Endoglucanase and its uses

Abstract

Provided is endoglucanase having excellent heat resistance.　This endoglucanase is characterized by the following: (A) a polypeptide has at least 80% amino acid identity with the amino acid sequence represented by SEQ ID NO: 1; (B) the molecular weight of the polypeptide portion is approximately 31 kDa or greater; and (C) the endoglucanase has a sugar chain, and the molecular weight as determined by SDS-PAGE is approximately 39 kDa or greater.

Description

[Technical field]

[0001] The art is disclosed relating to endoglucanases. [Background technology]

[0002] There are various methods for saccharifying cellulose, but enzymatic saccharification methods that use less energy and have high sugar yields are currently the mainstream. Cellulase, a cellulose-degrading enzyme, can be broadly divided into cellobiohydrolase, which acts on the crystalline regions of cellulose, and endoglucanase, which acts from within the cellulose molecular chain to reduce the molecular weight. β-Glucosidase is an enzyme that acts on water-soluble oligosaccharides or cellobiose, catalyzing the reaction of hydrolyzing the β-glycosidic bond.

[0003] Endoglucanases (endo β-1,4-glucanases (EC 3.2.1.4)) are enzymes that are effective in the hydrolysis of cellulose because they hydrolyze the β-1,4-glycosidic bonds between D-glucose units, which are the building blocks of cellulose. Endoglucanases catalyze the endo-type hydrolysis of β-1,4-bonds not only in cellulose but also in cellulose derivatives such as carboxymethylcellulose and hydroxyethylcellulose, lignin, mixed β-1,3-glucans such as cereal β-D-glucans, xyloglucans, and other plant materials that contain cellulose moieties. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4228073 [Non-patent literature]

[0005] [Non-Patent Document 1] Kishishita et al., J Ind Microbiol Biotechnol. 2015 42:137-141 Summary of the Invention [Problem to be solved by the invention]

[0006] When treating plant samples or textile products using endoglucanases, the hydrolysis efficiency is higher when the treatment is performed at high temperatures. Furthermore, endoglucanases that are not inactivated at high temperatures can not only hydrolyze cellulose at high temperatures, but also inactivate and denature other enzymes and other contaminants under high temperature conditions, thereby enabling the target product to be obtained with a high purity, and the endoglucanase itself can also be efficiently purified. Furthermore, such thermostable endoglucanases can be more efficiently recovered and reused after use. Therefore, one challenge is to provide endoglucanases with superior thermostability. [Means for solving the problem]

[0007] The following representative inventions are provided: Section 1. An endoglucanase having the following characteristics (A) to (C): (A) a polypeptide having an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO:1 (B) The molecular weight of the polypeptide portion is approximately 31 kDa. (C) It has a glycan and has a molecular weight of approximately 39 kDa or more as measured by SDS-PAGE. Section 2. The endoglucanase according to Item 1, further having the following characteristic (D): (D) The residual endoglucanase activity after heat treatment at 100°C for 60 minutes is 30% or more. Section 3. Item 3. The endoglucanase according to Item 1 or 2, in which the asparagine residues at positions 104, 175, 180, 221, 227, and 258 of the amino acid sequence of SEQ ID NO:1 are conserved. Section 4. A method for producing the endoglucanase according to any one of Items 1 to 3, comprising expressing a DNA encoding the endoglucanase according to any one of Items 1 to 3 in a strain of the genus Aspergillus as a host. Section 5. Item 4. A method for producing reducing sugars, comprising allowing the endoglucanase according to any one of Items 1 to 3 to act on a sample containing cellulose at a temperature of 70° C. or higher. Section 6. Exposing the endoglucanase according to any one of Items 1 to 3 to a temperature of 80°C or higher, and centrifuging the endoglucanase exposed to a temperature of 80°C or higher; Item 4. A method for isolating the endoglucanase according to any one of Items 1 to 3, comprising: Effect of the Invention

[0008] The present invention provides an endoglucanase having superior thermostability. In a preferred embodiment, a means for efficiently producing reducing sugars from cellulose is provided. In a preferred embodiment, a means for efficiently purifying endoglucanase is provided. [Brief description of the drawings]

[0009] [Figure 1] The molecular weight of endoglucanase was measured by SDS-PAGE. The left side shows the result of CBB staining, and the right side shows the result of PAS staining. Lane M is the molecular weight marker, lanes 1 and 2 show EGPf expressed in Aspergillus niger, 2 and 3 show EGPf expressed in Aspergillus luciensis, 4 and 5 show the culture supernatant of Aspergillus niger NS48, 4 and 5, respectively, and the arrows indicate the EGPf bands. [Diagram 2] The results of investigating the effect of glycans on the thermostability of endoglucanase are shown in the following figure. Lane M is a molecular weight marker, lane 1 is EGPf not heated, lane 2 is EGPf heated at 100°C for 1 hour, lane 3 is EGPf heated at 100°C for 5 hours, lane 4 is EGPf heated at 100°C for 6 hours, lane 5 is EGPf heated at 100°C for 7 hours, and lane 6 is EGPf heated at 100°C for 8 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 1. Endoglucanase The endoglucanase preferably has the following characteristics (A) to (C). (A) Having a polypeptide having an amino acid sequence that has 80% or more identity to the amino acid sequence of SEQ ID NO:1. (B) The molecular weight of the polypeptide portion is approximately 31 kDa. (C) It has a glycan and has a molecular weight of approximately 39 kDa or more as measured by SDS-PAGE.

[0011] The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence (not including the signal peptide) constituting the wild-type endoglucanase derived from the hyperthermophilic archaeon Pyrococcus furiosus. The identity between the amino acid sequence of the polypeptide possessed by the endoglucanase and the amino acid sequence of SEQ ID NO: 1 is preferably 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0012] The identity of amino acid sequences can be calculated using a commercially available analysis tool or an analysis tool available via an electric communication line (Internet), for example, ClustalW ver2.1 Pairwise Alignment (http: / / clustalw.ddbj.nig.ac.jp / index.php?lang=ja) using default (initial setting) parameters. Alternatively, it can be calculated using the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: / / www.ncbi.nlm.nih.gov / BLAST / using default parameters.

[0013] The molecular weight of the polypeptide portion of the endoglucanase is preferably about 31 kDa. The molecular weight calculated from the molecular weights of the constituent amino acid residues of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is 30,522 Da. In one embodiment, about 31 kDa means that the molecular weight calculated from the molecular weights of the amine residues constituting the polypeptide is in the range of 30 kDa to 32 kDa. In another embodiment, about 31 kDa means that the molecular weight of only the polypeptide portion measured by SDS-PAGE is about 31 kDa.

[0014] From the viewpoint of having excellent heat resistance, it is preferable that the endoglucanase has a sugar chain. It is considered that the presence of a sugar chain suppresses changes in three-dimensional structure caused by heat and aggregation of denatured polypeptides. The amount of sugar chain that the endoglucanase has is not particularly limited, but the molecular weight of the endoglucanase containing the sugar chain measured by SDS-PAGE is preferably about 35 kDa or more, about 36 kDa or more, about 37 kDa or more, about 38 kDa or more, about 39 kDa or more, or about 40 kDa or more. The upper limit of the molecular weight of the endoglucanase containing the sugar chain measured by SDS-PAGE is not particularly limited, but is, for example, about 300 kDa, about 200 kDa, about 150 kDa, about 100 kDa, about 90 kDa, about 80 kDa or less, about 70 kDa or less, or about 60 kDa or less. As described above, the molecular weight of the polypeptide portion of the endoglucanase is preferably about 31 kDa. Therefore, the molecular weight of the sugar chain possessed by the endoglucanase is preferably about 4 kDa or more, 5 kDa or more, 6 kDa or more, 7 kDa or more, 8 kDa or more, or 9 kDa or more.

[0015] The endoglucanase preferably has a residual endoglucanase activity of 30% or more after heat treatment at 100°C for 60 minutes. The residual activity rate (%) is measured by comparing the endoglucanase activity of the endoglucanase before heat treatment (activity after heat treatment / activity before heat treatment x 100). In one embodiment, the residual activity rate is preferably 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more. The heat treatment can be carried out by adding endoglucanase to a sodium phosphate buffer (pH 7.0) with a final concentration of 200 mM in an amount to give a concentration of 1 U / ml, dissolving or suspending the endoglucanase, setting the temperature in a thermostatic bath to a predetermined temperature, and maintaining the mixture for a predetermined time (e.g., 60 minutes).

[0016] Endoglucanase activity can be measured by any method, but in this document, unless otherwise specified, it is measured by the Somogyi-Nelson method. Specifically, 200 μl of 50 mM sodium acetate buffer (pH 5.0) containing carboxymethylcellulose sodium salt at a final concentration of 1% by weight as a substrate is prepared, a certain amount of endoglucanase is added to it to start the reaction, and the amount of reducing sugar generated at 70°C for 10 minutes is quantified by the Somogyi-Nelson method. The amount of enzyme that liberates reducing sugar equivalent to 1 μmol of glucose per minute is defined as 1 U, and endoglucanase activity per unit weight can be measured.

[0017] The endoglucanase may have any mutation in the amino acid sequence of SEQ ID NO: 1, so long as it has the desired properties as described above. In one embodiment, in terms of having the above-mentioned glycan, the endoglucanase preferably conserves at least one of the asparagine residues at positions 104, 175, 180, 221, 227, and 258 of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the endoglucanase preferably conserves at least two, three, four, five, or all of the aspartic acid residues. In a preferred embodiment, the endoglucanase preferably retains all of the aspartic acid residues. It is considered that the endoglucanase has a glycan when a glycan (N-glycan) is bound to these aspartic acid residues.

[0018] From the viewpoint of maintaining endoglucanase activity and / or higher-order structure of the protein, it is preferable that the endoglucanase retains at least one of the amino acid residues Trp at position 35, Trp at position 72, Glu at position 129, Glu at position 148, Tyr at position 194, and Glu at position 241 in the amino acid sequence of SEQ ID NO: 1. It is considered that Trp at position 35, Trp at position 72, Glu at position 129, and Tyr at position 194 in the amino acid sequence of SEQ ID NO: 1 are involved in the active center, and Glu at position 148 and Glu at position 241 are involved in substrate binding. In one embodiment, it is preferable that the endoglucanase retains at least two, three, four, five, or all of the amino acid residues. In a preferred embodiment, it is preferable that the endoglucanase retains all of the amino acid residues.

[0019] Any method can be used to introduce a mutation into a polypeptide, and any method known in the art can be appropriately selected, such as restriction enzyme treatment, treatment with exonuclease or DNA ligase, site-directed mutagenesis, random mutagenesis, etc.

[0020] Endoglucanase can be produced by genetic engineering techniques using the DNA described below. In addition, endoglucanase can also be produced by general protein chemical synthesis methods (e.g., liquid phase and solid phase methods) based on the information of the amino acid sequence shown in SEQ ID NO: 1.

[0021] 2. DNA encoding endoglucanase The base sequence of the DNA encoding the endoglucanase is not particularly limited. In one embodiment, the DNA preferably has a base sequence having a certain level of identity to the base sequence of SEQ ID NO: 2. The certain level of identity is, for example, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. SEQ ID NO: 2 is a base sequence that encodes the amino acid sequence of SEQ ID NO: 1.

[0022] The identity of nucleotide sequences can be calculated using an analytical tool that is commercially available or available via an electric communication line (Internet), for example, software such as FASTA, BLAST, PSI-BLAST, SSEARCH, etc. Specifically, the main initial conditions generally used for BLAST searches are as follows: In other words, in Advanced BLAST 2.1, the identity (%) of nucleotide sequences can be calculated by performing a search using the program blastn with various parameters set to default values.

[0023] In one embodiment, the DNA is preferably DNA that exists in an isolated state. Here, "isolated DNA" refers to a state in which it is separated from components such as other nucleic acids and proteins that coexist in the natural state. However, it may contain some other nucleic acid components such as adjacent nucleic acid sequences in the natural state (e.g., promoter region sequences and terminator sequences). In the case of DNA prepared by genetic engineering techniques such as cDNA molecules, the "isolated" state preferably does not substantially contain cellular components, culture medium, etc. Similarly, in the case of DNA prepared by chemical synthesis, "isolated DNA" preferably means that it does not substantially contain precursors (raw materials) such as dNTPs, chemicals used in the synthesis process, etc.

[0024] The DNA can be easily obtained based on the base sequence of SEQ ID NO:2 using chemical DNA synthesis methods (eg, the phosphoramidite method) or genetic engineering techniques.

[0025] 3. Vector The vector preferably contains the above DNA in an expressible manner. The type of vector can be appropriately selected in consideration of the type of host cell. For example, a plasmid vector, a cosmid vector, a phage vector, a virus vector (adenovirus vector, retrovirus vector, herpes virus vector, etc.) can be mentioned.

[0026] Examples of vectors that can be expressed in yeast include pBR322, pJDB207, pSH15, pSH19, pYepSec1, pMFa, pYES2, pHIL, pPIC, pAO815, and pPink. Examples of vectors that can be expressed in insects include pAc, pVL, and pFastbac. Examples of vectors that can be expressed in the genus Aspergillus include pSENS2512, pAUR316, pPTR I, and pPTR II.

[0027] When eukaryotic cells are used as host cells, the expression vector to be used may contain a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, etc., upstream of the polynucleotide to be expressed, and may further contain a replication origin, a secretion signal, an enhancer, and / or a selection marker, as necessary.

[0028] 4. Transformants The transformant is preferably one transformed with the above vector, and is preferably one capable of binding a sugar chain to the endoglucanase. In the transformant, the vector may exist autonomously in the host cell, or may be incorporated into the genome by homologous recombination or non-homologous recombination. From the viewpoint of binding a sugar chain to the endoglucanase, the host is preferably a eukaryotic cell, and examples of the host that can be mentioned include yeasts such as Saccharomyces, Piscia, and Kluyveromyces, fungal cells such as Aspergillus, Penicillium, Talaromyces, Trichoderma, Hypocrea, and Acremonium, insect cells such as Drosophila S2, Spodoptera Sf9, and silkworm cultured cells, and plant cells. In one embodiment, the preferred host is a fungus, more preferably a fungus of the genus Aspergillus, and even more preferably Aspergillus niger, Aspergillus luciensis, Aspergillus oryzae, Aspergillus sojae, Aspergillus nidulans, or Aspergillus aculeatus.

[0029] The recombinant expression vector can be introduced into a host cell by a conventional method, including, but not limited to, the competent cell method, the protoplast method, the electroporation method, the microinjection method, the liposome fusion method, and the like.

[0030] Since the transformant is capable of producing endoglucanase, it can be used to produce endoglucanase, and also, in its transformed state, it can be used to produce reducing sugars such as glucose, cellobiose, and cellooligosaccharides from a sample containing cellulose.

[0031] 5. Method for Producing Endoglucanase Using a Transformant The above-mentioned endoglucanase can be produced by culturing the above-mentioned transformant and recovering the endoglucanase from the culture. The culture can be subcultured or batch cultured using a medium suitable for the host. The culture can also be used to recover endoglucanase produced inside and outside the transformant. Using endoglucanase activity as an indicator, the process can be continued until an appropriate amount is obtained.

[0032] As the medium, various commonly used mediums can be appropriately selected and used depending on the type of host cell, and the culture can be performed under conditions suitable for the growth of the host cell. For example, for the culture of the genus Aspergillus, nutrient media such as PD medium and DP medium, or a minimal medium such as Czapek-Dox medium supplemented with a carbon source, a nitrogen source, a vitamin source, etc. can be used.

[0033] Culture conditions can be appropriately set depending on the type of host. Usually, the culture is performed at 16 to 42°C, preferably 25 to 37°C, for 5 to 168 hours, preferably 8 to 72 hours. Depending on the host, either shaking culture or stationary culture is possible, but stirring and / or aeration may be performed as necessary. When an inducible promoter is used for gene expression, the culture can also be performed by adding a promoter inducer to the medium.

[0034] Purification or isolation of endoglucanase from the culture supernatant can be carried out by appropriately combining known techniques, such as ammonium sulfate precipitation, precipitation with a solvent such as ethanol, dialysis, ultrafiltration, acid extraction, and techniques using various types of chromatography (e.g., gel filtration chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography, high performance liquid chromatography, etc.). Examples of carriers used in affinity chromatography include carriers to which an antibody against endoglucanase is bound, and when a peptide tag is added to endoglucanase, a carrier to which a substance having affinity for the peptide tag is bound can also be used.

[0035] When endoglucanase accumulates within the host cells, the transformed cells are disrupted, and endoglucanase can be purified or isolated from the centrifugal supernatant of the disrupted cells in the same manner as described above. For example, after completion of the culture, the cells collected by centrifugation are suspended in a cell disruption buffer (20 to 100 mM Tris-HCl (pH 8.0), 5 mM EDTA) and disrupted by ultrasonication, and the disrupted solution is centrifuged at 10,000 to 15,000 rpm for 10 to 15 minutes to obtain a supernatant. The precipitate after centrifugation can be solubilized with guanidinium hydrochloride or urea, etc., as necessary, and then further purified.

[0036] 6. Method for Producing Reducing Sugars Using Endoglucanase By allowing endoglucanase to act on a sample containing cellulose (e.g., a biomass resource), the cellulose can be decomposed to produce a sugar solution containing reducing sugars. Examples of reducing sugars include glucose, cellobiose, and cellooligosaccharides. When a biomass resource is used as the sample containing cellulose, it is preferable to use other enzymes such as cellulase in addition to the endoglucanase described above to produce a sugar solution more efficiently.

[0037] The type of cellulose-containing sample is not particularly limited as long as it can be decomposed by endoglucanase, and examples include bagasse, wood, bran, wheat straw, rice straw, pasture grasses such as grasses or legumes, corn cobs, bamboo grass, pulp, rice husks, wheat bran, soybean meal, soybean okara, coffee grounds, rice bran, etc.

[0038] The temperature at which endoglucanase is reacted with a sample containing cellulose is preferably 60°C or higher, 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, 90°C or higher, 95°C or higher, 98°C or higher, 99°C or higher, or 100°C or higher.

[0039] The production of sugar solution containing reducing sugar from a sample containing cellulose can be carried out according to a known method. The biomass resource to be used may be either dry or wet, but is preferably pulverized to a size of 100 to 10,000 μm in advance to improve the processing efficiency. The pulverization can be carried out using a device such as a ball mill, a vibration mill, a cutter mill, or a hammer mill. The pulverized biomass resource can be immersed in water, steam, or an alkaline solution and subjected to a high-temperature treatment or a high-temperature and high-pressure treatment at a temperature between 60 and 200°C to further improve the enzyme processing efficiency. For example, the alkaline treatment can be carried out using caustic soda, ammonia, or the like. The biomass sample thus pretreated can be suspended in an aqueous medium, endoglucanase and other cellulases are added, and the mixture is heated with stirring to decompose or saccharify the biomass resource.

[0040] When endoglucanase is allowed to act on a sample containing cellulose in an aqueous solution, the conditions of the reaction solution, such as pH, may be within a range in which endoglucanase is not inactivated.

[0041] The sugar solution containing reducing sugars may be used as it is, or may be used as a dried product after removing water, and may be further isomerized or decomposed by a chemical reaction or an enzymatic reaction depending on the purpose. The sugar solution or a fraction thereof may be used as a raw material for alcohols such as methanol, ethanol, propanol, isopropanol, butanol, and butanediol by fermentation, for example.

[0042] 7. Method for isolating endoglucanase During purification of endoglucanase, an endoglucanase-containing sample can be treated at 80°C or higher, thereby inactivating contaminating proteins and obtaining endoglucanase with high purity. In addition, by treating a solution containing endoglucanase and other contaminants (e.g., other enzymes and microorganisms), such as a solution obtained after allowing endoglucanase to act on a sample containing cellulose, at 80°C or higher, it is possible to inactivate the contaminating enzymes and microorganisms while maintaining the activity of endoglucanase. In one embodiment, the treatment temperature can be 80°C or higher, 85°C or higher, 90°C or higher, 95°C or higher, 98°C or higher, or 100°C or higher. The treatment time may be within a range in which endoglucanase is not inactivated.

[0043] The endoglucanase can be separated from contaminants by known techniques, such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, pressure filtration, cross-membrane microfiltration, cross-flow membrane microfiltration, or similar techniques. EXAMPLES

[0044] 1. Expression Vector Construction The endoglucanase gene shown in SEQ ID NO:2 was synthesized and inserted into the plasmid pSENSU previously prepared by the inventors (Takaya, T. et al. Appl Microbiol Biotechnol (2011) 90: 1171.). This plasmid contains a secretion signal derived from the α-amylase gene, and a gene of interest can be introduced between the secretion signal and the terminator by PmlI-XbaI treatment. The insertion procedure is as follows. pSENSU was digested with PmlI-XbaI and then subjected to agarose gel electrophoresis, and the pSENSU-PmlI-XbaI digested fragment was isolated and purified. Using the synthesized endoglucanase gene shown in SEQ ID NO:2 as a template, and primers having the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:4, the inserted fragment was amplified by PCR. The amplified fragment was digested with XbaI and then subjected to agarose gel electrophoresis, followed by isolation and purification. The resulting endoglucanase gene was ligated into the PmlI-XbaI site of pSENSU to construct an endoglucanase expression vector pSENSU-EGPf.

[0045] 2. Production of endoglucanase using Aspergillus sp. as a host Aspergillus niger NS48 (niaD, sC double deletion strain obtained by mutagenesis) and Aspergillus luciensis NS41 (niaD, sC double deletion strain obtained by mutagenesis) were transformed with pSENSU-EGPf by the protoplast-PEG method. Genomic DNA was extracted from the resulting transformants, and transformants with one or more copies of the plasmid were selected by real-time PCR. These transformants were cultured in dextrin-peptone-yeast extract medium (4% by weight dextrin, 2% by weight polypeptone, 2% by weight yeast extract, 0.5% by weight KH2PO4, 0.05% by weight MgSO4 7H2O) for 6 days, and the endoglucanase activity was measured using the culture supernatant as a crude enzyme solution. The activity was measured by adding 10 μl of the crude enzyme solution to 200 μl of 50 mM sodium acetate buffer (pH 5.0) containing 1% carboxymethylcellulose sodium salt as a substrate at a final concentration of 1 wt%, and then quantifying the amount of reducing sugars produced at 70°C for 10 minutes using the Somogyi-Nelson method. The amount of enzyme that liberates reducing sugars equivalent to 1 μmol of glucose per minute is defined as 1 U, and strains with endoglucanase activity of 0.1 U or more per ml of crude enzyme solution were selected as hyperthermostable endoglucanase-producing strains.

[0046] 3. Production of endoglucanase using E. coli as a host An expression vector for E. coli containing the endoglucanase gene shown in SEQ ID NO:2 was constructed by a standard method, and E. coli BL21 (DE3) strain was transformed with the vector. The obtained transformant was cultured in LB medium (1% by weight tryptone, 0.5% by weight yeast extract, 1% by weight sodium chloride, 50 μg / ml carbenicillin), and the cells collected by centrifugation were lysed with BugBuster Protein Extraction Reagent (Merck) to extract a crude enzyme solution. The cellulase activity of the extracted crude enzyme solution was measured by the method described in 2 above, and a strain having activity was obtained as a super thermostable cellulase expressing strain.

[0047] 4. Estimation of molecular weight by SDS-PAGE and detection of glycoproteins by PAS staining Each crude enzyme solution was added to a 200 mM sodium phosphate buffer (pH 7.0) at a final concentration of 1 U / ml of endoglucanase, heated at 80°C for 30 minutes, centrifuged at 13,000 rpm for 5 minutes, and the supernatant was obtained as a crude enzyme solution. The molecular weight of the hyperthermostable endoglucanase contained in the crude enzyme solution was measured by SDS-PAGE using molecular weight markers, and as shown in Figure 1 (left), the molecular weight of the enzyme produced by Aspergillus niger was 48,500 ± 6,500, that of the enzyme produced by Aspergillus luciensis was 44,500 ± 4,500, and that of the enzyme produced by E. coli was 31,000. In addition, the culture supernatant was subjected to SDS-PAGE and glycoproteins were detected by the PAS staining method. As shown in the right of Figure 1, the endoglucanases expressed in Aspergillus niger and Aspergillus lucuensis were detected as glycoproteins.

[0048] 5. Thermal Stability Assessment The crude enzyme solution was heated in a heat block at 100°C for 60 minutes, centrifuged at 13,000 rpm for 5 minutes, and the cellulase activity of the supernatant was measured by the method described above. As a control, the activity of a non-heated sample was also measured in the same manner, and the residual activity after heating was calculated as a relative value. The experiment was performed in triplicate, and the average value and standard error were calculated. As a result, as shown in Table 1, the activity of the endoglucanase expressed in E. coli decreased to 13.3% after heating at 100°C for 60 minutes, while the endoglucanase expressed in Aspergillus niger or Aspergillus lucuensis maintained more than 50% of its activity after heating.

[0049] [Table 1]

[0050] 6. Evaluation of the effect of glycans on thermal stability The crude enzyme solution obtained from Aspergillus niger as a host was heated in a heat block at 100°C for 1-8 hours, centrifuged at 13,000 rpm for 5 minutes, and the supernatant was subjected to SDS-PAGE. As a result, as shown in Figure 2, only the band around 30 kDa without glycosylation disappeared after prolonged heating, and the endoglucanase with glycosylation was not affected by prolonged heating.

[0051] From the above results, it was found that endoglucanase having a certain amount of sugar chains bound thereto has significantly higher thermostability than endoglucanase having substantially no sugar chains bound thereto.

Claims

1. An endoglucanase having all of the following characteristics (A) to (F): (A) a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:1; (B) the molecular weight of the polypeptide portion is 31 kDa; (C) having a sugar chain and a molecular weight of 39 kDa or more as measured by SDS-PAGE; (D) the residual endoglucanase activity after heat treatment at 100° C. for 60 minutes is 30% or more; (E) one or more asparagine residues selected from the group consisting of positions 104, 175, 180, 221, 227, and 258 of the amino acid sequence of SEQ ID NO:1 are conserved; and (F) Produced using Aspergillus as a host.

2. A method for producing the endoglucanase of claim 1, comprising expressing DNA encoding the endoglucanase of claim 1 in an Aspergillus host.

3. A method for producing reducing sugars, comprising reacting the endoglucanase according to claim 1 with a sample containing cellulose at a temperature of 70°C or higher.

4. exposing the endoglucanase of claim 1 to a temperature of 80° C. or higher; and Centrifuging the endoglucanase exposed to a temperature of 80° C. or higher; 2. A method for isolating the endoglucanase of claim 1, comprising:

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