KR20070053867A - Novel bacillus velezensis a-68 and use of the same - Google Patents

Abstract

The present invention relates to a novel microbial Bacillus Berethensis A-68 and its use, and more particularly, Bacillus Berethensis A-68 having a fibrinolytic enzyme activity identified from seawater, rice bran, rice husk, starch and CMC The present invention relates to a method for producing fibrinolytic enzymes and polysaccharides, the method comprising culturing in a medium containing at least one selected from the group consisting of carbon sources.

According to the present invention, since polysaccharides can be produced using agricultural by-products such as rice bran or rice hulls as carbon sources, polysaccharides can be produced at a lower cost than conventional methods of producing polysaccharides using expensive carbon sources, and environmental pollution. The use of rice bran and rice hull, one of the causes, in the production of polysaccharides is useful in establishing an environmentally friendly polysaccharide production process that reduces environmental pollution.

Bacillus subtilis subspis subtilis, marine microorganisms, fibrinolytic enzymes, polysaccharides, production

Description

Novel Bacillus Velezensis A-68 and Use of the Same

FIG. 1 shows the comparison of fibrin degradation capacity assayed using a paper disk after the supernatant from which the cells were removed by culturing the culture solution by centrifugation and dropping the supernatant onto a paper disk.

Figure 2 shows the phylogenetic tree between Bacillus berechensis A-68 and other strains based on the partial sequencing of the 16S rDNA and zyrease A gene.

Figure 3 shows the growth of Bacillus Berechesis A-68 according to the type of carbon source.

Figure 4 shows the fibrinase production amount of Bacillus Berechens A-68 according to the type of carbon source.

Figure 5 shows the polysaccharide production amount of Bacillus Berechesis A-68 according to the type of carbon source.

Field of invention

The present invention relates to a novel microbial Bacillus Berethensis A-68 and its use, and more particularly, Bacillus Berethensis A-68 having a fibrinolytic enzyme activity identified from seawater, rice bran, rice husk, starch and CMC The present invention relates to a method for producing fibrinolytic enzymes and polysaccharides, the method comprising culturing in a medium containing at least one selected from the group consisting of carbon sources.

Background of the Invention

Cellulose (cellulose) is a major component of the cell wall of higher plants and occupies most of the woody part, cellulose is an organic compound present in large quantities after coal in nature, and is an important industrial resource. In addition to higher plants, they are found in the shells of sea urchins, which are bacteria, sea horses, and marine animals. They are also contained in the extracellular secretions of acetic acid bacteria and in mucus of shellfish. Fibrin is degraded by cellulase, such as fungi, bacteria, or mollusks, and finally becomes starch (glucose).

Cululase is an enzyme that hydrolyzes cellulose, and the fibrin that is hydrolyzed by fibrinase is converted into cellobiose, which in turn is transformed by β-glucosidase. Hydrolyzed to Glucose.

Cellulase enzymes are known to be mainly produced by fungi, such as the Clostridium species T. viride, T. reesei Tricodema species, Aspergillus niger, such as Thermomonospora, reported to be produced also by Aspergillus, Penicillium, Clostrium, Sclerotium spp etc. It has been.

The conventional technique for producing fibrinase is a method of solid state fermentation of Tricoderma species, Aspergillus species, etc., which is inefficient compared to liquid culture, and molds have low productivity because of their low growth rate compared to bacteria. There is a problem that increases the price of cellulose enzymes.

In order to solve the problems with the prior art, including improving the production of fibrinase, fungal fibrinase genes are introduced into bacteria such as E. coli, and fibrinase gene-containing mutants are liquid cultured, Research is underway to produce degrading enzymes, but it is not yet applied to the industrialization stage.

The fibrinolytic enzyme produced can be used in various ways such as grain processing, ethanol fermentation from biological resources, liquor production, waste disposal, laundry or kitchen detergents. Existing laundry or kitchen detergents are mostly chemicals, which are difficult to be decomposed by microorganisms in a dissolved state, and bubbles are formed on the water to prevent oxygen from melting into the water. This prevents sunlight from entering the water, preventing the normal reproduction of plankton, as well as causing a number of problems that increase pollution of water resources. In addition, conventional laundry or kitchen detergents contain phosphorus to increase the cleaning power, causing eutrophication.

Polysaccharides include pectin in fruits, carrageenan in seaweed, Locust Bean Gum in seeds, Arabia Gum in sap, and collagen isolated from animal tissues and bones. It is produced by gellatin, which is partially hydrolyzed, starch and cellulose, which are condensates of glucose produced by plant carbonization, and algin, agar, and microorganisms extracted from algae. There are many kinds such as xanthan, gellan and pullulan. When these polysaccharides are dissolved in water, they are highly viscous or jellyy. These polysaccharides are widely used to improve the quality of foods because they have various functions to increase the viscosity of foods, to form gels, to improve emulsion stability, and to form food tissues and to improve texture. It is used.

Research into the production of chewing gum by microbial culture began in the late 1950s and early 1960s. Research is underway to discover and industrialize many types of microorganisms that produce polysaccharides such as xanthan, curdlan, microbial alginate, pullulan and microbial cellulose. It became. As a result, gellan, wellan and rhamsan have been developed, among which xanthan has been used in many areas of the food industry. It is being developed by. To replace the xanthan market. Curdlan is the third polysaccharide produced by cultivating microorganisms, followed by xanthan and gellan, and is commercialized by the US Food and Drug Administration (US FDA), and is also entering the market.

The culture medium of microorganisms for producing functional polysaccharides using microorganisms may be glucose, starch or sugar as a carbon source, yeast extract, peptone and tryptone or ammonium chloride (NH ₄ Cl) and nitric acid as organic nitrogen sources as nitrogen sources. Inorganic nitrogen sources such as ammonium ((NH ₄ ) ₂ NO ₃ ) are used. After inoculating a strain that produces polysaccharides into a sterile medium and incubating for a predetermined time under appropriate conditions, the polysaccharides biosynthesized by the microorganisms are accumulated in the culture medium.

Existing methods for producing polysaccharides using microorganisms on an industrial scale have relatively high production costs because they use excessive amounts of high cost glucose or sugar as a carbon source of the culture medium. Therefore, the unit price of the produced polysaccharides is also expensive, thereby increasing the price of food and the like.

Accordingly, the present inventors have made efforts to develop strains having the ability to produce cellulose degrading enzymes and excellent polysaccharides. As a result, the present inventors have isolated and identified Bacillus berechensis A-68 having cellulose degrading enzyme and polysaccharide generating ability from seawater. By generating fibrinase, it was confirmed that the produced fibrinase can produce polysaccharides using the carbon source of the medium, thereby completing the present invention.

After all, an object of the present invention is to provide a novel microbial Bacillus Berechens A-68 and a method for producing fibrinase and polysaccharides using the same.

Provides: (KACC Accession No. 91178P) In order to achieve the above object, the present invention is Bacillus Debrecen chopping system (B acillus velezensis) A-68 with a cellulolytic enzyme and a polysaccharide producing ability.

The present invention also comprises the steps of (a) culturing the microorganisms to produce and secrete fibrinase into the culture medium; And (b) provides a method for producing cellulose lyase comprising the step of recovering cellulose lyase from the culture. In the present invention, the carbon source of the culture solution may be any one or more selected from the group consisting of rice bran, rice hull, starch and CMC.

The present invention also comprises the steps of (a) culturing the microorganisms to secrete and produce a polysaccharide as a culture medium; And (b) recovering the polysaccharide from the culture solution. In the present invention, the carbon source of the culture may be any one or more selected from the group consisting of rice bran, rice hull, sugar and CMC.

Hereinafter, the present invention will be described in detail.

After liquid culture of the microorganisms isolated from seawater, the supernatant from which the cells were removed by centrifugation of the culture was dipped in a predetermined amount onto a paper disc of 1.0 cm in diameter, and then dried. It was placed on the solid medium to which the addition, and was cultured at a constant temperature to isolate a strain that decomposes carboxymethyl fiber. As a result of identifying the isolated strains by partially determining the base sequences of 16S rDNA and gyrase A and comparing the reported sequences with the reported strains, it was confirmed that Bacillus velezen is a bacterium. Berechens A-68 ( Bacillus velezensis A-68).

As a result of culturing Bacillus Berechens A-68 with a different carbon source, it was confirmed that the growth of the strain, the ability of fibrinase and the production of polysaccharide were excellent in the medium containing rice bran or rice hull as a carbon source.

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

Example 1 Microbial Isolation and Culture in Seawater

After properly diluting a certain amount of seawater and sterile saline solution (0.85% NaCl) collected from the East Sea and the South Sea in Gyeongsang Province, add 1.5% (w / v) agar to the composition of marine broth. ) Microorganisms were isolated by plating on a medium and incubating at 30 ° C. The isolated microorganisms were inoculated with platinum platinum in marine broth (requires substrate composition), and incubated at 25-35 ° C. for 150-200 rpm for 55-85 hours, and then the obtained culture solution was placed in fresh broth (marine broth). Inoculated at 5% (v / v), incubated for 3 to 5 days at 150 ~ 200rpm at 25 ~ 35 ℃. The culture and the culture was centrifuged for 10-30 minutes in the range of 5000 ~ 9000xg to obtain only the supernatant except the cells.

The composition of the marine broth 1L is as follows: 5g peptone, 1g yeast extract, 0.1g Ferric clitrate, 19.45 Sodium chloride, 5.9g Magnisium chloride, 3.24g Sodium sulfate, 1.8g Calcilum chloride, 0.55g Potsssium chloride, 0.16g Sodium bicarbonate, 0.08g Potassium bromide, 0.034g Strontium chloride, 0.022g Boric acid, 0.004g Sodium dilicate, 0.0024g Sodium fluoride, 0.0016g Ammonium nitrate and 0.008g Disodium phosphate.

Example 2 Isolation of Strains Having Fibrinase Production Ability

In order to isolate the strain having fibrinolytic activity, 40 μl of the supernatant obtained in Example 1 was dropped onto a paper disc having a diameter of 1 cm and dried. The dried paper disc was placed on agar medium containing 2% (w / v) CMC (carboxymethyl cellulose) and incubated at 37 ° C. for 3 days, and the fibrinase production ability of microorganisms isolated from seawater was observed (FIG. One).

As a result, as shown in Figure 1, it was confirmed that some microorganisms produce and secrete fibrinase. When microorganisms produce and secrete fibrinase, CMC in the medium is degraded to produce a pale red circle around the microorganisms present (FIG. 1). The radius of the circle and the degree of whiteness are proportional to the fibrinase generating ability of the microorganism.

Example 3 Identification of Bacillus Berechensis A-68 Strains

In order to identify strains which were determined to have excellent fibrinolytic ability in Example 2, the nucleotide sequences of 16S rDNA and zyrease A gene were analyzed.

3-1: Identification of Bacillus Berechesis A-68 Strains by 16S rDNA Sequencing

After culturing the strain determined to be excellent in fibrinolytic enzyme production in Example 2, the culture solution was crushed with a homogenizer and dissolved in 100 μl of TE buffer (pH 8.0), by adding lysozyme (lysozyme) The cell wall was digested by reacting at 37 ° C for 12 hours. 500 ul of guanidine-sarcosyl solution (Guanidine thiocyanate (Sigma) 60 g, 20 ml of 0.5 mM EDTA, and 20 ml of Deionized water) was added to the cell wall-decomposed cells, and the supernatant was obtained.

0.54 fold of isopropanol was added to the obtained supernatant to precipitate chromosomal DNA. The supernatant was removed to obtain precipitated chromosomal DNA, dissolved in 90 μl of TE buffer (pH 8.0), 10 μl of RNase A (10 mg / ml, Sigma) was added and reacted at 37 ° C. for 2 hours. A volume of ethanol was added to precipitate chromosomal DNA. The precipitated chromosomal DNA was dissolved in 30 μl of distilled water and used as a template for amplification of 16S rDNA.

27F (5'-AGA GTT TGA TCM TGG CTC AG-3 ') represented by SEQ ID NO: 1 and 1522R (5'-AAG GAG GTG WTC CAR CC-3') represented by SEQ ID NO: 2 were used as primers. PCR was performed under the same conditions to amplify the 16S rDNA gene.

Device used: GenAmp TM PCR System 9700 (Applied Biosystem)

Composition of reaction mixture for PCR (total volume: 50ul): template DNA 10ng, 200uM dNTP, 10mM Tris-HCl (pH 9.0), 40mM KCl, 0.15mM MgCl ₂ , 3mM MgSO ₄ , 20ug BSA, 0.005U Vent polymerase, 1 U Taq polymerase and primer (0.5 uM 27F and 1522R, respectively).

PCR reaction conditions for 16S rDNA amplification: 94 ° C, 3 minutes → [94 ° C, 30 seconds → 50 ° C, 30 seconds → 72 ° C, 5 minutes] 30 times → post-elongation: 72 ° C, 10 minutes

The amplified PCR product was purified using Wizard PCR Preps DNA Purification System (Promega), and electrophoresed on 1% agarose gel to confirm the size of 16S rDNA. The nucleotide sequence was analyzed using a BigDye TM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystem) labeled with fluorescent material on ddNTP, followed by PCR reaction, followed by analysis using ABI PRISM TM 310 Genetic Analyzer (Applied Biosystem). It was.

PCR reaction conditions for sequencing; 25 times of [96 degreeC 10 second-> 50 degreeC 5 second-> 60 degreeC 4 minutes].

As a result, as shown in Table 1, 16S rDNA nucleotide sequence of the selected A-68 strain could be partially determined.

16S rDNA sequence of the A-68 strain (874 bp): SEQ ID NO: 5 5'- AACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTA ACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTC - 3 '

3-2: Identification of Bacillus Berechensis A-68 Strains by Xyrease A Sequencing

P-gyrA-f (5′-CAG TCA GGA AAT GCG TAC GTC CTT-3 ′) as set forth in SEQ ID NO: 4, using the DNA obtained in Example 3-1 as a template, and p- Gyrase A gene was amplified by using gyrA-r (5′-CAA GGT AAT GCT CCA GGC ATT GCT-3 ′) as a primer and performing PCR under the following conditions.

Device used: GenAmp TM PCR System 9700 (Applied Biosystem)

Composition of reaction mixture for PCR (total volume: 50ul): template DNA 10ng, 200uM dNTP, 10mM Tris-HCl (pH 9.0), 40mM KCl, 0.15mM MgCl ₂ , 3mM MgSO ₄ , 20ug BSA, 0.005U Vent polymerase, 1U Taq polymerase and primers (0.5 uM for p-gyrA-f and p-gyrA-r, respectively).

PCR reaction conditions: 94 ℃, 3 minutes → [94 ℃, 30 seconds → 50 ℃, 30 seconds → 72 ℃, 5 minutes] 30 times → post-elongation: 72 ℃, 10 minutes

The amplified PCR product was purified using Wizard PCR Preps DNA Purification System (Promega), and electrophoresed on 1% agarose gel to confirm the size of 16S rDNA. The nucleotide sequence was analyzed using a BigDye TM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystem) labeled with fluorescent material on ddNTP, followed by PCR reaction, followed by analysis using ABI PRISM TM 310 Genetic Analyzer (Applied Biosystem). It was.

PCR reaction conditions for sequencing; 25 times of [96 degreeC 10 second-> 50 degreeC 5 second-> 60 degreeC 4 minutes].

As a result, as shown in Table 2, the zyrease A base sequence of the selected A-68 strain could be partially determined.

Zayase A nucleotide sequence of strain A-68 (963bp): SEQ ID NO: 6 5'- ATGCAATGAGCGTTATCGTATCCCGGGCGCTTCCGGATGTGCGTGACGGTCTGAAGCCGGTTC ACAGACGGATTTTGTACGCAATGAATGATTTAGGCATGACCAGTGACAAACCATATAAAAAATCTGCCCGTATCGTCGGTGAAGTTATCGGTAAGTACCACCCGCACGGTGACTCAGCGGTTTACGAATCAATGGTCAGAATGGCGCAGGATTTTAACTACCGCTACATGCTTGTTGACGGACACGGCAACTTCGGTTCGGTTGACGGCGACTCAGCGGCCGCGATGCGTTACACAGAAGCGAGAATGTCAAAAATCGCAATGGAAATTCTGCGTGACATTACGAAAGACACGATTGACTATCAAGATAACTATGACGGTTCAGAAAGAGAGCCTGCCGTCATGCCTTCGAGATTTCCGAATCTGCTCGTAAACGGGGCTGCCGGTATTGCGGTCGGAATGGCGACAAACATTCCCCCGCATCAGCTTGGGGAAGTCATTGAAGGCGTGCTTGCCGTAAGTGAGAATCCTGAGATTACAAACCAGGAGCTGATGGAATACATCCCGGGCCCGGATTTTCCGACTGCAGGTCAGATTTTGGGCCGGAGCGGCATCCGCAAGGCATATGAATCCGGACGGGGATCAATCACGATCCGGGCTAAGGCTGAAATCGAAGAGACTTCATCGGGAAAAGAAAGAATTATTGTCACGGAACTTCCTTATCAGGTGAACAAAGCGAGATTAATTGAAAAAATCGCGGATCTTGTCCGAGACAAAAAAATCGAAGGAATTACCGATCTGCGAGACGAATCCGACCGTAACGGAATGAGAATCGTCATTGAGATCCGCCGTGACGCCAATGCTCACGTCATTTTGAATAACCTGTACAAACAAACGGCCCTGCAGACGTCTTTCGGAATCAATCTGCTGGCGCTCGTTGACGGACAGCCGAAGGTACTAA-3 '

Based on the 16S rDNA of the selected A-68 strain and the partial sequencing of the zyrease A determined through the above experiment, the similarity with other strains is shown as Table 3, which is summarized in the schematic diagram of FIG. 2. It was.

Strain Accession No. Similarity nt differences / compared Bacillus velezensis LMG 22478T 98.77 11/896 Bacillus amyloliquefaciens KCTC 1660T AF272015 95.95 38/938 Bacillus mojavensis NRRL B-14698T AF272019 84.10 124/780 Bacillus subtilis subsp. spizizenii NRRL B-23049T AF272020 82.84 139/810 Bacillus subtilis subsp. subtilis KCTC 3135T AF272021 82.48 133/759 Bacillus atrophaeus KCTC 3701T AF272016 82.07 156/870 Bacillus vallismortis NRRL B-14890T AF272025 81.40 168/903 Bacillus licheniformis KCTC 1918T AF272017 78.25 186/855

Based on this result, the selected strain was identified as Bacillus velezensis , a kind of bacteria, and named Bacillus velezensis A-68. In addition, on October 17, 2005, it was deposited with the Korean Agricultural Culture Collection (KACC) under the deposit number KACC 91178P at the Rural Development Administration.

Example 4 Growth of Bacillus Berechens A-68 with Different Carbon Sources

Bacillus berechensis A-68 strain of the present invention was inoculated with a platinum tooth to marine broth, and incubated at 25-35 ° C. for 150-200 rpm for 55-85 hours. The seed culture was again cultured [0.25% (w / v) yeast extract, 0.5% K ₂ HPO ₄ , 0.1% NaCl, 0.02% MgSO ₄ 7H ₂ O (0.02%), 0.06% (NH ₄ ) ₂ SO ₄ And 2% carbon source] at 3 to 5% (v / v), and incubated at 25 to 35 ° C. for 150 days to 150 rpm for 3 to 5 days. OD of the culture was measured to observe the growth of the A-68 strain according to the carbon source and incubation time. Carbon sources used in the present invention are glucose (glucose), fructose (furctose), maltose (maltose), sugar (sucrose), starch (starch), CMC, rice bran and rice husk.

As a result, as shown in Figure 3, when the Bacillus berechensis A-68 strains were cultured in a medium containing starch, rice bran or chaff as a carbon source, it was confirmed that the growth is excellent.

Example 5: Production of Fibrinase by Bacillus Berechens A-68

The culture solution of Example 4 was centrifuged for 10 to 30 minutes in the range of 5000 to 9000xg to remove the cells and to obtain only the supernatant.

The amount of fibrinase in the supernatant was measured using CMC as a substrate. First, 0.5 ml of the supernatant and 0.5 ml of 1.0% (w / v) CMC solution were mixed and placed at 50 ° C. for 20 minutes, and then the production of reducing sugars was measured using the DNS method. The concentration of fibrinase present in the supernatant was measured using the Bradford method.

As a result, as shown in Figure 4, when the starch, rice bran or chaff as a carbon source was cultured Bacillus Berechesis A-68 strains, it was confirmed that the production of fibrinase increased.

Example 6: Production of Polysaccharides by Bacillus Berechens A-68

The culture solution of Example 4 was centrifuged for 10 to 30 minutes in the range of 5000 to 9000xg to remove the cells and to obtain only the supernatant. The obtained supernatant was mixed with isopropyl alcohol or ethanol at 1: 2 (v / v), stored at a low temperature of about 4 ° C. for 24 hours, and then centrifuged for 10-30 minutes in the range of 5000 to 9000xg. A precipitate was obtained. An appropriate amount of distilled water was added to the obtained precipitate, followed by dialysis to remove the salt component, thereby finally obtaining a polysaccharide. The amount of polysaccharide was measured using the phenol-sulfate method.

As a result, as shown in Figure 5, when Bacillus Berethensis A-68 was incubated for 72 hours using rice bran as the carbon source, the production of polysaccharides (5.99g / l) was the highest, the Bacillus Bacillus of the present invention It was confirmed that rice bran is an excellent carbon source in the production of polysaccharides using Rechensis A-68. The production of polysaccharides was 5.99 g / l for rice cultivation using rice bran as the carbon source, and the highest polysaccharides were produced. For rice cultivation using chaff and sugar as carbon source, the yield of polysaccharides was 4.61 g / l and 4.45, respectively. In g / l, chaff and sugar were also found to be excellent carbon sources. In addition, glucose, fructose, maltose, starch and CMC showed yields of 1.63 g / l, 1.67 g / l, 1.92 g / l, 1.61 g / l and 3.55 g / l, respectively.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention has the effect of providing a novel microbial Bacillus Berechens A-68 and a method for producing fibrinase and polysaccharides using the same. According to the present invention, it is possible to produce fibrinolytic enzymes and polysaccharides by using agricultural by-products such as rice bran or chaff as a carbon source, it is possible to produce fibrinolytic enzymes and polysaccharides at a lower cost than conventional methods using expensive carbon sources. In addition, by using rice bran and rice husk, one of the causes of environmental pollution, in the production of fibrinolytic enzymes and polysaccharides, it is useful to establish an environmentally friendly polysaccharide production process that reduces environmental pollution.

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> Novel Bacillus Velezensis A-68 and Use of the Same <130> P05-B318 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 aaggaggtgw tccarcc 17 <210> 3 <211> 874 <212> DNA <213> Bacillus velezensis <400> 3 aacgctggcg gcgtgcctaa tacatgcaag tcgagcggac agatgggagc ttgctccctg 60 atgttagcgg cggacgggtg agtaacacgt gggtaacctg cctgtaagac tgggataact 120 ccgggaaacc ggggctaata ccggatggtt gtctgaaccg catggttcag acataaaagg 180 tggcttcggc taccacttac agatggaccc gcggcgcatt agctagttgg tgaggtaacg 240 gctcaccaag gcgacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga 300 gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt 360 ctgacggagc aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag 420 ggaagaacaa gtgccgttca aatagggcgg caccttgacg gtacctaacc agaaagccac 480 ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggaattat 540 tgggcgtaaa gggctcgcag gcggtttctt aagtctgatg tgaaagcccc cggctcaacc 600 ggggagggtc attggaaact ggggaacttg agtgcagaag aggagagtgg aattccacgt 660 gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga ctctctggtc 720 tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt 780 ccacgccgta aacgatgagt gctaagtgtt agggggtttc cgccccttag tgctgcagct 840 aacgcattaa gcactccgcc tggggagtac ggtc 874 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cagtcaggaa atgcgtacgt cctt 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 caaggtaatg ctccaggcat tgct 24 <210> 6 <211> 963 <212> DNA <213> Bacillus velezensis <400> 6 atgcaatgag cgttatcgta tcccgggcgc ttccggatgt gcgtgacggt ctgaagccgg 60 ttcacagacg gattttgtac gcaatgaatg atttaggcat gaccagtgac aaaccatata 120 aaaaatctgc ccgtatcgtc ggtgaagtta tcggtaagta ccacccgcac ggtgactcag 180 cggtttacga atcaatggtc agaatggcgc aggattttaa ctaccgctac atgcttgttg 240 acggacacgg caacttcggt tcggttgacg gcgactcagc ggccgcgatg cgttacacag 300 aagcgagaat gtcaaaaatc gcaatggaaa ttctgcgtga cattacgaaa gacacgattg 360 actatcaaga taactatgac ggttcagaaa gagagcctgc cgtcatgcct tcgagatttc 420 cgaatctgct cgtaaacggg gctgccggta ttgcggtcgg aatggcgaca aacattcccc 480 cgcatcagct tggggaagtc attgaaggcg tgcttgccgt aagtgagaat cctgagatta 540 caaaccagga gctgatggaa tacatcccgg gcccggattt tccgactgca ggtcagattt 600 tgggccggag cggcatccgc aaggcatatg aatccggacg gggatcaatc acgatccggg 660 ctaaggctga aatcgaagag acttcatcgg gaaaagaaag aattattgtc acggaacttc 720 cttatcaggt gaacaaagcg agattaattg aaaaaatcgc ggatcttgtc cgagacaaaa 780 aaatcgaagg aattaccgat ctgcgagacg aatccgaccg taacggaatg agaatcgtca 840 ttgagatccg ccgtgacgcc aatgctcacg tcattttgaa taacctgtac aaacaaacgg 900 ccctgcagac gtctttcgga atcaatctgc tggcgctcgt tgacggacag ccgaaggtac 960 taa 963

Claims (5)

B acillus velezensis A-68 (accession number: KACC 91178P) having fibrinolytic enzyme and polysaccharide producing ability . Method for preparing fibrinolytic enzyme comprising the following steps: (a) culturing the microorganism of claim 1 to secrete and produce fibrinase into the culture medium; And (b) recovering fibrinase from the culture solution. The method of claim 2, wherein the carbon source of the culture medium is any one or more selected from the group consisting of rice bran, rice hull, starch and CMC. Method for preparing polysaccharides comprising the following steps: (a) culturing the microorganism of claim 1 to secrete and produce a polysaccharide as a culture medium; And (b) recovering the polysaccharide from the culture. The method of claim 4, wherein the carbon source of the culture medium is any one or more selected from the group consisting of rice bran, rice hull, sugar and CMC.

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