JP5415660B2 - Method for producing yeast thioredoxin - Google Patents

Description

  The present invention relates to a novel method for producing yeast thioredoxin, characterized in that yeast thioredoxin is quickly and easily prepared from a yeast extract containing a high TRX content. More specifically, an extract prepared from thioredoxin-rich yeast obtained by load culture or the like was further used to prepare a fraction containing yeast thioredoxin at a high concentration by using an ultrafiltration membrane. The present invention relates to a method for producing a novel yeast thioredoxin, which enables rapid and simple preparation.

  Thioredoxin (hereinafter referred to as “TRX”) is an approximately 12 kDa electron transfer protein with a highly conserved CXYC active center (especially X: Gly, Y: Pro), and is present in the cells of most organisms. It is known (Non-Patent Documents 1 to 4). TRX has the redox activity of disulfide bonds present in other proteins, and has the function of regulating the activity, function, and properties of proteins by its redox activity.

  Various stresses, especially oxidative stress, damages biological macromolecules such as lipids, genes, and proteins and causes damage to cells and tissues. It is known that the concentration of TRX in the body changes accordingly. That is, it is considered that an increase in TRX concentration in the body can improve or prevent allergies caused by various stresses, protect or repair mucous membranes, protect skin, and the like.

  Various techniques using the redox activity of TRX have been disclosed so far. For example, neutralization of various allergens by TRX (Patent Document 1), activation of transcription factor AP-1 by TRX (Patent Document 2), allergy prevention by TRX, cortical improvement, protection of mucosal disorders (Patent Document 3), inflammatory diseases Prevention or treatment (Patent Document 4) is disclosed.

Japanese National Patent Publication No. 10-510244 Japanese Patent Laid-Open No. 10-191977 JP 2000-103743 A JP 2002-179588 A Laurent, T. C., Moore, E. C., & Reichard, P. (1964) J. Biol. Chem. 239, 3436-3444 Holmgren, A. (1989) Ann. Rev. Biochem. 54, 237-271 Holmgren, A. (1989) J. Biol. Chem. 264, 13963-1366 Buchanan, B. B., Schurmann, P., Decottignies, P. & Lozano, R. M. (1994), Arch. Biochem. Biophys. 314, 257-60

  It is considered that TRX can be applied as a functional food material by utilizing the properties of TRX as described above. However, when manufacturing TRX on an industrial scale, there are challenges that must be overcome. That is, to purify TRX from yeast extract, it is usually performed by combining several types of chromatography. However, since chromatography requires a lot of equipment, raw materials, labor or time, it is simpler and more efficient. A method for purifying TRX was desired. That is, the problem to be solved by the present invention is to establish a method for simply and efficiently purifying TRX from yeast extract.

As a result of diligent research, the inventors of the present invention have included yeast TRX by filtering a yeast extract prepared by crushing bacterial cells obtained by culturing yeast with high TRX content using an ultrafiltration membrane. As a result, it was found that yeast TRX can be quickly and easily prepared, and the present invention has been completed. Means for solving the problems are as follows.
(1) The yeast cell extract obtained by crushing yeast cells containing a high thioredoxin content is centrifuged, and the resulting supernatant fraction is filtered at least once using an ultrafiltration membrane to obtain thioredoxin. A method for producing yeast thioredoxin, comprising a step of separating into a fraction containing and a fraction not containing.
(2) The method according to (1) above, wherein the step of filtering using an ultrafiltration membrane comprises a step of filtering through an ultrafiltration membrane having a molecular weight cut off of 5 to 6 kDa.
(3) The step of preparing a fraction containing thioredoxin using an ultrafiltration membrane is a step of filtering through an ultrafiltration membrane having a fractional molecular weight of 5 to 6 kDa, and a step of filtering through an ultrafiltration membrane having a fractional molecular weight of 100 kDa. The method according to (1) above, comprising a step.
(4) The step of preparing a fraction containing thioredoxin using an ultrafiltration membrane is a step of filtering through an ultrafiltration membrane having a fractional molecular weight of 1000 kDa, a step of filtering through an ultrafiltration membrane having a fractional molecular weight of 100 kDa, and The method according to (1) above, comprising a step of filtering through an ultrafiltration membrane having a molecular weight cut off of 5 to 6 kDa.
(5) After the step of preparing a fraction containing thioredoxin, the thioredoxin content in the total protein contained in the left fraction is 0.1 to 25% by mass, from (1) above (4) The method in any one of.

  The present invention is a method for producing yeast TRX, characterized in that the yeast extract prepared by crushing the cells obtained by culturing yeast with high TRX content is further separated using an ultrafiltration membrane. . According to the present invention, a fraction containing yeast thioredoxin at a high concentration is prepared, and as a result, yeast TRX can be prepared quickly and easily.

  TRX generally has a highly conserved active center of CXYC (especially X: Gly, Y: Pro), and is an electron present in the cells of most organisms involved in various redox reactions in vivo. It refers to the generic term for transfer proteins (TRX family).

  As long as the TRX protein of the present invention has the characteristics described in the present specification, its production method, supply source and the like are not particularly limited. That is to say, the TRX protein of the present invention can solve the problems of the present invention by using any naturally derived protein, recombinant protein produced by genetic engineering techniques, or chemically synthesized protein. However, TRX obtained from a natural source whose safety can be easily confirmed, preferably TRX derived from yeast, more preferably edible yeast such as baker's yeast, beer yeast, wine yeast, sake yeast and miso soy yeast, and more preferably baker's yeast TRX derived from yeast (Saccharomyces cerevisiae).

Further, preferably edible yeast as the yeast, for example Saccharomyces (Saccharomyces) genus Torulopsis (Torulopsis) genus Mikotorura (Mycotorula) genus Torulaspora (Torulaspora) genus Candida (Candida) genus Rhodotorula (Rhodotorula) genus Pichia ( Pichia ) genus etc. are mentioned. For these yeasts, a yeast in which stress is forcibly applied to the yeasts by peroxides such as green tea extract or hydrogen peroxide / t-butyl hydroperoxide to increase the TRX content in the yeasts is used.

  The yeast TRX protein is typically described in the yeast Saccharomyces cerevisiae TRX2 protein amino acid sequence (Genbank accession number AAA85584 (U40843), thioredox ... [gi: 1165214], Jae Mahn Song et al., 24-Jan-1996). Thus, it has a sequence composed of 104 amino acids. However, some natural proteins have one or more amino acid mutations due to differences in the varieties of species that produce them, genetic mutations due to differences in ecosystems, or the presence of similar isozymes. It is well known that mutant proteins exist, and for example, yeast belonging to other species of the genus Saccharomyces, or TRX derived from yeasts of other genera such as the genus belonging to the genus Pichia can also be used in the present invention. As used herein, the term “amino acid mutation” means substitution, deletion, insertion and / or addition of one or more amino acids. The yeast TRX protein of the present invention typically has the amino acid sequence described in the above Genbank accession number AAA85584 (U40843), but is not limited to the protein having that sequence, and the characteristics described herein. It is intended to include all homologous proteins as long as The homology is at least 70% or more, preferably 80% or more, more preferably 90% or more.

  Examples of yeast culture methods include known methods such as batch culture, fed-batch continuous culture, and fed-batch culture. The culture can be suitably performed using a jar fermenter, and the conditions in the jar fermenter at this time are not particularly limited and can be appropriately determined. For example, the culture temperature is about 28 to 33 ° C. The culture time is about 1 to 120 hours, the pH is about 4 to 7, the aeration amount is about 0 to 5 vvm, and the stirring speed is about 100 to 700 rpm. In the yeast culture, it is necessary to add various stress substances to the medium to increase the TRX content in the yeast cells to about twice (300 μg / g) or more than usual.

  The medium used for yeast culture and the fed-batch solution used during fed-batch culture are not particularly limited and can be appropriately selected from known ones according to the purpose. It is done. In addition, they may contain a carbon source, a nitrogen source and the like as appropriate. As a stress loading method performed during culture, there is usually a method of adding a green tea extract or a component thereof, and by doing so, the TRX content in the yeast cells can be increased by 2 times or more.

  As a method for disrupting yeast cells, it is sufficient that unbroken cells are eliminated under microscopic observation, and there is no particular limitation, and it can be appropriately selected according to the purpose. For example, repeated freeze-thawing, sonication, mechanical A suitable conventional method including disintegration or use of a cell lysing agent may be used, and specifically, a method using a dynomill in which 50% by volume of 0.5 mm-diameter beads are filled in a cylinder is preferable. .

  The dynomill is not particularly limited and may be appropriately selected according to the purpose. Examples thereof include Dynamyl Model Type KDL manufactured by WAB, and the capacity of the cylinder is not particularly limited and depends on the purpose. For example, it is about 0.6 liters experimentally. The flow rate of the copper-rich yeast suspension (30% (w / v)) into the cylinder in the dynomill is preferably, for example, about 2.16 liter / hour. Moreover, as a residence time in the said cylinder in the said dynomill of yeast, about 10 minutes are preferable. In addition, it is preferable to wash | clean the inside of the said cylinder in the said dynomill previously with ion-exchange water before the said crushing. In addition, the presence or absence of unbroken cells under the microscope observation can be confirmed by appropriately sampling and performing a microscope observation. Further, impurities may be removed using a deslagger or the like as necessary.

  Next, an extract containing TRX at a high concentration is prepared by filtering the yeast cell disrupted liquid obtained as described above using an ultrafiltration membrane. As the ultrafiltration membrane to be used, a flat membrane type, a tubular membrane type, a hollow fiber membrane type, etc. can be used. From the viewpoint of rapid TRX recovery, a flat membrane type ultrafiltration membrane should be used. desirable.

  In addition, when preparing an extract with a high TRX content using an ultrafiltration membrane, it is desirable to perform filtration by combining a plurality of ultrafiltration membranes having different fractional molecular weights. As for the ultrafiltration membrane to be combined, those having a molecular weight cut-off of 1,000 kDa, 100 kDa, and 5 to 6 kDa are desirably combined in that order. In addition, when there are one or two types of ultrafiltration membranes to be combined, a sufficient concentration effect cannot be obtained, and even when four or more types are combined, an effect commensurate with it cannot be obtained. By this method, the thioredoxin content in the total protein can be 0.1 to 25% by mass.

  After concentration by ultrafiltration membrane, if necessary, purification of yeast TRX is carried out by appropriately combining conventional methods of salting out, ion exchange, affinity purification or gel filtration purification. Alternatively, RP-HPLC may be used as the final purification step.

Yeast Culture In this example, whether or not the TRX content in yeast cells was increased by applying a stress load during yeast culture was examined at the laboratory level. Commercially available baker's yeast O-102 (Accession Number: FERM P-18869) (manufactured by Oriental Yeast Co., Ltd.) was inoculated with 1 platinum ear in SD medium (16 mm test tube 5 ml), cultured at 30 ° C. for 2 days, A medium (1% glucose, 0.2% yeast extract, 0.5% peptone, 0.03% K2HPO4, 0.03% KH2PO4, 0.01% MgCl2) was inoculated into a 100 mL Sakaguchi flask so that OD610≈0.05. After culturing at 30 ° C. for 14 hours (OD610≈4.5), a commercially available green tea extract “Sunphenon BG” (manufactured by Taiyo Chemical Co., Ltd.) was added to a final concentration of 2.0%, and then pH 7.6 using 5N NaOH. It adjusted so that it might become. Thereafter, shaking culture was performed at 30 ° C. for 90 minutes. 0.3 g of the obtained microbial cells were mixed with 0.3 ml of distilled water and approximately equal glass beads (φ0.5 mm), and the cells were disrupted by shaking vigorously with Fast Prep (Qbiogen) for 30 seconds. Further centrifugation was performed to obtain a supernatant. When the TRX concentration was measured by the method of Example 3 to be described later using the left supernatant, it was increased about twice as compared with the case where the culture was performed without applying stress.

Yeast disruption Under the same culture conditions as in Example 1, the culture was scaled up to a 30 L jar fermenter, and 1 kg of the obtained yeast cells were suspended in 4 L of distilled water (wet cells 20%). ) 5L was prepared. Cells were disrupted with a cell disruption device (Dynomill Model KDL, chamber capacity 0.6 L) in which the yeast suspension was filled with glass beads (φ0.5 mm) corresponding to 50% by weight of the chamber capacity. The residence time of the yeast suspension in the chamber was 10 minutes, and the cooling condition was a circulating cooling system (2 ° C.). Moreover, the yeast crushing liquid after the said crushing was centrifuged and the supernatant fraction 5L was prepared.

Concentration by flat membrane The supernatant fraction obtained in Example 2 was filtered using ultrafiltration membranes with fractional molecular weights of 1000 kDa, 100 kDa and 5 kDa. As the ultrafiltration device, “Pole TFF ultrafiltration cassette system” (manufactured by Nippon Pole Co., Ltd.) was used. FIG. 1 shows the rough flow of the work process, and the TRX content and purity in the extract at each stage of the work process.

The TRX concentration in the sample was measured according to the following procedure.
1) Mix R1 (300 μL) and sample (20 μL) 2) Preheat 5 min at 25 ° C. 3) Add R2 (6 μL) and mix 4) Incubate 1 min at 25 ° C., measure Abs 340 decrease during that time (R1 refers to 0.1 M Tris-HCl (pH 7.0), 2 mM EDTA, 0.8 mg / mL insulin, 0.16 mM NADPH solution. R2 refers to 0.3 U / mL NTR (NADP Dependent thioredoxin reductase), 0.1 M Tris-HCl (pH 7.0) solution.)

  Finally, a high TRX content extract having a content of 0.72% by mass in the dry residue and a purity of 1.13% by mass in the protein component was obtained. Since TRX is contained at a high concentration, the subsequent purification steps are simplified, and preparation of purified TRX products is quick and simple.

  Since the method for producing yeast TRX of the present invention includes a step of easily obtaining a yeast extract containing yeast TRX at a high concentration, yeast TRX can be produced quickly and easily. As a result, economic effects such as reduction in the production cost of yeast TRX and shortening of the production period can be expected.

The figure and table | surface which showed the rough flow of the preparation process of TRX high content extract, the TRX content in the extract in each step of the process, purity, etc.

Claims (1)

A yeast cell extract obtained by crushing yeast cells containing thioredoxin-rich yeast was centrifuged, and the resulting supernatant fraction was (1) an ultrafiltration membrane with a molecular weight cut-off of 1000 kDa; (2) a molecular weight cut-off 100 kDa ultrafiltration membrane; and (3) Separation into fractions containing thioredoxin and fractions not containing thioredoxin, consisting of filtration in this order by ultrafiltration membrane with a molecular weight cut off of 5-6 kDa Including steps , and
After the step of preparing a fraction containing thioredoxin, the thioredoxin content in the total protein contained in the left fraction is 0.1 to 25% by mass.
It is characterized by
A method for producing yeast thioredoxin.

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