JP2008017838A - Method for producing 4-halo-3-hydroxybutyronitrile - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a 4-halo-3-hydroxybutyronitrile in high concentration. <P>SOLUTION: In the method for producing the 4-halo-3-hydroxybutyronitrile from a 1, 3-dihalo-2-propanol and a cyanide donor by enzyme reaction, the concentration of the 1, 3-dihalo-2-propanol in a reaction system is brought to be ≤0.8 mol/kg after the concentration of the 4-halo-3-hydroxybutyronitrile in the reaction system has become 0.3 mol/kg since the reaction initiated. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a process for producing 4-halo-3-hydroxybutyronitrile by enzymatic reaction from 1,3-dihalo-2-propanol and a cyanide donor. 4-halo-3-hydroxybutyronitrile is a substance useful as a raw material for synthesizing various pharmaceuticals and physiologically active substances, and particularly useful as a raw material for synthesizing L-carnitine.

  A halohydrin epoxidase can be exemplified as an enzyme used in producing 4-halo-3-hydroxybutyronitrile from 1,3-dihalo-2-propanol and a cyanide donor by an enzymatic reaction.

  As halohydrin epoxidases, enzymes produced by microorganisms belonging to the genus Corynebacterium, Microbacterium, Agrobacterium, and Aureobacterium are known. ing.

  The present applicants 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 Document 1 and Patent Document 2) and a method for producing optically active 4-halo-3-hydroxybutyronitrile from epihalohydrin (see Patent Document 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 transformant introduced with this recombinant vector is used to produce 3-hydroxy Butyronitrile has been successfully produced (see Patent Document 4, Patent Document 5, and Non-Patent Document 1).

  Janssen et al., Agrobacterium radiobacter AD1 strain, Mycobacterium sp. GP1, Arthrobacter sp. A halohydrin epoxidase produced by AD2 has been found. In Agrobacterium radiobacter AD1 strain, the three-dimensional structure of the enzyme has been clarified. (Non-Patent Documents 2 and 3)

However, in the method for producing 4-halo-3-hydroxybutyronitrile using the above-mentioned enzyme, the accumulated concentration of the obtained nitrile is as low as about 0.1 mol / kg, and the productivity is low. It wasn't.
Japanese Patent No. 2840722 Japanese Patent Laid-Open No. 2001-25397 Japanese Patent No. 2840723 Japanese Patent No. 3073037 Japanese Patent No. 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 a production method of 4-halo-3-hydroxybutyronitrile having high productivity and industrially suitable.

  In order to solve the above-mentioned problems, the present inventors examined a method for producing 4-halo-3-hydroxybutyronitrile by an enzymatic method. In the process, it was found that when 4-halo-3-hydroxybutyronitrile was allowed to accumulate, the reaction suddenly stopped when the nitrile accumulated 0.3 mol / kg or more in the reaction system.

  As a result of intensive studies to solve this problem, the concentration of 1,3-dihalo-2-propanol was 0.8 mol after the formation of 0.3 mol / kg of 4-halo-3-hydroxybutyronitrile. By controlling not to exceed / kg, it was found that 4-halo-3-hydroxybutyronitrile can be accumulated at a high concentration without stopping the reaction, and the present invention has been completed.

That is, the present invention
(1) In a method for producing 4-halo-3-hydroxybutyronitrile from 1,3-dihalo-2-propanol and a cyanide donor by an enzymatic reaction, the enzymatic reaction is started, and 4-halo- After the concentration of 3-hydroxybutyronitrile reaches 0.3 mol / kg, the concentration of 1,3-dihalo-2-propanol in the reaction system should not exceed 0.8 mol / kg. A method for producing 4-halo-3-hydroxybutyronitrile, which is characterized.
(2) The method according to (1) above, wherein 1,3-dihalo-2-propanol is added so that 1,3-dihalo-2-propanol is 0.01 to 0.8 mol / kg.
(3) The method according to (1) or (2), wherein the cyanide donor is maintained at 0.05 mol / kg or more during the enzyme reaction.
(4) The method according to any one of (1) to (3), wherein the cyanide donor is maintained at 0.05 to 1.5 mol / kg during the enzyme reaction.
(5) The method according to any one of (1) to (4) above, wherein a cyanide donor is allowed to be present in the reaction system before the 1,3-dihalo-2-propanol and the enzyme contact each other.
(6) The method according to any one of (1) to (5), wherein the enzyme is halohydrin epoxidase.
(7) A halohydrin epoxidase is produced from Corynebacterium sp. The method according to (6) above, which is derived from a halohydrin epoxidase gene (hheB) of N-1074. It is.

  According to the present invention, 4-halo-3-hydroxybutyronitrile can be accumulated at a high concentration in the reaction system. Therefore, the productivity is high and industrially suitable 4-halo-3-hydroxybutyro is obtained. A method for producing a nitrile can be provided.

Hereinafter, the present invention will be described in detail.
The method for producing 4-halo-3-hydroxybutyronitrile according to the present invention (hereinafter, simply referred to as “the present production method”) refers to 1,3- In this method, 4-halo-3-hydroxybutyronitrile is produced from dihalo-2-propanol and a cyanide donor by an enzymatic reaction.

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

（式中、Ｘ_１はハロゲン原子を示す。具体的にはフッ素原子、塩素原子、臭素原子、ヨウ素原子であり、塩素原子、臭素原子が好ましく、塩素原子が最も好ましい。＊は不斉炭素を示し、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。）

(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 any of (R), (S), and racemic, but (R) 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 consists of only one of the enantiomers, the optical purity is 100%.

(2) Halohydrin epoxidase Examples of the enzyme used in this production method include halohydrin epoxidase. The halohydrin epoxidase is an activity of dehydrohalogenating 1,3-dihalo-2-propanol represented by the following general formula (2) to synthesize an epihalohydrin represented by the following general formula (3). And an enzyme having an activity of catalyzing the reverse reaction (EC number: 4.5.1.-).

（式中、Ｘ_１、Ｘ_２はハロゲン原子を示す。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−プロパノール等が挙げられ、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。）

(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.)

（式中、Ｘ_１はハロゲン原子を示し、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等が挙げられ、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。）

(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.)

  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 is published, for example, in GenBank, and the Accession number of the halohydrin epoxidase gene (hheB) derived from Corynebacterium sp. N-1074 is D90350.

  The halohydrin epoxidase can be prepared by extraction from the microorganism. It can also be produced by a recombinant microorganism produced by cloning a gene encoding halohydrin epoxidase and incorporating the gene. 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.

Extraction of the enzyme 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.
In addition, examples of host microorganisms used for transformation include microorganisms belonging to the genus Escherichia coli or Rhodococcus, but are not particularly limited thereto, 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.

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, 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.

(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 hydrogen cyanide (HCN), potassium cyanide (KCN), sodium cyanide ( Examples are NaCN) and acetone cyanohydrin (ACH). Hydrogen cyanide, potassium cyanide and sodium cyanide are preferably used.

(4) Enzymatic reaction The enzymatic reaction of this production method is started by adding cyanide donor, 1,3-dihalo-2-propanol and halohydrin epoxidase to water or a buffer solution to the reaction system. 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 solution etc. are illustrated. 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.

The order of introducing cyanide donor, 1,3-dihalo-2-propanol, and halohydrin epoxidase into the buffer solution is not particularly limited, but 1,3-dihalo-2-propanol and halohydrin epoxidase are not limited. Before contacting, it is preferable that a cyanide donor is present in the reaction system in that a high reaction rate can be obtained at the start of the reaction and epihalohydrin by-product can be suppressed. The concentration of the cyanide donor to be present is preferably 0.01 to 1.5 mol / kg, more preferably 0.8 to 1.5 mol / kg at the start of the reaction. If the concentration of the cyanide donor exceeds 1.5 mol / kg, it is not preferable from the viewpoint of enzyme stability.

  It is preferable that 1,3-dihalo-2-propanol is preliminarily present in the reaction system 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 not particularly limited from the start of the reaction until accumulation of 0.3 mol / kg of 4-halo-3-hydroxybutyronitrile, but from the viewpoint of enzyme stability. It is preferably 1 mol / kg or less.

  Here, the concentration of 1,3-dihalo-2-propanol exceeds 0.8 mol / kg when 4-halo-3-hydroxybutyronitrile accumulates 0.3 mol / kg in the reaction system. In such a case, water, cyanide donor, or the like is added to adjust the concentration of 1,3-dihalo-2-propanol to 0.8 mol / kg or less. Thereafter, it is more preferable to add 1,3-dihalo-2-propanol so as not to exceed 0.8 mol / kg from the viewpoint of improving reaction rate and high productivity. It is more preferable to add additionally so as to be 0.01 to 0.8 mol / kg.

  Further, since cyanide donors are consumed as the reaction proceeds, it is preferable to add them. In such a case, it is preferable to add it so that 0.05 mol / kg or more exists in the reaction system, but it is preferable not to exceed 1.5 mol / kg from the viewpoint of enzyme stability.

  The amount of the cyanide donor used is preferably 1 to 2 equivalents of 1,3-dihalo-2-propanol to be finally used from the viewpoint of improving the reaction rate and efficiently using the cyanide donor.

  Here, the amount of 1,3-dihalo-2-propanol used finally means the total amount of 1,3-dihalo-2-propanol used from the start of the reaction to the end of the reaction.

  The temperature during the enzyme reaction is preferably 0 to 30 ° C. from the viewpoint that the enzyme stability is good and the reaction proceeds smoothly.

  As the reaction proceeds, hydrogen halide is generated in the reaction solution, so that the pH is lowered. Therefore, it is preferable to adjust and maintain the pH in the system in a region where the enzyme activity is exhibited by additionally adding alkali into the reaction system. The pH is preferably 7.0 or more from the viewpoint of smooth progress of the reaction, and is preferably 8.5 or less from the viewpoint of suppressing side reactions.

  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, 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 liquid at the start of the reaction. Here, 1 U (unit) means an enzyme amount capable of producing 1 μmol of epichlorohydrin from 1,3-dichloro-2-propanol in 1 minute 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. The amount of 1,3-dihalo-2-propanol and cyanide donor to be added can be determined using these quantitative results as an indicator of the progress of the reaction, but 1,3-dihalo-2-propanol and The concentration of cyanide donor can be controlled more easily.

  That is, when the pH is kept constant using a pH controller after the start of the reaction, the generated hydrogen halide is theoretically equivalent to the generated hydrogen halide, ie, 1,3-dihalo-2-propanol and cyanide donor consumed by the reaction. An equimolar amount of alkali is added. Since the amount of alkali added serves as an indicator of the progress of the reaction, it can be used as a guide when adding 1,3-dihalo-2-propanol and cyanide donor to the reaction system.

  For example, when 1,3-dihalo-2-propanol is 0.5 mol / kg and cyanide donor is 1 mol / kg at the start of the reaction, it is introduced by maintaining the pH constant using a pH controller after the start of the reaction. By adding an equimolar amount of 1,3-dihalo-2-propanol and a cyanide donor, the concentration of 1,3-dihalo-2-propanol was adjusted so that the concentration of 1,3-dihalo-2-propanol did not exceed 0.5 mol / kg. The concentration can be maintained so as not to exceed 1 mol / kg.

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

  According to the above-described method, 4-halo-3-hydroxybutyronitrile can be accumulated in an amount exceeding 0.3 mol / kg in the reaction system without stopping accumulation on the way. The 4-halo-3-hydroxybutyronitrile produced and accumulated in the reaction solution can be collected and purified using a known method. For example, after removing bacterial cells from the reaction solution using a method such as centrifugation, extraction with a solvent such as ethyl acetate is performed, and the solvent is removed under reduced pressure to remove 4-halo-3-hydroxybutyronitrile. You can get syrup. Further, these syrups can be further purified by distillation under reduced pressure.

5. Unreacted cyanide donor and 1,3-dihalo-2-propanol can be reused in this reaction by distilling the reaction solution after completion of the enzyme reaction for reusing the substrate .

Specifically, as shown in the following step (3), unreacted cyanide donor and 1,3-dihalo-2-propanol are preferably recovered and reused. That is, it is a method for producing 4-halo-3-hydroxybutyronitrile including the following steps.
(1) 1,3-dihalo-2-propanol and an enzyme are added to an aqueous solution containing a cyanide donor, and the cyanide donor in the reaction system is 0.05 to 1.5 mol / kg, and 1,3-dihalo-2-propanol is While controlling to be 0.01 to 1 mol / kg, 4-halo-3-hydroxybutyronitrile was accumulated to 0.3 mol / kg,
(2) After accumulating 4-halo-3-hydroxybutyronitrile to 0.3 mol / kg in the step (1), the concentration of 1,3-dihalo-2-propanol in the reaction system is set to 0.01 to Managed to be 0.8 mol / kg,
(3) The reaction solution obtained in the step (2) is distilled, an aqueous solution mainly composed of 4-halo-3-hydroxybutyronitrile, an unreacted cyanide donor, and 1,3-dihalo-2-propanol. Each of the aqueous solutions containing is recovered.

Hereinafter, the present invention will be specifically described by way of examples. In addition, 1,3-dichloro-2-propanol, epichlorohydrin, and 4-chloro-3-hydroxybutyronitrile were quantified using high performance liquid chromatography (HPLC) under the analysis conditions shown in Table 1. It was.

[Preparation Example 1]
Culture of halohydrin epoxidase expressing microorganisms Escherichia coli JM109 / pST111 having halohydrin epoxidase activity was added to LB medium (1% bactotryptone, 0.5% bacto yeast extract, 0.5% (NaCl, 1 mM IPTG, 50 μg / ml ampicillin) 100 mL × 20 cells were inoculated and cultured at 37 ° C. for 20 hours with shaking.

  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. Add 0.25 g of this bacterial cell suspension to 100 mL of 50 mM Tris-sulfate buffer (pH 8.0), add 1,3-dichloro-2-propanol to 50 mM, and react at 20 ° C. for 10 minutes. did.

  The amount of epichlorohydrin in the reaction solution measured by HPLC was 11 mM. That is, 1100 μmol of epichlorohydrin was produced per 10 minutes, and it was found that the activity of this bacterial cell suspension was 440 U per 1 g of the bacterial cell suspension.

  This cell suspension was diluted with 50 mM Tris-sulfate buffer (pH 8.0) to 400 units per gram of cell suspension.

  PST111 is a plasmid in which a BamHI-PstI1.1 Kb fragment containing the halohydrin epoxidase gene (hheB) of Corynebacterium sp. N-1074 is linked to pUC118 (FIG. 1).

  PST111 is described in JP-A-5-317066, and JM109 / pST111 is FERM P-12065, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1-1-1 Tsukuba City East, Ibaraki Prefecture). Deposited on March 6, 1991 in the center No. 6).

<Examples 1-4>
The reaction was performed under the conditions shown in Table 2 below. In the reaction, a 300 mL four-necked flask equipped with an alkali charging pipe controlled by a pH electrode and a pH controller was charged with a predetermined amount of water and HCN, adjusted to a predetermined pH (I) with 30% NaOH, 1,3-Dichloro-2-propanol (DCP) was added and stirred until evenly dissolved.

  Next, 20.00 g (8.00 kU) of the cell suspension of Preparation Example 1 was added, and the reaction was started at a predetermined temperature and pH (II). After starting the reaction, a pH controller was set to control the pH in the reaction system, and the pH was controlled with 30% NaOH.

  DCP and HCN were additionally added at an approximately equimolar ratio with NaOH used for pH control, and the concentrations of DCP and HCN were maintained within a predetermined range.

The yield was calculated by the following method.
Yield (%) = DCP conversion rate × CHBN selectivity / DCP conversion rate (%) = 100 − {(total amount in system × DCP concentration) / (DCP charge amount + DCP cumulative additional amount)}
-CHBN selectivity (%) = {(total amount in system x CHBN concentration) / (DCP charge + DCP cumulative additional amount)} x DCP conversion rate

  Then, after adjusting a predetermined pH (III) with 35% HCl, a distillate was obtained using a cooling tube cooled to 2 ° C. by atmospheric distillation while blowing nitrogen gas at 5 mL / min.

The reaction profile of each example is shown in Table 3, Table 5, Table 7, and Table 9. The distillate / bottle composition of each example is shown in Table 4, Table 6, Table 8, and Table 10.

From the above results, 1,3-dichloro-2-propanol and HCN remaining in the reaction solution could be recovered at a recovery rate of 60 to 80%. It was found that almost all the product 4-chloro-3-hydroxybutyronitrile remained in the kettle residue and there was almost no loss.
<Example 5>

  Using 100.0 g of the distillate obtained in Example 1, 31.25 g of water and 3.41 g of HCN were added thereto, adjusted to pH 7.5 with 30% NaOH 0.65 g, and 1,3-dichloro-2- 7.3 g of propanol was added and stirred until evenly dissolved. Next, 20.00 g (8.00 kU) of the bacterial cell suspension of Preparation Example 1 was added, and the reaction was performed in the same manner as in Example 1.

23 hours after the start of the reaction, 22.0 g of 30% NaOH was added, and the total amount of the reaction solution was 200.78 g.
Thereafter, 0.8 g of 35% hydrochloric acid was added to adjust the pH to 2.0, and then distilled at 110 Torr and 50 ° C. under reduced pressure while blowing nitrogen gas at a rate of 5 mL per minute, and 129.92 g of distilled water was cooled to 2 ° C. A effluent was obtained. Table 11 shows the compositions of the reaction end liquid, distillate liquid and kettle residue.

From the above results, even when distilled water, unreacted 1,3-dichloro-2-propanol and HCN are recycled, there is no adverse effect on the enzyme reaction, and fresh water, HCN and 1,3-dichloro- The reaction results were the same as when 2-propanol was used.
Further, 1,3-dichloro-2-propanol remaining in the reaction solution was recovered at a recovery rate of 75%. 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 94.8% ee before and after distillation, and no decrease in optical purity due to distillation was observed.
<Comparative Example 1>

  In the four-necked flasks of Examples 1 to 4, 55.52 g of 0.1 mol / kg phosphate buffer (pH 7.5) was added, and 10.00 g (0.0775 mol) of DCP was added and stirred until evenly dissolved. .

20.00 g (8.00 kU) of the cell suspension of Preparation Example 1 was added, and the reaction was started at 20 ° C. A pH controller was set to maintain the pH in the system at 7.5 to 7.6, and the pH was controlled with 30% NaOH. After the reaction started, DCP was additionally added at a rate of 1.09 g at intervals of 20 minutes and HCN was added at a rate of 0.55 g at intervals of 10 minutes. The reaction results are shown in Table 12.

  From the above results, it can be seen that after 60 minutes after CHBN accumulated 0.302 mol / kg, when the concentration of DCP was continuously added exceeding 0.8 mol / kg, the reaction suddenly started after 120 minutes (2 hours). Stopped at.

After 180 minutes, CHBN was 0.298 mol / kg and its optical purity was 92.0% e.e. e. The R form was excessive, and the yield was 21.6%.
<Comparative example 2>

125 mol of 0.1 mol / kg phosphate buffer (pH 7.5) and 10.00 g (0.0775 mol) of DCP were put into the four-necked flasks of Examples 1 to 4 and stirred until they were uniformly dissolved.
After adding 20.00 g (8.00 kU) of the cell suspension of Preparation Example 1, 0.20 g (0.0369 mol) of HCN 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 is set so that 30% NaOH is added, and DCP and HCN are added at an approximately equimolar ratio with the added NaOH. Thus, the concentration of DCP in the system was not allowed to exceed 0.5 mol / kg. At this time, the cyanide concentration in the system did not exceed 0.05 mol / kg. The reaction results are shown in Table 13.

  From the above results, even when 180 minutes passed from the start of the reaction, CHBN accumulated only 0.145 mol / kg, and the reaction did not proceed smoothly. The optical purity is 92.0% e.e. e. The R form was excessive. The yield was 28.9%.

It is a block diagram of plasmid pST111.

Claims (7)

    In a method for producing 4-halo-3-hydroxybutyronitrile from 1,3-dihalo-2-propanol and a cyanide donor by an enzymatic reaction, the enzymatic reaction is started and 4-halo-3-hydroxy in the reaction system is started. After the butyronitrile concentration becomes 0.3 mol / kg, the concentration of 1,3-dihalo-2-propanol in the reaction system should not exceed 0.8 mol / kg. , 4-halo-3-hydroxybutyronitrile.   The method according to claim 1, wherein 1,3-dihalo-2-propanol is added so that 1,3-dihalo-2-propanol is 0.01 to 0.8 mol / kg.   The method according to claim 1 or 2, wherein the cyanide donor is maintained at 0.05 mol / kg or more during the enzymatic reaction.   The method according to any one of claims 1 to 3, wherein the cyanide donor is maintained at 0.05 to 1.5 mol / kg during the enzyme reaction.   The method according to any one of claims 1 to 4, wherein a cyanide donor is present in the reaction system before the contact of 1,3-dihalo-2-propanol with the enzyme.   The method according to any one of claims 1 to 5, wherein the enzyme is a halohydrin epoxidase. A halohydrin epoxidase is used in Corynebacterium sp. The method according to claim 6, which is derived from a halohydrin epoxidase gene (hheB) of N-1074.

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