Method for producing processed microbial cells containing nitrile hydratase and method for producing amide compound

Abstract

To provide a method for producing processed microbial cells containing nitrile hydratase that can improve the quality of the processed microbial cells, and a method for producing an amide compound including producing the processed microbial cells by the method.SOLUTION: A method for producing processed microbial cells containing nitrile hydratase, comprises the step of performing concentration of a liquid containing nitrile hydratase obtained from a microorganism that produces nitrile hydratase, the concentration being carried out with the pH of the liquid at 8.3 or higher.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a method for producing a treated microbial cell product containing nitrile hydratase and a method for producing an amide compound.

Background Art

[0002] Nitrile hydratase is an enzyme having nitrile hydration activity that converts a nitrile group of various compounds into an amide group by hydration. Nitrile hydratase is used in an industrial production process of an amide compound utilizing its enzymatic reaction.

[0003] As a method for producing nitrile hydratase, a method is known in which a microorganism capable of producing nitrile hydratase is cultured, and then the microorganism in the culture solution is disrupted to obtain a treated microbial cell product containing nitrile hydratase. The treated microbial cell product obtained by the above method may contain impurities derived from the microbial cells together with nitrile hydratase. The impurities can cause, for example, a decrease in the quality of the treated microbial cell product or an amide compound produced using the same. For this reason, attempts have been made to reduce the amount of impurities contained in the treated microbial cell product containing nitrile hydratase.

[0004] As a technique for reducing the amount of impurities contained in the treated microbial cell product, for example, Patent Document 1 describes a method of mixing a disrupted solution of a microorganism producing nitrile hydratase with a specific flocculant before filtering the disrupted solution. Patent Document 2 describes a method of filtering a disrupted solution of a microorganism producing nitrile hydratase with a specific filter aid added thereto.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] In the production technology of amide compounds using the enzymatic reaction of nitrile hydratase, there is still room for improvement in the quality of the treated microbial cells containing nitrile hydratase used in the production of amide compounds. In addition, newly finding a method capable of improving the quality of the treated microbial cells containing nitrile hydratase will greatly contribute to the development of the production technology of amide compounds using nitrile hydratase. In view of the above situation, an embodiment of the present disclosure aims to provide a method for producing a treated microbial cell capable of improving the quality of the treated microbial cell containing nitrile hydratase, and a method for producing an amide compound including producing the treated microbial cell by the above method.

Means for Solving the Problems

[0007] Means for solving the above problems include the following embodiments. <1> A method for producing a treated microbial cell containing nitrile hydratase, including a step of concentrating a liquid containing nitrile hydratase obtained from a microorganism producing nitrile hydratase, wherein the concentration is performed in a state where the pH of the liquid is 8.3 or higher. <2> The production method according to <1>, wherein the concentration is performed in a state where the pH of the liquid is 10.0 or lower. <3> The production method according to <1> or <2>, wherein the concentration is performed using an ultrafiltration membrane. <4> Before the step of concentrating the liquid containing nitrile hydratase, a step of obtaining a disrupted liquid containing a disrupted product of a microorganism producing nitrile hydratase and nitrile hydratase, and a step of removing at least a part of components other than nitrile hydratase contained in the disrupted liquid are further included. The production method according to any one of <1> to <3>. <5> The production method according to any one of <1> to <4>, wherein the microorganism is Escherichia coli. A process for producing a microbial cell-treated product containing nitrile hydratase by the production method according to any one of <6><1> to <5>, and a step of mixing the microbial cell-treated product and a nitrile compound, a method for producing an amide compound.

Effects of the Invention

[0008] According to an embodiment of the present disclosure, there is provided a method for producing a microbial cell-treated product capable of improving the quality of a microbial cell-treated product containing nitrile hydratase, and a method for producing an amide compound including producing a microbial cell-treated product by the method.

Modes for Carrying Out the Invention

[0009] In the present disclosure, the term "step" includes not only an independent step but also the present term if the intended purpose of the step is achieved even when it cannot be clearly distinguished from other steps. In the present disclosure, the numerical range indicated by using "~" represents a range including the numerical values described before and after "~" as the minimum value and the maximum value, respectively. 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 there are a plurality of substances corresponding to each component in the composition, unless otherwise specified. In the present disclosure, the notation "(meth)acrylamide" represents acrylamide or methacrylamide or both, the notation "(meth)acrylonitrile" represents acrylonitrile or methacrylonitrile or both, and the notation "(meth)acrylate" represents acrylate or methacrylate or both.

[0010] <Method for Producing Microbial Cell-Treated Product> The method for producing a microbial cell-treated product containing nitrile hydratase according to the present disclosure includes a step of concentrating a liquid containing nitrile hydratase obtained from a microorganism producing nitrile hydratase, and the concentration is performed in a state where the pH of the liquid is 8.3 or higher.

[0011] Hereinafter, the microbial cell-treated product containing nitrile hydratase is also referred to as "microbial cell-treated product", the liquid containing nitrile hydratase is also referred to as "nitrile hydratase-containing liquid", and the step of concentrating the nitrile hydratase-containing liquid is also referred to as "concentration step".

[0012] As shown in the examples described later, it was found that the microbial cell-treated product obtained by concentrating in a state where the pH of the nitrile hydratase-containing liquid is 8.3 or higher has higher clarity than the microbial cell-treated product obtained by concentrating in a state where the pH of the nitrile hydratase-containing liquid is less than 8.3.

[0013] In the method of the present disclosure, the quality of the microbial cell-treated product can be improved by adjusting the pH of the nitrile hydratase-containing liquid. The method of the present disclosure may be combined with other methods for improving the quality of the nitrile hydratase-containing liquid. For example, a flocculant, a filter aid, etc. may be used in the method of the present disclosure.

[0014] The method of the present disclosure includes a step of concentrating the nitrile hydratase-containing liquid. In the present disclosure, "concentration of the nitrile hydratase-containing liquid" means an operation of increasing the concentration of nitrile hydratase contained in the nitrile hydratase-containing liquid. The concentration of nitrile hydratase can be carried out, for example, using a membrane that allows the liquid component in the nitrile hydratase-containing liquid to pass through but does not allow nitrile hydratase to pass through. Examples of such a membrane include an ultrafiltration membrane (Ultra Filtration membrane, UF membrane). The type of ultrafiltration membrane is not particularly limited. For example, the Pencil type hollow fiber membrane AHP-0013 manufactured by Asahi Kasei Corporation can be used.

[0015] The nominal fractional molecular weight of the membrane used for concentration can be a molecular weight smaller than the molecular weight of nitrile hydratase. For example, a membrane having a nominal fractional molecular weight in the range of 40,000 to 100,000 can be used.

[0016] The pressure applied during concentration is not particularly limited and can be selected according to the type of membrane used, etc. For example, the pressure applied during concentration can be in the range of 0.01 MPa to 0.5 MPa, preferably 0.05 MPa to 0.3 MPa. The concentration degree of the nitrile hydratase-containing liquid can be set according to the time required for concentration and the required nitrile hydratase concentration. For example, the concentration degree of the nitrile hydratase-containing liquid can be 2 to 30 times, preferably 2.5 to 20 times, more preferably 3 to 10 times. The concentration degree of the nitrile hydratase-containing liquid is a value obtained by dividing the volume of the nitrile hydratase-containing liquid before concentration by the volume of the nitrile hydratase-containing liquid after concentration.

[0017] From the viewpoint of maintaining the enzyme activity of nitrile hydratase, the temperature of the nitrile hydratase-containing liquid during the concentration step is preferably carried out at 0°C to 40°C, more preferably at 10°C to 30°C.

[0018] The pH of the nitrile hydratase-containing liquid during the concentration step is not particularly limited as long as it is 8.3 or higher. From the viewpoint of enhancing the clarity of the treated microbial cells, the pH of the nitrile hydratase-containing liquid during the concentration step may be 8.4 or higher, or 8.5 or higher. From the viewpoint of maintaining the enzyme activity of nitrile hydratase contained in the treated microbial cells well, the pH of the nitrile hydratase-containing liquid during the concentration step may be 9.5 or lower, 9.3 or lower, or 9.0 or lower.

[0019] In the present disclosure, the pH of the liquid represents the value at 25°C. The adjustment of the pH of the liquid can be carried out using known basic substances or acidic substances without particular limitation as long as there is no particular notice. Examples of the basic substances include sodium hydroxide, potassium hydroxide, ammonia, etc. Examples of the acidic substances include inorganic acids such as hydrochloric acid and nitric acid, and organic acids such as acetic acid, citric acid, and tartaric acid.

[0020] The timing for adjusting the pH of the nitrile hydratase-containing solution to 8.3 or higher is not particularly limited as long as it is before the concentration step of the nitrile hydratase-containing solution. The pH of the nitrile hydratase-containing solution tends to decrease with the passage of time. Therefore, the adjustment of the pH of the nitrile hydratase-containing solution may be immediately before the concentration step of the nitrile hydratase-containing solution.

[0021] The nitrile hydratase-containing solution used in the method of the present disclosure contains nitrile hydratase obtained from a microorganism that produces nitrile hydratase. The type of microorganism that produces nitrile hydratase used in the method of the present disclosure is not particularly limited. Examples of microorganisms that produce nitrile hydratase include those belonging to the genera Nocardia, Corynebacterium, Bacillus, thermophilic Bacillus, Pseudomonas, Micrococcus, Rhodococcus represented by the rhodochrous species, Acinetobacter, Xanthobacter, Streptomyces, Rhizobium, Klebsiella, Enterobacter, Erwinia, Aeromonas, Citrobacter, Achromobacter, Agrobacterium, Pseudonocardia represented by the thermophila species, Bacteridium, or Brevibacterium.

[0022] The microorganism that produces nitrile hydratase may also be a transformant in which the nitrile hydratase gene cloned from the microorganism that produces nitrile hydratase is highly expressed in any host. As a representative example of the host, Escherichia coli can be mentioned. As hosts other than Escherichia coli, Bacillus genus bacteria such as Bacillus subtilis, yeast, actinomycetes, etc. can be mentioned. An example of the host is MT-10822. This strain was deposited on February 7, 1996, at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (currently located at 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan), under the accession number FERM BP-5785, based on the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0023] The microorganism that produces nitrile hydratase may also be a transformant that expresses a mutant nitrile hydratase obtained by using recombinant DNA technology. Examples of the mutant nitrile hydratase include those obtained by substitution, deletion, removal, or insertion of one or more amino acid residues included in the amino acid sequence constituting the nitrile hydratase of natural organisms.

[0024] Among the microorganisms that produce nitrile hydratase, from the viewpoints of enzyme activity and stability, microorganisms belonging to the genus Pseudonocardia, transformants in which the nitrile hydratase gene cloned from microorganisms belonging to the genus Pseudonocardia is highly expressed in any host, and transformants that express mutant nitrile hydratase are preferred. Rhodococcus rhodochrous J-1, which is a microorganism capable of highly expressing nitrile hydratase in the microorganism, and transformants in which the nitrile hydratase gene cloned from Rhodococcus rhodochrous J-1 is highly expressed in any host are also preferred.

[0025] The method for culturing a microorganism that produces nitrile hydratase is not particularly limited as long as it is a method by which the microorganism that produces nitrile hydratase can grow. As the culture solution used for culturing a microorganism that produces nitrile hydratase, a known medium containing appropriate amounts of glucose, a nitrogen source, inorganic substances, and other nutrients can be used without particular limitation. Specific examples of the medium include LB medium, M9 medium, and the like.

[0026] The cell density of the microbial cells in the culturing step is not particularly limited, and for example, it may be 10 6 cells / mL to 10 10 cells / mL. The volume of the culture is not particularly limited and can be set according to the desired amount of bacterial cells. For example, the volume of the culture may be 0.1 L to 10000 L. The culturing can be carried out using commonly used devices and equipment such as a culture tank. The culturing temperature can be set in consideration of the optimum growth temperature of the microorganism to be cultured. For example, the culturing temperature may be 15°C to 50°C, preferably 20°C to 40°C. The pH of the culture solution may be set to a pH appropriate for the growth of the microorganism. For example, when the microorganism is Escherichia coli, the pH of the culture solution may be 4.5 to 9.0, preferably 7.0 to 7.5. When the microorganism is a lactic acid bacterium, the pH can be 4.0 to 8.0, preferably 6.0 to 7.0.

[0027] The culturing time can be set in consideration of the initial cell density of the microorganism, the growth rate of the microorganism, the desired amount of bacterial cells, and the like. For example, the culturing time may be 5 hours to 100 hours. Whether to perform aeration during culturing can be determined based on the characteristics of the microorganism. When aeration is performed, the aeration amount (in the case of air) may be, for example, 0.02 vvm to 5.0 vvm, preferably 0.1 vvm to 2.0 vvm. In order to enhance the uniformity of the medium, the culture solution may be stirred using a stirring blade or the like. The rotation speed of the stirring by the stirring blade may be, for example, 100 rpm to 600 rpm, preferably 200 to 475 rpm. The cultivation can be carried out using ordinary cultivation methods such as shaking cultivation, aeration agitation cultivation, continuous cultivation, fed-batch cultivation, etc. In the case of continuous cultivation, the medium may be supplied continuously or intermittently.

[0028] In order to promote the growth of microorganisms, the cultivation process may include adding glucose to the culture solution. The addition of glucose to the culture solution may be carried out intermittently during the cultivation process or all at once at the initial stage of the cultivation process. From the perspective of the stability of the cultivation process, it is preferable to add glucose intermittently. In the present disclosure, "intermittent addition" means performing multiple additions at time intervals. That is, in intermittent addition, there is a period when no addition is made between the time when the addition is being made and the time when the next addition is being made. Examples of the form of glucose added to the medium include glucose powder and glucose solution, and glucose solution is preferred.

[0029] The glucose concentration of the glucose solution may be, for example, 10% to 80% by mass, preferably 40% to 70% by mass. The concentration of glucose in the culture solution can be set according to the properties of the microorganisms used. The concentration of glucose in the culture solution may be, for example, 0.01% to 10% by mass. When adding the glucose solution intermittently, the addition rate is not particularly limited. From the perspective of avoiding rapid dilution of the medium, for example, the addition rate of the glucose solution per liter of the culture solution may be 5 g / hour to 50 g / hour. The time required for one addition operation can be set in consideration of the concentration of glucose in the glucose solution and the addition rate of the glucose solution. For example, the time required for one addition operation may be 1 minute to 1.5 hours.

[0030] The culture process of a microorganism that produces nitrile hydratase may include measuring a parameter related to the amount of glucose in the culture solution and adding glucose based on the measurement result. A parameter related to the amount of glucose is a parameter that is affected by fluctuations in the amount of glucose and whose fluctuations in the amount of glucose can be indirectly estimated by measuring the parameter. Examples of such parameters include the pH of the culture solution, the dissolved oxygen concentration of the culture solution, the glucose concentration of the culture solution, the carbon dioxide concentration in the exhaust gas, the oxygen concentration in the exhaust gas, etc., and it is preferable that it is at least one selected from these examples.

[0031] For example, when the amount of glucose in the culture solution decreases, the pH of the culture solution increases due to, for example, a change in the balance between the glycolysis system and the downstream TCA cycle. Therefore, by monitoring the increase in the pH of the culture solution, it is possible to know the decrease in the amount of glucose. Also, since oxygen is consumed in the metabolism of glucose, it is possible to know the decrease in the amount of glucose through the dissolved oxygen concentration of the culture solution. The carbon dioxide concentration in the exhaust gas refers to the carbon dioxide concentration in the gas coming out of the culture medium after oxygen is fed into the culture medium, and similarly, the oxygen concentration in the exhaust gas refers to the oxygen concentration in the gas coming out of the culture medium after oxygen is fed into the culture medium. The consumption of oxygen due to glucose metabolism is also reflected in these parameters. These parameters may be measured individually, but by measuring two or more of them, the fluctuations in the amount of glucose can be determined more accurately.

[0032] For these parameters, set values may be determined in advance, and the addition of glucose in the culture process may be performed based on a comparison between the measured parameter and each predetermined set value. Specifically, the glucose addition operation may be started when the parameter reaches its set value. Also, the amount of glucose added may be set to an amount that adjusts the parameter to a preset target value.

[0033] Alternatively, the addition of glucose in the culturing step may be performed based on the amount of change in the parameter since the end of the previous addition of glucose. Specifically, when the amount of change in the parameter since the end of the previous addition reaches a preset value, the glucose addition operation may be started. Also, the amount of glucose added may be set to an amount that is adjusted to the value of the parameter at the end of the previous addition. For example, when using pH as the parameter, glucose addition may be started when the pH reaches a set value (e.g., 7.45) set within the range of 7.3 to 7.5. The culture solution is preferably mixed by a stirring blade or the like so that the added glucose and the culture solution are uniformly mixed.

[0034] In one embodiment of the present disclosure, the nitrile hydratase-containing solution is prepared by a method including a step of obtaining a disrupted solution containing disrupted cells of a microorganism that produces nitrile hydratase and nitrile hydratase (hereinafter also referred to as the disruption step), and a step of removing at least a part of components other than nitrile hydratase contained in the disrupted solution (hereinafter also referred to as the removal step). That is, the method of the present disclosure may further include a disruption step and a removal step before the concentration step.

[0035] The above method may include a step of performing heat treatment on the microorganism that produces nitrile hydratase after the disruption step (hereinafter also referred to as the heat treatment step). The above method may include a step of treating the disrupted cells of the microorganism with an acid after the disruption step (hereinafter also referred to as the acid treatment step). The above method may include a step of adding a flocculant to the disrupted solution after the disruption step (hereinafter also referred to as the flocculant addition step). The above method may include a step of adjusting the pH of the disrupted solution after the disruption step (hereinafter also referred to as the pH adjustment step of the disrupted solution). For the disrupted solution obtained in the disruption step, any one of the above steps may be performed, or two or more steps may be performed. The steps that may be included in the method for producing a nitrile hydratase-containing solution will be described below.

[0036] (Crushing step) In the crushing treatment, a crushed liquid containing the crushed product of the microorganism producing nitrile hydratase and nitrile hydratase is obtained. When the microorganism producing nitrile hydratase is crushed, the nitrile hydratase contained in the microbial cells is released, and a crushed liquid containing the crushed product of the microbial cells and nitrile hydratase is obtained.

[0037] The state of the microorganism to be crushed in the crushing step is not particularly limited. For example, it may be the culture solution itself containing the microorganism, the state of the cell mass obtained by centrifuging the culture solution containing the microorganism, the state of the cell mass washed with physiological saline, etc.

[0038] When performing a crushing treatment on a culture solution containing a microorganism, the temperature of the culture solution during the crushing treatment is not particularly limited, but is preferably 0°C to 50°C, more preferably 0°C to 25°C. The pH of the culture solution during the crushing treatment is not particularly limited, but is preferably 4.0 to 10.0, more preferably 6.0 to 8.0.

[0039] Examples of the device for crushing a microorganism include an ultrasonic crusher, a French press, a bead beater, a homogenizer, a dyno mill, a cool mill, etc. Among these, a homogenizer is preferable in terms of being able to be scaled up at low cost. A homogenizer is a device that adjusts the gap of a homovalve provided at the outlet of a plunger-type high-pressure pump that delivers liquid with a piston by screw or hydraulic pressure, and instantaneously generates a synergistic effect such as shearing, collision, and cavitation in the introduced fluid. Homogenizers are commercially available from Sanko Kikai Co., Ltd., Izumi Food Machinery Co., Ltd., etc. The pressure when crushing the cells using a homogenizer is not particularly limited as long as it is a pressure at which the cells are crushed, but is preferably 10 MPa or more and 300 MPa or less, more preferably 30 MPa or more and 100 MPa or less.

[0040] (Heat treatment process) In the heat treatment process, heat treatment is performed on the microorganism that produces nitrile hydratase. The conditions for heat treatment are not particularly limited as long as the enzyme activity of nitrile hydratase is not impaired and the microorganism is killed. For example, the temperature of heat treatment is preferably 45°C to 65°C, more preferably 50°C to 60°C. The time of heat treatment is preferably 5 minutes to 180 minutes, more preferably 5 minutes to 60 minutes.

[0041] (Acid treatment process) In the acid treatment process, the crushed product of the microorganism is treated with an acid. By treating the crushed product with an acid, the quality of the amide compound produced using the nitrile hydratase-containing solution can be maintained well. For example, components that have an adverse effect on the foaming of the amide compound can be made into insoluble substances. The acid treatment of the crushed product can be carried out, for example, by adding an acid to the crushed liquid obtained in the crushing process. As the acid used for the acid treatment, either a strong acid such as sulfuric acid or a weak acid can be used. From the viewpoint of suppressing the decrease in the enzyme activity of nitrile hydratase, a weak acid is preferred.

[0042] Examples of the weak acid used for the acid treatment include acrylic acid, acetic acid, phosphoric acid, etc. Among these weak acids, acetic acid and phosphoric acid are not highly toxic substances, so they are excellent in handleability. The pH of the crushed liquid after adding the acid is preferably 4.0 to 6.0, more preferably 4.5 to 5.5. Also, it is desirable to add the acid slowly so that the pH of the entire crushed liquid becomes as uniform as possible.

[0043] (Coagulant addition process) In the coagulant addition process, a coagulant is added to the crushed liquid. By adding a coagulant to the crushed liquid, impurities in the crushed liquid (for example, components that cause an increase in turbidity and foaming of the aqueous solution of the amide compound) can be removed more efficiently. When performing acid treatment on the crushed liquid, the addition of the coagulant may be carried out before or after the acid treatment.

[0044] Specific examples of the flocculant include amphoteric water-soluble polymers and polymers having cationic functional groups. More specific examples include amphoteric water-soluble polymers containing quaternary ammonium groups, amphoteric water-soluble polymer polyaminoalkyl (meth) acrylates containing tertiary amino groups, quaternary salt polymers of aminoalkyl (meth) acrylates, quaternary salt - acrylamide copolymers of aminoalkyl (meth) acrylates, polyvinyl - pyridinium - halide, polydiallyldimethylammonium chloride, polycondensates of hexamethylenediamine and epichlorohydrin, polyalkyleneimines (such as polyethyleneimine), and the like.

[0045] From the perspective of efficiently causing the aggregation of impurities, the amount of the flocculant added to the crushed liquid is preferably such that the concentration of the flocculant in the total mass of the mixed liquid of the flocculant and the crushed liquid is 0.01% by mass to 2.0% by mass, more preferably 0.02% by mass to 1.0% by mass, and even more preferably 0.05% by mass to 0.5% by mass. The addition of the flocculant may be carried out, for example, using a solution containing 5% by mass to 50% by mass, preferably 10% by mass to 40% by mass of the flocculant. From the perspective of maintaining the enzyme activity of nitrile hydratase, the addition of the flocculant is preferably carried out at 0°C to 40°C, and more preferably at 10°C to 30°C. After adding the flocculant to the crushed liquid, a step of stirring the crushed liquid may be carried out.

[0046] (pH adjustment step) In the pH adjustment step, the pH of the crushed liquid is adjusted. The pH of the crushed liquid is preferably adjusted so that the pH of the crushed liquid when filtered in the filtration step is 4.0 to 8.0, 4.0 to 6.8, or 4.5 to 6.8. When performing acid treatment or adding a flocculant to the crushed liquid, the pH adjustment is preferably carried out after these steps.

[0047] (Removal step) In the removal step, at least a part of the components other than nitrile hydratase contained in the crushed liquid obtained in the crushing step is removed. The crushed liquid from which at least a part of the components other than nitrile hydratase has been removed in the removal step is used as the nitrile hydratase-containing liquid. That is, the crushed liquid from which at least a part of the components other than nitrile hydratase has been removed in the removal step is subjected to a concentration treatment in a state where the pH is adjusted to 8.3 or higher.

[0048] The method for removing at least a part of the components other than nitrile hydratase contained in the crushed liquid is not particularly limited. Specific examples of the removal method include filtration, sedimentation separation, centrifugation, dialysis, etc. The removal step may be carried out by one method or a combination of two or more methods. From the viewpoints of being able to remove aggregates of a targeted particle size depending on the selection of the filter and the operation being relatively quick and simple, filtration is preferred as the removal method. The details and preferred embodiments of the removal step (hereinafter also referred to as the filtration step) carried out by filtering the crushed liquid are described below.

[0049] (Filtration step) In the filtration step, at least a part of the components other than nitrile hydratase contained in the crushed liquid is removed. By carrying out the filtration step of the crushed liquid, the content of impurities contained in the nitrile hydratase-containing liquid can be reduced.

[0050] The type of filter used in the filtration step is not particularly limited, and a wire mesh, filter cloth, membrane filter, etc. can be used. From the viewpoints of ease of scale-up and durability, the filter is preferably a wire mesh or filter cloth. The filter may be plain weave, twill weave, or reverse twill weave. The filter used in the filtration step may have, for example, a maximum diameter of passable spherical particles of 20 μm to 100 μm, and preferably a maximum diameter of passable spherical particles of 30 μm to 80 μm. Examples of such a filter include a wire mesh of 250 mesh to 1000 mesh, preferably 300 mesh to 700 mesh, and a filter cloth with an air permeability of 50 cc / min / cm 2 ~1000050 cc / min / cm 2 , preferably 100050 cc / min / cm2 ~8000 cc / min / cm 2 Examples include wire meshes such as those made of woven fabric. Such wire meshes are available, for example, from Manabe Kogyo Co., Ltd. and Okuya Wire Mesh Manufacturing Co., Ltd., and the filter cloth is available, for example, from Nakao Filter Industry Co., Ltd. and Shima Canvas Co., Ltd.

[0051] In the filtration step, a filter aid may be used. The filter aid is a porous body and can capture a part of the components contained in the crushing liquid in the pores. Therefore, the clarity of the filtrate after filtration can be further improved. In addition, by using a filter aid, effects such as suppression of filter clogging and reduction of filtration resistance can be obtained. As the filter aid, porous bodies such as diatomaceous earth, activated carbon, and perlite can be used. As the filter aid, diatomaceous earth is preferable in that the loss of nitrile hydratase activity is small, and celite (a mixture of sodium carbonate and calcined diatomaceous earth) is more preferable. Examples of the filter 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 Imerys (e.g., Hyflo Supercel (registered trademark)). Among these, products of the Radiolite (registered trademark) series, such as Radiolite #100, Radiolite #300, Radiolite #500, Radiolite #700, Radiolite #800, Radiolite #1500H, etc., are preferably used. Examples of the filter aid made of perlite include products of the Topco series manufactured by Showa Chemical Industry Co., Ltd., and products of the Rockhelp series available from Mitsui Mining & Smelting Co., Ltd.

[0052] The average particle size of the filter aid is preferably 12 μm to 100 μm, and more preferably 20 μm to 50 μm. When the average particle size of the filter aid is 12 μm to 100 μm, the clarity of the filtrate can be more effectively improved. In addition, filter clogging can be more effectively suppressed, and the filtration resistance can be more effectively reduced.

[0053] Examples of methods for using a filter aid in the filtration step include a method of adding a filter aid to the crushing liquid (body feed), a method of coating the surface of the filter with a filter aid (precoat), and the like. In the case of body feed, the cake formed by filtering the crushing liquid containing the filter aid has a mixture of impurity particles and the filter aid, a high porosity, and low filtration resistance. Therefore, the filtration rate is significantly improved, and the filter is less likely to be clogged. The amount of the filter aid added to the crushing liquid is not particularly limited. 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 crushing liquid before the addition of the filter aid. In the case of precoat, a filter aid is deposited on the filter surface to form a layer composed of the filter aid (precoat layer). When the crushing liquid passes through the precoat layer, part of the impurities are captured by the filter aid, and a filtrate with high clarity can be obtained. Also, clogging of the filter by impurities is suppressed, and the operation of peeling the cake after filtration from the filter becomes easier. The thickness of the precoat is not particularly limited. For example, the thickness of the precoat layer may be 2 mm to 6 mm.

[0054] The filtration step may be performed with or without applying pressure. From the viewpoint of increasing the filtration rate and improving the working efficiency, the filtration may be performed with pressure applied to the liquid (crushing liquid) on the upstream side of the filter. The pressure is not particularly limited, but for example, it may be 0.05 MPa to 1.0 MPa, or 0.1 MPa to 0.5 MPa.

[0055] <Method for producing an amide compound> The method for producing an amide compound according to the present disclosure includes a step of producing a microbial cell-treated product containing nitrile hydratase by the method for producing a microbial cell-treated product described above, and a step of mixing the microbial cell-treated product and a nitrile compound.

[0056] In the above method, the nitrile group of the nitrile compound is converted to an amide group by utilizing the nitrile hydrating activity of nitrile hydratase to produce an amide compound. In the present disclosure, the nitrile compound means a compound having a nitrile group, and the amide compound means a compound having an amide group. The nitrile compound is not particularly limited as long as it is a compound on which nitrile hydratase can act as a substrate. Specific examples of the nitrile compound include aliphatic saturated nitriles such as acetonitrile, propionitrile, succinonitrile, and adiponitrile; aliphatic unsaturated nitriles such as acrylonitrile and methacrylonitrile; aromatic nitriles such as benzonitrile and phthalodinitrile; and heterocyclic nitriles such as 3-cyanopyridine and 2-cyanopyridine. The nitrile group in the nitrile compound is converted to an amide group by hydration. For example, acrylonitrile is converted to acrylamide.

[0057] The production of the amide compound is usually carried out in an aqueous medium. The aqueous medium may be a solution obtained by dissolving additives such as a buffer containing phosphates, inorganic salts such as sulfates and carbonates, and hydroxides such as alkali metals in water. In the reaction solution obtained by mixing the aqueous medium, the nitrile compound as a raw material, and the treated bacterial cells as a catalyst, a production reaction of the amide compound is caused to occur.

[0058] The temperature of the reaction solution is not particularly limited as long as the nitrile hydration activity of nitrile hydratase is exhibited. The temperature of the reaction solution may be 0°C to 50°C, preferably 10°C to 40°C. The pH of the reaction solution is not particularly limited as long as the enzyme activity of nitrile hydratase is maintained. The pH of the reaction solution may be 6.0 to 10.0, preferably 7.0 to 9.0. The reaction time of the production reaction of the amide compound can be set in consideration of conditions such as the amount of catalyst used and the temperature. Usually, it is 1 hour to 120 hours, preferably 2 hours to 48 hours. The production reaction of the amide compound is usually carried out at normal pressure or near normal pressure, but it may be carried out under pressure in order to increase the solubility of the nitrile compound in the aqueous medium.

Examples

[0059] Hereinafter, the present disclosure will be described based on examples. However, the present disclosure is not limited to the following examples.

[0060] (1) Cultivation of microorganisms producing nitrile hydratase According to the method described in Example 1 of JP-A-2001-340091, using the pPT-DB1 plasmid DNA obtained by the method described in JP-A-09-275978 as a template, a microorganism producing nitrile hydratase shown as Clone No. 3 in Table 3 of JP-A-09-275978 was obtained. This Clone No. 3 is a transformant in which the 6th Leu from the N-terminus of the α subunit of nitrile hydratase, in which the nitrile hydratase gene derived from Pseudonocardia thermophila JCM3095 strain was modified and expressed, was substituted with Ala (the change in the codon on the nucleotide is from CTG to GTG), and was transformed into competent cells of Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.). Escherichia coli carrying the pPT-DB1 plasmid has been deposited with the above-mentioned Patent Organism Depositary as MT-10822 strain (Accession No. FERM BP-5785).

[0061] A pH 7.5 LB medium containing autoclaved yeast extract (15 g / L), sodium chloride (5 g / L), FeSO4 (0.1 g / L), CoCl2 (0.05 g / L), and Adekanol LG-126 (0.15 g / L) as a surfactant was prepared by adding ampicillin to a concentration of 0.1 mg / L to prepare 100 ml of the medium for preculture. The above-mentioned microorganism producing nitrile hydratase was inoculated into this medium in an amount of one loopful, and preculture was carried out at a temperature of 33°C. The growth amount of the microorganism during the preculture was grasped by the turbidity measurement method, and the preculture was terminated when the absorbance at 660 nm was in the range of 3.0 to 6.0.

[0062] The medium with the composition shown in Table 1 below was autoclaved to prepare the medium for this main culture. The culture solution obtained from the preculture was inoculated into 2 L of the medium for this main culture, and the main culture was carried out at 33 °C for 48 hours to obtain a culture solution (cell suspension). In this main culture, air was aerated into the culture solution at 1.0 vvm while stirring at 475 rpm. The pH of the culture solution was monitored from the start of the main culture, and 14.7 g of a 450 g / L aqueous glucose solution was added over 1 hour at immediately after the start of the culture, 3 hours after the start, 5 hours after the start, and 7 hours after the start. After 8 hours from the start of the culture, when the pH of the culture solution reached 6.8 or higher, 12 g of a 450 g / L aqueous glucose solution was added over 30 minutes. Through the above steps, a culture solution containing a microorganism producing nitrile hydratase was obtained.

[0063]

Table 1

[0064] (1) Crushing treatment The cells of the microorganism contained in 2 L of the culture solution after heat treatment were crushed to release the nitrile hydratase contained in the cells and a crushed solution was prepared. The crushing treatment was carried out using a homogenizer H11-H1 manufactured by Sanwa Engineering Co., Ltd. under the conditions of a temperature of 15 °C, a crushing pressure of 80 MPa, and a crushing time of 100 minutes.

[0065] (2) Heat treatment The crushed solution was transferred to a 10 L glass beaker and immersed in a water bath thermostat at 57 °C while stirring sufficiently. After the liquid temperature of the crushed solution reached 57 °C, the immersion was continued for another 27 minutes. Then, the beaker was taken out and cooled on ice.

[0066] (3) Acid treatment While stirring the crushed solution sufficiently, 2 M acetic acid was slowly added until the pH of the crushed solution reached 5.0.

[0067] (4) Addition of flocculant Polyethyleneimine (weight average molecular weight 4600) was dissolved in water to prepare a 30% by mass aqueous polyethyleneimine solution. This 30% by mass aqueous polyethyleneimine solution was added to the crushed liquid after acid treatment and stirred to obtain a mixed liquid. The concentration of polyethyleneimine in the mixed liquid was set to 0.1% by mass.

[0068] (5) pH adjustment of the crushed liquid While thoroughly stirring the mixed liquid of the flocculant and the crushed liquid, 2M acetic acid was slowly added until the pH of the mixed liquid reached 5.5.

[0069] (6) Filtration Diatomaceous earth (Radiolite (registered trademark) #1500H manufactured by Showa Chemical Industry Co., Ltd.) was added to the mixed liquid after pH adjustment as a filter aid and stirred at 40°C for 20 minutes. The addition amount of diatomaceous earth was 7% by mass based on the mass of the mixed liquid. After stirring, filtration was carried out while pressurizing at 0.1 MPa using a filter cloth with an air permeability of 2500 cc / min / cm 2 to obtain a filtrate.

[0070] (7) pH adjustment and concentration of the filtrate While thoroughly stirring the filtrate, an aqueous sodium hydroxide solution was added to adjust the pH of the filtrate to the values shown in Table 2. Then, the filtrate was concentrated using an ultrafiltration membrane. As the ultrafiltration membrane, a hollow fiber membrane AHP-0013 (manufactured by Asahi Kasei Corporation) was used, and the pressurization condition was 0.1 MPa. The concentration factor was set to 5 times. The concentrated liquid after concentration was stirred at 20°C for 72 hours.

[0071] (8) Evaluation of the clarity of the concentrated liquid As an index of the clarity of the concentrated liquid, the turbidity (OD660nm) of the concentrated liquid at 660 nm after 72 hours from the start of concentration was measured using a spectrophotometer. The lower the turbidity value, the higher the clarity of the concentrated liquid. The results are shown in Table 2.

[0072]

Table 2

[0073] As shown in Table 2, the concentrated liquids obtained in Example 1 and Example 2, where the pH during the concentration of the filtrate was set to 8.5 or 9.0, had lower turbidity values 72 hours after the start of concentration compared to the concentrated liquids obtained in Comparative Example 1 and Comparative Example 2, where the pH during the concentration of the filtrate was set to 7.4 or 8.0, and maintained high clarity. The above results suggest that a cell-treated product with high clarity can be obtained by performing concentration in a state where the pH of the nitrile hydratase-containing liquid is 8.3 or higher. By improving the clarity of the cell-treated product, effects such as improving the quality of the cell-treated product as a product and reducing the economic burden required for maintenance or replacement of the equipment and membranes used in the concentration process can be expected.

Claims

1. A method for producing a microbial cell-treated product containing nitrile hydratase, comprising a step of concentrating a liquid containing nitrile hydratase obtained from a microorganism producing nitrile hydratase, wherein the concentration is carried out in a state where the pH of the liquid is 8.3 or higher.

2. The production method according to claim 1, wherein the concentration is carried out in a state where the pH of the liquid is 10.0 or lower.

3. The production method according to claim 1, wherein the concentration is carried out using an ultrafiltration membrane.

4. Before the step of concentrating the liquid containing nitrile hydratase, the method further comprises a step of obtaining a disrupted liquid containing a disrupted product of a microorganism producing nitrile hydratase and nitrile hydratase, and a step of removing at least a part of components other than nitrile hydratase contained in the disrupted liquid.

5. The production method according to claim 1, wherein the microorganism is Escherichia coli.

6. A method for producing an amide compound, comprising a step of producing a microbial cell-treated product containing nitrile hydratase by the production method according to any one of claims 1 to 5, and a step of mixing the microbial cell-treated product and a nitrile compound.

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