KR20160133922A - - -xylosidase expression system for producing xylobiose - Google Patents

Abstract

The present invention provides a beta-xylosidase expression system for producing xylobiose. The beta-xylosidase expression system according to the present invention provides beta-xylosidase with a high conversion rate of xylobiose from xylose, and enables mass production of xylobiose with high yield in a short time. Accordingly, a large amount of new sugar supplement materials can be secured, and the beta-xylosidase expression system can be helpfully used not only in the functional sugar industry but also in health food materials, medicines and cosmetic materials.

Description

The present invention relates to a β-xylosidase expression system for producing xylobiose,

The present invention relates to a composition for producing xylobiose comprising β-xylosidase which converts xylose to xylobiose, an expression construct and a recombinant strain for producing the composition, a method for producing xylobiose from xylose .

Xylo-oligosaccharide (XOS) is a type of oligosaccharide in which xylose is bound by β- (1,4) -linkage, which is not degraded by the digestive enzymes of the human body Reaches the colon without being absorbed. Xylo-oligosaccharide (XOS) is known to be selectively degraded by Bifidobateria, a beneficial microorganism of the intestine, to induce the proliferation of Bifidobacteria. It is known that it exhibits health functions such as anticancer activity and bacteriostatic activity. In addition, with an excellent moisturizing effect, attempts have been made to apply it to cosmetics.

Commercial production of such xylo-oligosaccharides requires a physico-chemical pretreatment of wood, cotton wool, cornstarch and the like, followed by the decomposition of xylenes by endo-xylanase and? -Xylosidase . However, since the pretreatment process of the xylo-oligosaccharide production process requires the use of a large amount of chemical substances, a large amount of pollutants such as wastewater is generated in the process.

On the other hand, commercially available xylo-oligosaccharide products are generally provided as xylo-oligosaccharides in the form of mixtures. In other words, there are many difficulties in separating and purifying xylobiose from xylo-oligosaccharides, so there is no high-purity xylobiose product as a commercially available substance.

However, when the polymerization degree of xylo-oligosaccharide is high, the coloring tendency is low and the magnetization property by Bifidobateria is poor, so it is necessary to selectively produce a substance having low polymerization degree such as xylobiose.

In order to prevent the growth of microorganisms in the preservation and to prevent the inherent composition of the food from being impaired, syrup of high concentration as high as possible is preferable in order to reduce transportation costs. Further, it is preferable to use a concentrated liquid for spray drying when preparing a powdered xyloglucaride.

However, it is difficult to produce high purity xylobiose by the method known to date, and accordingly, various attempts have been made to produce high purity xylobiose syrup through enzyme treatment in xylan, but no related achievement has been made yet .

Under these circumstances, it is necessary to research and develop xylobiose to obtain high purity, which is known to be the main cause of all the physiological activities of xylo-oligosaccharides. In particular, xylobiose, a high- Research and development of an effective xylobiose production process for production should be made.

The present invention provides a composition for producing xylobiose comprising a novel? -Xylosidase having an activity of converting xylose to xylobiose.

The present invention also provides various methods for producing novel? -Xylosidase or for preparing novel? -Xylosidase.

The present invention also provides a method for producing xylobiose from xylose or various elements necessary for producing xylobiose from xylose.

The present inventors have made intensive efforts to develop commercially available β-xylosidase expression system and xylobiose production method technology for effective production of xylobiose, which is a health functional saccharide, and as a result, The present invention succeeded in mass conversion of xylose to xylobiose using sodium lauryl sulfate, thus completing the present invention.

The present invention provides a composition for producing xylobiose, which comprises? -Xylosidase consisting of the amino acid sequence of SEQ ID NO: 1.

The composition for producing xylobiose according to the present invention relates to a novel? -Xylosidase capable of converting xylose to xylobiose and has a maximum activity for the conversion of xylose to xylobiose, and has a short It is possible to mass-produce xylobiose with high yield during the time. The β-xylosidase is obtained by amplifying a specific DNA among DNA present in Bacillus pumilus genomic DNA by a polymerase reaction, inserting the amplified specific DNA into the expression construct, , Transforming the host strain with the recombinant expression construct to produce a recombinant strain, and culturing the recombinant strain to express the recombinant strain. More preferably, the Bacillus pumilus IPO xynB gene of SEQ ID NO: 12 is synthesized, and the β-xylosidase base sequence of SEQ ID NO: 2 is obtained therefrom and inserted into the expression construct, And then transforming the host strain with the recombinant expression construct to produce a recombinant strain, followed by culturing and expressing the recombinant strain.

The host strain of the recombinant strain is preferably a food-stable strain. The foodstuff-safe strains generally refer to GRAS (generally accepted as safe) strains which are considered to be safe. Examples include strains of Saccharomyces sp., Bacillus sp. Corynebacterium sp., And the like. The strains are industrial microorganisms that produce chemicals with diverse uses in fields such as feed, medicine, and food. These strains are easy to genetically manipulate and mass culture or have high stability under various process conditions. In addition, since these strains have a relatively rigid cell membrane structure as compared with other bacteria, they exhibit biological characteristics that the cells remain in a stable state even under the influence of osmotic pressure due to high sugar concentration and the like. Specific examples of the GRAS (generally accepted as safe) strain include Saccharomyces cerevisiae , bacillus subtilis , and Corynebacterium glutamicum . It is preferably Saccharomyces cerevisiae .

The? -Xylosidase according to the present invention preferably has a molecular weight of 55 to 60 kDa and an optimum activity temperature of 40 to 60 ° C, preferably 45 to 55 ° C, an optimum active pH of 5.0 to 8.0, 6.0 to 7.0. The? -Xylosidase according to the present invention comprises the amino acid sequence of SEQ ID NO: 1, but the equivalent range of? -Xylosidase according to the present invention is not limited thereto. For example, the homogeneous range of β-xylosidase according to the present invention is one in which some of the amino acids of SEQ ID NO: 1 are substituted, inserted and / or deleted as long as the activity of converting xylose to xylobiose is maintained .

The substitution of the amino acid is preferably carried out by conservative amino acid replacement in which the property of the protein is not changed. Further, the modification of the amino acid may be carried out by glycosylation, acetylation, phosphorylation, and the like. In addition, the equivalent range of β-xylosidase according to the present invention is a protein having increased structural stability against heat, pH or the like due to mutation or modification of amino acid sequence or increased activity for conversion of xylose to xylobiose . In addition, the equivalent range of? -Xylosidase according to the present invention is an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology with the amino acid sequence of SEQ ID NO: May include.

The present invention also provides a method for producing xylobiose comprising the step of reacting xylose with a composition comprising? -Xylosidase or? -Xylosidase consisting of the amino acid sequence of SEQ ID NO: 1.

Wherein the composition comprising the? -Xylosidase is transformed with a polynucleotide encoding? -Xylosidase consisting of the amino acid sequence of SEQ ID NO: 1 or a? -Xylosidase consisting of the amino acid sequence of SEQ ID NO: 1 A recombinant strain transformed by a recombinant construct comprising the polynucleotide encoding the recombinant strain, a culture of the recombinant strain, or a lysate of the recombinant strain. At this time, the reaction temperature of the composition comprising? -Xylosidase or? -Xylosidase is 40 to 60 ° C, preferably 45 to 55 ° C, and the reaction pH is 5.0 to 8.0, preferably 6.0 to 7.0 And the concentration of xylose for the reaction may be 400 g / L to 1000 g / L, preferably 500 to 800 g / L, but the concentration is not particularly limited.

The present invention also provides a β-xylosidase expression construct comprising the β-xyloεidase base sequence, the GPD promoter base sequence, the His-tag base sequence and the delta base sequence of SEQ ID NO: 2. The β-xylosidase expression construct of the present invention can express new β-xylosidase in a transformant with high efficiency, and can produce a large amount of β-xylosidase at low cost.

The β-xylosidase expression construct according to the present invention strongly expresses β-xyloidesidase through the GPD promoter and can express the gene in the cell including the delta sequence having the transposon function. The His-tag It is easy to separate and purify including sequence.

In the present invention, an expression construct means a nucleic acid molecule containing only a minimal element (elemnet) for protein expression in a cell. Preferably, the expression constructs of the present invention comprise the above-mentioned components as a minimum essential component.

The expression construct of the present invention may be a recombinant vector. Preferably, according to the vector recombinant method known in the art, the GPD promoter base sequence is ligated upstream of the β-xylosidase base sequence of SEQ ID NO: 2 to which the His-tag base sequence is added, and a recombinant vector containing the delta base sequence Lt; / RTI > Herein, the GPD promoter base sequence may include the nucleotide sequence of SEQ ID NO: 3, the His-tag nucleotide sequence may include the nucleotide sequence of SEQ ID NO: 4, and the delta nucleotide sequence may include the nucleotide sequence of SEQ ID NO: can do.

In addition, the expression construct according to the present invention comprises a 2-micron nucleotide sequence of SEQ ID NO: 6, an ampicillin resistance gene sequence of SEQ ID NO: 7, a CYC.1 terminator nucleotide sequence of SEQ ID NO: 8 and a TRP nucleotide sequence of SEQ ID NO: And a β-xylosidase expression construct further comprising one or more.

Since the above-mentioned nucleotide sequences are functionally equivalent, a nucleotide sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology with each of the above nucleotide sequences, Category.

For example, the β-xylosidase expression construct according to the present invention may be inserted into an expression vector having a 2-micron, an ampicillin resistance gene, a GPD promoter, a His-Tag, a CYC.1 terminator, a Delta and a TRP1 sequence, - xylosidase nucleotide sequence inserted therein, and has the recombinant expression vector structure described in Fig. A schematic diagram of the recombinant vector is shown in Fig. The construct for β-xylosidase insertion may have the nucleotide sequence of SEQ ID NO: 10. Also, according to one embodiment, the β-xylosidase expression construct has the nucleotide sequence of SEQ ID NO: 11.

The present invention also provides a transformant strain for the production of [beta] -xylosidase comprising a β-xylosidase expression construct. The host strain of the recombinant strain is preferably a food-stable strain. Food-stable strains generally refer to GRAS (generally accepted as safe) strains which are considered to be safe. Examples include strains of Saccharomyces sp., Bacillus sp. Corynebacterium sp., And the like. Specific examples include Saccharomyces cerevisiae , bacillus subtilis , Corynebacterium glutamicum , and the like, preferably Saccharomyces cerevisiae, cerevisiae . Most preferably, the transformant strain for producing [beta] -xylosidase according to the present invention is the transformed Saccharomyces cerevisiae deposited on Apr. 29, 2015 with the deposit number KCTC 18382P deposited at the microbiological resource center of the Korea Research Institute of Bioscience & Saccharomyces cerevisiae pTSY01-XynB.

In the present invention, the method for transforming the β-xylosidase expression construct into a strain may be a transformation method known in the art. For example, microinjection, calcium phosphate precipitation, Electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, acetic acid-lithium DMSO, and the like.

(A) a transformant strain for producing β-xylosidase, which comprises 0.5 to 2% by weight of yeast extract, 1 to 3% by weight of peptone and 1 To 4% by weight of glucose;

(b) culturing at a reaction temperature of from 25 to 35 DEG C and a reaction pH of from 7.5 to 8.5; And

(c) separating β-xylosidase from the culture medium of step (b).

The method for producing β-xylosidase according to the present invention comprises the steps of: (a) culturing a transformant strain for producing β-xylosidase in an amount of 0.5 to 2% by weight of yeast extract, To a YPD medium containing 1% to 4% by weight of peptone and 1 to 4% by weight of glucose. In the present invention, the YPD medium contains the yeast extract, the peptone and the glucose in the above-mentioned content range, and shows the effect on the rapid growth of the strain and the production of β-xylosidase when cultured. Transformant strain according to the invention is preferably a transgenic Saccharomyces my three Levy process jiae (Saccharomyces cerevisiae), and more preferably the deposit number KCTC 18382P of a transgenic Saccharomyces My process three Levy jiae (Saccharomyces cerevisiae ) pTSY01-XynB.

The method for producing beta -xylosidase according to the present invention comprises (b) culturing at a reaction temperature of 25 to 35 DEG C, preferably 28 to 32 DEG C, and a reaction pH of 7.5 to 8.5, preferably pH 7.8 to 8.2 . In the present invention, the culturing is preferably performed under the condition of 100 rpm to 400 rpm, and is effective for rapid growth and production of beta -xylosidase under the temperature and pH conditions.

The method for producing β-xylosidase according to the present invention provides (c) a method for producing β-xyloidesidase, which comprises separating β-xylosidase from the culture medium of step (b) . The step of isolating the β-xylosidase may be destroyed by various physical or chemical means such as freeze-thaw cycling, ultrasonication, mechanical destruction or cell disruption, and separation or purification by conventional biochemical separation techniques Is possible. For example, a method for separating or purifying a protein expressed by a strain includes electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immuno adsorption affinity chromatography, HPLC, gel permeation HPLC), isometric focus, and various variations or combinations thereof. On the other hand, in the present invention, the separation of β-xylosidase from the lysate of the transformant strain can be preferably carried out by affinity chromatography using a peptide tag. The peptide tag may be a known tag such as HA tag, FLAG tag, His tag, BCCP (biotin carboxyl carrier protein), c-myc tag, V5 tag, glutathione-S-transferase (GST) or MBP (maltose binding protein) , And a double His tag is preferable. The His-tagged protein is specifically trapped on a column of Ni-NTA (nickel-nitrilotriacetic acid) resin and is separable. Further, the separated? -Xylosidase may be further subjected to a step of concentrating after dialysis for use.

The β-xylosidase expression system according to the present invention provides β-xylosidase with a high conversion rate from xylose to xylobiose and enables mass production of xylobiose at a high yield in a short time do. Accordingly, it is possible to secure a large amount of new sugar supplement materials, and it can be used not only in the functional sugar industry but also in health food materials, medicines, cosmetic materials and the like.

Figure 1 shows a schematic diagram of a β-xylosidase expression construct.
FIG. 2 shows the results of β-xylosidase expression optimization conditions according to temperature conditions.
FIG. 3 shows the results of confirming the conditions for optimizing the expression of β-xylosidase according to pH conditions.
Fig. 4 shows the results of confirming conditions for optimizing expression of? -Xylosidase according to the carbon source condition.
Fig. 5 shows the results of xylobiose biosynthesis conversion results when treating 600 g / L xylose and 1-U-xylosidase.
Figure 6 shows the results of xylobiose biosynthesis conversion results at 600 g / L of xylose and 2 U of beta -xylosidase.
Fig. 7 shows the results of xylobiose biosynthesis conversion at the treatment of xylose 700 g / L and? -Xylosidase 1 U. Fig.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

≪ Example 1 > Synthesis of β-xylosidase ( XynB ) Expression vector cloning and expression system production

The DNA of Bacillus pumilus IPO xynB of SEQ ID NO: 12 was synthesized (2,201 bp), and a primer to which a restriction enzyme site was added was prepared, and β-xylosidase DNA was prepared by PCR.

A double-stranded β-xylosidase DNA synthesized to insert β-xylosidase into a vector (pTSY01) containing a GPD promoter that is strongly expressed in germ yeast was used as a template and a primer set PCR was performed. The vector comprising the GPD promoter has the nucleotide sequence of SEQ ID NO: 13.

Double enzyme digestion with Bam HI and Xho I was performed to insert XynB amplified by PCR in pTSY01. Beta -xylosidase < / RTI > The sequence has the nucleotide sequence of SEQ ID NO: 2. In addition, the pTSY01 vector was also cut with the same restriction enzyme. After cutting XynB and pTSY01 were purified by gel elution, ligation was performed using T4 ligase. After ligation of the DNA sample into E. coli , the ampicillin-resistant colony was screened and DNA sequencing using the following primer set was performed to ensure the sequencing of the nucleotide sequence of XynB .

In addition, for the continuous expression of XynB in the germline yeast cells expressing strongly by the GPD promoter, PCR was performed using the gDNA of germination yeast as a template and a primer set having the following sequence in order to insert a delta sequence having a transposon function .

The amplification delta sequence was amplified by PCR and inserted into pTSY01- XynB by enzyme digestion with Ngo MIV. At the same time, pTSY01- XynB was also cut with the same restriction enzyme. The cleaved delta sequence and pTSY01- XynB were purified by gel elution followed by ligation using T4 ligase. After ligation of the DNA sample into E. coli , the ampicillin-resistant colony was selected. DNA sequencing was performed using the following primer set to confirm the insertion of delta, and a β- xylosidase ( XynB ) expression vector Completed. A schematic diagram of the? -xylosidase expression vector is shown in FIG. 1 and has the nucleotide sequence of SEQ ID NO: 11.

≪ Example 2 > beta -xylosidase production Saccharomyces cerevisiae  Production of recombinant strains

The β- xylosidase ( XynB ) expression vector prepared above was transformed into Saccharomyces cerevisiae SK1 strain by the following method.

Saccharomyces cerevisiae SK1 strain was cultured on a YPG plate overnight at 30 ° C, and colonies were inoculated in 5 ml of YPD medium and cultured overnight at 30 ° C. Diluted in 10 ml of YPD medium at 1: 50 and cultured for 4 hours at 30 ° C. Then, the cells were harvested and centrifuged at 3,000 rpm for 5 minutes. The cell pellet was suspended in 1 ml of distilled water and centrifuged at 3,000 rpm for 5 minutes. The cell pellet was suspended in 1 ml of 10 mM LiAc and centrifuged again at 3,000 rpm for 5 minutes. The medium was removed and cells were supplemented with 480 ul of 50% PEG, 72 ul of 100 mM LiAc, 50 ul of 2 mg / ml single-stranded salmon sperm DNA and 1.0 ug of transforming plasmid DNA. Heat shock treatment was then performed at 42 ° C for 30 minutes and 0.7 ml of distilled water was added. The cells were centrifuged at 3,000 rpm for 5 minutes and collected. The medium was removed and the cell pellet was suspended in 1 ml YPD and transferred to a glass culture tube containing an additional 1 ml YPD. After further incubation at 30 ° C for 4 hours, the cells were harvested. Cells were suspended in 300 μl of distilled water and incubated on a selective plate at 30 ° C for 3 to 4 days.

The prepared strain was deposited on April 29, 2015 with the accession number KCTC18382P to the Microbiological Resource Center of the Korea Biotechnology Research Institute.

≪ Example 3 > S.cerevisiae  Optimization of β-xylosidase expression of recombinant strains Establishment of culture conditions

Optimization of Expression of β- xylosidase by the S. cerevisiae Recombinant Strain Culture was carried out under different conditions of temperature, pH and carbon source in order to establish the culture conditions.

The β-xylosidase activity assay was performed by the following method.

200 μL of 1 mM p-nitrophenyl-β-D-xylopyranoside and 200 μL of β-xylopyranoside were added and reacted at 37 ° C. for 10 minutes, and then 400 μL of 400 mM sodium bicarbonate was added to terminate the reaction. The activity of the enzyme was measured at 405 nm using p-nitrophenol as a standard, and 1 unit was used to measure 1 μM p-nitrophenol from p-nitrophenyl-β-D-xylopyranoside - Defined as the quantity of xylostadase.

The results of β-xylosidase expression changes according to temperature, pH and carbon source conditions in YPD medium (1% yeast extract, 2% peptone, 2% glucose) are shown in FIGS. 2 to 4.

Fig. 2 shows the results of the β-xylosidase expression optimization conditions according to the temperature conditions, and it was confirmed that the highest expression efficiency was obtained at about 30 ° C.

FIG. 3 shows the results of confirming the optimization conditions of β-xylosidase expression under pH conditions, and it was confirmed that the highest expression efficiency was obtained at about pH 8.

FIG. 4 shows the result of confirming the β-xylosidase expression optimization conditions according to the carbon source condition. At 4 hours after the culture, 1% carbon source was added to the basic medium to confirm the expression results. As a result, it was confirmed that the highest expression efficiency was obtained when glucose was used.

From the above results, 4% inoculation was performed on YPD medium (1% yeast extract, 2% peptone, 2% glucose) and the culture conditions were maintained at 30 ° C, 200 rpm, pH 8 for 22 hours.

<Example 4> S.cerevisiae  From the recombinant strain,? -Xylosidase purified

Β-xylosidase for xylobiose production was purified from the S. cerevisiae recombinant strain.

The cells cultured under the present culture conditions of Example 3 were collected and 50 mM Tri-HCl buffer (pH 7) was added to homogenize the cells. The cell wall was disrupted using an ultrasonic cell crusher and cell extract was obtained. Next, the enzyme was purified using a Ni-NTA column. The purified enzyme was dialyzed with 50 mM sodium phosphate buffer (pH 7) and concentrated to obtain β-xylosidase.

Example 5: Xylobiose biosynthesis reaction of? -Xylosidase

The efficiency of xylobiose biosynthesis was confirmed in comparison with xynB (E-BXSEBP), which is an enzyme of Megazyme, which commercially obtains β-xylosidase prepared in Example 4 above.

For comparison, the final concentration of the reaction solution was 1 U or 2 U, and the concentration of Xylose was 600 g / L and 700 g / L, respectively, and 50 mM sodium phosphate buffer (pH 7) Sampling was performed at 3, 6, 9, and 24 hours after incubation. After the sample was reacted, the enzyme was inactivated at 99 ° C for 10 minutes, and the concentration of xylobiose produced was quantified by HPLC using a Sugar-Pak I column (Waters, USA).

The results are shown in Figs. 5 to 7 and Tables 1 to 3.

Figure 5 and Table 1 show the results of treatment with 600 g / L xylose and 1 U of xylosidase.

[Table 1] Conversion rate of xylobiose biosynthesis (Xylose 600 g / L, treated with 1 U of enzyme)

The conversion rate of xylobiose biosynthesis was evaluated for 24 hours by treating the enzyme with 1 U of Xylose 600 g / L, and the conversion rate of pTSY01-XynB expression enzyme (Y_1U) was 8.1%. On the other hand, the commercial enzyme (M_1U), which is a commercial enzyme sold as a reagent, showed a conversion rate of only 6.3%, indicating that the β-xylosidase according to the present invention exhibited about 130% higher biosynthetic conversion rate than the commercial enzyme (M_1U) Respectively.

Figures 6 and Table 2 show the results of treatment with 600 g / L xylose and 2-U xylosidase.

[Table 2] Conversion rate of xylobiose biosynthesis (Xylose 600 g / L, treated with 2 U of enzyme)

The conversion rate of xylobiose biosynthesis was evaluated for 24 hours by treating the enzyme with 2 U at 600 g / L. As a result, the conversion rate of pTSY01-XynB expression enzyme (Y_2U) was 9.0%. On the other hand, it was confirmed that β-xylosidase according to the present invention exhibited about 156% higher biosynthetic conversion rate than the commercial enzyme (M_2U) sold as a reagent, while the commercial enzyme (M_2U) Respectively.

7 and Table 3 show the results of treatment with xylose 700 g / L and 1-xylosidase 1 U. FIG.

[Table 3] Conversion rate of xylobiose biosynthesis (Xylose 700 g / L, treated with 1 U of enzyme)

The conversion rate of xylobiose biosynthesis for 24 hours was evaluated by treating the enzyme with 1 U at 700 g / L. As a result, the conversion rate of pTSY01-XynB expression enzyme (Y_1U) was 10.5%. On the other hand, it was confirmed that β-xylosidase according to the present invention exhibited about 186% higher biosynthetic conversion rate than the commercial enzyme (M_1U), which is a commercial enzyme sold as a reagent (M_1U) Respectively.

From the above results, it was confirmed that the β-xylosidase of the present invention exhibits excellent xylobiose biosynthesis conversion ratio as compared with other enzymes.

Korea Biotechnology Research Institute KCTC18382P 20150429

<110> TS Corporation <120> Beta xylosidase expression system for producing xylobiose <130> P15-071-DHJ <160> 21 <170> Kopatentin 2.0 <210> 1 <211> 535 <212> PRT <213> Artificial Sequence <220> <223> beta xylosidase <400> 1 Met Lys Ile Thr Asn Pro Val Leu Lys Gly Phe Asn Pro Asp Pro Ser   1 5 10 15 Ile Cys Arg Ala Gly Glu Asp Tyr Tyr Met Ala Val Ser Thr Phe Glu              20 25 30 Trp Phe Pro Gly Val Gln Ile Tyr His Ser Lys Asp Leu Ile His Trp          35 40 45 Arg Leu Ala Ala Arg Pro Leu Gln Lys Thr Ser Gln Leu Asp Met Lys      50 55 60 Gly Asn Pro Asp Ser Gly Gly Val Trp Ala Pro Cys Leu Ser Tyr Ala  65 70 75 80 Asp Gly Gln Phe Trp Leu Ile Tyr Ser Asp Ile Lys Val Val Asp Gly                  85 90 95 Pro Phe Lys Asp Gly His Asn Tyr Leu Val Thr Ala Asp Ala Val Asp             100 105 110 Gly Glu Trp Ser Asp Pro Val Arg Leu Asn Ser Ser Gly Phe Asp Pro         115 120 125 Ser Leu Phe His Asp Pro Ser Gly Lys Lys Tyr Val Leu Asn Met Leu     130 135 140 Trp Asp His Arg Glu Lys His His Ser Phe Ala Gly Ile Ala Leu Gln 145 150 155 160 Glu Tyr Ser Val Ser Glu Lys Lys Leu Val Gly Glu Arg Lys Val Ile                 165 170 175 Phe Lys Gly Thr Pro Ile Lys Leu Thr Glu Ala Pro His Leu Tyr Tyr             180 185 190 Ile Asn Asp Val Tyr Tyr Leu Leu Thr Ala Glu Gly Gly Thr Arg Tyr         195 200 205 Glu His Ala Ala Thr Ile Ala Arg Ser Ser Arg Ile Asp Gly Pro Tyr     210 215 220 Glu Val His Pro Asp Asn Pro Ile Leu Thr Ala Phe His Ala Pro Ser 225 230 235 240 His Pro Leu Gln Lys Cys Gly His Ala Ser Ile Val Gln Thr His Thr                 245 250 255 Asn Glu Trp Tyr Leu Ala His Leu Thr Gly Arg Pro Ile His Ser Ser             260 265 270 Lys Glu Ser Ile Phe Gln Gln Arg Gly Trp Cys Pro Leu Gly Arg Glu         275 280 285 Thr Ala Ile Gln Lys Leu Glu Trp Lys Asp Gly Trp Pro Tyr Val Val     290 295 300 Gly Gly Lys Glu Gly Leu Leu Glu Val Glu Ala Pro Ala Met Ser Val 305 310 315 320 Lys Glu Phe Ser Pro Thr Tyr His Ile Val Asp Glu Phe Lys Asp Ser                 325 330 335 Ser Leu Asn Arg His Phe Gln Thr Leu Arg Ile Pro Phe Thr Asp Gln             340 345 350 Ile Gly Ser Val Thr Glu Asn Pro His His Leu Arg Leu Tyr Gly Gln         355 360 365 Glu Ser Leu Thr Ser Lys Phe Thr Gln Ala Phe Val Ala Arg Arg Trp     370 375 380 Gln Ser Phe Tyr Phe Glu Ala Glu Thr Ala Val Ser Phe Phe Pro Lys 385 390 395 400 Asn Phe Gln Gln Ala Ala Gly Leu Val Asn Tyr Tyr Asn Thr Glu Asn                 405 410 415 Trp Thr Ala Leu Gln Val Thr Tyr Asp Asp Ala Leu Gly Arg Ile Leu             420 425 430 Glu Leu Ser Val Cys Glu Asn Leu Ala Phe Ser Gln Pro Leu Ile Lys         435 440 445 Lys Ile Ile Pro Asp Glu Ile Pro Tyr Val Tyr Leu Lys Val Thr     450 455 460 Val Gln Arg Glu Thr Tyr Thr Tyr Ser Tyr Ser Phe Asp Gln Gln Glu 465 470 475 480 Trp Glu Lys Ile Asp Val Pro Leu Glu Ser Thr His Leu Ser Asp Asp                 485 490 495 Phe Ile Arg Gly Gly Gly Phe Phe Thr Gly Ala Phe Val Gly Met Gln             500 505 510 Cys Gln Asp Thr Ser Gly Glu Arg Leu Pro Ala Asp Phe Lys Tyr Phe         515 520 525 Arg Tyr Glu Glu Thr Thr Glu     530 535 <210> 2 <211> 1608 <212> DNA <213> Artificial Sequence <220> <223> beta xylosidase <400> 2 ttgaagatta ctaatccagt gctcaaagga tttaaccctg acccaagtat ttgccgtgcg 60 ggagaggact attacatggc ggtttctaca tttgaatggt tcccgggggt gcaaatttat 120 cactcgaagg atctcatcca ttggcggctc gccgcgcgtc ccttgcaaaa aacttctcaa 180 ctcgatatga aaggaaatcc tgattccggc ggggtatggg caccgtgttt aagttatgcg 240 gatggtcagt tttggctcat ctattcagat attaaagtag tagatggtcc gtttaaggat 300 ggtcataatt atttggtcac agctgatgca gtagatggtg agtggagcga tccggttcgg 360 ctcaatagtt ctgggtttga tccttccttg ttccatgatc caagcggaaa gaaatacgtg 420 ttgaatatgc tgtgggacca tcgagaaaaa catcactcct ttgcaggtat agccttgcag 480 gaatatagtg tatctgaaaa aaaactggtc ggtgagcgga aggtcatttt taaaggcacg 540 ccaatcaaac tcacagaagc cccgcatctt tattacatca atgatgtcta ttatttatta 600 acagctgaag ggggaacacg ttacgaacat gcagccacaa tcgcccgttc ctcgcgtatt 660 gatgggccgt acgaggttca tcctgacaac ccaattttaa cggcttttca cgcgcctagc 720 catccactac aaaaatgcgg gcatgcttcc attgtacaaa cgcatacaaa tgaatggtat 780 ttggctcatc tgactggccg cccaattcat tcaagtaagg aatccatttt tcagcaaaga 840 ggctggtgcc ctttaggaag agagacagcc atacaaaagc ttgaatggaa agatggctgg 900 ccgtatgtgg taggcggaaa agaggggctc ctagaggttg aagcgcctgc gatgagcgta 960 aaagagttct ctccaaccta tcacatagtc gatgaattta aagattcatc gttaaataga 1020 catttccaaa cattaagaat cccatttacg gatcagattg gttcagtgac ggaaaatcct 1080 catcatttaa ggctgtatgg gcaggaatct ttaacgtcta aatttaccca agcgtttgtt 1140 gcgaggcgct ggcaaagctt ttattttgaa gcagagacag cggtttcctt ctttccgaaa 1200 aactttcagc aagcggcagg tcttgtgaat tattataata cggaaaattg gactgcactt 1260 caggtgactt atgacgatgc gcttggccgt atccttgagt tatctgtgtg tgaaaacctg 1320 gccttttctc agccgctaat taaaaaaatc atcatcccag acgagattcc gtatgtgtat 1380 ttaaaagtga ccgttcagag agagacgtac acgtattctt attcttttga tcaacaagag 1440 tgggaaaaaa tagatgtacc gcttgaatcg acccatttgt cagatgattt tattcgagga 1500 ggggggttct ttacaggggc ttttgttgga atgcagtgcc aagacacaag cggtgaacgt 1560 ctgcctgccg attttaagta ttttcgatac gaagaaacaa ctgaataa 1608 <210> 3 <211> 673 <212> DNA <213> Artificial Sequence <220> <223> GPD promoter <400> 3 acaaaagctg gagctcagtt tatcattatc aatactcgcc atttcaaaga atacgtaaat 60 aattaatagt agtgattttc ctaactttat ttagtcaaaa aattagcctt ttaattctgc 120 tgtaacccgt acatgcccaa aatagggggc gggttacaca gaatatataa catcgtaggt 180 gtctgggtga acagtttatt cctggcatcc actaaatata atggagcccg ctttttaagc 240 tggcatccag aaaaaaaaag aatcccagca ccaaaatatt gttttcttca ccaaccatca 300 gttcataggt ccattctctt agcgcaacta cagagaacag gggcacaaac aggcaaaaaa 360 cgggcacaac ctcaatggag tgatgcaacc tgcctggagt aaatgatgac acaaggcaat 420 tgacccacgc atgtatctat ctcattttct tacaccttct attaccttct gctctctctg 480 atttggaaaa agctgaaaaa aaaggttgaa accagttccc tgaaattatt cccctacttg 540 actaataagt atataaagac ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa 600 cttcttaaat tctactttta tagttagtct tttttttagt tttaaaacac cagaacttag 660 tttcgacgga ttc 673 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> His tag <400> 4 atgggcagca gccatcatca tcatcatcac agcagcggcc at 42 <210> 5 <211> 332 <212> DNA <213> Artificial Sequence <220> <223> Delta <400> 5 gagaattggg tgaatgttga gataattgtt gggattccat ttttaataag gcaataatat 60 taggtatgta gaatgtacta gaagttctcc tcgaggattt aggaatccat aaaagggaat 120 ctgcaattct acacaattct ataaatatta ttatcatcat tttatatgtt aatattcatt 180 gatcctatta cattatcaat ccttgcgttt cagcttccac taatttagat gactatttct 240 catcatttgc gtcatcttct aacaccgtat atgataatat actagtaacg taaatactag 300 ttagtagatg atagttgatt tctattccaa ca 332 <210> 6 <211> 1337 <212> DNA <213> Artificial Sequence <220> <223> 2-micron <400> 6 caaaggaaat gatagcattg aaggatgaga ctaatccaat tgaggagtgg cagcatatag 60 aacagctaaa gggtagtgct gaaggaagca tacgataccc cgcatggaat gggataatat 120 cacaggaggt actagactac ctttcatcct acataaatag acgcatataa gtacgcattt 180 aagcataaac acgcactatg ccgttcttct catgtatata tatatacagg caacacgcag 240 atataggtgc gacgtgaaca gtgagctgta tgtgcgcagc tcgcgttgca ttttcggaag 300 cgctcgtttt cggaaacgct ttgaagttcc tattccgaag ttcctattct ctagaaagta 360 taggaacttc agagcgcttt tgaaaaccaa aagcgctctg aagacgcact ttcaaaaaac 420 caaaaacgca ccggactgta acgagctact aaaatattgc gaataccgct tccacaaaca 480 ttgctcaaaa gtatctcttt gctatatatc tctgtgctat atccctatat aacctaccca 540 tccacctttc gctccttgaa cttgcatcta aactcgacct ctacattttt tatgtttatc 600 tctagtatta ctctttagac aaaaaaattg tagtaagaac tattcataga gtgaatcgaa 660 aacaatacga aaatgtaaac atttcctata cgtagtatat agagacaaaa tagaagaaac 720 cgttcataat tttctgacca atgaagaatc atcaacgcta tcactttctg ttcacaaagt 780 atgcgcaatc cacatcggta tagaatataa tcggggatgc ctttatcttg aaaaaatgca 840 cccgcagctt cgctagtaat cagtaaacgc gggaagtgga gtcaggcttt ttttatggaa 900 gagaaaatag acaccaaagt agccttcttc taaccttaac ggacctacag tgcaaaaagt 960 tatcaagaga ctgcattata gagcgcacaa aggagaaaaa aagtaatcta agatgctttg 1020 ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca aaaaagaagt atagattctt 1080 tgttggtaaa atagcgctct cgcgttgcat ttctgttctg taaaaatgca gctcagattc 1140 tttgtttgaa aaattagcgc tctcgcgttg catttttgtt ttacaaaaat gaagcacaga 1200 ttcttcgttg gtaaaatagc gctttcgcgt tgcatttctg ttctgtaaaa atgcagctca 1260 gattctttgt ttgaaaaatt agcgctctcg cgttgcattt ttgttctaca aaatgaagca 1320 cagatgcttc gttcagg 1337 <210> 7 <211> 931 <212> DNA <213> Artificial Sequence <220> <223> Ampicillin resistance gene (amp) <400> 7 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 60 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 120 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 180 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 240 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 300 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 360 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 420 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 480 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 540 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 600 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 660 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 720 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 780 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 840 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 900 tttactcata tatactttag attgatttaa a 931 <210> 8 <211> 261 <212> DNA <213> Artificial Sequence <220> <223> CYC1 Terminator <400> 8 cgagtcatgt aattagttat gtcacgctta cattcacgcc ctccccccac atccgctcta 60 accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt 120 atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc 180 gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag 240 gctttaattt gcggccggta c 261 <210> 9 <211> 1015 <212> DNA <213> Artificial Sequence <220> <223> TRP <400> 9 tctgtgcggt atttcacacc gcatagatcg gcaagtgcac aaacaatact taaataaata 60 ctactcagta ataacctatt tcttagcatt tttgacgaaa tttgctattt tgttagagtc 120 ttttacacca tttgtctcca cacctccgct tacatcaaca ccaataacgc catttaatct 180 aagcgcatca ccaacatttt ctggcgtcag tccaccagct aacataaaat gtaagctttc 240 ggggctctct tgccttccaa cccagtcaga aatcgagttc caatccaaaa gttcacctgt 300 cccacctgct tctgaatcaa acaagggaat aaacgaatga ggtttctgtg aagctgcact 360 gagtagtatg ttgcagtctt ttggaaatac gagtctttta ataactggca aaccgaggaa 420 ctcttggtat tcttgccacg actcatctcc atgcagttgg acgatatcaa tgccgtaatc 480 attgaccaga gccaaaacat cctccttagg ttgattacga aacacgccaa ccaagtattt 540 cggagtgcct gaactatttt tatatgcttt tacaagactt gaaattttcc ttgcaataac 600 cgggtcaatt gttctctttc tattgggcac acatataata cccagcaagt cagcatcgga 660 atctagagca cattctgcgg cctctgtgct ctgcaagccg caaactttca ccaatggacc 720 agaactacct gtgaaattaa taacagacat actccaagct gcctttgtgt gcttaatcac 780 gtatactcac gtgctcaata gtcaccaatg ccctccctct tggccctctc cttttctttt 840 ttcgaccgaa ttaattctta atcggcaaaa aaagaaaagc tccggatcaa gattgtacgt 900 aaggtgacaa gctatttttc aataaagaat atcttccact actgccatct ggcgtcataa 960 ctgcaaagta cacatatatt acgatgctgt ctattaaatg cttcctatat tatat 1015 <210> 10 <211> 6836 <212> DNA <213> Artificial Sequence <220> <223> expression construct <400> 10 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg 120 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg 180 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac 240 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata 300 tatacgca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc 360 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt 420 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa 480 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga 540 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat 600 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct 660 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta 720 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag 780 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc 840 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct 900 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt 960 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg 1020 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa 1080 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa 1140 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta 1200 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt 1260 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt 1320 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt 1380 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc 1440 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 1500 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 1560 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 1620 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 1680 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 1740 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 1800 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1860 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 1920 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1980 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 2040 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 2100 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 2160 gctggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 2220 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 2280 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 2340 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 2400 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 2460 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 2520 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 2580 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 2640 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 2700 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 2760 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 2820 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2880 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2940 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 3000 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 3060 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 3120 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 3180 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 3240 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 3300 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 3360 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 3420 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc 3480 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa 3540 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa 3600 agggaacaaa agctggagct cagtttatca ttatcaatac tcgccatttc aaagaatacg 3660 taaataatta atagtagtga ttttcctaac tttatttagt caaaaaatta gccttttaat 3720 tctgctgtaa cccgtacatg cccaaaatag ggggcgggtt acacagaata tataacatcg 3780 taggtgtctg ggtgaacagt ttattcctgg catccactaa atataatgga gcccgctttt 3840 taagctggca tccagaaaaa aaaagaatcc cagcaccaaa atattgtttt cttcaccaac 3900 catcagttca taggtccatt ctcttagcgc aactacagag aacaggggca caaacaggca 3960 aaaaacgggc acaacctcaa tggagtgatg caacctgcct ggagtaaatg atgacacaag 4020 gcaattgacc cacgcatgta tctatctcat tttcttacac cttctattac cttctgctct 4080 ctctgatttg gaaaaagctg aaaaaaaagg ttgaaaccag ttccctgaaa ttattcccct 4140 acttgactaa taagtatata aagacggtag gtattgattg taattctgta aatctatttc 4200 ttaaacttct taaattctac ttttatagtt agtctttttt ttagttttaa aacaccagaa 4260 cttagtttcg acggattcta gaactagtgg atccatgggc agcagccatc atcatcatca 4320 tcacagcagc ggccatctcg agtcatgtaa ttagttatgt cacgcttaca ttcacgccct 4380 ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct 4440 atttattttt ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt 4500 ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag 4560 gtttgggac gctcgaaggc tttaatttgc ggccggtacc caattcgccc tatagtgagt 4620 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 4680 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 4740 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcgacg 4800 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 4860 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 4920 tcgccggctg agaattgggt gaatgttgag ataattgttg ggattccatt tttaataagg 4980 caataatatt aggtatgtag aatgtactag aagttctcct cgaggattta ggaatccata 5040 aaagggaatc tgcaattcta cacaattcta taaatattat tatcatcatt ttatatgtta 5100 atattcattg atcctattac attatcaatc cttgcgtttc agcttccact aatttagatg 5160 actatttctc atcatttgcg tcatcttcta acaccgtata tgataatata ctagtaacgt 5220 aaatactagt tagtagatga tagttgattt ctattccaac agccggcttt ccccgtcaag 5280 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 5340 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 5400 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 5460 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 5520 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 5580 cgtttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 5640 catagatcgg caagtgcaca aacaatactt aaataaatac tactcagtaa taacctattt 5700 cttagcattt ttgacgaaat ttgctatttt gttagagtct tttacaccat ttgtctccac 5760 acctccgctt acatcaacac caataacgcc atttaatcta agcgcatcac caacattttc 5820 tggcgtcagt ccaccagcta acataaaatg taagctttcg gggctctctt gccttccaac 5880 ccagtcagaa atcgagttcc aatccaaaag ttcacctgtc ccacctgctt ctgaatcaaa 5940 caagggaata aacgaatgag gtttctgtga agctgcactg agtagtatgt tgcagtcttt 6000 tggaaatacg agtcttttaa taactggcaa accgaggaac tcttggtatt cttgccacga 6060 ctcatctcca tgcagttgga cgatatcaat gccgtaatca ttgaccagag ccaaaacatc 6120 ctccttaggt tgattacgaa acacgccaac caagtatttc ggagtgcctg aactattttt 6180 atatgctttt acaagacttg aaattttcct tgcaataacc gggtcaattg ttctctttct 6240 attgggcaca catataatac ccagcaagtc agcatcggaa tctagagcac attctgcggc 6300 ctctgtgctc tgcaagccgc aaactttcac caatggacca gaactacctg tgaaattaat 6360 aacagacata ctccaagctg cctttgtgtg cttaatcacg tatactcacg tgctcaatag 6420 tcaccaatgc cctccctctt ggccctctcc ttttcttttt tcgaccgaat taattcttaa 6480 tcggcaaaaa aagaaaagct ccggatcaag attgtacgta aggtgacaag ctatttttca 6540 ataaagaata tcttccacta ctgccatctg gcgtcataac tgcaaagtac acatatatta 6600 cgatgctgtc tattaaatgc ttcctatatt atatatatag taatgtcgtt tatggtgcac 6660 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc 6720 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 6780 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcga 6836 <210> 11 <211> 8444 <212> DNA <213> Artificial Sequence <220> <223> beta xylosidase expression construct <400> 11 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg 120 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg 180 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac 240 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata 300 tatacgca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc 360 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt 420 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa 480 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga 540 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat 600 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct 660 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta 720 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag 780 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc 840 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct 900 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt 960 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg 1020 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa 1080 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa 1140 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta 1200 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt 1260 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt 1320 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt 1380 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc 1440 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 1500 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 1560 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 1620 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 1680 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 1740 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 1800 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1860 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 1920 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1980 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 2040 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 2100 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 2160 gctggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 2220 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 2280 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 2340 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 2400 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 2460 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 2520 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 2580 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 2640 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 2700 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 2760 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 2820 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2880 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2940 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 3000 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 3060 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 3120 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 3180 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 3240 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 3300 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 3360 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 3420 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc 3480 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa 3540 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa 3600 agggaacaaa agctggagct cagtttatca ttatcaatac tcgccatttc aaagaatacg 3660 taaataatta atagtagtga ttttcctaac tttatttagt caaaaaatta gccttttaat 3720 tctgctgtaa cccgtacatg cccaaaatag ggggcgggtt acacagaata tataacatcg 3780 taggtgtctg ggtgaacagt ttattcctgg catccactaa atataatgga gcccgctttt 3840 taagctggca tccagaaaaa aaaagaatcc cagcaccaaa atattgtttt cttcaccaac 3900 catcagttca taggtccatt ctcttagcgc aactacagag aacaggggca caaacaggca 3960 aaaaacgggc acaacctcaa tggagtgatg caacctgcct ggagtaaatg atgacacaag 4020 gcaattgacc cacgcatgta tctatctcat tttcttacac cttctattac cttctgctct 4080 ctctgatttg gaaaaagctg aaaaaaaagg ttgaaaccag ttccctgaaa ttattcccct 4140 acttgactaa taagtatata aagacggtag gtattgattg taattctgta aatctatttc 4200 ttaaacttct taaattctac ttttatagtt agtctttttt ttagttttaa aacaccagaa 4260 cttagtttcg acggattcta gaactagtgg atccatgggc agcagccatc atcatcatca 4320 tcacagcagc ggccatttga agattactaa tccagtgctc aaaggattta accctgaccc 4380 aagtatttgc cgtgcgggag aggactatta catggcggtt tctacatttg aatggttccc 4440 gggggtgcaa atttatcact cgaaggatct catccattgg cggctcgccg cgcgtccctt 4500 gcaaaaaact tctcaactcg atatgaaagg aaatcctgat tccggcgggg tatgggcacc 4560 gtgtttaagt tatgcggatg gtcagttttg gctcatctat tcagatatta aagtagtaga 4620 tggtccgttt aaggatggtc ataattattt ggtcacagct gatgcagtag atggtgagtg 4680 gagcgatccg gttcggctca atagttctgg gtttgatcct tccttgttcc atgatccaag 4740 cggaaagaaa tacgtgttga atatgctgtg ggaccatcga gaaaaacatc actcctttgc 4800 aggtatagcc ttgcaggaat atagtgtatc tgaaaaaaaa ctggtcggtg agcggaaggt 4860 catttttaaa ggcacgccaa tcaaactcac agaagccccg catctttatt acatcaatga 4920 tgtctattat ttattaacag ctgaaggggg aacacgttac gaacatgcag ccacaatcgc 4980 ccgttcctcg cgtattgatg ggccgtacga ggttcatcct gacaacccaa ttttaacggc 5040 ttttcacgcg cctagccatc cactacaaaa atgcgggcat gcttccattg tacaaacgca 5100 tacaaatgaa tggtatttgg ctcatctgac tggccgccca attcattcaa gtaaggaatc 5160 catttttcag caaagaggct ggtgcccttt aggaagagag acagccatac aaaagcttga 5220 atggaaagat ggctggccgt atgtggtagg cggaaaagag gggctcctag aggttgaagc 5280 gcctgcgatg agcgtaaaag agttctctcc aacctatcac atagtcgatg aatttaaaga 5340 ttcatcgtta aatagacatt tccaaacatt aagaatccca tttacggatc agattggttc 5400 agtgacggaa aatcctcatc atttaaggct gtatgggcag gaatctttaa cgtctaaatt 5460 tacccaagcg tttgttgcga ggcgctggca aagcttttat tttgaagcag agacagcggt 5520 ttccttcttt ccgaaaaact ttcagcaagc ggcaggtctt gtgaattatt ataatacgga 5580 aaattggact gcacttcagg tgacttatga cgatgcgctt ggccgtatcc ttgagttatc 5640 tgtgtgtgaa aacctggcct tttctcagcc gctaattaaa aaaatcatca tcccagacga 5700 gattccgtat gtgtatttaa aagtgaccgt tcagagagag acgtacacgt attcttattc 5760 ttttgatcaa caagagtggg aaaaaataga tgtaccgctt gaatcgaccc atttgtcaga 5820 tgattttatt cgaggagggg ggttctttac aggggctttt gttggaatgc agtgccaaga 5880 cacaagcggt gaacgtctgc ctgccgattt taagtatttt cgatacgaag aaacaactga 5940 ataactcgag tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc 6000 gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt 6060 atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac 6120 agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc 6180 tcgaaggctt taatttgcgg ccggtaccca attcgcccta tagtgagtcg tattacgcgc 6240 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 6300 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 6360 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcgacgcg ccctgtagcg 6420 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6480 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctgag 6540 aattgggtga atgttgagat aattgttggg attccatttt taataaggca ataatattag 6600 gtatgtagaa tgtactagaa gttctcctcg aggatttagg aatccataaa agggaatctg 6660 caattctaca caattctata aatattatta tcatcatttt atatgttaat attcattgat 6720 cctattacat tatcaatcct tgcgtttcag cttccactaa tttagatgac tatttctcat 6780 catttgcgtc atcttctaac accgtatatg ataatatact agtaacgtaa atactagtta 6840 gtagatgata gttgatttct attccaacag ccggctttcc ccgtcaagct ctaaatcggg 6900 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 6960 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 7020 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 7080 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 7140 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt 7200 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tagatcggca 7260 agtgcacaaa caatacttaa ataaatacta ctcagtaata acctatttct tagcattttt 7320 gacgaaattt gctattttgt tagagtcttt tacaccattt gtctccacac ctccgcttac 7380 atcaacacca ataacgccat ttaatctaag cgcatcacca acattttctg gcgtcagtcc 7440 accagctaac ataaaatgta agctttcggg gctctcttgc cttccaaccc agtcagaaat 7500 cgagttccaa tccaaaagtt cacctgtccc acctgcttct gaatcaaaca agggaataaa 7560 cgaatgaggt ttctgtgaag ctgcactgag tagtatgttg cagtcttttg gaaatacgag 7620 tcttttaata actggcaaac cgaggaactc ttggtattct tgccacgact catctccatg 7680 cagttggacg atatcaatgc cgtaatcatt gaccagagcc aaaacatcct ccttaggttg 7740 attacgaaac acgccaacca agtatttcgg agtgcctgaa ctatttttat atgcttttac 7800 aagacttgaa attttccttg caataaccgg gtcaattgtt ctctttctat tgggcacaca 7860 tataataccc agcaagtcag catcggaatc tagagcacat tctgcggcct ctgtgctctg 7920 caagccgcaa actttcacca atggaccaga actacctgtg aaattaataa cagacatact 7980 ccaagctgcc tttgtgtgct taatcacgta tactcacgtg ctcaatagtc accaatgccc 8040 tccctcttgg ccctctcctt ttcttttttc gaccgaatta attcttaatc ggcaaaaaaa 8100 gaaaagctcc ggatcaagat tgtacgtaag gtgacaagct atttttcaat aaagaatatc 8160 ttccactact gccatctggc gtcataactg caaagtacac atatattacg atgctgtcta 8220 ttaaatgctt cctatattat atatatagta atgtcgttta tggtgcactc tcagtacaat 8280 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc 8340 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 8400 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcga 8444 <210> 12 <211> 2201 <212> DNA <213> Artificial Sequence <220> <223> Bacillus pumilus IPO xynB <400> 12 ctgcagggag tattacagca ggtgcttaca tgtgggcact cattcctgaa accgttgaat 60 atggcgaata taaaacaggg aaaagaatgg gtggactgat ttacgcagtg attggcttct 120 tctttaaatt tgggatggcc ttaggcggca tcgttccagg gcttgtcctt gaccgatttg 180 gttatattgc aaatcaagtg caaacaccag aagctttact gggcatttta atcacgacaa 240 cggtgattcc tgtatgctta ctcattttag cgatggttga tattaatttt tataacctag 300 atgaagaaaa acataaaaaa gtcatacgag agcttgagaa tagagacaag gtgtatatcg 360 atcatatgga cgattttaaa atgtagttct ctgaaagagg aggaagttca attgaagatt 420 actaatccag tgctcaaagg atttaaccct gacccaagta tttgccgtgc gggagaggac 480 tattacatgg cggtttctac atttgaatgg ttcccggggg tgcaaattta tcactcgaag 540 gatctcatcc attggcggct cgccgcgcgt cccttgcaaa aaacttctca actcgatatg 600 aaaggaaatc ctgattccgg cggggtatgg gcaccgtgtt taagttatgc ggatggtcag 660 ttttggctca tctattcaga tattaaagta gtagatggtc cgtttaagga tggtcataat 720 tatttggtca cagctgatgc agtagatggt gagtggagcg atccggttcg gctcaatagt 780 tctgggtttg atccttcctt gttccatgat ccaagcggaa agaaatacgt gttgaatatg 840 ctgtgggacc atcgagaaaa acatcactcc tttgcaggta tagccttgca ggaatatagt 900 gtatctgaaa aaaaactggt cggtgagcgg aaggtcattt ttaaaggcac gccaatcaaa 960 ctcacagaag ccccgcatct ttattacatc aatgatgtct attatttatt aacagctgaa 1020 gggggaacac gttacgaaca tgcagccaca atcgcccgtt cctcgcgtat tgatgggccg 1080 tacgaggttc atcctgacaa cccaatttta acggcttttc acgcgcctag ccatccacta 1140 caaaaatgcg ggcatgcttc cattgtacaa acgcatacaa atgaatggta tttggctcat 1200 ctgactggcc gcccaattca ttcaagtaag gaatccattt ttcagcaaag aggctggtgc 1260 cctttaggaa gagagacagc catacaaaag cttgaatgga aagatggctg gccgtatgtg 1320 gtaggcggaa aagaggggct cctagaggtt gaagcgcctg cgatgagcgt aaaagagttc 1380 tctccaacct atcacatagt cgatgaattt aaagattcat cgttaaatag acatttccaa 1440 acattaagaa tcccatttac ggatcagatt ggttcagtga cggaaaatcc tcatcattta 1500 aggctgtatg ggcaggaatc tttaacgtct aaatttaccc aagcgtttgt tgcgaggcgc 1560 tggcaaagct tttattttga agcagagaca gcggtttcct tctttccgaa aaactttcag 1620 caagcggcag gtcttgtgaa ttattataat acggaaaatt ggactgcact tcaggtgact 1680 tatgacgatg cgcttggccg tatccttgag ttatctgtgt gtgaaaacct ggccttttct 1740 cagccgctaa ttaaaaaaat catcatccca gacgagattc cgtatgtgta tttaaaagtg 1800 accgttcaga gagagacgta cacgtattct tattcttttg atcaacaaga gtgggaaaaa 1860 atagatgtac cgcttgaatc gacccatttg tcagatgatt ttattcgagg aggggggttc 1920 tttacagggg cttttgttgg aatgcagtgc caagacacaa gcggtgaacg tctgcctgcc 1980 gattttaagt attttcgata cgaagaaaca actgaataaa aaaagacaac actctaaagg 2040 ctgtctctct ttaacatgta ttccatcatg gtgaaggcgt gacatcacgc aaaaaagctt 2100 cagtgacaag agatgtcatc cctaaggctg gtgcgttttg tcctaatgat gaaggaagca 2160 gctccacccg tgagctgaat tgtgcatcca ttctagaatt c 2201 <210> 13 <211> 6504 <212> DNA <213> Artificial Sequence <220> <223> pTSY01 expression construct <400> 13 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg 120 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg 180 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac 240 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata 300 tatacgca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc 360 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt 420 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa 480 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga 540 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat 600 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct 660 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta 720 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag 780 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc 840 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct 900 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt 960 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg 1020 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa 1080 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa 1140 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta 1200 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt 1260 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt 1320 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt 1380 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc 1440 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 1500 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 1560 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 1620 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 1680 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 1740 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 1800 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 1860 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 1920 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 1980 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 2040 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 2100 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 2160 gctggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 2220 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 2280 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 2340 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 2400 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 2460 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 2520 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 2580 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 2640 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 2700 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 2760 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 2820 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 2880 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 2940 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 3000 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 3060 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 3120 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 3180 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 3240 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 3300 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 3360 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 3420 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc 3480 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa 3540 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa 3600 agggaacaaa agctggagct cagtttatca ttatcaatac tcgccatttc aaagaatacg 3660 taaataatta atagtagtga ttttcctaac tttatttagt caaaaaatta gccttttaat 3720 tctgctgtaa cccgtacatg cccaaaatag ggggcgggtt acacagaata tataacatcg 3780 taggtgtctg ggtgaacagt ttattcctgg catccactaa atataatgga gcccgctttt 3840 taagctggca tccagaaaaa aaaagaatcc cagcaccaaa atattgtttt cttcaccaac 3900 catcagttca taggtccatt ctcttagcgc aactacagag aacaggggca caaacaggca 3960 aaaaacgggc acaacctcaa tggagtgatg caacctgcct ggagtaaatg atgacacaag 4020 gcaattgacc cacgcatgta tctatctcat tttcttacac cttctattac cttctgctct 4080 ctctgatttg gaaaaagctg aaaaaaaagg ttgaaaccag ttccctgaaa ttattcccct 4140 acttgactaa taagtatata aagacggtag gtattgattg taattctgta aatctatttc 4200 ttaaacttct taaattctac ttttatagtt agtctttttt ttagttttaa aacaccagaa 4260 cttagtttcg acggattcta gaactagtgg atccatgggc agcagccatc atcatcatca 4320 tcacagcagc ggccatctcg agtcatgtaa ttagttatgt cacgcttaca ttcacgccct 4380 ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct 4440 atttattttt ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt 4500 ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag 4560 gtttgggac gctcgaaggc tttaatttgc ggccggtacc caattcgccc tatagtgagt 4620 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 4680 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 4740 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcgacg 4800 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 4860 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 4920 tcgccggctg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 4980 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 5040 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 5100 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 5160 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 5220 tttaacgcga attttaacaa aatattaacg tttacaattt cctgatgcgg tattttctcc 5280 ttacgcatct gtgcggtatt tcacaccgca tagatcggca agtgcacaaa caatacttaa 5340 ataaatacta ctcagtaata acctatttct tagcattttt gacgaaattt gctattttgt 5400 tagagtcttt tacaccattt gtctccacac ctccgcttac atcaacacca ataacgccat 5460 ttaatctaag cgcatcacca acattttctg gcgtcagtcc accagctaac ataaaatgta 5520 agctttcggg gctctcttgc cttccaaccc agtcagaaat cgagttccaa tccaaaagtt 5580 cacctgtccc acctgcttct gaatcaaaca agggaataaa cgaatgaggt ttctgtgaag 5640 ctgcactgag tagtatgttg cagtcttttg gaaatacgag tcttttaata actggcaaac 5700 cgaggaactc ttggtattct tgccacgact catctccatg cagttggacg atatcaatgc 5760 cgtaatcatt gaccagagcc aaaacatcct ccttaggttg attacgaaac acgccaacca 5820 agtatttcgg agtgcctgaa ctatttttat atgcttttac aagacttgaa attttccttg 5880 caataaccgg gtcaattgtt ctctttctat tgggcacaca tataataccc agcaagtcag 5940 catcggaatc tagagcacat tctgcggcct ctgtgctctg caagccgcaa actttcacca 6000 atggaccaga actacctgtg aaattaataa cagacatact ccaagctgcc tttgtgtgct 6060 taatcacgta tactcacgtg ctcaatagtc accaatgccc tccctcttgg ccctctcctt 6120 ttcttttttc gaccgaatta attcttaatc ggcaaaaaaa gaaaagctcc ggatcaagat 6180 tgtacgtaag gtgacaagct atttttcaat aaagaatatc ttccactact gccatctggc 6240 gtcataactg caaagtacac atatattacg atgctgtcta ttaaatgctt cctatattat 6300 atatatagta atgtcgttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt 6360 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 6420 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 6480 caccgtcatc accgaaacgc gcga 6504 <210> 14 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> KK510 (BamH1) primer <400> 14 atatggatcc atgggcagca gccatcatca tcatcatcac agcagcggcc atttgaagat 60 tactaatcca gtgct 75 <210> 15 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> KK511 (Xho1) primer <400> 15 atatctcgag ttattcagtt gtttcttcgt at 32 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KK518 primer <400> 16 agccccgcat ctttattaca 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KK519 primer <400> 17 ctcttttccg cctaccacat 20 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> KK563 (NgoMIV) primer <400> 18 atatgccggc tgttggaata gaaatcaact atcat 35 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> KK564 (NgoMIV) primer <400> 19 atatgccggc tgagaattgg gtgaatgttg agata 35 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> KK484 primer <400> 20 atatagatct tgagaattgg gtgaatgttg agata 35 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> KK485 primer <400> 21 atatcatatg tgttggaata gaaatcaact atcat 35

Claims (14)

1. A composition for producing xylobiose comprising? -Xylosidase consisting of the amino acid sequence of SEQ ID NO: 1. 2. The composition for producing xylobiose according to claim 1, wherein said beta -xylosidase is encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2. Comprising reacting xylose with a composition comprising? -Xylosidase or? -Xylosidase consisting of the amino acid sequence of SEQ ID NO: 1. 4. The method for producing xylobiose according to claim 3, wherein the beta -xylosidase is encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2. The production method of xylobiose according to claim 3 or 4, wherein the reaction temperature is 40 to 60 DEG C and the reaction pH is 5.0 to 8.0. The production method of xylobiose according to claim 3 or 4, wherein the xylose concentration is 400 g / L to 1000 g / L. A β-xylosidase expression construct comprising the β-xyloεidase base sequence of SEQ ID NO: 2, the GPD promoter base sequence, the His-tag base sequence, and the delta base sequence. [Claim 7] The polynucleotide according to claim 7, wherein the GPD promoter base sequence comprises the nucleotide sequence of SEQ ID NO: 3, the His-tag nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 4 and the delta nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: β-xylosidase expression construct. 8. The recombinant vector according to claim 7, which comprises a 2-micron nucleotide sequence of SEQ ID NO: 6, an ampicillin resistance gene sequence of SEQ ID NO: 7, a CYC1 terminator nucleotide sequence of SEQ ID NO: 8 and a TRP nucleotide sequence of SEQ ID NO: Syidase expression construct. 9. A transformant strain for the production of beta -xylosidase comprising a beta -xylosidase expression construct according to any one of claims 7 to 9. 11. The method according to claim 10, wherein the strain is selected from the group consisting of Saccharomyces cerevisiae , bacillus subtilis , or Corynebacterium glutamicum , Transformational strains for producing cidase. 12. The method of claim 11, wherein the transformed strain is Saccharomyces &lt; RTI ID = 0.0 &gt; S. cerevisiae ) pTSY01-XynB-β-xylosidase transformant strain. (a) a transformant strain for producing β-xylosidase according to claim 10, comprising 0.5 to 2% by weight of yeast extract, 1 to 3% by weight of peptone and 1 To 4% by weight of glucose;
(b) culturing at a reaction temperature of from 25 to 35 DEG C and a reaction pH of from 7.5 to 8.5; And
(c) separating β-xylosidase from the culture medium of step (b). 14. The method of claim 13, let azepin transformant strain for the production of xylene is in the β- accession number KCTC 18382P saccharide as MY process three Levy jiae (Saccharomyces of cerevisiae ) pTSY01-XynB.

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