JPH0767669A - Production of natural type beta-phenethyl alcohol - Google Patents

Abstract

PURPOSE:To efficiently product beta-phenethyl alcohol by treating a styrenic compound with a bacterium belonging to the genus Corynebacterium, having ability of decomposing a styrenic compound. CONSTITUTION:A bacterium belonging to the genus Corynebacterium, having ability of decomposing a styrenic compound is inoculated into a medium containing a nitrogen source, an inorganic substance, etc., and a styrenic compound, etc., together with air is supplied to the medium. The cells are cultured at pH 5-8 at 25-35 deg.C for 48-72 hours. After the culture, styrene oxide is dissolved in an nonvolatile solvent such as n-hexadecane, shaken or stirred together with a cell suspension and extracted with the solvent to efficiently produce beta- phenethyl alcohol.

Description

Detailed Description of the Invention

[0001]

[Industrial application] β-phenethyl alcohol is currently produced as a rose-like fragrance in countries around the world at about 5000 tons per year, and is widely used as a versatile fragrance in detergents, foods, cosmetics and the like. However, as a natural fragrance, only a very small amount is used as a raw material for perfume and the like, and β-phenethyl alcohol is required as a natural fragrance.

[0002]

2. Description of the Related Art Currently, synthetic β-phenethyl alcohol is used which is produced as a by-product of the catalytic reduction method of styrene oxide or the petrochemical industry. However, such a method has a problem that the purification is very complicated. In addition, these products are chemically synthesized products and cannot be used as natural flavors. In addition, β present in plants
-Phenethyl alcohol is in very small quantities, cultivation, extraction,
It takes time and labor for refining, and it is hard to establish as an industry.
On the other hand, regarding the production of β-phenethyl alcohol by microorganisms, it was reported that β-phenethyl alcohol was detected as a styrene metabolite (Appl.Environ.Microbi
ol., 35,124 (1978)) and a method for producing β-phenethyl alcohol from styrene in Pseudomonas bacteria (Japanese Patent Publication No. 61-5394, Agric.Biol. It hasn't arrived yet.

[0003]

In the above-mentioned production of β-phenethyl alcohol, when fermentation production is carried out using a microorganism, the product is regarded as a natural flavor. Therefore, it has been desired to establish a technique for efficiently producing β-phenethyl alcohol from styrene or styrene oxide using a microorganism. In addition, in the case of using the Pseudomonas bacteria, the production amount is not always sufficient.

[0004]

Against this background, the present inventor has diligently searched for microorganisms that metabolize styrenic compounds, and newly discovered a bacterium of the genus Corynebacterium that metabolizes styrenic compounds. Furthermore, based on this discovery,
Method for decomposing and removing styrene compounds by microorganisms (Japanese Patent Application No. 4
No. 244444) and styrene oxide reductase and a production method (Japanese Patent Application No. 4-244445).
It was revealed that the styrene-assimilating Corynebacterium bacterium has the ability to convert styrene to β-phenethyl alcohol by styrene oxide and styrene oxide reductase (Japanese Patent Application No. 4-24444).
No. 5). Therefore, the present inventor has found that β-
By further searching for a microorganism having a high ability to produce phenethyl alcohol, it was discovered that some Corynebacterium bacteria produce β-phenethyl alcohol from styrene oxide, and completed the present invention.

The microorganism used in the present invention belongs to the genus Corynebacterium, has an ability to decompose styrene or styrene oxide, and can produce any β-phenethyl alcohol from styrene oxide. Also used in things. Of these, particularly preferred is a new bacterium A of the genus Corynebacterium.
C-5 strain, the same ST-5 strain (Microtechnology Research Institute, No. 13151), Corynebacterium pseudodifferiticum (Coryne
bacterium pseudodiphteriticum) ST-21 strain (Microtechnological Research Institute No. 13149), subspecies ST-10 strain (Microtechnical Research Institute No. 13)
No. 150).

Further, styrene oxide reductase can be obtained by irradiating these microorganisms with ultraviolet rays or treating them with a mutagenizing agent such as NTG (N-methyl-N'-nitro-N-nitrosoguanidine) or EMS (ethanemethanesulfonic acid). A mutant strain which enhances the activity or blocks the metabolism of the produced β-phenethyl alcohol can also be used.

The above-mentioned strains were newly isolated from the soil by the present inventor, and the above-mentioned patent (Japanese Patent Application No. 4-24).
No. 4444). The mycological properties of the new strain Corynebacterium sp. AC-5 and the deposit number to the National Institute of Advanced Industrial Science and Technology are as follows. [Strain AC-5] I. Cell morphology: Heteromorphic bacillus 1 to 1.5 μm × 5 to 15 μm Motility: None Gram stain: Positive Spore formation: None II. Growth state in medium (1) Standard agar plate medium Colony is dot-shaped, convex surface Shape, full-edged and ivory color. (2) Standard liquid medium No powdery precipitate or film is formed. III. Physiological Properties Catalase: Positive Cytochrome Oxidase: Negative Pigmentation: Not generated (King A and King B agar). OF test: acid production from undegraded sugar: glucose-xylose-maltose-lactose-mannitol-sucrose-starch-development: 5 ° C-37 ° C-41 ° C − MacConkey − Agar − Nitrate reduction − Urea decomposition + casein decomposition − Esculin decomposition − TSI − / − Oxygen demand obligatory aerobic From the above mycological properties, the barge manual (Bergey's
Manual of Determinative Bacteriology) 8th edition found that this bacterium was a new species of Corynebacterium. Therefore, this strain was identified as a new strain of Corynebacterium AC-5.
It was a stock. Then, this fungus was deposited at the Institute of Biotechnology, Institute of Biotechnology on August 19, 1993, and the deposit number is FERM P-1.
3807.

For culturing the microorganisms used in the present invention, if the medium contains a carbon source, a nitrogen source, an inorganic substance, etc., a synthetic medium,
Any natural medium can be used. As the carbon source, it is desirable to use a styrene compound since styrene oxide reductase that produces β-phenethyl alcohol is an inducing enzyme. Styrene, styrene oxide, etc. can be used as such a compound. The volatile styrene compound is sequentially added to the medium together with air. As the nitrogen source, natural media such as yeast extract, peptone and meat extract, ammonium sulfate, ammonium chloride and the like are used. As inorganic substances, potassium, sodium,
Metal salts such as magnesium, iron and zinc are optionally used. The culture temperature may be any temperature within the range in which the bacteria grow and β-phenethyl alcohol is produced, but it is preferably 25 to 35 ° C. Medium pH is usually 5
It is performed in the range of ~ 8. The culture time may be selected so that β-phenethyl alcohol can be most efficiently produced from styrene oxide, and is usually 48 to 72 hours.

Since the culture obtained as described above exhibits a high styrene oxide reductase activity, β-phenethyl alcohol can be produced using this culture. Usually, styrene oxide is easily decomposed into styrene glycol in an aqueous reaction. To prevent this decomposition, styrene oxide is dissolved in a non-volatile solvent such as n-hexadecane and shaken or stirred with the cell culture. Then, the reaction is carried out by bringing them into contact with each other. The reaction temperature is usually preferably 20 to 30 ° C., and the reaction time may be any as long as β-phenethyl alcohol is produced from styrene oxide, but it is preferably 48 to 96 hours.

After the reaction, the accumulated β-phenethyl alcohol can be purified by fractionating the reaction solution. If necessary, it can be purified by appropriately combining methods such as solvent extraction, dehydration, decolorization and column chromatography. According to the present invention, a natural β-phenethyl alcohol can be efficiently produced by a microbial reaction using a styrene compound as a raw material, which is an epoch-making method for producing a natural flavoring agent.

[0011]

The present invention will be described in detail with reference to the following examples. Example 1 0.3% ammonium sulfate, 0.3% monopotassium phosphate, 0.1% NaCl, 0.
On the surface of the agar medium containing 02 percent of MgSO ₄ · 7H ₂ O and 0.01% of yeast extract (pH 7.0) Corynebacterium new bacteria AC-5
The strain was inoculated on the entire surface and cultured at 30 ° C. for 72 hours. Styrene, which is an inducer of styrene oxide reductase and a carbon source,
A small Durham tube was charged with 200 μl and supplied in a gas phase system. After culturing, the cells were scraped off from the surface of the agar medium, suspended in 50 mM phosphate buffer (pH 7.0), and collected by centrifugation. The cells of two petri dishes were resuspended in 8 ml of 20 mM phosphate buffer (pH 7.0), and 2 ml of n-hexadecane, 60 μl, was added to this culture.
Styrene oxide was added, and the mixture was sealed in a 50 ml Erlenmeyer flask and then reacted at 30 ° C. for 3 days under shaking (130 times / min) conditions. After the reaction, the purified product was extracted with 10 ml of ethyl acetate and quantified by gas chromatography. As a result, it was confirmed that 18 μg of β-phenethyl alcohol was produced.

Example 2 The same operation as in Example 1 was carried out using the cells of 5 petri dishes, and it was confirmed that 40 μg of β-phenethyl alcohol was produced.

Example 3 Using a new Corynebacterium genus ST-5 strain, Example 2
When operated in the same manner as in, production of 25 μg of β-phenethyl alcohol was confirmed.

Example 4 A medium 15 obtained in Example 1 with a yeast extract concentration of 0.1%.
1 ml of Corynebacterium sp. AC-5 strain was inoculated into 1 ml and added to a previously sterilized porous cellulose carrier (about 3 mm square, pore size 100 µm, manufactured by Biomaterials). Using this medium-containing cellulose carrier, culturing was performed in the same manner as in Example 1 using a Durham tube. The cells grew well on this medium-containing cellulose carrier. 80 ml of buffer solution, 20 ml of n-hexadecane and 1000 μl of styrene oxide were added to the obtained culture, and the reaction was carried out in the same manner as in Example 1 in a 300 ml Erlenmeyer flask. The reaction solution was extracted with ethyl acetate, concentrated, and then purified by silica gel chromatography and concentrated under reduced pressure to completely remove the solvent.
200 μg of viscous β-phenethyl alcohol with rose-like aroma was obtained. The purity of this liquid was 99% by gas chromatography analysis. In addition, the NMR spectrum of this compound was completely in agreement with the authentic β-phenethyl alcohol.

[0015]

INDUSTRIAL APPLICABILITY According to the present invention, natural β-phenethyl alcohol can be efficiently produced by a microbial reaction using a styrene compound as a raw material.

Claims (1)

[Claims] 1. A microorganism having the ability to decompose styrene compounds belonging to the genus Corynebacterium or a culture thereof is brought into contact with styrene oxide, and β is converted from styrene oxide.
-Natural β- characterized by converting to phenethyl alcohol and obtaining β-phenethyl alcohol produced
Method for producing phenethyl alcohol.