JP4746084B2 - (R) -Hydroxynitrile lyase and method for using the same - Google Patents

Description

  The present invention relates to (R) -hydroxynitrile lyase and a method for producing (R) -cyanohydrin using the same.

  The optically active cyanohydrin is useful as an optically active organic synthetic intermediate, and is used for obtaining, for example, a pharmaceutical product.

  As one means for synthesizing optically active (R) -cyanohydrin from a cyanide compound and a carbonyl compound, a synthesis method using an enzyme called (R) -hydroxynitrile lyase is known. For example, when benzaldehyde and hydrogen cyanide are reacted with a (R) -hydroxynitrile lyase catalyst, (R) -mandelonitrile is produced (FIG. 1).

  Examples of (R) -hydroxynitrile lyase include those derived from cassava (Manihot esculenta) (EC 4.1.2.37) belonging to the family Euphorbiaceae, and those derived from almond (Prunus amygdalis) belonging to the family Rosaceae (EC). 4.1.2.10) or those derived from sorghum (Sorghum bicolor) (EC 4.1.2.11) are known, and gene sequences encoding these enzymes are also known.

  Usually, the synthesis of optically active cyanohydrin using the above-mentioned enzyme is based on the enzyme and the substrate cyanide compound and carbonyl compound as essential elements, aqueous system, water-organic solvent two-phase system, organic solvent-slightly water system, organic solvent system, etc. In which case an asymmetric cyanohydrin enantiomeric mixture is formed. In general, in a bioactive enantiomeric mixture, only one of the two enantiomers exhibits biological activity, so the (R) -enantiomer of the optically active cyanohydrin needs to be produced with as high purity as possible.

  However, the optical purity of the obtained (R) -cyanohydrin is not always satisfactory.

  An object of the present invention is to provide (R) -hydroxynitrile lyase. Another object of the present invention is to provide a method for producing (R) -cyanohydrin using the enzyme.

  As a result of earnest research to solve the above-mentioned problems, the present inventor obtained (R) -hydroxynitrile lyase composition from a suspension of a plant body such as passion fruit, thereby producing (R) -cyanohydrin. The present invention has been completed successfully.

That is, the present invention is as follows.
(1) (R) -hydroxynitrile lyase derived from passion fruit (Passiflora edulis) having the following properties.
(a) Action: catalyzes the synthesis of (R) -cyanohydrin from carbonyl compounds and cyanide compounds
(b) Molecular weight: Approximately 15,000 molecular weight determined by SDS-polyacrylamide gel electrophoresis, and approximately 18,000 molecular weight determined by gel filtration.
(c) The N-terminal sequence is the amino acid sequence represented by SEQ ID NO: 1 (2) Passion fruit (Passiflora edulis), loquat (Eriobotrya japonica), Karin (Chaenomeles siensis), Prunus persica and Solbus (R) -cyanohydrin comprising contacting a carbonyl compound and a cyanide with (R) -hydroxynitrile lyase activity derived from at least one plant selected from Sorbus aucuparia or a composition having the activity. Manufacturing method.
(3) A method for producing (R) -cyanohydrin, which comprises contacting (R) -hydroxynitrile lyase according to (1) with a carbonyl compound and a cyanide compound.
(4) The method for producing (R) -cyanohydrin according to (2) or (3), wherein the carbonyl compound is benzaldehyde and the cyanide compound is hydrogen cyanide.
(5) A method for producing an α-hydroxycarboxylic acid, comprising hydrolyzing (R) -cyanohydrin produced by any one of the methods (2) to (4) using a mineral acid.

  The present invention provides (R) -hydroxynitrile lyase. (R) -Hydroxynitrile hyase is industrially useful because it has a catalytic activity to produce an aldehyde into a nitrile by its catalytic action.

  Hereinafter, the present invention will be described in detail.

  The (R) -hydroxynitrile lyase of the present invention (hereinafter referred to as “(R) -hnl”) is an enzyme having a catalytic action to synthesize (R) -cyanohydrin from a cyanide compound and a carbonyl compound, It can be obtained from passion fruit, Karin, Purnus persica or Solvus occupia.

1. Enzyme Preparation Examples of the source of the enzyme composition of the present invention include passion fruit, loquat, quince, Prnus persica or Solvus occupia.

Passion fruit (Passiflora edulis) is a perennial passiflora plant native to South America and a tropical fruit tree.
Biwa (Eriobotrya japonica) is an evergreen tree of the Rosaceae native to the Yangtze River coast of China.
Karinome (Chaenomeles siensis) is a Rosaceae plant native to China.
Prunus persica is a plant belonging to the family Rosaceae and includes Broomberry and Gempeisidare.
Sorbus aucuparia is a Rosaceae plant.

  The plant body of the above plant may be directly harvested or commercially available, and stems, leaves (young leaves) and the like are used. In the present invention, leaves or seeds are preferable from the viewpoint of easy collection.

  The enzyme composition of the present invention can be obtained by the following preparation method. That is, the leaves or seeds of the plant are frozen with liquid nitrogen and ground and then suspended in a buffer solution such as a phosphate buffer.

  For this suspension, (1) fractionation by precipitation, (2) various chromatographies, (3) methods for removing low-molecular substances by dialysis, ultrafiltration, etc. Or it can obtain by using combining suitably.

  The substance used for the fractionation by precipitation of (1) is preferably ammonium sulfate (ammonium sulfate) from the viewpoint of high solubility in water and little temperature change in solubility. The concentration to be added is not particularly limited, but conditions where (R) -hnl can be recovered with good yield and can be separated from other protein components are preferable.

  Examples of the chromatography of (2) include gel filtration, ion exchange chromatography, isoelectric point chromatography, hydrophobic chromatography, affinity chromatography and the like.

  (I) The gel filtration method is a method for 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. Examples of the column used for gel filtration include, but are not limited to, Sephadex G25, Sephadex G50, Sephadex G100, Sephadex G200, S-100HR, S-200HR, and S-300HR (all of which are Pharmacia). It is not a thing.

  (Ii) 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.

  (Iii) Hydrophobic chromatography is a separation technique based on hydrophobic interaction between a protein and a ligand bound to a gel. For example, a phenyl-superose, TSK series column 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 also be used.

  (Iv) Affinity chromatography is characterized by attaching a substance that specifically binds to a protein or enzyme to a column and separating the target protein using a specific binding reaction. ), Dextran (Sephadex), cellulose, polyacrylamide (Biogel P), porous silica beads, and the like.

(3) Dialysis, ultrafiltration, etc.Dialysis is a technique in which a low molecular weight compound is permeated 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. is there.
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 and anion exchange chromatography.

2. Properties of enzyme (R) -hnl derived from passion fruit obtained by the above purification method is specified by the following physicochemical properties.
(a) Action It catalyzes the synthesis of (R) -cyanohydrin from a carbonyl compound and a cyanide compound.
(b) Substrate specificity It acts specifically on carbonyl compounds, especially benzaldehyde.
(c) Molecular weight It has a molecular weight of about 18000 according to the molecular weight determination method by gel filtration. It has a molecular weight of about 15000 by the molecular weight determination method by SDS-polyacrylamide gel electrophoresis.
(d) N-terminal sequence is the amino acid sequence represented by SEQ ID NO: 1.
(e) The specific activity of the enzyme composition after ammonium sulfate fractionation of a passion fruit leaf or seed suspension is 3.09 (U / mg), and then DEAE-Toyopearl, Butyl-Toyopearl, DEAE-Toyopearl , ConA-Sepharose, and Phenyl-Superose chromatography, the specific activities were 10 (U / mg), 10 (U / mg), 11 (U / mg), 69 (U / mg), respectively. Near, 136 (U / mg).

  As used herein, “specific activity” refers to the amount of enzyme (U / mg) that produces 1 μmol of optically active mandelonitrile per minute in 1 mg of enzyme from benzaldehyde under standard assay conditions.

  In addition, the enzyme composition of the present invention may be a mixture having the above-mentioned characteristics and containing a plurality of enzyme active substances, and an enzyme composition (or an individual active substance from which the active substances are isolated (or The enzyme itself).

3. Production of cyanohydrin In the present invention, a cyanohydrin is produced by treating a carbonyl compound and a cyanide compound, which are raw materials for producing (R) -cyanohydrin, using (R) -hnl of the present invention. “Treatment” means that a carbonyl compound and a cyanide are reacted to generate a cyanohydrin, and specifically, hydrogen cyanide is not reacted with the carbonyl compound in the presence of (R) -hnl. It means adding together.

Here, the carbonyl compound refers to an aldehyde or a ketone, specifically, the following formula (I):
R ¹ —CO—R ² (I)
The compound shown by these.

In Formula I, R ¹ and R ² are different from each other, and are each a hydrogen atom, a hydrocarbon group having 1 to 20 carbon atoms (C _{1 to} C ₂₀ ), or an optionally substituted carbon group having 1 to 20 carbon atoms ( C ₁ -C ₂₀ ) alkoxy group, optionally having ₆ to ₂₀ carbon atoms (C ₆ -C ₂₀ ) aryloxy group optionally having substituents, and optionally having 7 to 20 carbon atoms (C ₇ -C ₂₀ ) alkylaryloxy group, optionally having 2 to 20 carbon atoms (C ₂ -C ₂₀ ) alkoxycarbonyl group, optionally having amino group A silyl group or a hydroxyl group which may have a substituent. However, R ¹ and R ² are not simultaneously hydrogen atoms. Moreover, the alkylthio group which may have a substituent, the arylthio group which may have a substituent, the alkylsulfonyl group which may have a substituent, the arylsulfonyl which may have a substituent It may be a group.

During the hydrocarbon group, -CH ₂ - replaced by and CH ₂ carbonyl groups -CH _3, a sulfonyl group, may be replaced by -O- or -S-, = CH ₂ is = O or = S Or —CH ₂ —CH, —CH ₃ C—H,> CH—C—H, ═CH—C—H, and ═CH ₂ C—H are N Alternatively, it may be replaced by C-halogen, and R ¹ and R ² may jointly represent an asymmetric divalent group. Examples of the halogen include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

The hydrocarbon group may be saturated or unsaturated acyclic or saturated or unsaturated cyclic. When the hydrocarbon group is acyclic, it may be linear or branched. The hydrocarbon group, C ₁ -C ₂₀ alkyl group, C ₂ -C ₂₀ alkenyl group, C ₂ -C ₂₀ alkynyl group, C ₄ -C ₂₀ alkadienyl group, C ₆ -C ₁₈ aryl group, C ₆ -C ₂₀ alkylaryl group, C ₆ -C ₂₀ arylalkyl group, C ₄ -C ₂₀ cycloalkyl group, C ₄ -C ₂₀ cycloalkenyl group, (C ₃ ~C ₁₀ cycloalkyl) C ₁ -C ₁₀ alkyl group Etc. are included.

C ₁ -C ₂₀ alkyl group is preferably a C ₁ -C ₁₀ alkyl group. Examples of the alkyl group include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group, pentyl group, hexyl group, octyl group, nonyl group, decyl group, and undecyl group. And a dodecyl group.

C ₂ -C ₂₀ alkenyl group is preferably a C ₂ -C ₁₀ alkenyl group. Examples of the alkenyl group include a vinyl group, an allyl group, a propenyl group, an isopropenyl group, a 2-methyl-1-propenyl group, a 2-methylallyl group, and a 2-butenyl group.

C ₂ -C ₂₀ alkynyl group is preferably a C ₂ -C ₁₀ alkynyl group. Examples of the alkynyl group include ethynyl group, propynyl group, butynyl group and the like.
The C ₄ -C ₂₀ alkyl dienyl group is preferably a C ₄ -C ₁₀ alkyl dienyl group. Examples of the alkyldienyl group include a 1,3-butadienyl group.

The C ₆ -C ₁₈ aryl group is preferably a C ₆ -C ₁₀ aryl group. Examples of the aryl group include a phenyl group, a 1-naphthyl group, a 2-naphthyl group, an indenyl group, a biphenyl group, an anthryl group, and a phenanthryl group.
C ₆ -C ₂₀ alkylaryl group, preferably a C ₆ -C ₁₂ alkyl aryl group. 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.

The C ₆ -C ₂₀ arylalkyl group is preferably a C ₆ -C ₁₂ arylalkyl group. As the arylalkyl group, for example, benzyl group, phenethyl group, 1-naphthylmethyl group, 2-naphthylmethyl group, 1-phenylethyl group, phenylpropyl group, phenylbutyl group, phenylpentyl group, phenylhexyl group, methylbenzyl group Dimethylbenzyl group, trimethylbenzyl group, ethylbenzyl group, methylphenethyl group, dimethylphenethyl group, diethylbenzyl group and the like.

The C _{4 to} C ₂₀ cycloalkyl group is preferably a C _{4 to} C ₁₀ cycloalkyl group. Examples of the cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, and the like.
The C _{4 to} C ₂₀ cycloalkenyl group is preferably a C _{4 to} C ₁₀ cycloalkenyl 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.

C ₁ -C ₂₀ alkoxy group is preferably a C ₁ -C ₁₀ alkoxy group. Examples of the alkoxy group include a methoxy group, an ethoxy group, a propoxy group, a butoxy group, and a pentyloxy group.
The C ₆ -C ₂₀ aryloxy group is preferably a C ₆ -C ₁₀ aryloxy group. Examples of the aryloxy group include a phenyloxy group, a naphthyloxy group, and a biphenyloxy group.
The C ₇ -C ₂₀ alkylaryloxy group is preferably a C ₇ -C ₁₂ alkylaryloxy group. Examples of the alkylaryloxy group include a methylphenyloxy group, an ethylphenyloxy group, a propylphenyloxy group, a butylphenyloxy group, a dimethylphenyloxy group, a diethylphenyloxy group, a dipropylphenyloxy group, a dibutylphenyloxy group, and a methyl group. Examples thereof include an ethylphenyloxy group, a methylpropylphenyloxy group, and an ethylpropylphenyloxy group.

C ₂ -C ₂₀ alkoxycarbonyl group is preferably a C ₂ -C ₁₀ alkoxycarbonyl group. Examples of the alkoxycarbonyl group include methoxycarbonyl group, ethoxycarbonyl group, 2-methoxyethoxycarbonyl group, t-butoxycarbonyl group and the like.

  Examples of the amino group that may have a substituent include an amino group, a dimethylamino group, a methylamino group, a methylphenylamino group, and a phenylamino group.

  Examples of the silyl group which may have a substituent include a dimethylsilyl group, a diethylsilyl group, a trimethylsilyl group, a triethylsilyl group, a trimethoxysilyl group, a triethoxysilyl group, a diphenylmethylsilyl group, a triphenylsilyl group, A triphenoxysilyl group, a dimethylmethoxysilyl group, a dimethylphenoxysilyl group, a methylmethoxyphenyl group, etc. are mentioned.

  Examples of the carbonyl compound represented by the formula I include benzaldehyde, m-phenoxybenzaldehyde, p-methylbenzaldehyde, o-chlorobenzaldehyde, m-chlorobenzaldehyde, p-chlorobenzaldehyde, m-nitrobenzaldehyde, 3,4-methylene. Aromatic aldehydes such as dioxybenzaldehyde, 2,3-methylenedioxybenzaldehyde, phenylacetaldehyde, furfural; aliphatic aldehydes such as acetaldehyde, butyraldehyde, isobutyraldehyde, valeraldehyde, cyclohexanealdehyde; ethyl methyl ketone, butyl methyl ketone, Methyl propyl ketone, isopropyl methyl ketone, methyl pentyl ketone, methyl (2-methylpropyl) ketone, methyl (3-methyl Saturated aliphatic ketones such as (tilbutyl) ketone; unsaturated aliphatic ketones such as methyl (2-propenyl) ketone and (3-butenyl) methyl ketone; alkyl (haloalkyl) ketones such as (3-chloropropyl) methyl ketone; And 2- (protected amino) aldehydes such as alkoxycarbonylamino) -3-cyclohexylpropionaldehyde; and alkylthioaliphatic aldehydes such as 3-methylthiopropionaldehyde.

  In the present invention, an organic solvent can be used as the reaction solvent in order to increase the concentration of the reaction raw material and improve the productivity. As the organic solvent, for example, linear, branched or cyclic, saturated or unsaturated aliphatic hydrocarbon, saturated or unsaturated aromatic hydrocarbon, etc. can be used alone or in admixture of two or more. Specifically, for example, hydrocarbon solvents (eg, pentane, hexane, cyclohexane, benzene, toluene, xylene, etc.); halogenated hydrocarbon solvents (eg, methylene chloride, chloroform, etc.); alcohol solvents (eg, n-butanol) , Isobutanol, t-butanol, hexanol, cyclohexanol, n-amyl alcohol, etc.); ether solvents (eg diethyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, t-butyl methyl ether, dimethoxyethane, etc.); esters System solvents (for example, methyl formate, methyl acetate, ethyl acetate, butyl acetate, methyl propionate) and the like.

  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.

  Examples of the cyanide compound used in the present invention include hydrogen cyanide, potassium cyanide, sodium cyanide and the like.

  (R) -hnl of the present invention can be used in the form of a 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 an enzyme, for example, a method of absorbing an enzyme solution in a carrier, a method of adsorbing and immobilizing an enzyme by mixing the enzyme solution and a carrier, a method of comprehensively immobilizing an enzyme, a method of crosslinking an enzyme with a crosslinking agent Arbitrary methods, such as these, are employable.

  The usage amounts or reaction conditions of the immobilized enzyme and the substrate are appropriately determined according to the substrate used. Usually, the amount of the immobilized enzyme used is 0.1 to 500 units, preferably 1 to 100 units, based on 1 mmol of the substrate carbonyl compound. In the case of a carbonyl compound, the concentration of the substrate is usually set in the range of 1 to 500 g / L, and the cyan compound is added at a concentration of 0.1 to 10 times mol, preferably 0.5 to 5 times mol of the carbonyl compound used. The reaction time is appropriate until the conversion rate of the substrate carbonyl compound reaches 60% 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 0 to 50 ° C, preferably 10 to 40 ° C.

  The (R) -cyanohydrin produced by the method of the present invention can be measured or quantified by high performance liquid chromatography (HPLC) or the like, and if necessary, usual means such as extraction, vacuum distillation, column separation, etc. Can be separated and purified.

4). Production of α-hydroxycarboxylic acid In the present invention, 3. The α-hydroxycarboxylic acid can be produced by hydrolyzing the (R) -cyanohydrin produced by the above method using a mineral acid.

Examples of the mineral acid include hydrochloric acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, perchloric acid and the like, and hydrochloric acid is preferable. The usage-amount of a mineral acid is 1-10 equivalent with respect to cyanohydrin, and it is preferable that 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, the target α-hydroxycarboxylic acid can be isolated by extracting from the reaction solution using an organic solvent, washing with water if necessary, and then evaporating and drying the solvent. .

In this specification, when an amino acid is represented by an abbreviation, it is based on an abbreviation by the IUPAC-IUB Commission on Biochemical Nomenclature or an abbreviation commonly used in the field, and examples thereof are described below. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.
Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys Argin P : Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine SEQ ID NOs in the sequence listing of the present specification indicate the following sequences.
[SEQ ID NO: 1]
The amino acid sequence of the N terminal of (R) -hydroxynitrile lyase derived from passion fruit (Passiflora edulis) is shown.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1]
The seeds of loquat (Eriobotrya japonica), Karin (Chaenomeles sinensis), Prunus persica and Sorbus aucuparia were sampled in the amounts shown in Table 1 and subjected to the following treatment. Frozen in liquid nitrogen and homogenized with a mortar and pestle. This powder was suspended in 150 ml of 50 mM potassium phosphate buffer (pH 6.0) for about 3-4 hours and then filtered through several layers of gauze. The filtrate was centrifuged twice at 28,000 xg for 25 minutes. The resulting solution was dialyzed against 10 mM buffer for 4 hours (buffer was changed 3 times) to obtain 120 ml of enzyme solution. The enzyme solution was concentrated to 2 ml using an Amicon cell (PM 10 filter).

  Contains 300 mM potassium phosphate buffer (pH 6.0), 50 mM benzaldehyde and 100 mM cyanide solution in a final volume of 0.9 ml. The reaction was started immediately by adding 100 μl of each enzyme solution and incubated at 25 ° C. for 120 minutes. 900 μl of organic solvent (hexane: isopropanol = 9: 1) was added to stop the reaction, and the supernatant obtained by centrifugation (15,000 × g, 10 minutes) was assayed by HPLC.

  The amount of each component was measured at 254 nm using a CHIRALCEL OJ-H column with hexane: isopropanol = 90: 10 as a mobile phase at a flow rate of 1.0 ml / min, and optical purity was calculated using these standard curves.

* ey is an abbreviation for enantiomeric yield, and was calculated from the amount of produced mandelonitrile according to the following formula.

[Example 2] Preparation of enzyme solution and measurement of enzyme activity
(1) Preparation of crude enzyme solution Young leaves of passion fruit (708.8 g) were frozen in liquid nitrogen and homogenized with a mortar and pestle. This powder was suspended in 6,000 ml of 50 mM potassium phosphate buffer (pH 6.0) for about 10 hours and then filtered through several layers of gauze. The filtrate was centrifuged twice at 28,000 xg for 25 minutes. The resulting solution was dialyzed against 10 mM buffer for 4 hours (buffer was changed 3 times) to obtain 4,400 ml of crude enzyme solution.

  The protein (enzyme) concentration was determined using a Bio-Rad protein assay Kit (Bio-Rad, USA) with bovine serum albumin as a standard, or by measuring the absorbance at 595 nm.

(2) Activity measurement using benzaldehyde as substrate Next, the enzyme activity was assayed by measuring the production of (R) -mandelonitrile from benzaldehyde.
The standard assay solution contains 300 mM potassium phosphate buffer (pH 6.0), 50 mM benzaldehyde and 100 mM cyanide solution in a final volume of 0.9 ml. The reaction was started immediately by adding 100 μl of enzyme solution and incubated at 25 ° C. for 120 minutes. 900 μl of organic solvent (hexane: isopropanol = 9: 1) was added to stop the reaction, and the supernatant obtained by centrifugation (15,000 × g, 10 minutes) was assayed by HPLC.
The amount of each component was measured using a CHIRALCEL OJ-H column at a flow rate of 1.0 ml / min with hexane: isopropanol = 90: 10 as a mobile phase, measured at 254 nm, and calculated using these standard curves.
The amount of enzyme that produces 1 μmol of optically active mandelonitrile per minute in 1 mg of enzyme from benzaldehyde under standard assay conditions is defined as 1 unit of enzyme activity, the specific activity of the crude enzyme solution is 2.47 mg / mg.
The reaction results of (R) -hydroxynitrile lyase derived from the leaves of passion fruit are shown in FIGS. The optical purity of R-mandelnitrile produced after 20 minutes was 70% ee.

(3) Ammonium sulfate precipitation Solid ammonium sulfate was added to the above enzyme solution (4,400 ml) with stirring on ice until it was 20% saturated. After stirring for 30 minutes, the resulting precipitate was separated by centrifuging at 4 ° C. for 30 minutes and discarded. Thereafter, the ammonium sulfate concentration in the supernatant was increased to 30% saturation. The resulting precipitate was collected by centrifugation at 4 ° C. for 30 minutes and dissolved in 10 mM buffer. This solution was dialyzed against 10 mM buffer for 12 hours (buffer was changed 3 times).
When enzyme activity was measured in the same manner as in (2) above, activity was found in the 20-30% ammonium sulfate precipitate fraction.

(4) Various chromatography (i) DEAE-Toyopearl (1st)
Place the enzyme solution on a DEAE-Toyopearl column equilibrated with 10 mM phosphate buffer (pH 6.0), and elute the active fraction by applying a gradient of NaCl from 0 to 0.1 M based on the same buffer. It was. The active fraction was dialyzed against potassium phosphate buffer (pH 6.0).
(Ii) Butyl-Toyopearl
Place the enzyme solution on a Butyl-Toyopearl column equilibrated with 10 mM potassium phosphate buffer (pH 6.0) containing 30% saturated ammonium sulfate, and further increase the concentration gradient from 30% to 0% saturated ammonium sulfate based on the same buffer. The active fraction was eluted. The active fraction was dialyzed against potassium phosphate buffer (pH 6.0).
(Iii) DEAE-Toyopearl (second time)
It carried out on the same conditions as said (i).
(Iv) ConA-Sepharose
Place an enzyme solution containing 0.5M NaCl on a ConA-Sepharose column equilibrated with 20mM Tris-HCl buffer (pH 7.4) containing 0.5M NaCl, and start from 0 of α-D-methylglucoside based on the same buffer. Elution was performed using FPLC with a 0.1M gradient. The active fraction was dialyzed against potassium phosphate buffer (pH 6.0).
(V) Phenyl-Superose
Place the enzyme solution on a Phenyl-Superrose column equilibrated with 10 mM potassium phosphate buffer solution (pH 6.0) containing 30% saturated ammonium sulfate, and further increase the concentration gradient from 25% to 0% saturated ammonium sulfate based on the same buffer solution. Then, the active fraction was eluted using FPLC. The active fraction was dialyzed against potassium phosphate buffer (pH 6.0).

(5) Summary Table 2 shows the yield and activity of the enzyme composition in each purification step.

(6) Molecular weight determination by gel filtration The purified enzyme obtained by the above operation was separated by a G3000SW column (manufactured by Tosoh Corporation, 7.5 mm x 60 cm) attached to an HPLC apparatus. As the effluent, 10 mM sodium phosphate buffer (pH 7) containing 0.1 M Na ₂ SO ₄ was used. The logarithm of the molecular weight of the reference protein and the effluent amount were plotted, and the molecular weight corresponding to the effluent amount of the enzyme peak was estimated from the graph.
As a result, the enzyme was found to have a molecular weight of about 18,000.
(7) Determination of N-terminal sequence of (R) -hydroxynitrile lyase protein The purified enzyme was subjected to 12.5% polyacrylamide gel electrophoresis. A number of gels were cut at a width of about 1.5 cm in the migration direction and 4 to 5 mm in the length in the migration direction, and each gel fragment was extracted with 0.2 ml of 10 mM phosphate buffer (pH 6). The extract was concentrated with a membrane, and the enzyme activity of each fragment was measured. The active fragments were subjected to SDS-polyacrylamide gel (15%) electrophoresis and stained to confirm band identity and blotted onto PVDF membrane followed by gas-phase Edman degradation.
N-terminal sequence: the amino acid sequence represented by SEQ ID NO: 1

(8) Molecular weight determination by SDS-polyacrylamide gel electrophoresis
A gel fragment was cut out in the same manner as in (7) to obtain a sample having activity. The unity of the enzyme contained in the fraction was confirmed by SDS-polyacrylamide gel electrophoresis. The logarithm of the molecular weight of the reference protein and the mobility of the band were plotted on a graph, and the molecular weight corresponding to the actually measured value of the mobility of the enzyme was estimated.
As a result, it was shown to have a molecular weight of about 15,000.

[Example 3] Synthesis using aliphatic ketone as substrate This example shows the synthesis of chiral ketone cyanohydrin using (R) -hnl derived from leaves of passion fruit.

(1) Synthesis of (R) -methylpropylketone cyanohydrin from 2-pentanone
2-Pentanone (2 mmol) was added to potassium cyanide solution (1.0 M, 5.0 ml) in citrate buffer (400 mM, 39 ml, pH 4.0) and the mixture was stirred. The reaction was then started by adding 5.0 ml enzyme solution (14.5 U / ml enzyme solution per nmol substrate benzaldehyde) and incubated at 25 ° C. for 24 hours. After 24 hours, the reaction mixture was extracted three times with 50 ml of ethyl acetate. The organic layer was dried over Na ₂ SO ₄ and concentrated. The residue was dried in vacuum for 12 hours to obtain (R) -methylpropyl cyanohydrin in 44.5% yield (101 mg).

(2) Preparation of (R) -α-methyl-α-hydroxypentanoic acid 5 ml of concentrated hydrochloric acid was added to (R) -methylpropylcyanohydrin (0.92 mmol) synthesized in (1) and the mixture was stirred at room temperature. Stir for hours. Thereafter, it was heated at 60 ° C. for 12 hours, and then heated at 100 ° C. for 5 hours. After removing hydrochloric acid gas in vacuum using an aspirator, the residue was extracted three times using 15 ml of diethyl ether. NH ₄ Cl was removed from the organic layer, then the extract was dried over Na ₂ SO ₄ , concentrated and further dried in vacuo for 5 h. The residue was recrystallized from n-hexane.
As a result, (R) -2-methylhydroxypentanoic acid was obtained in a yield (89 mg) of 33.5%. The optical purity was 87.0% ee. The optical purity was measured according to Reference Example 1.

Example 4
The same operation as in Example 3 was performed, except that an enzyme solution derived from 3.4 U / ml loquat seeds was used.
As a result, (R) -methylpropyl cyanohydrin was obtained in a yield of 38.9% (89.0 mg), and (R) -2-methylhydroxypentanoic acid was obtained in a yield of 31.6% (83.6 mg). It was. The optical purity was 24.0% ee.

(Reference Example 1)
To a mixed solution consisting of 0.1 mmol of 2-methylhydroxypentanoic acid, 200 μl of methanol and 700 μl of toluene, 65 μl of (trimethylsilyl) diazomethane was slowly added. When the color of the mixed solution changed from yellow to colorless, the reaction was regarded as simple, and the mixed solution was concentrated and dried.
After drying, it was dissolved in ethyl acetate and analyzed by gas-liquid chromatography (GLC) (FIG. 4). The analysis conditions of GLC are as follows.

<GLC analysis conditions>
Column: Cyclodextrin-β-236M-19 (0.25mm × 25M, DF = 0.25)
Carrier gas made by GL Sciences: Helium (40ml / min), Hydrogen (40ml / min), Air (400ml / min)
Inlet temperature: 220 ° C
Oven temperature: 85 ° C (15 minutes)

FIG. 3 is a diagram showing a reaction for producing (R) -mandelonitrile from benzaldehyde and hydrogen cyanide using a (R) -hydroxynitrile lyase catalyst. It is a HPLC chromatogram of (R) -hnl derived from the passion fruit of the present invention. It is a figure which shows the (R) -mandelonitrile production | generation activity by (R) -hnl derived from the passion fruit of this invention. It is a GLC chromatogram of ethyl (R) -2-methylhydroxypentanoate synthesized from (R) -2-methylhydroxypentanoic acid obtained using (R) -hnl derived from passion fruit of the present invention.

Claims (2)

(R) -Hydroxynitrile lyase derived from seeds of chalin (Chaenomelessis) or a composition having the activity thereof, wherein the seeds are ground and suspended in a buffer , filtered and / or centrifuged. The (R) -hydroxynitrile lyase or a composition having an activity thereof, which is obtained by dialysis of the obtained solution. A method for producing (R) -cyanohydrin, which comprises contacting (R) -hydroxynitrile lyase derived from seeds of chalin (Chaenomenessiensis) or a composition having the activity thereof with a carbonyl compound and a cyanide compound,
The (R) -hydroxynitrile lyase or a composition having an activity thereof is obtained by filtering and / or centrifuging a suspension obtained by grinding the seeds and suspending them in a buffer solution, and dialyzing the obtained solution. Said method being obtained.