KR20070034822A - -1 Novel Bacillus sp. YN1 and method for producing poly gamma glutamic acid using the same - Google Patents

Abstract

The present invention relates to a novel Bacillus YN-1 strain producing polygamma glutamic acid and a method for producing polygamma glutamic acid using the same, wherein the strain is cultured in a medium containing a common carbon source and a nitrogen source, and the poly By providing a method for producing polygamma glutamic acid comprising obtaining gamma glutamic acid, it is possible to provide polygamma glutamic acid which is excellent in quality and inexpensive.

Polygamma glutamic acid, Bacillus YN-1 strain

Description

Novel Bacillus gen-1 strain producing polygamma glutamic acid and method for producing polygamma glutamic acid using the same {Novel Bacillus sp. YN-1 and method for producing poly gamma glutamic acid using the same}

1 is a graph showing the production of polygamma glutamic acid according to pH (A) and temperature (B) of the genus Bacillus YN-1 strain,

Figure 2 is a graph of the production of poly-gamma glutamic acid according to the carbon source of the genus Bacillus YN-1 strain,

Figure 3 is a graph of the production of polygamma glutamic acid according to fructose concentration of the genus Bacillus YN-1 strain,

Figure 4 is a graph of the production of polygamma glutamic acid according to the nitrogen source of the genus Bacillus YN-1 strain,

FIG. 5 is a schematic of the metabolic process in the body of L-glutamic acid independent polygammaglutamic acid producing strain,

Figure 6 is a photograph showing the change in the physical properties of the polygamma glutamic acid produced according to the incubation time of Bacillus YN-1 strain as a result of SDS-PAGE,

7 is a graph showing changes in PGA production and pH according to growth curve and growth time of Bacillus YN-1 strains.

Abe, K., Y. Ito, T. Ohmachi, and Y. Asada. 1997. Purification and properties of two isozymes of gamma-glutamyltranspeptidase from Bacillus subtilis TAM-4. Biosci . Biotechnol. Biochem . 61 : 1621-1625.

2.Ashiuchi, M., K, Tani, K, Soda, and H, Misono. 1998. Properties of glutamate racemase from Bacillus subtilis IFO 3336 producing poly-gamma-glutamate. J. Biochem . 123 : 1156-1163.

Ashiuchi, M., C. Nawa, T. Kamei, JJ Song, SP Hong, MH Sung, K. Soda, T. Yagi, and H. Misono. 2001. Physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of Bacillus subtilis. Eur . J. Biochem . 268 : 5321-5328.

4. Ashiuchi, M., T. Kamei, D.-H. Baek, S.-Y. Shin, M.-H. Sung, K. Soda, T. Yagi, and H. Misono. 2001. Isolation of Bacillus subtilis ( chungkookjang ), a poly-γ-glutamate producer with high genetic competence. Appl Microbiol Biotechnol . 5 7: 764-769.

5. Bovarnick, M. 1942.The formation of extracellular D (-) glutamic acid polypeptide by Bacillus subtilis . J. Biol . Chem . 145 : 415-424.

6. Cheng, C., Y. Asada, and T. Asida. 1989. Production of γ-polyglutamic acid by Bacillus subtilis A35 under denitrifying conditions. Agtic . Biol . Chem . 53 : 2369-2375.

7. Gota, A. and M. Kunioka. 1992. Biosynthesis and hydrolysis of poly (γ-glutamic acid) from Bacillus subtilis IFO3335. Biosci . Biotech. Biochem . 56 : 1031-1035.

8. Hara, T., Y, Fujio, and S. Ueda. 1982. Polyglutamate production by Bacillus subtilis ( natto ). J Appl Biochem . 4 : 112-120.

9.Holzer, H. 1969. Regulation of enzymes by enzyme-catalyzed chemical modification. Adv Enzymol . 32 : 297-326.

10. Ito, Y., T. Tanaka, T. Ohmachi, and Y. Asada. 1996. Glutamic acid independent production of poly (γ-glutamic acid) by Bacillus subtilis TAM-4. Biosci . Biotech. Biochem. 60 : 1239-1242.

11. Kubota, H., T. Matsunobu, K. Uotani, H. Takabe, A. Satoh. T. Tanaka, and M. Taniguchi. 1993. Production of poly (γ-glutamic acid) by Bacillus subtilis F-2-01. Biosci , Biotech. Biochem . 57 : 1212-1213.

12. Kunioka, M. 1997. Biosynthesis and chemical reactions of poly (amino acid) s from microorganisms. Appl Microbiol Biotechnol . 47 : 467-475.

13. Stadtman, ER 1966. Allosteric regulation of enzyme activity. Adv Enzymol . 28 : 41-154.

14. Thorne, CB and DM Molnar. 1955. D-Amino acid transamination in Bacillus anthracis . J. Bacteriol . 70 : 420-426.

15. Thorne, CB, CG Gomez, HE Noyes, and RD Housewright. 1954. Production of glutamyl polypeptide by Bacillus subtillis . J. Bacteriol . 68 : 307-315.

16. Yahata, K., J. Sadanobu and T. Endo, T. 1992. Preparation of poly-α-benzyl-γ-glutamate fiber. Polym . Prepr . Jpn . 41 : 1077.

The present invention relates to a novel Bacillus YN-1 strain producing polygamma glutamic acid and a method for producing polygamma glutamic acid using the same.

Recently, due to the seriousness of environmental problems and various environmental regulations, research on eco-friendly biodegradable polymers is being actively conducted. Among these, biodegradable polymers, which have attracted much research and attention, include polysaccharides and polysaccharides such as polyester-based polyhydroxyalkanoates (PHA), pullulan, and microbial cellulose. Peptide-based poly-gamma glutamic acid (poly-γ-glutamic acid; hereinafter referred to as PGA). Among these, PGA is well known as a slime substance of natto, a Korean traditional soup, and gamma-carboxylic acid and α-amino group of glutamic acid have amide linkages. It is a large polymer connected by the molecular weight, and its molecular weight is about 100 to 1000 kDa by the production bacteria [document information 1, 6, 7, 16]. In addition, the main production strain of PGA is Bacillus sp. , Bacillus (B. anthracis ), B. licheniformis ( B. licheniformis ), B. megaterium ( B. megaterium ) , B. subtilis - B. subtilis (natto)], B. subtilis [B. subtilis (chungkookjang)], etc. are known [Document information 2, 4].

PGA is a fermentation product of several strains of the genus Bacillus, such as Bacillus subtilis, which is secreted out of cells at the time of culture and is known as a water-soluble and biodegradable polymer that is not toxic to humans and the environment. Because of this feature, PGA can be used in various fields such as precipitation thickeners, wetting agents, cryoprotectants, drug carriers, biodegradable fibers, feed additives, etc. Has been recognized as a high value-added biomaterial with much interest.

In general, PGA-producing strains are classified into two types, which are classified into those which produce PGA only in the presence of L-glutamic acid and those which produce PGA regardless of the presence or absence of L-glutamic acid. Of these, L-glutamic acid dependent strain is Bacillus Subtilis [document 5], Bacillus Subtilis ATCC9945 [Documentation 15], Bacillus Subtilis IFO3335 [Ref. 7] and Bacillus Subtilis F-2-01 [document information 11] and the like have been reported. And L-glutamic acid independent strain, Bacillus Subtilis TAM-4 [Ref. 10] and Bacillus rickenformis A35 [Documentation 6] is reported.

However, the results of PGA studies reported to date are mainly related to synthetic pathways and genes, and there have been few studies on reports of mass production strains and methods of mass production that can be directly applied to industrialization.

Therefore, the present inventor isolates a new strain that produces large amounts of PGA in Cheonggukjang, and studies the biochemical properties of the strain so that it can be industrially used in the mass production process of PGA, thereby providing a high quality and low cost PGA to consumers. To provide.

Accordingly, an object of the present invention is to provide a novel strain having excellent production efficiency of polygamma glutamic acid from a medium containing a common carbon source and nitrogen source.

Another object of the present invention is to provide a method of culturing the novel strain in a medium containing a common carbon source and a nitrogen source, and producing polygammaglutamic acid from the culture.

In order to achieve the above object, the present invention provides a Bacillus sp. YN-1 strain ( Bacillus sp. YN-1) having a deposit number KACC 91170P for producing polygamma glutamic acid from a medium containing a common carbon source and nitrogen source. .

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 new strains of Bacillus spp. And apply them to industrial technology, new microorganisms suitable for the purpose of research were isolated from Cheonggukjang of various regions of the country and named Bacillus sp. YN-1. It was. And, as a result of identifying the strain, Bacillus subtilis MO4 ( Bacillus subtilis MO4), Bacillus sp . WL-3, Bacillus amyloliquefacien Ba-S13 ( Bacillus amyloliquefaciens) Ba-S13), and Bacillus licheniformis DSM13 ( Bacillus licheniformis DSM 13).

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 91170P.

2. Culture of Bacillus YN-1

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In order to increase the polygamma glutamic acid production of Bacillus YN-1 in the present invention, the carbon source is used as fructose, fructose, lactose, galactose, glycerol and citric acid. It is preferable to use at least 1 selected carbon source from the group which consists of, and a nitrogen source including ammonium chloride or L-glutamic acid. In particular, the carbon source is most preferably used 1 to 4% of the fructose by weight of the medium, which is less than 1%, the production of poly-gamma glutamic acid is reduced, when exceeding 4% compared to the excess This is because there is little increase effect. In the present invention, most preferably, 3% glutamic acid, 4% fructose, 1% citric acid, 1% ammonium chloride [(NH ₄ ) ₂ SO ₄ ], 0.1% potassium diphosphate (K ₂ HPO ₄ ), 0.05% magnesium sulfate (MgSO ₄ ), 0.005% iron dichloride (FeCl ₃ ), 0.02% calcium chloride (CaCl ₂ ), 0.002% manganese sulfate (MnSO ₄ ) are mixed and 50 μg of biotine per ml of the medium. A medium formed by adding is used.

Me, condition

The Bacillus YN-1 strain of the present invention exhibits the highest production of PGA at pH 8.0 and at 37 ° C.

3. Physical Properties of Polygammaglutamic Acid

The Bacillus YN-1 strain of the present invention produces polygamma glutamic acid having a molecular weight of 4,000 to 6,000 KDa when incubated for 12 hours or more under optimal conditions.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only intended to specifically describe the present invention, 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 ] PGA Separation, Purification and Molecular Weight Measurement

First, strains incubated in LB medium were inoculated in complete medium (0.5% polypeptone, 0.25% yeast extract, 0.5% NaCl, 0.05% MgSO ₄ · 7H ₂ O, pH7.0) and the culture absorbed at 660 nm. OD _{660 nm )} was incubated until 2.1, and the cells were collected by centrifugation of the culture solution, and the collected cells were suspended in 0.85% NaCl solution and centrifuged to collect the cells again. The collected cells were resuspended with NaCl, and the cells were inoculated in a PGA basal production medium so that the cells became 10% and incubated at 37 ° C. for 72 hours. At this time, the PGA basal production medium was 2% glutamic acid, 1% ammonium sulfate, 1% citric acid, 0.1% potassium diphosphate, 0.1% sodium hydrogen phosphate (Na ₂ HPO ₄ ), 0.05% magnesium sulfate, 0.005% ferric chloride, 0.02% 50 μg / ml of biotin was added to calcium chloride and 0.002% manganese sulfate, and the produced PGA was measured by using the cultured medium as a sample.

Then, the culture solution is centrifuged for 12,000 × g, 10 minutes to remove the cells and collect the supernatant [document 3]. PGA was precipitated by adding methanol four times the volume of the supernatant, and the precipitated PGA was dissolved in 2 ml of sterile water and dialyzed with sterile water so that only pure PGA remained. The separated and purified PGA was confirmed by performing SDS-PAGE.

In addition, PGA obtained by dialysis was dissolved in sterile water, and then 10 µl of PGA solution was analyzed by SDS-PAGE (5% acrylamide). PGA was confirmed by methylene blue staining [document 10]. The PGA separated by electrophoresis was quantified using a Fluorchem 5500 Image Analyzer.

[ Example  One] Polygamma Glutamic Acid (PGA) Production Strain Isolation and Identification

For the purpose of isolating PGA-producing strains, conventionally prepared Cheonggukjang was collected from each country in Korea, and 1g of each Cheonggukjang was suspended in 10ml of sterile water, followed by 5 minutes at 100 ° C to remove spores that did not form spores. Heat treatment. In addition, the strain was isolated by diluting the plate of Cheonggukjang which was heat-treated at 100 ° C. for 5 minutes in LB-agar medium (0.5% Yeast extract, 0.5% NaCl, 1% Bacto tryptone, 1.5% Agar) and incubated at 37 ° C. The isolated strain was PGA production medium [2% Glutamic acid, 1% (NH ₄ ) ₂ SO ₄ , 1% Citric acid, 0.1% K ₂ HPO ₄ , 0.1% Na ₂ HPO ₄ , 0.05% MgSO ₄ , 0.005% FeCl ₃ , 0.02% CaCl ₂ , 0.002% MnSO ₄ , 50 μg / ml biotin], and the presence of PGA in the culture was confirmed by SDS-PAGE to isolate the PGA producing strain.

Isolated PGA producing strains were identified according to 16s rRNA sequence analysis (Macrogen), API test (bioMetrieux) and Bergey's manual.

As a result, there were about 30 kinds of bacterial colonies that were purely separated through heat treatment, and their strains were pre-cultured in a complete medium, and then cultured for 72 hours in a PGA production medium to improve the production of PGA. In comparison, 12 strains producing PGA were selected from 30 kinds of bacteria isolated purely, and among them, the most productive strain 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 strain was Gram-positive bacillus, motionless, forming heat-resistant spores, negative in starch hydrolysis and lipase hydrolysis, and actively growing even at a temperature of 45 ° C. Biochemical properties were shown. In addition, the strain showed active growth even at high concentrations of NaCl (7%).

In addition, 97.7% of Bacillus subtilis MO4 was identified and identified using 16s rRNA sequencing. Bacillus WL-3 and 97.6%, Bacillus amyloliquefaciene Ba-S13 and 97.6%, It showed a homology of 88.6% with Bacillus licheniformis DFM13.

Therefore, the present inventor named the final selection strain Bacillus YN-1, and the strain was deposited on July 20, 2005 to the Agricultural Biotechnology Research Institute under the Agriculture Promotion Agency, was given the accession number KACC 91170P.

[ Example  2] Glutamic Acid of PGA-producing Strains Demand

Generally, PGA-producing strains are largely classified into two groups, one group is a glutamic acid dependent group requiring glutamic acid for PGA production and strain growth, and a glutamic acid independent group without glutamic acid. In this example, to determine the requirement of glutamic acid of the novel strain of the present invention, the presence of strain growth was confirmed by culturing except for glutamic acid in a PGA basal production medium.

As a result, even if glutamic acid was not required for growth of the strain of the present invention, it was confirmed that PGA production occurred smoothly (not shown). Therefore, Bacillus YN-1 strain of the present invention was found to belong to the glutamic acid independent group.

[ Example  3] Optimum Condition of PGA Production

In general, PGA-producing strains vary greatly in PGA production according to culture conditions, and it is known that culture conditions are very important for PGA production, such as requiring a carbon source and nitrogen source of about 2 to 20 times the production of PGA. 12].

Thus, this example was to determine the optimum PGA mass production conditions of the Bacillus YN-1 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 8.0.

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 and Nitrogen Sources

To determine the carbon and nitrogen sources for optimal PGA production, first, the carbon source is maltose, fructose, lactose, galactose, glucose, glycerol, and xylose. xylose) and saccharose were added to the PGA basal production medium to a final concentration of 2%. In addition, the PGA produced by culturing Bacillus YN-1 of the present invention in the medium to which the carbon source was added was identified by SDS-PAGE, and the medium of the carbon source in which the largest amount of PGA was produced was selected as the optimal carbon source for PGA production. The nitrogen sources were sodium nitrate (NaNO ₃ ), ammonium nitrate (NH ₄ NO ₃ ), ammonium chloride (NH ₄ Cl), ammonium sulfate [(NH ₄ ) ₂ SO ₄ ], peptone, yeast extract (yeast) extract was added to the PGA basal production medium to 1%, followed by cultivation, and the PGA production was examined to determine the optimal nitrogen source for PGA production.

First, in the case of the carbon source, to determine the carbon source required for PGA production, as shown in Figure 2, the final concentration of the carbon source was 2% and confirmed the change in PGA production of this strain using 10 carbon sources including fructose As a result, when maltose, xylose, glucose and saccharose were used as the carbon source, there was no significant difference in PGA production compared to the case where no carbon source was added. When lactose, galactose and glycerol were added as carbon sources, It was found that PGA production increased by 2.5 times and that PGA production increased by 4 times when fructose was used. In particular, in the case of fructose, as shown in Figure 3, the production of PGA gradually increased with the addition amount of fructose up to 1 to 4%, but the addition of 4% or more did not change the yield. Therefore, it was found that 4% of fructose is the most preferable carbon source of the genus Bacillus YN-1.

In the case of the nitrogen source, as shown in FIG. 4, as a result of adding various nitrogen sources at a concentration of 1%, various nitrogen sources including peptone did not increase PGA production, but ammonium chloride (NH ₄ Cl) When added, it was confirmed that more than 5 times the PGA production increased. Therefore, it was found that ammonium chloride was the most preferable nitrogen source of this strain.

In the PGA producing strain, L-glutamic acid-independent strains, as shown in FIG. 5, generally, L-glutamic acid is generated during metabolic processes in cells and L-glutamic acid is a tricarboxylic acid cycle. Is estimated to be produced from citric acid via 2-oxoglutaric acid and isocitric acid (Ref. 12). In addition, there are two known pathways for converting 2-oxoglutaric acid to L-glutamic acid, one of which is by the glutamate dehydrogenase pathway and α-, which is a substance of the TCA cycle. Ammonia (NH ₃ ) acts together with α-Ketoglutaric acid to produce L-glutamic acid [13], and another route is glutamine synthetase (GS) and glutamine 2--. It is known to produce L-glutamic acid by reaction produced by glutamine 2-oxoglutarate aminotransferase (GOGAT) [Ref. Information 9].

Thus, the Bacillus genus YN-1 strain of the present invention requires citric acid and a small amount of glutamic acid for mass production of PGA according to the results of the above-described embodiment and a well-known research paper, and both pathways are converted to L-glutamic acid. It is believed to pass via. In addition, the strain of the present invention requires fructose and ammonium chloride (ammonium chloride) for the production of PGA, which in the case of fructose glyceralde-D-phosphate by various enzymes, glycolysis It is supposed to enter the TCA cycle and to produce glutamic acid through citric acid and the like, and ammonium chloride is believed to provide ammonia to glutamate dehydrogenase or glutamine synthetase.

Based on this judgment, Bacillus subtilis IF03335, one of the PGA-producing strains, reported that isomer of glutamic acid L / D type was present at a ratio of 17/83 to generate PGA. ], D-glutamic acid is known to have many microorganisms that are indirectly produced rather than directly modified from L-glutamic acid. Specific examples are natto microorganisms (natto) and Bacillus anthracy ( B. anthracis ) D-glutamic acid production pathway, 2-oxoglutaric acid and L-alanine are produced by L-glutamic acid and pyruvic acid, D-alanine is produced by racemization in L-alanine, It has been reported that D-glutamic acid and pyruvic acid are synthesized from D-alanine and 2-oxoglutaric acid (Refs. 8 and 14).

Therefore, as a result of combining the results of the above-described examples and the known literature, the optimum PGA mass production medium of the Bacillus YN-1 strain of the present invention is 3% glutamic acid, 4% fructose, 1% citric acid, 1% Ammonium chloride, 0.1% potassium diphosphate (K 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 50 μg / ml biotin was determined.

[ Example  4] PGA production and molecular weight measurement according to growth curve

This example was to determine the molecular weight of PGA production and PGA according to the strain growth curve and growth time of the present invention.

As a result, when the strain of the present invention is inoculated in a large PGA production medium and cultured, unlike other strains, PGA is produced after 12 hours, and its size is about 4,000 to 6,000 kDa, which produces large molecular weight PGA from the beginning of production. It can be seen that there is a characteristic (see Fig. 6). Particularly, in the case of PGA production according to the growth curve of the Bacillus subtilis TAM-4 strain known as a glutamic acid-independent strain, when the incubation time of the strain is about 36 hours, the PGA having a molecular weight of 200 kDa or less is produced and 54 hours. In the case of the above culture, it can be seen that the efficiency is very excellent as compared with the production of PGA of 200 kDa or more.

In addition, in general, the strain producing PGA does not deviate significantly from the neutral pH range with growth, but the strain of the present invention starts producing PGA from 12 hours of culture regardless of the incubation time of the strain, and the yield according to the time. While increasing, the pH of the culture was gradually changed to pH toward the acid was found (see Figure 7).

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

<110> ORIENTAL BIOTECH CO, .LTD <120> Novel Bacillus subtilis YN-1 and Method for producing poly gamma          glutamic acid using the same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1418 <212> DNA <213> Bacillus sp. YN-1 <220> <221> rRNA (222) (1) .. (1418) <223> 16s rRNA partial sequence <400> 1 cggtaagatg ggagcttgnt ccctgatgtt agcggtcgga cgggtgagta acacgtgggt 60 aacctgcctg ctaagactgg gataactccg ggaaaccggg gctaataccg gatgnttgtt 120 tgaaccgcat ggttcagaca taaaaggtgg cttcggctac cacttacaga tggacccgcg 180 gcgcattagc tagttggtga ggtaacggct caccaaggcn acgatgcgta gccgacctga 240 gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 300 agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag tgatgaaggt 360 tttcggatcg taaagctctg ttgttaggga agaacaagtg ccgttcaaat agggcggcac 420 cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg 480 taggtggcaa gcgttgtccg gaattattgg gcgtaaaggg ctcgcaggcg gtttcttaag 540 tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg gaacttgagt 600 gcagaagagg agagtggaat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac 660 accagtggcg aaggcgactc tctggtctgt aactgacgct gaggagcgaa agcgtgggga 720 gcgaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttagg 780 gggtttccgc cccttagtgc tgcagctaac gcattaagca ctccgcctgg ggagtacggt 840 cgcaagactg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 900 taattcgaag caacgcgaag aaccttacca ggtcttgaca tcctctgaca atcctagaga 960 taggacgtcc ccttcggggg cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc 1020 gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt gccagcattc 1080 agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa 1140 atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacaga acaaagggca 1200 gcgaaaccgc gaggttaagc caatcccaca aatctgttct cagttcggat cgcagtctgc 1260 aactcgactg cgtgaagctg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata 1320 cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac acccgaagtc 1380 ggtgaggtaa cctttagagc cagccgccgg tgtgtcct 1418

Claims (6)

Bacillus YN-1 strain ( Bacillus sp. YN-1) having accession number KACC 91170P for producing polygammaglutamic acid from a medium containing a common carbon 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 a 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 fructose, lactose, galactose, glycerol and citric acid. The method of claim 3, wherein The medium is a method of producing polygamma glutamic acid, characterized in that the medium containing 1 to 4% of fructose by weight. The method of claim 2, The medium is ammonium chloride (NH ₄ Cl) or a method for producing polygamma glutamic acid, characterized in that the medium containing glutamic acid as a nitrogen source. The method according to any one of claims 2 to 5, The medium was 3% glutamic acid, 4% fructose, 1% citric acid, 1% ammonium chloride, 0.1% K2HPO4, 0.05% magnesium sulfate, 0.005% iron dichloride, 0.02% calcium chloride, 0.002% manganese sulfate and 50 μg / ml biotin and the remainder. The method for producing polygamma glutamic acid, characterized in that consisting of sterile water.

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