KR101503860B1 - cellulase cel45-KG80 gene from rumen microorganism of black goat and uses thereof - Google Patents

Abstract

The present invention relates to a cellulolytic enzyme cel45-KG80 gene derived from a black goat rumen microorganism and a use thereof. More specifically, the present invention provides a novel cellulolytic enzyme gene cel45-KG80 gene selected from a black goat rumen microorganism 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, cel45-KG80 gene, can be selected from a black goat rumen microorganism, which can be used for the development of a low-cost feed additive by providing a cellulolytic enzyme cel45-KG80 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

Cellulolytic cel45-KG80 gene derived from a black goat rumen microorganism and its use {Cellulase cel45-KG80 gene from rumen microorganism of black goat and uses thereof}

The present invention relates to a cellulolytic enzyme cel45-KG80 gene derived from a black goat rumen microorganism and a use thereof. More specifically, the present invention provides a novel cellulolytic enzyme gene cel45-KG80 gene selected from a black goat rumen microorganism 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 saccharin and cellulose using the same, as disclosed in Korean Patent Laid-Open Publication No. 2007-0116881 'Cellulases, nucleic acids encoding them, and methods of making and using them' are disclosed.

Korean Patent Publication No. 2011-0119961 Korean Patent Publication No. 2007-0116881

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

Finally, it is an object of the present invention to provide a fibrinolytic enzyme cel45-KG80 gene derived from a black goat rumen microorganism and its use.

According to one aspect of the present invention, a cellulolytic enzyme cel45-KG80 gene derived from a black goat rumen microorganism and consisting of the nucleotide sequence shown in SEQ ID NO: 1 can be provided.

According to another aspect of the present invention, there is provided a GH45 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, cel45-KG80 gene, can be selected from a black goat rumen microorganism, which can be used for the development of a low-cost feed additive by providing a cellulolytic enzyme cel45-KG80 gene and a protein product , Fiber softeners and detergents as well as the abundant resources of fiber, which can be used to produce biofuels.

Figure 1 shows nucleotide blast search results.
FIG. 2 shows the results of a major domain identification according to a protein blast search.
FIG. 3 shows the phylogenetic tree and homology of the highest homologous protein among the protein blast search results.
Figure 4 shows CMC degradation activity after inoculation on CMC-Agar plates.

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

As used herein, the term "rumen microorganism" refers to a microorganism in the rumen of ruminantia (cattle, goats, sheep, deer, etc.) The microorganisms that massively coexist in the microorganisms decompose 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.

As used herein, the term 'carbohydrate-binding module' (CBM) is a protein domain found in a carbohydrate-active enzyme, and the domain is a 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.

As used herein, the term "glycoside hydrolase (GH) domain" means a domain having an enzyme activity that acts on glycosides, oligosaccharides, and polysaccharides and hydrolyzes glycosidic bonds, It is classified into 127 families according to similarity of sequence.

The term " biomass " used in the present invention refers to a whole of a biological organism including a plant carrying out a carbon assimilation process for fixing carbon dioxide using sunlight and a living animal eating the carbon dioxide. 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" used in the present invention includes all the byproducts derived from metabolism activities such as living organisms as well as living organisms such as fuels, and is a renewable energy different from fossil fuels, Diesel and the like.

According to one aspect of the present invention, a cellulolytic enzyme cel45-KG80 gene derived from a black goat rumen microorganism and consisting of the nucleotide sequence (1,656 bp) shown in SEQ ID NO: 1 can be provided.

The fibrinolytic enzyme cel45-KG80 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 cel45-KG80 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 cel45-KG80 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 homologue (for example, 70% or more, preferably 80% or more, more preferably 90% or more homology with the fibrinolytic enzyme cel45- 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, there is provided a GH45 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 (551 amino acids) represented by SEQ ID NO: 2.

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 GH45 domain-based 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, pQE80L series, pET series and pUC19, etc.) commonly used in the technical field to which the present invention belongs, (E.g., gt4B, -Charon, z1, and M13) or viruses (e.g., SV40, etc.).

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. Detection of large-scale base sequence of a black goat rumen microorganism metagenome

DNA extracted from the feed small image (Ad) and gastric juice (Lq) was sequenced using 454 GS-FLX platform sequencing system. A total of 1.95 Gb of data was produced and the data was provided with good quality data using prinseq-lite.pl (version 0.14.2; minimum sequence length, 50 bp; repeat sequence removal parameter, 1245) prior to analysis See Table 1).

sample location gender Read Total base Minimum value Maximum value medium median Ad_A Small image cock 879,151 351, 697, 538 40 1,899 400 445 Ad_B Small image cock 837,109 332,000,114 40 2,020 396 440 Ad_C Small image female 858,051 347,688,619 40 1,587 405 445 Lq_A Liquid phase cock 772,580 305,470,209 40 1,749 395 456 Lq_B Liquid phase cock 742,853 294,733,416 40 1,873 396 455 Lq_C Liquid phase female 845,921 327,803,241 40 1,750 387 429

Table 1 shows the yield of the rumen microorganism metagenomic DNA base sequence decoded data extracted from the black goat. In the above data, genomic information of rice, cattle, and sheep was used to remove the DNA information of the goat's own DNA and the DNA information of the rice using the ssaha2 program (90% or more homology (eliminating sequences showing identity). The sequence information thus prepared was very strictly combined (minimal homology, 98%; minimum overlap length, 100 bp) with the de novo assembler of Newbler 2.5.3 (supplied by Roche).

Example 3: Screening of gene region and selection of gene related domain of fibrinolytic activity

We used the MetaGeneMark program to search for gene regions that have both start and stop codons to make a complete protein. In order to estimate their function, PFAMScan and HMMer3 program were used in the PFAM (protein family) database (version 25.0) to search for functional domains. Also, the clusters of orthologous groups of proteins (COG) Respectively. A gene having a glycoside hydrolase (GH) domain and a carbohydrate binding module (CBM) domain, which are essential domains for obtaining a fibrinolytic activity, is available from Carbohydrate Active Enzyme (CAZY) database (see Table 2), including genes specific to Pfam GH and CBM belonging to the genome sequence (www.cazy.org/).

In the contig In a singleton gun
gene* (CA gene **) Complete intestine gene (CA gene) gun
gene (CA gene) Complete intestine gene (CA gene) Sum of objects gun 198,396 (2,544) 69,171 (701) 2,417,798 (22,683) 252,056 (1,048) Small image
(Name) 116,908 (1,818) 38,524 (478) 1,292,970 (13,239) 127,155 (566) Ad_A 46,943 (765) 17,405 (237) 377,207 (3,896) 36,343 (146) Ad_B 34,964 (592) 10,956 (144) 432,904 (4,113) 44,807 (183) Ad_C 35,001 (461) 10,163 (97) 482,859 (5,230) 46,005 (237) Liquid phase
(Lq) 81,488 (726) 30,647 (223) 1,124,828 (9,444) 124,901 (482) Lq_C 29,726 (248) 11,893 (79) 358,599 (2,853) 39,969 (151) Lq_A 25,041 (244) 9,258 (81) 393,945 (3,435) 42,046 (144) Lq_B 26,721 (234) 9,496 (63) 372,284 (3,156) 42,886 (187) Merger Assembly gun 297,445 (3,673) 102,584 (1,097) 1,794,132 (16,426) 187,907 (765) Small image
(Name) 142,703 (2,111) 49,704 (644) 1,079,082 (10,761) 106,432 (464) Liquid phase
(Lq) 114,198 (1,111) 40,230 (304) 959,518 (8,003) 107,027 (417)

* The "total" gene means the total number of both structural and imperfect.

** "CA" gene means carbohydrate-degrading activity.

Table 2 above shows the number of gene and carbohydrate degrading activity genes deduced from ruminal microbial genome fragment sequences obtained through sequence combinations.

Example 4. Selection of Cellulolytic Activity Gene and Its Similarity and Activity

As shown in Table 2, in a total of 290,491 full-length gene sequence information, a gene having a glycoside hydrolase (GH) domain and a carbohydrate binding module (CBM) domain in its pfam domain database As a result, the cellulase domain was identified in the cel45-KG80 gene. BLAST searches for nucleotides and proteins were performed (as of June 2013) to confirm whether identical or similar sequences exist in the NCBI database. No nucleotide BLAST search results showed that no similar DNA sequence could be found under the default search conditions (see FIG. 1). Also, it was confirmed that the protein BLAST has a common cellulase domain (see FIG. 2), and the most similar gene is Fibrobacter succinogenes subsp. As a cellulase gene (Accession No. YP_003249506.1) having a GH45 domain in succinogenes S85, only 477 of the total 557 amino acids showed homology (86%) to each other (see FIG. 3).

The cel45-KG80 gene was amplified by polymerase chain reaction with the original metagenome extracted DNA, and the DNA fragment was obtained. The obtained DNA fragment was cloned into the pQE80L E. coli expression vector. This clone was cultured on an agar plate containing 0.5% CMC, and then it was confirmed that a clear zone was formed by Congo red staining. As a result, the cel45-KG80 gene And its product can be said to be a novel fibrinolytic enzyme (see Fig. 4).

As described above, according to the present invention, a novel cellulolytic enzyme gene, cel45-KG80 gene, can be selected from a black goat rumen microorganism, thereby providing a cellulolytic enzyme cel45-KG80 gene and a protein product, It can be used for the development of additives, as well as developing textile softeners and detergents, as well as decomposing the abundant resources of cellulose, and actively utilizing 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 cel45-KG80 gene from rumen microorganism of black goat          and uses <130> NPF-23705 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1656 <212> DNA <213> unidentified bacterium <400> 1 atgaagaata aactgtttaa aaccctcaca attttcgggc tttcctttat cgcatggaac 60 tgctccgatg accctgcatc tccttcggga gcaacctctg aattacctgc tatccagcaa 120 aagcagatgc tggaagtgga tcagcagagc tggttgctca ataccggaag tcaagttttc 180 ttgattgtac ccaacggcac aggcacgtac actgtcacgg atgcattcag caatccggtc 240 ggcacgttca atacggcaac aaagtccatt tcagacgcag ccggtaacac gactgttgtt 300 agcgttgatc tttcttcact cccgattgtc aatccggaca agaccatcac ctaccccgat 360 ggttccaaga ccactcttac gggtgaaccg atttcgagcg gcgtaacggt aaatagttcc 420 tcggcaacca ctccgggtac cgttgttgtc ttgagcagct cttctgtcta caatccgggt 480 actccggctg tgagctccag ctccgccaaa acatcgaccc cagttgcctc ttcctcttcg 540 cagaagaccc agcagcctgt cgcaagctcc tcctcgaagc aacagaccaa ctccggcacc 600 tccgacaaac agtgcaatgg ccagtgctat gacaatgctt cgggcaagtg cgttaactat 660 tacgaccaga tgaccggctc caagggcgaa aaatacgcct acgacaatga ttgcaaggta 720 aactgctact acgatcctga aaataagaat tgccagaaca tgaccggctc tacaccttct 780 cagcagccga agtccagctc tagcgtgaag tcttcttctt cacagcaaca gcagcaggca 840 aaatcttctt cgtcccagca gcagcaacag ccgaagtcct ccagcagcca gccgaaatct 900 tcgtctagcc agcagcaaca gcagaacaac cccaacgctt ccgccgaaga agccaagtac 960 ctcaatgcag gcgccggtgg acagcagggc tttgccaccc gttactggga ctgctgcatg 1020 cctcactgct cctggccgga acacggcggc gctgccaaaa cctgcgatgc taagggcaag 1080 accccgatcg gcaacaccaa cggaagcatc tgctccggcg gccagggcac cacttgcaca 1140 agccagattc ctataatcgt gagcgacaag ctcgcctacg cattcgctgc aactccgggt 1200 aacgacgcca catgcggcaa gtgcttcgcc ctcaccttca ccggtaccgg caagtacgaa 1260 accaaggcaa accacaaggc acttgcaggc aagactctcg tcgtgatggc atcgaacatc 1320 ggttacgacg tacagggcgg ccagttcgac atcatgatcc cgggtggcgg attcggtgca 1380 tttaatggtt gcagccagat gggctggaac atcccgagca actccacaac gtacggcggc 1440 ctcctctccg actgcgaaaa ggaagtcgga tacaatggcg accttttgac caaacgcaag 1500 gaatgcctca ccaagaagtg caacagcgct tttgctagcg acacacaggc caaggaaggc 1560 tgcctcttcc tcgctacatg gatggaagca gcaggcaacc cgaaccacac ttataaagaa 1620 gttgaatgcc cggctgcttt gaaggccaaa ttctaa 1656 <210> 2 <211> 551 <212> PRT <213> unidentified bacterium <400> 2 Met Lys Asn Lys Leu Phe Lys Thr Leu Thr Ile Phe Gly Leu Ser Phe   1 5 10 15 Ile Ala Trp Asn Cys Ser Asp Asp Pro Ala Ser Pro Ser Gly Ala Thr              20 25 30 Ser Glu Leu Pro Ala Ile Gln Gln Lys Gln Met Leu Glu Val Asp Gln          35 40 45 Gln Ser Trp Leu Leu Asn Thr Gly Ser Gln Val Phe Leu Ile Val Pro      50 55 60 Asn Gly Thr Gly Thr Tyr Thr Val Thr Asp Ala Phe Ser Asn Pro Val  65 70 75 80 Gly Thr Phe Asn Thr Ala Thr Lys Ser Ile Ser Asp Ala Ala Gly Asn                  85 90 95 Thr Thr Val Ser Val Asp Leu Ser Ser Leu Pro Ile Val Asn Pro             100 105 110 Asp Lys Thr Ile Thr Tyr Pro Asp Gly Ser Lys Thr Thr Leu Thr Gly         115 120 125 Glu Pro Ile Ser Ser Gly Val Thr Val Ser Ser Ser Ala Thr Thr     130 135 140 Pro Gly Thr Val Val Val Leu Ser Ser Ser Ser Val Tyr Asn Pro Gly 145 150 155 160 Thr Pro Ala Val Ser Ser Ser Ala Lys Thr Ser Thr Pro Val Ala                 165 170 175 Ser Ser Ser Gln Lys Thr Gln Gln Pro Val Ala Ser Ser Ser Ser             180 185 190 Lys Gln Gln Thr Asn Ser Gly Thr Ser Asp Lys Gln Cys Asn Gly Gln         195 200 205 Cys Tyr Asp Asn Ala Ser Gly Lys Cys Val Asn Tyr Tyr Asp Gln Met     210 215 220 Thr Gly Ser Lys Gly Glu Lys Tyr Ala Tyr Asp Asn Asp Cys Lys Val 225 230 235 240 Asn Cys Tyr Tyr Asp Pro Glu Asn Lys Asn Cys Gln Asn Met Thr Gly                 245 250 255 Ser Thr Pro Ser Gln Gln Pro Lys Ser Ser Ser Val Lys Ser Ser             260 265 270 Ser Ser Gln Gln Gln Gln Gln Ala Lys Ser Ser Ser Gln Gln Gln         275 280 285 Gln Gln Pro Lys Ser Ser Ser Gln Pro Lys Ser Ser Ser Ser Gln     290 295 300 Gln Gln Gln Gln Asn Asn Pro Asn Ala Ser Ala Glu Glu Ala Lys Tyr 305 310 315 320 Leu Asn Ala Gly Ala Gly Gly Gln Gln Gly Phe Ala Thr Arg Tyr Trp                 325 330 335 Asp Cys Cys Met Pro His Cys Ser Trp Pro Glu His Gly Gly Ala Ala             340 345 350 Lys Thr Cys Asp Ala Lys Gly Lys Thr Pro Ile Gly Asn Thr Asn Gly         355 360 365 Ser Ile Cys Ser Gly Gly Gln Gly Thr Thr Cys Thr Ser Gln Ile Pro     370 375 380 Ile Ile Val Ser Asp Lys Leu Ala Tyr Ala Phe Ala Ala Thr Pro Gly 385 390 395 400 Asn Asp Ala Thr Cys Gly Lys Cys Phe Ala Leu Thr Phe Thr Gly Thr                 405 410 415 Gly Lys Tyr Glu Thr Lys Ala Asn His Lys Ala Leu Ala Gly Lys Thr             420 425 430 Leu Val Val Met Ala Ser Asn Ile Gly Tyr Asp Val Gln Gly Gly Gln         435 440 445 Phe Asp Ile Met Ile Pro Gly Gly Gly Phe Gly Ala Phe Asn Gly Cys     450 455 460 Ser Gln Met Gly Trp Asn Ile Pro Ser Asn Ser Thr Thr Tyr Gly Gly 465 470 475 480 Leu Leu Ser Asp Cys Glu Lys Glu Val Gly Tyr Asn Gly Asp Leu Leu                 485 490 495 Thr Lys Arg Lys Glu Cys Leu Thr Lys Lys Cys Asn Ser Ala Phe Ala             500 505 510 Ser Asp Thr Gln Ala Lys Glu Gly Cys Leu Phe Leu Ala Thr Trp Met         515 520 525 Glu Ala Ala Gly Asn Pro Asn His Thr Tyr Lys Glu Val Glu Cys Pro     530 535 540 Ala Ala Leu Lys Ala Lys Phe 545 550

Claims (7)

A cellulolytic enzyme cel45-KG80 gene derived from a black goat rumen microorganism and consisting of the nucleotide sequence shown in SEQ ID NO: 1.
2. A glycoside hydrolase (GH) 45 domain-based protease protein from a black goat rumen microorganism, encoded by the gene according to claim 1 and consisting 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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