JPH0761259B2 - Extraction method of yeast intracellular substance - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for extracting intracellular substances such as proteins and ultraviolet absorbing substances contained in cells without mechanically crushing yeast. Proteins, nucleic acids produced by bacteria,
This is an effective pretreatment method for separating and purifying enzymes and the like. It can also be used as a method for extracting proteinaceous substances produced in yeast cells by gene recombination technology.

[Conventional technology]

It is generally known that yeast do not secrete proteins into the medium except for a few enzyme proteins. Therefore, in order to obtain proteins from yeast, shake the cells at high speed with glass beads to mechanically disrupt the cells, remove the disrupted cells by centrifugation, etc., and then remove the proteins contained in the supernatant. A method of separating and purifying is used.
In addition, in order to secrete the proteinaceous substance produced by the gene recombination technology to the outside of the bacterial cell, a DNA fragment is prepared by which the secretory peptide is combined with the target peptide to form a fusion peptide. , This D
Attempts have been made to secrete the fusion peptide outside the cells by expressing NA, and this method makes it possible to secrete a specific proteinaceous substance outside the cells.

In addition, hydrolyzing enzymes localized in the periplasmic space are extracted by the osmotic shock method developed by Heppel using E. coli as a material, and the amount of protein extracted here is the total protein of the cells. About 4
% Is also reported (Journal of Biological Chemistry).
gical Chemistry) Vol. 241, page 3685 (1965), Vol. 24.
Volume 1, page 3055 (1966), Vol. 242, page 2561, (1967)
Year)). Furthermore, recently, using genetic recombination technology,
Human interleukin-1 produced in E. coli cells
An experiment in which β is leaked into ice water by an osmotic shock method (Gene, Vol. 86, 291 (1990)) has been reported.

[Problems to be Solved by the Invention]

However, in the method of mechanically crushing bacterial cells,
Mechanically disrupted cell walls, cell membrane fragments, and intracellular granules coexist with proteins, making separation and purification difficult, and also in the method of secreting the fusion peptide extracellularly, Since it is secreted as a fusion peptide, it takes extra time to extract only the target peptide from the fusion peptide, and it cannot be said to be a simple method.

Furthermore, in the above-mentioned osmotic shock method, although there have been many reports of application to Escherichia coli, there are few reports on yeasts, and moreover, the method reported (European Journal of Biochemistry (European Journal)).
Journal of Biochemistry) Volume 21, Page 137 (1971)
Is based on the method used for E. coli. That is, after the cells that have been cultured once are collected and washed, they are further incubated in a medium containing a high concentration of saccharides, and then the cells are rapidly resuspended in ice water or an ice-cooled magnesium solution. It is a thing. However, the osmotic shock method does not show the effect of Escherichia coli and the like on yeasts because yeasts have morphologically and taxonomically different features such as having a thick cell wall as compared with Escherichia coli. Furthermore, this method
It is easy to handle as a method of treating a small amount of cells at the laboratory level, but to treat a large amount of cells at a practical level,
It is complicated in terms of operation such as temperature control.

Therefore, an object of the present invention is to provide a method for extracting an intracellular substance that is inexpensive and is not complicated to operate from yeast cells.

[Means for Solving the Problems]

The salt-tolerant yeast used for brewing miso and soy sauce is about
It can grow even in the presence of 18% salt (Advance
In Food Research (Advances in Food Researc)
h) Volume 12, page 53 (1963)). Moreover, since this yeast has been used as a food-producing strain for a long time, it does not produce toxic substances and is a highly safe yeast even when used as a host for genetic recombination. Therefore, using this yeast strain as a test strain, when extracting intracellular substances by osmotic shock, osmotic shock is applied by changing pH conditions, reaction temperature, addition of drugs, reaction time, etc. The present invention has been completed by thoroughly studying the method of extraction.

That is, the present invention has as its constituent feature a method for extracting an intracellular substance characterized by subjecting a yeast to an osmotic shock under mildly alkaline conditions without mechanical crushing.

The present invention will be described in detail below.

The type of yeast to which the osmotic shock method of the present invention is applied is not particularly limited, and in addition to the salt-tolerant yeast described above, it can be applied to yeast having low salt tolerance. For example, Saccharomyce cerevisiae (Saccharomyce) used for alcoholic fermentation, bread production, etc.
s cerevisiae) has lower salt tolerance than salt tolerant yeast,
Growth is suppressed in the presence of about 8% salt. However, intracellular substances can also be extracted from these yeasts by this method.

The osmotic shock in the present invention is, for example, culturing yeast by adding salt to a medium for yeast such as YPO medium, collecting the bacteria and washing with salt solution, and then in a weak alkaline buffer solution containing no salt. It is carried out by suspending the cells in and incubating.

The concentration of the salt added to the medium depends on the salt tolerance of the yeast, the upper limit must be a concentration that does not inhibit the growth of yeast, for example when using salt-resistant yeast such as T. saccharomyces lucii In the above, the salt concentration in the medium is preferably about 15 to 16%, and about 6 to 7% in the case of yeast having no salt tolerance such as Saccharomyces cerevisiae. The concentration of the saline solution used for washing should be adjusted to the same level as the salt concentration in the medium. Further, the pH of the buffer solution is preferably weak alkaline of about 9 to 10, and the kind of the buffer solution is not particularly limited, but for example, Tris / hydrochloric acid buffer solution, sodium hydrogen carbonate / sodium carbonate buffer solution,
A few% sodium hydrogen carbonate aqueous solution (aqueous sodium hydrogen carbonate) and the like can be mentioned.

[Effect of the present invention]

The method for extracting intracellular substances by osmotic shock of the present invention is free from contamination of cell walls, cell membrane fragments, etc., and can be carried out by a simple operation, as compared with conventional methods such as mechanical cell disruption. In addition, since it does not use any special chemicals, it can treat a large amount of cells inexpensively, and the extracted substance is highly safe in terms of food hygiene, etc. It is extremely useful as a processing means.

Next, the method will be specifically described with reference to examples.

Example 1 Salt-tolerant yeast Zygosaccha
romyces rouxii) IFO1876 strain in YPD medium (yeast extract 1
%, Peptone 2%, glucose 2%), cultured in a medium (pH 5) containing 15% sodium chloride, collected, washed with 15% saline, and then added with a buffer solution having a pH range of 4 to 11. Concentration is 5 × 1
Suspend at 0 ⁷ cells / ml. After incubating at 30 ° C. for 1 hour, the cells were removed by centrifugation, and the amount of protein extracted into the supernatant and the amount of ultraviolet absorbing substance were determined.
The amount of protein was determined by the dye binding method (Analytical Biochemistry, Vol. 79, p. 544 (1977)), and the amount of ultraviolet absorbing substance was determined from the absorbance at 260 nm. The three types of buffer used in the experiment were 100 mM citric acid / disodium hydrogen phosphate buffer (pH 4,5,6,7,
8), 100 mM Tris / hydrochloric acid buffer (pH 8,9), 100 mM sodium hydrogen carbonate / sodium carbonate buffer (pH 9,10,11).

As a result, a large amount of protein has been extracted from the cells under weakly alkaline conditions (pH 9, 10). Comparing the extraction effects of Tris / hydrochloric acid buffer (pH 9) and sodium hydrogen carbonate / sodium carbonate buffer (pH 9), the former was a little better. However, considering the use of the extract in foods, etc., sodium bicarbonate (sodium bicarbonate) and sodium carbonate have high safety, are inexpensive, and are easily available.
The extraction conditions were further examined using sodium carbonate buffer (pH 9) as the extraction solution. Although a buffer solution was used here, a few% sodium hydrogen carbonate aqueous solution (sodium bicarbonate solution, 5%
It is weakly alkaline (pH around 8 in aqueous solution) and is also used as a food additive, and its safety has been confirmed, but it can be used in any way even if it is substituted.

Example 2 The cells cultured, collected and washed in the same manner as in Example 1 were suspended in 100 mM sodium hydrogen carbonate / sodium carbonate buffer (pH 9) so that the final concentration was 5 × 10 ⁷ cells / m 1, and , 20,30,
After incubating at 40 ° C. for 1 hour, the amount of protein extracted into the supernatant and the amount of ultraviolet absorbing substance were determined by the same method as in Example 1).

Both protein and UV absorber were extracted well at 30 ° C. Furthermore, since the extraction is good at around 30 ° C, this indicates that the extraction can be performed at room temperature.

Example 3 In the same manner as in Example 1, the cells cultured, collected and washed were suspended in 100 mM sodium hydrogen carbonate / sodium carbonate buffer (pH 9) so that the final concentration was 5 × 10 ⁷ cells / m1, The amount of protein extracted into the supernatant and the amount of ultraviolet absorbing substance were compared with the total amount contained in the cells. That is, the cells were mechanically disrupted by the glass beads method, the disrupted bacterial cells were removed by centrifugation, and the amount of protein and the amount of ultraviolet absorbent contained in the supernatant were compared with the amount extracted by osmotic shock.
It is believed that nearly 100% of yeast cells are crushed by the glass bead method.

The amount of protein extracted by the osmotic shock method and the amount of UV absorbing substance reached about 34% and 33% of the amount obtained by the mechanical disruption method, respectively. This is a large amount compared to about 4% of the total protein in Escherichia coli by the osmotic shock method, and it is possible to obtain a considerable amount of protein and an ultraviolet absorbing substance without crushing the cells. It is considered to be effective as a method for extracting proteins, nucleic acids, enzymes, etc. produced in yeast cells.

Reference Example 1 In the same manner as in Example 1, the cells cultured, collected and washed were suspended in 100 mM sodium hydrogen carbonate / sodium carbonate buffer (pH 9) so that the final concentration was 5 × 10 ⁷ cells / m1, Extraction was examined by adding ethylenediaminetetraacetic acid disodium salt, which is a chelator used in the case of Escherichia coli, magnesium ions of metal ions, and 2-mercaptoethanol, which is an SH reagent, to the extraction solution. That is, 0.5 mM ethylenediaminetetraacetic acid disodium dihydrate, 0.5 mM magnesium chloride, or 1.0 mM 2-mercaptoethanol was added to 100 m.
It was added to M sodium hydrogen carbonate / sodium carbonate buffer (pH 9), and the effect of the addition on the extraction was examined.

With respect to protein extraction, a slight effect was observed with 0.5 mM disodium ethylenediaminetetraacetic acid dihydrate, but not with the ultraviolet absorber. As for the ultraviolet absorber alone, 0.5 mM magnesium chloride was effective, but the protein extraction was lower than that without addition. From these results, it was considered that the addition of a drug was not particularly necessary.

Example 4 Saccharomyces cerevisiae, which is used for alcoholic fermentation, bread production, etc., has lower salt tolerance than salt-tolerant yeast, and its growth is suppressed in the presence of about 8% salt. This yeast was mixed with YPD medium (yeast extract 1
%, Peptone 2%, glucose 2%), the cells were cultured in a medium (pH 5) containing 6% sodium chloride, and the cells were collected and washed with 6% saline, and the final concentration of the bacterial cells was 5 × 10 ⁷ cells / 100m to be m1
The suspension was suspended in M sodium bicarbonate / sodium carbonate buffer (pH 9), and the amount of protein extracted into the supernatant and the amount of ultraviolet absorbing substance were compared with the total amount contained in the cells. That is, the cells were mechanically disrupted by the glass beads method, the disrupted bacterial cells were removed by centrifugation, and the amount of protein and the amount of ultraviolet absorbent contained in the supernatant were compared with the amount extracted by osmotic shock.

Proteins and ultraviolet absorbing substances were also extracted from yeast cells used for alcoholic fermentation, bread making, etc. by osmotic shock under weak alkaline conditions. However, the difference in osmotic pressure is small compared to the case of using salt-tolerant yeast,
The amount of protein extracted and the amount of UV-absorbing substance extracted by the osmotic shock method are about 1% and about 1%, respectively, due to the difference in the structure of the cell wall and the cell membrane. It was 3%.

Claims (1)

[Claims] 1. A method for extracting an intracellular substance, which comprises subjecting a yeast strain to osmotic shock under mildly alkaline conditions without mechanical disruption.