JPH08126492A - Novel cellulase component NCE2 and breeding method using the same - Google Patents

Abstract

(57) [Summary] [Objective] To provide a novel cellulase and its gene. [Structure] The cellulase gene is isolated from a Humicola insolens strain, and the cellulase gene is introduced into a host microorganism and expressed to obtain the target cellulase.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel cellulase and its gene, and more particularly to a DNA sequence encoding the novel cellulase, said DNA.
It relates to an expression vector containing a sequence, a microorganism transformed with the vector, and a method for producing a novel cellulase by the microorganism.

[0002] Cellulose is the main constituent of the plant, present in widely naturally. And β with glucose as one unit
Since it is a high-molecular polysaccharide polymerized by -1,4-glycoside bond, it is essential for the effective utilization of cellulosic biomass to cleave the glycoside bond and efficiently extract sugars. However, the physicochemical method of producing soluble sugars by hydrolysis with acid, heat, etc. yields only a small amount of the product, and the purity of the product is remarkably heterogeneous due to lack of specificity. Cellulase is responsible for a series of reactions for efficiently hydrolyzing such hardly-decomposable cellulose and extracting energy therefrom. Therefore, it can be said that elucidation of the characteristics of this enzyme and efficient production of this enzyme form the basis of the technology related to effective utilization of cellulose resources.

Cellulase is commercially available as an industrial enzyme preparation and is used as a main component of various products such as detergents, textile processing preparations, feed additives and digestive agents. Therefore, the elucidation of the characteristics of cellulase can be expected to be very useful in such practical aspects.

On the other hand, the cellulase system is roughly divided into three enzyme systems, endoglucanase, exoglucanase, and β-glucosidase. These cellulases interact with each other and are responsible for the decomposition of cellulose by a synergistic effect. In addition to these cleavage modes, cellulase components having various properties such as action temperature and action pH are produced by a wide variety of microorganisms, and the enzyme system has been selected according to the purpose of use. However, depending on the purpose, the composition of the enzyme component obtained from the microorganism may not always be appropriate, and in order to optimize this, in addition to controlling the culture conditions of the microorganism, acquisition of mutant strains, fractionation of the enzyme, etc. Means are taken.

On the other hand, it has also been attempted to genetically isolate and utilize these enzyme components. According to this method, an enzyme having a very high degree of purification can be obtained, and it is considered that the characteristics of the enzyme can be elucidated and the effective use thereof can be achieved. However, as described above, since cellulase is a complex enzyme system, it is not easy to identify individual cellulase genes, and the current situation is that all cellulase genes have not been isolated yet.

[0006]

SUMMARY OF THE INVENTION The present inventors have succeeded in isolating a cellulase gene from Humicola insolens and obtaining a cellulase expressed from the gene. Therefore, the present invention aims at providing a cellulase and a DNA sequence encoding said cellulase. Another object of the present invention is to express the cellulase according to the present invention in a microorganism by a gene recombination operation.

The protein according to the present invention comprises a part of the amino acids shown in Sequence Listing 1 or the sequence from 1 to 453, and the sequence from -23 to -1 in its N sequence.
It is provided on the terminal side. Furthermore, the DN according to the invention
The A sequence is one that encodes the above-mentioned amino acid, particularly one that has a part or all of the base sequence described in Sequence Listing 2.

Deposit of microorganism pM14 containing cellulase gene according to the present invention
-1 transformed E. coli JM109 strain is FERM
It has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology under the deposit number of P-14585.

[0009]

DETAILED DESCRIPTION OF THE INVENTION Cellulase and Gene Thereof The protein according to the present invention is a part of the sequence shown in Sequence Listing 1 or consists of the 1st to 453rd sequences. Derivatives of the above-mentioned proteins are described in Sequence Listing 1 in which amino acid additions, insertions, deletions, substitutions, or other modifications have been made using methods commonly used in genetic engineering (eg, site-directed mutagenesis). Proteins having an amino acid sequence that still retain cellulase activity are included in the present invention.

The protein provided by the present invention is
In addition, 1-453 of the amino acid sequences described in Sequence Listing 1
It consists of arrays up to the turn. Also, the N of this array
The present invention also includes a protein having an amino acid sequence from -23 to -1 in Sequence Listing 1 at the end. These proteins according to the invention have cellulase activity, especially at pH in the neutral range.

These proteins are known to It is derived from insolence, and the protein having the sequence from 1 to 453 of Sequence Listing 1 is a mature protein,
Sequences -23 to -1 are signal peptides.

[0012] The amino acid sequences 1 to 453 of the amino acid sequence shown in Sequence Listing 1 will be referred to as cellulase NCE.
2. The gene is sometimes called cellulase NCE2 gene.

The present invention provides a DNA sequence encoding the amino acid sequence of the protein. A typical sequence of this DNA sequence has a part or all of the base sequence shown in Sequence Listing 2.

The nucleotide sequences shown in Sequence Listing 2 are H. It represents the chromosomal DNA of insolence. Sequence listing 2
The nucleotide sequence described in (1) has an open reading frame (reading frame) that starts with ATG of SEQ ID NO: 389 and ends with TGA of SEQ ID NOs: 2096-2098. The base sequences of SEQ ID NOs: 458 to 2095 correspond to the mature protein consisting of 453 residues. Furthermore, it was confirmed that four introns were present in the base sequence of Sequence Listing 2.

Given the amino acid sequence of a protein, the DNA sequence encoding the protein can be easily determined, and various base sequences encoding all or part of the amino acid sequence shown in Sequence Listing 1 can be selected. . Therefore,
The DNA sequence encoding a part or all of the amino acid sequence described in Sequence Listing 1 according to the present invention is a sequence encoding the same amino acid in addition to a part or all of the base sequence described in Sequence Listing 2. A sequence having a degenerate codon as a base sequence is also meant.

The nucleotide sequence of the gene encoding the protein according to the present invention is different from that of any cellulase gene which has been cloned and whose nucleotide sequence has been elucidated. This is the nucleic acid database Ge
It has already been confirmed by comparison with the cellulase gene registered in nBank®, R81.0, February, 1994.

The DNA of the present invention may be of natural origin or wholly synthetic. Alternatively, it may be synthesized using a part of a natural product. As a typical method for obtaining DNA, H. Methods commonly used in the field of genetic engineering from chromosome libraries or cDNA libraries derived from insolens, for example, a method of screening using an appropriate DNA probe prepared based on the information of the partial amino acid sequence, and the like can be mentioned. . It can also be obtained from the above-mentioned deposited bacteria.

The DNA sequence according to the present invention was determined as follows. (1) Preparation of DNA probe for cloning H. The insolens were cultured, and the cellulase was purified using various types of chromatography and two-dimensional electrophoresis using the cellulase activity as an index. Next, this purified cellulase was digested with various proteases, and the amino acid sequence was determined by a peptide sequencer. Based on this amino acid sequence, a mixture of oligonucleotides containing all possible nucleotide sequences was prepared and used as a primer.
As a result of amplification by the PCR method, a DNA of about 800 bp was synthesized, and this was used as a probe.

(2) Preparation of genomic DNA library H. The insolens were cultured and the cells were collected to obtain bacterial cells. From this bacterium, total DNA was collected from Hiroyuki Horiuchi et al., J. Bact.
eriol. 170: 272 to 278, 1988. The obtained DNA is digested with a restriction enzyme to give 6-10k
A DNA fragment mainly consisting of a bp fragment was obtained. Based on this DNA,
A genomic DNA library was prepared using a commercially available vector kit.

(3) Cloning of Cellulase Gene Escherichia coli was infected with the DNA library (for example, phage DNA library) obtained by the above (2), and the resulting plaque was blotted on a nylon membrane and alkali-denatured and fixed. After the hybridization, a positive plaque was selected by hybridizing with the probe obtained in (1) above.

(4) Determination of nucleotide sequence 1 of the clones (5 in total) selected as described above
DNA was prepared for each individual and the nucleotide sequence was determined using a DNA sequencer. In addition, H. MRN obtained from Insolence
CDNA was synthesized by making reverse transcriptase act on A, and a phage library was prepared based on this cDNA to obtain cDNA from positive plaques. The presence of introns and open reading frames was revealed by comparing the entire base sequence of this cDNA with the base sequence of the genome (see Example 6).

(5) Expression of cellulase gene A cellulase NCE2 gene for expression in a microorganism is made into a plasmid together with a selectable marker gene, and H. Transformed into insolence. As a result, the obtained transformant was endowed with resistance to the selectable marker and at the same time had improved cellulase activity (see Example 10).

Expression Vector and Transformed Microorganism According to the present invention, the above-mentioned DNA sequence according to the present invention is also contained in a state capable of replicating in a host microorganism and expressing a protein encoded by the DNA sequence. An expression vector consisting of This host-vector system is not particularly limited, and, for example, a system using Escherichia coli, actinomycetes, yeast, mold and the like, and a fusion protein expression system using other proteins using them can be used. As the procedure and method for constructing a vector according to the present invention, those conventionally used in the field of genetic engineering can be used.

In order to express the desired protein by actually introducing it into the host microorganism, the expression vector according to the present invention may include, in addition to the above-mentioned DNA sequence according to the present invention, a DNA sequence for controlling the expression or It may include a genetic marker for selecting a microorganism. Since it is considered that the base sequence in Sequence Listing 2 also includes these control sequences, it may be advantageous in some cases to use this base sequence as it is. Further, this expression vector may contain a DNA sequence encoding cellulase in a repeated form (tandem). These may be present in an expression vector according to a conventional method, and the method for transforming a microorganism with this vector may be one commonly used in this field.

This transformant can be obtained by culturing in a suitable medium and isolating the above-mentioned protein according to the present invention from the culture. Therefore, according to another aspect of the present invention, there is provided a method for producing the novel protein according to the present invention. The culture of the transformant and its conditions may be essentially the same as those for the microorganism used.
In addition, the novel protein of the present invention can be recovered from the culture medium and purified according to a conventional method.

[0026]

EXAMPLES Example 1 Purification of Cellulase NCE2 The insolens strain was cultured in (N) medium at 37 ° C. ((N) medium composition: 5.0% Avicel, 2.0% yeast extract,
0.1% polypeptone, 0.03% calcium chloride, 0.03% magnesium sulfate, pH 6.8). After culturing for 7 days, the obtained culture solution was centrifuged at 7,000 rpm for 20 minutes to remove bacterial cells. After freeze-drying the obtained culture supernatant, it was dissolved in 25 mM Tris-HCl buffer (pH 9.0) at 5% (w / v), and then anion-exchange column (monoQ (60/100), Pharmacia Biotech Co., Ltd.) was used. 25 mM Tris-HCl buffer (pH 9.0)
Eluted at. The fraction that did not adsorb to this column was lyophilized and then dissolved in 50 mM Tris-HCl buffer (pH 9.4) to a concentration of 10% (w / v).
enyl-superose HR5 / 5, made by Pharmacia Biotech)
Apply to 50mM Tris-HCl buffer (pH 9.4) 1.5 ~ 0M
The product was eluted with an ammonium sulfate solution having a concentration gradient of, and fractionated. Of these, it was confirmed that the fraction obtained when the concentration gradient was 0 M exhibited cellulase activity in the neutral pH range. Chromatographically treated cellulase NCE
2 was analyzed by SDS-PAGE. When the electrophoretically separated proteins were stained with Coomassie Blue R250, the gel showed a single stained band. The apparent molecular weight of this protein was calculated to be approximately 63000 daltons, as compared to the mobility of known marker proteins.

Example 2 Partial amino acid of cellulase NCE2
Acid Sequence (1) Identification of N-Terminal Amino Acid Residue After the protein purified in Example 1 was electrophoresed using a Multi-Four II electrophoresis apparatus (Pharmacia Biotech), the protein was applied to a PVDF membrane (Millipore). Was electrically copied. After staining this with Ponceau S dye, 6300
The portion on which 0 dalton protein was blotted was cut out, washed with water, and then air-dried. This was applied to a protein sequencer PSQ-1 (manufactured by Shimadzu Corp.) to try to determine the amino acid sequence on the N-terminal side, but no amino acid cleaved by Edman degradation was obtained and the amino acid on the N-terminal side was modified and protected. There was found. Therefore, the blotted protein was analyzed by the method of Podell, DN et al. (Podell, DN e
t al., Biochem. Biophys. Res. Commun. 81: 176,1978.
), The modified N-terminal residue was removed with calf liver pyroglutamate aminopeptidase (Boehringer Mannheim), washed with water, air-dried, and then the amino acid sequence of the N-terminal side was changed to 6 residues by the protein sequencer PSQ-1. Decided. The sequences obtained were in the order Asn- (Cys) -Ala-Ser-Thr-Trp.

(2) Peptide mapping In order to determine the amino acid sequence inside the protein, the protein of Example 1 was subjected to column chromatography using a Model 172μ preparative HPLC system (manufactured by Perkin Elmer) (column: C8 220 × 2.1 mm). , 0-70% acetonitrile gradient) The main peak was collected. After freeze-drying this, 50 mM ammonium bicarbonate buffer (pH 7.8)
Dissolved in. About 1/50 molar amount of V8 protease (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 200 pmol of protein, and the mixture was reacted overnight at 37 ° C. The same degradation product was subjected to column chromatography using the above-mentioned HPLC system (column: C8 220 x 2.1 m
m, 0-35% acetonitrile gradient) was performed to collect 3 kinds of peptides. The amino acid sequence of the obtained peptide fragment was determined by a protein sequencer Model 476A (manufactured by Perkin Elmer). The amino acid sequence of the obtained peptide is shown below. V8-2: Val-Gln-Leu-Trp-Ala-Asn-Asn-Tyr-Tyr-Arg-Ser-Glu (12 residues) * * * * * * V8-3: Phe-Ile-Val-Asp- Gln-Gly-Arg-Ser-Gly-Lys-Gln-Pro-Thr (13 residues) * * * * * * V8-8: Ala-Gly-Gln-Tyr-Phe-Gln-Ala-Tyr-Phe- Glu (10 residues) These amino acid sequences show the amino acid sequence of cbh-II obtained from Trichoderma reesei (Chung Mong Cen et al., Bio / Technology5: 274-2).
78,1987), the protein is
It was strongly suggested that it is a kind of cellulase. In addition, the above sequence is referred to as Protein Identification Resource (PIR) R44.
0, March, 1994, or SWISS-PROT R28.0, January, 1
When compared with the sequence registered in 994, the above-mentioned cbh-
Although there was a sequence showing homology with II, none were the same, which revealed that the protein was a novel protein.

Example 3 Preparation of genomic DNA library Isolation of genomic DNA was performed by the method of Horiuchi et al. (Hiroyuki Horiuc
hi et al., J. Bacteriol. 170: 272-278,1988). First, H. The insolens strain was cultured in the (N) medium of Example 1 for 2 days, and the cells were collected by centrifugation (3500 rpm, 10 minutes). The obtained bacterial cells were treated with phenol, proteinase K and ribonuclease A, and further subjected to PEG precipitation to obtain genomic DNA. Next, H. The insolence genomic DNA was digested with EcoRI. This was subjected to agarose gel electrophoresis, and a 6-10 kbp fragment was recovered from the gel using a Sephaglass bandprep kit (Pharmacia Biotech). The same DNA fragment was ligated to the EcoRI arm of a phage vector, λZAP II cloning kit (Stratagene) using T4 DNA ligase. After ethanol precipitation, it was dissolved in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Hohn, B., Methods in Enzymol
ogy 68: 299-309, 1979) and packaged in a lambda head using the package component (Gigapack II packaging kit manufactured by Stratagene) frozen and infected with E. coli XL1-Blue MRF 'strain. It was The target gene was cloned using the 7 × 10 ⁴ phage library obtained by this method.

Example 4 Production of long-chain probe by PCR method
The adult DNA probe is H. Using the total DNA of Insolence as a template, P
A long probe amplified by the CR method was prepared and used. The primers are the peptides V8-2 and V8 of Example 2, respectively.
A DNA corresponding to the amino acid indicated by * in -3 was synthesized. That is, the report of Chung Mong Cen et al.
Bio / Technology 5: 274-278, 1987), V8-2 was predicted to be located on the N-terminal side of V8-3, and a synthetic oligonucleotide having the following sequence was prepared. The base sequences of the prepared primers are as follows. V8-2U: 5'-TGG GC (ACGT) AA (CT) AA (CT) TA (CT) TA-3 'V8-3L: 5'-CC (CT) TG (AG) TC (ACGT) AC (AGT ) AT (AG) AA
The -3 'PCR reaction was first carried out in H. 1 μM of each primer was added to 1 μg of insolence genomic DNA, and heat denaturation was performed at 94 ° C. for 10 minutes in the presence of dNTPs. After that, Taq polymerase (recombinant Taq, manufactured by Takara Shuzo Co., Ltd.) was added, and 94 ° C for 1 minute and 50 ° C for 2 minutes.
Amplification was carried out by repeating the reaction condition of 72 ° C. for 3 minutes for 30 minutes. As a result, 800 bp of DNA was amplified. This was used as a probe for the subsequent screening.

Example 5 Cellulase component of NCE2 gene
Cloning (1) Screening by plaque hybridization 100 ng of a DNA fragment of 800 bp amplified by the PCR method was previously labeled with an ECL direct DNA / RNA labeling detection system (manufactured by Amersham). Example 3
The phage plaques prepared as above were transferred to Hybond N ⁺ nylon transfer membrane (manufactured by Amersham), denatured with 0.4N sodium hydroxide, and then 5 times concentrated SSC.
(15 mM trisodium citrate, 150 mM sodium chloride)
It was washed with and dried to fix the DNA. According to the method described in the above-mentioned kit, after pre-hybridization (42 ° C.) for 1 hour, the previously labeled probe was added, and hybridization was performed for 4 hours (42 ° C.). The label was washed according to the method described in the kit. First, 0.4% SDS,
Washing with 0.5M concentration SSC containing 6M urea at 42 ° C. for 20 minutes was repeated twice, and then washing with 2X concentration SSC at room temperature for 5 minutes was performed twice. After washing the probe with a nylon membrane, soak it in the attached detection solution for 1 minute, and then
It was exposed to ECL (Amersham) to obtain 5 positive clones.

(2) Preparation of Phage DNA The DNA from the positive clone was prepared as a plasmid DNA according to the method described in the kit. From E. coli SOLR ^TM showing resistance to ampicillin, plasmid (pMII)
Was prepared, digested with EcoRI, and subjected to agarose gel electrophoresis. As a result, the plasmids derived from all the positive clones contained the 7 kbp EcoRI fragment. This is H.
It was in agreement with the results of genomic Southern analysis of Insolence.

Example 6 Determination of Nucleotide Sequence Nucleotide Sequence Analysis of Genomic DNA An ALF DNA Sequencer II (Pharmacia Biotech) was used as a nucleotide sequence analyzer. Sequencing gel can be Readymix gel (Pharmacia Biotech) or Hydrolink long ranger (AT Bioch
An acrylamide carrier manufactured by em) was used. ALF grade (Pharmacia Biotech) reagents were used as the various reagents (N, N, N ', N'-tetramethylethylenediamine, urea, ammonium persulfate) for gel preparation. For the nucleotide sequence decoding reaction, an auto read sequencing kit or an auto cycle sequencing kit (Pharmacia Biotech) was used. The gel preparation conditions, reaction conditions, and electrophoresis conditions were set with reference to the details of each instruction.

(1) Preparation of template plasmid for nucleotide sequence decoding (hereinafter simply referred to as "temperate") The following subclones were prepared in order to facilitate nucleotide sequence decoding. First, plasmid pMII was digested with EcoRI-XbaI and cloned into EcoRI-XbaI digested pUC119 (pM14-
1, Figure 1). Furthermore, those ligated to pUC119 digested with EcoRI-BamHI or BamHI-XbaI and digested with the same restriction enzyme were also prepared (respectively pM14-1U, pM14-1L, FIG. 2).

(2) Decoding of nucleotide sequence 10 μg of each temperate of pM14-1, pM14-1U, pM14-1L
After alkaline denaturation with M sodium hydroxide, the primer was annealed with the universal and reverse primers attached to the AutoRead Sequencing Kit to carry out extension reaction.
When the reaction product was decoded with a sequencer, it was 826-1
The base sequence of 661 bp was revealed. From this result, WATA-3
FITC-labeled sequencing primer 1 and WATA-32 were prepared, reacted with pM14-1, and further decoded.
As a result, the sequence of 293 to 2276 bp was decoded. EcoR
The connection between the I recognition site and the XbaI recognition site is WATA-34, WA
This was performed by preparing a primer called TA-35 and reacting it with pM14-1. WATA-31: 5'-CTC AAG GCA GGG TCG GTG ATC TG-3 '(23mer) WATA-32: 5'-CTA CGA CGA GAA GCA CTA CAT CG-3' (23mer) WATA-34: 5'-TCC TTA ATT GAC GTC GCA CCG AGC-3 '(24mer) WATA-35: 5'-CTT GTT TGA CCG TGT GCC GTG AGG-3' (24mer)

(3) Determination of nucleotide sequence Based on the result of (2) above, FITC of WATA-41 to WATA-53
Labeled sequencing primer DNAs were synthesized, and the reaction between these primers and pM14-1 was performed using an auto-read sequencing kit. First, 10 μg of plasmid was denatured with alkali, annealed with each primer, and reacted with T7 polymerase. In addition, the unreadable part due to the secondary structure of the temperate was decoded by auto cycle sequencing. WATA-41: 5'-TTC CCA CAC GAA GCA TTC TCC AGC-3 '(24mer) WATA-42: 5'-CGA CTT CGA GCT CGT CGA CCA CC-3' (23mer) WATA-43: 5'-TCG AGA CCC TCT CTG AGA TCC GC-3 '(23mer) WATA-44: 5'-ATC GAG CCC GAC TCG CTG GCC AAC-3' (24mer) WATA-46: 5'-CCC GCT ACG ACT ACC ACT GCG GTC- 3 '(24mer) WATA-47: 5'-GAC GTC GCT TAA TGC ATT GAG GGG-3' (24mer) WATA-48: 5'-TCT TGT CCA ACA ATG GCA GAT GC-3 '(23mer) WATA-49 : 5'-CCT CAG GGG CGG GCT TGA GGG CG-3 '(23mer) WATA-50: 5'-CGA TGT AGT GCT TCT CGT CGT AG-3' (23mer) WATA-51: 5'-GGG CTC GAT AAC CAG AAT CGT GC-3 '(23mer) WATA-52: 5'-CGA CGA GCA GGG TGT CGA CCG TG-3' (23mer) WATA-53: 5'-ACC GGT CCT GGT GGT CGA GGT GGC-3 '( As a result, the 2409 bp base sequence of the EcoRI-XbaI fragment was determined as shown in Sequence Listing 2.

Example 7 Determination of Intron For determination of intron, H. MRNA was prepared from insolence, cDNA was synthesized by reverse transcriptase, and the same region was determined from this and the nucleotide sequence of the genome.

(1) Preparation of total RNA H. The insolens were cultured in a cellulase induction medium, preferably the (N) medium of Example 1, for 2 days, and the cells were recovered by centrifugation (3500 rpm, 10 minutes). 2 g of the cells were washed with sterilized water, pulverized with a blender (Nippon Seiki's homogenizer AM-3) while frozen in liquid nitrogen. 10 ml of a denaturing solution containing 4M guanidine thiocyanate (4M
It was suspended in guanidine thiocyanate, 25 mM trisodium citrate, 0.5% sodium N-lauryl sarcosinate, 0.1 M mercaptoethanol). After stirring at room temperature for a few minutes, neutralize with 1 ml of 2M sodium acetate (pH 4.5) and add 10 ml of
TE saturated phenol was added and further stirred. To this, 2 ml of chloroform-isoamyl alcohol (24: 1) was added, and after thoroughly stirring, the cell components denatured with phenol were removed by centrifugation (3500 rpm, 10 minutes). The upper layer (aqueous layer) was sucked up, and the nucleic acid was precipitated with 10 ml of isopropanol. Nucleic acids were recovered from the same precipitate by centrifugation (3500 rpm, 10 minutes), and the precipitate was washed by re-centrifugation with 70% ethanol-water. The precipitate was dissolved in 3.5 ml TE buffer and then 880μ
l of 10 M lithium chloride solution was added, and the mixture was refrigerated at 5 ° C. for 2 hours, and then the precipitate was recovered by centrifugation (12000 rpm, 10 minutes). The precipitate was washed with 70% -ethanol and used as the total RNA fraction. The yield was 2.7 mg, and the yield was 0.14%.

(2) Preparation of poly A tail + RNA (= mRNA) mRNA was prepared using mRNA Purification Kit (Pharmacia Biotech). First (1)
1 mg of the total RNA prepared in 1. was dissolved in 1 ml of elution buffer, and the mixture was subjected to heat denaturation treatment at 65 ° C. for 10 minutes. After quenching in ice, 0.2 ml of sample buffer was added. This total amount of RNA solution was applied to an oligo (dT) cellulose column, washed with a high salt buffer three times and with a low salt buffer three times, and then eluted with an elution buffer heated to 65 ° C. This column operation was repeated twice to obtain an mRNA fraction. The yield was 19.2 μg, and the yield was 2%.

(3) Synthesis of cDNA For the cDNA synthesis, a time saver cDNA synthesis kit (Pharmacia Biotech) was used. First, 20 μg of mRNA
It was dissolved in μl of sample buffer. After heat treatment at 65 ° C for 10 minutes, the dithiothreitol solution and oligo (dT) primer were added to the first strand synthesis mix, and the mixture was reacted at 37 ° C for 1 hour. Then add all of this to the second strand mix for 30 minutes at 12 ° C, then 22
The reaction was carried out at 0 ° C for 1 hour, and this was designated as cDNA.

(4) Cloning of Cellulase Component NCE2 cDNA The synthesized whole cDNA was end-phosphorylated and ligated with EcoRI-NotI adaptor. A phage library was prepared from this whole amount using a λgt11 cloning kit (manufactured by Stratagene). About 10 ⁵ plaques were transferred onto a nylon membrane, and the PCR amplified fragment of Example 4 was used as a probe for screening. As a result, 15 positive plaques were selected. E. coli Y1088 strain was infected with this phage, and 8 hours later, phage particles were collected, and D. Grossberger's method (Grossberger, D., Neucleic acid Res. 15: 6737,198
According to 7), after proteinase K and phenol treatment,
Phage DNA was isolated by ethanol precipitation. This was digested with EcoRI and analyzed by agarose gel electrophoresis. The two phage-derived DNAs showed the same restriction map, and the EcoRI fragment was 1.8kbp in size, so it was cloned into pUC118. (PPR2), a template for nucleotide sequence decoding.

(5) Nucleotide sequence analysis of cDNA The sequencing reaction was performed using the auto-read sequencing kit as described above. The plasmid pPR2 was alkali-denatured with 2M sodium hydroxide and precipitated with ethanol. This single-stranded plasmid was used as a template and reacted with T7 polymerase. The primers are universal and reverse attached to the kit, the above synthetic primers WATA-42, WA
TA-43, WATA-44, WATA-32, WATA-46, WATA-47, WATA-
48, WATA-49, WATA-50, WATA-51, WATA-52 and WATA-53 were used. As a result, 478-535bp (Intron I), 1
030-11411bp (Intron II), 1762-181
5bp (Intron III) and 1990-2044bp (Intron I
It was revealed that there are a total of four introns (V).

In Sequence Listing 2, the non-translation initiation sequence of the intron sequence, its termination sequence, and the regulatory sequence within the intron are as follows (the numbers are the sequence numbers in Sequence Listing 2). Intron I: 478-483, 533-535, 518
~ 524 Intron II: 1030 to 1035, 1139 to 114
1, 1124-1130 Intron III: 1762-1767, 1813-181
5, 1799-1805 Intron IV: 1990-1995, 2042-204
4, 2029-2035

Example 8 H. Transformation plate for insolence
Construction of H.C. In order to reintroduce the NCE2 gene into Insolens, first, Aspergillus nidulan (Aspergillus nidulan
s) derived trpC gene promoter and terminator (Mu
llaney EJ et al., Mol. Gen. Genet. 199: 37-45,1
Bialaphos resistance gene (bar: Thompson,
CJ et al., EMBO J. 6: 2519-2523, 1987). A gene that can be expressed by insolence was prepared. NC this
It was introduced into the XbaI site of the plasmid pM14-1 containing the E2 gene to prepare a recombinant plasmid pNCE4.

Example 9 H. Transformation of Insolens Insolens was cultured in (S) medium at 37 ° C., and after 24 hours, the cells were collected by centrifugation at 3000 rpm for 10 minutes. (S)
The composition of the medium was as follows: glucose (3.
0%) is added and Avicel is removed. The obtained bacterial cells were washed with 0.5 M sucrose and filtered with a 0.45 μm filter to give a protoplast-forming enzyme solution (5 mg / ml Nov
ozyme 234, 5 mg / ml Cellulase Onozuka R-10, 0.5 M sucrose). The hyphae were protoplasted by shaking at 30 ° C for 60 to 90 minutes. After this suspension was filtered, it was centrifuged at 2500 rpm for 10 minutes to recover protoplasts, which were washed with SUTC buffer (0.5 M sucrose, 10 mM calcium chloride, 10 mM Tris-HCl (pH 7.5)).
The protoplasts prepared as described above were suspended in 1 ml of SUTC buffer, and 10 μg (10 μl) of the DNA (TE) solution of the plasmid pNCE4 obtained in Example 8 was added to 100 μl of the suspension.
Let stand in ice for 5 minutes. Next, 400 μl of PEG solution (60%
PEG4000, 10 mM calcium chloride, 10 mM Tris-HCl (pH 7.
5)) was added and the mixture was allowed to stand in ice for 20 minutes, 10 ml of SUTC buffer was added, and the mixture was centrifuged at 2500 rpm for 10 minutes. After suspending the collected protoplasts in 1 ml of SUTC buffer, 4000 rpm
After centrifugation for 5 minutes, the cells were finally suspended in 100 μl of SUTC buffer. Bilaphos (200 μg / ml) added YMG medium (1% glucose, 0.4%
Yeast extract, 0.2% malt extract, 1% agar (pH 6.8)) was overlaid with YMG soft agar and incubated at 37 ° C for 5 days.
The formed colony was used as a transformant.

Example 10 H. Insolence transformant
Cellulase activity of H. 61 strains introduced into Insolence and resistant to bialaphos were selected. Of these, 5 strains with high bialaphos resistance (750-10
00 μg / ml) was cultured in the (N) medium of Example 1. When the culture supernatant was analyzed by SDS-PAGE, NCE2 was secreted 2 to 4 times as much as the parent strain. Cellulase activity is WA Wo
od et al. (WA Wood, ST Kellogg. "Methods in
Enzymology, vol. 160, Biomass part A, Cellulose a
nd Hemicellulose "(ACADEMIC PRESS, INC.): p97) and the avicel-degrading activity of the supernatant was measured.
The transformant had a specific activity of 141% of that of the parent strain.

[0047]

[Table 1]

[0048]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 476 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Ala Lys Phe Phe Leu Thr Ala Ala Phe Ala Ala Ala Ala Leu Ala -20 -15 -10 Ala Pro Val Val Glu Glu Arg Gln Asn Cys Ala Pro Thr Trp Gly Gln -5 1 5 Cys Gly Gly Ile Gly Phe Asn Gly Pro Thr Cys Cys Gln Ser Gly Ser 10 15 20 25 Thr Cys Val Lys Gln Asn Asp Trp Tyr Ser Gln Cys Leu Pro Gly Ser 30 35 40 Gln Val Thr Thr Thr Thr Thr Thr Thr Ser Thr Ser Ser Ser Ser Thr Thr Thr 45 50 55 Ser Arg Ala Thr Ser Thr Thr Arg Thr Gly Gly Val Thr Ser Ile Thr 60 65 70 Thr Ala Pro Thr Arg Thr Val Thr Ile Pro Gly Gly Ala Thr Thr Thr 75 80 85 Ala Ser Tyr Asn Gly Asn Pro Phe Glu Gly Val Gln Leu Trp Ala Asn 90 95 100 105 Asn Tyr Tyr Arg Ser Glu Val His Thr Leu Ala Ile Pro Gln Ile Thr 110 115 120 Asp Pro Ala Leu Arg Ala Ala Ala Ser Ala Val Ala Glu Val Pro Ser 125 130 135 Phe Gln Trp Leu Asp Arg Asn Val Thr Val Asp Thr Leu Leu Val Glu 140 145 150 Thr Leu Ser Glu Ile Arg Ala Ala Asn Gln Ala Gly Ala Asn Pro Pro 155 160 165 Tyr Ala Ala Gln Ile Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala 170 175 180 185 Ala Ala Ala Ser Asn Gly Glu Trp Ala Ile Ala Asn Asn Gly Ala Asn 190 195 200 Asn Tyr Lys Gly Tyr Ile Asn Arg Ile Arg Glu Ile Leu Ile Ser Phe 205 210 215 Ser Asp Val Arg Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn 220 225 230 Met Val Thr Asn Met Asn Val Ala Lys Cys Ser Gly Ala Ala Ser Thr 235 240 245 Tyr Arg Glu Leu Thr Ile Tyr Ala Leu Lys Gln Leu Asp Leu Pro His 250 255 260 265 Val Ala Met Tyr Met Asp Ala Gly His Ala Gly Trp Leu Gly Trp Pro 270 275 280 Ala Asn Ile Gln Pro Ala Ala Glu Leu Phe Ala Lys Ile Tyr Glu Asp 285 290 295 Ala Gly Lys Pro Arg Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn 300 305 310 Tyr Asn Ala Trp Ser Ile Ser Ser Pro Pro Pro Tyr Thr Ser Pro Asn 315 320 325 Pro Asn Tyr Asp Glu Lys His Tyr Ile Glu Ala Phe Arg Pro Leu Leu 330 335 340 345 Glu Ala Arg Gly Phe Pro Ala Gln Phe Ile Val Asp Gln Gly Arg Ser 350 355 360 Gly Lys Gln Pro Thr Gly Gln Lys Glu Trp Gly His Trp Cys Asn Ala 365 370 375 Ile Gly Thr Gly Phe Gly Met Arg Pro Thr Ala Asn Thr Gly His Gln 380 385 390 Tyr Val Asp Ala Phe Val Trp Val Lys Pro Gly Gly Glu Cys Asp Gly 395 400 405 Thr Ser Asp Thr Thr Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu Glu 410 415 420 425 Asp Ala Leu Lys Pro Ala Pro Glu Ala Gly Gln Trp Phe Gln Ala Tyr 430 435 440 Phe Glu Gln Leu Leu Arg Asn Ala Asn Pro Pro Phe 445 450

SEQ ID NO: 2 Sequence length: 2409 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: H. Characteristic of Humicola insolens sequence Characteristic symbol: sig peptide Location: 389. . 457 Method of determining feature: E Symbol representing feature: mat peptide Location: 458. . 2098 Method for determining characteristics: E Characteristic symbol: intron Location: 478. . 535 Method for determining feature: E Feature symbol: intron Location: 1030. . 1141 Method of determining feature: E Symbol representing feature: intron Location: 1762. . 1815 Method for determining feature: E Feature symbol: intron Location: 1990. . 2044 Method of determining feature: E Characteristic symbol: cleanage-site Location: 688. . 693 Other information: SmaI Characteristic symbol: cleanage-site Location: 1253. . 1259 Other information: BamHI Characteristic symbol: cleanage-site Location: 1505. . 1510 Other information: BglII Characteristic symbol: cleanage-site Location: 1643. . 1648 Other information: StuI sequence TGCTGGACCT TGGATGCGTC TGCCGAGCTG TGCGTGCGGA AGAGTCGAGC GTGATTCCGG 60 CATCACTGAA CACTCGCTGG TTGCTGGTTC TGGAAGCGGT ACGTCCGGCG CAAACCAGCA 120 AAAGCAGGTT TGCGCTGCCT TGGCCTCCGT GAGAGGCATG ATGCCAAGGA TGAATGGTTC 180 CTCTGCGGAC TCAACCATCC GCACTTCGAG CCCGACGATC CGGGCCCCCT GCTCCGGCGC 240 GGAGAGCCGT GGTGAGCTCC AAGTGATGCG GAATCGGTGA TGTGCAAGAT GCGGAGGGCA 300 TAAAAAGGCT GTTTCCCACA CGAAGCATTC TCCAGCTTGT TTCCTCACGG CACACGGTCA 360 AACAAGTCTG TGCAGTACCT GGGACAAG ATG GCC AAG TTC TTC CTT ACT GCT 412 Met Ala Lys Phe Phe Leu Thr Ala -20 GCC TTT GCG GCT GCC GCT CTC GCC GCT CCC GTT GTT GAG GAG CGC CAG 460 Ala Phe Ala Ala Ala Ala Prole Ala Leu Ala Pro Glu Glu Arg Gln -15 -10 -5 1 AAC TGT GCC CCG ACT TG GTGAGCAATG GTGTTTCATG GATCGTGTCT TTGGATGTGC 517 Asn Cys Ala Pro Thr Tr 5 GGCTAACAAC CATTCCAG G GGC CAG TGC GGT GGC ATC GGC TTC AAT GGC I 566 p Gly Gly Gly Gly Phe Asn Gly 10 15 CCG ACT TGC TGC CAG TCT GGT AGC ACC TGC GTG AAG CAG AAC GAC TGG 614 Pro Thr Cys Cys Gln Ser Gly Ser Thr Cys Val Lys Gln Asn Asp Trp 20 25 30 TAC TCC CAG TGC TTG CCC GGT AGC CAG GTC ACC ACG ACC TCG ACT ACG 662 Tyr Ser Gln Cys Leu Pro Gly Ser Gln Val Thr Thr Thr Ser Thr Thr Thr 35 40 45 TCG ACT TCG AGC TCG TCG ACC ACC TCC CGG GCC ACC TCG ACC ACC AGG 710 Ser Thr Ser Ser Ser Ser Thr Thr Ser Arg Ala Thr Ser Thr Thr Arg 50 55 60 65 ACC GGT GGT GTG ACC TCG ATC ACC ACT GCT CCC ACC CGC ACC GTC ACC 758 Thr Gly Gly Val Thr Ser Ile Thr Thr Ala Pro Thr Arg Thr Val Thr 70 75 80 ATC CCT GGC GGT GCC ACC ACC ACG GCC AGC TAC AAC GGC AAC CCC TTC 806 Ile Pro Gly Gly Ala Thr Thr Thr Ala Ser Tyr Asn Gly Asn Pro Phe 85 90 95 GAG GGT GTC CAG CTC TGG GCC AAC AAC TAC TAC CGC TCT GAG GTC CAC 854 Glu Gly Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg Ser Glu Val His 100 105 110 ACC CTC GCC ATT CCT CAG ATC ACC GAC CCT GCC TTG AGG GCT GCG GCC 902 Thr Leu Ala Ile Pro Gln Ile Thr Asp Pro Ala Leu Arg Ala Ala Ala 115 120 125 TCG GCC GTC GCT GAG GTC CCG AGC TTC CAG TGG CTC GAC CGC AAC GTC 950 Ser Ala Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp Arg Asn Val 130 135 140 145 ACG GTC GAC ACC CTG CTC GTC GAG ACC CTC TCT GAG ATC CGC GCC GCG 998 Thr Val Asp Thr Leu Leu Val Glu Thr Leu Ser Glu Ile Arg Ala Ala 150 155 160 AAC CAG GCG GGC GCG AAC CCC CCG TAT GCC G GTAAGTGCGG TGTCACCACC 1049 Asn Gln Ala Gly Ala Asn Pro Pro Tyr Ala A 165 170 ACCAACCCTA ACCCTGACCC CTGACCACCA CATCATCAAC CCCCGCATATCCACC Gln Ile Val Val Tyr Asp 175 CTT CCT GAC CGC GAC TGC GCT GCC GCG GCT TCG AAC GGC GAG TGG GCG 1209 Leu Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Trp Ala 180 185 190 ATC GCC AAC AAC GGC GCC AAC AAC TAC AAG GGA TAC ATC AAC CGG ATC 1257 Ile Ala Asn Asn Gly Ala Asn Asn Tyr Lys Gly Tyr Ile Asn Arg Ile 195 200 205 210 CGC GAG ATT CTC ATT TCG TTC TCG GAT GTC CGC ACG ATT CTG GTT ATC 1305 Arg Glu Ile Leu Ile Ser Phe Ser Asp Val Arg Thr Ile Leu Val Ile 215 220 225 GAG CCC GAC TCG CTG GCC AAC ATG GTC ACC AAC ATG AAC GTC GCC AAG 1353 Glu Pro Asp Ser Leu Ala Asn Met Val Thr Asn Met Asn Val Ala Lys 230 235 240 TGC AGC GGT GCC GCC TCG ACC TAC CGC GAG TTG ACC ATC TAT GCC CTC 1401 Cys Ser Gly Ala Ala Ser Thr Tyr Arg Glu Leu Thr Ile Tyr Ala Leu 245 250 255 AAG CAG CTC GAC CTC CCG CAC GTC GCC ATG TAC ATG GAC GCC GGC CAC 1449 Lys Gln Leu Asp Leu Pro His Val Ala Met Tyr Met Asp Ala Gly His 260 265 270 GCT GGC TGG CTT GGC TGG CCC GCC AAC ATC CAG CCC GCT GCT GAG CTC 1497 Ala Gly Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala Glu Leu 275 280 285 290 TTC GCC AAG ATC TAC GAG GAT GCC GGC AAG CCC CGC GCC GTC CGC GGT 1545 Phe Ala Ly Ile Tyr Glu Asp Ala Gly Lys Pro Arg Ala Val Arg Gly 295 300 305 CTC GCC ACC AAC GTC GCC AAC TAC AAC GCC TGG AGC ATC TCG AGC CCG 1593 Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Ile Ser Ser Pro 310 315 320 CCG CCG TAC ACC AGC CCC AAC CCC AAC TAC GAC GAG AAG CAC TAC ATC 1641 Pro Pro Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His Tyr Ile 325 330 335 GAG GCC TTC CGC CCT CTC CTC GAG GCC CGC GGC TTC CCC GCC CAG TTC 1689 Glu Ala Phe Arg Pro Leu Leu Glu Ala Arg Gly Phe Pro Ala Gln Phe 340 345 345 350 ATC GTC GAC CAG GGC CGC AGC GGC AAG CAG CCC ACC GGC CAG AAG GAA 1737 Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly Gln Lys Glu 355 360 365 370 TGG GGC CAC TGG TGC AAT GCC ATT GTACGTTAAG GTTAGGGTTA CATATTTGCG 1791 Trp Gly His Trp Cys Asn Ala Ile 375 TTCCCATGAC TAACATCCTT CCAG GGC ACC GGC CTC GGT ATG Phe Gly Met Arg Pro Thr 380 385 GCC AAC ACC GGC CAC CAG TAC GTC GAC GCC TTC GTC TGG GTC AAG CCC 1890 Ala Asn Thr Gly His Gln Tyr Val Asp Ala Phe Val Trp Val Lys Pro 390 395 400 GGC GGT GAG TGC GAC GGC ACC AGC GAC ACG ACC GCT GCC CGC TAC GAC 1938 Gly Gly Glu Cys Asp Gly Thr Ser Asp Thr Thr Ala Ala Arg Tyr Asp 405 410 415 TAC CAC TGC GGT CTC GAG GAC GCC CTC AAG CCC GCC CCT GAG GCC GGC 1986 Tyr His Cys Gly Leu Glu Asp Ala Leu Lys Pro Ala Pro Glu Ala Gly 420 425 430 435 CAG GTGAGCACCA AACCCGACCA CAACAAGAAA TGTACCAAAG GCTAACCAAC TCCAG 2044 Gln TGG TTC CAA GCC TAC TTT GAG CAA TTA CTT CGT AAT GCC AAT CCG CCG 2092 Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Arg Asn Ala Asn Pro Pro 440 445 450 TTC TGA GCGGTTTGAG GCGGGGCCCCGATGTTGGCC CGATGTTGGC GATGTTTAGG ATCAAAAAGG 2148 Phe *** 2208 ATCACGTGCT CCCCGCATCG GCACAGACGT CGCTTAATGC ATTGAGGGGG TTGACAAAAT 2268 TCAAGTCTTC TTCTGTAAAT AGTTGGCATC TGCCATTGTT GGACAAGATT TAGTCTTTCG 2328 AGTATATACA CTTTGTTCCA GTTT 2

SEQ ID NO: 3 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asn Cys Ala Ser Thr Trp 1 5

SEQ ID NO: 4 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Gln Leu Trp Ala Asn Asn Tyr Tyr Arg Ser Glu 1 5 10

SEQ ID NO: 5 Sequence length: 13 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Phe Ile Val Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr 1 5 10

SEQ ID NO: 6 Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Gly Gln Tyr Phe Gln Ala Tyr Phe Glu 1 5 10

SEQ ID NO: 7 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TGG GCN AAY AAY TAY TA 17

SEQ ID NO: 8 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CC YTG RTC NAC DAT RAA 17

SEQ ID NO: 9 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CTC AAG GCA GGG TCG GTG ATC TG 23

SEQ ID NO: 10 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CTA CGA CGA GAA GCA CTA CAT CG 23

SEQ ID NO: 11 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TCC TTA ATT GAC GTC GCA CCG AGC 24

SEQ ID NO: 12 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CTT GTT TGA CCG TGT GCC GTG AGG 24

SEQ ID NO: 13 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TTC CCA CAC GAA GCA TTC TCC AGC 24

SEQ ID NO: 14 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CGA CTT CGA GCT CGT CGA CCA CC 23

SEQ ID NO: 15 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence TCG AGA CCC TCT CTG AGA TCC GC 23

SEQ ID NO: 16 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence ATC GAG CCC GAC TCG CTG GCC AAC 24

SEQ ID NO: 17 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCC GCT ACG ACT ACC ACT GCG GTC 24

SEQ ID NO: 18 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GAC GTC GCT TAA TGC ATT GAG GGG 24

SEQ ID NO: 19 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TCT TGT CCA ACA ATG GCA GAT GC 23

SEQ ID NO: 20 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCT CAG GGG CGG GCT TGA GGG CG 23

SEQ ID NO: 21 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CGA TGT AGT GCT TCT CGT CGT AG 23

SEQ ID NO: 22 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGG CTC GAT AAC CAG AAT CGT GC 23

SEQ ID NO: 23 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CGA CGA GCA GGG TGT CGA CCG TG 23

SEQ ID NO: 24 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ACC GGT CCT GGT GGT CGA GGT GGC 24

[Brief description of drawings]

FIG. 1 is a diagram showing a restriction enzyme map of plasmid pM14-1.

FIG. 2 is a diagram showing the constitution of NCE2 gene and each plasmid.

Continuation of front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical display location // C12Q 1/68 A 9453-4B (C12N 1/11 C12R 1: 645) (72) Inventor Kaoru Aoyagi Kanagawa 788 Kayayama, Odawara-shi, Oita Prefecture Meiji Seika Co., Ltd., Pharmaceutical Technology Research Institute (72) Inventor, Koji Yanai 5-3-1, Chiyoda, Sakado, Saitama Prefecture, Meiji Seika Co., Ltd., Institute for Biological Sciences (72) Toru Hamaya, Chiyoda, Sakado, Saitama Prefecture 5-3-1 Meiji Seika Co., Ltd. Biological Sciences Research Institute (72) Inventor Ken Murakami 788 Kayayama, Odawara-shi, Kanagawa Meiji Seika Co., Ltd.

Claims (9)

[Claims] 1. A protein comprising a part of the amino acid sequence shown in Sequence Listing 1 or a sequence of 1 to 453 or a derivative thereof. 2. The protein according to claim 1, further comprising the sequences -23 to -1 of Sequence Listing 1 on the N-terminal side. 3. The protein according to claim 1, which has cellulase activity. 4. A DNA sequence encoding the amino acid sequence according to any one of claims 1 to 3. 5. The DNA sequence according to claim 4, wherein the DNA sequence has a part or all of the base sequence shown in Sequence Listing 2. 6. A DNA sequence having a sequence of 389 to 2098 in Sequence Listing 2.
The DNA sequence according to claim 5, which has a nucleotide sequence of No. 7. The DN according to claim 5 or 6.
An expression vector comprising an A sequence. 8. A microorganism transformed with the expression vector according to claim 7. 9. A method according to claim 1, which comprises culturing the microorganism according to claim 8 and collecting the protein according to any one of claims 1 to 3 from the culture. The method for producing the protein according to the item.