JP7208762B2 - A novel arabinofuranosidase - Google Patents

Description

The present invention relates to novel arabinofuranosidases.

In recent years, attention has been focused on research and development on the use of cellulosic biomass, which is produced as a by-product of agricultural production, such as post-harvest residues of crops and wood. Cellulosic biomass (hereinafter referred to as biomass) is composed of cellulose, hemicellulose and lignin, and these three components are strongly intertwined. Among these three components, sugar, which is a decomposition product of cellulose and hemicellulose, is used as a raw material for ethanol and chemical products.

Cellulose is a polysaccharide in which glucose is linked by β-1,4 bonds, hemicellulose is a heteropolysaccharide other than glucose, and arabinoxylan, which is the most abundant in hemicellulose, is mainly composed of xylose by β-1,4 bonds. Arabinose, galactose, glucuronic acid, ferulic acid, etc. are bound to the connected xylan skeleton as side chains. Enzymes that degrade cellulose and hemicellulose are cellulase and hemicellulase, and are collectively referred to as biomass saccharifying enzymes.

Biomass has a complexly entwined structure, which makes it difficult to decompose. Both cellulase and hemicellulase are essential for efficiently degrading cellulose and hemicellulose, and the type and composition of the enzyme have a great influence, so the development of saccharifying enzymes is an important issue.

In order to improve biomass decomposition, an enzyme composition has been developed in which hemicellulase such as xylanase derived from Trichoderma reesei or arabinofuranosidase is added to cellulase (Patent Document 1). Also Penicillium sp. A crude enzyme composition containing a xylanase having a high xylanase activity has been developed (Patent Document 2).

α-L-arabinofuranosidase, which is a glycoside hydrolase (GH), converts α-1,2 or α-1,3 or α-1,5-linked arabinose side chains to the non-reducing terminal side. and is classified under EC 3.2.1.55. It is also classified as GH43, 51, 54, and 62 in the classification based on amino acid sequence homology.
For example, Non-Patent Document 1 discloses the properties of α-L-arabinofuranosidase of 64.5 kDa and 62.7 kDa from Penicillium copsulatum, and Patent Document 3 discloses Aspergillus α-L-arabinofuranosidase from B. niger and its amino acid sequence are disclosed.

However, conventional arabinofuranosidases are not satisfactory as saccharifying enzymes capable of saccharifying biomass.

Japanese Patent Publication No. 2011-515089 Japanese Patent Application Laid-Open No. 2017-12006 WO2006/125438

Appl. Environ. Microbiol., 1996, 62, 168-173

The present invention relates to providing a novel arabinofuranosidase capable of efficiently saccharifying biomass.

The present inventors have found a novel arabinofuranosidase that has excellent arabinofuranosidase activity and high arabinoxylan-degrading activity, and that biomass can be efficiently saccharified by using this arabinofuranosidase. Found it.

That is, the present invention relates to the following 1) to 7).
1) A protein consisting of an amino acid sequence selected from the following (a) to (d) and having arabinofuranosidase activity:
(a) the amino acid sequence shown in SEQ ID NO: 2;
(b) an amino acid sequence represented by numbers 1 to 301 of SEQ ID NO: 2;
(c) an amino acid sequence having at least 90% identity with the amino acid sequence shown in (a) or (b);
(d) An amino acid sequence obtained by deleting, substituting or adding one or more amino acids in the amino acid sequence shown in (a) or (b).
2) A polynucleotide encoding the protein having arabinofuranosidase activity of 1).
3) A vector containing the polynucleotide of 2).
4) A method for producing a transformant, comprising introducing the polynucleotide of 2) or the vector of 3) into a host.
5) A transformant introduced with the polynucleotide of 2) or the vector of 3).
6) A biomass saccharifying agent containing the protein having arabinofuranosidase activity of 1).
7) A method for producing sugar from biomass using the protein having arabinofuranosidase activity of 1) or the biomass saccharifying agent of 6).

The present invention provides an arabinofuranosidase that has excellent arabinofuranosidase activity, high arabinoxylan-degrading activity, and a high biomass saccharification rate. By using the relevant arabinofuranosidase, more efficient biomass saccharification can be achieved as compared with the case of using a commercially available arabinofuranosidase or a composition containing it. Therefore, according to the present invention, sugar can be efficiently and inexpensively produced from biomass.

Structures of p-nitrophenyl-α-L arabinofuranoside and wheat arabinoxylan. Saccharification of wheat bran by addition of various α-L-arabinofuranosidases. Saccharification of wheat bran by saccharification enzyme expressing F81ABF. Multiple alignment of GH62 α-L-arabinofuranosidase. Molecular phylogenetic tree of GH62 α-L-arabinofuranosidase.

As used herein, the sequence identity of base sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing analysis using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win with Unit size to compare (ktup) set to 2.

As used herein, "at least 90% identity" with respect to amino acid sequences or nucleotide sequences means 90% or more, preferably 95% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more. Percent or more identity.

As used herein, "an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added" includes 1 or more and 30 or less, preferably 20 or less, more preferably 10 or less, and further Preferred amino acid sequences include deletions, insertions, substitutions or additions of 5 or less amino acids. Also, as used herein, "a nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted or added" includes 1 or more and 90 or less, preferably 60 or less, more preferably 30 or less, Nucleotide sequences in which 15 or fewer nucleotides, more preferably 10 or fewer nucleotides are deleted, inserted, substituted or added are more preferred.

As used herein, "biomass" refers to cellulosic and/or lignocellulosic biomass containing hemicellulose components produced by plants and algae. Specific examples of biomass include conifers such as larch and bald cedar, various types of wood obtained from broad-leaved trees such as oil palm (trunk) and cypress; processed or pulverized wood such as wood chips; wood pulp produced from wood. , Pulp such as cotton linter pulp obtained from fibers surrounding cotton seeds; Newspaper, cardboard, magazines, paper such as woodfree paper; bagasse (sugarcane pomace), empty palm bunch (EFB), rice straw , plant stems such as corn stems or leaves, leaves, fruit clusters, etc.; plant shells such as rice husks, palm shells and coconut shells, and algae. Among these, from the viewpoint of availability and raw material cost, wood, processed or pulverized wood, plant stems, leaves, fruit bunches, etc. are preferable, bagasse, EFB, oil palm (trunk) are more preferable, and bagasse is further preferable. preferable. You may use the said biomass individually or in mixture of 2 or more types. The biomass may also be dried.

As used herein, "arabinofuranosidase activity" refers to α-1,2 or α-1,3 linked side chains in arabinose-containing polysaccharides, or α-1,2 and α-1,3 linked It refers to the activity of cleaving side chains and hydrolyzing α-L-arabinose residues.
The arabinofuranosidase activity of a protein can be determined, for example, by reacting p-nitrophenyl-α-L arabinofuranoside with the protein as a substrate and measuring p-nitrophenol liberated. Specific procedures for measuring arabinofuranosidase activity are described in detail in Examples below.

(1. Arabinofuranosidase)
The arabinofuranosidase of the present invention is a protein having an amino acid sequence selected from (a) to (d) below and having arabinofuranosidase activity.
(a) the amino acid sequence shown in SEQ ID NO: 2;
(b) an amino acid sequence represented by numbers 1 to 301 of SEQ ID NO: 2;
(c) an amino acid sequence having at least 90% identity with the amino acid sequence shown in (a) or (b);
(d) An amino acid sequence obtained by deleting, substituting or adding one or more amino acids in the amino acid sequence shown in (a) or (b).
Here, the protein (b) is a mature protein obtained by removing the extracellular secretory signal peptide from the amino acid sequence of the protein (a), as shown in the sequence listing.

That is, in one embodiment, the arabinofuranosidase of the present invention is a protein consisting of the amino acid sequence (a) represented by SEQ ID NO: 2 or the amino acid sequence (b) represented by 1 to 301 of SEQ ID NO: 2. be. As the arabinofuranosidase, filamentous fungus Penicillium sp. A preferred example is arabinofuranosidase F81ABF (hereinafter also referred to as "F81ABF"). F81ABF is an arabinofuranosidase classified as GH62 from its amino acid sequence.

In another aspect, the arabinofuranosidase of the present invention is at least 90% identical to the amino acid sequence (a) set forth in SEQ ID NO:2 or the amino acid sequence (b) set forth in positions 1 to 301 of SEQ ID NO:2 and having arabinofuranosidase activity. In another aspect, the arabinofuranosidase of the present invention has one or more in the amino acid sequence (a) shown in SEQ ID NO: 2 or the amino acid sequence (b) shown in SEQ ID NO: 2 from 1 to 301. is a protein consisting of an amino acid sequence (d) in which amino acids of are deleted, substituted or added, and having arabinofuranosidase activity.
The protein includes, for example, the above-described amino acid sequence shown in SEQ ID NO: 2 or a naturally occurring or artificially produced mutant of F81ABF consisting of the amino acid sequence shown in 1 to 301 of SEQ ID NO:2. The mutant is obtained by, for example, introducing a mutation into the gene encoding the F81ABF by a known mutagenesis method such as ultraviolet irradiation or site-directed mutagenesis, and introducing the mutation. It can be produced by expressing a gene having the desired arabinofuranosidase activity and selecting a protein having the desired arabinofuranosidase activity. Procedures for making such mutants are well known to those of skill in the art.

The arabinofuranosidase of the present invention has an amino acid sequence that differs from previously isolated or purified arabinofuranosidases. For example, as a result of homology analysis of the amino acid sequences of F81ABF, EAK85771.1 and EAA59562.1 corresponding to the commercially available enzymes ABFUM and ABFAN, and CAM07245.1 described in Patent Document 3, respectively, EAK85771.1 and EAA59562.1 and CAM07245.1 show 66%, 72% and 82% sequence identity with F81AFB, respectively (Figure 4).
In addition, multiple alignment of the amino acid sequence of the α-L-arabinofuranosidase 67 protein of eukaryote-derived GH62 registered in CAZY (http://www.cazy.org/) was performed, and by the neighborhood binding method When a molecular phylogenetic tree is created, it is roughly classified into two groups. According to this, F81ABF belongs to the same group as EAK85771.1 and CAM07245.1, and belongs to a different group from EAA59562.1 (Fig. 5).

(2. Method for producing arabinofuranosidase)
The arabinofuranosidase of the present invention can be produced, for example, by expressing a polynucleotide encoding the arabinofuranosidase of the present invention. Preferably, the arabinofuranosidase of the present invention can be produced from a transformant into which a polynucleotide encoding the arabinofuranosidase of the present invention has been introduced. For example, after introducing a polynucleotide encoding the arabinofuranosidase of the present invention or a vector containing the same into a host to obtain a transformant, if the transformant is cultured in an appropriate medium, the transformant The arabinofuranosidase of the present invention is produced from the polynucleotide encoding the arabinofuranosidase of the present invention introduced into the body. The arabinofuranosidase of the present invention can be obtained by isolating or purifying the produced arabinofuranosidase from the culture.

A polynucleotide encoding an arabinofuranosidase of the present invention can be a polynucleotide comprising an amino acid sequence selected from (a) to (d) above and encoding a protein having arabinofuranosidase activity. Polynucleotides encoding the arabinofuranosidase of the present invention can also be in the form of single- or double-stranded DNA, RNA, or artificial nucleic acids, or can be cDNA, or chemically synthesized DNA that does not contain introns.
Preferable examples of the polynucleotide encoding the arabinofuranosidase of the present invention include a polynucleotide consisting of a nucleotide sequence selected from (e) to (h) below.
(e) the nucleotide sequence shown in SEQ ID NO: 1;
(f) the nucleotide sequence shown at positions 73 to 975 of SEQ ID NO: 1;
(g) a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in (e) or (f);
(h) A nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted or added to the nucleotide sequence shown in (e) or (f).
Here, the polynucleotide (f) above is a polynucleotide encoding a mature protein obtained by removing a polynucleotide encoding an extracellular secretion signal peptide from the nucleotide sequence of the polynucleotide (e), as shown in the sequence listing. be.

A polynucleotide encoding the arabinofuranosidase of the present invention can be obtained from the filamentous fungus Penicillium sp. It can be isolated from, etc., using any method used in the art. For example, the polynucleotide is Penicillium sp. After extracting the whole genomic DNA of SEQ ID NO: 1, the target gene is selectively amplified by PCR using primers designed based on the nucleotide sequence of SEQ ID NO: 1, and the amplified gene is purified. Alternatively, a polynucleotide encoding the arabinofuranosidase of the present invention can be chemically or genetically engineered based on the amino acid sequence of the arabinofuranosidase. For example, the polynucleotide can be chemically synthesized based on the arabinofuranosidase amino acid sequence of the invention described above. Nucleic acid synthesis contract services (for example, provided by Medical and Biological Laboratories, Inc., Genscript, etc.) can be used for chemical synthesis of polynucleotides. Furthermore, the synthesized polynucleotide can be amplified by PCR, cloning, or the like.

Alternatively, the polynucleotide encoding the arabinofuranosidase of the present invention can be obtained by subjecting the polynucleotide synthesized by the above procedure to a known mutagenesis method such as the above-described ultraviolet irradiation or site-directed mutagenesis. It can be produced by introducing mutations. For example, the polynucleotide encoding the arabinofuranosidase of the present invention is mutagenized into the polynucleotide of SEQ ID NO: 1 by a known method, the resulting polynucleotide is expressed, and the desired arabinofuranosidase activity is examined. can be obtained by selecting a polynucleotide that encodes a protein having arabinofuranosidase activity of

Site-directed mutagenesis into a polynucleotide, for example, any method such as inverse PCR method and annealing method (edited by Muramatsu et al., "Revised 4th Edition New Genetic Engineering Handbook", Yodosha, p.82-88) It can be done by If necessary, various commercially available site-directed mutagenesis kits such as QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit from Stratagene can be used.

The type of vector containing a polynucleotide encoding the arabinofuranosidase of the present invention is not particularly limited, and includes vectors commonly used for gene cloning, such as plasmids, cosmids, phages, viruses, YACs, BACs, and the like. . Plasmid vectors are preferred, and commercially available protein expression plasmid vectors such as shuttle vectors pHY300PLK, pUC19, pUC119, pBR322 (all manufactured by Takara Bio Inc.) can be suitably used.

The vector may contain a DNA region including a DNA replication origin or origin of replication. Alternatively, in the above vector, a promoter region, a terminator region, or a promoter region for initiating transcription of the gene, upstream of the polynucleotide encoding the arabinofuranosidase of the present invention (i.e., the arabinofuranosidase gene of the present invention) A control sequence such as a secretory signal region for extracellular secretion of the expressed protein may be operably linked. As used herein, the expression that a gene and a regulatory sequence are "operably linked" means that the gene and the regulatory region are arranged so that the gene can be expressed under the control of the regulatory region. Say.

The types of regulatory sequences such as the promoter region, terminator, and secretory signal region are not particularly limited, and commonly used promoters and secretory signal sequences can be appropriately selected and used according to the host into which the gene is introduced. For example, a suitable example of a control sequence that can be incorporated into the vectors of the present invention is the cbh1 promoter sequence from Trichoderma reesei (Curr, Genet, 1995, 28(1):71-79). Alternatively, promoters expressing other saccharifying enzymes such as cellobiohydrolase, endoglucanase, β-glucosidase, xylanase and β-xylosidase may be used. Alternatively, promoters of metabolic pathway enzymes such as pyruvate decarboxylase, alcohol dehydrogenase, pyruvate kinase may be used.

Alternatively, the vector of the present invention further incorporates a marker gene (for example, a drug resistance gene such as ampicillin, neomycin, kanamycin, chloramphenicol, etc.) for selecting a host into which the vector has been appropriately introduced. may be Alternatively, when an auxotrophic strain is used as the host, a gene encoding a required synthetase for nutrition may be incorporated into the vector as a marker gene. Alternatively, when using a selective medium that requires a specific metabolism for growth, a gene related to the metabolism may be incorporated into the vector as a marker gene. Examples of such metabolism-related genes include acetamidase genes for utilizing acetamide as a nitrogen source.

The ligation of the polynucleotide encoding the arabinofuranosidase of the present invention, the regulatory sequence and the marker gene can be performed by SOE (splicing by overlap extension)-PCR method (Gene, 1989, 77: 61-68). can be carried out by a known method. Procedures for introducing ligated fragments into vectors are well known in the art.

Examples of transformant hosts into which the vector is introduced include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include bacteria belonging to the genus Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus. Bacillus bacteria (eg, Bacillus subtilis or mutants thereof) are preferred. Examples of Bacillus subtilis mutant strains include J. Biosci. Bioeng. Biotechnol. Lett. , 2011, 33(9): 1847-1852, the D8PA strain with improved protein folding efficiency can be mentioned in the protease 8-fold deficient strain. Examples of filamentous fungi include Trichoderma, Aspergillus, Rhizopus, etc. Among them, Trichoderma is preferred from the viewpoint of enzyme productivity.

Methods commonly used in the art, such as the protoplast method and electroporation method, can be used to introduce the vector into the host. By selecting strains in which introduction has been appropriately performed using marker gene expression, auxotrophy, etc. as indicators, a desired transformant into which the vector has been introduced can be obtained.

Alternatively, a ligated fragment of a polynucleotide encoding an arabinofuranosidase of the invention, a regulatory sequence and a marker gene can be introduced directly into the genome of the host. For example, by the SOE-PCR method or the like, a DNA fragment is constructed by adding a sequence complementary to the genome of the host to both ends of the above-mentioned ligated fragment, and this is introduced into the host, so that the DNA fragment is placed between the host genome and the DNA fragment. A polynucleotide encoding the arabinofuranosidase of the present invention is introduced into the genome of the host by causing homologous recombination.

By culturing in an appropriate medium a transformant into which a polynucleotide encoding the arabinofuranosidase of the present invention or a vector comprising the same thus obtained is introduced, the arabinofuranosidase gene on the vector is Upon expression, an arabinofuranosidase of the invention is produced. A medium used for culturing the transformant can be appropriately selected by those skilled in the art according to the type of microorganism of the transformant.

Alternatively, an arabinofuranosidase of the invention may be expressed from a polynucleotide encoding an arabinofuranosidase of the invention or a transcript thereof using a cell-free translation system. "Cell-free translation system" refers to an in vitro transcription-translation system or an in vitro translation system by adding reagents such as amino acids necessary for protein translation to a suspension obtained by mechanically disrupting host cells. is configured.

The arabinofuranosidases of the present invention produced in the above cultures or cell-free translation systems can be purified by common methods used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion-exchange chromatography, affinity chromatography. It can be isolated or purified by using chromatography or the like alone or in combination as appropriate. At this time, when the polynucleotide encoding the arabinofuranosidase of the present invention and the secretory signal sequence are operably linked on the vector in the transformant, the produced arabinofuranosidase is secreted outside the cell. Therefore, it can be more easily recovered from the culture. Arabinofuranosidase recovered from the culture may be further purified by known means.

(3. Biomass saccharifying agent)
As shown in Examples below, the arabinofuranosidase of the present invention has significantly higher biomass saccharification activity than conventionally known arabinofuranosidases. Therefore, the arabinofuranosidase of the present invention can be used by being contained in a biomass saccharification agent for biomass saccharification.

The biomass saccharifying agent of the present invention contains the arabinofuranosidase of the present invention. The biomass saccharification agent is preferably an enzyme composition for biomass saccharification. The biomass saccharifying agent of the present invention contains the arabinofuranosidase of the present invention, and preferably further contains cellulase from the viewpoint of improving saccharification efficiency. Here, cellulase refers to an enzyme that hydrolyzes the glycosidic bond of β-1,4-glucan of cellulose, and is a general term for enzymes called endoglucanase, exoglucanase, cellobiohydrolase, β-glucosidase, and the like. is. Examples of the cellulase include commercially available cellulase preparations and cellulases derived from animals, plants and microorganisms. These cellulases may be used alone or in combination of two or more. From the viewpoint of improving saccharification efficiency, the cellulase preferably contains one or more selected from the group consisting of cellobiohydrolase and endoglucanase.

Specific examples of cellulase that can be used in combination with the arabinofuranosidase of the present invention in the biomass saccharifying agent of the present invention include, but are not limited to, cellulase derived from Trichoderma reesei; Trichoderma viride Bacillus sp. KSM-N145 (FERM P-19727), Bacillus sp. KSM-N252 (FERM P-17474), Bacillus sp. .) KSM-N115 (FERM P-19726), Bacillus sp. KSM-N440 (FERM P-19728), Bacillus sp. KSM-N659 (FERM P-19730), etc. cellulase derived from Bacillus strain; thermostable cellulase derived from Pyrococcus horikoshii; cellulase derived from Humicola insolens; Among these, cellulases derived from Trichoderma reesei, Trichoderma viride, or Humicola insolens are preferred from the viewpoint of improving saccharification efficiency. Recombinant cellulases obtained by expressing cellulase genes exogenously introduced into the above microorganisms may also be used. As a specific example, X3AB1 strain obtained by introducing a β-glucosidase gene derived from Aspergillus aculeatus into Trichoderma reesei (J. Ind. Microbiol. Biotechnol. (2012) 1741 -9) produces cellulase JN11. Specific examples of cellulase preparations containing the cellulase include Celluclast (registered trademark) 1.5L (manufactured by Novozymes), TP-60 (manufactured by Meiji Seika Co., Ltd.), Cellic (registered trademark) CTec2 (manufactured by Novozymes), AcceleraseTMDUET (manufactured by Genencor) and Ultraflo (registered trademark) L (manufactured by Novozymes) may be mentioned, and these cellulase preparations may be used in combination with the arabinofuranosidase of the present invention.

Further, specific examples of β-glucosidase, which is a type of cellulase, include Aspergillus niger-derived β-glucosidase (for example, Novozyme 188 manufactured by Novozymes and β-glucosidase manufactured by Megazymes), and Trichoderma reese. (Trichoderma reesei) or β-glucosidase derived from Penicillium emersonii. Among these, Novozym 188 and Trichoderma reese-derived β-glucosidase are preferred, and Trichoderma reese-derived β-glucosidase is more preferred, from the viewpoint of improving saccharification efficiency.

Specific examples of endoglucanase, which is a type of cellulase, include Trichoderma reesei, Acremonium celluloriticus, Humicola insolens, and Clostridium thermocellum. , Bacillus, Thermobifida, and Cellulomonas-derived enzymes. Among these, from the viewpoint of improving saccharification efficiency, endoglucanases derived from Trichoderma reese, Humicola insolens, Bacillus, and Cellulomonas are preferred, and endoglucanases derived from Trichoderma reese are more preferred.

The biomass saccharifying agent of the present invention may contain a hemicellulase other than the arabinofuranosidase of the present invention. Here, hemicellulase refers to an enzyme that hydrolyzes hemicellulose, and is a generic term for enzymes called xylanase, xylosidase, galactanase, and the like. Specific examples of hemicellulase other than arabinofuranosidase of the present invention include hemicellulase derived from Trichoderma reesei; xinalase derived from Bacillus sp. KSM-N546 (FERM P-19729); Xylanases from Aspergillus niger, Trichoderma viride, Humicola insolens, or Bacillus alcalophilus; , Streptomyces, Clostridium, Thermotoga, Thermoascus, Caldocellum, or Thermomonospora; pumilβ from Bacillus pumilus -xylosidase; β-xylosidase derived from Selenomonas ruminantium, and the like. Among these, from the viewpoint of improving saccharification efficiency, the enzyme composition of the present invention contains xylanase derived from Bacillus sp., Aspergillus niger, Trichoderma viride or Streptomyces, or β-xylosidase derived from Selenomonas luminantium. and more preferably xinalase derived from Bacillus sp. or Trichoderma viride, or β-xylosidase derived from Selenomonas luminantium.

The content of the arabinofuranosidase of the present invention in the total protein amount of the biomass saccharifying agent of the present invention may be in the range of 0.1% by mass or more and 70% by mass or less. The content of the cellulase in the total protein content of the enzyme composition of the present invention may be in the range of 10% by mass or more and 99% by mass or less. In addition, the content of the hemicellulase other than the arabinofuranosidase of the present invention in the total protein amount of the biomass saccharifying agent of the present invention may be in the range of 0.01% by mass or more and 30% by mass or less. The protein amount ratio between the arabinofuranosidase of the present invention and the cellulase (arabinofuranosidase/cellulase of the present invention) in the biomass saccharifying agent of the present invention may be in the range of 0.001 or more and 100 or less.

(4. Sugar production method)
The arabinofuranosidase of the present invention or a biomass saccharifying agent containing it is used to produce sugar from biomass. That is, the method for producing sugar of the present invention includes a step of saccharifying biomass with the arabinofuranosidase of the present invention or a biomass saccharifying agent containing the same.
Biomass is as described above, but from the viewpoint of availability, raw material cost, and improvement of saccharification efficiency, the biomass includes wood, processed or pulverized wood, plant stems, leaves, fruit bunches, etc. is preferred, bagasse, EFB, oil palm (trunk) and Erianthus are more preferred, and bagasse is even more preferred. The biomass may be used alone or in combination of two or more. The biomass may also be dried.

When saccharifying biomass, from the viewpoint of improving pulverization efficiency, improving saccharification efficiency, and improving production efficiency (that is, shortening the sugar production time), a step of pretreating the biomass is further added before the saccharification step. preferably included.

Examples of the pretreatment include one or more selected from the group consisting of alkali treatment, pulverization treatment and hydrothermal treatment. From the viewpoint of improving saccharification efficiency, alkali treatment is preferable as the pretreatment.

The above-mentioned alkali treatment means reacting biomass with a basic compound, which will be described later. Examples of the alkali treatment method include a method of immersing biomass in an alkaline solution containing a basic compound described later (hereinafter sometimes referred to as "immersion treatment"), and a method of mixing biomass and a basic compound, which is described later. and a method of applying a pulverization treatment (hereinafter sometimes referred to as "alkaline mixed pulverization treatment").

The pulverization treatment refers to mechanical pulverization of biomass into small particles. By making the biomass into small particles, the saccharification efficiency is further improved. Moreover, when the crystalline structure of the cellulose contained in the biomass is destroyed by the pulverization treatment, the saccharification efficiency is further improved. The pulverization treatment can be performed using a known pulverizer. There is no particular limitation on the crusher used, and any device capable of reducing biomass into small particles may be used. The pulverization treatment may be combined with the alkali treatment with the basic compound described above. The pulverization treatment may be performed before or after the alkali treatment, or may be performed in parallel with the pulverization treatment, such as the alkali mixed pulverization treatment described above. In the alkali mixed pulverization treatment, for example, biomass immersed in an alkaline solution may be pulverized (wet pulverization), or solid alkali and biomass may be pulverized together (dry pulverization). Grinding is preferred.

The above hydrothermal treatment means heat-treating biomass in the presence of moisture. The hydrothermal treatment can be performed using a known reactor, and the reactor used is not particularly limited.

The biomass saccharification treatment conditions are not particularly limited as long as the arabinofuranosidase of the present invention, the biomass saccharifying agent containing the arabinofuranosidase, and other enzymes used in combination are not deactivated. Appropriate conditions can be appropriately determined by those skilled in the art according to the type of biomass, the procedure of the pretreatment step, and the type of enzyme used.

In the saccharification treatment, it is preferable to add the arabinofuranosidase of the present invention or a biomass saccharifying agent containing the same to a suspension containing biomass. From the viewpoint of improving saccharification efficiency or sugar production efficiency (that is, shortening sugar production time), the content of biomass in the suspension is preferably 0.5% by mass or more, more preferably 3% by mass or more, and still more preferably It is 5% by mass or more, and preferably 30% by mass or less, more preferably 25% by mass or less, and even more preferably 20% by mass or less.

The amount of the arabinofuranosidase of the present invention or the biomass saccharifying agent containing the same for the suspension is appropriately determined according to the pretreatment conditions and the type and properties of the enzyme used in combination. , in terms of the mass of the arabinofuranosidase of the present invention, is preferably 0.001% by mass or more, more preferably 0.002% by mass or more, still more preferably 0.005% by mass or more, and 100% by mass or less , more preferably 50% by mass or less, and still more preferably 20% by mass or less.

The reaction pH of the saccharification treatment is preferably pH 3.5 or higher, more preferably pH 4.0 or higher, from the viewpoints of improving saccharification efficiency or sugar production efficiency (that is, shortening sugar production time) and reducing production costs. The pH is preferably 6.0 or less, more preferably 5.5 or less.

The reaction temperature for the saccharification treatment is 50 to 80° C. from the viewpoints of improving saccharification efficiency, improving saccharification efficiency or sugar production efficiency (that is, shortening sugar production time), reducing production costs, and optimal temperature for cellulase used at the same time. is preferred. The reaction time of the saccharification treatment can be appropriately set according to the type or amount of biomass, the amount of enzyme, etc., but from the viewpoint of improving saccharification efficiency or sugar production efficiency (that is, shortening sugar production time) and reducing production costs. , preferably 1 to 5 days, more preferably 1 to 4 days, still more preferably 1 to 3 days.

The following aspects are further disclosed in this invention regarding embodiment mentioned above.
<1> A protein consisting of an amino acid sequence selected from the following (a) to (d) and having arabinofuranosidase activity:
(a) the amino acid sequence shown in SEQ ID NO: 2;
(b) an amino acid sequence represented by numbers 1 to 301 of SEQ ID NO: 2;
(c) an amino acid sequence having at least 90% identity with the amino acid sequence shown in (a) or (b);
(d) An amino acid sequence obtained by deleting, substituting or adding one or more amino acids in the amino acid sequence shown in (a) or (b).
<2> A polynucleotide encoding a protein having the arabinofuranosidase activity of <1>.
<3> Polynucleotide of <2>, consisting of a nucleotide sequence selected from the following (e) to (h):
(e) the nucleotide sequence shown in SEQ ID NO: 1;
(f) the nucleotide sequence shown at positions 73 to 975 of SEQ ID NO: 1;
(g) a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in (e) or (f);
(h) A nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted or added to the nucleotide sequence shown in (e) or (f).
<4> A vector containing the polynucleotide of <2> or <3>.
<5> A method for producing a transformant, comprising introducing the polynucleotide of <2> or <3> or the vector of <4> into a host.
<6> A transformant introduced with the polynucleotide of <2> or <3> or the vector of <4>.
<7> A biomass saccharifying agent containing the protein having the arabinofuranosidase activity of <1>.
<8> A method for producing sugar from biomass using the protein having arabinofuranosidase activity of <1> or the biomass saccharifying agent of <7>.
<9> The production method of <5> or the transformant of <6>, wherein the host in <5> is a filamentous fungus belonging to the genus Trichoderma.
<10> The biomass saccharifying agent of <7>, wherein the content of the protein having arabinofuranosidase activity in the total protein amount of the biomass saccharifying agent is 0.1% by mass or more and 70% by mass or less.

<Example 1> Production of arabinofuranosidase Penicillium sp. derived arabinofuranosidase F81ABF was produced.

(1) Isolation of Arabinofuranosidase RNA and Synthesis of cDNA Filamentous fungus Penicillium sp. were cultured under inducing conditions with xylan. After 4 days of culture, mycelium and culture supernatant were obtained by filtration using Miracloth (Merck Millipore). The mycelium obtained was rapidly frozen in liquid nitrogen and crushed using a multi-bead shocker (Yasui Kikai Co., Ltd.). RNA solution was obtained from the obtained crushed cells using RNeasy Mini Kit (Qiagen). cDNA was synthesized from the RNA solution using SuperScript II Reverse Transcriptase (Life Technologies).

(2) Preparation of F81ABF expression vector Using pUC-cbh1-amdS constructed in JP 2017-12006 as a template, forward primer 1 (SEQ ID NO: 6) and reverse primer 1 (SEQ ID NO: 6) shown in Table 1 7), amdS (Aspergillus nidurans-derived acetamidase) was almost eliminated to amplify an approximately 7.2 kbp fragment (A). In addition, using pyr4 (SEQ ID NO: 3) derived from Trichoderma reesei as a template, PCR was performed using forward primer 2 (SEQ ID NO: 8) and reverse primer 2 (SEQ ID NO: 9) shown in Table 1, resulting in about 2.7 kbp Fragment (B) was amplified. The obtained DNA fragments (A) and (B) were treated according to the protocol of In-Fusion HD Cloning Kit (Takara Bio), and competent cell E. coli HB101 (Takara Bio). From among the transformants obtained as ampicillin-resistant strains, strains carrying the plasmid into which the gene of interest was inserted were selected by colony PCR. The selected transformants were cultured on LB agar medium (37° C., 1 day), and plasmids were recovered and purified from the obtained cells using High Pure Plasmid Isolation kit (Roche). The resulting vector was named pUC-cbh1-pyr4.

Using the above pUC-cbh1-pyr4 as a template, PCR was performed using forward primer 3 (SEQ ID NO: 10) and reverse primer 3 (SEQ ID NO: 11) shown in Table 1 to amplify an approximately 10 kbp fragment (C). Next, using the cDNA obtained in (1) above as a template, PCR is performed using forward primer 4 (SEQ ID NO: 12) and reverse primer 4 (SEQ ID NO: 13) shown in Table 1, so that the putative signal sequence is included. An approximately 1.0 kbp fragment (D) of the gene region (SEQ ID NO: 1) of the F81ABF preprotein (SEQ ID NO: 2) was amplified. The obtained DNA fragments (C) and (D) were incorporated into a vector in the same manner as above to prepare an expression vector pUC-Pcbh1-F81ABF-pyr4 containing the F81ABF gene.

(3) Preparation of transformant T. reeseiJN11 strain (X3AB1 strain shown in J. Ind. Microbiol. Biotechnol. (2012) 1741-9) was transformed with the vector constructed in (2) above. Introduction was performed by the protoplast PEG method. Transformants were grown in selective medium (2% glucose, 1.1 M sorbitol, ₂ % agar, 0.2% _{KH2PO4 (} pH 5.5), 0.06% _CaCl2.2H2O , 0.06% CsCl ₂ , 0.06% _MgSO4.7H2O , 0.06% (NH4) _2SO4 , 0.1% Trace element ₁ ; all percentages are w/v%). The composition of Trace element ₁ is as follows: 0.5g _FeSO4.7H2O , 0.2g _CoCl2 , _{0.16g MnSO4.H2O} , _{0.14g ZnSO4.7H2O} in distilled water. up to 100 mL. After stabilizing the selected transformants by passaging, strains stably retaining the target gene were further screened by colony PCR.

(4) Cultivation of Transformant Spores were inoculated into 50 mL of seed medium (500 mL Erlenmeyer flask) at 2×10 ⁵ spores/mL, and shake cultured at 28° C. with reciprocating shaking at 220 rpm for 2 days (PRECi : PRXYg-98-R type) was performed. Main culture: Performed in a 250 ml jar fermenter (manufactured by Biott). 10 mL of the preculture was inoculated to 100 mL of the main culture, and cultured for about 5 days at 28° C., pH 4.5, aeration rate of 0.5 vvm, and DO 3 ppm. 5% ammonia water was used for pH control. The culture solution was separated into a precipitate fraction and a culture solution supernatant by centrifugation, and filtered. The medium composition used is as follows.

Seed medium composition (conical flask culture)
₍ NH4) _{2SO4 0.14} %
_{KH2PO4 0.2} %
_{CaCl2.2H2O 0.03} %
_{MgSO4.7H2O 0.03} %
High polypeptone N 0.1%
Bact yeast extract 0.05%
Tween 80 0.1%
Trace element ^* 0.1%
Tartrate buffer (pH 4.0) ^** 10%
Glucose 1%

Trace element stock solution (100ml)
- _{H3BO3 6} mg
_{( NH4} ) _{6Mo7O24.4H2O 6} mg
- _FeCl3.6H2O 100 _mg
- _{CuSO4.5H2O 40} mg
- _{MnCl2.4H2O 8} mg
- 200 mg of _ZnCl2

Tartrate buffer (pH 4.0) tartaric acid buffer 1L
_A : _{0.5M C4H4Na2O6.2H2O 92.032g} / _800ml
B: _{0.5M C4H6O6 15.009g} /200ml
Adjust to pH 4 with A:B = 4:1

Main culture medium (1000 ml)
₍ NH4) _{2SO4 0.42} %
_{KH2PO4 0.2} %
_{CaCl2.2H2O 0.03} %
_{MgSO4.7H2O 0.03} %
High polypeptone N 0.1%
Bact yeast extract 0.05%
Tween 80 0.1%
Trace element ^* 0.1%
Antifoam PE-L 0.2%
Trace element ^** 0.1%
FD-101 10%

(5) Purification of F81ABF Using the culture supernatant obtained in (4) above, purification was performed under the following conditions.
Apparatus: HPLC preparative system PLC-561 (GL Science)
Column: TSKgel G2000SW 21.5x300mm (Tosoh)
Eluent: 20 mM acetate buffer (pH 5),
Flow rate: 4 mL/min,
Monitor wavelength: 280nm
Since it was confirmed that the peak eluting around 28 minutes contained F81ABF, fractions from 27 minutes to 28 minutes were collected. Furthermore, purification by gel filtration was performed under the following conditions.

Apparatus: HPLC preparative system PLC-561 (GL Science)
Column: POROS HS 20 μm column 4.6×100 mm
(Life Technologies)
Eluent A: 20 mM Tris-HCl buffer (pH 8)
Eluent B: 20 mM Tris-HCl buffer (pH 8) 1 M NaCl
Flow rate: 1 mL/min
Monitor wavelength: 280nm
Linear gradient: 5 min A: B = 100: 0,
15min A:B=20:80
Since it was confirmed that most of the pure F81ABF was contained in the peak eluting around 9 minutes, the fraction from 7 minutes to 11 minutes was collected, and the concentrated and desalted sample was used as the F81ABF crude enzyme solution. bottom.

(6) Protein concentration measurement Purified F81ABF is a mature arabinofuranosidase (25- 325 amino acid sequence). SignalP (Bendtsen et al., J. Mol. Biol. 340:783-795, 2004) was used to predict the signal sequence. The protein concentration was measured by the bradford method. In the bradford method, the Quick Start protein assay (BioRad) was used, and the protein amount was calculated based on a calibration curve using bovine gamma globulin as a standard protein.

<Example 2> α-L-arabinofuranosidase activity of F81ABF A comparison was made between F81ABF and the commercially available α-L-arabinofuranosidase activity. α-L-arabinofuranosidases derived from eukaryotic microorganisms such as fungi are classified as GH43, 51, 54 and 62. F81ABF belongs to GH62. AFAM2 (GH43), AFASE (GH51), ABFAN (GH62), and ABFUM (GH62) manufactured by Megazyme, which are commercially available, were used. Using p-nitrophenyl-α-L arabinofuranoside (FIG. 1A) and wheat arabinoxylan (FIG. 1B) as substrates, α-L-arabinofuranosidase activity was measured.

(1) p-nitrophenyl-α-L arabinofuranoside degradation activity 20 μL of 2.5 mM p-nitrophenyl-α-L arabinofuranoside (pNP-Ara, Sigma), 20 μL of 250 mM sodium acetate buffer 20 μL of appropriately diluted enzyme solution was added to 80 μL of substrate solution obtained by mixing liquid (pH 5) and 40 μL of deionized water, and reacted at 50° C. for 10 minutes. 100 μL of 0.4 M sodium carbonate solution was added to stop the reaction, and at the same time, the reaction solution was made alkaline to develop a yellow color from p-nitrophenyl liberated by enzymatic decomposition. The absorbance of the reaction solution was measured at 420 nm. One unit (U) was defined as the amount of enzyme that liberates 1 µmol of p-nitrophenol per minute.
Table 3 shows the results. Although F81ABF had a pNP-Ara degradation activity (pNPA) as low as 0.46 U/mg, it exhibited an activity equivalent to that of ABFAN.

(2) Wheat Arabinoxylan Degrading Activity 50 μL of 1.0% (w/v) wheat arabinoxylan, low viscosity (Megazyme), 20 μL of 50 mM sodium acetate buffer (pH 5), and 20 μL of deionized water were mixed. 10 μL of an appropriately diluted enzyme solution was added to 90 μL of the substrate solution and reacted at 50° C. for 10 minutes. After stopping the reaction by adding 100 μL of DNS solution (1.6% sodium hydroxide, 0.5% 3,5-dinitrosalicylic acid, 30% sodium potassium tartrate), heat treatment at 99.9° C. for 10 minutes. bottom. After cooling, the absorbance of the reaction solution was measured at 540 nm. As a control, 100 μL of DNS solution was added to 90 μL of substrate solution, and after mixing, 10 μL of enzyme solution was added. One unit (U) was defined as the amount of enzyme that liberates reducing sugars equivalent to 1 μmol of L-arabinose per minute. The results are also shown in Table 3 above. The wheat arabinoxylan degrading activity (WAX) of F81ABF was 121.3 U/mg, which was the highest value compared with commercially available enzymes.

<Example 3> Utilization of F81ABF in biomass saccharification From Example 2, since F81ABF has higher arabinoxylan degrading activity than commercially available enzymes, it was expected to exhibit an effect on biomass saccharification. Therefore, the effect of F81ABF on saccharification of wheat bran containing a large amount of arabinoxylan as biomass was investigated.

(1) Preparation of cellulase JN11 was used as cellulase. JN11 is a J.P. Ind. Microbiol. Biotechnol. (2012) 1741-9, which was prepared by culturing in the same manner as in Example 1 (4).

(2) Saccharification reaction In a screw tube with a lid (No. 3, φ21 x 45 mm, manufactured by Maruem Co., Ltd.), 50 mg of wheat bran as a substrate in terms of dry raw material and 0.1 mL of 0.5 M sodium acetate buffer (pH 5) was added, and JN11 prepared in (1) above (6 mg/g-substrate as the enzyme protein amount) and F81ABF enzyme solution prepared in Example 1 (5) (0.1 mg/g- as the enzyme protein amount- Substrate) was added, and then water was added so that the substrate concentration was 5 wt % to prepare a reaction solution. For comparison, a reaction solution was prepared by adding Megazyme's AFAM2 (GH43), AFASE (GH51), ABFAN (GH62) and ABFUM (GH62) (all 0.1 mg/g-substrate). As a control, a reaction solution containing only JN11 as an enzyme was prepared. Each reaction solution was saccharified for 2 days with reciprocating shaking at 50° C. and 150 rpm. The amount of each enzyme was based on the amount of protein measured by the Bradford method. After completion of the reaction, the supernatant was collected by centrifugation (15000 rpm, 5 min, 4° C.), and the concentrations of glucose, xylose and arabinose in the supernatant were measured using a DX500 chromatography system (manufactured by Nippon Dionex). Taking the amount of glucose, xylose, and arabinose sugar obtained when using JN11 as 100%, the amount of sugar obtained by adding various α-L-arabinofuranosidases was calculated as a relative value.

The results are shown in FIG. Addition of 0.1 mg/g of F81ABF greatly increased the amount of arabinose produced, showing 123%. Glucose and xylose production also increased by 106% and 105%. The production of three types of sugars, glucose, xylose and arabinose, was improved only when F81ABF was added, and was not observed with any of the commercially available enzymes. This result indicated that F81ABF is an enzyme suitable for biomass saccharification that promotes cellulose degradation through degradation of wheat bran hemicellulose.

<Example 4> Use of F81ABF-expressing saccharifying enzyme in biomass saccharification Example 3 showed that F81ABF is effective in saccharifying wheat bran. The F81ABF gene was introduced into S. reesei, a saccharifying enzyme expressing F81ABF was prepared, and the saccharifying properties of wheat bran were examined.

(1) Preparation of expression vector About 3.2 kbp fragment (A) by PCR using forward primer 5 (SEQ ID NO: 14) and reverse primer 5 (SEQ ID NO: 15) shown in Table 1 using pUC118 as a template was amplified. In addition, using the upstream to downstream region (SEQ ID NO: 4) of Cre1 derived from Trichoderma reesei as a template, PCR was performed using forward primer 6 (SEQ ID NO: 16) and reverse primer 6 (SEQ ID NO: 17) shown in Table 1. , amplified an approximately 2.9 kbp fragment (B). The obtained DNA fragments (A) and (B) were incorporated into a vector in the same manner as in Example 1 (2) to prepare pUC-cre1.

About 6.3 kbp fragment (C ) was amplified. Using SEQ ID NO: 4 as a template, PCR was performed using forward primer 8 (SEQ ID NO: 20) and reverse primer 8 (SEQ ID NO: 21) shown in Table 1 to amplify an approximately 500 bp fragment (D) downstream of the cre1 gene. bottom. Furthermore, using pyr4 (SEQ ID NO: 3) derived from Trichoderma reesei as a template, PCR was performed using forward primer 9 (SEQ ID NO: 22) and reverse primer 9 (SEQ ID NO: 23) shown in Table 1 to obtain an approximately 2.7 kbp fragment (E) was amplified. The obtained DNA fragments (D) and (E) were processed according to the protocol of In-Fusion HD Cloning Kit (Takara Bio) to prepare DNA fragment (F) by ligating (D)-(E) in order. The obtained DNA fragments (C) and (F) were incorporated into a vector in the same manner as in Example 1 (2) to prepare pUC-cre1-pyr4.

Using the above pUC-cre11-pyr4 as a template, PCR was performed using forward primer 10 (SEQ ID NO: 24) and reverse primer 10 (SEQ ID NO: 25) shown in Table 1 to amplify an approximately 9.5 kbp fragment (G). . Using the promoter region (SEQ ID NO: 5) of the eg1 gene as a template, PCR was performed using forward primer 11 (SEQ ID NO: 26) and reverse primer 11 (SEQ ID NO: 27) shown in Table 1 to obtain an approximately 1.5 kbp fragment. (H) was amplified. Using the cDNA obtained in Example 1 (1) as a template, PCR was performed using forward primer 12 (SEQ ID NO: 28) and reverse primer 12 (SEQ ID NO: 29) shown in Table 1 to obtain a putative signal sequence. An approximately 1.0 kbp fragment (I) of the gene region (SEQ ID NO: 1) of the F81ABF preprotein (SEQ ID NO: 2) was amplified. Furthermore, using pUC-cbh1-amdS constructed in JP 2017-12006 as a template, PCR is performed using forward primer 13 (SEQ ID NO: 30) and reverse primer 13 (SEQ ID NO: 31) shown in Table 1. , an approximately 0.6 kbp fragment (J) was amplified as the terminator region of cbh1. The obtained DNA fragments (H), (I) and (J) were treated according to the protocol of the In-Fusion HD Cloning Kit (Takara Bio), and the DNA fragments were ligated in the order of (H)-(I)-(J). (K) was prepared. The obtained DNA fragments (G) and (K) were incorporated into a vector in the same manner as in Example 1 (2) to prepare pUC-Peg1-F81ABF-Δcre1-pyr4.

(2) Preparation of Transformant Trichoderma reese JN11 strain (X3AB1 strain shown in J. Ind. Microbiol. Biotechnol. (2012) 1741-9) was transformed with the vector constructed in (1) above. rice field. The introduction was carried out as described in (3) of Example 1, and the resulting transformant was named TK16.

(3) Preparation of saccharifying enzyme TK16 Using the strain obtained in (2), it was prepared by culturing in the same procedure as in Example 1 (4).

(4) Saccharification Reaction Using the TK16 saccharifying enzyme obtained in (3), wheat bran was saccharified in the same manner as in Example 3(2). The results are shown in FIG. By saccharifying wheat bran with TK16, the sugar yields of glucose, xylose and arabinose were improved by 103%, 106% and 127%, respectively. This result showed that TK16 into which F81ABF was introduced is useful as a biomass saccharifying enzyme.

<Example 5> Comparison of α-L-arabinofuranosidase homology of GH62 (1) Alignment analysis EAK85771.1 and EAA59562.1 corresponding to F81ABF and commercially available enzymes ABFUM and ABFAN, CAM07245.1 of the above-mentioned Patent Document 3 A homology analysis of amino acid sequences was performed for each of the mature peptides. For the analysis, the fastp program of Genetyx Ver.12 (Genetics Co., Ltd.), which is genetic information processing software, was used. As a result, EAK85771.1, EAA59562.1 and CAM07245.1 showed 66, 72 and 82% identity with F81AFB, respectively. A multiple alignment of these four α-L-arabinofuranosidases is shown in FIG.

(2) Molecular phylogenetic tree analysis The amino acid sequence of the α-L-arabinofuranosidase 67 protein of eukaryote-derived GH62 registered in CAZY (http://www.cazy.org/) was multiplexed with clustalX. Alignment was performed, and a molecular phylogenetic tree was created by the Neighbor-Joining method (NJ method). Genetyx Ver. 12 included among the 67 proteins EAK85771.1 and EAA59562.1 corresponding to the commercial enzymes ABFUM and ABFAN, CAM07245.1 of WO2006125438. The results are shown in FIG. GH62 α-L-arabinofuranosidases were classified into two groups, and F81ABF belonged to Group 1. It was shown that CAM07245.1, which showed the highest homology in Example 5(1), belongs to the same Group 1. On the other hand, EAK85771.1 also had the lowest homology, but was shown to be in the same Group 1 from molecular phylogenetic tree analysis. On the other hand, EAA59562.1 belonged to group 2 although it had higher homology than EAK85771.1.

Claims (10)

A protein consisting of an amino acid sequence selected from the following (a) to (d) and having arabinofuranosidase activity:
(a) the amino acid sequence shown in SEQ ID NO: 2;
(b) an amino acid sequence represented by numbers 1 to 301 of SEQ ID NO: 2;
(c) an amino acid sequence having at least 95 % identity with the amino acid sequence shown in (a) or (b);
(d) An amino acid sequence obtained by deleting, substituting or adding 1 to 10 amino acids in the amino acid sequence shown in (a) or (b). 2. The protein having arabinofuranosidase activity according to claim 1, which promotes degradation of cellulose in the presence of cellulase. A polynucleotide encoding the protein having arabinofuranosidase activity according to claim 1 or 2 . The polynucleotide according to claim 3 , which consists of a nucleotide sequence selected from the following (e) to (h):
(e) the nucleotide sequence shown in SEQ ID NO: 1;
(f) the nucleotide sequence shown at positions 73 to 975 of SEQ ID NO: 1;
(g) a nucleotide sequence having at least 95 % identity with the nucleotide sequence shown in (e) or (f);
(h) A nucleotide sequence in which 1 to 30 nucleotides are deleted, inserted, substituted or added in the nucleotide sequence shown in (e) or (f). A vector comprising the polynucleotide of claim 3 or 4 . A method for producing a transformant, comprising introducing the polynucleotide according to claim 3 or 4 or the vector according to claim 5 into a host. A transformant into which the polynucleotide according to claim 3 or 4 or the vector according to claim 5 has been introduced. A biomass saccharifying agent comprising the protein having arabinofuranosidase activity according to claim 1 or 2 . The biomass saccharifying agent according to claim 8, further comprising cellulase. A method for producing sugar from biomass, using the protein having arabinofuranosidase activity according to claim 1 or 2 or the biomass saccharifying agent according to claim 8 .

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