# Patent

## Title
Xylanase

## Abstract

The purpose of the present invention is to provide a novel xylanase having various excellent enzymological properties, and that is suitable for industrial applications. The polypeptide is selected from the group consisting of (a), (b), and (c) below: (a) a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 7; (b) a polypeptide having xylanase activity and comprising an amino acid sequence selected from SEQ ID NOs: 1 to 7, the amino acid sequence having substitution, deletion, insertion, and/or addition of one or several amino acid residues; and (c) a polypeptide having xylanase activity and comprising an amino acid sequence having at least 80% identity with an amino acid sequence selected from SEQ ID NOs. 1 to 7.

## Background
TECHNICAL FIELD
The present invention relates to xylanase, its gene, and the use thereof.
BACKGROUND ART
Xylan is one of the ubiquitous, naturally occurring polysaccharides, and is a major constituent of plants. From a structural viewpoint, xylan is a macromolecular polysaccharide containing a main chain made from units of xylose, which is polymerized by β-1,4 xyloside linkage. Xylan is present in nature not only as a homoxylan consisting of only xylose, but also as a heteroxylan such as arabinoxylan containing branched arabinose attached to the main chain.
Xylanase is a generic term for a group of enzymes that hydrolyze xylan, and that are widely found in animals, plants, and microorganisms. There has been research largely focusing on xylanase derived from bacteria, actinomycetes, yeast, fungi, and the like. Xylanase is used in production of xylooligosaccharides or xylose from xylan, as well as in biomass processing. In particular, xylanase has recently been brought to attention as being useful from the point of view of enzyme utilization in biomass processing. For example, xylanase is used in enzymatic degradation of agricultural waste for alcohol fuels, enzymatic treatment for liberating saccharides in animal feed, enzymatic treatment for dissolving pulp in the step of obtaining cellulose, and enzymatic treatment for bleaching wood pulp.
In the paper and pulp industry, recent years have seen increasing promise of using xylanase for improvement in whiteness and quality of pulp, reduction in the amount of chemical bleaching agents, such as chlorine bleaching agents, used in the pulp-bleaching step, and increase in pulp freeness in the paper-recycling step. Xylanase is also used in animal feed and in the food-processing field (Patent Document 1).
An object of the present invention is to provide a novel highly-active xylanase that is useful for applications, for example, in biomass decomposition, food processing, pulp bleaching, animal feed, and silage.
CITATION LIST
Patent Documents
Patent Document 1: JPH06-261750A
SUMMARY OF INVENTION
Technical Problem
In view of the state of the art, an object of the present invention is to provide a novel xylanase having various excellent enzymological properties, and that is suitable for industrial applications.
Solution to Problem
The present inventors conducted extensive research to achieve the above object, and succeeded in obtaining a novel polypeptide having sufficient xylanase activity even in a high-temperature environment, and a DNA that encodes the polypeptide. The present inventors further analyzed the 3D-structure of the enzyme, and identified the key amino acid that is present in the catalytic site. The present inventors also confirmed the presence of other gene clusters and gene fragments for encoding xylanase in the genomic DNA of an organism from which the polypeptide is derived, and found from this study that there are a plurality of novel xylanases. On the basis of these findings, the inventors conducted further research, and made improvements to complete the present invention.
The following is the major part of the present invention.
Item 1.
A polypeptide selected from the group consisting of (a), (b), and (c) below:
(a) a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 7;
(b) a polypeptide having xylanase activity and comprising an amino acid sequence selected from SEQ ID NOs: 1 to 7, the amino acid sequence having substitution, deletion, insertion, and/or addition of one or several amino acid residues; and
(c) a polypeptide having xylanase activity and comprising an amino acid sequence having at least 80% identity with an amino acid sequence selected from SEQ ID NOs. 1 to 7.
Item 2.
A polynucleotide selected from the group consisting of (a), (b), and (c) below:
(a) a polynucleotide comprising a base sequence selected from SEQ ID NOs: 8 to 21;
(b) a polynucleotide comprising a base sequence that encodes an amino acid sequence selected from SEQ ID NOs. 1 to 7; and
(c) a polynucleotide that hybridizes with a polynucleotide comprising a base sequence complementary to a base sequence selected from SEQ ID NOs. 8 to 21 under stringent conditions, and that encodes a polypeptide having xylanase activity.
Item 3.
An expression vector comprising the polynucleotide according to Item 2.
Item 4.
A transformant obtained by transformation with the expression vector according to Item 3.
Item 5.
A process for producing xylanase, the process comprising the step of culturing the transformant according to Item 4.
Item 6.
A process for producing xylose and/or xylobiose, the process comprising the step of allowing the polypeptide according to Item 1 to act on a sample containing xylan.
Advantageous Effects of Invention
The polypeptide according to the present invention, when allowed to act on a sample comprising xylan, can produce xyloses and/or xylooligosaccharides on an industrial scale because of its xylanase activity. In a preferred embodiment, the polypeptide according to the present invention is suitably used in an environment where the temperature changes or is at a relatively high temperature (e.g., 40 to 80° C.) because of its high specific activity and excellent thermal stability. Moreover, since the polynucleotide according to the present invention encodes the polypeptide according to the present invention, the polynucleotide, when expressed in an appropriate host, can efficiently produce a large amount of the polypeptide (i.e., xylanase).



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows the results of SDS-PAGE on the protein purified in Example 1.
FIG. 2 shows the xylanase activity of the purified protein measured in Example 2.
FIG. 3 shows the temperature dependence of xylanase measured in Example 3.
FIG. 4 shows a photographic image of xylanase crystallized by the hanging drop technique in Example 5.
FIG. 5 shows a 3D-structure of xylanase according to the present invention obtained by a structural analysis.
FIG. 6 shows the alignment of the amino acid sequences of seven xylanases encoded in respective genomic DNA. The amino acid sequence of XYLC is provided in SEQ ID NO:1. The amino acid sequence of XYLA is provided in SEQ ID NO:2. The amino acid sequence of XYLB is provided in SEQ ID NO:3. The amino acid sequence of XYLD is provided in SEQ ID NO:4. The amino acid sequence of XYLE is provided in SEQ ID NO:5. The amino acid sequence of XYLF is provided in SEQ ID NO:6. The amino acid sequence of XYLG is provided in SEQ ID NO:7.



DESCRIPTION OF EMBODIMENTS
1. Polypeptide Having Xylanase Activity
The polypeptide according to the present invention is (a) a polypeptide comprising (an amino acid sequence selected from SEQ ID NOs: 1 to 7; (b) a polypeptide having xylanase activity and comprising an amino acid sequence selected from SEQ ID NOs: 1 to 7, the amino acid sequence having substitution, deletion, insertion, and/or addition of one or several amino acid residues; or (c) a polypeptide having xylanase activity and comprising an amino acid sequence having at least 80% identity with an amino acid sequence selected from SEQ ID NOs. 1 to 7.
The amino acid sequence of SEQ ID NO: 1 has been confirmed to have xylanase activity in the Examples described below. The results of structural analysis suggest that the glutamic acids residue at positions 119 and 210 of the amino acid sequence of SEQ ID NO: 1 correspond to the catalytic site, and these amino acids are conserved in the amino acid sequences of SEQ ID NOs: 2 to 7. The amino acid sequences around the amino acids at positions 119 and 210 are also highly conserved in amino acid sequences of SEQ ID NOs: 1 to 7. Furthermore, because the amino acid sequence of SEQ ID NO: 1 has identity of 37%, 42%, 35%, 37%, 47%, and 41% to amino acid sequences of SEQ ID NOs: 1 to 7, respectively, a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to 7 is considered to have xylanase activity. The amino acid sequence of SEQ ID NO: 22 is an amino acid sequence of SEQ ID NO: 1 to which a signal peptide is added. Each of the amino acid sequences of SEQ ID NOs: 2 to 7 also has a signal peptide added to its N-terminus.
In the polypeptide (b) above, the term “several” is not particularly limited, insofar as the polypeptide has xylanase activity, or preferably has excellent temperature dependence. For example, the term “several” refers to 50, 45, 30, 25, 20, 15, 10, 5, 3, or 2. The presence or absence of xylanase activity can be confirmed by a known technique such as the Somogyi-Nelson method. Specific examples of polypeptide (b) include polypeptides comprising an amino acid sequence selected from SEQ ID NOs: 2 to 7, and 22.
When the mutation is the substitution of one or several amino acid residues, the type of substitution is preferably, but not particularly limited to, conservative amino acid substitution from the standpoint that the higher-order structure, phenotype, or properties of the polypeptide is not adversely affected in a significant manner. The term “conservative amino acid substitution” refers to substitution of an amino acid residue with another amino acid residue having a side chain of similar nature. Amino acid residues can be classified into families of similar nature according to their side chains, such as basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Thus, substitution of an amino acid residue is preferably made such that an original amino acid residue is substituted with an amino acid residue belonging to the same family as that of the original amino acid residue of the original amino acid sequence.
In the polypeptide (b), mutations such as substitution, deletion, insertion and/or addition, or the like of one or several amino acid residues are made preferably in the regions that are not significantly essential for the higher-order structure of the polypeptide, or in the regions that are not directly associated with its catalytic activity. Examples of such regions include the N-terminal domain, the C-terminal domain, and poorly conserved regions between SEQ ID NOs: 1 to 7. For example, the N-terminus and/or the C-terminus may have a signal sequence added thereto. Examples of signal sequences include, but are not particularly limited to, those consisting of 3 to 50, preferably 5 to 30 amino acids.
Examples of relatively poorly conserved regions include positions 2 to 29, positions 34 to 39, positions 41 to 44, positions 46 to 52, positions 58 to 60, positions 70 to 73, positions 75 to 86, positions 111 to 117, and positions 142 to 149 of SEQ ID NO: 1. It thus appears that substitution, deletion, insertion and/or addition of one or several amino acid residues can be suitably made in these regions. The glutamic acid residues at positions 119 and 210 of SEQ ID NO: 1 appear to be directly associated with the catalytic site for xylanase activity. From the standpoint of prevention of decrease in xylanase activity, it is preferable that 10 amino acid residues on each side of the glutamic acid residues at positions 119 and 210 of SEQ ID NO: 1 remain unmutated; preferably 5 amino acid residues, more preferably 3 amino acid residues, still more preferably 2 amino acid residues, and particularly more preferably 1 amino acid residue on each side of the glutamic acid residues remains unmutated. An amino acid sequence selected from SEQ ID NOs: 1 to 7 may have a deletion of the first methionine.
Substitution, deletion, insertion, or addition of one or several amino acid residues may be made in any of the amino acid sequences of SEQ ID NOs: 1 to 7, but is preferably made in the amino acid sequence of, for example, SEQ ID NO: 1. It is known in the art how to make a mutation such as substitution, deletion, insertion, or addition of one or several amino acid residues of a specific amino acid sequence such as those of SEQ ID NOs: 1 to 7, and any technique can be used. Such a mutation can be made by using, for example, the restriction enzyme treatment, the treatment using exonuclease, DNA ligase, etc., directed mutagenesis, or random mutagenesis.
In the polypeptide (C), the amino acid sequence identity thereof to an amino acid sequence selected from SEQ ID NOs: 1 to 7 is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, further more preferably 93% or more, still more preferably 95% or more, even still more preferably 98% or more, and even still further more preferably 99% or more. In one embodiment, the polypeptide (c) preferably has the above degrees of identity to the amino acid sequence of SEQ ID NO: 1.
The amino acid sequence identity can be determined by using a commercially available analytical tool or an analytical tool available through telecommunication lines (Internet). For example, the amino acid sequence identity can be determined by using ClustalW Ver. 2.1 Pairwise Alignment (see the website clustalw.ddbj.nig.ac.jp/index.php?lang=ja) with default parameters. Alternatively, the amino acid sequence identity can be determined by using the BLAST (Basic Local Alignment Search Tool) homology algorithm available from of the National Center for Biotechnology Information (NCBI) at the website www.ncbi.nlm.nih.gov/BLAST/ with default parameters (default setting).
The polypeptide according to the present invention has xylanase activity, preferably with an optimum activity temperature of about 70° C., and it is preferable that the polypeptide exhibits sufficient activity at a temperature of about 40 to 80° C. As used herein, the term “sufficient activity” refers to 60% or more activity, and preferably 70% or more activity, when the activity at an optimum activity temperature is taken as 100%. The polypeptide according to the present invention preferably has an optimum pH of about 5 to 6.5.
The polypeptide according to the present invention can be produced in accordance with a genetic engineering procedure by using the later-described polynucleotide according to the present invention. The polypeptide according to the present invention can also be produced on the basis of the information of the amino acid sequences of SEQ ID NOs: 1 to 7 by using an ordinary protein chemical synthesis technique (e.g., liquid-phase method, and solid-phase method).
2. Polynucleotide
The polynucleotide according to the present invention encodes the polypeptide having xylanase activity described in section 1 above. A typical polynucleotide according to the present invention has a base sequence selected from SEQ ID NOs: 8 to 21. A polynucleotide having a base sequence selected from SEQ ID NOs: 8 and 9 encodes a polypeptide having the amino acid sequence of SEQ ID NO: 1. A polynucleotide having a base sequence selected from SEQ ID NOs: 10 and 11 encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2. A polynucleotide having a base sequence selected from SEQ ID NOs: 12 and 13 encodes a polypeptide having the amino acid sequence of SEQ ID NO: 3. A polynucleotide having a base sequence selected from SEQ ID NOs: 14 and 15 encodes a polypeptide having the amino acid sequence of SEQ ID NO: 4. A polynucleotide having a base sequence selected from SEQ ID NOs: 16 and 17 encodes a polypeptide having the amino acid sequence of SEQ ID NO: 5. A polynucleotide having a base sequence selected from SEQ ID NOs: 18 and 19 encodes a polypeptide having the amino acid sequence of SEQ ID NO: 6. A polynucleotide having a base sequence selected from SEQ ID NOs: 20 and 21 encodes a polypeptide having the amino acid sequence of SEQ ID NO: 7. The base sequences of SEQ ID NOs: 8, 10, 12, 14, 16, 18, and 20 comprise no regions corresponding to introns. The base sequences of SEQ ID NOs: 9, 11, 13, 15, 17, 19, and 21 comprise regions corresponding to introns.
In the polynucleotide (c), the term “stringent conditions” refers to the conditions in which a “specific” hybrid is formed, and a “non-specific” hybrid is not formed. Specific examples of stringent conditions include: hybridization at 42° C., 1×SSC, and washing at 42° C. with a buffer fluid containing 0.1% SDS; preferably hybridization at 65° C., 0.1×SSC, and washing at 65° C. with a buffer fluid containing 0.1% SDS. Besides the temperature conditions, a variety of parameters that affect the stringency of hybridization are known, and one of ordinary skill in the art can suitably combine the parameters to enable stringency equivalent to the above-described stringency of hybridization.
DNAs hybridized under the above-described conditions include those having a stop codon generated in the middle and those that lost activity because of the mutagenesis of the active center. However, such DNAs can be avoided by using site-specific mutagenesis, or can be easily removed by measuring the enzyme activity with a known technique.
Polynucleotides hybridized under stringent conditions typically have homology of above a certain level to a polynucleotide used as a probe. The homology is, for example, 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even still more preferably 98% or more, and particularly more preferably 99% or more. To the base sequence of SEQ ID NO: 8, the base sequences of SEQ ID NOs: 9 to 21 have homology of 99% (SEQ ID NO: 9), 58% (SEQ ID NO: 10), 60% (SEQ ID NO: 11), 62% (SEQ ID NO: 12), 63% (SEQ ID NO: 13), 57% (SEQ ID NO: 14), 62% (SEQ ID NO: 15), 63% (SEQ ID NO: 16), 62% (SEQ ID NO: 17), 64% (SEQ ID NO: 18), 67% (SEQ ID NO: 19), 59% (SEQ ID NO: 20), and 61% (SEQ ID NO: 21). These percentages have been determined by using ClustalW (see the website clustalw.ddbj.nig.ac.jp/index.php?lang=ja) with default parameters.
The base sequence homology can be determined by using a commercially available analytical tool, or an analytical tool available through telecommunication lines (Internet). For example, software such as FASTA, BLAST, PSI-BLAST, or SSEARCH can be used to determine the homology. The major initial conditions typically applied to a BLAST search are specifically as follows. In Advanced BLAST 2.1, a blastn program is used, and the parameters are set to default values to perform a search. The homology value (%) of a nucleotide sequence is then calculated.
The polynucleotide according to the present invention is preferably present in an isolated state. As used herein, “DNA in an isolated state” means that the DNA is separated from components such as other nucleic acids and proteins that naturally accompany it. However, the DNA may contain a portion of other nucleic acid components, such as nucleic acid sequences that naturally flank the DNA sequence (e.g., the promoter region sequence and terminator sequence). DNAs prepared by a genetic engineering procedure, such as cDNA molecules, are, when in an isolated state, preferably substantially free of other components such as cell components and culture media. Likewise, in DNAs prepared by a chemical synthesis, “DNA in an isolated state” means that the DNA is substantially free of precursors (starting materials) such as dNTP, as well as chemical substances, etc., used in the synthetic process.
The polynucleotide according to the present invention can easily be prepared on the basis of the base sequence information of SEQ ID NOs: 8 to 21 by using a chemical DNA synthesis technique (e.g., phosphoramidite method) or a genetic engineering technique.
3. Expression Vector
The recombinant vector according to the present invention comprises the polynucleotide according to the present invention. The expression vector according to the present invention may further comprise, in addition to the polynucleotide of the present invention, polynucleotides that encode other proteins without particular limitation, as long as the expression of the polynucleotide of the present invention is possible.
The type of the vector is suitably selected according to the type of the host cell. Examples of vectors include plasmid vectors, cosmid vectors, phage vectors, and virus vectors (e.g., adenovirus vectors, retroviral vectors, and herpes viral vectors).
Examples of vectors that enable expression in Escherichia coli include pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218, and pET. Examples of vectors that enable expression in yeast include pBR322, pJDB207, pSH15, pSH19, pYepSec1, pMFa, and pYES2. Examples of vectors that enable expression in insects include pAc and pVL.
For a eukaryotic host cell, suitably used expression vectors are those comprising, upstream of the polynucleotide to be expressed, a promoter, an RNA splicing site, a polyadenylation site, a transcription termination sequence, and the like. The expression vectors may further optionally comprise a replication origin, an enhancer, and/or a selection marker.
4. Transformant
The transformant according to the present invention is obtained by transformation with the above-described recombinant vector. The host cell used in transformation is not particularly limited as long as the polypeptide of the present invention can be produced, and either prokaryotic cells or eukaryotic cells can be used. Specific examples of host cells include prokaryotic cells including bacteria of genus Escherichia coli such as Escherichia coli (e.g., HB101, MC1061, JM109, CJ236, and MV1184), coryneform bacteria such as Corynebacterium glutamicum, actinomycetes such as bacteria of genus Streptomyces, bacteria of genus Bacillus such as Bacillus subtilis, bacteria of genus Streptococcus, and bacteria of genus Staphylococcus; yeast such as genus Saccharomyces, genus Pichia, and genus Kluyveromyces, and fungal cells such as genus Aspergillus, genus Penicillium, genus Trichoderma, and genus Acremonium; insect cells including Drosophila S2, Spodoptera Sf9, and silkworm-culturing cells; and plant cells. It is also possible to produce the polypeptide in a medium by exploiting the protein secretory capacity of Bacillus subtilis, yeast, Aspergillus oryzae, actinomycetes, and the like.
To introduce the recombinant expression vector into a host cell, a conventional method can be used. Examples include a variety of methods such as the competent cell method, the protoplast method, the electroporation method, the microinjection method, and the liposome fusion method. Specific examples of methods for introducing the recombinant expression vector into coryneform bacteria include, but are not limited to, the protoplast method (Gene, 39, 281-286, 1985) and the electroporation method (Bio/Technology, 7, 1067-1070, 1989).
The transformant according to the present invention is capable of producing the polypeptide of the present invention, and thus can be used for producing the polypeptide of the present invention. The transformant itself can also be used for producing xylose and/or xylooligosaccharides from a sample containing xylan.
5. Process for Producing Polypeptide Using Transformant
The polypeptide according to the present invention can be produced by culturing the transformant of the present invention, and collecting the polypeptide having xylanase activity from the culture. For culture, a passage culture or batch culture can be used with a medium suitable for the host cell. The culture can be carried out until a sufficient amount of the polypeptide is produced, with monitoring of the activity of the polypeptide produced inside and outside of the transformant as a guide.
The culture medium may be suitably selected from conventionally used media according to the host cell. The culture can be carried out under conditions suitable for growth of the host cell. Examples of media used for culturing Escherichia coli include nutrient media such as LB medium, and minimal media to which a carbon source, a nitrogen source, a vitamin source, and the like are added, such as M9 medium.
The culture conditions can also be suitably determined according to the type of the host cell. The culture is typically carried out at 16 to 42° C., preferably 25 to 37° C., for 5 to 168 hours, preferably for 8 to 72 hours. Depending on the host, either shaking culture or static culture can be used. Agitation may optionally be applied, and ventilation may optionally be provided. When an induction promoter is used for expressing xylanase, a promoter-inducing agent may be added to the medium to perform a culture.
Purification or isolation of xylanase from a culture supernatant can be carried out by suitably combining known techniques. Examples of techniques for use include ammonium sulfate precipitation, solvent precipitation (e.g., ethanol), dialysis, ultrafiltration, acid extraction, and a variety of chromatographic approaches (e.g., gel filtration chromatography, anion- or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography, and high-performance liquid chromatography). Examples of carriers used in affinity chromatography include carriers to which an antibody against xylanase is bound, and carriers to which a substance with affinity for a peptide tag is bound, when the peptide tag is added to xylanase.
When xylanase is accumulated inside the host cells, the transformed cells are disrupted, and xylanase is purified or isolated from the centrifuged supernatant of the disrupted product by the techniques described above. For example, after completion of culture, the cells collected by centrifugation are suspended in a buffer for cell disruption (20 to 100 mM Tris-HCl (pH 8.0), 5 mM EDTA), and disrupted by ultrasonication. The disruption-treated fluid is centrifuged at 10,000 to 15,000 rpm for 10 to 15 minutes to thereby obtain a supernatant. The precipitation after centrifugation can optionally be solubilized with guanidinium chloride, urea, or the like, and then further purified.
The xylanase activity of the obtained polypeptide can be measured by a saccharification test using a xylan-containing biomass (e.g., bagasse) as a substrate. For example, the activity of the purified enzyme can be measured by the method disclosed in Bailey et al. (J. Biotech. 23, 1992, pp. 257-270).
6. Process for Producing Xylose/Xylooligosaccharides Using Polypeptide of the Present Invention
The polypeptide having xylanase activity according to the present invention, when contacted with a sample containing xylan (e.g., biomass resources), decomposes the biomass resource to thereby produce molasses containing xylose and/or oligoxylose. When a biomass resource is used as a xylan-containing sample, molasses can be produced more efficiently by using the polypeptide of the present invention in combination with other enzymes such as cellulase.
The type of xylan-containing sample is not particularly limited, as long as the sample is decomposable by the polypeptide of the present invention. Examples of xylan-containing samples include bagasse, wood, bran, wheat straw, rice straw, chaff, soybean meal, soy pulp, coffee grounds, and rice bran. In the present invention, bagasse is preferable for use.
To produce molasses containing xylose and/or oligoxylose from a xylan-containing sample, a known method can be used. Biomass resources for use may be either dried materials or wet materials. The materials are preferably milled into particles of 100 to 1,000 μm in size beforehand to increase processing efficiency. Milling is performed by using a device such as a ball mill, a vibrational mill, a cutter mill, or a hammer mill. The milled biomass resource is suspended in an aqueous vehicle, and the xylanase of the present invention and cellulase are added thereto, followed by heating with stirring to thereby decompose or saccharize the biomass resource. The xylanase may be used singly, but is preferably added together with other enzymes such as cellulase to a sample from the standpoint of efficient decomposition of the biomass.
When the polypeptide of the present invention is contacted with a xylan-containing sample in an aqueous solution, the pH and the temperature of the reaction liquid can be suitably determined as long as the xylanase is not deactivated. Because of the relatively high optimum temperature and excellent temperature stability of the xylanase according to the present invention, it is preferable to carry out the reaction at or around the optimum temperature from the standpoint of efficient decomposition of the sample to obtain molasses. For example, the process according to the present invention can be carried out under ordinary pressure, a pH of 4 to 9, and a temperature of 5 to 90° C., preferably 15 to 80° C., more preferably 30 to 75° C., 50 to 70° C., or 50 to 65° C. There is no particular limitation to the amount of the polypeptide of the present invention to be added, and the amount can be within the range of, for example, 0.1 to 0.5% (w/w).
The molasses containing xylose and/or oligoxylose obtained by the process of the present invention may be used unmodified, or may be used as a dry product after removing water. It is also possible to further isomerize or decompose the molasses by a chemical reaction or enzymatic reaction depending on the intended use. The molasses or its fraction can be used, for example, as a starting material for alcohols such as methanol, ethanol, propanol, isopropanol, butanol, and butanediol by a fermentation process.
EXAMPLES
Example 1: Preparation of Xylanase
A polynucleotide comprising a base sequence of SEQ ID NO: 8 was introduced into an expression plasmid vector, pET11a, through a ligation reaction. A strain of Escherichia coli, BL21(DE3), was transformed by using this recombinant expression vector. The transformant was then inoculated into an LB medium containing 0.1 mg/mL of ampicillin, and grown in shake cultures at 37° C. until OD 0.6. After that, 0.1 mM of IPTG was added thereto, and the culture medium was shaken at 30° C. overnight, thereby inducing the expression of a protein. Upon completion of the culture, the cells were collected by centrifugation, and frozen at −20° C. The cells were then suspended and dissolved in 50 mL of 20 mM Tris-HCl at pH 8.0, followed by collection of a supernatant solution by centrifugation. The obtained supernatant was fractioned by anion chromatography (HiTrap Q HP column: GE Healthcare) eluting with a gradient of 0.0-1.0 M NaCl to thereby purify the polypeptide (XYLC-R1). The anion chromatography was carried out at a flow rate of 3 ml/min. The purified fraction was examined for the molecular weight and homogeneity by using SDS-PAGE (FIG. 1).
Example 2: Measurement of Activity of Purified Protein
0.5 μM of the purified protein (XYLC-R1) and 0.5% by mass of xylan (final concentration each) were added to 50 mM of a sodium acetate buffer (pH 5.5), and an enzymatic reaction was started at 50° C. After the reaction was started, the reducing sugar concentration was measured at the 0-, 10-, 20-, 30-, and 60-minute point by the Somogyi-Nelson method (FIG. 2). As shown in FIG. 2, the results of the measurement revealed that a sufficient amount of reducing sugar was liberated, and that the purified protein was xylanase.
Example 3: Temperature Dependence of Enzyme Activity
To examine the effects of the reaction temperature on the enzyme activity, the enzyme activity was measured in the same manner as in Example 2 by increasing the reaction temperature from 30° C. to 40° C., 50° C., 60° C., 70° C., and 80° C. As a comparable example, the activity of Trichoderma-derived xylanase of the same concentration was measured at a temperature in the range of 20 to 50° C. in the same manner. FIG. 3 shows the results (the symbols “▪” and “♦” respectively indicate the xylanase purified in Example 1, and Trichoderma-derived xylanase). FIG. 3 reveals that the xylanase of the present invention has outstandingly higher activity than Trichoderma-derived xylanase in the temperature range in which the measurement was carried out.
Example 4: PH Dependence of Enzyme Activity
The effects of pH on xylanase activity of the enzyme that was confirmed to be xylanase in Example 2 was examined by using buffers at a pH of 3 to 10, and the optimum pH was found to be 5 to 6.5.
Examples 5: Crystallization of Enzyme Protein
The protein purified in Example 1 was crystallized by using the hanging drop technique under the following conditions: enzyme concentration (10 mg/mL); crystal mother liquor (0.8 M NaH2PO4, 0.8 M KH2PO4, 0.1 M BisTris pH 5.5); crystallization temperature (25° C.); and crystallization period (about 3 days). FIG. 4 shows a photographic image of the obtained crystal. From the absence of the electron density of about 10 to 13 amino acid residues residing in the N-terminal domain, it is inferred that these amino acid residues (about 10 to 13 amino acid residues residing in the N-terminal domain) are not essential for the structure and function of the enzyme.
Example 6: Crystal Structure Analysis of Enzyme Protein
At SPring8, collection and integration of the reflection points of an X-ray reflected by the enzyme crystal was implemented by using HKL2000, and then phasing was performed by molecular replacement using scaled data with a homologous protein as a model. After refinement, the structure of the xylanase was determined. The results of the structural analysis revealed the helix and β sheet structures. The results also identified the glutamic acid residues at positions 119 and 210 as the amino acid residues present in the catalytic site (FIG. 5).
Example 7: Identification of Other Xylanases
Based on the DNA sequence that encodes the protein (XYLC-R1) determined as xylanase in the above-described Example, homology searching was carried out to make a comparison with the genomic DNA of the organism from which the DNA of the protein is obtained. The homology search was carried out using the commercially available software in silico Molecular Cloning. A multiple alignment analysis was performed by using ClustalW Ver. 2 Multiple Alignment with default parameters. The results revealed that there are six more amino sequences that are speculated to be xylanase. FIG. 6 shows the alignment of these amino acid sequences. In FIG. 6, the symbol “*” denotes completely conserved amino acids; the symbol “•” denotes moderately conserved amino acids; and the symbol “••” denotes highly conserved amino acids. The amino acid sequence of XYLA is indicated by SEQ ID NO: 2; the amino acid sequence of XYLB is indicated by SEQ ID NO: 3; the amino acid sequence of XYLD is indicated by SEQ ID NO: 4; the amino acid sequence of XYLE is indicated by SEQ ID NO: 5; the amino acid sequence of XYLF is indicated by SEQ ID NO: 6 and the amino acid sequence of XYLG is indicated by SEQ ID NO: 7.
Examples 8: Confirmation of Xylanase Activity
As shown in the table below, primers for obtaining, from the genomic DNA, base sequences that encode the proteins speculated to constitute xylanases (XYLA, XYLB, XYLD, XYLE, XYLF, and XYLG) in Example 7 were designed.











TABLE 1









SEQ



Enzyme
Primer
Base Sequence
ID NO:








XYLA
Forward
ATTGTTAACAAGATGAAGA
23





TCACATCAGTGTTCG



 




Reverse
AATCCTGCAGGTTAAGATA
24





CAGTAACAGTGGCACTTC



 



XYLB
Forward
ATTGTTAACATCATGGGCA
25





TCTCATCTATTCTTC



 




Reverse
AATCCTGCAGGCTATTGGC
26





ACTGACTGTAGTAAGCGT



 



XYLC
Forward
ATTGTTAACAAGATGAAGC
27





TCTCTCTGGCTGCAA



 




Reverse
AATCCTGCAGGCTAGGACA
28





CGGTGATGGTACTAGAAC



 



XYLD
Forward
ATTGTTAACAAGATGCGGT
29





CATTTGCTCGCCTTGTC



 




Reverse
AATCCTGCAGGTCAGCTAA
30





CAGTAAAATCCAGGTAAC



 



XYLE
Forward
ATTGTTAACAAGATGATTT
31





ATTTCCCTCAGCTCATG



 




Reverse
AATCCTGCAGGCTATTGAG
32





TGGCAGTCTGCTGGGCA



 



XYLF
Forward
ATTGTTAACAAGATGTTCT
33





CTTTCAGTACTGCCTT



 




Reverse
AATCCTGCAGGCTACAAGC
34





ATTGATAGTAGTACGGGT



 



XYLG
Forward
ATTGATATCAAGATGGTTG
35





CTTTCTCGAGCTTATTTAC



 




Reverse
AATCCTGCAGGGTCCAACA
36





TCAATGCTACTTACAGC









These primers comprise a restriction enzyme site, HpaI (GTTAAC), or SbfI (CCTGCAGG), in a 5′ end region. In accordance with an ordinary technique, polynucleotide fragments to encode XYLA, XYLB, XYLC, XYLD, XYLE, XYLF, and XYLG were amplified using the genomic DNA as a template with these primer sets. Each sequence was cleaved with HpaI or SbfI, and introduced into a plasmid having a restriction enzyme site (EcoRV-SbfI). Accordingly, seven recombinant plasmids for expressing the proteins were obtained.
The recombinant plasmids were individually incorporated non-homologously into chromosomes of a uracil-requiring strain of a microorganism by using the protoplast-PEG technique in accordance with a known method, thereby giving seven types of transformants. Each of the transformants was cultured in a medium containing 2% starch as a carbon source, thereby giving a culture fluid. The xylanase activity was observed in the same manner as in Example 2. Table 2 below shows the results.










TABLE 2







Enzyme
Xylanase Activity In a Culture Fluid (U/mL)



















XYLA
98.12



XYLB
57.59



XYLC
1186



XYLD
—



XYLE
143.7



XYLF
103.67



XYLG
27.3










As seen in the results above, all of the proteins except for XYLD showed xylanase activity. Each of the recombinantly expressed proteins was homogeneously purified by column chromatography, and the N-terminal sequence of each protein was determined. This revealed that the recombinantly expressed proteins were the desired recombinase. Table 3 below shows the N-terminal sequence of each of the confirmed proteins.









TABLE 3






Enzyme
N-terminal Sequence








XYLA
AGGIN (SEQ ID NO: 37)


 



XYLB
AEAIN (SEQ ID NO: 38)


 



XYLC
QSITT (SEQ ID NO: 39)


 



XYLE
ATNYI (SEQ ID NO: 40)


 



XYLF
NTPNS (SEQ ID NO: 41)


 



XYLG
SAINY (SEQ ID NO: 42)

## Claims

1. A polypeptide having xylanase activity and comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence of SEQ ID NO:1, wherein the polypeptide does not have the amino acid sequence of SEQ ID NO: 1.

2. A polynucleotide selected from the group consisting of
(a) a polynucleotide comprising a base sequence of SEQ ID NO:8; and
(b) a polynucleotide having at lease 95% sequence identity with the base sequence of SEQ ID No:8 and that encodes a polypeptide having xylanase activity.

3. An expression vector comprising the polynucleotide according to claim 2.

4. A transformant obtained by transformation with the expression vector according to claim 3.

5. A process for producing xylanase, the process comprising the step of culturing the transformant according to claim 4.

6. A process for producing xylose and/or xylobiose, comprising:
(a) preparing an isolated polypeptide having an amino acid sequence with at least 99% sequence identity with the amino acid sequence of SEQ ID NO:1 and that exhibits xylanase activity, and
(b) allowing the polypeptide of (a) to act on a sample containing xylan.