JP2001120289A - Method for producing optically active cyanohydrin with hydroxynitrile lyase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing an optically active cyanohydrin, which stabilizes the activity of hydroxynitrile lyase, does not cause the deterioration of the activity, when the lyase is repeatedly used, and is therefore very useful for the industrial production of the optically active cyanohydrin, in a reaction system for synthesizing the optically active cyanohydrin from a carbonyl compound and a cyan compound in the presence of the hydroxynitrile lyase. SOLUTION: This method for producing an optically active cyanohydrin comprises synthesizing the optically active cyanohydrin from a carbonyl compound and a cyan compound in the presence of the hydroxynitrile lyase. Therein, either or both of (i) a treatment for reducing the concentration of oxygen in the reaction system and (ii) a treatment for reducing the concentration of hydroquinone and compounds derived from the hydroquinone in the reaction system are performed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a reaction system for synthesizing optically active cyanohydrin from a carbonyl compound and a cyanide compound using hydroxynitrile lyase as a catalyst, wherein the optically active cyanohydrin is incorporated with a treatment for stabilizing the hydroxynitrile lyase activity. And a method for producing the same.

[0002]

2. Description of the Related Art Optically active cyanohydrins are useful as intermediates for optically active organic synthesis. As one of means for directly synthesizing optically active cyanohydrin from hydrogen cyanide and a carbonyl compound, various synthesis methods using an enzyme called hydroxynitrile lyase have been proposed. This enzyme has an activity of producing R-cyanohydrin,
An R-hydroxynitrile lyase and an S-hydroxynitrile lyase having an activity of producing S-form cyanohydrin are known. The former R- hydroxynitrile lyase, for example, Rosaceae plants, specifically almonds (Prunus amygdalus) derived from R- hydroxynitrile lyase (EC 4.1.2.10), flax (Linum usitatissi
mum) -derived R-hydroxynitrile lyase and the like. Examples of the latter S-hydroxynitrile lyase include, for example, S-hydroxynitrile lyase (EC 4.1.2.37) derived from cassava ( Manihot esculenta) derived from a plant belonging to the family Euphorbiaceae, and Hevea brasiliensis ( H
S-hydroxynitrile lyase (EC 4.1.2.39) derived from Evea brasiliensis ) or S-hydroxynitrile lyase (EC 4.1.2.11) derived from sorghum ( Sorghum bicolor ), which is a gramineous plant, is known. Gene sequences encoding enzymes are also known. These enzymes can be obtained by extracting from a plant tissue containing the enzyme, or by introducing the enzyme gene into another cell and expressing the enzyme gene in the obtained transgenic cells. .

[0003] Usually, the synthesis of optically active cyanohydrin using the enzyme is carried out in an aqueous system, a water-organic solvent two-phase system, an organic solvent-microaqueous system, an organic solvent containing the enzyme and substrates hydrogen cyanide and a carbonyl compound as essential elements. Is implemented in the system. In consideration of productivity, this reaction is more advantageously performed in a reaction system containing an organic solvent in terms of increasing the product concentration and separating the reaction product. As an organic solvent used in the reaction, an organic solvent that is hardly soluble or insoluble in water is preferably used in many cases, and particularly, an ether solvent is often used.

However, when synthesizing optically active cyanohydrin in a reaction system containing an organic solvent, the present inventors have proposed:
It has been found that the stability of the enzyme is poor and the activity is extremely reduced when the same enzyme is used repeatedly.
Therefore, in industrial production of optically active cyanohydrin, it is desired to improve the stability of the enzyme in a reaction system containing an organic solvent.

[0005]

An object of the present invention is to stabilize the activity of hydroxynitrile lyase in a reaction system for synthesizing optically active cyanohydrin from a carbonyl compound and a cyanide using hydroxynitrile lyase as a catalyst. It is in.

[0006]

Means for Solving the Problems The inventors of the present invention have made intensive studies to solve the above problems, and as a result, have found that a reaction system for synthesizing optically active cyanohydrin from a carbonyl compound and a cyanide compound using hydroxynitrile lyase as a catalyst. By focusing on the oxygen concentration in the reaction system and hydroquinone contained as a stabilizer in the organic solvent that is the reaction solvent, by performing a treatment to reduce these from the reaction system,
They have found that the hydroxynitrile lyase activity of the reaction system can be stabilized, and have completed the present invention.

That is, the present invention provides a reaction system for synthesizing optically active cyanohydrin from a carbonyl compound and a cyanide compound using hydroxynitrile lyase as a catalyst, wherein (i) a treatment for reducing the oxygen concentration in the reaction system, A process for reducing the concentration of hydroquinone and a compound derived from hydroquinone in the reaction system, or both of them, and a method for producing an optically active cyanohydrin. Hereinafter, the present invention will be described in detail.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION A reaction system to which the method of the present invention is applied is a reaction system for synthesizing optically active cyanohydrin from a carbonyl compound and a cyanide using hydroxynitrile lyase as a catalyst. In the present invention, the stabilization of the hydroxynitrile lyase activity is performed by either or both of the treatment for reducing the oxygen concentration in the reaction system, the treatment for reducing hydroquinone and the compound derived from hydroquinone in the reaction system, or both. Performed by

In the present invention, the treatment for reducing the oxygen concentration in the reaction system is, specifically, by bringing the reaction solvent into contact with a gas (such as nitrogen, argon or helium) which does not affect the reaction, It refers to a process of reducing dissolved oxygen concentration by replacing dissolved oxygen with the above gas.

The treatment for reducing the oxygen concentration in the reaction system is as follows:
It may be carried out by a conventional method, for example, by putting a reaction solvent in a container having a stirring function, and passing the above-mentioned inert gas through the liquid under stirring. Specifically, the above-mentioned inert gas is used in an amount of 1 ml to 10 L per minute per 1 L of the reaction solvent.
For 1 minute to 1 hour, preferably for 10 to 5 L per minute for 1 L of the reaction solvent.

Alternatively, the reaction can be carried out by distilling the reaction solvent under the atmosphere of the above inert gas. It can also be carried out by adding an oxygen scavenger such as sodium sulfite or hydrosulfite. Further, the gas phase of the reaction vessel is filled with an inert gas, or the reaction is performed while the inert gas is passed through the reaction solution and / or the gas phase of the reaction system during the reaction at the above-described flow rate. It can also be performed by causing

In the present invention, the treatment for reducing the concentration of hydroquinone and a compound derived from hydroquinone (benzoquinone, quinhydrone) in the reaction system is performed by distilling the reaction solvent and deriving from hydroquinone or hydroquinone contained in the reaction solvent. It is performed by separating from the compound. Hydroquinone and the concentration of the compound derived from hydroquinone is less than 40 ppm, preferably 1 ppm
It is preferred to reduce it to less than. Distillation may be carried out under normal or reduced pressure at a temperature at which a compound derived from hydroquine and hydroquinone remains and only the reaction solvent is separated by distillation.

Alternatively, the above treatment can also be carried out by adsorbing and removing hydroquinone and a compound derived from hydroquinone using an adsorbent such as activated carbon. The adsorption can be carried out by charging the adsorbent into the reaction solvent, passing the solvent through a column filled with the adsorbent, or contacting the reaction solvent with the adsorbent for a certain period of time by other methods. The input amount of the adsorbent is appropriately determined according to the adsorption capacity of the adsorbent. In the above reaction system, the carbonyl compound refers to an aldehyde or a ketone, and is specifically represented by the following formula (I).

[0014]

Embedded image

In the above formula (I), R ₁ and R ₂ are (i)
A hydrogen atom, (ii) a substituted or unsubstituted linear or branched saturated alkyl group having 1 to 18 carbon atoms, or (iii) a substituted or unsubstituted aromatic group having 5 to 22 ring members. . However,
R ₁ and R ₂ do not simultaneously represent a hydrogen atom.

In the above (ii), when R ₁ and R ₂ are substituted alkyl groups, the substituent may be one or more of an amino group, an imino group, a hydroxy group, an alkoxy group having 1 to 8 carbon atoms,
A halogen, a carboxyl group, a cycloalkyl group having 3 to 20 carbon atoms, or an aromatic group having up to 22 carbon atoms which may be substituted by a heteroatom of N, O or S (where the substituent is a cyclic substituent In the case of a group, it may be one or more halogen, a hydroxy group, a linear or branched alkyl group having 1 to 8 carbon atoms, a linear or branched alkyl group having 2 to 8 carbon atoms. May be substituted with an alkenyl group.).

In the above (iii), the aromatic group may be a heteroaromatic group in which up to four ring members are substituted by N, O and / or S. When R ₁ and R ₂ are substituted aromatic groups, the substituent may be one or more of an amino group, an imino group, a hydroxy group, an alkoxy group having 1 to 8 carbon atoms, an allyloxy group, a halogen, and a carboxyl group. ,
It is a linear or branched saturated or unsaturated alkyl group having up to 22 carbon atoms (where one aromatic group may be substituted with at least two substituents).

In order to convert the above aldehyde or ketone into optically active cyanohydrin, a cyanide compound is used as a raw material. For example, when hydrogen cyanide is used,
As a supply method, any of a method of supplying as a liquid and a method of supplying as a gas can be adopted. Further, not only hydrogen cyanide but also hydrocyanic acid (that is, hydrocyanic acid) which is an aqueous solution of hydrogen cyanide can be used in exactly the same manner. In addition, any substance that generates cyanide ions (CN ⁻ ) by adding it to the reaction system can be used. Examples thereof include hydrogen cyanide salts such as sodium cyanide and potassium cyanide, and cyanohydrins such as acetone cyanohydrin. Can be

Hydroxynitrile lyase, which is an enzyme used in the reaction, is an enzyme obtained by extracting from a plant tissue containing the enzyme by a conventional method, or a gene encoding the enzyme, followed by cloning of the gene into which the gene is incorporated. Any of the enzymes produced by the recombinant cells can be used and is not particularly limited.

The above enzymes extracted from plant tissues include:
Rosaceae-derived R-hydroxynitrile lyases such as almonds ( Prunus amygdalus) , apples ( Malus d
omestica) , apricot ( Prunus armeniaca) , peach ( Prunus
persica) , an enzyme derived from plum ( Prunus mume ); R-hydroxynitrile lyase derived from flax, such as flax ( L
inum usitatissimum) ; S from grasses
-Hydroxynitrile lyases, such as sorghum ( Sorg
hum bicolor) ; S from Euphorbiaceae
-Hydroxynitrile lyases such as cassava ( Ma
nihot esculenta) , Hevea brasiliensis
S-hydroxynitrile lyase derived from a plant of the ragwood plant, for example, Ximenia america
The enzyme of na) can be exemplified.

[0021] The gene encoding the enzyme may be a protein produced by the gene expression, a protein produced by the gene expression may be a protein having an activity of producing an optically active cyanohydrin using a carbonyl compound and a cyanide compound as substrates. Means the gene sequence containing the genetic information of

The microorganism to be transformed by incorporating the enzyme gene is not particularly limited as long as the microorganism can incorporate the enzyme gene and can express the enzyme gene to produce the enzyme. For example, eukaryotic microorganisms such as yeast and prokaryotic microorganisms such as Escherichia coli can be mentioned.

The form of use of the enzyme obtained by any of the above may be a purified enzyme powder, an enzyme solution dissolved in an aqueous solvent, or an immobilized enzyme obtained by immobilizing the enzyme on a suitable immobilization carrier. Either form may be used. As the reaction system to which the method of the present invention is applied, water-
Examples include an organic solvent two-phase system, an organic solvent-small water system, and an organic solvent system.

A water-organic solvent two-phase system is a system formed by mixing water or an aqueous buffer with an organic solvent that is hardly soluble or insoluble in water. In particular, the reaction system is characterized in that a substrate as a carbonyl compound and a cyanohydrin as a product are distributed to an organic solvent phase. The organic solvent-microaqueous system refers to a reaction system formed by adding water or an aqueous buffer to an organic solvent in excess of a saturated amount. The organic solvent system is a reaction system using only an organic solvent as a reaction solvent.

As the organic solvent, any organic solvent that is hardly soluble or insoluble in water and that does not affect the synthesis reaction of optically active cyanohydrin by an enzymatic reaction can be used without particular limitation. It can be appropriately selected according to the physical properties of the starting material aldehyde or ketone used for the reaction and the physical properties of the product cyanohydrin. Specifically, aliphatic or aromatic linear or branched or cyclic saturated or unsaturated hydrocarbon solvents which may be halogenated, for example, pentane, hexane, toluene, xylene, methylene chloride and the like An optionally halogenated aliphatic or aromatic linear or branched or cyclic saturated or unsaturated alcoholic solvent, for example,
Isopropyl alcohol, n-butanol, isobutanol, t-butanol, hexanol, cyclohexanol, n-amyl alcohol and the like; an optionally halogenated aliphatic or aromatic linear or branched or cyclic Saturated or unsaturated ether solvents, for example, diethyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, methyl-t-butyl ether and the like; optionally halogenated aliphatic or aromatic linear or branched Or cyclic saturated or unsaturated ester solvents, for example, methyl formate, methyl acetate,
Ethyl acetate, butyl acetate, isopropyl acetate, methyl propionate and the like, even when used alone,
Further, a plurality of them may be mixed and used.

The above-mentioned aqueous buffer is an aqueous buffer having a pH of 7 or less and has a pH within a range that does not impair the enzyme activity.
Salts and salt concentrations can be employed. For example, citrate buffer,
Phosphate buffer, acetate buffer and the like can be mentioned. The concentration of the aldehyde or ketone in the reaction solvent is preferably in the range of 0.01 mM to 5 M, and the concentration of aldehyde or ketone is 1 to 20 moles of hydrogen cyanide or a substance which generates cyanide ion in the reaction system per mole of aldehyde or ketone. Use an amount of enzyme that shows an enzyme activity of 1 unit / mmol or more.

The enzyme activity can be calculated by using DL-mandelonitrile as a substrate and measuring the change in absorbance at the time when the substrate is decomposed by the enzyme to produce benzaldehyde at a wavelength of 249.6 nm. The pH of the reaction solvent is
When the organic solvent is used without being saturated with the aqueous buffer, there is no need to adjust the pH. However, when the organic solvent is used with being saturated with the aqueous buffer, the pH of the aqueous buffer is in the range of 3 to 7, preferably 3 Adjust to the range of ~ 6. The reaction temperature is to suppress the racemic cyanohydrin by-product that is not due to the enzyme reaction.
It is preferably as low as possible within the range in which the enzyme activity is exhibited, and is usually 0 to 40 ° C, preferably 0 to 30 ° C.

[0028]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. [Example 1] Synthetic reaction in which treatment for reducing the oxygen concentration in the reaction system was performed (1) S-hydroxynitrile lyase produced by a transgenic yeast into which an S-hydroxynitrile lyase gene derived from cassava ( Manihot esculenta) was introduced. (1950unit / ml)
25 μl, 0.15 M sodium citrate buffer (pH5) 25 μl
And 50 mg of powdered cellulose (W200G, made by Nippon Paper)
An immobilized enzyme was prepared.

Then, 2.5 ml of diisopropyl ether, 3-
Phenoxybenzaldehyde (99 μl) and hydrocyanic acid (56.6 μl) were added, and the mixture was stirred at room temperature. One of the diisopropyl ethers was replaced with nitrogen by adding a nitrogen gas at a flow rate of 1 L / min for 1 minute while stirring for 15 minutes.

As shown in FIG. 1, it was found that the synthesis reaction rate was higher when nitrogen-substituted diisopropyl ether was used than when air-substituted diisopropyl ether was used. Table 1 shows the conversion ratio of each reaction system at the end of the reaction (after 50 hours) and the optical purity of the generated S-3-phenoxybenzaldehyde cyanohydrin.

[0031]

[Table 1]

Example 2 Synthesis reaction in which the oxygen concentration in the reaction system was reduced (2) 1 ml of cassava-derived S-hydroxynitrile lyase (50 units / ml) and a ceramic-based immobilized carrier as in Example 1 (To
0.1 g of yonite-200 (manufactured by Toyo Denka Kogyo) was mixed to immobilize the enzyme. This was filtered and dried to prepare an immobilized enzyme.

Then, 2.5 ml of diisopropyl ether, 3-
Phenoxybenzaldehyde (99 μl) and hydrocyanic acid (56.6 μl) were added, and the mixture was stirred at room temperature. One diisopropyl ether used under the same conditions as in Example 1 was subjected to a nitrogen substitution treatment by bubbling nitrogen before the reaction, and the other was used without treatment.

As shown in FIG. 2, as in the case of cellulose immobilization in Example 1, the synthesis reaction rate was higher when diisopropyl ether substituted with nitrogen was used than when diisopropyl ether substituted without nitrogen was used. Turned out to be large. In addition, Table 2 shows the change over time in the conversion ratio and the optical purity of the generated S-3-phenoxybenzaldehyde cyanohydrin in each reaction system.

[0035]

[Table 2]

Example 3 Synthetic Reaction Treated to Reduce Hydroquinone Concentration in Reaction System Commercially available diisopropyl ether was distilled under reduced pressure to prepare hydroquinone and an ether free of hydroquinone-derived products. On the other hand, hydroquinone,
Benzoquinone and quinhydrone were added to 100 ppm each. Example 1 was performed using each of these solvents.
The reaction was carried out using an immobilized enzyme in the same manner as described above. Table 3 shows the conversion ratio in each reaction system and the optical purity of the generated S-3-phenoxybenzaldehyde cyanohydrin.

[0037]

[Table 3]

As shown in Table 3, under the conditions in which benzoquinone and quinhydrone were added, the conversion rate decreased and the optical purity also decreased, indicating that the enzyme reaction was inhibited. Hydroquinone is easily oxidized to benzoquinone by oxygen, and quinhydrone is generated by the presence of benzoquinone and hydroquinone.Hydroquinone does not contain hydroquinone because the presence of hydroquinone in the reaction system would potentially generate benzoquinone and quinhydrone. It may be preferable to use a solvent.

Example 4 Synthetic Reaction Treated to Reduce the Oxygen Concentration and Hydroquinone Concentration in the Reaction System Based on the results of each of the above Examples, after removing hydroquinone by distillation, nitrogen was replaced by nitrogen aeration. S-3-phenoxybenzaldehyde cyanohydrin was synthesized using diisopropyl ether as a solvent.

The immobilized enzyme prepared by the method of Example 2
235.8 ml of diisopropyl ether treated above, 3-
The reaction was carried out by adding 11.9 g of phenoxybenzaldehyde and 64 ml of a hydrogen cyanide-diisopropyl ether solution (37.85 g HCN / 500 ml) and stirring at 25 ° C.

At the time when the reaction was almost completed, the immobilized enzyme was separated, and the same amount of solvent and substrate were added as above, and the reaction was repeated. As shown in FIG. 3, no decrease in the reaction rate was observed even when the reaction was repeated four times. Table 4 shows the conversion ratio at the end of each reaction and the optical purity of the generated S-3-phenoxybenzaldehyde cyanohydrin.
From this, it can be said that the optical purity does not decrease.

[0042]

[Table 4]

[0043]

According to the present invention, there is provided a reaction system for synthesizing optically active cyanohydrin from a carbonyl compound and a cyanide compound using hydroxynitrile lyase as a catalyst.
Hydroxynitrile lyase activity is stabilized, and the activity does not decrease even after repeated use, so that it is very useful for industrial production of optically active cyanohydrin.

[Brief description of the drawings]

FIG. 1 shows the change in conversion rate in Example 1 when nitrogen replacement was performed and when nitrogen replacement was not performed.

FIG. 2 shows the change in the conversion ratio in Example 2 when nitrogen replacement is performed and when nitrogen replacement is not performed.

FIG. 3 shows the change in conversion rate in each reaction round.

Claims (1)

[Claims] 1. A reaction system for synthesizing optically active cyanohydrin from a carbonyl compound and a cyanide compound using hydroxynitrile lyase as a catalyst, wherein (i) a treatment for reducing the oxygen concentration in the reaction system, and (ii) a treatment for reducing the oxygen concentration in the reaction system. A method for producing optically active cyanohydrin, which comprises performing one or both of hydroquinone and a treatment for reducing the concentration of a compound derived from hydroquinone.