KR20130068007A - Cel10-kg41 endo-xylanase gene from ruminant stomach microorganism of black goat and uses thereof - Google Patents

Abstract

PURPOSE: A product of cel10-KG41 gene, endoxylanase, is provided to be used for a feed additive and a detergent composition, and to develop biofuel with high efficiency. CONSTITUTION: An endoxylanase protein derived from a ruminant stomach microorganism of a black goat has an amino acid sequence of sequence number 2. A gene encodes endoxylanase protein. A recombinant vector contains the gene. A host cell is transformed by the recombinant vector. A method for producing a large amount of endoxylanase from the host cell comprises a step of overexpressing endoxylanase by transducing the recombinant vector to the host cells.

Description

Endocylanase derived from black goat ruminant microorganisms and its use {Cel10-KG41 endo-xylanase gene from ruminant stomach microorganism of black goat and uses approximately}

The present invention relates to an endocylanase cel10 - KG41 gene derived from a black goat rumen microorganism and its use, and more particularly, to an endocylanase protein derived from a black goat rumen microorganism (endo-xylanase) protein, A step of overexpressing endozylanase by introducing a gene encoding the recombinant vector, the recombinant vector containing the gene, the host cell transformed with the recombinant vector, the antibody against the endocylanase protein, and the recombinant vector into the host cell. A method for mass-producing endocylanase in a host cell comprising, a recombinant xylanase protein prepared by the method, the xylan degradation comprising the endocylanase protein or the recombinant endocylanase protein as an active ingredient Feed additives or detergent compositions for promotion and biomass materials It relates to a method for producing a biofuel (Biofuel) comprising the step of hydrolysis by adding the endozylanase protein or the recombinant endozylanase protein to.

The major components of plant cell walls are cellulose, hemi-cellulose and pectin, which are digested by complex and effective microbial degradation processes in the rumen of herbivores. Rumen microorganisms are extremely anaerobic and consist of fungi, protozoa and bacteria, and fibrinolysis is known to occur mainly in bacteria and fungi. However, most of these microorganisms are not well known because they are difficult to culture. To date, among the ruminant bacteria, the main ones involved in digesting plant cell walls are Fibrobacter ( Fibrobacter). succinogenes , Ruminococcus flavefaciens , Ruminococcus, albus ), butyrivibrio fibrisolvens ( Butyrivibrio fibrisolvens ), Eubacterium cellulosolvens , Prevotella and some bacteria belonging to the genus Clostridium . To date, most of ruminant fibrinase genes have been studied to study the total microbial DNA or metagenome of the digestive system of cattle, rabbits, and kangaroos, and many of these glues. Glucanase, xylanase and cellulosome complexes have been found. However, most of them have evolved by feeding soft plant fibrinogen, and it is required to decompose the harder fibrinogen among fibrinase.

Black goats have evolved to the present day to better digest diets of coarser fibrinogens such as twigs, bark and vegetation. This means that the ruminal microbial population of the black goat evolved differently from other herbivores. Techniques for searching for novel substances using fosmid, cosmid, and bacterial artificial chromosome (GAC) gene banks have been widely used for gene search for promiscuous microorganisms. In this way, many fibrinase genes have been identified to date, but the black goat-derived fibrinase gene of the present invention is novel and different from any genes so far identified.

On the other hand, Korean Patent Publication No. 2010-0021175 discloses a novel penivacillus sp. HPL-001 strain, a novel xylanase enzyme isolated therefrom and a production method thereof are disclosed. One xylanase 'is disclosed. However, as in the present invention, there is no disclosure about the novel endocylanase protein derived from the black goat rumen microorganism having excellent xylase activity.

The present invention was derived from the above-mentioned demands, and the present inventors focused on the fibrinolytic ability of the black goat rumen microorganisms, and established a fosmid gene bank for the black goat rumen microbial metagenome, and from which the CMC ( A phosphid clone having an activity of degrading a carboxymethyl cellulose substrate was selected, and genetic information was obtained by decoding the plasmid DNA of the clone. The DNA genetic information obtained is an endozylanase coding gene having a glycoside hydrolase (GH) domain and a carbohydrate binding module (CBM) domain, which are essential domains for fibrinolytic activity. By confirming, the present invention was completed.

In order to solve the above problems, the present invention provides an endo-xylanase protein derived from black goat rumen microorganisms.

In addition, the present invention provides a gene encoding the endozylanase protein.

The present invention also provides a recombinant vector comprising the gene.

The present invention also provides a host cell transformed with the recombinant vector.

In addition, the present invention provides an antibody against the endozylanase protein.

In another aspect, the present invention provides a method for mass-producing endozylanase in a host cell comprising the step of introducing the recombinant vector into the host cell and overexpressing the endozylanase.

The present invention also provides a recombinant endocylanase protein produced by the above method.

In another aspect, the present invention provides a feed additive for xylan degradation enhancement comprising the endozylanase protein or the recombinant endozylanase protein as an active ingredient.

The present invention also provides a detergent composition comprising the above-mentioned endo xylanase protein or said recombinant endo xylanase protein as an active ingredient.

In addition, the present invention provides a method for producing a biofuel (Biofuel) comprising the step of hydrolysis by adding the endozylanase protein or the recombinant endozylanase protein to a biomass material.

Cel10 - KG41 according to the invention Endozylanase, a product of the gene, was obtained through the discovery of fibrinase genes from the rumen microorganism of the black goat, which has excellent digestibility, even for coarse fibrin feed, and because of its high ability to decompose hard fibrinogen, Protein can be used in feed additives and detergent compositions, and furthermore, it is highly useful in the field of biofuel development, which is emerging as an energy countermeasure against fossil fuel depletion.

1 shows the results of establishing the metagenome DNA cleavage conditions and insert DNA cleavage for constructing the Fosmid gene bank.
Figure 2 shows the results of confirming the average insertion size of the phosmid gene bank for the Ad (Ad; adherent phase) fraction and Lq (liquid phase) fraction.
3 shows an example of phosphide clone selection with CMC degrading activity after inoculation on CMC-Agar plates (clear zone confirmation by Congo red / NaCl).
4 shows the results of the nucleotide blast search.
Figure 5 shows the results of confirming the main domain according to the protein blast (protein blast) search.
6 shows the phylogenetic tree and homology and estimates for the top homologous proteins in the protein blast search results.

In order to achieve the object of the present invention, the present invention provides an endozylanase protein derived from black goat rumen microorganisms.

Endocylanase protein of the present invention was fed to three black goats (two males, one female) from the National Institute of Animal Science, Animal Rural Development Institute, after one month of feeding with rice straw feed and mineral supplements. It was obtained from rumen microbial samples obtained by slaughter.

The range of endozylanase proteins according to the present invention includes proteins having an amino acid sequence represented by SEQ ID NO: 2 derived from a black goat rumen microorganism and functional equivalents of the proteins. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 90% or more, more preferably 90% or more, more preferably 90% or more, Refers to a protein exhibiting substantially the same physiological activity as the protein represented by SEQ ID NO: 2 and having a sequence homology of 95% or more. "Substantially homogeneous physiological activity" refers to xylobiose or xyl by random hydrolysis of β-1,4-D-xyllan, a major component of hemi-cellulose of biomass materials such as straw and corn stalks, which are agricultural wastes. It refers to the activity of xylanase which can catalyze the reaction of xyloligosaccharides such as rotilos (xylotriose).

The present invention also includes fragments, derivatives and analogues of the endozylanase protein. As used herein, the terms “fragment”, “derivative” and “analogue” refer to a polypeptide that retains a biological function or activity substantially the same as the endozylanase polypeptide of the invention. Fragments, derivatives, and analogs of the present invention comprise (i) polypeptides substituted with one or more conservative or nonconservative amino acid residues (preferably conservative amino acid residues), wherein the substituted amino acid residues are encoded by a genetic code. Or (ii) a polypeptide having substituent (s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of a polypeptide, such as polyethylene glycol) A polypeptide derived from a bound mature polypeptide, or (iv) an additional amino acid sequence (e.g., a leader sequence, a secretory sequence, a sequence used to purify the polypeptide, a proteinogen sequence or a fusion protein) and It may be a polypeptide derived from said polypeptide bound. Such fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

Polynucleotides encoding a mature polypeptide represented by SEQ ID NO: 2 may include a coding sequence encoding only a mature polypeptide; Sequences encoding mature polypeptides and various additional coding sequences; Mature polypeptides (and any additional coding sequences) and sequences encoding noncoding sequences.

The term "polynucleotide encoding a polypeptide" refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising additional coding and / or noncoding sequences.

The invention also relates to variants of said polynucleotides encoding polypeptides comprising the same amino acid sequences as described herein, or fragments, analogs, and derivatives thereof. Polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. The nucleotide mutant includes a substitution mutant, a deletion mutant, and an insertion mutant. As is known in the art, allelic variants are alternatives to polynucleotides, which may include one or more substitutions, deletions, or inserted nucleotides, which do not result in substantial functional changes in the polypeptide encoded by the variant. Do not.

The present invention also provides a gene encoding an endozylanase protein. The gene of the present invention may be DNA or RNA encoding an endozylanase protein. DNA includes genomic DNA, cDNA or artificial synthetic DNA. The DNA may be single stranded or double stranded. DNA may be a coding strand or a non-coding strand.

Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1. Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene comprises a base sequence having at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% homology with each of the nucleotide sequences of SEQ ID NO: 1. can do. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In addition, the present invention provides a poly-hybridized hybridization with a sequence having at least 50%, preferably at least 70%, more preferably at least 80% identity with a nucleotide sequence of SEQ ID NO. It relates to nucleotides. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present invention, "stringent conditions" are (1) hybridization and washing under lower ionic strength and higher temperature such as 0.2 x SSC, 0.1% SDS, 60 ° C; Or (2) hybridization in the presence of denaturing agents such as 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficoll and 42 ° C .; Or (3) at least 80%, preferably at least 90%, more preferably at least 95%. In addition, the biological function and activity of the polypeptide encoded by the hybridizable polynucleotide are identical to the biological function and activity of the mature polypeptide represented by SEQ ID NO: 2.

According to an embodiment of the invention, it should be understood that the endozylanase gene is preferably derived from a black goat rumen microorganism. However, embodiments of the present invention have a high degree of homology (e.g., at least 60%, ie 70%, 80%, 85%, 90%, 95%, even 98% sequence identity) with the endozylanase gene. Also included are genes derived from other organisms. Sequencing methods and means for determining sequence identity or homology (eg, BLAST) are well known in the art.

In another aspect, the present invention provides a recombinant vector comprising the endocylanase gene.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been re-introduced into the cell by an artificial means as modified.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism.

The vector of the present invention can typically be constructed as a vector for cloning or expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pL? Promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) It is common to include a ribosome binding site and a transcription / translation termination sequence for initiation of translation. When E. coli is used as a host cell, a promoter and an operator site of the E. coli tryptophan biosynthetic pathway, and a phage λ left promoter (pLλ promoter) can be used as regulatory sites.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, etc.) which are often used in the art, phage (e.g. λgt4.λB , λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the mammalian cell genome (for example, metallothionine promoter) or a promoter derived from a mammalian virus (for example, adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be a selection marker and may include an antibiotic resistance gene commonly used in the art, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

The present invention also provides a host cell transformed with the recombinant vector.

The host cell capable of continuously cloning and expressing the vector of the present invention in a stable manner can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. strains of the genus Bacillus, such as coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas species. Enterobacteria and strains.

When the vector of the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae), and the like insect cells, human cells (e.g., CHO cells (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cell may be used.

The method of carrying the vector of the present invention into a host cell is performed by the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973), when the host cell is a prokaryotic cell). ), Hanahan method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and electroporation method (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like. In addition, when the host cell is a eukaryotic cell, microinjection method (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 (1973)), Electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), and gene balm (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) The vector can be injected into the host cell.

The present invention also provides an antibody against the endo xylanase protein.

As used herein, the term “antibody” refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single chain Fvs (scFv), single chain antibodies, Single domain antibodies, Fab fragments, F (ab) fragments, disulfide-binding Fvs (sdFv), and antiidiotype (anti-Id) antibodies, and any epitope binding fragment described above. In particular, antibodies include immunoglobulin molecules and immunologically active immunoglobulin molecule fragments, ie molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ ) or subclass Can be.

The antibody of the present invention can be produced by a conventional method from the obtained protein by cloning the gene of the present invention in an expression vector according to a conventional method to obtain an endozylanase protein. Also included are partial peptides that can be made from the protein, and the partial peptides of the invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibody, monoclonal antibody or antigen-binding. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

In addition, the present invention provides a method for mass-producing an endozylanase protein in a host cell comprising the step of introducing the recombinant vector into the host cell and overexpressing the endozylanase. The method for isolating and purifying the recombinant endocylanase protein produced in the host cell may use any method known in the art.

The present invention also provides a recombinant endocylanase protein produced by the above method.

In another aspect, the present invention provides a feed additive for xylan degradation enhancement comprising the endozylanase protein or the recombinant endozylanase protein produced by the host cell as an active ingredient.

The feed additive of the present invention includes organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechins and alpha. It may further include any one or more of natural antioxidants such as tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid.

Feed additives of the present invention include adjuvant components such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes, microbial agents in other live bacterial forms, and the like; Cereals, such as ground or shredded wheat, oats, barley, corn and rice; Vegetable protein feedstuffs, for example, based on rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; A dry component consisting of sugar and dairy products such as various powdered milk and whey powder; Lipids such as animal fat and vegetable fat optionally arbitrarily liquefied by heating; Additives such as nutritional supplements, digestion and absorption enhancers, growth promoters and disease prevention agents may be included.

Feed additives of the present invention may be in the form of a dry or liquid formulation, and may include excipients for feed addition. Feed additives include, for example, zeolite, jade powder or rice bran, but is not limited thereto.

Feed additives of the present invention may further comprise one or more enzyme preparations. For example, alpha-1,4-glycosidic bonds (α-1), which are contained in lipolytic enzymes such as lipases, phytases that break down phytic acid to form phosphates and inositol phosphates, and starch and glycogen. Amylase, an enzyme that hydrolyzes and 4-glycoside bonds, phosphatase, an enzyme that hydrolyzes organic phosphate esters, maltase, which hydrolyzes maltose into two molecules of glucose, and glucose to hydrolysis by saccharose Conversion enzymes to make a toss mixture, and the like.

Livestock feed additives of the present invention may be administered to animals alone or in combination with other feed additives in an edible carrier. In addition, the composition for adding livestock feed can be easily administered as a top dressing or mixed directly with the livestock feed or separately from the feed, in a separate oral dosage form, or in combination with other ingredients. Typically, a single daily intake or divided daily intake may be used, as is well known in the art.

Animals that may be used in the feed additives of the present invention include, for example, cattle, chicks, chickens, domestic chickens, roosters, such as edible cattle, cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats, etc. Poultry such as ducks, geese, turkeys, quails, small birds and the like.

The amount of the endozylanase of the present invention included in the feed additive of the present invention is not particularly limited, but is included in an amount suitable for decomposing fibrous and semifibrous substances while remaining in the digestive tract of livestock for a long time.

In another aspect, the present invention provides a detergent composition comprising an endozylanase protein or a recombinant endozylanase protein produced by the host cell as an active ingredient.

The detergent compositions of the present invention may be in the form of one and two part aqueous detergent compositions, non-aqueous liquid detergent compositions, cast solids, granules, particles, compressed tablets, gels and / or pastes and slurries. Using the detergent composition as described above, when the food is steamed, the food residual film and other small amount of the food composition can be removed quickly. The detergent composition of the present invention may be effective in removing dried stains by catalytic hydrolysis of starch polysaccharides.

The detergent composition of the present invention may provide a detergent composition including a detergent composition for cleaning a hard surface, a detergent composition for cleaning a fabric, a detergent composition for washing dishes, a detergent composition for oral cleaning, a denture cleaning detergent, and a contact lens cleaning solution. .

In another aspect, the present invention is a method for producing a biofuel (Biofuel) characterized in that it comprises the step of hydrolysis by adding the endocylanase protein or recombinant endocylanase protein produced by the host cell to a biomass material. To provide.

The biomass material of the present invention is a biomass material containing a high molecular weight carbohydrate, the method for producing a biofuel of the present invention comprises the steps of pre-treating a biomass material containing a high molecular weight carbohydrate, pre-treated bio Hydrolysis of the mass with an enzyme and fermentation of the hydrolyzed biomass material may be carried out through a method for producing a biofuel commonly used in the art.

In the biofuel production method of the present invention, the biomass is starch-based (grains, potatoes, etc.), cellulose-based (herbaceous, tree, chaff, etc.), sugar-based (sugarcane, sugar beet, etc.) and protein-based (manure manure) Carbohydrates of high molecular weight, such as carcasses, microbial cells, and organic resources such as paper and food waste derived from them, but are not limited thereto.

The present invention also provides a biofuel produced by the above method. The biofuel may include, but is not limited to, bioethanol, biodiesel, biogas, and the like.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1: Isolation of Black Goat Rumen Microorganism and DNA  extraction

Two males and one female black goat were raised for only one month with rice straw and mineral supplements, and then slaughtered. The rumen was obtained from the four rumen, and the contents were obtained. The obtained contents were first divided into gastric liquid phase (Lq; liquid phase) and feed digestive phase (Ad) using gauze to prepare a sample. Feed digestion ad was suspended in 0.15% (volume / volume) Tween-80, PBS solution after filtering with gauze to recover the microorganisms attached to rice straw digest. For each of them, 5 g of precipitate samples were prepared and total DNA was extracted using CTAB (hexadecylmethylammonium bromide) and SDS (sodium dodecyl sulfate).

Example  2: black goat rumen microorganism Phosmid  ( fosmid Gene Bank Construction

By adjusting the amount of various HindIII restriction enzymes for any partial cleavage condition to an appropriate size for constructing a phosmid gene bank on the DNA extracted from Ad and Lq, Ad was used to use HindIII 5 units and Lq was 2.5 units. Was determined (FIG. 1). The cleaved DNA fragments were cut into regions between 10 and 50 kb using Pulse-Field Gel electrophoresis to prepare the inserted DNA fragments therefrom. The prepared DNA was subjected to terminal smoothing reaction and linked to the pCC1FOS vector by ligation reaction. After the Ligation ligation reaction, it was transformed into E. coli DH5α by electric shock method to build a gene bank. As a result of electrophoresis, NotI enzyme treatment was performed to confirm the average insertion size of the constructed phosphide gene bank, and it was found to be 30 kb for Ad and 31 kb for Lq (FIG. 2). Each of them was inoculated in cryopreservable medium in 150 plates of 384 well plates, respectively, and stored in an cryogenic freezer (-70 ℃).

Example  3: CMCase  Active Phosmid  Clone selection

All 115,200 clones of all Ad and Lq phosphide clones were inoculated into 0.25% CMC LB chloramphenicol agar medium in 384 clone units using a MultiMek 96 instrument and incubated at 37 ° C. for 18 hours. The cultured plate was poured into 0.2% Congo red solution and left for 30 minutes, and then washed three times or more with primary distilled water, and again put 1M NaCl solution and left for 5 minutes. At the end of this step, the plate was placed on the light box and the address of the clone in which the clear zone was confirmed (Fig. 3) was obtained.

Example  4: CMCase  activation Phosmid  Sequence Decoding for Clones

Phosmid plasmid extraction was performed using the Qiagen plasmid midi prep kit for the phosphid clone Ad_142N07 identified in Example 3, and the extracted plasmid DNA was subjected to Sanger shotgun sequencing and unidirectional pyro sequencing. In parallel, sequence information of the DNA inserted into the plasmid of the clone was obtained. Sanger shotgun sequencing was performed by random cleavage of the extracted plasmid DNA, followed by ligation reaction with dephosphorylated pUC19 vector prepared with SmaI restriction enzyme, which was transformed into DH5α Escherichia coli to generate a single clone 192. Plasmid DNA was extracted from dogs and subjected to sequencing using a Dye termination sequencing kit using M13_forward primer (GTAAAACGACGGCCAGT: SEQ ID NO: 3) and M13_reverse primer (AACAGCTATGACCATG: SEQ ID NO: 4) in both directions. After the reaction product was purified by ethanol, detoxification was performed using ABI Genome Analyzer 3730XL. Decoded sequencing data were assembled using the phredphrap program to obtain common sequence information. In addition, 7200 sequences were obtained using GS Junior model using Pyro sequencing technique for fragments randomly cut using a nebulizer for DNA of the same clone, and using the Newbler2.5 de novo assembler program. Combination work was carried out. Sanger sequencing data and pyro sequencing data were integrated using Lasergene's Seqman program to obtain the longest sequence.

Example  5: Gene search and similarity investigation

For the DNA sequencing information of the obtained phosmid clone, the gene region was searched using the MetaGeneMark program. For the found genetic information primarily self pfam Hyde article lycopene seed from the domain database roller dehydratase (GH; glycoside hydrolase) domain and a carbohydrate binding unit (CBM; carbohydrate binding module) After a search a gene having a domain, GH10 from KG41 gene The domains were identified, and then nucleotide blasts and protein blasts operated by NCBI were used to determine whether the same sequences exist. As of October 2011, no similar DNA sequences were found in the baseline search conditions (Figure 4). In addition, protein blasting confirmed that it had a GH10 domain commonly seen in endocylanase enzyme (FIG. 5), and the most similar protein was 436 amino acids as endocylanase of Cellulosilyticum ruminicola . Only 185 amino acids showed similarity (47%) (FIG. 6). As a result, the cel10 - KG41 gene and its product disclosed in the present invention can be referred to as novel fibrinolytic endocylanase .

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Cel10-KG41 endo-xylanase gene from ruminant stomach microorganism of          black goat and uses <130> PN11270 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1311 <212> DNA <213> Ruminant stomach microorganism of blackgoat <400> 1 ttgtcaataa gcaaattcag cggaaaacag tggatttttt catggaatta tggtatcatc 60 cttgtgcaac aatccgcaaa ggaggacaat gaatccatga agaaaatgat cgcctggctt 120 tccgtgatca cgctgcttct cctctcccct gcggccgcgc tggcgcagga ggaagaggac 180 tatgagagcc tcgcttccct cgccgccgcc tatggcttca agctgggcgc gccgctgtcc 240 tatgggcaga tggtggatca cgcgtatcag gattttgtgg ccaagcattt caacagcatc 300 accaccacca acgaaatgaa ggcctattcc ctgctggatc agtccgcctc ccgcaagacc 360 gaggacggca tgccggccat ggactatcgc aaggccgatc agatggtggc ctgggcgcgg 420 aaaaacggca tcgccgtgcg cggccacacg ctggtgtggg acgcgtatat gaacgactgg 480 ttcttccgcg agggctacag catgaacgcg ccctacgccg atcaggaaac catcaaggcc 540 cgcacggagt cgtatatcac ccaggtgatc acgcactttg aggagaaatt ccccggcgtg 600 gtgtactgct gggatgtggt gaacgaagcc gtgggcgata acgccggcga gtacaagccc 660 gccgacgccc gccatctgcg caccaagcgc aacggcggcg acaacctgtt ccagaaatac 720 atgggcgacg attacgtgga gctggccttc ctgtacgcca agaacgcggt ggaagccctg 780 ggcgccgaca ccaagctgta ttacaacgat tataacgcct acttcggcga caaggccaag 840 gccatccgcg ccctggctaa gagcgtgaac agctacgccg ccgacgagga gggcaatccc 900 cggcaattga tggacggcat cggcatgcag ggctacatcg gcggctacgg cacgcaggac 960 ggctgcctga tcgacagcca cgtggacatg atcaagaatg agatcctcaa ttacgcggcg 1020 atgggcctgg aagtgcagat caccgaaatg gccgtgcgca attttgatcc ggcgcaggcc 1080 gataagcaca cggcgttcta cgccaagctg tttaccatgt tcacttcgct ctgccaggac 1140 ggcgaaacgc ccctcaaggc cgtttccatc tggggcctga ccgacaatcc caacgagccc 1200 aagggcacct acgtctacaa cctgaacagc ccctacggcg gcctggtgac cgaaacgtat 1260 cagtacaaag acgcgttcaa gcaggtttac gccgtgctga agcaagagta a 1311 <210> 2 <211> 436 <212> PRT <213> Ruminant stomach microorganism of blackgoat <400> 2 Leu Ser Ile Ser Lys Phe Ser Gly Lys Gln Trp Ile Phe Ser Trp Asn   1 5 10 15 Tyr Gly Ile Ile Leu Val Gln Gln Ser Ala Lys Glu Asp Asn Glu Ser              20 25 30 Met Lys Lys Met Ile Ala Trp Leu Ser Val Ile Thr Leu Leu Leu Leu          35 40 45 Ser Pro Ala Ala Ala Leu Ala Gln Glu Glu Glu Asp Tyr Glu Ser Leu      50 55 60 Ala Ser Leu Ala Ala Ala Tyr Gly Phe Lys Leu Gly Ala Pro Leu Ser  65 70 75 80 Tyr Gly Gln Met Val Asp His Ala Tyr Gln Asp Phe Val Ala Lys His                  85 90 95 Phe Asn Ser Ile Thr Thr Thr Asn Glu Met Lys Ala Tyr Ser Leu Leu             100 105 110 Asp Gln Ser Ala Ser Arg Lys Thr Glu Asp Gly Met Pro Ala Met Asp         115 120 125 Tyr Arg Lys Ala Asp Gln Met Val Ala Trp Ala Arg Lys Asn Gly Ile     130 135 140 Ala Val Arg Gly His Thr Leu Val Trp Asp Ala Tyr Met Asn Asp Trp 145 150 155 160 Phe Phe Arg Glu Gly Tyr Ser Met Asn Ala Pro Tyr Ala Asp Gln Glu                 165 170 175 Thr Ile Lys Ala Arg Thr Glu Ser Tyr Ile Thr Gln Val Ile Thr His             180 185 190 Phe Glu Glu Lys Phe Pro Gly Val Val Tyr Cys Trp Asp Val Val Asn         195 200 205 Glu Ala Val Gly Asp Asn Ala Gly Glu Tyr Lys Pro Ala Asp Ala Arg     210 215 220 His Leu Arg Thr Lys Arg Asn Gly Gly Asp Asn Leu Phe Gln Lys Tyr 225 230 235 240 Met Gly Asp Asp Tyr Val Glu Leu Ala Phe Leu Tyr Ala Lys Asn Ala                 245 250 255 Val Glu Ala Leu Gly Ala Asp Thr Lys Leu Tyr Tyr Asn Asp Tyr Asn             260 265 270 Ala Tyr Phe Gly Asp Lys Ala Lys Ala Ile Arg Ala Leu Ala Lys Ser         275 280 285 Val Asn Ser Tyr Ala Ala Asp Glu Glu Gly Asn Pro Arg Gln Leu Met     290 295 300 Asp Gly Ile Gly Met Gln Gly Tyr Ile Gly Gly Tyr Gly Thr Gln Asp 305 310 315 320 Gly Cys Leu Ile Asp Ser His Val Asp Met Ile Lys Asn Glu Ile Leu                 325 330 335 Asn Tyr Ala Ala Met Gly Leu Glu Val Gln Ile Thr Glu Met Ala Val             340 345 350 Arg Asn Phe Asp Pro Ala Gln Ala Asp Lys His Thr Ala Phe Tyr Ala         355 360 365 Lys Leu Phe Thr Met Phe Thr Ser Leu Cys Gln Asp Gly Glu Thr Pro     370 375 380 Leu Lys Ala Val Ser Ile Trp Gly Leu Thr Asp Asn Pro Asn Glu Pro 385 390 395 400 Lys Gly Thr Tyr Val Tyr Asn Leu Asn Ser Pro Tyr Gly Gly Leu Val                 405 410 415 Thr Glu Thr Tyr Gln Tyr Lys Asp Ala Phe Lys Gln Val Tyr Ala Val             420 425 430 Leu Lys Gln Glu         435 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gtaaaacgac ggccagt 17 <210> 4 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aacagctatg accatg 16

Claims (11)

An endo-xylanase protein derived from a black goat rumen microorganism, consisting of the amino acid sequence of SEQ ID NO: 2. The gene encoding the endocylanase protein of claim 1. 3. The gene according to claim 2, wherein the gene consists of the nucleotide sequence of SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2. A host cell transformed with the recombinant vector of claim 4. An antibody against the endocyanalase protein of claim 1. A method for mass-producing endozylanase in a host cell comprising the step of introducing the recombinant vector of claim 4 into the host cell to overexpress endozylanase. Recombinant endocylanase protein produced by the method of claim 7. A feed additive for improving xylan degradation, comprising the endozylanase protein of claim 1 or the recombinant endozylanase protein of claim 8 as an active ingredient. A detergent composition comprising the endocylanase protein of claim 1 or the recombinant endocylanase protein of claim 8 as an active ingredient. A method of producing a biofuel, comprising the step of hydrolyzing the biomass material by adding the endocylanase protein of claim 1 or the recombinant endocylanase protein produced by the host cell of claim 8. .

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