JPH0588112B2 - - Google Patents

Description

[Detailed Description of the Invention] (Field of Industrial Application) POA is one of the unsaturated fatty acids that widely exist in the biological world. Recently, it has been reported that this substance protects against vascular disorders caused by hypertensive diseases (Japanese Patent Application Laid-open No. 175425/1983), and it has been revealed that this substance exhibits excellent pharmacological and physiological effects. Development for pharmaceutical use is regulated. (Prior art) As an industrial method for producing POA, it is known to extract POA-containing oil from plants such as macadamia nuts and rice bran, and fish such as whales and sardines. Little is known about fermentation production methods using microorganisms. Regarding POA in microorganisms such as yeast, there are research reports on species, species, fatty acid composition of bacterial lipid components under various culture conditions, membrane structure and function, and the influence of culture temperature on POA content in S. cerevisiae [ Biochim. Biophys. Acta, 260 , 639 (1972)], [J.
Biol.Chem., 254 , 12281 (1979)], Change in POA content under anaerobic and aerobic culture conditions [J.Brew.Soc.Japan,
72, 735 (1977)], [J.Cell.Biol, 37 , 221 (1968)
],
[Arch. Mikrobiol. 66 , 34 (1969)], [Arch.
Microbiol., 117 , 239 (1978)], fatty acid composition of cytoplasmic membrane and cell wall [Chem.Phys.Lipids, 4 , 247
(1970)], on the relationship between the structure and function of fatty acids [Exp.Mycol., 8 , 55 (1984)], on the relationship between cell membrane lipid composition and EtOH resistance [J.Inst.Brew., 82 ,
218 (1976)], and lipid composition under anaerobic and aerobic culture conditions in S. uvarum [J.Inst.Brew., 88 ,
367 (1982)], Effect of culture temperature on POA content [ASBC Journal, 40 , 26 (1982)], Effect of thiamine pyridoxine on fatty acid composition [Biochem.
Biophys.Res.Commun. 59 , 777 (1974)], and
Fatty acid composition in S. rouxii (Agriculture, 46 , 657
(1972)], but there are no reports on the collection of POA from microorganisms such as yeast. (Problem to be solved by the invention) Because the POA content in microorganisms such as yeast is low, it is extremely difficult to obtain high-purity, high-quality POA-containing oil or pure POA products, making it difficult to industrially produce POA. was considered disadvantageous. However, if a fermentation production method using microorganisms such as yeast is perfected, natural product-derived
Compared to POA-containing oils, it can be stably supplied at a low cost and in large quantities, and pure POA products can also be easily supplied, making it widely applicable for cosmetics, pharmaceuticals, etc. (Means and Effects for Solving the Problems) As a result of intensive studies on the production method of POA by fermentation, the present inventors found that yeast belonging to the genus Satucharomyces, which has the ability to produce POA, has anticancer, antibiotic, and antibacterial properties. The present inventors have discovered that mutant strains resistant or sensitive to at least one fatty acid and sterol metabolism-related inhibitor, such as drugs, produce a significant amount of POA than the parent strain, and have completed the present invention. As mentioned above, it is already known that yeast of the genus Satucharomyces produces and accumulates POA, but as mentioned above, it is completely unknown that the ability to produce POA increases by imparting drug resistance or sensitivity. First, the findings by the present inventors are the first. The present invention will be explained in detail below. The bacteria used in the present invention include anti-cancer yeast that belongs to the genus Satucharomyces and has POA-producing ability;
Any mutant strain that is resistant or sensitive to at least one of antibiotics, fatty acids such as antibacterial agents, and sterol metabolism-related inhibitors can be used. Polyene antibiotics such as nystatin, amphotericin B, trichomycin, filipin, and pimacillin are used as fatty acid and sterol metabolism-related inhibitors to induce mutant strains; cerulenin, primaquine, quinacrine, and chloroquine are the only antibiotics that inhibit fatty acid synthesis. styrene synthesis inhibitors such as; imidazole antibacterial agents such as miconazole, metrazole, econazole, and clotrimazole; and sterol biosynthesis-related substances such as 5-mevalonic acid, lanosterol, and 7-ketocholesterol. Regarding the mechanism of action of polyene antibiotics, there are reports such as damage to membrane permeation function due to specific binding with sterols (Biochem. J. 108 (1968)). POA is suggested as a fatty acid that exists in an ester bond with. Furthermore, regarding the mechanism of action of imidazole antibacterial agents, the effect of econazole on intramembrane phospholipid unsaturated fatty acid residues and unsaturated fatty acids [J.
Electron Microsc., 30 (1981)], Unsaturated fatty acid antagonism in the antibacterial membrane action mechanism of clotrimazole and miconazole [Antimicrob.Agents
Chemother.12 (1977)], suggesting a relationship with POA, an unsaturated fatty acid. To exemplify the method of obtaining various drug-resistant or sensitive mutant strains used in the method of the present invention, first, mutation treatment is carried out by ordinary mutagenesis means. For example, methods include treatment with ultraviolet rays, gamma rays, etc., and chemical treatments such as nitroliganidine, EMS, etc. There are no particular limitations on the mutation processing means or processing conditions. To obtain the drug-resistant or sensitive mutant strains from among the mutant-treated strains, for example, a suspension of the mutant-treated bacteria diluted at each stage is applied onto a nutrient agar medium supplemented with amphotericin B and cultured. . It is possible to obtain a bacterial strain having amphotericin B resistance by collecting a relatively large colony from among the resulting colonies. Sensitive mutant strains can be obtained by carrying out replicas in a conventional manner on minimal media with and without the addition of various drugs, and selecting strains that are difficult to grow on the supplemented media. Mutant strains resistant or sensitive to various drugs obtained by such methods include the mutant strain KE276 (see Table 1), which is derived from S. cerevisiae IFO 2229 (FeRM BP No. 1054). −1054)]. As carbon sources for the culture medium in the present invention, carbohydrates such as glucose, sucrose, and molasses, organic acids such as lactic acid and acetic acid, ethanol, and the like are used. As the nitrogen source, ammonium sulfate, ammonium chloride, ammonium phosphate, urea, ammonia, etc., or organic nitrogen sources such as casamino acid, peptone, corn staple liquor, etc. are used. Furthermore, phosphate,
Sulfate, magnesium salt, potassium salt, iron salt, manganese salt, zinc salt, and other inorganic salts are added as necessary. Organic nitrogen sources such as yeast extract and malt extract are also appropriately used as growth promoters. For auxotrophic strains, the relevant nutritional substances are added.
The culture temperature is 10 to 45°C, and the pH during culture is 3.0 to 7.5. A known method can be used to collect POA from the obtained yeast cells. For example, as shown in Japanese Patent Publication No. 59-33354, yeast cells separated from a culture solution are previously treated with acid and alkali, and then fatty acids are extracted with an organic solvent to obtain a crude POA-containing oil. To further increase POA purity, more conventional methods such as molecular distillation, countercurrent distribution,
Isolation and purification by chromatography, etc.
You can get pure POA products. POA obtained in the present invention is cis-9-hexadecenoic acid (palmitoleic acid) according to high performance liquid chromatography, gas chromatography, thin layer chromatography, infrared absorption spectrum, nuclear magnetic resonance spectrum, elemental analysis, etc. It was confirmed. (Example) The following will be described in detail with reference to Examples. Example: A suspension of Saccharomyces cerevisiae IFO 2229 cells in physiological saline was irradiated with ultraviolet rays at a residual rate of approximately 99.9% (the irradiation conditions were determined through a preliminary test).
A diluted solution of the treated bacteria was applied onto a solid medium plate shown in Table 2, and cultured at 30°C. Colony formation was observed 1 to 2 days after the start of culture, relatively large colonies were marked, and after 3 to 4 days of culture, drug-resistant mutants were collected from the colonies. The obtained bacterial strain was inoculated into two Sakaguchi flasks containing 400 ml of the medium shown in Table 3, cultured with shaking at 30°C for 72 hours, and the obtained culture solution was used as a seed mother for the following jar fermenter. there was. The bacteria were inoculated into a 30-volume jar fermenter containing medium 15 shown in Table 4, stirring number 350 rpm, aeration rate 12/min, temperature 30°C, glucose as a carbon source, and ethanol concentration.
Culture was carried out for 32 hours while controlling the concentration to 0.1%. For pH fluctuations, add ammonia or hydrochloric acid.
The pH was kept at 5.0. Dry the bacterial strain centrifuged from the culture solution, add 20 times the amount of chloroform:methanol mixture to 680 g of dried bacterial cells, repeat the extraction three times for 4 hours at room temperature, and extract the POA-containing oil.
Obtained 54g. Table 1 shows the results of measuring the POA content in this oil by saponification and methyl esterification gas chromatography analysis in a conventional manner. Table 1 also shows the results of culturing the parent strain Saccharomyces cerevisiae IFO 2229 in the same manner as in the above example and dividing the POA content. [Table] [Table] * Malt syrup manufactured by Nippon Malt Co., Ltd. [Table] [Table] [Table] [Table]

Claims (1)

[Claims] 1. Resistant or sensitive mutant strain of yeast belonging to the genus Satucharomyces and capable of producing palmitoleic acid (hereinafter abbreviated as POA) to at least one type of anti-cancer, antibiotic, antibacterial, or other fatty acid or sterol metabolism-related inhibitory agent. 1. A method for producing palmitoleic acid by a fermentation method, which comprises obtaining the same, culturing it in a nutrient medium, producing and accumulating POA in the bacterial cells, and collecting POA from the culture solution.