KR20140065118A - Noble xylanase, gene coding for the xylanase, and use thereof - Google Patents

Abstract

The present invention relates to xylanase represented by amino acid sequences of SEQ ID NO: 1, polynucleotide coding the xylanase, an expression vector comprising the polynucleotide, a transformant having the expression vector introduced therein, feed additives comprising the transformant, and a method for producing biofuel by using the xylanase or the transformant. A novel xylanase of the present invention has high activity in a near-neutral condition thereby having high value as yeast for feed additives and has xylosidase activity at the same time thereby being widely used for bioethanol production.

Description

[0001] The present invention relates to a novel xylanase, a gene coding therefor and a use thereof,

The present invention also provides a method for producing a recombinant vector comprising the steps of: preparing a recombinant vector comprising the xylanase comprising the amino acid sequence of SEQ ID NO: 1, the gene encoding the xylanase, the expression vector containing the xylanase, And a method of producing biofuel using the xylanase or the transformant.

Xylanase is one of the various enzymes produced by microorganisms and is currently highly available and is expected to be developed in various applications in the future. The most important possibility of using this enzyme is decomposing and saccharifying the vegetable fiber material, the most abundant and renewable biomass on the planet, to energize it. However, this is a field that can be put to practical use only in the future. Further, there is a practical application for the production and recycling of high-quality paper, the removal of turbidity of fruit drinks and beer, the extraction of coffee and vegetable oil and starch, These include industrial applications such as increased feed utilization of unit animals and xyloglucose production.

In recent years, interest in producing bio-energy from biomass resources including crops has been increasing in preparation for depletion of petroleum resources. It has been reported that non-oligosaccharides of woody and grain cell walls including xylan can be used as monosaccharides The usefulness of xylanase for decomposing into a low sugar has been increased. Xylen consists of 7-10% of soft-walled cell walls, 30-35% of hard-walled cell walls, and 30-35% of annual cell walls, with the greatest amount of biomass resources, It is therefore the main carbohydrate for feed and can also be used as a major carbon source for bioethanol production.

In order to use xylan as a carbon source for bioethanol production, it must be completely decomposed and saccharified. It is generally known that two kinds of enzymes, endo-β-xylanase and β-xylosidase, must act together to completely glycanize xylan, and they are referred to as xylanase enzymes do.

      On the other hand, the use of crops that can be used as feed grains in the production of bioenergy has led to a decline in the quality of livestock feeds or a rise in prices. The addition of xylanase to grain feeds hydrolyzes the hemicellulose layer surrounding the grains of the feed grains to increase the availability of nutrients in the grains, as well as decomposing non-aggregated polysaccharides present in the grain feed, Lowering the condition of intestinal parasites in the livestock can be improved. Especially, when wheat products with high hemicellulose content are used as feeds, it is easy for the livestock to cause Yanbian or Fertilizing phenomenon. Xylanase can prevent this phenomenon and not only increase the feed value, but also improve the quality deviation of the wheat product, It is possible to use the product as a stable feed material, so it is highly valuable as an enzyme for feed additive.

The microorganisms used for the production of xylanase for feed are fungi, and among the fungi, strains belonging to Trichoderma genus are mainly used. Their enzymatic productivity is generally superior to the enzyme productivity of xylanase-producing bacteria and exhibits maximum activity mainly in acidic conditions. However, pH in the digestive organs of pigs and chickens is around 6.5 after the small intestine that xylan or antibiotics should act on. Therefore, it is known that xylanase, which is active in the condition of acidity near neutrality rather than xylanase produced by Trichoderma sp. Strains and produces xylooligosaccharide, which is a growth factor of intestinal microorganism, as a reaction product, is an enzyme for feed addition High value.

Therefore, after cloning a gene encoding a novel xylanase, the present inventor has found that a novel xylanase is stable in an acidic state and has high activity in a neutral state, and thus has high utility as an enzyme for feed addition. In addition, The present inventors completed the present invention by confirming that they have the ability to decompose xylan effectively.

Korean Registered Patent No. 10-0729938 (Registered on June 14, 2007)

Li, R., R. Kibblewhite, W. L. Orts, and C. C. Lee, World J. Microbiol. Biotechnol. 25, 2071-2078, (2009)

It is an object of the present invention to provide a novel xylanase.

Another object of the present invention is to provide a gene encoding the xylanase.

It is still another object of the present invention to provide an expression vector containing the xylanase.

It is still another object of the present invention to provide a transformant into which the above expression vector has been introduced.

It is still another object of the present invention to provide a method of using the xylanase.

In one aspect of the present invention, the present invention provides a xyloranase comprising an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 1.

The term "xylanase" in the present invention refers to a xylan hydrolytic enzyme, and is one of various enzymes produced by microorganisms. An enzyme which hydrolyzes and cleaves xylan, the main component of hemicellulose, into xylose is commonly referred to as xylanase enzyme.

Preferably, the xylanase of the present invention may be derived from the Fanny Bacillus sp. Strain YB-45.

In one embodiment of the present invention, Fannibacillus sp. ≪ RTI ID = 0.0 > YB-45 < / RTI > (Lee, J.-C. and K.-H. Yoon, International Journal of Systematic and Evolutionary Microbiology , 612-616 (2008)) was prepared, and an E. coli transformant containing xylanase gene was selected, and the production, purification and xylanase activity of xylanase were examined, It was confirmed that a protein having an active activity was secreted.

The xylanases according to the present invention exhibit maximum activity at pH 5.5-6.5 and are not rendered non-volatile at pH 3.0-6.0.

In a specific example of the present invention, the xylanase activity of the present xylanase was measured by varying the reaction temperature and the reaction pH. As a result, as shown in FIG. 3, the highest enzyme activity was observed at 55-60 ° C and pH 6, and more than 90% activity was observed at pH 5.5-6.5. Especially, at pH 6.5, which is the physiological condition in the intestines of the animals, it was confirmed that the activity corresponding to more than 97% of the highest activity

In order to analyze the stability of xylanase according to pH, it is important to not inactivate in the process of passing through the stomach when using as a feed additive. The purified enzyme is left in different buffers in the range of pH 3.0-6.0 for 1 hour The residual activity of the enzyme was measured. As a result, as shown in Table 1 below, residual activity was 90% or more at pH 4.0-6.0 and remained about 73% at pH 3.0.

In another aspect, the present invention provides a polynucleotide encoding said xylanase.

The term "polynucleotide" in the present invention is a polymer of a nucleotide in which a nucleotide monomer is linked in a long chain by covalent bonds, and is a DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) Is a polynucleotide encoding a xylanase of the invention.

Preferably, the polynucleotide may comprise any one of the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence having at least 90% homology with the nucleotide sequence of SEQ ID NO: 2, and the nucleotide sequence or sequence of SEQ ID NO: Or a polynucleotide encoding a polynucleotide comprising a sequence complementary to any of the nucleotide sequences having 90% or more homology with the nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the present invention, plasmids were isolated from E. coli transformants having xylenolytic activity to determine the nucleotide sequence of the gene encoding xylanase. The recombinant plasmids contained a cloned DNA fragment of about 2.5 kb in size. To determine the nucleotide sequence of the cloned 2.5 DNA fragment, various restriction enzymes were used to fragment the fragment into small fragments , And inserted into pUC19. The nucleotide sequence of 2,430 bp was determined by dideoxynucleotide sequencing. It was confirmed that a total of 1,443 bp open reading frame corresponding to the xylanase structural gene was present in this nucleotide sequence (SEQ ID NO: 2). The amino acid sequence of the xylanase enzyme deduced from the nucleotide sequence of this structural gene is a protein consisting of a total of 481 amino acids as shown in SEQ ID NO: As a result of investigating the similarity between the deduced amino acid sequence of xylanase and the previously found amino acid sequence of xylanase, the highest degree of homology was 76%. As a result, It is a novel gene that shows a complete difference from what is reported.

In another embodiment, the present invention provides an expression vector comprising a polynucleotide encoding a xylanase of the present invention.

In the present invention, the term "vector" means a nucleic acid molecule capable of carrying other nucleic acid to which it is linked. One type of vector, "plasmid" refers to a circular double-stranded DNA loop in which additional DNA fragments can be ligated.

The vector of the present invention can direct expression of a gene encoding a target protein operatively linked, which is referred to as an "expression vector. &Quot; Generally, in the use of recombinant DNA technology, the expression vector is in the form of a plasmid. Depending on the host cell, the type of the expression vector that can be used can be determined. Therefore, the recombinant vector of the present invention can use a commercially available vector, but the present invention is not limited thereto, and a recombinant vector suitable for a person skilled in the art may be prepared and used.

According to the present invention, the gene inserted into the expression vector is a polynucleotide encoding the xylanase, preferably a nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having 90% or more homology with the nucleotide sequence of SEQ ID NO: 2 Or a polynucleotide comprising a sequence complementary to any one of the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of 90% or more homology with the nucleotide sequence of SEQ ID NO: 2, or a polynucleotide comprising a sequence complementary to any of the nucleotide sequences of SEQ ID NO: .

More preferably a polynucleotide encoding a xylanase represented by the amino acid sequence of SEQ ID NO: 1 derived from Fanny Bacillus Wort and Sis YB-45 strain.

In another embodiment, the present invention provides a transformant into which a recombinant vector of the present invention has been introduced into a host cell.

The term "transformed" in the present invention means that DNA is introduced into a host cell so that the DNA can be replicated as an extrachromosomal factor or by chromosome integration completion.

The host cell used for the transformation according to the present invention may be any host cell well known in the art. However, the present invention is not limited to the use of the polynucleotide encoding the xylanase of the present invention, Hosts with high expression efficiency can be used, for example, bacteria, fungi, yeast, plant or animal (e.g., mammalian or insect) cells.

In one embodiment of the present invention, E. coli is transformed using the recombinant plasmid pXY37, which is an expression vector containing the xyllanase gene according to the present invention, and then xylanase is produced and purified from the transformant, ≪ / RTI >

In another embodiment of the present invention, the present invention provides a feed additive comprising the xylanase. Also provided is a feed additive comprising xylanase, characterized in that the feed material is wheat.

In one embodiment of the present invention, hydrolytic relative activities of arabinogylan, oat xylan, and birch xylan were compared in order to measure the enzyme activity according to the type of xylan using purified xylanase. As shown in Table 4, it was found that the hydrolysis power of arabinozylan, which is a wheat-derived xylan used as a feed grain, was higher than that of oat xylan. These results indicate that the xylanase according to the present invention is suitable for the purpose of feed addition.

In another embodiment, a method for producing bioenergy using the xylanase or the transformant is provided. Specifically, the present invention provides a method for degrading xylan comprising the step of adding the xylanase or a transformant containing the xylanase gene.

In the above method, the addition amount of the xylanase or the transformant may be adjusted by a person skilled in the art.

In an embodiment of the present invention, in order to analyze xylan hydrolysates of zyla lyase produced by expression of an Escherichia coli transformant containing the zyla lyase gene of the present invention, it has been reported that oat xylan and birch xylan and xylobiose, The hydrolysis reaction was carried out under optimal reaction conditions using xylitriose, xylotetraose and xylopentose as substrates, and the final products were confirmed by thin layer chromatography (TLC). As a result, as shown in Fig. 4, xylose was produced along with xylobiose as a final product. Therefore, it can be seen that the xylanase according to the present invention decomposes the xylan substance and produces xylose, which is a monosaccharide, which is easily available to microorganisms, along with xylobiose having excellent function.

In addition, in order to investigate whether beta-xylosidase activity is present, the hydrolytic activity of para-nitrophenyl-beta-galactopyranoside was examined. As a result, the resolving ability of xylanase was confirmed, Lt; RTI ID = 0.0 > beta-xylocidase activity. ≪ / RTI >

This shows that the xylanase according to the present invention can be utilized for the production of bioethanol.

The novel xyllanase according to the present invention not only has an excellent effect on the hydrolysis of xylenes derived from wheat, oats and own products, but also has a beta-xylocidase activity, and its hydrolysis products are useful for the growth of useful intestinal bacteria It can be used extensively in the feed industry or the bio-energy industry because it produces xylobiose, which acts as a factor, and xylose, which can be used as a fermentation sugar.

1 is a restriction map of a recombinant plasmid pXY37 containing a gene coding for xylanase of Fanny Bacillus Wort and YS-45 strain.
Fig. 2 is an amino acid sequence at the amino acid terminal determined by purifying xylanase produced by an E. coli transformant containing the recombinant plasmid pXY37.
FIG. 3 is a graph showing the activity of xylanase produced and purified from an E. coli transformant containing the recombinant plasmid pXY37 according to reaction temperature and reaction pH.
4 is a thin layer chromatography photograph of a final degradation product decomposed from a sample by a xylanase produced and purified from an E. coli transformant containing the recombinant plasmid pXY37.
X1: Xyloose, X2: Xylobiose, X3: Xylotriose, X4: Gyrotetraose, X5: Xylopentose
1: sample of xylose, 2: sample before enzymatic hydrolysis of xylobeads, 3: reaction product after xylarin hydrolysis of xylobiose, 4: sample before enzymatic hydrolysis of xylotriose, 5 : Reaction product after xylanase hydrolysis of xylotriose, 6: sample before enzymatic hydrolysis of xylotetraose, 7: reaction product after xylanase hydrolysis of xylotetraose, 8: 10: xylose and sugar samples, 11: enzyme hydrolysis of oat xylan, and reaction product, 12: 1, The reaction product after enzymatic hydrolysis of birch xylan.

Hereinafter, the present invention will be described in detail with reference to examples. However, these embodiments are for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Cloning of xylanase gene of Fanny Bacillus Wort and Sis YB-45 strain

Step 1  : Fanny Bacillus Mail and cis YB -45 gene library production

The strain Fanny Bacillus Wortensis YB-45, which was found to be a novel strain having excellent xylan-resolving ability, was inoculated into 200 mL of Tryptic Soy Broth (TSB) liquid medium, shake cultured at 37 ° C to logarithmic growth phase, and the bacterial culture broth was recovered by centrifugation The cells were washed with TEN buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 10 mM NaCl) and then treated with SET buffer (20% sucrose, 50 mM Tris-HCl, pH 7.6, 50 mM EDTA) and allowed to react at 37 DEG C for 30 minutes. Then, 2 mL of 10% sodium dodecyl sulfate (SDS) was added to dissolve the cells, and an appropriate amount of protease K was treated and allowed to stand at 37 DEG C for 1 hour. Phenol was added and centrifuged. The supernatant was collected, and double volume of cold ethanol was added. The resulting cholesteric DNA was washed with 70%, 80%, and 90% ethanol sequentially with a glass rod. Solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

50 쨉 g of the separated chromosomal DNA was partially digested with restriction enzyme Sau3AI and then subjected to agarose gel electrophoresis to recover a DNA fragment of 1.5 to 8 kb in size. Equal amounts of the plasmid pUC19 digested with restriction enzyme BamHI and chromosomal DNA were mixed in the same amount, and T4 DNA ligase was added thereto, followed by reaction at 14 DEG C for 15 hours to effect a linkage reaction, thereby preparing a gene library of Fanny Bacillus Wort and YS-45.

Step 2  : Xylanase  Selection of Escherichia coli Transformants Having the Gene

Escherichia coli DH5α was transformed with the gene library of Fanny Bacillus Wurf & Sys YB-45 obtained in Step 1, and about 10,000 transformants were obtained by streaking on LB-xylan agar medium. An E. coli transformant containing a xylanase gene was obtained by selecting a transformant expressing a xylenolytic ring around the colonies of these transformed hosts.

Example  2: invention Xylanase To code  Determination of gene base sequence

The transformants obtained in step 2 of Example 1 were cultivated in LB liquid medium supplemented with ampicillin (50 mg / L) and cultured in the alkaline digestion method (Birnboim, HC and J. Doly, Nucleic Acid Res. -1523 (1979)). The plasmid was isolated and treated with various restriction enzymes. As a result, the recombinant plasmid contained a cloned DNA fragment of about 2.5 kb size, and this recombinant plasmid was named pXY37. The restriction map of pXY37 is shown in Fig. In Fig. 1, Xyn is a DNA fragment containing the xylanase gene of the present invention.

In order to determine the nucleotide sequence of the cloned 2.5 kb DNA fragment in plasmid pXY37, the fragment was divided into small pieces using various restriction enzymes and inserted into pUC19, followed by dideoxynucleotide sequencing to obtain a total of 2,430 bp The nucleotide sequence was determined. In this base sequence, a total of 1,443 bp open reading frame corresponding to the xylanase structural gene was present (SEQ ID NO: 2). The amino acid sequence of the xylanase enzyme deduced from the nucleotide sequence of such a structural gene is shown in SEQ ID NO: 1 and is a protein consisting of a total of 481 amino acids. The amino acid sequence of xylanase deduced from the nucleotide sequence of the Xyllanase gene of Fanny Bacillus Wort and Sis YB-45 revealed that the amino acid sequence of the xylanase of the Bacillus sp. Strain was high, Paenibacillus < / RTI > of Fanny Bacillus basinonensis, barrenonensis xylanase A) (GenBank accession number CBA13561) and a xylanase gene (Li, R (SEQ ID NO: 1)) encoded by a xylanase gene found in a gene library that has a homology of about 76% The amino acid sequence homology was found to be 69% with 14 amino acids in size, and the amino acid sequence homology with R. Kibblewhite, WL Orts, and CC Lee, World J. Microbiol. Biotechnol. 25, 2071-2078 (2009) appear.

Example  3: invention Xylanase  Sequence of amino terminal of protein

Expression of E. coli transformants containing the recombinant plasmid pXY37 prepared to examine which part of the amino acid sequence of the deduced xylanase protein functioned based on the xylanase gene sequence revealed in Example 2 above The amino acid sequence of the amino terminal of the xylanase produced was examined. For this purpose, the purified xylanase was subjected to sodium dodecyl sulfate gel electrophoresis, and the proteins present in the gel were transferred to a PVDF membrane, followed by automatic Edman degradation using a Procise 491 protein sequencer (Applied Biosystems, USA), and analyzed as shown in FIG. Compared with the amino acid sequence deduced from the xylanase gene, it corresponds to the 33-40th amino acid portion in the xylanase precursor protein, so that the 32 amino acid sequence of the precursor protein in front of this portion was confirmed as the lead peptide.

Example  4: The < RTI ID = 0.0 > transgenic < / RTI > Xylanase  Investigation of activity

Ampicillin (50 mg / L) was added to investigate the hydrolytic activity of xylanase produced by expressing E. coli transformants containing the recombinant plasmid pXY37 prepared in Example 3 according to reaction temperature and pH. One LB liquid medium was inoculated with pXY37 transformed E. coli transformants and cultured at 37 ° C. for 16 hours. The cultured cells were ultrasonically disrupted, and the lysate was subjected to ammonium sulfate fractionation, ion chromatography, and concentration The enzyme was purified and then the purified enzyme was reacted with oat xylan by varying the reaction temperature and pH to measure the xylanase activity. As shown in FIG. 3, the enzyme showed the highest activity at 55 to 60 ° C and pH 6.0, and showed an activity of more than 90% at pH 5.5 to 6.5. Especially, at pH 6.5, which is a physiological condition in the intestines of the animals, it showed activity equivalent to more than 97% of the highest activity. These results indicate that the homologous amino acid homology with the present xylanase is 69%, and similar fibrin-derived xylanase (Li, R., Kibblewhite, WL Orts, and CC Lee, World (J. Microbiol. Biotechnol. 25, 2071-2078, (2009)) and the reaction temperature and the reaction pH, and the xylanase of the present invention was found to have a favorable effect on neutral pH and excellent thermal stability .

In order to analyze the stability of xylanase according to pH, it is important to avoid inactivation in the process of passing through the stomach when used as a feed additive. The purified enzyme is left in different buffer solution of pH 3.0 to 6.0 for 1 hour The residual activity of the post-enzyme was measured. As shown in Table 1, the residual activity was 90% or more at pH 4.0 to 6.0, and about 73% at pH 3.0. In order to investigate this, it is necessary to leave the purified xylanase at a temperature ranging from 30 ° C. to 65 ° C. for 1 hour, Residual activity was measured. As a result of the test, as shown in Table 2, 95% or more was inactivated at 65 ° C, but the enzyme activity was not maintained at all at 60 ° C or lower. Therefore, the enzyme was allowed to stand at a temperature ranging from 40 ° C to 60 ° C for 6 hours, and the residual activity of the enzyme was measured. As a result of the test, as shown in Table 3, even after standing at 60 ° C for 6 hours, the activity remained at 100%, indicating that it was very stable at room temperature.

      In addition, hydrolytic relative activities of arabinoxylan, oat xylan, and birch xylan were compared in order to measure enzyme activity according to the type of xylan using purified xylanase. At this time, the reaction was carried out at pH 6.0 and 50 ° C. As a result of the experiment, it was confirmed that the hydrolysis power of arabinose-ylan, a xylen derived from wheat, which is frequently used as a feed material, is higher than that of oat xylan, as shown in Table 4.

These results indicate that the xylanase produced by the E. coli transformant containing the xylanase gene of the Fanny Bacillus strain YS-45 of the present invention is suitable for feed addition.

       On the other hand, in order to investigate the activity of beta-xylosidase that degrades xylose sugar from the non-reducing end of oligosaccharide having low polymerization degree of xylose, the resolution of para-nitrophenyl-beta-xylopyranoside This result indicates that xylanase of the present invention has an activity to degrade xylan to xylose unlike other xylanases and thus has a value as a xylanase in the production of bioenergy Could know.

The residual activity of xylanase produced by expression of the transformed Escherichia coli according to the present invention pH 3.0 4.0 5.0 6.0 Relative activity (%) of residual activity 73.2 93.3 100 100

The residual activity of the xylanase produced by the expression of the transformed Escherichia coli of the present invention after being allowed to stand at each incubation temperature for 1 hour Allowable temperature (℃) 40 50 60 65 Relative activity (%) of residual activity 100 100 100 5

The residual activity of the xylanase produced by the expression of the transformed Escherichia coli according to the present invention, Allowable temperature (℃) 40 50 60 Time left (hours) 2 4 6 2 4 6 2 4 6 Relative activity (%) of residual activity 100 100 100 100 100 100 100 100 100

Comparison of activity of xylanase produced by the expression of the transformed Escherichia coli according to the present invention Type of substrate Relative activity (%) Arabinozaylan 100 Birch tree 70 Oat xylan 73

Example  5: Production of the present invention by expression of the transformed Escherichia coli Xylanase Xyl Is a hydrolysis product

In order to analyze the final reaction product of the purified xylanase obtained in Example 3, the optimum reaction was performed using oat xylan, xylan xylan, xylotriose, xylotrothose and xylopentose as substrates Hydrolysis reaction was carried out under the conditions and the final hydrolysis product was subjected to thin layer chromatography (TLC). As a result, xylose and xylobiose were produced as major end products as shown in Fig. In particular, it is unique that this enzyme has the ability to decompose xylobiose, and thus, xylanase decomposes the xylenic substance to produce xylobiose having excellent functionality and also produces xylose which can be used as a fermentation sugar appear.

<110> Woosong University Corporation of Industrical Educational Programs <120> Noble xylanase, Gene coding for the xylanase, and use thereof <130> P12110271394 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 481 <212> PRT <213> Amino acid of novel xylanase <400> 1 Met Leu Lys Ser Lys Ser Lys Leu Ala Val Trp Leu Thr Thr Val Ala   1 5 10 15 Ile Met Ala Ser Leu Phe Ser Ser Ala Leu Val Lys Asp Ala His Ala              20 25 30 Gly Ile Ala Asn Gly Ser Lys Phe Leu Gly Asn Ile Ile Ala Asn Asn          35 40 45 Val Pro Ser Asn Phe Ala Thr Tyr Trp Asp Gln Val Thr Pro Glu Asn      50 55 60 Ser Thr Lys Trp Gly Ala Val Glu Ser Thr Arg Asn Ser Met Asn Trp  65 70 75 80 Ser Ala Ala Asp Thr Ala Tyr Asn Tyr Ala Lys Ser Arg Gly Met Pro                  85 90 95 Phe Lys Phe His Thr Leu Val Trp Gly Ser Gln Glu Pro Ser Trp Ile             100 105 110 Ser Gly Leu Ser Ala Ser Glu Gln Lys Ala Glu Val Thr Gln Trp Ile         115 120 125 Gln Ala Gly Gln Arg Tyr Gly Asn Ser Glu Phe Val Asp Val Val     130 135 140 Asn Glu Pro Leu His Ala Lys Pro Ser Tyr Arg Asn Ale I Gly Gly 145 150 155 160 Asp Gly Ala Thr Gly Trp Asp Trp Val Ile Trp Ser Phe Gln Gln Ala                 165 170 175 Arg Gln Ala Phe Pro Asn Ser Lys Leu Leu Ile Asn Asp Tyr Gly Ile             180 185 190 Ile Ser Asp Pro Asn Ala Ala Gln Tyr Val Gln Ile Ile Asn Leu         195 200 205 Leu Lys Ala Arg Gly Leu Val Asp Gly Ile Gly Ile Gln Cys His Tyr     210 215 220 Phe Asn Met Asp Asn Val Ser Ile Ser Thr Met Asn Ser Val Leu Asn 225 230 235 240 Thr Leu Ala Ala Thr Gly Leu Pro Ile Tyr Val Ser Glu Leu Asp Ile                 245 250 255 Thr Gly Asp Asp Asn Thr Gln Leu Gln Arg Tyr Gln Gln Lys Phe Pro             260 265 270 Val Leu Trp Glu His Pro Ser Val Lys Gly Ile Thr Leu Trp Gly Tyr         275 280 285 Ile Gln Gly Gln Thr Trp Ala Ser Asn Thr His Leu Val Thr Ser Ser     290 295 300 Gly Ala Glu Arg Pro Ala Met Gln Trp Leu Lys Gln Tyr Leu Gly Gly 305 310 315 320 Gly Gly Gly Gln Pro Gly Thr Gly Thr Gly Ser Gly Ala Tyr Ala Asn                 325 330 335 Phe Glu Ser Gly Thr Asp Gly Trp Ser Ala Ser Asn Val Val Ser Gly             340 345 350 Pro Ser Ser Ser Ala Asp Trp Ser Ser Lys Asp Thr Arg Ser Leu Lys         355 360 365 Ser Thr Ile Asn Met Ser Ser Gly Ser Gly His Tyr Met Phe Lys Ser     370 375 380 Gly Asn Ala Asp Phe Thr Gly Tyr Asn Gln Leu Arg Ala Thr Val Lys 385 390 395 400 Gly Ala Asn Ser Gly Asn Tyr Gly Ser Gly Leu Gly Val Lys Leu Tyr                 405 410 415 Val Lys Tyr Gly Asn Asn Tyr Ala Trp Ala Asp Ser Gly Trp Lys Thr             420 425 430 Ile Ser Ala Gly Gly Gln Thr Asp Leu Thr Leu Asn Leu Ser Gly Val         435 440 445 Asp Lys Ala Asn Ile Lys Glu Tyr Gly Val Gln Phe Ile Gly Ala Ser     450 455 460 Asn Ala Ser Gly Gln Thr Ser Val Tyr Val Asp Asn Val Tyr Leu Trp 465 470 475 480 Asn     <210> 2 <211> 1446 <212> DNA <213> novel xylanase <400> 2 atgttaaagt caaaatcgaa attggcggtt tggctgacta cagttgcaat tatggcttcc 60 ttgttttcct cagccttggt taaggacgct catgcgggga tagctaacgg cagcaagttt 120 ttgggcaaca tcattgcaaa caacgttcct tccaactttg ctacgtattg ggaccaggtt 180 acacctgaga attcaaccaa atggggtgcg gtggaatcga cccgcaacag tatgaactgg 240 agtgcggcgg atacagcgta caactatgcg aaaagccgag gaatgccttt caaattccat 300 accctggtct ggggcagcca agagccctcc tggattagcg gcctctccgc ctccgagcaa 360 aaggcagagg tgacccagtg gattcaagcc gccggccaga gatatggaaa ttcagaattt 420 gtcgatgtcg ttaatgaacc gctgcatgcg aagccctcat atagaaacgc catcggcggg 480 gatggagcta caggctggga ttgggttatc tggtcctttc agcaggcaag acaagccttt 540 cctaactcta aattgctgat caatgattac ggaattatca gcgaccccaa cgcagcggct 600 caatatgtac agatcatcaa tctgctcaag gccagagggc tggttgacgg cattgggatc 660 cgtgtcact attttaatat ggataatgta tcgataagta cgatgaacag cgttctgaat 720 acgctggcgg cgacaggact tccgatttat gtatccgagc tggatataac gggtgatgac 780 aacacccaat tgcagcggta ccaacagaag tttcctgtac tgtgggagca cccatccgtt 840 aaaggcatca ccttgtgggg atatattcaa ggacagacct gggcgtcgaa tacgcatctg 900 gtgaccagct ccggtgctga gcggccagcc atgcaatggc tgaagcagta tttaggcgga 960 ggaggaggcc agccaggaac aggcacgggt tcgggcgcct atgcaaactt tgaatcgggt 1020 acggatggat ggtccgccag caacgtcgta tccggaccat cttcctcagc cgattggagc 1080 tcgaaggata caagatcgct gaagtcgacg attaacatgt ccagcggcag cgggcattac 1140 atgttcaaga gcggaaacgc agatttcacc ggctacaacc agcttagagc aacagtaaag 1200 ggcgctaatt ctggtaatta cggctccgga ttaggcgtga agctgtatgt gaaatatggc 1260 aacaactatg cctgggcaga cagcggctgg aagacaatca gcgccggcgg ccaaaccgat 1320 ctgaccttga atttatccgg cgtagacaag gccaatatta aggaatatgg cgttcaattc 1380 atcggcgcaa gcaatgcgtc cggccaaacc tccgtatatg tggataacgt atatttgtgg 1440 aattaa 1446

Claims (11)

1 or an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 1. 2. The xylanase according to claim 1, wherein the xylanase exhibits maximum activity at pH 5.5-6.5. 2. The xylanase according to claim 1, wherein the xylanase contains beta-xylocidase activity. A polynucleotide encoding the xylanase of claim 1. 5. The polynucleotide encoding a polynucleotide according to claim 4, which comprises a nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having 90% or more homology with the nucleotide sequence of SEQ ID NO: 2. 5. The polynucleotide of claim 4, which comprises a nucleotide sequence of SEQ ID NO: 2 or a sequence complementary to any one of nucleotide sequences having 90% or more homology with the nucleotide sequence of SEQ ID NO: 2. A recombinant vector comprising the polynucleotide of any one of claims 4 to 6. A transformant into which the recombinant vector of claim 7 has been introduced. A feed additive comprising the xylanase of claim 1. The feed additive of claim 9, wherein the feed material is wheat. A method for producing bioenergy using the xylanase of claim 1 or the transformant of claim 8.

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