KR20120131066A - Novel strain Cellulophaga sp. degrading polysaccharides and a method for degrading polysaccharides using the same - Google Patents

Abstract

PURPOSE: A novel cellulophage sp. which is isolated from seawater and mud flat is provided to ensure various polysaccharide decomposition activities. CONSTITUTION: A composition for decomposing polysaccharides contains Cellulophaga sp.(deposit number KCTC11940BP or KCTC11941BP) or a supernatant of a culture thereof. The polysaccharides are agarose, carrageenan, starch, pllulan, carboxylmethylcellulose(CMC), xylan, chitin, or pectin. A method for producing bioethnaol comprises: a step of isolating polysaccharides from a biomass; a step of contacting the strain or supernatant with the polysaccharides to prepare monosaccharides; and a step of fermenting monosaccharides.

Description

Cellulpaga genus strain with 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 wave (Cellulophaga a novel cellulose having polysaccharide-degrading activity sp . In particular, the present invention relates to a novel strain of cellulopa, which has various polysaccharide degrading activities isolated from seawater, tidal flats, and shore.

Biofuels are renewable and environmentally friendly. This is because biofuel is produced from biomass produced by photosynthesis by carbon dioxide fixation. In particular, with the ever-increasing concern for the supply of chemical fuels and the need for clean renewable energy, the need for biofuels is on the rise.

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

Algae in aquatic life multiply through spores 'asexual reproduction method and spouses' sexual reproduction method. They can multiply faster than on land plants if appropriate conditions are met. They also live in the oceans, which make up more than 70% of the world's supply, so they are very likely to be supplied. Thus, extracellular polysaccharides derived from algae that can grow rapidly and have a larger cultivation area than land plants may provide higher efficiency in producing biofuels than polysaccharides derived from land plants.

Cellulose refers to a polysaccharide composed of D-glucose units of β- (1, 4) -bonds. It is a major component of plant cell walls and the most abundant source of carbon on earth. Degrading cellulose by microorganisms is an important issue for global carbon recycling. Cellulose Degrading Microorganisms produce endoglucanase, exoglucanase, beta-glucosidase, and coenzyme to synergistically degrade β-1,4-glucosidic bonds of cellulose and effectively break down glucose into glucose. These enzymes often include an accessory module called the carborhydrate binding domain (CBD). The main role of CBD is to bring about effective hydrolysis by transferring the catalytic domain of cellulase closer to the cellulose surface.

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

Thus, the present inventors, in order to screen new microorganisms capable of degrading algae and plant-derived polysaccharides, isolate various microorganisms by collecting various samples, and then isolate microorganisms capable of degrading carboxymethyl cellulose (CMC). The present invention was completed by screening, confirming that the microorganism belongs 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 a polysaccharide decomposition activity, a composition for polysaccharide decomposition using the strain and a method for degrading polysaccharide using the strain.

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

The present invention also provides a composition for degrading polysaccharide comprising a cellulose genus strain having a polysaccharide degrading activity and a culture supernatant thereof.

The present invention also provides a polysaccharide decomposition method comprising the step of contacting the cellulose genus strain having a polysaccharide degradation activity and its culture supernatant with the polysaccharide.

In addition,

1) separating the polysaccharides from the biomass;

2) contacting the polysaccharide of step 1) with the strain of claim 1 or 2, or a culture supernatant thereof to produce monosaccharides; 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 Cellulophaga genus strain having a polysaccharide degrading activity. The microorganisms capable of degrading were selected, and 16S rRNA analysis confirmed that the microorganisms were novel strains of cellulose, and then the strains and their culture supernatants were confirmed to have degrading activity of various polysaccharides.

1 is a diagram showing a phylogenetic analysis of 16S rRNA of Cellulophaga genus PDB-1 and PDB-2. 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. Bacteroides fragilis ATCC 25285T (GenBank accession number, X83935) was used as the outgroup. Scale bar, 0.01 substitutions per nucleotide position.
FIG. 2 is a diagram showing CMC-SDA-PAGE analysis and gyography of extracellular cellulase of Cellulopa genus PDB-1 and PDB-2; FIG.
The left panel shows SDS-PAGE analysis via Coomassie Brilliant Blue R-250 stained 0.1% CMC of proteins precipitated in 60% and 70% ammonium sulfate; And
The right panel shows zymography for cellulase activity staining of precipitated proteins.
Lanes: M: molecular weight marker protein
60 is the precipitated extracellular protein of PDB-1 and PDB-2 in 60% ammonium sulfate;
70 is the precipitated extracellular protein of PDB-1 and PDB-2 in 70% ammonium sulfate; And
Arrows indicate 35 kDa protein with N-terminal sequencing run.
3 is a diagram showing the galactan (Galactan) resolution of the secreted protein of the genus PDB-1 cellulose. The x-axis shows the monosaccharides and polysaccharides contained on the culture, and the y-axis shows the low-temperature soluble agarose and carrageenan, the polysaccharides contained on the reaction solution, when the culture medium containing the secreted protein of 1 mL of PDB-1 was added. ) Shows the amount of reducing sugar free per hour.
4 is a diagram showing the glucan resolution of the secreted protein of the genus PDB-1 cellulose. The x-axis shows the monosaccharides and polysaccharides contained on the culture, and the y-axis shows the starch, CMC, and laminarin, which are the polysaccharides contained on the reaction solution when the culture medium containing the secreted protein of 1 mL of PDB-1 was added. ) 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 genus PDB-1. The x-axis shows the monosaccharides and polysaccharides contained on the culture, and the y-axis shows the polysaccharides, xylan, alginate and colloid chitin, included in the reaction solution when the culture medium containing 1 mL of PDB-1 secreted protein was added. (Chitin, Pectin) shows the amount of reducing sugar released per hour.
6 is a diagram showing the galactan (Galactan) resolution of the secreted protein of the genus PDB-2 cellulose. The x-axis shows the monosaccharides and polysaccharides contained on the culture, and the y-axis shows the low-temperature soluble agarose and carrageenan, the polysaccharides included in the reaction solution when the culture medium containing 1 mL of PDB-2 secreted protein was added. Shows the amount of reducing sugar liberated per hour from.
7 is a diagram showing the glucan resolution of the secreted protein of the genus PDB-2 cellulose. The x-axis shows the monosaccharides and polysaccharides contained on the culture, and the y-axis shows the starch, CMC, and laminarin, which are the polysaccharides contained in the reaction solution when the culture medium containing the secreted protein of 1 mL of PDB-1 was added. , Shows the amount of reducing sugar freed from Pullulan per hour.
FIG. 8 is a diagram showing the resolution of xylan and other polysaccharides of the secreted protein of genus PDB-2. The x-axis shows the monosaccharides and polysaccharides contained on the culture, and the y-axis shows the polysaccharides, xylan, alginate and colloid chitin, included in the reaction solution when the culture medium containing 1 mL of PDB-1 secreted protein was added. (Chitin, Pectin) shows the amount of reducing sugar released per hour.

Hereinafter, the present invention will be described in detail.

The present invention is an accession number KCTC11940BP Cellulophaga sp. Provided is a Cellulophaga genus strain having polysaccharide degradation activity deposited with PDB-1.

In addition, the present invention accession number KCTC11941BP Cellulophaga sp. Provided is a Cellulophaga genus strain having polysaccharide degradation activity deposited with PDB-2.

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

In one embodiment of the present invention, in order to isolate microorganisms that break down polysaccharides, the inventors take samples from seawater, tidal flats and coastal areas of the South Sea to separate microorganisms, and then decompose carboxymethyl cellulose (CMC). Possible microorganisms were selected.

In addition, in one embodiment of the present invention, the inventors performed sequencing and phylogenetic analysis of the 16S rRNA gene to identify the selected microorganisms, and these microorganisms are represented by SEQ ID NO: 1 and SEQ ID NO: 2. Like C. It was confirmed that this corresponds to the classification of the clade containing the genus Cellulophaga ( Cellulopaga ) which forms a consistent cluster in lytica ATCC 23178 ^T and C. fucicola NN015860 ^T (see FIG. 2).

In addition, in one embodiment of the present invention, the present inventors performed a zymogram analysis on SDS-PAGE with CMC to confirm the cellulolytic activity of the selected microorganisms. It was confirmed (see FIG. 2).

Further, in one embodiment of the present invention, the inventors performed N-terminal sequencing of the 35 kDa protein of PDB-1, and found that the N-terminal sequence of the 35 kDa protein did not match other known cellulose degrading enzymes. It was.

In addition, in one embodiment of the present invention, the inventors of the present invention provide for analyzing the degradation activity of the secreted protein of PDB-1. As a result of confirming the cellulolytic activity of the secreted protein of PDB-1, the secreted protein of PDB-1 hydrolyzed the CMC well, while the crystal substrate could not be effectively hydrolyzed. This confirmed that these enzymes are more active in β-1,4 binding of the amorphous site of cellulose (see Table 1).

In addition, in one embodiment of the present invention, the present inventors, in order to confirm the polysaccharide degradation activity of cellulose is PDB-1 and PDB-2, after culturing in a minimal medium culture containing polysaccharides, polysaccharide degradation of the secreted protein extracellularly 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, pectin (FIGS. See FIG. 8).

Therefore, a novel microorganism having degradation activity for the various polysaccharides was identified as Cellulopa spp ., Cellulophaga sp. PDB-1 and Cellulophaga sp. It was named PDB-2 and deposited on May 24, 2011 to the Korea Biotechnology Research Institute (KRIBB) Biological Resources Management Center (KBRC) Microbial Resource Center (KCTC) (Accession No .: KCTC11940BP and KCTC11941BP).

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

In one embodiment of the present invention, the inventors have performed the assay for the degradation activity of the secreted protein of PDB-1. As a result of confirming the cellulolytic activity of the secreted protein of PDB-1, the secreted protein of PDB-1 hydrolyzed the CMC well, while the crystal substrate could not be effectively hydrolyzed. This confirmed that these enzymes are more active in β-1,4 binding of the amorphous site of cellulose (see Table 1).

In addition, in one embodiment of the present invention, the present inventors, in order to confirm the polysaccharide degradation activity of cellulose is PDB-1 and PDB-2, after culturing in a minimal medium culture containing polysaccharides, polysaccharide degradation of the secreted protein extracellularly 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, pectin (FIGS. 8).

Therefore, PDB-1 and PDB-2, or the culture supernatant thereof of the present invention have a degradation activity for a variety of polysaccharides can be usefully used as a composition for degradation of polysaccharides.

The present invention also provides a polysaccharide degradation method comprising the step of contacting a polysaccharide comprising PDB-1 and PDB-2, or a culture supernatant thereof.

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

PDB-1 and PDB-2, or the culture supernatant thereof of the present invention can be used as a polysaccharide decomposition method comprising the step of contacting the polysaccharide because it has a degradation activity for a variety of polysaccharides.

In addition,

1) separating the polysaccharides from the biomass;

2) contacting the polysaccharide of step 1) with the strain of claim 1 or 2, or a culture supernatant thereof to produce monosaccharides; 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 stem, agricultural by-product of cassava stalk, waste paper, solid waste of fluff, thin lumber, waste wood, wood of processed by-products, algae, seaweed 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 one selected from the group consisting of agarose, carrageenan, starch, pullulan, carboxymethyl cellulose, xylan, laminarin, chitin and pectin, but is not limited thereto.

The microorganism of step 3) is Saccharomyces cerevisiae, Sarcina ventriculum, Kluyveromyces pragilis, Zygomonas mobilis, Kluyveromyces maxianas IMB3, Bretanomyces custersii, Clos It is preferably, but not limited to, any one selected from the group consisting of tridium acetobutylicum, clostridium biyeringki, clostridium aurantibutylricum and clostridium tetanomorphium.

PDB-1 and PDB-2, or the culture supernatant thereof of the present invention has degradation activity for various polysaccharides, so that the bioethanol is effectively reacted by reacting microorganisms capable of producing biofuels with the polysaccharide degradation products produced through such degradation. It can manufacture.

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

However, the following examples are only for exemplifying 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 cellulase-producing microorganisms, the microorganisms growing in Marine Agar 2216 (MA; Difco, USA) were isolated by dilution plating method in the sandy coast of Namhae, Korea.

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

Specifically, the isolated microorganism was transferred to a method of smearing and spreading on a CMC agar medium (1.0% of carboxymethyl cellulose (Sigma, USA) and MA), incubated at 25 ° C. for 3 days, and then the plates were 0.1% After staining with Congo red solution for 15 minutes and 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>, according to the method described in Yoon et al., Chromosomal DNA extraction and purification are performed on the isolated bacteria, and the modification of RNase T1 102 is combined with RNase A. Combination use minimizes contamination by RNA (Yoon et al., 1996). 16S rRNA gene amplification using the two universal primers mentioned above (Yoon et al., 1998), followed by sequencing and lineage of the amplified 16S rRNA gene according to the method described in Yoon et al. Embryological analysis was performed (Yoon et al., 2003).

As a result, according to the phylogenetic analysis of 16S rRNAs of PDB-1 and PDB-2 as shown in FIG. 1, these microorganisms were identified as C as shown in SEQ ID NO: 1 and SEQ ID NO: 2. It was confirmed that this corresponds to the classification of clade including the genus Cellulophaga ( Cellulopaga ) forming coherent clusters on lytica ATCC 23178 ^T and C. fucicola NN015860 ^T. In addition, strain PDB-2 is strain PDB-1, C. fucicola NN015860 ^T , And C. lytica ATCC 23178 ^T , having 16S rRNA gene homology 99.0, 98.5, and 97.6% for each, except for 92.2 to 93.6% for other Cellulophaga genera (FIG. 1), Cellulophaga sp . PDB-1 and Cellulophaga sp. It was named PDB-2 and deposited on May 24, 2011 to the Korea Biotechnology Research Institute (KRIBB) Biological Resources Management Center (KBRC) Microbial Resource Center (KCTC) (Accession No .: KCTC11940BP and KCTC11941BP).

< Example  3> microbial Cellulose  ( cellulose ) Decomposition activity confirmation

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

Specifically, SDS-PAGE gel of 0.1% CMC was prepared, followed by 70 V electrophoresis, and then refolding buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% Triton-X100). Incubated at room temperature for 1 hour and replaced with fresh refolding buffer. After an additional incubation for 1 hour, the gel was kept overnight in 20 mM Tris-HCl, pH 8.0 with 150 mM NaCl, and then stained with 0.1% Congo Red.

As a result, as shown in FIG. 2, a Zymogram analysis of SDS-PAGE was performed with CMC, and it was confirmed that each bacterium had cellulose degrading enzymes having various molecular weights and similar clear bands. , They confirmed that they had a similar cellulose decomposition system (FIG. 2).

< Example  4> Activity analysis of microorganism-derived protein

<4-1> N-terminal sequencing ( sequencing )

N-terminal sequencing of 35 kDa protein of PDB-1 was performed. SDS-PAGE analysis of precipitated fractions of extracellular proteins of PDB-1 in 70% saturated ammonium sulfate followed by electrotransferred onto polyvinylidene difluoride membrane (PVDF). After 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 is NTAQTVV. Recently, the genome sequence of C. algicola DSM 14237 has been reported and cellulase (GenBank Accession No. ADV51156, ADV50035, and ADV 47383) and Glucoside Hydrolysis Series 5 (GenBank Accession No. 4) for four ORFs. ADV50845). However, the N-terminal sequence of the 35 kDa protein was not matched 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 degradation system.

<4-2> PDB Degradation Activity Analysis of Secretory Proteins of -1

To determine the cellulolytic activity of the secreted protein of PDB-1, pH 5.0 at 0.5% concentration using 450 μL of carboxymethylcellulose (CMC), 20 μm of microcrystalline cellulose and Avicel PH-101, cellulose fiber. It was suspended in 25 mM citrate buffer and 10 mM phosphate buffer at pH 7.0. 50 μg of PDB-1 secretion protein was added to each reaction solution, followed by reaction 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, the secreted protein of PDB-1 hydrolyzed the CMC well, while the crystalline substrate could not be effectively hydrolyzed. This confirmed that these enzymes are more active in β-1,4 binding of the amorphous region of cellulose. It was also confirmed that the specific activities at pH 5.0 and pH 7.0 were similar. Thus, the cellulose degradation system of PDB-1 is active at acidic and neutral pH. Since the marine environment is generally weakly alkaline, most of the characterized cellulase obtained in the marine environment exhibits maximum activity in neutral and weakly alkaline conditions, and in acidic conditions, the cellulose system of PDB-1 It is interesting that the CMC can be hydrolyzed well at this pH 5.0. In addition, total extracellular protein and precipitated protein in 50-70% saturated ammonium sulfate showed similar specific activity. Thus, the cellulose degradation system of PDB-1 remained active even under acidic conditions. These properties have been found to be useful in biotechnological applications such as degradation of pre-treated cellulose materials under acidic conditions.

< Example  5> Cellulopaga PDB -1 Polysaccharide Degradation Activity

In order to measure the polysaccharide degradation ability of the cellulopa PDB-1, the cellulopa PDB-1 was cultured using the following medium. Each medium contains 0.2% carbon source of 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 g 1.3 g / L), 1.0 g / L yeast extract, 0.5 g / L ammonium chloride (NH ₄ Cl), 6.05 g / L trisma base, and then using hydrochloric acid Titrated to pH 7.4. The monosaccharides used were glucose, galactose, N-acetylglucosamine (NAG) and xylose. The polysaccharides were low temperature soluble agarose (Agarose, Lonza) and alginic acid. (Alginate, Wako), Carrageenan (Sigma), Carboxymethylcellulose (Sigma), Colloidal chitin (Sigma), Laminarin (Sigma), Pectin from apple peel (Sigma), Pulu Eggs (Pullulan, Sigma), starch (Soluble starch, Difco), xylan (Birchwood xylan, Sigma) were used and cultured with a medium containing no added monosaccharides and polysaccharides. Colloidal chitin was prepared by conventional methods using concentrated hydrochloric acid. As a carbon source for spawn culture, 0.1% of Bacto-Trypton (Difco) was used.

Specifically, after 3 mL seed culture of the strain (seed culture), 30 mL seed culture and then 50 mL medium containing monosaccharides and polysaccharides were incubated at 30 ℃ in the main culture. Then, after removing the cells by centrifugation, the medium solution was stored frozen. The amount of protein secreted out of the cells was measured by the Lowey method. The polysaccharide used in the reaction for measuring 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 ℃. After diluting each reaction solution by 1/10, 40 μL of the diluted solution was mixed with 120 μL of a 1: 1 mixture of 2% (w / v) pHBH dissolved in 0.5 M hydrochloric acid and 2M NaOH, and then heated at 100 ° C. for 7 minutes. The absorbance at 415 nm was then measured using microplate analysis (Moretti and Thorson, 2008).

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

< Example  6> Cellulopaga PDB -2 Polysaccharide Degradation Activity

Cellulopagar PDB-2 was incubated using the following media to measure the ability of cellulolytic PDB-2 to degrade polysaccharides. Each medium contains 0.2% carbon source in 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 g 1.3 g / L), 1.0 g / L yeast extract, 0.5 g / L ammonium chloride (NH ₄ Cl), 6.05 g / L trisma base, and then using hydrochloric acid Titrated to pH 7.4. The monosaccharides used were glucose, galactose, N-acetylglucosamine (NAG) and xylose, and the polysaccharides were low temperature soluble agarose (Agarose, Lonza), alginic acid ( Alginate, Wako, Carrageenan (Sigma), Carboxymethylcellulose (Sigma), Colloidal chitin (Sigma), Laminarin (Sigma), Pectin from apple peel (Sigma), Pullulan (Pullulan, Sigma), starch (Soluble starch, Difco), xylan (Birchwood xylan, Sigma) were used and cultured with a medium containing no added monosaccharides and polysaccharides. Colloidal chitin was prepared by conventional methods using concentrated hydrochloric acid. As a carbon source for spawn culture, 0.1% of Bacto-Trypton (Difco) was used.

Specifically, after 3 mL seed culture of the strain (seed culture), 30 mL seed culture and then 50 mL medium containing monosaccharides and polysaccharides were incubated at 30 ℃ in the main culture. Then, after removing the cells by centrifugation, the medium solution was stored frozen. The amount of protein secreted out of the cells was measured by the Lowey method. The polysaccharide used in the reaction for measuring the activity of 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 ℃. After diluting each reaction solution by 1/10, 40 μL of the diluted solution was mixed with 120 μL of a 1: 1 mixture of 2% (w / v) pHBH dissolved in 0.5 M hydrochloric acid and 2M NaOH, and then heated 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 Figures 6 to 8, it was confirmed that the cellulopa PDB-2 has a significant polysaccharide decomposition activity against galactan, glucan, xylan and other polysaccharides (Figs. 6 to 8).

Korea Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC11940BP 20110524 Korea Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC11941BP 20110524

<110> ONCOTHERAPY SCIENCE, INC. <120> RAB6KIFL / KIF20A EPITOPE PEPTIDE AND VACCINES CONTAINING THE SAME <130> 11fpi-04-06 <150> US61 / 197,106 <151> 2008-10-22 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 3471 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (497) .. (3166) <400> 1 tttttcccct taagacaaag caagcaccct aaaccagtta ccctgtgcac tcctgttaag 60 attgttgcta aggaaggaca ggagttggct gctgaagcct caagatttcc tttaggctct 120 taggtaagaa atgtctaagg ttcaaggaaa aaggttaagt tggaagaatc ccaggcaaaa 180 taagtgcgaa tccacgacag ttggtaaccc ggacccacat tagaactcag aggtcaagca 240 gaagcgaacg actggaattc cagtcaggcc cgcccccttt ccttacgcgg attggtagct 300 gcaggcttcc ctatctgatt ggccgaacga acgcagcgcg taatttaaaa tattgtatct 360 gtaacaaagc tgcacctcgt gggcggagtt gtgctctgcg gctgcgaaag tccagcttcg 420 gcgactaggt gtgagtaagc cagtatccca ggaggagcaa gtggcacgtc ttcggaccta 480 ggctgcccct gccgtc atg tcg caa ggg atc ctt tct ccg cca gcg ggc 529                       Met Ser Gln Gly Ile Leu Ser Pro Pro Ala Gly                         1 5 10 ttg ctg tcc gat gac gat gtc gta gtt tct ccc atg ttt gag tcc aca 577 Leu Leu Ser Asp Asp Asp Val Val Val Ser Pro Met Phe Glu Ser Thr              15 20 25 gct gca ttg ggg tct gtg gta cgc aag aac ctg cta tca gac tgc 625 Ala Ala Asp Leu Gly Ser Val Val Arg Lys Asn Leu Leu Ser Asp Cys          30 35 40 tct gtc gtc tct acc tcc cta gag gac aag cag cag gtt cca tct gag 673 Ser Val Val Ser Thr Ser Leu Glu Asp Lys Gln Gln Val Pro Ser Glu      45 50 55 gac agt atg gag aag gtg aaa gta tac ttg agg gtt agg ccc ttg tta 721 Asp Ser Met Glu Lys Val Lys Val Tyr Leu Arg Val Arg Pro Leu Leu  60 65 70 75 cct tca gag ttg gaa cga cag gaa gat cag ggt tgt gtc cgt att gag 769 Pro Ser Glu Leu Glu Arg Gln Glu Asp Gln Gly Cys Val Arg Ile Glu                  80 85 90 aat gtg gag acc ctt gtt cta caa gca ccc aag gac tct ttt gcc ctg 817 Asn Val Glu Thr Leu Val Leu Gln Ala Pro Lys Asp Ser Phe Ala Leu              95 100 105 aag agc aat gaa cgg gga att ggc caa gcc aca cac agg ttc acc ttt 865 Lys Ser Asn Glu Arg Gly Ile Gly Gln Ala Thr His Arg Phe Thr Phe         110 115 120 tcc cag atc ttt ggg cca gaa gtg gga cag gca tcc ttc ttc aac cta 913 Ser Gln Ile Phe Gly Pro Glu Val Gly Gln Ala Ser Phe Phe Asn Leu     125 130 135 act gtg aag gag atg gta aag gat gta ctc aaa ggg cag aac tgg ctc 961 Thr Val Lys Glu Met Val Lys Asp Val Leu Lys Gly Gln Asn Trp Leu 140 145 150 155 atc tat aca tat gga gtc act aac tca ggg aaa acc cac acg att caa 1009 Ile Tyr Thr Tyr Gly Val Thr Asn Ser Gly Lys Thr His Thr Ile Gln                 160 165 170 ggt acc atc aag gat gga ggg att ctc ccc cgg tcc ctg gcg ctg atc 1057 Gly Thr Ile Lys Asp Gly Gly Ile Leu Pro Arg Ser Leu Ala Leu Ile             175 180 185 ttc aat agc ctc caa ggc caa ctt cat cca aca cct gat ctg aag ccc 1105 Phe Asn Ser Leu Gln Gly Gln Leu His Pro Thr Pro Asp Leu Lys Pro         190 195 200 ttg ctc tcc aat gag gta atc tgg cta gac agc aag cag atc cga cag 1153 Leu Leu Ser Asn Glu Val Ile Trp Leu Asp Ser Lys Gln Ile Arg Gln     205 210 215 gag gaa atg aag aag ctg tcc ctg cta aat gga ggc ctc caa gag gag 1201 Glu Glu Met Lys Lys Leu Ser Leu Leu Asn Gly Gly Leu Gln Glu Glu 220 225 230 235 gag ctg tcc act tcc ttg aag agg agt gtc tac atc gaa agt cgg ata 1249 Glu Leu Ser Thr Ser Leu Lys Arg Ser Val Tyr Ile Glu Ser Arg Ile                 240 245 250 ggt acc agc acc agc ttc gac agt ggc att gct ggg ctc tct tct atc 1297 Gly Thr Ser Thr Ser Phe Asp Ser Gly Ile Ala Gly Leu Ser Ser Ile             255 260 265 agt cag tgt acc agc agt agc cag ctg gat gaa aca agt cat cga tgg 1345 Ser Gln Cys Thr Ser Ser Ser Gln Leu Asp Glu Thr Ser His Arg Trp         270 275 280 gca cag cca gac act gcc cca cta cct gtc ccg gca aac att cgc ttc 1393 Ala Gln Pro Asp Thr Ala Pro Leu Pro Val Pro Ala Asn Ile Arg Phe     285 290 295 tcc atc tgg atc tca ttc ttt gag atc tac aac gaa ctg ctt tat gac 1441 Ser Ile Trp Ile Ser Phe Phe Glu Ile Tyr Asn Glu Leu Leu Tyr Asp 300 305 310 315 cta tta gaa ccg cct agc caa cag cgc aag agg cag act ttg cgg cta 1489 Leu Leu Glu Pro Pro Ser Gln Gln Arg Lys Arg Gln Thr Leu Arg Leu                 320 325 330 tgc gag gat caa aat ggc aat ccc tat gtg aaa gat ctc aac tgg att 1537 Cys Glu Asp Gln Asn Gly Asn Pro Tyr Val Lys Asp Leu Asn Trp Ile             335 340 345 cat gtg caa gat gct gag gag gcc tgg aag ctc cta aaa gtg ggt cgt 1585 His Val Gln Asp Ala Glu Glu Ala Trp Lys Leu Leu Lys Val Gly Arg         350 355 360 aag aac cag agc ttt gcc agc acc cac ctc aac cag aac tcc agc cgc 1633 Lys Asn Gln Ser Phe Ala Ser Thr His Leu Asn Gln Asn Ser Ser Arg     365 370 375 agt cac agc atc ttc tca atc agg atc cta cac ctt cag ggg gaa gga 1681 Ser His Ser Ile Phe Ser Ile Arg Ile Leu His Leu Gln Gly Glu Gly 380 385 390 395 gat ata gtc ccc aag atc agc gag ctg tca ctc tgt gat ctg gct ggc 1729 Asp Ile Val Pro Lys Ile Ser Glu Leu Ser Leu Cys Asp Leu Ala Gly                 400 405 410 tca gag cgc tgc aaa gat cag aag agt ggt gaa cgg ttg aag gaa gca 1777 Ser Glu Arg Cys Lys Asp Gln Lys Ser Gly Glu Arg Leu Lys Glu Ala             415 420 425 gga aac att aac acc tct cta cac acc ctg ggc cgc tgt att gct gcc 1825 Gly Asn Ile Asn Thr Ser Leu His Thr Leu Gly Arg Cys Ile Ala Ala         430 435 440 ctt cgt caa aac cag cag aac cgg tca aag cag aac ctg gtt ccc ttc 1873 Leu Arg Gln Asn Gln Gln Asn Arg Ser Lys Gln Asn Leu Val Pro Phe     445 450 455 cgt gac agc aag ttg act cga gtg ttc caa ggt ttc ttc aca ggc cga 1921 Arg Asp Ser Lys Leu Thr Arg Val Phe Gln Gly Phe Phe Thr Gly Arg 460 465 470 475 ggc cgt tcc tgc atg att gtc aat gtg aat ccc tgt gca tct acc tat 1969 Gly Arg Ser Cys Met Ile Val Asn Val Asn Pro Cys Ala Ser Thr Tyr                 480 485 490 gat gaa act ctt cat gtg gcc aag ttc tca gcc att gct agc cag ctt 2017 Asp Glu Thr Leu His Val Ala Lys Phe Ser Ala Ile Ala Ser Gln Leu             495 500 505 gtg cat gcc cca cct atg caa ctg gga ttc cca tcc ctg cac tcg ttc 2065 Val His Ala Pro Pro Met Gln Leu Gly Phe Pro Ser Leu His Ser Phe         510 515 520 atc aag gaa cat agt ctt cag gta tcc ccc agc tta gag aaa ggg gct 2113 Ile Lys Glu His Ser Leu Gln Val Ser Pro Ser Leu Glu Lys Gly Ala     525 530 535 aag gca gac aca ggc ctt gat gat gat att gaa aat gaa gct gac atc 2161 Lys Ala Asp Thr Gly Leu Asp Asp Asp Ile Glu Asn Glu Ala Asp Ile 540 545 550 555 tcc atg tat ggc aaa gag gag ctc cta caa gtt gtg gaa gcc atg aag 2209 Ser Met Tyr Gly Lys Glu Glu Leu Leu Gln Val Val Glu Ala Met Lys                 560 565 570 aca ctg ctt ttg aag gaa cga cag gaa aag cta cag ctg gag atg cat 2257 Thr Leu Leu Leu Lys Glu Arg Gln Glu Lys Leu Gln Leu Glu Met His             575 580 585 ctc cga gat gaa att tgc aat gag atg gta gaa cag atg caa cag cgg 2305 Leu Arg Asp Glu Ile Cys Asn Glu Met Val Glu Gln Met Gln Gln Arg         590 595 600 gaa cag tgg tgc agt gaa cat ttg gac acc caa aag gaa cta ttg gag 2353 Glu Gln Trp Cys Ser Glu His Leu Asp Thr Gln Lys Glu Leu Leu Glu     605 610 615 gaa atg tat gaa gaa aaa cta aat atc ctc aag gag tca ctg aca agt 2401 Glu Met Tyr Glu Glu Lys Leu Asn Ile Leu Lys Glu Ser Leu Thr Ser 620 625 630 635 ttt tac caa gaa gag att cag gag cgg gat gaa aag att gaa gag cta 2449 Phe Tyr Gln Glu Glu Ile Gln Glu Arg Asp Glu Lys Ile Glu Glu Leu                 640 645 650 gaa gct ctc ttg cag gaa gcc aga caa cag tca gtg gcc cat cag caa 2497 Glu Ala Leu Leu Gln Glu Ala Arg Gln Gln Ser Val Ala His Gln Gln             655 660 665 tca ggg tct gaa ttg gcc cta cgg cgg tca caa agg ttg gca gct tct 2545 Ser Gly Ser Glu Leu Ala Leu Arg Arg Ser Gln Arg Leu Ala Ala Ser         670 675 680 gcc tcc acc cag cag ctt cag gag gtt aaa gct aaa tta cag cag tgc 2593 Ala Ser Thr Gln Gln Leu Gln Glu Val Lys Ala Lys Leu Gln Gln Cys     685 690 695 aaa gca gag cta aac tct acc act gaa gag ttg cat aag tat cag aaa 2641 Lys Ala Glu Leu Asn Ser Thr Thr Glu Glu Leu His Lys Tyr Gln Lys 700 705 710 715 atg tta gaa cca cca ccc tca gcc aag ccc ttc acc att gat gtg gac 2689 Met Leu Glu Pro Pro Pro Ser Ala Lys Pro Phe Thr Ile Asp Val Asp                 720 725 730 aag aag tta gaa gag ggc cag aag aat ata agg ctg ttg cgg aca gag 2737 Lys Lys Leu Glu Glu Gly Gln Lys Asn Ile Arg Leu Leu Arg Thr Glu             735 740 745 ctt cag aaa ctt ggt gag tct ctc caa tca gca gag aga gct tgt tgc 2785 Leu Gln Lys Leu Gly Glu Ser Leu Gln Ser Ala Glu Arg Ala Cys Cys         750 755 760 cac agc act ggg gca gga aaa ctt cgt caa gcc ttg acc act tgt gat 2833 His Ser Thr Gly Ala Gly Lys Leu Arg Gln Ala Leu Thr Thr Cys Asp     765 770 775 gac atc tta atc aaa cag gac cag act ctg gct gaa ctg cag aac aac 2881 Asp Ile Leu Ile Lys Gln Asp Gln Thr Leu Ala Glu Leu Gln Asn Asn 780 785 790 795 atg gtg cta gtg aaa ctg gac ctt cgg aag aag gca gca tgt att gct 2929 Met Val Leu Val Lys Leu Asp Leu Arg Lys Lys Ala Ala Cys Ile Ala                 800 805 810 gag cag tat cat act gtg ttg aaa ctc caa ggc cag gtt tct gcc aaa 2977 Glu Gln Tyr His Thr Val Leu Lys Leu Gln Gly Gln Val Ser Ala Lys             815 820 825 aag cgc ctt ggt acc aac cag gaa aat cag caa cca aac caa caa cca 3025 Lys Arg Leu Gly Thr Asn Gln Glu Asn Gln Gln Pro Asn Gln Gln Pro         830 835 840 cca ggg aag aaa cca ttc ctt cga aat tta ctt ccc cga aca cca acc 3073 Pro Gly Lys Lys Pro Phe Leu Arg Asn Leu Leu Pro Arg Thr Pro Thr     845 850 855 tgc caa agc tca aca gac tgc agc cct tat gcc cgg atc cta cgc tca 3121 Cys Gln Ser Ser Thr Asp Cys Ser Pro Tyr Ala Arg Ile Leu Arg Ser 860 865 870 875 cgg cgt tcc cct tta ctc aaa tct ggg cct ttt ggc aaa aag tac taag 3170 Arg Arg Ser Pro Leu Leu Lys Ser Gly Pro Phe Gly Lys Lys Tyr                 880 885 890 gctgtgggga aagagaagag cagtcatggc cctgaggtgg gtcagctact ctcctgaaga 3230 aataggtctc ttttatgctt taccatatat caggaattat atccaggatg caatactcag 3290 acactagctt ttttctcact tttgtattat aaccacctat gtaatctcat gttgttgttt 3350 ttttttattt acttatatga tttctatgca cacaaaaaca gttatattaa agatattatt 3410 gttcacattt tttattgaat tccaaatgta gcaaaatcat taaaacaaat tataaaaggg 3470 a 3471 <210> 2 <211> 890 <212> PRT <213> Homo sapiens <400> 2 Met Ser Gln Gly Ile Leu Ser Pro Pro Ala Gly Leu Leu Ser Asp Asp   1 5 10 15 Asp Val Val Val Ser Pro Met Phe Glu Ser Thr Ala Ala Asp Leu Gly              20 25 30 Ser Val Val Arg Lys Asn Leu Leu Ser Asp Cys Ser Val Val Ser Thr          35 40 45 Ser Leu Glu Asp Lys Gln Gln Val Pro Ser Glu Asp Ser Met Glu Lys      50 55 60 Val Lys Val Tyr Leu Arg Val Arg Pro Leu Leu Pro Ser Glu Leu Glu  65 70 75 80 Arg Gln Glu Asp Gln Gly Cys Val Arg Ile Glu Asn Val Glu Thr Leu                  85 90 95 Val Leu Gln Ala Pro Lys Asp Ser Phe Ala Leu Lys Ser Asn Glu Arg             100 105 110 Gly Ile Gly Gln Ala Thr His Arg Phe Thr Phe Ser Gln Ile Phe Gly         115 120 125 Pro Glu Val Gly Gln Ala Ser Phe Phe Asn Leu Thr Val Lys Glu Met     130 135 140 Val Lys Asp Val Leu Lys Gly Gln Asn Trp Leu Ile Tyr Thr Tyr Gly 145 150 155 160 Val Thr Asn Ser Gly Lys Thr His Thr Ile Gln Gly Thr Ile Lys Asp                 165 170 175 Gly Gly Ile Leu Pro Arg Ser Leu Ala Leu Ile Phe Asn Ser Leu Gln             180 185 190 Gly Gln Leu His Pro Thr Pro Asp Leu Lys Pro Leu Leu Ser Asn Glu         195 200 205 Val Ile Trp Leu Asp Ser Lys Gln Ile Arg Gln Glu Glu Met Lys Lys     210 215 220 Leu Ser Leu Leu Asn Gly Gly Leu Gln Glu Glu Glu Leu Ser Thr Ser 225 230 235 240 Leu Lys Arg Ser Val Tyr Ile Glu Ser Arg Ile Gly Thr Ser Thr Ser                 245 250 255 Phe Asp Ser Gly Ile Ala Gly Leu Ser Ser Ile Ser Gln Cys Thr Ser             260 265 270 Ser Ser Gln Leu Asp Glu Thr Ser His Arg Trp Ala Gln Pro Asp Thr         275 280 285 Ala Pro Leu Pro Val Pro Ala Asn Ile Arg Phe Ser Ile Trp Ile Ser     290 295 300 Phe Phe Glu Ile Tyr Asn Glu Leu Leu Tyr Asp Leu Leu Glu Pro Pro 305 310 315 320 Ser Gln Gln Arg Lys Arg Gln Thr Leu Arg Leu Cys Glu Asp Gln Asn                 325 330 335 Gly Asn Pro Tyr Val Lys Asp Leu Asn Trp Ile His Val Gln Asp Ala             340 345 350 Glu Glu Ala Trp Lys Leu Leu Lys Val Gly Arg Lys Asn Gln Ser Phe         355 360 365 Ala Ser Thr His Leu Asn Gln Asn Ser Ser Arg Ser His Ser Ile Phe     370 375 380 Ser Ile Arg Ile Leu His Leu Gln Gly Glu Gly Asp Ile Val Pro Lys 385 390 395 400 Ile Ser Glu Leu Ser Leu Cys Asp Leu Ala Gly Ser Glu Arg Cys Lys                 405 410 415 Asp Gln Lys Ser Gly Glu Arg Leu Lys Glu Ala Gly Asn Ile Asn Thr             420 425 430 Ser Leu His Thr Leu Gly Arg Cys Ile Ala Ala Leu Arg Gln Asn Gln         435 440 445 Gln Asn Arg Ser Lys Gln Asn Leu Val Pro Phe Arg Asp Ser Lys Leu     450 455 460 Thr Arg Val Phe Gln Gly Phe Ph Thr Gly Arg Gly Arg Ser Cys Met 465 470 475 480 Ile Val Asn Val Asn Pro Cys Ala Ser Thr Tyr Asp Glu Thr Leu His                 485 490 495 Val Ala Lys Phe Ser Ala Ile Ala Ser Gln Leu Val His Ala Pro Pro             500 505 510 Met Gln Leu Gly Phe Pro Ser Leu His Ser Phe Ile Lys Glu His Ser         515 520 525 Leu Gln Val Ser Pro Ser Leu Glu Lys Gly Ala Lys Ala Asp Thr Gly     530 535 540 Leu Asp Asp Asp Ile Glu Asn Glu Ala Asp Ile Ser Met Tyr Gly Lys 545 550 555 560 Glu Glu Leu Leu Gln Val Val Glu Ala Met Lys Thr Leu Leu Leu Lys                 565 570 575 Glu Arg Gln Glu Lys Leu Gln Leu Glu Met His Leu Arg Asp Glu Ile             580 585 590 Cys Asn Glu Met Val Glu Gln Met Gln Gln Arg Glu Gln Trp Cys Ser         595 600 605 Glu His Leu Asp Thr Gln Lys Glu Leu Leu Glu Glu Met Tyr Glu Glu     610 615 620 Lys Leu Asn Ile Leu Lys Glu Ser Leu Thr Ser Phe Tyr Gln Glu Glu 625 630 635 640 Ile Gln Glu Arg Asp Glu Lys Ile Glu Glu Leu Glu Ala Leu Leu Gln                 645 650 655 Glu Ala Arg Gln Gln Ser Val Ala His Gln Gln Ser Gly Ser Glu Leu             660 665 670 Ala Leu Arg Arg Ser Gln Arg Leu Ala Ala Ser Ala Ser Thr Gln Gln         675 680 685 Leu Gln Glu Val Lys Ala Lys Leu Gln Gln Cys Lys Ala Glu Leu Asn     690 695 700 Ser Thr Thr Glu Glu Leu His Lys Tyr Gln Lys Met Leu Glu Pro Pro 705 710 715 720 Pro Ser Ala Lys Pro Phe Thr Ile Asp Val Asp Lys Lys Leu Glu Glu                 725 730 735 Gly Gln Lys Asn Ile Arg Leu Leu Arg Thr Glu Leu Gln Lys Leu Gly             740 745 750 Glu Ser Leu Gln Ser Ala Glu Arg Ala Cys Cys His Ser Thr Gly Ala         755 760 765 Gly Lys Leu Arg Gln Ala Leu Thr Thr Cys Asp Asp Ile Leu Ile Lys     770 775 780 Gln Asp Gln Thr Leu Ala Glu Leu Gln Asn Asn Met Val Leu Val Lys 785 790 795 800 Leu Asp Leu Arg Lys Lys Ala Ala Cys Ile Ala Glu Gln Tyr His Thr                 805 810 815 Val Leu Lys Leu Gln Gly Gln Val Ser Ala Lys Lys Arg Leu Gly Thr             820 825 830 Asn Gln Glu Asn Gln Gln Pro Asn Gln Gln Pro Pro Gly Lys Lys Pro         835 840 845 Phe Leu Arg Asn Leu Leu Pro Arg Thr Pro Thr Cys Gln Ser Ser Thr     850 855 860 Asp Cys Ser Pro Tyr Ala Arg Ile Leu Arg Ser Arg Arg Ser Pro Leu 865 870 875 880 Leu Lys Ser Gly Pro Phe Gly Lys Lys Tyr                 885 890 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artifical peptide sequence <400> 3 Leu Leu Ser Asp Asp Asp Val Val Val   1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificial peptide sequence <400> 4 Cys Ile Ala Glu Gln Tyr His Thr Val   1 5 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> An artificial peptide sequence <400> 5 Ala Gln Pro Asp Thr Ala Pro Leu Pro Val   1 5 10 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> An artificially synthesized primer for PCR <400> 6 ctacaagcac ccaaggactc t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> An artificially synthesized primer for PCR <400> 7 agatggagaa gcgaatgttt 20 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> An artificially synthesized primer for PCR <400> 8 catccacgaa actaccttca act 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> An artificially synthesized primer for PCR <400> 9 tctccttaga gagaagtggg gtg 23 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> An artificially synthesized peptide <400> 10 Ser Leu Tyr Asn Thr Tyr Ala Thr Leu   1 5 <210> 11 <211> 9 <212> PRT <213> mouse <400> 11 Leu Leu Ser Asp Glu Asp Val Val Asp   1 5 <210> 12 <211> 10 <212> PRT <213> mouse <400> 12 Ala Gln Pro Asp Thr Val Pro Val Ser Val   1 5 10

Claims (9)

Cellulophaga genus strain having polysaccharide degrading activity deposited with accession number KCTC11940BP.
Cellulophaga genus strain having polysaccharide degrading activity deposited with accession number KCTC11941BP.
The method according to claim 1 or 2, wherein the polysaccharide is any one selected from the group consisting of agarose, carrageenan, starch, pullulan, carboxylmethylcellulose (CMC), xylan, laminarin, chitin and pectin. Characterized in the Cellulophaga genus strain.
The composition for degradation of polysaccharide comprising the strain of claim 1 or 2, or a culture supernatant thereof.
The method of claim 1 or 2, wherein the method of polysaccharide degradation comprising the step of contacting the polysaccharide with the culture supernatant thereof.
1) separating the polysaccharides from the biomass;
2) contacting the polysaccharide of step 1) with the strain of claim 1 or 2, or a culture supernatant thereof to produce monosaccharides; And
3) A method for producing bioethanol comprising the step of fermenting the monosaccharides of step 2) by microorganisms.
7. The biomass of claim 6, wherein the biomass comprises polysaccharides of rice straw, barley straw, sweet potato stalk, rapeseed stem, cassava stalk, agricultural by-products, waste paper, fluff solid wastes, thinning trees, waste wood, processing by-products, wood A method for producing a bioethanol, characterized in that any one selected from the group consisting of algae, seaweed marine plants and combinations thereof.
The bioethanol according to claim 6, wherein the polysaccharide of step 1) is any one selected from the group consisting of agarose, carrageenan, starch, pullulan, carboxymethyl cellulose, xylan, laminarin, chitin and pectin. Manufacturing method.
The method of claim 6, wherein the microorganism of step 3) is Saccharomyces cerevisiae, Sarcina ventriculum, Kluyveromyces pragilis, Zygomonas mobilis, Kluyveromyces maximaus IMB3, Bretanomay Bio-characterized in the present invention, which is any one selected from the group consisting of sescusterssei, clostridium acetobutylicum, clostridium bayeringki, clostridium aurantibutyricum and clostridium tetanomorphium. Ethanol manufacturing method.

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