KR100652186B1 - -53 Novel Bacillus Subtilis Subsp. Subtilis A-53 and Method for Preparing Cellulase Using the Same - Google Patents

Abstract

A novel Bacillus subtilis subspecies subtilis A-53 derived from seawater is provided to show excellent cellulase formation capability, thereby being used for mass-producing the cellulase. The novel Bacillus subtilis subspecies subtilis A-53 is deposited as a deposit number of KACC 91179P. The method for preparing cellulase comprises the steps of: (a) culturing the Bacillus subtilis subspecies subtilis A-53 to secret and generate cellulase to a culture solution; and (b) recovering the cellulase from the culture solution.

Description

Novel Bacillus Subtilis Subsp. Subtilis A-53 and a method for producing fibrinase using the same [Novel Bacillus Subtilis Subsp. Subtilis A-53 and Method for Preparing Cellulase Using 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 subtilis subspis subtilis A-53 and other strains based on determining partial sequencing of 16S rDNA and zyrease A gene.

3 shows the growth of Bacillus subtilis subspis subtilis A-53 according to the type of carbon source.

Figure 4 shows the protein production amount of Bacillus subtilis subspis subtilis A-53 according to the type of carbon source.

Figure 5 shows the fibrinolytic activity of Bacillus subtilis subspis subtilis A-53 according to the type of carbon source.

Field of invention

The present invention provides a novel microorganism Bacillus subtilis subspis subtilis A-53 ( Bacillus subtilis subsp. Subtilis A-53) and a method for producing fibrinase using the same, and more specifically, Bacillus subtilis subspecosis subtilis A-53 identified from seawater and fibrinase, characterized by culturing the same. It relates to a manufacturing method of.

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, hindering the normal reproduction of plankton, and causing a number of problems that increase pollution of water resources. In addition, conventional laundry or kitchen detergents contain phosphorus to increase the washing power, causing eutrophication.

Accordingly, the present inventors have made intensive efforts to develop strains having an excellent ability to produce cellulose degrading enzymes. As a result, the present inventors have isolated and identified Bacillus subtilis subspis subtilis A-53 having the ability to produce cellulose lyase from seawater, It was confirmed that a large amount of high quality fibrinase can be obtained through the culturing, and the present invention was completed.

After all, it is an object of the present invention to provide a novel microbial Bacillus subtilis subspis subtilis A-53 and a method for producing fibrinase using the same.

In order to achieve the above object, the present invention provides a Bacillus subtilis subspis subtilis A-53 (Accession Number: KACC 91179P) having a fibrinolytic enzyme production 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, maltose, starch and CMC.

Hereinafter, the present invention will be described in detail.

After liquid culture of the microorganisms separated from seawater, the supernatant from which the cells were removed by centrifugation of the culture solution was dipped in a predetermined amount onto a paper disc of 1.0 cm in diameter and dried, followed by carboxymethyl cellulose, a derivative of cellulose. CMC) was added to the solid medium added, and the strains decomposing carboxymethyl fiber were isolated while incubating at a constant temperature. 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 the strain is a genus of Bacillus, a bacterium, and Bacillus subtilis subsp. system subtilis A-53 (Bacillus subtilis subsp. subtilis A-53).

As a result of culturing Bacillus subtilis subspis subtilis A-53 with different carbon sources, the growth of the strain was excellent when rice bran, rice hull, maltose, CMC (carboxymethyl cellulose) or starch was used as a carbon source. When CMC, or starch was used as the carbon source, it was confirmed that the cellulose-enzyme generating ability of the strain was excellent.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, 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. ) The microorganisms were separated by plating on a plate and incubating at 30 ° C. The isolated microorganisms were inoculated with platinum platinum in marine broth, incubated at 25-35 ° C. for 150-200 rpm for 55-85 hours, and then the obtained broth was in 3-5% (v / v) in fresh broth (marine broth). ) Was inoculated and incubated at 25-35 ° C. at 150-200 rpm for 3-5 days. 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 fibrinolytic enzymes, CMC contained in the medium is degraded to generate 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 subtilis subspis subtilis A-53 strain

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 subtilis subspis subtilis A-53 strain 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-53 strain could be partially determined.

16S rDNA sequence of the A-53 strain (871 bp): SEQ ID NO: 3 5 '- GCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAAC ACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTC - 3'

3-2: Identification of Bacillus subtilis subspis subtilis A-53 strain by zyrease 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-53 strain could be partially determined.

Zayase A base sequence of the A-53 strain (962 bp): SEQ ID NO: 6 5 '- ATGCAATGAGCGTTATCGTGTCCCGTGCTCTTCCGGATGTTCGAGACGGTTTAAAACCAGTTCATAGACGGATTTTGTATGCA ATGAATGATTTAGGCATGACAAGTGACAAGCCTTATAAAAAATCCGCGCGTATCGTTGGAGAAGTTATCGGGAAATACCACCCGCACGGTGATTCAGCGGTATATGAATCCATGGTCAGAATGGCTCAGGATTTCAACTACCGTTATATGCTCGTTGACGGTCACGGAAACTTCGGTTCTGTTGACGGAGACTCAGCGGCGGCCATGCGTTATACAGAAGCAAGAATGTCTAAAATCTCAATGGAGATTCTTCGTGACATCACAAAAGACACAATCGATTACCAGGATAACTATGACGGGTCAGAAAGAGAACCTGTCGTTATGCCTTCAAGGTTCCCGAATCTGCTCGTGAACGGTGCTGCCGGCATTGCGGTAGGTATGGCAACAAACATTCCTCCGCACCAGCTGGGAGAAATCATTGACGGAGTACTTGCTGTCAGTGAGAATCCGGACATTACCATTCCAGAGCTTATGGAAGTCATTCCAGGACCTGATTTCCCGACTGCGGGTCAAATCTTGGGCCGCAGCGGTATCCGGAAAGCATACGAATCAGGCCGAGGCTCTATCACGATCCGGGCAAAAGCTGAGATCGAACAAACATCTTCGGGTAAAGAAAGAATTATCGTTACAGAGTTACCTTACCAAGTAAATAAGGCGAAATTAATTGAGAAAATTGCTGATCTCGTAAGGGACAAAAGATAGAGGGTATCACAGATCTGCGTGATGAGTCAGATCGTACAGGTATGAGAATTGTCATTGAAATCAGACGCGATGCCAATGCGAATGTCATCTTAAACAATCTGTACAAACAAACTGCTCTACAAACATCTTTTGGCATCAACCTCCTTGCACTTGTTGATGGCCAGCCGAAAGTTTTAA - 3'

Based on the 16S rDNA of the selected A-53 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 subtilis subsp. subtilis KCTC 3135T AF272021 98.94 8/758 Bacillus subtilis subsp. spizizenii NRRL B-23049T AF272020 95.06 40/809 Bacillus vallismortis NRRL B-14890T AF272025 93.02 63/902 Bacillus mojavensis NRRL B-14698T AF272019 83.70 127/779 Bacillus amyloliquefaciens KCTC 1660T AF272015 83.03 159/937 Bacillus velezensis LMG 22478T 83.02 152/895 MD 1713 82.33 170/962 Bacillus atrophaeus KCTC 3701T AF272016 81.93 157/869 Bacillus licheniformis KCTC 1918T AF272017 78.45 184/854

Based on this result, the selected strain was identified as Bacillus subtilis subsp. Subtilis , and Bacillus subtilis subsp. Subtilis A-53 ( Bacillus subtilis subsp. Subtilis ) was identified. A-53). And on October 26, 2005, it was deposited with the Korean Agricultural Culture Collection (KACC) under the deposit number KACC 91179P at the Rural Development Administration.

Example 4: Growth of Bacillus subtilis subspis subtilis A-53 according to carbon source

Bacillus subtilis subspis subtilis A-53 strain of the present invention was inoculated with platinum on Marine broth and incubated at 25-35 ° C. for 150-200 rpm for 55-85 hours. The spawn culture was again fibrinase production medium [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] was inoculated at 3 to 5% (v / v), and incubated at 25 to 35 ° C. at 150 to 200 rpm for 3 to 5 days. OD of the culture was measured to observe the growth of the A-53 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 A-53 strain was cultured in the medium added with rice bran, rice hull, maltose, CMC or starch as a carbon source, it was confirmed that the growth is excellent.

Example 5: Fibrinase by Bacillus subtilis subspis subtilis A-53

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 concentration of protein in the supernatant was measured using the Bradford method (FIG. 4).

As a result, as shown in FIG. 4, Bacillus subtilis subspis subtilis A-53 strains were cultured using rice bran, chaff, CMC, or starch as a carbon source than glucose, fructose, maltose or sugar. In this case, it was confirmed that the amount of protein produced increased.

In order to measure the activity of fibrinolytic enzymes in the supernatant, first, 0.5 ml of 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 reduction of The degree of production was measured (FIG. 5). Since fibrinase converts the carbon source in the medium to sugars, an increase in the amount of reducing sugar produced means that the activity of fibrinase is high.

As a result, as shown in Figure 5, the activity of fibrinase was the highest when the strain was cultured for 72 hours using maltose as a carbon source, the activity at this time was about 6,000 unit / ml. In addition, fructose, grapevine, sugar and starch have also been found to be excellent carbon sources for the production of fibrinase. One unit represents the amount of enzyme that hydrolyzes cellulose to produce 1 μmol of reducing sugar for 10 minutes.

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 subtilis subspis subtilis A-53 and a method for producing fibrinase using the same. According to the present invention, it is possible to produce fibrinase by using agricultural by-products such as rice bran or rice hulls as a carbon source, and thus, at a lower cost than conventional methods of producing fibrinase using expensive glucose or sugar. Enzymes can be produced, and rice bran and rice husk, one of the causes of environmental pollution, are useful for establishing environmentally friendly cellulose degrading enzyme production processes that reduce environmental pollution.

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Claims (3)

Bacillus subtilis subspis subtilis A-53 with fibrinase generating ability (Accession No .: KACC 91179P). 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, maltose, starch and CMC.

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