KR102010836B1 - Protease-KG01 gene from rumen microorganism of black goat and uses thereof - Google Patents

Abstract

The present invention relates to a protease-KG01 gene derived from a black goat rumen microorganism and its use, and more particularly to a novel protease-KG01 gene selected from a black goat rumen microorganism and a protein product thereof, using the same as a feed additive and a detergent composition. And it relates to a method for producing food such as dairy products, pharmaceuticals.

Description

Protease-KG01 gene from rumen microorganism of black goat and uses approximately}

The present invention relates to a protease-KG01 gene derived from a black goat rumen microorganism and its use, and more particularly to a novel protease-KG01 gene selected from a black goat rumen microorganism and a protein product thereof, using the same as a feed additive and a detergent composition. And it relates to a method for producing food such as dairy products, pharmaceuticals.

The major components of plant cell walls are cellulose (celluloase), hemi-cellulose and pectin, which are degraded and taken up by microorganisms through complex and effective processes in the rumen of herbivores.

Rumen microorganisms are extremely anaerobic and include fungi, protozoa and bacteria, and the breakdown of cellulose is known to be primarily responsible for bacteria and fungi.

However, most of these microorganisms are difficult to cultivate and are not well known. Just known to date, with the main bacteria involved to digest plant cell walls from the rumen microorganisms Ness (Fibrobacter succinogenes) I bakteo succinonitrile to five, luminometer Caucus Playa Pacifica beret Enschede (Ruminococcus flavefaciens ) , Ruminococcusalbus , Butyrivibrio fibrisolvens ( Butyrivibrio fibrisolvens), has been identified as a bacterium belonging to cellulite Solarium Te oil cake in Sol Bence (Eubacterium cellulosolvens), frame Beam telra (Prevotella), and some of Clostridium (Clostridium). To date, most of ruminant fibrinase genes have been studied to investigate the total microbial DNA or metagenome of the digestive system of cattle, rabbits and kangaroos. A number of glucanases and cellulosome complexes have been identified.

However, most of these cases have evolved to feed soft plant fibrinogen. Industrially, the ability to decompose lighter fibrinogen in fibrinase is required.

Black goats have evolved to better digest diets of coarser fibrinogens such as twigs, bark, and vegetation. This means that the rumen microbial community of black goats evolved symbioticly from other herbivores. In order to search for genes for a non-cultured microbial population, a technique for searching for new materials using phosmid, cosmid and bacterial artificial chromosome (BAC) gene banks is widely used. In this way, many fibrinase genes have been identified.

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

Under such technical background, the present inventors have completed the present invention as a result of diligent efforts.

After all, it is an object of the present invention to provide a protease-KG01 gene derived from a black goat rumen microorganism and its use.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, a protease KG01 gene derived from a black goat rumen microorganism, which consists of a nucleotide sequence represented by SEQ ID NO: 1, may be provided.

According to another aspect of the invention, the Dis_P_Dis superfamily and peptidase A24 superfamily domain derived from the black goat ruminant microorganism, which is encoded from the gene and consists of the amino acid sequence represented by SEQ ID NO: 2 Protease proteins that are family can be provided.

According to another aspect of the present invention, a recombinant vector comprising 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, a feed additive for improving protein degradation including the protease protein may be provided.

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

According to another aspect of the present invention, a food comprising the protease protein may be provided.

The present invention provides a protease derived from a novel black goat rumen microorganism, through which a wide variety of substrates can be degraded. By using the present invention can be developed a low-cost feed additive in preparation for rising feed grain prices, can be used in food processing, such as dairy products and pharmaceuticals, can be applied to various aspects, such as stain removal and the development of detergents using the same.

1 shows the results of establishing the metagenome DNA cleavage conditions and inserting DNA cleavage for constructing a Fosmid gene bank according to an embodiment of the present invention.
Figure 2 shows the results of confirming the average insertion size of the phosmid gene bank for the Ad fraction and Lq fraction according to an embodiment of the present invention.
3 shows a clone selection result in which a transparent ring is formed after skim milk digestion after inoculation onto skim milk-Agar plate according to an embodiment of the present invention.
4 shows nucleotide blast results of protease-KG01 according to an embodiment of the present invention.
Figure 5 shows the main domain identification results according to the protease-KG01 protein search (protein blast) according to an embodiment of the present invention.
Figure 6 shows the main analogous protein according to the result of protease-KG01 protein blast (protein blast) according to an embodiment of the present invention.
FIG. 7 shows a similarity (phylogenetic tree) and homology and estimated value between the highest homologous protein and another protease protein among protease-KG01 protein blast results according to an embodiment of the present invention.

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

As used herein, the term "superfamily" refers to a group of proteins similar in amino acid sequence, protein function, and conformation of proteins.

The present invention focuses on the proteolytic ability of the black goat ruminant microorganism, and builds a fosmid gene bank for the black goat rumen microorganism metagenome, and uses skim milk which is used to verify the normal proteolytic activity therefrom. As a substrate, a fosmid clone having a degrading activity was selected, and genetic information was obtained by decoding the plasmid DNA of the clone. The plasmid DNA of the phosphid clone having activity was extracted using the Qiagen Midi Prep Kit, and the extracted DNA produced 100 times of shotgun sequencing data with an average length of 400 bp. The produced data was converted to fasta format and combined using a Newbler de novo assembler to obtain sequencing information. The obtained active phosphmid plasmid DNA sequence information was searched for genes using the Metagenemark program, and the protease-related genes were discovered by searching for the genes similar to the major domains through NCBI blastp search.

The present invention has been identified as a novel protease gene that is very different from the protease gene sequences published to date.

According to an aspect of the present invention, a protease-KG01 gene derived from a black goat rumen microorganism may be provided, which consists of a nucleotide sequence represented by SEQ ID NO: 1.

The protease-KG01 gene according to the present invention may be DNA or RNA encoding a protease protein. DNA includes cDNA, genomic DNA or artificial synthetic DNA. DNA can be single stranded or double stranded. DNA may be a coding strand or a non-coding strand.

Preferably, the protease-KG01 gene according to the present invention may include a nucleotide sequence represented by SEQ ID NO: 1. In addition, variants of the above nucleotide sequences are included within the scope of the present invention. More specifically, the gene may include a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1. Can be.

The '% sequence homology' for a polynucleotide is determined by comparing the two optimally arranged sequences with the comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

According to an embodiment of the present invention, the protease-KG01 gene is preferably derived from a black goat rumen microorganism. However, the present invention is not necessarily limited thereto, and the exemplary embodiments of the present invention have a high homology with protease-KG01 (eg, 70% or more, preferably 80% or more, more preferably 90% or more, most preferably Genes derived from rumen microorganisms of organisms other than black goats. Sequencing methods and means for determining sequence homology (eg, BLAST, etc.) are well known to those of ordinary skill in the art.

According to another aspect of the invention, the Dis_P_Dis superfamily and peptidase A24 superfamily domain derived from the black goat ruminant microorganism, which is encoded from the gene and consists of the amino acid sequence represented by SEQ ID NO: 2 Protease proteins that are family can be provided.

The range of the protease protein according to the present invention is the Dis_P_Dis superfamily and peptidase A24 superfamily domain family proteins and the protein comprising an amino acid sequence represented by SEQ ID NO: 2 isolated from a black goat ruminant microorganism. Includes functional equivalents of

The term 'functional equivalent' is at least 70%, preferably at least 80%, more preferably at least 90%, of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of amino acids. Most preferably, it refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity with a protein consisting of the amino acid sequence represented by SEQ ID NO: 2. The term 'substantially homogeneous physiological activity' means the same protease activity.

The invention also includes fragments, derivatives and analogues of protease proteins. The terms 'fragment', 'derivative' and 'analogue' refer to a polypeptide having substantially the same biological function or activity as the protease protein according to the present invention.

Fragments, derivatives and analogs of the protease protein according to the present invention may comprise (i) a polypeptide substituted with one or more conservative or non-conservative or non-conservative amino acid residues (preferably conservative amino acid residues) wherein said substituted amino acid residues are genetic Or (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 a polypeptide, for example A polypeptide derived from a mature polypeptide bound to polyethylene glycol), or (iv) an additional amino acid sequence (e.g., a leader sequence, a secretion sequence, a sequence used to purify the polypeptide, a proteinogen sequence or Fusion protein) and a polypeptide derived from said polypeptide. Such 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, ie, the polynucleotide encoding a mature polypeptide, includes a coding sequence encoding only a mature polypeptide; Sequences encoding mature polypeptides and various additional coding sequences; Mature polypeptides (and any additional coding sequences) and sequences encoding noncoding sequences.

The term 'polynucleotide encoding a polypeptide' refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising an additional coding and / or noncoding sequence.

The 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 may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitutional variants, deletional variants, and insertional variants.

As is well known to those skilled in the art, allelic variants are alternatives to polynucleotides, which may include one or more substituted, deleted or inserted nucleotides, It does not result in a substantial functional change in the polypeptide encoded by it.

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

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

Here, the vectors that can be used in the present invention are plasmids commonly used in the art (eg pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX, pQE80L series, pET series and pUC19, etc.), Phage (eg gt4B, -Charon, z1 and M13 etc.) or viruses (eg SV40 etc.) can be produced.

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 the mammalian cell (e.g., metallothionine promoter) or a promoter derived from a mammalian virus (e.g., adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The vector according to the present invention is a marker, and antibiotic resistance genes commonly used in the art to which the present invention belongs, such as ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin It may further comprise resistance genes for 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 prokaryotic vector and expressing the vector according to the present invention is a host cell commonly used in the art to which the present invention belongs, for example, E. coli. JM109 , E. coli BL21 , E. coli RR1 , E. coli LE392 , E. coli B , E. coli X 1776 , E. coli Strains of the genus Bacillus, such as W3110, Bacillus subtilis, Bacillus thuringiensis, or enterobacteria and strains such as Salmonella typhimurium, Serratia marcensons and various Pseudomonas species.

In addition, when transforming a vector according to the present invention to eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines), plant cells, and the like.

The method of transporting the vector according to the present invention into a host cell is performed by using the CaCl ₂ method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983) when the host cell is a prokaryotic cell. ) And the electroporation method. In addition, 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, or the like. have.

According to another aspect of the present invention, a feed additive for improving protein degradation including the protease protein may be provided.

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 (polyphosphate) and the like; Natural antioxidants such as polyphenols, catechins, alpha-tocopherols, rosemary extracts, vitamin C, green tea extracts, licorice extracts, chitosan, tannic acid, phytic acid, and the like; have.

Feed additives according to the present invention include adjuvant components such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes and other microbial agents in the form of live bacteria; Cereals such as milled or crushed wheat, oats, barley, corn and rice; Vegetable protein feed, such as rape, soybeans and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Dry ingredients consisting of sugars and dairy products such as various powdered milk and whey powders; Main components such as lipids, for example animal fats and vegetable fats, optionally liquefied by heating; It may further include one or more selected from additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

Feed additives according to the invention may be in the form of a dry or liquid formulation, and may include excipients for feed addition. The feed additive excipients include, for example, zeolite, jade powder or rice bran, and are not necessarily limited to the above examples.

The feed additive according to the present invention is contained in an enzyme preparation, for example, a lipase such as lipase, a phytase which decomposes phytic acid to make phosphate and inositol phosphate, starch and glycogen, etc. Amylase, an enzyme that hydrolyzes α-1,4-glycoside bonds, phosphatase, an enzyme that hydrolyzes organic phosphate esters, and maltose as two molecules of glucose It may further include one or more selected from hydrolase hydrolyzing maltase and saccharose to convert to a glucose-fructose mixture.

The feed additive according to the present invention may be administered to an animal alone or in combination with other feed additives in an edible carrier. In addition, the feed additives can be easily administered as top dressings or directly mixed with livestock feed or separately from the feed, in a separate oral formulation, or in combination with other ingredients.

As is commonly known in the art, single daily or divided daily intakes may be used.

Animals that may be used in the feed additives according to the present invention include, for example, cattle, chicks, chickens, domestic chickens, roosters such as edible cattle, cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats, etc. And poultry such as ducks, geese, turkeys, quails, birds, and the like, and are not necessarily limited to the above examples.

The amount of the protease according to the present invention, which is included in the feed additive according to the present invention, is not particularly limited, but as is generally known in the art to which the present invention belongs, it is an amount suitable for degrading protein source material while prolonged survival in the digestive tract of livestock. Included.

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

The detergent compositions according to the invention may be in the form of one and two part aqueous detergent compositions, non-aqueous liquid detergent compositions, cast solids, granules, particles, compressed tablets, gels, pastes or slurries. When the food composition is used, the food residue film and other small amount of the food composition can be quickly removed.

The detergent composition according to the present invention may be effective for removing dried stains through catalyst-mediated hydrolysis of proteins.

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

According to another aspect of the present invention, a food comprising the protease protein may be provided. The protease protein may be available in liquid or solid form. In addition, the food may be an additive of powder, pill, beverage, tea or general food.

Food according to an embodiment of the present invention is not limited thereto, but may be a food group requiring a protease, such as dairy products, food for bowel movement or diet, functional food for preventing hypertension.

The amount of the protease according to the present invention, which is included in the food according to one embodiment of the present invention, is not particularly limited, but is suitable for decomposing protein source material while prolonged survival in the digestive tract as is commonly known in the art. It is included.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely to illustrate the present invention is not limited to the contents of the present invention.

Example

Example  1: Isolation of Black Goat Ruminant Microorganism and DNA Extraction

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

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

By adjusting the amount of various HindIII restriction enzymes for arbitrary partial cleavage conditions to an appropriate size for constructing a fosmid gene bank for the DNA extracted from Ad and Lq, Ad was HindIII 5 units and Lq was 2.5 units. Decided to use (FIG. 1).

The cleaved DNA fragments were cut out from 10 kb to 50 kb using Pulse-Field Gel electrophoresis to obtain insert DNA fragments from them. The obtained DNA was subjected to terminal smoothing reaction and linked to the pCC1FOS vector by ligation reaction. After ligation ligation, DH5alpha Escherichia coli was transformed by electric shock to construct a gene bank.

As a result of electrophoresis, NotI enzyme treatment was performed to confirm the average insertion size of the constructed fosmid gene bank, and it was found to be 30 kb for Ad and 31 kb for Lq (FIG. 2). Each of them was inoculated in cryopreservable medium in 150 plates of 384 well plates, respectively, and stored in an cryogenic freezer (-70 ℃).

Example  3: skim milk degrading activity Phosmid ( fosmind Clone selection

Inoculate 0.5 μl of LB-chloramphenicol agar plate containing 1% skim milk substrate in 384 clones using the VIAFLO 384 instrument from INTEGRA for 115,200 clones of all Ad and Lq phosphid clones, Incubated for 48 hours at 37 ℃. When a transparent ring was observed around the clone in the cultured agar plate, the clone was judged to have proteolytic activity and was selected (FIG. 3).

Example  4: skim milk degradation activity Phosmid  Sequence Decoding for Clones

Phosmid plasmid extraction was performed using the Qiagen plasmid midi prep kit for the phosphid clone LQ_039M10 identified in Example 3, and the extracted plasmid DNA was obtained by using the GS-Junior platform, which is a pyro-sequencing method. 100 times as many shotgun sequencing data were produced. The produced data was converted to fasta format and assembled using the Newbler 2.5 de novo assembler program. A total of five contiguous sequences were generated, and lengths of 11,786 bp, 6.061 bp, 5,873 bp, 5,837 bp, and 2,461 bp, respectively, resulted in a total of 32,018 bp metagenome sequence information.

-Place of discovery: National Institute of Animal Science, Rural Development Administration

 Sequence-derived black goat: ruminant microorganism obtained by slaughtering three months of black goats (two males, one female) from the National Institute of Animal Science, Rural Development Administration, slaughtered by feeding with straw straw and mineral supplements for one month after transfer to the animal genome division. sample

Phosmid gene bank vector: pCC1Fos

Gene-derived phosphide clone name: LQ039M10

DNA sequence of the gene (SEQ ID NO: 1) length: 1,116 bp

Amino acid sequence of the gene (SEQ ID NO: 2) length: 371 amino acids

DNA and protein sequences of genes: Table 1, Table 2 and Sequence Listing attached

Example  5: Gene search and similarity investigation

For the DNA sequencing information of the obtained phosmid clone, the gene region was searched using the MetaGeneMark program. For the searched gene information, similar sequence information of each gene was obtained through protein blast of NCBI. In this process, protease-KG01, a new protease gene, was referred to the main domain search results and similar proteins. Genes were excavated.

Protease-KG01 according to the present invention, as of November 2016 (Nucleotide blast) results (Fig. 4), it was found that there is no similar sequence for the gene of the entire 1,116bp size. In addition, as a result of protein blast, DiS-P-DiS domain is located in the 49-129 region from the N-terminal, and peptidase A24 domain is located in the 153-260 region. In addition, it was confirmed that the PulO domain of the prepilin signal peptidase was located in the 49-293 region as a whole. It can be said that it has a prepilin peptidase activity that cleaves Gly-Phe bond (FIG. 5). The search for a similar protein showed 39% homology with the peptidase A24 of Desulfobacterales bacterium S7086C20 at an overall 67% level (FIG. 6). Compared to other similar peptidase protein sequences in the BLASTP results, the product of the protease-KG01 gene is a protease with novel peptidase activity (FIG. 7).

The product of the KG01 gene, which is a kind of protease protease containing the Dis_P_Dis superfamily and the peptidase A24 superfamily domain according to the present invention, can be used as a feed additive, It is also used in detergents and food processing can be very useful industrially.

As described above in detail specific parts of the present invention, it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (7)

Protease-KG01 gene derived from a black goat rumen microorganism, consisting of the nucleotide sequence represented by SEQ ID NO: 1. Protease protein of the Dis_P_Dis superfamily and Peptidase A24 superfamily derived from the Black Goat rumen microorganism, which is encoded by the gene according to claim 1 and consists of the amino acid sequence represented by SEQ ID NO: 2 . Recombinant vector comprising a gene according to claim 1. A host cell transformed with the recombinant vector according to claim 3. delete delete delete

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