KR100700910B1 - Saccharomyces cerevisiae mutant producing alkali-soluble ?-glucan - Google Patents

Abstract

A novel Saccharomyces cerevisiae mutant is provided to be able to produce alkali-soluble beta-glucan having immuno-activity with high yield, thereby being applied to various products having immuno-activity such as functional foods and cosmetics. The Saccharomyces cerevisiae mutant producing alkali-soluble beta-glucan is deposited in a Korean Culture Center of Microorganisms(KCCM) as a deposition number of KFCC-11359P. The method for preparing the Saccharomyces cerevisiae mutant comprises the steps of: (a) applying UV to yeast; (b) firstly screening the mutant yeast survived after the UV radiation; (c) after adding lyticase to the firstly screened yeast to cleave cell wall, performing the osmotical lysis using distilled water; and (d) secondly screening the lyticase resistant yeast survived after the lysis. The method for producing the alkali-soluble beta-glucan comprises the steps of: (a) culturing the Saccharomyces cerevisiae mutant KFCC-11359P; (b) treating the cultured mutant with NaOH to perform an alkali extraction; and (c) treating the product obtained from the step(b) with cetavlon and borate to selectively precipitate mannoprotein.

Description

Saccharomyces cerevisiae mutant producing alkali-soluble β-glucan

1 is a micrograph (X1,000) of wild type JH and mutant JUL7.

2 is a graph showing Lyticase-resistance of wild type and mutant strains.

3 is a graph showing the activity of the immunoactive substance Nitric Oxide (NO) according to the injection amount of Alkali-soluble and Water-solube β-glucan.

The present invention relates to a new yeast mutant strain, and more particularly to a yeast mutant strain for producing an alkali-soluble β-glucan having high immunological activity and a method for producing an alkali-soluble β-glucan using the same.

β-glucan is one of the most abundant polysaccharides in the yeast cell wall and exists as a homopolymer of glucose linked via β- (1,3)-or β- (1,6) -D-glycosidic bonds (Manners et al. 1973; Sentandreu et al. 1975). In yeast ( Saccharomyces cerevisiae ) the cell wall contains β- (1,3) -D-glucan, β- (1,6) -D-glucan, chitin and mannoprotein. The four cell wall structural components are all connected to each other by a double bond. Mannoproteins are linked to β- (1,6) -glucan via the remainder of the glycosyl-phosphatidyl-inositol (GPI) anchor having a protein portion of about 100 kDa and containing five α-linked mannosyl residues ( Klis et al. 1997; Lu et al. 1995; Shahinian and Bussey 2000). The reducing end of β- (1,6) -glucan is linked to the non-reducing terminal glucose of β- (1,3) -glucan. The link between mannoproteins and β- (1,6) -glucans plays a central role in organizing yeast cell walls. Here, chitin is attached directly to the branch of β- (1,6) -glucan (Jamas et al. 1986; Kollar et al. 1997; Lipke and Ovalle 1998; Shahinian et al. 2000).

Glucans are generally grouped into several fractions (Jamas et al. 1986; Kuliche et al. 1997). Alkali-soluble glucans are the minor component [15-20% (w / w)] of total glucans. This glucan fraction consists of a high molecular weight (240,000-550,000) β- (1,3) -linked backbone containing 3% β- (1,6) -linkage and is responsible for the structural integrity of the cell wall (Jamas et al. 1986; Kiho et al. 1991; Shahinian et al. 2000).

Recently, there has been increasing interest in β-glucans isolated from the cell walls of yeast. This compound exhibits a variety of biological activities by stimulating the immune system, including adjuvant, anticancer and radioprotective agents (Bohn and BeMiller 1995; Muller et al. 1996; Munzberg et al. 1995; Sakurai et al. 1996; Vetvicka et al. 1997). Alkali-soluble β-glucans act as nonspecific regulators of the immune system (Kiho et al. 1991; Kulicke et al. 1997; Mullet et al. 1996; Williams et al. 1992).

Accordingly, the present inventors have made extensive efforts to produce high yields of alkali-soluble β-glucans having immunological activity, and screened cell wall mutants after non-specifically mutating yeast, resulting in high yields of alkali-soluble β-glucans. It was confirmed that the mutant yeast to produce can be produced, the present invention was completed.

Therefore, the main object of the present invention is to provide a yeast ( Saccharomyces cerevisiae ) mutant that produces a high yield of alkali-soluble β-glucan.

Another object of the present invention to provide a method for producing the yeast mutant strain.

It is still another object of the present invention to provide a method for preparing an alkali-soluble β-glucan using the yeast mutant strain.

According to one aspect of the invention, the invention provides a yeast ( Saccharomyces cerevisiae ) mutant KFCC-11359P, which produces an alkali-soluble β-glucan.

According to another aspect of the present invention, the present invention comprises the steps of irradiating ultraviolet (UV) to the yeast, primary screening the mutant yeast surviving the ultraviolet irradiation, Lyticase (Lyticase) to the primary screened yeast Osmotic lysis with distilled water after cell wall cleavage reaction by addition; It provides a method for producing the yeast mutant strain KFCC-11359P of the present invention comprising the step of secondary screening the yeast-resistant yeast surviving the lysis.

According to another aspect of the present invention, the present invention provides a method for producing an alkali-soluble β-glucan using a known method of purifying β-glucan after culturing the yeast strain KFCC-11359P of the present invention. To provide, preferably the present invention comprises the steps of culturing the yeast mutant strain KFCC-11359P of the present invention; Alkali extraction by treating the cultured yeast with sodium hydroxide (NaOH); And it provides a method for producing an alkali-soluble β-glucan comprising the step of selectively precipitate the mannoprotein by treating the cetavlon (cetavlon) and borate (borate).

In the method for producing the alkali-soluble β-glucan of the hrxk invention, more preferably, the remaining mannose protein that has not been removed by cetavlon by concanavalin-A chromatography. It characterized in that it further comprises the step of completely removing.

Hereinafter, the present invention will be described in more detail.

The present inventors specifically cleave β-1,3-glucan in the yeast cell wall to screen cell wall mutants after nonspecific mutation of Saccaromyces cerevisiae , so that the general yeast cells use an enzyme called lyticase that causes cell lysis to die. The mutant strain JUL7 in the cell wall was found. The yeast mutant JUL7 can produce an alkali-soluble glucan 1.72 times higher than the wild type. The present inventors have deposited the yeast strain JUL7 of the present invention to the Korean Culture Center of Microorganisms (KCCM) on November 18, 2005 with accession number KFCC-11359P.

The bacteriological properties of the yeast mutant strain KFCC-11359P had a morphologically microscopic observation and had an oval shape and a size of 2 to 5 μm in length, had a sporulation ability, a proliferative form of germination, and biochemical sugar fermentation. , Had higher glucan content than wild type, had 100 times higher cell wall degrading enzyme resistance than wild type, and had higher viscosity of cell wall particles than wild type.

Purification of β-glucan from yeast has been extensively studied and various methods are known. Most of these methods are due to the insolubility of β (1-3) -glucan in alkali or organic solvents. The main known methods are:

(a) hot extracts containing concentrated sodium hydroxide obtained by hot extracts containing acids and precipitates containing ethanol (Manners, DJ et al., Biochem. J. 135 19-30 (1973), U.S. Patent No. 4810646 No. 5028703 and 5250436).

(b) Hot acid extracts and extracts containing concentrated sodium hydroxide obtained by enzymes for modifying or purifying glucans (Czech Patent Application No. 890038 'Alkali-acid extracts obtained by preparations containing enzymes that act as amylases) Purification of β-D-Glucan by

(c) Yeast cell wall extract produced as a result of degrading enzyme function of yeast by diluting autolyzed or concentrated phenol in a ratio of 1: 1 with water (US Pat. No. 4138479).

(d) Extracts containing organic solvents such as isopropaol, ethanol, acetone or methanol added with sole methanol or alkali (Japanese Patent Publication Nos. 7051081, 6340701, 5295003, 3002202)

Current methods of isolating β-glucans generally use a multistage alkali-acid extraction process (Manners, D. J., et al., J. Gen. Micro. 80 411-417 (1974)). The alkaline extraction step removes most of the amorphous mannoprotein and glucan preparation, and the subsequent acid extraction step comprises glycogen and most of the β (1-6)-from glucans that are predominantly fibrillar bound to β (1-3). Remove the side branches. The final solvent extraction step sometimes removes lipids.

Β-glucan prepared according to the present invention has various functions. Looking at this, β-glucan has the function of preventing the attack of pathogens by enhancing the immunity of macrophage cells, and recently, studies on anti-tumor activity Is being reported. In addition to activating the cytostatic function of macrophage cells, β-glucan has the function of increasing the function of other immune systems. β-glucans cause common main resistance actions, including serum supplementation, lysozyme effects in the system, division and proliferation of bone marrow, and division of cytotoxin T cells. It also induces antibody secretion of plasma cells that divide from B lymphocytes. All immune actions are linked with two cytokines called interleukin-1 (IL-1) and interleukin-2 (IL-2). In 1987, Tulan University School of Medicine found that β-glucan induced the release of these two cytokines in rats and increased their protective function for 12 days after administration.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Selected strains and medium (strains & media)

In the yeast Saccaromyces cerevisiae , JH (Hansen1883, Mat a / Mat α) (KCCM 11201), which isolated only mate type a, was used as a standard strain, and mutant JUL7 which mutated JH was used together. Experiment. The growth temperature was 30 ℃ and incubated in YPD medium (yeast extract 2%, bactopeptone 4% adenine solution (× 100) 2%, uracilsolution (× 100) 2%, glucose 8%).

Example 2: random mutation

Ethylene sulfonate (EMS) causes base transitions at most GC sites in DNA, and UV can obtain a wider range of base substitutions than EMS and bases and bases in pyrimidine, especially TT pairs. It causes both translations. The two mutation methods received below were optionally used to obtain the high yield mutants of the invention.

1) UV mutagenesis

After incubation for 12 hours in YPD medium (10 ml) to 1 × 108 cells / ml, 200 μl each plate, and UV irradiation (irradiation) for 30 minutes with the plate lids open. After UV irradiation, the cells were incubated for about 24 hours in a dark place. The lethal rate of yeast by UV irradiation varies with the irradiation time, but approximately 70-90% of the cells were killed when exposed to fluence of 50 J / m2.

2) Mutation using EMS (ethylmethane sulfonate) mutagenesis

Incubate yeast in YPD medium (4ml) for 12 hours to 3 * 10 ⁷ cells / ml, and then wash 500µl in tubes and wash twice with pH 7.0 and 125µl of 50mM potassium phosphate buffer. This was resuspended in 500 µl of potassium phosphate buffer, and 30 µl of EMS was added thereto and reacted at 30 ° C for 30 minutes. To stop mutagenesis, add the same amount (500 µl) sodium thiosulfonate (10%) and mix well. After centrifugation for 5 minutes at 10000 rpm, washed twice with sterile water and incubated at 30 ℃ for more than 48 hours. (Catility rate: about 60-90%)

Example 3 Screening of Lyticase Resistant Strains (Cell Wall Mutant)

After culturing the mutant Yeast surviving in Example 2 to the beginning of the YPD medium (10ml) log phase, the cells were precipitated by centrifugation and washed twice with 0.1M phosphate buffer, pH 7.0. Solution I [0.1M phosphate buffer; 0.01 M EDTA; After resuspension in 100 μl of 1% v / v 2-mercaptoethanol], the mixture was reacted at 30 ° C. for 30 minutes and centrifuged. This was accomplished by solution II [1M sorbitol; 0.01 M EDTA; 0.01 M phosphate buffer] was added to 300 μl of lyticase and reacted at 37 ° C. for 30 minutes, followed by 3 times washing with 1 M sorbitol. After osmotic lysis with distilled water, the cells are incubated at 30 ° C for 24 hours. After incubation, strains showing higher survival rates than the wild type were selected as Lyticase-resistant strains. Among them, JUL7 derived from UV mutants with the highest survival rate was estimated and tested as alkali-soluble β-glucan high productivity yeast. 1 is a micrograph (X1,000) of wild type JH and mutant strain JUL7 (Bar 5 μm).

UV mutagenesis randomly mutates genes, causing cells to die. Survival of the random mutated yeast was screened first, and only screened secondary to those mutated in the cell wall using lyticase, a cell wall degrading enzyme. When the lyticase is put in the yeast growing media, the wild type is 99.9% killed by the degradation of the matrix in the cell wall, but the death rate is greatly reduced when glucan binding or changes in their composition occur in the cell wall. Among the mutants isolated from JH, JUL7 showed no significant difference in morphology and the survival rate increased up to 400 times (Fig. 2). Therefore, they were selected as experimental subjects.

Example 4 Extraction of Alkali-Soluble β-glucan

1) Alkali extraction

1 g of Yest cells were washed and then suspended in 20 ml of 5% (w / v) NaOH and heated to 90 ° C. for 8 hours. The pellet was suspended in 20 ml of 3% (w / v) NaOH and then heated at 90 ° C. for 5 hours. The suspension was centrifuged and the supernatant combined with the former. The alkaline extract was neutralized with 2M acetic acid and precipitated with three volumes of EtOH containing 0.3M sodium acetate.

2) Cetavlon extraction

The Cetavlon method (Jozef et al. 1999; Lloyd et al. 1971) was used to remove mannoproteins. 1 g of alkaline extract was dissolved in distilled water and dialyzed against sterile water. To separate mannoproteins from β-glucan, 10 ml of Cetavlon solution (1 g of hexadecyl trimethyl ammonium bromide dissolved in 5 ml of water) was mixed with alkaline extract for 5 minutes. The mixture was left at room temperature overnight and then centrifuged to remove the precipitate. However, in order to precipitate the mannoprotein, because the mannoprotein remains in the solution, 20 ml of 1% (w / v) boric acid solution was added to the supernatant, the pH was adjusted to 8.8 with 2M NaOH, and then left at room temperature overnight, followed by Was precipitated with three volumes of EtOH containing 1% (w / v) sodium acetate.

3) Concanavalin-A chromatography

The binding properties of lectins were used to remove mannan or mannoprotein still remaining in the extract. Concanavalin-A (Con A) binds to α-D-glucosyl and α-D-mannosyl residues but has no affinity for β-D-glucosyl groups (Roman et al. 1997). 20 ml of Con A-Sepharose 4B gel was charged to a polypropylene column (Bio-Rad Laboratories) and then equilibrated with 200 ml of wash buffer (20 mM Tris-HCl, pH 7.4; 0.5 M NaCl; 1 mM MnCl 2, MgCl 2, CaCl 2). . After applying the extract (a mixture of β-glucan and mannoprotein), the solution was eluted with a washing buffer of 2-3 times the volume of β-glucan until it was released. Eluted β-glucan was dialyzed against sterile water and lyophilized. Alkali- and water-soluble β-glucans were separated from the water-insoluble fraction by centrifugation.

Example 5 Yield of Alkali-soluble β-glucan in Mutant JUL7

Table 1 shows the yield of JH (wild type) and JUL7 (mutant). As a result of measuring the yield of Alkali-soluble β-glucan by HPLC and gas chromatography, the amount of Alkali-soluble β-glucan was 1.72 times higher than that of wild type in JUL7. In particular, fractions having water solubility in Alkali-soluble β-glucan were separated again using affinity chromatography. These fractions have water solubility and are easy to use as functional food or cosmetic raw materials. Alkali-soluble and water-soluble β-glucan were 7.27 times more efficient in JUL7.

TABLE 1 Yield of Alkali-soluble β-glucan of JH and mutant JUL7

JH (Wild Type) JUL7 (Mutant) Alkali-soluble β-glucan Alkali-soluble and Water-soluble β-glucan Alkali-soluble β-glucan Alkali-soluble and Water-soluble β-glucan Yield (mg / g of cell dry mass) 80.5 3.6 138.2 26.2

Example 6: Immune Activity Experiment of β-glucan

To test the immunological activity of the isolated β-glucan, 0, 10, 20, 50, 100 and 200 μg of glucan were treated in RAW 264.7 (Murine Macrophage, 1 × 10 ⁶ cells / each well), respectively. After cell culture for a time, the amount of NO released from the cells was measured. 3 is a graph showing the activity of the immunoactive substance Nitric Oxide (NO) according to the injection amount of Alkali-soluble and Water-solube β-glucan. Both wild type JH and mutant JUL7 showed immunological activity, and the mutant JUL7 showed 100-fold higher immunological activity than JH at 100 μg.

As described above, according to the present invention, the overall anticancer effect can be seen through activation of the human immune system of high yield alkali-soluble β-glucan, and immunization using alkali-soluble β-glucan produced from safe yeast Development of a variety of active products such as functional foods and cosmetics may be developed.

Claims (4)

Saccharomyces cerevisiae strain deposited with the accession no. KFCC-11359P to the Korean Culture Center of Microorganisms (KCCM) producing alkali-soluble β-glucan KFCC-11359P. Irradiating ultraviolet light (UV) to the yeast, first screening the mutant yeast surviving the ultraviolet light irradiation, adding Lyticase to the first screened yeast and performing a cell wall cleavage reaction followed by osmotic cells with distilled water. Osmotical lysis; A method for preparing a yeast strain KFCC-11359P according to claim 1, comprising the step of secondary screening the yeast-resistant yeast surviving the lysis. Culturing the yeast mutant strain KFCC-11359P according to claim 1; Alkali extraction by treating the cultured yeast with sodium hydroxide (NaOH); And, treating cetavlon and borate to selectively precipitate the mannoprotein. 12. A method for producing an alkali-soluble β-glucan, comprising: 4. The method of claim 3, further comprising the step of removing the remaining mannoprotein by concanavalin-A chromatography.

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