KR101455720B1 - KRICT PXD2 Enzyme Having Multifunctional β-D-xylosidase Activity - Google Patents

Abstract

The present invention provides a xyloidase having an amino acid sequence set forth in Sequence Listing 2. More specifically, the xylosidase of the present invention has a multienzyme function. By replacing the existing chemical method, it is possible to reduce waste and expensive refining costs, and because it exhibits excellent xylan decomposition activity, it is utilized in the saccharification process of fibrous biomass as well as in the feed industry, paper and detergent industry, and is an alternative to petroleum replacement raw materials. It can be usefully used to produce raw materials such as functional materials and bio polymers.

Description

New Xylosidase with Multi-Enzyme Function ＩＲＩＣＴ ＰＸＤ２{KRICT PXD2 Enzyme Having Multifunctional β-D-xylosidase Activity}

The present invention relates to a novel xylosidase KRICT PXD2 having a multienzyme function.

Humanity living in the 21st century has eight fields of renewable energy (solar heat, solar power, biomass, wind power, small hydro power, geothermal energy, marine energy, and waste energy) and three fields to solve the big task of developing fossil fuel alternative resources. Was designated as new energy (fuel cell, coal liquefied gasification and hydrogen energy). Of these, biomass is the only carbon resource, so biorefinery processes that produce biofuels and chemical raw materials using biomass raw materials must be developed.

Although xylan, which exists in the natural world, is a very important carbon source, which accounts for 10-30% of the dry weight of biomass, it has not been directly available to date, so most are treated as waste resources. The hydrolysis of xylan has been mainly carried out by chemical methods to date. The method of decomposing xylan by chemical method adds sulfuric acid to fibrin-based biomass and decomposes by pressing with steam at 130℃, which consumes a large amount of energy and requires expensive production equipment capable of withstanding acid and high temperature. Do. In addition, the over-reaction products and wastes generated at this time cause environmental pollution or increase the overall production cost due to separation and purification costs. On the other hand, endoxylanase and beta-xylosidase are enzyme systems that break down xylan. Xyloligosaccharides are attacked at the ends to produce xylose. The method of decomposing xylan by the biological method is very economically economical because it consumes less energy and generates a small amount of waste compared to chemical decomposition method and is easy to process. However, the problem of the xylanase enzyme system is that the enzyme titer of endozylanase and xylosidase is low, so that the reaction rate and decomposition rate are low, and the properties of the known enzymes are sufficient for heat resistance and alkali resistance suitable for industrial use. They do not have it, and in Korea, these enzymes depend entirely on imports. Therefore, it is necessary to solve these problems in order to use the xylanase system.

In order to solve this problem, basically, the development of a domestic enzyme having alkali resistance and heat resistance should be preceded first, and a mass production system should be equipped for economical xylosidase production.

Throughout this specification, a number of papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated by reference into this specification as a whole, and the level of the technical field to which the present invention pertains and the content of the present invention are more clearly described.

The present inventors tried to develop a method for decomposing xylan to industrially use xylan, which accounts for 10-30% of the dry weight of biomass present in nature. As a result, the present invention was completed by developing a novel xyllosidase having heat resistance and alkali resistance, which can replace the existing chemical method that consumes a lot of energy.

Accordingly, it is an object of the present invention to provide xyloxidase.

Another object of the present invention is to provide a nucleic acid molecule encoding xylosidase.

Another object of the present invention is to provide a recombinant vector.

Another object of the present invention is to provide transformed cells.

Another object of the present invention is to provide a method for decomposing oligosaccharides or polysaccharides.

Another object of the present invention is to provide a composition for processing xylan in food.

Another object of the present invention is to provide a composition for feed additives.

Another object of the present invention is to provide a composition for a paper-making process.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a xyloidase having an amino acid sequence set forth in Sequence Listing 2.

The present inventors tried to develop a method for decomposing xylan to industrially use xylan, which accounts for 10-30% of the dry weight of biomass present in nature. As a result, a novel xylosidase has been developed that can replace the existing chemical method that consumes a lot of energy.

The xyloxidase of the present invention is interpreted to also include an amino acid sequence showing substantial identity to the amino acid sequence described above. The above substantial identity is at least 80% when the amino acid sequence of the present invention is aligned with any other sequence as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Means an amino acid sequence that exhibits homology, more preferably at least 90% homology, and most preferably at least 95% homology.

According to a preferred embodiment of the present invention, the xyloxidase of the present invention exhibits maximum enzymatic activity at 35-45°C, more preferably the optimum temperature is 37-43°C, even more preferably 38-42°C to be.

According to a preferred embodiment of the present invention, the xylosidase of the present invention exhibits maximum enzymatic activity at pH 3.5-10.5, more preferably optimal pH 4.0-9.0, even more preferably pH 4.0-7.0, more Even more preferably, it is pH 4.0-5.0.

According to a preferred embodiment of the invention, the xylosidase of the invention has a value of 3-5 for the Michaelis-Menten constant ( Km ), more preferably a value of 3.8-4.3.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding a xylosidase having the amino acid sequence set forth in Sequence Listing 2.

As used herein, the term “nucleic acid molecule” has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, are modified with natural nucleotides as well as sugar or base sites. Also includes analogues (Scheit, Nucleotide) Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

Some mutations in nucleotides do not change in proteins. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (e.g., by degeneracy of codons, there are six codons for arginine or serine), or codons that encode biologically equivalent amino acids It contains a nucleic acid molecule.

In addition, mutations in the nucleotides can also lead to changes in the xylosidase itself. Even in the case of a variation that causes a change in the amino acid of the xylosidase, one exhibiting almost the same activity as the xylosidase of the present invention can be obtained.

It is clear to those skilled in the art that biological functional equivalents that may be included in the xyloxidase of the present invention will be limited to variations in the amino acid sequence that exerts equivalent biological activity with the xyloxidase of the present invention.

These amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically equivalent.

In introducing mutations, the hydrophobic index of amino acids (hydropathic idex) can be considered. Each amino acid is assigned a hydrophobicity index according to hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine/cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in conferring the protein's interactive biological function. It is well known that amino acids with similar hydrophobicity indexes must be replaced to retain similar biological activity. When introducing a variation with reference to the hydrophobic index, substitution is performed between amino acids showing hydrophobic index difference, preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+3.0±1); Glutamate (+3.0±1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5±1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When introducing a variation with reference to a hydrophilicity value, substitution is performed between amino acids showing a difference in hydrophilicity values of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

Amino acid exchange in proteins that do not alter the overall activity of the molecule is known in the art (H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/ Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

In view of the above-described variations with biologically equivalent activity, the xyloxidase of the present invention or a nucleic acid molecule encoding the same is interpreted to also include a sequence showing substantial identity with the sequence described in the sequence listing. The substantial identity above aligns the above-described sequence of the present invention with any other sequence as much as possible, and when the aligned sequence is analyzed using an algorithm commonly used in the art, for example, at least 98%. It means a sequence showing homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment can be found in Smith and Waterman, Adv . Appl . Math . 2:482 (1981) ; Needleman and Wunsch, J. Mol . Bio . 48:443 (1970); Pearson and Lipman, Methods in Mol . Biol . 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc . Acids Res. 16:10881-90 (1988); Huang et al., Comp . Appl . BioSci . 8:155-65 (1992) and Pearson et al., Meth. Mol . Biol . 24:307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol . Biol . 215:403-10 (1990)) can be accessed from the National Center for Biological Information (NCBI), etc., and blastp, blastm, It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. The BLSAT can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/. The sequence homology comparison method using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Preferably, the nucleic acid molecule is a nucleic acid molecule characterized by having the nucleotide sequence described in Sequence 1 of the Sequence Listing.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this can be found in Sambrook et al., Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

The vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using prokaryotic cells as hosts. The vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression.

For example, when the vector of the present invention is an expression vector, and a prokaryotic cell is a host, a strong promoter (eg, T7 promoter, tac promoter, lac) that can progress transcription Promoter, lac UV5 promoter, lpp promoter, p _L ^λ promoter, p _R ^λ promoter, rac 5 promoter, amp promoter, recA promoter, SP6 promoter and trp promoter, etc.), ribosome binding sites and transcription/ It is common to include a translation termination sequence. When E. coli is used as a host cell, E. coli The promoter and operator site of the tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol . , 158:1018-1024 (1984)) and the phage λ leftward promoter (p _L ^λ promoter, Herskowitz, I. and Hagen, D., . Ann Rev Genet, 14:. . 399-445 (1980)) may be used as a control region.

On the other hand, vectors that can be used in the present invention include plasmids often used in the art (e.g., pIVEX, pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19, pET, etc.), phage (e.g., λgt4λB, λ-Charon , λΔz1 and M13) or a virus (eg, SV40, etc.).

On the other hand, the vector of the present invention, as a selection marker, includes antibiotic resistance genes commonly used in the art, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin And resistance to tetracycline.

According to another aspect of the present invention, cells transformed with the recombinant vector are provided.

Host cells capable of continuously cloning and expressing the vector of the present invention in a stable manner are known in the art, and any host cell can be used. For example, E. coli JM109, E. coli Bacillus strains such as BL21(DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium Enterococci and strains such as Um, Ceratia Marcesons and various Pseudomonas species.

The method of transporting the vector of the present invention into a host cell includes a CaCl ₂ method (Cohen, SN et al., Proc . Natl . Acac. Sci . USA , 9:2110-2114 (1973)), one method (Cohen, SN et al., Proc . Natl . Acac . Sci . USA , 9:2110-2114 (1973); and Hanahan, D., J. Mol . Biol . , 166:557-580 (1983)) and electroporation method (Dower, WJ et al., Nucleic . Acids Res . , 16:6127-6145 (1988).

The vector injected into the host cell can be expressed in the host cell, in which case a large amount of xylosidase is obtained. For example, when the expression vector includes the lac promoter, gene expression can be induced by treating the host cell with IPTG.

According to a preferred embodiment of the present invention, the host cell used in the present invention is E. coli (BL21).

According to another aspect of the invention. Xylose-containing oligosaccharides or polysaccharides, arabinose-containing oligosaccharides or polysaccharides of the present invention include xylose, transformed cells or HPL-1 strains (KCTC11356BP). A method for decomposing oligosaccharides or polysaccharides comprising contacting a saccharide, or a glucose-containing oligosaccharide or polysaccharide.

In this specification, the'xylose-containing oligosaccharide or polysaccharide' includes various xylose-containing oligosaccharides or polysaccharides known in the art. For example, various oligosaccharides or polysaccharides including xylose include, but are not limited to, xylan, arabinoxylan, hemicellulose, and xylopyranoside. .

In the present specification, the'arabinose-containing oligosaccharide or polysaccharide' includes various arabinose-containing oligosaccharides or polysaccharides known in the art. For example, arabinose-containing oligosaccharides or polysaccharides include hemicellulose, pectin, arabinan, arabinoxylan, arabinogalactan, and arabinofuranoside. ), but is not limited thereto.

In the present specification, the'glucose-containing oligosaccharide or polysaccharide' includes various glucose-containing oligosaccharides or polysaccharides known in the art. For example, glucose-containing oligosaccharides or polysaccharides include, but are not limited to, starch, cellulose, sucrose, lactose and glucopyranoside. no.

Preferably, the step further comprises a metal ion.

According to a preferred embodiment of the present invention, when additionally including Ca ⁺² , Mn ⁺² , Cu ⁺² or Zn ⁺² , the activity of the xylosidase is increased by 140-180%.

According to another aspect of the present invention, the composition for processing xylan in a food product comprising the xylosidase of the present invention, transformed cells, or HPL-1 strain (KCTC11356BP).

Preferably, it can be used to improve the quality through softening and purification efficiency of food materials, extracting reduced viscosity and increasing filtration efficiency.

According to another aspect of the present invention, the composition for feed additives comprising the xylosidase, transformed cell or HPL-1 strain (KCTC11356BP) of the present invention is included.

Preferably, it can be used to reduce feed non-starch carbohydrates in livestock, improve intestinal viscosity, and increase digestion and absorption rates of proteins and starch.

According to another aspect of the present invention, the present invention comprises a composition for a papermaking process comprising the xylosidase, transformed cell or HPL-1 strain (KCTC11356BP).

Preferably, it can be used for biological whitening process of papermaking process, shortening process, deinking effect, separation of starch and gluten, and chemical fuel production.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a novel xylosidase having a multi-enzyme function.

(b) The xyloxidase of the present invention can reduce waste and expensive purification costs by replacing conventional chemical methods.

(c) Since the present invention exhibits excellent xylan decomposition activity, it is useful not only in the feed industry, papermaking and detergent industry, but also in the saccharification process of fibrous biomass, useful for producing raw materials such as petroleum substitute raw materials, special functional materials, bio polymers, etc. Can be used.

1 is an electron micrograph of HPL-1 of the genus Penibacillus , showing a gram-positive bacterium of a rod having a cell size of 1.1 μm and a cell length of 2.5 to 4.0 μm.
2 shows the results of screening for active clones from 1,248 gDNA libraries.
Figure 3 shows the activity after separation and purification of xylosidase from transformants.
Figure 4 shows the xylosidase activity according to temperature.
Figure 6 shows the enzyme reaction rate (kinetics) after separation and purification of xylosidase.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example  1: Strain separation and strain selection

Strain isolation

The strain of the present invention was isolated from the soil samples collected from the Jangsu scarab breeding farm located in Gunseo-myeon, Okcheon-gun, Chungbuk. Soil samples were collected from the 2-5 cm layer from the surface layer of the soil, and then dried and passed through 2 mm poles to select the soil. 30 g of selected soil was suspended in 270 ml of sterilized physiological saline (NaCl 8.0 g/l), shaken with a shake incubator (37°C, 200 rpm) for about 20 minutes, and then left at room temperature for about 30 minutes to coarse soil particles And impurities to precipitate to the bottom. The supernatant was transferred to a sterilized container and used as the primary dilution. After stirring well, 10 ml was taken, and 90 ml of physiological saline was added thereto to prepare 100 ml of the second dilution, and 10 ml was taken in the same manner while sufficiently stirring the second diluent, and 90 ml of physiological saline was added and the third dilution was performed. 100 ml was prepared. Thereafter, a sixth dilution was prepared in the same manner. After dispensing 3, 4, 5, and 6 dilutions of 3, 4, 5, and 6 dilutions into TSA (Tryptic Soy Agar, Difco Co.) medium for strain separation three times in duplicates, stir uniformly and incubate for 2 days in a 37°C normal incubator. Colonies of the formed microorganisms were selected. At this time, considering the various factors such as the shape, size, and color of the colonies, the separated colonies were subcultured again in TSA medium to isolate pure strains and stored at -70°C for use as a parent strain.

Strain selection

Selection of active strains with xylosidase activity among purely isolated strains was made by inoculating strain with soft agar double medium containing 0.5-1.0% of birch xylan (Fluka Bio Chemika. Co.) in TSA medium overnight. The following day after cultivation , around the colonies cultured by Congo-red staining (Theater RM, PJ. Wood. Appl Environ Microbiol 43, 777-780, 1982; Beguin P. Analytical Biochemistry, 131(2):333-336, 1983) Strains and active clones forming a transparent ring (Halo) were selected. The xylan resolution of the selected strain was measured once again to confirm reproducibility, and among them, the strain having the best xylan decomposition ability was selected and finally selected as a microorganism producing xylosidase.

Example 2: Identification of strain

The present inventors cultured the strain producing the highest activity xylosidase isolated in Example 1 at 30° C., and then performed gram staining and spore staining, resulting in spores. Was confirmed as Gram-positive bacilli. As a result of observing the shape with an electron microscope, as shown in FIG. 1, it was a rod having a cell size of 1.1 µm and a cell length of 2.5 µm to 4 µm, and appeared to be a motility bacillus without flagella. In addition, the strain has a 16S rRNA described in the third sequence of the Sequence Listing, and homology analysis results for rRNA, Penicillus genus, Phenibacillus favisporus (Paenibacillus favisporus (GenBank accession number AY308758)) strain and 97.7%, Penicillus Since it was confirmed that it has homology with 97.2% of Pabisporus [Paenibacillus favisporus (GenBank registration number AY208751)] and Penicillus favisporus (Paenibacillus favisporus (GenBank registration number EU798300)), this strain was identified as HPL- in the genus Penicillus. Named 1, and deposited with the Korea Research Institute of Bioscience and Biotechnology's Life Resources Center on July 17, 2008, and the deposit number is KCTC11365BP.

Example 3: Isolation of new xylosidase

Genetic library preparation and activity assay of Penicillus strains

The present inventors extracted the genomic DNA in order to separate the gene encoding the enzyme protein having xylosidase activity from the HPL-1 strain of the genus Penicillus identified in Examples 1 and 2, and having a size of 5 kb or less A gDNA library with gene fragments was constructed. In the preparation of the library, the DNA extracted from the strain was prepared as a DNA fragment of 1-6 kb size through a random cutting method, and the size was selected by electrophoresis on an agarose gel to obtain a DNA fragment having a desired size of about 5 kb. This was inserted into the pCB31 plasmid vector and transformed into E. coli DH10B cells. 1,248 clones of the library thus prepared were tested for xylosidase activity under solid or liquid conditions.

Xylosidase  Activity test

Measurement of xylosidase activity (xylan decomposition ability) of isolated strains, active clones, transformants, and separated and purified enzymes was performed using one or both of the following two methods. The first method is a solid culture measurement method. A soft agar double medium containing 0.5-1.0% of birch xylan (Fluka Bio Chemika. Co.) in LB medium was made, inoculated with a strain, incubated overnight, and then cultured through Congo Red staining the next day. Strains and active clones forming a halo around the colonies were selected. In the second method, a xylosidase assay was used as a method for measuring the enzyme activity of liquid culture, specifically 10 μl p-nitrophenyl β-D-xylopyranoside (100 mM) and 90 μl After culturing the Pinyphacilus sp. library at 37° C. for 2 days in a potassium phosphate buffer (100 mM, pH 7.0), 100 μl enzyme solution digested by sonication was mixed. After reacting at 40° C. for 10 minutes, absorbance (400 nm) was measured. One unit of enzyme was defined as an enzyme activity in which 1 g of xyloxidase produces 1 mmol of reducing sugar (xylose) in 1 minute. The top 1 clone Peani- PXD2 was selected through liquid culture and solid culture activity tests (FIG. 2).

Xylosidase  Active clone selection and genetic analysis

The nucleotide sequence of the fragment DNA inserted into the clone selected in the'Xylosidase Activity Test' of Example 3 was analyzed. As a result of analyzing the nucleotide sequence, the size of the DNA fragment inserted into the plasmid was 4,795 bp (SEQ ID NO: 4), and the NCBI's Blast P and Blast N programs (//www.ncbi.nlm.nih.gov/) were used. Through the analysis, an open reading frame (ORF) that has been encrypted was analyzed. Among the ORFs analyzed, four ORFs having a size of 100 amino acids or more were named PXD2-O1, PXD2-O2, PXD2-O3, and PXD2-O4. Based on the homology search result of the protein encoded by the gene among the ORFs, targets PXD2-O1 (sequence list 2nd sequence) having 86% homology to XylC, and based on the base sequence, Nde I (NEB) and Bam HI ( NEB) A primer was added with a restriction enzyme recognition site, and after PCR amplification, it was inserted into a pGEM-T-Easy vector (Promega, USA) vector to produce a recombinant plasmid.

Specifically, 1 ng of the recombinant plasmid template is a primer pair consisting of a forward primer of the fifth sequence (5'-ACATGCCATATGATGAAAAAACAAGGG-3') and a reverse primer of the sixth sequence (5'-ACATGCGGATCCCTTACTCTAAAATAAACGAAG-3') After mixing with 10 pmol), PCR amplification conditions were performed by denaturation at 94°C for 5 minutes using PCR premix (GenetBio, Korea), followed by denaturation at 94°C for 30 seconds, 55°C at 30°C. Annealing for 2 seconds and extension for 2 minutes at 72°C were repeated 30 times, finally, the extension was completed for 7 minutes at 72°C, and the reaction was terminated after maintaining at 4°C. The product amplified through PCR was purified using a GENCLEAN II kit (Q-Biogene, USA), and recombinant DNA was prepared using T4 ligase (RBC, Taiwan) for the pGEM-T-Easy vector. Genetically transformed E. coli was prepared by transforming the recombinant plasmid into E. coli JM109. After transforming the transformed E. coli in LB liquid medium, the plasmid DNA was extracted using HiYield™ plasmid mini kit (RBC, Taiwan) and cut with restriction enzymes Nde I (NEB, UK) and Bam HI (NEB, UK). , It was confirmed by electrophoresis that the desired DNA fragment was inserted. In addition, it was confirmed that the transformed Escherichia coli showed xyloxidase activity as a result of testing the xylosidase activity under liquid conditions as in the'xyloxidase activity test' of Example 3.

The base sequence of the target DNA fragment of the transformed E. coli was confirmed to confirm the ORF of the new xylosidase gene of the base sequence described in Sequence Listing 1 (Table 1).

Xylosidase Overexpression  making( pIVEX - GST - PXD2 )

PCR was performed using the Peani- PXD2 plasmid as a template and using the fifth sequence of the sequence list and the sixth sequence of the sequence list to prepare a transformed E. coli that overexpresses the new xylose. PCR reaction solution and reaction conditions were carried out in the same manner as in the'xyloxidase activity test' of Example 3. The amplified product was purified, cut with Nde I (NEB) and Bam HI (NEB), and inserted into the protein overexpression vector pIVEX-GST (Roche, Switzerland) to produce a recombinant overexpression plasmid, which was E. coli BL21 (RBC) , Taiwan) to produce a transfected E. coli overexpressing xylosidase. The transformed E. coli was cultivated, extracted with plasmid DNA, and then cut using the restriction enzyme to confirm whether the vector and the target DNA were successfully recombined through electrophoresis, and the identified cells in LB liquid medium (ampicillin 100 mg/L Addition) After incubation for 18 hours (37°C, 250 rpm, A ₆₀₀ =1.0), re-inoculation into fresh LB liquid medium to treat 1 mM IPTG when the A ₅₉₅ absorbance value is 0.4-0.6 and further 18 hours at 18°C After cultivation, the cells were harvested. The harvested cells were suspended and ultrasonically crushed, followed by centrifugation at 10,000 g to separate into supernatant and sediment, and the desired protein was overexpressed in the supernatant through SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) and the molecular weight was weak. It was confirmed that it was 52 kD (Fig. 3).

Xylosidase  Analysis of active expression conditions

' Xylosidase of Example 3 The overexpression and the activity of xylosidase overexpressed and isolated from the overexpressed transformed E. coli produced in'Overexpression Production ' by the method of'Xylosidase Activity Test' in Example 3 by pH, temperature and metal ion Was investigated. The pH of the reaction solution is adjusted to pH 3-7 with citric acid buffer, pH 6-8 with phosphoric acid buffer, and pH 8-11 with glycine/NaOH buffer. Was performed. As metal ions, 1 mM CaCl ₂ , MgCl ₂ , MgCl ₂ , CuCl ₂ , ZnCl ₂ Or FeCl ₃ was added to investigate the effect on xylosidase activity, and other NaCl, LiCl, KCl, NH ₄ Cl, EDTA, 2-ME (2-Mercaptoethanol), DTT (Dithiothreitol) or PMSF (Penylmethylsulfonyl fluoride) ) Was added to investigate the effect on xyloxidase activity expression.

As a result, the novel xylosidase showed maximum activity at pH 4 and 40° C. as shown in FIG. 4. In addition, as shown in Table 2, 1 mM Ca ⁺² , Mn ⁺² , Cu ⁺² or Xylosidase activity on p-nitrophenyl β-D-xylpyranoside was increased to 156, 178, 156 and 144%, respectively, by heavy metals or additives such as Zn ⁺² .

additive Relative activity according to additive concentration (%) 1 mM Negative control 100 NaCl 112 LiCl 97 KCl 98 NH4Cl 101 CaCl ₂ 156 MgCl ₂ 125 MnCl ₂ 178 CuSO ₄ 156 ZnSO ₄ 144 FeCl ₃ 108 EDTA 115 2-ME 117 DTT 106 PMSF 97

Example  4: New Xylosidase  massive production

The pIVEX GST-xylosidase recombinant vector (Bioprogen Co., Ltd., Korea) containing the gene encoding the new xylosidase of SEQ ID NO:2 (Bioprogen Co., Ltd., Republic of Korea) is coliform BL21-Gold(DE) ( Stratagene, USA), and then inoculated into a liquid medium (LB 25 g/L) with ampicillin added (50 µg/ml) and OD ₅₉₅ The mixture was stirred and cultured at 37°C under conditions of 150 rpm until the value became 0.4-0.6. To induce intracellular expression of the target protein, IPTG (isopropyl-D-thiogalactoside) was added to the suspension to a final concentration of 1 mM, followed by further incubation at 18°C for 18 hours. The culture was centrifuged at 10,000 rpm for 10 minutes, and the recovered precipitate was washed twice with PBS. After resuspending the washed precipitate in PBS again, the cells were crushed using an ultrasonic crusher (Cosmo Bio Co., LTD), and then centrifuged (12,000 rpm, 10 minutes) to recover the supernatant. A glutathione-transferase column (GST binding resin column, Novagen) was used for pure separation of xyloxidase in the recovered supernatant. At this time, the pure separation of xylosease is a glutathione-transferase column (GST binding resin column, Novagen) equilibrated with the prepared supernatant with a washing buffer solution (50 mM Tris-HCl, 100 mM NaCl; pH 7.0). After attaching to, the factor Xa protease (NEB, UK) was treated and purified using a buffer solution (50 mM Tris-HCl, 100 mM NaCl; pH 7.0). Xylosidase enzyme activity was measured from each sample collected in the purification step, and the purification of the enzyme active fraction was confirmed by SDS-PAGE. Protein content was measured using the Bradford method (Bradford, Sigma Aldrich), and the standard protein was used as a BSA (bovine serum albumin).

As a result, glutathione resin column chromatography was able to easily produce a large amount of xylosidase. In addition, it can be seen that it can be applied to a high-efficiency conversion process through an enzyme immobilization method because it showed a characteristic that the xyloxidase activity does not change even in the state of xyloxidase bound to the resin. The enzyme purified by the above method showed 5 times more activity than the enzyme before purification. Enzyme activity is 0.10, 15.15, 2.97, 3.79, 4.57, 2.44, 2.64, 1.48 at pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, pH 11.0 at 40°C, respectively. And 0.19 units (myl xylose produced by 1 g of enzyme per minute).

Characteristic contents are p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl(N)-α-L-arabino at pH 4.0 and 40℃. Furanoside (p-nitrophenyl(N)-α-L-arabinofuranoside), pN-β-D-glucopyranoside and pN-β-D-cellobioside (pN When using a substrate such as -β-D-cellobioside), it showed saccharification activity, indicating 5, 12, 4 and 3 inactive units. That is, it could be expected to be used as a multifunctional enzyme. However, pN-α-D-glucopyranoside, pN-α-L-arabinopyranoside, pN-β-L-arabinopyranoside, pN-β-L-arabino Pyranoside (pN-β-L-arabinopyranoside), pN-β-D-mannopyranoside and pN-β-D-galactopyranoside (pN-β-D -galactopyranoside) and the like.

Example  5: Mass-produced new Xylosidase  Enzyme properties

In order to examine the properties of the enzyme, 50 ml of potassium phosphate buffer (pH 7.0) containing purified xyloxidase in a test tube and containing various amounts of p-nitrophenyl-β-D-xylpyranoside Was added. The reaction mixture was reacted at 40° C. for 10 minutes to investigate the enzyme reaction rate (Hanes-woolf).

As a result, as shown in FIG. 6, the affinity K _m value for the xylan substrate was 4.091, and most of the hydrolysis products were xylose. Xylosidase activity against xylan (beech, oat, etc.) other than birch xylan was similar.

Since the specific parts of the present invention have been described in detail above, it is obvious that for those skilled in the art, this specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> KOREA RESEARCH INSTITUTE OF CHEMICAL TECHNOLOGY <120> Novel Enzyme PXD2-01 Having Xylase Activity <130> PN120303 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 1425 <212> DNA <213> PXD2-01 <400> 1 atgatgaaaa aacaagggct aaacccctat ctcccatcgt gggaatacat acctgacgga 60 gaaccgtacg tttttaacga cagagtttat gtatatggct cgcatgaccg ctttaacgga 120 catgtgtttt gtttaaacga ctacgcttgc tggtcggcac ccgttaatga tctaggcgac 180 tggcggtatg agggcgtgat ctacaccaaa acggatgacc cgctaaatcc tgatggcagg 240 atgtgtcttt atgcgccgga cgtcaccgtc ggaccggatg gtcgttatta cctttactat 300 gtcctggata aggttccggt tgtttcggtt gccgtctgcg atacacctgc tggaaagtat 360 gaattttacg gatacgtaaa atacgcggac ggtacacgct tgggcgaaag agaaggagat 420 gagccccagt ttgatccggg tgtattgaca gaagggaaga agacctactt atataccggc 480 ttctgtgcac ccaaggataa atccagacat ggcgcaatgg ccacggtgct tggtgaagat 540 atgcttacca ttatggagga acctgtattt gtcgccccaa gtgaacctta cagtgcggga 600 agcgggtttg aagggcatga attttttgag gctccctcca tccgtaagaa gggtgatatt 660 tattatcttg tctattcatc gattgtaatg catgaattat gttatgcgac cagtaaattt 720 ccgacaaaag gcttcattta tcagggcgtc atcataagca acaatgatct tcacatcgat 780 tcctacaaac cggcggacaa gccgatgtat tatggcggta ataaccatgg cagcattgtg 840 gaaatgaacg atagatggtt cattttttat catcgccata ccaacggtac ggcttttagc 900 cggcagggct gcatcgaacc gatcgtcatt cgggaggatg gaacgattcc tcaagtggaa 960 atgacctctt gcggtcccaa tgacggcccg cttgtgggtc gcggtgaata ccacgcctat 1020 ttggcatgta atctgttctg taaagacgaa gaattgtata caggcggctt cggctcaggg 1080 gtatggatgg acagccgttt tccgaagatc acgcaggagg gaagagacgg ggacgaagag 1140 atcggataca tcgcaaacat gacagactcc gctactgccg gtttcaagta tttcgactgc 1200 caaggaatac gtaaggtgaa aatcaaagtg cgtggttatt gccagggcaa ctttgaaatt 1260 aaaacggctt gggacggccc ggctctcgga aaaataaccg ttaactttac aaatatatgg 1320 acggaatatt caacggatct cgtcattccg gatggcatac aggctctata ttttacgtac 1380 acaggcagag gaagtgcgga tctggcttcg tttattttag agtaa 1425 <210> 2 <211> 474 <212> PRT <213> PXD2-01 <400> 2 Met Met Lys Lys Gln Gly Leu Asn Pro Tyr Leu Pro Ser Trp Glu Tyr   1 5 10 15 Ile Pro Asp Gly Glu Pro Tyr Val Phe Asn Asp Arg Val Tyr Val Tyr              20 25 30 Gly Ser His Asp Arg Phe Asn Gly His Val Phe Cys Leu Asn Asp Tyr          35 40 45 Ala Cys Trp Ser Ala Pro Val Asn Asp Leu Gly Asp Trp Arg Tyr Glu      50 55 60 Gly Val Ile Tyr Thr Lys Thr Asp Asp Pro Leu Asn Pro Asp Gly Arg  65 70 75 80 Met Cys Leu Tyr Ala Pro Asp Val Thr Val Gly Pro Asp Gly Arg Tyr                  85 90 95 Tyr Leu Tyr Tyr Val Leu Asp Lys Val Pro Val Val Ser Val Ala Val             100 105 110 Cys Asp Thr Pro Ala Gly Lys Tyr Glu Phe Tyr Gly Tyr Val Lys Tyr         115 120 125 Ala Asp Gly Thr Arg Leu Gly Glu Arg Glu Gly Asp Glu Pro Gln Phe     130 135 140 Asp Pro Gly Val Leu Thr Glu Gly Lys Lys Thr Tyr Leu Tyr Thr Gly 145 150 155 160 Phe Cys Ala Pro Lys Asp Lys Ser Arg His Gly Ala Met Ala Thr Val                 165 170 175 Leu Gly Glu Asp Met Leu Thr Ile Met Glu Glu Pro Val Phe Val Ala             180 185 190 Pro Ser Glu Pro Tyr Ser Ala Gly Ser Gly Phe Glu Gly His Glu Phe         195 200 205 Phe Glu Ala Pro Ser Ile Arg Lys Lys Gly Asp Ile Tyr Tyr Leu Val     210 215 220 Tyr Ser Ser Ile Val Met His Glu Leu Cys Tyr Ala Thr Ser Lys Phe 225 230 235 240 Pro Thr Lys Gly Phe Ile Tyr Gln Gly Val Ile Ile Ser Asn Asn Asp                 245 250 255 Leu His Ile Asp Ser Tyr Lys Pro Ala Asp Lys Pro Met Tyr Tyr Gly             260 265 270 Gly Asn Asn His Gly Ser Ile Val Glu Met Asn Asp Arg Trp Phe Ile         275 280 285 Phe Tyr His Arg His Thr Asn Gly Thr Ala Phe Ser Arg Gln Gly Cys     290 295 300 Ile Glu Pro Ile Val Ile Arg Glu Asp Gly Thr Ile Pro Gln Val Glu 305 310 315 320 Met Thr Ser Cys Gly Pro Asn Asp Gly Pro Leu Val Gly Arg Gly Glu                 325 330 335 Tyr His Ala Tyr Leu Ala Cys Asn Leu Phe Cys Lys Asp Glu Glu Leu             340 345 350 Tyr Thr Gly Gly Phe Gly Ser Gly Val Trp Met Asp Ser Arg Phe Pro         355 360 365 Lys Ile Thr Gln Glu Gly Arg Asp Gly Asp Glu Glu Ile Gly Tyr Ile     370 375 380 Ala Asn Met Thr Asp Ser Ala Thr Ala Gly Phe Lys Tyr Phe Asp Cys 385 390 395 400 Gln Gly Ile Arg Lys Val Lys Ile Lys Val Arg Gly Tyr Cys Gln Gly                 405 410 415 Asn Phe Glu Ile Lys Thr Ala Trp Asp Gly Pro Ala Leu Gly Lys Ile             420 425 430 Thr Val Asn Phe Thr Asn Ile Trp Thr Glu Tyr Ser Thr Asp Leu Val         435 440 445 Ile Pro Asp Gly Ile Gln Ala Leu Tyr Phe Thr Tyr Thr Gly Arg Gly     450 455 460 Ser Ala Asp Leu Ala Ser Phe Ile Leu Glu 465 470 <210> 3 <211> 1404 <212> RNA <213> HPL-1 16S rRNA <400> 3 tagagtttga tcatggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgag 60 cggacttgat ggagagcttg ctctcctgat ggttagcggc ggacgggtga gtaacacgta 120 ggcaacctgc ctgcaagacc gggataaccc acggaaacgt gagctaatac cggatatctc 180 atttcctctc ctgagggaat gatgaaagac ggagcaatct gtcacttgcg gatgggcctg 240 cggcgcatta gctagttggt gaggtaacgg ctcaccaagg cgacgatgcg tagccgacct 300 gagagggtga acggccacac tgggactgag acacggccca gactcctacg ggaggcagca 360 gtagggaatc ttccgcaatg ggcgaaagcc tgacggagca acgccgcgtg agtgatgaag 420 gttttcggat cgtaaagctc tgttgccagg gaagaacgtc cggtagagta actgctatcg 480 gagtgacggt acgtgagaag aaagccccgg ctaactacgt gccagcagcc gcggtaatac 540 gtagggggca agcgttgtcc ggaattattg ggcgtaaagc gcgcgcaggc ggtcatttaa 600 gtctggtgtt taaggccaag gctcaacctt ggttcgcact ggaaactggg tgacttgagt 660 gcagaagagg agagtggaat tccacgtgta gcggtgaaat gcgtagatat gtggaggaat 720 cnccagtggc gaaggcgact ctctgggctg taactgacgc tgaggcgcga aagcgtgggg 780 agcaaacagg attagatacc ctggtagtcc acgccgtaaa cgatgaatgc taggtgttag 840 gggtttcgat acccttggtg ccgaagttaa cacattaagc attccgcctg gggagtacgg 900 tcgcaagact gaaactcaaa ggaattgacg gggacccgca caagcagtgg agtatgtggt 960 ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac atccctctga ccggtctaga 1020 gatagacctt tccttcggga cagaggagac aggtggtgca tggttgtcgt cagctcgtgt 1080 cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgattttagt tgccagcact 1140 tcgggtgggc actctagaat gactgccggt gacaaaccgg aggaaggcgg ggatgacgtc 1200 aaatcatcat gccccttatg acctgggcta cacacgtact acaatggcca gtacaacggg 1260 aagcgaagcc gcgaggtgga gccaatccta tcaaagctgg tctcagttcg gattgcaggc 1320 tgcaactcgc ctgcatgaag tcggaattgc tagtaatcgc ggatcagcat gccgcggtga 1380 atacgttccc gggtcttgta caca 1404 <210> 4 <211> 4795 <212> DNA <213> Peani-PXD2 <400> 4 gtatcgataa gcttgatatt ttcgctggct aatccggcgg ttgcagccgt cattcccggt 60 gcaagtcgtc ctgagcggat tgcagaagac aaagctgcat tgaacacagt cattccggca 120 gctttctggg aagaaatgcg cgaacaaaaa ctggtagcac cccatgcacc gctgcctatc 180 gacatgaaat caatagggga gtaatgaaca tggcacatac ggctgtctgg cagttttacc 240 ccggcagggg tcagtgatca agaggctatt gatctgttcc atgggatcta tcaagacggc 300 ttagaggcat tacgacaagc atttttagat tgaagattta ataagtaaaa gaggcgtgta 360 tccgaggcga aatttgaact tggatacact cttttttttg ttcgctcaaa tacagaagcg 420 ttaataaaat atgcccaagt attatttgct gtgctcaagg ttcgaacttg tgggtcatat 480 catcctttcc ataaaagcag gaaagtgcat gaagcaagag gaatacatag acaaatcatc 540 aagctaagga ggtcatgaat tggcgtttcc gacacatatt gtatctgcag gcggtattgt 600 agaagatgga aagggaaata tccttttagt aaaagcgcat gacgacggtt gggtatatcc 660 tgggggaatc actgaagttg gcgaaaacct gatggatggc gtgatccgtg aaatcaaaga 720 ggaaagtgga atagacgcta cggttagcca tttaatcagt gtggtttcaa atacagcgat 780 ccataagtgg tatgacggtg taaccgatgt tcctacgaag gtcatgtttg atttcgtgtg 840 tacagccgtg gggggagagt tagcgacctc tgaggagacg agcgaatgca ggtgggttcc 900 caaagaaaat gttctggatt ggatcacctt acccgcaatc cgcatgcgct acgaagctta 960 tttgaacttt aacggctctg tgaattatat cgagtacgtt actgcaacaa cgtctgaatg 1020 tcaggttaaa ctgcaaagga agatgtagtt cgcttgtcac ctccaagaga tatgggttag 1080 gaaattttcg ctagcgtgaa ggcatattta aaggagtgaa cgtttatgat gaaaaaacaa 1140 gggctaaacc cctatctccc atcgtgggaa tacatacctg acggagaacc gtacgttttt 1200 aacgacagag tttatgtata tggctcgcat gaccgcttta acggacatgt gttttgttta 1260 aacgactacg cttgctggtc ggcacccgtt aatgatctag gcgactggcg gtatgagggc 1320 gtgatctaca ccaaaacgga tgacccgcta aatcctgatg gcaggatgtg tctttatgcg 1380 ccggacgtca ccgtcggacc ggatggtcgt tattaccttt actatgtcct ggataaggtt 1440 ccggttgttt cggttgccgt ctgcgataca cctgctggaa agtatgaatt ttacggatac 1500 gtaaaatacg cggacggtac acgcttgggc gaaagagaag gagatgagcc ccagtttgat 1560 ccgggtgtat tgacagaagg gaagaagacc tacttatata ccggcttctg tgcacccaag 1620 gataaatcca gacatggcgc aatggccacg gtgcttggtg aagatatgct taccattatg 1680 gaggaacctg tatttgtcgc cccaagtgaa ccttacagtg cgggaagcgg gtttgaaggg 1740 catgaatttt ttgaggctcc ctccatccgt aagaagggtg atatttatta tcttgtctat 1800 tcatcgattg taatgcatga attatgttat gcgaccagta aatttccgac aaaaggcttc 1860 atttatcagg gcgtcatcat aagcaacaat gatcttcaca tcgattccta caaaccggcg 1920 gacaagccga tgtattatgg cggtaataac catggcagca ttgtggaaat gaacgataga 1980 tggttcattt tttatcatcg ccataccaac ggtacggctt ttagccggca gggctgcatc 2040 gaaccgatcg tcattcggga ggatggaacg attcctcaag tggaaatgac ctcttgcggt 2100 cccaatgacg gcccgcttgt gggtcgcggt gaataccacg cctatttggc atgtaatctg 2160 ttctgtaaag acgaagaatt gtatacaggc ggcttcggct caggggtatg gatggacagc 2220 cgttttccga agatcacgca ggagggaaga gacggggacg aagagatcgg atacatcgca 2280 aacatgacag actccgctac tgccggtttc aagtatttcg actgccaagg aatacgtaag 2340 gtgaaaatca aagtgcgtgg ttattgccag ggcaactttg aaattaaaac ggcttgggac 2400 ggcccggctc tcggaaaaat aaccgttaac tttacaaata tatggacgga atattcaacg 2460 gatctcgtca ttccggatgg catacaggct ctatatttta cgtacacagg cagaggaagt 2520 gcggatctgg cttcgtttat tttagagtaa gccgcccgcc cgggaaccag cttcccaaaa 2580 aaatacgcaa gcagttccga gcattttccg gaactgcttt ttggtttgtg tgatctttta 2640 atgataaagc catggaggcc gagtattaaa aaatgacaac gctttcttaa aaatgttggc 2700 ttacgagcag atgggtcaag aggatagact agcaataaag gaggagcgca tatggatttg 2760 gatgcacagt ttacgagaac tgcggatcac ggtggtcagc agcaccaatt atggaagcgg 2820 ttcaaaaaac aaaaagtact gcatgtattt gtaggactgg gtatgatctt tctgctgatt 2880 ttctcgtaca cgccgatgtt cggtattctt atggcgttta aagactacag tatttcaaat 2940 ggtattaagg ggatctttac cagcgagtgg gtcggtttga gatatttcga tgaattcatc 3000 catgattatc agtttgccac gattgtacgc aacacgcttg tcctgagctt attgaaggtc 3060 attttcgctt ttcccgcacc gattttgctt gcgatcctgt tgaatgaagt gaaaaatatg 3120 gcgttcaagc gattcgttca gacgatcagc tacctgccgc attttatttc ctgggttgtt 3180 gtcgtcggag tatcctacgc cttcctgtct gcggatgttg gcatggttaa ccgggccctg 3240 gtcgaaaccg ggctgattga caaaccgctc aacattttga cgagtccgaa ctatttctgg 3300 gggcttgccg tcggcagtgc aatttggaag gaaatgggct ggtggacgat catcttcctg 3360 gccgccatca cggggattaa tccttcgctt tacgaagctg ccgagatgga tggtgccgga 3420 aggctggcac ggatccgata tatcacactt ccgggaatca gaggaacgat cgtcgtcgtg 3480 ctggtgttga ccattggcag tattctcgga ggcggcctgg tcggctccaa cttcgaacaa 3540 gcctacttat acggcaacag tattaacaat ccgacatcgg aaattgttca gacgtacgca 3600 ttcaaggttg gtctgagtga cgggcgattc tcctacgcgg cagcaatcga tctcattcag 3660 tccgtcatct ccgtcatttt gatattttcc agtaacttca ttgccaagcg ggtgtcaggg 3720 tcaagcttat tctaaaaagg agggctggtg gtgcttaaga gccaaagaca gaaggatgtt 3780 atttttgaca gctttattta catcatgttg tttatattga tgctcatcat gctgtatcca 3840 ttctattatg tattcatcgc ctcgtttaac aaaggctccg atacgctcct gggaggcatt 3900 tatttgtggc cacgaaacgt aactctggaa aactataaaa tttttctgga agatccgaag 3960 tggtatcgag cctttttggt cacggttgcc cggacgatat cgggtacggc attgggactg 4020 ctgctgacaa gcctggtcgc ttacgccctt tcacaccgtg atttgttgtt cagcaaaacg 4080 tattttacgg tcatcatttt cgcgatgtac ttttccggcg gattgattcc ttattatgtt 4140 gtgctgcgct cgatcggatt gcttaattca tttgcagtgt acatcgtgcc gtccatgctc 4200 aacacgtttt tcctgctcat tgccatctcg ttcttccgcg aaatcccggg cgaactgaaa 4260 gaatcggcgc atatcgacgg cgctggcgaa ctcaagatct tttttcggat catcctgccg 4320 gtctcgacgc cggtactcgc cacgatggcg ttatttatgg gcgtcggcca atggaattca 4380 tggctggact ccgcctattt cgtgcaatcg gaaaacttgc gaacgcttac cttccggatg 4440 atggaagtga tcaacaagag caacacgccg ctggattcca ttgccgtagc aaacagtgcc 4500 tcggcttcgg ccggagtgac gagcttctcg ctgcaggtga cggcgatggt catctccatc 4560 gtgccgatca tatgcgtata cccgtttttg caaaaatatt ttgtgcatgg aattatgtta 4620 ggatctgtga aagggtaaat cgttgtttct atatttaacc gcttacatta aaataccagt 4680 aaatccattg gaggggtaca cttgaaaaca atcaagagct ataagctgct cgctgcggca 4740 ctgtctgcga tcgaattcct gcagcccggg ggatccacta gttctagagc ggccg 4795 <210> 5 <211> 27 <212> DNA <213> foward primer <400> 5 acatgccata tgatgaaaaa acaaggg 27 <210> 6 <211> 33 <212> DNA <213> reverse primer <400> 6 acatgcggat cccttactct aaaataaacg aag 33

Claims (8)

Xylosidase consisting of the amino acid sequence set forth in Sequence Listing 2nd Sequence.
A nucleic acid molecule encoding a xylosidase consisting of the amino acid sequence set forth in SEQ ID NO:2.
A recombinant vector comprising the nucleic acid molecule of claim 2.
Cells transformed with the recombinant vector of claim 3.
The xylose-comprising oligosaccharide or polysaccharide, arabinose-comprising oligosaccharide or poly of the xylose-containing oligosaccharide of claim 1 or the transformed cell of claim 4 A method for degrading an oligosaccharide or polysaccharide comprising contacting a saccharide, or a glucose-containing oligosaccharide or polysaccharide.
A composition for processing xylan in a food product comprising the xylosidase of claim 1 or the transformed cell of claim 4.
A composition for feed additives comprising the xylosidase of claim 1 or the transformed cell of claim 4.
A composition for a paper-making process comprising the xylosidase of claim 1 or the transformed cell of claim 4.

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