JPS6317437B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing all-trans polyprenyl alcohol using microorganisms. More specifically, the present invention is based on the general formula of bacterial cells obtained by culturing bacteria belonging to the genus Pseudomonas. The present invention relates to a method for collecting all-trans polyprenyl alcohol represented by the formula (where n is an integer of 9 to 11). The general names of all-trans polyprenyl alcohol represented by the general formula are nonaprenol (n=9) and decaprenol (n=9), respectively.
10) and undecaprenol (n=11), which is an important raw material for the synthesis of coenzyme Q. Among the above substances, for example, nonaprenol (also known as solanesol) is known to exist in plants such as tobacco leaves, but it is difficult to obtain raw materials in large quantities. In this respect, the production method using microorganisms can be an economical method. Regarding microorganisms, all-trans decaprenol and undecaprenol have been detected from Bacillus acidocardalius, and Micrococcus lysodeikticus.
There is a known method for producing all-trans nonaprenol using the enzyme obtained. However, in the former case, the culture conditions for the bacteria are different from normal culture conditions, with an optimum pH of 2.6 to 2.8 and an optimum temperature of 58 to 58.
60°C, which is not preferable for fermentation production on an industrial scale. Furthermore, the latter method requires special operations such as enzyme purification, and is far from being able to produce all-trans polyprenyl alcohol on an industrial scale. An object of the present invention is to provide a method for easily obtaining all-trans polyprenyl alcohol using bacteria of the genus Pseudomonas. The culture medium used in the present invention is not particularly limited. For example, as carbon sources, hydrous carbon such as glucose, organic acids such as citric acid, alcohols such as methanol and glycerin can be used, and as nitrogen sources, ammonium sulfate, urea, ammonium nitrate, ammonium phosphate, ammonium chloride,
Ammonia gas, etc. can be used, inorganic substances such as phosphates, magnesium salts, iron salts, manganese salts, and other trace metal salts as necessary, and growth-promoting substances such as amino acids, vitamins, soybean protein hydrolysate, yeast extract, etc. Peptone, casamino acid, etc. can be used. For culturing, culture is carried out aerobically with shaking or stirring at pH 5-8 and 20-40°C for about 2-10 days. Next, the bacterial cells are separated from the culture solution by conventional methods such as centrifugation or filtration. The bacterial cells that are the starting material for the separation of all-trans polyprenyl alcohol can be any of live bacterial cells, dried bacterial cells, or processed bacterial cells. All-trans polyprenyl alcohol can be extracted from bacterial cells by extraction with an organic solvent such as methanol, ethanol, or acetone, or by adding a mixture of methanol, caustic soda, and pyrogallol to a liquid containing bacterial cells, and heating at 60 to 90°C.
A method such as heating extraction for 1 to 2 hours can be adopted. The extracted all-trans polyprenol can be purified by silica gel column chromatography, high porous polymer column chromatography, or the like. In the present invention, the bacteria are cultured at an appropriate temperature (20 to 40°C) and pH (PH5 to 5) for industrial scale culture.
8), and the produced all-trans alcohol can be easily extracted using an organic solvent or the like. Therefore, the method of the present invention does not require any special operations and is suitable for industrial production purposes. The mycological properties of representative strains suitable for use in the method of the present invention are as follows.

[Table] Um, sodium sulfate
um, sodium sulfate

[Table] Acetic acid, propionic acid,
Serine, gut, maleic acid, oxalic acid, apple
Lysine, formaldehyde, beric acid, propionic acid, glycine,
p-hydroxybenzoic acid omaldehyde, propionia

Rudehyde, benzene, lactose The mycological properties of Pseudomonas N842-M16 strain are similar to Pseudomonas except that it produces less carotenoid than Pseudomonas N842.
It is almost the same as N842. Examples will be shown next, but the present invention is not limited thereto. Example 1 Pseudomonas N842-M16 (Feikoken Bacterial Serial No. 3155) was added to an aqueous medium (PH6.5) containing 2% glucose, 1% peptone and 1% yeast extract.
) was cultured for 48 hours in 300 ml of a medium with the same composition as a seed, and cultured with stirring at 30°C for 4 days using a 30-volume jar fermenter with 15 air per minute. A wet bacterial cell paste was obtained by centrifugation. Wet bacterial cells from four jar fermenters were combined and dried with a spray dryer to obtain 320 g of dried bacterial cells. The bacterial cells were extracted twice with acetone 3 with stirring at 60°C for 1 hour. Acetone was distilled off under reduced pressure, and the residue was dissolved in hexane. This solution is developed using an ether/n-hexane mixture using a silica gel column. ether/
The coenzyme Q fraction was eluted with n-hexane (10:90), and the all-trans polyprenol fraction containing nonaprenol, decaprenol, and undecaprenol was eluted with ether/n-hexane (15:85). The solvent was distilled off from this all-trans polyprenol-containing fraction, and the resulting residue was added to a small amount of n
- Dissolve in hexane, leave the solution in the cold and remove the precipitated crystals by filtration. The remaining 2-hexane solution was concentrated and further purified by thin layer chromatography using benzene/dichloroethane/acetone (48.25:48.25:3.5) as a developing solvent to obtain a mixture of all-trans nonaprenol, decaprenol, and undecaprenol. Obtained 64 mg. The obtained all-trans polyprenol was further mixed with acetone/
The product was purified by reverse phase thin layer chromatography using water (90:10) as a developing solvent to obtain 1 mg of all-trans nonaprenol, 45 mg of all-trans decaprenol, and 5 mg of all-trans undecaprenol. Identification of each compound was performed by thin layer chromatography using benzene/dichloroethane/acetone (48.25:48.25:3.5) as the developing solvent and acetone/acetone/
The behavior, NMR, IR, and mass spectra on reversed-phase thin layer chromatography using water (90:10) as the developing solvent were analyzed for standard all-trans nonaprenol (solanesol), all-trans decaprenol, and all-trans undeca. This was done by direct comparison with those of prenol. Example 2 Using the same medium as in Example 1, Pseudomonas
N842 (Feikoken Bacterial Serial No. 3154, JP-A-52-47990 (Japanese Patent Application No. 122-452)) was cultured and treated in the same manner as in Example 1 to obtain 300 g of dry bacterial cells. Ta. All-trans polyprenol was extracted and purified from the dried bacterial cells in the same manner as in Example 1 to obtain 0.05 mg of all-trans nonaprenol, 10 mg of all-trans decaprenol, and 0.1 mg of all-trans undecaprenol. Example 3 Pseudomonas C1-36 (Feikoken Bacteria Serial No. 5209,
Japanese Patent Application Laid-Open No. 56-51986 (Patent Application No. 124727-1987)
) was cultured and treated in the same manner as in Example 1 to obtain 330 g of dried bacterial cells. All-trans polyprenol was extracted and purified from the dried bacterial cells in the same manner as in Example 1 to obtain all-trans nonaprenol 1.
65 mg of all-trans decaprenol and 7 mg of all-trans undecaprenol were obtained.

Claims (1)

[Claims] 1. General formula (wherein n is an integer from 9 to 11) is obtained from bacterial cells produced by culturing in a medium a bacterium belonging to the genus Pseudomonas that has the ability to produce all-trans polyprenyl alcohol represented by the general formula 3. 1. A method for producing all-trans polyprenyl alcohol, which comprises collecting different types of all-trans polyprenyl alcohol individually or as a mixture.