JP2679739B2 - Method for producing aldehydes - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing aldehydes. More specifically, the present invention uses a styrene derivative which is produced by a specific fungus belonging to the genus Pseudomonas to selectively and oxidatively oxidize an ethylene bond conjugated to a benzene ring in the styrene derivative. The present invention relates to a method for decomposing and producing aldehydes.

<Prior Art> To the best knowledge of the present inventor, it has not been known that an aldehyde is produced by selectively and oxidatively decomposing an ethylene bond conjugated to a benzene ring in a styrene derivative using a conventional enzyme.

<Object of the Invention> An object of the present invention is to provide a method for producing an aldehyde by selectively and oxidatively decomposing an ethylene bond conjugated to a benzene ring in a styrene derivative with an enzyme.

Another object of the present invention is to provide a method capable of producing an aldehyde by oxidatively decomposing an ethylene bond conjugated to a benzene ring in a styrene derivative by using an enzyme produced by a strain belonging to the genus Cichudomonas. It is in.

According to the study by the present inventor, the above-mentioned object of the present invention is In the formula, R ₁ is a linear or branched alkyl group having 1 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, a cycloalkyl group having 5 to 15 carbon atoms, a group —COOR ¹ (wherein R ¹ is a hydrogen atom Or an alkyl group having 1 to 10 carbon atoms), a formyl group or Is shown. However, the alkyl group, cycloalkyl group and aryl group may have a substituent.

R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are the same or different from each other, and each represent a hydrogen atom, a hydroxyl group or a glycoside thereof, and a carbon number of 1 to 1.
10 linear or branched alkyl groups or 1 to 1 carbon atoms
Two alkoxy groups which represent 10 alkoxy groups or which are adjacent to each other are taken together to form CH _{2 n} , —OCH _2
n or optionally form a -OCH _{2 n} O-(where n is an integer of 1 to 4).

A method for producing aldehydes, which comprises contacting a styrene derivative represented by the following formula with an enzyme produced by a strain that belongs to the genus Cichudomonas and is capable of producing a dioxygenase-activating enzyme for the styrene derivative under aerobic conditions: It

According to the present invention, the styrene derivative [I] is treated with an enzyme produced by a strain capable of producing an enzyme having a dioxygenase activity with respect to the conjugated ethylene bond of the benzene ring in the styrene derivative, and the styrene derivative [I] is reacted with the styrene derivative [I]. The ethylene bond in the derivative [I] is oxidatively decomposed and converted into an aldehyde group. The reaction formula is schematically shown below.

Thus, in the present invention, the dioxygenase activity means an action in which an ethylene bond conjugated to a benzene ring in a styrene-derived pair is selectively decomposed by an oxygen addition reaction as shown in the above reaction formula to be converted into two aldehyde groups. To do.

As far as the present inventor knows, it is a completely new finding that the ethylene bond in the styrene derivative undergoes the above dioxygenase activity by the enzyme produced by a strain of the genus C. pseudomonas.

Thus, according to the present invention, the styrene derivative [I] is subjected to the dioxygenase activity of the above-mentioned enzyme to obtain an aldehyde.

 Some of the reaction examples are shown below.

According to the method of the present invention, as shown in the above reaction example (2), when the starting styrene derivative contains a group -OGlu, the group -OGlu in the produced aldehydes is a culture product of the strain. When glycosidase is contained in the enzyme separated from, the group OGlu may be obtained as a form converted to a hydroxyl group which is considered to be due to its action.

Preferred R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ as the styrene derivative represented by the above general formula [I] of the present invention are as described below.

R ₁ is a linear or branched alkyl group having 1 to 6 carbon atoms; an aryl group having 6 to 8 carbon atoms, particularly a phenyl group;
A cycloalkyl group having 5 to 10 carbon atoms, particularly a cyclohexyl group; a group -COOR ¹ (wherein R ¹ is a hydrogen atom or a carbon number of 1 to
4 alkyl groups); formyl groups or It is preferred that The above alkyl group and aryl group each have a hydroxyl group or a glycoside thereof or a carbon number of 1
It may be substituted with an alkoxy group of ~ 4.

On the other hand, R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are the same or different and each is a hydrogen atom; a hydroxyl group or a glycoside thereof; an alkyl group having 1 to 6 carbon atoms, particularly 1 to 3 carbon atoms; 6, particularly 1 to 3 alkoxy groups; R ₃ and R ₄ or R ₄ and R ₅ are preferably a ring which is bonded to each other to form —OCH ₂ O—.

The compound of the above formula [I] is particularly preferably one in which 1 to ₂ of R _{2 to} R ₆ are substituted with a hydroxyl group or a glycoside thereof or an alkoxy group having 1 to 3 carbon atoms.

Hereinafter, specific examples of the styrene derivative [I] that can be decomposed using the enzyme having the dioxygenase activity in the present invention include the following.

In the formulas below, Me represents a methyl group and the group OGlu represents a glucosyl group.

Next, in the method of the present invention, an enzyme having the above-mentioned dioxygenase activity, which is used in the styrene-induced oxidative decomposition represented by the above-mentioned general formula [I], a strain of the genus Cleudomonas producing the enzyme, and a method for obtaining the enzyme will be described. .

The enzyme having a dioxygenase activity used in the method of the present invention is produced by a bacterium belonging to the genus C. pseudomonas and has the following physicochemical properties.

(A) Action and Substrate Specificity: The present dioxygenase has the action of selectively acting on the ethylene bond conjugated to the benzene ring in the styrene derivative represented by the above general formula [I] to convert it into aldehydes.

(B) Optimum pH and stable pH range; Optimum pH 6-10 Stable pH range 6.5-9.5 (c) Optimum temperature range of action; pH 8.0 at 20-50 ° C (d) Inactivation condition; pH; 5 Below or 12 or higher Temperature: 60 ° C. or higher (e) Measurement of titer Described in a of Example 2 described later (f) Molecular weight; Molecular weight measured by SDS-PAGE electrophoresis is about 5
The molecular weight of the dioxygenase was about 2,000, and the molecular weight determined by gel filtration was about 94,000.
It is presumed to form a homodimer consisting of 00 identical subunits.

(G) Purification method: Purification is carried out by subjecting the crude enzyme solution to hydroxylapatide treatment, and combining ion exchange chromatography (DEAE, Toyopearl) and hydrophobic chromatography (butyl Toyopearl).

(H) Inhibition, Activation and Stabilization; Each of the following inhibitors was added to the enzyme reaction solution to examine the effect on the enzyme activity. The enzyme reaction conditions are pH 8.0, 50 ° C for 10 minutes,
Inhibition was evaluated by the relative activity, which was set to 100 when no inhibitor was added.

In the present invention, the dioxygenase is produced from a strain having the above activity and belonging to the genus Cichudomonas, and particularly preferably a bacterium belonging to the genus Cichudomonas.
It is an enzyme produced by the TMY1009 strain.

This bacterium of the genus C. pseudomonas TMY1009 has been deposited with the Institute of Microtechnology as Microorganism Research Institute No. 10362.

The bacterium TMY1009 strain has the following mycological properties.

(I) Morphology 1. Cell shape and size: Bacillus, 0.8 × 1.1 μm 2. Motility and presence of flagella epithelial state, 1 polar flagella 3. Gram stainability; negative (ii) in various media Growth condition LB medium plate culture Round and smooth colony formation, yellowish brown broth agar plate culture Colony is round, surface is smooth, color is pale yellow,
Glossy broth agar slant culture The surface was smooth, the color was pale yellow, glossy, and no pigment diffusion was observed.

No growth was observed on the surface of the broth broth. The whole liquid was a little cloudy. Bacteria were settled on the bottom.

Small bacterial mass and gelatin liquefaction were not observed in the whole broth gelatin stab culture.

Litmus milk No bacterial sedimentation on the bottom, milk coagulation, liquefaction, and no change in pH.

(Iii) Physiological properties (1) Nitrate reduction: negative (2) Pigment formation: carotenoid maximum absorption 479, 450
(425) nm (3) Oxidase: Positive (4) Catalase: Positive (5) Attitude toward enzyme: Aerobic (6) OF test: Organic acid production under oxidative conditions (7) Denitrification reaction: Negative ( 8) MR test: negative (9) VP test: negative (10) Indole formation: negative (11) Hydrogen sulfide formation: negative (12) Starch hydrolysis: positive (13) Use of citric acid Koser's medium: Negative Chvistensen medium: Positive (14) Utilization of inorganic nitrogen source Nitrate: Negative Ammonium salt: Positive (15) Urease: Negative (16) Production of acid and gas from saccharide Acid production Gas production L-arabinose + -D-xylose + -D-glucose + -D-mannose + -D-fructose + -D-galactose + -maltose + -sucrose + -lactose + -trehalose + -D-sorbit --- D-mannitol − − Inosit − − Glycerin − − Starch − − (17) Other characteristics Gluconic acid oxidation: Negative Arginine grouping: Negative Lysine decarboxylation: Negative Ornithine decarboxylation: Negative Protocatechin acid decomposition: Meta cleavage (18) Other Tween degradation Tween40: Negative Tween60: Negative Tween80: Negative DNase: Positive 3-Ketolactic acid production: Negative (iv) Growth range Temperature 20-33 ℃ pH 5.5-10 Doubling Time 27 ℃ LB medium (pH 7. 4) 2.5 hrs (v) assimilation of carbon compounds catechol-benzene-benzoic acid-ferulic acid + p-coumaric acid + protocatechuic acid + parahydroxybenzoic acid + vanillic acid + sodium glutamate + to the genus Cichudomonas in the present invention To obtain the enzyme of interest from a strain that belongs to the group and is capable of acid-producing an enzyme having the dioxygenase activity, particularly from the strain TMY1009
It suffices to culture the cells in a medium in which a strain of the genus Cleudomonas known per se grows, and separate the enzyme from the obtained culture.

Hereinafter, the composition of the medium and the method for extracting the enzyme that can be used for growing the above-mentioned strain and for producing the acid of the enzyme will be described, but this is merely for explanation, and the present invention extracts the medium and the enzyme of this composition. It is not limited to law.

Examples of the carbon source include carbohydrates such as glucose and fructose, organic compounds such as ethanol, and organic acids such as succinic acid. These may be one or more carbon compounds that can be used by the strain of the present invention. Can be used as a carbon source.

Further, the nitrogen source is not particularly limited, for example,
Inorganic nitrogen compounds such as ammonium sulfate, ammonium sulfate, and organic nitrogen sources such as peptone can be used.

As the inorganic salts, various phosphates, magnesium sulfate, etc. can be used. Further, a trace amount of metal (iron salt, calcium salt, etc.) may be contained in the medium.

As a culture method, a shaking culture method, a deep aeration stirring culture method or the like can be used. The culture temperature is preferably 20 ° C. to 33 ° C., and the pH is preferably in the range of 6 to 10. The number of days for culturing is not particularly limited, but, for example, it is usually in the range of 1 to 7 days.

In order to remove the low-molecular substances in the cell-free extract obtained by separating the cell-free extract obtained by destroying the bacterial cells in the culture in which the enzyme is produced and accumulated in this way by ultrasonication An enzyme solution is obtained by dialysis treatment with a sodium phosphate buffer solution.

Thus, in the method of the present invention, the enzyme solution itself or an enzyme purified from the enzyme solution is used in the formula [I]
It is possible to obtain the desired aldehydes by bringing them into contact with the styrene derivative (1) under aerobic conditions.

At that time, the temperature is advantageously in the range of 20 to 50 ° C, preferably 30 to 40 ° C, and the pH is preferably in the range of 6 to 10, preferably 6.5 to 9.5.

In addition, the reaction may be carried out in either a batch system or a continuous system, and the enzyme may be immobilized and used.

 The present invention will be described in detail below with reference to examples.

Example (1) Preparation of enzyme solution TMY1009 strain was aerobically shake-cultured at 27 ° C. for about 22 hours in the nutrient medium (LB) of Example 1 to obtain about 3.5 g of wet bacterial cells. The cells were collected, washed, suspended in 15 ml of a 50 mM sodium phosphate buffer solution, sonicated to destroy the cells, and centrifuged (10,000 xg, 20 min). In order to remove low-molecular substances in the obtained cell-free extract, dialysis was performed with a 20 mM sodium phosphate buffer (pH 7.5) to obtain a crude enzyme solution (protein concentration: 15.4 mg / ml).

Example (2) Method for manufacturing vanillin a. 450 μm buffer containing 1 μmol isoeugenol (pH
Add 50μ of enzyme solution (15.4mg / ml) to 7.5) and incubate at 26 ℃ for 30 minutes. After adding 50μ of 1N HCl to make the mixture acidic, extract with 500μ of ethyl acetate. HPL the extract
As a result of quantitative analysis with C, 0.16 μmol of vanillin was found.
From this result, the titer of this enzyme was calculated to be 0.11 unit. Here, one unit refers to the amount of enzyme that produces 1 μmol of vanillin per minute.

HPLC measurement conditions Model: Shimadzu LC-6A column; LiChrosorb RP-18 (4.6 x 250 mm) Leaching: 0.05% phosphoric acid water (A) / acetonitrile (B)
= 70/30… (5 minutes)… (A) / (B) = 70/30… (incr
easing 5% per min) ... (A) / (B) = 0/100 The retention time of vanillin in the above is 6.1 minutes b. Isolabotene 1 μ as a substrate under the same conditions as in Example a.
As a result of carrying out with mol, 0.16 micromol vanillin was recognized. In addition, 3,5-dihydroxybenzaldehyde was also recognized.

Example (3) Method for Producing Vanillin a. To 950 μ sodium phosphate buffer (pH 7.5) containing 1.2 μmol of 5- [2 ′-(4 ″ -hydroxy-3 ″ -methoxyphenyl) vinyl] ferulic acid. Enzyme solution (15.4mg / m
l) 50 μm was added and incubated at 27 ° C. for 30 minutes. The reaction was stopped when 1 μmol of the enzyme had been consumed. This reaction product was extracted with 1000 μl of ethyl acetate and quantitatively analyzed by HPLC. As a result, 1 μmol of vanillin and 1 μmol of 5-formylferulic acid were found. The HPLC measurement conditions are shown in Example (2).
Measured under the same conditions as in a.

b. 1,2-Bis- (4-
Hydroxy-3-methoxyphenyl) ethylene 1 μmol
The reaction was stopped when 0.54 μmol of the enzyme had been consumed. The reaction product was extracted with 1000 μl of ethyl acetate and quantitatively analyzed by HPLC. As a result, 1.1 μmol of vanillin was found.

Example (4) Production of p-hydroxybenzaldehyde This was carried out under the same conditions as in Example (2) a, using 1 μmol of psoid as a substrate. As a result of quantitative analysis by HPLC, 0.1
6 μmol of p-hydroxybenzaldehyde was observed.

The HPLC measurement conditions were the same as in Example (2) a.

Example (5) Production of dihydroxybenzaldehyde It was carried out using 1 μmol of astridine as a substrate under the same conditions as in Example (2) a. As a result of quantitative analysis by HPLC, 0.026 μmol of dihydroxybenzaldehyde was found. The HPLC measurement conditions were the same as in Example (2) a.

Claims (2)

(57) [Claims] 1. A compound represented by the following general formula [I] In the formula, R ₁ is a linear or branched alkyl group having 1 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, a cycloalkyl group having 5 to 15 carbon atoms, a group —COOR ¹ (wherein R ¹ is hydrogen. Atom or carbon number 1
~ 10 alkyl groups), formyl groups or However, the alkyl group, the cycloalkyl group and the aryl group may have a substituent, R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are the same or different, and each is a hydrogen atom or a hydroxyl group. Or its glycosides, carbon number 1-10
Or a linear or branched alkyl group of 1 to 10 or an alkoxy group having 1 to 10 carbon atoms, or two groups adjacent to each other are combined to form CH _{2 n} , —OCH ₂
(where n is an integer of 1 to 4) _n or -OCH _{2 n} O-may form, in a styrene derivative represented, the following physicochemical properties and produced by a bacterium belonging to Shiyudomonasu genus : (A) Action and Substrate Specificity It has the action of selectively acting on the ethylene bond conjugated to the benzene ring in the styrene derivative represented by the above general formula [I] to convert it into an aldehyde. (B) Optimum pH and stable pH range Optimum pH: 6 to 10 Stable pH range: 6.5 to 9.5 (c) Optimum temperature range of pH 8.0 to 20 to 50 ° C (d) Deactivation condition pH 5 or less or Deactivates at a temperature of 12 or higher. Deactivates at a temperature of 60 ℃ or higher. (E) Molecular weight The molecular weight measured by SDS-PAGE electrophoresis is 52,00.
0, and the molecular weight determined by the gel filtration method is 94,00.
0 (f) Inhibition, activation and stabilization The enzyme activity after adding each inhibitor to the enzyme reaction solution and reacting at pH 8.0 and 50 ° C for 10 minutes is the enzyme activity when no inhibitor is added. Is shown as relative activity with A method for producing aldehydes, which comprises contacting a dioxygenase having a salt with an aerobic condition. 2. The method according to claim 1, wherein the strain is TMY1009 strain.