KR20120040696A - Novel gene encoding mannanase from cow rumen metagenome - Google Patents

Abstract

PURPOSE: A novel mannanase gene derived from ruminant stomach metagenome is provided to produce a large amount of recombinant glycosyl hydrolase. CONSTITUTION: A mannanase gene contains base sequence of sequence number 7. A mannanase protein contains an amino acid sequence of sequence number 8. The mannanase gene is isolated from a ruminant stomach sample. A recombinant expression vector, pEREng16, contains the mannanase gene. An E.coli HIT-21/pEREng16 transformant(deposit number:KCTC 11656BP) is prepared by the recombinant expression vector.

Description

Novel gene encoding mannanase from cow rumen metagenome {Novel gene encoding mannanase from cow rumen metagenome}

The present invention relates to a novel metase gene derived from a bovine rumen metagenome, a recombinant vector containing the gene, a transformant using the recombinant vector, and a metase protein produced by the transformant, in more detail Is produced a bovine rumen metagenome library to select a clone having cellulase activity, and a novel metase gene identified from the clone, a recombinant vector containing the gene, a transformant using the recombinant vector, and the transformant It relates to a protein produced by the met nanase.

It is known that only about 0.1% to 1% of microorganisms that can be cultured in nature are cultivable. Metagenome is an effective means for discovering useful genes from microorganisms that cannot be cultured. After being used for new drug discovery at the University of Wisconsin in 1998 using unknown soil microorganisms, various environmental samples such as oceans, deserts, rivers, and hot springs. It is applied to the field. In the case of using the metagenome, it is difficult to obtain information on the microorganism from which the gene is derived, but it has the advantage of securing the product of the microorganism and the gene at the same time.

Cellulose is a linear polysaccharide of a polymer in which glucose is linked by a beta-1,4-glycosidic bond. It is the most abundant organic material in nature and is valuable as an energy resource that can be recycled. Very high. Cellulose is broken down into glucose, a constituent component, by a hydrolytic enzyme called cellulase. Cellulases known to date are largely endo-beta-1,4-glucanase (EC 3.2.1.4), exo-beta-1,4-glucanase (exo-beta- It can be classified into three types: 1,4-glucanase, EC 3.2.1.91), and beta-glucosidase (EC 3.2.1.21). Endo-beta-1,4-glucanase randomly hydrolyzes beta-1,4 bonds in the inner chain of cellulose, and exo-beta-1,4-glucanase is endo-beta-1,4-glucanase. The reduced and non-reduced ends of the cellulose polymer produced by kanase are sequentially hydrolyzed to produce cellobiose to which two molecules of glucose are bound. Cellobiose is hydrolyzed by beta-glucosidase to finally produce glucose.

Cellulase is widely used in various fields such as detergent, pulp and paper industry, feed and food for livestock, and recently, as a next-generation energy source to replace fossil fuels, it is a key in the industry that produces ethanol by decomposing plant biomass. It is being used as a technology. Cellulase is currently forming the third-largest market among industrial enzymes in the world, and it is predicted that the market size can be further expanded if biomass-derived transport bioenergy is commercialized.

Although a lot of research and development has been made to secure excellent cellulase, there is still a need for the development of a new cellulase having desirable properties and activities that can be used for sugar conversion of cellulose derived from lignocellulosic biomass to obtain bioethanol. Accordingly, the present inventors conducted research to discover a new excellent cellulase, and the present invention by cloning a multifunctional glycosyl hydrolase having a hemicellulase function as well as a cellulase not previously identified in the rumen metagenome of cattle. Completed

It is an object of the present invention to provide a novel metase gene derived from a cow rumen metagenome and a novel metase protein encoded therefrom.

Another object of the present invention is to provide a recombinant vector containing the gene and a transformant into which the recombinant vector has been introduced.

In order to achieve the above object, the present invention is characterized by providing nucleotide sequences and amino acid sequences of novel metase-coding genes derived from bovine rumen metagenome.

In addition, the present invention is characterized in that it provides a recombinant vector containing the gene and a transformant into which the recombinant vector has been introduced.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of extracting metagenomic DNA from a bovine rumen sample and cloning it into a fosmid vector to prepare a metagenomic library; Screening for cellulase in the metagenomic library; Subcloning and sequencing a metagenome clone having cellulase activity; Obtaining three different lengths of transcriptional reading frames (ORFs) using polymerase chain reaction (PCR) and then introducing them into expression vectors, respectively; Transforming the expression vectors into which the genes have been introduced into host cells; Expressing a recombinant protein using the above transformants and confirming hydrolytic activity for various polysaccharides; It consists of a step of purifying the recombinant enzyme expressed in the expression vector into which the finally selected transcriptional decoding frame was introduced and determining the properties of the enzyme.

In the case of mass production of recombinant glycosyl hydrolase using a novel glycosyl hydrolase gene derived from ruminant bacteria, which is difficult to cultivate from a bovine rumen metagenome library, detergent, pulp and paper industries , It can be used for commercial purposes in various fields such as feed and food for livestock, and bioenergy.

1 is a diagram of selecting a transformant having a recombinant plasmid having cellulase activity from a metagenome library on a plate medium by the Congo red staining method (A: confirmation of cellulase activity in the metagenome library, B: selected traits Reconfirmation of the cellulase activity of the transformant).
2 is a schematic representation of the gene structure of ORF-E secured by the present invention (GH: glycoside hydrolase).
FIG. 3 is a diagram showing a comparison of homology between ORF-E and prevotella bryontii xylanase (AAC97596) and cellulase isolated from ruminants (ABB46200).
4 is a schematic diagram of genes encoding transcriptional and decoding frames according to the present invention and recombinant expression vectors into which they are inserted.
5 is a schematic diagram of a recombinant vector pEREng13 containing reng 13, which is a glycosyl hydrolase-encoding gene.
6 is a schematic diagram of a recombinant vector pEREng43 containing reng 43, a glycosyl hydrolase-encoding gene.
7 is a schematic diagram of a recombinant vector pEREng16 containing reng 16, which is a glycosyl hydrolase-encoding gene.
Figure 8 is a metagenome-derived E. coli transformed with a novel glycosyl hydrolase gene E.coli HIT-21/pEREng13, E.coli HIT-21/pEREng43, E.coli HIT-21/pEREng63, E.coli HIT As a picture of analyzing the protein expressed in -21/pEREng16 by SDS-PAGE,
lane M is a molecular weight standard marker;
lane 1, lane 3, lane 6 and lane 8 are cell extracts of the negative control group transformed with pET21a;
lane 2 is a cell extract of E.coli HIT-21/pEREng13 treated with 0.5mM IPTG;
lane 4 is a cell extract of E.coli HIT-21/pEREng43 not treated with IPTG;
lane 5 is a cell extract of E.coli HIT-21/pEREng43 treated with 0.5mM IPTG;
lane 7 is a cell extract of E.coli HIT-21/pEREng63 treated with 0.5mM IPTG;
lane 9 is a cell extract of E.coli HIT-21/pEREng16 not treated with IPTG;
Lane 10 represents the cell extract of E.coli HIT-21/pEREng16 treated with 0.5mM IPTG.
9 is a diagram of proteins expressed in E. coli HIT-21/pEREng13, E.coli HIT-21/pEREng43, E.coli HIT-21/pEREng63, E.coli HIT-21/pEREng16 according to the present invention. It is a figure showing the hydrolytic activity of polysaccharides.
10 is a result of confirming the expression and purification of the recombinant proteins REng13 and REng43 by SDS-PAGE,
lane M is a molecular weight standard marker;
lane C is the cell extract of the negative control transformed with pET21a;
lane CL is a cell extract of E.coli HIT-21/pEREng13 or E.coli HIT-21/pEREng43 treated with 0.5mM IPTG;
Lanes E3 to E7 represent fractions that were eluted after adding the cell extract of E.coli HIT-21/pEREng13 or E.coli HIT-21/pEREng43 treated with 0.5mM IPTG to a HIS*Bind column and washing several times.
11 is a graph showing the effect of temperature on the activity of the recombinant proteins REng13 and REng43.
12 is a graph showing the effect of pH on the activity of the recombinant proteins REng13 and REng43 (blue: REng13, red: REng43).
13 is a diagram showing the results of TLC analysis of the cellulooligosaccharide hydrolyzate of the recombinant proteins REng13 and REng43.
Lane 1 is a cellooligosaccharide mixture (G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose);
Lane 2 is a hydrolyzate obtained by reacting cellobiose with recombinant REng13 or REng43;
Lane 3 is a hydrolyzate obtained by reacting cellotriose with recombinant REng13 or REng43;
Lane 4 is a hydrolyzate obtained by reacting cellotetraose with recombinant REng13 or REng43;
Lane 5 shows a hydrolyzate obtained by reacting cellopentaose with recombinant REng13 or REng43.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples do not limit the scope of the present invention, and conventional changes by those skilled in the art are possible within the scope of the technical idea of the present invention.

<Example 1>

Construction of metagenomic library of bovine rumen samples

1-1. Isolation of metagenomic DNA from bovine rumen samples

The rumen sample was secured using an automatic sample extraction device cannula. The rumen sample was naturally precipitated at 4°C to remove large-sized impurities first, and then centrifuged at a low speed of less than 1,000 rpm to further remove impurities other than the cells to collect microorganisms. After extracting DNA using a metagenomic DNA isolation kit (Metagenomic DNA isolation kit for water, Epicentre, USA), the extracted DNA ends with a DNA end-repair enzyme mixture (Epicentre, USA) are blunt end. It was repaired with.

1-2. Rumen metagenome library construction

The DNA repaired with the blunt end of 1-1 was electrophoresed with 0.4% agarose gel, and only about 40 kb of DNA was purified with a gel extraction kit (QIAEX II gel extraction kit, Qiagen, Germany), followed by force. After linking to the fosmid pCC1FOS vector using a mid library production kit (Copy Control Fosmid Library Production Kit, Epicentre, USA), packing it in a λDNA packaging kit, and transduced into E.coli EPI300-T1, and the rumen metagenome library Was secured.

<Example 2>

Screening and isolation of recombinant plasmids with cellulase activity

In order to find a recombinant plasmid having cellulase activity in the metagenome library, the transformants were inoculated on an LB plate medium containing 0.5% of CM-cellulose and cultured at 37°C for 24 hours. Thereafter, the plate medium was stained with 0.5% (w/v) Congo red solution for 15 minutes and then bleached with 1M NaCl for 15 minutes. Cellulase enzyme activity can be seen as a transparent ring formed around the colony, where it was confirmed that one transformant had cellulase activity (FIG. 1). The transformant exhibiting the above cellulase activity was named R1-4-G9.

<Example 3>

Cellulase gene sequence determination from recombinant plasmid with cellulase activity

3-1. Construction of shotgun library of recombinant plasmid with cellulase activity

Recombinant plasmid pC1R14G9 was isolated from the transformant R1-4-G9 having cellulase activity of Example 2 using a plasmid midi kit (QIAGEN, Germany). The isolated recombinant plasmid was sheared using a hydroshear device, and then the DNA end was repaired with a blunt end. The repaired DNA fragments were electrophoresed to cut out only the gel corresponding to the 2-5 kb position, purified with a gel extraction kit, and then subcloned into pC31A21 vector to create a shotgun library.

3-2. Sequence analysis of recombinant plasmid with cellulase activity

The nucleotide sequence analysis of the shotgun library prepared in 3-1 was performed using an automatic base sequence analyzer (ABI 3730 DNA analyzer). As a result of combining the sequence analysis results of the shotgun library, it was found that a DNA fragment consisting of 32,747 bp was introduced into the recombinant plasmid pC1R14G9. As a result of analyzing the nucleotide sequence of the DNA fragment with an ORF finder of the National Center for Biotechnology Information (NCBI), an open reading frame of 2,760 bp, which is estimated to be one of the cellulases, endo-beta-1,4-glucanase, ORF). This transcription and decoding frame was named ORF-E.

<Example 4>

Analysis of new cellulase sequence

The transcriptional decoding frame ORF-E estimated to be endo-beta-1,4-glucanase found in Example 3 consisted of 920 amino acids, had a transmembrane domain at the N-terminal region, and had two glycosylated proteins. Seed hydrolase (glycoside hydrolase) had an active site (catalytic domain) (Fig. 2). Glycoside hydrolase is an enzyme that hydrolyzes the glycosidic bond between two or more carbohydrates or between carbohydrates and non-hydrates. It is divided into groups of dogs. The glycoside hydrolase family 26 present in ORF-E has beta-mannanase and beta-1,3-xylanase activities. It is known to show a wide range of activities such as chitosanase, beta-mannosidase, and cellulase in the case of glycoside hydrolase family 5. It is known to be. In addition, as a result of amino acid homology analysis using the nucleotide sequence of ORF-E, it was confirmed that it was 54% and 62% identical to the previously known Xylanase of Prevotella bryantii or cellulase isolated from the stomach of ruminants, respectively (Fig. 3).

<Example 5>

Functional expression of new cellulase in E. coli

5-1. Preparation of transformants

In order to express the novel cellulase of the present invention, a DNA fragment encoding four types of transcription and decoding frames according to the location of the initiation codon (ATG) in the nucleotide sequence of ORF-E of Example 3 and the catalytic domain it contains Were amplified by PCR to include Bam HI and Xho I restriction sites. DNA fragments amplified by the above PCR were reng 13 (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2), reng 43 (base sequence: SEQ ID NO: 3, amino acid sequence: SEQ ID NO: 4), reng 63 (base sequence : SEQ ID NO: 5, amino acid sequence: SEQ ID NO: 6), named reng 16 (base sequence: SEQ ID NO: 7, amino acid sequence: SEQ ID NO: 8). These DNA fragments were digested with the above restriction enzymes and then introduced into pET21a (Novagen, USA) vector digested with the same enzyme, and these were designated as pEREng13, pEREng43, pEREng63, and pEREng16, respectively (FIG. 4). The cleavage map for the recombinant vector pEREng13 is shown in FIG. 5, the cleavage map for the recombinant vector pEREng43 is shown in FIG. 6, and the cleavage map for the recombinant vector pEREng16 is shown in FIG. 7. The above vectors were introduced into E. coli HIT-21 (RBC, USA), an E. coli strain used as an expression host, and transformed. These were named E.coli HIT-21/pEREng13, E.coli HIT-21/pEREng43, E.coli HIT-21/pEREng63, E.coli HIT-21/pEREng16, respectively.

5-2. Expression of cellulase enzyme

The transformants were suspended in liquid LB medium to have an OD value of 0.4 to 0.6, and then 0.5 mM IPTG was added to the medium to induce protein expression at 30° C. and 200 rpm for 4 hours. The above cultures were centrifuged at 3,000 rpm for 15 minutes to collect only the cells, and then a Bugs Buster Master Mix solution (Novagen, USA) was added to destroy the cells, followed by centrifugation to remove insoluble residues. The cell extracts obtained here were used to confirm the enzyme activity. On the other hand, cell extracts were analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), transformants E.coli HIT-21/pEREng13, E.coli HIT-21/pEREng43, E.coli HIT-21/pEREng63 , E. coli HIT-21/pEREng16, it was confirmed that recombinant proteins of about 103, 63, 54, and 40 kDa sizes were expressed, respectively (FIG. 8 ). Recombinant proteins expressed from the genes reng 13, reng 43, and reng 16 were designated as REng13, REng43, and REng16, respectively.

5-3. Confirmation of hydrolytic activity of recombinant glycosyl hydrolases

Using the cell extracts of 5-2, the hydrolytic activity of polysaccharides was confirmed. The enzyme reaction was carried out at 37° C. for 1 hour by adding 100 mM Sodium Acetate buffer solution pH 5.5, 1% (w/v) polysaccharide and 10% (v/v) cell extract. The activity of the enzyme was measured by quantifying the reducing sugar produced by the decomposition of polysaccharides, and the known DNS method (Miller GL. Anal. Chem., (1959) 31: 426-428) was used to quantify the reducing sugar. As a result of the analysis, the recombinant protein REng13 hydrolyzed all of CM-cellulose, xylan, beta-glucan, and beta-mannan, and the recombinant protein REng43 was CM-cellulose, xylan, beta- Glucan could be hydrolyzed. Recombinant protein REng16 had an activity to degrade only beta-mannan, and the recombinant protein REng63 did not show any activity (FIG. 9).

Through the above results, it was found that the recombinant protein REng13 according to the present invention is a multifunctional glycosyl hydrolase capable of decomposing not only cellulose but also hemicellulose such as xylan and beta-mannan.

On the other hand, through the difference in substrate specificity between the recombinant proteins REng43 and REng16, among the two active sites, the glycoside hydrolase family 26 series exhibits beta-mannanase activity, and the glycoside hydrolase family 5 It was found that the family showed cellulase and xylanase activities.

The transformants E.coli HIT-21/pEREng43 were deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on September 16, 2009, and the transformants E.coli HIT-21/pEREng13 and E.coli HIT-21 were deposited with the Korea Research Institute of Bioscience and Biotechnology. /pEREng16 was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on March 15, 2010. The accession number of the transformant E.coli HIT-21/pEREng43 is KCTC 11556BP, the accession number of the transformant E.coli HIT-21/pEREng13 is KCTC 11655BP, and the transformant E.coli HIT-21 The accession number of /pEREng16 is KCTC 11656BP.

<Example 6>

Characterization of recombinant proteins

6-1. Expression and purification of recombinant proteins REng13 and REng43

The recombinant proteins REng13 and REng43 according to the present invention were expressed, respectively, and then purified to examine the characteristics. After expressing proteins from transformants E.coli HIT-21/pEREng13 and E.coli HIT-21/pEREng43 in the same manner as in 5-2, HIS*Bind column (Novagen, USA) and HIS*Bind buffer kit (Novagen, USA) was used to purify the recombinant proteins (FIG. 10).

6-2. Characterization of temperature and pH

In order to investigate the effect of temperature on the activity of the recombinant proteins REng13 and REng43 according to the present invention, CM-cellulose hydrolysis reaction was performed between 30 and 80°C. As a result, both the recombinant proteins REng13 and REng43 showed optimal activity at 43°C. (Fig. 11).

In order to investigate the effect of pH on the activity of recombinant proteins, as a result of conducting CM-cellulose hydrolysis in buffer solutions of various pHs, it was confirmed that both the recombinant proteins REng13 and REng43 of the present invention exhibited optimal activity at pH 3.5. There was (Fig. 12).

6-3. Confirmation of cellooligosaccharide decomposition patterns of recombinant proteins

Cello-oligosaccharide degradation patterns of the recombinant proteins according to the present invention were confirmed. 5 μL of enzyme solution is mixed with 20 μL of 100 mM sodium acetate buffer (pH 5.3) each containing 5 mg/mL of cellopentaose, cellotetraose, cellotriose, and cellobiose. After reacting at 43° C. for 1 hour, the hydrolyzed product was confirmed by TLC. As the mobile phase, 1-butanol:acetic acid:water (v/v/v, 2:1:1) was used. After development, a methanol solution containing 5% sulfuric acid was sprayed and heated to 100°C to confirm the reaction. As a result of TLC development, there was no difference between the recombinant proteins REng13 and REng43, and cellopentaose was almost completely degraded, whereas cellotetraose was partially degraded (FIG. 13). Since cellotriose or cellobiose hardly degrades, the recombinant proteins according to the present invention seem to be able to use four or more cellulooligosaccharides bound to them as a substrate.

Korea Research Institute of Bioscience and Biotechnology KCTC11556BP 20090916 Korea Research Institute of Bioscience and Biotechnology KCTC11655BP 20100225 Korea Research Institute of Bioscience and Biotechnology KCTC11656BP 20100225

<110> KOREA YAKULT CO., LTD <120> Novel gene encoding multifunctional glycosyl hydrolase from cow rumen metagenome <130> P10-10 <150> KR 10-2009-0107596 <151> 2009-11-09 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 2760 <212> DNA <213> Unknown <220> <223> DNA isolated from cow remen directly <400> 1 atgaagaaca tgaaatcatt actcacaatg gttgtgttgc tgctgacggc aagtgccatg 60 tatgcagaca tccccacaat gcctcaaacg gcatcgaccg atgctgctaa gaagctgtat 120 ggctatttcg ttgagcagta tggtcagaag accatctcca gcgtgatggc tgatgtcaac 180 tggaacaata cgattgctga gaaggtgaag accttcacag gcaagtatcc cgccatgaac 240 tgctatgact ttatccatat ctacgtgccc aatcagggca gcaatgggtg ggttaactac 300 tcggatatca cgcctgttag cgagtggcat aatgctggcg gtattgtaca gttgatgtgg 360 cacttcaatg ttcctctaat ggagactacc gagatcaagg atgatggttc gggtgttacc 420 tgttcacccg acaagaccac gttcaaagca cagaacgctt tggtcagtgg cacgtgggag 480 aacaaatggt tctacgaaca gatggacaag gtcgttgccg tagtcctgaa actgcaggag 540 gctggcatcg ctgccacgtg gcgtccgttt cacgaggctg ccggcaatgc tacgctgaag 600 tcgggggcct catggggcaa gtcttggttc tggtggggct acgatggtgc cgataccttt 660 aaaaaactgt gggtggctat gttcgactac ttcaagcaga agggtgtcca gaacctcatc 720 tggatatgga ccacgcagaa ctacaatggt gacagcatgt cgtacaatca ggacacagac 780 tggtatcctg gtgatgccta cgttgatatg gtggcccgcg atctctatgg ctacgatgct 840 gacaagaata agctggagtt cacagaaatc caggctgctt atccaaacaa gatggttgta 900 ttgggcgagt gtggcaaggg cgacagcggc gagcaaggta cgatgagcga ctgttggact 960 aagggtgcca agtggggcca ctttttggtg tggtatcagg gcgaacaggg ttctaccaac 1020 accatgtgca gcgatgcttg gtggaaggat gctatgagta gtgccaacgt cgtgacccgt 1080 gaccagttgc ctaacctcgt tccaggtaag gtggagtttg agactgctac tgatgctgtc 1140 aagaatatgg gactgggttg gaatcttggc aacacgcttg aggccaacag ccagacggtg 1200 accgatatga cgcaggacgg ctattggggt cagcaggact tgacatccga gacctgctgg 1260 ggacagttct ataccaagcc cgaactgctg cagatgatga agaatgcagg cttcggcgcc 1320 atccgtgtac ccgtcacgtg gtataaccac atggataaag acggtaacgt cgatgctgca 1380 tggatgcgtc gtgtccatga ggttgtcgac tatgtcctca acactggcat gtactgtatc 1440 atcaacgtcc atcacgacac aggtgccgac agcgaaagct tcaaatcgtg gattaaggcc 1500 gatgctacca gctacagcaa caataaggtc cgctacgaga acctgtggcg ccagatagcc 1560 caggaattca aggactatga ccagcgcctg ctctttgaga gctataacga gatgctcgac 1620 gcacagagcg catggaacta tccctcgtcg aaggccactg gcggctacaa cgctgccgaa 1680 gccctgaagg cctacaacgc catcaacagc tatgcccaaa gttttgtcga cgtagtacgt 1740 gcctcaggtg gctataacga ccagcgcaac atcattgtca acacctatgc agccagcaac 1800 ggctatggca cgtggtctac acacctgagc gaggtgctga cgaagatgaa gaagcctaca 1860 ggcgagacca accacctcat cttcgaggtt catgcttatc ctaatatcca gaatctggcc 1920 tctgctaaga ctgaggtcga caacatgatt agtggtctga acaccaatct cgttacacgt 1980 ctgggcgctc ccgtcatatt tggtgagtgg ggaaccagta acgttgatgc aggagcaggt 2040 aaaaatgact atgacgtccg tcgtgacgat atgctagagt tcgttgacta ttttgtcaag 2100 aagaccaagg agaacggtat tggtaccttc tactggatgg gcctgaccga tggcttcgca 2160 cgccagttgc ctgcctttac tcagcccgac ttggcgctga agatgctgca ggcctggtat 2220 ggcaccagct ataatcctac cttgcccgtc aagtccgact atggtgatgc caccctgtcg 2280 tgtaccgtca actataccca gcagtatggt gagttcaatc tgttctcagg tgccatcagt 2340 gcctctgact acaagtacct gcgcctggag ttgggtgctg tccccaccag tggtaccctg 2400 caaataaaga tttacgctga cgtggataag cagcagtcga taaatgccaa gaacaataac 2460 gtgcagttta cggggacagg caatatcaac cgcatcaccc tgcagtggct tggtagcact 2520 ttgggaagtg tcaagatcaa caaagcctat ctcgtcaagc aggacggctc gaaggaaatc 2580 atagaaccca gcgtgttttg gggctgcaac tattctgacc tcaatattgt tactggtatt 2640 caggatgtgg tcttcaaacc tgtccaaagt gatgccatct acaatctcca tgggcaacgt 2700 gtgctgtcgc gcgccaaggg tatctatatt cgagatggta aaaaaatcat gatgaagtaa 2760 2760 <210> 2 <211> 919 <212> PRT <213> Unknown <220> <223> Obtained from cow rumen <400> 2 Met Lys Asn Met Lys Ser Leu Leu Thr Met Val Val Leu Leu Leu Thr 1 5 10 15 Ala Ser Ala Met Tyr Ala Asp Ile Pro Thr Met Pro Gln Thr Ala Ser 20 25 30 Thr Asp Ala Ala Lys Lys Leu Tyr Gly Tyr Phe Val Glu Gln Tyr Gly 35 40 45 Gln Lys Thr Ile Ser Ser Val Met Ala Asp Val Asn Trp Asn Asn Thr 50 55 60 Ile Ala Glu Lys Val Lys Thr Phe Thr Gly Lys Tyr Pro Ala Met Asn 65 70 75 80 Cys Tyr Asp Phe Ile His Ile Tyr Val Pro Asn Gln Gly Ser Asn Gly 85 90 95 Trp Val Asn Tyr Ser Asp Ile Thr Pro Val Ser Glu Trp His Asn Ala 100 105 110 Gly Gly Ile Val Gln Leu Met Trp His Phe Asn Val Pro Leu Met Glu 115 120 125 Thr Thr Glu Ile Lys Asp Asp Gly Ser Gly Val Thr Cys Ser Pro Asp 130 135 140 Lys Thr Thr Phe Lys Ala Gln Asn Ala Leu Val Ser Gly Thr Trp Glu 145 150 155 160 Asn Lys Trp Phe Tyr Glu Gln Met Asp Lys Val Val Ala Val Val Leu 165 170 175 Lys Leu Gln Glu Ala Gly Ile Ala Ala Thr Trp Arg Pro Phe His Glu 180 185 190 Ala Ala Gly Asn Ala Thr Leu Lys Ser Gly Ala Ser Trp Gly Lys Ser 195 200 205 Trp Phe Trp Trp Gly Tyr Asp Gly Ala Asp Thr Phe Lys Lys Leu Trp 210 215 220 Val Ala Met Phe Asp Tyr Phe Lys Gln Lys Gly Val Gln Asn Leu Ile 225 230 235 240 Trp Ile Trp Thr Thr Gln Asn Tyr Asn Gly Asp Ser Met Ser Tyr Asn 245 250 255 Gln Asp Thr Asp Trp Tyr Pro Gly Asp Ala Tyr Val Asp Met Val Ala 260 265 270 Arg Asp Leu Tyr Gly Tyr Asp Ala Asp Lys Asn Lys Leu Glu Phe Thr 275 280 285 Glu Ile Gln Ala Ala Tyr Pro Asn Lys Met Val Val Leu Gly Glu Cys 290 295 300 Gly Lys Gly Asp Ser Gly Glu Gln Gly Thr Met Ser Asp Cys Trp Thr 305 310 315 320 Lys Gly Ala Lys Trp Gly His Phe Leu Val Trp Tyr Gln Gly Glu Gln 325 330 335 Gly Ser Thr Asn Thr Met Cys Ser Asp Ala Trp Trp Lys Asp Ala Met 340 345 350 Ser Ser Ala Asn Val Val Thr Arg Asp Gln Leu Pro Asn Leu Val Pro 355 360 365 Gly Lys Val Glu Phe Glu Thr Ala Thr Asp Ala Val Lys Asn Met Gly 370 375 380 Leu Gly Trp Asn Leu Gly Asn Thr Leu Glu Ala Asn Ser Gln Thr Val 385 390 395 400 Thr Asp Met Thr Gln Asp Gly Tyr Trp Gly Gln Gln Asp Leu Thr Ser 405 410 415 Glu Thr Cys Trp Gly Gln Phe Tyr Thr Lys Pro Glu Leu Leu Gln Met 420 425 430 Met Lys Asn Ala Gly Phe Gly Ala Ile Arg Val Pro Val Thr Trp Tyr 435 440 445 Asn His Met Asp Lys Asp Gly Asn Val Asp Ala Ala Trp Met Arg Arg 450 455 460 Val His Glu Val Val Asp Tyr Val Leu Asn Thr Gly Met Tyr Cys Ile 465 470 475 480 Ile Asn Val His His Asp Thr Gly Ala Asp Ser Glu Ser Phe Lys Ser 485 490 495 Trp Ile Lys Ala Asp Ala Thr Ser Tyr Ser Asn Asn Lys Val Arg Tyr 500 505 510 Glu Asn Leu Trp Arg Gln Ile Ala Gln Glu Phe Lys Asp Tyr Asp Gln 515 520 525 Arg Leu Leu Phe Glu Ser Tyr Asn Glu Met Leu Asp Ala Gln Ser Ala 530 535 540 Trp Asn Tyr Pro Ser Ser Lys Ala Thr Gly Gly Tyr Asn Ala Ala Glu 545 550 555 560 Ala Leu Lys Ala Tyr Asn Ala Ile Asn Ser Tyr Ala Gln Ser Phe Val 565 570 575 Asp Val Val Arg Ala Ser Gly Gly Tyr Asn Asp Gln Arg Asn Ile Ile 580 585 590 Val Asn Thr Tyr Ala Ala Ser Asn Gly Tyr Gly Thr Trp Ser Thr His 595 600 605 Leu Ser Glu Val Leu Thr Lys Met Lys Lys Pro Thr Gly Glu Thr Asn 610 615 620 His Leu Ile Phe Glu Val His Ala Tyr Pro Asn Ile Gln Asn Leu Ala 625 630 635 640 Ser Ala Lys Thr Glu Val Asp Asn Met Ile Ser Gly Leu Asn Thr Asn 645 650 655 Leu Val Thr Arg Leu Gly Ala Pro Val Ile Phe Gly Glu Trp Gly Thr 660 665 670 Ser Asn Val Asp Ala Gly Ala Gly Lys Asn Asp Tyr Asp Val Arg Arg 675 680 685 Asp Asp Met Leu Glu Phe Val Asp Tyr Phe Val Lys Lys Thr Lys Glu 690 695 700 Asn Gly Ile Gly Thr Phe Tyr Trp Met Gly Leu Thr Asp Gly Phe Ala 705 710 715 720 Arg Gln Leu Pro Ala Phe Thr Gln Pro Asp Leu Ala Leu Lys Met Leu 725 730 735 Gln Ala Trp Tyr Gly Thr Ser Tyr Asn Pro Thr Leu Pro Val Lys Ser 740 745 750 Asp Tyr Gly Asp Ala Thr Leu Ser Cys Thr Val Asn Tyr Thr Gln Gln 755 760 765 Tyr Gly Glu Phe Asn Leu Phe Ser Gly Ala Ile Ser Ala Ser Asp Tyr 770 775 780 Lys Tyr Leu Arg Leu Glu Leu Gly Ala Val Pro Thr Ser Gly Thr Leu 785 790 795 800 Gln Ile Lys Ile Tyr Ala Asp Val Asp Lys Gln Gln Ser Ile Asn Ala 805 810 815 Lys Asn Asn Asn Val Gln Phe Thr Gly Thr Gly Asn Ile Asn Arg Ile 820 825 830 Thr Leu Gln Trp Leu Gly Ser Thr Leu Gly Ser Val Lys Ile Asn Lys 835 840 845 Ala Tyr Leu Val Lys Gln Asp Gly Ser Lys Glu Ile Ile Glu Pro Ser 850 855 860 Val Phe Trp Gly Cys Asn Tyr Ser Asp Leu Asn Ile Val Thr Gly Ile 865 870 875 880 Gln Asp Val Val Phe Lys Pro Val Gln Ser Asp Ala Ile Tyr Asn Leu 885 890 895 His Gly Gln Arg Val Leu Ser Arg Ala Lys Gly Ile Tyr Ile Arg Asp 900 905 910 Gly Lys Lys Ile Met Met Lys 915 <210> 3 <211> 1707 <212> DNA <213> Unknown <220> <223> DNA isolated from cow rumen directly <400> 3 atgagtagtg ccaacgtcgt gacccgtgac cagttgccta acctcgttcc aggtaaggtg 60 gagtttgaga ctgctactga tgctgtcaag aatatgggac tgggttggaa tcttggcaac 120 acgcttgagg ccaacagcca gacggtgacc gatatgacgc aggacggcta ttggggtcag 180 caggacttga catccgagac ctgctgggga cagttctata ccaagcccga actgctgcag 240 atgatgaaga atgcaggctt cggcgccatc cgtgtacccg tcacgtggta taaccacatg 300 gataaagacg gtaacgtcga tgctgcatgg atgcgtcgtg tccatgaggt tgtcgactat 360 gtcctcaaca ctggcatgta ctgtatcatc aacgtccatc acgacacagg tgccgacagc 420 gaaagcttca aatcgtggat taaggccgat gctaccagct acagcaacaa taaggtccgc 480 tacgagaacc tgtggcgcca gatagcccag gaattcaagg actatgacca gcgcctgctc 540 tttgagagct ataacgagat gctcgacgca cagagcgcat ggaactatcc ctcgtcgaag 600 gccactggcg gctacaacgc tgccgaagcc ctgaaggcct acaacgccat caacagctat 660 gcccaaagtt ttgtcgacgt agtacgtgcc tcaggtggct ataacgacca gcgcaacatc 720 attgtcaaca cctatgcagc cagcaacggc tatggcacgt ggtctacaca cctgagcgag 780 gtgctgacga agatgaagaa gcctacaggc gagaccaacc acctcatctt cgaggttcat 840 gcttatccta atatccagaa tctggcctct gctaagactg aggtcgacaa catgattagt 900 ggtctgaaca ccaatctcgt tacacgtctg ggcgctcccg tcatatttgg tgagtgggga 960 accagtaacg ttgatgcagg agcaggtaaa aatgactatg acgtccgtcg tgacgatatg 1020 ctagagttcg ttgactattt tgtcaagaag accaaggaga acggtattgg taccttctac 1080 tggatgggcc tgaccgatgg cttcgcacgc cagttgcctg cctttactca gcccgacttg 1140 gcgctgaaga tgctgcaggc ctggtatggc accagctata atcctacctt gcccgtcaag 1200 tccgactatg gtgatgccac cctgtcgtgt accgtcaact atacccagca gtatggtgag 1260 ttcaatctgt tctcaggtgc catcagtgcc tctgactaca agtacctgcg cctggagttg 1320 ggtgctgtcc ccaccagtgg taccctgcaa ataaagattt acgctgacgt ggataagcag 1380 cagtcgataa atgccaagaa caataacgtg cagtttacgg ggacaggcaa tatcaaccgc 1440 atcaccctgc agtggcttgg tagcactttg ggaagtgtca agatcaacaa agcctatctc 1500 gtcaagcagg acggctcgaa ggaaatcata gaacccagcg tgttttgggg ctgcaactat 1560 tctgacctca atattgttac tggtattcag gatgtggtct tcaaacctgt ccaaagtgat 1620 gccatctaca atctccatgg gcaacgtgtg ctgtcgcgcg ccaagggtat ctatattcga 1680 gatggtaaaa aaatcatgat gaagtaa 1707 <210> 4 <211> 568 <212> PRT <213> Unknown <220> <223> Obtained from cow rumen <400> 4 Met Ser Ser Ala Asn Val Val Thr Arg Asp Gln Leu Pro Asn Leu Val 1 5 10 15 Pro Gly Lys Val Glu Phe Glu Thr Ala Thr Asp Ala Val Lys Asn Met 20 25 30 Gly Leu Gly Trp Asn Leu Gly Asn Thr Leu Glu Ala Asn Ser Gln Thr 35 40 45 Val Thr Asp Met Thr Gln Asp Gly Tyr Trp Gly Gln Gln Asp Leu Thr 50 55 60 Ser Glu Thr Cys Trp Gly Gln Phe Tyr Thr Lys Pro Glu Leu Leu Gln 65 70 75 80 Met Met Lys Asn Ala Gly Phe Gly Ala Ile Arg Val Pro Val Thr Trp 85 90 95 Tyr Asn His Met Asp Lys Asp Gly Asn Val Asp Ala Ala Trp Met Arg 100 105 110 Arg Val His Glu Val Val Asp Tyr Val Leu Asn Thr Gly Met Tyr Cys 115 120 125 Ile Ile Asn Val His His Asp Thr Gly Ala Asp Ser Glu Ser Phe Lys 130 135 140 Ser Trp Ile Lys Ala Asp Ala Thr Ser Tyr Ser Asn Asn Lys Val Arg 145 150 155 160 Tyr Glu Asn Leu Trp Arg Gln Ile Ala Gln Glu Phe Lys Asp Tyr Asp 165 170 175 Gln Arg Leu Leu Phe Glu Ser Tyr Asn Glu Met Leu Asp Ala Gln Ser 180 185 190 Ala Trp Asn Tyr Pro Ser Ser Lys Ala Thr Gly Gly Tyr Asn Ala Ala 195 200 205 Glu Ala Leu Lys Ala Tyr Asn Ala Ile Asn Ser Tyr Ala Gln Ser Phe 210 215 220 Val Asp Val Val Arg Ala Ser Gly Gly Tyr Asn Asp Gln Arg Asn Ile 225 230 235 240 Ile Val Asn Thr Tyr Ala Ala Ser Asn Gly Tyr Gly Thr Trp Ser Thr 245 250 255 His Leu Ser Glu Val Leu Thr Lys Met Lys Lys Pro Thr Gly Glu Thr 260 265 270 Asn His Leu Ile Phe Glu Val His Ala Tyr Pro Asn Ile Gln Asn Leu 275 280 285 Ala Ser Ala Lys Thr Glu Val Asp Asn Met Ile Ser Gly Leu Asn Thr 290 295 300 Asn Leu Val Thr Arg Leu Gly Ala Pro Val Ile Phe Gly Glu Trp Gly 305 310 315 320 Thr Ser Asn Val Asp Ala Gly Ala Gly Lys Asn Asp Tyr Asp Val Arg 325 330 335 Arg Asp Asp Met Leu Glu Phe Val Asp Tyr Phe Val Lys Lys Thr Lys 340 345 350 Glu Asn Gly Ile Gly Thr Phe Tyr Trp Met Gly Leu Thr Asp Gly Phe 355 360 365 Ala Arg Gln Leu Pro Ala Phe Thr Gln Pro Asp Leu Ala Leu Lys Met 370 375 380 Leu Gln Ala Trp Tyr Gly Thr Ser Tyr Asn Pro Thr Leu Pro Val Lys 385 390 395 400 Ser Asp Tyr Gly Asp Ala Thr Leu Ser Cys Thr Val Asn Tyr Thr Gln 405 410 415 Gln Tyr Gly Glu Phe Asn Leu Phe Ser Gly Ala Ile Ser Ala Ser Asp 420 425 430 Tyr Lys Tyr Leu Arg Leu Glu Leu Gly Ala Val Pro Thr Ser Gly Thr 435 440 445 Leu Gln Ile Lys Ile Tyr Ala Asp Val Asp Lys Gln Gln Ser Ile Asn 450 455 460 Ala Lys Asn Asn Asn Val Gln Phe Thr Gly Thr Gly Asn Ile Asn Arg 465 470 475 480 Ile Thr Leu Gln Trp Leu Gly Ser Thr Leu Gly Ser Val Lys Ile Asn 485 490 495 Lys Ala Tyr Leu Val Lys Gln Asp Gly Ser Lys Glu Ile Ile Glu Pro 500 505 510 Ser Val Phe Trp Gly Cys Asn Tyr Ser Asp Leu Asn Ile Val Thr Gly 515 520 525 Ile Gln Asp Val Val Phe Lys Pro Val Gln Ser Asp Ala Ile Tyr Asn 530 535 540 Leu His Gly Gln Arg Val Leu Ser Arg Ala Lys Gly Ile Tyr Ile Arg 545 550 555 560 Asp Gly Lys Lys Ile Met Met Lys 565 <210> 5 <211> 1467 <212> DNA <213> Unknown <220> <223> DNA isolated from cow rumen directly <400> 5 atgatgaaga atgcaggctt cggcgccatc cgtgtacccg tcacgtggta taaccacatg 60 gataaagacg gtaacgtcga tgctgcatgg atgcgtcgtg tccatgaggt tgtcgactat 120 gtcctcaaca ctggcatgta ctgtatcatc aacgtccatc acgacacagg tgccgacagc 180 gaaagcttca aatcgtggat taaggccgat gctaccagct acagcaacaa taaggtccgc 240 tacgagaacc tgtggcgcca gatagcccag gaattcaagg actatgacca gcgcctgctc 300 tttgagagct ataacgagat gctcgacgca cagagcgcat ggaactatcc ctcgtcgaag 360 gccactggcg gctacaacgc tgccgaagcc ctgaaggcct acaacgccat caacagctat 420 gcccaaagtt ttgtcgacgt agtacgtgcc tcaggtggct ataacgacca gcgcaacatc 480 attgtcaaca cctatgcagc cagcaacggc tatggcacgt ggtctacaca cctgagcgag 540 gtgctgacga agatgaagaa gcctacaggc gagaccaacc acctcatctt cgaggttcat 600 gcttatccta atatccagaa tctggcctct gctaagactg aggtcgacaa catgattagt 660 ggtctgaaca ccaatctcgt tacacgtctg ggcgctcccg tcatatttgg tgagtgggga 720 accagtaacg ttgatgcagg agcaggtaaa aatgactatg acgtccgtcg tgacgatatg 780 ctagagttcg ttgactattt tgtcaagaag accaaggaga acggtattgg taccttctac 840 tggatgggcc tgaccgatgg cttcgcacgc cagttgcctg cctttactca gcccgacttg 900 gcgctgaaga tgctgcaggc ctggtatggc accagctata atcctacctt gcccgtcaag 960 tccgactatg gtgatgccac cctgtcgtgt accgtcaact atacccagca gtatggtgag 1020 ttcaatctgt tctcaggtgc catcagtgcc tctgactaca agtacctgcg cctggagttg 1080 ggtgctgtcc ccaccagtgg taccctgcaa ataaagattt acgctgacgt ggataagcag 1140 cagtcgataa atgccaagaa caataacgtg cagtttacgg ggacaggcaa tatcaaccgc 1200 atcaccctgc agtggcttgg tagcactttg ggaagtgtca agatcaacaa agcctatctc 1260 gtcaagcagg acggctcgaa ggaaatcata gaacccagcg tgttttgggg ctgcaactat 1320 tctgacctca atattgttac tggtattcag gatgtggtct tcaaacctgt ccaaagtgat 1380 gccatctaca atctccatgg gcaacgtgtg ctgtcgcgcg ccaagggtat ctatattcga 1440 gatggtaaaa aaatcatgat gaagtaa 1467 <210> 6 <211> 488 <212> PRT <213> Unknown <220> <223> Obtained from cow rumen <400> 6 Met Met Lys Asn Ala Gly Phe Gly Ala Ile Arg Val Pro Val Thr Trp 1 5 10 15 Tyr Asn His Met Asp Lys Asp Gly Asn Val Asp Ala Ala Trp Met Arg 20 25 30 Arg Val His Glu Val Val Asp Tyr Val Leu Asn Thr Gly Met Tyr Cys 35 40 45 Ile Ile Asn Val His His Asp Thr Gly Ala Asp Ser Glu Ser Phe Lys 50 55 60 Ser Trp Ile Lys Ala Asp Ala Thr Ser Tyr Ser Asn Asn Lys Val Arg 65 70 75 80 Tyr Glu Asn Leu Trp Arg Gln Ile Ala Gln Glu Phe Lys Asp Tyr Asp 85 90 95 Gln Arg Leu Leu Phe Glu Ser Tyr Asn Glu Met Leu Asp Ala Gln Ser 100 105 110 Ala Trp Asn Tyr Pro Ser Ser Lys Ala Thr Gly Gly Tyr Asn Ala Ala 115 120 125 Glu Ala Leu Lys Ala Tyr Asn Ala Ile Asn Ser Tyr Ala Gln Ser Phe 130 135 140 Val Asp Val Val Arg Ala Ser Gly Gly Tyr Asn Asp Gln Arg Asn Ile 145 150 155 160 Ile Val Asn Thr Tyr Ala Ala Ser Asn Gly Tyr Gly Thr Trp Ser Thr 165 170 175 His Leu Ser Glu Val Leu Thr Lys Met Lys Lys Pro Thr Gly Glu Thr 180 185 190 Asn His Leu Ile Phe Glu Val His Ala Tyr Pro Asn Ile Gln Asn Leu 195 200 205 Ala Ser Ala Lys Thr Glu Val Asp Asn Met Ile Ser Gly Leu Asn Thr 210 215 220 Asn Leu Val Thr Arg Leu Gly Ala Pro Val Ile Phe Gly Glu Trp Gly 225 230 235 240 Thr Ser Asn Val Asp Ala Gly Ala Gly Lys Asn Asp Tyr Asp Val Arg 245 250 255 Arg Asp Asp Met Leu Glu Phe Val Asp Tyr Phe Val Lys Lys Thr Lys 260 265 270 Glu Asn Gly Ile Gly Thr Phe Tyr Trp Met Gly Leu Thr Asp Gly Phe 275 280 285 Ala Arg Gln Leu Pro Ala Phe Thr Gln Pro Asp Leu Ala Leu Lys Met 290 295 300 Leu Gln Ala Trp Tyr Gly Thr Ser Tyr Asn Pro Thr Leu Pro Val Lys 305 310 315 320 Ser Asp Tyr Gly Asp Ala Thr Leu Ser Cys Thr Val Asn Tyr Thr Gln 325 330 335 Gln Tyr Gly Glu Phe Asn Leu Phe Ser Gly Ala Ile Ser Ala Ser Asp 340 345 350 Tyr Lys Tyr Leu Arg Leu Glu Leu Gly Ala Val Pro Thr Ser Gly Thr 355 360 365 Leu Gln Ile Lys Ile Tyr Ala Asp Val Asp Lys Gln Gln Ser Ile Asn 370 375 380 Ala Lys Asn Asn Asn Val Gln Phe Thr Gly Thr Gly Asn Ile Asn Arg 385 390 395 400 Ile Thr Leu Gln Trp Leu Gly Ser Thr Leu Gly Ser Val Lys Ile Asn 405 410 415 Lys Ala Tyr Leu Val Lys Gln Asp Gly Ser Lys Glu Ile Ile Glu Pro 420 425 430 Ser Val Phe Trp Gly Cys Asn Tyr Ser Asp Leu Asn Ile Val Thr Gly 435 440 445 Ile Gln Asp Val Val Phe Lys Pro Val Gln Ser Asp Ala Ile Tyr Asn 450 455 460 Leu His Gly Gln Arg Val Leu Ser Arg Ala Lys Gly Ile Tyr Ile Arg 465 470 475 480 Asp Gly Lys Lys Ile Met Met Lys 485 <210> 7 <211> 1134 <212> DNA <213> Unknown <220> <223> DNA isolated from cow rumen directly <400> 7 atgaagaaca tgaaatcatt actcacaatg gttgtgttgc tgctgacggc aagtgccatg 60 tatgcagaca tccccacaat gcctcaaacg gcatcgaccg atgctgctaa gaagctgtat 120 ggctatttcg ttgagcagta tggtcagaag accatctcca gcgtgatggc tgatgtcaac 180 tggaacaata cgattgctga gaaggtgaag accttcacag gcaagtatcc cgccatgaac 240 tgctatgact ttatccatat ctacgtgccc aatcagggca gcaatgggtg ggttaactac 300 tcggatatca cgcctgttag cgagtggcat aatgctggcg gtattgtaca gttgatgtgg 360 cacttcaatg ttcctctaat ggagactacc gagatcaagg atgatggttc gggtgttacc 420 tgttcacccg acaagaccac gttcaaagca cagaacgctt tggtcagtgg cacgtgggag 480 aacaaatggt tctacgaaca gatggacaag gtcgttgccg tagtcctgaa actgcaggag 540 gctggcatcg ctgccacgtg gcgtccgttt cacgaggctg ccggcaatgc tacgctgaag 600 tcgggggcct catggggcaa gtcttggttc tggtggggct acgatggtgc cgataccttt 660 aaaaaactgt gggtggctat gttcgactac ttcaagcaga agggtgtcca gaacctcatc 720 tggatatgga ccacgcagaa ctacaatggt gacagcatgt cgtacaatca ggacacagac 780 tggtatcctg gtgatgccta cgttgatatg gtggcccgcg atctctatgg ctacgatgct 840 gacaagaata agctggagtt cacagaaatc caggctgctt atccaaacaa gatggttgta 900 ttgggcgagt gtggcaaggg cgacagcggc gagcaaggta cgatgagcga ctgttggact 960 aagggtgcca agtggggcca ctttttggtg tggtatcagg gcgaacaggg ttctaccaac 1020 accatgtgca gcgatgcttg gtggaaggat gctatgagta gtgccaacgt cgtgacccgt 1080 gaccagttgc ctaacctcgt tccaggtaag gtggagtttg agactgctac tgat 1134 <210> 8 <211> 352 <212> PRT <213> Unknown <220> <223> Otained from cow rumen <400> 8 Met Pro Gln Thr Ala Ser Thr Asp Ala Ala Lys Lys Leu Tyr Gly Tyr 1 5 10 15 Phe Val Glu Gln Tyr Gly Gln Lys Thr Ile Ser Ser Val Met Ala Asp 20 25 30 Val Asn Trp Asn Asn Thr Ile Ala Glu Lys Val Lys Thr Phe Thr Gly 35 40 45 Lys Tyr Pro Ala Met Asn Cys Tyr Asp Phe Ile His Ile Tyr Val Pro 50 55 60 Asn Gln Gly Ser Asn Gly Trp Val Asn Tyr Ser Asp Ile Thr Pro Val 65 70 75 80 Ser Glu Trp His Asn Ala Gly Gly Ile Val Gln Leu Met Trp His Phe 85 90 95 Asn Val Pro Leu Met Glu Thr Thr Glu Ile Lys Asp Asp Gly Ser Gly 100 105 110 Val Thr Cys Ser Pro Asp Lys Thr Thr Phe Lys Ala Gln Asn Ala Leu 115 120 125 Val Ser Gly Thr Trp Glu Asn Lys Trp Phe Tyr Glu Gln Met Asp Lys 130 135 140 Val Val Ala Val Val Leu Lys Leu Gln Glu Ala Gly Ile Ala Ala Thr 145 150 155 160 Trp Arg Pro Phe His Glu Ala Ala Gly Asn Ala Thr Leu Lys Ser Gly 165 170 175 Ala Ser Trp Gly Lys Ser Trp Phe Trp Trp Gly Tyr Asp Gly Ala Asp 180 185 190 Thr Phe Lys Lys Leu Trp Val Ala Met Phe Asp Tyr Phe Lys Gln Lys 195 200 205 Gly Val Gln Asn Leu Ile Trp Ile Trp Thr Thr Gln Asn Tyr Asn Gly 210 215 220 Asp Ser Met Ser Tyr Asn Gln Asp Thr Asp Trp Tyr Pro Gly Asp Ala 225 230 235 240 Tyr Val Asp Met Val Ala Arg Asp Leu Tyr Gly Tyr Asp Ala Asp Lys 245 250 255 Asn Lys Leu Glu Phe Thr Glu Ile Gln Ala Ala Tyr Pro Asn Lys Met 260 265 270 Val Val Leu Gly Glu Cys Gly Lys Gly Asp Ser Gly Glu Gln Gly Thr 275 280 285 Met Ser Asp Cys Trp Thr Lys Gly Ala Lys Trp Gly His Phe Leu Val 290 295 300 Trp Tyr Gln Gly Glu Gln Gly Ser Thr Asn Thr Met Cys Ser Asp Ala 305 310 315 320 Trp Trp Lys Asp Ala Met Ser Ser Ala Asn Val Val Thr Arg Asp Gln 325 330 335 Leu Pro Asn Leu Val Pro Gly Lys Val Glu Phe Glu Thr Ala Thr Asp 340 345 350

Claims (4)

Mannanase gene comprising a nucleotide sequence of SEQ ID NO: 7.
Mannanase protein comprising the amino acid sequence of SEQ ID NO: 8.
Recombinant expression vector pEREng16 having a cleavage map as described in FIG. 7 comprising a mannase gene of SEQ ID NO.
E. coli HIT-21 / pEREng16 transformants transformed with the recombinant expression vector of claim 3 (Accession No .: KCTC 11656BP).

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