JP2011206020A - Method of producing n-alkyl succinimide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of producing N-alkyl pyrrolidone at low cost by efficiently recovering N-alkyl succinimide being an intermediate when producing N-alkyl pyrrolidone from succinic acid and its salts.SOLUTION: In a method of producing N-alkyl succinimide by making a composition containing a succinic acid derivative derived from a biomass resource and its salt react with an alkylating agent, a method of producing a composition containing N-alkyl succinimide includes adjusting pH of a reaction mixture, after the end of reaction, to 9.8 or less.

Description

  The present invention relates to a method for producing an N-alkyl succinimide.

Succinic acid and its derivatives are important precursors of resin raw materials such as polyester, solvents and other chemicals, and derivatization into various resin raw materials and chemical precursors has been studied. Among them, pyrrolidones that are cyclic amide compounds are used in the fields of metals and electronic materials as cleaning solvents and extractants.
Specifically, as a synthesis method of (N-alkyl) pyrrolidone, a method of synthesizing N-alkylpyrrolidone from N-alkylsuccinimide and hydrogen in the presence of a reduction catalyst is known (Patent Documents 1 and 2). ), Succinic acid and its salts, acid anhydrides, or succinic acid esters via (N-alkyl) succinimide or directly obtaining (N-alkyl) pyrrolidone (Patent Documents 3 and 4) Is common. In addition, N-alkylpyrrolidone can be obtained from succinic acid and its salts and acid anhydrides via succinimide and 2-pyrrolidone (Patent Documents 5 and 6).
In the past, succinic acid was generally produced from maleic anhydride synthesized from butane by a petrochemical method, but in recent years, research on fermentation production methods using biomass resources as raw materials has progressed. The possibility of producing succinic acid on an industrial scale is also expected (Non-Patent Documents 1 and 2).
Patent Documents 4 and 6 describe a method for synthesizing N-methylpyrrolidone using succinic acid as a raw material by a fermentation production method.

Japanese Patent Laid-Open No. 62-289557 JP-A 63-27474 International Publication WO2002 / 102772 Pamphlet International Publication WO2004 / 058708 Pamphlet JP-A 63-27476 Special table 2008-531067 gazette

Chemical Reviews, 2007, Vol. 107, no. 6, 2417-2421 Green Chem. , 2009, 11, 13-16

  In a method for producing pyrrolidones from succinic acid and its derivatives, for example, N-alkyl succinimide synthesized from alcohol of succinic acid ammonium salt or succinic acid and ammonia and alcohol is hydrogenated in the presence of a catalyst to produce N-alkyl pyrrolidone. In order to carry out the catalytic reaction satisfactorily and produce at low cost, it is preferable to efficiently recover the N-alkyl succinimide as an intermediate.

The intermediate recovery method can be selected from crystallization, distillation, extraction, chromatography, etc., but when succinic acid and its salts are used as a raw material by fermentation, separation from the water used in the fermentation process is possible. It becomes a problem. When N-alkyl succinimide is recovered by crystallization or distillation, a large amount of water must be distilled off in the concentration step after the reaction, which requires energy costs. Chromatographic separation also requires concentration and is not suitable for large-scale industrial production.

Here, when extracting N-alkyl succinimide with an organic solvent, since it is not necessary to distill off water, energy in water separation can be reduced and it is suitable for large-scale industrial production. Conceivable.
However, when N-alkyl succinimide is produced from succinic acid and its ammonium salts, particularly ammonium succinate aqueous solution by fermentation production method, the concentration of N-alkyl succinimide in the reaction mixture is determined by hydrolysis reaction. Was found to decrease with time. N-alkyl succinamic acid, which is a hydrolysis product of N-alkyl succinimide, has a high affinity with water and is difficult to extract into an organic solvent, so the recovery rate is lowered, resulting in an increase in product cost. Become.

  The present invention provides a method for producing N-alkylpyrrolidone at low cost by efficiently recovering N-alkylsuccinimide as an intermediate in producing N-alkylpyrrolidone from succinic acid and its salts. The purpose is to do.

  As a result of intensive studies to achieve the above object, the present inventor obtained N-alkyl succinimide from succinic acid and its ammonia salts, acid anhydrides, succinic acid esters, other succinic acid derivatives, and mixtures thereof with ammonia. It has been found that N-alkylsuccinimide can be efficiently recovered by adjusting the pH of the reaction mixture to a specific range after the reaction to be produced is completed.

That is, the gist of the present invention is as follows.
[1] In a method for producing an N-alkyl succinimide by reacting a composition containing succinic acid represented by the following general formula (I) and a salt thereof with an alkylating agent, The manufacturing method of the composition containing N-alkyl succinimide represented by the following general formula (II) characterized by adjusting pH to 9.8 or less.

(In the formula, X and Y represent a hydrogen atom, NH ₄ ⁺ or another monovalent cation, and at least one of X and Y represents NH ₄ ⁺ .)

(In the formula, R ¹ is an optionally substituted linear, branched or cyclic C ₁ -C ₆ alkyl group.)
[2] The method according to [1], wherein the composition containing succinic acid and a salt thereof is derived from biomass resources.
[3] The method according to [1] or [2], wherein R ¹ is a methyl group or an ethyl group.

  According to the present invention, N-alkylpyrrolidone can be produced at low cost. In particular, N-alkyl succinimide as an intermediate can be efficiently recovered using succinic acid and its salts derived from a biomass resource-derived fermentation production method as raw materials, and the production cost of N-alkyl pyrrolidone can be reduced.

Hereinafter, embodiments of the present invention will be described in detail.
The description of the constituent elements described below is an example (representative example) of an embodiment of the present invention, and is not specified in these contents unless it exceeds the gist of the present invention.
<Composition containing succinic acid and its salt>
The succinic acid and the salt thereof according to the present invention are compounds represented by the following formula (I).

X and Y represent a hydrogen atom, NH ₄ ⁺ or another monovalent cation, and at least one of X and Y is NH ₄ ⁺ .
Other monovalent cations according to the present invention include alkali metal ions such as Na ⁺ , K ⁺ , and Li ⁺ .
Specific examples of succinic acid and salts thereof according to the present invention include ammonium salts such as diammonium succinate, monoammonium succinate, and ammonium sodium succinate. Among these, ammonium salts containing no other monovalent cation such as diammonium succinate and monoammonium succinate are preferable. A mixture of these compounds may be used.

The succinic acid and the salt thereof according to the present invention include succinic acid not containing NH ₄ ⁺ such as disodium succinate, monosodium succinate, dipotassium succinate, monopotassium succinate, magnesium succinate, calcium succinate and the like. It can also be prepared by adding a compound containing NH ₄ ⁺ to the salt. The compound containing NH ₄ ⁺ is not particularly limited, and examples thereof include aqueous ammonia and ammonium carbonate. The NH ₄ ⁺ content of the composition containing compound (I) is usually 1 mol or more, more preferably 1.2 mol or more, on the other hand, usually 5 mol or less, preferably 3 mol, relative to 1 mol of succinic acid. It is as follows. If the amount is too small, the reaction yield tends to decrease. If the amount is too large, undesirable side reactions such as hydrolysis tend to occur.

The composition containing succinic acid and a salt thereof according to the present invention may be produced by petrochemical means using petroleum or coal as a raw material, but is preferably derived from biomass resources from the viewpoint of environmental protection.
The concentration of succinic acid and its salt in the composition according to the present invention is not particularly limited, but the succinic acid concentration is usually 1% by weight or more, preferably 10% by weight or more, and usually 80% by weight or less, preferably Is 70% by weight or less. If the concentration is too low, there may be a problem in operability and efficiency in the reaction process, and if the concentration is too high, a problem in which succinic acid and a salt thereof precipitate may occur.

  Biomass resources include, for example, wood, rice straw, rice husk, rice bran, old rice, corn, sugar cane, cassava, sago palm, okara, corn cob, tapioca cass, bagasse, vegetable oil scum, persimmon, buckwheat, soybean, fat, waste paper, papermaking residue , Marine product residue, livestock excrement, sewage sludge, food waste and the like. Among them, plant resources such as wood, rice straw, rice husk, rice bran, old rice, corn, sugar cane, cassava, sago palm, okara, corn cob, tapioca cass, bagasse, vegetable oil residue, buckwheat, soy, oil, waste paper, paper residue More preferred are wood, rice straw, rice husk, old rice, corn, sugar cane, cassava, sago palm, straw, oil and fat, waste paper, papermaking residue, and most preferred are corn, sugar cane, cassava, sago palm. These biomass resources generally contain a large amount of alkali metals and alkaline earth metals such as nitrogen element, Na, K, Mg, and Ca.

  These biomass resources are not particularly limited, but are induced to carbon sources through known pretreatment and saccharification processes such as chemical treatment with acids and alkalis, biological treatment with microorganisms, physical treatment, and the like. Is done. The process is not particularly limited, but includes, for example, a refinement process by pretreatment such as chipping, scraping, or crushing biomass resources. If necessary, a grinding step with a grinder or a mill is further included. The biomass resources thus refined are further guided to a carbon source through pretreatment and saccharification processes. Specific methods include acid treatment with strong acids such as sulfuric acid, nitric acid, hydrochloric acid, and phosphoric acid, and alkali treatment. Chemical methods such as treatment, ammonia freezing steam explosion method, solvent extraction, supercritical fluid treatment, oxidizing agent treatment, physical methods such as fine grinding, steam explosion method, microwave treatment, electron beam irradiation, microorganism and enzyme treatment Biological treatments such as hydrolysis by

Carbon sources derived from the above biomass resources usually include hexoses such as glucose, mannose, galactose, fructose, sorbose and tagatose, pentoses such as arabinose, xylose, ribose, xylulose and ribulose, maltose, sucrose, lactose and trehalose. , Starch, cellulose and other disaccharides / polysaccharides, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, monoctinic acid, Fatty acids such as arachidic acid, eicosenoic acid, arachidonic acid, behenic acid, erucic acid, docosapentaenoic acid, docosahexaenoic acid, lignoceric acid, ceracolic acid, glycerin, mannitol, xylitol, ribitol, etc. Fermentable carbohydrates such as polyalcohols are used, and among these, hexoses such as glucose, mannose, galactose, fructose, sorbose, tagatose, pentoses such as arabinose, xylose, ribose, xylulose, ribulose, maltose, sucrose, Disaccharides and polysaccharides such as lactose, trehalose, starch, and cellulose are preferable, and among these, glucose, maltose, fructose, sucrose, lactose, trehalose, and cellulose are preferable.

  In the present invention, an aliphatic dicarboxylic acid is derived from a carbon source derived from these biomass resources. Specifically, for example, using these carbon sources, fermentation methods by microbial conversion, chemical conversion methods including reaction steps such as hydrolysis, dehydration reaction, hydration reaction, oxidation reaction, reduction reaction, and these fermentation methods A dicarboxylic acid is synthesized by a combination of a chemical conversion method and a chemical conversion method, and among these, a fermentation method by microbial conversion using a microorganism having an ability to produce an aliphatic dicarboxylic acid is preferable.

The microorganism having the ability to produce an aliphatic dicarboxylic acid is not particularly limited as long as it is a microorganism having an ability to produce an aliphatic dicarboxylic acid, and examples include enterobacteria such as Escherichia coli, Bacillus bacteria, coryneform bacteria, and yeast. It is preferable to use aerobic microorganisms, facultative anaerobic microorganisms or microaerobic microorganisms.
Examples of aerobic microorganisms include Coryneform Bacterium, Bacillus genus bacteria, Rhizobium genus bacteria, Arthrobacter genus bacteria, Mycobacterium genus bacteria, Rhodococcus (cc) Examples include genus bacteria, Nocardia bacteria, and Streptomyces bacteria, and coryneform bacteria are more preferable.

  The coryneform bacterium is not particularly limited as long as it is classified as such, and examples thereof include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium, and bacteria belonging to the genus Arthrobacter. Examples include those belonging to the genus Corynebacterium or Brevibacterium, more preferably, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes (Brevibaterium). bacteria classified as Ammoniagenes) or Brevibacterium lactofermentum It is below.

When a succinic acid-producing bacterium is used as the aliphatic dicarboxylic acid-producing bacterium, it is preferable to use a strain having enhanced pyruvate carboxylase activity and reduced lactate dehydrogenase activity, as described in Examples below.
Reaction conditions such as reaction temperature and pressure in microbial conversion will depend on the activity of microorganisms such as selected cells and molds, but suitable conditions for obtaining dicarboxylic acids should be selected in each case. That's fine.

  In microbial conversion, neutralizing agents are usually used because the metabolic activity of microorganisms decreases or the activity of microorganisms stops when pH decreases, and the production yield deteriorates or the microorganisms die. To do. Usually, the pH in the reaction system is measured by a pH sensor, and the pH is adjusted by adding a neutralizing agent so as to be in a predetermined pH range. The pH value is adjusted to a range where the activity is most effectively exhibited according to the type of microorganisms such as fungus bodies and molds to be used. There is no restriction | limiting in particular about the addition method of a neutralizing agent, Continuous addition or intermittent addition may be sufficient.

Examples of the neutralizing agent include ammonia, ammonium carbonate, urea, alkali metal hydroxide, alkaline earth metal hydroxide, alkali metal carbonate, and alkaline earth metal carbonate. Ammonia, ammonium carbonate and urea are preferred. Examples of the alkali (earth) metal hydroxide include NaOH, KOH, Ca (OH) ₂ , Mg (OH) ₂ , or a mixture thereof. Examples of the alkali (earth) metal carbonate include _{is, Na 2 CO 3, K 2} CO 3, CaCO 3, MgCO 3, NaKCO 3 etc., or the like and mixtures thereof.

In the present invention, the fermentation broth after microbial conversion may be appropriately concentrated in consideration of operability and efficiency in the subsequent purification step. Although it does not specifically limit as a concentration method, The method of distribute | circulating an inert gas, the method of distilling off water by heating, the method of distilling off water by pressure reduction, the method of combining these, etc. are mentioned.
In addition, when using a fermented liquid in the method of this invention, it is preferable to use the fermented liquid after removing microorganisms. As a microorganism separation method, an existing method such as an ultrafiltration membrane or centrifugation can be arbitrarily used. Moreover, you may sterilize the fermented liquor after microorganism conversion by autoclave sterilization, heat sterilization, or pasteurization.

The composition containing succinic acid and its salt according to the present invention can use succinic acid and its salt contained in the fermentation broth obtained as described above as it is, but succinic acid contained in the fermentation broth described later. In addition, succinic acid obtained using a method for converting a salt thereof into succinic acid and a purification method thereof may be used together with the compound containing NH ₄ ⁺ as necessary.
Although the method of converting the salt of the succinic acid contained in a fermented liquor to the succinic acid and the method of refine | purifying below are illustrated, it is not limited to this. Moreover, you may take out and use from arbitrary processes among the refinement | purification processes after converting into succinic acid.

<Succinic acid conversion method>
The succinic acid may be converted to succinic acid by a reaction crystallization method using a weakly acidic organic acid having a pH higher than that of the intended succinic acid. An example of such an organic acid is acetic acid. Alternatively, the succinic acid and its salt obtained by the fermentation may be converted to succinic acid with an inorganic acid. Here, examples of the inorganic acid used include sulfuric acid, hydrochloric acid, carbonic acid, phosphoric acid, and nitric acid. More specifically, in the case of succinic acid fermentation, when fermentation is performed while neutralizing the produced succinic acid with ammonia or magnesium hydroxide, ammonium succinate or magnesium succinate is produced in the fermentation broth. When such a fermented solution containing ammonium succinate or magnesium succinate is treated with sulfuric acid or the like, an aqueous solution containing succinic acid is obtained.

  The solution or aqueous solution containing succinic acid is a solution or aqueous solution mainly containing succinic acid. Accordingly, a solution or an aqueous solution mainly containing a succinate such as ammonium succinate or magnesium succinate is referred to as a solution or aqueous solution containing succinate. The term “contained as the main component” as used herein means that the weight of the component relative to the weight of all components excluding the solvent is usually 50% by weight or more, preferably 60% by weight or more, more preferably 70% by weight or more, and particularly preferably The state of 90% by weight or more is shown.

<Purification of succinic acid>
The aqueous solution converted into succinic acid with the above-mentioned inorganic acid is not particularly limited, but succinic acid may be extracted from the aqueous solution using an organic solvent, or concentrated, crystallized, and salted out.
Furthermore, the succinic acid obtained above can be subjected to a drying treatment or a further advanced purification treatment depending on its use. For example, a decolorization process using an adsorbent such as activated carbon, an ion exchange process for removing coexisting ions with an ion exchange resin, a process for hydrogenating coexisting unsaturated dicarboxylic acid, and a crystallization process for further refinement. Conceivable.

<Alkylating agent>
Alkylating agents of the present invention is not particular limitation, straight chain, alkyl alcohol or alkyl amines C ₁ -C ₆ branched or cyclic and the like. Among them, preferably
Alkyl alcohols or alkyl amines C ₁ -C ₄ straight or branched. Specific examples include methanol, ethanol, propanol or butanol, methylamines, ethylamines, propylamines or butylamines, and a mixture thereof. More preferably, methanol or ethanol is used, and mixtures of these alcohols with the corresponding alkylamines are also advantageously used.

  In the present invention, a stoichiometric or exceeding stoichiometric alkylating agent is used with respect to succinic acid and its salt in the composition containing succinic acid and its salt. Preferably, the alkylating agent is used in an amount of usually 1-fold mol or more, usually 100-fold mol or less, preferably 50-fold mol or less, more preferably 25-fold mol or less, relative to the succinic acid and its salt. If the amount of the alkylating agent is too small, the reaction yield tends to decrease, and if the amount of the alkylating agent is too large, side reactions tend to occur. A catalyst may also be used.

<N-alkyl succinimide>
The N-alkyl succinimide according to the present invention is a compound represented by the following formula (II).

R ^{1 according} to the present invention is a linear, branched or cyclic C ₁ -C ₆ alkyl group which may be substituted.
The linear, branched or cyclic alkyl group of R ^{1 according to} the present invention includes a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tert-butyl group, a pentyl group, a hexyl group, a cyclopropyl group, and a cyclobutyl group. , Cyclopentyl group, cyclohexyl group and the like. Among them, preferably a straight-chain or alkyl group of C ₁ -C ₄ branch, specifically, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tert- butyl group. Most preferably, they are a methyl group and an ethyl group. Alkyl group of the C ₁ -C ₆ may be substituted.

The substituent which may be substituted is an alkyl group, alkenyl group, alkynyl group, cycloalkyl group, aryl group, ether group, thioether group, halogen atom and the like.
<Imidation reaction conditions>
In the present invention, in the method for producing an N-alkyl succinimide by reacting the composition containing the succinic acid and a salt thereof with the alkylating agent, the pH of the reaction mixture after the reaction is reduced to 9.8 or less. It is characterized by adjusting.

<PH control of reaction mixture after imidation reaction>
The pH of the reaction mixture after completion of the reaction in the production process of N-alkyl succinimide, which is an intermediate for producing N-alkyl pyrrolidone using succinic acid and its salts and alkylating agent as raw materials, is preferably 9.8 or less, preferably 9.5 or less, more preferably 9 or less, still more preferably 8.5 or less, particularly preferably 8 or less. On the other hand, there is no particular limitation on the lower limit, but usually 2 or more, preferably 3
More preferably, it is adjusted to 4 or more, more preferably 5 or more, and particularly preferably 5.5 or more.

  If the pH of the reaction mixture is too low, the problem of corrosion of the reactor tends to occur. If the pH of the reaction mixture is too high, the hydrolysis reaction of the N-alkyl succinimide in the reaction mixture proceeds and the hydrolysis proceeds. N-alkyl succinamic acid, which is a decomposition product, has a high affinity with water and is difficult to extract into an organic solvent, so that the recovery rate of N-alkyl succinimide decreases, resulting in an increase in product cost. Arise.

The pH control of the reaction mixture after completion of the reaction is performed, for example, as follows, but is not limited thereto. Moreover, you may control pH during reaction.
Examples of the method for controlling the pH after completion of the reaction include a method of quickly removing a basic component generated by the reaction after the completion of the reaction. Specifically, a method of distilling off a low-boiling basic component under standard pressure or reduced pressure, a method of blowing an inert gas into the reaction mixture and volatilizing and removing the low-boiling component are effective. . It is also effective to add a neutralizing agent so that the reaction mixture is in a predetermined pH range after completion of the reaction.

  Examples of the method for controlling pH during the reaction include a method of reacting while removing a basic component having a low boiling point generated as the reaction proceeds from the reaction system. Specifically, using a pressure-resistant reactor equipped with a cooling pipe, only low-boiling gas components are distilled off, and other components are agglomerated and reacted while returning to the reaction system. A method in which an inert gas is blown, a low-boiling component is distributed to the gas phase, and a reaction is performed while purging a part of the gas phase is effective. It is also effective to monitor the pH of the reaction mixture during the reaction and react while sequentially adding a neutralizing agent so that the reaction system is maintained in a predetermined pH range. Alternatively, the reaction may be performed in the presence of a buffer capable of maintaining the reaction mixture at a predetermined pH.

  Here, the neutralizing agent used for controlling the pH of the reaction mixture during or after the reaction is not limited as long as it can maintain the reaction mixture in a predetermined pH range, and is an inorganic or organic acid compound. Can be used. The form of the acid compound may be a gas, a liquid, or a solid. For example, examples of the inorganic acid include sulfuric acid, hydrochloric acid, carbonic acid, phosphoric acid, and nitric acid. Examples of organic acids include aliphatic carboxylic acids such as acetic acid, propionic acid, butanoic acid, and lactic acid, sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid, succinic acid, fumaric acid, maleic acid, apple Examples thereof include aliphatic dicarboxylic acids such as acid, adipic acid and cyclohexanedicarboxylic acid. Examples of the solid acid include a sulfonic acid type ion exchange resin and an inorganic solid acid having a sulfonic acid group supported on an inorganic support.

For example, in the production of N-alkyl succinimide, when succinic acid fermentation liquor or diammonium succinate is used as the raw material succinic acid and its salts, the pH of the raw material mixture at the start of the reaction is usually about 6-8. However, as the reaction proceeds, the basic component is liberated, and the pH of the reaction mixture during the reaction and immediately after the completion of the reaction rises to a range exceeding 10. When the pH of the reaction mixture shows high basicity, the hydrolysis reaction of N-alkyl succinimide in the reaction mixture proceeds, and the yield of N-alkyl succinimide decreases. An efficient method for recovering N-alkyl succinimide, which is an intermediate for producing N-alkyl pyrrolidone, includes an extraction method using an organic solvent. N-alkyl succinimide is a hydrolysis product of N-alkyl succinimide. Alkyl succinamic acid has a high affinity with water and is difficult to extract into an organic solvent, so that the recovery rate of N-alkyl succinimide is lowered, resulting in an increase in product cost. By adjusting the pH of the reaction mixture during reaction or after completion of the reaction to 10 or less, hydrolysis reaction from N-alkyl succinimide to N-alkyl succinamic acid is suppressed, and N-alkyl is more efficiently produced. N-alkyl succinimide, which is an intermediate for producing pyrrolidone, can be recovered, which is industrially advantageous.

<Imidation reaction temperature>
The imidation reaction temperature according to the present invention is not particularly limited, but is usually 50 ° C. or higher, preferably 100 ° C. or higher, more preferably 150 ° C., while it is usually 500 ° C. or lower, preferably 450 ° C., More preferably, it is 400 degrees C or less. If the temperature is too low, there is a tendency that the reaction rate is slow and it takes time to complete the reaction, and if the temperature is too high, an undesirable side reaction occurs or the organic matter is burnt in the reactor. Tend to occur.

<Imidation reaction pressure>
The imidation reaction pressure according to the present invention is not particularly limited, but is usually atmospheric pressure or higher, preferably 0.5 MPa or higher, more preferably 1 MPa or higher, and usually 30 MPa or lower, preferably 25 MPa or lower, more preferably. Is 20 MPa or less. It may be the vapor pressure of the raw materials and reactants at the reaction temperature, or may be pressurized by other gas components, and is carried out in the gas phase and / or liquid phase.

<Imidation reaction method>
The imidation reaction method according to the present invention is performed, for example, by the following procedure, but is not limited thereto.
a) A composition containing succinic acid and a salt thereof is reacted with an alkylating agent at a predetermined reaction temperature and pressure.
b) After reacting the composition containing succinic acid and its salt with the alkylating agent at a predetermined reaction temperature, the reaction is performed again at the predetermined reaction temperature and pressure, and the reaction temperature and pressure may be the same or different. Good.
c) After treating only the composition containing succinic acid and its salt at a predetermined reaction temperature and pressure in advance, an alkylating agent is added and reacted at the predetermined reaction temperature and pressure, and each reaction temperature and pressure is the same. But it can be different.

  When the reaction is carried out in two stages at different reaction temperatures and pressures, particularly when the first stage is reacted with only the succinic acid composition and the second stage is added with an alkylating agent, the reaction conditions for the second stage are usually 1 It is preferable to carry out the reaction at a higher temperature and pressure than the first stage, and it is advantageous to carry out the first stage reaction at a temperature of 100 ° C. to 300 ° C. and the second stage reaction at a temperature of 150 ° C. to 400 ° C. . In the process of producing an N-alkyl succinimide from a composition containing succinic acid and its ammonium salt and an alkylating agent, the succinic imide structure, which is a five-membered ring containing a nitrogen atom, is produced from the succinic acid skeleton. This is because a higher reaction temperature is required in the latter than in the former in the process of conversion and in the process of alkylating nitrogen atoms with an alkylating agent such as alcohol. The higher the reaction temperature, the more advantageous for cyclization and alkylation, but the above temperature range is more advantageous from the viewpoints of suppressing side reactions and saving energy.

<Purification of N-alkyl succinimide>
When N-alkylpyrrolidone is produced by hydrogenation in the presence of a catalyst, which will be described later, the composition containing succinic acid and its salt is reacted with an alkylating agent in order to carry out the catalytic reaction satisfactorily and at low cost. Thus, it is necessary to efficiently recover the N-alkyl succinimide from the mixture in which the N-alkyl succinimide is synthesized. The N-alkyl succinimide to be used for the hydrogenation reaction can be used as it is, or the light boiling component in the reaction mixture can be distilled off and concentrated, but the desired catalytic reaction is good. It is preferable to purify and use in order to carry out to manufacturing at low cost. In particular, when the composition containing succinic acid and its salt is derived from biomass resources, the recovery and purification method of N-alkyl succinimide is selected from known methods such as crystallization, distillation, extraction, chromatography, etc. A plurality of methods may be combined. Of these, extraction with an organic solvent is preferred. This is because when N-alkyl succinimide is extracted with an organic solvent, it is not necessary to distill off water, so it is possible to reduce energy in water separation and is suitable for large-scale industrial production. It is. Moreover, since most of the water-soluble impurities derived from fermentation can be separated together with water, the purification effect is also high.

In the case where the unreacted portion of the alcohol used as the alkylating agent deteriorates the separation between the organic solvent and water after the N-alkyl succinimide extraction, the alcohol may be separated before the extraction.
Examples of the organic solvent for extraction according to the present invention include aliphatic hydrocarbon solvents such as hexane, heptane, and octane, unsaturated hydrocarbon solvents such as hexene and octene, and aromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene. Solvents, diethyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, methyl tert-butyl ether, tetrahydrofuran, tetrahydropyran, dioxane and other ether solvents, methyl ethyl ketone, methyl isobutyl ketone and other ketone solvents, methyl acetate, ethyl acetate , Ester solvents such as propyl acetate, butyl acetate, propyl formate and methyl propionate, alcohol solvents such as butanol, hexanol, octanol and 2-ethylhexanol, dimethyl carbonate, diethyl carbonate Carbonate solvents such as Boneto like. Preferably, aromatic hydrocarbon solvents such as toluene, ether solvents such as diethyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydropyran, dioxane, ketone solvents such as methyl isobutyl ketone, ethyl acetate, propyl acetate And ester solvents such as butyl acetate, propyl formate and methyl propionate, alcohol solvents such as butanol and hexanol, and carbonate solvents such as dimethyl carbonate and diethyl carbonate.

Extraction may be performed in one stage or in multiple stages. Further, the solvent may be flowed in a cross flow or a counter flow with respect to the reaction solution. If sufficient yield cannot be obtained by one-stage extraction, the residual phase and a new solvent may be mixed by counter-current multistage extraction to improve the yield.
When subjected to the subsequent reduction step, as long as the extraction solvent does not adversely affect the hydrogenation reaction, the extract may be used as it is, or the extraction solvent may be separated and used. The organic solvent used as the extraction solvent can be separated, for example, by distillation. Since the organic solvent has a lower latent heat than water, the thermal energy required for the distillation separation is small and the environmental load is also small.

  By combining the pH control step and the extraction step described above, N-alkyl succinimide can be efficiently extracted. The selectivity of N-alkyl succinimide is not particularly limited, but in the case of N-methyl succinimide, it is usually 83.5% or more, preferably 84% or more, more preferably 84.5% or more. is there. The ratio of N-alkyl succinimide to N-alkyl succinamic acid is not particularly limited, but in the case of the ratio of N-methyl succinimide to N-methyl succinamic acid at 25 ° C., it is usually 10 0.5 or more, preferably 11 or more, more preferably 12 or more, still more preferably 13 or more, and particularly preferably 14 or more. On the other hand, the upper limit is not particularly limited, but is usually 200 or less, preferably 100 or less, more Preferably it is 50 or less, More preferably, it is 25 or less.

<Reduction process to composition containing N-alkylpyrrolidone>
The reduction step from the composition containing N-alkyl succinimide according to the present invention to the composition containing N-alkylpyrrolidone includes a catalytic hydrogenation reaction using hydrogen in the presence of a catalyst, a hydrogenation reaction using a composite hydride, Alternatively, for example, it is carried out by a hydrogen transfer reaction for supplying hydrogen while alcohol such as methanol or isopropanol is converted into aldehyde or ketone. Among them, the catalytic hydrogenation reaction is preferable, and can be performed in a gas phase or a liquid phase. Either a batch system or a continuous reaction system may be used, and the reaction can be carried out according to a conventionally known method. The composition containing an N-alkyl succinimide to be subjected to the hydrogenation reaction may be used as it is, or may be diluted with a solvent that does not adversely influence the reaction. In order to carry out the desired catalytic reaction satisfactorily and to produce at low cost, it is preferable to purify and use the N-alkyl succinimide.

  The hydrogenation reaction temperature according to the present invention is not particularly limited, but is usually 50 ° C or higher, preferably 100 ° C or higher, more preferably 150 ° C or higher, and usually 400 ° C or lower, preferably 350 ° C or lower, more preferably Is 300 ° C. or lower. If the temperature is too low, there is a tendency that the reaction rate is slow and it takes time to complete the reaction. If the temperature is too high, undesirable side reactions such as hydrogenolysis of carbon-nitrogen bonds proceed. This tends to cause problems.

  The hydrogenation reaction pressure according to the present invention is not particularly limited, but is usually at least atmospheric pressure, preferably at least 0.5 MPa, more preferably at least 1 MPa, and usually at most 30 MPa, preferably at most 25 MPa, more preferably Is 20 MPa or less. If the pressure is too low, there is a tendency that the reaction rate is slow and it takes time to complete the reaction. If the pressure is too high, the hydrogenation reaction of the target product proceeds and the amount of by-products by hydrogenation increases. This tends to cause problems.

Even if it is the vapor pressure of the raw material and reactant at the reaction execution temperature, it may be pressurized by other gas components, and it is carried out in the gas phase or liquid phase.
As a specific method of the hydrogenation reaction, a composition containing N-alkyl succinimide and a hydrogenation catalyst are allowed to coexist in a pressure reactor, and hydrogen treatment is performed by introducing hydrogen gas while stirring the mixture. , A method of separating a reaction mixture after treatment from a hydrogenation catalyst and removing it from a reactor, a composition containing N-alkyl succinimide and a hydrogen gas using a fixed bed multitubular or single tube reactor The hydrogen treatment is carried out while circulating from the upper part or the lower part of the reactor, and the reaction mixture after the treatment is taken out, or the hydrogen gas is passed from the lower part of the reactor and the composition containing N-alkyl succinimide is passed from the upper part to perform the hydrogen treatment. And a method of taking out the reaction mixture after the treatment, a method using a fluidized bed reactor, and the like.

<Contact hydrogenation catalyst>
As the catalytic hydrogenation catalyst according to the present invention, known homogeneous and heterogeneous hydrogenation catalysts can be used. Specifically, although not particularly limited, preferably one or more metals such as ruthenium, rhodium, palladium, rhenium, platinum, copper, nickel, cobalt, iron, manganese, chromium, zinc, tin, indium and antimony are used. A hydrogenation catalyst containing one or more metals such as ruthenium, rhodium, palladium, rhenium, platinum, copper, nickel, cobalt, chromium, zinc, and tin is more preferably used.

These hydrogenation catalysts can be used as they are, or they can be used in the presence of ligands such as organic phosphines. However, heterogeneous metal-containing catalysts are used because of the ease of catalyst separation. Is preferred.
A compound containing a metal serving as a catalytically active component is used in the presence of a metal oxide such as silica, titanium, zirconia, activated alumina, or a composite metal oxide thereof, or activated carbon, or the above metal is used in silica, titanium, A metal oxide such as zirconia or activated alumina or a composite metal oxide thereof, or a supported catalyst supported on a support such as activated carbon is preferably used. The carrier used here may have a unique structure such as montmorillonite, silicate and zeolite. Moreover, you may use as a precipitation catalyst containing the metal used as said catalyst active component as an oxide or composite oxide, and the precipitation catalyst produced | generated in the presence of a support | carrier component can also be used. The content of the catalytically active component metal is usually about 0.1 to 90% by weight or less based on the whole catalyst. In the noble metal supported catalyst, the content is preferably about 0.1 to about 50% by weight, and in the metal oxide precipitation catalyst, the content is preferably about 10 to about 90% by weight.

In the present invention, the kind of the catalytically active metal, the combination of various metals, the combination with the carrier, the content of the catalytically active metal and the preparation method thereof as long as the above hydrogenation catalyst does not adversely affect the target hydrogenation reaction. Any combination is possible and is not limited.
In the use of a heterogeneous catalyst, it can be used by being activated in the reaction system by hydrogen present at the start of the reaction, but a catalyst activated in the presence of hydrogen before use may be used. The catalyst activation prior to use is a method of hydrotreating in a batch reactor in the presence or absence of a solvent that does not adversely affect the catalyst, a continuous fixed bed reactor or a fluidized bed reactor. And a hydrogen treatment method by circulating hydrogen. The temperature and pressure in hydroprocessing are selected from ranges suitable for each catalyst.

<Hydrogen>
Hydrogen used may be pure hydrogen, but hydrogen diluted with an inert gas such as nitrogen, helium or argon can also be used. The carbon monoxide concentration in the hydrogen gas is usually 10000 ppm or less, preferably 2000 ppm or less, more preferably 1000 ppm or less because there is concern about the influence on the hydrogen treatment efficiency.

EXAMPLES Hereinafter, although the Example of this invention is shown and this invention is demonstrated more concretely, this invention is not limited to a following example, unless it deviates from the summary.
Quantitative analysis of acids, saccharides, N-methylsuccinimide (MSI) and N-methylsuccinamic acid (NMSA) in the following examples was performed using LC, and amino acid quantitative analysis was performed using an amino acid analyzer under the following conditions. Went.

<Analysis of acids and sugars>
Column: ULTRON PS-80H 8.0 mmI. D. × 30cm
Eluent: Water (perchloric acid) (60% aqueous solution of perchloric acid 1.8 ml / 1 L-H ₂ O)
Temperature: 60 ° C

<Amino acid analysis>
Apparatus: Hitachi Amino Acid Analyzer L-8900
Analysis conditions: biogenic amino acid separation condition-ninhydrin coloring method (570 nm, 440 nm)
Standard product: PF (Wako amino acid mixture ANII type 0.8 ml + B type 0.8 ml → 10 ml)
Injection volume: 10 μl
Quantitative calculation: Pro is calculated from the peak area of 440 nm for other amino acids and 570 nm for other amino acids by the single point external standard method

<Quantitative analysis of succinic acid, N-methylsuccinimide (MSI) and N-methylsuccinamic acid (NMSA)>
Column: Ion exchange column MCI GEL CK08EH 8mm x 300mm
Temperature: 50 ° C
Eluent: 0.1% -trifluoroacetic acid aqueous solution Flow rate: 1.0 ml / min
Injection volume: 20 μl
Detector: RI detector

<Preparation of pyruvate carboxylase (PC) -enhanced strain>
(A) Extraction of genomic DNA of Brevibacterium flavum MJ233 strain Brevibacterium flavum MJ233 was established on April 28, 1975 at the Institute of Microbial Industrial Technology, Ministry of International Trade and Industry (currently National Institute of Advanced Industrial Science and Technology). Patent Biological Deposit Center) (deposited as FERM P-3068 at Tsukuba 1-1-1 Higashi 1-chome, Ibaraki, Japan 305-8856 Japan) and deposited on May 1, 1981 as an international deposit under the Budapest Treaty It has been transferred and deposited with a deposit number of FERM BP-1497.

A medium [urea 2 g, (NH ₄ ) ₂ SO ₄ 7 g, KH ₂ PO ₄ 0.5 g, K ₂ HPO ₄
0.5 g, MgSO ₄ · 7H ₂ O 0.5 g, FeSO ₄ · 7H ₂ O 6 mg, MnSO
_4. 4-5H ₂ O 6 mg, biotin 200 μg, thiamine 100 μg, yeast extract 1 g, casamino acid 1 g, glucose 20 g, dissolved in 1 L of distilled water] In 10 mL, Brevibacterium flavum MJ233 strain is cultured until the late logarithmic growth phase The cells were collected by centrifugation (10000 g, 5 minutes). The obtained cells were suspended in 0.15 mL of a 10 mM NaCl / 20 mM Tris buffer solution (pH 8.0) / 1 mM EDTA · 2Na solution containing lysozyme at a concentration of 10 mg / mL. Next, proteinase K was added to the suspension so that the final concentration was 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C. for 6 hours for lysis. To this lysate, add an equal amount of phenol / chloroform solution, shake gently at room temperature for 10 minutes, and then centrifuge (5,000 × g, 20 minutes, 10-12 ° C.) to obtain a supernatant. The fraction was collected and sodium acetate was added to a concentration of 0.3 M, and then twice as much ethanol was added and mixed. The precipitate collected by centrifugation (15,000 × g, 2 minutes) was washed with 70% ethanol and then air-dried. To the obtained DNA, add 5 mL of 10 mM Tris buffer (pH 7.5) / 1 mM EDTA · 2Na solution, and let stand at 4 ° C. overnight.
The template DNA was used.

(B) Construction of plasmid for replacing PC gene promoter The DNA fragment of the N-terminal region of the pyruvate carboxylase gene derived from Brevibacterium flavum MJ233 strain was obtained using the DNA prepared in (A) above as a template, This was performed by PCR using synthetic DNA (SEQ ID NO: 1 and SEQ ID NO: 2) designed based on the reported sequence of the gene of Corynebacterium glutamicum ATCC13032 strain (Cg0689 of GenBank Database Accession No. BA00000036). The DNA of SEQ ID NO: 1 used was phosphorylated at the 5 ′ end. Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentrated buffer, 0.3 μM each primer, 1 mM MgSO ₄ , 0.25 μM dNTPs were mixed to make a total volume of 20 μL. Reaction temperature conditions:
Using a DNA thermal cycler PTC-200 (manufactured by MJ Research), a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds and 72 ° C. for 1 minute was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 4 minutes. Confirmation of the amplified product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 0.9 kb. Recovery of the target DNA fragment from the gel was performed using the QIAQuick Gel Extraction Kit (QIAGEN
This was used as a PC gene N-terminal fragment.

On the other hand, a TZ4 promoter fragment constitutively highly expressed from Brevibacterium flavum MJ233 strain is a PCR using plasmid pMJPC1 (Japanese Patent Laid-Open No. 2005-95169) as a template and the synthetic DNAs described in SEQ ID NO: 3 and SEQ ID NO: 4. It was prepared by. The DNA of SEQ ID NO: 4 was phosphorylated at the 5 ′ end. Reaction solution composition: Template DNA 1 μ
L, Pfx DNA polymerase (manufactured by Invitrogen) 0.2 μL, 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MgSO ₄ , 0.25 μM dNTPs were mixed to make a total volume of 20 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 25 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 3 minutes. The amplification product was confirmed by separation by 1.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.5 kb was detected. Recovery of the target DNA fragment from the gel is performed using QIAQuick Ge
1 Extraction Kit (manufactured by QIAGEN) was used as a TZ4 promoter fragment.

The PC gene N-terminal fragment prepared above and the TZ4 promoter fragment were mixed, and ligation kit ver. 2 (manufactured by Takara Shuzo Co., Ltd.), cleaved with restriction enzyme PstI, separated by 1.0% agarose (SeaKem GTG agarose: FMC BioProducts) gel electrophoresis, and a DNA fragment of about 1.0 kb was isolated from QIAQuick Gel Extraction Kit (Manufactured by QIAGEN) was used as a TZ4 promoter :: PC gene N-terminal fragment. Further, this DNA fragment and E. coli plasmid pHSG299 (Takara Shuzo) were mixed with DNA prepared by cutting with PstI, and ligation kit ver. 2 (Takara Shuzo) were used for connection. Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kana machine and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was cleaved with the restriction enzyme PstI, whereby an inserted fragment of about 1.0 kb was recognized, and this was named pMJPC17.1.

The DNA fragment in the 5 ′ upstream region of the pyruvate carboxylase gene derived from Brevibacterium flavum MJ233 strain was obtained using the DNA prepared in Example 1 (A) as a template and the entire genome sequence reported. It was carried out by PCR using synthetic DNA (SEQ ID NO: 5 and SEQ ID NO: 6) designed based on the sequence of the gene of Glutamicum ATCC13032 strain (GenBank Database Accession No. BA00000036). Composition of reaction solution: template DNA 1 μL, Pfx DNA polymerase (manufactured by Invitrogen) 0.2 μL, 1-fold concentration buffer, 0.3 μM each primer,
1 mM MgSO ₄ and 0.25 μM dNTPs were mixed to make a total volume of 20 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes. The amplification product was confirmed by separation by 1.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.7 kb was detected. Recovery of the target DNA fragment from the gel was performed using the QIAQuick Gel Extraction Kit (QI
AGEN). The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo), and then ligated kit ver. 2 (Takara Shuzo) was used to bind to the SmaI site of E. coli vector pUC119 (Takara Shuzo), and Escherichia coli (DH5α strain) was transformed with the resulting plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was subjected to a PCR reaction using the synthetic DNAs shown in SEQ ID NO: 7 and SEQ ID NO: 6 as primers. Reaction solution composition: 1 ng of the above plasmid, Ex
-Taq DNA polymerase (Takara Shuzo) 0.2 μL, 1 × concentration attached buffer, 0
. 2 μM of each primer and 0.25 μM dNTPs were mixed to make a total volume of 20 μL. Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds and 72 ° C. for 50 seconds was repeated 20 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes. As a result of confirming the presence or absence of the inserted DNA fragment in this way, a plasmid that recognized an amplification product of about 0.7 kb was selected and named pMJPC5.1.

Next, the above-mentioned pMJPC17.1 and pMJPC5.1 were cleaved with restriction enzyme XbaI and mixed, and ligation kit ver. 2 (Takara Shuzo) were used for connection. The DNA fragment cleaved with restriction enzyme SacI and restriction enzyme SphI was separated by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis, and a DNA fragment of about 1.75 kb was prepared by QIAQuick Gel Extraction Kit (manufactured by QIAGEN Gel Extraction Kit). ). A DNA fragment in which the TZ4 promoter is inserted between the 5 ′ upstream region and the N-terminal region of the PC gene, and a DNA prepared by cleaving the plasmid pKMB1 (JP 2005-95169) containing the sacB gene with SacI and SphI Mixed and ligation kit ver. 2 (Takara Shuzo) were used for connection. Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kana machine and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes SacI and SphI, an insert fragment of about 1.75 kb was recognized, which was designated as pMJPC17.2.

(C) Preparation of PC-enhanced strain Plasmid DNA used for transformation of Brevibacterium flavum MJ233 / ΔLDH (lactate dehydrogenase gene-disrupted strain: Japanese Patent Laid-Open No. 2005-95169) is a calcium chloride method using plasmid DNA of pMJPC17.2. (Journal of Molecular Biology, 53, 159, 1970) was re-prepared from E. coli JM110 strain transformed. Transformation of Brevibacterium flavum MJ233 / ΔLDH strain is carried out by the electric pulse method (Res. Microbiol., Vol. 144, p.181-
185, 1993), and the obtained transformant was treated with kanamycin 25 μg / mL.
-Containing LBG agar medium [tryptone 10 g, yeast extract 5 g, NaCl 5
g, 20 g of glucose, and 15 g of agar were dissolved in 1 L of distilled water]. The strain grown on this medium is a plasmid in which pMJPC17.2 is a plasmid that cannot be replicated in Brevibacterium flavum MJ233 strain, so the PC gene of the plasmid and the same gene on the genome of Brevibacterium flavum MJ233 strain As a result of homologous recombination with the kanamycin, the kanamycin resistance gene and sacB gene derived from the plasmid should be inserted on the genome. Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 25 μg / mL kanamycin. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, sacB gene was removed by the second homologous recombination and sucrose-insensitive sucrose strains were obtained. Among the strains thus obtained, those in which the TZ4 promoter derived from pMJPC17.2 is inserted upstream of the PC gene and those that have returned to the wild type are included. Confirmation of whether the PC gene is promoter-substituted type or wild type can be easily confirmed by subjecting the cells obtained by liquid culture in LBG medium to direct PCR reaction and detecting the PC gene. . When analyzed using primers (SEQ ID NO: 8 and SEQ ID NO: 9) for PCR amplification of the TZ4 promoter and PC gene, a 678 bp DNA fragment should be observed in the promoter-substituted form. As a result of analyzing the sucrose-insensitive strain by the above method, a strain into which the TZ4 promoter was inserted was selected, and the strain was named Brevibacterium flavum MJ233 / PC-4 / ΔLDH.

(D) Measurement of pyruvate carboxylase enzyme activity The transformant Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain obtained in (C) above was cultured overnight in 100 mL of A medium containing 2% glucose. The obtained cells were collected, washed with 50 mL of 50 mM potassium phosphate buffer (pH 7.5), and resuspended in 20 mL of the same composition. The suspension was SONIFIER 350 (BRANSO
The supernatant obtained by crushing and centrifuging with N) was used as a cell-free extract. The pyruvate carboxylase activity was measured using the obtained cell-free extract. Enzyme activity was measured with 100 mM Tris / HCl buffer (pH 7.5), 0.1 mg / 10 ml biotin, 5 mM magnesium chloride, 50 mM.
Reaction is carried out at 25 ° C. in a reaction solution containing sodium hydrogen carbonate, 5 mM sodium pyruvate, 5 mM adenosine triphosphate, 0.32 mM NADH, 20 units / 1.5 ml malate dehydrogenase (manufactured by WAKO, derived from yeast) and the enzyme. Was done. 1 U was defined as the amount of enzyme that catalyzes the decrease of 1 μmol NADH per minute. The specific activity in the cell-free extract with enhanced pyruvate carboxylase expression was 0.1 U / mg protein. In addition, in the cells cultured in the same manner as the parent strain MJ233 / ΔLDH strain, it was below the detection limit of this activity measurement method.
Hereinafter, the Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain was used as an organic acid-producing bacterium for microbial cell preparation culture and organic acid production reaction.

[Preparation of succinate-containing culture medium]
<Seed culture>
Urea: 4 g, ammonium sulfate: 14 g, monopotassium phosphate: 0.5 g, dipotassium phosphate 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate heptahydrate: 20 mg, sulfuric acid Manganese hydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 5 g, casamino acid: 5 g, and distilled water: 100 mL of a medium of 1000 mL are placed in a 500 mL Erlenmeyer flask at 120 ° C. for 20 minutes. Heat sterilized. This was cooled to room temperature, 4 mL of a 50% aqueous glucose solution sterilized in advance was added, and Brevibacterium flavum MJ233 / PC-4 / ΔLDH constructed above was inoculated and seed-cultured at 30 ° C. for 24 hours.

<Main culture>
Ammonium sulfate: 1.0 g, monopotassium phosphate: 1.5 g, dipotassium phosphate 1.5 g, potassium chloride: 1.67 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate, heptahydrate Product: 40 mg, manganese sulfate / hydrate: 40 mg, D-biotin: 1.0 mg, thiamine hydrochloride: 1.0 mg, yeast extract 10 g, antifoaming agent (CE457: manufactured by NOF Corporation): 1.0 g and distilled water: 400 mL of 1000 mL of medium was placed in a 1 L fermentor and sterilized by heating at 120 ° C. for 20 minutes. After cooling to room temperature, 20 mL of 72% aqueous glucose solution sterilized in advance was added, and 20 mL of the above seed culture solution was added thereto, and the mixture was kept at 30 ° C. The pH was kept at 7.0% or less using 9.3% aqueous ammonia, main culture was performed at aeration of 300 mL / min, and stirring at 600 rpm for 24 hours. The dissolved oxygen concentration gradually decreased immediately after the start of the culture, and became almost zero 4 hours after the start of the culture. Thereafter, since the dissolved oxygen concentration increased 15 hours after the start of culture, when 380 μL of 72% glucose aqueous solution sterilized in advance was added, it rapidly decreased again and became almost zero. Since an increase in dissolved oxygen concentration was observed after about 13 minutes, 380 μL of a 72% aqueous glucose solution sterilized in advance was added and lowered again. Thereafter, a similar increase was observed about every 13 minutes, but it was reduced by the same method each time. The OD660 after 24 hours of culture was 87.3.

<Succinic acid production>
Monoammonium phosphate: 84.4 mg, diammonium phosphate: 75.8 mg, potassium chloride 149.1 mg, magnesium sulfate heptahydrate: 0.2 g, ferrous sulfate heptahydrate: 8 mg, manganese sulfate Hydrate: 8 mg, D-biotin: 80 μg, thiamine hydrochloride: 80 μg and distilled water: 200 mL of medium was placed in a 500 mL Erlenmeyer flask and sterilized by heating at 120 ° C. for 20 minutes. After cooling to room temperature, it was placed in a 1 L jar fermenter. To 200 mL of this suspension, 90 mL of the culture solution obtained by the above main culture, 72% glucose solution sterilized in advance: 40 mL, and sterilized water: 125 mL were added and mixed, and kept at 35 ° C. The pH was maintained at 7.6 using an aqueous solution of ammonium carbonate: 154 g, 28% aqueous ammonia: 239 ml, distilled water: 650 mL, and an organic acid production reaction was performed while stirring at 200 rpm. At 21 hours after the start of the reaction, the produced succinic acid concentration was 34.8 g / L, the produced malic acid concentration was 3.1 g / L, the produced fumaric acid concentration was 1.1 g / L, the produced acetic acid concentration was 5.0 g / L, and alanine. The concentration was 2.1 g / L and the valine concentration was 1.4 g / L.
The main culture contained trehalose 0.27 g / L and unreacted glucose 0.37 g / L.
Bacteria were separated from the succinic acid fermentation broth prepared as described above by an ultrafiltration membrane having a molecular weight cut off of 1300, concentrated under reduced pressure and heated, and used in the following Examples and Comparative Examples.

[Example 1]
A 70 mL Hastelloy pressure-resistant container containing a magnetic stir bar was charged with 10.025 g of a succinic acid fermentation solution having a succinic acid concentration of 25.06 wt% obtained in the organic acid production culture and 15.016 g of methanol, and sealed. The inside of the reactor was replaced with nitrogen gas, placed in an electric furnace preheated to 250 ° C., and heated and stirred for 5 hours. After completion of the heating, the reactor was cooled to ambient temperature, the reactor was opened, the reaction solution was collected, and the product composition was analyzed by the above analysis method. The succinic acid conversion rate was 97.3%, the N-methylsuccinimide (MSI) yield was 83.8%, and the N-methylsuccinamic acid (NMSA) yield was 5.56%. The reaction solution was adjusted to pH 9.5 with 1N hydrochloric acid, and the product composition was analyzed after 5 days. N-methylsuccinimide selectivity / N-methylsuccinamic acid selectivity at each pH The ratio (MSI / NMSA ratio) was determined and shown in Table 1.

[Example 2]
The same procedure as in Example 1 was performed except that the pH after the reaction was adjusted to 9.04. The results are shown in Table 1.
[Example 3]
The same procedure as in Example 1 was performed except that the pH after the reaction was adjusted to 8.50. The results are shown in Table 1.

[Example 4]
The same procedure as in Example 1 was performed except that the pH after the reaction was adjusted to 7.9. The results are shown in Table 1.
[Example 5]
The same procedure as in Example 1 was performed except that the pH after the reaction was adjusted to 7.53. The results are shown in Table 1.

[Example 6]
The same procedure as in Example 1 was performed except that the pH after the reaction was adjusted to 7.06. The results are shown in Table 1.
[Example 7]
The same procedure as in Example 1 was performed except that the pH after the reaction was adjusted to 6.51. The results are shown in Table 1.

[Example 8]
The same procedure as in Example 1 was performed except that the pH after the reaction was adjusted to 6.00. The results are shown in Table 1.
[Comparative Example 1]
The same procedure as in Example 1 was performed except that the pH was not adjusted after the reaction. The results are shown in Table 1.

[Comparative Example 2]
The same procedure as in Example 1 was performed except that the pH after the reaction was adjusted to 10.1. The results are shown in Table 1.

  As shown in Table 1, when the reaction mixture was stored at the post-reaction pH (Comparative Example 1) and when the reaction mixture was adjusted to a pH exceeding 10 and stored (Comparative Example 2), the pH of the reaction mixture Was adjusted to 9.8 or less (Examples 1 to 8), it was found that the MSI / NMSA ratio was maintained high.

Claims (3)

In the method for producing an N-alkyl succinimide by reacting a composition containing succinic acid represented by the following general formula (I) and a salt thereof with an alkylating agent, the pH of the reaction mixture after the reaction is 9 A method for producing a composition containing an N-alkylsuccinimide represented by the following general formula (II):

(In the formula, X and Y represent a hydrogen atom, NH ₄ ⁺ or another monovalent cation, and at least one of X and Y represents NH ₄ ⁺ .)

(In the formula, R ¹ is an optionally substituted linear, branched or cyclic C ₁ -C ₆ alkyl group.)   The method according to claim 1, wherein the composition containing succinic acid and a salt thereof is derived from a biomass resource. The method according to claim 1 or 2, wherein R ¹ is a methyl group or an ethyl group.