Enzyme composition for biomass saccharification

Abstract

To provide an enzyme composition for biomass saccharification that can efficiently saccharify biomass.SOLUTION: Provided is an enzyme composition for biomass saccharification, containing cellulase and arabinofuranosidase, the arabinofuranosidase being an enzyme composition consisting of an amino acid sequence selected from the following (a) to (c): (a) a specific amino acid sequence; (b) an amino acid sequence having at least 90% identity with the specific amino acid sequence; and (c) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added to the specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to an enzyme composition for biomass saccharification and a method for producing sugar from biomass using the same. [Background technology]

[0002] In recent years, research and development on the utilization of cellulosic biomass, which is a by-product of agricultural production such as residues after harvesting crops and timber, has attracted attention. Cellulosic biomass (hereinafter referred to as biomass) is composed of cellulose, hemicellulose, and lignin, and these three components are tightly intertwined. Of these three components, sugars, which are decomposition products of cellulose and hemicellulose, are used as raw materials for ethanol and chemical products.

[0003] Cellulose is a polysaccharide in which glucose molecules are linked by β-1,4 bonds, while hemicellulose is a heteropolysaccharide containing sugars other than glucose. Arabinoxylan, the most abundant component in hemicellulose, consists mainly of a xylan skeleton linked by β-1,4 bonds, with arabinose, galactose, glucuronic acid, ferulic acid, etc., attached as side chains. The enzymes that break down cellulose and hemicellulose are cellulase and hemicellulase, respectively, and are collectively called biomass saccharifying enzymes.

[0004] Biomass has a complex, interwoven structure, making it difficult to decompose. Cellulase and hemicellulase are both essential for the efficient breakdown of cellulose and hemicellulose. Since the type and composition of enzymes greatly influence biomass decomposition, the development of biomass saccharifying enzymes is a crucial issue.

[0005] To improve biomass decomposition, enzyme compositions have been developed in which hemicellulases such as xylanase derived from Trichoderma reesei or arabinofuranosidase are added to cellulase (Patent Document 1). Furthermore, enzyme compositions have been developed in which xylanase with high xylanase activity from Penicillium sp. is added (Patent Document 2).

[0006] α-L-arabinofuranosidase is a glycoside hydrolase (GH) that can hydrolyze arabinose side chains linked by α-1,2, α-1,3, or α-1,5 bonds from the non-reducing end. α-L-arabinofuranosidase is classified under EC3.2.1.55, and based on amino acid sequence homology, it is classified as GH43, GH51, GH54, or GH62. Non-patent document 1 discloses the properties of 64.5 kDa and 62.7 kDa α-L-arabinofuranosidase from Penicillium copsulatum. Patent document 3 discloses α-L-arabinofuranosidase classified as GH62 from Penicillium sp. Patent document 4 discloses α-L-arabinofuranosidase and its amino acid sequence from Aspergillus niger. Regarding Aspergillus section Nigri (black koji mold), which includes Aspergillus niger, genome analysis of carbohydrate-related enzymes and interspecies genome sequencing comparisons have been conducted in recent years (Non-patent Literature 2). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Special Publication No. 2011-515089 [Patent Document 2] Japanese Patent Publication No. 2017-12006 [Patent Document 3] Japanese Patent Publication No. 2020-065514 [Patent Document 4] International Publication No. 2006 / 125438 [Non-patent literature]

[0008] [Non-Patent Document 1] Appl. Environ. Microbiol., 1996, 62:168-173 [Non-Patent Document 2] Nature Genetics, 2018, 50:1688-1695 [Overview of the project] [Problems that the invention aims to solve]

[0009] This invention provides an enzyme composition for biomass saccharification that can efficiently saccharify biomass, and a method for producing sugar from biomass using the same. [Means for solving the problem]

[0010] The inventors have discovered an arabinofuranosidase with excellent arabinofuranosidase activity, and have also found that biomass can be efficiently saccharified by using this arabinofuranosidase in combination with cellulase.

[0011] The present invention relates to an enzyme composition for biomass saccharification, comprising cellulase and arabinofuranosidase, wherein the arabinofuranosidase comprises an amino acid sequence selected from (a) to (c) below: (a) Amino acid sequence of Sequence ID No. 1; (b) an amino acid sequence having at least 90% identity with the amino acid sequence of Sequence ID No. 1; (c) An amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence of Sequence ID No. 1, To provide. The present invention also provides a method for producing sugar from biomass using the enzyme composition for biomass saccharification. [Effects of the Invention]

[0012] The arabinofuranosidase contained in the enzyme composition of the present invention has excellent arabinofuranosidase activity and promotes the decomposition of hemicellulase contained in biomass, thereby improving the saccharification rate of biomass. Therefore, according to the present invention, sugar can be efficiently produced from biomass. [Brief explanation of the drawing]

[0013] [Figure 1] The optimum temperature of F5054ABF. It shows the relative enzyme activity with respect to the enzyme activity at 60°C. [Figure 2] The optimum pH of F5054ABF. It shows the relative enzyme activity with respect to the enzyme activity at pH 3.0. [Figure 3] The temperature stability of F5054ABF. It shows the relative enzyme activity with respect to the activity of the enzyme pretreated at 50°C. [Figure 4] The pH stability of F5054ABF. It shows the relative enzyme activity with respect to the activity of the enzyme pretreated at pH 4.0. [Figure 5] The amount of sugar produced in the saccharification of biomass by using F5054ABF in combination with a cellulase agent. The relative production amount when the amount of sugar produced in the case of using cellulase alone is set to 100%. [Figure 6] The amounts of glucose, xylose, and arabinose produced in the saccharification of biomass by using F5054ABF in combination with a cellulase agent. The relative production amounts when the amount of sugar produced in the case of using cellulase alone is set to 100%.

Modes for Carrying Out the Invention

[0014] In this specification, the sequence identity of a base sequence and an amino acid sequence is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435 - 1441). Specifically, it is calculated by performing an analysis using the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win with Unit size to compare (ktup) being 2.

[0015] In this specification, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means an identity of 90% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, still more preferably 99% or more, and still more preferably 99.5% or more.

[0016] In this specification, "amino acid sequences in which one or more amino acids are deleted, substituted, inserted, or added" preferably include amino acid sequences in which one to ten amino acids are deleted, 1 to five amino acids are deleted, more preferably one to three amino acids are deleted, 1 or two amino acids are inserted, or added. Also in this specification, "nucleotide sequences in which one or more nucleotides are deleted, substituted, inserted, or added" preferably include nucleotide sequences in which one to thirty nucleotides are deleted, 1 to fifteen nucleotides are deleted, more preferably one to nine nucleotides are deleted, 1 to six nucleotides are deleted, 1 to three nucleotides are inserted, or added. In this specification, "addition" of nucleotides includes the addition of nucleotides to one end and both ends of a sequence.

[0017] In this specification, the "corresponding position" or "corresponding region" on an amino acid sequence or nucleotide sequence can be determined by aligning the target sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 1) to give the greatest possible homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. Det. al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Clustal W can be used, for example, on the websites of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) and the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]), operated by the National Institute of Genetics. The position of the target sequence aligned to any position in the reference sequence by the alignment described above is considered a "corresponding position" to that arbitrary position. Furthermore, the region enclosed by the corresponding positions, or the region consisting of the corresponding motifs, is considered a corresponding region.

[0018] In this specification, "operable linkage" between a gene and a regulatory region such as a promoter means that the gene and the regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operable linkage" between a gene and a regulatory region are well known to those skilled in the art.

[0019] In this specification, "upstream" and "downstream" with respect to a gene refer to the upstream and downstream directions of the gene's transcription. For example, the "upstream sequence" and "downstream sequence" of a gene refer to the sequences located at the 5' and 3' ends of the gene on the DNA sense strand, respectively. For example, "promoter ligated upstream of a gene" means that the promoter is located at the 5' end of the gene on the DNA sense strand.

[0020] In this specification, the term "inherent" used with respect to the function, properties, or characteristics of a cell is used to indicate that the function, property, or characteristic is inherently present in the cell. In contrast, the term "external" is used to indicate a function, property, or characteristic that is not inherently present in the cell but has been introduced from outside. For example, "external" nucleotide or DNA is a nucleotide or DNA that has been introduced into a cell from outside. The external nucleotide or DNA may originate from the same species of organism as the cell into which it was introduced, or from a different species of organism (i.e., a different nucleotide or different DNA).

[0021] In this specification, "biomass" refers to cellulosic and / or lignocellulosic biomass containing hemicellulose components produced by plants and algae. Specific examples of biomass include one or more selected from the group consisting of: various types of wood obtained from coniferous trees such as larch and cypress, broad-leaved trees such as oil palm (trunk) and cypress; processed or crushed wood products such as wood chips; pulps such as wood pulp produced from wood and cotton linter pulp obtained from the fibers surrounding cotton seeds; paper products such as newspapers, cardboard, magazines, and fine paper; plant stems, leaves, and fruit clusters such as bagasse (sugarcane residue), empty palm fruit clusters (EFB), rice straw, Erianthus, and corn stalks or leaves; plant shells such as rice husks, palm husks, and coconut shells; and algae. Of these, wood, processed or crushed wood, plant stems, leaves, fruit clusters, etc. are preferred from the viewpoint of availability and raw material cost, bagasse, EFB, oil palm (trunk), and Erianthus are more preferred, and bagasse is even more preferred. The above biomass may be used individually or in mixtures of two or more types. The above biomass may also be dried.

[0022] In this specification, "arabinofuranosidase" refers to a protein having arabinofuranosidase activity. In this specification, "arabinofuranosidase activity" refers to the activity of hydrolyzing α-1,2-linked or α-1,3-linked side chains in arabinose-containing polysaccharides, or side chains with both α-1,2-linked and α-1,3-linked structures, to produce α-L-arabinose residues. The arabinofuranosidase activity of a protein can be determined, for example, by reacting the protein with p-nitrophenyl-α-L-arabinofuranoside as a substrate and measuring the released p-nitrophenol (pNP method). The specific procedure for measuring arabinofuranosidase activity is described in detail in the examples below.

[0023] (1. Arabinofuranosidase) In one embodiment, the present invention provides an arabinofuranosidase. The arabinofuranosidase of the present invention is a protein having an amino acid sequence selected from (a) to (c) below and having arabinofuranosidase activity. (a) Amino acid sequence of Sequence ID No. 1; (b) an amino acid sequence having at least 90% identity with the amino acid sequence of Sequence ID No. 1; (c) An amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence of Sequence ID No. 1. An example of a protein consisting of the amino acid sequence of Sequence ID No. 1 in (a) above is the mature protein of arabinofuranosidase derived from Aspergillus aculeatus No. F-50. An example of a protein consisting of the amino acid sequences of (b') and (c') above is a naturally occurring or artificially produced mutant of the arabinofuranosidase of Sequence ID No. 1. The arabinofuranosidase of the present invention is classified into the carbohydrate hydrolase family 54 (GH54) based on its amino acid sequence.

[0024] In another embodiment, the present invention provides an arabinofuranosidase preprotein comprising an amino acid sequence selected from (a') to (c') below. (a') Amino acid sequence of SEQ ID NO: 2; (b') An amino acid sequence having at least 90% identity with the amino acid sequence of Sequence ID No. 2; (c') An amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added to the amino acid sequence of Sequence ID No. 2. The above preproteins include an extracellular secretion signal peptide region and the amino acid sequence of the mature arabinofuranosidase protein, and are converted to the mature arabinofuranosidase protein by cleavage of the secretion signal peptide. For example, the amino acid sequence of SEQ ID NO: 2 in (a') above includes the amino acid sequence of the extracellular secretion signal peptide region (positions 1-26 of SEQ ID NO: 2) and the amino acid sequence of arabinofuranosidase in SEQ ID NO: 1 (positions 27-501 of SEQ ID NO: 2). The amino acid sequences in (b') and (c') above include the amino acid sequence of the extracellular secretion signal peptide region corresponding to positions 1-26 of SEQ ID NO: 2 and the amino acid sequence of arabinofuranosidase corresponding to positions 27-501 of SEQ ID NO: 2. An example of a protein consisting of the amino acid sequence of Sequence ID No. 2 in (a') above is the arabinofuranosidase preprotein derived from Aspergillus acritus No. F-50. An example of a protein consisting of the amino acid sequences in (b') and (c') above is a naturally occurring or artificially produced variant of the arabinofuranosidase preprotein of Sequence ID No. 2.

[0025] The aforementioned variants of the arabinofuranosidase and its preprotein can be produced, for example, by introducing a mutation into the gene encoding the protein of SEQ ID NO: 1 or 2 using known mutagenesis methods such as ultraviolet irradiation or site-directed mutagenesis, expressing the gene with the mutation, and selecting from the expressed protein those having the desired amino acid sequence and arabinofuranosidase activity. Such procedures for producing variants are well known to those skilled in the art.

[0026] (2. Method for producing arabinofuranosidase) The arabinofuranosidase or its preprotein of the present invention can be produced by expressing a polynucleotide encoding the arabinofuranosidase or its preprotein of the present invention (hereinafter collectively referred to as the polynucleotide of the present invention). For example, the arabinofuranosidase of the present invention can be produced from a transformant into which the polynucleotide of the present invention has been introduced. More specifically, after introducing the polynucleotide of the present invention or a vector containing it into a host to obtain a transformant, the transformant is cultured in an appropriate medium, and the arabinofuranosidase or its preprotein of the present invention is expressed from the polynucleotide introduced into the transformant. Furthermore, the expressed preprotein can be secreted extracellularly as mature arabinofuranosidase via the action of its secretory signaling peptide. The arabinofuranosidase of the present invention can be obtained by isolating or purifying the obtained arabinofuranosidase from the culture.

[0027] The polynucleotide of the present invention may be a polynucleotide comprising an amino acid sequence selected from (a) to (c) above and encoding a protein having arabinofuranosidase activity. Preferred examples of the polynucleotide of the present invention include polynucleotides comprising a nucleotide sequence selected from (d) to (g) below. (d) Nucleotide sequence shown in Sequence ID No. 3; (e) The nucleotide sequence shown in sequence number 79 through 1506 of sequence number 3; A nucleotide sequence having at least 90% identity with the nucleotide sequence shown in (f), (d), or (e); A nucleotide sequence in which one or more nucleotides are deleted, inserted, substituted, or added in the nucleotide sequence shown in (g)(d) or (e). The polynucleotide in (d) above encodes the preprotein of the arabinofuranosidase of SEQ ID NO: 2. When the preprotein expressed from the polynucleotide in (d) is secreted extracellularly via the secretory signal peptide it possesses, it becomes the arabinofuranosidase of SEQ ID NO: 1. The polynucleotide in (e) above consists of the nucleotide sequence obtained by removing the sequence encoding the extracellular secretory signal peptide from the nucleotide sequence in (d), and encodes the arabinofuranosidase of SEQ ID NO: 1.

[0028] The polynucleotides of the present invention may be in the form of single-stranded or double-stranded DNA, RNA, or artificial nucleic acids, or they may be cDNA or chemically synthesized DNA that does not contain introns.

[0029] The polynucleotides of the present invention can be synthesized chemically or genetically based on the amino acid sequence of the arabinofuranosidase or its preprotein of the present invention. For example, the polynucleotides of the present invention can be isolated from Aspergillus species such as Aspergillus acritus No. F-50 using any method used in the field. For example, the polynucleotides of the present invention can be obtained by extracting the whole genomic DNA of Aspergillus acritus No. F-50, selectively amplifying the target gene by PCR using primers designed based on the base sequence of Sequence ID No. 3, and purifying the amplified gene. Alternatively, the polynucleotides of the present invention can be synthesized chemically based on the amino acid sequence of the arabinofuranosidase or its preprotein of the present invention as described above. For the chemical synthesis of polynucleotides, nucleic acid synthesis contract services (for example, those provided by Medical & Biological Laboratories, Inc., Genscript, Inc., etc.) can be used. Furthermore, the synthesized polynucleotides can be amplified by PCR, cloning, etc.

[0030] Furthermore, for example, the polynucleotides of the present invention can be produced by introducing mutations into the polynucleotides synthesized in the above procedure using known mutagenesis methods such as ultraviolet irradiation or site-directed mutagenesis. For example, the polynucleotide encoding arabinofuranosidase of the present invention can be obtained by introducing mutations into the polynucleotide of Sequence ID No. 3 using a known method, expressing the resulting polynucleotide and examining its arabinofuranosidase activity, and selecting a polynucleotide encoding a protein having the desired arabinofuranosidase activity.

[0031] Site-directed mutagenesis into polynucleotides can be performed using any method, such as inverse PCR or annealing (Muramatsu et al., eds., "Revised 4th Edition New Genetic Engineering Handbook," Yodosha, pp. 82-88). Various commercially available site-directed mutagenesis kits, such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit, can also be used if necessary. Alternatively, methods for deleting, substituting, adding, or inserting nucleotides into nucleotide sequences are described, for example, by Dieffenbach et al. (Cold Spring Harbor Laboratory Press, New York, 581-621, 1995).

[0032] If necessary, the polynucleotides of the present invention may be codon-optimized to suit the host into which they are introduced. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0033] The type of polynucleotide-containing vector of the present invention is not particularly limited and includes vectors commonly used for gene cloning, such as plasmids, cosmids, phages, viruses, YACs, BACs, etc. Of these, plasmid vectors are preferred, and examples include commercially available protein expression plasmid vectors such as shuttle vectors pHY300PLK, pUC19, pUC119, pBR322 (all manufactured by Takara Bio Inc.) and pNAN8142 (Minetoki et al., J Biol Macromol, 2003, 3(3):89-96), which can be suitably used.

[0034] The vector may include a DNA region containing a DNA replication initiation region or replication origin. Alternatively, in the vector, a regulatory sequence such as a promoter region for initiating transcription of the polynucleotide of the present invention (i.e., the arabinofuranosidase gene of the present invention) or a secretion signal sequence encoding a secretion signal peptide for secreting the expressed protein outside the cell may be operably ligated upstream of the polynucleotide of the present invention (i.e., the arabinofuranosidase gene of the present invention).

[0035] The types of regulatory sequences such as the promoter region, terminator, and secretion signal sequence are not particularly limited, and commonly used promoters and secretion signal sequences can be appropriately selected and used depending on the host to which the gene is introduced. For example, a suitable example of a regulatory sequence that can be incorporated into the vector of the present invention is the Trichoderma reesei-derived cbh1 promoter sequence (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, and pyruvate kinase may be used.

[0036] Alternatively, the vector may further incorporate marker genes (e.g., resistance genes for drugs such as ampicillin, neomycin, kanamycin, and chloramphenicol) for selecting a host into which the vector has been appropriately introduced. Alternatively, if a nutrient-requiring strain is used as the host, a gene encoding an enzyme for synthesizing the required nutrients may be incorporated into the vector as a marker gene. Or, if a selective medium is used that requires specific metabolism for growth, a gene related to that metabolism may be incorporated into the vector as a marker gene. An example of such a metabolism-related gene is the acetamidase gene for utilizing acetamide as a nitrogen source.

[0037] The polynucleotide of the present invention can be linked to the regulatory sequence and marker gene by methods known in the art, such as SOE (splicing by overlap extension)-PCR (Gene, 1989, 77:61-68). The procedure for introducing the linked fragment into a vector is well known in the art.

[0038] Examples of hosts for transformants into which the vector is introduced include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus, among which Escherichia coli and Bacillus bacteria (e.g., Bacillus subtilis or its mutants) are preferred. Examples of Bacillus subtilis mutants include the KA8AX protease 9-fold knockout strain described in J.Biosci.Bioeng.,2007,104(2):135-143, and the D8PA strain, which is an improved version of the protease 8-fold knockout strain with enhanced protein folding efficiency, described in Biotechnol.Lett.,2011,33(9):1847-1852. Examples of filamentous fungi include the genera Trichoderma, Aspergillus, and Rizhopus, with Aspergillus being preferred among them.

[0039] Methods commonly used in this field, such as protoplast synthesis and electroporation, can be used to introduce the vector into the host. By selecting strains in which the vector has been properly introduced based on indicators such as marker gene expression and nutritional requirements, the desired transformants into which the vector has been introduced can be obtained.

[0040] Alternatively, a DNA fragment containing the polynucleotide of the present invention can be directly introduced into the host genome. For example, a DNA fragment containing the operably linked polynucleotide of the present invention, a regulatory sequence, and a marker gene, and having sequences complementary to the host genome at both ends, can be constructed by SOE-PCR or the like. This fragment can then be introduced into the host to induce homologous recombination between the host genome and the DNA fragment, thereby introducing the polynucleotide of the present invention into the host genome.

[0041] When a transformant into which the obtained polynucleotide of the present invention or a vector containing it has been introduced is cultured in a suitable medium, the arabinofuranosidase gene on the polynucleotide or vector is expressed, and the arabinofuranosidase or its preprotein of the present invention is produced. The medium used to culture the transformant can be appropriately selected by a person skilled in the art according to the type of microorganism of the transformant. Alternatively, the arabinofuranosidase or its preprotein of the present invention may be expressed from the polynucleotide of the present invention or its transcript using a cell-free translation system. A "cell-free translation system" is an in vitro transcription-translation system or in vitro translation system constructed by adding reagents such as amino acids necessary for protein translation to a suspension obtained by mechanically disrupting host cells.

[0042] The arabinofuranosidase or its preprotein produced in the culture or cell-free translation system of the present invention can be isolated or purified by common methods used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combinations. In this case, if the polynucleotide encoding the arabinofuranosidase of the present invention introduced into the transformant is operably linked to a secretion signal sequence, the generated arabinofuranosidase is secreted extracellularly and can be more easily recovered from the culture. The arabinofuranosidase recovered from the culture may be further purified by known means.

[0043] (3. Enzyme compositions for biomass saccharification) As shown in the examples described below, the arabinofuranosidase of the present invention exhibits excellent arabinofuranosidase activity and improves the biomass saccharification rate by promoting hemicellulase degradation. Therefore, the arabinofuranosidase of the present invention can be used for biomass saccharification. Thus, in a further embodiment, the present invention provides an enzyme composition for biomass saccharification containing the arabinofuranosidase of the present invention.

[0044] The enzyme composition for biomass saccharification provided in the present invention (hereinafter also referred to as the enzyme composition of the present invention) contains the arabinofuranosidase of the present invention as described above, and further contains cellulase from the viewpoint of improving the biomass saccharification rate. 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 such as endoglucanase, exoglucanase, cellobiohydrolase, and β-glucosidase. From the viewpoint of improving the biomass saccharification rate, it is preferable that the cellulase contained in the enzyme composition of the present invention contains one or more selected from the group consisting of cellobiohydrolase and endoglucanase. The cellulase contained in the enzyme composition of the present invention may be selected from cellulases derived from animals, plants, and microorganisms, or commercially available cellulases may be used. These cellulases may be used alone or in combination of two or more types.

[0045] Examples of cellulases contained in the enzyme composition of the present invention are not limited to these, but include: cellulase derived from Trichoderma reesei; cellulase derived from Trichoderma viride; cellulase derived from various Bacillus strains such as 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), and Bacillus sp. KSM-N659 (FERM P-19730); heat-resistant cellulase derived from Pyrococcus horikoshii; and Humicola insolence. Examples include cellulase derived from *Trichoderma rhizon*, *Trichoderma viride*, or *Humicola insolens*. Among these, cellulase derived from *Trichoderma rhizon*, *Trichoderma viride*, or *Humicola insolens* is preferred from the viewpoint of improving the biomass saccharification rate. Recombinant cellulase obtained by expressing a cellulase gene exogenously introduced into the above microorganisms may also be used. A specific example is cellulase JN11 produced by strain X3AB1 (J.Ind.Microbiol.Biotechnol.(2012)1741-9), which was obtained by introducing a β-glucosidase gene derived from *Aspergillus acritus* into *Trichoderma rhizon*.

[0046] Examples of cellulases that may be contained in the enzyme composition of the present invention include cellobiohydrolases derived from Trichoderma liize, Trichoderma viride, Acremonium celluloriticus, Humicola insolence, Clostridium thermocellum, Bacillus, Thermobifida, and Cellulomonas.

[0047] Examples of endoglucanases include those derived from Trichoderma liese, Acremonium ceruloticus, Fumicola insolence, Clostridium thermoserum, Bacillus, Thermobifida, and Cellulomonas. Of these, from the viewpoint of improving the biomass saccharification rate, endoglucanases derived from Trichoderma liese, Fumicola insolence, Bacillus, and Cellulomonas are preferred, with endoglucanase derived from Trichoderma liese being more preferred.

[0048] Examples of β-glucosidases include β-glucosidase derived from Aspergillus niger (e.g., Novozyme 188 and Megazyme β-glucosidase from Novozymes), β-glucosidase derived from Aspergillus acretus, and β-glucosidase derived from Trichoderma liese or Penicillium emersonii. Of these, Novozyme 188 and β-glucosidase derived from Trichoderma liese are preferred from the viewpoint of improving the biomass saccharification rate, and β-glucosidase derived from Trichoderma liese is more preferred.

[0049] Examples of commercially available cellulases include Cellclast® 1.5L (manufactured by Novozymes), TP-60 (manufactured by Meiji Seika Co., Ltd.), Cellic® CTec2 (manufactured by Novozymes), Accellerase™DUET (manufactured by Genencor), and Ultraflo® L (manufactured by Novozymes).

[0050] The enzyme composition of the present invention may further contain the hemicellulase of the arabinofuranosidase of the present invention described above. Here, hemicellulase refers to an enzyme that hydrolyzes hemicellulose, and is a general term for enzymes such as xylanase, xylosidase, and galactanase. Examples of hemicellulases other than arabinofuranosidase in the present invention include: hemicellulase derived from Trichoderma liese; xylanase derived from Bacillus sp. KSM-N546 (FERM P-19729); xylanase derived from Aspergillus niger, Trichoderma viride, Humicola insolens, or Bacillus alcalophilus; xylanase derived from Thermomyces, Aureobasidium, Streptomyces, Clostridium, Thermotoga, Thermoascus, Caldocellum, or Thermomonospora species; and Bacillus pumila. Examples include β-xylosidase derived from Bacillus pumilus; β-xylosidase derived from Selenomonas ruminantium, etc. Of these, from the viewpoint of improving saccharification efficiency, the enzyme composition of the present invention preferably contains xylanase derived from Bacillus sp., Aspergillus niger, Trichoderma viride or Streptomyces, or β-xylosidase derived from Selenomonas ruminantium, and more preferably contains xynarase derived from Bacillus sp. or Trichoderma viride, or β-xylosidase derived from Selenomonas ruminantium.

[0051] The content of the arabinofuranosidase of the present invention in the enzyme composition of the present invention may be in the range of 0.1% by mass or more and 70% by mass or less, preferably in the range of 0.5% by mass or more and 50% by mass or less, of the total protein amount of the composition. The content of the cellulase of the present invention in the enzyme composition of the present invention may be in the range of 10% by mass or more and 99% by mass or less, preferably in the range of 15% by mass or more and 95% by mass or less, of the total protein amount of the composition. The content of hemicellulase other than the arabinofuranosidase of the present invention in the enzyme composition of the present invention may be in the range of 0.01% by mass or more and 30% by mass or less, preferably in the range of 0.1% by mass or more and 20% by mass or less, of the total protein amount of the composition. The protein ratio of the arabinofuranosidase of the present invention to the cellulase of the present invention (arabinofuranosidase / cellulase of the present invention) in the enzyme composition of the present invention may be in the range of 0.001 or more and 7 or less, preferably in the range of 0.005 or more and 1 or less.

[0052] (4. Methods for producing sugar) The enzyme composition of the present invention is used to produce sugar from biomass. Accordingly, the present invention provides a method for producing sugar from biomass using the enzyme composition of the present invention. The method for producing sugar from biomass according to the present invention (hereinafter also referred to as the method of the present invention) includes the step of saccharifying biomass with the enzyme composition of the present invention (i.e., a combination of arabinofuranosidase and cellulase of the present invention).

[0053] As described above, the biomass used in the method of the present invention is as follows, but from the viewpoint of availability, raw material cost, and improvement of biomass saccharification rate, wood, processed or crushed wood, plant stems, leaves or fruit clusters are preferred as the biomass, bagasse, EFB, oil palm (trunk), and Erianthus are more preferred, and bagasse is even more preferred. The biomass may be used alone or in a mixture of two or more types. The biomass may also be dried.

[0054] When saccharifying biomass, it is preferable to further include a step of pre-treating the biomass before the saccharification process, from the viewpoint of improving crushing efficiency, improving the saccharification rate, and improving production efficiency (i.e., shortening the sugar production time).

[0055] Examples of the aforementioned pretreatment include one or more selected from the group consisting of alkaline treatment, pulverization, and hydrothermal treatment. From the viewpoint of improving the biomass saccharification rate, alkaline treatment is preferred as the pretreatment.

[0056] The aforementioned alkali treatment refers to reacting biomass with a basic compound, which will be described later. Methods of this alkali treatment include immersing the biomass in an alkaline solution containing the basic compound, which will be described later (hereinafter sometimes referred to as "immersion treatment"), and mixing the biomass with the basic compound and subjecting it to a grinding treatment, which will be described later (hereinafter sometimes referred to as "alkaline mixed grinding treatment").

[0057] The aforementioned pulverization process refers to mechanically crushing biomass to reduce it into smaller particles. Reducing the biomass into smaller particles further improves the saccharification rate. Furthermore, if the crystalline structure of cellulose contained in the biomass is destroyed by the pulverization process, the saccharification rate will improve even further. This pulverization process can be carried out using a known pulverizer. There are no particular restrictions on the pulverizer used; any device capable of reducing biomass into smaller particles is acceptable. This pulverization process may be combined with the alkali treatment using the basic compound described above. This pulverization process may be carried out before or after the alkali treatment, or the alkali treatment, such as the alkali mixed pulverization process described above, may be carried out in parallel with the pulverization process. In the alkali mixed pulverization process, for example, biomass immersed in an alkaline solution may be subjected to the pulverization process (wet pulverization), or solid alkali and biomass may be subjected to the pulverization process together (dry pulverization), but dry pulverization is preferred.

[0058] The aforementioned hydrothermal treatment refers to the heating treatment of biomass in the presence of water. This hydrothermal treatment can be carried out using a known reactor, and there are no particular restrictions on the reactor used.

[0059] The conditions for the saccharification treatment of biomass are not particularly limited, as long as the enzymes contained in the enzyme composition of the present invention are not deactivated. Appropriate conditions can be appropriately determined by those skilled in the art depending on the type of biomass, the procedure of the pretreatment step, and the type of enzyme used.

[0060] In the saccharification treatment described above, the enzyme composition of the present invention is added to the pre-treated biomass as needed to induce a saccharification reaction. It is preferable to add the enzyme composition of the present invention to a suspension containing biomass. At this time, the components of the enzyme composition of the present invention, namely the arabinofuranosidase and cellulase of the present invention, and other enzymes as needed, may be added to the biomass simultaneously, sequentially, or separately. The biomass content in the suspension is preferably 0.5% by mass or more, more preferably 3% by mass or more, even more preferably 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, from the viewpoint of improving the biomass saccharification rate or sugar production efficiency (i.e., shortening the sugar production time). For example, 0.5 to 30% by mass, 0.5 to 25% by mass, or 0.5 to 20% by mass is preferred, 3 to 30% by mass, 3 to 25% by mass, or 3 to 20% by mass is more preferred, and 5 to 30% by mass, 5 to 25% by mass, or 5 to 20% by mass is even more preferred.

[0061] The initial concentration of biomass in the reaction solution for the saccharification treatment is preferably 1% by mass or more, more preferably 2% by mass or more, even more preferably 3% by mass or more, and preferably 30% by mass or less, more preferably 20% by mass or less, and even more preferably 15% by mass or less, from the viewpoint of improving the biomass saccharification rate or sugar production efficiency (i.e., shortening the sugar production time). For example, 1 to 30% by mass, 1 to 20% by mass, or 1 to 15% by mass is preferred, 2 to 30% by mass, 2 to 20% by mass, or 2 to 15% by mass is more preferred, and 3 to 30% by mass, 3 to 20% by mass, or 3 to 15% by mass is even more preferred.

[0062] The amount of the enzyme composition of the present invention used in the saccharification treatment can be appropriately determined depending on the pretreatment conditions and the type and properties of the enzyme used in combination. For example, the initial concentration of the enzyme composition of the present invention in the reaction solution for the saccharification treatment is preferably 0.0001% by mass or more, more preferably 0.0002% by mass or more, even more preferably 0.0005% by mass or more, and preferably 100% by mass or less, more preferably 50% by mass or less, and even more preferably 20% by mass or less, when converted to the mass of cellulase, or preferably 0.1% by mass or more, more preferably 0.2% by mass or more, even more preferably 0.3% by mass or more, and 100% by mass or less, more preferably 50% by mass or less, and even more preferably 20% by mass or less. For example, the initial concentration of the arabinofuranosidase of the present invention in the reaction solution is preferably 0.0001 to 100% by mass, 0.0001 to 50% by mass, or 0.0001 to 20% by mass, more preferably 0.0002 to 100% by mass, 0.0002 to 50% by mass, or 0.0002 to 20% by mass, and even more preferably 0.0005 to 100% by mass, 0.0005 to 50% by mass, or 0.0005 to 20% by mass, based on the biomass mass (100% by mass). Alternatively, the initial concentration of cellulase in the reaction solution is preferably 0.01 to 100% by mass, 0.01 to 50% by mass, or 0.01 to 20% by mass, more preferably 0.02 to 100% by mass, 0.02 to 50% by mass, or 0.02 to 20% by mass, and even more preferably 0.03 to 100% by mass, 0.03 to 50% by mass, or 0.03 to 20% by mass, based on the biomass mass (100% by mass). The protein ratio of the arabinofuranosidase to cellulase of the present invention in the reaction solution (arabinofuranosidase / cellulase of the present invention) may be in the range of 0.001 to 7, and preferably 0.005 to 1.

[0063] The pH of the reaction solution for the saccharification treatment is preferably 2.0 or higher, more preferably 3.0 or higher, and more preferably 5.5 or lower, from the viewpoint of improving biomass sugar efficiency or sugar production efficiency (i.e., shortening sugar production time) and reducing production costs. For example, pH 2.0 to 5.5 is preferred, and pH 3.0 to 5.0 is more preferred.

[0064] The reaction temperature for the saccharification treatment is preferably 40 to 60°C from the viewpoint of improving the biomass saccharification rate or sugar production efficiency (i.e., shortening the sugar production time), reducing production costs, and ensuring the optimal temperature and temperature stability of the cellulase used simultaneously. The reaction time for 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 (i.e., shortening the sugar production time) and reducing production costs, it is preferably 1 to 5 days, more preferably 1 to 4 days, and even more preferably 1 to 3 days.

[0065] Examples of sugars produced by the aforementioned saccharification process include glucose, xylose, mannose, and arabinose. The produced saccharified solution can be recovered and purified from the reaction solution by known methods such as solid-liquid separation using a screw press, screen, filter press, belt press, rotary press, etc., or by distillation separation. [Examples]

[0066] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0067] <Example 1> Production of Arabinofuranosidase Arabinofuranosidase F5054ABF was produced from Aspergillus aculeatus No. F-50.

[0068] (1) Preparation of chromosomal DNA Aspergillus aculeatus No. F-50 was cultured at 30°C for approximately 2 days. The cells were collected by filtration, washed with distilled water, and freeze-dried. This was then crushed in a mortar with a small amount of crushed sea sand until it became a powder. A sufficient amount of the cells to be suspended was placed in a 50 mL centrifuge tube with a cap, and 2,160 μL of extraction buffer (0.1 M Tris-HCl (pH 8.0), 0.1 M EDTA, 0.25 M NaCl) was added and the mixture was thoroughly suspended. 240 μL of 10% Sarkosyl and several mg of powdered Proteinase K were added to the resulting suspension, and the mixture was incubated at 55°C for at least 2 hours, stirring every 30 minutes, until cell lysis was observed. When the suspension became viscous, an additional 280 μL of 5M NaCl solution was added and mixed well. Then, 300 μL of CTAB / NaCl solution (10% Hexadecyltrimethyl ammonium bromide in 0.7M NaCl), which had been preheated to 65°C, was added and incubated at 65°C for 10 minutes. The resulting solution was chloroform extracted, and the aqueous layer was transferred to a microtube after centrifugation (4°C, 13,500 rpm, 10 minutes). After further phenol / chloroform extraction, the aqueous layer was collected by centrifugation (4°C, 13,500 rpm, 5 minutes). The collected aqueous layer was left to stand on ice for 5 minutes, then approximately equal volumes of ice-cold isopropanol were added, and the mixture was gently shaken until a DNA precipitate appeared. The DNA precipitate was lifted from the solution after shaking, washed with 1 mL of 70% EtOH, vacuum-dried, and suspended in 500 μL of TE buffer. To the resulting suspension, 1 μL of RNase solution (10 mg / mL) was added and incubated at 37°C for 30 minutes. Then, phenol / chloroform extraction was repeated until no precipitate appeared at the interface between the two layers. The upper layer obtained from the final extraction was placed in a separate tube, 1 / 10 volume of 3M NaOAc was added, and it was allowed to stand on ice for 5 minutes. Then, approximately an equal volume of ice-cold isopropanol was added, and the tube was gently shaken until a white DNA precipitate appeared. The resulting DNA precipitate was lifted off the tube, washed several times with 1 mL of 70% EtOH, transferred to an empty tube, lightly dried, and then suspended in an appropriate amount of TE buffer.

[0069] (2) Preparation of arabinofuranosidase DNA Using the obtained Aspergillus aculeatus No. F-50 chromosomal DNA as a template, a fragment of approximately 1.5 kbp containing the gene sequence encoding F5054ABF (SEQ ID NO: 3) was amplified by PCR using the primer sets (SEQ ID NOs: 4 and 5) shown in Table 1. PrimeSTAR was used for PCR. TM HS DNA Polymerase (manufactured by Takara Bio Inc.) and the accompanying buffer were used. Based on its gene sequence, F5054ABF was presumed to be an enzyme classified as belonging to the carbohydrate hydrolase family 54 (GH54).

[0070] (3) Preparation of the F5054ABF expression vector pNAN-54a The gene fragment obtained in (2) above was purified by agarose electrophoresis and subcloned into the Hind III / Xba I site within the multicloning site of pUC119. Subcloning of the target gene was confirmed by treating the constructed plasmid with Hind III / Xba I. The constructed plasmid was prepared in large quantities, treated with Hind III / Xba I, and purified the target gene fragment by agarose gel electrophoresis and gel recovery. The purified target gene fragment was subcloned into the Hind III / Xba I site within the multicloning site of the filamentous fungus high expression vector pNAN8142 (Minetoki et al., 2003, J Biol Macromol, 3(3):89-96), and it was confirmed that the fragment was correctly subcloned by Hind III / Xba I treatment. The constructed plasmid was named pNAN-54a.

[0071] [Table 1]

[0072] (4) Preparation of transformed organisms Aspergillus oryzae niaD300 (J Biosci Bioeng, 2001;92(2):131-137) was transformed with the vector pNAN-54a constructed in (3) above. Vector introduction was performed by protoplast PEG method, referring to the method of Gomi et al. (Agric Biol Chem, 1987, 51(9):2549-2555). Transformants were selected using the selective medium RM plate (5% Salts solution, 1% glucose, 0.3% NaNO3, 4.68% NaCl, 0.1% Trace element; % is w / v%, pH 6.5). The composition of the Salts solution was as follows: 2.6% KCl, 2.6% MgSO4·7H2O, 7.6% KH2PO4 (% is w / v%). The composition of the trace element is as follows: 0.5% FeSO4·7H2O, 0.16% CoCl2·6H2O, 0.16% CuSO4·5H2O, 0.11% Mo7O 24 · 4H2O, 0.11% H3BO3, 5.0% EDTA (% is w / v%). After culturing in selective medium at 30°C for 3-5 days, the resulting strains were isolated. The isolated strains were spore-formed and dispersed in Tween / Saline solution to prepare a spore suspension. A small amount of this suspension was spread onto minimal medium containing 0.01% Triton X-100 (see (5) below for the composition of the minimal medium) and cultured. Monospore formation of the transformants was performed by separating the colonies formed from the mononuclear spores.

[0073] (5) Culture of transformed organisms Minimal medium (NO3 - The bacterial strains grown on the plates were cut out with a spatula and inoculated into a 500 mL baffled Erlenmeyer flask containing 200 mL of minimal medium (NO3) adjusted to 3% glucose and 0.9% NaNO3. The cells were then cultured at 30°C and 160 rpm for approximately 3 days. - The composition of the solution is as follows: 5% Salts solution, 0.1% Trace element, 1% glucose, 0.3% NaNO3, pH 6.5 (% is w / v%). The composition of the Salts solution and Trace element is as described in (4) above.

[0074] (6) Purification of F5054ABF The culture supernatant obtained in (5) above was filtered by suction using a Buchner funnel lined with filter paper (No. 2, manufactured by ADVANTEC), and the filtrate was collected as crude enzyme solution. The enzyme was purified from the crude enzyme solution using the following procedure.

[0075] Ammonium sulfate was added to the crude enzyme solution to prepare a 30% saturated ammonium sulfate solution. The resulting crude enzyme solution was passed through Phenyl-TOYOPEARL 650M (Tosoh), which had been pre-equilibriumized with a 30% saturated ammonium sulfate solution (in 20mM Acetate buffer (pH 5.0)), to adsorb the enzyme, and eluted using a reverse linear gradient of 1 L of 30-0% saturated ammonium sulfate solution (in 20mM Acetate buffer (pH 5.0)). Fractions in which the A280 value overlapped with the activity for the final concentration of 1mM pNP-Ara were subjected to SDS-PAGE, and fractions in which a band of approximately 60 kDa was confirmed were collected.

[0076] Ammonium sulfate was added to the recovered fraction to make an 80% saturated ammonium sulfate solution, and ammonium sulfate salting was performed. After recovering the precipitate by centrifugation (4°C, 9,000 rpm, 45 minutes), it was dissolved in a small amount of 20 mM Acetate buffer (pH 5.0). This was dialyzed overnight in 20 mM Acetate buffer (pH 5.0) using a dialysis membrane (Sanko Pure Chemical Industries). The collected sample was passed through a DEAE-TOYOPEARL 650 (Tosoh) pre-equilibriumized with 20 mM Acetate buffer (pH 5.0) to adsorb the enzyme, and eluted with a linear gradient of 1 L of 0-0.3 M NaCl solution (in 20 mM Acetate buffer (pH 5.0)) to recover the fraction showing activity against pNP-Ara. The recovered fraction was again precipitated with ammonium sulfate, and the precipitate was collected by centrifugation (4°C, 12,000 rpm, 45 minutes). This precipitate was then dissolved in a small amount of 20 mM Acetate buffer (pH 5.0). This was dialyzed overnight to obtain a purified enzyme solution.

[0077] F5054ABF contained in the purified enzyme solution is mature arabinofuranosidase (SEQ ID NO: 1), consisting of the amino acid sequence obtained by removing the predicted signal sequence (positions 1-26) from the amino acid sequence of the arabinofuranosidase preprotein (SEQ ID NO: 2). The signal sequence was predicted using signalP (Bendtsen et al., J Mol Biol, 2004, 340:783-795).

[0078] <Example 2> Enzyme properties of F5054ABF (1) Protein concentration measurement The protein concentration in the purified enzyme solution prepared in Example 1 was measured by extinction coefficient and the Bradford method. For extinction coefficient measurement, the protein concentration was determined by measuring at OD280nm. The enzyme extinction coefficient was retrieved using ProtParam (web.expasy.org / protparam / ) (Gasteiger et al., The Proteomics Protocols Handbook, (ed) John M. Walker, Humana Press, 2005, pp. 571-607). For the Bradford method, a protein assay reagent (Bio-Rad) was used, and the protein amount was calculated based on a calibration curve using bovine γ-globulin as the standard protein.

[0079] (2) p-nitrophenyl-α-L-arabinofuranoside degradation activity The α-L-arabinofuranosidase activity of F5054ABF was measured. Activity was measured by the pNP method. A 1 mM p-Nitrophenyl-α-L-arabinofuranoside (pNP-Ara) solution (in 20 mM Acetate buffer (pH 5.0)) was used as the substrate solution. The purified enzyme solution was diluted to an appropriate concentration with 20 mM Acetate buffer (pH 5.0) to prepare the enzyme solution. 100 μL of the enzyme solution, pre-incubated at 37°C for 5 minutes, was added to 100 μL of the substrate solution, which had also been pre-incubated, and mixed thoroughly. The reaction was then incubated at 37°C for 10 minutes. 2 mL of 1 M Na2CO3 was added to the reaction mixture to stop the reaction, and the 405 nm value, representing the concentration of free p-nitrophenol, was measured. A blank was used where Acetate buffer (pH 5.0) was added instead of the enzyme solution. The enzyme activity of the purified enzyme solution was determined from the measured absorbance and the extinction coefficient of pNP using the following formula. 1 unit (U) was defined as the amount of enzyme that releases 1 μmol of p-nitrophenol per minute. Enzyme activity (unit / mL) = (ΔA 405 (×Reaction volume (mL) × Dilution ratio of purified enzyme sample) / (ε 405nm × Sample volume (mL) × Reaction time (min) ΔA 405 : The value obtained by subtracting the blank value from the 405nm absorbance of the reaction solution. ε 405nm : 1.85 μL / μmol / cm Based on the calculated enzyme activity of the purified enzyme solution and the protein concentration of the purified enzyme solution obtained in (1) above, the enzyme activity per unit mass was determined using the following formula. Specific activity (U / mg) = Enzyme activity (unit / mL) / Enzyme weight (mg / mL)

[0080] As shown in Table 2, the pNP-Ara degradation activity of F5054ABF was 53.4 U / mg.

[0081] (3) Substrate specificity The substrate specificity of F5054ABF was evaluated by reacting the purified enzyme solution with various pNP glycosides or natural substrates.

[0082] The following pNP glycosides were used: p-Nitrophenyl-β-D-xylopyranoside (pNP-Xyl,Sigma-Aldrich), p-Nitrophenyl-α-L-arabinopyranoside (pNP-α-Arap,Sigma-Aldrich), or p-Nitrophenyl-β-L-arabinopyranoside (pNP-β-Arap,Sigma-Aldrich). The enzymatic activity against the pNP glycosides was measured by the pNP method according to the procedure in (2) above. A final concentration 1 mM pNP glycoside-20 mM acetate buffer solution was used as the substrate solution. The enzymatic reaction was carried out at 37°C for 10 minutes.

[0083] The following were used as natural substrates: Wheat arabinoxylan (Megazyme), Rye arabinoxylan (Megazyme), Beech wood xylan (Sigma - Aldrich), Oat spelt xylan (Tokyo Chemical Industry), Sugar beet arabinan (Megazyme), or Debranched arabinoxylan (Megazyme). The enzyme activity against the natural substrate was determined according to the method of Dubois et al. (Dubois et al., Analytical Chemistry, 1956, 28: 350 - 356) by the Somogyi - Nelson method. A solution of the above natural substrate adjusted to a final concentration of 0.1% (w / v) (in 20 mM Acetate buffer (pH 5.0)) was used as the substrate solution. The purified enzyme solution was appropriately diluted with 20 mM Acetate buffer (pH 5.0). 100 μL of the substrate solution was added to 100 μL of the diluted solution, and the reaction was carried out at 37 °C for 16 hours or 10 minutes. 500 μL of Somogyi solution was added to the reaction solution to stop the reaction, then 300 μL of ion - exchanged water was added, and it was boiled at 100 °C for 15 minutes, cooled in running water for 5 minutes, 500 μL of Nelson solution was added and stirred, and left at room temperature for 30 minutes. 3.5 mL of ion - exchanged water was added to the obtained solution and stirred, and the absorbance at 500 nm was measured with a spectrophotometer. A solution obtained by adding 500 μL of Somogyi solution to the diluted solution of the purified enzyme solution was used as a blank. From the measured absorbance, the reducing equivalent in the reaction solution was calculated based on the calibration curve prepared using an Ara solution of known concentration. From the calculated amount of reducing sugar, the enzyme activity of the purified enzyme solution against the natural substrate was determined by the following formula. 1 unit was defined as the amount of enzyme that produces 1 μmol of reducing sugar per minute. Enzyme activity (unit / mL) = {amount of reducing sugar × reaction time (min)} × (1 / 0.1 mL) × dilution factor of the purified enzyme sample Calibration curve (Ara): y = 337.52 × A 500 + 0.967 A 500 : the value obtained by subtracting the blank value from the absorbance at 500 nm of the reaction solution Molecular weight of Ara: 150.13 Amount of reducing sugar (μg / mL) = y / 150.13 Based on the calculated enzyme activity of the purified enzyme solution and the protein concentration of the purified enzyme solution, the enzyme activity per unit mass was determined using the following formula. Specific activity (U / mg) = Enzyme activity (unit / mL) / Enzyme weight (mg / mL)

[0084] Table 2 shows the enzymatic activity against various substrates. F5054ABF showed the highest activity against pNP-Ara and also showed some activity against arabinose-containing polysaccharides such as wheat arabinoxylan, rye arabinoxylan, sugar beet arabinan, and debranched arabinan. On the other hand, F5054ABF showed almost no activity against the pyranose forms of pNP-Ara, such as pNP-α-Arap and pNP-β-Arap, as well as xylose-containing sugars such as pNP-Xyl, beech wood xylan (Sigma-Aldrich), and oat spelt xylan. Arabinofuranosidase B from Aspergillus niger, which, like F5054ABF, belongs to GH54 and has 69% amino acid sequence homology, has been reported to show the highest activity against pNP-Ara (147 U / mg) and also activity against arabinan (1.6 U / mg) (Rombouts et al., Carbohydrate Polymers, 1988, 9(1):25-47). The specific activity trend of F5054ABF against various substrates was similar to that of A. niger arabinofuranosidase B.

[0085] [Table 2]

[0086] (4) Effects of temperature and pH The optimal temperature, optimal pH, temperature stability, and pH stability of F5054ABF were investigated. Enzyme activity was measured using the pNP method as described in (1) above, with pNP-Ara (in 20mM Acetate buffer (pH 5.0)) as the substrate. The final substrate concentration was 1mM. For the evaluation of the optimal temperature, a diluted solution of the purified enzyme (in 20mM Acetate buffer) was pre-incubated at temperatures from 30 to 70°C for 5 minutes, and then the substrate solution pre-incubated at the same temperature was added, and the activity was measured after a reaction time of 10 minutes. For the evaluation of the optimal pH, the activity was examined when a diluted solution of the purified enzyme was reacted with a substrate solution prepared using 100mM Britton-Robinson buffer (Briton et al., Journal of the Chemical Society, 1931, 0:1456-1462) with a pH of 2.2 to 11.7 at 37°C for 10 minutes. For temperature stability evaluation, diluted purified enzyme solutions were pretreated at various temperatures from 30 to 70°C for 30 minutes, rapidly cooled on ice for 5 minutes, and then the residual activity against the substrate was examined. For pH stability evaluation, diluted purified enzyme solutions were diluted 3 to 10 times with 100 mM Britton-Robinson buffer (pH 2.2 to 11.7) (Briton et al., 1931, mentioned above), pretreated at room temperature for 1 hour, and then diluted 10 times with 100 mM Acetate buffer (pH 5.0) to return to pH 5.0, after which the residual activity against the substrate was measured.

[0087] Figures 1-4 show the optimal temperature, optimal pH, temperature stability, and pH stability of F5054ABF. Figure 1 shows the relative enzyme activity relative to the enzyme activity at 60°C. The optimal temperature for F5054ABF is 50-60°C, and it showed a relative activity of over 90% around 50°C. Figure 2 shows the relative enzyme activity relative to the enzyme activity at pH 3.0. The optimal pH for F5054ABF is pH 3.0-5.0, and it showed a relative activity of over 80% within this range. Regarding temperature stability, Figure 3 shows the relative enzyme activity relative to the enzyme activity pretreated at 50°C. F5054ABF showed a relative activity of over 80% at temperatures up to 60°C, demonstrating excellent thermal stability. Regarding pH stability, Figure 4 shows the relative enzyme activity relative to the enzyme activity pretreated at pH 4.0. F5054ABF showed a relative activity of over 80% around pH 2.5-6.0.

[0088] (5) Sugars produced by F5054ABF The sugars produced by polysaccharide decomposition using F5054ABF were analyzed. Wheat arabinoxylan, rye arabinoxylan, sugar beet arabinan, debranched arabinoxylan, beech wood xylan, and oat spelt xylan, as used as substrates, were the same as those used in (3) above. 100 μL of purified enzyme solution moderately diluted with 20 mM Acetate buffer (pH 5.0) was added to 100 μL of a 0.2% (w / v) substrate solution prepared with 20 mM Acetate buffer (pH 5.0), and the mixture was reacted at 50°C for 10 minutes or 1 hour. After stopping the reaction by boiling for 5 minutes, twice the volume of acetone was added to the reaction solution, and the mixture was vortexed and centrifuged (4°C, 13,500 rpm, 10 minutes) to remove the precipitate and vacuum-dried. The resulting dry material was dissolved in purified water to obtain the sample solution. The sugars in the sample solution were analyzed by high-performance anion chromatography (HPAEC-PAD) using pulsed amperometry detection. The HPAEC-PAD system used was either a Dionex ICS-3000 Ion Chromatography System or a Dionex ICS-5000 Ion Chromatography System, along with a CarboPac PA100 (guard column, 4×50mm; analytical column, 4×250mm). 30 μL or 25 μL of sample solution was injected into the sample loop, and separation was performed at a flow rate of 1 mL / min using 20 mM NaOH as the eluent. Arabinose or xylose was used as the standard. The Chromeleon Chromatography Data System was used for data analysis. The analysis results are shown in Table 3.

[0089] [Table 3]

[0090] F5054ABF showed significant Ara release from sugar beet arabinan. Ara release was also observed when wheat arabinoxylan, rye arabinoxylan, and debranched arabinan were used as substrates, although not to the same extent as with sugar beet arabinan. This trend was similar to the specific activity trend shown in Table 2. α-L-AFase II from Aspergillus awamori, which, like F5054ABF, belongs to GH54 and has 72.6% amino acid sequence homology, has been reported to show high hydrolytic activity against arabinoxylan, debranched arabinan, and arabinan, with hydrolysis efficiencies of 16.0%, 23.3%, and 67.4% respectively (Kaneko et al., Appl Environ Microbiol, 1998, 64(10):4021-4027). Similarly, F5054ABF also degraded arabinan in particular among polysaccharides. Furthermore, this suggests that F5054ABF is an enzyme that degrades alpha-1,2 or alpha-1,3 linked arabinose side chains.

[0091] <Example 3> Usability of F5054ABF in biomass saccharification

[0092] (1) Preparation of biomass Bagasse was used as the biomass. The bagasse was treated in a 1% NaOH aqueous solution at 120°C for 20 minutes and then washed to obtain alkali-treated bagasse (composition: glucan 62.7%, xylan 17.9%). The obtained alkali-treated bagasse was used as a substrate for the saccharification reaction.

[0093] (2) Glycation reaction Cellic® Ctec2 (Novozymes) was used as the cellulase agent. The saccharification reaction was carried out based on the method of Kawai et al. (Kawai et al., Journal of Industrial Microbiology and Biotechnology, 2012, 39(12):1741-1749). 3 mg / g of cellulase agent (dry weight) and 0.1 mg / g of purified enzyme F5054AGF (dry weight) were added to 5 w / v% (dry weight) substrate (in 100 mM Acetate buffer (pH 5.0)). A reaction solution containing only the cellulase agent as the enzyme was prepared as a control. The reaction solution was shaken at 150 rpm for 72 hours at 50°C to carry out the saccharification reaction. The reaction was stopped by boiling for 5 minutes, and the supernatant was collected after centrifugation (4°C, 13,500 rpm, 10 minutes). The sugars produced in the reaction solution were analyzed using the Somogyi-Nelson method and the HPAEC-PAD method described in Example 2(5). The amount of glucose, xylose, and arabinose produced when using only the cellulase agent was set to 100%, and the amount of sugars obtained when using F5054ABF in combination was calculated as a relative value.

[0094] The relative sugar production amounts are shown in Figures 5 and 6. When F5054ABF was used in combination with a cellulase agent, the total amount of reducing sugars produced from biomass increased by 20% compared to when the cellulase agent was used alone (Figure 5). Furthermore, when F5054ABF was used in combination with a cellulase agent, glucose production increased by 2%, xylose production by 12%, and arabinose production by 400% compared to when the cellulase agent was used alone (100%) (Figure 6). These results indicate that F5054ABF is an enzyme suitable for biomass saccharification, promoting cellulose degradation through hemicellulose degradation, and that its use in combination with cellulose enables efficient sugar production from biomass.

Claims

1. An enzyme composition for biomass saccharification, comprising cellulase and arabinofuranosidase, wherein the arabinofuranosidase consists of an amino acid sequence selected from (a) to (c) below: (a) Amino acid sequence of Sequence ID No. 1; (b) an amino acid sequence having at least 90% identity with the amino acid sequence of Sequence ID No. 1; (c) An amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 1 An enzyme composition wherein the biomass is alkali-treated bagasse.

2. The enzyme composition according to claim 1, wherein the content of the arabinofuranosidase is 0.1% by mass or more and 70% by mass or less of the total protein amount.

3. The enzyme composition according to claim 1 or 2, wherein the content of the cellulase is 10% by mass or more and 99% by mass or less of the total protein amount.

4. The enzyme composition according to any one of claims 1 to 3, wherein the protein ratio of arabinofuranosidase to cellulase (arabinofuranosidase / cellulase) is 0.001 or more and 7 or less.

5. The process involves saccharifying biomass with the enzyme composition described in any one of claims 1 to 4, The biomass in question is alkali-treated bagasse. A method for producing sugar from biomass.

6. The method according to claim 5, wherein the initial concentration of the biomass in the reaction solution for the saccharification treatment is 1 to 30% by mass.

7. The method according to claim 5 or 6, wherein the initial concentration of arabinofuranosidase contained in the enzyme composition in the reaction solution for the saccharification treatment is 0.0001 to 100% by mass relative to the mass of the biomass.

8. The method according to any one of claims 5 to 7, wherein the initial concentration of cellulase contained in the enzyme composition in the reaction solution for the saccharification treatment is 0.1 to 100% by mass relative to the mass of the biomass.

9. The method according to any one of claims 5 to 8, wherein the reaction solution for the saccharification treatment has a pH of 2.0 to 5.

5.

10. The method according to any one of claims 5 to 9, wherein the reaction temperature of the saccharification treatment is 40 to 60°C.

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