JP2009000097A - Method for producing 4-halo-3-hydroxybutyramide and its derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a 4-halo-3-hydroxybutyramide, completing reaction in a short time and not requiring a lot of nitrile hydratase. <P>SOLUTION: The method for producing the 4-halo-3-hydroxybutyramide comprises a step I of subjecting an epihalohydrin and/or a 1,3-dihalo-2-propanol and a cyanide donor to enzymatic reaction to synthesize a 4-halo-3-hydroxybutyronitrile and a step II of bringing the 4-halo-3-hydroxybutyronitrile into contact with nitrile hydratase so that the product of the concentration (mass%) of the nitrile with salt concentration (total of a salt of an alkali metal salt with a buffer agent salt and salts of metals composed of nickel, zinc and cobalt) (mass%) becomes ≤160. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing 4-halo-3-hydroxybutyramide. Optically active 4-halo-3-hydroxybutyramide is a substance useful as a raw material for synthesizing various pharmaceuticals and physiologically active substances, and the (R) -form is particularly useful as a raw material for synthesizing L-carnitine.

Examples of the method for producing 4-halo-3-hydroxybutyramide include a method for hydrating 4-halo-3-hydroxybutyronitrile. In general, as a method for producing an amide by hydration of nitrile, a method using a mineral acid such as hydrochloric acid or an alkali, a method using an alkali and hydrogen peroxide in combination, and the like, the process becomes complicated. There is a problem that the corresponding acid is by-produced and the yield is reduced (Patent Document 1). In addition, as a biological reaction, a method of producing an amide compound by hydrating a nitrile compound by the action of nitrile hydratase is known, and the present applicants first described 4-halo-3-hydroxybutyl. As a method for producing amides, a method of causing nitrile hydratase to act on 4-halo-3-hydroxybutyronitrile (Patent Document 2) has been proposed.

  As a method for producing 4-halo-3-hydroxybutyronitrile, a method of reacting an epihalohydrin and an alkali metal salt of cyanic acid (Patent Document 3), a method of reacting an epihalohydrin and hydrocyanic acid (Patent Document 4), 1,3- A method (Patent Document 5) of producing by enzymatic reaction from dihalo-2-propanol and hydrocyanic acid or an alkali metal salt of hydrocyanic acid can be mentioned. As an enzyme used in the enzymatic reaction, halohydrin epoxidase can be exemplified, and the method of producing by enzymatic reaction is particularly useful because 4-halo-3-hydroxybutyronitrile to be produced is an optically active substance.

  Unreacted hydrocyanic acid and raw materials remain in the reaction solution containing 4-halo-3-hydroxybutyronitrile produced by the above production method. Further, when 1,3-dihalo-2-propanol and hydrocyanic acid or alkali metal salt of hydrocyanic acid are used as raw materials, and when epihalohydrin and alkali metal salt of hydrocyanic acid are used as raw materials, 4-halo-3-hydroxybutyro Along with the formation of nitrile, theoretically an equimolar or more inorganic salt is formed and remains in the reaction solution. Subsequently, when the amidation reaction is carried out with nitrile hydratase, since the nitrile hydratase is deactivated by the remaining cyanate, 4-halo-3-hydroxybutyronitrile produced by the above-mentioned production method is usually reacted. The solution is extracted with an organic solvent, and the solvent is removed together with hydrocyanic acid under reduced pressure. If necessary, it is recovered by a known method of obtaining a purified fraction by distillation, and 4-halo-3-hydroxy with nitrile hydratase. It is used for the butyramide production process.

  However, the above-mentioned method has a large cost / environmental burden for the use, extraction and distillation purification of organic solvents, and is not necessarily industrially suitable. In addition, when the amidation reaction is carried out with nitrile hydratase, the reaction rate of the amidation reaction is very slow even if the cyanic acid is removed to 1 ppm or less by a method such as distillation. Nitrile hydratase may be required.

The present applicants have previously proposed a method (Patent Document 6) for reducing hydrocyanic acid contained in a very small amount in a nitrile compound by a chemical method such as metal addition, but the amount of metal addition is insufficient. In the case of excess, some nitrile hydratase was deactivated, and it was difficult to sufficiently increase the reaction rate of the amidation reaction.
Japanese Patent No. 2588930 Japanese Patent No. 3014171 Japanese Patent Publication No. 5-69818 JP 2002-241357 A Japanese Patent No. 3073037 Japanese Patent No. 3428404

  An object of the present invention is to provide a method for producing 4-halo-3-hydroxybutyramide having high productivity and suitable for both cost and environment.

  In order to solve the above problems, the present inventors have obtained a reaction solution containing 4-halo-3-hydroxybutyronitrile obtained from an epihalohydrin and a cyanide donor, or 1,3-dihalo-2-propanol and a cyanide donor. A 4-halo-3-hydroxybutyronitrile fraction obtained by purifying a reaction solution containing 4-halo-3-hydroxybutyronitrile obtained by the action of halohydrin epoxidase from an organic solvent by distillation and distillation with an organic solvent The production method of 4-halo-3-hydroxybutyramide by the action of nitrile hydratase was examined without passing through the recovery step.

  In the process, it was found that nitrile hydratase was inactivated under conditions where a high concentration of 4-halo-3-hydroxybutyronitrile and a high concentration of inorganic salt existed. As a result of diligent studies to solve this problem, the product of the concentration (mass%) of 4-halo-3-hydroxybutyronitrile and the concentration (mass%) of inorganic salt was 160 or less, and 4-halo- It has been found that by reacting 3-hydroxybutyronitrile with nitrile hydratase, it is possible to produce 4-halo-3-hydroxybutyramide in a short time without requiring complicated purification operations and a large amount of nitrile hydratase. Completed the invention.

  That is, the present invention is a method for producing 4-halo-3-hydroxybutyramide including the following steps.

Step I: Step of synthesizing 4-halo-3-hydroxybutyronitrile by subjecting epihalohydrin and / or 1,3-dihalo-2-propanol and cyanide donor to an enzymatic reaction,
Step II: The product of the concentration (mass%) of nitrile and the salt concentration (total of alkali metal salt and buffer salt and metal salt made of nickel, zinc, cobalt) (mass%) is 160 or less. Contacting with nitrile hydratase.

  According to the present invention, 4-halo-3-hydroxybutyramide can be obtained without going through complicated 4-halo-3-hydroxybutyronitrile extraction / distillation purification steps, so that the productivity is high and the cost is reduced. A method for producing 4-halo-3-hydroxybutyramide suitable for both environmental aspects can be provided.

  Hereinafter, the present invention will be described in detail.

Step I: Synthesis of 4-halo-3-hydroxybutyronitrile The method for producing 4-halo-3-hydroxybutyronitrile is not particularly limited, but epihalohydrin and / or 1,3-dihalo-2 described below is used. A method in which -propanol and cyanide donor are subjected to an enzymatic reaction (hereinafter sometimes referred to as a cyanating enzyme reaction) is preferable in that optically active 4-halo-3-hydroxybutyronitrile is obtained.

  The cyanation enzyme reaction means an enzyme conversion reaction using epihalohydrin and / or 1,3-dihalo-2-propanol and cyanide donor as a substrate, using an enzyme called halohydrin epoxidase.

(Step I-1) 4-halo-3-hydroxybutyronitrile 4-halo-3-hydroxybutyronitrile refers to a compound represented by the following general formula (1).

(In the formula, X ₁ represents a halogen atom. Specifically, they are a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a chlorine atom and a bromine atom, and most preferably a chlorine atom. * Represents an asymmetric carbon. The configuration may be either (R) or (S), but (R) is preferred.
Moreover, optically active 4-halo-3-hydroxybutyronitrile is 4-halo-3-containing one enantiomer (for example, R form) more than the other enantiomer (for example, S form). It refers to hydroxybutyronitrile or 4-halo-3-hydroxybutyronitrile consisting of only one of the enantiomers.

  Specifically, (R) -4-fluoro-3-hydroxybutyronitrile, (S) -4-fluoro-3-hydroxybutyronitrile, (R) -4-chloro-3-hydroxybutyronitrile, S) -4-chloro-3-hydroxybutyronitrile, (R) -4-bromo-3-hydroxybutyronitrile, (S) -4-bromo-3-hydroxybutyronitrile, (R) -4- Examples include iodo-3-hydroxybutyronitrile and (S) -4-iodo-3-hydroxybutyronitrile, preferably (R) -4-chloro-3-hydroxybutyronitrile, (S) -4- Chloro-3-hydroxybutyronitrile, (R) -4-bromo-3-hydroxybutyronitrile, (S) -4-bromo-3-hydroxybutyronitrile, especially (R) -4-chloro- 3 Hydroxybutyronitrile is preferable.

(Step I-2) A halohydrin epoxidase halohydrin epoxidase is a dehalogenated 1,3-dihalo-2-propanol represented by the following general formula (2), and the following general formula (3 ) And an enzyme (EC number: 4.5.1.-) having an activity of synthesizing an epihalohydrin and an activity of catalyzing the reverse reaction.

(In the formula, X ₁ and X ₂ represent a halogen atom. Fluorine, chlorine, bromine and iodine are preferred, and chlorine and bromine are particularly preferred. Specifically, 1,3-difluoro-2-propanol, 1,3- Examples include dichloro-2-propanol, 1,3-dibromo-2-propanol, 1,3-diiodo-2-propanol, and preferably 1,3-dichloro-2-propanol, 1,3-dibromo-2 -Propanol.)

(Wherein X ₁ represents a halogen atom, preferably fluorine, chlorine, bromine or iodine, particularly preferably chlorine or bromine. Specifically, epifluorohydrin, epichlorohydrin, epibromohydrin, epiiodo (Hydrin and the like can be mentioned, and epichlorohydrin and epibromohydrin are particularly preferable.)
The microorganisms that produce this enzyme include Agrobacterium sp., Arthrobacter sp., Aureobacterium sp., Corynebacterium sp., Examples include microorganisms belonging to the genus Leifsonia sp., Microbacterium sp., Mycobacterium sp., Pseudomonas sp., Rhizobium sp. . Preferably, Aureobacterium sp., Corynebacterium sp., Leifsonia sp., Microbacterium sp., Mycobacterium sp. Aureobacterium sp. DH095 (FERM P-12360), Corynebacterium sp. N-1074 (FERM BP-2643), Leifsonia sp. MRC03 (FERM AP-20453), Microbacterium sp. N-4701 (FERM BP-2644), Mycobacterium sp. GP1 is (R) -form 4-halo-3-hydroxybutyro Nitrile is more preferable, and Corynebacterium sp. N-1074 (FERM BP-2643) is particularly preferable.

Among the halohydrin epoxidases whose amino acid sequences have been identified, the GenBank database (http: //www.ncbi.nlm) provided by the National Center for Biotechnology Information (NCBI) .nih.gov / entrez / query.fcgi? CMD = search & DB = protein), it is registered with the following Accession No.

・ Accession No. BAA14361 (amino acid sequence of HheA derived from Corynebacterium sp. N-1074)
・ Accession No. AAK92100 (amino acid sequence of HheA _AD2 derived from Arthrobacter sp. AD2 strain)
・ Accession No. BAA14362 (amino acid sequence of HheB derived from Corynebacterium sp. N-1074)
・ Accession No. AAK73175 (amino acid sequence of HheB _GP1 derived from Mycobacterium sp. GP1 strain)
・ Accession No. AAK92099 (amino acid sequence of HheC derived from Agrobacterium radiobacter AD1 strain)
Accession No. AAD34609 (Amino acid sequence of HalB derived from Agrobacterium tumefaciens)
HheB derived from Corynebacterium sp. N-1074 strain, HheB _GP1 derived from Mycobacterium sp. GP1 strain is (R) -form 4-halo-3-hydroxybutyronitrile. HheB derived from the Corynebacterium sp. N-1074 strain is particularly preferable in terms of the obtained point.

  The halohydrin epoxidase can be produced by culturing the microorganism. It can also be produced by a recombinant microorganism produced by cloning a gene encoding halohydrin epoxidase and incorporating the gene. A halohydrin epoxidase obtained by culturing a genetically modified microorganism prepared by incorporating a halohydrin epoxidase gene is preferably used because a desired halohydrin epoxidase can be obtained in a large amount.

  Further, examples of the host microorganism used for transformation include microorganisms belonging to the genus Escherichia coli or Rhodococcus. However, the present invention is not particularly limited thereto, and other host microorganisms can also be used.

  Furthermore, a halohydrin epoxidase obtained by modifying a natural halohydrin epoxidase gene and modifying the enzyme function can also be used in the present invention.

  As a medium for culturing microorganisms producing the halohydrin epoxidase, any medium can be used as long as these microorganisms can usually grow. As the carbon source, for example, sugars such as glucose, sucrose, maltose and fructose, organic acids such as acetic acid, citric acid and fumaric acid or salts thereof, alcohols such as ethanol and glycerol can be used. As the nitrogen source, for example, various inorganic and organic acid ammonium salts can be used in addition to general natural nitrogen sources such as peptone, meat extract, yeast extract and amino acids. In addition, inorganic acids such as sulfuric acid, hydrochloric acid, phosphoric acid and boric acid or salts thereof, inorganic salts containing sodium, magnesium, potassium, calcium, etc. that can be used by microorganisms used, iron, manganese, zinc, cobalt, nickel trace metal salts In addition, vitamins such as vitamins B1, B2, C, and K are appropriately added as necessary as a microorganism growth promoter. The culture of the microorganism of the present invention is usually performed by liquid culture, but can also be performed by solid culture.

  The culture is performed at a temperature of 10 to 50 ° C. and in the range of pH 2 to 11. In order to promote the growth of microorganisms, aeration and agitation may be performed. Microorganisms obtained by culturing are microbial cells obtained from the culture solution as it is or by collection operations such as centrifugation from the culture, or treated cells (for example, microbial cell disruption solution, crude enzyme, purified enzyme) or It can be used as a halohydrin epoxidase in the form of a microbial cell immobilized by a conventional method or a processed microbial cell product.

(Step I-3) Cyanide Donor A cyanide donor means a compound that generates cyanide ion (CN ⁻ ) or hydrogen cyanide when added to a reaction solution, and is not particularly limited, but may be referred to as hydrogen cyanide (hereinafter referred to as cyanide or HCN). ), Potassium cyanide (hereinafter sometimes referred to as KCN), sodium cyanide (hereinafter sometimes referred to as NaCN), and acetone cyanohydrin (hereinafter sometimes referred to as ACH). Hydrogen cyanide, potassium cyanide and sodium cyanide are preferably used. In particular, hydrogen cyanide is preferable because the amount of by-produced salt is small when adjusting the pH to the enzymatic reaction.

(Step I-4) Cyanation enzyme reaction The cyanation enzyme reaction is carried out by using cyanide donor, epihalohydrin or 1,3-dihalo-2-propanol, and halohydrin epoxidase in water or a buffer solution. Start by adding. As the buffer solution, for example, a buffer solution composed of a salt such as hydrocyanic acid, phosphoric acid, boric acid, citric acid, glutaric acid, malic acid, malonic acid, o-phthalic acid, succinic acid or acetic acid, Tris buffer Or Good buffer etc. can be illustrated and used. Among them, a buffer solution composed of hydrocyanic acid and a salt thereof and a Tris buffer solution are preferable. A buffer solution composed only of hydrocyanic acid and a salt thereof is particularly preferable in that impurities derived from the buffer agent are not mixed.

  In the enzyme reaction using the halohydrin epoxidase, the concentration of epihalohydrin or 1,3-dihalo-2-propanol in the reaction solution is preferably 0.1 to 10 wt%. Moreover, the usage-amount of a cyanide donor is usually 1-3 times mole with respect to epihalohydrin or 1,3-dihalo-2-propanol. Epihalohydrin or 1,3-dihalo-2-propanol and cyanide donor can be added to the reaction solution all at once or in divided portions.

  The reaction temperature is preferably 0 to 40 ° C. and the pH is preferably 4 to 10. In addition, when 1,3-dihalo-2-propanol is used as the raw material, as the reaction proceeds, hydrogen halide is generated in the reaction solution and the pH is lowered, so an alkali is additionally added to the reaction system. Thus, it is preferable to adjust and maintain the pH in the system in a region where the enzyme activity is exhibited.

  The alkali to be used is not particularly limited as long as it forms a salt with hydrogen halide and the aqueous solution of the salt is in a pH range where the activity of the enzyme is exerted. For example, an alkali metal or an alkaline earth metal or Examples thereof include salts of ammonia with hydroxides or weak acids. Sodium hydroxide, potassium hydroxide, sodium cyanide and potassium cyanide are preferred. The form of alkali used is not particularly limited, but an aqueous solution is preferable from the viewpoint of ease of handling.

  The reaction time varies depending on the concentration of the substrate, the amount of halohydrin epoxidase, and the reaction conditions, but it is preferable to set the conditions so that the reaction time is usually completed in 1 to 120 hours.

(Step I-5) Separation of 4-halo-3-hydroxybutyronitrile and unreacted cyanide donor In the synthesis of 4-halo-3-hydroxybutyronitrile, it is produced by an enzymatic reaction using halohydrin epoxidase. Regardless of whether or not 1,3-dihalo-2-propanol and hydrocyanic acid or alkali metal salt of hydrocyanic acid are used as raw materials, and when epihalohydrin and alkali metal salt of hydrocyanic acid are used as raw materials, 4-halo-3 -In theory, an equimolar or more salt is formed with the formation of hydroxybutyronitrile. Even when epihalohydrin and hydrocyanic acid are used as raw materials, a salt is produced by adjusting the pH of the reaction solution. Further, unreacted cyanide donor and 1,3-dihalo-2-propanol or epihalohydrin remain in the reaction solution.

  For the above reasons, 4-halo-3-hydroxybutyronitrile generated and accumulated in the reaction solution is extracted with a solvent such as ethyl acetate, and the solvent is removed under reduced pressure to remove 4- Syrups of halo-3-hydroxybutyronitrile can be obtained, and these syrups are distilled under reduced pressure, recovered as a further purified fraction, and hydrated with nitrile hydratase (hereinafter referred to as amide). From the viewpoint of cost and environment for the use of organic solvents, extraction and distillation purification, after the completion of the 4-halo-3-hydroxybutyronitrile synthesis reaction. The reaction solution was concentrated by distillation, and a fraction containing an unreacted cyanide donor and epihalohydrin or 1,3-dihalo-2-propanol was obtained. And release, it is advantageous to subject the amidation enzymatic reaction.

  In the concentration, the 4-halo-3-hydroxybutyronitrile synthesis reaction solution is distilled in order to increase the separation efficiency of the unreacted cyanide donor from the reaction solution and prevent the decomposition of 4-halo-3-hydroxybutyronitrile. It is preferred to keep the bottom neutral or acidic. The pH of the distillation bottom is preferably 7 or less, and more preferably 5 or less. The lower limit of the pH is not particularly limited, but it prevents corrosion of the apparatus due to vapors such as cyanic acid and hydrogen halide generated during concentration, or when adjusting the pH to near neutral in the subsequent Step I 'and Step II. From the viewpoint of reducing the amount of salt produced, the pH is preferably 0 or more, and more preferably 2 or more.

  When the pH of the 4-halo-3-hydroxybutyronitrile synthesis reaction solution exceeds 7, the pH can be increased by adding an acid stronger than hydrocyanic acid to the reaction solution or heating the reaction solution to 30 ° C. or higher and below the boiling point. Can be reduced to 7 or less. In addition, when the pH of the cyanation enzyme reaction solution is controlled by adding an alkali, the pH can be lowered to 7 or less by blocking the alkali and continuing the reaction.

  When the reaction liquid is heated to 30 ° C or more and below the boiling point, the addition of an alkali is blocked if an acid stronger than hydrocyanic acid is added to the reaction liquid or the pH of the cyanation enzyme reaction liquid is controlled by the addition of an alkali. Then, by continuing the reaction and lowering the pH to 7 or less, heating is preferably performed to obtain a desired pH.

  The acid is not particularly limited as long as it generates a hydrocyanic acid gas by mixing with an acid stronger than hydrocyanic acid, that is, an aqueous solution of a salt composed of hydrocyanic acid and a strong base, but organic acids such as p-toluenesulfonic acid and acetic acid. And inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid.

  When the pH is adjusted to a value lower than the desired pH, it can be adjusted to the desired pH by adding an alkali. The alkali to be used is not particularly limited as long as it forms a salt with hydrogen halide and the aqueous solution of the salt is in a pH range where the activity of the enzyme is exerted. For example, an alkali metal or an alkaline earth metal or Examples thereof include salts of ammonia with hydroxides or weak acids. Sodium hydroxide and potassium hydroxide are preferred. Metal cyanide salts such as sodium cyanide and potassium cyanide are not preferred because the cyan ion concentration of the 4-halo-3-hydroxybutyronitrile synthesis reaction solution increases. The form of alkali used is not particularly limited, but an aqueous solution is preferable from the viewpoint of ease of handling. When the temperature of the reaction solution is 30 ° C. or higher, pH adjustment by adding alkali induces decomposition of optically active 4-halo-3-hydroxybutyronitrile, which is not preferable.

  Further, when there is a solid derived from the halohydrin epoxidase used in the enzyme reaction, it may be removed before concentration or not. In the case of removing the solid matter, it may be carried out by a conventional method, and examples thereof include methods such as filtration and centrifugation.

  The pressure and temperature can be appropriately determined according to the type of apparatus used and the purpose of distillation. Usually, the distillation temperature is 0 to 110 ° C., preferably 40 to 110 ° C., and the pressure is 1 to 760 Torr, preferably 50 to 760 Torr. When it is desired to increase the concentration rate, the concentration rate can be increased by setting the distillation temperature to 40 to 80 ° C., more preferably 40 to 60 ° C., and the pressure to 1 to 300 Torr, more preferably 50 to 150 Torr. .

  It is preferable to reduce the cyanide donor remaining in the distillation bottom as much as possible because it significantly inhibits the activity of nitrile hydratase in the subsequent Step II. The concentration is preferably 100 ppm or less, more preferably 10 ppm or less, and particularly preferably 1 ppm or less.

  Distillation can also be carried out while blowing air or inert gas in order to increase the separation efficiency of unreacted cyanide donor and epihalohydrin or 1,3-dihalo-2-propanol from the reaction solution. When reducing the concentration of the cyanide donor to 100 ppm or less, it is preferable to distill while blowing air or an inert gas.

  As an inert gas, a gas that does not substantially react with 4-halo-3-hydroxybutyronitrile, an unreacted cyanide donor, and epihalohydrin or 1,3-dihalo-2-propanol, which are products during distillation. If it is, it will not restrict | limit in particular, Water vapor | steam, nitrogen, a carbon dioxide, helium, and argon are illustrated. These gases may be used alone, or two or more kinds thereof may be mixed, or the composition may be constant or change during distillation. Although it does not restrict | limit especially as air, It is preferable to use by diluting with said inert gas so that excess oxygen may not be introduce | transduced in the system which performs distillation. The gas flow rate is not particularly limited, but is preferably set so that the gas phase in the reaction vessel is replaced in 1 to 120 hours.

  The distilled cyanide donor and the fraction containing epihalohydrin or 1,3-dihalo-2-propanol were optionally cooled to a temperature sufficient to collect them, for example, -10 ° C to 10 ° C. Can be collected using a cooling tube.

The concentration time is not particularly limited, but it is preferable to carry out the concentration so that the distillation bottom 4-halo-3-hydroxybutyronitrile has a desired concentration in 1 to 120 hours. The concentration of 4-halo-3-hydroxybutyronitrile in the distillation bottom is not particularly limited, but when it exceeds 30% by weight, depending on the 4-halo-3-hydroxybutyronitrile synthesis reaction conditions, a salt of hydrogen halide In some cases, it may be difficult to handle due to precipitation, and therefore it is preferable not to exceed 30% by weight.
Even if the concentration of 4-halo-3-hydroxybutyronitrile is concentrated to 30% by weight or more, the recovery rate of cyanide donor and 1,3-dihalo-2-propanol and the concentration in the distillation bottom do not reach the desired values. In addition, water can be added to the distillation bottom to further concentrate.

  In this step, the concentration of epihalohydrin or 1,3-dihalo-2-propanol in the reaction system, the concentration of optically active 4-halo-3-hydroxybutyronitrile, and the optical purity are determined by, for example, gas chromatography (GLC) or It can be measured and quantified by high performance liquid chromatography (HPLC). The concentration of cyanide donor in the reaction system can be quantified by titration, for example.

  The solution containing 4-halo-3-hydroxybutyronitrile obtained as described above is subjected to Step I ′ described below.

Step I ′: Amidation enzyme reaction pretreatment step I ′ is a heat treatment of a solution containing 4-halo-3-hydroxybutyronitrile and / or a metal salt selected from the group consisting of nickel, zinc and cobalt. It is a step of adding one or more types. By carrying out this step, the step of obtaining 4-halo-3-hydroxybutyramide by the action of nitrile hydratase from 4-halo-3-hydroxybutyronitrile in the subsequent Step II can be smoothly advanced. .

  Although it does not restrict | limit especially about the preparation method of the solution containing 4-halo-3-hydroxybutyronitrile, From a viewpoint with a low cost side in the 4-halo-3-hydroxybutyronitrile synthesis | combination process, and an environmental load, it is the above-mentioned. A solution containing 4-halo-3-hydroxybutyronitrile obtained in Step I is preferred.

(Step I′-1) 4-halo-3-hydroxybutyramide 4-halo-3-hydroxybutyramide refers to a compound represented by the following general formula (4).

(In the formula, X ₁ represents a halogen atom. Specifically, they are a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a chlorine atom and a bromine atom, and most preferably a chlorine atom. * Represents an asymmetric carbon. The configuration may be either (R) or (S), but (R) is preferred.
Optically active 4-halo-3-hydroxybutyramide is 4-halo-3-hydroxy in which one enantiomer (for example, R-form) is contained more than the other enantiomer (for example, S-form). It refers to butyramide or 4-halo-3-hydroxybutyramide consisting of only one of the enantiomers.

  Specifically, (R) -4-fluoro-3-hydroxybutybutyramide, (S) -4-fluoro-3-hydroxybutyramide, (R) -4-chloro-3-hydroxybutyramide, (S) -4-chloro-3-hydroxybutyramide, (R) -4-bromo-3-hydroxybutyramide, (S) -4-bromo-3-hydroxybutyramide, (R) -4-iodo-3-hydroxy Butyramide, (S) -4-iodo-3-hydroxybutyramide, and (R) -4-chloro-3-hydroxybutyramide, (S) -4-chloro-3-hydroxybutyramide, (R) -4-bromo-3-hydroxybutyramide, (S) -4-bromo-3-hydroxybutyramide, especially (R) -4-chloro-3-hydroxybutyramide Soil is preferred.

(Step I′-2) Heat treatment The heat treatment of 4-halo-3-hydroxybutyronitrile refers to heating a solution containing optically active 4-halo-3-hydroxybutyronitrile. The heating method is not particularly limited as long as the object of the present invention is achieved, and any of pressurized, reduced pressure, and normal pressure conditions can be selected. Moreover, the method etc. which heat the solution containing 4-halo-3-hydroxybutyronitrile obtained by the said process I above the boiling point, and recirculate | reflux or distill off are mentioned.

  The concentration of the solution containing 4-halo-3-hydroxybutyronitrile at the time of heating is not particularly limited, but it is preferable to reduce it as much as possible because cyan ions significantly inhibit the activity of nitrile hydratase. The concentration is preferably 100 ppm or less, more preferably 10 ppm or less, and particularly preferably 1 ppm or less.

  The heating time is about 0.1 to 100 hours, preferably 0.5 to 50 hours, and more preferably 1 to 10 hours. The heating temperature can be processed in a range from about 40 ° C. to a temperature at which 4-halo-3-hydroxybutyronitrile does not decompose, preferably 60 to 150 ° C., more preferably 80 to 100 ° C.

  In order to prevent the decomposition of 4-halo-3-hydroxybutyronitrile, the pH is preferably 7 or less, more preferably 5 or less. The lower limit of the pH is not particularly limited, but it prevents corrosion of the apparatus due to steam such as hydrogen halide during heating, or a salt generated when adjusting the pH to near neutral in the subsequent step of adding metal salt and Step II. From the viewpoint of reducing the production amount of, the pH is preferably 0 or more, and more preferably 2 or more.

  In addition, after the heat treatment, it is preferable that the nitrile hydratase is adjusted again to an optimum pH at which high activity is exhibited, and then the process proceeds to the amidating enzyme reaction in Step II.

  The method for adjusting the pH is not particularly limited unless cyanic acid and / or metal cyanide is used, but pH adjustment by adding an alkali is 0 to 30 from the viewpoint of preventing decomposition of 4-halo-3-hydroxybutyronitrile. It is preferable to carry out at ° C.

(Step I'-3) Addition of metal salt A trace amount of metal salt is added to a solution containing 4-halo-3-hydroxybutyronitrile to add a trace amount of 4-halo-3-hydroxybutyronitrile. It can reduce by making the cyanide contained into a metal complex. Thereby, the amidation enzyme reaction of the process II can be advanced smoothly.

  As the metal, cobalt, nickel, and zinc are preferable, and the form of the salt is not particularly limited, but examples thereof include nitrates, halide salts, sulfates, and carboxylates.

  The amount of the metal salt to be added is not particularly limited, but 4-halo-3-hydroxybutyronitrile is usually used from the viewpoint of sufficiently reducing the influence of cyanide ions and preventing the deactivation of nitrile hydratase in Step II. An amount corresponding to the concentration of cyanide ions contained in the solution to be contained can be added.

  The cyanide concentration before adding the metal salt to the aqueous solution containing 4-halo-3-hydroxybutyronitrile is preferably 100 ppm or less, more preferably 10 ppm or less, and particularly preferably 1 ppm or less. preferable. The concentration of the metal salt to be added is preferably 10,000 ppm or less in the reaction solution in Step II from the viewpoint of the stability of nitrile hydratase. It is more preferably 1,000 ppm or less, and particularly preferably 10 ppm or less. About the minimum of the said metal salt density | concentration, it is preferable to set it as 0.1 ppm or more in the reaction liquid in process II from a viewpoint that it is a density | concentration which can reduce the influence of a cyanide ion.

  When the concentration of the metal salt to be added is higher than 10,000 ppm in the reaction solution, partial deactivation of nitrile hydratase may occur. In Step II, before contacting the nitrile hydratase with the solution containing 4-halo-3-hydroxybutyronitrile and the metal salt, the solution is diluted, or the metal salt and the metal cyano complex are mixed with an adsorbent or the like. The concentration of the metal salt is preferably 10,000 ppm or less by a method such as removal. As the adsorbent, for example, activated carbon, activated alumina, zeolite, silica gel, ion exchange resin, or the like can be used.

  The pH after adding the metal salt is preferably 6 or more, more preferably 7 or more, from the viewpoint of smoothly forming the metal cyano complex. Moreover, it is preferable to set it as pH 9 or less from a viewpoint of preventing decomposition | disassembly of 4-halo-3-hydroxybutyronitrile, and it is more preferable to set it as pH 8.5 or less. The timing of pH adjustment is not particularly limited before and after the addition of the metal salt.

  The method for adjusting the pH is not particularly limited unless cyanic acid and / or metal cyanide is used, but pH adjustment by adding an alkali is 0 to 30 from the viewpoint of preventing decomposition of 4-halo-3-hydroxybutyronitrile. It is preferable to carry out at ° C.

  Although the temperature at the time of adding a metal salt and after adding is not specifically limited, it is preferable to set it as 0-40 degreeC from a stability viewpoint of 4-halo-3-hydroxybutyronitrile.

  In addition, with respect to the treatment time from the addition of the metal salt to the time of being subjected to the amidating enzyme reaction in Step II, the metal salt is added from the viewpoint of sufficiently forming the metal cyano complex and preventing the deactivation of the nitrile hydratase. It is preferable that the reaction time is 5 minutes or more, more preferably 10 minutes or more, and then the amidation enzyme reaction in Step II is used. Moreover, after adding a metal salt, after adjusting pH to 6-8.5, it is preferable to use for the amidating enzyme reaction of the process II, maintaining pH in this range. After the metal salt is added, if the pH drops to 6 or less, the pH is adjusted to 6 to 8.5 by adding an alkali, and the process is maintained for 1 second or longer, more preferably 5 minutes or longer. It is preferable to use for II amidation enzyme reaction. It is possible to add nitrile hydratase in the state where the pH is less than 6 to start the amidating enzyme reaction in Step II. In that case, the pH is adjusted to 6 to 8.5 within 1 hour after the start of the reaction. The pH is preferably adjusted within 30 minutes, more preferably within 15 minutes, and the pH is adjusted to 6 to 8.5 within 15 minutes. Although the upper limit of processing time is not specifically limited, It can be set as 1 hour-1 year. Further, stirring is possible during the treatment, but there is no particular limitation.

  In addition, the process by addition of a metal salt in this process and said heat processing can also be used in combination as needed. In the case of combination, it is preferable that after the above heat treatment, a metal salt addition treatment is performed and used for the amidating enzyme reaction in the following Step II.

Step II: The amidating enzyme reaction step II is a step in which the treatment liquid obtained in the above step I is brought into contact with nitrile hydratase to obtain 4-halo-3-hydroxybutyramide by an enzymatic reaction.

(Step II-1) Nitrile hydratase Examples of the enzyme used in this step include nitrile hydratase. Nitrile hydratase refers to an enzyme that catalyzes the formation of an amide compound by a hydration reaction of a nitrile compound, and is an enzyme belonging to lyase according to international enzyme classification. The nitrile hydratase used in the present invention may be any enzyme that catalyzes the reaction of converting 4-halo-3-hydroxybutyronitrile nitrile into amide.

  For example, the genus Achromobacter, Acidoborax, Agrobacterium, Arthrobacter, Bacillus, Brevibacterium, Burkhol Burkholderia genus, Candida genus, Caseobacter genus, Comamonas genus, Corynebacterium genus, Dietzia genus, Enterobacter genus, Erwinia (Erwinia) ) Genus, Geobacillus genus, Gordona genus, Klebsiela genus, Microascus Morganella genus, Pantoea genus, Proteus genus, Pseudomonas genus , Pseudonocardia, Rhodococca A nitrile hydratase produced by microorganisms belonging to the genera Rhodococcus, Rhizobium, Serratia, Syctalidium, and Tukamurella.

  Specifically, Arthrobacter globiformis IFO 12138, Brevibacterium helvolum ATCC11822, Corynebacterium flavescens IAM 1642, Rhodococcus O12 539, Rhodococcus Oery 12 Streptomyces alboglycelus (Streptomyces aLBogriseolus) HUT 6045, Streptomyces chrysomallus HUT 6141, Streptomyces cinereouruber (Streptomyces cinereouruber) HUT6142 Streptomyces olivaceus) HUT 6061, Streptomyces rubrocyanodiastaticus HUT 6117, Klebsiella nuumonia Klebsiella pneumoniae IFO 12019, IFO 3319, IFO12059, IAM 1063, Klebsiella pneumoniae subsp. Pneumoniae NH-36T2 strain, Serratia plymus Erwinia carotovora IFO 3057, Tukamurella paurometabolum JCM 3226, Gordona rubropertinctus JCM 3227, Morganella morganii IFO 3848, Prote vulis I 3167, Enterobacter aerogenes IFO 12010, Microascus desmosporus IFO6761, Candida guilliermondii NH-2 (FERM P-11350), Pantoe A nitrile hydratase produced by Pantoea agglomerans NH-3 strain (FERM P-11349).

  In addition, the microorganism strain provided with the ATCC number can be easily obtained from the American Type Culture Collection (ATCC). Microorganisms with IFO numbers are described in "List of Cultures, 8th Edition, Volume 1 (1988)" published by the Fermentation Research Institute (IFO), and are currently independent administrative corporation product evaluation technology. It can be obtained from Bioresources Division, Biotechnology Headquarters, Biotechnology Headquarters. Microorganisms with IAM numbers can be obtained from the Institute for Applied Microbiology, the University of Tokyo. Microorganisms with JCM numbers are described in “Catalogue of strains 4th edition (1989)” issued by the Institute of Physical and Chemical Research, RIKEN, and can be obtained from the Institute of Microorganisms of RIKEN. Microorganisms with HUT numbers are described in “Catalogue of Cultures, Fourth Edition (1987)” issued by the Japan Microbial Conservation Federation (JFCC), and can be obtained from the Faculty of Engineering, Hiroshima University. Microorganisms with FERM numbers can be obtained from the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

  Furthermore, Rhodococcus rhodochrous J-1 [FERM BP-1478] isolated from soil by Yamada et al., And Arthrobacter sp. SK103 [FERM P], which some of the present applicants isolated from soil. -11300], Caseobacter sp. BC23 [FERM P-11261], Pseudomonas sp. BC15-2 [FERM BP-3320], Pseudomonas sp. SK31 [FERM P-11310] ], Pseudomonas sp. SK87 [FERM P-11311], Pseudomonas sp. SK13 [FERM BP-3325], Rhodococcus sp. SK70 [FERM P-11304], Rhodococcus ) Nitrile hydratase produced by sp. HR11 [FERM P-11306] and Rhodococcus sp. SK49 [FERM P-11303]. These microorganisms are deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, using the above deposit numbers.

  In addition, a microorganism obtained by cloning and transforming (introducing) a nitrile hydratase gene of the above microorganism is also included as a microorganism that produces the above nitrile hydratase. For example, E. coli strain MT-10822 (FERM BP-5785) transformed with a nitrile hydratase gene belonging to the genus Pseudonocardia described in US Pat. No. 5,807,730, Achromobacter described in Japanese Patent No. 3531997 Examples include Escherichia coli MT-10770 strain (FERM P-14756) transformed with a nitrile hydratase belonging to the genus and microorganisms transformed with a nitrile hydratase of Rhodococcus rhodochrous species described in Japanese Patent No. 3162091.

  Among them, a nitrile hydratase derived from a microorganism belonging to the genus Rhodococcus is preferable, more preferably a Rhodococcus rhodochrous species, and particularly preferably a Rhodococcus rhodochrous J-1 strain. belongs to.

  The composition of the medium for culturing a microorganism that produces nitrile hydratase is not particularly limited as long as the microorganism can grow and produce a desired enzyme. For example, sugars such as glucose, fructose, sucrose and maltose as carbon sources, organic acids such as acetic acid and citric acid, alcohols such as ethanol and glycerol, peptone, meat extract, yeast extract and protein hydrolyzate as nitrogen sources In addition to natural nitrogen sources such as amino acids, various inorganic and organic acid ammonium salts can be used. In addition, inorganic salts, trace metals, vitamins, and the like are used as needed. In some cases, in order to induce enzyme activity, various nitrile compounds such as 4-chloro-3-hydroxybutyronitrile, propionitrile, isobutyronitrile, benzylcyanide, 4-chloro-3-hydroxybutyramide Various amide compounds such as propionamide and isobutyramide may be added to the medium. The microorganism may be cultured by a conventional method, for example, aerobically cultured for 10 to 180 hours at a pH of 4 to 10 and a temperature of 10 to 45 ° C. Culturing can be performed by either liquid culture or solid culture.

  The microbial cells obtained as described above can be obtained by subjecting the culture solution as it is, a suspension of the microbial cells obtained by centrifugation or the like, a processed microbial cell product (for example, crushed microbial cell extract, microbial cell extract, etc.) As immobilized microbial cells or treated cells, roughly purified enzymes, and purified enzymes, nitrile hydratase can be used for amidating enzyme reactions.

(Step II-2) Amidating enzyme reaction The treatment liquid obtained in the above step I ′ includes 4-halo-3-hydroxybutyronitrile as well as 4-halo-3-hydroxybutyronitrile. Includes inorganic salts such as hydrogen halide salts. Since this salt partially inhibits the activity of nitrile hydratase depending on the concentration of 4-halo-3-hydroxybutyronitrile in the amidation reaction solution in this step, 4-halo-3-hydroxybutyronitrile is used. It is preferable to adjust the concentration so that the product of the concentration (mass%) and the salt concentration (mass%) is 160 or less, preferably 120 or less, and contact with nitrile hydratase. Further, the concentration of the metal salt added in the step I ′ may be adjusted in addition to the salt concentration.

  The method for adjusting the concentration (the concentration is adjusted so that the product of the concentration of 4-halo-3-hydroxybutyronitrile (mass%) and the salt concentration (mass%) is 160 or less) is not particularly limited, It can be carried out by dilution of the treatment liquid obtained in the above step I ′, desalting operation or the like. Examples of the desalting operation include, for example, a method of removing a salt deposited by concentration by filtration, an aqueous solution containing 4-halo-3-hydroxybutyronitrile, and the treatment liquid obtained in the above step I ′. It is performed by a method of removing salt by dialysis treatment or treatment with an ion exchange resin or the like.

  The solution of 4-halo-3-hydroxybutyronitrile, the concentration of which has been adjusted by the above method, is adjusted so that the nitrile hydratase activity can be sufficiently exerted before bringing the solution into contact with the nitrile hydratase prepared as described above. It is preferable from the standpoint of enzyme stability.

  In this step, a buffer solution can also be used to facilitate pH adjustment. Examples of the buffer solution include a buffer solution composed of phosphoric acid, boric acid, citric acid, glutaric acid, malic acid, malonic acid, o-phthalic acid, succinic acid, acetic acid, or the like, Tris buffer, or Good. Buffers and the like can be exemplified and used. However, a buffer composed only of hydrocyanic acid and its salt remarkably inhibits the activity of nitrile hydratase, and must not be used in this step. When a buffer solution is used in the step of producing 4-halo-3-hydroxybutyronitrile and when a buffer solution is used in this step, the concentration of 4-halo-3- It is preferable to adjust the concentration by calculating the product with the concentration (% by mass) of hydroxybutyronitrile.

  The method for contacting the nitrile aqueous solution with the nitrile hydratase is not particularly limited. For example, a method of batch-adding nitrile hydratase to the aqueous solution of nitrile, or a method of dividingly adding nitrile hydratase to the aqueous solution of nitrile And an aqueous solution containing nitrile hydratase in a divided manner. Since the amidation reaction is an exothermic reaction, a method in which nitrile hydratase is added in portions to the aqueous solution of nitrile and a method in which the aqueous solution of nitrile is added in portions to an aqueous solution containing nitrile hydratase are preferred.

  The reaction temperature is appropriately selected depending on the nitrile hydratase to be used, but is preferably maintained at about 0 to 30 ° C., more preferably from the viewpoint of enzyme stability, smooth progress of the reaction, and suppression of side reactions. 0-20 ° C.

  The pH of the reaction solution is also appropriately selected depending on the microorganism to be used, and can be carried out in the range of pH 4 to 10. However, the pH is 6 to 8.5 in terms of enzyme stability, smooth progress of reaction, and suppression of side reactions. The pH is preferably 7 to 8.5.

  Although the reaction time varies depending on the nitrile concentration, the amount of enzyme, or other reaction conditions, it is preferable to set the conditions so that the reaction is usually completed in 0.5 to 120 hours. More preferably, the setting is made so that the process is completed in about 5 to 24 hours.

  The concentration and optical purity of optically active 4-halo-3-hydroxybutyronitrile and optically active 4-halo-3-hydroxybutyramide in the reaction system should be measured and quantified by high performance liquid chromatography (HPLC). Can do. The concentration of the salt in the reaction system can be measured and quantified by, for example, titration, ion chromatography, ICP emission spectroscopy, or atomic absorption analysis.

Step III: Utilization of 4-halo-3-hydroxybutyramide 4-halo-3-hydroxybutyramide synthesized as described above is a method of removing, extracting, and extracting solid contents derived from bacterial cells and the like by filtration and centrifugation. Isolation and purification can be performed according to conventional methods such as column separation and recrystallization.

  Carnitine amide can be obtained by contacting trimethylamine with an aqueous solution containing 4-halo-3-hydroxybutyramide obtained in this step. This contact may be carried out according to a conventional method. A method of dropping an aqueous solution of trimethylamine into an aqueous solution containing 4-halo-3-hydroxybutyramide obtained in this step, a method of blowing gaseous trimethylamine, an aqueous solution of trimethylamine Examples include a method of dropping an aqueous solution containing 4-halo-3-hydroxybutyramide obtained in this step.

  The reaction between 4-halo-3-hydroxybutyramide and trimethylamine can be carried out at 0 to 100 ° C., but it can be carried out at 0 to 50 ° C. from the viewpoint of smooth progress of the reaction and suppression of side reactions. 0-40 degreeC is preferable and especially preferable. The amount of trimethylamine used is preferably 1 to 3 equivalents, particularly preferably 1.1 to 1.5 equivalents, based on 4-halo-3-hydroxybutyramide.

  While the reaction time varies depending on the temperature, the amount of trimethylamine used, and other reaction conditions, it is usually preferable to set the conditions so that the reaction is completed in 0.5 to 120 hours.

  Carnitine amide synthesized as described above can be isolated and purified in accordance with conventional methods such as removal of solids derived from bacterial cells and the like by filtration and centrifugation, desalting with ion exchange resin, and recrystallization. Carnitine can also be obtained by hydrolysis under basic conditions.

  Hereinafter, the present invention will be specifically described by way of examples.

[Analysis method]
HPLC analysis: chemical purity analysis 1,3-dichloro-2-propanol (hereinafter sometimes referred to as DCP) and 4-chloro-3-hydroxybutyronitrile (hereinafter referred to as CHBN) in the cyanation reaction of Step I of the present invention. Quantification was carried out under the analysis conditions shown in Table 1 using high performance liquid chromatography (HPLC).

In the amidation reaction of Step II of the present invention, 1,3-dichloro-2-propanol, epichlorohydrin, 4-chloro-3-hydroxybutyronitrile, 4-chloro-3-hydroxybutyramide ( The quantitative determination of CHBA (hereinafter sometimes referred to as CHBA) was performed under the analysis conditions shown in Table 2 using high performance liquid chromatography (HPLC).

  In the above HPLC quantitative analysis, a calibration curve is prepared in advance using a solution of DCP, ECH, CHBN and CHBA of known concentrations, and the concentration of DCP, ECH, CHBN and CHBA in the reaction solution is determined using the calibration curve. Asked. The DCP and ECH preparations can be purchased and obtained from Wako Pure Chemical Industries, Ltd. Regarding the CHBN standard, (R) -CHBN can be purchased from AMAX Co., Ltd. Racemic CHBN can be obtained by manufacturing according to the description of JP-B-5-69818. The CHBA preparation can be obtained by using the above CHBN as a raw material according to the description in JP-B-53-13611.

In addition, the quantification of carnitine amide and carnitine in the above-mentioned step III was performed under the analysis conditions shown in Table 3 using high performance liquid chromatography (HPLC).

  In the above HPLC quantitative analysis, a calibration curve was prepared in advance using a solution of carnitine amide and carnitine with known concentrations, and the concentration of carnitine amide and carnitine in the reaction solution was determined using the calibration curve. The preparation of carnitine amide was produced according to the method described in JP-A-57-140749, using ethyl 4-chloroacetoacetate, etc., which can be purchased and obtained from Wako Pure Chemical Industries, Ltd. as a raw material. It can be obtained by doing. Carnitine preparations can be purchased and obtained from Wako Pure Chemical Industries, Ltd.

HPLC analysis: optical purity analysis The optical purity of (R) -CHBN and (R) -CHBA was measured as follows. About 3 mL of an aqueous solution containing (R) -CHBN and / or (R) -CHBA was collected and extracted by adding an equal amount of ethyl acetate. The ethyl acetate layer was separated, a small amount of anhydrous sodium sulfate was added and stirred, and then the supernatant was concentrated with an evaporator (bath temperature 40 ° C., 30 Torr) to distill off the ethyl acetate, and (R) -CHBN and / or Or the oil which has (R) -CHBA as a main component was collect | recovered. Collect 2 μL of the above oil in a 1.5 mL microtube, add 20 μL of dichloromethane and 20 μL of pyridine, and then add (R) -α-methoxy-α- (trifluoromethyl) phenylacetyl chloride (hereinafter referred to as (R)- 2 μL (which may be referred to as MTPA) was added, and the mixture was shaken for about 10 seconds and allowed to stand at room temperature for 5 to 24 hours to carry out an esterification reaction. About 300 μL of diisopropyl ether (hereinafter sometimes referred to as “IPE”) was added to the reaction-terminated liquid, followed by washing with about 300 μL of 1N aqueous hydrochloric acid, and the IPE layer was collected. Further, the IPE layer was washed with about 300 μL of a saturated aqueous sodium hydrogen carbonate solution, and the collected IPE layer was concentrated under reduced pressure by an aspirator. The residue was dissolved in a mixed solution of n-hexane: 2-propanol = 4: 1, and this was used by HPLC. The analysis was performed under the analysis conditions shown in Table 4.

  From the area ratio of (R) -CHBN (or CHBA)-(R) -MTPA ester and (S) -CHBN (or CHBA)-(R) -MTPA ester, the optical properties of (R) -CHBN (or CHBA) The purity was calculated as follows. In addition, about the said area, it analyzed on the analysis conditions of the said Table 2 about the (R) -MTPA ester of racemic CHBN (or CHBA) beforehand, and obtained (R) -CHBN (or Correction was made by the area area ratio of CHBA)-(R) -MTPA ester and (S) -CHBN (or CHBA)-(R) -MTPA ester.

ここで [R]は被測定試料における（Ｒ）−ＣＨＢＮ（またはＣＨＢＡ）−（Ｒ）−ＭＴＰＡエステルの、[S]は被測定試料における（Ｓ）−ＣＨＢＮ（またはＣＨＢＡ）−（Ｒ）−ＭＴＰＡエステルのエリア面積を示す。 <Formula 1>

Here, [R] is (R) -CHBN (or CHBA)-(R) -MTPA ester in the sample to be measured, and [S] is (S) -CHBN (or CHBA)-(R)-in the sample to be measured. The area area of MTPA ester is shown.

  r is defined as r = [R] rac / [S] rac, and is a calculated correction coefficient. [R] rac is (R) -CHBN (or CHBA)-(in racemic CHBN (or CHBA). R) -MTPA ester area area, [S] rac indicates (S) -CHBN (or CHBA)-(R) -MTPA ester area area in racemic CHBN (or CHBA).

Analysis by titration: Cyanide analysis For the concentration of cyanide in the reaction solution, refer to “Ministerial Ordinance for Determination of Substances Containing Toxic Substances or Deleterious Substances” It was measured by titration as described below with reference to the titration method using a silver nitrate test solution using p-dimethylaminobenzylidene rhodanine as an indicator described in the section “Measurement of Absorbance of Cyanide Ion Standard Solution”.

  Weigh accurately the liquid to be measured in a 200 mL Erlenmeyer flask and add ion-exchanged water or distilled water as necessary to make about 100 mL (no need to add if 100 mL of measurement liquid), then caustic ammonia Add 10 mL of water (5% aqueous ammonia containing 4% sodium hydroxide) (increase until pH> 9 if the solution to be measured is strongly acidic) to 0.02 g of p-dimethylaminobenzylidene rhodanine. 0.5 mL of a solution made up to 100 mL by adding acetone is added, titrated with an aqueous silver nitrate solution (0.1 N, or 0.01 N if necessary) until the liquid turns red, and the amount of aqueous silver nitrate solution required for titration ( mL) from the following formula.

滴定による分析：塩化物イオン分析
反応液中における塩化ナトリウム濃度については、ホルハルト法における滴定結果から、上記により測定したシアンイオンの濃度を差し引いて塩化物イオンの濃度を算出し、塩化ナトリウム濃度に換算することにより求めた。具体的には下記のようにして行った。 <Formula 2>

Analysis by titration: Chloride ion analysis The sodium chloride concentration in the reaction solution is calculated by subtracting the cyan ion concentration measured above from the titration result in the Forhardt method, and converted to the sodium chloride concentration. Was determined by Specifically, it was performed as follows.

  Into a 200 mL Erlenmeyer flask, accurately take 20 mL of 0.1N silver nitrate aqueous solution and accurately weigh and add the liquid for measurement. Add 50 mL of ion exchange water or distilled water and 10 mL of 15% nitric acid aqueous solution, and then add 10 mL of 20% nitric acid aqueous solution and ion exchange water or distilled water to 10 g of iron (III) ammonium sulfate solution (iron (III) ammonium sulfate dodecahydrate). 80 mL was added and dissolved) 5 mL and nitrobenzene 5 mL were added, and titrated with 0.1 N ammonium thiocyanate aqueous solution until the solution became reddish brown. From the amount (mL) of ammonium thiocyanate aqueous solution required for titration, ( The weight molar concentration (mmol / mg) of cyan ion + chloride ion is calculated by the following formula.

ここで、ブランクとは、０．１Ｎ硝酸銀水溶液２０ｍＬを上記方法にて滴定（つまり、測定に係る液を加えないで滴定）した際の、滴定に要したチオシアン酸アンモニウム水溶液の量（ｍＬ）である。 <Formula 3>

Here, the blank is the amount (mL) of ammonium thiocyanate aqueous solution required for titration when titrating 20 mL of a 0.1N silver nitrate aqueous solution by the above method (that is, titrating without adding the liquid for measurement). is there.

  The concentration of sodium chloride was calculated based on the cyanide ion concentration calculated in the above-described “analysis by titration” from the molality (mmol / mg) of (cyan ion + chloride ion) calculated by the above formula 3. / Mg) is subtracted to obtain the molar weight concentration (mmol / mg) of chloride ions, and this is multiplied by the molecular weight of sodium chloride (58.44). Specifically, it is expressed by the following equation.

［調製例１］
ハロヒドリンエポキシダーゼ発現形質転換微生物JM109/pST111の培養
ハロヒドリンエポキシダーゼ活性を持つ大腸菌形質転換体JM109/pST111（FERM BP-10922）の培養を以下のように行った。なお、pST111は、コリネバクテリウム（Corynebacterium）sp.N-1074のハロヒドリンエポキシダーゼ遺伝子（ＨｈｅＢ）を含むBam H I - Pst I １．１ｋｂｐ断片をｐＵＣ１１８に結合させたプラスミドであり、ｐＳＴ１１１は、特許第３０２６３６７号公報に記載されている。 <Formula 4>

[Preparation Example 1]
Cultivation of halohydrin epoxidase-expressing transformed microorganism JM109 / pST111 The E. coli transformant JM109 / pST111 (FERM BP-10922) having halohydrin epoxidase activity was cultured as follows. PST111 is a plasmid obtained by binding a Bam HI-Pst I 1.1 kbp fragment containing a halohydrin epoxidase gene (HheB) of Corynebacterium sp. N-1074 to pUC118. This is described in Japanese Patent No. 3026367.

  20 colonies of JM109 / pST111 were prepared in 100 mL of LB medium (1% bactotryptone, 0.5% bactoeast extract, 0.5% NaCl, 1 mM IPTG, 50 μg / mL ampicillin sodium) prepared in a 500 mL Erlenmeyer flask. And incubating at 37 ° C. for 20 hours with shaking. The LB medium is prepared by dissolving the necessary amounts of ingredients other than ampicillin sodium and IPTG in water and making up to 100 mL, followed by heat sterilization (121 ° C., 20 minutes), cooling to room temperature, and using a 0.45 μm filter in advance. 50 μL of ampicillin sodium aqueous solution (100 g / L) that had been sterilized by filtration and 100 μL of IPTG aqueous solution (240 g / L) that had been previously sterilized by filtration with a 0.45 μm filter were added under aseptic conditions. Prepared.

  The cultured cells were washed with 50 mM Tris-sulfate buffer (pH 8), and 50 mM Tris-sulfate buffer (pH 8) was added to 20 g and suspended. The activity of this cell suspension was 440 U / g. This bacterial cell suspension was diluted with 50 mM Tris-sulfate buffer (pH 8) to 400 U per 1 g of the bacterial cell suspension.

  The activity of the cell suspension obtained above was calculated by measuring the amount of chloride ion desorption from DCP as described below.

  100 mL of a reaction solution for activity measurement (50 mM DCP, 50 mM Tris-sulfuric acid (pH 8)) was prepared, and the temperature was adjusted to 20 ° C. A diluted cell suspension was added to the reaction solution to start the reaction. The decrease in pH accompanying the release of chloride ions due to the halohydrin epoxidase activity was continuously adjusted using a pH automatic controller to maintain the pH at 8 using a 0.01N aqueous sodium hydroxide solution. From the amount of 0.01N aqueous sodium hydroxide solution added to keep the pH at 8 during the 10-minute reaction, the amount of chloride ion produced is calculated, and the halohydrin epoxidase activity (dechlorination activity) is calculated. (U) was calculated. 1 U was defined to correspond to the amount of enzyme from which 1 μmol of chloride ion was eliminated per minute from DCP under the above conditions.

[Preparation Example 2]
Culture of halohydrin epoxidase-expressing transformed microorganism JM109 / pST111
A colony of JM109 / pST111 was prepared in 100 mL of a preculture medium prepared in a 500 mL flask (polypeptone N 20 g / L, yeast extract 5 g / L, potassium dihydrogen phosphate 1.5 g / L, ampicillin sodium 0.1 g / L; pH 7.2) was inoculated, and shaking culture was performed at a temperature of 37 ° C. and a rotation speed of 210 rpm for 6 hours.

  The pre-culture medium is prepared by dissolving the necessary amounts of ingredients other than ampicillin sodium in water and making up to 100 mL, followed by heat sterilization (121 ° C., 20 minutes), cooling to room temperature, and filtering through a 0.45 μm filter in advance. An ampicillin sodium aqueous solution (100 g / L) that had been sterilized was added by adding 100 μL under aseptic conditions.

  20 mL of the obtained preculture solution was prepared in 1.8 L of main culture medium (fructose 40 g / L, polypeptone N 20 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 1.5 g prepared in a 3 L jar fermenter. / L, magnesium sulfate heptahydrate 2.375 g / L, manganese sulfate pentahydrate 0.2 g / L, calcium chloride dihydrate 0.02 g / L, zinc sulfate heptahydrate 0.02 g / L , Ampicillin sodium 0.1 g / L, IPTG 0.24 g / L, Pluronic L-61 (ADEKA, Japan) 0.5 g / L) Inoculated into two, temperature 37 ° C., rotation speed 750 rpm, aeration rate Culturing was performed for 24 hours under 2 L / min, normal pressure, pH 6.8-7.2 control (using 25% aqueous sodium hydroxide and 24% aqueous sulfuric acid).

  The main culture medium is prepared by dissolving the required amount of fructose in water and making up to 500 mL and then heat sterilizing (121 ° C., 20 minutes), and dissolving each component required amount other than fructose and ampicillin sodium in water. Ampicillin sodium aqueous solution that has been sterilized by heating after being sterilized by heating (121 ° C, 20 minutes) to 1.3 L, cooled to room temperature, and filtered and sterilized with a 0.45 µm filter in advance. (100 g / L) was prepared by adding 1.8 mL, and 1.8 mL of an IPTG aqueous solution (240 g / L) that had been previously sterilized by filtration with a 0.45 μm filter.

After completion of the culture, the cells were collected and washed. 3565 g (OD ₆₃₀ = 35) was taken from the mixture of the two jar fermenter cultures and centrifuged at 12,000 rpm (14,100 G) for 10 minutes to obtain 142 g of wet cells. 278 g of water was added to the wet cells and resuspended uniformly to obtain 420 g (OD ₆₃₀ = 300) of the cell suspension. The activity of the cell suspension was 2000 U / g.

220 g of the obtained cell suspension was cooled to about 10 ° C., and then crushed at about 100 MPa using a high-pressure homogenizer PA2K (NiroSoavi, Italy). After cooling to about 10 ° C. again, crushing was performed again at about 100 MPa to obtain 170 g of a microbial cell disruption solution. To this was added 17 mL of 10% alkyldiaminoethylglycine hydrochloride aqueous solution (final concentration 0.91%), and after stirring for about 10 minutes, 17.0 g of Celite Hyflo Super Cel (World Minerals, USA) was added. The mixture was further stirred for about 5 minutes. No. No. with a filtration area of about 163 cm ² pre-coated with 9 g of Celite Hyflo Super Cel. Using a 5A filter paper (Advantech Co., Ltd., Japan) and a pressure filter (Advantech Co., Ltd., Japan), pressure filtration was performed at a pressure of 0.2 MPa to obtain a crude enzyme solution. The activity of the crude enzyme solution was 1750 U / g.

[Preparation Example 3]
A nitrile hydratase-expressing microorganism Rhodococcus rhodochrous J-1 strain Rhodococcus rhodochrous J-1 strain (FERM BP-1478) having nitrile hydratase activity was prepared using a 30 L jar fermenter with 2% glucose and urea. Cultured aerobically for 60 hours at 30 ° C. in a medium (pH 7.0) containing 1%, 0.5% peptone, 0.3% yeast extract, 0.05% cobalt chloride, and this was incubated with 50 mM phosphate buffer The solution was washed with a liquid (pH 7.7) to obtain 500 g of a cell suspension (20% in terms of dry cells).

<Examples 1-6, Comparative Examples 1-4>
Step IA: Cyanation enzyme reaction A 300 mL four-necked flask equipped with an alkaline charging pipe controlled by a pH electrode and a pH controller was charged with 127.55 g of water and 4.41 g (0.1632 mol) of HCN and 0.65 g of 30% NaOH. (0.0049 mol) and adjusted to pH 7.5. 1,3-Dichloro-2-propanol (10.00 g, 0.0775 mol) was added and stirred until it was uniformly dissolved.

20.00 g of the JM109 / pST111 cell suspension of Preparation Example 1 was added, and the reaction was started at 20 ° C. In order to maintain the pH in the system at 7.5 to 7.6, the pH controller was set so as to add 30% NaOH, and 1,3-dichloro-2- By adding propanol and HCN, the concentration of 1,3-dichloro-2-propanol in the system should not exceed 0.5 mol / kg, and the cyanide concentration in the system should be 1.1 mol / kg. Not to exceed. The reaction profile at this time is shown in Table 5.

  After 23 hours, 4-chloro-3-hydroxybutyronitrile was able to accumulate 0.753 mol / kg (9.0%) with an optical purity of 94.8% e.e. e. The (R) -form was excessive. The yield based on 1,3-dichloro-2-propanol consumed by the reaction was 96.3%.

  The reaction solution was adjusted to pH 5.0 with hydrochloric acid, and reduced in pressure at 60 ° C. and 140 torr for 4 hours to remove HCN. By titration, it was confirmed that the HCN in the reaction system was 1 ppm or less.

Step IB: Pretreatment of amidating enzyme reaction The (R) -CHBN aqueous solution obtained in the above cyanation reaction step was decompressed at 140 torr and heat-treated at 60 ° C. for 11 hours. This (R) -CHBN aqueous solution was concentrated to 20%, and the aqueous solution was adjusted to pH 7.5 using an aqueous sodium hydroxide solution.

Step IC: Amidating enzyme reaction The (R) -CHBN concentration, salt concentration, and the product of the CHBN concentration and the salt concentration of the (R) -CHBN 20% aqueous solution subjected to the above treatment are adjusted to the predetermined values shown in Table 6. Thereafter, 5 g was collected, and a predetermined amount of Rhodococcus rhodochrous J-1 strain suspension of Preparation Example 3 was added dropwise as shown in Table 6 and the reaction was started at 2 ° C. The results are shown in Table 6.

<Example 7>
The (R) -CHBN aqueous solution obtained in Step IA of Example 1 was concentrated as it was without heat treatment, and adjusted to pH 7.5 using an aqueous sodium hydroxide solution. An aqueous solution having 14.0% and a salt concentration of 7.7%, ie, a product of CHBN concentration and salt concentration of 108 was prepared. Cobalt acetate was added to this aqueous solution so that it might become 120 ppm, and it stirred at room temperature for 5 minutes. Otherwise, the reaction was carried out in the same manner as in Example 1. The results are shown in Table 6.

<Example 8>
The (R) -CHBN aqueous solution obtained in Step IA of Example 1 was concentrated as it was without heat treatment, and adjusted to pH 7.5 using an aqueous sodium hydroxide solution. An aqueous solution having 14.0% and a salt concentration of 8.1%, that is, a product of the CHBN concentration and the salt concentration of 114 was prepared. Nickel nitrate was added to this aqueous solution so that it might become 190 ppm, and it stirred at room temperature for 1 hour. Otherwise, the reaction was carried out in the same manner as in Example 1. The results are shown in Table 6.

  In Comparative Examples 1 to 4, the conversion rate of 100% was not reached even after a sufficient time had elapsed, whereas in Examples 1 to 8, the conversion rate was 100% 2 hours after the start of the reaction.

<Example 9>
The (R) -CHBN aqueous solution obtained in Step IA of Example 1 was concentrated as it was without heat treatment, and adjusted to pH 7.5 using an aqueous sodium hydroxide solution. An aqueous solution having 13.5% and a salt concentration of 6.9%, that is, a product of CHBN concentration and salt concentration of 93 was prepared. Cobalt chloride was added to this aqueous solution so that it might become 8.1 ppm, and it stirred at room temperature for 5 minutes. Otherwise, the reaction was carried out in the same manner as in Example 1. The results are shown in Table 7.

<Example 10>
The (R) -CHBN aqueous solution obtained in Step IA of Example 1 was concentrated as it was without heat treatment, and adjusted to pH 7.5 using an aqueous sodium hydroxide solution. An aqueous solution having 13.5% and a salt concentration of 6.9%, that is, a product of CHBN concentration and salt concentration of 93 was prepared. Cobalt chloride was added to this aqueous solution so that it might be 8.1 ppm, and it stirred at room temperature for 30 minutes. Otherwise, the reaction was carried out in the same manner as in Example 1. The results are shown in Table 7.

<Example 11>
The (R) -CHBN aqueous solution obtained in Step IA of Example 1 was concentrated as it was without heat treatment, and adjusted to pH 7.5 using an aqueous sodium hydroxide solution. An aqueous solution having 13.5% and a salt concentration of 6.9%, that is, a product of CHBN concentration and salt concentration of 93 was prepared. Cobalt chloride was added to this aqueous solution so that it might be 8.1 ppm, and it stirred at room temperature for 2 hours. Otherwise, the reaction was carried out in the same manner as in Example 1. The results are shown in Table 7.

<Example 12>
The (R) -CHBN aqueous solution obtained in Step IA of Example 1 was concentrated as it was without heat treatment, and adjusted to pH 7.5 using an aqueous sodium hydroxide solution. An aqueous solution having 13.5% and a salt concentration of 6.9%, that is, a product of CHBN concentration and salt concentration of 93 was prepared. Cobalt chloride was added to this aqueous solution so that it might be 8.1 ppm at pH 4, and it stirred at room temperature for 5 minutes, and reaction was started with pH 4. Otherwise, the reaction was carried out in the same manner as in Example 1. The results are shown in Table 7.

<Example 13>
Step II-A: Cyanide enzyme reaction pH 45 and 10L separable flask equipped with an alkali charging pipe controlled by a pH controller were charged with 5542 g of water and 169 g of HCN, and then adjusted to pH 7.8 with 29 g of 30% NaOH. did. Thereafter, 400 g of DCP was added and stirred until evenly dissolved. Next, 183 g (320 kU) of the crude enzyme solution of Preparation Example 2 was added, and the reaction was started at 18 ° C. and pH 7.8 to 7.9.

  After starting the reaction, in order to control the pH in the reaction system, a pH controller was set, and the pH was controlled from 7.8 to 7.9 with 30% NaOH. Immediately after the start of the reaction, the pH dropped to 7.0, but after that, the pH was maintained between 7.8 and 7.9. In parallel, 400 g of DCP and 99 g of HCN were additionally added at a ratio of approximately equimolar to the charged 30% NaOH.

  At the time when 820 g of 30% NaOH was added 23 hours after the start of the reaction, the pH control was terminated to obtain 7552 g (a) of a cyanation enzyme reaction end solution. The yield of CHBN from DCP was 86.8%.

  24 g of 35% hydrochloric acid was added to 7500 g of the above-mentioned cyanation enzyme reaction completion liquid (a), and the pH was adjusted to 2.0. Concentration under reduced pressure was carried out at a temperature in the kettle of 80 ° C. and 400 to 500 Torr, and 144 g of distillate (b) was obtained using a cooling tube cooled at −10 ° C. Subsequently, while nitrogen gas was blown at 20 mL / min, the pressure in the kettle was reduced to 50 to 60 ° C. and 80 to 100 Torr, and 3267 g of distillate (c) was added using a cooling tube cooled at −2 ° C. Obtained. At this time, the residue (d) in the kettle was 3981 g. The recovery rates of unreacted DCP, HCN, and CHBN in the cyanation enzyme reaction-terminated liquid (a) obtained in the distillates (b) and (c) were 69.0%, 53.1%, and 0.3%, respectively. 3%.

Step II-B: Pretreatment of amidating enzyme reaction Of the residue (d) obtained in Step II-A above, 2000 g was charged into a 3 L separable flask, and 20 g of 0.1% cobalt chloride (II) aqueous solution was added. After stirring, the pH was adjusted to 8.2 with 2.7 g of 30% NaOH, and 257 g of water was added to dilute the reaction solution, followed by stirring at 20 ° C. for 1 hour. During stirring, the pH remained 8.2.

Step II-C: amidating enzyme reaction The treatment solution obtained in the above Step II-B was cooled to 2 ° C., and 6 g of Rhodococcus rhodochrous J-1 cell suspension of Preparation Example 3 was added. The reaction was started at composition (e). During the reaction, 4% NaOH was added from time to time to maintain the pH between 7.8 and 8.2. Four hours after the start of the reaction, disappearance of CHBN and production of CHBA were confirmed by HPLC. At this time, the amount of 4% NaOH added was 2 g, the composition of the reaction solution was (f), and the yield of CHBA from CHBN was 99.9%.

In addition, Table 8 shows the composition of each solution in the above Steps II-A to II-C.

According to Table 8, in this step B, the product of CHBN concentration (% by weight) and salt concentration (% by weight) is
It was 14 (%) x (8.04 (%) + 0.0008 (%)) = 112.57.

Step II-D: Synthesis of carnitine amide 633 g of a 30% trimethylamine aqueous solution was added to the reaction solution obtained in the above Step II-C, and the reaction was performed while stirring at 30 ° C. Five hours after the start of the reaction, disappearance of CHBA and formation of carnitine amide were confirmed by HPLC. At this time, the yield of carnitine amide from CHBA was 95%.

<Example 14>
Step III-B: Pretreatment of amidating enzyme reaction Of the residue (d) obtained in Step II-A of Example 13, 1000 g was charged into a 2 L separable flask and heated to reflux at 100 ° C. for 12 hours under normal pressure. Thereafter, the pH was adjusted to 8.2 with 1.3 g of 30% NaOH.

Step III-C: amidating enzyme reaction The treatment solution obtained in Step III-B above was cooled to 2 ° C., and 3 g of Rhodococcus rhodochrous J-1 cell suspension in Preparation Example 7 was added to initiate the reaction. . During the reaction, 4% NaOH was added from time to time to maintain the pH between 7.8 and 8.2. Ten hours after the start of the reaction, disappearance of CHBN and generation of CHBA were confirmed by HPLC. At this time, the amount of 4% NaOH added was 1 g, and the yield of CHBA from CHBN was 99.5% (optical purity 91.9% ee). In Step II, the product of the CHBN concentration (wt%) and the salt concentration (wt%) was 15.93 (%) × 9.15 (%) = 145.76.

Step III-D: Synthesis of carnitine amide 316.5 g of 30% trimethylamine aqueous solution was added to the reaction solution obtained in the above Step III-C, and the reaction was carried out with stirring at 30 ° C. Five hours after the start of the reaction, disappearance of CHBA and formation of carnitine amide were confirmed by HPLC. At this time, the yield of carnitine amide from CHBA was 95%.

<Example 15>
Step IV-A: Synthesis of 4-chloro-3-hydroxybutyronitrile In a 3 L separable flask equipped with a stirrer, a thermometer, and a Dimroth cooling tank, 274.93 g (2.97 mol) of epichlorohydrin, 1271. 3 g and 191.75 g (1.35 mol) of sodium sulfate were charged, and 72.3 g (2.70 mol) of HCN was directly fed into the reaction solution over one hour at an internal temperature of 50 to 60 ° C. while stirring in a warm water bath. Thereafter, aging was performed at an internal temperature of 50 to 60 ° C. for 6 hours. While blowing nitrogen gas at a rate of 10 mL / min, vacuum concentration was performed at an internal temperature of 50 to 60 ° C. and 80 to 100 Torr, and 550 g of a distillate was obtained using a cooling tube cooled at −2 ° C. At this time, the residue in the kettle was 1260 g, and in the residue in the kettle, the concentration of CHBN was 16%, the concentration of sodium sulfate was 15.21%, and the concentration of HCN was 1 ppm.

Step IV-B: Pretreatment of amidating enzyme reaction Among the residues obtained in the above step IV-A, 500 g was charged into a 1 L separable flask, and 5 g of 0.1% cobalt chloride (II) aqueous solution was added and stirred. The pH was adjusted to 8.2 with 0.5 g of 30% NaOH, and 293 g of water was added to dilute the reaction solution, followed by stirring at 20 ° C. for 1 hour. During stirring, the pH remained 8.2.

Step IV-C: amidating enzyme reaction The treatment solution obtained in the above step IV-B is cooled to 2 ° C., 1.5 g of Rhodococcus rhodochrous J-1 cell suspension of Preparation Example 3 is added, and the reaction is carried out. Started. During the reaction, 4% NaOH was added from time to time to maintain the pH between 7.8 and 8.2. Four hours after the start of the reaction, disappearance of CHBN and production of CHBA were confirmed by HPLC. At this time, the amount of 4% NaOH added was 0.5 g, and the yield of CHBA from CHBN was 99.9%. In this step, the product of CHBN concentration (% by weight) and salt concentration (% by weight) at the start of the reaction was 10 (%) × (9.5 (%) + 0.0006 (%)) = 95. .

<Example 16>
Step VB: Pretreatment of amidating enzyme reaction Of the residue obtained in Step IV-A of Example 15, 500 g was charged into a 1 L separable flask, heated to reflux at 100 ° C. for 12 hours under normal pressure, and then 30% The pH was adjusted to 8.2 with 0.5 g of NaOH, and water was added to make the concentration of CHBN 10.05%.

Step VC: Amidating enzyme reaction The treatment solution obtained in the above step VB was cooled to 2 ° C., 4 g of Rhodococcus rhodochrous J-1 cell suspension in Preparation Example 3 was added, and the reaction was started. During the reaction, 4% NaOH was added from time to time to maintain the pH between 7.8 and 8.2. Ten hours after the start of the reaction, disappearance of CHBN and generation of CHBA were confirmed by HPLC. At this time, the amount of 4% NaOH added was 1 g, and the yield of CHBA from CHBN was 99.5%. In this step, the product of the CHBN concentration (% by weight) and the salt concentration (% by weight) was 10 (%) × 9.5 (%) = 95.

Claims (14)

The manufacturing method of 4-halo-3-hydroxybutyramide including the following processes.
Step I: Step of synthesizing 4-halo-3-hydroxybutyronitrile by subjecting epihalohydrin and / or 1,3-dihalo-2-propanol and cyanide donor to an enzymatic reaction,
Step II: The product of the concentration (mass%) of nitrile and the salt concentration (total of alkali metal salt and buffer salt and metal salt made of nickel, zinc, cobalt) (mass%) is 160 or less. Contacting with nitrile hydratase.   The method according to claim 1, wherein, in Step II, the nitrile is produced by Step I, and then the solvent is completely or partially distilled off.   In Step II, before bringing the nitrile into contact with the nitrile hydratase, the solution containing 4-halo-3-hydroxybutyronitrile is heat-treated and / or one or more metal salts composed of nickel, zinc, and cobalt are added. The method according to claim 1, comprising a step (step I ′). A method for producing 4-halo-3-hydroxybutyramide, comprising the following steps I ′ and II.
Step I ′: a step of heat-treating a solution containing 4-halo-3-hydroxybutyronitrile and / or adding one or more kinds of metal salts composed of nickel, zinc and cobalt,
Step II: The solution obtained in the above step I ′ is composed of the 4-halo-3-hydroxybutyronitrile concentration (mass%) and salt concentration (alkaline metal salt and buffer salt, nickel, zinc and cobalt). A step of obtaining 4-halo-3-hydroxybutyramide by an enzymatic reaction by contacting with nitrile hydratase under the condition that the product of (total of metal salts) (% by mass) is 160 or less.   The method according to any one of claims 1 to 4, wherein in step II, the pH of the reaction solution is maintained at 6 to 8.5.   The process according to any one of claims 1 to 5, wherein in step II, the temperature of the reaction solution is maintained at 0 to 30 ° C.   The method according to any one of claims 1 to 6, wherein in Step II, the nitrile hydratase is a nitrile hydratase derived from a Rhodococcus microorganism.   The method according to any one of claims 3 to 7, wherein in Step I ', the concentration of cyanide ion in the solution containing 4-halo-3-hydroxybutyronitrile is 100 ppm or less.   The total concentration of the salt of the metal selected from the group consisting of nickel, zinc and cobalt added in step I 'is 0.1 to 10,000 ppm in the reaction solution of step II. The method of any one of these.   The liquid after adding one or more kinds of metal salts composed of nickel, zinc and cobalt in Step I 'is brought into contact at pH 6 to 8.5 for 5 minutes or more, and then subjected to the enzyme reaction in Step II. The method of any one of -9.   In step II, in a solution containing 4-chloro-3-hydroxybutyronitrile and sodium chloride, the concentration (mass%) and salt concentration (alkali metal salt and buffer salt of 4-chloro-3-hydroxybutyronitrile) And the sum of the metal salts of nickel, zinc and cobalt) (mass%) is brought into contact with nitrile hydratase under the condition that the product is 120 or less, and 4-chloro-3-hydroxybutyramide is obtained by the enzymatic reaction. The method according to any one of claims 3 to 10.   The total concentration of salts of metals selected from the group consisting of nickel, zinc, and cobalt added in step I 'is 0.1 to 1,000 ppm in the reaction solution of step II. The method of any one of these.   The manufacturing method of a carnitine amide which makes trimethylamine contact the aqueous solution containing 4-halo-3-hydroxybutyramide obtained by the method of any one of Claims 1-12.   The method of Claim 13 whose temperature at the time of making it contact with a trimethylamine is 0-50 degreeC.