JP2686439B2 - Method for producing squalene by microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing squalene, and more particularly to a method for producing squalene using a microorganism. More specifically, it is intended to efficiently produce squalene by growing a yeast of the genus Saccharomyces having a high ergosterol content in a culture medium having a remarkable effect on squalene productivity.

[0002]

2. Description of the Related Art Squalene is a raw material for squalane used as a base oil for cosmetics. It is also used as a health food and has high blood pressure, stomach pain, liver disease, rheumatism, duodenal ulcer, arthritis, stomatitis, hemorrhoids, various dermatitis,
It is said to be effective against stiff shoulders, burns, and hangovers. In addition, it has industrial applications such as resin plasticizers, magnetic tapes, and precision machine lubricants.

Squalene is found in a variety of animal and vegetable foods, most often in the liver of deep-sea sharks. Therefore, most of squalene has conventionally been extracted and purified from shark liver oil.

[0004]

In the production of squalene from shark liver oil, the instability is inevitable because the production amount and price of shark liver oil depend on the balance between supply and demand as well as the weather peculiar to natural products. The current situation. Furthermore, since shark liver oil is used as the main raw material, the absolute price is high, and it is the actual situation that it has not been used for industrial purposes. Therefore,
There is a demand for a method for producing squalene that does not use such a high-priced natural product whose supply is unstable as a raw material.

[0005] Therefore, so far a method of producing natural squalene microbial is invented, for example (JP 58-165791 JP) method according filamentous fungus Mortierella and methods Bacterial (JP 61-212290) However, the productivity of squalene is low and it has not been put to practical use.

[0006]

As a result of intensive studies, the inventors of the present invention have invented a method by which a large amount of squalene can be easily produced by culturing a specific yeast in a special medium.

That is, the present invention uses ergosterol-metabolizing mutant yeast that accumulates squalene to produce squalene, in which sterols are added as medium components, and yeast extract and urea are added to a nitrogen source, followed by culturing. This is a method for producing squalene whose basic technical idea is to collect squalene from an object.

The present invention will be described in detail below.

With the aim of establishing an industrial mass production method for squalene by microorganisms, the present inventors have studied the metabolic pathways, and as a result, squalene is widely used as an intermediate metabolite of eukaryotes such as molds and yeasts in organisms. I found that it exists in. Based on this finding, we focused on the microorganisms in which squalene accumulates in the cells by mutating the microorganisms that have reduced or lost the activity of the enzyme squalene epoxidase, which converts squalene in the cells, and mass-culture such microorganisms. After that, the microorganisms were collected, and squalene was extracted from the cells of the collected microorganisms to solve the purpose.

As a result of repeated screening of various microorganisms, the present inventors have succeeded in mutagenizing microorganisms having a dramatically high content of squalene per bacterium, for example, the yeast Saccharomyces diastaticus.
s), derived by artificial mutation treatment, moy
We have succeeded in developing various ergosterol-metabolizing mutant yeasts that accumulate squalene, such as strain 500-A-6 (Microtechnology Research Institute No. 12085).

Further, in order to further enhance the squalene productivity by using these mutant strains and to significantly reduce the production cost for industrialization, the method for producing squalene by the mutant strains will be further improved. Various studies were conducted.

[0012] As a result, it was found that the effect can be improved by devising a combination of nitrogen sources in the culture medium, and further investigation revealed that a nitrogen source that specifically produces squalene as a yeast culture medium was identified and produced. We have developed a manufacturing method that can produce squalene at high cost and at low cost.

Here, the ergosterol-metabolizing mutant yeast is a yeast in which the activity of the enzyme squalene epoxidase in the organism is reduced or lost in order to accumulate squalene existing in the organism as an intermediate metabolite of yeast. Mutagenesis treatment of yeast may be performed to produce such yeast. As the mutagenesis treatment, a conventional method is used. For example, in addition to physical methods such as ultraviolet rays, X-rays, γ-rays, electromagnetic waves such as temperature changes, chloroethylamine compounds such as nitrogen mustard, ethyleneimine compounds, sulfonic acid, etc. Ester compounds, diepoxy butane,
A chemical method using an alkylating agent such as colchicine, a peroxide, or a purine compound and other agents is appropriately used. Inducing mutations by such a method,
The target yeast is screened.

The present invention is characterized not only by using ergosterol-metabolizing mutant yeast as described above in mass production of squalene, but also by its medium composition, particularly the particular combination of nitrogen sources. In addition, it has a remarkable effect of further increasing the production efficiency of squalene.

In the production of squalene using the ergosterol-metabolizing mutant yeast according to the present invention, urea or a combination of ammonium sulfate and yeast extract is effective as a nitrogen source having an action of specifically improving squalene productivity. It was also confirmed from the examples described later that the extremely effective combination is urea and yeast extract.

[0016] Generally, YPD medium (components: yeast extract 10 g / l, peptone 10 g / l, glucose 20 g / l) is often used for culturing yeast.
As a result of investigating a medium component that can obtain a higher squalene production amount instead of the medium, as a carbon source, glucose, sucrose, molasses, nitrogen source, yeast extract, corn steve liquor, NH ₄ NO ₃ , Among (NH ₄ ) ₂ SO ₄ and urea, the combination of urea and yeast extract showed extremely remarkable squalene accumulation.

In the combination of urea and yeast extract according to the present invention, peptone is not necessary, the amount of urea added is about 1 to 5 times that of yeast extract, and the total amount of nitrogen in the medium is 2 g / l to 6 g. At 1 / l, significant ergosterol accumulation was observed in the cells. In addition, the amount of peptone can be halved by adding about 3 g / l of ammonium sulfate to the medium.

In order to carry out the present invention, the presence of sterols is necessary in the culture of the ergosterol-metabolizing mutant yeast in addition to the above-mentioned limiting conditions relating to the nitrogen component. That is, in the present invention, it is necessary to add ergosterol, cholesterol, lanosterol, campesterol, and sitosterol. These sterols may be used alone or as a mixture. Further, a substance containing these, such as soybean oil, may be used. Addition of this sterol to the medium blocks or narrows the ergosterol metabolic pathway of yeast to accumulate squalene, resulting in the lack or deficiency of essential ergosterol in the organism to supplement the necessary amount. It is a thing.

After culturing the mutagenized yeast, squalene is recovered from the culture (bacteria, culture broth). The means of recovery may be a known solid-liquid separation method such as filtration or centrifugation. The bacterial cells thus obtained are directly or dried or extracted from the culture solution. When extracting from dry cells, a solvent with a low polarity such as n-hexane or petroleum ether is used for extraction efficiency, but squalene is extracted by adding the solvent to a mixture of water and cells. In this case, extraction efficiency is poor unless a mixed solvent such as methylene chloride / methanol or ethanol / n-hexane is used. To remove squalene dissolved in this solvent alone, distillation is performed to remove the solvent, and the resulting crude squalene is distilled under reduced pressure to obtain high-purity squalene. If necessary, the cells may be destroyed by ultrasonic waves or the like before the extraction.

Identification of squalene can be carried out by mass spectrometry and nuclear magnetic resonance spectroscopy, and quantification of squalene can be carried out by gas chromatography.

[0021]

EXAMPLES Examples of the present invention will be shown below.
It is not restricted by this.

[0022]

Example 1 Yeast Saccharomyces diastaticus moy500-A-6 strain (FERMP-12085) was used to compare the squalene productivity by changing the type, amount and combination of nitrogen sources.

The YPD medium was used as a reference medium, and the medium used for the test was replaced with urea and ammonium sulfate, which corresponded to the total amount or half amount in terms of the nitrogen amount of peptone and yeast extract contained in the reference medium as nitrogen sources. Was used and the total nitrogen content in each medium was constant.

Here, the components of 1 liter of YPD (standard) medium are carbon source: glucose 20 g / l, nitrogen source: peptone 1
The nitrogen content of nitrogen source was 0 g / l, the nitrogen content of nitrogen source was 10 g / l, and the nitrogen content of the nitrogen source was 13.3% by weight of peptone and 10.0% by weight of yeast extract from the analysis values. It becomes 2.33 g / l. The nitrogen contents of urea and ammonium sulfate used in the test are 46.7% by weight of urea and 21.2% by weight of ammonium sulfate. (Note that glucose (anhydrous), urea, and ammonium sulfate all used reagent special grade (Wako Pure Chemical Industries, Ltd.), and polypeptone and yeast extract used for microbial culture medium (Nippon Pharmaceutical Co., Ltd. and DIFCO). .)

Therefore, the amount of urea corresponding to 10 g of peptone in 1 L of YPD (standard) medium is 2.1 g and ammonium sulfate is 4.7 g.

The medium used in the examples was adjusted to 1 liter by using a combination of 1 liter of YPD (standard) medium, in which nitrogen equivalent to half or the whole amount of peptone was replaced with urea. 100 ml of each adjusted medium was collected, 0.4 mg of ergosterol (special grade reagent, Wako Pure Chemical Industries, Ltd.) was added thereto, and shake culture at 30 ° C. and 200 rpm was performed in a 500 ml Erlenmeyer flask for 144 days. I went on time. After culturing, the cells were separated by a conventional method and dried, and then squalene was extracted by solvent extraction and quantified by gas chromatography. That is, cells were separated and dried by a conventional method, extracted with about 20 times amount of methanol: chloroform (2: 1), the obtained extract was concentrated under a nitrogen blow, and then subjected to gas chromatography (Shimadzu GC .14A) to quantify the squalene content.

The results of squalene production of each medium are shown in Table 1 (Samples 1 to 3) below.

[0028]

[Table 1]

From the results of the sample 3 shown in Table 1, as compared with the standard medium (standard 1), the combination of yeast extract 10 g / l and urea 2.9 g / l as a nitrogen source was used for the production of squalene. It can be seen that it shows a remarkable effect.

[0030]

[Comparative Example] For comparison, YPD medium was used as a standard medium (standard 1), and comparative sample mediums (comparisons 1 to 9) were prepared by changing the amount of peptone in the standard medium and the amount of yeast extract, and using urea or ammonium sulfate. Was added to bring the total nitrogen content of the comparative sample medium to the same level as the standard medium (2.3 g / l), and the strain and other culture conditions were the same as in Example 1. The results of squalene production of each medium are shown in Table 1 (Comparative 1 to Comparative 9).

[0031]

[Example 2] In Example 1, it was revealed that the combination of the nitrogen source with yeast extract and urea showed high squalene productivity. Therefore, yeast extract and urea were added without adding peptone to the medium. Yeast extract 10
The medium was adjusted by changing the amount of urea added to 0 to 10 g / l while keeping the g / l constant.

As in Example 1, moy 500-A-6
Using the strain (FERM P-12085), 100 ml of each adjusted medium was collected, and ergosterol 0.
Add 4 mg and add 30 in a 500 ml Erlenmeyer flask.
Shaking culture at 200 ° C. and 144 ° C. was performed for 144 hours.
After culturing, the bacterial cells were separated by a conventional method and dried, and then squalene was extracted by solvent extraction and quantified by gas chromatography.

The results are shown in Table 2 below.

[0034]

[Table 2]

From the results shown in Table 2, the amount of urea added was 6.0.
It was found that the amount of squalene produced was the highest when g / l was used.

From these results, the effective amount of urea added was in the range of the equivalent amount of the yeast extract nitrogen (urea 2 g / l) to 5 times the equivalent amount (urea 10 g / l) and the amount of urea in the medium. The total amount of nitrogen is suitably in the range of 2 g / l to 6 g / l.
It was also found that when the amount of urea added was further increased, the amount of bacterial cells was decreased and the amount of squalene produced was decreased.

[0037]

INDUSTRIAL APPLICABILITY In a squalene fermentation production from a newly created ergosterol-metabolizing mutant yeast, by using a medium which is a combination of yeast extract and urea,
Squalene production by microorganisms can be carried out efficiently and stably.

Claims (5)

(57) [Claims] 1. When producing squalene using an ergosterol-metabolizing mutant yeast that accumulates squalene, the yeast is cultured in a culture medium containing sterols and yeast extract and urea as nitrogen sources, A method for producing squalene, which comprises collecting squalene. 2. The method for producing squalene according to claim 1, wherein the ergosterol-metabolizing mutant yeast is of the genus Saccharomyces. 3. The method for producing squalene according to claim 1, wherein the ergosterol-metabolizing mutant yeast is Saccharomyces diastaticus moy 500-A-6. 4. The production method according to claim 1, wherein the amount of urea added is in the range of 1 to 5 times the yeast extract nitrogen equivalent amount, and the total nitrogen amount of the culture medium is 2 g / l to 6 g / l. The manufacturing method of squalene characterized by being in the range. 5. The method for producing squalene according to claim 1, wherein the sterols are one kind or two or more kinds selected from ergosterol, cholesterol, lanosterol, campesterol and sitosterol.