KR20120018986A - NOVEL GENE ENCODING ß-GLUCOSIDASE FROM COW RUMEN METAGENOME - Google Patents

Abstract

PURPOSE: A novel beta-glucosidase gene derived from cow rumen metagenome is provided to produce a large amount of recombinant beta-glucosidase. CONSTITUTION: A beta-glucosidase gene is denoted by sequence number 1. A beta-glucosidase protein is denoted by amino acid sequence number 2. The beta-glucosidase protein has a signal sequence of sequence number 3. A recombinant vector contains the beta-glucosidase gene of sequence number 1. A method for isolating beta-glucosidase comprises: a step of collecting a sample from a cow rumen; and a step of isolating a metagenome from the collected sample; a step of analyzing the gene.

Description

Novel gene encoding β-glucosidase from cow rumen metagenome

The present invention relates to a novel beta-glucosidase gene derived from metagenome isolated from rumen, the rumen of cow, and a recombinant vector comprising the gene, and more particularly to rumen of cow. A metagenome library was prepared to select a clone having beta-glucosidase activity, and to a novel beta-glucosidase gene identified from the clone and a recombinant vector comprising the gene. It is about.

Cellulose is the most abundant organic substance on earth and is a renewable resource that you don't have to worry about, like coal or oil. These resources also serve as major environmental pollutants in the current flooding of agricultural waste. Therefore, the development of efficient saccharification process for these polysaccharides will be a great help for food, energy and environmental problems. Therefore, research has been made for glycosylation for a long time, and a lot of research has been made, especially focusing on the development of glycosylase. As a result, many uses of glycosylase have been developed in various ways and commercialized in textile, detergent, feed industry, etc., and research on new application fields is being actively conducted.

Cell wall of the plant is composed of polymers such as cellulose (Cellulose), hemicellulose (Hemicellulose), lignin (Lignin), of which the most cellulose is present.

Cellulose is a homogeneous conjugate in which glucose units are linked by β-1,4 bonds, and in order to completely decompose cellulose, endo β-1,4-glucanase, exo β-1,4- Three enzymes are required: glucanase (exo β-1,4-glucanase, cellobiohydrolase) and beta-glucosidase (β-glucosidase, cellobiase). Endo β-1,4-glucanase randomly cleaves β-1,4 glucose bonds from the inside, and Cellobiose, in which exo β-1,4-glucanase is a glucose disaccharide at the non-reducing end Cut off. Cellobiose is finally degraded into glucose by beta-glucosidase. Beta-glucosidase is an enzyme that releases glucose by hydrolyzing the beta-1,4-glucosidic bond between two glucoses or other substituted molecules. It is a kind of exocellulase that has specificity for various beta D-glucoside family of substrates. In general, beta-glucosidase has a hydrolysis effect on cellobiose, cellotriose, cellotetraose, etc., in which about one to four glucoses are polymerized, and cellopentaose which has been polymerized further. It is known that there is no enzymatic activity for substances such as (Cellopentaose). In addition, Celobiase, which has a specific activity on the hydrolysis of two glucose cells, and Lysozyme, which is secreted into the tears to prevent bacterial infection in the eye, also belong to the range of beta-glucosidase. do.

In the past, the production of these enzymes has been mainly carried out by Aspergillus, Trichoderma. In particular, the cellulase production strain is Trichoderma reesei (Trichoderma reesei) is representative. However, the production and activity of enzymes are not enough to satisfy the industrialization, and expensive materials such as lactose (Lactose) as a carbon source has a high production cost problems. This contributes to the largest cost (about 60%) in the process of producing ethanol from biomass.

Therefore, the present inventors completed the present invention for economical enzyme production and provision through the selection of new high productivity enzymes and maximizing the production using E. coli.

An object of the present invention is to provide a novel beta-glucosidase gene derived from bovine rumen metagenome and a recombinant vector comprising the beta-glucosidase gene.

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

In addition, the present invention is characterized by providing a recombinant vector comprising a novel beta-glucosidase gene.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of collecting a sample from the bovine lumen, obtaining a metagenome (Metagenome) from the collected sample, genetic determination and analysis of the obtained enzyme, step of measuring the activity of the enzyme.

The present invention uses a novel beta-glucosidase gene of a rumen-derived bacterium that is difficult to cultivate by conventional techniques from a cow's rumen metagenomic library when mass-producing recombinant beta-glucosidase. It can be used for commercial purposes in various fields such as detergents, pulp and paper industry, livestock feed and food, and bioenergy.

1 is a schematic representation of the structure of the beta-glucosidase gene of the present invention.
2 is a homology comparison between the beta-glucosidase gene of the present invention and the beta-glucosidase gene of the known prevotella and bacteroids. Figure shown.
Figure 3 shows a schematic representation of the recombinant expression vector to find the optimal active site of the beta-glucosidase (β-glucosidase) of the present invention.
Figure 4 shows the expression of beta-glucosidase (β-glucosidase) by the recombinant expression vector of Figure 3
5 shows the expression of beta-glucosidase cloned into the pMAL-c2 vector.
Figure 6 shows the activity of beta-glucosidase (β-glucosidase) against cellobiose (Cellobiose (black bar graph) and para-nitrophenyl glucoside (pNPG, gray bar graph).
Figure 7 shows the amount of glucose produced by the enzymatic reaction of beta-glucosidase (gray bar graph) from cellobiose, arbutin, salicycin, para-nitrophenyl glucoside (pNPG). It is shown. For comparison of glucose production, an empty vector (pET21a, black bar graph), Cytophaga jhonsonii chu_2268 (dark gray bar graph), and chu_2273 (light gray bar graph) were used as controls.
8 is a schematic diagram of a recombinant vector prbgl containing rbgl, a beta-glucosidase coding gene.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are not intended to limit the scope of the present invention, and ordinary changes by those skilled in the art are possible within the scope of the technical idea of the present invention.

≪ Example 1 >

Construction of a Metagenome Library of Bovine Rumen Samples

1-1. Securing Rumen Samples and Extracting Metagenomes

After obtaining a rumen sample by using an automatic sample extractor cannula in the lumen, the first place of cow rumen, spontaneously settled at 4 ° C to remove large impurities first, and then centrifuged at a low speed of less than 1,000 rpm to remove extracellular bacteria. Were further removed. DNA extracted from the rumen liquid by using a metagenomic DNA isolation kit (Metagenomic DNA isolation kit for water, Epicentre, USA), and then blunt end the DNA end extracted with DNA end-repair enzyme mixture (Epicentre, USA). Repaired).

1-2. Building the rumen metagenome library

DNA repaired to the blunt end of 1-1 was electrophoresed with 0.4% agarose gel to purify only about 40 kb of DNA with a gel extraction kit (QIAEX II gel extraction kit, Qiagen, Germany) Linked to a phosmid pCC1FOS vector using a Copy Control Fosmid Library Production Kit (Epicentre, USA), packaged in a λDNA packaging kit and transduced into E. coli EPI300-T1 to the rumen metagenomic library Secured.

<Example 2>

Sequence analysis of metagenome

The prepared metagenome of Example 1 was fragmented using a hydroshear device, and the DNA end was repaired with a blunt end. The repaired DNA fragments were electrophoresed to remove only the gel corresponding to the 2-5kb position, purified by gel extraction kit, subcloned into the pC31A21 vector, and a shotgun library was prepared. ABI 3730 DNA analyzer).

As a result of combining the sequence analysis results of the shotgun library, it was found that the metagenome introduced a DNA fragment consisting of 32,747 bp. The DNA sequence was analyzed by ORF finder of the National Center for Biotechnology Information (NCBI), and a 2,469 bp open reading frame (ORF) estimated to be beta-glucosidase, one of cellulase. It was confirmed to have. This transcription decoding frame was named 'rbgl'.

<Example 3>

Analysis of β-glucosidase Gene Sequences

Transcription detoxification frame rbgl estimated to be beta-glucosidase found in Example 2 was composed of 822 amino acids, has a transmembrane domain at the N-terminal region, and has one glycoside hydride. Glycoside hydrolase had a catalytic domain (FIG. 1). Glycoside hydrolase is an enzyme that hydrolyzes glycosidic bonds between two or more carbohydrates or between carbohydrates and non-carbohydrates, based on the amino acid homology and structural properties of each enzyme. It is divided into two groups. Glycoside hydrolase family 3 in rbgl was generally found to be widely distributed in beta-glucosidase (EC 3.2.1.21). In addition, the amino acid homology analysis using the rbgl sequence showed that the beta-glucosidase of Prevotella bryantii and Prevotella oris were 61% and 62% respectively. (FIG. 2).

<Example 4>

Expression of Novel Beta-glucosidase in Escherichia Coli

4-1. Preparation of Transformant

In order to express the novel beta-glucosidase of the present invention, the sequence of the signal sequence (Signal sequence) in the base sequence of rbg1 (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2) of Example 2 To confirm the presence, rbg1-42 having 42 amino acids deleted at the N-terminus of rbg1 and rbg1-66 having 66 amino acids deleted at the N-terminus were amplified by a polymerase amplification reaction (PCR). Polymerization amplification of rbg1-267 containing 267 amino acids at the N-terminus, rbg1-391 having 391 amino acids at the N-terminus and N-terminal to determine the minimum ORF having Amplification via reaction (PCR).

The amplified DNA fragments were digested with restriction enzymes BamHI and XhoI, and then inserted into the pET21a (Novagen, USA) vector digested with the same enzymes, respectively, prbgl-42, prbgl-66, prbg1-267, prbg1-391, prbg1, respectively. Named -n (FIG. 3).

Further, the base sequence of rbg1 (base sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2) was amplified by a polymerase amplification reaction, cleaved with restriction enzymes BamHI and XhoI, and then pET21a (Novagen, USA). ) Was inserted into the vector and named prbg1 (see FIG. 8).

The vectors were introduced and transformed into E. coli HIT-21 (RBC, USA), which was used as an expression host. These were E. coli HIT-21 / prbg1-42, E. coli HIT-21 / prbg1-66, E. coli HIT-21 / prbg1-267, E. coli HIT-21 / prbg1-391, and E.coli HIT, respectively. Named -21 / prbg1-n.

4-2. Expression of β-glucosidase

The transformants were suspended in culture so that the OD value was 0.4-0.6 in a liquid LB medium and 0.5 mM IPTG was added to the medium to induce protein expression at 30 ° C. and 200 rpm for 4 hours. The cultures were centrifuged at 3,000 rpm for 15 minutes to collect only the cells and then added to the Bugs Buster Master Mix solution (Novagen, USA) to destroy the cells and centrifuged to remove insoluble residues. The cell extracts obtained here were used to confirm the enzyme activity. Cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transformants E. coli HIT-21 / prbg1-42, E. coli HIT-21 / prbg1-66, and E. coli HIT. Recombinant proteins of about 85 kDa, 82 kDa, 61 kDa, 46 kDa, and 42 kDa were expressed in -21 / prbg1-267, E. coli HIT-21 / prbg1-391, and E. coli HIT-21 / prbg1-n, respectively. (FIG. 4).

Most of the proteins were found to be present in the membrane region of the membrane in an insoluble state. It was confirmed that the same expression pattern was maintained even in the change of expression conditions through the low culture temperature (16 ℃) and the replacement of the host, which was determined to remain unstable in E. coli. Therefore, in order to stably induce expression and increase solubility in E. coli, an rbgl / pMAL-c2 expression vector having a fusion tag such as maltose binding protein was prepared. After cutting the BamHI and SalI sites of pMAL-c2 using restriction enzymes, rbg1-42, rbg1-66, rbg1-267, rbg1-391, and rbg1-n fragments of rbgl PCR products were digested with BamHI and XhoI. Then, it was linked to complete the rbgl / pMAL-c2 expression vector (expression vector). When the beta-glucosidase was expressed using the pMAL-c2 vector, it was confirmed that the protein was present in the cytosol in the form of a tagging of maltose binding protein at the N-terminus (FIG. 5). ).

The mechanism of Membrane attachment or Membrane insertion of beta-glucosidase has not been known to date. In addition, the hydrophobicity analysis of the protein showed no specific hydrophobic region (Hydrophobic region) to explain the membrane localization. However, although not common, a few membrane proteins are known to have cell membrane distribution even in the absence of hydrophobicity. The consensus signal sequence of bacterial lipoprotein is known as Leu-Leu-Ala-Gly-Cys-Ser-Asn-Ala. The glyceryl moiety of lipoproteins is known to be posttranslationally attached to cysteine residues so that the lipoproteins can be anchored to bacterial membranes. Analysis of the putative β-glucosidase of the present invention using the signal sequence data base of the lipoprotein showed no consensus signal sequence, but Leu-Leu- in the N-terminal region. The amino acid sequence of Met-Pro-Ser-His-Asn-Glu-Met-Cys-Cys-Asn-Trp-Thr was confirmed to be similar to the bacterial consensus signal sequence. In addition, the length of the hydrophobic core and carboxy terminal of the signal sequence of most of the known lipoproteins is known as 14.7 amino acids, and the putative β-glucosidase of the present invention also has a similar length. Was analyzed.

In order to confirm the presence of the predicted signal sequence, a beta-glucosidase expression vector from which the signal sequence was removed was constructed. As a result of transforming the produced expression vectors (prbgl-42, prbgl-66, prbgl-267) into E. coli, the transformant E. coli HIT removing the signal sequence of 41 amino acid (SEQ ID NO 3: amino acid sequence) was removed. It was confirmed that -21 / prRbg1-42 and E. coli HIT-21 / prbg1-66 expressed proteins are present in the cytoplasm (FIG. 4). Specifically, the predicted beta-glucosidase (β-glucosidase) N-terminal region of about 40 amino acid signal sequence seems to confirm that plays an important role in the localization of proteins.

Example 5

Activity measurement of beta-glucosidase

The activity of beta-glucosidase was measured by the amount of hydrolyzed glucose using the glucose assay kit of Sigma (USA) after reacting the isolated protein with cellobiose at 40 ° C for 2 hours. In addition, p -nitrophenyl β-D-1,4-glucopyranoside (pNPG), β-naphthyl-β-D-glucopyranoside and the like for 2 hours at 40 ℃, the absorbance at a specific wavelength (400nm) Measured and analyzed (FIG. 6).

In general, beta-glucosidase exhibits high specificity for substrates present in nature. Cellodextrins in nature are β-1,4-linked soluble cellodextrin (cellotriose, cellotetraose and cellopentaose) and beta-linked aryl-glucoside (pNPG, arbutin and prunasin), β-1,6, depending on their chemical properties. It is divided into linked glucosides (gentibiose, amygdalin). Beta-glucosidase of the present invention showed that the activity of pNPG is higher than the hydrolytic activity of cellobiose as a result of activity measurement.

In detail, when the reaction was performed at 40 ° C. for 5 hours using 5 mM pNPG, glucose 1.4 mg / ml / hr was produced, and about 0.5mg / ml / hr of glucose was also produced in 1% cellobiose, arbutin, and salicin ( 7).

<110> KOREA YAKULT CO., LTD <120> Novel gene encoding beta-glucosidase from cow rumen metagenome <130> P10-23 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 2469 <212> DNA <213> Unknown <220> <223> DAN isolated from cow rumen directly <400> 1 atgcttctga tgccgtcaat ggacatgtgc tgtatgtgga cggaggtatt cttgcataca 60 tcggaaaaca gccctgatat gaagaccaaa ctattcatta catgtctggc agctaccatg 120 cttatgacga caacgacgaa ggcgcagcag gcgccacagc tacgtgccga taatatcgac 180 gaggtgctga aggcgatgac acttgaagaa aaagccaagc tattggttgg tggtgcaaac 240 catttcttca gtgccaatgc cgtcgtgggt agtgaggccc acctcgttgc cggtgctgcc 300 ggaacctcac cagagattcc ccgtttgggt attcctgcca cggtactgac tgacggccct 360 gccggtgtcc gtatcgatcc gacgcgcaaa ggtgacacac agacttacta tgcgacggca 420 ttccccatcg gtacctgtct ggcatcgaca tggaacacca aactggtaca gaaggtgggt 480 gaggccattg gcaatgagac caaagagtat cgctgtgacg tgctgctggg tccgggtatg 540 aacctgcacc gcaatccgct gtgtggacgt aactttgagt actatagcga agatccattg 600 gtgacgggta agatagctgc cgcctatatc aagggtgtgc agagtcaggg cgctggcgta 660 tctgccaagc actttgccgt taactcgcag gaaaccaacc gtaccagtgt tgacgaacgt 720 gtctcacagc gtgcagcccg tgagctctac ctgaagggct tcgagattgc cgtccgtgag 780 agtgaccctt ggacgattat ggcttcgtat aacaaaatca atggtcagta ttcgatgggt 840 aaccacgacc tgctgacgaa gattctgcgt gacgactggg gctataaagg tatcgtgatg 900 actgactgga ttggtattcg tgagggtctg cccaccatca ctgaggtgta tgcaggtaat 960 gacctcatgg aacccggaca gcctgcccag gtgcaggaaa tcattgccgg tgtgaagaat 1020 ggtaagctgg atatcaagga tgtggaccgt aatgtgcgcc gcatgctgga gtatatcgtg 1080 aagacgccaa gcttcaacaa gtatccgtac accaacaagc ctgacctgaa ggcacatgcc 1140 gccatcacgc gccagagtgc tacagaaggt atcgtcctcc tgaagaataa cggagcatta 1200 cccttcaaga ctgaaggcaa tctctcaccc tctcacctct cacctctgat taagactgtg 1260 gcattgttcg gtgagaatag ctatgaattc ctctctggtg gtacgggttc aggatgcgta 1320 catcctccct atgttgtcga tatgctgacg ggtctgaaga atgccggtat cgcctcgacg 1380 gagacgctga cggacattta ccgtaaatat atcgcgtatg cgaaggtgaa gttccaggct 1440 gagcgccatc ccgccaagtg gttccagact gagatgatgg gacagcagaa atacccggag 1500 atagctattg caccgattgc cgtcaacaaa gaggcgaatc aggctgatgc tgctatcatc 1560 accattggtc gtcaggctgg tgagggtatc gaccgcgata ttgagaccga gtttaacctc 1620 gttcccgagg aacgtgcact cattacggat gtctgcaatg ctttccatgc tgtcggcaaa 1680 cctgtcatcg tcatcatcaa tagcggttcc gtcatcgaga cggcatcgtg gagagactat 1740 cccgatgcca tcctctgtgc ctggcagcca ggtgaggaag gtggtaactc catcgccgac 1800 atcctgacag gtaaggtgaa cccctctggt aagctgacca tgacgtggcc tatcgctgct 1860 accgaccatg cttcgacgaa gaatttcccg ggaacactcg acgcttactc gttcgagatg 1920 atggttggca acaagtcgca gataccagga ctcgactata ccaaccacga tgaggatatc 1980 tatgtaggct atcgttactt tgacaccttc ggtcgtcagg tggcttaccc cttcggctac 2040 ggtctgagct ataccacttt tgcctactca cagcctaagg tcaaggtgaa cggtgatgca 2100 gtcactgttt ccgtcactgt caagaacaca ggcagtgtgg ctggcaagga gatagcacag 2160 gtgtatatca gtgcccccaa gggacagatc gagaaacccg ccaaggagtt gaaggccttt 2220 ggcaagacac gtgagctgaa acctggcgag agtgagacgc tgaccatgac catggcagta 2280 cgcgacttgg cgtcgtttga cgaagccaac agccagtggc tctcggaggc gggtgcctat 2340 gatgtcctga ttggcagcaa tattgccgac atacgtgcta cggcacagtt caaactggct 2400 aagacctata ccgaaaagac cagccaggct ttgcagccta agcagaagct gacgctgctg 2460 aggaaatga 2469 <210> 2 <211> 822 <212> PRT <213> Unknown <220> <223> Obtained from cow rumen <400> 2 Met Leu Leu Met Pro Ser Met Asp Met Cys Cys Met Trp Thr Glu Val   1 5 10 15 Phe Leu His Thr Ser Glu Asn Ser Pro Asp Met Lys Thr Lys Leu Phe              20 25 30 Ile Thr Cys Leu Ala Ala Thr Met Leu Met Thr Thr Thr Thr Lys Ala          35 40 45 Gln Gln Ala Pro Gln Leu Arg Ala Asp Asn Ile Asp Glu Val Leu Lys      50 55 60 Ala Met Thr Leu Glu Glu Lys Ala Lys Leu Leu Val Gly Gly Ala Asn  65 70 75 80 His Phe Phe Ser Ala Asn Ala Val Val Gly Ser Glu Ala His Leu Val                  85 90 95 Ala Gly Ala Ala Gly Thr Ser Pro Glu Ile Pro Arg Leu Gly Ile Pro             100 105 110 Ala Thr Val Leu Thr Asp Gly Pro Ala Gly Val Arg Ile Asp Pro Thr         115 120 125 Arg Lys Gly Asp Thr Gln Thr Tyr Tyr Ala Thr Ala Phe Pro Ile Gly     130 135 140 Thr Cys Leu Ala Ser Thr Trp Asn Thr Lys Leu Val Gln Lys Val Gly 145 150 155 160 Glu Ala Ile Gly Asn Glu Thr Lys Glu Tyr Arg Cys Asp Val Leu Leu                 165 170 175 Gly Pro Gly Met Asn Leu His Arg Asn Pro Leu Cys Gly Arg Asn Phe             180 185 190 Glu Tyr Tyr Ser Glu Asp Pro Leu Val Thr Gly Lys Ile Ala Ala Ala         195 200 205 Tyr Ile Lys Gly Val Gln Ser Gln Gly Ala Gly Val Ser Ala Lys His     210 215 220 Phe Ala Val Asn Ser Gln Glu Thr Asn Arg Thr Ser Val Asp Glu Arg 225 230 235 240 Val Ser Gln Arg Ala Ala Arg Glu Leu Tyr Leu Lys Gly Phe Glu Ile                 245 250 255 Ala Val Arg Glu Ser Asp Pro Trp Thr Ile Met Ala Ser Tyr Asn Lys             260 265 270 Ile Asn Gly Gln Tyr Ser Met Gly Asn His Asp Leu Leu Thr Lys Ile         275 280 285 Leu Arg Asp Asp Trp Gly Tyr Lys Gly Ile Val Met Thr Asp Trp Ile     290 295 300 Gly Ile Arg Glu Gly Leu Pro Thr Ile Thr Glu Val Tyr Ala Gly Asn 305 310 315 320 Asp Leu Met Glu Pro Gly Gln Pro Ala Gln Val Gln Glu Ile Ile Ala                 325 330 335 Gly Val Lys Asn Gly Lys Leu Asp Ile Lys Asp Val Asp Arg Asn Val             340 345 350 Arg Arg Met Leu Glu Tyr Ile Val Lys Thr Pro Ser Phe Asn Lys Tyr         355 360 365 Pro Tyr Thr Asn Lys Pro Asp Leu Lys Ala His Ala Ala Ile Thr Arg     370 375 380 Gln Ser Ala Thr Glu Gly Ile Val Leu Leu Lys Asn Asn Gly Ala Leu 385 390 395 400 Pro Phe Lys Thr Glu Gly Asn Leu Ser Pro Ser His Leu Ser Pro Leu                 405 410 415 Ile Lys Thr Val Ala Leu Phe Gly Glu Asn Ser Tyr Glu Phe Leu Ser             420 425 430 Gly Gly Thr Gly Ser Gly Cys Val His Pro Pro Tyr Val Val Asp Met         435 440 445 Leu Thr Gly Leu Lys Asn Ala Gly Ile Ala Ser Thr Glu Thr Leu Thr     450 455 460 Asp Ile Tyr Arg Lys Tyr Ile Ala Tyr Ala Lys Val Lys Phe Gln Ala 465 470 475 480 Glu Arg His Pro Ala Lys Trp Phe Gln Thr Glu Met Met Gly Gln Gln                 485 490 495 Lys Tyr Pro Glu Ile Ala Ile Ala Pro Ile Ala Val Asn Lys Glu Ala             500 505 510 Asn Gln Ala Asp Ala Ala Ile Ile Thr Ile Gly Arg Gln Ala Gly Glu         515 520 525 Gly Ile Asp Arg Asp Ile Glu Thr Glu Phe Asn Leu Val Pro Glu Glu     530 535 540 Arg Ala Leu Ile Thr Asp Val Cys Asn Ala Phe His Ala Val Gly Lys 545 550 555 560 Pro Val Ile Val Ile Ile Asn Ser Gly Ser Val Ile Glu Thr Ala Ser                 565 570 575 Trp Arg Asp Tyr Pro Asp Ala Ile Leu Cys Ala Trp Gln Pro Gly Glu             580 585 590 Glu Gly Gly Asn Ser Ile Ala Asp Ile Leu Thr Gly Lys Val Asn Pro         595 600 605 Ser Gly Lys Leu Thr Met Thr Trp Pro Ile Ala Ala Thr Asp His Ala     610 615 620 Ser Thr Lys Asn Phe Pro Gly Thr Leu Asp Ala Tyr Ser Phe Glu Met 625 630 635 640 Met Val Gly Asn Lys Ser Gln Ile Pro Gly Leu Asp Tyr Thr Asn His                 645 650 655 Asp Glu Asp Ile Tyr Val Gly Tyr Arg Tyr Phe Asp Thr Phe Gly Arg             660 665 670 Gln Val Ala Tyr Pro Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Ala         675 680 685 Tyr Ser Gln Pro Lys Val Lys Val Asn Gly Asp Ala Val Thr Val Ser     690 695 700 Val Thr Val Lys Asn Thr Gly Ser Val Ala Gly Lys Glu Ile Ala Gln 705 710 715 720 Val Tyr Ile Ser Ala Pro Lys Gly Gln Ile Glu Lys Pro Ala Lys Glu                 725 730 735 Leu Lys Ala Phe Gly Lys Thr Arg Glu Leu Lys Pro Gly Glu Ser Glu             740 745 750 Thr Leu Thr Met Thr Met Ala Val Arg Asp Leu Ala Ser Phe Asp Glu         755 760 765 Ala Asn Ser Gln Trp Leu Ser Glu Ala Gly Ala Tyr Asp Val Leu Ile     770 775 780 Gly Ser Asn Ile Ala Asp Ile Arg Ala Thr Ala Gln Phe Lys Leu Ala 785 790 795 800 Lys Thr Tyr Thr Glu Lys Thr Ser Gln Ala Leu Gln Pro Lys Gln Lys                 805 810 815 Leu Thr Leu Leu Arg Lys             820 <210> 3 <211> 41 <212> PRT <213> Unknown <220> <223> Obtained from cow rumen <400> 3 Met Leu Leu Met Pro Ser Met Asp Met Cys Cys Met Trp Thr Glu Val   1 5 10 15 Phe Leu His Thr Ser Glu Asn Ser Pro Asp Met Lys Thr Lys Leu Phe              20 25 30 Ile Thr Cys Leu Ala Ala Thr Met Leu          35 40

Claims (4)

Beta-glucosidase gene represented by the nucleotide sequence of SEQ ID NO: 1.
Beta-glucosidase protein represented by the amino acid sequence of SEQ ID NO: 2.
Beta-glucosidase protein, characterized in that it comprises a signal sequence (Signal sequence) of SEQ ID NO: 3.
A recombinant vector having a cleavage map as described in FIG. 8 comprising a beta-glucosidase gene of SEQ ID NO: 1.

Images