by weight. When a powdery activated carbon is used as the activated carbon, the activated carbon may be directly added to the amide compound-containing liquid as it is, or once the activated carbon is dispersed in a medium such as water to form a slurry. It may be added to or supplied to the amide compound-containing liquid.

In the present invention, the temperature for purifying the amide compound-containing solution with activated carbon is not particularly limited as long as the amide compound crystals do not precipitate and do not affect the stability. Usually, it is performed in the range of 0 to 80 ° C. In particular, when purifying a solution containing an amide compound having an unsaturated bond, such as a solution containing acrylamide or methacrylamide, the temperature is preferably 60 ° C. or less, and more preferably 10 to 50 ° C., in order to prevent gelation due to polymerization reaction. It is preferable to contact with activated carbon. The time required for the contact treatment with activated carbon depends on the treatment type and the amount of activated carbon, but is usually in the range of 0.5 to 20 hours.

Next, in the present invention, activated carbon is separated from the contact-treated amide compound-containing liquid to obtain a purified liquid of the amide compound-containing liquid. As a method of separating activated carbon,
There is no particular limitation as long as it is a method using a generally used solid-liquid separation device, and examples thereof include a pressure filter, a reduced pressure filter, and a centrifugal separator, and further, any of a batch type and a continuous type I do not care. Further, in the present invention, the amide compound-containing liquid after separating the activated carbon is cooled,
By adopting a method of crystallizing the target amide compound from the liquid, it is possible to obtain a further purified amide compound.

In this example, the amino acid substitution having the nitrile hydratase activity is obtained by site-specific mutation. However, based on the type of base to be replaced with the mutation point disclosed in the examples, to construct a recombinant plasmid by a method other than site-specific mutation,
Even if it is introduced into a host cell, it is possible to obtain the same results as in this example. 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 separated separately from the obtained fragment. By substituting the region corresponding to the fragment of pPT-DB1 which has been set in advance, a desired recombinant plasmid can be obtained.

[0032]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples. In the following, HPLC analysis of the reaction solution was performed using a column of ULRON 80HG (50 × 8φ).
mm) as a developing solution, and acrylamide is detected by absorbance at 220 nm. Further, in order to confirm the effects of the present invention, proteins contained in the obtained amide compound-containing solution were analyzed.
The protein concentration was determined by removing the amide compound contained in the amide compound-containing solution by dialysis using a semipermeable membrane, and then quantifying the protein concentration using a protein analysis kit manufactured by Biorat, to determine the protein removal rate.

Example 1 In a 500 ml Erlenmeyer flask with a baffle, 100 ml of a medium having the following composition was prepared, and sterilized by autoclaving 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 sulfate heptahydrate 40.0 mg / L pH 7.0 5

1.5 g of the wet cells obtained above was mixed with 98.5.
g of 0.3 mM NaOH aqueous solution, 36 g of acrylonitrile was added to the suspension at a time, and the mixture was reacted at 10 ° C. while stirring. The reaction solution was analyzed by HPLC analysis 24 hours after the start of the reaction. As a result, only acrylamide was present in the reaction solution (concentration = 3).
5% by weight), and no acrylonitrile was observed. The pH of this reaction solution was 8.0. This reaction solution was adjusted to pH 5 with a 10% sulfuric acid aqueous solution, and 2% by weight of activated carbon (Powdered activated carbon PM manufactured by Mikura Kasei Co., Ltd.)
-SX) and stirred at 25 ° C. for 5 hours.
Filtration was performed with filter paper. When the protein concentration in the obtained filtrate was measured, the removal rate was 99% or more. Also,
Even when 100 ml of methanol was added to 10 ml of the filtrate, the solution did not become cloudy, and no polymer was observed.

Example 2 A filtrate was obtained in the same manner as in Example 1 except that the pH of the reaction solution obtained in Example 1 was adjusted to 5 with a 10% aqueous solution of acrylic acid. When the protein concentration in the filtrate was measured, the removal rate was 99% or more. In addition, this filtrate 1
Even when 100 ml of methanol was added to 0 ml, the mixture did not become cloudy and no polymer was observed.

Example 3 Obtaining 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 by autoclaving at 121 ° C. for 20 minutes. Ampicillin was added to this medium to a final concentration of 100 μg / ml.
The T-10822 strain was inoculated with a monobacterial ear and incubated at 37 ° C. for 300
The culture was performed at pm for about 20 hours. After dispensing 1 ml of the culture solution into an appropriate centrifuge tube, centrifugation (15,000 rpm)
(m × 5 minutes). Then alkali S
A plasmid DNA of pPT-DB1 was prepared from the cells by DS 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), the composition being based on the conditions described in the kit). (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. 1 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 was added to prepare 50 μl of each solution. A total volume of 47.5 containing each 0.5 μl of the TE solution.
A 1 μl annealing solution (composition according to the conditions described in the kit) was prepared, subjected to a heat denaturation treatment (98 ° C.) for 10 minutes, cooled to 37 ° C. for 60 minutes at a constant rate, and then cooled to 37 ° C. An annealing treatment was performed by holding at 15 ° C. for 15 minutes. TAKA for annealing treatment solution
0.5 μl of RALATaq was added and heat treatment was performed at 72 ° C. for 3 minutes to complete a heteroduplex. M13
After adding 50 pmol of each of primer M4 (sequence described in SEQ ID NO: 2 in the sequence listing) and M13 primer RV (sequence described in SEQ ID NO: 4 in the sequence listing) to a total volume of 50 μl, heat denaturation (98 ° C.) for 15 seconds , Annealing (55 ° C)
The PCR reaction No. by repeating the conditions of 30 seconds and extension reaction (72 ° C.) 120 seconds for 25 cycles. 3 was performed.
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.
The presence of the NA product 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 finely pulverized and mixed with 1 ml of TE.
After suspending in the solution, the temperature 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 dissolved in 10 μl of TE. 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, pPT-DB1
The only restriction enzyme sites above are EcoRI and Hi
pPT-DB1 was cleaved with ndIII 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, which was finally dissolved in 10 μl of TE. The thus obtained amplified DNA product and pPT-D
DNA ligation kit for B1 fragment (Takara Shuzo)
After ligation using E. coli, competent cells of Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) were transformed to prepare an Escherichia coli bank.

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 each white ear 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 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.

[0041]

[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 a monobacterial ear 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 white fungus ear 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.

[0046]

[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 a monobacterial ear 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 white fungus ear 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.

[0051]

[Table 3]

This clone NO. The cells of No. 3 were cultured in the same manner as in Example 1 to obtain cells necessary for the reaction. Further, 1.5 g of the obtained wet cells was mixed with 98.5 g of 0.3 mM NaOH.
Acrylonitrile is suspended in an aqueous solution and acrylonitrile is added to the suspension.
g was added all at once and reacted while stirring at 10 ° C. The reaction solution was analyzed by HPLC analysis 24 hours after the start of the reaction. As a result, only acrylamide was present (concentration = 50% by weight) in the reaction solution, and acrylonitrile was not observed. The pH of this reaction solution was 8.0. This reaction solution was p-pulped with 10% aqueous sulfuric acid.
The reaction solution was adjusted to H5, and 2% by weight of activated carbon (Powdered activated carbon PM-SX manufactured by Mikura Kasei Co., Ltd.) was added to the reaction solution.
After stirring for 5 hours, the mixture was filtered with filter paper. When the protein removal rate in the obtained filtrate was measured, the removal rate was 99% or more. Even when 100 ml of methanol was added to 10 ml of the filtrate, the solution did not become cloudy and no polymer was observed.

Example 4 A filtrate was obtained by performing the same treatment as in Example 1 except that the pH of the hydration reaction solution obtained in Example 3 was adjusted to 3 with a 10% aqueous sulfuric acid solution. The removal rate of the protein in the filtrate was measured and found to be 75%. In addition, this filtrate 10m
Even when 100 ml of methanol was added to 1 l, the mixture did not become cloudy and no polymer was observed.

Comparative Example 1 A filtrate was obtained in the same manner as in Example 1 except that the hydration reaction solution obtained in Example 3 was adjusted to pH 7 with a 10% aqueous sulfuric acid solution. The protein removal rate of the filtrate was measured and found to be 25%.

[0055]

As is clear from the above description, particularly from the results of the above Examples and Comparative Examples, according to the method of the present invention, compared with the conventional purification method using activated carbon, the contact with activated carbon is performed under acidic conditions. Thereby, the amide compound can be purified much more effectively. In particular, when acrylamide purified by the method of the present invention is polymerized, polyacrylamide having a high molecular weight, excellent storage stability, and high water solubility can be obtained.

[0056]

[Sequence list]

<110> Mitsui Chemicals In
c. <120> Process for purifyin
gamide compound <130> 31000788 <150> JP 2000-007993 <151> 2000-1-17 <160> 6 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 aacatcatgc gcaagtcg 18 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <400> 2 caggaaacag ctatgac 17 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 ggccagtgcc tagcttacat 20 <210 > 4 <211> 17 <212> DNA <213> Artificial Sequence <400> 4 gttttcccag tcacgac 17 <210> 5 <211> 18 <212> DNA <213 > Artificial Sequence <400> 5 aactggtaca aggagccg 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <400> 6 ccgaactaca gcgtctac 18

 ──────────────────────────────────────────────────続 き 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 Person Tamotsu Asano 1144 Togo, Mobara-shi, Chiba F-term (reference) 4H006 AA02 AC53 AD15 AD17

Claims (13)

[Claims] 1. A method for purifying an amide compound, comprising contacting an amide compound-containing liquid with activated carbon under acidic conditions. 2. The purification method according to claim 1, wherein the amide compound-containing liquid is a product liquid obtained by a hydration reaction of a corresponding nitrile compound. 3. The purification method according to claim 1, wherein the amide compound has 2 to 20 carbon atoms. 4. The purification method according to claim 1, wherein the amide compound has an unsaturated bond. 5. The method according to claim 4, wherein the amide compound is acrylamide or methacrylamide. 6. The purification method according to claim 2, wherein the amide compound is synthesized using a microorganism cell containing nitrile hydratase or a processed product of the microorganism cell. . 7. The purification method according to claim 6, wherein the microbial cell is a transformant in which a nitrile hydratase gene cloned from a microorganism is expressed in any host. 8. The purification method according to claim 1, wherein the pH of the amide compound-containing liquid at the time of contact with activated carbon is 3.5 to 6.5. 9. The method according to claim 1, wherein the amide compound-containing liquid is prepared acidic by using an organic acid having an acid dissociation index of 3.5 to 5.5 or an organic acid and a base. Or the purification method described in 10. The purification method according to claim 9, wherein the organic acid is acrylic acid or methacrylic acid. 11. The method according to claim 1, wherein the activated carbon is activated carbon obtained from wood or coconut shell. 12. The temperature at the time of contact with activated carbon is 10 to 50.
The purification method according to any one of claims 1 to 11, wherein the temperature is ° C. 13. The method according to claim 1, wherein, after the amide compound-containing liquid is brought into contact with activated carbon, the activated carbon is separated, and the remaining liquid is brought to a saturation temperature or lower to precipitate crystals. Purification method.