JP2003070475A - Cellulase gene derived from protozoan symbiotically living with termite - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a cellulase by taking out a cellulase gene from a protozoan symbiotically living with a termite the culture of which is impossible, and by using a genetic engineering technique using the gene. SOLUTION: An mRNA is extracted from a content of the hindgut of Coptotermes formosanus, and a cDNA library is constructed from the mRNA. The cDNA library is screened by the cellulase activity to obtain a recombinant having the cellulase activity, and the new recombinant protein having the cellulase activity is obtained from the transformant transformed by using a recombinant vector including the obtained gene. The recombinant cellulase is derived from Spirotrichonympha leidyi of a protozoan, and the optimum pH, the optimum temperature, the Km value and the Vmax are 6.0, 70 deg.C, 1.9 mg/mL, and 148.2 units/mg-protein, respectively.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protein having cellulase activity derived from termite symbiotic protozoa, and a gene encoding the protein.

[0002]

2. Description of the Related Art Cellulose contains β-1,4 in glucose.
-A polymeric polysaccharide linked by glycosidic bonds. Therefore, when this is hydrolyzed, glucose is obtained, and cellulose can be effectively used as a glucose supply source. However, cellulose is a persistent substance,
The utilization of cellulosic biomass has not been put to practical use. Cellulase is responsible for a series of reactions for efficiently hydrolyzing this cellulose and extracting energy from it. Therefore, elucidation of the characteristics of cellulase and efficient production of cellulase can be said to be the basis of the technology related to effective utilization of cellulose resources, and isolation of cellulase genes derived from microorganisms has hitherto been performed. Is being done. For example, JP-A-8-5666.
Japanese Patent Laid-Open No. 20-34187 discloses that a cellulase gene is isolated from a Humicola insolens strain, the cellulase gene is introduced into a host microorganism and expressed to obtain a target cellulase.
No. 00-210081 discloses Bacillus.
The thermostable alkaline cellulase genes from the genus are each described.

On the other hand, termites that use cellulose as an energy source, such as wood, have cellulolytic enzymes (cellulases) in their digestive tracts, and protozoa coexist in the hindgut of some termites such as house termites. And
Since termites cannot survive without protozoa, they are thought to play a major role in the breakdown of cellulose. This symbiotic protozoa is very difficult to isolate and culture, and isolation of cellulase derived from protozoa has not been successful so far. On the other hand, regarding cellulase genes derived from symbiotic protozoa, sequences having high homology with known cellulases have been obtained, but it is not known whether they actually function as active cellulases.

The cellulase of the termite itself such as Yamato termite or Takasago termite and the gene of the cellulase are disclosed in JP-A-11-46764.
The gazette discloses that, as its enzymatic chemistry, it decomposes cellulose that is made in the salivary glands or intestines of insects and taken in as a bait, has a molecular weight of 40,000 to 50,000, a thermal stability of -60 ° C, and an optimum temperature. It is described that the pH is 5.0 to 6.0 and the specific activity to carboxymethyl cellulose is 70 to 1300 units / mg.

[0005]

The symbiotic system of termites and protozoa is one of the most efficient utilization systems of cellulose in the natural world. The root of the symbiotic system is the symbiotic protozoa, but a cellulase gene capable of producing an active protein from the symbiotic protozoa has not been extracted so far. The object of the present invention is to
It is an object of the present invention to provide a method for producing a cellulase by a genetic engineering method using a cellulase gene extracted from a symbiotic protozoan of a termite that cannot be cultured.

[0006]

In order to solve the above problems, the present inventor extracted mRNA from the contents of the hindgut of 250 termites, constructed a cDNA library from the mRNA, and confirmed cellulase activity. Based on the screening, obtain recombinants with cellulase activity,
Furthermore, it was found that a novel recombinant protein having cellulase activity was obtained from the transformant transformed with the recombinant vector containing the obtained gene, its enzymatic chemistry was determined, and it was obtained by screening. It was confirmed that the novel cellulase gene is derived from protozoa, and the present invention has been completed.

That is, the present invention relates to (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or (b) an amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added. From a DNA consisting of a selected amino acid sequence and encoding a protein having cellulase activity (claim 1), the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence, or a sequence containing a part or all of these sequences. DNA (Claim 2)
Or a DNA that hybridizes with the DNA of claim 2 under stringent conditions and that encodes a protein having cellulase activity (claim 3), or SEQ ID NO: 2
In the protein consisting of the amino acid sequence shown in (claim 4) or the amino acid sequence shown in SEQ ID NO: 2,
The present invention relates to a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added and having cellulase activity (claim 5).

Further, the present invention is specific to a fusion protein (claim 6) in which the protein according to claim 4 or 5 is bound to a marker protein and / or a peptide tag, and the protein according to claim 4 or 5. An antibody that binds to (claim 7), the antibody according to claim 7 (claim 8) characterized in that the antibody is a monoclonal antibody, and claim 1.
Host cell comprising an expression system capable of expressing the recombinant vector (claim 9) containing the DNA according to any one of (1) to (3) or the protein according to claim 4 (claim 1).
0) or a host cell comprising the expression system according to claim 10 is cultured in a medium, and a recombinant protein having cellulase activity is collected from the resulting culture (a method for producing a recombinant protein). Claim 11).

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The DNA which is the subject of the present invention includes a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 and one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2. Is deleted,
DNA consisting of a substituted or added amino acid sequence and encoding a protein having cellulase activity
Alternatively, any DNA may be used as long as it is composed of the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence containing a part or all of these sequences. Is a protein that qualitatively has an activity of hydrolyzing cellulose, specifically, an activity of cleaving β-1,4-glycoside bond in an amorphous region of cellulose.

Under the stringent condition against various DNA libraries, a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence and a sequence containing a part or all of these sequences is used as a probe under stringent conditions. It is also possible to obtain a DNA encoding a protein having a novel cellulase activity by hybridizing and isolating a DNA that hybridizes to the probe. The DNA thus obtained is also included in the present invention. The hybridization conditions for obtaining the DNA of the present invention include, for example, hybridization at 42 ° C., 1 × SSC, and 0.1% SD.
Examples include washing treatment with a buffer containing S at 42 ° C., hybridization at 65 ° C., and 0.
65 with 1 × SSC, buffer containing 0.1% SDS
A more preferable example is a washing treatment at ° C. There are various factors other than the above temperature conditions as factors that affect the stringency of hybridization, and those skilled in the art can appropriately combine various factors and are equivalent to the stringency of hybridization described above. It is possible to achieve the stringency of.

Based on the DNA sequence information disclosed in the present invention, these DNAs of the present invention are publicly known, such as PCR or hybridization using a DNA fragment as a probe, from a genomic gene of a protozoan such as Spirotrichonympha leidyi. It can be prepared by the method of. In addition, it can be prepared by chemical synthesis. In addition, from these DNAs of the present invention, the DN of the present invention encoding an artificial mutant protein having cellulase activity
In order to obtain A, that is, the artificially mutated DNA of the present invention at the nucleotide sequence level or the amino acid sequence level, known mutation means may be used. For example, a commercially available mutagenesis kit is used to easily introduce the mutation. be able to. In addition, the first methionine (Me
Those in which t) is deleted are also included in the protein due to mutation of the amino acid sequence.

The protein of the present invention includes SEQ ID NO: 2
In particular, a protein having the amino acid sequence shown in SEQ ID NO: 2 or a protein having the amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added and having cellulase activity The protein of the present invention is not limited and can be prepared by a known method based on the DNA sequence information and the like, and the origin thereof is not particularly limited.

The fusion protein of the present invention may be any one as long as the protein of the present invention is bound to the marker protein and / or peptide tag, and the marker protein is conventionally known. The marker protein is not particularly limited, and specific examples thereof include alkaline phosphatase, Fc region of antibody, HRP, GFP, and the like.
Further, as the peptide tag in the present invention, conventionally known peptide tags such as Myc tag, His tag, FLAG tag and GST tag can be specifically exemplified. Such a fusion protein can be prepared by a conventional method, for purification of a protein having cellulase activity utilizing the affinity of Ni-NTA and His tag, for quantification of an antibody against the protein having cellulase activity, etc., It is also useful as a research reagent in the field.

Specific examples of the antibody which specifically binds to the protein of the present invention include immunospecific antibodies such as monoclonal antibody, polyclonal antibody, chimeric antibody, single chain antibody and humanized antibody, These can be prepared by a conventional method using all or part of the above-mentioned protein of the present invention, fusion protein, etc. as an antigen,
Among them, the monoclonal antibody is more preferable in terms of its specificity. Antibodies such as such monoclonal antibodies are useful, for example, for clarifying the characteristics of cellulase and the production mechanism thereof.

The above-mentioned antibody of the present invention is produced by administering a protein or a fragment thereof containing the epitope of the present invention to an animal (preferably non-human) using a conventional protocol, for example, for preparing a monoclonal antibody. Is a hybridoma method (Nature 256, 495-497, 197) that results in antibodies produced by cultures of continuous cell lines.
5), trioma method, human B cell hybridoma method (Imm
unology Today 4, 72, 1983) and EBV-hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY,
pp.77-96, Alan R. Liss, Inc., 1985).

In addition, the above-mentioned monoclonal antibody and the like,
For example, FITC (fluorescein isocyanate) or
Is a fluorescent substance such as tetramethylrhodamine isocyanate
Or ¹²⁵I,³²P,¹⁴C,³⁵S or³Radio iso such as H
Taupe, alkaline phosphatase, peroxidase
Enzyme, β-galactosidase, phycoerythrin, etc.
Labeled with a green fluorescent protein or green fluorescent protein (GF
P) and other fluorescent proteins, etc.
By using the protein, the protein of the present invention can be produced.
Capability analysis can be performed. In addition, using the antibody of the present invention
As the immunological measurement method, the RIA method and the ELISA method are used.
Method, fluorescent antibody method, plaque method, spot method, hemagglutination reaction
Examples of such methods include a reaction method and an Ouchterlony method.

The recombinant vector of the present invention is not particularly limited as long as it is a vector containing the above-mentioned DNA of the present invention, but preferably one containing an expression system capable of expressing the protein of the present invention in a host cell, For example, expression systems derived from chromosomes, episomes and viruses, more specifically bacterial plasmid derived, yeast plasmid derived,
Vectors derived from papovavirus such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, retrovirus derived, bacteriophage derived, transposon derived and combinations thereof, for example, plasmids such as cosmid and phagemid. And those derived from genetic elements of bacteriophage. The expression system may include control sequences that regulate as well as direct expression.

The present invention also relates to a host cell comprising an expression system capable of expressing the above-mentioned protein of the present invention. Introduction of a recombinant vector containing such a DNA encoding the protein of the present invention or the above expression system into a host cell is carried out by Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1
986) and Sambrook et al. (MOLECULAR CLONING: A LABORATO
RY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, 1989) and many other standard laboratory manual methods such as calcium phosphate transfection, DEAE.
-Dextran mediated transfection, transvection, microinjection,
Cationic lipid-mediated transfection, electroporation, transduction, scrape loading (sc
rape loading, ballistic introduction, infection, etc. The host cells include bacterial prokaryotic cells such as Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus, Staphylococcus, fungal cells such as yeast and Aspergillus, and Drosophila S.
2, insect cells such as Spodoptera Sf9, L cells, CH
O cells, COS cells, HeLa cells, C127 cells, B
ALB / c3T3 cells (including mutant strains lacking dihydrofolate reductase and thymidine kinase), BHK
21 cells, HEK293 cells, animal cells such as Bowes malignant melanoma cells, plant cells and the like can be mentioned.

The method for producing the recombinant protein of the present invention may be a method of culturing a host cell containing the above expression system in a medium and collecting a recombinant protein having cellulase activity from the obtained culture. Any method may be used, and examples of the method for recovering and purifying the protein of the present invention from cell culture include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, and hydrophobic interaction. Known methods including chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, preferably, high performance liquid chromatography can be used. In addition, as the column used in the affinity chromatography,
For example, by using a column to which an antibody against the protein of the present invention is bound or a column to which the above-mentioned protein of the present invention is added with a normal peptide tag, a column to which a substance having an affinity for this peptide tag is bound is used. The protein of the invention can be obtained. The protein purification method of the present invention can be applied to peptide synthesis.

[0020]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples. (1. Isolation of mRNA) In order to isolate mRNA,
The hindgut was removed from 250 house termites and the contents were tested using Solution U (Trager 1934, Biological Bulletin, Vo.
l66: 182-190) and gently centrifuge (300
g, 1 minute) to obtain a protozoan fraction. Further, the protozoan fraction was washed twice with Solution U to obtain a protozoan fraction for RNA extraction. Under the microscope,
This protozoa fraction contains three types of protozoa (Spirotrichon
ympha leidyi, Pseudotrichonympha grassi, Holomasti
The presence of gotoides hartmanni) was confirmed. Isolation of RNA was performed using ISOGEN (Nippon Gene) according to the instructions, and purification and isolation of mRNA was performed using Oligotex-dT30 <Supe.
r> (Nippon Roche) and follow the instructions, and proceed to about 3.
9 μg of mRNA was obtained.

(2. Construction of cDNA library) cD
In order to construct an NA library, mRNA was used as a template, oligo dT was used as a primer, and Superscript II was used.
An RNA-DNA complex was formed by reverse transcription (Gibco BRL), and a second cDNA chain was synthesized using the recovered first cDNA chain as a template. A cDNA library was prepared from the obtained double-stranded cDNA using λZAPII (STRATAGENE) according to the instructions. The packaging lysate for cloning is GIGAPAK III Glod
(STRATAGENE) was used according to a conventional method. As a result, 3 × 10 ⁵ recombinant phages could be obtained, and the titer after amplification was 3 × 10 ⁹ pfu / ml.

(3. Expression Screening) Using 2 × YT medium containing 0.5% of carboxymethyl cellulose (CMC), the recombinant phages were incubated for 8 to 12 hours.
The plaques were formed by culturing at ℃. Then, 10 ml of 0.1% Congo red was poured and stained on the plate on which plaque was formed, and further 10 mM of 1M NaCl was added.
Decolorization with 1 was performed. After staining with Congo red, 11 colonies giving halo (circular portion not stained with Congo red due to CMC decomposition) were obtained. Of the 11 clones that gave halos in the screening, three clones were bluescript (pBluescr) by superinfection with the helper phage EXASSIST and E. coli SOLR strains contained in the λZAPII vector kit.
ipt) converted to phagemid. When the nucleotide sequences of the converted clones were determined, the nucleotide sequences of the three clones were the same. The nucleotide sequence of the obtained DNA is shown in SEQ ID NO: 1, and the amino acid sequence of the protein that is the translation product thereof is shown in SEQ ID NO: 2.

(4. Identification of Origin of Cellulase Gene) The above cDNA library is a mixed mR of three protozoa.
Since it was produced from NA, it is not possible to specify which protozoan the cellulase gene is derived from. Then, based on the obtained DNA nucleotide sequence of the cellulase gene, a primer specific to this cellulase, 5'-CTTCAAGGTAT
TGTTGATAC-3'Forward (SEQ ID NO: 3) and 5'-TTAAGCAATG
CTGATCATTA-3'Reverse (SEQ ID NO: 4) was designed and fractionated by microcapillary.
PCR was tried using 0 to 30 individuals as a template.
PCR conditions are 94 ° C for 30 seconds, 59 ° C for 45 seconds, 72 ° C.
There were 35 cycles in 2 minutes. As a result of confirming the PCR product by agarose gel electrophoresis (Fig. 1), among the three protozoa, only from the sponge tree (Spirotrichonympha leidyi), D
The amplification of NA could be confirmed. In FIG. 1, lane M is a marker, lane 1 is Pseudotrichonympha grassi, lane 2 is Holomastigotoides hartmanni, and lane 3 is Spirotr.
ichonympha leidyi, lane 4 is a control for the results of PCR using DNA extracted from a mixture of three protozoa as templates. Therefore, it can be concluded that the origin of the cellulase gene is this kecamuri.

(5. Expression and Purification of Cellulase by Escherichia coli) In order to prepare an expression vector of cellulase, a DNA fragment containing the cellulase gene was salI and sacI from the pBluescript vector containing the cellulase gene.
Cut out with the vector pET21b (Novagen)
Was cloned into the corresponding site. E. coli (BL21 (DE3)) strain was transformed with the vector and cultured in an LB liquid medium at 37 ° C for 12 hours with shaking. After collecting E. coli, the BugBuster Protein Extraction Reagent (Novagen
The protein was recovered according to the instructions. Furthermore, T7 / Tag Affinity Purification Kit (Novagen)
Was used to purify the recombinant protein according to the instructions.
The recombinant protein was detected by SDS-PAGE as a single band with a molecular weight of approximately 36 kDa. Furthermore, the cellulase activity of this recombinant protein was measured using CMC as a substrate. The optimum pH of this recombinant cellulase is 6.0 (FIG. 2), the optimum temperature is 70 ° C. (FIG. 3), the Km value is 1.9 mg / ml, and the Vmax is 148.
It was 2 units / mg protein (1 unit is the amount of enzyme that produces a reducing sugar equivalent to 1 μM glucose per minute).

[0025]

INDUSTRIAL APPLICABILITY The novel cellulase derived from termite symbiotic protozoa provided by the present invention is capable of efficiently decomposing cellulose when used alone or in combination with already known cellulase. , It is useful as various industrial enzyme agents such as textile processing preparations, feed additives, and digestive agents. In addition, the novel cellulase and its gene of the present invention are expected to be very useful in elucidating the characteristics of cellulase.

[0026]

[Sequence list]                                SEQUENCE LISTING <110> JAPAN SCIENCE AND TECHNOLOGY CORPORATION       RIKEN <120> Coptotermes-symbiosis protozoa-derived from cellulase       genes <130> TC-13-1 <140> <141> <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 938 <212> DNA <213> Spirotrichonympha leidyi <220> <221> CDS <222> (1) .. (894) <400> 1 ctt caa ggt att gtt gat act tat ggt gcc ctc agc gta tct gga agc 48 Leu Gln Gly Ile Val Asp Thr Tyr Gly Ala Leu Ser Val Ser Gly Ser   1 5 10 15 aaa gtt gtt ggc aag tct ggt tca cct gca gcg ttg cat ggt gtt tca 96 Lys Val Val Gly Lys Ser Gly Ser Pro Ala Ala Leu His Gly Val Ser              20 25 30 ttt ggt tgg cat aat tgg tgg cct gag ttc tac aca gca gac aca gtt 144 Phe Gly Trp His Asn Trp Trp Pro Glu Phe Tyr Thr Ala Asp Thr Val          35 40 45 aaa cat ctt gct gaa gat tgg aaa gcg act gtt ctt cgt gca gca att 192 Lys His Leu Ala Glu Asp Trp Lys Ala Thr Val Leu Arg Ala Ala Ile      50 55 60 ggt gtt gag cct gac gga gga tat ctt caa gat tct tct tta ggt gat 240 Gly Val Glu Pro Asp Gly Gly Tyr Leu Gln Asp Ser Ser Leu Gly Asp  65 70 75 80 caa tgt gca aca act gtg gct gat gct gca att gca aat gga att tat 288 Gln Cys Ala Thr Thr Val Ala Asp Ala Ala Ile Ala Asn Gly Ile Tyr                  85 90 95 gtt att ttg gat tgg cat cag cat cgt att aat caa gat gct gct att 336 Val Ile Leu Asp Trp His Gln His Arg Ile Asn Gln Asp Ala Ala Ile             100 105 110 aaa ttt ttc act aaa ttt gtt acc aag tat aaa ggt gtg cct aat gtt 384 Lys Phe Phe Thr Lys Phe Val Thr Lys Tyr Lys Gly Val Pro Asn Val         115 120 125 atc tat gaa ata ttc aat gag cca gaa tca gct aca tgg cca gag gtc 432 Ile Tyr Glu Ile Phe Asn Glu Pro Glu Ser Ala Thr Trp Pro Glu Val     130 135 140 aaa caa tat gct aat gca gtg atc aaa gtg att aga gat att gat cct 480 Lys Gln Tyr Ala Asn Ala Val Ile Lys Val Ile Arg Asp Ile Asp Pro 145 150 155 160 gac gca tta gta ttg gta gga tgt gca aat tgg gat caa aag atc aca 528 Asp Ala Leu Val Leu Val Gly Cys Ala Asn Trp Asp Gln Lys Ile Thr                 165 170 175 gaa cca gca gct gat cca ttg gtt ggt ttt gga aat gtt gca tat aca 576 Glu Pro Ala Ala Asp Pro Leu Val Gly Phe Gly Asn Val Ala Tyr Thr             180 185 190 ttg cat ttt tat gca gca aca cat act cag tgg ttg aga gat gat gct 624 Leu His Phe Tyr Ala Ala Thr His Thr Gln Trp Leu Arg Asp Asp Ala         195 200 205 caa aag gca att gat gct gga ttg cct ata ttt gtg agt gaa tgt ggt 672 Gln Lys Ala Ile Asp Ala Gly Leu Pro Ile Phe Val Ser Glu Cys Gly     210 215 220 gga atg gaa tct agt ggt gat gga gct att aat caa aat gag tgg aat 720 Gly Met Glu Ser Ser Gly Asp Gly Ala Ile Asn Gln Asn Glu Trp Asn 225 230 235 240 aat tgg ata tca ttc tta gac aag aat tca att tca tgg gtt gct tgg 768 Asn Trp Ile Ser Phe Leu Asp Lys Asn Ser Ile Ser Trp Val Ala Trp                 245 250 255 tca att tct aat aag gct gaa aca tgt tca atg att act aca aca ggt 816 Ser Ile Ser Asn Lys Ala Glu Thr Cys Ser Met Ile Thr Thr Thr Gly             260 265 270 cct gtc aat cct cca tgg cct gat agt ggc tta tct gaa tgg ggc aaa 864 Pro Val Asn Pro Pro Trp Pro Asp Ser Gly Leu Ser Glu Trp Gly Lys         275 280 285 tta gtc aag aaa tta atg atc agc att gct taataaacac tgcctccctt 914 Leu Val Lys Lys Leu Met Ile Ser Ile Ala     290 295 tgaaaaaaaa aaaaaaaaaa aaaa 938 <210> 2 <211> 298 <212> PRT <213> Spirotrichonympha leidyi <400> 2 Leu Gln Gly Ile Val Asp Thr Tyr Gly Ala Leu Ser Val Ser Gly Ser   1 5 10 15 Lys Val Val Gly Lys Ser Gly Ser Pro Ala Ala Leu His Gly Val Ser              20 25 30 Phe Gly Trp His Asn Trp Trp Pro Glu Phe Tyr Thr Ala Asp Thr Val          35 40 45 Lys His Leu Ala Glu Asp Trp Lys Ala Thr Val Leu Arg Ala Ala Ile      50 55 60 Gly Val Glu Pro Asp Gly Gly Tyr Leu Gln Asp Ser Ser Leu Gly Asp  65 70 75 80 Gln Cys Ala Thr Thr Val Ala Asp Ala Ala Ile Ala Asn Gly Ile Tyr                  85 90 95 Val Ile Leu Asp Trp His Gln His Arg Ile Asn Gln Asp Ala Ala Ile             100 105 110 Lys Phe Phe Thr Lys Phe Val Thr Lys Tyr Lys Gly Val Pro Asn Val         115 120 125 Ile Tyr Glu Ile Phe Asn Glu Pro Glu Ser Ala Thr Trp Pro Glu Val     130 135 140 Lys Gln Tyr Ala Asn Ala Val Ile Lys Val Ile Arg Asp Ile Asp Pro 145 150 155 160 Asp Ala Leu Val Leu Val Gly Cys Ala Asn Trp Asp Gln Lys Ile Thr                 165 170 175 Glu Pro Ala Ala Asp Pro Leu Val Gly Phe Gly Asn Val Ala Tyr Thr             180 185 190 Leu His Phe Tyr Ala Ala Thr His Thr Gln Trp Leu Arg Asp Asp Ala         195 200 205 Gln Lys Ala Ile Asp Ala Gly Leu Pro Ile Phe Val Ser Glu Cys Gly     210 215 220 Gly Met Glu Ser Ser Gly Asp Gly Ala Ile Asn Gln Asn Glu Trp Asn 225 230 235 240 Asn Trp Ile Ser Phe Leu Asp Lys Asn Ser Ile Ser Trp Val Ala Trp                 245 250 255 Ser Ile Ser Asn Lys Ala Glu Thr Cys Ser Met Ile Thr Thr Thr Gly             260 265 270 Pro Val Asn Pro Pro Trp Pro Asp Ser Gly Leu Ser Glu Trp Gly Lys         275 280 285 Leu Val Lys Lys Leu Met Ile Ser Ile Ala     290 295 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer Forward <400> 3 cttcaaggta ttgttgatac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer Reverse <400> 4 ttaagcaatg ctgatcatta 20

[Brief description of drawings]

FIG. 1 is a diagram showing the results of PCR using 20 to 30 individual protozoa of each of the three types of microcapillaries as templates, using primers specific to the cellulase gene.

FIG. 2 pH of activity of recombinant cellulase on CMC
It is a figure which shows the influence by.

FIG. 3 is a graph showing the effect of temperature on the activity of recombinant cellulase on CMC.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12N 9/42 5/10 C12P 21/08 9/42 C12N 15/00 ZNAA // C12P 21 / 08 5/00 A (72) Inventor Moriya Okuma 1-1-24 Mizutani, Fujimi-shi, Saitama (72) Inventor Toshiaki Kudo 1-2-1-20-606 Hiramachi, Meguro-ku, Tokyo (72) Moriya Shigeharu 4-4-22 Haneda, Ota-ku, Tokyo Avion Haneda 303 F term (reference) 4B024 AA01 AA03 BA12 BA42 CA04 CA07 DA02 DA05 DA11 EA02 EA03 EA04 FA02 GA01 GA11 HA01 HA03 HA11 4B050 CC01 CC03 CC05 DD20 LL04 LL05 4B064 AG27 CA10 CA10 CA27 CA10 CA10 CC24 DA13 4B065 AA01X AA57X AA86Y AA90X AB01 AB02 BA01 BA08 CA25 CA31 CA43 CA44 CA46 CA57 4H045 AA11 AA20 AA30 BA10 BA41 DA89 EA07 EA20 EA36 EA50 FA72 FA74

Claims (11)

[Claims] 1. A DNA encoding the following protein (a) or (b). (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) an amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added, and cellulase activity Protein with 2. A DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence, or a sequence containing a part or all of these sequences. 3. A DNA which hybridizes with the DNA according to claim 2 under stringent conditions and which encodes a protein having cellulase activity. 4. A protein consisting of the amino acid sequence shown in SEQ ID NO: 2. 5. A protein comprising an amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added, and having cellulase activity. 6. The protein according to claim 4 or 5,
A fusion protein having a marker protein and / or a peptide tag bound thereto. 7. An antibody that specifically binds to the protein according to claim 4 or 5. 8. The antibody according to claim 7, wherein the antibody is a monoclonal antibody. 9. A recombinant vector containing the DNA according to claim 1. 10. A host cell comprising an expression system capable of expressing the protein of claim 4 or 5. 11. A method for producing a recombinant protein, which comprises culturing a host cell containing the expression system according to claim 10 in a medium and collecting the recombinant protein having cellulase activity from the resulting culture. .