KR20160017797A - Polyglutamic Acid production method use Bacillus subtillis MJ80 strain - Google Patents

Abstract

The present invention relates to isolation and identification of a soil-derived novel microorganism B.subtillis MJ80 strain producing polyglutamic acid from soil, and to a high efficiency method for producing and purifying polyglutamic acid using the same.

Description

[0001] The present invention relates to a novel Bacillus subtilis MJ80 strain and a method for producing the same using polyglutamic acid production method using Bacillus subtillis MJ80 strain,

The present invention relates to a novel strain of Bacillus subtilis MJ80 and a method for producing flurbactamic acid using the same.

Polyglutamic acid is an anionic polypeptide polymer composed of polymerized D- and L-glutamic acid units through (-PGA) -amide linkages (Ashiuchi and Misono 2002). The polyglutamic acid molecules produced by the microorganisms have various molecular weights ranging from 10 to 1000 kDa, which are homopolymers of D-glutamic acid (-D-PGA), homopolymers of L-glutamic acid (-L-PGA) / -L-glutamic acid (-DL-PGA) (Ashiuchi and Misono, 2003).

Polyglutamic acid is a non-toxic, biodegradable, eco-friendly edible amino acid. Polyglutamic acid has a wide range of applications such as food, cosmetics, medicine, wastewater treatment (Shih et al., 2001), and products using polyglutamic acid and polyglutamic acid include sedimentation concentrators, wetting agents, Freezing prevention, bitter taste reducing food additives, and drug carriers.

The microorganisms producing polyglutamic acid belong mainly to the Bacillus species, and the polyglutamic acid was the first to be found in Bacillus anthracis and was a capsule component and an important toxic factor. Polyglutamic acid is also known to exist in the mucus of Japanese traditional fermented soybean natto (Fuji 1963, Makino et al., 1989), which is secreted into the culture medium of B. subtilis .

Recently, we have been able to find polyglutamic acid production sources from various soybean fermented foods containing a large amount of salt (Ashiuchi et al., 2001). During the search for polyglutamic acid - producing microorganisms, B. subtilis C1 strains resistant to high salt were identified and this strain produced polyglutamic acid derivatives combined with glycerol, a biopolymer. The novel archaeobacteria species Natrialba aegyptiaca survived extreme environmental stresses with a NaCl concentration above 20% due to their strong hydrophilic properties.

Polyglutamic acid producing strains are divided into two groups according to nutritional requirements. In the first group, L-glutamic acid-dependent strains require glutamic acid for polyglutamic acid production and glucose, citric acid and glycerol as carbon sources for the production of polyglutamic acid or the activation of polyglutamic acid synthase system in need. This group includes B. anthracis, B. subtilis IFO 3335, B. licheniformis ATCC 9945A and B. subtilis MR-141 (Goto and Kunioka, 1992), B. licheniformis ATCC 9945A in the case of glutamic acid, citric acid And a culture medium containing glycerol are used. In addition, citric acid and glutamic acid are used as precursor substrates for polymer production (Cromwick et al., 1995b).

Production of the second group, L-glutamic acid-independent polyglutamic acid, requires an organic / inorganic nitrogen source and a variety of sugar components under aerobic conditions. This group includes B. subtilis (Natto) 5E, B. subtilis TAM-4, B. licheniformis A35, and B. licheniformis S173.

Productive strains of polyglutamic acid affect the productivity of polyglutamic acid in addition to the carbon source by factors such as nitrogen source, ionic strength (Cromwick et al., 1995a) and acidity of the culture medium (Cromwick et al., 1996) . Although organic nitrogen sources such as yeast extract and peptone do not affect the production of polyglutamic acid, inorganic nitrogen sources such as ammonium sulfate and ammonium chloride affect the production of polyglutamic acid (Kunioka, 1995) .

As a prior art relating to a production strain of polyglutamic acid, Korean Patent Application No. 10-2004-0083455 discloses a technique for producing a polyglutamic acid derived from Bacillus subtilis BS621, 10-1998-0011707 discloses a method for producing Bacillus subtilis TB11 strain and an aggregated polymer material having excellent heat resistance from the strain.

Accordingly, an object of the present invention is to isolate and identify novel microorganisms originating from soil producing polyglutamic acid.

Another object of the present invention is to provide a method for efficiently producing polyglutamic acid from glutamic acid or soybean powder using the novel microorganism derived from the soil and a method for purifying the same.

The object of the present invention is to provide a method for detecting a polyglutamic acid strain from soil and analyzing a genetic characteristic of a polyglutamic acid producing strain; The polyglutamic acid producing strain in which the genetic characteristic was analyzed in the above step was identified as a strain of B. subtillis species, named B. subtillis MJ80, deposited with the Institute of Agricultural Biotechnology and received the deposit number KACC91728P; Establishing optimal culture conditions for the production of polyglutamic acid of B. subtillis strain MJ80 ; Establishing a production method of polyglutamic acid using soybean powder as a substrate to replace glutamic acid in the optimal culture condition; Through the step of establishing a purification method capable of obtaining polyglutamic acid with high efficiency from B. subtillis MJ80 culture obtained through the above optimal culture conditions and using soybean powder.

The present invention relates to a novel microorganism derived from soil, which produces polyglutamic acid. It is effective to provide subtillis MJ80.

In addition, there is an excellent effect of providing a purification method capable of increasing the yield of polyglutamic acid produced by the novel microorganism B. subtillis MJ80.

Brief Description of the Drawings Fig. 1 is a diagram showing the results of analysis of the strain Bacillus subtilis MJ80 of the present invention and its genetic system.
FIG. 2 is a photograph of the molecular weight of polyglutamic acid produced by the Bacillus subtilis MJ80 strain of the present invention, which was confirmed by SDS-PAGE. FIG.
FIG. 3 is a graph showing the results of measurement of the amount of polyglutamic acid produced and the acidity of the culture of Bacillus subtilis MJ80 according to the present invention, depending on the culture medium and the number of days of culture.
FIG. 4 is a graph showing the results of measurement of the amount of polyglutamic acid produced and the acidity of the culture of Bacillus subtilis MJ80 according to the present invention.
FIG. 5 is a graph showing the results of measurement of polyglutamic acid production and acidity of cultured Bacillus subtilis MJ80 strain according to the amount of L-glutamic acid added.
FIG. 6 is a graph showing the results of measurement of polyglutamic acid production amount and acidity of cultured Bacillus subtilis MJ80 strain according to carbon source.
FIG. 7 is a graph showing the results of measuring the amount of polyglutamic acid produced and the acidity of the culture of Bacillus subtilis MJ80 according to the present invention.
FIG. 8 is a graph showing the results of measurement of polyglutamic acid production amount and acidity of the culture of Bacillus subtilis MJ80 according to the present invention.
FIG. 9 is a graph showing the results of measurement of polyglutamic acid production amount and acidity of culture of Bacillus subtilis MJ80 strain according to the present invention in accordance with the amount of Urea and NH ₄ Cl added.
FIG. 10 is a graph showing the results of measurement of polyglutamic acid production amount and acidity of culture of Bacillus subtilis MJ80 strain according to the present invention according to the amount of glycerol added.
11 is a graph showing the results of measurement of polyglutamic acid production amount and acidity of culture of Bacillus subtilis MJ80 strain of the present invention according to the amount of citric acid added.
FIG. 12 is a graph showing the results of measuring the amount of polyglutamic acid produced and the acidity of the culture of Bacillus subtilis MJ80 according to the present invention.
13 is a graph showing the results of measurement of polyglutamic acid production amount and acidity of culture of Bacillus subtilis MJ80 strain according to the present invention in accordance with the addition amount of MgSO ₄ 7H ₂ O. FIG.
FIG. 14 is a graph showing the results of measurement of the amount of polyglutamic acid produced and the acidity of the culture of Bacillus subtilis MJ80 according to the present invention.
FIG. 15 is a photograph showing the result of SDS-PAGE of the purification effect of the polyglutamic acid derived from the Bacillus subtilis MJ80 strain according to the present invention with the protease K treatment time.
FIG. 16 is a photograph showing the result of analysis of the polyglutamic acid hydrolyzate derived from the Bacillus subtilis MJ80 strain of the present invention through TLC. FIG.
17 is an FT-IR spectrum graph of polyglutamic acid derived from the Bacillus subtilis MJ80 strain of the present invention cultured in an SB culture medium according to the present invention.
18 is an FT-IR spectrum graph of polyglutamic acid derived from the Bacillus subtilis MJ80 strain of the present invention cultured in an SF culture medium according to the present invention.
19 is an FT-IR spectrum graph of polyglutamic acid derived from the Bacillus subtilis MJ80 strain of the present invention cultured in a GA culture medium according to the present invention.
FIG. 20 is a diagram illustrating an entire process of a method for identifying the Bacillus subtilis MJ80 strain of the present invention and a method for purifying polyglutamic acid produced from the MJ80 strain of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples and experimental examples. However, these examples and experimental examples are only for the sake of understanding the present invention, and the scope of the present invention is not limited in any sense in any sense.

< Example  1> invention PGA Search for production strains

Soil samples were collected from Jeollanam - do rice fields, dried at room temperature, and then dried. Soil samples were uniformly ground in a mortar and sifted through 150 .mu.m sieve. 1 g of the soil sample was mixed with 50 mL of 0.9% (w / v) NaCl aqueous solution and stirred at 150 rpm and 37 DEG C for 1 hour. Then, 1 mL of the supernatant of the above-mentioned stirred mixture was plated on LB agar (Merck cooperation) and cultured at 37 ° C for 24 hours.

After the incubation, only single colonies were taken from the microbial communities grown on the LB agar medium and inoculated into the modified SB (Soybean) and glutamic acid culture medium described in Table 1, respectively, To obtain an inoculum. The obtained inoculum was inoculated into the modified SB (soybean) and glutamic acid culture medium of Table 1, respectively, and cultured at 37 DEG C for 96 hours.

The present invention provides a culture medium for the search for a producing strain of fl glutamic acid Component Amount (g / L) Soybean 100g Citric acid 12g Glycerol 80ml NH ₄ Cl 7g K ₂ HPO ₄ 0.5 g MgSO ₄ .7H ₂ O 0.5 g MnSO ₄ 0.1 g CaCl ₂ 0.15 g FeCl ₃ 0.04 g

The cultured microorganisms were cultivated in the modified SB (Soybean) and glutamic acid culture medium shown in Table 1 below in the form of cluster of each microorganism, spore formation, Respectively.

The characteristics of the microorganisms were observed through Bergey's Manual. For the identification of the microorganisms, all the microbial cultures were stored at 4 ° C and stored at -80 ° C for 2 months.

As a result of the experiment, about 1,500 bacterial populations were obtained from the soil, and 73 strains were selected by observing the growth pattern such as colony shape, spore formation and color of each microorganism. Among them, as shown in Table 2, the best MJ80 strain was selected at a final yield of 15 to 20 g / L of plglutamic acid.

Measurement of flurbactamic acid production of MJ80 strain of the present invention GA-medium (g / L) SB-medium (g / L) The present invention (MJ80) 6.71 17.82 A comparative strain 0.58 1.83

< Example  2> invention PGA production MJ80  Analysis of growth and morphological characteristics of strains

 The morphology of the inventive flurbu- tamic acid high production-producing strain MJ80 isolated according to Example 1 was as follows: GS-SS agar plate medium, which is a production medium of fl glutamic acid, and LB agar plate culture medium, The MJ80 strain of the present invention was observed while culturing.

The growth characteristics of the strain MJ80 according to the present invention were evaluated by comparing the strain MJ80 of the present invention and the strain B.J. subtilis , B. licheniformis , B. vallismortis were inoculated and cultured under the microscope. The effects of temperature, salt tolerance, plasmid content, spore formation ability, nitrate reduction ability, indole production ability, gelatin / starch decomposition ability, B Whether or not the use of glycerol, galactose, glucose, sucrose, maltose, starch, etc. was determined by API 50CH identification kit (Biomerieux, France) And the experimental results are shown in Table 3 below.

Biochemical characterization of MJ80 strain of the present invention oTest Result No. Test Result Characteristics MJ80 28 Amygdalin - Morphology shape Rod 29 Oxidase - Gram stain + 30 Arbutin - Cell dimension (탆) 0.5 to 0.8x2 to 5 31 Esculin ferric citrate + Catalase + 32 Salicin + Spore + 33 Cellobiose + Glycerol + 34 Maltose + Erythritol - 35 Lactose - D-Arabinose - 36 Melibiose + L-Arabinose + 37 Sucrose + D-Ribose + 38 Trehalose + D-Xylose + 39 Inulin + L-Xylose - 40 Melezitose - D-Adonitol - 41 Raffinose + beta methyl-D-xyloside - 42 Starch + D-Galactose - 43 Glycogen + D-Glucose + 44 Xylitol - D-Fructose + 45 Gentiobiose - D-Mannose + 46 D-Turanose - L-Sorbose - 47 D-Lyxose - L-Rhamnose - 48 D-Tagatose - Dulcitol - 49 D-Fructose - Inositol + 50 L-Fructose - D-Manitol + 51 D-arabitol - D-Sorbitol + 52 L-Arabitol - Methyl-aD-manopyranoside - 53 Potassium Gluconate - Methyl-aD-glucopyranoside - 54 Potassium 2-ketogluconate - N-Acetyl-glucosamine - 55 Potassium 5-ketogluconate -

< Example  3> invention MJ80  Gene analysis of strains

 Genetic analysis of the MJ80 strain of the present invention was performed by performing gene sequencing of 16S rDNA. The oligonucleotide primer sequences used for PCR were 8F (5'-AGA GTT TGA TCC TGG CTC AG-3 ') and 1492R (5'-ACG GCT ACC TTG TTA CGA GTT-3 ') was used to amplify the 16S rDNA gene by PCR. The PCR product obtained by the PCR was purified (PCR purification kit, Bioneer Inc.), ligated to a colonizing vector pCRII, and transformed using DH5a component cell. The transformed E. coli DH5a was inoculated into LB culture medium, cultured for 18 hours, and the recombinant plasmid was extracted and purified from the transformed DH5a. The recombinant plasmid was treated with a restriction enzyme enzyme (EcoR I) to confirm that the PCR product was inserted into the cloning vector. Then, the gene sequence of the insert was sequenced and the 16S rDNA sequence of the MJ80 strain of the present invention inserted into the cloning vector Decrypted and displayed in Sequence Listing 1. The 16S rDNA nucleotide sequence of the present invention strain MJ80 having the nucleotide sequence of SEQ ID NO: 1 was compared with 16S rDNA nucleotide sequences of various microorganisms reported through the NCBI homology gene and the homology was examined.

As a result of the experiment, as shown in Fig. Subtilis was identified as a strain. subtillis MJ80 and deposited it with the National Institute of Agricultural Science and Technology Center of the National Academy of Agriculture on July 30, 2012 and received the deposit number KACC91728P.

< Example  4> invention MJ80  Production efficiency of flourglutamic acid from strain

The MJ80 strain of the present invention was inoculated into 100 mL of SB medium containing 5 g / 100 mL of soybean powder and cultured at 37 DEG C for 72 hours. The MJ80 culture of the present invention was centrifuged at 6,000 rpm for 20 minutes, and then a culture supernatant was obtained. The culture supernatant and ethanol were mixed in a ratio of 1: 3 (v / v) 3 times, and the mixture was allowed to stand at 4 ° C for 10 hours and centrifuged to obtain a plglutamic acid precipitate. The plglutamic acid precipitate was dissolved in sterile distilled water Then, the undissolved solids were removed by centrifugation and only the supernatant was taken. Ethanol was added to the volume of the mixture obtained by mixing the same volume of sterilized distilled water as that of the supernatant and reprecipitated at 4 캜 for 10 hours. The resulting mixture was centrifuged and dried, and then purified with pure water Glutamic acid was obtained.

Experimental Results As shown in Table 4, 0.2 mL of flurbutamic acid was obtained by inoculating the MJ80 strain of the present invention into 100 mL of SB medium containing 5 g / 100 mL of soybean powder.

Production efficiency of flurbactamic acid of the present invention strain MJ80 cultured in SB culture medium Name PGA (g) % MJ80 2.14 4.28

< Example  5> Present invention MJ80 From strain  Produced flourglutamic acid of  Investigate characteristics

SDS-PAGE was used to measure the average molecular weight of the flurbylic acid obtained according to Example 4 above.

The purified purified glutamic acid obtained according to Example 4 above was electrophoresed on a 10% SDS-PAGE with a high molecular weight marker (Amersham Biosciences). In order to visualize the plglutamic acid and the high molecular weight marker on the electrophoretic SDS-PAGE, it was stained with Coomassie Brilliant Blue R-250.

2. Experimental Results As shown in FIG. 2, the molecular weight of the plglutamic acid produced by the MJ80 strain of the present invention was found to have a molecular weight of 200 kDa or more.

< Example  6> The present invention MJ80  Seeking optimal culture conditions of strains

GA culture medium according to the present invention, L-glutamic acid 40 g / L , citric acid 12 g / L, glycerol 80 g / L, yeast extract 7 g / L, MgSO 4 and _{7H 2 O 0.5 g / L,} FeCl 3 and _{6H 2 O 0.04 g / L,} K 2 HPO 4 0.5 g / L, CaCl 2 and 2H ₂ O 0.15 g / L, and MnSO ₄ and H ₂ was prepared by O 0.1 g / L, SB ( Soybean Boiling) culture medium Was prepared by adding 100 g / L of soybean powder 40 to 60 mesh in place of L-glutamic acid in the GA culture medium composition. Then, the two kinds of medium were used as a culture medium of the present invention strain MJ80 by high pressure sterilization.

The culture medium of SF (Soybean Fresh) was prepared in the same composition as the SB culture medium but without high pressure sterilization.

The optimal culture conditions of the MJ80 strain of the present invention were inoculated into the above three culture media adjusted to pH 7.0, and then cultured at 37 ° C and 150 rpm.

Experimental Example 1: Inoculation number of the MJ80 strain of the present invention , soybean powder and L- glutamic Determination of flurbactamic acid production rate according to the amount of acid added

The present invention B. subtilis MJ80 was inoculated into SB, SF and GA culture medium, respectively, and then cultured for 7, 11 and 7 days. At this time, the inoculation number of the MJ80 strain of the present invention inoculated into the above three culture media was 1, 2, 3, 4, 5 and 7% (v / v) in which the CFU was adjusted to 1 x 10 ⁷ / Respectively.

Experimental Results As shown in Fig. 3, when the inoculum was inoculated with 5% (v / v) and cultured in a GA culture medium for 6 to 7 days, the production rate of plughlutamic acid was the best at 20 to 45 g / L .

The optimal culture conditions of the present invention strain MJ80 were 0, 2, 4, 6, 8, 10, 12.5, 15, 17.5 and 20% (w / v) Respectively.

As shown in FIG. 4, when soybean powder was added to the SB or SF culture medium at a concentration of 125 and 175 g / L, respectively, the flourglutamic acid produced by the MJ80 strain of the present invention was produced at 15 to 45 g / L It was confirmed that the added concentration of soybean powder was preferably 125 to 175 g / L.

As shown in FIG. 5, the optimal culture conditions for the high-efficiency production of the plglutamic acid of the MJ80 strain of the present invention according to the concentration of L-glutamic acid in the GA culture medium were as follows: L-glutamic acid was added at a concentration of 70 g / , The production efficiency of plaglutamic acid produced by the MJ80 strain of the present invention was the best at 50 g / L, and the concentration of glutamic acid was 60 to 90 g / L.

Experimental Example  2: On carbon  According to the present invention MJ80 Production rate of flourglutamic acid in strain

The fructose, galactose, glucose, lactose, maltose, soluble starch and sucrose were added to each culture medium of SB, SF and GA prepared according to the present invention in an amount of 2% (w / v) And the MJ80 strain of the present invention was cultured.

Experimental Results As shown in FIG. 6, B. subtilis MJ80 showed the highest production efficiency of plubl glutamic acid when starch was added to SB, SF, and GA culture media and used as a carbon source. In particular, when starch was added to SB or GA culture medium, 40 to 60 g / L of flig glutamic acid was produced. When starch was added to the SF culture medium, 30 g / L of fl glutamic acid was produced.

Starch was added to each culture medium of SB, SF and GA at concentrations of 0, 10, 20, 30, 40, and 50 g / L, respectively, in order to confirm the desirable concentration of starch added to increase the production rate of flourglutamic acid The production of the plul glutamic acid was measured. As shown in FIG. 7, when the starch of 10 to 30 g / L was added to each culture medium, it was confirmed that the production efficiency of plubl glutamic acid was increased.

Experimental Example  3: invention according to nitrogen source MJ80 Production rate of flourglutamic acid in strain

In order to examine the preferred nitrogen source of the MJ80 strain of the present invention, ammonium chloride, ammonium sulfate, beef extract, casein peptone, malt extract, tryptone, urea and yeast extract were added to each culture medium of SB, SF and GA prepared according to the present invention. % (w / v), respectively, and the MJ80 strain of the present invention was cultured.

Experimental Results As shown in FIG. 8, B. subtilis MJ80 was found to have the highest production efficiency of plubl glutamic acid when ammonium chloride or urea was added to each culture medium of B, SF and GA and used as a nitrogen source.

3, 5, 7, 9, and 12 g / ml were added to each culture medium of SB, SF, and GA to confirm the amount of ammonium chloride or urea added to increase the production efficiency of the plglutamic acid of the MJ80 strain of the present invention, L of ammonium chloride or urea were added to the flourglutamic acid.

As shown in FIG. 9, when the ammonium chloride or urea of 5 to 12 g / L was added to each culture medium, it was confirmed that the production efficiency of the flurbactamic acid was increased.

Experimental Example 4: Determination of flurbactamic acid production rate of the MJ80 strain of the present invention by glycerol addition

(W / v) soybean powder, 2% (w / v) soluble starch and 0.7% (w / v) starch in order to evaluate the production efficiency of the plglutamic acid of the present invention strain MJ80 under glycerol addition conditions (w / v) soybean, 2% (w / v) soluble starch and 0.7% (w / v) urea, 20, 40, 80, 120 and 160 g / L of glycerol were added to each of the GA culture medium consisting of L-glutamic acid, 2% (w / v) soluble starch and urea and then the MJ80 strain of the present invention was cultured.

As shown in FIG. 10, when glycerol was added to each culture medium of SB, SF and GA at a concentration of 120 g / L, the production of flourglutamic acid was as high as 30 to 55 g / L.

Experimental Example  5: Citric acid  According to the present invention MJ80 Production rate of flourglutamic acid in strain

In order to evaluate the production efficiency of the flurbactamic acid in the citric acid addition condition of MJ80 strain of the present invention, 15% (w / v) soybean, 2% (w / v) soluble starch, and 0.7% (W / v) soy broth, 2% (w / v) soluble starch, and 0.7% (w / v) urea, and 7% (w / v) L-glutamic Citric acid was added at 0, 4, 8, 12, 16, 20, and 24 g / L to each GA culture medium consisting of 2% (w / v) soluble starch and 0.7% (w / v) urea The MJ80 strain of the present invention was cultured.

As shown in FIG. 11, when 12 or 24 g / L of citric acid was added to citric acid and culture medium of SB and GA, the production of flourglutamic acid was as high as 30 to 55 g / L.

Experimental Example 6: Production rate of flurbactamic acid of MJ80 strain of the present invention by addition of NaCl

0.5, 1, 2, 3, and 4 were added to each of the same SB, SF, and GA culture mediums as in Experimental Example 5 to evaluate the production efficiency of the strain and the flurbumatic acid under the NaCl addition conditions of the MJ80 strain of the present invention % (w / v) NaCl was further added thereto, followed by culturing the MJ80 strain of the present invention.

Experimental Results As shown in FIG. 12, the addition of NaCl to SB, SF, and GA culture mediums resulted in 20 to 30 g / L of fl glutamic acid produced in SB, SF, and GA culture medium, When the molecular weight of each of the produced polyglutamic acid was measured by SDS-PAGE, it was confirmed that the higher the concentration of NaCl added, the larger the molecular weight of the plughlutamic acid was.

Experimental Example 7: MgSO ₄ or MnSO ₄ Production rate of flurbactamic acid of MJ80 strain of the present invention by addition

In order to evaluate the production efficiency of the plglutamic acid under the conditions of adding MgSO ₄ or MnSO ₄ of the MJ80 strain of the present invention, 0, 0.25, 0.5, 0.75 , 1 g / L MgSO ₄ .7H ₂ O or 0, 0.05, 0.1, 0.15, and 0.2 g / L MnSO ₄ were further added, respectively, and then the MJ80 strain of the present invention was cultured.

Experimental Results As shown in FIGS. 13 to 14, the addition of MgSO ₄ .7H ₂ O or MnSO ₄ did not significantly affect the production efficiency of the flurbumatic acid.

According to the present invention, the culture conditions for producing the MJ80 strain of the present invention at a high efficiency are as shown in the following Tables 5 and 6, and the MJ80 strain produces SB, SF and GA culture medium, respectively Plyglutamic acid is 35-40, 16-20 and 55-60 g / L.

The optimal medium composition of the MJ80 strain of the present invention established according to the present invention
Condition
Glutamic acid-independent (g / L)
Glutamic acid-dependent (g / L)
Soybean Powder Boiled  ( SB )
125
-
- Soybean Powder Fresh  ( SF ) - 175 - Glutamic acid  ( GA ) - - 70 Carbon  ( Starch ) 50 30 50 Nitrogen 12 (NH ₄ Cl) 9 (Urea) 7 (Urea) Citric acid 12 24 12 Glycerol 120 180 120 NaCl 10 10 10 MgSO4 ₄ 7H ₂ O 0.25 0.25 0.25 MnSO ₄ 0.05 0.05 0.05 K ₂ HPO ₄ 0.5 0.5 0.5 CaCl ₂ 0.15 0.15 0.15 FeCl ₃ 0.04 0.04 0.04 Temperature  (° C) 37 37 37 Agitation  ( rpm ) 150 150 150 pH 7 7 7 Incubation time  ( Day ) 7 11 5

Optimal culture conditions of the MJ80 strain of the present invention established according to the present invention Medium composition Culture conditions Production of γ-PGA (g / L) Glutamic acid-dependent γ-PGA producers Lecithin (70 g / L), Starch (50 g / L), Urea (7 g / L), Citric acid (12 g / L), Glycerol 37 ℃, 5 days 55-60 Glutamic acid-independent γ-PGA producers Lecithin, Glycerol (120 g / L), NaCl, * Trace element (125 g / L), Starch (50 g / L), NH ₄ Cl 37 ℃, 5 days 35-40 Liposomes were prepared by adding trace elements such as soybean fresh (175 g / L), Starch (30 g / L), Urea (9 g / L), Citric acid (24 g / L), Glycerol 37 ℃, 11 days 16-20

_{* Trace elements: MgSO 4 · 7H} 2 O (0.25g / L), MnSO 4 (0.05g / L), K 2 HPO 4 (0.5g / L), CaCl 2

< Example 7> Invention MJ80 High-Efficiency Purification Method of Strain-derived Fliglutamic Acid

Experimental Example 8: Effect of the present invention on pH MJ80 Strain derived Purification efficiency measurement of poly-gamma glutamic acid

Optimum Culture Conditions for Producing B. glutamicum MJ80 Strain of High Efficiency of B. subtilis MJ80 According to Table 5, strains MJ80 of the present invention were inoculated into SB, SF and GA culture medium, Lt; / RTI &gt; days. After the culture was completed, each culture was centrifuged at 2,000 rpm at 6,000 rpm, and the pH of the culture was adjusted to 3, 4, 5, and 6 using 6N H ₂ SO ₄ , respectively. The purification efficiency of Crude γ-PGA produced from this was measured by the following method. The crude Crude γ-PGA was dissolved in distilled water and centrifuged to recover the insoluble fraction, which was then dried and the dry weight of the first precipitate was measured. The solubilized material (supernatant) recovered in this process was neutralized again with 6N NaOH, and the precipitated material (insoluble; neutralization) was centrifuged to collect only the precipitate, dried, and the dry weight of the precipitate was measured. The neutralized material (supernatant) was obtained by using ethanol to obtain a precipitated substance (? -PGA), followed by drying and measuring the dry weight of? -PGA.

Experimental Results As shown in Table 7, when the culture liquid was titrated to pH 3.0 before the purification of the plul glutamic acid, the highest purity and yield of the plglutamic acid were observed when the plglutamic acid was precipitated.

Purification efficiency of the plglutamic acid derived from the MJ80 strain of the present invention by pH adjustment pH Crude  (g) water - Insoluble (g) Insoluble after neutralization (g) γ- PGA weight  (g) Purity
(%) Purity Impurity 3 2.82 0.32 0.118 2.382 0.438 84.5 4 2.15 0.18 0.215 1.755 0.395 81.6 5 2.26 0.22 0.349 1.691 0.569 74.8 6 1.53 0.26 0.053 1.217 0.313 79.5

Experimental Example 9: Invention of MJ80 according to Proteinase K treatment Determination of purification efficiency of strains derived from pseudolactic acid

The crude glutamic acid obtained in Example 7 was dialyzed 2 to 3 times with 10 mM Tris-HCl buffer (pH 8.0), treated with 25 μg / mL of Proteinase K, And 10 hours, respectively, and electrophoresed on SDS-PAGE to confirm the purification and purity of the fl glutamic acid.

Proteinase K was purified by electrophoresis on purified glutamic acid by the treatment time. As shown in FIG. 15, when pure glutamic acid was obtained for 4 to 6 hours, pure glutamic acid was obtained.

Experimental Example 10: Trichloroacetic acid acid &lt; RTI ID = 0.0 & gt; ( TCA ) &lt; / RTI & Strain derived Measuring efficiency of purified glutamic acid purification

The present invention B. subtilis Optimal Culture Conditions for Producing MJ80 Strain with High Efficiency of Plurultamic Acid [0254] The strains of the present invention were inoculated into SB, SF and GA culture medium according to the above Table 5, respectively, and cultured for 5, 11, and 5 days This study was carried out to investigate the purification efficiency of γ-PGA. 250 mg of purified γ-PGA was treated with 25 mL of 2.5% Trichloroacetic acid (TCA). The precipitate (impurity) obtained by centrifuging this solution was dried at 50 캜 and its weight was measured. In contrast, the dissolved material (supernatant) was adjusted to pH 7.0 by treatment with 1 N NaOH. The solution was centrifuged again, and the dry weight of the precipitate was measured. The precipitate was recovered by adding ethanol at a ratio of 1: 3 (v / v) to the supernatant. The precipitate was rinsed with 80% ethanol and finally purified by pure water using 100% ethanol to measure the dry weight.

Experimental Results As shown in Table 8, when the respective purification ratios were compared, it was found that all of the produced plughlutamic acids were purified more than 85% purely, indicating that the produced plublactamic acid was purely purified I could.

Purification efficiency of plglutamic acid derived from MJ80 strain of the present invention by TCA treatment With TCA With Distilled Water SB SF GA SB SF GA ^(A) -PGA (mg) 264 251 266 262 256 254 ^(B) insoluble content (mg) 10 35 One 27 28 2 ^(C) After adjust pH 7.0 with 6N NaOH (mg) 3 4 0 2 4 0 ^(D) Impurity amount (B + C) 13 39 One 29 32 2 ^(E) Impurity  (%) 4.9 15.5 0.4 11.1 12.5 0.8 ^(F) Purified-PGA (mg) 232 200 245 225 205 223

Calculation: (E)% = (D) / (A) X 100, (G)

(H)% = 100 - (E)

< Example  8> The present invention MJ80 Strain derived  Flourglutamic acid of  Character analysis

Experimental Example 11: TLC analysis of the hydrolyzed product of the MJ80 strain-derived flurbumatic acid of the present invention analysis

To hydrolyze γ-PGA, 0.2 mL of a 12.5 mg / mL γ-PGA solution and 0.8 mL of 7.5 N HCl were mixed and hydrolyzed by sealing at 105 ° C. for 24 hours. After hydrolysis, the vial was opened and the remaining HCl was evaporated. The remaining vial was dissolved in 1 mL of distilled water and used for TLC analysis.

As shown in FIG. 16A, the purified glutamic acid (line 1 in FIG. 16A) and its hydrolyzate (line 2 in FIG. 16A) and glutamic acid Acetic acid: H ₂ O = 4: 1: 3 (v: v) was prepared by dropping 0.5 mM, 5 mM, and 50 mM (lines 3, : v) as a developing solvent, the Rf values of the hydrolyzate of glutamic acid and the glutamic acid were the same on the TLC.

As a result of the experiment, as shown in FIG. 16B, the hydrolyzate of the purified glutamic acid according to Experimental Example 10 (line 4 in FIG. 16B) and 50 mM, 5 mM, and 0.5 mM 16 (B) lines 1, 2 and 3) were dispersed in Silica gel 60 (Merck) and analyzed using EtOH: H 2 O = 7: 3 (v: v) as a developing solvent, glutamic acid and glutamic acid Showed the same Rf values on TLC.

Therefore, it was confirmed that the hydrolyzed product of the flurbylamic acid obtained from the culture of strain MJ80 of the present invention was composed of only glutamic acid.

Experimental Example 12: FT - IR of the hydrolyzed product of the MJ80 strain-derived flurbumatic acid of the present invention

The culture medium of the present invention strain MJ80 was cultured in the medium composition shown in the above [Table 5]. These cultures were analyzed for COO-, C = O and N-H contained in the fl glutamic acid through FTIR / KBr (Perkinelmer, Korea) analyzer according to Experimental Example 10 above.

As a result of the experiment, peaks were confirmed at the absorption wavelengths of 3312, 1730 and 1630 cm ^-1 , which represent COO-, C═O and NH, respectively, as shown in Table 9 and FIG. 17 to FIG.

The wavenumber of purified glutamic acid according to the MJ80 strain culture medium of the present invention, Wavenumber (cm ^-1 ) Medium type (Graph) -COO ^- stretchpeak C = O stretch peak N-H stretch peak SB (Fig. 17) 3278.62 1713.31 1631.62 SF (Fig. 18) 3312.57 1743.30 1639.08 GA (Figure 19) 3284.40 1729.13 1630.03

Therefore, the present invention was isolated and identified in accordance with FIG. 20, and conditions for producing and purifying flurbumatic acid with high efficiency were established.

Industrial Applicability As described above, the present invention relates to a novel microorganism derived from soil, which produces plyglutamic acid. It is a very useful invention in the biomaterial industry because it has an excellent effect of providing a subtillis MJ80 strain and providing a highly efficient production and purification method of flourglutamic acid using the same.

Claims (6)

Bacillus subtilis MJ80 strain KACC91728P which has a 16S rDNA sequence represented by SEQ ID NO: 1 and produces glutamic acid in a medium containing glutamic acid or soybean powder at a concentration of 60 to 90 g / L. Culturing the Bacillus subtilis MJ80 strain KACC91728P having the 16S rDNA sequence represented by SEQ ID NO: 1 in a medium containing glutamic acid or soybean powder at a concentration of 60 to 90 g / L. The method according to claim 2, wherein the soybean powder is sterilized. 4. The method of claim 3, wherein the medium further comprises starch. The method according to any one of claims 2 to 4, wherein the medium comprises a nitrogen source such as ammonium chloride or urea, glycerol, citric acid, and NaCl.
4. The method of claim 3, further comprising: purifying the culture obtained from the culturing step to obtain a plglutamic acid; And
And adding trichloroacetic acid to the obtained plglutamic acid. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;

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