JP7020615B2 - A method for producing a cell-treated product containing a nitrile hydratase and a method for producing an amide compound. - Google Patents

Description

The present disclosure relates to a method for producing a cell-treated product containing nitrile hydratase and a method for producing an amide compound.

Nitrile hydratase is an enzyme having a nitrile hydration activity that converts a nitrile group of various compounds into an amide group by hydration, and is used in an industrial process for producing an amide compound using an enzymatic reaction.

In recent years, a process for producing an amide compound from a nitrile compound using a cell-treated product obtained by disrupting the cells by ultrasonic treatment or the like has been known. For example, Patent Document 1 discloses a method for producing a cell-treated product containing a nitrile hydratase, which comprises a step of acid-treating the crushed cell product, then removing the insoluble material by centrifugation, and then treating the cell with an alkali. Has been done.

International Publication No. 2011/007725

In the production of a cell-treated product containing a nitrile hydratase, it is required to be a clear cell-treated product with reduced impurities other than the nitrile hydratase. In Patent Document 1, Celite such as High Flow Supercell is used as a filtration aid, but the method of Patent Document 1 has room for improvement in that a clear cell-treated product can be obtained.

In view of the above circumstances, one embodiment of the present disclosure is a method for producing a clear cell-treated product containing a nitrile hydratase containing reduced impurities other than the nitrile hydratase, and an amide compound using the cell-treated product. It is an object to provide a method for manufacturing a hydra.

The disclosure includes the following aspects:
<1> A method for producing a cell-treated product containing nitrile hydratase, which comprises a step of treating a crushed solution of a nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 12 μm to 100 μm and filtering the solution.
<2> The step of treating the crushed solution of the nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 12 μm to 100 μm and filtering the crushed solution of the nitrile hydratase-producing microorganism is filtered with an average particle size of 12 μm to 100 μm. The production method according to <1>, which comprises adding an auxiliary agent and filtering the crushed liquid.
<3> The step of treating the crushed solution of the nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 12 μm to 100 μm and filtering the crushed solution of the nitrile hydratase-producing microorganism having an average particle size of 12 μm to 100 μm. The production method according to <1>, which comprises filtering with a filter medium precoated with a filtration aid.
<4> The production method according to any one of <1> to <3>, wherein the average particle size of the filtration aid is 20 μm to 50 μm.
<5> The production method according to any one of <1> to <4>, wherein the filtration aid is diatomaceous earth.
<6> The production method according to <5>, wherein the diatomaceous earth is radiolite (registered trademark).
<7> The production according to any one of <1> to <6>, wherein the pH of the crushed solution treated with a filtering aid having an average particle size of 12 μm to 100 μm is 4.5 to 6.8. Method.
<8> Of <1> to <7>, which comprises a step of treating the crushed liquid with a filtering aid having an average particle size of 12 μm to 100 μm and adding a flocculant to the crushed liquid before the step of filtering. The manufacturing method according to any one.
<9> The production method according to any one of <1> to <8>, wherein the nitrile hydratase-producing microorganism is a pseudonocardia thermophila or a recombinant Escherichia coli having a nitrile hydratase-producing ability. ..
<10> The production method according to any one of <1> to <9>, wherein the cell-treated product is used for producing an amide compound.
<11> The production method according to <10>, wherein the amide compound is (meth) acrylamide.
<12> To produce a cell-treated product containing nitrile hydratase by the production method according to any one of <1> to <9>, and to mix the cell-treated product with a nitrile compound. ,
A method for producing an amide compound, which comprises.
<13> The production method according to <12>, wherein the nitrile compound is (meth) acrylonitrile and the amide compound is (meth) acrylamide.

According to the present disclosure, there is provided a method for producing a clear cell-treated product containing a nitrile hydratase containing reduced impurities other than nitrile hydratase, and a method for producing an amide compound using the cell-treated product. To.

The present disclosure relates to a method for producing a cell-treated product containing nitrile hydratase, which comprises a step of treating a disrupted solution of a nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 12 μm to 100 μm and filtering the lysate. Also referred to as "a method for producing a treated bacterial cell product according to the disclosure").

By using the method for producing a treated cell product according to the present disclosure, a clear treated cell product containing nitrile hydratase with reduced impurities can be produced. The reason for this is not always clear, but it can be estimated as follows. The lysate of nitrile hydratase-producing microorganisms contains a large amount of cell-derived components of microorganisms such as nucleic acids, cell walls, and cell membranes. Among these, those having a particle size larger than the wavelength of visible light scatter light to increase the turbidity of the crushed liquid, and those having a larger particle size precipitate in the crushed liquid. Although nitrile hydratase is contained in such a crushed solution, when this crushed solution is used for the production of an amide compound or the like, an amide compound (for example, (meth) acrylamide) produced by a large amount of impurities is produced. When the aqueous solution containing) is polymerized without purification, quality problems such as improper polymerization and foaming occur. In the present disclosure, the term "contaminant" refers to, in particular, the foaming of an aqueous solution containing an amide compound (for example, (meth) acrylamide) when a contaminating substance that increases turbidity and a cell-treated product are used for producing an amide compound. Means the contaminants that give rise to. Therefore, in order to utilize the nitrile hydratase contained in the crushed liquid for a chemical reaction, it is necessary to reduce impurities. When impurities are reduced, it is indicated as a decrease in turbidity of the solution.

The filtration aid is porous and can capture contaminant particles having a particle size equal to or larger than a predetermined size in the pores. Therefore, at least a part of the particles of the contaminants that cause the decrease in turbidity and the foaming of the aqueous solution containing the amide compound is captured in the pores of the filtration aid, and the turbidity of the solution decreases due to the reduction of the impurities. However, the effect of removing contaminant particles from the solution depends on the average particle size of the filter aid. When the average particle size of the filtration aid is large, the filtration flow rate becomes large when the filtration aid is used for filtration, so that the effect of removing impurities tends to decrease. On the other hand, when the average particle size of the filter aid is small, the effect of removing the particles of the contaminants tends to increase, but when the average particle size of the filter aid is too small, the impurities tend to increase. The effect of removing particles tends to be smaller. It is considered that this is because if the average particle size is too small, the pores of the size required for efficiently capturing the particles of contaminants cannot be secured.
Therefore, the nitrile hydratase has reduced impurities by the production method according to the present disclosure, which comprises a step of treating a crushed solution of a nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 12 μm to 100 μm and filtering the solution. It becomes possible to produce a clear cell-treated product containing. The above reasons are presumed.

In the present disclosure, the microbial crushing liquid is a liquid obtained by crushing a microorganism or one of the steps of heat treatment, acid treatment, centrifugation, addition of a flocculant, alkali treatment, pH adjustment, etc., which will be described later. Means a liquid that has been subjected to more than one. In microbial lysates, the intracellular components of the microbial cells are no longer confined within the cell membrane, but are dispersed in the lysate. The crushed liquid of the microorganism treated with the filtration aid having an average particle size of 12 μm to 100 μm may be the liquid itself obtained by crushing the microorganism (hereinafter, also referred to as “raw crushed liquid”), and may be used as the raw crushed liquid. On the other hand, one or a plurality of steps such as heat treatment, acid treatment, centrifugation, addition of flocculant, alkali treatment, and pH adjustment may be added.

In the present disclosure, the cell-treated product means a composition containing a component derived from a microorganism obtained by treating the microorganism. Even in the cell-treated product, the components in the cells of the microorganism are no longer confined in the cell membrane, but are dispersed in the cell-treated product. It is not necessary that the cell-treated product contains all of the components derived from microorganisms, and depending on the components, the whole amount or a partial amount thereof may be removed. The method for producing a cell-treated product containing nitrile hydratase according to the present disclosure may be referred to as a method for producing a composition containing nitrile hydratase. The composition may be a liquid or a solid.
In the cell-treated product containing nitrile hydratase in the present disclosure, it is preferable that the content of contaminants such as proteins and nucleic acids other than nitrile hydratase is low from the viewpoint of use in the process for producing an amide compound. The presence of impurities tends to facilitate the foaming of aqueous solutions containing the amide compound when an amide compound (eg, (meth) acrylamide) is produced.

In the present disclosure, the term "process" is included in this term not only as an independent process but also as long as the intended purpose of the process is achieved even if it cannot be clearly distinguished from other processes.
The numerical range indicated by using "-" in the present disclosure indicates a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively.
In the present disclosure, each element described may be present or present, unless otherwise specified in terms of the number thereof.
In the present disclosure, the amount of each component in the composition means the total amount of the plurality of substances present in the composition when a plurality of substances corresponding to each component in the composition are present, unless otherwise specified. do.

<Nitrile hydratase>
The nitrile hydratase in the method for producing a cell-treated product according to the present disclosure may be a nitrile hydratase of a natural organism, or a modified nitrile hydratase obtained by modifying the nitrile hydratase of a natural organism with an amino acid sequence modification. May be. Examples of the nitrile hydratase of a natural organism include nitrile hydratase of a microorganism. More specifically, the genera Nocardia, Corynebacterium, Bacillus, thermophilic Bacillus, Pseudomonas, Micrococcus, rhodochrous. Species such as Rhodococcus, Acinetobacter, Xanthobacter, Streptomyces, Rhizobium, Klebsiella, Enterobacter Pseudonocardia represented by the genera, Erwinia, Aeromonas, Citrobacter, Achromobacter, Agrobacterium, and thermophila species. Examples include nitrile hydratase of microorganisms belonging to the genus (Pseudonocardia), the genus Bacteridium, or the genus Brevibacterium.

The modified nitrile hydratase includes amide compound resistance and nitrile compound resistance by substituting, deleting, deleting or inserting one or more amino acid residues into the amino acid sequence of natural nitrile hydratase. , Modified nitrile hydratase with further improved temperature tolerance.

Nitrile hydratase of a microorganism belonging to the genus Pseudonocardia (for example, Pseudonocardia thermophila) and modified nitrile hydratase modified thereto are preferable from the viewpoint of high activity and high stability. Further, a nitrile hydratase of Rhodococcus rhodochrous J-1 and a modified nitrile hydratase modified thereto are also preferable.

<Nitrile hydratase-producing microorganisms>
The nitrile hydratase-producing microorganism in the present disclosure is a microorganism capable of producing nitrile hydratase. The nitrile hydratase-producing microorganism may be a natural microorganism, for example, Nocardia, Corynebacterium, Bacillus, thermophilic Bacillus, Pseudomonas ( Genus Pseudomonas, genus Micrococcus, genus Rhodococcus represented by rhodochrous species, genus Acinetobacter, genus Xanthobacter, genus Streptomyces, lysovium ( Rhizobium, Klebsiella, Enterobacter, Erwinia, Aeromonas, Citrobacter, Achromobacter, Agrobacterium Examples include the genus, microorganisms belonging to the genus Pseudonocardia represented by the thermophila species, the genus Bacteridium, or the genus Brevibacterium.

Alternatively, the nitrile hydratase-producing microorganism may be a host microorganism having a foreign nitrile hydratase gene (hereinafter, “nitrile hydratase-producing recombinant microorganism”). The foreign nitrile hydratase gene may be a nitrile hydratase gene of a natural organism, for example, a natural microorganism, or a modified nitrile hydratase gene obtained by modifying the nucleotide sequence of the nitrile hydratase gene of such a natural organism. There may be. By introducing an expression vector incorporating a nitrile hydratase gene into a host microorganism, a nitrile hydratase-producing recombinant microorganism can be obtained.

As a host microorganism of the nitrile hydratase-producing recombinant microorganism, Escherichia coli is a typical example as described later, but the present invention is not particularly limited to Escherichia coli, and Bacillus subtilis and the like. Other microbial strains such as Bacillus spp., Yeast and Escherichia coli can also be mentioned. As an example of a nitrile hydratase-producing recombinant microorganism, MT-10822 (this strain was introduced on February 7, 1996, 1-1, Higashi, Tsukuba City, Ibaraki Prefecture) in which the nitrile hydratase gene of Pseudonocardia thermophila was introduced into E. coli. No. 3 National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology and Industrial Technology (currently 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Tsukuba Center Central No. 6 Incorporated Administrative Agency, Industrial Technology Research Institute, Patented Biological Deposit Center) 5785 is deposited under the Budapest Treaty on International Approval of the Deposit of Microorganisms in Patent Procedures).

As nitrile hydratase-producing recombinant microorganisms, microorganisms belonging to the genus Pseudonocardia (for example, Pseudonocardia thermophila) and the genus Pseudonocardia in that they have highly active and highly stable nitrile hydratase. Recombinant microorganisms (transformers) in which the nitrile hydratase gene cloned from a microorganism is highly expressed in any host microorganism, and recombinants expressing modified nitrile hydratase modified from the nitrile hydratase of a microorganism belonging to the genus Pseudonocardia. Microorganisms are preferred. These recombinant microorganisms are preferable from the viewpoint of further increasing the stability of the nitrile hydratase and further increasing the activity per cell.

Further, Rhodococcus rhodochrous J-1, which can highly express nitrile hydratase in a microorganism, and a recombinant microorganism in which a nitrile hydratase gene cloned from the microorganism is highly expressed in an arbitrary host are also preferable.
It is also preferred that the nitrile hydratase-producing recombinant microorganism is a pseudonocardia thermophila or recombinant Escherichia coli capable of producing nitrile hydratase.

By culturing the nitrile hydratase-producing microorganism by a general method known in the fields of molecular biology, biotechnology, and genetic engineering, a desired amount of the nitrile hydratase-producing microorganism can be prepared. For example, after inoculating a nitrile hydratase-producing microorganism in a normal liquid medium such as LB medium or M9 medium, an appropriate culture temperature (generally 20 ° C to 50 ° C, but in the case of thermophiles) By culturing at 50 ° C. or higher), nitrile hydratase-producing microorganisms can be grown. The nitrile hydratase-producing microorganism thus grown can be subjected to the disruption described below.

<Crushing liquid>
The crushed solution of the nitrile hydratase-producing microorganism may be the original crushed solution itself prepared by crushing the nitrile hydratase-producing microorganism, or may be subjected to heat treatment, acid treatment, centrifugation, addition of flocculant, alkali treatment, pH adjustment, etc. It may be obtained by performing one or more of the steps.

The existence form of the nitrile hydratase-producing microorganism to be crushed is not particularly limited, but for example, the culture solution itself containing the nitrile hydratase-producing microorganism may be used, or the culture solution may be collected by centrifugation. It may be a body, or the collected cells may be washed with physiological saline or the like. When the collected cells are crushed, an appropriate solution such as physiological saline may be added to the crushed material to prepare a raw crushed solution.

The device for crushing the nitrile hydratase-producing microorganism is not particularly limited as long as it can crush the nitrile hydratase-producing microorganism, but for example, an ultrasonic crusher, a French press, a bead shocker, a homogenizer, a dyno mill, a cool mill or the like. Examples include a crusher. Among these, the homogenizer is preferable in that it can be scaled up at low cost. Homogenizers are commercially available from Sanwa Machinery Co., Ltd. and Izumi Food Machinery Co., Ltd.

The temperature at which the nitrile hydratase-producing microorganism is disrupted is not particularly limited, but is preferably 0 ° C. or higher and 50 ° C. or lower, and more preferably 0 ° C. or higher and 25 ° C. or lower.

The pH at the time of disrupting the nitrile hydratase-producing microorganism is not particularly limited, but is preferably pH 4 or more and 10 or less, and more preferably pH 6 or more and 8 or less.

The pressure when crushing the nitrile hydratase-producing microorganism using a homogenizer is not particularly limited as long as the pressure is such that the nitrile hydratase-producing microorganism is crushed, but is preferably 10 MPa or more and 300 MPa or less, more preferably 30 MPa or more and 100 MPa or less. Is.

The obtained raw crushed liquid may be further subjected to one or more of steps such as heat treatment, acid treatment, centrifugation, addition of flocculant, alkali treatment, and pH adjustment. The pH of the crushed liquid when treated with a filtration aid having an average particle size of 12 μm to 100 μm (for example, a filtration aid having an average particle size of 12 μm to 100 μm is added) is not particularly limited, and is, for example, 4.0 to 8. Although it may be 0.0, it is preferably 4.0 to 6.8, preferably 4.5 to 6.8, from the viewpoint of further improving the clarity of the obtained bacterial cell treatment product and improving the filtration rate. It is more preferably 6.8. The pH of the crushed liquid when treated with a filtration aid having an average particle size of 12 μm to 100 μm may be, for example, 4.5 to 5.5.

In the conventional method such as the method described in International Publication No. 2011/007725 (Patent Document 1), the pH of the crushed liquid at the time of filtration is neutralized by neutralizing as necessary before filtration. I was trying to be. However, in the method for producing a treated cell product according to the present disclosure, it is preferable that the pH of the crushed solution is weakly acidic as in the above range rather than neutral. By making the pH of the crushed liquid weakly acidic, it is possible to more efficiently capture the contaminant particles that increase the turbidity and the contaminant particles that clog the filter medium in the pores of the filtration aid having an average particle size of 12 μm to 100 μm. Because it can be done.

<Filtration aid>
The filtration aid generally refers to a substance having one or more functions of reducing filtration resistance, reducing clogging of the filter medium, and improving the clarity of the filtrate. The type of the filtration aid having an average particle size of 12 μm to 100 μm in the method for producing a treated cell product according to the present disclosure is not particularly limited, and examples thereof include diatomaceous earth, pearlite derived from volcanic rock, and cellulose derived from wood.
Diatomaceous earth is a fossil of diatom, which is a phytoplankton. Unlike other algae, diatoms are covered with a porous, hard cell wall made of amorphous hydrated silicic acid (SiO ₂ · nH ₂ O). This is called a silica shell and has innumerable holes with a diameter of about 0.1 μm to 1 μm. The mined raw material is refined through operations such as drying, classification, and firing. Filtration aids made of diatomaceous earth are roughly classified into calcined products and flux-fired products, and there are various products having different particle sizes. As the flux, for example, sodium carbonate is used. In the method for producing the treated bacterial cell product according to the present disclosure, either a calcined product or a flux-fired product may be used, but from the viewpoint of further improving the clarity of the treated bacterial cell product, the calcined product may be used. preferable.
Perlite is a general term for vitreous volcanic rocks. Magma is rapidly cooled near the surface of the earth, forming volcanic rocks mainly composed of amorphous silica. Perlite contains 2% to 5% of bound water, and when it is finely crushed and rapidly heated to the melting temperature, the solid part melts and at the same time the water gasifies, foams and expands. Have. The foamed pearlite can be crushed into flaky particles, and the particle size adjusted can be used as a filtration aid.

Examples of the filtration aid made of diatomaceous earth include products of the Radiolite (registered trademark) series manufactured by Showa Chemical Industry Co., Ltd. and products of the Celite (celite) series manufactured by Imeris (for example, Hyflo Supercell (registered trademark)). Can be mentioned. Among these, it is preferable to use products of the Radiolite (registered trademark) series, for example, Radiolite # 100, Radiolite # 300, Radiolite # 500, Radiolite # 700, Radiolite # 800, Radiolite # 1500H and the like. Examples of the filtration aid made of pearlite include products of the Topco series manufactured by Showa Chemical Industry Co., Ltd. and products of the Rocahelp series available from Mitsui Mining & Smelting Co., Ltd.

The average particle size of the filtration aid in the method for producing a treated cell product according to the present disclosure is 12 μm to 100 μm. In the present disclosure, the average particle size of the filtration aid is a 50% average particle size measured by a laser diffraction type particle size distribution measuring device. That is, it is the particle size of the particle corresponding to the (total number of particles × 0.5) th particle in the total number of particles when measured by the laser diffraction type particle size distribution measuring device. As the laser particle size distribution measuring device, a laser diffraction type particle size distribution measuring device SALD-2300 manufactured by Shimadzu Corporation is used for measurement.

In the method for producing a treated cell product according to the present disclosure, the average particle size of the filtration aid is more preferably 20 μm to 80 μm, preferably 20 μm to 50 μm, from the viewpoint of further improving the clarity of the treated cell product. It is more preferable to have.

<Step of treating the crushed liquid with a filtration aid having an average particle size of 12 μm to 100 μm and filtering it>
As described above, the filtration aid is porous, and at least a part of the contaminant particles having a particle size equal to or larger than the particle size determined by the pore size of the filtration aid can be captured in the pores. Therefore, the clarity of the filtrate can be improved by treating the crushed liquid with a filtration aid having an average particle size of 12 μm to 100 μm and filtering the crushed liquid. In addition, clogging of the filter medium can be suppressed, and further, filtration resistance can be reduced.

In the step of treating the crushed solution of the nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 12 μm to 100 μm and filtering, the filtration aid having an average particle size of 12 μm to 100 μm is added to the crushed solution of the nitrile hydratase-producing microorganism. , May include filtering the crushed liquid (added with a filtration aid having an average particle size of 12 μm to 100 μm). This corresponds to the body feed of a filtration aid having an average particle size of 12 μm to 100 μm. In the case of body feed, the cake formed by filtering the crushed liquid containing the filtration aid having an average particle size of 12 μm to 100 μm contains contaminant particles and the filtration aid, and has a high porosity. , The filtration resistance is low. Therefore, the filtration rate is remarkably improved, and the clogging of the filter medium tends to be suppressed. The amount of the filtration aid having an average particle size of 12 μm to 100 μm (preferably a filtration aid having an average particle size of 20 μm to 80 μm, more preferably a filtration aid having an average particle size of 20 μm to 50 μm) added to the crushed liquid is particularly limited. Although not, for example, it may be 3% by mass to 30% by mass, or 5% by mass to 20% by mass, based on the mass of the crushed solution before the addition of the filtration aid having an average particle size of 12 μm to 100 μm. May be good.

Alternatively, a filtration aid having an average particle size of 12 μm to 100 μm may be precoated on the surface of the filter medium. In the case of precoating, a filtration aid having an average particle size of 12 μm to 100 μm is deposited on the surface of the filter medium to form a layer (precoat layer) composed of a filtration aid having an average particle size of 12 μm to 100 μm having a thickness of 2 mm to 6 mm, for example. When the crushed liquid is passed through this precoat layer, at least a part of the particles of the contaminants are trapped in the pores of the filter aid having an average particle size of 12 μm to 100 μm, and a highly clear filtrate can be obtained. .. In addition, clogging of the filter medium due to the particles of impurities is suppressed, and the operation of peeling the cake after the filtration operation from the filter medium is easy. Therefore, in the step of treating the crushed solution of the nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 12 μm to 100 μm and filtering, the crushed solution obtained by crushing the nitrile hydratase-producing microorganism has an average particle size of 12 μm to 100 μm. It may include filtering with a filter medium pre-coated with a filtration aid.

Filtration aids with an average particle size of 12 μm to 100 μm are used in combination with filtration. The method for producing a treated cell product according to the present disclosure includes filtering a crushed liquid treated with a filtration aid having an average particle size of 12 μm to 100 μm.

The filter (filter medium) for filtering may be, for example, a filter having a maximum diameter of sphere particles that can pass through 20 μm to 100 μm. The maximum diameter of the ball particles that can pass through is preferably 30 μm to 80 μm. Examples of such a filter include wire mesh and filter cloth. For example, a wire mesh of 250 mesh to 1000 mesh, preferably 300 mesh to 700 mesh, or an air permeability of 50 cc / min / cm ² to 1000050 cc / min / cm ² , preferably 100050 cc / min / cm ² to 8000 cc / min / cm ² . Examples include the filter cloth. The wire mesh, filter cloth, or the like may be a flat tatami mat, a twill weave, or a reverse twill weave. Such wire nets can be obtained from Manabe Kogyo Co., Ltd., Okutani Wire Net Mfg. Co., Ltd., and filter cloths can be obtained from Nakao Filter Kogyo Co., Ltd., Shikishima Kambus Co., Ltd., etc.
The filter may be a membrane filter, but a wire mesh filter or a filter cloth is preferable from the viewpoint of increasing the area for mass processing and durability.

When precoating with a filtration aid having an average particle size of 12 μm to 100 μm, a candle filter, a leaf filter, a filter press, a precoat filter (drum filter), a bag filter, etc. can also be used as the usage form of the precoated filter. ..

Filtration may be performed by gravity without applying pressure, but in order to increase the filtration rate and improve the process efficiency, for example, the pressure is about 0.05 MPa to 1.0 MPa or 0.1 MPa to 0.5 MPa. May be added to the liquid on the upstream side of the filter for filtration. The solution containing the nitrile hydratase obtained by filtration (hereinafter, also referred to as "nitrile hydratase-containing solution"; the nitrile hydratase-containing solution may be a filtrate obtained by further concentrating the filtrate as described below) is nitrile. It is a cell-treated product containing hydratase and can be used for the production of amide compounds and the like.

<Other processes>
In the method for producing a cell-treated product according to the present disclosure, in addition to the step of treating a disrupted solution of a nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 12 μm to 100 μm and filtering, one or more other methods are used. It may include a step. In addition to the above-mentioned filtration treatment, an example of a step that may be optionally included in the method for producing a treated cell product according to the present disclosure is described below, but the method for producing a treated cell product according to the present disclosure includes. The processes that may be performed are not limited to these.

<Acid treatment>
Crushed liquids of nitrile hydratase-producing microorganisms (eg, raw crushed liquids) are preferably acid-treated before being treated with a filtration aid having an average particle size of 12 μm to 100 μm. That is, in the method for producing a bacterial cell-treated product according to the present disclosure, preferably, the disrupted liquid of the nitrile hydratase-producing microorganism is treated with a filtration aid having an average particle size of 12 μm to 100 μm, and the nitrile hydra is before the step of filtering. It may include a step (hereinafter, also referred to as an acid treatment step) of acid-treating the crushed solution of the tase-producing microorganism. The acid treatment is a step of treating the crushed liquid with an acid, and the acid treatment promotes the insolubilization of contaminants. By forming such an insoluble matter in advance, the effect of removing contaminants by the method for producing a bacterial cell-treated product according to the present disclosure is further improved.

As the acid, any strong acid such as sulfuric acid or a weak acid can be used without particular limitation as long as the effect of the method for producing the cell-treated product according to the present disclosure is not impaired. Weak acids are preferred in that the enzyme activity does not decrease during acid treatment.

Examples of the weak acid include acrylic acid, acetic acid, and phosphoric acid. Among these weak acids, acetic acid and phosphoric acid are not deleterious substances and are easy to handle.

In the acid treatment, the pH of the crushed liquid is preferably in the range of 4 or more and 6 or less, and more preferably in the range of 4.5 or more and 5.5 or less.

When treating the crushed solution with an acid, an acid (preferably an acid solution) may be added to the crushed solution of the nitrile hydratase-producing microorganism, or the nitrile hydratase-producing microorganism may be crushed into the acid (preferably an acid solution). A liquid may be added, or both a disrupted liquid of a nitrile hydratase-producing microorganism and an acid (preferably an acid solution) may be gradually poured into the container. Further, when treating the crushed liquid with an acid, it is desirable to slowly add an acid (preferably an acid solution) to the crushed liquid so that the pH of the entire crushed liquid becomes as uniform as possible.

Further, prior to the acid treatment, the crushed liquid (for example, the original crushed liquid) may be heat-treated within a range in which the activity of the enzyme does not decrease. That is, the method for producing a cell-treated product according to the present disclosure may include a step of heat-treating the disrupted solution of the nitrile hydratase-producing microorganism before the step of acid-treating the disrupted solution of the nitrile hydratase-producing microorganism. .. When the heat treatment is performed, the insoluble matter generated when the crushed liquid is treated with an acid can be more easily precipitated. The heat treatment temperature of the crushed liquid is preferably 45 ° C. or higher and 65 ° C. or lower, and more preferably 50 ° C. or higher and 60 ° C. or lower. The heat treatment time of the crushed liquid is preferably 5 minutes or more and 180 minutes or less, and more preferably 5 minutes or more and 60 minutes or less.
The heat treatment may be performed before crushing the nitrile hydratase-producing microorganism.

<Alkaline treatment>
The method for producing a bacterial cell-treated product according to the present disclosure may include a step of subjecting a crushed solution of a nitrile hydratase-producing microorganism to an alkali treatment (hereinafter, also referred to as an alkali treatment step). Alkaline treatment promotes insolubilization of impurities. By forming such an insoluble matter in advance, the effect of removing contaminants by the method for producing a bacterial cell-treated product according to the present disclosure is further improved. The time for alkaline treatment is prior to the step of treating the crushed solution of the nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 12 μm to 100 μm and filtering it from the viewpoint of preventing new impurities from being generated in the filtrate. Is preferable. When the above acid treatment step is performed, the alkali treatment may be performed before or after the acid treatment step.

The alkali used for this alkali treatment is not particularly limited as long as the effect of the method for producing a cell-treated product according to the present disclosure is not impaired, but sodium hydroxide is preferable in that it is an inexpensive alkali. The alkaline treatment may include, for example, adding an aqueous solution of sodium hydroxide in an amount necessary for adjusting the pH of the crushed solution to a desired pH to the mixed solution.

The alkaline treatment is preferably a treatment for adjusting the pH of the crushed liquid to a range of 7.5 or more and 10 or less, and more preferably a treatment for adjusting the pH to a range of 8.0 or more and 9.5 or less. Further, when the alkali treatment is performed, the crushed liquid may be treated with a filtration aid having an average particle size of 12 μm to 100 μm, and a pH adjustment step described later may be performed before the step of filtering.

<pH adjustment>
The method for producing a treated cell product according to the present disclosure may include a step of adjusting the pH of the crushed solution to 4.0 to 8.0 (hereinafter, also referred to as a pH adjusting step). This step is a step aimed at adjusting the pH of the crushed liquid when it is treated with a filtration aid. Therefore, when one or more of the above acid treatment and alkali treatment is performed, it is preferable to perform the pH adjustment step after those steps. As described above, from the viewpoint of further improving the clarity of the obtained bacterial cell treatment product and improving the filtration rate, the pH of the crushed liquid when treated with a filtration aid having an average particle size of 12 μm to 100 μm is It is preferably 4.0 to 6.8, and more preferably 4.5 to 6.8. Therefore, the pH adjusting step is preferably a step of adjusting the pH of the crushed liquid to 4.0 to 6.8, and more preferably a step of adjusting the pH to 4.5 to 6.8. The pH adjusting step may be a step of adjusting the pH of the crushed liquid to, for example, 4.5 to 5.5.

The acid treatment step described above may also serve as a pH adjusting step. The pH can be adjusted by adding an acidic substance, a basic substance, or the like to the crushed solution as needed. Examples of acidic substances include inorganic acids such as hydrochloric acid and nitric acid, and organic acids such as acetic acid, citric acid and tartaric acid. Examples of the basic substance include sodium hydroxide, potassium hydroxide, ammonia and the like. The step of adjusting the pH of the crushed liquid can be performed by adding an aqueous solution of an appropriate acidic substance or basic substance selected according to the target pH to the crushed liquid while monitoring the pH of the crushed liquid. In the present disclosure, the pH of the crushed liquid represents the pH at 25 ° C.

<Addition of coagulant>
In the method for producing a treated bacterial cell product according to the present disclosure, preferably, a crushed solution of a nitrile hydratase-producing microorganism is treated with a filtration aid having an average particle size of 12 μm to 100 μm, and a flocculant is added before the step of filtering. (Hereinafter, also referred to as a flocculant addition step) may be included. When the acid treatment step is performed, the step of adding the flocculant may be performed after the acid treatment step or before the acid treatment step. When the alkali treatment step is performed, the step of adding the flocculant may be performed after the alkali treatment step or before the alkali treatment step. By including the step of adding the flocculant, it becomes possible to more efficiently remove the contaminants that increase the turbidity, the contaminants that foam the aqueous solution of the amide compound, and the like. When the pH adjustment step is performed, the step of adding the flocculant may be performed after the pH adjustment step or before the pH adjustment step, but the crushed liquid when treated with the filtration aid. From the viewpoint of enabling more precise adjustment of the pH of the pH, it is preferable to perform the pH adjustment step before the pH adjustment step.

The flocculant is not particularly limited, and examples of the flocculant include an amphoteric water-soluble polymer and a polymer having a cationic functional group, and more specific examples thereof include a quaternary ammonium group-containing amphoteric water-soluble polymer. Molecular, tertiary amino group-containing amphoteric water-soluble polymer polyaminoalkyl (meth) acrylate, aminoalkyl (meth) acrylate quaternary salt polymer, aminoalkyl (meth) acrylate quaternary salt-acrylamide copolymer, polyvinyl-pyridinium- Examples thereof include halide, polydiallyldimethylammonium chloride, polycondensate of hexamethylenediamine and epichlorohydrin, polyalkyleneimine and the like. In the present disclosure, the notation of (meth) acrylate represents acrylate and / or methacrylate, the notation of (meth) acrylonitrile represents acrylonitrile and / or methacrylonitrile, and the notation of (meth) acrylamide represents acrylamide or methacrylamide or both thereof. Represents both.

As for the amount of the flocculant to be added to the crushed liquid, the concentration of the flocculant with respect to the total mass of the crushed liquid after mixing with the flocculant is 0.01% by mass to 2. The amount is preferably 0% by mass, more preferably 0.02% by mass to 1.0% by mass after mixing, and 0.05% by mass to 0.5% by mass. It is more preferable that the amount is as high as possible. In the step of adding the flocculant to the crushed solution of the nitrile hydratase-producing microorganism, for example, a flocculant solution of 5% by mass to 50% by mass, preferably 10% by mass to 40% by mass is added to the crushed solution with stirring. May be done by. From the viewpoint of maintaining the activity of the nitrile hydratase, the addition of the flocculant is preferably carried out at 0 ° C to 40 ° C, more preferably at 10 ° C to 30 ° C. The step of adding the flocculant to the disrupted solution of the nitrile hydratase-producing microorganism may include a step of stirring the disrupted solution after the addition of the flocculant.

<Centrifugation>
In the method for producing a treated cell product according to the present disclosure, the above-mentioned filtration aid is used as a clear cell for the disrupted solution of the nitrile hydratase-producing microorganism without performing centrifugation before the step of filtering. A processed product can be obtained. Therefore, in the method for producing a bacterial cell-treated product according to the present disclosure, the disrupted liquid of a nitrile hydratase-producing microorganism is treated with a filtration aid having an average particle size of 12 μm to 100 μm, and nitrile hydratase is produced before the step of filtering. There is no need to centrifuge the microbial disruption. However, if desired, the disrupted solution of the nitrile hydratase-producing microorganism is treated with a filtration aid having an average particle size of 12 μm to 100 μm, and before the step of filtering, the disrupted solution of the nitrile hydratase-producing microorganism is centrifuged. Treatment may be performed to remove insoluble matter present in the crushed liquid (hereinafter, also referred to as a centrifugation step). Centrifugal separation treatment makes it possible to separate and remove insoluble matter present in the disrupted liquid of the nitrile hydratase-producing microorganism. Here, the insoluble matter removed by the centrifugation may be at least a part of the insoluble matter present in the disrupted solution of the nitrile hydratase-producing microorganism. By performing the centrifugation treatment, it becomes possible to more efficiently remove impurities that increase turbidity, impurities that foam an aqueous solution of an amide compound, and the like.

When the acid treatment step is performed, it is preferable that the centrifugation step is performed after the acid treatment step is performed. When the alkali treatment step is performed, it is preferable that the centrifugation step is performed after the alkali treatment step is performed. Insoluble matter generated by one or more of the acid treatment and the alkali treatment can also be removed by the centrifugation step, so that the impurities that increase the turbidity and the impurities that foam the aqueous solution of the amide compound are more efficient. This is because it can be removed as a target.

When the pH adjustment step is performed, the centrifugation step may be performed after the pH adjustment step or before the pH adjustment step, but the pH of the crushed liquid when treated with a filtration aid may be adjusted. From the viewpoint of enabling more precise adjustment, it is preferable to perform the pH adjustment step. When the coagulant addition step is performed, the centrifugation step may be performed before the coagulant addition step or after the coagulant addition step.

If the aqueous solution containing the amide compound has a high foaming property, the foaming becomes remarkable when a gas such as nitrogen is blown into the aqueous solution, and in some cases, the aqueous solution overflows from the inside of the reaction device, which makes subsequent operation difficult. Will be. The blowing of such a gas is performed for the purpose of removing substances that adversely affect the reaction.

The centrifugation may be carried out immediately after the acid treatment, preferably 1 hour to 72 hours after the acid treatment, and more preferably 3 hours to 48 hours after the acid treatment. The treatment time of the centrifugation is not particularly limited as long as the insoluble matter can be removed, but is preferably 72 hours or less, more preferably 48 hours or less.

The temperature of the crushed liquid during the centrifugation is preferably 0 ° C. or higher and 50 ° C. or lower, and more preferably 0 ° C. or higher and 25 ° C. or lower.

<Concentration of filtrate>
The filtrate obtained by treating the above-mentioned crushed solution of the nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 12 μm to 100 μm and filtering the filtrate is subjected to a concentration step for further increasing the concentration of nitrile hydratase. May be. By subjecting to the concentration step, a cell-treated product having an increased concentration of nitrile hydratase can be obtained. Concentration can be performed by removing the liquid component in the filtrate. Concentration can be performed by, for example, a membrane concentration method such as evaporation or reverse osmosis. When the membrane concentration method is used, the nominal molecular weight of the membrane can be set to be larger than the molecular weight of the nitrile hydratase produced by the nitrile hydratase-producing microorganism, for example, the nominal molecular weight of the fraction is in the range of 40,000 to 100,000. A membrane that is a value within can be used. As such a film, for example, a pencil-type hollow fiber membrane AHP-0013 manufactured by Asahi Kasei Corporation can be used. The pressure applied at the time of concentration may be selected according to the film to be used, and may be, for example, in the range of 0.01 MPa to 0.5 MPa, preferably 0.05 MPa to 0.3 MPa.
The concentration of the filtrate in the concentration step may be set according to the time required for concentration and the required nitrile hydratase concentration, and may be, for example, 2 to 30 times, preferably 5 to 20 times. be able to. The degree of enrichment can be determined by dividing the volume of the solution after concentration by the volume of the solution before concentration.

<Manufacturing of amide compounds>
An amide compound can be produced from a nitrile compound by using a nitrile hydratase-containing solution which is a cell-treated product obtained by the method for producing a cell-treated product according to the present disclosure described above. The method for producing an amide compound according to the present disclosure includes, for example, mixing a nitrile compound with a cell-treated product obtained by the method for producing a cell-treated product according to the present disclosure. The details of the reaction may be appropriately adjusted according to the type of the nitrile compound as a raw material and the target amide compound. As an example, the process for producing (meth) acrylamide from (meth) acrylonitrile is described below. Other amide compounds can be similarly prepared from the corresponding nitrile compounds. The nitrile hydratase-containing solution may be a solution prepared by further performing a known purification treatment after filtration.

The production of (meth) acrylamide is usually carried out in an aqueous medium. The aqueous medium dissolves one or more selected from a buffer containing a phosphate and the like, an inorganic salt such as a sulfate and a carbonate, an alkali metal hydroxide, an amide compound and the like in water at an appropriate concentration. An aqueous medium, (meth) acrylonitrile as a raw material, and a nitrile hydratase-containing solution (bacterial treatment product) as a catalyst are mixed to prepare a reaction solution. By keeping the reaction solution at a temperature of about 10 ° C. to 20 ° C., the reaction for producing (meth) acrylamide proceeds.

The amount of the nitrile hydratase-containing solution to be mixed may be set so as to include the required number of units of nitrile hydratase in consideration of the scale of the reaction and the like. The concentration of (meth) acrylonitrile in the reaction solution at the start of the reaction is not particularly limited, but if the concentration of the nitrile compound at the start of the reaction is set too high, a large amount of catalyst will be used to complete the reaction. It becomes necessary, the volume of the required reactor becomes large, the size of the heat exchanger etc. required for heat removal becomes large, and the financial burden on the equipment becomes large.

Therefore, when acrylonitrile is used, the concentration of acrylonitrile in the reaction solution at the start of the reaction is that of the acrylamide produced relative to the total mass of the reaction solution when it is converted to acrylamide with a conversion efficiency of 100%. The concentration is preferably such that the concentration is 40% by mass to 80% by mass.

Also, when methacrylnitrile is used, the concentration of methacrylnitrile in the reaction solution at the start of the reaction is relative to the total mass of the reaction solution when it is converted to methacrylamide with a conversion efficiency of 100%. It is preferable that the concentration of the produced methacrylamide is 10% by mass to 40% by mass.

The reaction time of the (meth) acrylamide production reaction is usually 1 hour to 120 hours, preferably 2 hours to 48 hours, although it depends on conditions such as the amount of catalyst used and the temperature.

The reaction mode of the (meth) acrylamide production reaction is not particularly limited, and may be a batch type, a semi-batch type, or a continuous type. Further, it may be any of a suspension bed, a fixed bed, a moving bed and the like. Further, a plurality of types of reactors may be used in combination.

The hydration reaction catalyzed by nitrile hydratase 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 an aqueous medium. The reaction temperature is not particularly limited as long as it is above the freezing point of the aqueous medium, but it is usually preferably 0 ° C to 50 ° C, more preferably 10 ° C to 40 ° C. The reaction can also be carried out in a slurry state in which the product is crystallized in the reaction solution. The pH of the reaction solution in the hydration 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, and more preferably in the range of pH 7 to 9. Is.

As described above, according to the method for producing a treated cell product according to the present disclosure, a clear treated cell product containing nitrile hydratase containing reduced impurities other than nitrile hydratase can be produced. Further, this cell-treated product can be used for producing an amide compound such as (meth) acrylamide.

Hereinafter, embodiments will be described in more detail based on examples, but the present disclosure is not limited thereto. Hereinafter, unless otherwise specified,% and ppm are both based on mass.

[Analysis method of acrylamide]
For HPLC analysis in each Example and Comparative Example, Finepak SIL C18-5 (250 × 4.6φ mm) manufactured by JASCO Corporation was used as a column, and a 10 mM phosphoric acid aqueous solution containing 4% by volume acetonitrile was used as a developing solution. did. In addition, acrylamide was detected by the absorbance at 220 nm.

[Preparation of cultures of nitrile hydratase-producing microorganisms]
According to the method described in Example 1 of JP-A-2001-3400091, the pPT-DB1 plasmid DNA obtained in JP-A-09-275978 was used as a template, and clone No. 3 was shown in Table 3 of JP-A-09-275978. The nitrile hydratase-producing microorganism shown as 3 was obtained. This clone No. Reference numeral 3 is a gene in which Leu, which is the sixth from the N-terminal of the α-subunit of nitrile hydratase expressed by modifying the nitrile hydratase gene derived from Pseudonocardia thermophila JCM3095 strain, is replaced with Ala (codon on nucleotide). The change is a transformant obtained by transforming (change from CTG to GTG) into competent cells of Escherichia coli HB101 (manufactured by Toyo Spinning Co., Ltd.). Escherichia coli carrying the pPT-DB1 plasmid has been deposited as MT-10822 strain (accession number FERM BP-5785) at the Patent Organism Depositary as described above.

5 L of the medium having the following composition was adjusted, 500 mL each was added to 10 Erlenmeyer flasks with 2 L baffles, and the mixture was sterilized by autoclaving at 121 ° C. for 20 minutes. After adding ampicillin to this medium so that the final concentration becomes 50 μg / mL, the above clone No. The cells of 3 (nitrile hydratase-producing microorganisms) were inoculated with a single platinum loop and cultured at 37 ° C. at 130 rpm for 20 hours.

Medium composition Yeast extract 5.0 g / L
Polypeptone 10.0g / L
NaCl 5.0g / L
Cobalt Chloride Hexahydrate 10.0mg / L
Ferric sulfate / heptahydrate 40.0 mg / L
pH 7.5

[Preparation of raw crushed solution of nitrile hydratase-producing microorganism]
(Crushing nitrile hydratase-producing microorganisms with a homogenizer)
Using a homogenizer H50 manufactured by Sanwa Engineering Co., Ltd., the cells of the nitrile hydratase-producing microorganism contained in 5 L of the culture solution prepared in the preparation of the culture of the above-mentioned nitrile hydratase-producing microorganism were crushed at a temperature of 15 ° C. and a crushing pressure of 50 MPa. The cells were crushed under the condition of 100 minutes, and the nitrile hydratase contained in the cells was released into the solution to prepare a raw crushed solution.

[Heat treatment]
The raw crushed liquid was transferred to a 10 L glass beaker, and the glass beaker was immersed in a water bath constant temperature bath at 50 ° C. while sufficiently stirring the raw crushed liquid. After the temperature of the original crushed liquid reached 50 ° C., the immersion was continued for another 15 minutes, and then the beaker was taken out and air-cooled.

[Addition of coagulant]
Polyethyleneimine (weight average molecular weight 1800) manufactured by Wako Pure Chemical Industries, Ltd. was dissolved in water to prepare a 30% by mass aqueous solution. This 30% by mass polyethyleneimine aqueous solution was added to the acid-treated crushed solution so that the concentration of polyethyleneimine in the added crushed solution was 0.1% by mass, and the mixture was stirred for 30 minutes.

[PH adjustment]
While sufficiently stirring the crushed solution after the addition of the 30 mass% polyethyleneimine aqueous solution, 2N acetic acid was slowly added until the pH of the crushed solution reached 5.0.

[Addition of filtration aid to crushed liquid and filtration]
Diatomaceous earth (Radiolite (registered trademark) # 1500H manufactured by Showa Chemical Industry Co., Ltd.) was added to the crushed liquid after neutralization in an amount of 10% by mass based on the mass of the crushed liquid, and the mixture was stirred at 20 ° C. After stirring, pressure filtration was performed at 0.2 MPa using a 360 mesh wire mesh to obtain a filtrate. The time from the start of filtration until 25 g of the filtrate was obtained was measured and used as the time required for passing 25 g. The clarity of the obtained filtrate was evaluated by measuring the absorbance at a wavelength of 660 nm using a Shimadzu purple-visible spectrophotometer UV1650PC.

[Concentration of filtrate]
Using a pencil-type hollow fiber membrane AHP-0013 manufactured by Asahi Kasei Corporation, the filtrate was concentrated 15 times under the conditions of an average pressure of 0.1 MPa and a circulation speed of 1.5 mm / sec to obtain a cell-treated product.

[Manufacturing of acrylamide]
A 1L glass flask equipped with a stirrer as the first reactor and a Teflon (registered trademark) tube 20 m having an inner diameter of 5 mm as the second reactor were prepared. The first reactor was charged with 400 g of water in advance.

The cell-treated product (filter solution after 15-fold concentration) obtained above was suspended in a 0.3 mM NaOH aqueous solution to a concentration of 0.03% by mass to obtain a suspension. The suspension and acrylonitrile were continuously fed to the first reactor with stirring at a rate of 49 g / h (suspension) and 31 g / h (acrylonitrile), respectively. Further, the reaction solution was continuously withdrawn from the first reactor at a rate of 80 g / h so as to keep the liquid level of the first reactor constant. The extracted reaction solution was continuously fed to the second reactor at a rate of 80 g / h to further proceed the reaction in the second reactor.

During the above reaction, both the first reactor and the second reactor are immersed in a water bath having a temperature of 10 ° C to 20 ° C, and the temperature is controlled so that the liquid temperature inside each reactor becomes 15 ° C. Was done.

The amount of the cell-treated product added to the 0.3 mM NaOH aqueous solution was such that the conversion rate to acrylamide at the outlet of the first reactor was 90% or more, and the concentration of acrylonitrile at the outlet of the second reactor was below the detection limit (100 ppm). The following) was adjusted. The conversion rate to acrylamide was determined from the results of HPLC analysis. 2M acrylic acid was added to the obtained aqueous acrylamide solution to adjust the pH to 7.

[Effervescence test of acrylamide aqueous solution (measurement of foam height)]
300 g of the acrylamide aqueous solution obtained above was placed in a 500 ml graduated cylinder. Kinoshita type glass ball filter 503G was introduced into this acrylamide aqueous solution, and air was blown from the bottom of the measuring cylinder through the glass ball filter at 900 ml / min, and the height of foaming after 5 minutes was measured.

<Example 2>
A cell-treated product was obtained in the same manner as in Example 1 except that radiolite (registered trademark) # 1500H as a filtration aid was replaced with radiolite (registered trademark) # 700 manufactured by Showa Chemical Industry Co., Ltd. having the same mass. Using the obtained cell-treated product, the production of acrylamide and the foaming test of the aqueous acrylamide solution were carried out in the same manner as in Example 1.

<Example 3>
A cell-treated product was obtained in the same manner as in Example 1 except that radiolite (registered trademark) # 1500H as a filtration aid was replaced with radiolite (registered trademark) # 500 manufactured by Showa Chemical Industry Co., Ltd. having the same mass. Using the obtained cell-treated product, the production of acrylamide and the foaming test of the aqueous acrylamide solution were carried out in the same manner as in Example 1.

<Example 4>
A cell-treated product was obtained in the same manner as in Example 1 except that the pH of the crushed solution was adjusted to 6.0 instead of 5.0 when the pH of the crushed solution was adjusted. In the same manner as in Example 1, the production of acrylamide and the foaming test of the aqueous acrylamide solution were carried out.

<Example 5>
A cell-treated product was obtained in the same manner as in Example 1 except that the pH of the crushed solution was adjusted to 7.0 instead of 5.0 when the pH of the crushed solution was adjusted. In the same manner as in Example 1, the production of acrylamide and the foaming test of the aqueous acrylamide solution were carried out.

<Comparative Example 1>
A cell-treated product was obtained in the same manner as in Example 1 except that radiolite (registered trademark) # 1500H as a filtration aid was replaced with radiolite (registered trademark) # 3000 manufactured by Showa Chemical Industry Co., Ltd. having the same mass. Using the obtained cell-treated product, the production of acrylamide and the foaming test of the aqueous acrylamide solution were carried out in the same manner as in Example 1.

<Comparative Example 2>
A cell-treated product was obtained in the same manner as in Example 1 except that radiolite (registered trademark) # 1500H as a filtration aid was replaced with Celite Hyflo Supercell (registered trademark) manufactured by Imeris Co., Ltd. having the same mass. Using the cell-treated product, the production of acrylamide and the foaming test of the aqueous acrylamide solution were carried out in the same manner as in Example 1.

The results in Examples and Comparative Examples are summarized in Table 1. The average particle size of diatomaceous earth is a 50% average particle size measured using a laser diffraction type particle size distribution measuring device SALD-2300 manufactured by Shimadzu Corporation as described above.

As shown in Table 2, in Examples 1 to 5 in which the crushed liquid was treated with a filtration aid having an average particle size of 12 μm to 100 μm and filtered, good filtrate turbidity was obtained, and the acrylamide aqueous solution was foamed. The sex was also low. On the other hand, in Comparative Example 1 and Comparative Example 2 in which the average particle size of the filter aid was outside the range of 12 μm to 100 μm, the turbidity of the filtrate was high and a large amount of contaminant particles were present. Further, in Comparative Example 1 and Comparative Example 2, the effervescent property of the acrylamide aqueous solution was also high.
Comparing Example 1, Example 4 and Example 5, the filtrate is adjusted by adjusting the pH of the crushed solution treated with the filtration aid having an average particle size of 12 μm to 100 μm to 4.5 or more and 6.8 or less. It can be seen that the turbidity of is further reduced.

Claims (12)

A method for producing a cell-treated product containing nitrile hydratase, which comprises a step of treating a crushed solution of a nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 20 μm to 50 μm and filtering the solution. The step of treating the crushed solution of the nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 20 μm to 50 μm and filtering the crushed solution of the nitrile hydratase-producing microorganism is added to the crushed solution having an average particle size of 20 μm to 50 μm . The production method according to claim 1, which comprises adding and filtering the crushed liquid. The step of treating the crushed liquid of the nitrile hydratase-producing microorganism with a filtration aid having an average particle size of 20 μm to 50 μm and filtering the crushed liquid obtained by crushing the nitrile hydratase-producing microorganism is a filtration aid having an average particle size of 20 μm to 50 μm . The production method according to claim 1, which comprises filtering with a filter medium precoated with. The production method according to any one of claims 1 to 3 , wherein the filtration aid is diatomaceous earth. The production method according to claim 4 , wherein the diatomaceous earth is Radiolite (registered trademark). The production method according to any one of claims 1 to 5 , wherein the pH of the crushed liquid treated with a filtration aid having an average particle size of 20 μm to 50 μm is 4.5 to 6.8. One of claims 1 to 6 , which comprises a step of treating the crushed liquid with a filtering aid having an average particle size of 20 μm to 50 μm and adding a flocculant to the crushed liquid before the step of filtering. The manufacturing method described in the section. The production method according to any one of claims 1 to 7 , wherein the nitrile hydratase-producing microorganism is a pseudonocardia thermophila or a recombinant Escherichia coli having a nitrile hydratase-producing ability. The production method according to any one of claims 1 to 8 , wherein the cell-treated product is used for producing an amide compound. The production method according to claim 9 , wherein the amide compound is (meth) acrylamide. To produce a cell-treated product containing nitrile hydratase by the production method according to any one of claims 1 to 8 , and to mix the cell-treated product with a nitrile compound.
A method for producing an amide compound, which comprises. The production method according to claim 11 , wherein the nitrile compound is (meth) acrylonitrile and the amide compound is (meth) acrylamide.