JP2003164285A - New endoglucanase and endoglucanase gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new endoglucanase, a gene encoding the enzyme, a recombinant vector containing the gene and a transformant transformed by the vector and to provide a method for producing the endoglucanase by using the transformant. <P>SOLUTION: This invention provides a protein containing a specific amino acid sequence, the endoglucanase gene encoding the protein, the endoglucanase gene containing DNA containing a specific base sequence, the recombinant vector containing the gene, the transformant containing the recombinant vector and the method for producing the endoglucanase by collecting the endoglucanase protein from culture products obtained by culturing the transformant. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an endoglucanase, a gene encoding the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and an endoglucanase using the transformant. It relates to a manufacturing method.

[0002]

2. Description of the Related Art Endo-β-1,4-glucanase (EC
3.2.1.4, referred to herein as endoglucanase)
Is 1,4 of cellulose, lichenin, and β-D-glucan.
An enzyme that hydrolyzes a β-D-glycoside bond,
It is one of the enzymes called cellulase. Cellulose is a major component of plant cell walls, and endoglucanase, which cleaves glycoside bonds inside the cellulose, plays an important role in the decomposition of plant components. Therefore, endoglucanase is used in a wide range of industrial fields such as decomposition of plant biomass, detergents, food processing, and the like, and active research has been conducted. There are numerous reports on endoglucanase. For example, from Ascomycetes, Aspergillus niger (Table 1-11-502112), Aspergillus acuriatas (WO93 / 20193), Trichoderma reesei (JP-A-7-51071), and basidiomycetes, Macrofomia
Among the chytrids, such as Phaseolina (Gene, 158 (1): 125-8, 1995), those derived from the rumen fungus Orpinomyces joyonii (Gene, 245 (1), 119-126, 2000) are known. . Among those derived from bacteria, for example, Bacillus lautas (Gene, 93 (1): 55-60, 1990), Clostridium cellulovorans (Mol Gen Genet, 231 (3):
472-9, 1992) and the like are known. The above endoglucanases have also been reported for genes.

Regarding the endoglucanase derived from Aspergillus kawachii, the nucleotide sequence (Ito et al., Transla
tions from annotated coding regions in GenBank, BA
B62317, 2001) ・ Amino acid sequence (Ito et al., GenBank, AB05
5431, 2001) has been reported.

Regarding the filamentous fungus Trichoderma reesei, for which research on endoglucanase has been advanced, four types of endoglucanase and genes thereof have been reported in addition to the above (Gene, 63 (1): 11-22, 1988). ; Mol. Microbi
ol., 13 (2): 219-228, 1994; Eur. J. Biochem., 249
(2): 584-591, 1997; Appl. Environ. Microbiol., 64:
55-563, 1998).

Aspergillus oryzae and Aspergillus soya that are Aspergillus oryzae have been used for a long time in the production of brewed foods in Japan such as miso, soy sauce and sake, and have high enzyme productivity and long-term use. It is a microorganism that is particularly important in industry because of its high reliability in safety. Regarding these Aspergillus oryzae, only two types of cellulases (CelA and CelB) of Aspergillus oryzae and their genes are known (Appl Microbiol Biotech
nol, 46 (5-6): 538-44, 1996). Like Trichoderma reesei, filamentous fungi produce a wide variety of endoglucanases, and these are considered to act synergistically, and it has been expected to obtain novel endoglucanases and their genes in Aspergillus oryzae. Further, the endoglucanase known in Aspergillus oryzae was of a type having no cellulose binding domain which is considered to contribute to the decomposition of crystalline cellulose.

[0006]

DISCLOSURE OF THE INVENTION The present invention uses a novel endoglucanase, a novel gene encoding the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and the transformant. The present invention aims to provide a method for producing the endoglucanase.

[0007]

[Means for Solving the Problems] The present inventors have conducted extensive studies to solve the above problems, succeeded in cloning a novel endoglucanase gene from Aspergillus oryzae, and completed the present invention. That is, the present invention is as follows.
It is the protein of (a), (b) or (c). (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence represented by SEQ ID NO: 2 containing an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and Protein having glucanase activity (c) A protein having an amino acid sequence showing 85% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or a partial fragment thereof, and having endoglucanase activity

Furthermore, the present invention provides the following (a), (b) or
It is an endoglucanase gene encoding the protein of (c). (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence represented by SEQ ID NO: 2 containing an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and Protein having glucanase activity (c) A protein having an amino acid sequence showing 85% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or a partial fragment thereof, and having endoglucanase activity

Furthermore, the present invention provides the following (a), (b), (c)
Alternatively, it is an endoglucanase gene containing the DNA of (d). (a) DNA containing the base sequence represented by SEQ ID NO: 1 (b) Hybridizing with a nucleic acid containing the base sequence represented by SEQ ID NO: 1 or a complementary strand thereof under stringent conditions and exhibiting endoglucanase activity DNA encoding a protein having (c) A protein which hybridizes with a nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 2 or a nucleic acid containing a complementary strand thereof under stringent conditions and which has an endoglucanase activity DNA (d) A DNA which shows a sequence identity of 85% or more with the DNA of the nucleotide sequence represented by SEQ ID NO: 1 and which encodes a protein having an endoglucanase activity.

Further, the present invention is a recombinant vector containing the above gene. Furthermore, the present invention is a transformant containing the above recombinant vector. Further, the present invention provides
A method for producing an endoglucanase, which comprises culturing the transformant and collecting an endoglucanase protein from the resulting culture. Hereinafter, the present invention will be described in detail.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION 1. Endoglucanase of the present invention The endoglucanase of the present invention is a protein containing the amino acid sequence represented by SEQ ID NO: 2. The enzyme is, for example,
It can be purified from a culture of Aspergillus oryzae such as Aspergillus soja or Aspergillus oryzae. The enzyme can be obtained by expressing the endoglucanase gene cloned from Aspergillus oryzae in an appropriate host-vector system.

The endoglucanase of the present invention may have one or several amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 as long as it has enzymatic activity. Furthermore, as long as it has an endoglucanase activity, a protein containing an amino acid sequence having at least 85% or more, preferably 90% or more, and most preferably 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, or a protein thereof It may be a partial fragment.

In order to determine the sequence identity between two amino acid sequences or base sequences, the sequences are pre-conditioned for optimal comparison. For example, by inserting a gap in one sequence, the alignment with the other sequence is optimized. Then, the amino acid residues or bases at each site are compared. At a site in the first sequence,
If the same amino acid residue or base is present at the corresponding site in the second sequence, then those sequences are identical at that site. Sequence identities in two sequences are shown as a percentage of the number of identical sites between sequences with respect to the total number of sites (all amino acids or all bases).

In accordance with the above principle, the sequence identities in two amino acid sequences or base sequences are determined by Karlin and Al.
tschul's algorithm (Proc. Natl. Acad. Sci. USA 8
7: 2264-2268, 1990 and Proc. Natl. Acad. Sci. USA
90: 5873-5877, 1993). The BLAST program using such an algorithm was developed by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990).
Further, Gapped BLAST is a program for determining sequence identity with higher sensitivity than BLAST (Nucleic Acids Res. 25: 3389).
-3402, 1997). The above program is mainly used to search a database for a sequence showing high sequence identity to a given sequence. These are available, for example, on the website of the National Center for Biotechnology Information on the Internet.

Sequence identity between sequences is herein referred to as BLAST developed by Tatiana A. Tatusova et al.
2 Sequences software (FEMS Microbiol Lett., 17
4: 247-250, 1999). This software is National Center for Biotechnology
It is available and available on the Information website on the Internet. The programs and parameters used are as follows. In the case of amino acid sequence, the parameters are Open gap: 11 and extension gap: 1 penalties, using the blastp program.
gap x_dropoff: 50, expect: 10, word size: 3, Filte
r: Use ON. In the case of nucleotide sequences, the parameters are Reward for a match: 1, P using the blastn program.
enalty for amismatch: -2, Strand option: Both stra
nds, Open gap: 5 and extension gap: 2 penalties,
gap x_dropoff: 50, expect: 10, word size: 11, Fil
Use ter: ON. Both parameters are used as default values on the website.

However, if a sequence showing significant sequence identity is not found by the above BLAST software, FASTA software (WR Pearson and DJ Lipm
An, Proc. Natl. Acad. Sci., 85: 2444-2448, 1988) can be used to search the database for sequences showing sequence identity. FASTA software is available, for example, on the GenomeNet website. In this case as well, the default values are used for the parameters. For example, when searching for a nucleotide sequence, nr-nt is used for the database and a ktup value of 6 is used. However, in any case, if there is no overlap of 30% or more, 50% or more, or 70% or more of the whole, it is not necessarily presumed that they are functionally correlated, and therefore the sequence between the two sequences It is not used as a value indicating identity.

2. Cloning of endoglucanase gene The endoglucanase gene of the present invention can be obtained, for example, from Aspergillus oryzae such as Aspergillus soja or Aspergillus oryzae, other filamentous fungi, or other fungi. More specifically, for example, Aspergillus oryzae RIB40 strain can be mentioned. Total RNA is recovered by a conventional method from a culture of these cells in a medium that produces endoglucanase. As the medium, for example, a bran medium (2.78 g wheat bran added with 2.22 g deionized water and autoclaved at 121 ° C. for 50 minutes) can be used. Suitable time in the above medium, for example 30
After culturing for an hour, transfer an appropriate amount (eg, 1g) to a mortar filled with liquid nitrogen, crush with a pestle, and use Cathala.
Total RNA is prepared by the method described above (DNA, 2 (4): 329-335, 1983).

RT-PCR is performed using the obtained total RNA as a template. As the primer, any combination may be used as long as it is a combination capable of amplifying the endoglucanase gene of the present invention. For example, use of oligonucleotides having the sequences of SEQ ID NO: 3 and SEQ ID NO: 4 You can RT-PCR can be performed by a conventional method using a commercially available kit such as RNA LA-PCR Kit (manufactured by Takara Shuzo). The obtained DNA containing the endoglucanase gene of the present invention can be incorporated into a plasmid by a conventional method, for example. The nucleotide sequence of the DNA thus obtained can be determined by the Sanger method using a commercially available reagent and a DNA sequencer. Examples of the DNA containing the endoglucanase gene of the present invention thus obtained and the endoglucanase encoded thereby are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

In addition to the above, the endoglucanase gene of the present invention has an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 as long as it has endoglucanase activity. It may be one encoding a protein containing Such a gene can be obtained by various known mutagenesis methods in addition to the selection by hybridization described below.

The endoglucanase gene of the present invention can also be obtained using a selection method by hybridization as follows. Examples of gene sources include Aspergillus oryzae such as Aspergillus soja and Aspergillus oryzae. RNA from these organisms
Alternatively, genomic DNA is prepared and integrated into a plasmid or phage to prepare a library. Then, the nucleic acid used as a probe is labeled by a method suitable for the detection method.
The nucleic acid used as a probe may have a length that allows sufficient specificity, and for example, at least 100 bases or more, preferably 200 bases or more, and most preferably 450 bases or more of the sequence shown in SEQ ID NO: 1 or The thing including the whole is mentioned. Then, a clone that hybridizes to the labeled probe under stringent conditions is selected from the above library. Hybridization can be performed by colony hybridization for a plasmid library and plaque hybridization for a phage library. The stringent condition is a condition under which a signal of a specific hybrid is clearly distinguished from a signal of a non-specific hybrid, and varies depending on the hybridization system used, the type of probe, the sequence and the length. Such conditions can be determined by changing the temperature of hybridization, the temperature of washing and the salt concentration. For example, when even a non-specific hybrid signal is strongly detected, the specificity can be increased by raising the temperature of hybridization and washing and, if necessary, lowering the salt concentration of washing. Further, when no specific hybrid signal is detected, the hybrid can be stabilized by lowering the temperature of hybridization and washing and, if necessary, increasing the salt concentration of washing. Such optimization can be easily performed by a researcher in this technical field.

Specific examples of the stringent conditions include, for example, hybridization of 5 × SSC, 1.
Using 0% (W / V) blocking reagent for nucleic acid hybridization (manufactured by Boehringa Mannheim), 0.1% (W / V) N-lauroylsarcosine, 0.02% (W / V) SDS overnight (8-
About 16 hours), wash 0.5 × SSC, 0.1% (W /
V) SDS, preferably 0.1 x SSC, 0.1% (W / V) SDS, 15
Do twice a minute. The temperature of hybridization and washing is 52 ° C or higher, preferably 57 ° C or higher, more preferably
It is 62 ° C or higher, most preferably 67 ° C or higher. In addition, a nucleotide sequence showing 85% or more, preferably 90% or more, and most preferably 95% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1 is substantially equivalent to the endoglucanase of the present invention. It is considered to encode a protein having activity.

A DNA showing the sequence identity of the above-mentioned base sequence or the sequence identity of the encoded amino acid sequence can be obtained by using the hybridization as an index as described above. Among the obtained DNA groups of unknown function and public databases, for example, the above-mentioned B
It is also easy to find by searching using LAST software. Such a search is a method usually used by researchers in this technical field.

The DNA thus obtained encodes a protein having an endoglucanase activity,
As described below, it can be confirmed by incorporating into an appropriate vector, transforming an appropriate host, culturing the transformant, and measuring the endoglucanase activity.

2. Preparation of Recombinant Vector The recombinant vector of the present invention can be obtained by ligating the endoglucanase gene of the present invention on a suitable vector. As the vector, any vector can be used as long as it can produce endoglucanase in the host to be transformed. For example, a vector such as a plasmid, cosmid, phage, virus, chromosome-integrated type, or artificial chromosome can be used.

The above-mentioned vector may contain a marker gene for enabling the selection of transformed cells. Examples of marker genes include UR
Examples include genes that complement host auxotrophy, such as A3 and niaD, and resistance genes to drugs such as ampicillin, kanamycin, and oligomycin. Further, it is desirable that the recombinant vector contains a promoter or other control sequences capable of expressing the gene of the present invention in a host cell (for example, enhancer sequence, terminator sequence, polyadenylation sequence, etc.). Specific examples of the promoter include GAL1 promoter and amyB.
Examples thereof include a promoter and a lac promoter. You can also add a tag for purification. For example, by appropriately connecting a linker sequence downstream of the endoglucanase gene and connecting a base sequence encoding histidine for 6 codons or more, purification using a nickel column can be enabled.

3. Obtaining Transformant The transformant of the present invention can be obtained by transforming a host with the recombinant vector of the present invention. As a host,
There is no particular limitation as long as it can produce the endoglucanase of the present invention. For example, Saccharomyces
Yeasts such as cerevisiae and C. saccharomyces lucii
Aspergillus Sawyer, Aspergillus Orize,
It may be a filamentous fungus such as Aspergillus niger or a bacterium such as Escherichia coli or Bacillus subtilis. Transformation can be performed by a known method depending on the host. In the case of yeast, for example, using lithium acetate, Me
The method of thods Mol. Cell. Biol., 5, 255-269 (1995) can be used. In the case of a filamentous fungus, for example, polyethylene glycol and calcium chloride are used after protoplast formation, Mol. Gen. Genet., 218, 99-104 (19
The method of 89) can be used. When using bacteria, for example, by methods such as electroporation, Methods Enzy
The method of mol., 194, 182-187 (1990) can be used.

4. Production of endoglucanase The method for producing the endoglucanase of the present invention comprises culturing the transformant of the present invention and collecting the endoglucanase protein from the resulting culture. A suitable medium and culture method are selected depending on the type of host and the expression control sequence in the recombinant vector. For example, when the host is Saccharomyces cerevisiae and the expression control sequence is the GAL1 promoter, for example, cells pre-cultured in a liquid minimal medium containing raffinose as a carbon source, a liquid minimal medium containing galactose and raffinose as a carbon source. The endoglucanase of the present invention can be produced by diluting, inoculating, and culturing. Further, for example, when the host is Aspergillus soja and the expression control sequence is the amyB promoter, for example, the endoglucanase of the present invention can be produced by culturing in a liquid minimal medium containing maltose as a carbon source. .

When the host is Escherichia coli and the expression control sequence is the lac promoter, the endoglucanase of the present invention can be produced by culturing in a liquid medium containing IPTG. When the endoglucanase of the present invention is produced in the microbial cells or the cell surface,
The endoglucanase of the present invention can be obtained by separating the cells from the medium and treating the cells appropriately. When the endoglucanase of the present invention is produced in the culture solution, the endoglucanase of the present invention can be obtained by removing the bacterial cells by centrifugation, filtration or the like. In any case, according to a conventional method using ammonium sulfate fractionation, various chromatography, alcohol precipitation, ultrafiltration, etc.
The endoglucanase of the present invention can also be obtained as a highly pure product.

The activity of the endoglucanase of the present invention can be measured, for example, by the following method. [Method for measuring endoglucanase activity] The solution used is as follows. Substrate solution: 2% Azo-CM-cellulose (Megazyme) in 100
mM Sodium acetate, pH4.5 Substrate suspension: 2% Azo-alpha cellulose (Megazyme) in
100 mM sodium acetate, pH4.5 (insoluble) Substrate suspension: 2% Azo-avicel (Megazyme) in 100 mM sodium acetate, pH4.5 (insoluble) Stop solution: 4% sodium acetate trihydrate, Adjust to 0.4% zinc acetate, 80% ethanol, pH 5.0 with hydrochloric acid Mix 50 μl of substrate solution or substrate suspension with 50 μl of culture supernatant, and incubate at 37 ℃. The reaction time is adjusted depending on the intensity of activity. Add 250 μl of reaction stop solution, mix, and then at room temperature.
Leave for 10 minutes (A). In the blank, the reaction stop solution is added to the substrate solution, left for 10 minutes, and then the culture supernatant is added (B). Centrifuge at 10,000 rpm for 5 minutes at room temperature and remove the supernatant.
Measure the absorbance at 590 nm. The value obtained by subtracting the absorbance of the supernatant of (B) from the absorbance of the supernatant of (A) is defined as ΔOD, and the magnitude of this value is used as an index of activity.

[0030]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present description is not limited thereto. 1. Cloning of Aspergillus oryzae endoglucanase gene Aspergillus oryzae RIB40 conidia Approximately 100,000
Each of them was inoculated into 5 g of a bran medium (described above) in a 150 ml Erlenmeyer flask, and statically cultured at 30 ° C. for 30 hours. The culture was put into a mortar cooled by pouring liquid nitrogen, liquid nitrogen was added, and then the culture was carefully crushed using a pestle cooled with liquid nitrogen. Total RNA was extracted from the crushed culture by the method of Cathala et al. (DNA, 2 (4): 329-335, 1983). In addition, DNase using DNA-free kit (Amibion)
I treatment was performed to decompose the contaminated DNA. RT-PCR was carried out using 0.5 ug of the obtained total RNA as a template and an RNA LA-PCR kit (Takara Shuzo).

As the primer for the reverse transcription reaction, an adapter sequence attached to the 3'side of oligo dT, which is attached to the kit, is used. 30 ° C for 10 minutes, 50 ° C for 30 minutes, 99 ° C for 5 minutes, 5 ° C for 5 minutes went.
Then, a primer, a thermostable polymerase and the like were added to the reverse transcription reaction product according to the instructions attached to the kit, and PCR was performed. As a primer, an adapter primer attached to the kit and the following were used, and 5 ′ side was amplified immediately before the start codon and 3 ′ side was amplified from poly A part. C31FH: 5'-GGC AAA GCT TCA TTC AAA ATG AGA A-3 '(SEQ ID NO: 3) However, the start codon upstream part was modified in order to insert the recognition sequence of the restriction enzyme HinDIII.

The PCR reaction was conducted at 94 ° C for 2 minutes, then at 94 ° C for 10 seconds, and at 53 ° C.
Fifteen cycles of 20 seconds at 72 ° C and 1 minute 45 seconds were performed, and 72 ° C for 3 minutes. As a thermal cycler, DNA Engine P
CT-200 (manufactured by MJ Research) was used, and the temperature control method was based on the calculate control (the same applies to the following PCR).

Subsequently, PCR was carried out using the above-mentioned RT-PCR amplification product as a template and the above-mentioned C31FH and the following primers. As a thermostable DNA polymerase, Tbr EXT DNA
Polymerase (manufactured by Fynnzymes) was used, and the composition of the reaction solution was in accordance with the instructions attached to the polymerase. C31RX: 5'- TCT CTA GAT TCA ACA ACT CAC CAC ATC-3 '(SEQ ID NO: 4) However, the sequence downstream of the stop codon was modified so that the recognition sequence for the restriction enzyme XbaI could be inserted. The PCR reaction was carried out at 94 ° C. for 2 minutes, then 94 ° C. for 10 seconds, 56 ° C. for 20 seconds, 72 ° C. for 1 minute and 20 seconds for 30 cycles, and 72 ° C. for 5 minutes. When a part of the amplified product was electrophoresed on a 0.7% agarose gel, a band of about 1.3 kb was confirmed.

Next, TOPO TA Cloning Kit with TOP10
Cell (Invitrogen) was used to pCR2.
The E. coli TOP10 strain was transformed by incorporating it into the 1-TOPO vector, and 16 transformant clones were obtained. A plasmid was extracted from each transformant, digested with the restriction enzyme EcoRI (Takara Shuzo), and electrophoresed on a 0.7% agarose gel. As a result, a fragment of approximately 1.5 kb was confirmed in all clones. It was determined.

The base sequence is determined by CEQ DTCS as a reagent.
Using the Quick Start Kit (Beckman Coulter), as a sequencer CEQ 2000XL (Beckman Coulter
The Sanger method was used. Comparing the nucleotide sequences of the 16 clones obtained, 1 of them was
The clone was found to be free of PCR mutations. p
The nucleotide sequence from the start codon to the stop codon of the clone contained in C31EF is shown in SEQ ID NO: 1.

When the base sequence of SEQ ID NO: 1 was analyzed,
This DNA was found to encode a protein consisting of 490 amino acids. This amino acid sequence is shown in SEQ ID NO: 2. Furthermore, the amino acid sequence of this amino acid sequence was searched for a sequence having high sequence identity with a known amino acid sequence database. Search for NCBI blastp (http://www.ncbi.nlm.ni
h.gov/BLAST/) was used and nr was designated as the database. As a result, there was no matching sequence, and the highest sequence identity was found to be endoglucanase A (Ito et al., Translations from annotate) of Aspergillus kawachii.
d coding regions in GenBank, BAB62317, 2001) 334
There was 78% identity to the residues. Also, BLAST 2 Seque
Sequence identity was determined using ncep software blastp and was found to be 55% of 519 residues. In addition, NCBI BLAST
A homology search for the conserved sequence of (http://www.ncbi.nlm.nih.gov/BLAST/) revealed that it has a cellulose-binding domain in addition to the cellulase domain and is degraded to crystalline cellulose. It was suggested that it also showed activity.

A sequence having a high sequence identity was searched for with the nucleotide sequence of SEQ ID NO: 1. Search for NCBI blastn
(http://www.ncbi.nlm.nih.gov/BLAST/) was used and nr was designated as the database. As a result, the highest sequence identity was found to be 80% identity with the 282 base portion of the gene encoding the above protein (Ito et al., GenBank, AB055431, 2001). Also, BLAST 2 Seq
When the sequence identity over the entire length of the coding region was determined using blastp of uences software, it showed sequence identity in two parts: 75% in 993 bases and 79% in 67 bases.

2. Preparation of Endoglucanase Gene Recombinant Vector The endoglucanase gene on pC31EF was transformed into the expression vector pYES2 (Invitrogen) for Saccharomyces cerevisiae.
Sub-cloning). Specifically, the following method was used. First, pC31EF was digested with restriction enzymes HindIII and XbaI.
Cut with (Takara Shuzo) and electrophorese on agarose gel,
A band of about 1.5 kb containing the endoglucanase gene was cut out. From the cut band, QIAquick Gel Ext
The DNA fragment was extracted using the raction Kit (manufactured by Qiagen). On the other hand, pYES2 was cleaved with restriction enzymes HindIII and XbaI (Takara Shuzo), and electrophoresed on agarose gel to cut out a band of about 5.9 kb containing the vector portion. A DNA fragment was extracted from the cut out band using QIAquick Gel Extraction Kit (manufactured by Qiagen). Mix the above two types of DNA and use DNA Ligation Kit Ver.2 (Takara Shuzo)
Ligation was performed at 16 ° C for 1 hour. XL1-Blue Competent cells (manufactured by STRATA GENE) were transformed with the ligation product, and 3 transformants were obtained. A plasmid was extracted and purified from each transformant. All clones showed the same mobility on agarose gel electrophoresis, so we decided to use one of them, pYC31, as a representative.
"PYC31" was deposited at the Japan Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, under the deposit number P-18640. pYC31 has an autonomously replicating region in E. coli and an ampicillin resistance gene, an autonomously replicating region in Saccharomyces cerevisiae and the URA3 gene, and has a structure in which the endoglucanase gene is inserted between the GAL1 promoter and the CYC1 terminator. Therefore, it becomes possible to induce and produce endoglucanase by galactose in Saccharomyces cerevisiae cells.

3. Obtaining the transformant The INVSc1 strain carrying the uracil-requiring ura3-52 mutation was transformed with pYC31 by the transformation method using lithium acetate (described above), and the trait was obtained by selecting on the minimal agar medium containing no uracil. A transformant INV Sc1 / pYC31 strain was obtained. Further, as a negative control, INVSc1 / pYES2 strain was obtained by similarly transforming INVSc1 with the pYES2 vector plasmid.

4. Production of endoglucanase INVSc1 / pYC31 strain and negative control INVSc1
The / pYES2 strain was cultured to obtain endoglucanase. First,
Colonies of each strain were inoculated into 15 ml of SC medium (minimal medium) containing 2% raffinose but not uracil at 30 ° C.
It was shaken and cultured overnight. The OD at 600 nm of each overnight culture was measured and OD when inoculated into 50 ml of induction medium.
An amount of the culture solution of 0.4 was placed in a sterile centrifuge tube, and the supernatant was removed by centrifugation. Each of the obtained bacterial cells was suspended in 50 ml of SC medium (induction medium) containing 2% galactose and not containing uracil, and cultured at 30 ° C. with shaking. After 4 hours, 8 hours, and 24 hours, sample 10 ml each, centrifuge, and
The supernatant was collected. (Cryopreservation at -80 ℃) Endoglucanase activity was measured (Table 1)
Azo-CM- was added to the culture supernatant of the Sc1 / pYC31 strain for 4 hours and 8 hours.
It has a hydrolytic activity against cellulose, and although it is weak, A
It also showed hydrolytic activity against zo-avicel.

[0041]

[Table 1]

As described above, endoglucanase could be produced from the transformant transformed with the recombinant vector incorporating the DNA of SEQ ID NO: 1. Also,
SEQ ID NO: 2 encoded by the nucleotide sequence of SEQ ID NO: 1
It was also revealed that the protein containing the described amino acid sequence has endoglucanase activity.

[0043]

INDUSTRIAL APPLICABILITY The present invention provides a novel endoglucanase, an endoglucanase gene, a recombinant vector containing the gene, and a transformant. The present invention also provides a method for producing endoglucanase. According to the present invention, the above-mentioned endoglucanase can be improved in protein engineering. The present invention can also be used for enzyme production for food processing and improvement of microorganisms used for brewed food production. Further, the endoglucanase of the present invention shows a hydrolytic activity against Azo-avicel,
It is also expected to show a synergistic effect with other endoglucanases in the degradation of crystalline cellulose.

[0044]

[Sequence list] SEQUENCE LISTING <110> Kikkoman Corporation <120> Endoglucanase and Endoglucanase Gene <160> 4 <210> 1 <211> 1473 <212> DNA <213> Aspergillus oryzae (RIB40) <400> 1 ATGAGAATCG GCAACTTGAT CGTGGCTGCA AGTGCTGCAA GCCTGGTGCA TGCGTATCCC 60 ACGCGTGATA TCAAAAAGCG TGGCTCGGGA TTCACCTGGG TCGGCGTTAG CGAATCTGGC 120 GCTGAGTTCG GATCCAGCAT CCCAGGAACA CTGGGCACCG ACTACACTTG GCCTGATACA 180 TCTAAGATTC AGGTTCTGAG GGATGATGGA ATGAACGTCT TCCGTATTCC CTTCCTCATG 240 GAGCGTTTGG CACCCAACCA GATGACTGGA TCTCTGGACG CGACCTATTT GAAGGACCTG 300 AAATCTACTG TCCAAGCCGT CACTGATAGC GGTGCTTACG TCGTTCTCGA CCCCCACAAC 360 TACGGACGAT ACTCTGGAAG TATCATTTCT TCTACTTCTG ACTTTAAAAC ATTCTGGAAG 420 ACCGTCGCTG GAGAATTTGC ATCGAACGAG AAGGTCATCT TCGATACTAA CAACGAGTAC 480 CACGATATGG AACAGTCTCT CGTCCTTAGC CTAAACCAGG CCGCCATTGA CGGCATTCGC 540 GCGGCTGGAG CCACGACCCA ATACATTTTT GTTGAGGGCA ATTCGTACTC GGGTGCTTGG 600 AAGTGGGCCG ATACCAACGA CAACCTCAGT CAATTGACTG ACCCGCAAGA CAAGATTGTC 660 TACGAGATGC ACCAGTATCT CGATTCGGAT GGCTCTGGTA CCTCCGAGAC CTGTGCCAGC 720 TCAACCATCG GCAAGGAGAG GCTGCAGACC GCCACGGAGT GGTTGAAGAC CAACAACAAG 780 AAGGGTTTCC TGGGTGAATT CGCCGGCGGT GTCAACGAGC AGTGCGAGCA GGCTGTCGAG 840 GGTCTCCTCT CTTACATGTC AGACAACTCC GACGTCTGGA TGGGTGCCGA ATGGTGGTCG 900 GCTGGCCCCT GGTGGGGTTC CTACATGTAC AGCATGGAGC CGACTGACGG AACTGCTTAC 960 TCTACATACC TCCCTATCCT GAAGAAATAC TTCGTGGACG GCGCTGGCGC TAGCACCAGC 1020 TCATCGGCCA CTTCCGCAGC CCCCTCAACT GCAGCTGCCA GCACCTCTAC CTCTGTCAGT 1080 GCCTCGACTT CCTCTGCATC CTCGACTACC ATCAGTGCTG TCGAGTCCTC CTCCACTAGT 1140 AGTGTTGCCG AAGCCCCCTC AACCACCTCG GGAGTTGTCA CTGCCACGCC TACCCCATCT 1200 CACCCAGCCC CTCAGCCAAC CTCCAACTCG TCCAGCGCTT CATCAGGTGC GCCCACAAGC 1260 AGCGCCCCGA CGACTCTTGC GACTTCCCCG GCCTGTGGAT ACCAAACCAC TGTCACTGTG 1320 ACCGCCAGCA GATCAACCGC AGCACCCAGC AGCTCCGCAG GCGCCGTCGC CCACTACTAC 1380 CAGTGCGGTG GAATCAACTA CAGCGGACCC ACCACCTGCG AGAGCGGCTA CACCTGCGTC 1440 AAGCAGAACC CCTACTACAG CCAGTGCCTG TAA 1473 <210> 2 <211> 490 <212> PRT <213> Aspergillus oryzae (RIB40) <400> 2 Met Arg Ile Gly Asn Leu Ile Val Ala Ala Ser Ala Ala Ser Leu Val       1 5 10 15     His Ala Tyr Pro Thr Arg Asp Ile Lys Lys Arg Gly Ser Gly Phe Thr                  20 25 30     Trp Val Gly Val Ser Glu Ser Gly Ala Glu Phe Gly Ser Ser Ile Pro              35 40 45     Gly Thr Leu Gly Thr Asp Tyr Thr Trp Pro Asp Thr Ser Lys Ile Gln          50 55 60     Val Leu Arg Asp Asp Gly Met Asn Val Phe Arg Ile Pro Phe Leu Met      65 70 75 80     Glu Arg Leu Ala Pro Asn Gln Met Thr Gly Ser Leu Asp Ala Thr Tyr                      85 90 95     Leu Lys Asp Leu Lys Ser Thr Val Gln Ala Val Thr Asp Ser Gly Ala                 100 105 110     Tyr Val Val Leu Asp Pro His Asn Tyr Gly Arg Tyr Ser Gly Ser Ile             115 120 125     Ile Ser Ser Thr Ser Asp Phe Lys Thr Phe Trp Lys Thr Val Ala Gly         130 135 140     Glu Phe Ala Ser Asn Glu Lys Val Ile Phe Asp Thr Asn Asn Glu Tyr     145 150 155 160     His Asp Met Glu Gln Ser Leu Val Leu Ser Leu Asn Gln Ala Ala Ile                     165 170 175     Asp Gly Ile Arg Ala Ala Gly Ala Thr Thr Gln Tyr Ile Phe Val Glu                 180 185 190     Gly Asn Ser Tyr Ser Gly Ala Trp Lys Trp Ala Asp Thr Asn Asp Asn             195 200 205     Leu Ser Gln Leu Thr Asp Pro Gln Asp Lys Ile Val Tyr Glu Met His         210 215 220     Gln Tyr Leu Asp Ser Asp Gly Ser Gly Thr Ser Glu Thr Cys Ala Ser     225 230 235 240     Ser Thr Ile Gly Lys Glu Arg Leu Gln Thr Ala Thr Glu Trp Leu Lys                     245 250 255     Thr Asn Asn Lys Lys Gly Phe Leu Gly Glu Phe Ala Gly Gly Val Asn                 260 265 270     Glu Gln Cys Glu Gln Ala Val Glu Gly Leu Leu Ser Tyr Met Ser Asp             275 280 285     Asn Ser Asp Val Trp Met Gly Ala Glu Trp Trp Ser Ala Gly Pro Trp         290 295 300     Trp Gly Ser Tyr Met Tyr Ser Met Glu Pro Thr Asp Gly Thr Ala Tyr     305 310 315 320     Ser Thr Tyr Leu Pro Ile Leu Lys Lys Tyr Phe Val Asp Gly Ala Gly                     325 330 335     Ala Ser Thr Ser Ser Ser Ala Thr Ser Ala Ala Pro Ser Thr Ala Ala                 340 345 350     Ala Ser Thr Ser Thr Ser Val Ser Ala Ser Thr Ser Ser Ala Ser Ser             355 360 365     Thr Thr Ile Ser Ala Val Glu Ser Ser Ser Thr Ser Ser Val Ala Glu         370 375 380     Ala Pro Ser Thr Thr Ser Gly Val Val Thr Ala Thr Pro Thr Pro Ser     385 390 395 400     His Pro Ala Pro Gln Pro Thr Ser Asn Ser Ser Ser Ala Ser Ser Gly                     405 410 415     Ala Pro Thr Ser Ser Ala Pro Thr Thr Leu Ala Thr Ser Pro Ala Cys                 420 425 430     Gly Tyr Gln Thr Thr Val Thr Val Thr Ala Ser Arg Ser Thr Ala Ala             435 440 445     Pro Ser Ser Ser Ala Gly Ala Val Ala His Tyr Tyr Gln Cys Gly Gly         450 455 460     Ile Asn Tyr Ser Gly Pro Thr Thr Cys Glu Ser Gly Tyr Thr Cys Val     465 470 475 480     Lys Gln Asn Pro Tyr Tyr Ser Gln Cys Leu                 485 490     <210> 3 <211> 25 <212> DNA <213> Artifical Sequence <400> 3 GGCAAAGCTT CATTCAAAAT GAGAA 25 <210> 4 <211> 27 <212> DNA <213> Artifical Sequence <400> 4 TCTCTAGATT CAACAACTCA CCACATC 27

Front page continued (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/42 C12N 5/00 A (72) Inventor Osamu Hatamoto 399 Noda, Chiba Prefecture Foundation Noda Industrial Science Research Foundation In-house (72) Riki Masuda, 399 Noda, Chiba Prefecture, Noda City, Institute of Industrial Science, Noda Foundation (72) Inventor, Taiji Koyama, 250 Noda, Noda, Chiba Prefecture, Kikkoman Co., Ltd. (72) Inventor, Toshifumi Matsuda Chiba Prefecture Noda City 250 Noda Kikkoman Co., Ltd. (72) Inventor Kotaro Ito 250 Noda City Chiba Prefecture Noda 250 Kikkoman Co., Ltd. (72) Inventor Osamu Takahashi Noda City Chiba Prefecture Noda 250 Kikkoman Co., Ltd. (72) Inventor Kenichiro Matsushima 250 Noda, Noda, Chiba Prefecture Kikkoman Co., Ltd. F-term in the company (reference) 4B024 AA03 AA05 AA17 BA12 CA02 GA11 HA20 4B050 CC03 DD03 EE10 LL02 LL04 LL10 4B065 AA63Y AB01 AC14 BA02 BD14 CA31 CA41 CA41 CA41 CA41 CA41

Claims (6)

[Claims] 1. A protein according to the following (a), (b) or (c). (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added, and Protein having glucanase activity (c) A protein comprising an amino acid sequence showing 85% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or a partial fragment thereof, and having endoglucanase activity 2. An endoglucanase gene encoding the following protein (a), (b) or (c). (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added, and Protein having glucanase activity (c) A protein comprising an amino acid sequence showing 85% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or a partial fragment thereof, and having endoglucanase activity 3. An endoglucanase gene containing the following DNA (a), (b), (c) or (d): (a) DNA containing the base sequence represented by SEQ ID NO: 1 (b) Hybridizing with a nucleic acid containing the base sequence represented by SEQ ID NO: 1 or a complementary strand thereof under stringent conditions and exhibiting endoglucanase activity DNA encoding a protein (c) that encodes a protein that hybridizes with a nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 2 or a nucleic acid containing a complementary strand thereof under stringent conditions and has an endoglucanase activity DNA (d) A DNA that shows a sequence identity of 85% or more with the DNA of the nucleotide sequence represented by SEQ ID NO: 1 and encodes a protein having an endoglucanase activity. 4. A recombinant vector containing the gene according to claim 2 or 3. 5. A transformant containing the recombinant vector according to claim 4. 6. A method for producing an endoglucanase, which comprises culturing the transformant according to claim 5 and collecting an endoglucanase protein from the resulting culture.