JPH10234363A - New enzyme and production of para-hydroxybenzaldehyde derivative, etc., using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new enzyme capable of efficiently producing conifenyl alcohol, hydroxyphenylpropenol derivatives, etc. SOLUTION: This new enzyme is obtained from mycelia of a strain belonging to the genus Byssochlamys fluva V107 (FERM P-16024), etc., cultured in a culture medium containing vanillyl alcohol and expresses the following characteristics; action: produces conifenyl alcohol by shifting the double bond at the α-position of eugenol to the adjacent position and adding a hydroxy group at the α-position; substrate specificity: acts on compounds of formulas I-III in the presence of water and produces compounds of formulas IV-VI respectively; substrate affinity: the Michaelis constant for eugenol or vanillyl alcohol is 15.6, 214μmole concentration respectively; optimum pH: around pH7.5; optimum temperature: 60 deg.C; stable pH range: stable in pH4-9 at 30 deg.C for 30 minutes treatment; influence of various inhibitors: activities are inhibited with p- chloromercuribenzoic acid and phenylhydrazine; molecular weight: 58000 (SDS method), 110000 (ultracentrifuge analysis), a dimer consisting of the two same subunits.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel catalyzing reaction for oxidizing a substrate such as a p-allylphenol derivative, a p-hydroxytoluene derivative or a p-alkylphenol derivative, which is obtained from the cells of a strain belonging to the genus Bissoclamis. Using the enzyme and the enzyme, 3- (p-
(Hydroxyphenyl) -2-propenol derivative, p-
The present invention relates to a method for producing a hydroxybenzaldehyde derivative, a 1- (4-hydroxyphenyl) alcohol derivative and the like.

[0002]

2. Description of the Related Art In the presence of an electron donor and an electron acceptor, a double bond at the α-position of a substrate of a p-allylphenol derivative represented by eugenol is moved to the next position.
There has been no report of an oxidoreductase that catalyzes a reaction for adding a hydroxyl group to a position, and production of a 3- (p-hydroxyphenyl) -2-propenol derivative represented by coniferyl alcohol using such an enzyme. The law was not yet known. Furthermore, there have been no reports of various reactions catalyzed by such enzymes. In addition, coniferyl alcohol, which is one of the conventional 3- (p-hydroxyphenyl) -2-propenol derivatives, is widely used as a raw material for pharmaceuticals, cosmetics, agricultural chemicals, food additives, feed additives, liquid crystals, and the like. It is a promising substance, and it is known that the coniferyl alcohol can be formed as a by-product of the vanillin production process. However, since the amount of coniferyl alcohol produced in this step is small, coniferyl alcohol is expensive and its widespread use has been hindered. Attempts have been made to produce vanillin-related substances by allowing inexpensive eugenol to act on microorganisms,
(JP-A-5-227980, Agricultural Biological Chemistry, Vol. 41, 925-929)
(1977) and Vol. 47, pp. 2639-2640 (1983)) produce vanillin or vanillic acid, but have not succeeded in the efficient production of coniferyl alcohol. Therefore, the present inventors isolated from nature the microorganism Pseudomonas fluorescens E118 (FERM P-15185) that produces ferulic acid on eugenol as a substrate, and in the microorganism, produces coniferyl alcohol using eugenol as a substrate. An enzyme, eugenol dehydrogenase, was found. A patent application was filed after confirming that coniferyl alcohol could be produced using this enzyme. However, there is a need for a more efficient method for producing coniferyl alcohol than that method. Because, when eugenol is applied to the cells, most of the eugenol is converted to ferulic acid and the amount of coniferyl alcohol produced is small, and only the coniferyl alcohol is produced when the enzyme extracted from the cells and purified solely is used. Although,
The problem is that it takes time and cost to extract and purify from the cells. In WO95 / 02062, Penicilliu grown in a culture solution using veratryl alcohol as a single carbon source was used.
A method for producing coniferyl alcohol by reacting eugenol with vanillyl alcohol oxidase of m simplicissimum as a substrate is described. However, in this case, expensive veratryl alcohol must be added to the culture medium, which is not an industrial production method.
Further, there has been no report of an oxidoreductase that catalyzes a reaction for efficiently producing an optically active 1- (4-hydroxyphenyl) alcohol derivative by oxidizing a p-alkylphenol derivative. Optically active 1- (4-hydroxyphenyl) alcohol derivatives are used in pharmaceuticals, cosmetics, agricultural chemicals,
It is a substance that is expected to be widely used as a raw material for synthesizing liquid crystals and the like. Particularly, it is used as a chiral synthon of a vasodilator.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a novel oxidoreductase which catalyzes a reaction in which a double bond at the α-position of a substrate such as eugenol is moved to the next position and a hydroxyl group is added to the α-position. An object of the present invention is to provide a method for efficiently producing useful compounds such as coniferyl alcohol using an enzyme. Furthermore, 3- (p-hydroxyphenyl) -2-yl is utilized by utilizing various organic reactions catalyzed by the enzyme.
An object of the present invention is to provide a method for producing a compound such as a propenol derivative, a p-hydroxybenzaldehyde derivative, and an optically active 1- (4-hydroxyphenyl) alcohol derivative.

[0004]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have searched for strains capable of assimilating vanillyl alcohol as a single carbon source more widely in nature, and one fungus strain of eugenol was used. When produced, it was found that it produced coniferyl alcohol without producing ferulic acid. This strain was identified as Bissoclamis fulva based on morphological features such as lacking the ascocarp and naked asci on the hypha. The present inventors have conducted intensive studies on this strain and completed the inventions shown in 1 to 7 below.

[0005] 1. Obtained from the cells of a strain belonging to the genus Bissoclamis cultivated in a culture solution containing vanillyl alcohol, and in the presence of water, at least the following reaction formula A,
An enzyme that catalyzes the reactions shown in B and C.

[0006]

Embedded image

(Wherein, R ¹ represents a hydrogen atom, a hydroxyl group or a methoxy group, R ² represents a hydroxyl group or a methoxy group, R ³ represents a hydrogen atom or a hydroxyl group, and R ⁴ represents a carbon atom having 1 carbon atom.
And 10 to 10 alkyl groups. However, in reaction formula C, R ² can be a hydrogen atom other than the above-mentioned groups, but in this case, R ¹ and R ² are not hydrogen atoms at the same time. )

[0008] 2. 2. The enzyme according to the above 1, wherein the reaction represented by the reaction formula C is an asymmetric induction reaction.

3. The strain belonging to the genus Bissoclamis fulva (Byssochlamysfulva) V107 FERM P
The enzyme according to the above 1 or 2, which is -16024.

[0010] 4. The p-allylphenol derivative represented by the general formula (1-a) is reacted with the enzyme according to any one of the above 1 to 3 as a catalyst in the presence of water in the presence of water to give a compound represented by the general formula (2) A method for producing a 3- (p-hydroxyphenyl) -2-propenol derivative represented by -a).

[0011]

Embedded image (In the formula, R ¹ represents a hydrogen atom, a hydroxyl group or a methoxy group, and R ² represents a hydroxyl group or a methoxy group.)

5. The p-hydroxytoluene derivative represented by the general formula (1-b) is converted from the p-hydroxytoluene derivative represented by the general formula (1-b) by a reaction represented by the following reaction formula B in the presence of water, using the enzyme according to any one of the above 1 to 3 as a catalyst. A method for producing a p-hydroxybenzaldehyde derivative represented by -b).

[0013]

Embedded image

(Wherein, R ¹ represents a hydrogen atom, a hydroxyl group or a methoxy group, R ² represents a hydroxyl group or a methoxy group, and R ³ represents a hydrogen atom or a hydroxyl group.)

6. The p-alkylphenol derivative represented by the general formula (1-c) is reacted by the reaction represented by the following reaction formula C in the presence of water, using the enzyme according to any one of the above 1 to 3 as a catalyst, and the general formula (2- Optical activity 1 represented by b)
A method for producing a (4-hydroxyphenyl) alcohol derivative.

[0016]

Embedded image

[0017] (wherein, R ¹ represents a hydrogen atom, a hydroxyl group or a methoxy group, R ² represents a hydrogen atom, a hydroxyl group or a methoxy group, R ⁴ is an alkyl group having 1 to 10 carbon atoms, R ¹ And R ² are not hydrogen atoms at the same time.)

[7] A strain belonging to the genus Bissock Ramis is cultured in a culture solution containing vanillyl alcohol, and then the substrate represented by the general formula (1-a) is added to the culture solution, or the culture is cultured in the culture solution. Wherein the obtained strain is transplanted to another culture solution containing the substrate represented by the general formula (1-a) and further cultured.
A method for producing the compound represented by a).

8. A strain belonging to the genus Bissock Ramis is cultured in a culture solution containing vanillyl alcohol, and then a substrate represented by the general formula (1-b) is added to the culture solution, or the culture is cultured in the culture solution. Wherein the obtained strain is transplanted to another culture solution containing the substrate represented by the general formula (1-b) and further cultured.
A method for producing the compound represented by b).

9. A strain belonging to the genus Bissock Ramis is cultured in a culture solution containing vanillyl alcohol, and then a substrate represented by the general formula (1-c) is added to the culture solution, or the culture is cultured in the culture solution. Wherein the obtained strain is transplanted to another culture solution containing the substrate represented by the general formula (1-c) and further cultured.
A method for producing an optically active compound represented by c).

In order to obtain the enzyme of the present invention, first, a strain belonging to the genus Bissoclamis, which is a cell producing the enzyme, is aerobically cultured in a culture solution containing vanillyl alcohol. The culture solution may be any as long as it contains vanillyl alcohol and the bacterium grows. Preferably, the culture solution is 0.1 to 0.4% vanillyl alcohol.
(W / v), glycerin 0.5 to 3% (w / v), ammonium sulfate 0.1 to 0.5% (w / v), dipotassium hydrogen phosphate 0.1 to 0.5% (w / v) / V), magnesium sulfate heptahydrate 0.01 to 0.1% (w / v),
Yeast extract consisting of 0.01 to 0.1% (w / v) and metal salt mixture 0.05 to 0.2% (v / v)
H3.5 to 7.0 are used. The mixed solution of metal salt is 0.4 g of calcium chloride dihydrate, 0.3 g of boric acid, 0.04 g of copper sulfate pentahydrate, 0.1 g of potassium iodide, 0.4 g of manganese sulfate heptahydrate, 0.4 g of molybdenum in 1 L of deionized water. What consists of the solution which melt | dissolved 0.2 g of sodium acid dihydrate is preferable. The culture is carried out at 20 to 35 ° C for 24 to 72 hours with aeration and stirring. The cells obtained by centrifuging or filtering the resulting culture are suspended in a 1M potassium phosphate buffer (pH 7.0). The cell suspension is passed through an ultrasonic crusher or a cell crusher to crush the cells to obtain a crude enzyme solution.

The obtained crude enzyme solution is subjected to salting out treatment with ammonium sulfate and / or various column chromatography methods such as ion exchange, gel filtration, and hydrophobicity to obtain a high purity enzyme preparation. In this way, a purified enzyme preparation that is recognized as single by SDS polyacrylamide gel electrophoresis is obtained. The enzymatic properties of this enzyme are described below.

(1) Function: In the presence of water, the double bond at the α-position of eugenol is moved to the next position, and a hydroxyl group is added to the α-position to produce coniferyl alcohol. (2) Substrate specificity: General formula (1-a) or (1-b)
Acting on the compound represented by the general formula (2-a)
Alternatively, a compound represented by (2-b) is produced. Further, it acts on the compound represented by the general formula (1-c) to generate a compound represented by the general formula (2-c). (3) Substrate affinity: Michaelis constant for eugenol or vanillyl alcohol was 15.6 or 21 respectively.
4 μM. (4) Optimum pH: around pH 7.5. (5) Optimal temperature: 60 ° C. (6) Stable pH range: at 30 ° C. for 30 minutes, p
It is stable from H4 to 9. (7) Influence by various metal salts: As shown in Table 1, copper,
Its activity is significantly inhibited by silver and mercury salts.

[0024]

[Table 1]

(8) Effect of various inhibitors: As shown in Table 2, the activity is inhibited by p- (chloromercuri) benzoic acid and phenylhydrazine.

[0026]

[Table 2]

(9) Molecular weight: 58,000 by SDS-polyacrylamide gel electrophoresis, 110 by ultracentrifugation analysis
000. It is a dimer composed of two identical subunits. Because of these properties, the enzyme of the present invention is useful in the European Journal of Biochemistry 208: 65.
Penicill, described in pages 1-657 (1992).
It is clear that this is quite different from vanillyl alcohol oxidase induced by veratryl alcohol of ium simplicissimus. In particular, the Penicillium enzyme has a Michaelis constant for vanillyl alcohol of 730 μmol, an optimum pH of around 10, an optimum temperature of 38 ° C., and a molecular weight of 6
It differs in that it is an octamer consisting of eight 5,000 subunits.

In the present invention, the enzyme activity is measured as follows. 6. 1 molar potassium phosphate buffer pH7.
50 μL of the enzyme solution was added to a test tube containing a reaction solution for enzyme activity measurement consisting of 20 μL of 0 and 930 μL of deionized water, and the mixture was incubated at 30 ° C. for 5 minutes. , And the reaction was stopped by adding 1 mL of methanol.
The resulting reaction solution is centrifuged, and the supernatant is subjected to high performance liquid chromatography to detect and quantify the peak of coniferyl alcohol. In the high performance liquid chromatography, an ODS C18 column having a size of 4.6 × 150 mm was used, and an eluate having a component ratio of methanol: deionized water: acetic acid = 45: 52: 3 was flowed at a flow rate of 1.0 mL / min. Develop and detect absorbance at 280 nm. The peak around 3.6 minutes, which is the retention time of standard coniferyl alcohol, is defined as coniferyl alcohol. One unit of the activity of the present enzyme is an enzyme activity that produces 1 μmol of coniferyl alcohol per minute.

Using the enzyme, a 3- (p-hydroxyphenyl) -2-propenol derivative represented by the general formula (2-a), a p-hydroxybenzaldehyde derivative represented by the general formula (2-b), 1 represented by the formula (2-c)
A method for producing a-(4-hydroxyphenyl) alcohol derivative will be described by taking a method for producing coniferyl alcohol as an example. From the reaction solution for measuring the enzyme activity, the pH was adjusted to 5 to 8 by dropping dilute sulfuric acid or aqueous ammonia with the exception of the buffer solution, and the pH was adjusted to 1 to 25 ° C to 70 ° C.
Stir for 5 to 180 minutes. After completion of the reaction, coniferyl alcohol is extracted from the reaction solution with an organic solvent such as ethyl acetate, chloroform, methylene chloride or ethyl ether, and purified by means such as silica gel chromatography, reverse phase liquid chromatography, or vacuum distillation.
If necessary, decolorize with activated carbon and dry under vacuum to obtain a colorless coniferyl alcohol powder. 3- (p-hydroxyphenyl) -2-propenol derivative represented by general formula (2-a), p-hydroxybenzaldehyde derivative represented by general formula (2-b) using the enzyme of the present invention, general formula 1- (4-hydroxyphenyl) represented by (2-c)
Water used in the method for producing an alcohol derivative,
Acts as a solvent and a source of oxygen atoms. Water is required at least in equimolar amount with the substrate, but the reaction is usually carried out in a large excess amount of an aqueous solution.

Using the enzyme of the present invention, general formula (1-a)
From the substrates represented by (1-b) and (1-c), general formula (2)
-(P-hydroxyphenyl) -2 represented by -a)
-Propenol derivative, p represented by the general formula (2-b)
-Hydroxybenzaldehyde derivative, a compound represented by the general formula (2-
In the case of producing each of the 1- (4-hydroxyphenyl) alcohol derivatives represented by c), an isolated and purified enzyme may be used, but vanillyl alcohol can be used without isolating and purifying the enzyme. A strain belonging to the genus Bissock Ramis is cultured in a culture medium containing the same, and then the substrate represented by any of the general formulas (1-a) to (1-c) is added to the culture medium, or When the cultured strain is transplanted to another culture solution containing the substrate represented by any of the general formulas (1-a) to (1-c) and further cultured, the corresponding general formula (2-a) is obtained. ~ The compound represented by (2-c) can be produced. In this case, the step of isolating and purifying the enzyme can be omitted because a living strain is used, which is advantageous.

[0031]

Next, the present invention will be described in detail with reference to examples. The present invention is not limited only to these examples.

Example 1 Bissoclamis Fulva V107 FERM P-16024 was prepared using vanillyl alcohol 0.3% (w / v), glycerin 1.5.
% (W / v), ammonium sulfate 0.25% (w / v)
v), potassium dihydrogen phosphate 0.2% (w / v), magnesium sulfate heptahydrate 0.05%, yeast extract 0.05
% (W / v), and a metal salt mixture 0.1% (v / v), and the pH was adjusted to 4.5, and the pH was adjusted to 4.5 with 20 L of a culture solution for 54 hours.
The cells were cultured with aeration and stirring. The mixed solution of metal salt is 0.4 g of calcium chloride dihydrate, 0.3 g of boric acid, 0.04 g of copper sulfate pentahydrate, 0.1 g of potassium iodide, 0.4 g of manganese sulfate heptahydrate, 0.4 g of molybdenum in 1 L of deionized water. What consisted of a solution in which 0.2 g of sodium acid dihydrate was dissolved was used. The bacterial cells obtained by filtering the culture solution were washed with physiological saline, and 10 m
The suspension was suspended in 300 ml of M potassium phosphate buffer (pH 7.0). The enzyme was extracted and purified from the cell suspension as described below. Table 3 shows a purification scheme.

The cell suspension was treated with a cell disrupter to disrupt the cells, and 621 mg of the supernatant obtained by centrifugation (total activity: 7%).
5.2 units) was taken as the cell-free extract. Ammonium sulfate was added to this to obtain 174 mg of a sulfuric acid saturated concentration fraction of 50 to 80% (w / v) (total activity: 63.7 units). This fraction was applied to a column of phenyl sepharose CL-4B (manufactured by Pharmacia) to obtain 48.7 mg of an active fraction (total activity: 38.9 units). Octyl Sepharose CL-6B
(Product of Pharmacia) and hydroxyapatite, and chromatography was performed to obtain 3.77 mg (total activity: 33.4 units) of the enzyme preparation. This preparation was single on SDS-polyacrylamide gel electrophoresis. The specific activity was finally increased by 73.2 times from the cell-free extract, and the recovery rate was 44.4%.
Met.

[0034]

[Table 3]

Example 2 The ammonium sulfate salting-out prepared according to Example 1 was completed in a reaction vessel equipped with a reflux condenser containing 50 mL of a 200 mM n-octane solution of eugenol and 725 mL of deionized water. 25 mL (20 units) of the enzyme solution was added, and the mixture was stirred at 50 ° C. for 2 hours while maintaining the pH at 5.5 by automatically adding dilute sulfuric acid dropwise. Ethyl acetate was added to the reaction mixture.
The ethyl acetate layer extracted by adding 00 mL was concentrated to 20 mL with a rotary evaporator and applied to a silica gel column. Fractions eluted with a solvent of methanol: chloroform (1: 1) were collected and dried in vacuo at room temperature to obtain 1.6 g of a colorless powder having a melting temperature of 74 ° C. The yield was 89%. This powder was subjected to GCMS analysis, and the molecular weight peak was measured. The result was 180 m / z, which was consistent with that of standard coniferyl alcohol. The infrared absorption spectrum was also consistent with the standard coniferyl alcohol, and was identified as coniferyl alcohol.

Example 3 In the same manner as in Example 2, corresponding products were produced from the respective substrates as shown in Table 4. Each product was identified by GCMS analysis, infrared absorption spectrum analysis and the like. Optical activity is high performance liquid chromatography using a chiral cell OB column,
The mobile phase was n-hexane: n-propanol (9: 1), and the peak was detected by using an ultraviolet detector and an optical rotation detector. The activity for each substrate was shown as a relative activity with respect to the case where eugenol was used as a substrate.

[0037]

[Table 4]

Example 4 3 g (wet weight) of Bissoclamis fulva V107 cells obtained by culturing according to the method described in Example 1 were mixed with 2 mL of a 1 molar potassium phosphate buffer (pH 7.0) and a substrate. Eugenol (525 μL) was added as the total volume and the substrate concentration was 40 mL.
The millimolar suspension was shaken at 28 ° C. 35
After an hour, 0.54 g of 74 mM coniferyl alcohol and 0.05 g of 7 mM coniferyl aldehyde were formed in the suspension.

[0039]

The present invention relates to an enzyme which catalyzes a reaction for producing an unsaturated alcohol by adding a hydroxyl group to the α-position accompanied by transfer of a double bond at the α-position of a substrate. Various 3- (p
(Hydroxyphenyl) -2-propenol derivative, p-hydroxybenzaldehyde derivative and 1- (4-hydroxyphenyl) alcohol derivative can be efficiently produced.

Claims (10)

[Claims] An enzyme having the following properties (1) to (9): (1) Action: Coniferyl alcohol is generated by moving the double bond at the α-position of eugenol to the next position and adding a hydroxyl group to the α-position. (2) Substrate specificity: In the presence of water, as shown in the following reaction formulas A, B and C, the general formulas (1-a), (1-b) and (1-
c) acts on the compound represented by the general formula (2-
The compounds represented by a), (2-b) and (2-c) are produced. Embedded image (Wherein, R ¹ represents a hydrogen atom, a hydroxyl group or a methoxy group, R ² represents a hydroxyl group or a methoxy group, R ³ represents a hydrogen atom or a hydroxyl group, and R ⁴ represents an alkyl group having 1 to 10 carbon atoms. . However, although in Scheme C may also be R ² is hydrogen atom in addition to the groups described above, in this case R ¹
And R ² are not simultaneously hydrogen atoms. (3) Substrate affinity: Michaelis constants for eugenol or vanillyl alcohol are 15.6 and 214μ, respectively.
It is a molar concentration. (4) Optimum pH: around pH 7.5. (5) Optimal temperature: 60 ° C. (6) Stable pH range: at 30 ° C. for 30 minutes, p
It is stable from H4 to 9. (7) Influence by various metal salts: The activity is remarkably inhibited by copper, silver and mercury salts. (8) Effect of various inhibitors: p- (chloromercuri) benzoic acid and phenylhydrazine inhibit the activity. (9) Molecular weight: 58,000 as measured by SDS-polyacrylamide gel electrophoresis, 110, as measured by ultracentrifugation analysis
000, which is a dimer composed of two identical subunits. 2. A method according to claim 1, wherein said cells are obtained from the cells of a strain belonging to the genus Bissoclamis cultivated in a culture solution containing vanillyl alcohol and contain at least the following reaction formulas A, B,
An enzyme that catalyzes the reaction shown in C. Embedded image (Wherein, R ¹ represents a hydrogen atom, a hydroxyl group or a methoxy group, R ² represents a hydroxyl group or a methoxy group, R ³ represents a hydrogen atom or a hydroxyl group, and R ⁴ represents an alkyl group having 1 to 10 carbon atoms. . However, although in Scheme C may also be R ² is hydrogen atom in addition to the groups described above, in this case R ¹
And R ² are not simultaneously hydrogen atoms. ) 3. The enzyme according to claim 2, wherein the reaction represented by the reaction formula C is an asymmetric induction reaction. 4. A strain belonging to the genus Bissoclamys fulva (Byssochlamys fulva) V107 FERM P-16.
The enzyme according to claim 2 or 3, which is 024. 5. A p-type compound represented by the general formula (1-a) by a reaction represented by the following reaction formula A in the presence of water, using the enzyme according to any one of claims 1 to 4 as a catalyst. A method for producing a 3- (p-hydroxyphenyl) -2-propenol derivative represented by the general formula (2-a) from an allylphenol derivative. Embedded image (In the formula, R ¹ represents a hydrogen atom, a hydroxyl group or a methoxy group, and R ² represents a hydroxyl group or a methoxy group.) 6. A p-type compound represented by the general formula (1-b) by a reaction represented by the following reaction formula B in the presence of water using the enzyme according to any one of claims 1 to 4 as a catalyst. A method for producing a p-hydroxybenzaldehyde derivative represented by the general formula (2-b) from a hydroxytoluene derivative. Embedded image (In the formula, R ¹ represents a hydrogen atom, a hydroxyl group or a methoxy group, R ² represents a hydroxyl group or a methoxy group, and R ³ represents a hydrogen atom or a hydroxyl group.) 7. A p-alkylphenol derivative represented by the general formula (1-c) from the p-alkylphenol derivative represented by the general formula (1-c) by a reaction represented by the following reaction formula C, using the enzyme according to any one of claims 1 to 4 as a catalyst. A method for producing an optically active 1- (4-hydroxyphenyl) alcohol derivative represented by (2-b). Embedded image (Wherein, R ¹ represents a hydrogen atom, a hydroxyl group or a methoxy group, R ² represents a hydrogen atom, a hydroxyl group or a methoxy group,
R ⁴ represents an alkyl group having 1 to 10 carbon atoms, and R ¹ and R ²
Are not simultaneously hydrogen atoms. ) 8. A method for culturing a strain belonging to the genus Bissock Ramis in a culture solution containing vanillyl alcohol, and subsequently adding a substrate represented by the general formula (1-a) to the culture solution, or culturing the culture. The strain cultivated in the solution is represented by the general formula (1)
A method for producing a compound represented by the general formula (2-a), wherein the compound is transplanted to another culture solution containing the substrate represented by -a) and further cultured. Embedded image 9. A method for culturing a strain belonging to the genus Bissock Ramis in a culture solution containing vanillyl alcohol, and subsequently adding a substrate represented by the general formula (1-b) to the culture solution, or culturing the culture. The strain cultivated in the solution is represented by the general formula (1)
A method for producing a compound represented by the general formula (2-b), wherein the compound is transplanted to another culture solution containing the substrate represented by -b) and further cultured. Embedded image 10. A method for culturing a strain belonging to the genus Bissock Ramis in a culture solution containing vanillyl alcohol, and subsequently adding a substrate represented by the general formula (1-c) to the culture solution, or culturing the culture. The method according to the general formula (2-), wherein the strain cultured in the liquid is transplanted to another culture liquid containing the substrate represented by the general formula (1-c) and further cultured.
A method for producing an optically active compound represented by c). Embedded image