JP2011223962A - New cellulase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new cellulase that has much improved cellulose degradability in order to achieve efficient saccharification of plant-derived biomass.SOLUTION: A cellulose binding domain of cellobiohydrolase I derived from A. aculeatus, preferably, a strain of A. aculeatus No.F-50 is added via a linker to a catalytic domain of β-glucosidase 1 derived from A. aculeatus, preferably, a strain of A. aculeatus No.F-50 or a catalytic domain of carboxymethylcellulase 1 and the C-terminal side and/or the N-terminal side of the catalytic domain.

Description

  The present invention relates to a novel cellulase.

  Fuel produced from biomass has a low environmental impact, is abundant as a resource, and is a renewable resource even if it is consumed as long as plants are present on the earth. Therefore, it attracts attention as a powerful alternative to fossil fuels. ing. In particular, the method of producing ethanol (bioethanol) from cellulose present in significant amounts in plant-derived biomass is abundant in resources, available worldwide as a biomass resource, and sugars produced in the process of conversion to ethanol There is an advantage that a polymer to replace petroleum products can be synthesized by using or by-products.

  In the case of producing ethanol from plant-derived biomass, it is common to saccharify the cellulose possessed by the plant using a strong acid or cellulase, followed by alcohol fermentation with yeast or the like.

  Cellulase is a general term for enzymes that hydrolyze cellulose, and cellulases are roughly classified into three types depending on their properties. The first is cellulobiohydrolase (CBH), which acts on crystalline cellulose and breaks down at the disaccharide unit from the end of the cellulose chain. The second cannot break down crystalline cellulose, but randomly breaks amorphous cellulose chains. Endoglucanase (EG), and the third is β-glucosidase (BGL) that acts on solubilized cellobiose and cellooligosaccharide to produce glucose. Cellulose degradation by these enzymes is a very slow reaction when each enzyme reacts. However, when these enzymes act in a complex manner, each enzyme acts in concert and efficient degradation. (Non-patent Document 1).

By the way, cellulose is a linear polymer in which glucose is linked by β-1,4 bonds, and molecules are bonded to each other by hydrogen bonds to form a strong crystal structure. Very slow. In addition, it is known that the crystal structure has a two-phase structure of I _α and I _β and also includes an amorphous region (amorphous) that is not a complete crystal. Different. Furthermore, the property that cellulose has low water solubility, and cellulose having a degree of polymerization of glucose of about 6 to 7 hardly dissolves in water is one of the factors that make it difficult to decompose by cellulase.

  The filamentous fungus Trichoderma. Reesei has long been studied as an enzyme that degrades cellulose, and the cellulase that it produces is said to be the strongest cellulase. Although this cellulase is excellent in the degradation of crystalline cellulose, it has a weak ability to produce monosaccharides, and a large amount of cellooligosaccharide (β-glucan oligosaccharide) remains in the saccharified solution. In addition, natural cellulose such as contained in plant biomass is rarely present alone, and is accompanied by hemicellulose, lignin, etc., and therefore it is necessary to first remove them in order to decompose the cellulose. However, since T. reesei cellulase has a weak hemicellulase activity, it cannot efficiently degrade natural cellulose.

  A. aculeatus No. F-50 strain is known as a microorganism that compensates for these weaknesses of T. reesei cellulase and produces a cellulase enzyme group exhibiting a strong synergistic effect with T. reesei cellulase (non-patent document). 1). The cellulase enzyme group produced by this bacterium has a very strong ability to produce monosaccharides and also has a strong hemicellulase activity, which is very effective for saccharification of plant biomass including not only cellulose but also hemicellulose. A. aculeatus produces nine types of cellulases including the above three types (three categories) of cellulases (CBH, EG, BGL). A. aculeatus has three types of BGL, all of which have very excellent features for cellulose degradation in which only a monosaccharide is produced without causing a transglycosylation reaction (Non-patent Document 2). Among them, BGL1 has a strong degrading activity not only on cellobiose but also on relatively long-chain cellooligosaccharides such as cellopentaose and cellohexaose, and plays an important role in cellulose saccharification because it produces only monosaccharides. . BGL3 is less active against cellooligosaccharide substrates such as cellobiose and cellotriose than BGL1, but, like BGL1, does not cause a transglycosylation reaction and produces only a monosaccharide. At present, BGL2 is expressed from the same gene as BGL1, but is considered to have been obtained as a separate enzyme due to a difference in sugar chain modification.

  On the other hand, carboxymethylcellulase 1 (CMC1), which is a kind of EG, is highly expressed in A. aculeatus cellulase and is thought to play an important role in saccharification of cellulose. However, it does not have a cellulose binding domain (CBD), so it is crystalline cellulose. Can not act on. On the contrary, CMC2 plays an important role in decomposing the non-crystalline part partially contained in crystalline cellulose because it has a cellulose-binding domain on the C-terminal side although it is expressed in a small amount and can act on crystalline cellulose. It is thought that there is.

  For CBH, which is an Exo-glucanase, A. aculeatus has one more type of hydrocellulase (HCase) in addition to two types of CBH (CBHI, CBHII). Although HCase hardly decomposes Avicel, it exhibits high activity against amorphous cellulose such as alkali swollen cellulose (ASC) and phosphate swollen cellulose (PSC). Both CBHI and CBHII have CBD and decompose crystalline cellulose from the end in cellobiose units, whereas CBHI decomposes from the reducing end side of the cellulose chain, whereas CBHII decomposes from the non-reducing end side.

  Thus, in the degradation of cellulose by the cellulase enzymes, even in A. aculeatus No. F-50 strain, which is said to be more advantageous than T. reesei, which is said to be the strongest cellulase producing bacterium, only monosaccharides BGL1 that plays an important role in the hydrolysis of cellulose and CMC1 with a high expression level are inferior in binding to cellulose. On the other hand, CBHI and CBHII, which have excellent binding properties to cellulose, hydrolyze cellulose in cellobiose units, so that even monosaccharides are not decomposed, and the production efficiency of monosaccharides (glucose) required for alcoholic fermentation is poor. .

  Under such circumstances, in recent years, the optimum mixing ratio of each cellulase in the cellulase enzyme group is determined, the production amount of each cellulase is increased using molecular biological techniques, the resolution and stability of each cellulase itself is increased, Various studies have been conducted on improving cellulase functions, such as fusing different cellulase proteins. As one of the methods, for example, improvement and modification of cellulase properties, that is, cellulase is adsorbed to insoluble cellulose by using cellulose binding domain (CBD) is disclosed. For example, Japanese Patent Application Laid-Open No. 2009-142260 (Patent Document 1), Japanese Patent Application Laid-Open No. 2008-193990 (Patent Document 2), Japanese Translation of PCT International Publication No. 2007-530054 (Patent Document 3), Japanese Patent Application Publication No. 2004-536593 (Patent Document). Document 4), publication of Japanese translations of PCT publication No. 2003-522517 (patent document 5), and Japanese translations of PCT publication No. 2001-504352 (patent document 6).

  CBD is produced as a protein linked to a cellulose hydrolysis domain (catalytic domain), and plays an important role in cellulose hydrolysis. CBD itself has no degrading activity but has the ability to bind to cellulose alone. As a function of CBD, by adsorbing to an insoluble substrate, the concentration of the catalytic domain associated with the CBD around the substrate is increased to improve the decomposition rate of cellulose, or the hydrogen bond between cellulose chains is separated by CBD binding. It is known that the crystal structure is destroyed (Non-Patent Documents 3 and 4). In addition, when CBD is removed from cellobiohydrolase (CBH) that degrades crystalline cellulose, although the reactivity to soluble substrates does not change, the decomposition activity and affinity for crystalline cellulose are extremely reduced. It is considered to be a domain necessary for the enzyme to act on crystalline cellulose (Non-patent Document 5). Conversely, there is also a report that the affinity and resolution to crystalline cellulose increase by adding CBD to a kind of EG that does not originally have CBD (Non-patent Document 6).

  However, in view of the fact that many CBDs have been found so far, and are classified into 17 types of Family as Carbohydrate-binding module (CBM) family by the homology of their amino acid sequences (Non-patent Document 7). Many combinations of CBD and cellulase are conceivable, and it has not always been expected that specific cellulase activity will be improved by adding a certain CBD.

JP 2009-142260 A JP 2008-193990 A Special table 2007-530054 gazette JP-T-2004-536593 Special table 2003-522517 published JP-T-2001-504352

Murao S. Kanamoto J. Arai M. Isolation and identification of a cellulolytic enzyme producing microorganisms. J. Ferment Techol., 57, 151, 1979 Soichiro Sakamoto Study on cellulase system of Aspergillus aculeatus No. F-50, Doctoral dissertation, Osaka Prefecture University, 1984 Bolam DN, Ciruela A, McQueen-Mason S, Simpson P, Williamson MP, Rixon JE, Boraston A, Hazlewood GP, Gilbert HJ Pseudomonas cellulose-binding domains mediate their effects by increasing enzyme substrate proximity. Biochem J 331 (Pt3) 775- 781, 1998 DIN N, DAMUDE HG, GILKES NR, MILLER RC, WARREN RAJ, KILBURN DG: C1-Cx, revisited: Intramolecular synergism in a cellulose. Proc. Nati. Acad. Sci. USA Vol. 91, pp. 11383-11387, 1994 Riedel K, Ritter F, Bauer S, Bronnenmeier K: The modular cellulase CelZ of the thermophilic bacterium Clostridium stercorarium contains a thermostabilizing domain. FEMS Microbiology Letters, 164 261-267, 1998 Mahadevan SA, Wi SG, Lee DS, Bae HJ: Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima). FEMS Microbiol Lett 287: 205-211, 2008 http://www.cazy.org/index.html

  The present invention has been made in view of the above-mentioned background art, and aims to provide a novel cellulase with further improved degradability of cellulose aiming at efficient saccharification of plant-derived biomass resources. .

  The cellulase of the present invention includes a region containing the catalytic domain of β-glucosidase 1 (BGL1) derived from Aspergillus aculeatus (A. aculeatus) or a region containing the catalytic domain of carboxymethyl cellulase 1 (CMC1) derived from A. aculeatus; It is a cellulase having a cellulose-binding domain (CBD) derived from A. aculeatus added to a region via a linker or not via a linker.

  According to the present invention, β-glucosidase having a high decomposing activity for insoluble cellooligosaccharide and carboxymethyl cellulase having a high degrading activity for CMC, to which CBD is added, are provided. By using either or both of these, cellulose, which is the center of plant-derived biomass resources, does not undergo a transglycosylation reaction and produces degradation products such as cellooligosaccharides that are not assimilated by microorganisms in the fermentation process. Without breaking down to glucose, which is a simple sugar.

It is a part of figure which shows the construction method of the vector which integrated the gene which codes the protein which couple | bonded CBD with BGL1. FIG. 2 is a continuation diagram of FIG. 1. It is a figure which shows the gene structure of the high expression vector pNAN8142 for filamentous fungi. A shows the restriction enzyme map, and B shows the structure of the modified promoter. It is an image which shows the result by electrophoresis of a PCR amplification product. Lane 1 is cbhl-LC, lane 2 is cmc2-LC, lane 3 is bgl1, lane 4 is cbhII-CL, and lane M is λDNA Pst I digest. A and B are diagrams showing the enzyme activity of crude protein (enzyme) released from cells. It is an image which shows the result by electrophoresis of the produced crude protein (enzyme). Lane 1 is BGL1-CBD _CBHI , Lane 2 is BGL1-CBD _CMC2 , Lane 3 is pNAN8142vector, Lane 4 is BGL1, Lane 5 is CBD _CBHI- BGL1, Lane 6 is BGL1, Lane 7 is purified BGL1, Lane M is Molecular weight marker. It is a column chromatogram in the refinement | purification of BGL1 crude protein (enzyme), Comprising: A is a chromatogram using DEAE-TOYOPEARL650M, B is a chromatogram using Butyl-TOYOPEARL650M. It is a column chromatogram in the refinement _| purification of BGL1-CBD _CBHI crude protein (enzyme), A is a chromatogram using DEAE-TOYOPEARL650M, B is a chromatogram using Butyl-TOYOPEARL650M. It is a column chromatogram in the case of _refinement | purification of CBD _CBHII- BGL1 crude protein (enzyme), A is a chromatogram using DEAE-TOYOPEARL650M, B is a chromatogram using Butyl-TOYOPEARL650M. CBD _CBHII -BGL1-CBD _CBHI A column chromatogram in the purification of crude protein (enzyme), A is a chromatogram using DEAE-TOYOPEARL650M, and B is a chromatogram using Butyl-TOYOPEARL650M. It is an image which shows the result by electrophoresis of the produced purified protein (enzyme). Lanes 1 and 3 are CBD _CBHII -BGL1, lanes 2, 4, and 7 are BGL1-CBD _CBHI , lanes 5 and 6 are BGL1, and lane 8 is BGL1-CBD _CMC2 . It is a graph which shows adsorption | suction to the ASC of the produced purified protein (enzyme). It is a graph which shows the substrate decomposition activity of the produced purified protein (enzyme). A shows the decomposition of ASC and B shows the decomposition of ICOS. It is a graph which shows stability of the produced purified protein (enzyme). A shows thermal stability and B shows pH stability. It is a graph which shows the ASC degradation activity of the produced purified protein (enzyme). It is a figure which shows the construction method of the vector for E. coli expression which integrated the gene which codes the protein which CBD couple | bonded with CMC1. It is a figure which shows the construction method of the vector for A.oryzae expression which integrated the gene which codes the protein which CBD couple | bonded with CMC1. It is an image which shows the result by electrophoresis of a PCR amplification product. Lane 1 is cmc1g, Lane 2 is cmc1c, and Lane 3 is λDNA Pst I digest. It is an image which shows the result by electrophoresis (IPTG 0 mM) of the protein (enzyme) produced by colon_bacillus | E._coli. S is a soluble fraction, I is an insoluble fraction, and M is a molecular weight marker. It is an image which shows the result by electrophoresis (IPTG 1mM) of the protein (enzyme) produced by colon_bacillus | E._coli. S is a soluble fraction, I is an insoluble fraction, and M is a molecular weight marker. It is an image which shows the result of electrophoresis of the product by A.oryzae. Lane 1 is CMC1-CBD _CBHI and lane 2 is _A.oryzae8142 . It is the result of having analyzed the product by E. coli and A. oryzae, A is a figure which shows the enzyme activity, B is the image which shows the result by the electrophoresis. No. _1-5 is CMC-CBD _CBHI , No. 6-9 is CMC1-CBD _CMC2 , No10 is pNAN8142vector, and M is a molecular weight marker. It is a column chromatogram in the case of refinement _| purification of CMC1-BCD _CBHI crude protein (enzyme), A is a chromatogram using DEAE-TOYOPEARL650M, B is a chromatogram using Butyl-TOYOPEARL650M. It is an image which shows the result by electrophoresis of CMC1 purified protein (enzyme) and CMC1-BCD _CBHI purified protein (enzyme). Lane 1 is CMC1-BCD _CBHI , lane 2 is CMC1, and lane M is a molecular weight marker. It is a graph which shows adsorption _| suction to the substrate of CMC1 purified protein and CMC1-BCD _CBHI purified protein (enzyme). A shows adsorption of ASC, and B shows adsorption of ICOS. It is a graph which shows the decomposition activity of CMC1-purified protein and CMC1-BCD _CBHI purified protein (enzyme). A shows the decomposition of ASC and B shows the decomposition of ICOS.

  The cellulase of the present invention comprises (1) a region containing the catalytic domain of β-glucosidase 1 (BGL1) derived from Aspergillus aculeatus and a cellulose-binding domain derived from Aspergillus aculeatus (hereinafter referred to as “CBD”) indirectly or directly added to the region. Or (2) a cellulase having a CBD derived from Aspergillus aculeatus and a region containing the catalytic domain of carboxymethyl cellulase 1 derived from Aspergillus aculeatus and indirectly or directly added to the region. In the present invention, the catalytic domain means a minimum polypeptide that exhibits a so-called enzyme activity or function (enzyme activity or binding ability to cellulose). CBD means a minimum polypeptide that does not have degradation activity by itself but has the ability to bind to cellulose alone. Therefore, in the present invention, a polypeptide corresponding to a linker associated with CBD is not included in CBD.

  The cellulase of the present invention is a cellulase produced by adding a CBD cellulose-binding domain to a cellulase having no CBD among cellulases produced by A. aculeatus to improve the degradability of crystalline cellulose or insoluble cellooligosaccharide. A. aculeatus produces three types of β-glucosidase (hereinafter sometimes referred to as “BGL”), all of which have very excellent characteristics of producing only a monosaccharide without causing a transglycosylation reaction. Among them, β-glucosidase 1 (BGL1) has high activity not only for cellobiose but also for relatively long-chain oligosaccharides such as cellopentaose and cellohexaose, and produces only monosaccharides. The cellulase (1) of the present invention utilizes the characteristics of BGL1 derived from this A. aculeatus and provides BGL having no transglycosylation reaction and having high activity against relatively long-chain oligosaccharides. .

  A. aculeatus produces three types of endo-glucanases, carboxymethyl cellulase (hereinafter sometimes referred to as “CMC”), among which carboxymethyl cellulase 1 (CMC1) is produced by A. aculeatus. It is thought that cellulase has a high expression level and plays an important role in cellulose saccharification. The cellulase (2) of the present invention focuses on this cellulase with a high expression level, and is a CMC with improved degradability for crystalline cellulose.

  A strain of A. aculeatus is appropriately selected. In the present invention, the strain A. aculeatus No. F-50 is most preferably used. Compared to other strains, this strain produces a group of enzymes with strong hemicellulase activity in addition to having a very strong ability to produce monosaccharides, which makes it extremely useful for saccharification of plant biomass containing not only cellulose but also hemicellulose. This is because it is considered effective.

  BGL1 and CMC1 produced by A. aculeatus each do not have a cellulose binding domain (hereinafter sometimes referred to as “CBD”). The cellulase of the present invention is obtained by adding CBD to these BGL1 and CMC1. Although the origin of CBD used in the present invention is not limited, CBD possessed by hydrocellulase (hereinafter sometimes referred to as “CBH”) which is an exo-glucanase produced by A. aculeatus is preferably used as CBD. A. aculeatus produces two types of CBH, both of which (CBHI, CBHII) have CBD and decompose crystalline cellulose from the end in cellobiose units, but CBHI decomposes from the reducing end of the cellulose chain. On the other hand, CBHII is decomposed from the non-reducing terminal side. However, in the enzymatic degradation of cellulose, since it is desired that each enzyme acts in concert in a complex system consisting of three types of enzymes, BGL, CMC, and CBH, focusing on the high cellulase activity of A. aculeatus, The CBD of CBH, which is the only CBD that A. aculeatus has, is desirable. As long as it is CBD of this CBH, either CBD of CBHI decomposing from the reducing end side or CBD of CBHII decomposing from the non-reducing end side may be used. From this point of view, the A. aculeatus No. F-50 strain is most preferably used as the strain of A. aculeatus from which CBD is derived.

  The amino acid sequences of BGL1 and CMC1 used in the present invention and the base sequences encoding these amino acid sequences have already been clarified (BGL1: Kawaguchi T, et al., Gene. 1996) ep 16; 173 (2): 287-8., CMC1: Ooi T, et al., Nucleic Acids Res., 18 (19), 5884, 1990). In addition, the amino acid sequences of CBD of CBHI and CBHII and the base sequences encoding these amino acid sequences have already been clarified (Takada G, Kawaguchi T, Sumitani J, Arai M, Cloning, nucleotide sequence, and transcriptional analysis of Aspergillus J Ferment Bioeng 85: 1-9, 1998 31, Atsushi Naito, Cloning and high expression of cbhII type cellulase gene from Aspergillus aculeatus. Osaka Prefecture University, Bachelor thesis, 2009). Aculeatus No. F-50 cellobiohydrolase I (cbhI) gene. The cellulase of the present invention is not necessarily provided with all the amino acid sequences possessed by these polypeptides, and may be provided with a domain that exhibits so-called activity or function (enzyme activity or binding ability to cellulose). Further, as long as the activity or function is exhibited, a part of the amino acid may be deleted, added, substituted, or modified. In addition, the base sequence need not have the entire base sequence as long as it exhibits the activity or function, and has 80% homology, preferably 85%, more preferably 95% or more. It only has to be.

  The cellulase (1) of the present invention is a BGL having a region containing the catalytic domain of β-glucosidase 1 derived from A. aculeatus and a CBD derived from A. aculeatus indirectly or directly added to the region. Cellulase (2) of the present invention is a CMC having a domain region of carboxymethyl cellulase 1 derived from A. aculeatus and a cellulose binding domain derived from A. aculeatus added indirectly or directly to the region. Here, indirect means having a linker between the region containing the catalytic domain of BGL1 or CMC1 and CBD. The linker is composed of about 1 to 100 amino acids. The linker is a portion having no enzyme activity or function such as cellulase activity, and has a function of preventing steric hindrance generated between the bound catalytic domain and the CBD. The amino acid sequence of the linker is not limited, but an amino acid sequence that increases the degree of freedom of steric arrangement between domains is preferably used. A CBD found in nature is associated with a linker. Such a linker, for example, a linker (peptide) attached to the CBD of CBHI or CBHII is used as it is. A peptide in which a peptide functioning as a linker is bound to a linker (peptide) associated with CBD can also be used as a linker. Direct means that no linker is interposed between the region containing the catalytic domain of BGL1 or CMC1 and CBD. Further, the binding position of CBD may be any of the C-terminal side and N-terminal side of BGL1 and CMC1, and may be added to both the C-terminal side and N-terminal side positions thereof.

  The cellulase of the present invention is produced using various known genetic engineering techniques. That is, in the production of cellulase (1), at least a base sequence encoding the catalytic domain of BGL1 and at least a base sequence encoding the domain region of CBD to be added are included, and if necessary, a base sequence encoding a linker is interposed. Thus, both are combined to produce a polynucleotide having a base sequence encoding the target cellulase. In the production of cellulase (2), at least a base sequence encoding the catalytic domain of CMC1 and at least a base sequence encoding the domain of CBD to be added are included, and if necessary, a base sequence encoding a linker is interposed. Are combined to produce a polynucleotide having a base sequence encoding the target cellulase.

  The obtained polynucleotide is incorporated into an expression vector to which a promoter, terminator and the like are added by a conventional method. Then, the vector is introduced into a host cell, the cellulase gene is expressed, and cellulase is produced. In this case, when a prokaryotic cell such as Escherichia coli is used as a host cell, a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11 may be used. In addition, when a eukaryotic cell is used as a host cell, such as an Aspergillus genus filamentous fungus, an intron region may be inserted into the polynucleotide. This is because, when the host cell is a eukaryotic cell, a messenger RNA with an intron spliced is formed, and protein expression is easily performed. The intron has a consensus sequence and may be a sequence that functions as an intron, and a specific sequence is determined as appropriate. Furthermore, a signal sequence for secreting the enzyme produced in the host cell outside the cell is added as necessary. The signal sequence need not be a specific base sequence, and may be any sequence as long as the function is exhibited. For example, a signal sequence provided in BGL1 derived from A. aculeatus is used.

  The base sequences shown in SEQ ID NOs: 1 to 4 are examples of the base sequence of the polynucleotide for producing the cellulase of the present invention. The polynucleotides shown in SEQ ID NOs: 1 to 4 encode cellulases (polypeptides) having the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12, respectively. It is a polynucleotide used for producing eukaryotic cells such as fungi using host cells. The polynucleotides shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11 do not contain a signal sequence. A polynucleotide having the base sequence shown in SEQ ID NO: 1 comprises a linker containing a recognition site for the restriction enzyme Nsi I and a linker (peptide) associated with CBHI derived from A. aculeatus on the C-terminal side of BGL1 derived from A. aculeatus A cellulase in which CBD of A. aculeatus-derived CBHI is bound is encoded. The base sequence shown in SEQ ID NO: 2 encodes a cellulase in which the CBD of A.aculeatus-derived CBHII is bound to the N-terminal side of BGL1 derived from A.aculeatus via a linker (peptide) attached to CBHII derived from A.aculeatus To do. In the base sequence shown in SEQ ID NO: 3, CBD associated with CBHII derived from A.aculeatus binds to the N-terminal side of BGL1 derived from A.aculeatus via a linker (peptide) attached to CBHII derived from A.aculeatus, It encodes a cellulase in which the CBD of A. aculeatus-derived CBHI is bound to the C-terminal side via a linker containing a recognition site for restriction enzyme Nsi I and a linker (peptide) associated with A. aculeatus-derived CBHI. The base sequence shown in SEQ ID NO: 4 is formed on the N-terminal side of A. aculeatus-derived CMCI via a linker containing a restriction enzyme Nsi I recognition site and a linker (peptide) associated with A. aculeatus-derived CBHI. It encodes a cellulase bound to CBD of CBHI derived from .aculeatus. All of the polynucleotides shown in SEQ ID NO: 1 to SEQ ID NO: 4 contain introns.

  The vectors and host cells used in the present invention are not limited, but as host cells, prokaryotic cells such as bacteria such as Escherichia coli and Bacillus subtilis, and eukaryotic cells as yeasts or filamentous fungi of the genus Aspergillus or Trichoderma. For example, A.aculeatus or A.oryzae is exemplified. In the present invention, filamentous fungi of the genus Aspergillus are preferred. This is because filamentous fungi of the genus Aspergillus are of the same genus as A. aculeatus from which BGL1, CMC1 and CBH are derived, and the cellulase of the present invention is easily expressed. A. aculeatus is more preferred as a host because it can produce not only the cellulase of the present invention but also other cellulase enzymes necessary for saccharification of cellulose. Known vectors for bacteria, filamentous fungi, and yeast can be used. Examples of known vectors include pBR322 and pKK233-2 for E. coli, pAUR316 for filamentous fungi, Yip5, Yrp19 and the like for yeast.

  As a transformation method, a known method such as permeation of a cell wall using a particle or gene gun, alkali, or the like, a protoplast method, or an electroporation method can be used.

  The enzyme produced in the host cell is extracted from the bacterial cells or the culture solution by a conventional method. In addition, when enzymes are secreted outside the cells, the cells or the culture solution can be used directly for saccharification of plant biomass.

  The method for saccharifying plant biomass of the present invention is a method for saccharifying plant biomass using cellulase (1) and / or cellulase (2) obtained above. In addition, plant biomass may be saccharified using a transformant obtained by transforming a vector having the base sequence encoding cellulase (1) and / or the base sequence encoding cellulase (2) in the present invention. it can. For example, when an Aspergillus filamentous fungus, particularly A. aculeatus, is transformed with a polynucleotide having the base sequences shown in SEQ ID NOs: 1 to 4, it can be saccharified using the transformed filamentous fungus.

  The plant-based biomass is exemplified by biomass such as so-called trash such as grass, wood, agricultural waste, paper and food waste, and is not particularly limited as long as it contains cellulose. Biomass saccharification may be carried out by a known method, and is saccharified to glucose using the above-mentioned cellulase or transformant after various pretreatments such as biomass pulverization, pretreatment with acid or alkali, pretreatment with high temperature and pressure. The

  Since the cellulases (1) and (2) of the present invention have a cellulose-binding domain for crystalline cellulose, they can be sufficiently saccharified even under milder conditions such as pretreatment at a low temperature. Further, depending on the biomass, it is possible to perform saccharification without performing pretreatment. In particular, the cellulase (1) of the present invention is very advantageous for the alcoholic fermentation process performed after the saccharification treatment because it has a small transglycosylation reaction and has the ability to sufficiently decompose even a monosaccharide.

  Next, the present invention will be described in detail based on the following examples. The following examples are merely illustrative, and the present invention is not limited to the following examples.

[Production of cellulase (1)]
First, a cellulase was prepared by binding A. aculeatus-derived β-glucosidase 1 to CBD of A. aculeatus-derived cellobiohydrase I (CBHI) or II (CBHII) via a linker.

  As β-glucosidase 1 derived from A. aculeatus, β-glucosidase 1 derived from A. aculeatus F-50 strain whose nucleotide sequence has already been determined was used. As the cellulose-binding domain, CBHI CBD and / or CBHII CBD and CMC2 CBD derived from F-50 strains whose base sequences were already determined were used.

As cellulase conjugated with CBD, cellulase conjugated with CBD of CBHI on the C-terminal side of β-glucosidase 1 (BGL1-CBD _CBHI ), cellulase conjugated with CBD of CBHII on the N-terminal side of β-glucosidase 1 (CBD _CBHII − Three cellulases (1) of BGL1) and a cellulase (CBD _CBHII -BGL1-CBD _CBHI ) in which CBD of _CBHI was bound to the C-terminal side of β-glucosidase 1 and CBD of CBHII were bound to the N-terminal side were prepared. The amino acid sequence of cellulase (BGL1-CBD _CBHI ) is SEQ ID NO: 6, the amino acid sequence of cellulase (CBD _CBHII -BGL1) is SEQ ID NO: 8, and the amino acid sequence of cellulase (CBD _CBHII -BGL1-CBD _CBHI ) is SEQ ID NO: 10. Indicated. A cellulase (BGL1-CBD _CMC2 ) in which CBD of _CMC2 was bound to the N-terminal side of CMC was also prepared.

1. Construction of expression plasmids To add CBD to BGL1, cloning vector of A. aculeatus bgl1 gene amplified by PCR and DNA sequence encoding linker region and CBD from cbhI, cbhII, cmc2 The genes were constructed by binding on pBluescript II KS (+) so that CBD and Linker derived from CBHII bind to the N-terminal side of BGL1 and Linker and CBD derived from CBHI or CMC2 bind to the C-terminal side of BGL1 ( 1 and 2).

  Next, each constructed gene has a bgl1 gene fragment inserted into the promoter downstream region of the high-expression vector pNAN8142 for filamentous fungi (see FIG. 3; according to Ozeki Co., Ltd.), on the chromosome of the host Aspergillus oryzae niaD300 strain. Homologous recombination was performed at the niaD site.

A. Preparation of competent cells
The method of Inoue et al. (Inoue H, Nojima H, Okayama H: High efficiency transformation of Escherichia coli with plasmids. Gene 96: 23-28, 1990) was followed. Escherichia coli (E. coli DH5αF ′ strain) was streaked on an LB medium plate (not containing Ampicillin) and then cultured at 37 ° C. overnight. Several colonies were inoculated into 250 mL SOB medium in a 2 L Erlenmeyer flask and cultured at 18 ° C. with shaking until OD ₆₆₀ = 0.4 to 0.6. The Erlenmeyer flask was cooled on ice for 10 minutes, and then centrifuged (3000 rpm, 10 min, 4 ° C.). After removing the supernatant by decantation, the precipitated E. coli was transformed into 1/3 volume (about 84 mL) of transformation buffer (TB) cooled to 0 ° C. (10 mM PIPES, 15 mM CaCl ₂ , 15 mM KCl, 55 mM MnCl _2). ) And cooled on ice for 10 minutes, centrifuged again (3000 rpm, 10 min, 4 ° C.), and the supernatant was removed. The precipitated Escherichia coli was suspended in 20 mL of TB cooled to 0 ° C., DMSO (Dimethyl Sulfoxide) was added to a final concentration of 7.5%, and the mixture was cooled on ice for 10 minutes. Thereafter, 200 μL each was dispensed into an Eppendorf tube, frozen in liquid nitrogen, and stored at −80 ° C.

B. Transformation into E. coli
Cohen et al. (Cohen SN, Chang AC, Hsu L: Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl AcadSci USA 69: 2110-2114, 1972). The competent cell prepared as described above was dissolved on ice, an appropriate amount of plasmid solution or ligation reaction solution which was also allowed to stand on ice was added, and the mixture was allowed to stand on ice for about 30 minutes. Then, after heat shock at 42 ° C. for 45 seconds, the mixture was allowed to stand on ice for about 2 minutes. After adding 1000 μL of 2 × TY medium and shaking culture at 37 ° C. for 45 minutes, it was spread on an LB medium plate containing 100 μg / mL Ampicillin and cultured at 37 ° C. overnight.

C. Preparation of plasmid DNA
The Alkalinelysis method (Sambrook J., Russell DW: Molecular Cloning: a laboratory manual. 1.31-1.34, 2001) was followed. A single colony grown on the LBplate was inoculated into 1.7 mL of 2 × TY medium (including Ampicillin) using a toothpick and cultured overnight at 37 ° C. with shaking. The culture solution was put into an Eppendorf tube, and the cells were collected by centrifugation (10000 rpm, 1 min). After removing the culture supernatant, 100 μL of Solution I (50 mM Glucose, 10 mM EDTA, 25 mM Tris-HCl (pH 8.0)) was added to suspend the cells. 200 μL of Solution II (02N NaOH, 1.0% SDS) was added thereto and gently stirred. After leaving still for 5 minutes, 150 μL of Solution III (3M Potassium acetate (pH 4.8)) was added, and after stirring, allowed to stand on ice for 10 minutes. The supernatant was collected by centrifugation (15000 rpm, 10 min), extracted with phenol / chloroform, and centrifuged (15000 rpm, 5 min) at room temperature. The supernatant was separated into a separate eppen, 2.5 volumes of EtOH (ice cold) was added, and the mixture was allowed to stand on ice for 10 minutes. Centrifugation (15000 rpm, 5 min) at 4 ° C., removal of the supernatant, washing with 70% EtOH (ice cold), and centrifugation (15000 rpm, 5 min) at 4 ° C. Was removed and vacuum-dried. The precipitate was dissolved in about 100 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), 2 μL of 10 mg / mL RNase A was added, and the mixture was allowed to stand at 37 ° C. for 30 minutes to obtain a plasmid solution.

D. Agarose gel electrophoresis In order to analyze DNA fragments digested with restriction enzymes and the like, they were subjected to agarose electrophoresis. Agarose-LE Classic Type was heated and melted in TAE buffer (0.04Tris, 0.02MAcetic acid, 1mM EDTA, pH 8.0) to a concentration of 0.7 to 1.5% (w / v), and edidium bromide was added to 0.5%. Agarose gel was prepared by adding 5 μg / mL. This gel was placed in a Mupid mini gel electrophoresis apparatus (Advance) filled with TAE buffer, and 1/10 amount of 10 × Loading Buffer (0.1% Bromophenol blue (BPB), 10% Glycerol, 0.5% sodium dodecyl sulfate (SDS), volume adjusted with TE) was added to the agarose gel. After electrophoresis under a constant voltage of 50V or 100V, DNA was observed on a transilluminator (manufactured by VILVERLOURMAT).

E. Restriction enzyme treatment and modification enzyme treatment Restriction enzyme treatment and modification enzyme treatment were basically performed according to the attached protocol. When necessary, phenol extraction, phenol / chloroform extraction, and ethanol precipitation were performed.

F. Recovery of DNA fragments from agarose gel
According to the method of Wang et al. (Wang Z., Rossman TG: Isolation of DNA fragments from agarose gel by centrifugation. Nucleic Acids Research, Vol. 22, No. 14, 2862-2863, 1994). A DNA fragment obtained by restriction enzyme treatment or PCR was subjected to agarose gel electrophoresis, and a gel containing the target fragment was excised and recovered from it in the following steps.

  A hole is made in the bottom of the Eppendorf tube, the hole is closed with 4 mm diameter filter paper (FILTER PAPER GA-100, manufactured by ADVANTEC), 300 μL of Sephadex G-10 (swollen in TEbuffer) is overlaid, and another Eppendorf tube is formed. Then, centrifugation (15000 rpm, 1 min) was performed to remove TEbuffer. The cut agarose gel was put into the upper Eppendorf tube, placed on a new Eppendorf tube and centrifuged (15000 rpm, 10 min, 4 ° C.), and the DNA solution remaining in the lower Eppendorf was subjected to phenol extraction and phenol / chloroform extraction. Then, ethanol precipitation (2.5 times amount EtOH, 1/10 times amount 3M Sodium acetate (pH 5.2) was added) was performed. After standing for 10 minutes on ice, centrifugation (15000 rpm, 10 min, 4 ° C.) was performed. The precipitate was washed with 70% EtOH and then vacuum dried, and dissolved in an appropriate amount of TE buffer to obtain a DNA solution.

G. Ligation reaction After treating the plasmid with a restriction enzyme, it was recovered from an agarose gel after electrophoresis to obtain a plasmid vector. As the ligation reaction conditions, the molar ratio of the DNA fragment to the vector was set to 3: 1, 1/10 of the total amount of 10 × ligation buffer was added and stirred, and then 1 μL of T4 DNA ligase (500 units / μL) was added. The reaction was performed at 16 ° C. for 3 hours.

H. Amplification of DNA fragment using PCR method For the PCR method, Prime STARHS DNA polymerase (manufactured by Takara Bio Inc.) and each attached buffer were used. As a reaction apparatus, TaKaRa Thermal cycler (manufactured by Takara Bio Inc.) was used. Preparation of the reaction solution and reaction conditions are based on the following conditions, and combinations of Template and primer pairs are shown in Table 1. However, the extension time was set to 30 seconds only when the DNA sequence encoding cbhII or CBD and Linker was amplified. Each PCR product was named bgl1, cbhI-LC, cmc2-LC, and cbhIICL-1.

(PCRreaction mixture)
Template (0.5 ng / μL) 2
Fprimer (5 μM) 2
Rprimer (5 μM) 2
dNTP Mixture (2.5 mMeach) 4
5 x Prime STAR buffer 10
Prime STARHS DNA polymerase (0.625 U / μL) 2
Distilled water 28
Total 50 (μL)
(PCRcondition)
Denature 98 ℃ 0min 10sec
Anneal 55 ℃ 0min 5sec 30cycle
Extend 72 ℃ 3min 0sec
72 ℃ 10min 0sec 1cycle

I. Construction of Expression Plasmid PCR products cbhI-LC and cmc2-LC obtained by the above PCR method are digested with EcoR I and BamH I, and EcoR located in the multiple cloning site of pBluescript II KS (+) (pBs) They were inserted into the I site and the BamH I site (pBs-CBD _cbhI , pBs-CBD _cmc2 ). Furthermore, the PCR product bgl1 was digested with Nsi I and Xho I, and added to the 5 ′ end of the CBD of pBs-CBD _cbhI and pBs-CBD _cmc2 , and further inside the multiple cloning site of pBs upstream of it. Inserted into the Sal I site. This is cleaved at the Not I site added to the 5 ′ end side of bgl1 and the Nde I site added to the 3 ′ end side of each CBD to cut out the target gene, and within the multi-cloning site of the high expression vector pNAN8142 for filamentous fungi Inserted into the Not I and Nde I sites. The plasmids thus obtained were designated as pNAN-bgl1-CBD _cbhI and pNAN-bgl1-CBD _cmc2 . On the other hand, in order to add CBD to the N-terminal side of BGL1, the PCR product cbhII-CL1 digested with Xho I was converted to the Xho I site 64 bp upstream from the translation start point of the bgl1 gene at pH 3K. It was inserted into the Eco47 III site 54 bp downstream from the translation start point. As a result, the signal sequence (57 bp) of bgl1 was removed, and CBD was added to the N-terminal side of BGL1 by inserting only one amino acid residue (alanine) between the linker and the catalytic domain. This was excised at the Xho I site added to the 5 'end side of CBD and the Sph I site present 319 bp downstream from the stop codon of bgl1, and inserted into the Xho I site and Sph I site within the multi-cloning site of pNAN8142. . The thus obtained plasmid was designated as pNAN-CBD _cbhII- bgl1, and a total of three plasmids constructed respectively were used for transformation of A. oryzae.

In order to bind CBD to the N-terminal and / or C-terminal of BGL1, 2937 bp (see FIG. 1) excluding the stop codon from the start codon of bgl1, 240 bp (see FIG. 1) from 1384 bp to the stop codon of cbhI, cmc 2 The 327 bp from the 1140 bp to the stop codon (see FIG. 1) and the cbhII start codon to the 377 bp (see FIG. 1) were amplified by PCR. The PCR product was subjected to agarose gel electrophoresis, and it was confirmed that a fragment of the correct length was amplified (FIG. 4). Moreover, and Not I and Nde I was used when inserting expression was constructed by combining these plasmids _{pNAN-bgl1-CBD cbhI, pNAN} -bgl1-CBD cmc2, pNAN-CBD cbhII -bgl1 is a gene of interest into an expression vector, It was confirmed that the target gene was correctly inserted using Pst I, Sal I, etc. (data not shown).

J. Reagents used, etc. The strains, plasmids, media, primers, etc. used for the construction of the expression plasmid are as follows.
a) Strains used
Escherichia coli DH5αF´F '/ endA1 hsdR17 (r _K ^- m _K ⁺ ) supE44, thi-1,
recA1, gyrA (Nal ^r ), relA1,
Δ (lacZYA-argF) U169 (m80lacZΔM15)
b) Plasmid used
pNAN8142
pBluescript II KS (+)
pH3K
pUC118, bgl1
pNANBGL1 pNAN8142, bgl1
pGA1 pSL1180 / 1190, cbhI
pNANcbhII pNAN8142, cbhII
pGC2 pUC118 / 119, cmc2 genome
c) Medium used The following medium was used for the culture of E. coli. Agar with 1.5% concentration was added to the plate medium. If necessary, Ampicillin was added so that the final concentration was 100 μg / mL.
i) LB medium (pH 7.0)
Polypepton 1.0%
Bacto-yeast extract (Difco) 0.5%
NaCl 0.5%
ii) 2 × TY medium (pH 7.0)
Bacto-tryptone (Difco) 1.6%
Bacto-yeast extract (Difco) 1.0%
NaCl 0.5%
iii) SOB medium (pH 7.0)
Bacto-tryptone (Difco) 2.0%
Bacto-yeast extract (Difco) 0.5%
NaCl 10 mM
KCl 2.5 mM
MgCl ₂ 5 mM
MgSO ₄ 5 mM
d) Reagents used i) Restriction enzymes and modification enzymes Restriction enzymes and modification enzymes are manufactured by Nippon Gene, Toyobo, Takara Bio, or NEW ENGLAND BIOLABS (NEB). Each company's protocol was followed.
Restriction enzymes: BamHI, Eco47III, EcoRI, NdeI, NotI, NsiI, SalI, XhoI
Modification enzyme: T4 DNA ligase, Prime STARHS DNA polymelase
ii) Primer
The primers shown in Table 2 were used for amplification of the target gene.
iii) Other reagents Unless otherwise indicated, special grade reagents manufactured by Wako Pure Chemical Industries or Nacalai Tesque were used.

2. Enzyme expression in A.oryzae The expression plasmid constructed above was transformed into A.oryzae to express the enzyme.
A. Protoplast preparation Add 12 mL of Tween 80 / Saline solution (0.01% Tween 80, 0.9% NaCl) to A. oryzae niaD300 strain grown on MM (NH ₄ ⁺ ) plate, and collect spores using a spreader. It became cloudy. 5 mL of this was added to 150 mL of MM (NH ₄ ⁺ ) in a 500 mL baffled Erlenmeyer flask, and the mixture was shaken at 30 ° C. and 160 min ⁻¹ for 16 to 20 h. After completion of the culture, the cells were collected on Miracloth and washed with Protoplasting Buffer (PB, 0.8M NaCl, 10 mM NaH ₂ PO ₄ ). A part of the collected cells was suspended in 10 mL of PB containing 40 mg of Yatalase and 30 mg of Lysing Enzyme in a 50 mL centrifuge tube, and incubated at 30 ° C. for 120 minutes with gentle shaking. During this time, the cells were gently loosened by pipetting every 30 minutes. After the reaction, the reaction solution was filtered with Miracloth to remove debris such as mycelia, and the filtrate was centrifuged at 4 ° C. and 2000 rpm for 5 minutes. The precipitate was suspended in about 10 mL of Aspergillus Transformation Buffer (ATB, 0.8 M NaCl, 10 mM Tris-HCl (pH 7.5), 50 mM CaCl ₂ ), and centrifuged at 4 ° C. and 2000 rpm for 5 minutes. The precipitate was suspended in an appropriate amount of ATB to obtain a protoplast solution.

B. Transformation of A. oryzae Various plasmid solutions (pNAN8142, pNAN-bgl1-CBD _cbhI , pNAN-bgl1-CBD) in which equal amounts of 2 × ATB were added to 100 μL of protoplast solution (1 × 10 ^-9 plopplast / mL) _cmc2 , pNAN-CBD _cbhII- bgl1 10 μg-DNA each). A 20% amount of PEG solution (60% PEG 4000, 10 mM Tris-HCl (pH 7.5), 50 mM CaCl ₂ ) was added to this solution and mixed gently. After standing for 10 minutes on ice, 1 mL of PEG solution was added and gently mixed. After leaving still at room temperature for 10 minutes, 10 mL ATB was added and mixed well, and it centrifuged at 4 degreeC and 2000 rpm for 5 minutes. The precipitate was suspended in an appropriate amount of ATB, mixed with 5 mL of Top Agar (0.7% Agarose dissolved in RM) kept at 45 ° C., and overlaid on the RM plate. Each transformant was obtained by culturing at 30 ° C. for 3 days. The obtained strains were named as follows.
Plasmid strain name
pNAN8142 A.oryzae8142
pNAN-bgl1-CBD _{cbh1 A.oryzaeBGL1} -CBD _CBHI1
pNAN-bgl1-CBD _{cmc2 A.oryzaeBGL1} -CBD _CMC1
pNAN-CBD _cbhII -bgl1 A oryzaeCBD _CBHII -BGL1

C. Culture In order to confirm the expression of the target protein, the obtained transformant was cultured in MM (NO ₃ ⁻ ). One platinum loop was inoculated into a thick test tube containing 10 mL of medium containing 5% MM (NO ₃ ⁻ ) lucose and 1% NaNO _3, and cultured at 30 ° C. and 170 min ⁻¹ for 3 days.

D. Release of BGL1 and CBD-bound BGL1 It is known that BGL1 binds to the cell surface after secretion. Therefore, the cultured transformants were treated as follows in order to release BGL1 and various CBD-bound BGL1 bound to the surface of the cells.
The culture solution was poured into a Buchner funnel covered with filter paper together with the cells and suction filtered to remove the medium. The cells remaining on the filter paper were washed with 150 mL of 20 mM acetate buffer (pH 5.0) to completely remove the medium components. The obtained bacterial cells were added to a free buffer (Cycloheximide 20 μg / mL, 1 mM phenylmethylsulfonyl fluoride, 0.2% Triton X-100, 20 mM acetate buffer (pH 5.0)) equivalent to the medium and shaken at 30 ° C. and 160 min ⁻¹ for 4 days. did.

E. Activity measurement The enzyme activity of BGL1 was measured by the pNP method. A 3 mM p-Nitrophenyl-β-D-glucopyranoside (pNP-Glc) solution (in 100 mM Acetate buffer pH 5.0) was used as a substrate, and the enzyme solution was diluted to an appropriate concentration with 100 mM Acetate buffer (pH 5.0). In addition, 1 mg / mL ovalbumin was added so that 10 μg of ovalbumin was contained in the reaction solution when the enzyme solution was diluted to stabilize BGL1. The enzyme reaction was performed by adding 100 μL of the same pre-incubated substrate solution to 100 μL of the enzyme solution preincubated for 5 minutes at 37 ° C., mixing well, and reacting at 37 ° C. for 10 minutes. After the reaction, 2 mL of 1M Na ₂ CO ₃ was added to stop the reaction, and the released pNP concentration was calculated from the absorbance at 405 nm using the extinction coefficient of p-Nitrophenol (pNP) to determine the activity. As a blank, 100 μL of 100 mM acetate buffer (pH 5.0) was used instead of the enzyme solution, and 100 μL of substrate solution and ₂ mL of 1M Na ₂ CO ₃ were added.

1 unit (1 U) was defined as the amount of enzyme that liberates 1 μmol of pNP per minute, and the enzyme activity was calculated by the following formula. As the extinction coefficient, ε (405) = 0.0185 mL / nmol · cm ⁻¹ was used.
Enzyme activity (units / mL) =
x / 18.5 × 2.2 mL / (10 min) × (1 / 0.1 mL) × dilution rate
x: A ₄₀₅ (however, the value obtained by subtracting the blank value from the test value)

F.SDS-PAGE (SDS-polyacrylamide gel electrophoresis)
SDS-PAGE followed the method of Leammli (Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970). A polyacrylamide gel (Acrylamide: N, N′-Methylene bisacrylamide = 29.2: 0.8) consisting of two layers of 9.0% separation gel and 5% concentration gel was prepared. Add equal volume of 2x Sample buffer (0.125M Tris-HCl, 20% Glycerol, 2% SDS, 2% 1-Mercaptoethanol, 0.001% Bromophenol blue, pH 6.8) to the sample enzyme solution at 100 ° C. Samples were processed for 10 minutes. Using a vertical slab electrophoresis apparatus (manufactured by ATTO), electrophoresis was performed in a buffer for electrophoresis (0.1% SDS, 25 mM Trisbase, 192 mM Glycine) at a constant current of 20 mA.

G. Reagents etc. The strains and culture media used for enzyme expression are as follows.
a) Strains used
A.oryzae niaD300 (derived from RIB40) ΔniaD
A.oryzae BGL1 bgl1
A.oryzae BGL1 strain is a strain in which bgl1 of A.aculeatus is incorporated into A.oryzae using pNAN8142 vector, and was obtained by Saijo et al. (Toru Nishijo, b-derived from Aspergillus aculeatus High expression of glucosidase 1 gene in Aspergillus oryzae, Master's thesis, Osaka Prefecture University, 2003).
b) Medium used
The following media were used for the culture of A.oryzae. Agar with 1.5% concentration was added to the plate medium.
^{_{i) Minimum medium (NO 3 -}} ) (MM (NO 3 -)) (pH6.5)
Salts solution ^* 5.0%
Trace element mixture ^** 0.10%
Glucose 1.0%
NaNO ₃ 0.3%
ii) Minimum medium (NH ₄ ⁺ ) (MM (NH ₄ ⁺ )) (pH 6.5)
MM (NO ₃ ^-) in place of NaNO ₃ of those using Ammonium tartrate 0.18%.
iii) Regeneration Medium (RM) (pH 6.5)
Salts solution ^* 5.0%
Trace element mixture ^** 0.10%
Glucose 1.0%
NaNO ₃ 0.3%
NaCl 4.68%
* Salts solution
KCl 2.6%
MgSO ₄ · 7H ₂ O 2.6%
KH ₂ PO ₄ 7.6%
※※ Trace element mixture
Mo ₇ O ₂₄・ 4H ₂ O 0.11%
H ₃ BO ₃ 0.11%
CoCl ・ 6H ₂ O 0.16%
CuSO ₄ · 5H ₂ O 0.16%
EDTA 5.0%
FeSO ₄・ 7H ₂ O 0.50%
MnCl ₂ · 4H ₂ O 0.50%
ZnSO ₄ · 7H ₂ O 2.2%
c) Reagents used The following enzymes were used for the preparation of protoplasts.
Yatalase (manufactured by Takara Bio Inc.), Lysing Enzyme (manufactured by Sigma)
d) Other reagents Unless otherwise specified, special grade reagents manufactured by Wako Pure Chemical Industries, Ltd. or Nacalai Tesque were used.
H. Results pNAN-bgl1-CBD _cbhI constructed _{above, pNAN-bgl1-CBD cmc2,} pNAN-CBD cbhII -bgl1, and was transformed into A.oryzae using pNAN8142 vector containing no insert. Monospore formation was repeated 2-3 times, and the final strains were 16 _A.oryzae8142 strains, 8 _A.oryzae BGL1-CBD _CBHI strains, 7 _A.oryzae BGL1-CBD _CMC2 strains, A .oryzae CBD _CBHII -BGL1 stock became 8 stocks.

When each strain was cultured in multiple strains and released, and the activity against pNP-Glc was followed, almost no activity was detected in A. _oryzae8142 , but _A.oryzae BGL1-CBD _CBHI and A. oryzae strains were detected. In the oryzae CBD _CBHII- BGL1 strain, an activity equivalent to that of the A.oryzae BGL1 strain was detected (FIG. 5). Furthermore, the presence of proteins that were slightly larger than BGL1 and considered to be BGL1-CBD _CBHI and CBD _CBHII -BGL1, respectively, was confirmed by SDS-PAGE (FIG. 6). On the other hand, in the A. oryzae BGL1-CBD _CMC2 strain, the activity per free buffer was low and the amount of enzyme produced was small compared to other CBD-bound BGL1 (FIG. 6).

3. Purification of enzyme A. Culture Seed culture was carried out by the method of 1-2 above C), and the whole culture solution was added to a 500 mL baffled Erlenmeyer flask containing 200 mL of the same medium, and further 30 ° C., 160 min. ^-1 for 3 days.

B. Preparation of Crude Enzyme Solution As shown in 2.D. above, after recovering the cultured cells, free buffer without Triton X-100 (Cycloheximide 20 μgmL, 1 mM phenylmethylsulfonyl fluoride, 20 mM Acetate buffer (pH 5.0)) ) Was used to release the enzyme from the cell surface (30 ° C., 160 min ⁻¹ , 4 days). Next, this solution was filtered with stockings together with stockings to roughly remove the cells, and the filtrate was centrifuged (4 ° C., 10800 rpm, 30 min) to obtain a supernatant to completely remove the cells. . This was used as a crude enzyme solution for the following purification.

C. Purification Each enzyme was purified as follows.
The crude enzyme solution was adsorbed on DEAE-TOYOPEARL650M equilibrated with 20 mM Acetate buffer (pH 5.0) and eluted with 1 L linear gradient of 0-0.3 M NaCl solution (in 20 mM Acetate buffer (pH 5.0)), then pNP The fraction showing the degradation activity against -Glc was collected. Next, ammonium sulfate is added to the collected active fraction so as to be 30% saturated, and adsorbed on Butyl-TOYOPEARL650M equilibrated in advance with a 30% saturated ammonium sulfate solution (in 20 mM Acetate buffer (pH 5.0)). Elution was performed with 1 L of a reverse linear gradient of a saturated ammonium sulfate solution (in 20 mM Acetate buffer (pH 5.0)). The active fraction was collected and added with ammonium sulfate to 80% saturation, and ammonium sulfate salting out was performed. The precipitate was recovered by centrifugation (4 ° C., 10800 rpm, 30 min), and then dissolved in a small amount of 20 mM Acetate buffer (pH 5.0). This was dialyzed overnight in a 20 mM acetate buffer (pH 5.0) using a dialysis membrane (manufactured by Sanko Junyaku Co., Ltd.) to obtain a purified sample. On the other hand, BGL1-CBD _CBHI , BGL1-CBD _CMC2 , and CBD _CBHII -BGL1 _were subjected to ethanol precipitation by adding an equal amount of 99.5% ethanol. The precipitate was collected by centrifugation (4 ° C., 8000 rpm, 30 min), washed with 50% ethanol solution (in 20 mM Acetate buffer (pH 5.0)), and the precipitate was separated again by centrifugation (4 ° C., 8000 rpm, 30 min). . After performing this washing twice, the supernatant was completely removed and dried under reduced pressure using a desiccator, and the precipitate was dissolved in an appropriate amount of 20 mM Acetate buffer (pH 5.0) to obtain a purified sample.

D. Activity measurement The measurement was performed in the same manner as 2.E.

E.SDS-PAGE
The same measurement as in 2.F.

F. Measurement of protein amount The protein amount was calculated from the absorbance at 280 nm using the extinction coefficient of each enzyme.
Extinction coefficient of each enzyme Gill et al. Method (. Gill SC, Hippel PH: Calculation of protein extinction coefficients from amino acid sequence data Anal Biochem 182: 319-326, 1989) with reference to, the molar extinction coefficient of Trp 5690cm ^{- 1 · M -1, 1280cm -1 ·} M -1 and Tyr, the Cys and 120cm ^-1 · M ^-1, and deduced from the primary sequence of amino acids.

G. Results
BGL1 and CBD-bound BGL1 produced in A. oryzae were purified using DEAE-TOYOPEARL650M, Butyl-TOYOPEARL650M, or Phenyl-TOYOPEARL650S (FIGS. 7 to 9). Were subjected to various purified enzyme to SDS-PAGE, BGL1 about 130kDa, BGL1-CBD _CBHI about 140kDa, CBD _CBHII -BGL1 about 145kDa, BGL1-CBD _CMC2 each observed single band at approximately 130 kDa It was confirmed that the protein was purified as an electrophoretically uniform protein. The results are shown in FIG.

4. Properties of enzyme Next, the effect of CBD binding to BGL1 was examined using the enzyme purified above.
A. Preparation of Alkaline Swelling Cellulose (ASC) ASC is a funacell using Hash's method (Hash JH, King KW: On the nature of the b-glucosidases of Myrothecium verrucaria. J Biol Chem, 232: 381-393, 1958). (Manufactured by Funakoshi). 6 g of funacel was gradually added to 200 mL of ice-cooled 35% NaOH while stirring and allowed to stand for 30 min. Thereafter, 3 L of ice-cooled distilled water was added, and HCl was added to adjust to pH 3.0. After pH adjustment, ASC was precipitated by allowing to stand at 4 ° C. for 30 to 40 minutes, the supernatant was removed by decantation, and 3 L of distilled water cooled in advance at 4 ° C. was added. After repeating this 5 times, ASC was separated by centrifugation (10800 rpm, 30 min, 4 ° C.), and the precipitate was suspended again in distilled water. After repeating this 5 times, it was suspended again in distilled water and subjected to ultrasonic crushing using ULTRASONIC DISRUPTOR UD-201 (manufactured by TOMY) (output 8, duty 50, 10 min, interval 10 min, 6 sets). Distilled water was added to make a total volume of about 400 mL to prepare an ASC stock solution. Measure the average amount of reducing sugar and total sugar (Dubois M, Gilles K., Hamilton JK, Rebers PA, Smith F: A colorimetric method for the determination of sugars. Anal Chem 28 (3), 350-356, 1956) The degree of polymerization was calculated. The enzyme reaction substrate was used as a 1% (w / v) ASC suspension (in 20 mM Acetate buffer (pH 5.0)) based on the concentration of the stock solution determined from the total amount of sugar.

B. Examination of substrate specificity a) Activity against pNP-Glc
The activity against pNP-Glc was carried out in the same manner as in 2.E.
b) Activity against Salicin
This was performed by the Somogyi-Nelson method (Sakuzo Fukui: Biochemical Experimental Method 1, Reducing Sugar Quantification Method, Second Edition, 1990). A moderately diluted enzyme solution (100 μL, 1% Salicin (in 100 mM Acetate buffer (pH 5.0)) (100 μL) was mixed and reacted at 37 ° C. for 10 minutes. The reaction was stopped by adding 500 μL of Somogyi solution, and distilled water was added to make a total volume of 1 mL, followed by boiling in boiling water for 15 minutes. After boiling, it was cooled with running water for 5 minutes, and 500 μL of Nelson solution was quickly added and mixed well. After allowing to stand for 20 minutes, 3.5 mL of ion exchange water was added and mixed well, and the absorbance at a wavelength of 500 nm was measured. The obtained value was substituted into a standard curve obtained using glucose, and the amount of reducing sugar was calculated. One unit was defined as the amount of enzyme that produced 1 μmol of reducing sugar per minute.

Activity against C. Cellobiose
Glucose-oxidase method was used. 100 μL of 1% Cellobiose (in 100 mM Acetate buffer (pH 5.0)) was mixed with 100 μL of an appropriately diluted enzyme solution and reacted at 37 ° C. for 10 minutes. The reaction was stopped by adding 50 μL of 1M HCl, and allowed to stand for 5 minutes, and then 50 μL of neutralizing solution (1MTris: 2MNaOH = 8: 2) was added. 100 μL was taken from this solution, mixed with 100 μL of a color-developing reagent (glucose CII-Test Wako (manufactured by Wako Pure Chemical Industries)), and allowed to develop color at 30 ° C. for 15 minutes, and the absorbance at a wavelength of 500 nm was measured. The obtained value was substituted into a standard curve obtained using glucose, and the amount of produced glucose was calculated. One unit was defined as the amount of enzyme that produces 2 μmol of glucose per minute.

D. Time Course of ICOS Decomposition 1% (w / v) ICOS suspension (in 100 mM Acetate buffer (pH 5.0)) Each reaction was started by dispensing 100 μL of an appropriately diluted enzyme solution. After 30 minutes, 2 hours, 4 hours, 8 hours and 16 hours after the start of the reaction, 50 μL of 1M HCl was added to stop the reaction, and the amount of glucose produced was determined by the Glucose-oxidase method in the same manner as the activity against C. cellobiose. However, since ICOS is insoluble, the neutralizing solution was added, the reaction solution was taken in an eppen, and 100 μL of the supernatant obtained by centrifugation (15000 rpm, 4 ° C., 10 min) was mixed with 100 μL of the coloring reagent. 1 unit is defined as the amount of enzyme that produces 1 μmol of glucose per minute, and in the ICOS decomposition reaction solution, sodium azide is used to prevent the growth of miscellaneous bacteria. The reaction was performed by adding 0.02%.

E. Time Course of ASC Decomposition ASC was decomposed in the same manner as the time course of D.ICOS decomposition. However, the base mass was 300 μL of 1% ASC suspension (in 100 mM Acetate buffer (pH 5.0)), and the reaction time was 30 minutes, 8 hours, 24 hours, and 48 hours. In all enzyme reactions, 10 μg of ovalbumin was added to the reaction solution in order to stabilize GBL1 and CBD-bound BGL1.

F. Adsorption to ASC Each enzyme sample of known concentration is diluted at an arbitrary ratio, mixed with 1 mg of ASC in an eppen (in 100 mM Acetate buffer (pH 5.0), total volume 0.6 mL) and adsorbed on ice for 120 minutes. I let you. However, the reaction solution was lightly shaken 30 minutes after the start of adsorption and 1 hour later. After completion of the reaction, centrifugation (15000 rpm, 4 ° C., 1 min) was performed to precipitate ASC and the enzyme bound thereto, and 500 μL of supernatant was obtained. From the absorbance A ₂₈₀ of the obtained supernatant, the enzyme concentration in the supernatant was calculated, and the amount of the adsorbed enzyme was calculated by calculating the difference from the initial enzyme concentration.

G. Thermostability 200 μL of enzyme (0.264 μM, in 100 mM Acetate buffer (pH 5.0)) was taken in an eppen and incubated in a water bath set at an arbitrary temperature of 30 to 75 ° C. for 30 minutes. After incubation, the mixture was quenched in ice and the residual activity against pNG-Glc was measured. The measurement was performed in the same manner as the above 2.E. activity measurement.

H. pH stability The pH was adjusted by diluting the enzyme solution 10 times using various 60 mM buffers shown in Table 4 below. 100 μL (1.32 μM) of the enzyme solution whose pH was adjusted was taken in an eppen and allowed to stand in an air incubator at 25 ° C. for 15 hours. After allowing to stand, 100 mM Acetate buffer (pH 5.0) was quickly added to dilute it 10 times to return to pH 5.0, and the residual activity against pNP-Glc was measured. The measurement was performed in the same manner as the above 2.E. activity measurement.

I. Reagents used The reagents used for examining the properties of the enzyme are as follows.
i) Reagents used a) Insoluble cellooligosaccharide (ICOS)
ICOS was prepared from Cellulosepowder D (manufactured by ADVANTEC) by Nakaya et al. Using the method of Sakamoto et al. (Shinichiro Sakamoto, Research on Cellulase System of Aspergillus aculeatus No. F-50, Osaka Prefecture University Graduate School Doctoral Dissertation, 1984). It was used. The average degree of polymerization is determined to be 26.7 from the ratio of (total sugar amount) / (reducing sugar amount).
b) Somogyi-Nelson reagent
Somogyi liquid
Na ₂ SO ₄ 18%
Na ₂ CO ₃ 2.4%
CuSO ₄ · 5H ₂ O 0.4%
NaHCO ₃ 1.6%
(CH (OH) COO) ₂ KNa · 4H ₂ O 1.2%
Nelson liquid
(NH ₄ ) ₆ Mo ₇ O ₂₄・ 4H ₂ O 5.0%
_{Na 2 HAsO 4 · 7H 2 O} 0.6%
H ₂ SO ₄ 4.6%

J. Results When an adsorption assay for ASC was performed using a purified enzyme, BGL1 did not adsorb to ASC, but BGL1-CBD _CBHI and CBD _CBHII -BGL1 confirmed adsorption (FIG. 12). Both reciprocal plot (Seki H, Suzuki A, Maruyama H:. Adsorption of egg albumin onto methylated yeast biomass Journal of Colloid and Interface Science 270: 304-308, 2004) adsorption constants takes (K _a) and the maximum adsorption amount (B was determined to _max), BGL1-CBD _CBHI and CBD _CBHII K _a is respectively 6.0 × 10 ⁶ M ^-1 of -BGL1, slightly better of 8.7 × 10 ⁶ M ^-1 and CBD _CBHII -BGL1 B _max was 0.78 × 10 ⁻⁸ mol / mg-ASC, 0.64 × 10 ⁻⁹ mol / mg-ASC and BGL1-CBD _CBHI , respectively, slightly higher (Table 5). ). Since BGL1-CBD _CMC2 was not adsorbed on ASC, other properties were not examined (data not shown).

BGL1, BGL1-CBD _CBHI , CBD _CBHII -BGL1 were examined for degradation activity against soluble substrates (Cellobiose, pNP-Glc, Salicin), and no difference was found between BGL1 and various CBD-bound BGL1 (Table 6). ). On the other hand, in the degradation of insoluble substrates (ASC, ICOS), a significant difference was observed between BGL1 and CBD-bound BGL1. The amount of glucose produced at the time of reaction with ASC at 37 ° C. for 48 hours was measured to be 11.1 μg for BGL1, 18.1 μg for BGL1-CBD _CBHI , and 14.7 μg for CBD _CBHII -BGL1, and BGL1- CBD _CBHI and CBD _CBHII -BGL1 produced 1.6 times and 1.3 times the amount of glucose compared to BGL1, respectively (FIG. 13A). In the degradation to ICOS, 24.3 μg of glucose was produced in BGL1 in a reaction at 37 ° C. for 16 hours, whereas in BGL1-CBD _CBHI and CBD _CBHII -BGL1, _37.0 μg and _36.1 μg, respectively, with respect to BGL1 Both produced 1.5 times as much glucose (FIG. 13B).

    The stability of various purified enzymes to heat and pH was examined. When 200 μL of an enzyme solution with an equal concentration was incubated at an arbitrary temperature of 30 to 75 ° C. for 30 minutes and rapidly cooled, and the residual activity against pNP-Glc was measured, there was a difference between wild type BGL1 and CBD-bound BGL1. There was no (FIG. 14A). Both wild type and CBD binding type retained 80% or more of activity up to 65 ° C, but lost about 50% of activity at 67.0 ° C and almost completely inactivated at 70 ° C. Moreover, when the residual activity against pNP-Glc was measured after exposure to an arbitrary pH of 2.3 to 10.1 at 25 ° C. for 15 hours, both the wild type and CBD binding types had pH of 2.7 to 9.1. While the activity of 80% or more was maintained in the range, the activity decreased rapidly outside the range (FIG. 14B, Table 5).

    The size of BGL1 purified this time was about 130 kDa as confirmed by SDS-PAGE. This was reported in a previous report (Toru Nishizaki, β-glucosidase 1 derived from Aspergillus aculeatus 1 in A. oryzae, Master's thesis, Osaka Prefecture University, 2003). The size of BGL1 purified from A. oryzae BGL1 strain was about 130 kDa. It was also agreed that the authentic BGL1 was 133 kDa. Therefore, it was concluded that BGL1 purified this time was produced from the Bgl1 gene of A. aculeatus.

In contrast, various CBD-bound BGL1s were expected to increase in molecular size as much as Linker and CBD were bound. As a result, BGL1-CBD _CBHI and CBD _CBHII- BGL1 were larger than BGL1, being about 140 kDa and 145 kDa, respectively, and it was estimated that they were purified in a form in which Linker and CBD were correctly bound. Moreover, when the specific activity with respect to the soluble substrate was calculated | required about these enzymes, the difference was not seen in all the substrates used for the measurement, and it turned out that the catalytic activity of BGL1 is not prevented by CBD addition. On the other hand, BGL1-CBD _CMC2 has a specific activity for pNP-Glc that is equivalent to that of BGL1, but a band was seen at about 130 kDa as BGL1 by SDS-PAGE, suggesting that Linker and CBD are not bound. It was broken. Therefore, when BGL1-CBD _CMC2 was adsorbed on ASC, BGL1-CBD _CBHI was adsorbed at a concentration (A ₂₈₀ = 0.256) at which sufficient binding could be confirmed, but A ₂₈₀ representing the adsorbed amount _. the difference ((initial a ₂₈₀₎ - (a ₂₈₀ of non-adsorbed fraction)) is just 0.025 mm was less than 20% of other CBD linked BGL1. In addition, since this enzyme is a crude enzyme solution at the initial stage of the purification step and a band is already shown at about 130 kDa by SDS-PAGE (data not shown), the gene is not expressed correctly, or It is _{thought that the} CBD _CMC2 or Linker region is susceptible to protease action and was cleaved during culture or release treatment. Based on the above, it was determined that BGL1-CBD _CMC2 was not purified in a state where CBD was bound, and the following examination was not performed.

When BGL1 and BGL1-CBD _CBHI and CBD _CBHII -BGL1 were adsorbed to ASC, BGL1 did not adsorb at all, whereas both CBD-bound BGL1 adsorbed to ASC. As a result, it was confirmed that BGL1-CBD _CBHI and CBD _CBHII -BGL1 were purified in a CBD-bound form. Regarding the adsorption constant and the maximum amount of adsorption, K _a = 6.0 × 10 ⁶ M ⁻¹ , 8.7 × 10 ⁶ M ⁻¹ , B _max = 0.78 × 10 ⁻⁹ mol / mg-ASC, 0.7 It became 64 * 10 < ^-9 > mol / mg-ASC. CBHI and CBHII of Ka of T. reesei, referring to B _max, CBHI under adsorption temperature 4 ° C., K _a, respectively 0.93 × 10 ⁶ M ^-1 for Avicel of CBHII, 1.92 × 10 ⁶ M ^{- 1} and B _max were 0.74 × 10 ⁻⁹ mol / mg and 0.52 × 10 ⁻⁹ mol / mg (Medve J, St_hlberg J, Tjerneld F: Isotherms for adsorption of cellobiohydrolase I and II from Trichoderma reesei on microcrystalline cellulose. Applied Biochemistry and Biotechnology 66: 39-56, 1997). Also, K _a of adsorption to BMCC of the CBHI is ^{8.33 × 10 6 M -1, B} max is ^{6.0 × 10 -9 mol / mg (} Reinikainen T, Teleman O, Teeri TT: Effects of pH and high ionic strength on the adsorption and activity of native and mutated cellobiohydrolase I from Trichoderma reesei. 22: 392-403, 1995). Compared with these values, BGL1-CBD _CBHI and CBD _CBHII -BGL1 show the same affinity and maximum adsorption amount as _T. reesei CBHI adsorbed on Avicel, and CBD functions inferior. It was considered.

Regarding degradation to insoluble substrates, it was expected that differences were also observed among CBD-bound BGL1 due to the difference in affinity of each CBD-bound BGL1. However, when measured with the same amount of enzyme and the same reaction time, BGL1-CBD _CBHI and CBD _CBHII- BGL1 both produced 1.5 times as much glucose as BGL1 compared to ICOS. There was also a difference between -CBD _CBHI and CBD _CBHII -BGL1. This is because the K _a is greater in BGL1-CBD _CBHI in the adsorption test of the ASC, it was contrary to the expectation that it will decompose more ASC. In consideration of this, in order to decompose the portion of CBD-bound BGL1 that cannot be decomposed by BGL1 alone, the stepwise reaction in which adsorption to the insoluble substrate first occurs and then the catalytic domain binds to the substrate and decomposes. FEBS J 273: 2869-2878, Igarashi K, Wada M, Hori R, Samejima M: Surface density of cellobiohydrolase on crystalline celluloses. A critical parameter to evaluate dynamic kinetics at a solid-liquid interface. 2006). Therefore, it is considered that whether or not the transition from the adsorption to the substrate to the substrate binding of the catalytic domain is performed smoothly also affects the decomposition rate of the insoluble substrate. In this study, it is expected that the spatial positional relationship between the CBD and the catalytic site is three-dimensionally different due to the difference of CBD addition to the N-terminal or C-terminal. In addition, the length of Linker is also different (CBD _CBHII- BGL1 is 14 amino acid residues longer). From these differences, it _seems that BGL1-CBD _CBHI transitioned more smoothly from adsorption to decomposition and showed higher activity against ASC. On the other hand, ICOS has a lower average degree of polymerization than ASC (DP = 165) (DP = 26.7), and BGL1 is a substrate that is more easily decomposed than ASC, so it can be considered that there is no difference.

K. Degradation test of ASC In order to evaluate the effect of CBD binding more strictly, the following test was further performed. Here, CBD _CBHII- BGL1-CBD _CBHI (FIG. 10) prepared and purified by the same procedure as described above was also evaluated.
100 μl of a 6.60 μMBGL1 solution was dispensed into 300 μl of 1% ASC suspension (in 100 mM Acetate buffer (pH 5.0)) and reacted at 37 ° C. for 16 hours. Subsequently, 100 μl of 6.60 μM BGL1 solution or 100 μl of CBD-bound BGL1 (0.0985-4.8 μM) to which various CBDs diluted in stages were added and reacted at 37 ° C. for another 24 hours, 125 μl of 1M HCl To stop the reaction. Hereinafter, the amount of produced glucose was measured by the Glucose-oxidase method. After stopping the reaction, add 125 μl of neutralizing solution (1M Tris: 2M NaOH = 8: 2 mixed solution), take the whole amount of the reaction solution in an eppen, obtain the supernatant obtained by centrifugation at 13500 rpm, 4 ° C., 10 min, 100 μl of which is 96 holes The plate was mixed with 100 μl of a coloring reagent (glucose CII test Wako), reacted at 30 ° C. for 15 minutes, and the absorbance at a wavelength of 500 nm was measured. The obtained value was substituted into a standard curve obtained using glucose, and the amount of produced glucose was calculated. For blank, the reaction was stopped by adding 100 μl of 1M HCl at the time of 16 hours in the previous reaction (using only BGL1), and the amount of produced glucose was calculated in the same manner as described above. However, the addition of the neutralizing solution was 100 μl. The amount of glucose produced in blank (16 hours) was subtracted from the amount of glucose produced in each sample (16 hours + 24 hours), and the amount of newly produced glucose was determined from the time when a new enzyme was added. The amount of newly produced glucose is plotted against the amount of newly added enzyme, and the amount of glucose equivalent to BGL1 is calculated from a linear expression between the two nearest points sandwiching the amount of newly produced glucose (6.97 μg) of the sample to which BGL1 is added. The amount of CBD-bound BGL1 required for production was calculated. In the hydrolysis reaction, 10 μg of ovalbumin was added to the reaction solution in order to stabilize BGL1 and CBD-bound BGL1. Further, in order to prevent the propagation of various bacteria, the reaction was carried out by adding 0.02% sodium azide to the ASC decomposition reaction solution. The results are shown in FIG.

BGL1 and BGL1-CBD _CBHI and BGL1-CBD _CBHI were examined for degradation activity against soluble substrates (Cellobiose, pNP-Glc, Salicin). As shown in Table 6 above, between BGL1 and various CBD-binding BGL1 There was no difference. On the other hand, in the degradation of insoluble substrates (ASC, ICOS), a significant difference was observed between BGL1 and CBD-bound BGL1. As a pre-reaction, AGL was decomposed at 37 ° C. for 16 hours using BGL1, BGL1 or various CBD-bound BGL1 was added, and the mixture was further reacted at 37 ° C. for 24 hours, and the amount of newly formed glucose was measured. . As a result, the addition of BGL1 newly produced 6.97 μg of glucose, and as a result of calculating the amount of enzyme required to produce the same amount of glucose, BGL1-CBD _CBHI was 0.0299 nmol, BGL1-CBD _CBHI was 0.0437 nmol and CBD _CBHII -BGL1-CBD _CBHI was 0.0504 nmol, which was 22.2 times, 15.0 times and 13.1 times compared to BGL1 (0.662 nmol), respectively.

    Thus, by adding a CBHI-derived Linker and CBD to the C-terminal side of A. aculeatus-derived BGL1, or a CBHII-derived CBD and Linker to the N-terminal side, the insoluble cellulose remains while maintaining the original properties of BGL1. It was possible to increase the affinity for and dramatically improve the degradation activity.

[Production of cellulase (2)]
Next, a cellulase was prepared by binding A. aculeatus-derived carboxymethyl cellulase 1 to a cellulose binding domain of A. aculeatus-derived cellobiohydrase I or II via a linker region.

  As carboxymethyl cellulase 1 derived from A. aculeatus, carboxymethyl cellulase 1 derived from A. aculeatus F-50 strain already determined was used. For the cellulose-binding domain, CBD of cellobiohydrase I or CBD of cellobiohydrase II derived from the F-50 strain whose nucleotide sequence has already been determined was used.

As cellulases conjugated with CBD, cellulases (CMC1-CBD _CBHI ) in which CBD of _CBHI is bound to the C-terminal side of carboxymethyl cellulase 1 and cellulases ( _CMC1-- ) in which CBD of _CBHII is bound to the C-terminal side of β-carboxymethyl cellulase 1 Two cellulases of CBD _CBHI were made. These cellulases were expressed in separate hosts of E. coli and A. oryzae.

1. Construction of expression plasmid For E. coli, 666 bp from the cDNA of cmc1 except for the signal sequence and the stop codon is used. For A. oryzae, 895 bp (SEQ ID NO: 5) from the start codon of the cmc1 genomic gene to immediately before the stop codon is used. Amplified by The PCR product was subjected to agarose gel electrophoresis, confirming that a fragment of the correct length was amplified (FIG. 18). In addition, the PCR products cmc1g and cmc1c are inserted into the 5 ′ side of each cbd of pBs-CBD _cbhI and pBs-CBD _CMC2 constructed in the section 1. Expression plasmid of Example 1, so that CBHI or The gene was constructed so that CMC2-derived Linker and CBD were bound. By incorporating this into the expression vector pET20b (+) or pNAN8142, the expression plasmids pET-cmc1c-CBD _cbhI and pET-cmc1c-CBD _cmc2 for E. coli and the expression plasmids pNAN-cmc1g-CBD _cbhI and pNAN-cmc1g-CBD for filamentous fungi _cmc2 was obtained. Moreover, it was confirmed that the target gene was correctly inserted using Not I, Nde I, EcoR I, Hinc II and the like used when inserting the insert into the expression vector (data not shown).

A. Amplification of DNA fragment using PCR method The procedure was the same as in Example 1. 1. Construction of expression plasmid. Each PCR product was named cmc1g and cmc1c, respectively. Table 8 shows combinations of Template and primer pairs.

B. Construction of Expression Plasmid In order to express CBD-bound CMC1 in two hosts, E. coli and A. oryzae, each expression plasmid was constructed in the following manner.
a) Construction of expression plasmid for E. coli The PCR product cmc1c obtained by the PCR method described above was digested with Xho I and Nsi I, and inserted into pBs-CBD _cbhI and pBs-CBD _cmc2 digested with Sal I and Nsi I. This is cleaved at the Nde I site added to the 5 'end of cmc1c and the Bgl II site added to the 3' end of each CBD to cut out the target gene, and the Nde within the multiple cloning site of pET20b (+) Inserted into I site and BamH I site. The plasmids thus obtained were designated as pET-cmc1c-CBD _cbhI and pET-cmc1c-CBD _cmc2 and used for transformation of E. coli JM109 (DE3) (FIG. 16).
b) Construction of high-expression plasmid for filamentous fungi PCR product cmc1g obtained by the above PCR method was digested with Xho I and Nsi I and inserted into pBs-CBD _cbhI and pBs-CBD _cmc2 digested with Sal I and Nsi I did. This is cleaved at the Not I site added to the 5 'end of cmc1g and the Nde I site added to the 3' end of each CBD, and the target gene is excised, within the multi-cloning site of the high expression vector pNAN8142 for filamentous fungi. Inserted into the Not I and Nde I sites. The thus obtained plasmids were used as pNAN-cmc1g-CBD _cbhI and pNAN-cmc1g-CBD _cmc2 for transformation of A. oryzae (FIG. 17).

C. Other genetic engineering procedure The same procedure as in Example 1, 1. Construction of expression plasmid was performed.

D. Reagents used, etc. The strains, plasmids, media, primers, etc. used for the construction of the expression plasmid are as follows.
a) Strain used The same as used in Example 1. 1. Construction of expression plasmid.
b) Plasmid used
pNAN8142
pET20b (+)
pCMG14 pUC18, cmc1 (genome)
pCMC31 pUC13, cmc1 (cDNA)
And pBs-CBD _cbhI and pBs-CBD _cmc2 constructed in Example 1
c) Medium used The same as that used in 1. Construction of expression plasmid in Example 1.
d) Reagents used i) Restriction enzymes and modification enzymes Restriction enzymes and modification enzymes are manufactured by Nippon Gene, Toyobo, Takara Bio, or NEW ENGLAND BIOLABS (NEB). Each company's protocol was followed.
Restriction enzymes: BamH I, Bgl II, Nde I, Not I, Nsi I, Sal I, Xho I
Modification enzyme: T4 DNA ligase, PrimeSTAR HS DNA polymelase
ii) Primers Primers shown in Table 9 were used for amplification of the target gene.
iii) Other reagents Unless otherwise indicated, special grade reagents manufactured by Wako Pure Chemical Industries or Nacalai Tesque were used.

2. Enzyme Expression 2-1. Enzyme Expression in Escherichia coli Transformation of A. E. coli JM 109 (DE3) Strain It was carried out in the same manner as the transformation of B. A. oryzae in Example 1. However, pET20b (+), pET-cmc1c-CBD _cbhI , and pET-cmc1c-CBD _cmc2 were used as the plasmid solution. The resulting strains were designated as E. coli pET strain, E. coli CMC1c-CBD _CBHI strain, and E. coli CMC1c-CBD _CMC2 strain, respectively, and used in the following experiments.

B. Other genetic engineering operations The same procedure as in the transformation of B. A. oryzae in Example 1 was performed.

C. Culture A single colony of E. coli grown on LBplate was inoculated into 1.7 mL of 2 × TY medium (including Ampicillin) using a toothpick, and seed culture was performed at 30 ° C. and 130 min ⁻¹ for 15 hours. Inoculate 17 μL of each culture solution into 1.7 mL / tube x 6 of fresh 2 × TY medium (including Ampicillin), 3 at 37 ° C, and 3 at 30 ° C for another 6 hours. It was. Six hours later, 0.1 M Isopropyl-β-D-thiogalactopyranoside (IPTG) was added in an amount of 0 μL (no addition), 1.7 μL, and 17 μL, respectively, to the culture medium at three temperatures, and the culture was continued at the same temperature for another 6 hours. It was.

D. Preparation of bacterial cell extract A total of 18 culture solutions with different strains, culture temperatures, and IPTG concentrations were transferred to Eppens, centrifuged (4 ° C, 10,000 rpm, 1 min) to remove the supernatant, The body was recovered. Thereto, 1 mL of 20 mM Acetate buffer (pH 5.0) was added to suspend the cells, and ultrasonic crushing was performed using Handy Sonic UR-20P (TOMY SEIKO) while cooling well in ice water (30 sec, Power7, 4 sets). After crushing, the supernatant obtained by centrifugation (4 ° C., 15000 rpm, 10 min) was obtained as a soluble fraction. The precipitate was suspended in 1 mL of 20 mM Acetate buffer (pH 5.0) to obtain an insoluble fraction.

E. Confirmation of expression by SDS-PAGE It was carried out in the same manner as the transformation of B. A. oryzae in Example 1. However, the separation gel concentration was 15%.

F. Reagents used The strains and culture media used for transformation are as follows.
a) Strains used
Escherichia coli JM109 (DE3)
endA1, recA1, gyrA96, thi, hsdR17 (r _k ^- m _k ⁺ ), relA1, supE44,
Δ (lac-proAB), [F ', traD36, proAB, lacI ^q ZΔM15], λ (DE3)
b) Plasmid used
pET20b (+), pET-cmc1c-CBD _cbhI , pET-cmc1c-CBD _cmc2
c) Medium used The same as that used in the transformation of B. A. oryzae in Example 1.
d) Reagents used Unless otherwise indicated, special grade reagents manufactured by Wako Pure Chemical Industries or Nacalai Tesque were used.

G. Results E. coli JM109 (DE3) was transformed with the above-constructed pET-CMC1c-CBD _cbhI , pET-CMC1c-CBD _cmc2 and the vector pET20b (+) containing no insert. It was confirmed by carrying out plasmid extraction from the cultured transformant that the obtained strains had the target gene, by restriction enzyme treatment and agarose gel electrophoresis (data not shown). In order to confirm the production of the target protein, each transformant was cultured at different culture temperatures and IPTG concentrations, and the soluble and insoluble fractions of the cell disruption solution were each subjected to SDS-PAGE (FIG. 19 and FIG. 19). FIG. 20). The average molecular weights estimated for the amino acids of the proteins CMC1-CBD _CBHI and CMC1-CBD _CMC2 were 32100 and 35200, respectively, but no production of the target protein was observed in CMC1-CBD _{CBHI under} any culture conditions. . On the other hand, in CMC1-CBD _CMC2 , a band (about 35 kDa) that seems to be the target protein was observed at all temperatures and IPTG concentrations, but none was found in the soluble fraction and the insoluble fraction was not found at all. Only the presence was confirmed. This is because many O-linked sugar chains are usually added to the Linker region of enzymes derived from filamentous fungi (Reinikainen T, Teleman O, Teeri TT: Effects of pH and high ionic strength on the adsorption and activity of native and mutated cellobiohydrolase I from Trichoderma reesei. 22: 392-403, 1995), it is considered that the protein was relatively agglomerated due to the increased relative hydrophobicity of the protein because glycosylation was not performed. In FIG. 21, the result of having analyzed the product by colon_bacillus | E._coli and A.oryzae is shown.

2-2 Expression in Aspergillus oryzae Transformation of A.A.oryzae It was carried out in the same manner as the transformation of B.A.oryzae in Example 1. The obtained strains were named as follows.
Plasmid strain name
pNAN-cmc1g-CBD _{cbhI A.oryzae} CMC1-CBD _CBHI
pNAN-cmc1g-CBD _{CMC2 A.oryzae} CMC1-CBD _CMC2

B. Culture The culture was carried out in the same manner as the transformation of B. A. oryzae in Example 1. However, the culture was performed for 6 days, and 1 mL of 20% Glucose solution was added on the third day of culture.

C. Activity measurement The substrate used was a 0.625% Carboxymethyl cellulose (CMC) solution (in 100 mM Acetate buffer (pH 5.0)). 100 μL of enzyme solution was added to 400 μL of the substrate, mixed, and reacted at 37 ° C. for 10 minutes. Hereinafter, the amount of produced reducing sugar was measured by the Somogyi-Nelson method as shown in the section of 4.B.b) Activity for Salicin in Example 1. However, the OD ₅₀₀ was measured on the supernatant obtained by adding and mixing 3.5 mL of ion exchange water and then separating the insoluble components by centrifugation (4 ° C., 2000 rpm, 10 min).

D. SDS-PAGE (SDS-polyacrylamide gel electrophoresis)
This was carried out in the same manner as 2.E.SDS-PAGE in Example 1. However, the separation gel concentration was 15%.

E. Reagents used The strains, media, etc. used for enzyme expression are as follows.
a) Strain and plasmid used: A.oryzae niaD300 ΔniaD
Plasmid pNAN-cmc1g-CBD _cbhI , pNAN-cmc1g-CBD _cmc2
b) Medium used The same as that used in the enzyme expression in 2.A.oryzae of Example 1.
c) Reagent used The same as that used in the enzyme expression in 2.A.oryzae of Example 1.
d) Other Reagents Unless otherwise indicated, special reagents manufactured by Wako Pure Chemical Industries and Nacalai Tesque were used.

F. Results A. oryzae was transformed with pNAN-cmc1g-CBD _cbhI and pNAN-cmc1g-CBD _cmc2 constructed as described above. Monosupoa the repeated 2-3 times, finally strain obtained A.oryzae CMC1-CBD _CBHI 5 strain, A. oryzae CMC1-CBD _CMC2 was 7 strain. In order to confirm the production of the target protein, each of the obtained strains was cultured several times, and the activity against CMC and SDS-PAGE were performed using the culture supernatant. As a result, of the A. oryzae CMC1-CBD _CBHI strains, One of the strains showed about 4 times the activity (20.8 units / mL on the 6th day) per culture compared to the other strains, and a band with an intensity proportional to the enzyme activity was detected at a position of about 45 kDa (Fig. 21). Therefore, this strain was used for the following experiments on behalf of the _A.oryzae CMC1-CBD _CBHI strain. On the other hand, the _A.oryzae CMC1-CBD _CMC2 strains all have low activity per culture (2.82 units / mL on the 6th day), and the production of a protein that seems to be CMC1-CBD _CMC2 is confirmed by SDS-PAGE. Since it was not possible (data not shown), only CMC1-CBD _CBHI, which was confirmed to be mass-produced this time, was purified and used for the examination of the following properties.

4. Purification of enzyme A. Preparation of crude enzyme solution Seed culture was carried out for 3 days in the same manner as 3.A. Enzyme purification in Example 1, followed by main culture for 4 days. However, in order to suppress protease production due to depletion of nutrients on the third day of the main culture, a 1/4 volume of 20% Glucose / 5% NaNO ₃ solution was added. After culturing, the culture broth was filtered through stockings together with the stockings to roughly remove the cells, and the filtrate was centrifuged (4 ° C., 10800 rpm, 30 min) to obtain a supernatant to completely remove the cells. This was used as a crude enzyme solution for the following purification.

B. Purification CMC1-CBD _CBHI was purified as follows.
Ammonium sulfate is added to the crude enzyme solution to make it 30% saturated, adsorbed on Butyl-TOYOPEARL 650 M previously equilibrated with 30% saturated ammonium sulfate solution (in 20 mM Acetate buffer (pH 5.0)), and 30 to 0% saturated ammonium sulfate The solution was eluted with a 1 L reverse linear gradient. Fractions having activity against CMC were collected, and ammonium sulfate salting out was performed on the collected active fractions with 80% ammonium sulfate saturation. The precipitate was recovered by centrifugation (4 ° C., 10800 rpm, 30 min), dissolved in a small amount of 20 mM Glycine-HCl buffer (pH 3.5), and pre-equilibrated with 20 mM Glycine-HCl buffer (pH 3.5). Desalted through -2. After desalting, the active fraction was recovered, adsorbed on SP-TOYOPEARL 650M previously equilibrated with 20 mM Glycine-HCl buffer (pH 3.5), and eluted with a linear gradient of 0 to 0.3 M NaCl 1 L. After collecting the active fraction, ammonium sulfate salting out is performed again with 80% ammonium sulfate saturation, and the precipitate is collected by centrifugation (4 ° C., 10800 rpm, 30 min), and then dissolved in a small amount of 20 mM acetate buffer (pH 5.0). 20 for desalting. The solution thus obtained was used as a purified sample.

C. Activity measurement The measurement was performed in the same manner as the 2.E. activity measurement in Example 1.

  D.SDS-PAGE

    It carried out like 2.F.SDS-PAGE of Example 1. However, the concentration of the separation gel was 12.5%.

E. Measurement of protein amount It was carried out in the same manner as 3.F. protein amount in Example 1.

F. Reagents used The strains used for the purification of the enzyme are as follows.
a) Strains used
_A.oryzae CMC1-CBD _CBHI , _A.oryzae 8142
b) Medium used It is the same as that used in 2. Enzyme Expression in Example 2.
c) Reagents used Unless otherwise indicated, special grade reagents manufactured by Wako Pure Chemical Industries or Nacalai Tesque were used.

5. Properties of enzyme A. Examination of substrate specificity The amount of reducing sugar produced after the enzyme reaction was measured by the Somogyi-Nelson method in the same manner as in 5.A. Substrate specificity in Example 2 above. One unit was defined as the amount of enzyme that produced 1 μmol of reducing sugar per minute.
a) Activity against CMC The activity was the same as in Example 2, section 2-2, C. Activity measurement.
b) Activity against ASC 100 μL of enzyme solution was added to 100 μL of 0.5% ASC suspension (in 100 mM Acetate buffer (pH 5.0)), and reacted at 37 ° C. for 1 hour.
c) Activity against ICOS 100 μL of enzyme solution was added to 100 μL of 0.5% ICOS suspension (in 100 mM Acetate buffer (pH 5.0)) and reacted at 37 ° C. for 4 hours.
d) Activity against Avicel To 200 μL of 1% Avicel suspension, 500 μL of enzyme solution diluted so that each ΔOD500 value shows the same value was added and reacted at 37 ° C. for 24 hours.

B. Adsorption to Insoluble Substrate a) Adsorption to ASC The same method as in Example 2, 5.A. Substrate specificity was examined.
b) Adsorption on Avicel The same as the adsorption on ASC. However, the amount of Avicel used for adsorption was 10 mg.

C. Thermal stability test The test was performed in the same manner as in Example 2, 5.A. However, 200 μL of an enzyme solution having a concentration of 0.685 μM was used, and the residual activity was the same as that in Example 2-2, C. Activity measurement section, with respect to CMC.

D. pH stability The pH was adjusted by diluting the enzyme solution 10 times using the buffer shown in Table 10 below in addition to the buffer shown in 5. H. pH stability of Example 1. . 200 μL (1.37 μM) of the enzyme solution whose pH was adjusted was taken in an eppen and allowed to stand in an air incubator at 25 ° C. for 4 hours. After standing, 100 mM Acetate buffer (pH 5.0) was added to those having a pH of 2.3 to 9.6, and the pH was returned to 5.0 by diluting 10 times. On the other hand, those having a pH of 10.1 or more are returned to pH 5.0 by adding 200 mM Acetate buffer (pH 5.0) and diluted 7-fold, and the residual activity against CMC is the same as in Example 2 above. It measured by the method of.

E. Reagents Used The strains used for the properties of the enzyme are as follows.
a) Enzymes used CMC1, CMC1-CBD _CBHI
CMC1 was expressed in A.oryzae using A.aculeatus cmc1 using the pNAN8142 vector. Kobayashi et al. (Kebayashi Eriko: Aspergillus aculeatus No. F-50 cellulase-derived cellulase component derived from synergistic effects. Verification, Osaka Prefectural University Bachelor thesis, 2005).
b) Other reagents Same as those used in Example 4, 4. Enzyme Properties.

F. Results
CMC1-CBD _CBHI produced in A. oryzae was purified using Butyl-TOYOPEARL 650M and SP-TOYOPEARL 650M (FIG. 23, Table 11). When purified CMC-CBD _CBHI was subjected to SDS-PAGE, a single band was observed at a position of about 45 kDa, confirming that it was purified as an electrophoretically uniform protein (FIG. 24). This size is considerably larger than the average molecular weight estimated from the amino acid sequence of 32100, which is thought to be due to the addition of a sugar chain to the Linker region (Srisodsuk M, Reinikainen T, Penttila M, Teeri TT: Role of the interdomain linker peptide of Trichoderma reesei cellobiohydrolase I in its interaction with crystalline cellulose. Journal of Biological Chemistry 268: 20756-20761, 1993). Therefore, this was _designated as purified CMC1-CBD _CBHI and already purified CMC1 (Eriko Kobayashi: Verification of synergistic effect by reconstitution of cellulase components derived from Aspergillus aculeatus No. F-50, Osaka Prefecture University Bachelor thesis, 2005) The following properties were examined using

To examine adsorption to insoluble cellulose, adsorption tests were performed on ASC and Avicel. As a result, wild-type CMC1 showed only slight adsorption to ASC, but CMC1-CBD _CBHI clearly increased the amount of adsorption compared to CMC1. Further, in the adsorption to Avicel, almost no adsorption of CMC1 was observed, but CMC1-CBD _CBHI showed either higher adsorption activity (FIG. 25). Taking both reciprocal plot, adsorption constant was calculated K _a and adsorption maximum amount B _max, respectively K _a and B _max for ASC, CMC1 in ^{0.77 × 10 6 M -1, 0.32} × 10 - ^In contrast to ⁹ mol / mg-ASC, CMC1-CBD _CBHI has 27 × 10 ⁶ M ⁻¹ and 0.97 × 10 ⁻⁹ mol / mg-ASC, and the maximum adsorption is about 35 times. The amount increased about 3 times. Furthermore, the adsorption to Avicel whereas was observed almost the CMC1 adsorption, CMC1-CBD in _CBHI K _a is ^{1.2 × 10 6 M -1, B} max is 0.13 × 10 ^-9 mol / It became mg-Avicel. From these results, it was confirmed that the affinity to an insoluble substrate was improved by addition of CBD (Table 12).

Next, the substrate specificity of both enzymes was examined. There was little difference in the specific activity of both enzymes relative to the soluble substrate CMC. On the other hand, there was a significant difference between CMC1 and CMC1-CBD _{CBHI in} activity against insoluble substrates ICOS, ASC, and Avicel. When the enzyme activity was calculated by reacting at 37 ° C. for 1 hour using ASC as a substrate, the activities of CMC1 and CMC1-CBD _CBHI were 5.79 unit / μmol and 6.65 unit / μmol, respectively, and CMC1-CBD _CBHI was 1 of CMC1. Doubled. When enzyme activity was calculated by reacting with ICOS at 37 ° C. for 4 hours, the activities of CMC1 and CMC1-CBD _CBHI were 1.54 units / μmol and 2.78 units / μmol, respectively, which was 1.8 times higher. . Furthermore, _when the enzyme concentrations of CMC1 and CMC1-CBD _CBHI were adjusted to produce equal amounts of reducing sugars with respect to Avicel, which is crystalline cellulose, and reacted at 37 ° C. for 24 hours, 0.005 7 unit / Whereas it was μmol, CMC1-CBD _CBHI was 0.0263 nit / μmol, and 4.4 times the activity was obtained (Table 13). The improvement of the degradation activity for these insoluble substrates was thought to be due to the increase in the region where the catalytic domain can act on the substrate due to the increased affinity for the insoluble substrate due to the addition of CBD.

When the enzyme stability of the purified CMC1-CBD _CBHI and CMC1 was examined, the thermal stability of CMC1-CBD _CBHI was slightly lower than that of CMC1 (FIG. 26A). Both enzymes prepared at equimolar concentrations were incubated at an arbitrary temperature of 30 to 67.5 for 30 minutes, and after rapid cooling, the activity against CMC was measured. As a result, wild-type CMC1 retained 80% or more activity up to 62.5 ° C., whereas CMC1-CBD _CBHI had a residual activity of 32% at the same temperature. This decrease in thermal stability is due to the addition of CBD to the C-terminal of the catalytic domain, and the binding of the N-terminal and C-terminal and the loose-end structure often found in thermostable enzymes (Voutilainen SP, Boer H, Alapuranen M, Janis J , Vehmaanpera J, Koivula A: Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B. Appl Microbiol Biotechnol 83: 261-272, 2009). It is thought that it decreased. On the other hand, there was no difference between the two enzymes in terms of pH stability. When the residual activity against CMC was measured by incubating at 25 ° C. for 4 hours at an arbitrary pH, returning to pH 5.0, both were stable between pH 2 and 9.5 and destabilized at pH 10 or higher ( FIG. 26B, Table 12).

    From the above results, the addition of CBHI-derived Linker and CBD to CMC1 achieves an improvement in affinity for insoluble substrates and an improvement in degradation activity, thereby achieving a higher function of CMC1.

  According to the present invention, a novel cellulase having a cellulose binding domain and high cellulase activity is provided. By using this cellulase, saccharification of biomass can be performed more efficiently than before.

Claims (11)

  A region containing the catalytic domain of β-glucosidase 1 derived from A. aculeatus or a region containing the catalytic domain of carboxymethyl cellulase 1 derived from A. aculeatus, and a region derived from A. aculeatus directly or indirectly added to the region Cellulase having a cellulose binding domain.   The cellulase according to claim 1, wherein the cellulose binding domain is a cellulose binding domain of cellobiohydrolase I derived from A. aculeatus or cellobiohydrolase II derived from A. aculeatus.   The cellulose-binding domain is added to the C-terminal side and / or the N-terminal side of the region containing the catalytic domain of β-glucosidase 1 derived from A. aculeatus or the region containing the catalytic domain of carboxymethyl cellulase 1 derived from A. aculeatus. The cellulase according to claim 1 or 2.   The cellulase according to any one of claims 1 to 3, wherein the A. aculeatus is A. aculeatus No. F-50 strain.   A polypeptide having the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, or an amino acid sequence having 1 to several deletions / substitutions / additions in the amino acid sequence and a cellulase A polypeptide having activity. A polynucleotide encoding the polypeptide of claim 5.   A polynucleotide having the base sequence represented by any one of SEQ ID NOs: 1-4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11.   A vector containing a polynucleotide having the sequence according to claim 6 or 7.   A transformed cell transformed with the vector according to claim 8.   The transformed cell according to claim 9, wherein the transformed cell is any one of Escherichia coli, Bacillus subtilis, filamentous fungus, and yeast.   A method for saccharifying plant biomass using the cellulase according to any one of claims 1 to 4 or the transformed cell according to claim 9 or 10.