JP4669304B2 - Optically active cyanohydrin synthase derived from ume fruit and method for producing optically active substance using the same - Google Patents

Description

  The present invention relates to a novel optically active cyanohydrin synthase derived from ume fruit and a method for producing an optically active substance using the same. More specifically, the present invention relates to a method for synthesizing an optically active cyanohydrin from a carbonyl compound and a cyanide using the enzyme, and a method for synthesizing an optically active α-hydroxycarboxylic acid by hydrolyzing the obtained cyanohydrin.

Conventionally, it is known that enzymes obtained from almonds, apples, cherry apricots, plum seeds and the like have an action of synthesizing optically active cyanohydrin compounds from carbonyl compounds and cyanide compounds. The enzyme is generally called hydroxynitrile lyase, for example, one derived from cassava (EC-No.4.1.2.37), a plant belonging to the family Euphorbiaceae (EC-No.4.1.2.37), almond (Prunus amygdalis), a plant belonging to the family Rosaceae Derived from EC-No.4.1.2.10, sorghum (Sorghum bicolor) (EC-No.4.1.2.11), and passion fruit (Passiflora) edulis) and the like are known (Patent Document 1). For example, the enzyme obtained from almonds acts as an enzyme that catalyzes the reversible reaction of benzaldehyde (carbonyl compound) and hydrogen cyanide (cyanide compound) with (R) -mandelonitrile ((R) -cyanohydrin) (described below). (See Formula (I)).

An optically active cyanohydrin compound such as (R) -mandelonitrile is useful as an optically active organic synthesis intermediate, and is used, for example, to obtain pharmaceuticals. In certain plants, a cyanide compound such as HCN is produced from an optically active cyanohydrin compound by the enzymatic action of (R) -hydroxynitrile lyase. The produced HCN acts as a repellent factor for the plant to repel carnivores or as a sensitive component of the plant to fungi.
In order to obtain an optically active cyanohydrin compound, it is necessary to react with a substrate such as a carbonyl compound or a cyanide compound to efficiently produce an optically active cyanohydrin compound. That is, a catalyst or enzyme having excellent enantioselectivity (stereoselectivity) is required. Enzymes used for the synthesis of such optically active cyanohydrin compounds have substrate specificity that specifically reacts with specific carbonyl compounds, can catalyze reactions under a wide range of reaction conditions, and react quickly. It is desirable to be able to proceed.
However, hydroxynitrile lyases obtained from the above almonds and the like have not always had sufficient enantioselectivity, substrate specificity, broad adaptability to reaction conditions, and the like.
And until now, the synthesis of optically active cyanohydrins using the above-mentioned enzymes is extremely limited, so that water-based, water-organic solvent two-phase systems, organic solvents are used for the purpose of obtaining high optical purity cyanohydrin compounds. -Studies have been carried out by improving reaction conditions such as fine water systems and organic solvent systems.

JP 2004-248598 A

The first object of the present invention is to provide a novel optically active cyanohydrin synthase that exhibits excellent enantioselectivity, broad substrate specificity, etc. in the synthesis reaction of an optically active cyanohydrin compound from a carbonyl compound and a cyanide compound. is there.
A second object of the present invention is to provide a method for synthesizing an optically active cyanohydrin compound from a carbonyl compound and a cyanide compound using the enzyme.
A third object of the present invention is to provide a method for synthesizing α-hydroxycarboxylic acid by hydrolyzing the optically active cyanohydrin compound.

As a result of intensive studies to solve the above problems, the present inventor has found that it is possible to catalyze a synthesis reaction of optically active cyanohydrin from a carbonyl compound and a cyanide compound from ume (Prunus mume) fruit. It came to.
That is, the present invention
1. An optically active cyanohydrin synthase derived from Prunus mume fruit and having the following properties.
(1) Action: Converts a carbonyl compound into optically active cyanohydrin in the presence of a cyanide compound.
(2) Substrate specificity: Conversion to R-mandelonitrile using benzaldehyde as a substrate. When chloro-substituted benzaldehyde is used as a substrate, the optical selectivity of the optically active cyanohydrin obtained from the para-substituted product of the substrate is higher than that of the meta-substituted product of the substrate.
(3) Molecular weight: about 60,000 when measured by gel filtration.
2. A method for synthesizing an optically active cyanohydrin from a carbonyl compound and a cyanide compound using the enzyme of 1 above as a catalyst.
3. A method for synthesizing an α-hydroxycarboxylic acid, comprising hydrolyzing an optically active cyanohydrin obtained by the method described in 2 above.

  According to the present invention, a novel optically active cyanohydrin synthase is provided. The optically active cyanohydrin synthase has excellent enantioselectivity and substrate specificity, and is extremely useful industrially. In particular, the catalytic action of the enzyme composition of the present invention can be applied to a wider range of reaction conditions (e.g., temperature, pH) than oxynitrile lyase ((R) -mandelonitrilase) derived from almond (Prunus amygdalis), And it is excellent in that it has high enantioselectivity. Moreover, it is excellent in that it has a substrate specificity different from that of conventional hydroxynitrilase.

(1) Optically active cyanohydrin synthase
(1-1) Preparation of enzyme The source of the optically active cyanohydrin synthase of the present invention is ume fruit. Ume is a beautiful tree about 6m (20 feet) high in the end. Ume's scientific name is Prunus mume, a member of the family Rosaceae. There are two types of ume: “fruit plums” for fruit trees and “flower plums” for viewing. Examples of varieties of real plums include Juro, Shirakaga, Yoro, and Tamume. Examples of flower plum varieties include oolong plum, yellow gate, cherry blossom mirror, and cold clothes. The ume fruit is round in shape, has a diameter of about 1.3 to 12.7 cm (0.5 to 5 inches), preferably about 2.5 to 7.6 cm (1 to 3 inches), and weighs 10 to 35 g. Preferably it is 15-30g, More preferably, it is about 20-25g. In particular, ripe ume fruit and yellowish red are preferable, and those covered with succulent are more preferable. The fruit may be directly collected or commercially available, and the seeds contained therein are particularly preferred as the enzyme supply source, and the white portion called nin contained in the seeds is particularly preferred.

The optically active cyanohydrin synthase of the present invention contains, as an active ingredient, an extract obtained by extracting the ume fruit as described above, for example, by the following method.
(a) crushing the fruit or seed of the plant;
(b) a step of suspending and extracting the pulverized product in an extraction solvent; and
(c) optionally purifying the resulting extract.
(a) The step of crushing the fruit or seed of the plant is performed by crushing the fruit or seed of the plant using a general crushing method. Examples of the method include crushing with a hammer or a mallet or pulverization with a grinder, and a method in which seeds are frozen with liquid nitrogen at about −196 ° C.

  (b) The extraction solvent used in the step of suspending and extracting the pulverized product in an extraction solvent is not particularly limited, but water or an aqueous buffer is suitable. Preferably, it is composed of a buffer solution having a buffer capacity in the range of weakly acidic to weakly basic, such as a salt of phosphoric acid, citric acid, glutaric acid, malic acid, malonic acid, o-phthalic acid, succinic acid, acetic acid, etc. A buffer solution or the like is preferable. The pH of the buffer is usually 2-9, preferably 3-7. In addition, as an extraction solvent, an organic solvent can be used in combination for the purpose of improving the efficiency or for the purpose of preventing extraction of unnecessary substances. Any organic solvent can be used without particular limitation as long as it does not adversely affect the enzyme activity. Specifically, hydrocarbon solvents which may be halogenated (for example, linear, branched or cyclic saturated or unsaturated aliphatic hydrocarbons, aromatic hydrocarbons), for example, pentane, hexane, cyclohexane , Benzene, toluene, xylene, methylene chloride, chloroform and the like; an alcohol solvent which may be halogenated (for example, linear, branched or cyclic saturated or unsaturated aliphatic alcohol, aralkyl alcohol), for example, n -Butanol, isobutanol, t-butanol, hexanol, cyclohexanol, n-amyl alcohol and the like; ether solvents which may be halogenated (for example, linear, branched or cyclic saturated or unsaturated aliphatic ethers) Aromatic ether), for example, diethyl ether, dipropyl ether, diisopropyl Ether, dibutyl ether, t-butyl methyl ether, dimethoxyethane, etc .; ester solvents which may be halogenated (for example, linear, branched or cyclic saturated or unsaturated aliphatic esters, aromatic esters), Examples thereof include methyl formate, methyl acetate, ethyl acetate, butyl acetate, methyl propionate and the like, and these may be used alone or in combination of two or more. It is also possible to use it in a state where it is substantially dissolved in water or in an immiscible state. Further, an organic solvent can be used in advance for the purpose of degreasing. Such an organic solvent can be appropriately selected according to the purpose.

  The extraction is performed using a general extraction method. For example, the extraction is performed at -10 to 50 ° C, preferably 0 to 40 ° C by adding the pulverized product to the extraction solvent. The amount of ume fruit, seeds or seeds is, for example, 0.1 to 300 g, preferably 1 to 50 g, in 100 ml of the extraction solvent. Thereafter, the aqueous phase portion is recovered as an extract by separation means such as filtration or centrifugation.

The obtained plant fruit or seed extract can be used as it is, but (c) optionally, the obtained extract can be purified and used.
Commonly used for the purification of extracts;
(x) fractionation by precipitation,
(y) various chromatography,
(z) It can be obtained by using a method for removing low-molecular substances by dialysis, ultrafiltration or the like alone or in appropriate combination. Hereinafter, these steps will be described in more detail. In addition, the following processes are an example which manufactures the enzyme of this invention, Comprising: It is not limited to these.
(x) Fractionation by precipitation Fractionation by precipitation is performed by adding a precipitation aid to the above extract to produce a precipitate. The substance used for the precipitation aid is a salt containing a polyvalent anion such as phosphate and sulfate. Ammonium sulfate (ammonium sulfate) is preferred because of its high solubility in water and low temperature change in solubility. . The concentration of the precipitation aid to be added is not particularly limited, but it is preferable that the optically active cyanohydrin synthase of the present invention can be recovered with good yield and can be separated from other protein components. For example, when ammonium sulfate is used, it is appropriate to add 10 to 80% (w / v), preferably 20 to 60% (w / v) of ammonium sulfate saturated solution per liter of the above extract. . The sedimented sediment is appropriately collected using centrifugation or the like.

(y) Chromatography Examples of the chromatography include gel filtration, ion exchange chromatography, isoelectric point chromatography, hydrophobic chromatography, affinity chromatography and the like.
Among these, the gel filtration method is a method of separating proteins according to molecular weight, and can be selected according to the molecular weight of the target protein depending on the network structure of the gel particles. Columns used for gel filtration include TSK series columns (TSKgel SuperSW2000, SuperSW3000, TSK-GelG3000SW, TSKgel G4000SW, etc. manufactured by Tosoh Corporation), Sephadex G25, Sephadex G50, Sephadex G100, Sephadex G200, S-100HR, S- Examples include, but are not limited to, 200HR and S-300HR (both are Pharmacia).
Ion exchange chromatography is a separation method that utilizes the fact that proteins are ampholytes, and includes cation exchange chromatography and anion exchange chromatography. Carboxymethyl (CM) is used as an exchange group for cation exchange chromatography, and examples thereof include CM-Toyopearl (Tosoh), CM-Cellulofine (Seikagaku), and CM-Sephadex (Pharmacia) column. Diethylaminoethyl (DEAE) is used as an exchange group for anion exchange chromatography. For example, DEAE-Toyopearl (Tosoh), DEAE-Cellulofine (Seikagaku), DEAE-Sephadex (Pharmacia), MonoQ column, etc. Is mentioned. However, it is not limited to these columns.
Hydrophobic chromatography is a separation technique based on hydrophobic interaction between a protein and a ligand bound to a gel. For example, a column of Butyl-TOYOPEARL, Phenyl-TOYOPEARL, or Phenyl-Superose is used. Alternatively, reverse phase chromatography (for example, PEP-PRC, TSK-ODS, etc.) in which the mobile phase is a polar organic solvent, the stationary phase is a long nonpolar ligand, and the solute is distributed between the two phases can be used.
Affinity chromatography is characterized by attaching a substance that specifically binds to a protein or enzyme to a column and using a specific binding reaction to separate the target protein, etc., and is characterized by agarose (Sepharose) and dextran. (Sephadex), cellulose, polyacrylamide (Biogel P), porous silica beads and the like are used as the support.

(z) Dialysis, ultrafiltration Dialysis is a method in which a low molecular weight compound permeates outside the cellophane tube by placing the sample solution in a cellophane dialysis tube and immersing it in a large amount of pure water or a low concentration buffer solution. .
The ultrafiltration method is a method of concentrating a target protein using an ultrafiltration membrane (flat membrane, hollow fiber holofiber). The cut-off molecular weight is in the range of about 500 to 500,000. As a method, there are a pressure stirring method, a forced circulation type concentration method (holofiber), and a centrifugal method.
In the present invention, among the above purification methods, it is preferable to use a combination of ammonium sulfate fractionation, ion exchange chromatography and gel filtration.

(1-2) Enzyme Properties The optically active cyanohydrin synthase derived from ume fruit obtained by the above purification method is specified by the following physicochemical properties (1) to (3). In the present invention, “optically active cyanohydrin synthase” refers to a substance mainly composed of proteins and having the following physicochemical properties, including a holoenzyme to which a cofactor (coenzyme) is bound. It is a concept to get.
(1) Action Converts a carbonyl compound into optically active cyanohydrin in the presence of a cyanide compound.
(2) Substrate specificity Converts to R-mandelonitrile using benzaldehyde as a substrate. When chloro-substituted benzaldehyde is used as a substrate, the optical selectivity of the optically active cyanohydrin obtained from the para-substituted product of the substrate is higher than that of the meta-substituted product of the substrate.
(3) Molecular weight It is about 60,000 when measured by gel filtration. It has a molecular weight of about 58,000 according to the molecular weight determination method by SDS-polyacrylamide gel electrophoresis.
Moreover, the optically active cyanohydrin synthase of the present invention may be further specified by the following physicochemical properties (4).
(4) Reaction adaptation temperature
In the R-mandelonitrile synthesis activity, a relative activity of 50% or more is maintained over a wide range of 0 ° C to 40 ° C.
Hereinafter, the properties (1) to (4) will be described in detail.

(1-2-1) Action The optically active cyanohydrin synthase of the present invention has a catalytic action of converting carbonyl compounds and cyanide compounds as raw materials into optically active cyanohydrins. Specifically, a cyanide compound can be asymmetrically added to a carbonyl compound in the presence of an optically active cyanohydrin synthase.


Here, the carbonyl compound refers to an aldehyde or a ketone, specifically, the following formula (i):
R ¹ —CO—R ² (i)
(Wherein R ¹ and R ² are different from each other and each represents a hydrogen atom or a hydrocarbon group).
The hydrocarbon group is, for example, a hydrocarbon group having 1 to 30 carbon atoms, preferably 1 to 20 carbon atoms. Part -CH ₂ - and CH ₂ of -CH ₃ represents a carbonyl group, a sulfonyl group, may be replaced by -O- or -S-, = CH ₂ may be replaced with = O or = S and -CH ₂ - of CH, CH of -CH _3,> CH- of CH, = CH- of CH and = of CH ₂ CH may be replaced by N or halogen It may be. Examples of the halogen include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

The hydrocarbon group may have a substituent and may be a saturated or unsaturated acyclic group or a saturated or unsaturated cyclic group. When the hydrocarbon group is acyclic, it may be linear or branched. Hydrocarbon groups include alkyl groups, alkenyl groups, alkynyl groups, alkyldienyl groups, aryl groups, alkylaryl groups, arylalkyl groups, arylalkenyl groups, cycloalkyl groups, cycloalkenyl groups, (cycloalkyl) alkyl groups, Bicycloalkyl groups and the like are included.
More specifically, examples of the linear or branched alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a 1-methylpropyl group, a pentyl group, a 1-methylbutyl group, a hexyl group, 1-methylpentyl group, heptyl group, 1-methylhexyl group, 1-ethylpentyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, 2-methylpropyl group, 2- Methylbutyl group, 3-methylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, methylhexyl group, methylheptyl group, methyloctyl group, methylnonyl group, 1,1-dimethylethyl group, 1 , 1-dimethylpropyl group, 2,2-dimethylpropyl group, 2,6-dimethylheptyl group, 3,7 Dimethyl octyl group, 2-ethylhexyl group. Examples of linear or branched alkenyl groups include vinyl, allyl, crotyl (2-butenyl), isopropenyl (1-methylvinyl), propenyl, and 2-methyl-1-propenyl groups. , Etc. Examples of the linear or branched alkynyl group include an ethynyl group, a propynyl group, and a butynyl group. Examples of the alkyldienyl group include a 1,3-butadienyl group. Examples of the aryl group include phenyl group, 1-naphthyl group, 2-naphthyl group, 2-phenylphenyl group, 3-phenylphenyl group, 4-phenylphenyl group, 9-anthryl group, indenyl group, biphenyl group, and methylphenyl. Group, dimethylphenyl group, trimethylphenyl group, ethylphenyl group, methylethylphenyl group, diethylphenyl group, propylphenyl group, butylphenyl group and the like.

Examples of alkylaryl groups include o-tolyl, m-tolyl, p-tolyl, 2,3-xylyl, 2,4-xylyl, 2,5-xylyl, o-cumenyl, m- Examples thereof include cumenyl group, p-cumenyl group, mesityl group and the like. Examples of the arylalkyl group include benzyl group, phenethyl group, 1-naphthylmethyl group, 2-naphthylmethyl group, 1-phenylethyl group, phenethyl group (2-phenylethyl group), phenylpropyl group, phenylbutyl group, phenyl Examples include pentyl group, phenylhexyl group, methylbenzyl group, dimethylbenzyl group, trimethylbenzyl group, ethylbenzyl group, methylphenethyl group, dimethylphenethyl group, diethylbenzyl group and the like. Examples of the arylalkenyl group include a styryl group, a methyl styryl group, an ethyl styryl group, a dimethyl styryl group, and a 3-phenyl-2-propenyl group. Examples of the cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a methylcyclopentyl group, a cyclohexyl group, a methylcyclohexyl group, a cycloheptyl group, and a cyclooctyl group. Examples of the cycloalkenyl group include a cyclopropenyl group, a cyclobutenyl group, a cyclopentenyl group, a cyclopentadienyl group, a cyclohexenyl group, and the like. Examples of the cycloalkylalkyl group include a cyclopentylmethyl group and a cyclohexylmethyl group. Examples of the bicycloalkyl group include a norbornyl group, a bicyclo [2.2.2] octyl group, and an adamantyl group.

Examples of the group in which CH ₂ in the hydrocarbon group is a carbonyl group, a sulfonyl group, O or S, or C—H is replaced by N or C-halogen include ketone, aldehyde, carboxylic acid, ester, sulfone, And groups containing one or more structures such as ether, thioether, amine, alcohol, thiol, halogen, and heterocyclic ring (for example, oxygen-containing heterocyclic ring, sulfur-containing heterocyclic ring, and nitrogen-containing heterocyclic ring). The oxygen-containing heterocycle, sulfur-containing heterocycle, and nitrogen-containing heterocycle mean that the carbon of the ring skeleton of the cyclic hydrocarbon group is replaced with oxygen, sulfur, and nitrogen, respectively. It may be a heterocyclic ring as described above. Examples of the hydrocarbon group having the above-described substitution include an acetylmethyl group and an acetylphenyl group having a ketone structure; an acetoxy group and a propoxy group having an ester structure; a methanesulfonylmethyl group having a sulfone structure; a methoxymethyl group having an ether structure, and a methoxyethyl group. Group, ethoxyethyl group, methoxypropyl group, butoxyethyl group, ethoxyethoxyethyl group, methoxyphenyl group, dimethoxyphenyl group, phenoxymethyl group; thioether structure methylthiomethyl group, methylthiophenyl group; amine structure aminomethyl group, 2 -Aminoethyl group, 2-aminopropyl group, 3-aminopropyl group, 2,3-diaminopropyl group, 2-aminobutyl group, 3-aminobutyl group, 4-aminobutyl group, 2,3-diaminobutyl group 2,4-diaminobutyl group, 3 , 4-Diaminobutyl group, 2,3,4-triaminobutyl group, methylaminomethyl group, dimethylaminomethyl group, methylaminoethyl group, propylaminomethyl group, cyclopentylaminomethyl group, aminophenyl group, diaminophenyl group , Aminomethylphenyl group; oxygen-containing heterocyclic tetrahydrofuranyl group, tetrahydropyranyl group, morpholylethyl group; oxygen-containing heteroaromatic furyl group, furfuryl group, benzofuryl group, benzofurfuryl group; sulfur-containing heteroaromatic ring thienyl Group: pyrrolyl group, imidazolyl group, oxazolyl group, thiadiazolyl group, pyridyl group, pyrimidinyl group, pyridazinyl group, pyrazinyl group, tetrazinyl group, quinolyl group, isoquinolyl group, pyridylmethyl group of nitrogen-containing heteroaromatic ring; Hydroxyethyl 2-hydroxypropyl group, 3-hydroxypropyl group, 2,3-dihydroxypropyl group, 2-hydroxybutyl group, 3-hydroxybutyl group, 4-hydroxybutyl group, 2,3-dihydroxybutyl group, 2,4 -Dihydroxybutyl group, 3,4-dihydroxybutyl group, 2,3,4-trihydroxybutyl group, hydroxyphenyl group,

Dihydroxyphenyl group, hydroxymethylphenyl group, hydroxyethylphenyl group; thiol structure 2-mercaptoethyl group, 2-mercaptopropyl group, 3-mercaptopropyl group, 2,3-dimercaptopropyl group, 2-mercaptobutyl group, 3-mercaptobutyl group, 4-mercaptobutyl group, mercaptophenyl group; halogenated hydrocarbon group 2-chloroethyl group, 2-chloropropyl group, 3-chloropropyl group, 2-chlorobutyl group, 3-chlorobutyl group, 4-chlorobutyl, fluorophenyl, chlorophenyl, bromophenyl, difluorophenyl, dichlorophenyl, dibromophenyl, chlorofluorophenyl, trifluorophenyl, trichlorophenyl, fluoromethylphenyl, trimethyl Fluoromethylphenyl group; 2-amino-3-hydroxypropyl group, 3-amino-2-hydroxypropyl group, 2-amino-3-hydroxybutyl group, 3-amino-2-hydroxy having an amine structure and an alcohol structure Butyl group, 2-amino-4-hydroxybutyl group, 4-amino-2-hydroxybutyl group, 3-amino-4-hydroxybutyl group, 4-amino-3-hydroxybutyl group, 2,4-diamino-3 -Hydroxybutyl group, 3-amino-2,4-dihydroxybutyl group, 2,3-diamino-4-hydroxybutyl group, 4-amino-2,3-dihydroxybutyl group, 3,4-diamino-2-hydroxy Butyl group, 2-amino-3,4-dihydroxybutyl group, aminohydroxyphenyl group; Hydrocarbon substituted with halogen and hydroxyl group Fluoro-hydroxyphenyl group is a group, chloro hydroxyphenyl group; and carboxyphenyl groups for carboxylic structure. The asymmetric divalent group represented by R ¹ and R ² is not particularly limited, and examples thereof include norbornane-2-ylidene and 2-norbornene-5-ylidene.

  Specific examples of the carbonyl compound represented by the formula (i) include benzaldehyde, chlorobenzaldehydes at 2,3,4 positions, bromobenzaldehydes at 2,3,4 positions, and 2,3,4 positions. Each fluorobenzaldehyde, each phenoxybenzaldehyde at 2,3,4 position, each methylbenzaldehyde at 2,3,4 position, each nitrobenzaldehyde at 2,3,4 position, each methylbenzaldehyde at 2,3,4 position, 2, 3rd and 4th ethylbenzaldehyde, 2nd and 3rd position methoxybenzaldehyde, 2nd and 3rd position ethoxybenzaldehyde, 2nd and 3rd position trifluoromethylbenzaldehyde, 2nd and 3rd position Each hydroxybenzaldehyde, 2,3,4 position benzyloxybenzaldehyde, 2,3,4 position allyloxybenzaldehyde, 2,3,4 position t-butyldimethylsilyloxybenzaldehyde 3,4-methylenedi Oxybe Aldehyde, 2,3-methylenedioxybenzaldehyde, phenylacetaldehyde, furfural, 2-pyridinecarbaldehyde, 3-pyridinecarbaldehyde, 4-pyridinecarbaldehyde, 2,3,4-trimethoxybenzaldehyde, 2,4,5 -Trimethoxybenzaldehyde, 3,4,5-trimethoxybenzaldehyde, 2,3,4,5-tetrafluorobenzaldehyde, 2,3,5,6-tetrafluorobenzaldehyde, 2,3,4,5,6-penta Fluorobenzaldehyde, 2,3,4,5,6-pentabromobenzaldehyde, 2,3-dichlorobenzaldehyde, 2,4-dichlorobenzaldehyde,

2,5-dichlorobenzaldehyde, 2,6-dichlorobenzaldehyde, 3,4-dichlorobenzaldehyde, 3,5-dichlorobenzaldehyde, 2,3-dimethoxybenzaldehyde, 2,4-dimethoxybenzaldehyde, 2,5-dimethoxybenzaldehyde, 2 , 6-Dimethoxybenzaldehyde, 3,4-dimethoxybenzaldehyde, 3,5-dimethoxybenzaldehyde, 2,4-dimethylbenzaldehyde, 4-N, N-dimethylaminobenzaldehyde, piperonal, 1-naphthaldehyde, 2-naphthaldehyde, 9 -Aromatic aldehydes such as anthral; acetaldehyde, propanal, butanal, isobutyraldehyde, pivalaldehyde, pentanal, hexanal, cyclopentanecarbaldehyde, cyclohexanecarbaldehyde, 4-hydroxy-3,3-dimethylbutyraldehyde Aliphatic aldehydes such as 2-butanone, 2-pentanone, 3-methyl-2-butanone, 2-hexanone, 4-methyl-2-pentanone, 3,3-dimethyl-2-butanone, 2-heptanone, 5- Methyl-2-hexanone, 2-octanone, 2-nonanone, 2-decanone, 2-undecanone, 2-dodecanone, cyclopropylmethylketone, trimethylsilylmethylketone ethylmethylketone, methyl (2-propenyl) ketone, (3-butenyl ) Aliphatic ketones such as methyl ketone; alkyl (haloalkyl) ketones such as (3-chloropropyl) methyl ketone; 2- (protected amino) aldehydes such as 2- (alkoxycarbonylamino) -3-cyclohexylpropionaldehyde; 3-methylthiopropion Alkylthioaliphatic aldehydes such as aldehydes; 2-furaldehyde, 2-thiophenecarbaldehyde , 2-pyridinecarboxaldehyde, 3-pyridinecarboxaldehyde, 4-pyridine carbaldehyde, 2-quinoline-carbaldehyde and a heterocyclic aldehyde, such as 4-quinoline-carbaldehyde.

The cyanide compound that can be used in the present invention may be any compound that can be condensed with the carbonyl compound to obtain the corresponding cyanohydrin. Preferably, cyanide which can generate HCN or HCN is suitable. Examples of the cyan compound include HCN, KCN, NaCN, acetone cyanohydrin ((CH ₃ ) ₂ C (OH) CN), and the like.
These carbonyl compounds and cyanide compounds, which are raw materials, can be used for this reaction after purification (by acidic aqueous solution or alkaline aqueous solution), distillation, recrystallization and the like, if necessary.

The reaction for synthesizing an optically active cyanohydrin from a carbonyl compound and a cyanide using the optically active cyanohydrin synthase of the present invention is performed, for example, under the following conditions.
The reaction proceeds by adding the carbonyl compound and cyanide compound to the reaction solvent and maintaining the temperature for a predetermined time. The reaction conditions and the amounts of the optically active cyanohydrin synthase and the carbonyl compound and cyanide compound used as substrates are appropriately determined according to the substrate used. Usually, the amount of enzyme used is 0.1 to 1000 units, preferably 1 to 500 units, per 1 mmol of the carbonyl compound as a substrate. The concentration of the carbonyl compound is usually in the range of 1 to 500 g / L. The cyan compound is added at a concentration of 0.1 to 10 times mol, preferably 0.5 to 3 times mol of the carbonyl compound used. The reaction time is appropriate until the conversion rate of the substrate carbonyl compound reaches 40% or more, preferably 80% or more. However, the reaction time is not limited to these. The reaction temperature is not particularly limited as long as the enzyme activity is sufficiently exerted, and is usually −10 to 70 ° C., preferably 0 to 40 ° C. Further, a metal ion, a coenzyme (FAD, FMN, NAD, etc.) and the like may coexist in the reaction system as necessary. By adding these, enzyme activity may be increased and the yield of the target compound may be improved.
The optically active cyanohydrin synthase of the present invention may be used in the form of powder, liquid, or an immobilized enzyme formed by immobilization on an appropriate carrier. Examples of the immobilization carrier include a porous inorganic carrier, a fibrous carrier such as cellulose, and a carrier made of a polymer compound. Specifically, porous ceramic particles, porous silica gel particles, zeolites, etc. Examples thereof include natural polymer gels such as system particles, agar, calcium alginate, and chitosan, and synthetic polymer gels such as polyacrylic acid, polyacrylamide, and polyvinyl alcohol. In order to immobilize the enzyme, for example, a method in which the enzyme solution is absorbed in a carrier, a method in which the enzyme solution and the carrier are mixed and adsorbed and immobilized, a method in which the enzyme is immobilized and immobilized, and the enzyme is crosslinked with a crosslinking agent Any method such as a method can be adopted.

As a reaction solvent, an organic solvent is used as a reaction solvent in order to improve productivity by increasing the concentration of the reaction raw material alone or a buffer solution containing sodium citrate, potassium citrate, sodium phosphate, potassium phosphate, sodium acetate, etc. It can also be used.
The pH of the buffer solution is not particularly limited as long as it does not adversely affect the enzyme reaction, and is pH 2-9, preferably pH 3-7.
Any organic solvent can be used without particular limitation as long as it is substantially immiscible with water, sufficiently dissolves the substrate and product, and does not adversely affect the enzyme reaction. Specifically, hydrocarbon solvents which may be halogenated (for example, linear, branched or cyclic saturated or unsaturated aliphatic hydrocarbons, aromatic hydrocarbons), for example, pentane, hexane, cyclohexane , Benzene, toluene, xylene, methylene chloride, chloroform and the like; an alcohol solvent which may be halogenated (for example, linear, branched or cyclic saturated or unsaturated aliphatic alcohol, aralkyl alcohol), for example, n -Butanol, isobutanol, t-butanol, hexanol, cyclohexanol, n-amyl alcohol and the like; ether solvents which may be halogenated (for example, linear, branched or cyclic saturated or unsaturated aliphatic ethers) Aromatic ether), for example, diethyl ether, dipropyl ether, diisopropyl Ether, dibutyl ether, t-butyl methyl ether, dimethoxyethane, etc .; ester solvents which may be halogenated (for example, linear, branched or cyclic saturated or unsaturated aliphatic esters, aromatic esters), Examples thereof include methyl formate, methyl acetate, ethyl acetate, butyl acetate, methyl propionate and the like, and these may be used alone or in combination of two or more. Such an organic solvent can be appropriately selected according to the physical properties of the raw material aldehyde or ketone and the physical properties of the product cyanohydrin.

The organic solvent may be saturated with water or an aqueous buffer, or may be in a solution state in which an excess of water or an aqueous buffer is added to form a two-phase system of an organic solvent phase and an aqueous phase. Good. As a method of saturating the organic solvent with water or an aqueous buffer solution, for example, the organic solvent and water or an aqueous buffer solution are mixed and stirred at a ratio of forming two phases, and allowed to stand. The method to use is mentioned.
The organic solvent is preferably saturated with water or an aqueous buffer. Here, the aqueous buffer solution is not particularly limited, but a buffer solution that exhibits a buffering ability near the optimum pH (pH 4 to 7) of the enzyme activity, such as phosphoric acid, citric acid, glutaric acid, malic acid, malon. A buffer composed of a salt of acid, o-phthalic acid, succinic acid, acetic acid or the like is preferably used.
The optically active cyanohydrin obtained as described above can be measured or quantified by high performance liquid chromatography (HPLC) or the like, and separated by usual means such as extraction, vacuum distillation, column separation, etc., if necessary. Can be purified. The yield can be determined using 1H-NMR.

(1-2-2) Substrate specificity The optically active cyanohydrin synthase of the present invention has substrate specificity for converting benzaldehyde as a substrate into R-mandelonitrile. When chloro-substituted benzaldehyde is used as a substrate, the optical selectivity for the meta-position and para-position substitution is higher than that of the ortho-position substitution. Further, when the meta-position and para-position substitution products are compared, the optical selectivity to the para-position substitution product is high. The substrate specificity of these enzymes is common to all substitutions such as methyl (-Me), methoxy (-OMe), trifluoromethyl (-CF ₃ ), nitro (-NO ₂ ), etc. It is characterized by higher optical selectivity for para-substituted products than for position-substituted products. Here, for example, when a substrate such as benzaldehyde is converted into a product such as mandelonitrile, the optical selectivity is the ratio (optical purity) in which the R- optical isomer is present in the product. It can be expressed in enantioselectivity (% ee). For example, if all of the products are R- optical isomers, it is 100% ee, and if all of the products are racemic, it is 0% ee. The high optical selectivity of the present invention is
(Enantioselectivity of ortho-substituted product) <(optical selectivity for meta-substituted product) × α
(Optical selectivity for meta-substituted products) <(Optical selectivity for para-substituted products) × β
Here, α is 1 or more, preferably 1.2 or more, and more preferably 1.5 or more. Further, β is 1 or more, preferably 1.05 or more, and more preferably 1.2 or more.
The optically active cyanohydrin synthase of the present invention has high optical selectivity for aliphatic aldehydes and monosubstituted aromatic aldehydes (enantioselectivity of aliphatic aldehydes: 70% ee or higher, preferably 75% ee or higher; Enantioselectivity of substituted aromatic aldehydes: 50% ee or more, preferably 60% ee or more). In addition, when an electron withdrawing group is present in the aromatic nucleus of the substrate, the reactivity of the substrate is improved. When benzaldehyde substituted with halogen at the meta and para positions is used as a substrate, the optically active cyanohydrin synthase of the present invention can synthesize optically active cyanohydrin exhibiting excellent optical selectivity (enantioselectivity: 90% ee or more). , Preferably 95% ee or more). In addition, when a substrate having an electron donating group (for example, -Me, -OMe; mainly at the meta position and the para position) is used, the optically active cyanohydrin synthase of the present invention has an optical selectivity that exhibits excellent optical selectivity. An active cyanohydrin can be synthesized (enantioselectivity: 80% ee or more, preferably 85% ee or more). Furthermore, when an aliphatic ketone, particularly methyl ketone having no branch at the α-position, is used as a substrate, an optically active cyanohydrin exhibiting excellent optical selectivity can be synthesized (enantioselectivity: 50% ee or more, preferably 60% ee or higher).

(1-2-3) Molecular Weight The molecular weight of the optically active cyanohydrin synthase of the present invention is about 60,000 when measured by gel filtration. More specifically, they are 60,000 ± 5,000 to 10,000, 60,000 ± 3,000 to 4000, and 60,000 ± 1000 to 2,000. Here, the molecular weight measurement by the gel filtration method can be performed by a general method. The active fraction fractionated by chromatography or the like is collected and concentrated, and it is eluted by gel filtration, and the molecular weight can be determined from a calibration curve using a protein having a known molecular weight.

  The molecular weight of the optically active cyanohydrin synthase of the present invention has a molecular weight of about 58,000 when measured by a molecular weight determination method by SDS-polyacrylamide gel electrophoresis. More specifically, it is 58,000 ± 5,000 to 10,000, 58,000 ± 3,000 to 4000, and 58,000 ± 1000 to 2,000. Here, SDS-polyacrylamide gel electrophoresis can be performed by a general technique such as the method of Davis and Laemmli. The active fraction gel-filtered as described above is subjected to SDS-polyacrylamide gel electrophoresis, and compared with a calibration curve using a protein with a known molecular weight, the molecular weight of the obtained fraction can be determined.

(1-2-4) Reaction Adaptation Temperature The optically active cyanohydrin synthase of the present invention has a wide temperature range of -10 to 70 ° C, preferably -5 to 50 ° C, more preferably 0 to 40 ° C. In the presence of the compound, the carbonyl compound can be converted to an optically active cyanohydrin. The optical activity of the optically active cyanohydrin in the temperature range of 0 to 40 ° C. is 40% or more, preferably 50% or more as a relative activity when the enantioselectivity value at a reaction temperature of 20 ° C. is 100%.

(2) Method for synthesizing α-hydroxycarboxylic acid An optically active α-hydroxycarboxylic acid can be obtained by hydrolyzing the obtained optically active cyanohydrin. In the hydrolysis, the reaction can be rapidly advanced by using hydrochloric acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, perchloric acid, etc., preferably hydrochloric acid as a catalyst. The usage-amount of a mineral acid is 1-10 equivalent with respect to cyanohydrin, Preferably it is 1.5-5 equivalent. In the hydrolysis reaction, the temperature during the reaction is 10 to 100 ° C. (reflux temperature), preferably 20 to 90 ° C. The reaction time is preferably 1 to 50 hours when the reaction temperature is 20 to 40 ° C, and preferably 0.5 to 30 hours when the reaction temperature is 40 to 90 ° C. After completion of the reaction, isolation and purification are carried out by a known method. For example, the reaction solution is extracted with a solvent and further washed with water as necessary, and then the solvent is evaporated, dried, recrystallized, etc. α-Hydroxycarboxylic acids can be isolated.

[Example 1]
(1) Preparation of enzyme solution The seed (64.3 g) was taken out from commercially available ripe plum fruits (30), and the shell was split from the plum seed to take out the seed (16.6 g). Using a mortar and pestle, 50 mM phosphate buffer (KPB) pH 6.0, 40 ml was added and homogenized in ice with a homogenizer. The enzyme extract was recovered by filtration through four layers of gauze and further centrifugation (28,000 × g, 15 minutes, 4 ° C.).
The supernatant was subjected to ammonium sulfate fractionation, and the 0-30% saturated fraction was dialyzed (2 l × 3) against 10 mM KPB. The enzyme solution was concentrated with an ultrafiltration system (Centriprep YM-10 Millipore Corporation, USA) and used as it was for the activity measurement of optically active cyanohydrin synthase.
In addition, the optically active cyanohydrin synthase solution in Example 2 and after was used after this dialysis.

(2) Measurement of enzyme activity Next, enzyme activity was assayed by measuring the production of (R) -mandelonitrile from benzaldehyde.
To 760 μl of 400 mM citrate buffer (pH 4.0), 40 μl of 1.0 M benzaldehyde in dimethyl sulfoxide (DMSO), 100 μl of enzyme solution and 100 μl of 1.0 M potassium cyanide solution are added in order (final volume 1.0 ml), and 5 minutes at 25 ° C. Incubated.
100 μl of the reaction solution was taken, 900 μl of an organic solvent (hexane: isopropanol = 9: 1) was added to stop the reaction, and the supernatant was analyzed by HPLC shown below. As a blank, an enzyme-free system (10 mM KPB was added instead of the enzyme solution) was performed, and the produced mandelonitrile was measured. The enzyme activity was calculated based on the value obtained by subtracting the blank value.

[HPLC analysis conditions]
Column: CHIRALCEL OJ-H (manufactured by Daicel Chemical Industries)
4.6mm ID x 250mm
Mobile phase: n-hexane: 2-propanol = 90:10
Flow rate: 1.0 mL / min Detection: UV 245 nm

The amount of enzyme that produced 1 μmol of optically active mandelonitrile per minute under standard assay conditions was defined as 1 unit (U) of enzyme activity.
The protein was quantified using the Bradford method (Biorat).

(3) Purification by various chromatographies
(i) Purification by anion exchange chromatography (DEAE-Toyopearl 650M)
The enzyme solution was placed on a DEAE-Toyopearl column 650M equilibrated with 10 mM phosphate buffer (pH 6.0), washed with 100 mM phosphate buffer (pH 6.0), and then 100 mM phosphate buffer containing 100 mM NaCl (pH 6.0). The active fraction was eluted at pH 6.0). The active fraction was dialyzed against 10 mM potassium phosphate buffer (pH 6.0).
(ii) Purification by gel filtration (TSK-GelG3000SW)
After concentrating the above hydrophobic chromatographic active fraction, it was applied to TSK-GelG3000SW equilibrated with 0.1 M phosphate buffer + 0.2 M NaCl buffer (pH 6.0) to elute the active fraction (shown as Hnl in FIG. 1). Is the active fraction). Molecular weight of optically active cyanohydrin synthase calculated from a calibration curve (Figure 2) of a known protein (thyroglobulin (669,000); ferritin (443,000); lactate dehydrogenase (139,850); albumin (66,267); trypsin inhibitor (21,000)) Was 60KD.

(iii) SDS polyacrylamide electrophoresis
According to the method of Davis and Laemmli, 12.5% polyacrylamide gel was used, and the gel filtration (TSK-GelG3000SW) active fraction was subjected to SDS polyacrylamide electrophoresis (FIG. 3, the one labeled Hnl is the active fraction). From a calibration curve (Fig. 4) of known proteins (phosphorylase b (95,000), bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100) and m-lactoalbumin (14,400)), The calculated molecular weight of the optically active cyanohydrin synthase was 58 KD.

(4) Optimum reaction temperature Using the optically active cyanohydrin synthase solution obtained in Example 1 (1), the activity was measured at each reaction temperature (reaction for 10 minutes) using benzaldehyde as a substrate (the method described above). It was. Graph 1 shows the results in terms of relative activity, assuming that the value at a reaction temperature of 20 ° C. is 100%. It was shown that relative activity of 50% or more was maintained over a wide range of 0 ° C to 40 ° C.
Graph 1

[Example 2] Synthesis of aromatic cyanohydrin
To 760 μl of 400 mM citrate buffer (pH 4.0), 40 μl of a solution in which the carbonyl compounds described in Tables 1 to 4 were dissolved in DMSO to a concentration of 1.25 M was mixed (final concentration 50 mM), and optically active cyanohydrin was mixed. Synthetic enzyme solution 100 μl and 1.0 M potassium cyanide solution 100 μl (citrate buffer (pH 4.0) solution, final concentration 100 mM) were sequentially added (final volume 1.0 ml) and reacted at 25 ° C.
25 μl of the reaction solution was taken, ethyl acetate was added, the reaction product was extracted, and the ethyl acetate layer was analyzed by high performance liquid chromatography (HPLC) to measure its optical purity.

[HPLC analysis conditions]
Column; CHIRALCEL OJ-H (manufactured by Daicel Chemical Industries)
4.6mm ID x 250mm
Mobile phase: n-hexane: 2-propanol = 90:10 (basic conditions)
Flow rate: 1 mL / min
Detection; UV 245 nm

The enzyme activity of the obtained aromatic cyanohydrin was evaluated based on the ratio (optical purity) in which the R- or S- optical isomer was present in the obtained cyanohydrin, that is, enantioselectivity (% ee). For example, if all of the obtained cyanohydrins are (R) -cyanohydrin, it is 100% ee, and if all of the obtained cyanohydrins are racemic cyanohydrins, they are 0% ee.
The results of evaluation are shown in Tables 1 to 4 below.

Table 1 Unsubstituted or monosubstituted benzaldehyde
a) Analysis by moving bed composition (n-hexane: 2-propanol = 19: 1)
b) Analysis with moving bed composition (n-hexane: 2-propanol = 80:20)

Table 2. Disubstituted benzaldehydes
c) Analysis with moving bed composition (n-hexane: 2-propanol = 39: 1)

Table 3. Multi-substituted benzaldehyde

Table 4 Heteroaromatic aldehydes and polycyclic aldehydes

As shown in Table 1, with respect to unsubstituted or monosubstituted benzaldehydes, the enzyme of the present invention showed very good enantioselectivity except for a part. In particular, the optical selectivity of the para-substituted product is higher than that of the meta-substituted product. All methoxy benzaldehydes showed high optical selectivity compared to benzaldehyde. 4-Hydroxybenzaldehyde has poor enantioselectivity as it is, but when the hydroxyl group of 4-hydroxybenzaldehyde is protected as an ether functionality (benzyl, allyl, terbutyldimethylsilyl), all the protected aldehydes are excellent. It had enantioselectivity (99%).
As shown in Table 2, with respect to the disubstituted benzaldehyde, the enzyme of the present invention showed excellent enantioselectivity, especially 3,4-dichlorobenzaldehyde and 3,5-dimethoxybenzaldehyde. From 3,4-dichlorobenzaldehyde, (R) -cyanohydrin having high optical purity was obtained. On the other hand, aldehydes having a halogen group or a methoxy group at the ortho position adjacent to the aldehyde group tended to have reduced enantioselectivity.
Among the bicyclic aldehydes, 1-naphthal and 2-naphthal are very good substrates. All aldehydes were superior in enantioselection compared to benzaldehyde.

Example 3 Synthesis of Aliphatic Cyanohydrin 40 μl (final concentration 50 mM), 400 mM citrate buffer (pH 4.0) of an aliphatic carbonyl compound solution 1.25 M solution (dissolved in DMSO) described in Tables 5-6 760 μl (final concentration of about 300 mM) and 1.0 M of KCN solution (100 μl (final concentration of 100 mM) were added (total volume: 1 ml)) were reacted at 25 ° C.
The reaction solution was extracted with ethyl acetate or ether in the same manner as in Example 2 and analyzed by gas chromatography (GC) to measure its optical purity. For some aliphatic aldehydes and aliphatic ketones, the obtained cyanohydrin was derivatized as the corresponding trimethylsilyl ether (—OTMS) (trimethylsilyl ether derivative (TMS-ether derivative)) and measured by GC. That is, it was treated with excess imidazole and TMS-Cl in anhydrous dichloromethane, purified with a silica gel column (eluent: hexane: ethyl acetate = 9: 1), and measured under the GC conditions shown below.

[GC analysis condition 1]
Column: β-Dex325 (30m x 0.25mm x 0.25μm thickness, manufactured by SUPELCO)
Carrier gas; helium detection; FID
Detector temperature: 230 ° C
Injection temperature: 220 ℃
Column temperature: Varies by compound.

[GC analysis condition 2]
Column: β-Dex120 (30m x 0.25mm x 0.25μm thickness, manufactured by SUPELCO)
Carrier gas; helium detection; FID
Detector temperature: 230 ° C
Injection temperature: 220 ℃
Column temperature: Varies by compound.

Table 5 Aliphatic aldehydes
a) Measured under GC analysis condition 1.
b) The corresponding TMS-ether derivative was prepared and the optical purity was determined.

Table 6 Aliphatic methyl ketone
b) The corresponding TMS-ether derivative was prepared and the optical purity was determined.
c) Measured under GC analysis condition 2.
As shown in Table 5, for aliphatic aldehydes, the enzyme of the present invention generally showed excellent enantioselectivity.
As shown in Table 6, for the aliphatic ketone, the enzyme of the present invention showed good enantioselectivity for methyl ketone having no branch at the α-position.

[Example 4] Synthesis of cyanohydrin in organic solvent 50 mL of diisopropyl ether and 5 mL of 400 mM citrate buffer (pH 4.0) were mixed to prepare water-saturated diisopropyl ether. To this was further added the carbonyl compound (5 g). To the obtained solution, an enzyme solution containing the optically active cyanohydrin synthase of the present invention obtained in Example 1 (1) was further added so as to be 50 U / mmol with respect to the carbonyl compound. Thereafter, 1.5 equivalents of acetone cyanohydrin was added to initiate the reaction. After completion of the reaction, ethyl acetate was added, the reaction product was extracted, and the ethyl acetate layer was subjected to HPLC analysis by the method described in Example 2, and its optical purity and reaction yield were calculated.

Table 7 Synthesis of cyanohydrins in organic solvents

Example 5
Synthesis of α-hydroxycarboxylic acid To R-mandelonitrile obtained in Example 2, 5 equivalents of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 7 hours. Then, it heated at 60 degreeC for 12 hours, and then heated at 100 degreeC for 5 hours. After removing hydrochloric acid gas in vacuum using a suction device, the residue was extracted three times using twice the amount of ethyl acetate. The extract was dried over Na ₂ SO ₄ and concentrated.
As a result, R-mandelic acid was obtained with a yield of 85%.

Shows the elution pattern by TSK-GelG3000SW column. The molecular weight measurement result of optically active cyanohydrin synthase is shown. Shows SDS polyacrylamide electrophoresis pattern. The molecular weight measurement result of optically active cyanohydrin synthase is shown.

Claims (3)

An optically active cyanohydrin synthase derived from Prunus mume fruit and having the following properties.
(1) A carbonyl compound is converted into an optically active cyanohydrin in the presence of a cyanide compound.
(2) Substrate specificity: Conversion to R-mandelonitrile using benzaldehyde as a substrate. When chloro-substituted benzaldehyde is used as a substrate, the optical selectivity of the optically active cyanohydrin obtained from the para-substituted product of the substrate is higher than that of the meta-substituted product of the substrate.
(3) Molecular weight: about 60,000 when measured by gel filtration.
(4) Reaction adaptive temperature: R-mandelonitrile synthesis activity maintains a relative activity of 50% or more over a wide range of 0 ° C to 40 ° C. A method for synthesizing optically active cyanohydrin from a carbonyl compound and a cyanide using the enzyme according to claim 1 as a catalyst. A method for synthesizing an α-hydroxycarboxylic acid, comprising hydrolyzing an optically active cyanohydrin obtained by the method according to claim 2.