KR101463899B1 - Novel strain Cellulophaga sp. degrading polysaccharides and a method for degrading polysaccharides using the same - Google Patents

Abstract

The present invention relates to a novel strain of Cellulophaga sp. Having a polysaccharide-decomposing activity . More particularly, the present invention relates to a method for isolating microorganisms from seawater, tidal flats and coastal waters of the South Sea region, The microorganisms capable of degrading methylcellulose (CMC) were selected, and 16S rRNA analysis was performed to confirm that the microorganism was a new strain of cellulase. Then, the strain and its culture supernatant were subjected to various polysaccharide decomposing activities Respectively.

Description

Cellulophaga genus strain having a novel polysaccharide degrading activity and its use {Novel strain Cellulophaga sp. degrading polysaccharides and a method for degrading polysaccharides using the same}

The present invention relates to a novel strain of Cellulophaga sp. having polysaccharide decomposition activity, and more particularly, to a novel cellulopaga genus having various polysaccharide decomposition activities isolated from seawater, tidal flats, and coastal areas. It relates to strains.

Biofuel is renewable and environmentally friendly. This is because biofuels are produced from biomass produced by photosynthesis by fixing carbon dioxide. In particular, the need for biofuels is on the rise due to the ever-declining concerns over the supply of chemical fuels and the demand for clean renewable energy.

Currently, two biofuels, bioalcohol (bioethanol) and biodiesel, are being proposed and used as alternative fuels to gasoline and diesel, respectively. Not only do they have a positive effect on reducing carbon dioxide emissions in the atmosphere and global warming, but they can be used without changing the existing automotive system. An example of biofuel is the production of bioethanol using cellulose resources such as grass, hay and switchgrss. Recently, the annual worldwide production of bioethanol is estimated at approximately 1 billion gallons. Most of these are produced from starches and sugars in existing established systems. Current technology is limited to the production of biofuels using polysaccharides derived from land plants, the most abundant carbon source in nature. In particular, land plants vary according to their type, but take time to grow and respond easily to changes in climatic conditions. Therefore, there is a limit to commercializing biofuels with polysaccharides derived from land plants.

Seaweeds living in aquatic life proliferate through asexual reproduction by spores and sexual reproduction by spores. They can grow at a faster rate compared to land plants if appropriate conditions are met. In addition, they live in the oceans, which account for more than 70% of the planet, so there is a high possibility of supplying them. Therefore, extracellular polysaccharides derived from seaweeds that can grow at a fast rate and have a large cultivation area compared to land plants may provide higher efficiency in producing biofuels compared to polysaccharides derived from land plants.

Cellulose refers to a polysaccharide composed of β-(1, 4)-linked D-glucose units. It is a major component of plant cell walls and is the most abundant carbon source on Earth. The decomposition of cellulose by microorganisms is an important issue for global carbon recycling. Cellulose-degrading microorganisms produce endoglucanase, exoglucanase, beta-glucosidase, and auxiliary enzymes, thereby synergizing the β-1,4-glucoside bond of cellulose and effectively decomposing it into glucose. These enzymes contain an accessory module often referred to as a carborhydrate binding domain (CBD). The main role of CBD is in causing effective hydrolysis by transporting the catalytic domains of cellulase more closely to the cellulose surface.

Currently, cellulase is widely used in the animal feed, textile, waste treatment and brewing industries (Beguin, 1983; Mandels, 1985). In addition, biofuels such as ethanol and butanol through fermentation using glucose, obtained by decomposing cellulose biomass by cellulase, are increasingly being regarded as attractive sustainable and alternative energy sources. Therefore, effective cellulose degradation using cellulase becomes an important issue in the use of cellulose biomass. Large amounts of cellulolytic enzymes produced by fungi and bacteria have been isolated and characterized from various sources such as compost soil, decaying plant material, sewage debris, ruminant feces and harsh environments (Blumer-Schuette et al., 2008; Doi, 2008; Maki et al., 2009; Viikari et al., 2007). However, relatively few studies have been conducted on the cellulose degradation system obtained from marine bacteria (Ekborg et al., 2007; Fu et al., 2010; Gao et al., 2010; Hirasawa et al., 2006; Ryu et al. al., 2001; Shanmughapriya et al., 2010; Taylor et al., 2006; Yin et al., 2010; Zeng et al., 2006). Recently, it ^{has been reported for Saccharophagus degradans 2-20 T} and Teredinibacter turnerae T7902 ^T (Taylor et al., 2006; Yang et al., 2009). S.degradans 2-20 ^T and T. turnerae T7902 ^T possess multiple endoglucanases and exoglucanases, as well as polysaccharide degradation systems of other marine algae and plants. Thus, marine bacteria are likely to be a rich source for novel cellulosic biomass degradation systems.

Accordingly, in order to select a new microorganism capable of decomposing seaweed and plant-derived polysaccharides, the present inventors collect various samples and separate the microorganisms, and then remove a microorganism capable of decomposing carboxymethyl cellulose (CMC). The present invention was completed by selecting and confirming that it is a novel microorganism belonging to the genus Cellulophaga through 16S rRNA analysis, and then confirming that the novel microorganism can effectively degrade various polysaccharides.

It is an object of the present invention to provide a novel strain having polysaccharide degrading activity, a composition for decomposing polysaccharides using the strain, and a method for decomposing polysaccharides using the strain.

In order to achieve the above object, the present invention provides a strain of the genus Cellulophaga having polysaccharide decomposition activity.

In addition, the present invention provides a composition for decomposing polysaccharides comprising a cellulopaga genus strain having a polysaccharide decomposing activity and a culture supernatant thereof.

In addition, the present invention provides a polysaccharide decomposition method comprising the step of contacting a cellulosic strain having a polysaccharide decomposing activity and a culture supernatant thereof with a polysaccharide.

In addition, the present invention

1) separating polysaccharides from biomass;

2) producing a monosaccharide by contacting the polysaccharide of step 1) with the strain of claim 1 or 2, or a culture supernatant thereof; And

3) It provides a method for producing bioethanol comprising the step of fermenting the monosaccharide of step 2) by a microorganism.

An object of the present invention relates to a novel strain of Cellulophaga genus having polysaccharide decomposition activity, and after separating microorganisms by collecting samples from seawater, tidal flats, and coastal areas in the South Sea region, carboxymethylcellulose (CMC) After selecting a microorganism capable of decomposing the cellulose, and confirming that the microorganism is a novel strain of the genus Cellulopa through 16S rRNA analysis, it was confirmed that the strain and its culture supernatant have decomposition activity of various polysaccharides.

1 is a diagram showing a schematic analysis of 16S rRNA of PDB-1 and PDB-2 in Cellulophaga genus. The adjacent phylogenetic tree along the 16S rRNA gene shows the location of PDB-1 and PDB-2 among other related taxa. Bootstrap values (expressed as a percentage of 1000 replicates)> 50% at the branching point are indicated. Bacteroides fragilis ATCC 25285T (GenBank accession number, X83935) was used as an outgroup. Scale bar, 0.01 substitutions per nucleotide position.
FIG. 2 is a diagram showing CMC-SDA-PAGE analysis and gyrography for extracellular cellulase of PDB-1 and PDB-2 of the genus Cellulophas;
The left panel shows SDS-PAGE analysis of proteins precipitated in 60% and 70% ammonium sulfate through Coomassie Brilliant Blue R-250 stained 0.1% CMC; And
The right panel shows the zymography of the cellulase activity staining of the precipitated protein.
Lanes: M: molecular weight marker protein
60 is the precipitated extracellular protein of PDB-1 and PDB-2 at 60% ammonium sulfate;
70 is the precipitated extracellular protein of PDB-1 and PDB-2 at 70% ammonium sulfate; And
Arrows indicate 35 kDa proteins subjected to N-terminal sequencing.
Figure 3 is a diagram showing the galactan (Galactan) resolution of the secreted protein of the cellulopaga genus PDB-1. The x-axis represents monosaccharides and polysaccharides contained in the culture medium, and the y-axis represents low-temperature soluble agarose and carrageenan, which are polysaccharides contained in the reaction solution when a culture medium containing 1 mL of PDB-1 secreted protein is added. ) Shows the amount of reducing sugar released per hour.
Figure 4 is a diagram showing the glucan (Glucan) resolution of the secreted protein of the genus PDB-1 Cellulopa. The x-axis represents monosaccharides and polysaccharides contained in the culture medium, and the y-axis represents the polysaccharides Starch, CMC, and Laminarin contained in the reaction solution when the culture medium containing 1 mL of PDB-1 secreted protein is added. ), it shows the amount of reducing sugar released per hour from pullulan.
5 is a diagram showing the resolution of xylan and other polysaccharides of the secreted protein of the genus PDB-1 of cellulose. The x-axis represents the monosaccharides and polysaccharides contained in the culture medium, and the y-axis represents the polysaccharides xylan, alginate, and colloidal chitin contained in the reaction solution when the culture medium containing 1 mL of PDB-1 secreted protein is added. It shows the amount of reducing sugar released per hour from (Chitin) and pectin.
Figure 6 is a diagram showing the galactan (Galactan) resolution of the secreted protein of the cellulopaga genus PDB-2. The x-axis represents monosaccharides and polysaccharides contained in the culture medium, and the y-axis represents low-temperature soluble agarose and carrageenan, which are polysaccharides contained in the reaction solution when a culture medium containing 1 mL of PDB-2 secreted protein is added. It shows the amount of reducing sugar released per hour from
Figure 7 is a diagram showing the glucan (Glucan) resolution of the secreted protein of the genus cellulose PDB-2. The x-axis represents the monosaccharides and polysaccharides contained in the culture medium, and the y-axis represents the polysaccharides Starch, CMC, and Laminarin, which are polysaccharides contained in the reaction solution when a culture medium containing 1 mL of PDB-1 secreted protein is added. , It shows the amount of reducing sugar released per hour from pullulan.
8 is a diagram showing the resolution of xylan and other polysaccharides of the secreted protein of the genus PDB-2 of cellulose. The x-axis represents the monosaccharides and polysaccharides contained in the culture medium, and the y-axis represents the polysaccharides xylan, alginate, and colloidal chitin contained in the reaction solution when a culture medium containing 1 mL of PDB-1 secreted protein is added. It shows the amount of reducing sugar released per hour from (Chitin) and pectin.

Hereinafter, the present invention will be described in detail.

The present invention accession number KCTC11940BP Cellulophaga sp. It provides a strain of the genus Cellulophaga having a polysaccharide degrading activity deposited as PDB-1.

In addition, the present invention accession number KCTC11941BP Cellulophaga sp. It provides a strain of the genus Cellulophaga having a polysaccharide degrading activity deposited as PDB-2.

The polysaccharide is preferably any one selected from the group consisting of agarose, carrageenan, starch, pullunan, carboxylmethylcellulose (CMC), xylan, laminarin, chitin, and pectin, but is not limited thereto.

In one embodiment of the present invention, in order to separate the microorganisms that degrade polysaccharides, the present inventors separate the microorganisms by collecting samples from seawater, tidal flats and coastal areas in the southern sea area, and then decompose carboxymethyl cellulose (CMC). The capable microorganisms were selected.

In addition, in one embodiment of the present invention, the present inventors performed sequencing and phylogenetic analysis of the 16S rRNA gene in order to identify the selected microorganism. As a result, these microorganisms were shown in SEQ ID NO: 1 and SEQ ID NO: 2. Together with C. lytica ATCC 23178 ^T and C. fucicola NN015860 ^{T It} was confirmed that it corresponds to the classification of a clade including the genus Cellulophaga (Cellolopaga) forming a consistent cluster (see Fig. 2).

In addition, in one embodiment of the present invention, the present inventors performed a zymogram analysis on SDS-PAGE with CMC in order to confirm the cellulose-degrading activity of the selected microorganism. As a result, the microorganism has a cellulose-degrading enzyme. Confirmed (see Fig. 2).

In addition, in one embodiment of the present invention, the present inventors performed N-terminal sequencing of the 35 kDa protein of PDB-1, and as a result, it was confirmed that the N-terminal sequence of the 35 kDa protein did not match other known cellulose degrading enzymes. I did.

In addition, in one embodiment of the present invention, in order to analyze the degradation activity of the secreted protein of PDB-1, the present inventors As a result of confirming the cellulolytic activity of the secreted protein of PDB-1, the secreted protein of PDB-1 hydrolyzed CMC well, whereas the crystalline substrate could not be effectively hydrolyzed. Through this, it was confirmed that these enzymes showed more activity in the β-1,4 binding of the amorphous region of cellulose (see Table 1).

In addition, in one embodiment of the present invention, in order to confirm the polysaccharide degrading activity of PDB-1 and PDB-2, the present inventors decompose polysaccharides of proteins secreted to the outside of cells after culturing under a minimal medium containing polysaccharides. As a result of confirming the ability, it was confirmed that PDB-1 and PDB-2 have a decomposition system of various polysaccharides such as agarose, carrageenan, starch, pullulan, CMC, xylan, laminarin, chitin, and pectin (Fig. 3 to See Fig. 8).

Accordingly, a novel microorganism having decomposition activity for the various polysaccharides was identified into the genus Cellulopha , and Cellulophaga sp. PDB-1 and Cellulophaga sp. It was named PDB-2 and was deposited with the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Biological Resource Management Division (KBRC) Microbial Resource Center (KCTC) on May 24, 2011 (Accession numbers: KCTC11940BP and KCTC11941BP).

In addition, the present invention provides a composition for decomposing polysaccharides comprising PDB-1 and PDB-2, or a culture supernatant thereof.

In one embodiment of the present invention, in order to analyze the degradation activity of the secreted protein of PDB-1, the present inventors As a result of confirming the cellulolytic activity of the secreted protein of PDB-1, the secreted protein of PDB-1 hydrolyzed CMC well, whereas the crystalline substrate could not be effectively hydrolyzed. Through this, it was confirmed that these enzymes showed more activity in the β-1,4 binding of the amorphous region of cellulose (see Table 1).

In addition, in one embodiment of the present invention, in order to confirm the polysaccharide degrading activity of PDB-1 and PDB-2, the present inventors decompose polysaccharides of proteins secreted to the outside of cells after culturing under a minimal medium containing polysaccharides. As a result of confirming the ability, it was confirmed that PDB-1 and PDB-2 have a decomposition system of various polysaccharides such as agarose, carrageenan, starch, pullulan, CMC, xylan, laminarin, chitin, and pectin (Fig. 3 to See Fig. 8).

Therefore, PDB-1 and PDB-2 of the present invention, or a culture supernatant thereof, has a decomposing activity for various polysaccharides, and thus can be usefully used as a composition for decomposing polysaccharides.

In addition, the present invention provides a method for decomposing a polysaccharide comprising the step of contacting a polysaccharide comprising PDB-1 and PDB-2, or a culture supernatant thereof.

In the step of contacting the polysaccharide, the polysaccharide and PDB-1 and PDB-2, or a culture supernatant thereof, are mixed at 55° C.-85° C., preferably 65° C., at a pH of 5.0 to 5.5 or 7.5 to 8.0 for 10 to 30 minutes. It is preferable to contact, but is not limited thereto.

Since PDB-1 and PDB-2 of the present invention, or a culture supernatant thereof, has a decomposing activity for various polysaccharides, it can be used as a polysaccharide decomposition method including the step of contacting a polysaccharide.

In addition, the present invention

1) separating polysaccharides from biomass;

2) producing a monosaccharide by contacting the polysaccharide of step 1) with the strain of claim 1 or 2, or a culture supernatant thereof; And

3) It provides a method for producing bioethanol comprising the step of fermenting the monosaccharide of step 2) by a microorganism.

The biomass is rice straw, barley straw, sweet potato stalk, rapeseed stalk, cassava stalk agricultural by-products, waste paper, solid waste of fluff, thinning wood, waste wood, wood of processed by-products, brown algae, marine algae And it is preferably any one selected from the group consisting of a combination thereof, but is not limited thereto.

The polysaccharide of step 1) is preferably any one selected from the group consisting of agarose, carrageenan, starch, pullunan, carboxymethylcellulose, xylan, laminarin, chitin, and pectin, but is not limited thereto.

The microorganisms of step 3) are Saccharomyces cerevisiae, Sarsina ventriculi, Kluyveromyces prazilis, Zygomonas mobilis, Kluyveromyces maximus IMB3, Bretanomyces custersi, Clos. Tridium acetobutylicum, Clostridium Bayerlinki, Clostridium aurantibutylicum, and Clostridium thetanomorphium are preferably any one selected from the group consisting of, but is not limited thereto.

Since PDB-1 and PDB-2 of the present invention, or a culture supernatant thereof, has decomposition activity for various polysaccharides, bioethanol is effectively produced by reacting microorganisms capable of producing biofuels with the polysaccharide decomposition products generated through such decomposition. Can be manufactured.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples and experimental examples.

<Example 1> Isolation of microorganisms

In order to isolate microorganisms that produce cellulase, microorganisms growing in Marine Agar 2216 (MA; Difco, USA) were isolated using a dilution plating method on the sandy shores of the South Sea of Geoje Island, Korea.

In order to select the microorganisms that produce cellulase among the isolated microorganisms, microorganisms capable of decomposing carboxymethyl cellulose (CMC) were selected.

Specifically, the isolated microorganisms were transferred by dipping and smearing on CMC agar medium (1.0% carboxymethyl cellulose (Sigma, USA) and MA), followed by incubation at 25° C. for 3 days, and then 0.1% of the plates. After staining with congo red solution for 15 minutes, washing with 1M NaCl (Beguin, 1983), two strains with CMC degrading activity showing large halos were selected. .

<Example 2> Identification of microorganisms

In order to classify the microorganisms selected in <Example 1>, chromosomal DNA extraction and purification were performed on the isolated bacteria according to the method described in Yoon et al., and the modification of RNase T1 102 was performed with RNase A. By using in combination, contamination by RNA was minimized (Yoon et al., 1996). After performing 16S rRNA gene amplification using the two universal primers mentioned above (Yoon et al., 1998), sequencing and lineage of the amplified 16S rRNA gene according to the method described in Yoon et al. Embryologic analysis was performed (Yoon et al., 2003).

As a result, according to the phylogenetic analysis for 16S rRNA of PDB-1 and PDB-2, as shown in FIG. 1, these microorganisms were C lytica ATCC 23178 ^T and C as shown in SEQ ID NO: 1 and SEQ ID NO: 2. It was confirmed that it corresponds to the classification of the clade including the genus Cellulophaga (Celolopega) that forms a cluster consistent with fucicola NN015860 ^T. In addition, strain PDB-2 is strain PDB-1, C. fucicola NN015860 ^T , And C. lytica ATCC 23178 ^T , 16S rRNA gene homology for each of 99.0, 98.5, and 97.6%, but after confirming that it has a value of 92.2 to 93.6% for other Cellulophaga genus (Fig. 1), Cellulophaga sp . PDB-1 and Cellulophaga sp. It was named PDB-2 and was deposited with the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Biological Resource Management Division (KBRC) Microbial Resource Center (KCTC) on May 24, 2011 (Accession numbers: KCTC11940BP and KCTC11941BP).

<Example 3> Confirmation of cellulose decomposition activity of microorganisms

In order to confirm the cellulose-degrading activity of the microorganisms selected in <Example 2>, the extracellular proteins of PDB-1 and PDB-2 cultured for two days at 30° C. in 0.2% CMC MA medium at 4° C. Salting out was performed. Most of the active cellulase fraction was precipitated between 50-70% of saturated ammonium sulfate and the desalted fraction was analyzed using a zymography technique.

Specifically, after preparing a 0.1% CMC SDS-PAGE gel, 70 V electrophoresis, and then refolding buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% Triton-X100) Incubated for 1 hour at room temperature, and replaced with a new refolding buffer. After additional incubation for 1 hour, the gel was maintained overnight at 20 mM Tris-HCl, pH 8.0 with 150 mM NaCl, and then stained with 0.1% Congo Red.

As a result, as a result of performing a zymogram analysis on SDS-PAGE with CMC as shown in FIG. 2, it was confirmed that each bacterium had a cellulose-degrading enzyme having various molecular weights and similar clear bands, and , It was confirmed that they have a similar cellulosic decomposition system (Fig. 2).

<Example 4> Analysis of the activity of microbial-derived proteins

<4-1> N-terminal sequencing

N-terminal sequencing of the 35 kDa protein of PDB-1 was performed. After SDS-PAGE analysis of the precipitated fraction of the extracellular protein of PDB-1 in 70% saturated ammonium sulfate, it was electrotransferred onto a polyvinylidene difluoride membrane (PVDF). After that, the N-terminal amino acid sequencing of the 35 kDa protein was performed using an Automatic Protein Sequencer (Applied Biosystems).

As a result, it was confirmed that the N-terminal sequence of the protein was NTAQTVV. Recently, the genomic sequence of C. algicola DSM 14237 has been reported, cellulase (GenBank accession numbers. ADV51156, ADV50035, and ADV 47383) and glucoside hydrolysis line 5 (GenBank accession number. ADV50845). However, the N-terminal sequence of the 35 kDa protein did not match with C. algicola DSM 14237 cellulase as well as other known cellulolytic enzymes. Therefore, it was confirmed that PDB-1 of the genus Cellulophaga possesses a novel cellulose decomposition system.

<4-2> Analysis of the degradation activity of secreted proteins of PDB-1

To investigate the cellulose decomposition activity of the secreted protein of PDB-1, use 450 μL of carboxymethylcellulose (CMC), 20 μm of microcrystalline cellulose and Avicel PH-101, and cellulose fiber at a pH of 5.0 at a concentration of 0.5%. It was suspended in 25 mM citrate buffer solution and 10 mM phosphate buffer solution at pH 7.0. After 50 μg of the secreted protein of PDB-1 was added to each reaction solution, the reaction was carried out at 30° C. for 1 hour. After heating to 100 ℃ to inactivate the enzyme, the released reducing sugar para-hydroxybenzoic acid hydrazine Zaid (p -hydroxybenzoic acid hydrazide; pHBH) using the assay was measured by the change in absorbance at 415 nm (Moretti and Thorson, 2008 ).

As a result, as shown in Table 1, while the secreted protein of PDB-1 hydrolyzed CMC well, the crystalline substrate could not be effectively hydrolyzed. Through this, it was confirmed that these enzymes showed more activity in the β-1,4 binding of the amorphous region of cellulose. In addition, it was confirmed that the specific activities at pH 5.0 and pH 7.0 are similar. Thus, the cellulose degradation system of PDB-1 is active at acidic and neutral pH. Since the marine environment is generally weakly alkaline, considering the fact that most of the characterized cellulase obtained in the marine environment exhibits maximum activity under neutral and weakly alkaline conditions, and decreases in acidic conditions, the cellulose system of PDB-1 It is an interesting fact that CMC can be hydrolyzed well under this pH of 5.0. In addition, the total extracellular protein and the precipitated protein in 50 to 70% saturated ammonium sulfate showed similar specific activity. Thus, the cellulose decomposition system of PDB-1 retained its activity even under acidic conditions. It has been confirmed that these properties can be usefully used in bioengineering applications such as decomposition of pretreated cellulose materials under acidic conditions.

<Example 5> Confirmation of polysaccharide decomposition activity of cellulose PDB-1

In order to measure the ability of cellulosic PDB-1 to decompose polysaccharides, cellulosic PDB-1 was cultured using the following medium. Each medium contains 0.2% carbon source and artificial sea water (Artificial sea water: NaCl 23.6 g/L, KCl 0.64 g/L, MgCl ₂ 6H ₂ O 4.53 g/L, MgSO ₄ H ₂ O 5.94 g/L, CaCl ₂ H ₂ O 1.3 g/L), add 1.0 g/L of yeast extract _{, 0.5 g/L of ammonium chloride (NH 4} Cl), 6.05 g/L of Trizma Base, and use hydrochloric acid. Then, it was titrated to pH 7.4. Monosaccharides used were glucose, galactose, N-acetylglucosamine (NAG), and xylose, and polysaccharides were low-temperature soluble agarose (Agarose, Lonza), alginic acid. (Alginate, Wako), Carrageenan (Sigma), Carboxymethylcellulose (Sigma), Colloidal Chitin (Sigma), Laminarin (Sigma), Pectin (Pectin from apple peel, Sigma), Pullul Eggs (Pullulan, Sigma), starch (Soluble starch, Difco), and xylan (Birchwood xylan, Sigma) were used, and cultured including a medium to which monosaccharides and polysaccharides were not added. Colloidal chitin was prepared by a conventional method using concentrated hydrochloric acid. As a carbon source for seed culture, 0.1% Bacto-Trypton (Difco) was used.

Specifically, 3 mL of the strain was seeded, followed by 30 mL of seed culture, and then a 50 mL medium containing monosaccharides and polysaccharides was cultured at 30°C in the main culture. Then, after removing the cells through centrifugation, the medium solution was frozen and stored. The amount of protein secreted out of the cell was measured by the Lowey method. The polysaccharide used in the reaction to measure the activity of the secreted protein was the same as the polysaccharide used as a carbon source in the medium, and the concentration of the stock solution was 0.5%. After mixing the polysaccharide stock solution and the medium, it was reacted at 400 rpm for 24 hours at 30°C. After diluting each reaction solution to 1/10, 40 μL of the diluted solution was mixed with 120 μL of a 1:1 mixture of 2% (w/v) pHBH and 2M NaOH dissolved in 0.5M hydrochloric acid, followed by heating at 100° C. for 7 minutes. Then, 415 nm absorbance was measured using microplate analysis (Moretti and Thorson, 2008).

As a result, as shown in Figs. 3 to 5, it was confirmed that cellulopaga PDB-1 has significant polysaccharide decomposition activity against galactan, glucan, xylan, and other polysaccharides (Figs. 3 to 5).

<Example 6> Cellulopa confirmed polysaccharide decomposition activity of PDB-2

In order to measure the ability of cellulosic PDB-2 to decompose polysaccharides, cellulosic PDB-2 was cultured using the following medium. Each medium contains 0.2% carbon source and artificial sea water (NaCl 23.6 g/L, KCl 0.64 g/L, MgCl ₂ 6H ₂ O 4.53 g/L, MgSO ₄ H ₂ O 5.94 g/L, CaCl ₂ H ₂ O 1.3 g/L), add 1.0 g/L of yeast extract _{, 0.5 g/L of ammonium chloride (NH 4} Cl), 6.05 g/L of Trizma Base, and use hydrochloric acid. Then, it was titrated to pH 7.4. Monosaccharides used were glucose, galactose, N-acetylglucosamine (NAG), and xylose, and polysaccharides were low-temperature soluble agarose (Agarose, Lonza), alginic acid ( Alginate, Wako), Carrageenan (Sigma), Carboxymethylcellulose (Sigma), Colloidal Chitin (Sigma), Laminarin (Sigma), Pectin (Pectin from apple peel, Sigma), Pullulan (Pullulan, Sigma), starch (Soluble starch, Difco), and xylan (Birchwood xylan, Sigma) were used, and cultured including a medium to which monosaccharides and polysaccharides were not added. Colloidal chitin was prepared by a conventional method using concentrated hydrochloric acid. As a carbon source for seed culture, 0.1% Bacto-Trypton (Difco) was used.

Specifically, 3 mL of the strain was seeded, followed by 30 mL of seed culture, and then a 50 mL medium containing monosaccharides and polysaccharides was cultured at 30°C in the main culture. Then, after removing the cells through centrifugation, the medium solution was frozen and stored. The amount of protein secreted out of the cell was measured by the Lowey method. The polysaccharide used in the reaction to measure the activity of the secreted protein was the same as the polysaccharide used as a carbon source in the medium, and the concentration of the stock solution was 0.5%. After mixing the polysaccharide stock solution and the medium, it was reacted at 400 rpm for 24 hours at 30°C. After diluting each reaction solution to 1/10, 40 μL of the diluted solution was mixed with 120 μL of a 1:1 mixture of 2% (w/v) pHBH and 2M NaOH dissolved in 0.5M hydrochloric acid, followed by heating at 100° C. for 7 minutes. Then, 415 nm absorbance was measured using microplate analysis (Moretti and Thorson, 2008).

As a result, as shown in Figs. 6 to 8, it was confirmed that cellulopaga PDB-2 has significant polysaccharide decomposition activity against galactan, glucan, xylan, and other polysaccharides (Figs. 6 to 8).

Korea Research Institute of Bioscience and Biotechnology Biological Resource Management Division Microbial Resource Center (KCTC) KCTC11941BP 20110524

<110> Korea research institute of bioscience and biotechnology <120> Novel strain Cellulophaga sp. degrading polysaccharides and a method for degrading polysaccharides using the same <130> 14p-01-42 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1441 <212> DNA <213> cellulophaga sp. <400> 1 gatgaacgct agcggcaggc ttaacacatg caagtcgagg ggtaacagag gagcttgctt 60 tgctgacgac cggcgcacgg gtgcgtaacg cgtatacaat ctgccttgca ctaagggata 120 gcccagagaa atttggatta atatcttatg gtttataaat atggcatcat atttataata 180 aaggttacgg tgtaagatga gtatgcgtcc cattagtttg ttggtaaggt aacggcttac 240 caagactacg atgggtaggg gccctgagag ggggatcccc cacactggta ctgagacacg 300 gaccagactc ctacgggagg cagcagtgag gaatattgga caatggagga gactctgatc 360 cagccatgcc gcgtgcagga agacggtcct atggattgta aactgctttt atacaggaag 420 aataaggact acgtgtagtc tggtgacggt actgtaagaa taaggaccgg ctaactccgt 480 gccagcagcc gcggtaatac ggagggtccg agcgttatcc ggaattattg ggtttaaagg 540 gtccgtaggc gggctgttaa gtcaggggtg aaagtttgca gctcaactgt agaattgcct 600 ttgatactga cggtcttgaa ttattgtgaa gtggttagaa tatgtagtgt agcggtgaaa 660 tgcatagata ttacatagaa taccgattgc gaaggcagat cactaacaat tgattgacgc 720 tgatggacga aagcgtgggt agcgaacagg attagatacc ctggtagtcc acgccgtaaa 780 cgatggatac tagctgtttg gatttcgatc tgagcggcca agcgaaagtg ataagtatcc 840 cacctgggga gtacgttcgc aagaatgaaa ctcaaaggaa ttgacggggg cccgcacaag 900 cggtggagca tgtggtttaa ttcgatgata cgcgaggaac cttaccaggg cttaaatgta 960 gattgacagg tttagagata gactttcctt cgggcaattt acaaggtgct gcatggttgt 1020 cgtcagctcg tgccgtgagg tgtcaggtta agtcctataa cgagcgcaac ccctgttgtt 1080 agttaccagc acattatggt ggggactcta gcaagactgc cggtgcaaac cgtgaggaag 1140 gtggggatga cgtcaaatca tcacggccct tacgtcctgg gccacacacg tgctacaatg 1200 gtaggtacag agagcagcca cttagcgata aggagcgaat ctataaaacc tatcacagtt 1260 cggatcggag tctgcaactc gactccgtga agctggaatc gctagtaatc ggatatcagc 1320 catgatccgg tgaatacgtt cccgggcctt gtacacaccg cccgtcaagc catggaagct 1380 gggggtacct gaagttcgtc accgcaagga gcgacctagg gtaaaactgg taactagggc 1440 t 1441 <210> 2 <211> 1442 <212> DNA <213> cellulophaga sp. <400> 2 gatgaacgct agcggcaggc ttaacacatg caagtcgagg ggtaacagag gagcttgctc 60 ttgctgacga ccggcgcacg ggtgcgtaac gcgtatacaa tctgccttac actaagggat 120 agcccagaga aatttggatt aacaccttat agtttatttt catggcatca tttaaataat 180 aaagtttacg gtgtaagatg agtatgcgtc ccattagttt gttggtaagg taacggctta 240 ccaagactac gatgggtagg ggccctgaga gggggatccc ccacactggt actgagacac 300 ggaccagact cctacgggag gcagcagtga ggaatattgg acaatggagg ggactctgat 360 ccagccatgc cgcgtgcagg aagacggtcc tatggattgt aaactgcttt tatacaggaa 420 gaataaggac tacgtgtagt ctggtgacgg tactgtaaga ataaggaccg gctaactccg 480 tgccagcagc cgcggtaata cggagggtcc gagcgttatc cggaattatt gggtttaaag 540 ggtccgtagg cgggctgtta agtcaggggt gaaagtttgc agctcaactg tagaattgcc 600 tttgatactg atggtcttga attattgtga agtggttaga atatgtagtg tagcggtgaa 660 atgcatagat attacataga ataccgattg cgaaggcaga tcactaacaa ttgattgacg 720 ctgatggacg aaagcgtggg tagcgaacag gattagatac cctggtagtc cacgccgtaa 780 acgatggata ctagctgttt ggatttcgat ctgagcggcc aagcgaaagt gataagtatc 840 ccacctgggg agtacgttcg caagaatgaa actcaaagga attgacgggg gcccgcacaa 900 gcggtggagc atgtggttta attcgatgat acgcgaggaa ccttaccagg gcttaaatgt 960 agattgacag gtttagagat agactttcct tcgggcaatt tacaaggtgc tgcatggttg 1020 tcgtcagctc gtgccgtgag gtgtcaggtt aagtcctata acgagcgcaa cccctgttgt 1080 tagttaccag cacattatgg tggggactct agcaagactg ccggtgcaaa ccgtgaggaa 1140 ggtggggatg acgtcaaatc atcacggccc ttacgtcctg ggccacacac gtgctacaat 1200 ggtaggtaca gagagcagcc acttagcgat aaggagcgaa tctataaaac ctatcacagt 1260 tcggatcgga gtctgcaact cgactccgtg aagctggaat cgctagtaat cggatatcag 1320 ccatgatccg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaag ccatggaagc 1380 tgggggtacc tgaagttcgt caccgcaagg agcgacctag ggtaaaactg gtaactaggg 1440 ct 1442

Claims (4)

A cellulosic substance having a polysaccharide-decomposing activity selected from the group consisting of agarose, carrageenan, starch, pullulan, carboxymethylcellulose (CMC), xylan, laminarin, chitin and pectin deposited with accession number KCTC11941BP Cellulophaga strains.
delete A composition for degrading a polysaccharide comprising the strain of claim 1, or a culture supernatant thereof.
A method for degrading a polysaccharide, comprising contacting the strain of claim 1, or a culture supernatant thereof, with a polysaccharide.

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