JPS59232095A - Production of phenylacetic acid - Google Patents

Abstract

PURPOSE:To obtain phenylacetic acid from the cultured product, by culturing a microbial strain belonging to Streptomyces genus and capable of producing phenylacetic acid. CONSTITUTION:A microbial strain belonging to Streptomyces genus and capable of producing phenylacetic acid [e.g. Streptomyces lavendofoliae S26608 (FERM-P No.7077)] is cultured in a conventional nutrient medium aerobically at 20-33 deg.C, preferably 25-30 deg.C for 1-7 days, preferably 2-5 days, and the objective phenylacetic acid is separated from the cultured product. The amount of the phenylacetic acid can be increased remarkably by adding 0.5-5.0%, preferably 1.0 -3.0% of phenylalanine to the medium and carrying out the cultivation for >=2 days.

Description

[Detailed description of the invention] The present invention relates to a method for producing phenylacetic acid by fermentation.

Phenylacetic acid occupies an important position as an industrial raw material, and in addition to being produced by synthetic methods, it is also known that some microorganisms other than Streptomyces produce phenylacetic acid.However, these wax organisms The production amount of phenylacetic acid is extremely small; for example, in the case of Noeardla bacteria, for which production amount has been reported, the production amount was only 0.49 F per 1 medium (Appl.
Mlerobiol, s 9 e 383 (1961)
).

While conducting research on the production of phenylacetic acid by a fermentation method, the present inventor discovered that microorganisms belonging to the genus Streptomyces have the ability to produce phenylacetic acid, and that adding phenylalanine to the medium for culturing the microorganisms produces phenylacetic acid. They discovered that the production amount of acetic acid can be significantly increased, and completed the present invention.

That is, the present invention provides a method for producing phenylacetic acid, which is characterized by culturing phenylacetic acid-producing bacteria belonging to the genus Streptomyces and collecting phenylacetic acid from the culture.

Microorganisms that can be used in the present invention may belong to the genus Streptomyces and have the ability to produce phenylacetic acid. Good too. An example of a microorganism that belongs to the genus Streptomyces and has the ability to produce phenylacetic acid is Streptomyces lavendofoli, which was isolated from a soil sample collected in Shisui Town, Chiba Prefecture.
ae) 826608 strains are listed.

The mycological properties of this strain are as follows.

The branches of type 1 aerial hyphae are simple branches, and the tips are loop-like to spiral-like (Retlt+acult+m -ap@rtum
), and no axle branching is permitted. The mature conidia are 50 or more in chains, the shape of the spores is oval to cylindrical, and the size is 3-0, 7-0.8 x O, 9-1.3 pm. . The spore surface is smooth.

2 Growth status in each medium The growth status indicates the state when cultured in each medium at 27°C for 14 days, and the color tone is shown according to "Color Name Dictionary J (first edition, 1981)" published by Nippon Shikiken Business Co., Ltd. Ta.

Blank space below 4-3 Physiological properties (1) Growth temperature range Possible growth temperature 12-36℃ Optimum growth temperature 26-33℃ (2) Liquefaction of gelatin; positive (3) Hydrolysis of starch; positive (4) a Coagulation of membrane pores; positive Peptonization of skim milk, positive (5) Production of melanin-like pigments; positive (6) Reduction of nitrates; negative (7) Decomposition of cellulose: negative 4 Availability of disaster sources (Pridham-Gotzlieb Agar medium, 27° C., 14-day culture) From the above properties and the fact that LL-diaminopimelic acid is contained in all bacterial cells, it is clear that strain 826608 is a strain belonging to the genus Streptomyces. The properties of this strain are summarized as follows. The tips of aerial hyphae are loop-shaped to spiral-shaped (Re-tinaeulum -ip@rtum)
Fifty or more spores are chained together, and the spore surface is smooth. The color of the aerial mycelia is in the red color series, and the underside does not show any distinctive color and does not produce soluble pigments. Produces melanin-like pigment.

826608 strain was extracted from Waxman's ``The Actinomycetes J (The Aetlnomyet*s)''.
Volume 2 (1961), ISP report by Scherling et al.
Bacteriology” (International Journal of
8y■t@matieBacteriology)
Vol. 18, pp. 69, 279 (1968), Vol. 19, pp. 3
91 (1969), Vol. 22, p. 265 (1972) and [Bsrgerys Manual of Determinative Bacteriology J (BsrgerysM
annual of Determinative Ba
cteriology) 8th edition (1974), a search reveals that Streptomyces lapendophoriae (Str@ptomyees 1avsndo) is a closely related species.
follae ) Streptomyces goshikiensis (
S, goshlklensim), Streptomyces
S, vlrginims. Furthermore, in order to identify the species, we added the 826608 strain and the above 3 strains.
A comparative experiment with species was conducted under the same conditions. As a result, 82
6608 strain is Streptomyces Φ goshikiensis 18
P5190 differs from this strain in that it reduces nitrate, does not utilize L-arabinose and D-xylose, and has poor growth and formation of aerial mycelia on Shuk9-rose/nitrate agar and glucose/asparagine agar. , while Streptomyces virginiae x
sp s 094 has weak starch hydrolysis, lacks the use of L-arabinose and D-xylose, poor growth and aerial mycelium formation on sucrose/nitrate agar, and poor growth on glucose/asparagine agar. The bad things were different.

On the other hand, streptomycetes and lapendophoriae IBP 5
It differs from No. 217 in that wild boar was used and growth was poor on sucrose/nitrate agar medium, but the other properties were in good agreement. Therefore, 826608
The strain was determined to be a strain belonging to 10- Streptomyces lavendofoliae and was distinguished from known strains. For Streptomyces labendfoliae 826608 (8treptomyce
s Javsndofolla@826608) and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FEBMP-7077. .

The medium used for the production of phenylacetic acid is not particularly limited as long as it is used for culturing actinomycetes. Any commonly used nutrient medium can be used, and typical medium compositions are as follows.・ Nutrient medium
Glucose 1.0% glycerol 1. O
X Soluble starch 1.0% Polypeptone 0.5% Meat extract 0.5% Salt 0.5% Calcium carbonate 0.3% pH 7.0 ± 0.2 In addition, the culture conditions were aerobic as usual. For example, 20 to 33°C, preferably 25 to 3°C.
Incubate at a temperature of 0° C. for 1 to 7 days, preferably 2 to 5 days.

Even when phenylalanine is added to the medium, the medium composition and culture conditions other than phenylalanine are exactly the same as above. And phenylalanine 0.5-5.0%
, preferably "・0..~3..X,, □* *
Kf! When cultured using fsml, 2B, the amount of phenylacetic acid produced increases significantly.

Thus, the phenylacetic acid produced is the culture F? The extract is extracted with an appropriate solvent such as benzene, chloroform or ethyl acetate under acidic conditions, made basic by adding an alkaline aqueous solution to the extract, and transferred to the aqueous layer by stirring. Furthermore, the aqueous layer is extracted again under acidic conditions with a suitable solvent such as benzene, chloroform or ethyl acetate.
By dehydrating the extract and concentrating it under reduced pressure, a crude phenylacetic acid product can be obtained. By column purifying this using an appropriate developing solution, phenylacetic acid can be obtained as white crystals, and further, by recrystallization, white needle-like crystals can be obtained.

The white needle-like crystals that were present were identified as phenylacetic acid because the melting point and ultraviolet absorption spectrum and infrared absorption spectrum as shown in Figures 1 and 2 completely matched those of the standard substance of phenylacetic acid.

Next, examples will be given to illustrate the present invention. Example 1 Glucose 10f1 Glycerol 10f.

Soluble starch 10t1 polypeptone 5f, meat extract 5
Dissolve f, 5 f of common salt, and 3 t of calcium carbonate in 1 t of water. Dispense 60ml of this solution into a 500-capacity shake flask, and adjust the pH to 7.0 with 5N hydrochloric acid or 5N sodium hydroxide solution. This is sterilized at 121°C for 14-20 minutes and then rapidly cooled. Streptomyces labendoforiae strain 826608 (FERM,
One platinum loop of P-7077) was inoculated. The shake flask was incubated aerobically for 48 hours in a shaker at 27°C. Thereafter, the bacterial cells were removed by centrifugation, and the resulting separated liquid was acidified with hydrochloric acid (pH 2 to 3) and extracted twice with an equal amount of ethyl acetate. Next, separate the ethyl acetate layer, add 1/2 volume of 0.1N sodium aqueous solution,
After making it basic and stirring and transferring it to the aqueous layer, the aqueous layer was acidified with hydrochloric acid (p
H2-3) and extracted twice with an equal amount of ethyl acetate.

This extract was dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure at 40°C to obtain a crystalline substance. This crystalline substance was prepared with chloroform using 10 times the weight of silica gel (manufactured by Merck & Co., Ltd., Kieselgel 60) and applied to a column. Thereafter, by developing the product in chloroform, it was easily separated from other impurities and phenylacetic acid was obtained as white crystals. Furthermore, this white crystal was recrystallized from a mixed solution of benzene and hexane to obtain approximately 1209 highly pure phenylacetic acid as white needle-like crystals.

The melting points of the phenylacetic acid (C) obtained for confirmation, the standard phenylacetic acid (B), and the mixture of these (A+B)
) was investigated. The results are shown in Table 1.

Table 1 From these results, it is clear that the melting point decreases even when mixed.
It was confirmed that the phenylacetic acid obtained by the method of the present invention has high purity.

Example 2 Sterile nutrient medium 60- prepared in exactly the same manner as in Example 1
Phenylalanine 1.8 sterilized at 115°C for 30 minutes
Add 2. Thereafter, the cells were inoculated and cultured in exactly the same manner as in Example 1, and after 120 hours of culture, the bacterial cells were removed by centrifugation to obtain 1t of separated liquid. Again in Examples 1 and 17, phenylacetic acid was separated and purified in exactly the same manner as in Example 1 and 17.
00 tons of white needle-like crystals were obtained.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectra of the product of the present invention and the phenylacetic acid standard substance in methanol solution. In the figure,
Human shows the spectrum of the invention product, and B shows the spectrum of the standard substance. FIG. 2 shows the infrared absorption spectra of potassium bromide tablets of the product of the present invention and the phenylacetic acid standard substance. In the figure, person shows the spectrum of the invention product, and B shows the spectrum of the standard substance. Above 18-

Claims (1)

[Scope of Claims] 1. A method for producing phenylacetic acid, which comprises culturing phenylacetic acid-producing bacteria belonging to the genus Streptomyces and collecting phenylacetic acid from the culture. 2. The method for producing phenylacetic acid according to claim 1, wherein the culture is carried out in a phenylalanine-containing medium. 3. A phenylacetic acid-producing bacterium belonging to the genus Streptomyces is Streptomyces lapendophoriae (Strept.
myces 1avendofoliae) 826
608 strain, the method for producing phenylacetic acid according to claim 1.