KR101444277B1 - cellulase cel5-KG65 gene from rumen microorganism of black goat and uses thereof - Google Patents

Abstract

The present invention relates to a cellulase cel5-KG65 gene derived from rumen microorganisms of a black goat, and uses thereof. More specifically, provided are a cel5-KG65 gene which is a novel cellulase gene separated from rumen microorganisms of a black goat and a protein product of the cel5-KG65 gene. The present invention relates to a feed additive using the cel5-KG65 gene, a detergent composition using the cel5-KG65 gene, and a biofuel manufacturing method using the cel5-KG65 gene. According to the present invention, a cel5-KG65 gene, which is a novel cellulase gene, can be separated from rumen microorganisms of a black goat, thereby providing a cellulase cel5-KG65 gene and a protein product of the cellulase cel5-KG65 gene. Therefore, the cellulase cel5-KG65 gene can be used to develop a low-priced feed additive, and can positively be used even for the production of biofuel using a decomposition process of cellulose which is an abundant resource as well as the development of a fabric conditioner and a detergent.

Description

Cellulase cel5-KG65 gene derived from a black goat rumen microorganism and its use {Cellulase cel5-KG65 gene from rumen microorganism of black goat and uses thereof}

The present invention relates to a cellulolytic enzyme cel5-KG65 gene derived from a black goat rumen microorganism and a use thereof. More specifically, the present invention provides a novel cellulolytic enzyme gene cel5-KG65 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 cellulolytic enzyme cel5-KG65 gene derived from a black goat rumen microorganism and its use.

According to one aspect of the present invention, a cellulolytic enzyme cel5-KG65 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 cellulase superfamily domain-based fibrinolytic enzyme protein encoded by the gene and consisting of the amino acid sequence of SEQ ID NO: 2, which is derived from a black goat rumen microorganism .

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, cel5-KG65 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 cel5-KG65 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 and xylan degradation activity after inoculation on CMC-Agar and Xylan-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 " superfamily " used in the present invention means a group of proteins having similar amino acid sequences, protein functions, and three-dimensional structures of proteins.

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 cel5-KG65 gene derived from a black goat rumen microorganism and consisting of the nucleotide sequence (1,434 bp) shown in SEQ ID NO: 1 can be provided.

The fibrinolytic enzyme cel5-KG65 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 cel5-KG65 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 cel5-KG65 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 cel5- 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 cellulase superfamily domain-based cellulolytic enzyme protein (hereinafter referred to as " cellulase superfamily ") comprising the amino acid sequence (477 amino acids) encoded by the gene and represented by SEQ ID NO: Can 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 a cellulase superfamily domain-based protein consisting of the amino acid sequence shown in SEQ ID NO: 2 isolated from the black goat rumen microorganism, and functional equivalents 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 of cel5-KG65 gene was confirmed. 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). As a result of protein BLAST search, it was confirmed that it has a common cellulase domain (see FIG. 2). The most similar gene has a cellulase superfamily domain in the uncultured bacterium Of the total 405 amino acids, only 198 of the putative cellulase (Accession No. AEV59736.1) showed homology (49%) to each other (see FIG. 3).

The cel5-KG65 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 0.5% xylan, and then confirmed to have a clear zone by Congo red staining. As a result, The cel5-KG65 gene and its product can be said to be novel cellulolytic enzymes (see Fig. 4).

As described above, according to the present invention, a novel cellulolytic enzyme gene, cel5-KG65 gene, can be selected from a black goat rumen microorganism, thereby providing a cellulolytic enzyme cel5-KG65 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 cel5-KG65 gene from rumen microorganism of black goat          and uses <130> NPF-23709 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1434 <212> DNA <213> unidentified bacterium <400> 1 atgccgaaaa tgaaccgtgt aatgaccatc attaccgcag tgctgatcgt ggcgaccgca 60 ctgctcgggg tcctgatctg gagcaacgaa tccaagcctg tacaggctcc tgtggaaact 120 actacggcgg taccgaccga agcaccgacc gagattccga ctgagatccc gacggaggag 180 ccgaccgaga tcccaacgga gatcccgaca gaggttccga cggaggagcc gacagccacg 240 ccgacggaag cgcctacggc gacgccgact gagaagccga cagctgtacc gaccgacaca 300 cctgtgccca ttaccgttcc ggtgatcgac gacatcaaaa cgtttgagat cccggataac 360 gaagcgatgg cactgatgcg agacatgaag agcggctgga acctggggaa tacgtttgac 420 gcctataacg gatacagcat ccatgccagc gggatcagca tggagacaag ctgggtcaac 480 gccaagacca cccagaagct gatcgctgcc gtcaaggacg cgggattcaa taccatccgc 540 attcctgtca gctggcacaa ccatgtggat gagaacgacg tcattgaccg ggaatggatc 600 gaacgcgtca gagaagtggc agactgggcg cttgacctgg gcatgtacgt gatcgtgaac 660 gtccatcatg accacgacgt aaaatacctg tatccggatt cggcccatta cgaccggtcc 720 gcggcatacc tgacctccgt atggacgcag atggcggaga tgttcaggga ctgcgatgat 780 cacctgatcc tcgagtcgat gaacgagccc cggctggtgg gtacacagta tgagtggaac 840 tggaacgcgg gcagcgcggc atgcaaggaa gcggctgaat gcatcaacag gctgaaccag 900 ctgtttgtgg acacagtccg ttcttccggc ggaaacaacg ccacgcggta cctggcggta 960 cccgcatact gcgcctctcc ctggtatgcc agcgacgcag cctttacgct gcctgcagac 1020 agtgcggaga accggatcat cgtatccgca cacgcctata cgccttacga tttcgccctg 1080 aacctcaaca gcaaagaccg tacgtttgac ctggaaaagg accagggaaa gaaaaacgag 1140 atctccggtt tcctggacaa gctgtacaac aaattcgtca aaaacggcat accggtcatg 1200 atggacgaat tcggtgcgct cgacaagcag ggaaacctgc aggcccgtgt gaatttcacc 1260 gcatattacg ttgctgccgc cagcgcgcgg ggcattacct gcgtatggtg ggacaatcat 1320 ggcttcagcg ggaacgggga acgcttcggc ctgatcaggc gtatctcggt cgaatgggta 1380 ttccctgata tcgtccgggc gatccaggaa aactgccttt ataaccgtaa ataa 1434 <210> 2 <211> 477 <212> PRT <213> unidentified bacterium <400> 2 Met Pro Lys Met Asn Arg Val Met Thr Ile Ile Thr Ala Val Leu Ile   1 5 10 15 Val Ala Thr Ala Leu Leu Gly Val Leu Ile Trp Ser Asn Glu Ser Lys              20 25 30 Pro Val Gln Ala Pro Val Glu Thr Thr Thr Ala Val Pro Thr Glu Ala          35 40 45 Pro Thr Glu Ile Pro Thr Glu Ile Pro Thr Glu Glu Pro Thr Glu Ile      50 55 60 Pro Thr Glu Ile Pro Thr Glu Val Pro Thr Glu Glu Pro Thr Ala Thr  65 70 75 80 Pro Thr Glu Ala Pro Thr Ala Thr Pro Thr Glu Lys Pro Thr Ala Val                  85 90 95 Pro Thr Asp Thr Pro Val Ile Thr Val Pro Val Ile Asp Asp Ile             100 105 110 Lys Thr Phe Glu Ile Pro Asp Asn Glu Ala Met Ala Leu Met Arg Asp         115 120 125 Met Lys Ser Gly Trp Asn Leu Gly Asn Thr Phe Asp Ala Tyr Asn Gly     130 135 140 Tyr Ser Ile His Ala Ser Gly Ile Ser Met Glu Thr Ser Trp Val Asn 145 150 155 160 Ala Lys Thr Thr Gln Lys Leu Ile Ala Ala Val Lys Asp Ala Gly Phe                 165 170 175 Asn Thr Ile Arg Ile Pro Val Ser Trp His Asn His Val Asp Glu Asn             180 185 190 Asp Val Ile Asp Arg Glu Trp Ile Glu Arg Val Glu Val Ala Asp         195 200 205 Trp Ala Leu Asp Leu Gly Met Tyr Val Ile Val Asn Val His His Asp     210 215 220 His Asp Val Lys Tyr Leu Tyr Pro Asp Ser Ala His Tyr Asp Arg Ser 225 230 235 240 Ala Ala Tyr Leu Thr Ser Val Trp Thr Gln Met Ala Glu Met Phe Arg                 245 250 255 Asp Cys Asp Asp His Leu Ile Leu Glu Ser Met Asn Glu Pro Arg Leu             260 265 270 Val Gly Thr Gln Tyr Glu Trp Asn Trp Asn Ala Gly Ser Ala Ala Cys         275 280 285 Lys Glu Ala Ala Glu Cys Ile Asn Arg Leu Asn Gln Leu Phe Val Asp     290 295 300 Thr Val Arg Ser Ser Gly Gly Asn Asn Ala Thr Arg Tyr Leu Ala Val 305 310 315 320 Pro Ala Tyr Cys Ala Ser Pro Trp Tyr Ala Ser Asp Ala Ala Phe Thr                 325 330 335 Leu Pro Ala Asp Ser Ala Glu Asn Arg Ile Ile Val Ser Ala His Ala             340 345 350 Tyr Thr Pro Tyr Asp Phe Ala Leu Asn Leu Asn Ser Lys Asp Arg Thr         355 360 365 Phe Asp Leu Glu Lys Asp Gln Gly Lys Lys Asn Glu Ile Ser Gly Phe     370 375 380 Leu Asp Lys Leu Tyr Asn Lys Phe Val Lys Asn Gly Ile Pro Val Met 385 390 395 400 Met Asp Glu Phe Gly Ala Leu Asp Lys Gln Gly Asn Leu Gln Ala Arg                 405 410 415 Val Asn Phe Thr Ala Tyr Tyr Val Ala Ala Ala Ser Ala Arg Gly Ile             420 425 430 Thr Cys Val Trp Trp Asp Asn His Gly Phe Ser Gly Asn Gly Glu Arg         435 440 445 Phe Gly Leu Ile Arg Arg Ile Ser Val Glu Trp Val Phe Pro Asp Ile     450 455 460 Val Arg Ala Ile Gln Glu Asn Cys Leu Tyr Asn Arg Lys 465 470 475

Claims (7)

A cellulolytic enzyme cel5-KG65 gene derived from a black goat rumen microorganism and consisting of the nucleotide sequence shown in SEQ ID NO: 1.
A cellulase superfamily domain-based fibrinolytic enzyme protein derived from a black goat rumen microorganism, which is encoded by the gene according to claim 1, and is composed of the amino acid sequence represented by 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.
A feed additive for promoting fibrin degradation comprising the fibrinolytic enzyme protein according to claim 2.
A detergent composition comprising the fibrinolytic enzyme protein according to claim 2.
A method for producing a biofuel comprising the step of hydrolyzing a biodegradable material with a fibrinolytic enzyme protein according to claim 2.

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