KR20130067998A - Cel-kg02 cellulase gene from ruminant stomach microorganism of black goat and uses thereof - Google Patents

Abstract

PURPOSE: Cellulase which is a product of cel-KG02 gene is used as a feed additive and to industrially produce a detergent and biofuel. CONSTITUTION: A cellulase protein derived from a ruminant stomach microorganism of a black goat has an amino acid sequence of sequence number 2. A gene encodes the cellulase protein. A recombinant vector contains the gene. A host cell is transformed by the recombinant vector. A method for producing a large amount of cellulase from the host cell comprises a step of transducing the recombinant vector to the host cell and overexpresing cellulase.

Description

Cellulase gene derived from black goat ruminant microorganisms cÉｌ-ＫＧ010 and its use {Cel-KG02 cellulase gene from ruminant stomach microorganism of black goat and uses approximately}

The present invention relates to a cellulase gene cel - KG02 derived from a black goat rumen microorganism and its use, and more particularly, a cellulase protein derived from a black goat rumen microorganism, a gene encoding the cellulase protein, and a recombinant vector comprising the gene. A method for mass-producing cellulase in a host cell comprising the step of transforming the host cell transformed with the recombinant vector, an antibody against the cellulase protein, and introducing the recombinant vector into the host cell to overexpress the cellulase. The cellulase protein or the recombinant cellulase to a feed additive or detergent composition for promoting cellulolysis and a detergent composition and a biomass material comprising the recombinant cellulase protein, the cellulase protein or the recombinant cellulase protein as an active ingredient A method of producing biofuels (Biofuel) to be added to comprise the step of hydrolysis.

The major constituents 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 difficult to cultivate and are not well known. 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 part of Clostridium It is known as bacteria belonging to the genus. To date, we have studied the total microbial DNA or metagenome of the digestive system of cattle, rabbits and kangaroos in order to discover the fibrinolytic genes of most ruminants. Glucanase, xylanase and cellulosome complexes have been found. However, most of them have evolved by feeding soft plant fibrinogen, and among the fibrinase, it is required to decompose the hard fibrinogen.

Black goats have evolved to the present day to digest foods from poor sources of fiber, 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.

Meanwhile, Korean Patent Publication No. 2011-0119961 discloses' Nectria sinabarina producing cellulase and glycosylation method using the same ', and Korean Patent Publication No. 2007-0116881 discloses' Cellulase, which encodes it. Nucleic acids and methods of making and using them.

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. By confirming that the obtained DNA genetic information is a cellulase coding gene having a glycoside hydrolase (GH) domain and a carbohydrate binding module (CBM) domain, which are essential domains for fibrinolytic activity, The present invention has been completed.

In order to solve the above problems, the present invention provides a cellulase protein (cellulase) derived from the black goat rumen microorganism.

The present invention also provides a gene encoding the cellulase 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.

The present invention also provides an antibody against the cellulase protein.

The present invention also provides a method of mass-producing cellulase in a host cell comprising the step of introducing the recombinant vector into the host cell to overexpress the cellulase.

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

In addition, the present invention provides a feed additive for cellulose degradation enhancement comprising the cellulase protein or the recombinant cellulase protein as an active ingredient.

The present invention also provides a detergent composition comprising the cellulase protein or the recombinant cellulase 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 cellulase protein or the recombinant cellulase protein to a biomass material.

Cellulase, a product of the cel - KG02 gene according to the present invention, can be used not only as a feed additive, but also used in detergent and biofuel production, and can be very useful industrially.

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 phosphide gene bank mean insertion size for the Ad (Ad (ad; adherent phase) fraction and Lq (Lq) liquid phase) fraction.
Figure 3 shows an example of phosphide clone selection (clear zone confirmation by Congo Red / NaCl) with CMC degrading activity after inoculation on CMC-Agar plates.
4 shows the results of the nucleotide blast search.
Figure 5 shows the main domain identification results according to 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 a cellulase protein derived from the black goat rumen microorganism.

The cellulase protein of the present invention was obtained by slaughtering three months of black goats (two males and one female) from the National Institute of Animal Science, Animal Rural Development Institute, after feeding for one month only with rice straw feed and mineral supplements after administration in the animal genome. Obtained from rumen microbial samples.

The range of cellulase proteins according to the present invention includes proteins having an amino acid sequence represented by SEQ ID NO: 2 isolated 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, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogeneous physiological activity" means cellulase activity.

The invention also includes fragments, derivatives and analogues of cellulase proteins. As used herein, the terms “fragment”, “derivative” and “analogue” refer to a polypeptide having biological function or activity substantially the same as the cellulase polypeptide of the present 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 (eg, a leader sequence, a secretion 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 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 coding for 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 sequence 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, an allelic variant is an alternative to a polynucleotide, which may comprise one or more substituted, deleted or inserted nucleotides and which does not result in a substantial functional change in the polypeptide encoded by the variant Do not.

The present invention also provides a gene encoding a cellulase protein. The gene of the present invention may be DNA or RNA encoding a cellulase protein. DNA includes cDNA, genomic DNA, or artificial synthetic DNA. The DNA may be single stranded or double stranded. The 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 has a nucleotide sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1 . "% 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) in the presence of a denaturant 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 is the same as the biological function and activity of the mature polypeptide represented by SEQ ID NO: 2.

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

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

Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, for example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

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 using the CaCl ₂ method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation method. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

The present invention also provides an antibody against the cellulase protein. Antibodies of the present invention can be produced by conventional methods from the proteins obtained by cloning each gene into an expression vector according to conventional methods to obtain the protein encoded by the cellulase gene. This includes partial peptides that can be made from the protein, and the partial peptide of the present invention includes 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.

The present invention also provides a method of mass-producing cellulase in a host cell comprising the step of introducing the recombinant vector into the host cell to overexpress the cellulase. The method for isolating and purifying recombinant cellulase produced in the host cell may use any method known in the art.

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

The present invention also provides a feed additive for cellulose degradation enhancement comprising the cellulase protein or recombinant cellulase 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) contained in lipases 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 that hydrolyzes maltose into two molecules of glucose, and glucose to fructose by hydrolysis of 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, birds, and the like.

The amount of cellulase 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 materials while prolonged survival in the digestive tract of livestock.

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

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 (grains, potatoes, etc.), cellulose (herbaceous, tree, chaff, etc.), sugar (sugarcane, sugar beet, etc.) and protein (livestock 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

The amount of various HindIII restriction enzymes was adjusted for arbitrary partial cleavage to the appropriate size for constructing the phosmid gene bank on the DNA extracted from Ad and Lq. Ad decided to use HindIII 5 units and Lq 2.5 units (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  Sequence Decoding for Active Phosmid Clones

Phosmid plasmid extraction was performed using the Qiagen plasmid midi prep kit for the phosphid clone Ad_10C07 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 the dog 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. The decoded sequencing data was assembled using a 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. To proceed with the assembly. 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 domain database article lycopene seed hydrolase azepin at (GH; glycoside hydrolase) domain and a carbohydrate binding unit (CBM; carbohydrate binding module) After a search a gene having a domain, the cellulase in KG02 gene It was confirmed that the domain, and then using the nucleotide blast (protein blast) and protein blast (protein blast) operated by NCBI to determine whether the same sequence exists. As of October 2011, no similar DNA sequences were found in the baseline search conditions (Figure 4). In addition, it was confirmed that the protein blasting had a cellulase domain (FIG. 5), and the most similar gene was Clostridium phytofermentans ( Clostridium). phytofermentans ) was most similar to the cellulase of ISDg , but only 164 of 309 amino acids showed homology (53%) with each other. As a result, the cel- KG02 gene and its products disclosed in the present invention may be referred to as novel fibrinolytic cellulase (FIG. 6).

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Cel-KG02 cellulase gene from ruminant stomach microorganism of          black goat and uses <130> PN11262 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 921 <212> DNA <213> Ruminant stomach microorganism of blackgoat <400> 1 gtgttcgagg cgctgaaggc ccagggcttc ggcacggtgc gcattcccgt gtcgtggcat 60 aaccacgtga gcggcgagga tttcgtcatc aacgaggcat ggctggaccg cgttcaggag 120 ctcgttgact gggccctcga gtgcggcctg cacgtgatcg tgaacacgca tcacgacaac 180 ttcaaggcgt actactatcc ggacgaggag cacctggacg gctccatcag atatgtcacc 240 gccatctgga cgcaggtcgc cgcgcgcttc gcggattacg acgagcggtt gatcttcgaa 300 tcgctcaacg agccgcgcct ggcgggcacc aaccacgaat ggtggtttga agcggacagc 360 gccgagtgca tggccgcggc cgagtgcatc aacaggctga accaggcctt tgtggacacc 420 gtgcgcgcca gcggcggcgt gaacgccacg cgctacctga tggtgccggg ctacgacgcc 480 gccccggcca ataccgttcc ggagatcttc cgcctgccgg aggacagcgc ggacaaccgc 540 atcatcgttt ccgcgcacgc ctacctgccg tattccttcg cgctggacac caagggcgga 600 tcggaattca gcatcaggaa cctgcagcag accggcgaga tcgccatggc catgaacggc 660 ctgtatgagc gcttcatcag ccagggcgtt ccggtggtga tgggcgagtt cggcgcgctc 720 aacaaggaca acctgcaggc ccgcgttgac ctgacggcct attacgtggc cagtgccagc 780 gcgcgcggca ttccctgcgt gtggtgggac aaccacgcct tccacggcaa gggcgagaac 840 ttcgggctgc tggatcgcag gacgctcgag tggccctgtc cggagatcgt ggaggccgcc 900 atgcgctacg cccttcactg a 921 <210> 2 <211> 306 <212> PRT <213> Ruminant stomach microorganism of blackgoat <400> 2 Val Phe Glu Ala Leu Lys Ala Gln Gly Phe Gly Thr Val Arg Ile Pro   1 5 10 15 Val Ser Trp His Asn His Val Ser Gly Glu Asp Phe Val Ile Asn Glu              20 25 30 Ala Trp Leu Asp Arg Val Gln Glu Leu Val Asp Trp Ala Leu Glu Cys          35 40 45 Gly Leu His Val Ile Val Asn Thr His His Asp Asn Phe Lys Ala Tyr      50 55 60 Tyr Tyr Pro Asp Glu Glu His Leu Asp Gly Ser Ile Arg Tyr Val Thr  65 70 75 80 Ala Ile Trp Thr Gln Val Ala Ala Arg Phe Ala Asp Tyr Asp Glu Arg                  85 90 95 Leu Ile Phe Glu Ser Leu Asn Glu Pro Arg Leu Ala Gly Thr Asn His             100 105 110 Glu Trp Trp Phe Glu Ala Asp Ser Ala Glu Cys Met Ala Ala Ala Glu         115 120 125 Cys Ile Asn Arg Leu Asn Gln Ala Phe Val Asp Thr Val Arg Ala Ser     130 135 140 Gly Gly Val Asn Ala Thr Arg Tyr Leu Met Val Pro Gly Tyr Asp Ala 145 150 155 160 Ala Pro Ala Asn Thr Val Pro Glu Ile Phe Arg Leu Pro Glu Asp Ser                 165 170 175 Ala Asp Asn Arg Ile Ile Val Ser Ala His Ala Tyr Leu Pro Tyr Ser             180 185 190 Phe Ala Leu Asp Thr Lys Gly Gly Ser Glu Phe Ser Ile Arg Asn Leu         195 200 205 Gln Gln Thr Gly Glu Ile Ala Met Ala Met Asn Gly Leu Tyr Glu Arg     210 215 220 Phe Ile Ser Gln Gly Val Pro Val Val Gly Glu Phe Gly Ala Leu 225 230 235 240 Asn Lys Asp Asn Leu Gln Ala Arg Val Asp Leu Thr Ala Tyr Tyr Val                 245 250 255 Ala Ser Ala Ser Ala Arg Gly Ile Pro Cys Val Trp Trp Asp Asn His             260 265 270 Ala Phe His Gly Lys Gly Glu Asn Phe Gly Leu Leu Asp Arg Arg Thr         275 280 285 Leu Glu Trp Pro Cys Pro Glu Ile Val Glu Ala Ala Met Arg Tyr Ala     290 295 300 Leu his 305 <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)

Cellulase protein derived from the black goat rumen microorganism, consisting of the amino acid sequence of SEQ ID NO: 2. Gene encoding the cellulase 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 cellulase protein of claim 1. A method of mass-producing cellulase in a host cell comprising introducing the recombinant vector of claim 4 into the host cell to overexpress the cellulase. A recombinant cellulase protein prepared by the method of claim 7. Claim 1 cellulase protein or a recombinant cellulase protein of claim 8 as an active ingredient cellulose degradation feed additives. A detergent composition comprising the cellulase protein of claim 1 or the recombinant cellulase protein of claim 8 as an active ingredient. A method of producing a biofuel, comprising the step of hydrolysis by adding the cellulase protein of claim 1 or the recombinant cellulase protein of claim 8 to a biomass material.

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