JP5001631B2 - Industrial production method of 4-halo-3-hydroxybutyronitrile - Google Patents

Description

  The present invention relates to an industrial method for producing 4-halo-3-hydroxybutyronitrile, which is a substance useful as a raw material for synthesizing various pharmaceuticals and physiologically active substances.

  A halohydrin epoxidase can be exemplified as an enzyme used in producing 4-halo-3-hydroxybutyronitrile by an enzymatic reaction from 1,3-dihalo-2-propanol and a cyanide donor. As halohydrin epoxidases, those of the genus Corynebacterium, Microbacterium, Agrobacterium, and Aureobacterium are known. The present applicants have previously described the optically active 4-halo-3-hydroxylase from 1,3-dihalo-2-propanol by the action of dehalogenating enzymes of the genus Corynebacterium, Microbacterium, and Aureobacteria. A method for producing hydroxybutyronitrile (see Patent Literature 1 and Patent Literature 2) and a method for producing optically active 4-halo-3-hydroxybutyronitrile from epihalohydrin (see Patent Literature 3) are proposed. Furthermore, using a gene recombination technique, a recombinant vector having a halohydrin epoxidase enzyme gene DNA derived from the genus Corynebacterium is obtained, and a 4-halo compound using a transformant introduced with this recombinant vector is obtained. It has succeeded in producing -3-hydroxybutyronitrile (see Patent Document 4, Patent Document 5, and Non-Patent Document 1). Janssen et al. Also found the halohydrin epoxidase of Agrobacterium radiobacter AD1 strain, Mycobacterium sp. GP1, Arthrobacter sp. AD2, and Agrobacterium radiobacter. In AD1 strain, the three-dimensional structure of the enzyme has been clarified. (Non-Patent Documents 2 and 3)

  4-halo-3-hydroxybutyronitrile produced by such an enzyme reaction is usually recovered by performing extraction from the reaction solution with an organic solvent and then distilling off the solvent under reduced pressure. At this time, the distilled-off organic solvent contains unreacted 1,3-dihalo-2-propanol and cyanide donor. Considering the efficiency of the reaction, it is preferable to reuse these unreacted raw materials. However, since the enzyme reaction is usually carried out in an aqueous system, further distillation of an organic solvent or the like is required to reuse the unreacted raw materials. Complicated operations are required. In addition, the cost and environmental burden of waste liquid treatment are large. Therefore, it cannot be said that it is industrially suitable. Therefore, a method for efficiently recovering and reusing these unreacted raw materials and reaction solvent and industrially producing 4-halo-3-hydroxybutyronitrile has been desired.

Patent 2840722 Patent 3168202 Patent No.2840723 Patent 3073037 Patent 3026367 Biosci. Biotech. Biochem., 58 (8), 1451-1457, 1994 The EMBO Journal., 22 (19), 4933-4944, 2003 J. Bacteriology 183 (17), 5058-5066, 2001

An object of the present invention is to provide an industrially advantageous production method of 4-halo-3-hydroxybutyronitrile by reusing unreacted 1,3-dihalo-2-propanol and cyanide donor.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventors distilled unreacted cyanide donor and 1,3-dihalo-2-propanol into 4-halogen by distilling the reaction solution after completion of the enzyme reaction. It has been found that it can be efficiently separated and recovered from -3-hydroxybutyronitrile and can be easily reused, and the present invention has been completed.

That is, the present invention includes the following inventions.
In a method for producing 4-halo-3-hydroxybutyronitrile from 1,3-dihalo-2-propanol and a cyanide donor by an enzymatic reaction in an aqueous system,
(1) A step of distilling the reaction aqueous solution after completion of the enzyme reaction to recover unreacted cyanide donor and 1,3-dihalo-2-propanol;
(2) a step of repeatedly using the recovered cyanide donor and 1,3-dihalo-2-propanol for the enzymatic reaction at least once,
A process for producing 4-halo-3-hydroxybutyronitrile.

  According to the present invention, unreacted cyanide donor and 1,3-dihalo-2-propanol can be efficiently separated and recovered and reused. Therefore, it is possible to provide a production method of 4-halo-3-hydroxybutyronitrile having high productivity and industrially suitable. Furthermore, since this distillation can be carried out below the boiling point of 1,3-dihalo-2-propanol at the pressure during distillation, energy for distillation can be saved.

The present invention relates to a method for producing 4-halo-3-hydroxybutyronitrile from 1,3-dihalo-2-propanol and a cyanide donor by an enzymatic reaction in an aqueous system.
(1) A step of distilling the reaction aqueous solution after completion of the enzyme reaction to recover unreacted cyanide donor and 1,3-dihalo-2-propanol;
(2) a step of repeatedly using the recovered cyanide donor and 1,3-dihalo-2-propanol for the enzymatic reaction at least once,
Is a process for producing 4-halo-3-hydroxybutyronitrile.

（式中、Ｘ₁、Ｘ₂はハロゲン原子を示す。）
式１において、ハロゲン原子としては、フッ素、塩素、臭素、ヨウ素が好ましく、塩素、臭素が特に好ましい。具体的には１，３−ジフルオロ−２−プロパノール、１，３−ジクロロ−２−プロパノール、１，３−ジブロモ−２−プロパノール、１，３−ジヨード−２−プロパノールが挙げられ、好ましくは、１，３−ジクロロ−２−プロパノール、１，３−ジブロモ−２−プロパノールである。 1. Explanation of Terms (1) 1,3-Dihalo-2-propanol
1,3-dihalo-2-propanol refers to a compound represented by the following general formula (1).

(In the formula, X ₁ and X ₂ represent halogen atoms.)
In Formula 1, as the halogen atom, fluorine, chlorine, bromine and iodine are preferable, and chlorine and bromine are particularly preferable. Specific examples include 1,3-difluoro-2-propanol, 1,3-dichloro-2-propanol, 1,3-dibromo-2-propanol, and 1,3-diiodo-2-propanol. 1,3-dichloro-2-propanol and 1,3-dibromo-2-propanol.

(2) Cyanide Donor Cyanide donor means a compound that generates cyanide ion (CN ⁻ ) or hydrogen cyanide when added to a reaction solution, and is not particularly limited. NaCN) and acetone cyanohydrin (ACH). Cyanic acid, potassium cyanide, and sodium cyanide are preferably used. In particular, cyanic acid is preferably used because it can be easily recovered and recycled.

(式中X₁はハロゲン原子を示す。＊は不斉炭素を示す。)
式２において、X₁のハロゲン原子としては、フッ素、塩素、臭素、ヨウ素が好ましく、塩素、臭素が特に好ましい。その立体配置は(R)-体、(S)-体、ラセミ体のいずれでも構わないが、(R)-体が好ましい。 (3) 4-halo-3-hydroxybutyronitrile
4-halo-3-hydroxybutyronitrile refers to a compound represented by the following general formula (2).

(In the formula, X ₁ represents a halogen atom. * Represents an asymmetric carbon.)
In Formula 2, as the halogen atom of X ₁ , fluorine, chlorine, bromine and iodine are preferable, and chlorine and bromine are particularly preferable. The configuration may be any of the (R) -isomer, (S) -isomer, and racemate, but the (R) -isomer is preferred.

  Specific examples include 4-fluoro-3-hydroxybutyronitrile, 4-chloro-3-hydroxybutyronitrile, 4-bromo-3-hydroxybutyronitrile, 4-iodo-3-hydroxybutyronitrile. 4-chloro-3-hydroxybutyronitrile and 4-bromo-3-hydroxybutyronitrile are preferable. 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. When 4-halo-3-hydroxybutyronitrile is composed of only one of the enantiomers, the optical purity is 100%.

(4) Halohydrin epoxidase A halohydrin epoxidase is dehalogenated from 1,3-dihalo-2-propanol represented by the above general formula (1) and represented by the following general formula (3). An enzyme (EC number: 4.5.1.-) having an activity to synthesize epihalohydrin and an activity catalyzing the reverse reaction.

（図中、X₁はハロゲン原子を示す。）
式３において、X₁は、フッ素、塩素、臭素、ヨウ素が好ましく、塩素、臭素が特に好ましい。具体的にはエピフルオロヒドリン、エピクロロヒドリン、エピブロモヒドリン、エピヨードヒドリンが挙げられ、特に好ましくはエピクロロヒドリン、エピブロモヒドリンである。

(In the figure, X ₁ represents a halogen atom.)
In Formula 3, X ₁ is preferably fluorine, chlorine, bromine or iodine, particularly preferably chlorine or bromine. Specific examples include epifluorohydrin, epichlorohydrin, epibromohydrin, and epiiodohydrin, with epichlorohydrin and epibromohydrin being particularly preferred.

  Microbes that produce this enzyme include Corynebacterium sp. N-1074 (FERM BP-2643), Microbacterium sp. N-4701 (FERM BP-2644), Agrobacterium radiobacter (Agrobacterium radiobacter) AD1, Mycobacterium sp.GP1, Arthrobacter sp.AD2, etc. are mentioned. A particularly preferred microorganism is Corynebacterium sp. N-1074 (FERM BP-2643). The halohydrin epoxidase gene (hheB) derived from Corynebacterium sp. N-1074 has been published in GenBank and its Accession number is D90350.

  The halohydrin epoxidase can be prepared by extraction from the microorganism, but can also be produced by a recombinant microorganism prepared by cloning a halohydrin epoxidase gene and incorporating the gene. In addition, 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 long as it has the above activity. Extraction may be carried out by a conventional method, and even if the preparation contains components other than halohydrin epoxidase, it does not need to be purified if it does not adversely affect the reaction. A halohydrin epoxidase obtained by culturing a genetically modified microorganism prepared by incorporating a halohydrin epoxidase gene is preferably used because it is easy to prepare. Examples of host microorganisms used for transformation include microorganisms belonging to the genus Escherichia coli or Rhodococcus, but are not particularly limited to these, and other host microorganisms can also be used.

  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. Culturing is performed at a temperature of 10 to 50 ° C. and in a pH range of 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, disrupted cells, 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.

2. Enzyme reaction In this production method, an enzyme reaction using 1,3-dihalo-2-propanol and a cyanide donor as a substrate by halohydrin epoxidase is in the vicinity of an optimum pH of 4 to 10 for the enzyme used. It is preferably carried out in the presence of water or a buffer. 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 Alternatively, Good buffer, etc. are exemplified, and a buffer composed of hydrocyanic acid and its salt is preferable, and a Tris buffer is preferable. Particularly, a buffer composed only of hydrocyanic acid and its salt is not mixed with impurities derived from the buffer. preferable.

  Regarding the order of introduction of cyanide donor, 1,3-dihalo-2-propanol and halohydrin epoxidase into the reaction system containing the above buffer solution, 1,3-dihalo-2-propanol and halohydrin epoxidase are in contact with each other. Before starting the reaction, it is possible to obtain a high reaction rate at the start of the reaction by allowing the cyanide donor in an amount such that the concentration at the start of the reaction is 0.01 to 1.5 mol / kg in the reaction system. And it is preferable at the point which can suppress the byproduct of epihalohydrin. More preferably, 0.8 to 1.5 mol / kg cyanide donor is present.

  It is preferable that 1,3-dihalo-2-propanol is added in advance so as to be 0.01 mol / kg or more at the start of the reaction because a high reaction rate can be obtained at the start of the reaction.

  The concentration of 1,3-dihalo-2-propanol is preferably 1 mol / kg or less from the viewpoint of enzyme stability. Moreover, it is preferable to add 1,3-dihalo-2-propanol so that it may not exceed 1 mol / kg from the viewpoint of improving the reaction rate and high productivity.

  Since the cyanide donor in the reaction solution is consumed by the reaction after the start of the reaction, it is preferable to add a cyanide donor. In that case, it is preferable to add additionally so that 0.05 mol / kg or more exists in the reaction system. From the viewpoint of enzyme stability, it is more preferable not to exceed 1.5 mol / kg.

  In addition, the amount of cyanide donor used is preferably 1.2 equivalents or more of 1,3-dihalo-2-propanol to be used finally from the viewpoint of smooth progress of the reaction, and is preferably 2 equivalents or less. It is preferable from the viewpoint of efficient use. The amount of 1,3-dihalo-2-propanol finally used means the total amount of 1,3-dihalo-2-propanol used from the start of the reaction to the end of the reaction.

  The reaction temperature is preferably 10 to 30 ° C. from the viewpoint of good enzyme stability and smooth reaction.

  As the reaction proceeds, hydrogen halide is generated in the reaction solution and the pH is lowered. By sequentially adding alkali to the reaction system, the pH in the system is brought into a region where the enzyme activity is exerted. It is preferable to maintain. The pH is particularly preferably 7.0 to 8.5 from the viewpoint of smooth progress of the reaction and suppression of side reactions.

  The alkali 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, an alkaline earth metal, or ammonia is used. And salts with hydroxides or weak acids. Sodium hydroxide, potassium hydroxide, aqueous ammonia, 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 amount of halohydrin epoxidase used is not particularly limited, and an amount that allows the reaction to proceed smoothly may be used. For example, 1 U to 1000 U can be used per 1 g of the reaction solution at the start of the reaction. Here, 1U (unit) is the amount of enzyme that can produce 1 μmol of epichlorohydrin per minute from 1,3-dichloro-2-propanol in a buffer solution at 20 ° C. and pH 8.0. Means that.

  The concentration of 1,3-dihalo-2-propanol and 4-halo-3-hydroxybutyronitrile in the reaction system can be quantified by, for example, high performance liquid chromatography (HPLC). The concentration of cyanide donor in the reaction system can be quantified by titration, for example. Using these quantitative results as an indicator of reaction progress, the amount of 1,3-dihalo-2-propanol and cyanide donor to be added can be determined.

The reaction time is appropriately selected depending on the concentration of the substrate and the like, the bacterial cell concentration, or other reaction conditions, but it is preferable to set the conditions so that the reaction is completed in 1 to 120 hours.

3. The reaction solvent, unreacted cyanide donor and 1,3-dihalo-2-propanol are separated and recovered from the reaction solution containing 4-halo-3-hydroxybutyronitrile after completion of the distillation / reuse enzyme reaction. By doing. Moreover, when the solid substance derived from the halohydrin epoxidase used by the enzyme reaction exists in the reaction liquid used as distillation object, you may remove a solid substance before distillation. The removal of the solid matter may be performed by a conventional method, and examples thereof include methods such as filtration and centrifugation.

  The distillation is preferably performed while blowing air or inert gas in order to increase the separation efficiency of unreacted cyanide donor and 1,3-dihalo-2-propanol from the reaction solution. The inert gas is a gas that does not substantially react with 4-halo-3-hydroxybutyronitrile, which is a product during distillation, or an unreacted cyanide donor and 1,3-dihalo-2-propanol. Often, water vapor, nitrogen, carbon dioxide, helium, and argon are exemplified. These gases may be used alone or in combination of two or more, or the composition may be constant or change during distillation. The air is preferably used after being diluted with the above inert gas so that excessive oxygen is not introduced into the system in which distillation is performed. The gas flow rate is preferably set so that the gas phase in the reaction vessel is replaced in 1 to 120 hours.

  In this distillation operation, in order to increase the separation efficiency of the unreacted cyanide donor from the reaction solution and prevent decomposition of 1,3-dihalo-2-propanol and 4-halo-3-hydroxybutyronitrile, It is preferable to add an acid stronger than hydrocyanic acid to keep the distillation bottom acidic. The pH of the distillation bottom is preferably 8 or less, and more preferably 5 or less. The acid to be added is not particularly limited as long as it is an acid stronger than hydrocyanic acid, that is, hydrocyanic acid gas is generated by mixing with an aqueous solution of a salt composed of hydrocyanic acid and a strong base. Examples thereof include organic acids such as p-toluenesulfonic acid and acetic acid, and inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid.

  The degree of decompression and the temperature can be appropriately determined according to the type of apparatus used. Since 1,3-dihalo-2-propanol is obtained even at a temperature lower than the boiling point of 1,3-dihalo-2-propanol at the pressure during distillation, the temperature of distillation is 1,3-dihalo-2-propanol. A temperature lower than the boiling point of is preferable from the viewpoint of energy saving. Usually, the distillation temperature is 0 to 110 ° C, preferably 40 to 110 ° C, and the degree of vacuum is 1 to 760 torr, preferably 50 to 760 torr. When the distillation temperature is high, the optically active 4-halo-3-hydroxybutyronitrile is reacted with unreacted 1,3-dihalo-2-propanol and cyanide donor non-enzymatically, resulting in racemic 4- A part of halo-3-hydroxybutyronitrile is generated, and the optical purity is lowered.

  Therefore, the concentration of cyanide donor in the distillation bottom is appropriately measured, and the distillation temperature is preferably 40 to 80 ° C. until it is less than 1000 ppm, preferably less than 500 ppm, more preferably less than 100 ppm. The distilled reaction solvent, cyanide donor, and 1,3-dihalo-2-propanol can be collected using a cooling tube cooled to a temperature sufficient to collect each, for example, 10 ° C. or less.

The solution containing the unreacted cyanide donor and 1,3-dihalo-2-propanol recovered in this way can be used as a solvent for the next enzyme reaction.

In the present production method, the recovered unreacted 1,3-dihalo-2-propanol can be quantified by, for example, high performance liquid chromatography (HPLC) in the same manner as the reaction solution during the enzyme reaction. In addition, the cyanide donor in the reaction system can be quantified by titration, for example, in the same manner as the reaction solution during the enzyme reaction.

Hereinafter, the present invention will be specifically described with reference to examples, but the scope of the present invention is not limited thereto. In addition, 1,3-dichloro-2-propanol, epichlorohydrin, and 4-chloro-3-hydroxybutyronitrile were quantified using high performance liquid chromatography (HPLC) under the following analysis conditions.

[HPLC analysis conditions]
Sample preparation method: Dissolve reaction solution in mobile phaseColumn: Inertsil ODS-3V, 4.6mm ID × 250mm, particle size 5μm
(GL Science)
Column oven temperature: 40 ℃
Mobile phase: water / acetonitrile / phosphoric acid = 70/30 / 0.1, 1mL / min
Detection: Differential refractometer (RI)
Retention time: 1,3-dichloro-2-propanol 15.7min
: Epichlorohydrin 11min
: 4-chloro-3-hydroxybutyronitrile 7.2min

<Reference Example 1> Preparation of cell suspension Escherichia coli JM109 / pST111 (FERM P-12065, see JP-A-5-317066) having halohydrin epoxidase activity was added to LB medium (1% Bactertripton, 0.5% Bacto yeast extract, 0.5% NaCl, 1 mM IPTG, 50 μg / ml ampicillin) 100 mL × 20 cells were inoculated and cultured with shaking at 37 ° C. for 20 hours. The cultured cells were washed with 50 mM Tris-sulfate buffer (pH 8.0), and 50 mM Tris-sulfate buffer (pH 8.0) was added to 20 g and suspended. 0.25 g of this bacterial cell suspension was added to 100 mL of 50 mM Tris-sulfate buffer (pH 8.0), and 1,3-dichloro-2-propanol was further added to adjust to 50 mM, followed by reaction at 20 ° C. for 10 minutes. When the amount of epichlorohydrin in the reaction solution was measured by HPLC, it was 11 mM. That is, 1100 μmol of epichlorohydrin was produced per 10 minutes, and the activity of this cell suspension was found to be 440 U per 1 g of cell suspension. This cell suspension was diluted with 50 mM Tris-sulfate buffer (pH 8.0) to 400 U per gram of cell suspension.

<Example 1>
(1) First synthesis of 4-chloro-3-hydroxybutyronitrile
31 L of water and 10.9 g of HCN were placed in a 1 L flask equipped with an alkaline charging pipe controlled by a pH electrode and a pH controller, and adjusted to pH 7.5 with 30 g NaOH 1.0 g. 1,5-dichloro-2-propanol (25.0 g) was added and stirred until evenly dissolved.
In order to maintain the pH in the system at 7.5 to 7.6, the pH controller was set so that 30% NaOH was added, and 43.2 g (19.0 kU) of the cell suspension prepared in Reference Example 1 was added. The reaction was started.
Immediately after the start of the reaction, 25.0 g of 1,3-dichloro-2-propanol and 6.5 g of HCN were added dropwise evenly over 2 hours. Thereafter, the reaction was carried out for 22 hours (24 hours from the start of the reaction) while maintaining the pH in the system at 7.5 to 7.6 at 20 ° C. At the end of the reaction, 47.1 g of 30% NaOH was added, and the total amount of the reaction solution was 478.1 g. The composition of the reaction completion liquid is shown in Table 1.

(2) Distillation of 4-chloro-3-hydroxybutyronitrile synthesis reaction liquid To the reaction completion liquid obtained in (1) above, 1.1 g of 35% hydrochloric acid was added to adjust the pH to 4.6. Distilling at atmospheric pressure while blowing 10 mL per minute, 341.5 g of distillate was obtained using a cooling tube cooled to 2 ° C. Table 1 shows the composition of the distillate and the residue in the kettle.

(3) Synthesis of 4-chloro-3-hydroxybutyronitrile-2nd time Add 6.6 g of HCN to 329.2 g of the distillate obtained by the above distillation, and adjust to pH 7.5 with 1.2 g of 30% NaOH. After adjusting and adding 21.2 g of 1,3-dichloro-2-propanol and stirring until it was uniformly dissolved, the reaction was carried out in the same manner as described above. At the end of the reaction, 47.5 g of 30% NaOH was added, and the total amount of the reaction solution was 480.4 g. The composition of the reaction completion liquid is shown in Table 1.

  From the above results, 1,3-dichloro-2-propanol and HCN remaining in the reaction solution could be recovered at a recovery rate of about 80%. It was found that almost all the product 4-chloro-3-hydroxybutyronitrile remained in the kettle residue and there was almost no loss. In addition, recycling of distilled water, unreacted 1,3-dichloro-2-propanol and HCN has no adverse effect on the enzymatic reaction, and fresh water, HCN and 1,3-dichloro-2-propanol The reaction results were the same as when using.

<Example 2> Distillation of 4-chloro-3-hydroxybutyronitrile synthesis reaction solution (2)
To the reaction solution obtained in Example 1 (3), 1.1 g of 35% hydrochloric acid was added to adjust the pH to 4.6, and then distilled under reduced pressure at 110 Torr and 50 ° C. while blowing 10 mL of nitrogen gas per minute. 336.4 g of distillate was obtained using a cooling tube cooled to ° C.

  From the above results, 1,3-dichloro-2-propanol remaining in the reaction solution could be recovered at a recovery rate of 70%. It was found that almost all the product 4-chloro-3-hydroxybutyronitrile remained in the kettle residue and there was almost no loss. The optical purity of 4-chloro-3-hydroxybutyronitrile was 92.0% ee before and after distillation, and no decrease in optical purity due to distillation was observed.

Claims (1)

In a method for producing 4-halo-3-hydroxybutyronitrile from 1,3-dihalo-2-propanol and a cyanide donor by an enzymatic reaction in an aqueous system,
(1) The reaction aqueous solution after completion of the enzymatic reaction was distilled at an acidic pH, temperature of 0 ° C. to 110 ° C., pressure of 1 to 760 Torr (133.322 to 101325 Pa), and unreacted cyanide donor and 1,3-dihalo-2- Recovering propanol,
(2) a step of repeatedly using the recovered cyanide donor and 1,3-dihalo-2-propanol for the enzyme reaction at least once,
A process for producing 4-halo-3-hydroxybutyronitrile.