KR100670166B1 - -10 Novel Bacillus subtilis CH10 and method for producing poly gamma glutamic acid using the same - Google Patents

Abstract

A novel Bacillus is provided to be able to produce high quality poly gamma glutamic acid(PGA) with high yield in a culture medium including a conventional carbon source and nitrogen source, thereby being able to provide inexpensive PGA with high quality. The Bacillus subtilis CH-10 for producing PGA from culture medium including a carbon source and a nitrogen source is deposited as a deposition number of KACC91169P. To produce the PGA, the Bacillus subtilis CH-10 is cultured in the culture medium including a conventional carbon source such as sucrose, glucose, fructose, galactose and maltose, and nitrogen source such as glutamic acid and glutamine at a temperature of 37 deg.C under pH of 7.5, and the PGA is isolated from the culture material.

Description

Novel Bacillus subtilis CH-10 and method for producing poly gamma glutamic acid using the same}

1 is a graph of the production of poly gamma glutamic acid according to pH (A) and temperature (B) of Bacillus subtilis CH-10 strain,

FIG. 2 is a graph showing comparison of pH change and growth of medium (-○-) and polygamma glutamic acid production (-●-) according to incubation time of B. subtilis CH-10 strain,

Figure 3 is a photograph showing the change in physical properties and yield of the polygamma glutamic acid produced according to the culture time of the Bacillus subtilis CH-10 strain as a result of SDS-PAGE.

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The present invention relates to a novel Bacillus subtilis CH-10 strain producing polygamma glutamic acid and a method for producing polygamma glutamic acid using the same.

In general, poly-gamma glutamic acid (hereinafter referred to as PGA) is an amino acid polymer material in which the γ-carboxyl group of glutamic acid and the α-amino group of glutamic acid are amide bonds [3] and have high water solubility and biodegradability. Known as a sex polymer.

Polygamma glutamic acid is an electrically viscous fluid, an aqueous electrolyte solution, a moisture absorbent, a moisture-absorbing resin, a material of a fiber or film, a mineral absorption accelerator, a calcium ion dissolving agent, a calcium absorption accelerator, a pharmaceutical carrier, a feed additive. Its use range is wide, such as food freshness and maintenance agents [Ref. Information 10], and it can be applied to various fields as high value-added materials for food, medicine, environment, cosmetics, and industrial use [Ref. Information 18].

Then, the poly-gamma-glutamic acid is so far or tree alba aejip thiazol car (Natrialba aegyptiaca), Bacillus anthranilic cyano (Bacillus anthracis), Bacillus mesen Terry kusu (Bacillus mesentericus), Bacillus piece you formate miss (Bacillus licheniformis) to the producing microorganism, Bacillus magaterium ( Bacillus magaterium ), Bacillus subtilis ( Bacillus subtilis ) and other strains have been reported to produce [document information 1,2,11,17], it is known to show slightly different properties depending on the microorganism. . Polygamma glutamic acid producing strains are classified into two types according to their dependence on glutamic acid. First, glutamic acid-dependent strains ( B. subtilis) that require addition of L-glutamic acid to produce polygamma glutamic acid are used. chungkookjang, B. anthracis , B. licheniformis ACTC 9945A , B. subtilis IFO 3335, B. subtilis F-2-01) [document 9,15], the second is glutamic acid independent strain ( B. subtilis 5E, B. subtilis) that does not require the addition of L-glutamic acid in the medium. TAM-4, B. subtilis A35) [document information 8, 13]. At present, the production of poly-gamma-glutamic acid is mainly in Bacillus subtilis Soybean Bacillus strains (B. subtilis chungkookjang), Bacillus subtilis natto (B. subtilis natto) and Bacillus licheniformis, etc. The production process from the culture medium of the microorganisms is the majority.

In addition, as the application range of polygamma glutamic acid is diversified recently, researches on the development of strains having excellent productivity of polygamma glutamic acid are being actively conducted. Since the physical properties of polygamma glutamic acid largely depend on the molecular weight, biomolecules of various molecular weights are produced. This situation is required [document information 7,14,16,21,22,23].

Therefore, the present inventors have discovered a new strain of Bacillus genus that mass-produces polygammaglutamic acid in the compost, and studies the biochemical properties from the strain, so that it can be industrially used in the mass production process of PGA, PGA quality and low cost It was provided to the consumer to complete the present invention.

Accordingly, an object of the present invention is to provide a novel microorganism capable of producing a large amount of polygamma glutamic acid from a medium containing a common carbon source and a nitrogen source.

Another object of the present invention is to provide a method for producing polygamma glutamic acid using the novel microorganism.

In order to achieve the above object, the present invention provides a Bacillus subtilis CH-10 strain having the accession number KACC 91169P for producing polygamma glutamic acid from a medium containing a common carbon source and nitrogen source. do.

In addition, to achieve another object, the present invention provides a method for producing polygamma glutamic acid comprising culturing the strain in a medium containing a common carbon source and a nitrogen source, and obtaining polygamma glutamic acid from the culture.

Hereinafter, the present invention will be described in detail.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art.

In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

Hereinafter, the present invention will be described in detail.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art.

In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

1. Isolation and Identification of New Microorganisms

The present invention is a method of producing poly-gamma glutamic acid (Poly-γ-glutamic acid; PGA) using a microorganism in a medium containing a common carbon source and nitrogen source, the production efficiency is very excellent to replace the conventionally used strains In order to study the novel strains of the genus Bacillus and to apply them to industrial techniques, new microorganisms suitable for the purpose of the study were isolated from compost and named Bacillus subtilis . CH-10. The strains were identified as Gram-positive bacillus, non-motile and heat-resistant spores, and analyzed using the Bacillus strain identification kit, and belong to Bacillus subtilis , and also 16s rRNA. The sequence was analyzed to find homology with other strains. As a result, Bacillus subtilis BFAS ( Bacillus subtilis BFAS) and 99.4%, Bacillus vallismortis and 99.1%, It has a homology of 98.8% with Bacillus licheniformis .

2. Incubation of Bacillus subtilis CH-10

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In order to increase the polygamma glutamic acid production of Bacillus subtilis CH-10 in the present invention, the carbon source is a carbon source selected from the group consisting of sucrose, glucose, fructose, galactose and maltose. , It is preferable to use a nitrogen source containing L- glutamic acid or L- glutamine (glutamime). Of these, it is most preferable to use sucrose as the carbon source and L-glutamic acid as the nitrogen source. And most preferably in the present invention 2% sucrose (sterucrose), 3% L-glutamic acid (L-glutamic acid), 1% ammonium sulfate [(NH ₄ ) ₂ SO ₄ ], 0.1% potassium diphosphate in sterile water (K ₂ HPO ₄ ), 0.1% sodium hydrogen phosphate (Na ₂ HPO ₄ ), 0.05% magnesium sulfate (MgSO ₄ ), 0.005% ferric chloride (FeCl ₃ ), 0.02% calcium chloride (CaCl ₂ ), 0.002% manganese sulfate ( MnSO ₄ ) and a medium consisting of 0.00005% biotine are added.

In addition, the strain was deposited with the Agricultural Biotechnology Research Institute under the Agriculture Promotion Agency on July 20, 2005, and received the accession number KACC 91169P.

Me, condition

The Bacillus subtilis CH-10 strain of the present invention exhibits the highest production of PGA at pH 7.5 and temperature 37 ° C.

3. Physical Properties of Polygammaglutamic Acid

The Bacillus subtilis CH-10 strain of the present invention produces polygamma glutamic acid having a molecular weight of 200 to 1,000 kDa when incubated for 12 hours or more under optimal conditions, and polygamma glutamic acid having an average molecular weight of 1,100 kDa when incubated for 96 hours or longer. Is produced in an amount of 18 mg / ml.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[ Experimental Example ]

1. Isolation and Purification of PGA

50 ml of the cell culture was centrifuged (12,000 x g, 30 minutes) to remove the cells and the supernatant was recovered. Four times the amount of methanol was added to the supernatant, and the resulting precipitate was collected, dissolved in 10 ml of 10 mM Tris-HCl buffer (pH 8.0), and dialyzed three times with 2 L of copper buffer. The dialysate was lyophilized and then dissolved in 1 ml of 10 mM Tris-HCl buffer (pH 8.0) and protease K (Proteinase K, 100 μg / ml) was removed at 37 ° C., 12 for removal of α-polypeptide. Time was processed. The treated solution was dialyzed with 10 mM Tris-HCl buffer (pH 8.0) and then lyophilized.

And, the amount of glutamate in the solution was measured after hydrolysis at 105 ° C. for 24 hours with the same amount of 6M HCl and neutralized with 6M NaOH for quantification of PGA. The neutralized glutamate solution was reacted with ninhydrin at 37 ° C. for 10 minutes, the absorbance was measured at A ₅₇₀ , and the standard curve of L-glutamate was prepared and quantified.

2. Molecular Weight Measurement of PGA

The molecular weight of PGA was measured using SDS-PAGE and FDNB methods. Purified PGA was electrophoresed with high molecular weight marker (Amersham Biosciences) on SDS-PAGE at 10% concentration. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 and bleached with a destaining solution consisting of 7% acetic acid / 10% methanol. After dyeing again with a dye solution consisting of methylene blue / 3% acetic acid, it was decolorized using distilled water. At this time, the average molecular weight of PGA was measured according to the FDNB method [document information 6].

[ Example  1] Isolation and Identification of Strains of the Invention

Aerobic and spore-forming microorganisms producing polygamma glutamic acid (PGA) were intended to be separated from compost in the South Sea of Gyeongsangnam-do.

The compost was suspended in sterile water, boiled for 10 minutes, inoculated in LB agar medium and incubated at 37 ° C. for 2-3 days. Among the colonies formed on the medium, the viscous colonies were inoculated in LB liquid medium and cultured one day before, followed by PGA production medium (2% sucrose, 3% L-glutamic acid, 1% (NH ₄ ) ₂ SO ₄ , 0.27% KH). ₂ PO ₄ , 0.42% Na ₂ HPO ₄ , 0.5% MgSO ₄ · 7H ₂ O, and 0.5 μg / ml biotin, pH 7.5) was inoculated 1% (v / v) and incubated for 5 days at 37 ℃.

The isolates were analyzed for 16s rRNA sequences and examined for physiological and biochemical properties using API 50CH identification kit (Biomerieux, France), and the results were identified according to Bergy's Manual of Systematic Bacteriology [Ref. 20]. .

As a result, first, about 50 kinds of strain colonies isolated purely by heat treatment were pre-cultured in each LB medium (LB medium), and then cultured in PGA production medium for 72 hours to produce PGA. In comparison, 10 strains producing PGA were selected from 50 kinds of bacteria isolated purely, and among them, the strain having the highest productivity was selected.

In addition, the morphological and physiological characteristics of the most productive strains, the availability of carbon sources, and the measurement results of the 16s rRNA sequences are shown in Table 1 below.

As a result, the final selected strains were Gram-positive bacilli, motile and heat-resistant spores, and Bacillus was identified and identified by 16s rRNA sequencing using the Bacillus strain identification kit (API 50CH). Subtilis BFAS ( Bacillus subtilis BFAS) and 99.4%, Bacillus vallismortis and 99.1%, It showed 98.8% homology with Bacillus licheniformis (see SEQ ID NO: 1).

Accordingly, the present inventor named the final selection strain Bacillus subtilis CH-10, and the strain was deposited with the Agricultural Biotechnology Research Institute under the Agricultural Promotion Agency on July 20, 2005, and was given accession number KACC 91169P.

[ Example  3] Optimum Condition of PGA Production

In this example, to determine the PGA mass production conditions of the Bacillus subtilis CH-10 strain of the present invention, to find the optimal culture conditions such as pH, temperature, carbon source and nitrogen source.

PH and temperature

Changes in PGA production with pH and temperature were measured.

As a result, as shown in (A) of FIG. 1, it was confirmed that PGA production was significantly weak in an acidic environment, and PGA production was increased in an alkaline environment. Was determined to be 7.5.

In addition, according to (B) of Figure 1, the difference in PGA production according to the temperature was shown, it can be seen that the production of the highest amount of PGA at 37 ℃.

2. Carbon source  Nitrogen source

(1) carbon source

To determine the carbon and nitrogen sources for optimal PGA production, first, the carbon source is glucose, fructose, galactose, sucrose, maltose, glycerol, and xyl. Loss of xylose and citric acid were added to the PGA production medium to a final concentration of 2%. In addition, the Bacillus subtilis CH-10 of the present invention was cultured in a medium to which each carbon source was added, and the amount of PGA generated was determined by a ninhydrin reaction, and the nitrogen source was L-glutamate, L-glutamine, sodium nitrate (NaNO ₃ ), ammonium nitrate (NH ₄ NO ₃ ), ammonium chloride (NH ₄ Cl), ammonium sulfate [(NH ₄ ) ₂ SO ₄ ], casamino acid ), The production of PGA was examined by adding peptone to 1% PGA production medium.

As a result, in the case of a carbon source, as shown in Table 2, when xylose and citric acid were added, the strain hardly grew and therefore did not produce PGA, which means that other PGA producing strains used citric acid as a carbon source. In contrast to Information 9], the strain of the present invention did not use citric acid, but when glycerol was added, the strain grew but produced little PGA, whereas glucose, fructose, galactose, sucrose, maltose PGA production increased significantly, and sucrose produced 18.84 mg / ml PGA as the most useful carbon source for PGA production of B. subtilis CH-10.

(2) nitrogen source

On the other hand, as a result of measuring the change in PGA production of this strain using a variety of nitrogen sources in addition to sucrose as a carbon source, as shown in Table 3, sodium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate as the strain is grown N-glutamate and L-glutamine produced 12 and 6.43 mg / ml of PGA, respectively.

Therefore, it was found that the strain of the present invention is a glutamate dependent strain that requires glutamate for PGA production and strain growth.

And, in general, a large amount of glutamate is required for PGA synthesis, which is known to be produced from citric acid and ammonium sulfate, and generally two different routes are required for the synthesis of L-glutamate, one of which is glutamate dehydro The glutamate dehydrogenase pathway (GD) [Ref. 19] proceeds to the reaction of formula (1), and another route is glutamine synthetase (GS) and glutamine 2-oxoglutarate amino. It is reported that the transferase (Glutamine 2-oxoglutarate aminotransferase; GOGAT) [Ref. 12] proceeds to the reaction scheme as shown in the following formulas (2) and (3).

[expression]

Therefore, since the strain of the present invention necessarily requires L-glutamate for PGA production, and also L-glutamine also activates PGA synthesis, L-glutamate is produced according to the schemes of Formulas (2) and (3) above. I think that.

3. Bacillus Subvitlis  Changes in PGA Production According to Incubation Time of CH-10

For the strain of the present invention, to determine the PGA production and molecular weight according to the incubation time.

As a result, as shown in Figure 2, the strain of the present invention began to produce PGA from 12 hours of cultivation, PGA production increased with time and the maximum yield of PGA (18 mg / culturing 96 hours) ml).

In addition, the change in pH according to the culture gradually changed to acidic until 36 hours of culture, and then changed to neutral again, but there was no significant pH change.

In addition, as shown in Figure 3, using the SDS-PAGE to confirm the molecular weight of the PGA produced according to the culture time, after 84 hours of incubation, the low molecular weight PGA of 200 kDa or less increased, After 120 hours of incubation the amount of PGA produced decreased.

As a result, the strain of the present invention is estimated to degrade PGA produced by secretion of PGA depolymerase [document information 5] extracellularly as the culture time passes.

In addition, the average molecular weight of PGA after 96 hours of cultivation by the FDNB method showed an average molecular weight of 1100 kDa (not shown). Among the PGA producing strains, Bacillus subtilis (chungkookjang) and Natrialba agyattiaka strains are significantly different compared to producing PGA having a molecular weight of 10 ⁶ Da or more, the strain of the present invention is Bacillus subtilis (natto ) And Bacillus Richenformis It was found to be similar to the molecular weight of 10 ⁵ ~ 10 ⁶ Da of IFO 3335 [Document 4].

On the other hand, Zhao et al. Recently isolated Bacillus mesentericus MJM1, which produces PGA, and in this strain the gamma-glutamyltranspeptidase involved in PGA production. It was reported that the gene was present on plasmid pMMH1 [plasma 24], the strain of the present invention was found to have no plasmid as a result of examining the presence or absence of plasmid, thus producing PGA The gene involved in is thought to be present on the chromosome.

In addition, it is expected that the production of PGA of various sizes will be possible if a detailed physical property study of PGA produced by the strain of the present invention and the cutting of PGA by PGA degrading enzyme are studied.

As described above, the novel Bacillus genus YN-1 strain of the present invention has excellent production efficiency of high quality polygamma glutamic acid in a medium containing a common carbon source and nitrogen source, and when applied to the production process of polygamma glutamic acid using microorganisms, High quality and low cost polygammaglutamic acid can be provided.

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Claims (6)

Bacillus subtilis CH-10 strain having accession number KACC 91169P for producing polygammaglutamic acid from a medium containing a common carbon source and nitrogen source. A method for producing polygamma glutamic acid comprising culturing the strain of claim 1 in a medium containing a common carbon source and nitrogen source, and obtaining polygamma glutamic acid from the culture. The method of claim 2, The medium is a method for producing polygamma glutamic acid, characterized in that the medium containing at least one carbon source selected from the group consisting of sucrose, glucose, fructose, galactose and maltose. The method of claim 2, The medium is a method for producing polygamma glutamic acid, characterized in that the medium containing glutamic acid or glutamine as a nitrogen source. The method according to any one of claims 2 to 4, The culture medium is 2% sucrose, 3% L- glutamate, 1% ammonium sulfate, 0.1% potassium phosphate, 0.1% of sodium hydrogen phosphate _{(Na 2 HPO 4 · 12H 2} O), 0.05% magnesium sulfate (MgSO _4), 0.005% ferric chloride, 0.02% calcium chloride, 0.002% manganese sulfate and 0.00005% biotin and the remainder is made of sterile water. The method according to any one of claims 2 to 4, The culturing is a method for producing polygamma glutamic acid, characterized in that the culture at a temperature of 37 ℃ and pH 7.5.

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