KR101437542B1 - cellulase cel-KG21 gene from rumen microorganism of black goat and uses thereof - Google Patents

Abstract

The present invention relates to a cellulolytic enzyme cel-KG21 gene derived from a black goat rumen microorganism and its use, more specifically, to a novel cellulolytic enzyme gene selected from a black goat rumen microorganism and a cel-KG21 gene and a protein product thereof, To a feed additive, a detergent composition, and a method for producing biofuel.
According to the present invention, a novel cellulolytic enzyme gene, cel-KG21 gene, can be selected from a black goat rumen microorganism, which can be used in the development of a low-cost feed additive by providing a cellulolytic enzyme cel-KG21 gene and a protein product , Fiber softeners and detergents as well as the abundant resources of fiber, which can be used to produce biofuels.

Description

Cellulase CEL-KG21 gene derived from a black goat rumen microorganism and its use {Cellulase cel-KG21 gene from rumen microorganism of black goat and uses thereof}

The present invention relates to a cellulolytic enzyme cel-KG21 gene derived from a black goat rumen microorganism and its use, more specifically, to a novel cellulolytic enzyme gene selected from a black goat rumen microorganism and a cel-KG21 gene and a protein product thereof, To a feed additive, a detergent composition, and a method for producing biofuel.

The main components of plant cell walls are cellulose, hemi-cellulose and pectin, which are digested by complex and effective microbial degradation processes in herbivore rumen. The rumen microorganisms are extreme anaerobic microorganisms, consisting of fungi, protozoa, and bacteria, which are known to be composed mainly of bacteria and fungi. However, most of these microorganisms are difficult to cultivate and are not well known.

To date, among the ruminal bacteria, bacteria that are mainly involved in digesting plant cell walls include Fibrobacter succinogenes , Ruminococcus flavefaciens , Ruminococcus albus , Butyrivibrio fibrisolvens , Eubacterium cellulosolvens , Prevotella , And a portion of Clostridium < RTI ID = 0.0 > It is known as bacteria belonging to the genus.

To date, we have studied total microbial DNA of the digestive organs of cattle, rabbits, kangaroos, or the metagenome (called the total DNA of environmental microorganisms) in order to identify the most abundant enzymes in the rumen, Glucanase, xylanase, and cellulosome complexes have been identified. However, most of them have evolved from soft fungal fibrinogen to food, and among fibrin degrading enzymes, they are required to have a strong fibrinolytic ability.

Black goats are generally known to have evolved to the point where they can adapt and digest to the feed of coarse fibrinous materials such as twigs, bark, and vegetation. Although the research on the rumen microbes of the black goats is relatively inefficient compared with the cattle, the rumen microbes of the black goats are likely to be more efficient environmental microbes than the rumen rumen like cattle It is expected. 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. The present invention also attempts to identify a high-efficiency fibrinolytic enzyme gene by applying a recently developed metagenomic genome analysis technique. In this way, a large number of fibrinolytic enzyme genes have been found so far, but the gene of the goat-derived fibrinolytic enzyme according to the present invention is novel and different from any genes revealed to date.

In the meantime, Korean Patent Laid-Open Publication No. 2011-0119961 discloses a method of saccharifying saccharides and cellulose using the same, and Korean Patent Laid-Open No. 2007-0116881 'Cellulases, nucleic acids encoding them, and methods of making and using them' are disclosed.

Under these technical backgrounds, the present inventors have made intensive efforts to accomplish the present invention.

It is an object of the present invention to provide a cellulolytic enzyme cel-KG21 gene derived from a black goat rumen microorganism and its use.

According to one aspect of the present invention, there can be provided a cellulolytic enzyme cel-KG21 gene derived from a black goat rumen microorganism comprising the nucleotide sequence shown in SEQ ID NO: 1.

According to another aspect of the present invention, there is provided a GH2C domain-based fibrinolytic enzyme protein derived from a black goat rumen microorganism, which is encoded by the gene and is composed of the amino acid sequence represented by SEQ ID NO: 2.

According to another aspect of the present invention, a recombinant vector containing the gene may be provided.

According to another aspect of the present invention, a host cell transformed with the recombinant vector may be provided.

According to another aspect of the present invention, there can be provided a feed additive for promoting fibrin degradation comprising the fibrinolytic enzyme protein.

According to another aspect of the present invention, a detergent composition comprising the protease protein can be provided.

According to another aspect of the present invention, there is provided a method for producing a biofuel comprising the step of hydrolyzing the biodegradable material with the fibrinolytic enzyme protein.

According to the present invention, a novel cellulolytic enzyme gene, cel-KG21 gene, can be selected from a black goat rumen microorganism, which can be used in the development of a low-cost feed additive by providing a cellulolytic enzyme cel-KG21 gene and a protein product , Fiber softeners and detergents as well as the abundant resources of fiber, which can be used to produce biofuels.

FIG. 1 shows the results of cleavage of the meta genomic DNA fragment and insertion of the inserted DNA fragment for construction of the phosphide gene bank.
Figure 2 shows the results of the average insertion size of the fosmid gene bank for Ad (Ad; adherent phase) fraction and Lq (liquid phase) fraction.
FIG. 3 shows a selection example of a phosphide clone having cleavage activity of CMC after inoculation on a CMC-Agar plate (cleaved zone by Congoled / NaCl).
Figure 4 shows nucleotide blast search results.
FIG. 5 shows a result of identifying a major domain according to a protein blast search.
FIG. 6 shows the phylogenetic tree and homology of the highest-order homologous proteins in the protein blast search results.
Figure 7 shows CMC degradation activity after inoculation on CMC-Agar plates.

Hereinafter, the present invention will be described in more detail.

The term "rumen microorganism" as used in the detailed description of the present invention refers to microorganisms in the rumen of ruminantia (cattle, goats, sheep, deer, etc.) And microorganisms that massively coexist in the rumen are decomposed into volatile fatty acids and the like. Representative examples include, but are not limited to, Fibrobacter succinogenes , Ruminococcus flavefaciens , Ruminococcus albus , Butyrivibrio fibrisolvens , Eubacterium cellulosolvens , Prevotella, < RTI ID = 0.0 > And a portion of Clostridium < RTI ID = 0.0 > Bacteria belonging to the genus.

The term 'carbohydrate-binding module' (CBM) as used in the detailed description of the present invention is a protein domain found in a carbohydrate-active enzyme, and the domain is a carbohydrate-binding activity carbohydrate-binding activity. The CBM is mainly known as a cellulose-binding domain. It is also found in cellulosomal scaffoldin protein, and is classified into 64 families according to the similarity of the amino acid sequence.

The term "glycoside hydrolase (GH) domain" as used in the detailed description of the present invention means a domain having an enzyme activity that acts on glycosides, oligosaccharides, and polysaccharides and hydrolyzes glycosidic bonds , And are classified into 127 families according to the degree of similarity of the present amino acid sequence.

The term " biomass " used in the detailed description of the present invention refers to a whole of an organism including a plant carrying a carbon assimilation process for fixing carbon dioxide by using sunlight and an animal living there. Various fuels such as alcohol, diesel, and hydrogen produced from the biomass are called biofuels, and biomass such as biomass such as fibrin is proposed as a substance capable of replacing chemicals obtained from petroleum have. Plant-based biomass is more advantageous in terms of energy balance than petroleum as raw material in producing various chemical products. The reason for this is that, in the case of the plant-based biomass, various functional groups including oxygen are included, so that it is possible to convert to a lower activation energy when various chemical products with functional groups are produced.

The term "biofuel" as used in the detailed description of the present invention includes all the byproducts derived from metabolism activities such as living organisms as well as animal organisms as fuel, Ethanol and biodiesel.

According to one aspect of the present invention, a cellulolytic enzyme cel-KG21 gene derived from a black goat rumen microorganism and consisting of the nucleotide sequence (1,206 bp) represented by SEQ ID NO: 1 can be provided.

The fibrinolytic enzyme cel-KG21 gene according to the present invention may be DNA or RNA encoding a fibrinolytic enzyme 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 fibrinolytic enzyme cel-KG21 gene according to 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. More specifically, the gene comprises a nucleotide sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, from the nucleotide sequence of SEQ ID NO: 1 .

The 'percentage of sequence homology to polynucleotides' is identified 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 (addition or deletion) (I. E., A gap) relative to the < / RTI >

According to an embodiment of the present invention, the fibrinolytic enzyme cel-KG21 gene may preferably be derived from a black goat rumen microorganism. However, the present invention is not necessarily limited thereto. Examples of the present invention may include a homologous recombinant vector having high homology (for example, 70% or more, preferably 80% or more, more preferably 90% or more, Most preferably at least 95% sequence homology) and may comprise a gene derived from a rumen microorganism of another organism other than a black goat. Methods for sequencing and means for determining sequence homology (e.g., BLAST, etc.) are well known to those skilled in the art.

According to another aspect of the present invention, a GH2C domain-based fibrinolytic enzyme protein derived from a black goat rumen microorganism, which is encoded by the gene and is composed of the amino acid sequence (401 amino acids) represented by SEQ ID NO: 2, may be provided.

The fibrinolytic enzyme protein according to the present invention was administered to three animal goats (2 males and 1 female) born in the livestock genetic resource test center of the National Livestock Research Institute of RDA and fed with rice straw feed and mineral supplement for one month Were obtained from microbial samples that were symbiotic to rumen obtained from slaughter.

The range of the fibrinolytic enzyme protein according to the present invention includes the GH2C domain family protein consisting of the amino acid sequence shown in SEQ ID NO: 2 isolated from the black goat rumen microorganism and the functional equivalent of the protein.

The term "functional equivalent" means that at least 70% or more, preferably 80% or more, more preferably 90% or more, or more preferably 90% or more, of the amino acid sequence shown in SEQ ID NO: 2 as a result of addition, 2, and most preferably at least 95%, of the amino acid sequence shown in SEQ ID NO: 2, and has substantially the same physiological activity as the protein consisting of the amino acid sequence shown in SEQ ID NO: 2. The term " substantially homogenous physiological activity " means the same enzymatic activity of fibrinolytic enzymes.

The present invention also includes fragments, derivatives and analogues of fibrinolytic enzymes proteins. The terms "fragment", "derivative" and "analog" refer to polypeptides having substantially the same biological function or activity as the protease protein according to the present invention.

Fragments, derivatives and analogs of the fibrinolytic enzyme proteins according to the present invention may be prepared by (i) replacing one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) with a substituted polypeptide (Ii) a polypeptide having a substituent (s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of the polypeptide, e.g., (Iii) a polypeptide derived from a mature polypeptide conjugated to a poly (ethylene glycol), or (iv) a polypeptide derived from a polypeptide having additional amino acid sequence (e.g., a sequence, sequence, sequence used to purify the polypeptide, ≪ RTI ID = 0.0 > a < / RTI > sequence or fusion protein). The fragments, derivatives and analogs defined in accordance with the present invention are well known to those skilled in the art.

The amino acid sequence represented by SEQ ID NO: 2, that is, the polynucleotide encoding the mature polypeptide, is a coding sequence that encodes only the 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 the polypeptide " refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising additional coding and / or noncoding sequences.

The present invention also relates to variants of said polynucleotides encoding polypeptides comprising the same amino acid sequence as described above, 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 to those of ordinary skill in the art, an allelic variant is an alternative to a polynucleotide, which may comprise one or more substituted, deleted or inserted nucleotides, Lt; RTI ID = 0.0 > polypeptide < / RTI >

According to another aspect of the present invention, a recombinant vector containing the gene may be provided.

The vector according to the invention can typically be constructed as a vector for cloning or expression. In addition, the vector according to the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts. For example, when the vector according to the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter (for example, pL promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) , Ribosome binding sites for initiation of detoxification, and transcription / translation termination sequences. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway and the left promoter of the phage (pL promoter) can be used as the regulatory region.

Herein, the vectors that can be used in the present invention include plasmids (e.g., pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, etc.) commonly used in the technical field to which the present invention belongs, For example, gt4B, -Charon, z1 and M13, or viruses such as SV40.

In addition, when the vector according to the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter) or a mammalian virus (e.g., Adeno Virus late 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 vectors according to the present invention may be used as selectable markers and include antibiotic resistance genes commonly used in the art to which the present invention belongs, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, , ≪ / RTI > neomycin, and tetracycline.

According to another aspect of the present invention, a host cell transformed with the recombinant vector may be provided.

The host cell for stably transforming the vector according to the present invention into a prokaryotic cell and then expressing the host cell may be a host cell commonly used in the technical field of the present invention, for example, E. coli JM109, E. coli BL21, E Bacillus strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, and Bacillus tulifene, or Salmonella typhimurium, And various enterococci such as Pseudomonas species and strains.

When the vector according to the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (for example, Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells.

The method of delivering a vector according to the present invention into a host cell can be carried out by the CaCl ₂ method, Hanahan, D., J. MoI. Biol., 166: 557-580 (1983) ) And electric drilling method. When the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, have.

According to another aspect of the present invention, there can be provided a feed additive for promoting fibrin degradation comprising the fibrinolytic enzyme protein.

Feed additives according to the present invention include organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid; Phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymerized phosphate) and the like; Natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid and the like have.

The feed additive according to the present invention may include adjuvant components such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes and other microbial agents in the form of live cells; Grain, for example, ground or crushed 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, for example various powdered milk and whey powders; Lipids such as animal fat and vegetable fat optionally liquefied by heating; Nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents, and the like.

The feed additive according to the present invention may be in the form of a dry or liquid preparation, and may contain an excipient for feed addition. Examples of the excipient for adding the feed include zeolite, corn or rice bran, and the present invention is not limited to the above examples.

The feed additive according to the present invention may be used as an enzyme preparation, for example, a lipase such as lipase, a phytase which decomposes phytic acid to form phosphate and inositol phosphate, starch and glycogen Amylase, which is an enzyme that hydrolyzes the -1,4-glycoside bond, phosphatase, which is an enzyme that hydrolyzes the organic phosphate ester, and hydrolyzes maltose into two molecules of glucose And a conversion enzyme which hydrolyzes maltase and saccharose to produce a glucose-fructose mixture, and the like.

The feed additive according to the present invention may be administered to animals alone or in combination with other feed additives in edible carriers. The feed additives can also be administered as top dressing or they can be mixed directly with the livestock feed or separately from the feed, in separate oral formulations, or in combination with other ingredients.

A single daily intake or divided daily intake can be used as is commonly known in the art.

The animal in which the feed additive according to the present invention can be used can be, for example, an animal such as a cow, a cow, a calf, a pig, a pig, a sheep, a goat, a horse, a rabbit, a dog, , Poultry such as ducks, geese, turkeys, quails, small birds, and the like, and are not necessarily limited to the above examples.

The amount of the fibrotic degrading enzyme according to the present invention contained in the feed additive according to the present invention is not particularly limited. However, as is commonly known in the art to which the present invention pertains, long-term survival in the digestive tract of livestock, And is included in an amount suitable for decomposition.

According to another aspect of the present invention, a detergent composition comprising the protease protein can be provided.

The detergent composition according to the present invention may be in the form of a part and a part of an aqueous detergent composition, a non-aqueous liquid detergent composition, a cast solid, a granular form, a granular form, a compressed tablet, a gel, a paste or a slurry. The detergent composition can be used to quickly remove food stains, food residue films, and other small amounts of food compositions.

The detergent compositions according to the present invention may be effective in removing dried stains through the catalytic-mediated hydrolysis of starch polysaccharides.

The detergent composition according to the present invention may be provided in the form of a detergent composition for cleaning hard surfaces, a detergent composition for cleaning fabrics, a detergent composition for washing dishes, a detergent composition for oral cleaning, a detergent for cleaning denture or a contact lens cleaning solution .

According to another aspect of the present invention, there is provided a method for producing a biofuel comprising the step of hydrolyzing the biodegradable material with the fibrinolytic enzyme protein.

The biomass material according to the present invention is a biomass material containing a high molecular weight carbohydrate. The method for preparing a biofuel according to the present invention comprises: pre-treating a biomass material containing a high molecular weight carbohydrate; Hydrolyzing the pretreated biomass with an enzyme; And a step of fermenting the hydrolyzed biomass material. The method of manufacturing the biofuel may be performed by a method of manufacturing a biofuel commonly known in the art.

In the method for producing a biofuel according to the present invention, the biomass may be selected from the group consisting of starchy (cereals, potatoes), cellulose (herb, wood, chaff) Organic material such as manure, carcass, microbial cells and paper and food waste derived therefrom), and the like, and the present invention is not limited to the above examples.

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

Example 1. Isolation and DNA extraction of black goat rumen microorganism

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 separated into a liquid phase (Lq) and an adherent phase (Ad) by using gauze. Feed scoop Ad was obtained by suspending the residue after filtering with gauze in 0.15% (volume / volume) Tween-80, PBS solution, and recovering the microorganisms attached to rice straw. A total of 5 g of the precipitated sample was prepared and extracted with CTAB (hexadecylmethylammonium bromide) and SDS (sodium dodecyl sulfate).

Example 2. Construction of a black goat rumen microbial phosphide gene bank

For the DNA extracted from Ad and Lq, the amount of various HindIII restriction enzymes was adjusted for arbitrary fragmentation conditions at an appropriate size to construct the phosphide gene bank. Ad was determined to use HindIII 5 units and Lq 2.5 units (Fig. 1). The partially cleaved DNA fragments were clipped in a region between 10 kb and 50 kb using a Pulse-Field Gel electrophoresis method to prepare an insert DNA fragment therefrom. The prepared DNA was subjected to terminal smoothing reaction and ligated to the pCC1FOS vector by ligation reaction. After the ligation reaction, the gene was transformed into E. coli DH5α by an electric shock method to construct a gene bank.

In order to confirm the average insertion size of the constructed phosphide gene bank, NotI enzyme treatment was carried out and it was confirmed by electrophoresis that 30 kb for Ad and 31 kb for Lq (FIG. 2). To each of them, 150 plates of 384 well plates were inoculated into a cryopreservation medium and stored in a cryogenic freezer (-70 ° C).

Example 3. Fosmid clone selection with CMCase activity

For the 115,200 clones of Ad and Lq all of the phosphide clones, 384 clones were inoculated into 2.5% CMC LB chloramphenicol agar medium using MultiMek 96 instrument and incubated at 37 ° C for 18 hours. The cultured plate was poured 0.2% Congo Red solution and allowed to stand for 30 minutes. Then, it was washed with primary distilled water more than 3 times, 1M NaCl solution was added thereto, and left for 5 minutes. At the end of this step, the plate was placed in a light box and the address of the clone identified for the clear zone was determined (FIG. 3).

Example 4. Detection of base sequence for CMCase-activated phosmid clone

The phosphide plasmid extraction was carried out using the Qiagen plasmid midi prep kit for the phasmid clone Ad_91K22 identified in Example 3. The extracted plasmid DNA was used in combination with the Sanger shotgun base sequencing method and the uni-directional pie base sequencing method To obtain the sequence information of the DNA inserted into the plasmid of the clone.

Sanger shotgun DNA sequencing was performed by arbitrarily truncating the extracted plasmid DNA and ligating it with a dephosphorylated pUC19 vector prepared by SmaI restriction enzyme and then transforming it into DH5? E. coli to produce a single clone 192 The plasmid DNA was extracted in both directions and sequenced using a Dye termination sequencing kit using M13_ forward primer (GTAAAACGACGGCCAGT) and M13_ reverse primer (AACAGCTATGACCATG). The reaction products were purified through ethanol purification, We used the ABI Genome Analyzer 3730XL to decode.

The decoded nucleotide sequence data were assembled using the phredphrap program to secure the common sequence information. In addition, for the same clone DNA, 7200 sequences were obtained using a GS Junior model using a Pyrosequencing Detection technique for randomly digested fragments using a navelizer, and using the Newbler2.5 de novo assembler program And the combination work was carried out. Sanger sequencing data and pyrosequencing data were integrated using Lasergene's Seqman program to ensure the longest sequence.

Example 5. Genetic search and similarity investigation

For the inserted DNA sequence information of the acquired phosphodiester clones, the gene region was searched using the MetaGeneMark program. As a result of searching for a gene having a glycoside hydrolase (GH) domain and a carbohydrate binding module (CBM) domain in its pfam domain database, cel-KG21 gene And the nucleotide blast and protein blast run by NCBI were used to confirm the presence of the same sequence.

As of September 2012, the nucleotide blast results showed no similar DNA sequences in the basic search conditions (FIG. 4). In addition, it was confirmed that the protein blast resulted in a common fibrinolytic enzyme domain (Fig. 5), and the most similar gene was the endoglucanase gene (Accession) having the GH2C domain in Cellulosilyticum ruminicola No. ACZ98609), only 194 of the 401 amino acids showed homology (47%) with each other. Thus, the cel-KG21 gene and its product revealed in the present invention can be said to be a novel cellulolytic enzyme (Fig. 6).

As described above, according to the present invention, a novel cellulolytic enzyme gene, cel-KG21 gene, can be selected from a black goat rumen microorganism. By providing a cel-KG21 gene and a protein product, It can be used for the development of additives, as well as the development of fabric softener and detergent, as well as the decomposition of the abundant fiber, which can be actively used for the production of biofuels.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> cellulase cel-KG21 gene from rumen microorganism of black goat          and uses <130> NPF-22661 <160> 2 <170> PatentIn version 3.2 <210> 1 <211> 1206 <212> DNA <213> unidentified bacterium <400> 1 cctgcggcc cttggggaag cggcccggga aaacgccgtc tccgttccgg agcttaccat cgcgcaccgt 120 cccatccccg aaaacgaggc catggcgttt ttgaaaaaaa tgggcgtcgg ctggaacctg 180 ggcaacacct tcgacgccac caagggggac tggaaccgca acgccgacga aatgacggtg 240 gaaacctcct ggggcaaccc caaaaccact ttggcgctgt ttgacgccct ccaggcggcg 300 ggcttttcca ccgtgcgcat ccccgtgtcc tggcacgacc atgtggacga agcgtacaat 360 atcagcgaaa aatggctttc ccgggtggaa gaagtggtgg attgggccct gagccgggac 420 ctttacgtga ttttgaacat ccatcacgac gaggaacagt tcctgcccac cctggcccat 480 gaaagcgaat ccacccgcta tgtgagcgcc gtctggacgc agctggcggc ccggttccgg 540 gaccgggacg accatctgat ctttgaaagc ctgaacgagc cccgggtgct gggcgcgtcc 600 tacgagtggt ggtacgacgc caacaacccc ggctgcgtgg aagcggccca gtgcatcaac 660 cggctgaacc agctgtttgt ggacatcgtg cgcgcttccg gcggcaacaa cgcggacagg 720 tacctgatgg tgcccggcta cgacgccgcg ccgggcggcg cgctgaacaa cgtgtttgaa 780 ttgccccagg acaccgcgga caataagctc atcgtgtcca tccacgccta cacgccctac 840 gatttcgctt tgaacgtgtc cggcggcaaa accttcggca cccaggccca gaagcaggaa 900 atcgtgtcct ttatgaacag cctgtacaac cggtatatcg taaacggcgt ccccgtggtc 960 atcggcgagt acggggccct gatcaaaggc ggcaatttgc aggaccgggt gaactggacc 1020 gccttctacg tggccacggc cagcgcccgg aacatcccct gcgtgtggtg ggataacggc 1080 gcgttcaaag gcagcggcga gctgttcggc ctcgtcgacc gccgcgccgc ttccgtgtac 1140 gatcccgaaa tcgtggaagc catcatgacc tacggcggct gggataagct gccggacgcg 1200 aactaa 1206 <210> 2 <211> 401 <212> PRT <213> unidentified bacterium <400> 2 Met Lys Lys Thr Phe Ala Leu Leu Leu Ala Leu Ala Leu Cys Leu Gln   1 5 10 15 Cys Leu Thr Ala Leu Gly Glu Ala Ala Arg Glu Asn Ala Val Ser Val              20 25 30 Pro Glu Leu Thr Ile Ala His Arg Pro Ile Pro Glu Asn Glu Ala Met          35 40 45 Ala Phe Leu Lys Lys Met Gly Val Gly Trp Asn Leu Gly Asn Thr Phe      50 55 60 Asp Ala Thr Lys Gly Asp Trp Asn Arg Asn Ala Asp Glu Met Thr Val  65 70 75 80 Glu Thr Ser Trp Gly Asn Pro Lys Thr Thr Leu Ala Leu Phe Asp Ala                  85 90 95 Leu Gln Ala Gly Phe Ser Thr Val Arg Ile Pro Val Ser Trp His             100 105 110 Asp His Val Asp Glu Ala Tyr Asn Ile Ser Glu Lys Trp Leu Ser Arg         115 120 125 Val Glu Glu Val Val Asp Trp Ala Leu Ser Arg Asp Leu Tyr Val Ile     130 135 140 Leu Asn Ile His His Asp Glu Glu Gln Phe Leu Pro Thr Leu Ala His 145 150 155 160 Glu Ser Glu Ser Thr Arg Tyr Val Ser Ala Val Trp Thr Gln Leu Ala                 165 170 175 Ala Arg Phe Arg Asp Arg Asp Asp His Leu Ile Phe Glu Ser Leu Asn             180 185 190 Glu Pro Arg Val Leu Gly Ala Ser Tyr Glu Trp Trp Tyr Asp Ala Asn         195 200 205 Asn Pro Gly Cys Val Glu Ala Gln Cys Ile Asn Arg Leu Asn Gln     210 215 220 Leu Phe Val Asp Val Val Arg Ala Ser Gly Gly Asn Asn Ala Asp Arg 225 230 235 240 Tyr Leu Met Val Pro Gly Tyr Asp Ala Ala Pro Gly Gly Ala Leu Asn                 245 250 255 Asn Val Phe Glu Leu Pro Gln Asp Thr Ala Asp Asn Lys Leu Ile Val             260 265 270 Ser Ile His Ala Tyr Thr Pro Tyr Asp Phe Ala Leu Asn Val Ser Gly         275 280 285 Gly Lys Thr Phe Gly Thr Gln Ala Gln Lys Gln Glu Ile Val Ser Phe     290 295 300 Met Asn Ser Leu Tyr Asn Arg Tyr Ile Val Asn Gly Val Pro Val Val 305 310 315 320 Ile Gly Glu Tyr Gly Ala Leu Ile Lys Gly Gly Asn Leu Gln Asp Arg                 325 330 335 Val Asn Trp Thr Ala Phe Tyr Val Ala Thr Ala Ser Ala Arg Asn Ile             340 345 350 Pro Cys Val Trp Trp Asp Asn Gly Ala Phe Lys Gly Ser Gly Glu Leu         355 360 365 Phe Gly Leu Val Asp Arg Arg Ala Ala Ser Val Tyr Asp Pro Glu Ile     370 375 380 Val Glu Ala Ile Met Thr Tyr Gly Gly Trp Asp Lys Leu Pro Asp Ala 385 390 395 400 Asn

Claims (7)

A cellulolytic enzyme cel-KG21 gene derived from a black goat rumen microorganism and consisting of the nucleotide sequence shown in SEQ ID NO: 1.
A GH2C domain-based fibrinolytic protein derived from a black goat rumen microorganism, which is encoded from the gene according to claim 1 and consists of the amino acid sequence shown in SEQ ID NO: 2.
A recombinant vector comprising the gene according to claim 1.
A host cell transformed with the recombinant vector according to claim 3.
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