JPH02200192A - Production of aldehydes - Google Patents

Abstract

PURPOSE:To make it possible to readily and selectively obtain aldehydes by bringing a specific styrene derivative into contact with a dioxygenase-active enzyme derived from bacterial strain of the genus Pseudomonas in aerobic conditions. CONSTITUTION:A cultured product obtained by culturing a bacterium TMY 1009 strain (FERM P-10362) of the genus Pseudomonas is separated and purified to afford a dioxygenase-active enzyme. Then a styrene derivative expressed by formula I [R1 is 1-10C straight-chain or branched alkyl, 6-10C aryl, 5-15C cycloalkyl, the group -COOR<1> (R1 is H or 1-10C alkyl), formyl, group expressed by formula II, provided that the above-mentioned alkyl, cycloalkyl and aryl may have substituent groups; R2-6 are mutually same or different and R, OH or glycoside thereof, 1-10C straight chain or branched alkyl or 1-10C alkoxy or two groups thereof which are adjacent each other are groups expressed by formula III (n is 1-4), formula IV or formula V] is subjected to catalytic reaction with the above-mentioned enzyme at 20-50 deg.C and pH6-10 under aerobic conditions to provide aldehydes such as vanillin.

Description

[Detailed description of the invention] <Industrial application field> The present invention relates to a method for producing aldehydes.

More specifically, the present invention uses an enzyme produced by a specific bacterium belonging to the genus Pseudomonas to selectively and oxidatively oxidize the ethylene bond conjugated to the benzene ring in the styrene derivative. This invention relates to a method for decomposing aldehydes to produce aldehydes.

<Prior Art> To the best of the present inventor's knowledge, it has not been known to selectively and oxidatively decompose the ethylene bond conjugated to the benzene ring in a styrene derivative using an enzyme to produce aldehydes.

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

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

According to the research of the present inventors, the object of the present invention is to provide the following general formula [I], where R 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' (where R' is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms), a formyl group, a cycloalkyl group and an aryl group have a substituent. You can leave it there.

R3, R8, R4, R1 and R6 are the same or different and each represents a hydrogen atom, a hydroxyl group or a glycoside thereof, a linear or branched alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms. The two groups shown or adjacent to each other are (CHz'+-1-o +
CH! ) or -0+CH,)O-n
It may be n (here, n represents an integer of 1 to 4).

A method for producing aldehydes characterized by bringing a styrene derivative represented by the following into contact with an enzyme produced by a strain belonging to the genus Pseudomonas and capable of producing an enzyme with dioxygenase activity against styrene derivatives under aerobic conditions. Ru.

According to the present invention, the styrene derivative [I] is treated with an enzyme produced by a strain of Pseudomonas that is capable of producing an enzyme having dioxygenase activity against the conjugated ethylene bond of the benzene ring in the styrene derivative. The ethylene bond in derivative [11] is oxidatively decomposed and converted to an aldehyde group. This is schematically represented by the reaction formula as follows.

O!・'-・CHO+OHC~ ・-・CH-CH~-> Thus, in the present invention, dioxygenase activity is
This refers to the action in which the ethylene bond conjugated to the benzene ring in the styrene derivative is selectively decomposed by an oxygen addition reaction and converted into two aldehyde groups as shown in the above reaction formula.

As far as the present inventors know, it is a completely new finding that ethylene bonds in styrene derivatives undergo the dioxygenase activity described above by enzymes produced by strains of the genus Pseudomonas.

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

Some of the reaction examples are shown below.

Preferred examples of the styrene derivative of the present invention represented by the above general formula [I] include R,, R,, R3, R, and R.
, is as explained below.

R1 is a straight chain or branched alkyl group having 1 to 6 carbon atoms; an aryl group having 6 to 8 carbon atoms, especially a phenyl group;
A cycloalkyl group having 5 to 10 carbon atoms, especially a cyclohexyl group; -GOOR' (where RI is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms [vanillin]) According to the method of the present invention, in the reaction example (2), As shown, when the raw material styrene derivative contains the group -0C1u, the group -0GQu in the generated aldehydes is
When glycosidase is contained in the enzyme isolated from the culture product of the bacterial strain, this group 0GQu may be obtained in the form converted to a hydroxyl group, which is believed to be due to its action.

preferable. Further, the above alkyl group and aryl group may be substituted with a hydroxyl group or a glycoside thereof, or an alkoxy group having 1 to 4 carbon atoms.

On the other hand, R3, R8, R1, R1 and R6 are the same or different, hydrogen atom; hydroxyl group or glycoside thereof; alkyl group having 1 to 6 carbon atoms, especially 1 to 3 carbon atoms; carbon number 1 to 6;
6, especially 1 to 3 alkoxy groups; R1 and R, or R4
and R, are preferably rings that combine with each other to form -0CR1O-.

In the compound of the above formula [I], one in which 1 to 2 of R3-R1 are substituted with a hydroxyl group, a glycoside thereof, or an alkoxy group having 1 to 3 carbon atoms is particularly preferable.

In the present invention, the following can be exemplified as specific examples of the styrene derivative [I] that can be decomposed using the enzyme having dioxygenase activity.

In the following formula, Me represents a methyl group, and the group 0C1u represents a glucosyl group.

Isoeugenol Coni 7Eryl Alcohol Insa 70-Ruin Cavicol Anethole 0Me Cinavir Alcohol (P-Propenylcatechol) P-Coumaryl Alcohol Ma MeO Asalon Me Syringen OMe Me HO” Inlabotenin Abiol Diphenyl) Ethylene Sylaviolゝ'c-c Next, in the method of the present invention, the enzyme having the dioxygenase activity used for the oxidative decomposition of styrene derivatives represented by the general formula [I], the Pseudomonas strain that produces the enzyme, and the acquisition of the enzyme Explain the method.

The enzyme having dioxygenase activity used in the method of the present invention is produced by bacteria belonging to the genus Pseudomonas and has the following physical and chemical properties.

(a) Action and substrate specificity: This dioxygenase is conjugated to the benzene ring in the styrene derivative represented by the above general formula [I] and has the action of selectively acting on the ethylene bond and converting it into aldehydes.

011) Optimal pH and stable pH range; optimal pH 6~
10 Stable pH range 6.5-9.5 (c) Suitable temperature range for action; 20-50°C at pH 8.0 (d) Deactivation conditions; pH: 5 or less or 12 or more, temperature -60°C or more (e) (f) Molecular weight: The molecular weight measured by 5DS-PAGE electrophoresis was about 52,000. Moreover, the number of molecules 1 determined by gel filtration was about 94,000. Therefore, this dioxygenase is considered to be a dimeric enzyme consisting of identical subunits with a molecular weight of about 52,000.

Purification method: Crude enzyme solution is treated with hydroxylapatite and purified by a combination of ion exchange chromatography (DEAE, Toyovar) and hydrophobic chromatography (Putyl Toyovar).

M) Inhibition, activation, and stabilization; The following inhibitors were added to the enzyme reaction solution to examine their effects on enzyme activity. Enzyme reaction conditions are pH 8, 0, 1 at 50°C.
0 minutes. Inhibition was evaluated by relative activity, with the case where no inhibitor was added being set as 100.

KCN l
93Na-azide l
97 Dipyridile
l 97 Ascorbate
8 77 In the present invention, the dioxygenase has the above activity and is produced by a strain belonging to the genus Pseudomonas, and particularly preferably,
This enzyme is produced by the TMY I O09 strain of bacteria belonging to the genus Pseudomonas.

This bacterial strain TMY1009 of the genus Pseudomonas has been deposited with the National Institute of Fine Arts and Technology under the title No. 10362.

The bacterium TMYIO09 strain is new and has the following mycological properties.

ω-亙厘■, Cell shape and size: Bacillus, 0.8X1. lpm2,
Presence of motility and epiphytic status of flagella, 1 polar flagellum 3, Durham staining: negative Growth status on various media ■ LB medium plate culture round, smooth colony formation, yellowish brown■ Flesh liquid culture Colonies are round, The surface was smooth, the color was pale yellow, and there was a gloss ■ The surface of the broth agar slant culture was smooth, the color was a pale yellow, and there was a gloss, and no pigment diffusion was observed.

■ No growth was observed on the surface of the meat juice liquid culture solution. The entire liquid was slightly cloudy. Bacterial cells had settled at the bottom.

■ Meat juice gelatin puncture culture Small borders and no gelatin liquefaction were observed throughout.

■ There was no sedimentation of bacterial cells at the bottom of the lidmus milk, no coagulation or liquefaction of the milk, and no change in pH.

Hook Physiological properties (1) Nitrate reduction: negative (2) Pigment production Dicarotenoid maximum absorption 479.45
0 (425) nm (3) Oxidase: Positive (4) Catalase: Positive (5) Attitude towards oxygen: Aerobic (6) O-F test Organic acid production under oxidative conditions ω Denitrification reaction: Negative (8) MR test: Negative (9) VP test: Negative (10) Indole production: Negative (11) Hydrogen sulfide production: Negative (12) Starch hydrolysis: Positive (13) Utilization of citric acid Koser's medium: Negative Chvistensen Medium: Positive (14) Utilization of inorganic nitrogen sources Nitrate: Negative Ammonium salt: Positive (15) Urease: Negative (16) Generation of acid and gas from sugars Acid production ■L-arabinose + ■D-xylose + ■D- Glucose + ■D-mannose + ■D-fructose + ■D-galactose + ■maltose 10■sucrose 10■lactose + [phase] Trehalose + gas production ■D-sorbitol ■D-mannite@inozit ■glycerin ■starch (17) Other characteristics ■ Oxidation of gluconic acid: negative ■ Subdivision of arginine: negative ■ Decarboxylation of lysine: negative ■ Decarboxylation of ornithine: negative ■ Decomposition of protocatechuic acid Dimeta cleavage (18) Other Tween Decomposition Tween 40: Negative Tween 60: Negative Tween 80: Negative DNase: Positive 3-keto-lactic acid production: Negative trowel-is town! Temperature 20-33°C pH 5,5-IO Doubling Time 27°C LB medium (pH
7,4) 2,5 hrs V Assimilation of carbon compounds Catecholbenzenebenzoic acid ferulic acid Tenp-fumaric acid Tenprotocatechinic acid Tenbarahydroxybenzoic acid Tenvanillic acid Sodium tenglutamate + Belongs to the genus Pseudomonas in the present invention In order to obtain the desired enzyme from a strain capable of producing an enzyme having dioxygenase activity, particularly the strain TMY I OO9, the bacterial cells are grown in a medium in which a known strain of the Pseudomonas genus grows. The enzyme may be separated from the culture obtained by culturing.

Hereinafter, the composition of a medium that can be used to grow the above-mentioned bacterial strain and the enzyme extraction method will be described, but this is merely for the sake of explanation, and the present invention describes the medium with this composition and the enzyme extraction method. It is not limited to.

Examples of carbon sources include carbohydrates such as glucose and fructose, organic compounds such as ethanol, and organic acids such as succinic acid. can be used as a carbon source.

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

Moreover, various phosphates, magnesium sulfate, etc. can be used as inorganic salts. In addition, trace amounts of metals (iron salts,
Calcium salts, etc.) may be included in the medium.

The culturing method may be a shaking culture method, a deep aeration agitation culture method, or the like. The culture temperature is preferably in the range of, for example, 20'' to 33°C, and the pH is preferably in the range of about 6 to IO.Although the number of days of culture is not particularly limited, it is usually carried out, for example, in the range of 1 to 7 days.

In order to separate the cell-free extract obtained by destroying the bacterial cells in the culture in which enzymes have been produced and accumulated by ultrasonication, and to remove low-molecular substances from the obtained cell-free extract. An enzyme solution is obtained by dialysis with a sodium phosphate buffer.

Thus, in the method of the present invention, the enzyme solution itself or the enzyme purified from the enzyme solution is brought into contact with the styrene derivative of the general formula [1] under aerobic conditions, thereby obtaining the target aldehyde. becomes possible.

The temperature at that time is 20-50℃, preferably 30-40℃
Advantageously, the pH ranges from 6 to 1O1, preferably 6.5.
A range of ~9.5 is desirable.

Further, the reaction may be carried out either batchwise or continuously, and the enzyme may be used in an immobilized state.

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

Example (1) Preparation of enzyme solution If (7) Nutrient medium (LB) "c'TMy l O
About 3.5 g of wet bacterial cells were obtained by culturing O9 strain aerobically at 27° C. with shaking for about 22 hours. The bacterial cells were collected, washed, suspended in 15 ml of 50 mM sodium phosphate buffer, destroyed by ultrasonication, and centrifuged. f: (10,ooOXg, 20mln)
. 20 to remove low molecular weight substances in the obtained cell-free extract.
Dialysis was performed with w+M sodium phosphate buffer (pH 7.5) to obtain a crude enzyme solution (protein concentration: l 5.4
mg/m(1).

Example (2) Vanillin production method a, enzyme solution (15.4 mg/raQ) in 450 μα buffer (pH 7.5) containing isoeugenol 1 praoQ
) and incubate at 26°C for 30 minutes. After adding 50 μa of IN H (l) to make it acidic,
Extract with 00 μa ethyl acetate. As a result of quantitative HPLC analysis of the extract, 0.16 μmoa of vanillin was detected.

HPLC measurement conditions Model; 5hin+adzu L C-6A column; LiChrosorb RP-18 (4,6X25
Oram) Elution; 0.05% phosphoric acid water (A)/acetonitrile (
B) -70/30 --- (5 minutes") --- (A)
/ (B) -70/30---(increasi
ng 5% per m1n)-(A)/(B)-0/
100 The retention time of vanillin in the above is 6.1 minutes.
As a result, O, 16*mod vanillin was observed. 3,5-dihydroxybenzaldehyde was also observed.

Example (3) Preparation of vanillin 8.5-[2'-(4''-Hydroxy-31-methoxyphenyl)vinyl]7 erulic acid 1.2 μa IOQ containing 950 μa sodium phosphate buffer (pH 7.5)
Enzyme solution (15,4+++g/wQ) 50 ttQ was added to the mixture and incubated at 27°C for 30 minutes. oxygen 1ptmo
The reaction product was extracted with 1000 μa of ethyl acetate and quantitatively analyzed by HPLC. As a result, 1 μ+10Q of vanillin and 1 μ-Oa of 5-formylferulic acid were observed. The HPLC measurement conditions were the same as in Example (2) a.

b, 1,2-bis-(
4-hydroxy-3-methoxyphenyl)ethylene lp
react using moQ, oxygen 0.54. The reaction was stopped when umoI2 was consumed. This reaction product is 100
As a result of extraction with 0 μg of ethyl acetate and quantitative analysis by HPLC, vanillin of 1.1 μm〇α was observed.

Example (4) Production of p-hydroxybenzaldehyde Under the same conditions as in Example (2) a, p-hydroxybenzaldehyde was used as a substrate.
It was performed using μl1OQ. As a result of quantitative analysis by HPLC, p-hydroxybenzaldehyde of 0.16 μm O+2 was observed.

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

Example (5) Production of dihydroxybenzaldehyde This was carried out under the same conditions as in Example (2) a using astridine 1 μmoQ as a substrate. As a result of quantitative HPLC analysis, 0.026 μraoQ of dihydroxybenzaldehyde was observed. The HPLC measurement conditions are as in Example (2).
It was carried out under the same conditions as in a.

Claims (1)

[Claims] 1. The following general formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] In the formula, R_1 is a straight chain or branched alkyl group having 1 to 10 carbon atoms, or a straight chain or branched alkyl group having 6 to 10 carbon atoms. aryl group, carbon number 5-15
cycloalkyl group, group -COOR^1 (where R^1 is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms), formyl group or group ▲ formula,
There are chemical formulas, tables, etc. Showing ▼. However, the above alkyl group, cycloalkyl group and aryl group may have a substituent. R_3, R_3, R_4, R_5 and R_6 are the same or different and each represents a hydrogen atom, a hydroxyl group or a glycoside thereof, a linear or branched alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms. The two groups shown or adjacent to each other are ▲mathematical formula, chemical formula,
There are tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (Here, n indicates an integer from 1 to 4). A method for producing aldehydes, which comprises bringing a styrene derivative represented by the following formula into contact with an enzyme produced by a strain belonging to the genus Pseudomonas and capable of producing an enzyme with dioxygenase activity against styrene derivatives under aerobic conditions. 2. The production method according to claim 1, wherein the bacterial strain is TMY1009 strain.