KR101472993B1 - Novel xylanase containing a novel alkali resistance glycoside hydrolase family, GH family 10 from Microbacterium sp. strain HY-17 - Google Patents

Abstract

The present invention relates to a novel alkali-resistant xylanase corresponding to glycoside hydrolase family 10 (GH10) isolated from a Microbacterium sp. HY-17 strain. In particular, the xylanase has stability in an alkali environment and effectively hydrolyzes various kinds of xylans to hydrolyze β-glucan and decompose cellobiose. Accordingly, the xylanase of the present invention can be usefully used as an enzyme for the food industry, an enzyme for a fodder, a biomass pre-treating enzyme, and an enzyme for various industries including the pulp industry.

Description

A novel alkali-tolerant glycoside hydrolase family produced from a microbacterium genus HY-17, comprising a novel alkaline-resistant glycoside hydrolase family comprising 10 to 30 microbacterium sp. strain HY-17}

The present invention relates to xylene Rana claim having the novel properties of the alkali-resistant for the micro tumefaciens in (Microbacterium sp.) HY-17 10 The glycoside dihydro raise group isolated from strains (glycoside hydrolase family 10, GH10) .

 Many studies have been conducted to use various carbon sources as raw materials. Starch polysaccharides contained in these raw materials are mainly used in the food industry because they are easily hydrolyzed. Therefore, the need for non starch polysaccharides is emerging for carbon sources as raw materials in various industries.

Xylan is a non-aggregated polysaccharide, present in woody, plant, and grain by-products. Non-starch polysaccharides can be classified into cellulose and hemicellulose, and an important component of hemicellulose is xylan. Xylanase is an enzyme that hydrolyzes such xylan to xylose, xylobiose, and the like.

Xylanase can be used for improving the physical properties and chromaticity of high viscous polysaccharides in the food industry and is being used in the production process of xylooligosaccharides. In addition, by improving the hydrolysis efficiency of the feed, improving the intestinal flow of the animal and increasing the digestion efficiency, various cereals or cereal by-products can be used as feed materials.

Recently, industrial enzymes are gradually changing from chemical use to biological use environment based on green technology. Industrial xylanases are used in bleaching processes to remove the lignin polysaccharide complex of hemicellulose from pulp. The bleaching process of pulp traditionally used organic solvents to effectively remove lignin, but this process is harmful to the environment. In addition, in the bio-energy industry, the first-generation bioenergy using sugarcane or corn is increasingly used as an essential xenlanase for second-generation bioenergy using non-wood degradable hemicellulose such as woody or woody. Therefore, an effective eco-friendly process can be developed using xylanase.

Xylan is a major component of plant biomass and can be hydrolyzed using xylanase. The glycoside hydrolase family (GH family), which hydrolyzes the xylan bound to the beta-1,4-xylose structure, is known as 6, 5, 8, 10, 11, 30 and 43. (http://www.cazy.org, Luo et al., 2010). Among them, a representative group of xylanase is the glycoside hydrolase family (GH family) 10 and 11.

The biochemical properties of beta-1,4-xylanase (beta-1,4-xylanase) involved in depolymerization of carbohydrate polymers are important for their utility value. In particular, since the amount of the enzyme used is increased or decreased depending on the pH in the process, a stable enzyme is required in various pH ranges.

Thus, the inventors of the present invention have found that a novel endo-beta-1,4-xylanase resistant to the glycoside hydrolase-10 (GH-10 domain) from microbacterium sp. HY- (Endo-β-1,4-xylanase) was identified, and its gene sequence and amino acid sequence were analyzed. Further, the present invention has been completed by confirming that the above-described xylanase enzyme is useful for industrial purposes by analyzing the characteristics of the enzyme and the temperature and pH.

An object of the present invention is to provide a micro tumefaciens in producing the xylene Rana (Microbacterium sp.) HY-17 strain (KCTC 12338BP).

Yet another object of the present invention is to provide xylanase having the amino acid sequence of SEQ ID NO: 7 produced from said strain.

A further object of the present invention is to provide

1) culturing a transformant into which a recombinant expression vector comprising a polynucleotide having the nucleotide sequence of SEQ ID NO: 8 encoding the xylanase is introduced into a host cell, followed by centrifuging to obtain a crude enzyme solution; And

2) purifying the xylanase in the crude enzyme solution obtained in the step 1).

It is still another object of the present invention to provide a feed additive comprising xylanase as an active ingredient.

It is still another object of the present invention to provide a food additive containing xylanase as an active ingredient.

Another object of the present invention is to provide a composition for papermaking process comprising xylanase as an active ingredient.

In order to achieve the foregoing object, the present invention provides a micro tumefaciens in producing the xylene Rana (Microbacterium sp.) HY-17 strain (KCTC 12338BP).

In addition, the present invention provides xylanase having the amino acid sequence of SEQ ID NO: 7 produced from said strain.

The xylanase preferably has the nucleotide sequence of SEQ ID NO: 8, but is not limited thereto.

In addition,

1) culturing a transformant into which a recombinant expression vector comprising a polynucleotide having the nucleotide sequence of SEQ ID NO: 8 encoding the xylanase is introduced into a host cell, followed by centrifuging to obtain a crude enzyme solution; And

2) purifying xylanase in the crude enzyme solution obtained in the step 1).

The present invention also provides a feed additive comprising xylanase as an active ingredient.

The present invention also provides a food additive comprising xylanase as an active ingredient.

The present invention also provides a composition for paper making comprising xylanase as an active ingredient.

The present invention is a novel Giles Lana having a micro tumefaciens in the enzyme and the homologous corresponding to (Microbacterium sp.) HY-17 a-glycoside dihydro-raised Group 10 (glycoside hydrolase family 10, GH10 ) isolated from strain zero, specifically The xylanase is a novel enzyme that has stability in an alkaline environment, effectively hydrolyzes various xylans, hydrolyzes beta-glucan, and decomposes cellobiose. Accordingly, the xylanase of the present invention can be used as an enzyme useful in various industries such as enzyme for food industry, enzyme for feed, biomass pretreatment enzyme, and pulp process.

Brief Description of the Drawings Fig. 1 is a diagram showing the gene sequence of a novel xylanase (XylH) isolated in the present invention.
2 is a diagram showing the amino acid sequence of a novel xylanase (XylH) isolated in the present invention.
FIG. 3 is a graph showing the effect of the birchwood xylan on the reaction pH and temperature in order to investigate the optimum reaction conditions of xylanase (XylH) of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a microbacterium sp. HY-17 ( Microbacterium sp. HY-17) strain (KCTC 12338BP) producing xylanase.

In addition, the present invention provides a xylanase having an amino acid sequence represented by SEQ ID NO: 7 produced from said strain.

The xylanase preferably has the nucleotide sequence of SEQ ID NO: 8, but is not limited thereto.

The xylanase preferably has a molecular weight of 42 kda, but is not limited thereto.

The xylanase preferably exhibits the maximum activity at pH 5.5 to 10.0, but is not limited thereto.

The xylanase preferably exhibits the maximum activity at 60 ° C, but is not limited thereto.

The xylanase preferably exhibits endo-beta-1,4-xylanase activity, but is not limited thereto.

The xylanase is preferably a glycoside hydrolase-10 domain (GH-10 domain), but is not limited thereto.

In a particular embodiment of the present invention, the inventors have found that micro tumefaciens in (Microbacterium sp.) HY-17 strain: gene sequence (accession No. KCTC 12338BP) genomic DNA from GH10 of (glycoside hydrolase family 10) of the series Giles Rana claim (GenBank accession number: KF233593) was amplified by using a primer constructed based on the genomic DNA encoding the polynucleotide encoding the xylanase protein. Then, the gene sequence was analyzed and the protein blast survey was performed in the NCBI database , It was confirmed that xylanase (XylH) was an active domain corresponding to GH10. In the gene sequence analysis, it was confirmed that it consisted of 1167 bp ORF (open reading frame) or 388 amino acids, and the molecular weight was 41,584 Da, which was confirmed to be pI 4.84. As a result of examining the secretion signal region in the SignalP 3.0 database, it was confirmed that the xylanase was composed of the final 358 amino acids, and the molecular weight was found to be 38,632 Da and pI 4.67 (see FIGS. 1 and 2).

In order to confirm the optimal reaction conditions of xylanase, the effect of pH and temperature on the reaction pH was examined using birchwood xylan as a substrate. As a result, the xylanase (XylH) And showed activity over 75% for 1 hour at pH 5.5 ~ 10.0. Further, it was confirmed that the activity was maintained at 50% or more in the pH range of 4.5 to 11, and the remaining activity was maintained at 90% or more for 1 hour at 37 to 50 ° C at the reaction temperature (see FIG. 3).

In addition, the resolving power of xylanase to various xylan and sugar substrates was confirmed by DNS quantitation. As a result, it was found that among the evaluated xylan substances, olyth-spelled xylan was most efficiently hydrolyzed by xylanase, Xylan were hydrolyzed in this order. In addition, it is also possible to use the barley-derived glucan (β-1,3 / β-1,4-d-glucan from barley), PNP-cellobioside and PNP-xylopyranoside (See Table 1).

The hydrolysis product of the xylen disaccharides was verified by high performance liquid chromatography (HPLC) using X2 (71.2% (w / v)) and X3 (28.8 % (w / v)). In addition, it was confirmed that the xylanase had a transxylosylation activity (see Table 2).

Accordingly, the present invention relates to a method for producing a microorganism belonging to the genus Microbacterium sp. HY-17 ( Microbacterium sp. HY-17) (KCTC 12338BP) The endo-β-1,4-xylanase was identified, and its gene sequence and amino acid sequence analysis, the characteristics of the xylanase enzyme and the temperature and pH The inventors have confirmed that the xylanase can be usefully used in various industries.

In addition,

1) introducing a recombinant expression vector containing a polynucleotide having the nucleotide sequence of SEQ ID NO: 8 encoding xylanase into a host cell, and then transforming the transformant to obtain a crude enzyme solution; And

2) purifying xylanase in the crude enzyme solution obtained in the step 1).

The host cell is preferably selected from the group consisting of prokaryotes including E. coli, yeast, animal cells, and eukaryotic cells, but is not limited thereto.

Since the present invention has confirmed the nucleotide sequence of a novel gene encoding a novel xylanase isolated from a strain of microbacterium HY-17, a recombinant vector containing the gene can be produced using conventional methods known in the art . The recombinant vector of the present invention may use a commercially available vector, but the present invention is not limited thereto, and a recombinant vector suitable for a person skilled in the art may be prepared and used.

In this method, step 2) comprises: 1) inducing precipitation of a soluble protein by adding a precipitant to the supernatant obtained by centrifuging the culture solution of the transformant;

2) In the above method, the insoluble precipitate is removed from the precipitate of step 1), followed by dialysis to obtain a crude enzyme solution; And

3) In the above method, it is preferable, but not limited, to carry out the method including the step of purifying the crude enzyme solution of step 2) by column chromatography.

In the above method, it is preferable to use a medium suitable for the Mycobacterium sp. Strain HY-17 of the present invention or the transformant of the present invention in a commercial medium known to those skilled in the art.

In the above method, any one selected from the group consisting of ammonium sulfate, acetone, isopropanol, methanol, ethanol, and polyethylene glycol may be used as the precipitating agent in step 1). The precipitation can be carried out by a method of concentration by replacing with ultrafiltration using membranes having various pore sizes.

In this method, column chromatography in step 3) can be carried out by performing column chromatography using a filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide, Toyo Pearl and XAD resin. The column chromatography can be carried out several times by selecting an appropriate filler as necessary.

The present invention also provides a feed additive comprising xylanase as an active ingredient.

The novel xylanase of the present invention can be effectively used as a feed additive by confirming that it has specificity and efficiently decomposes xylan.

The feed additive of the present invention is added to feeds of non-ruminants such as pigs and chickens which have no enzymes capable of decomposing plant cell walls, It is possible to improve the value of the feed by saccharifying the component xylan.

It is preferable that 0.01 to 10 parts by weight of the xylanase is added to the feed to be added as the active ingredient of the feed additive of the present invention, more preferably 0.05 to 5 parts by weight of the xylanase, and 0.12 parts by weight of the xylanase .

In addition, the feed additive may additionally contain a carrier that is acceptable to the unit animal. In the present invention, the feed additive may be added as it is or a known carrier, stabilizer and the like may be added. Various nutrients such as vitamins, amino acids and minerals, antioxidants and other additives may be added as needed, Powders, granules, pellets, suspensions, and the like. When the feed additive of the present invention is supplied, it can be supplied to the unit animal singly or mixed with the feed.

The present invention also provides a food additive composition comprising xylanase as an active ingredient.

The novel xylanase of the present invention has specificity and confirms efficient decomposition of xylan, so that the xylanase can be effectively used as a food additive composition.

The present invention also provides a composition for the papermaking process comprising xylanase as an active ingredient.

The novel xylanase of the present invention has specificity and confirms efficient decomposition of xylan, so that the xylanase can be effectively used as a composition for papermaking process.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention in detail, and the present invention is not limited to the examples.

Example 1 Isolation of Microbacterium sp. Strain Producing Xylanase

The present inventors have isolated microorganisms having enzymatic activity for hydrolyzing xylan among intestinal symbiotic microorganisms of the dogs.

In order to selectively isolate the microorganisms hydrolyzing xylan, 5 g / L yeast extract (Difco) and 2 g / L azo-xylan (Megazyme ) Was used. The microbial suspension in the selective medium was diluted with physiological saline by a serial dilution method, and 100 μl of each suspension was dispensed into each of the selective mediums and cultured at 25 to 35 ° C for 18 to 36 hours. In each medium, the microorganism colony forming the transparent ring in which the azo-xylan was decomposed was selectively isolated. PCR was performed on the 16S rDNA sequence as a pair of 27F primer (SEQ ID NO: 1) and 1492R primer (SEQ ID NO: 2) to identify the isolated microorganism. The 16S rDNA sequence obtained by PCR was analyzed using ABI prism BigDye Terminator Cylcle Sequencing Ready Reaction kit and ABI 3730x1 DNA analyzer (Applied Biosystems). The finally obtained 16S rDNA sequence was analyzed in the Blast program of the NCBI database and in the Genebank of the EzTaxon server. The analysis of the 16S rDNA sequence of the strain that degrades xylan micro tumefaciens in (Microbacterium sp.) Has been identified as micro tumefaciens in (Microbacterium sp.) HY-17 named by the international deposit agency genes within the Korea Research Institute of Bioscience and Biotechnology to (Accession No .: KCTC 12338BP).

SEQ ID NO: 1: 5`-AGACTTTGATCMTGGCTCAG-3`; And

SEQ ID NO: 2: 5'-AAGTCGTAACAAGGTAACC-3`.

< Example  2> Xylanase ( Xylanase )of Cloning

A primer based on the genomic sequence of GY10 (glycoside hydrolase family 10) in the genomic DNA of Microbacterium sp. HY-17 (accession number: KCTC 12338BP) A polynucleotide sequence encoding the xylanase protein was amplified and cloned.

Specifically, the genomic DNA was isolated from the strain, and then the genomic DNA was used as a template in 10 × buffer (MgCl 2), 2.5 mM dNTPs, 5 × buffer, FastStart Taq DNA polymerase (Roche), and MF-10 forward primer (SEQ ID NO: 3) and MR-10 reverse primer (SEQ ID NO: 4) were used to perform PCR on the xylanase gene. The PCR conditions were as follows: denaturation at 95 DEG C for 5 minutes, denaturation at 95 DEG C for 30 seconds, annealing at 50 DEG C for 30 seconds, extension at 72 DEG C for 40 seconds, and then final extension at 72 DEG C for 7 minutes Lt; / RTI &gt; The PCR product of 375 bp of xylanase obtained through the above PCR was subjected to genome walking and nested-PCR using a DNA Walking SpeedUp premix kit (Seegene, Korea) to obtain total xy1K2 PCR products were obtained. The entire sequence obtained using the DNA Wakling technique was amplified by PCR using the pair of FMF primer (SEQ ID NO: 5) and RMF primer (SEQ ID NO: 6) for 5 minutes at 95 ° C, 30 seconds at 95 ° C, 30 seconds at 50 ° C The PCR was performed under the condition of annealing, repeating 35 times at 72 캜 for 40 seconds under the condition of extension, and then performing final extension at 72 캜 for 7 minutes to obtain the xylH gene.

The PCR product of the whole XylH gene and the pET-28a (+) vector (Novagen, USA) were digested with NdeI and HindIII restriction enzymes and purified. About 100 ng of each of the purified vector and the PCR product was used, and 1 unit of a ligase of TaKaRa was added thereto, followed by reaction at 16 ° C for 16 hours. After ligation reaction, BL21 (Novagen) was transformed and screened with plates containing kanamycin. The plasmid was digested with appropriate restriction enzymes to obtain a plasmid having the desired DNA fragment. DNA sequencing was performed to finally obtain a clone Respectively. Then, the prepared expression vector was named 'pET-XylH'. In addition, the above xylanase gene sequence was registered in the gene bank (GenBank accession number: KF233593).

SEQ ID NO: 3: 5`-TGGGACGTCSTCAACGAG-3`;

SEQ ID NO: 4: 5'-GACGTCSGCCTCSGTGAC-3`;

SEQ ID NO: 5: 5'-CATATGGCGCCTCCCGGATTCAGC-3`; And

SEQ ID NO: 6: 5`-AAGCTTTCAGCCGCGTCGGGGC-3`.

< Example  3> Xylanase ( Xylanase ) Tablets

The recombinant XylH (rXylH) in which the pET-XylH expression vector prepared in Example 2 was over-expressed in E. coli was isolated.

Specifically, Escherichia coli transformed with each expression vector was inoculated into a liquid LB medium and cultured at 37 DEG C with shaking. When the OD ₆₀₀ value of each E. coli culture reached 0.4-0.5, 1.0 mM IPTG was added, followed by further shaking at 30 ° C for 5 hours. The culture broth was centrifuged, and the cells were pulverized with a sonicator. As a result, rXylH was detected by incubating inclusion bodies with 20 mM imidazole, 20 mM sodium phosphate (pH 7.4) containing 0.5 M sodium chloride buffer pH 7.4). The solubilized rXylH cell lysate was refolded and purified using a His-Trap HP (GE Healthcare, Sweden) (5-ml) column and then subjected to a liquid chromatography (LC) system (Amersham Pharmacia Biotech, Sweden). The electrophoretic homology of the purified rXylH protein was confirmed by gel permeation chromatography using a HiLoad 26/60 Superdex 200 prep-grade (Amersham Biosciences, Sweden) column, a known method. Protein was quantified using the Bradford reagent (Bio-Rad, USA), purified from the above rXylH protein, and lyophilized and stored at -20 ° C.

< Example  4> Xylanase ( Xylanase ) Activity measurement

Din (dinitrosalicylic acid) assay (Miller GL, Anal. Chem., 55: 952-959, 1959) was used to measure the activity of xylanase.

Specifically, 100 μl of the enzyme solution diluted stepwise was added to 50 mM glycine sodium hydroxide buffer (pH 9.0) containing 1% (w / v) beechwood xylan, After reacting for a minute, 750 μl of a solution of 3,5-dinitrosalicylic acid (DNS) was added, and the solution was allowed to stand at 100 ° C. for 5 minutes, and the absorbance was measured at 540 nm. One unit of enzyme was defined as the amount of enzyme that released 1 mu mol of reducing sugar per minute.

< Example  5> Xylanase ( Xylanase )

<5-1> Sequence analysis

The inventors analyzed the amino acid sequence using the protein blast survey in the NCBI database.

As a result, as shown in FIG. 1 and FIG. 2, it was confirmed that the xylanase (XylH) of the present invention was an active domain corresponding to GH10. In the gene sequence analysis, it was confirmed that it consisted of 1167 bp ORF (open reading frame) or 388 amino acids, and the molecular weight was 41,584 Da, which was confirmed to be pI 4.84. As a result of examining the secretion signal region in the SignalP 3.0 database, it was confirmed that the xylanase was composed of the final 358 amino acids and the molecular weight was 38,632 Da and pI 4.67 (FIGS. 1 and 2).

<5-2> Biochemical properties of xylanase

In order to confirm the optimal reaction conditions of the xylanase (XylH) of the present invention, the effect on the reaction pH and temperature was confirmed using birchwood xylan as a substrate.

Specifically, the optimal enzyme activity was determined by measuring the activity of the enzyme activity in 50 mM sodium citrate buffer (pH 3.5-5.5), 50 mM sodium phosphate buffer (pH 5.5-7.5), 50 mM tris hydrochloride buffer solution (Tris-HCl buffer) (pH 7.5-9.0) and 50 mM glycine-NaOH buffer (pH 9.0-10.5) at 55 ° C for 15 hours.

In order to confirm the stability of the enzyme against pH, the enzyme activity was measured under the conditions of Example 4 after standing at 4 ° C for 1 hour in a buffer solution prepared for each pH, and the optimum reaction of the enzyme The enzyme activity was measured at 5 ° C intervals in the temperature range of 30 ~ 70 ℃. The stability of the enzyme was measured at 37, 45, 50, 55, 60 ℃ for 15, 30, Residual activity was measured.

As a result, as shown in FIG. 3, xylanase (XylH) of the present invention showed the highest activity at pH 9.0 and 60 ° C, and showed activity over 75% for 1 hour at pH 5.5 to 10.0. Further, it was confirmed that the activity was maintained at 50% or more in the pH range of 4.5 to 11, and it was confirmed that the residual activity was maintained at 90% or more for 1 hour at 37 to 50 ° C at the reaction temperature (FIG.

<5-3> Substrate specificity of xylanase

The resolving ability of the xylanase (XylH) of the present invention against various xylan and sugar substrates was confirmed by the DNS assay of Example 4 above. (W / v) xylose polymers such as beechwood xylan, birchwood xylan, and oatspelt xylan) and starch, pectin, glucan, (w / v) polymer such as glucan, and 5 mM PNP (p-nitrophenyl) derivative were used to confirm the substrate specificity. The standard assay mixture (0.5 ml) containing the enzyme solution (0.05 ml) diluted in 50 mM glycine sodium hydroxide buffer (pH 9.0) containing each substrate was subjected to enzymatic reaction at 45 ° C for 10 minutes Respectively. One unit of xylanase activity for xylan or PNP-sugar derivatives was defined as the amount of enzyme required to produce 1 μmol of reducing sugar or PNP per minute under standard analytical conditions.

As a result, as shown in Table 1, among the evaluated xylan substances, the oat spelled xylan was hydrolyzed most efficiently by xylanase (XylH), and showed hydrolytic activity in the order of beech woody xylan and birchwood xylan. In addition, it is also possible to use the barley-derived glucan (β-1,3 / β-1,4-d-glucan from barley), PNP-cellobioside and PNP-xylopyranoside (Table 1). &Lt; tb &gt;&lt; TABLE &gt;

Substrate Specific activity (IU / mg) ^a Birch wood Xylan 72.2 ± 0.3 Beech wood Xylan 82.7 ± 1.3 Oat spelts Xylan 92.3 ± 1.0 Millarabinozaylan 52.6 ± 5.0 Xyloglucan ND Soluble starch (Soluble starch) ND Pectin ND Locust bean gum ND Glucan from barley (b-1,3 / b-1,4-d-Glucan from barley) 2.3 ± 0.5 Carboxy methylcellulose (Carboxy methylcellulose) ND PNP-cellobioside &lt; RTI ID = 0.0 &gt; 118.5 ± 0.6 PNP-glucopyranoside (PNP-glucopyranoside) ND PNP-xylopyranoside (PNP-xylopyranoside) 30.4 ± 1.1 PNP-mannopyranoside (PNP-mannopyranoside) ND PNP-galactopyranoside &lt; RTI ID = 0.0 &gt; (PNP-galactopyranoside) ND

<5-4> Characteristics of Xylen Decomposition Products

In the same manner as that of Example 4, a mixture of birchwood xylan, BX, xylobiose (X2), xylotriose (X3), xylotetraose (X4), and The enzyme reaction was stopped by heating the reaction mixture at 100 ° C for 5 minutes using xylopentose (X5) as a substrate, and the hydrolyzate was dissolved in the eluting solution A (0.05% formalin / sterilized water) eluting solution B High Performance Liquid Chromatography (HPLC) using an Asahipak NH2P-50 2D column (5 탆, 2.0 x 150 mm, Shodex) under the moving phase conditions of 0.05% Folic acid / sterilized water: acetonitrile / methanol = 6: , HPLC) assay (Kim DY et al., 2011).

As a result, as shown in Table 2, it was confirmed that the hydrolysis product for birchwood xylenes was represented by X2 (71.2% (w / v)) and X3 (28.8% (w / v)). The hydrolysis products for xylotriose were X2 (45.8% (w / v)) and X3 (38.9% (w / v)) in the hydrolytic activity of xylooligosaccharides such as xylotriose and xylotriose The hydrolysis products for Xylotetraose were X2 (34.1% w / v), X4 (12.3% (w / v)) and X5 (3.1% ), X3 (38.3% (w / v), X4 (22.7% (w / v)) and X5 (5.0% (w / v)), xylopentose hydrolysis products were X2 (W / v)), X3 (35.9% (w / v)), X4 (23.4 (w / v)), X5 (12.1% ). As a result, it was confirmed that the xylanase had a transxylosylation activity (Table 2).

Substrate
Composition (%) of product formed by hydrolysis reaction
(Composition (%) of products formed by hydrolysis reaction) X2 X3 X4 X5 X6 X2 100.0 X3 45.8 38.9 12.3 3.1 X4 34.1 38.3 22.7 5.0 X5 25.9 35.9 23.4 12.1 2.8 Birchwood Jaylan
(birchwood Xylan) 71.2 28.8

Micro tumefaciens in (Microbacterium sp.) Of the present invention as HY-17 a GH10 (glycoside hydrolase family 10) isolated from the strain of the novel alkali-resistant having an enzyme and the homology sequence xylene Rana agent properties improved and oligosaccharide production process The same food industry, the feed industry for the utilization of feed grains, pulp for replacing chemical processes, and pretreatment processes for pretreatment of raw materials for bioenergy. Therefore, it is an enzyme that can be developed as a material that can improve the health of livestock through the improvement of digestion efficiency, the added value of various grains or by-products through processes using enzymes, development of more environmentally friendly processes.

Korea Biotechnology Research Institute KCTC12338BP 20121217

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel xylanase containing a novel alkali resistance glycoside          hydrolase family, GH family 10 from Microbacterium sp. strain          HY-17 <130> 13P-07-24 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27 F <400> 1 agactttgat cmtggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R <400> 2 aagtcgtaac aaggtaacc 19 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> MF-10 forward <400> 3 tgggacgtcs tcaacgag 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> MF-10 reverse <400> 4 gacgtcsgcc tcsgtgac 18 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FMF <400> 5 catatggcgc ctcccggatt cagc 24 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> RMF <400> 6 aagctttcag ccgcgtcggg gc 22 <210> 7 <211> 388 <212> PRT <213> Mycobacterium <400> 7 Met Arg Arg Ser Ile Arg Ser Leu Leu Val Ala Thr Ala Ala Ala Thr   1 5 10 15 Ala Leu Val Leu Pro Ala Ala Ala Ala Ala Ala Ala Ala Pro              20 25 30 Pro Gly Phe Ser Asn Ala Asp Lys Asp Ala Leu Arg Asn Gln Ala Pro          35 40 45 Arg Asp Leu Ala Ile Gly Ser Ala Val Trp Ala Gln Gln His Leu Val      50 55 60 Gly Tyr Asp Ala Ser Ala Pro Thr Glu Phe Gln Gln Val Leu Ala Gly  65 70 75 80 Gln Phe Ser Ser Leu Thr Pro Glu Asn Asp Met Lys Trp Asp Ala Val                  85 90 95 His Pro Ala Pro Gly Val Tyr Asp Phe Thr Ser Ala Asp Ala Leu Ile             100 105 110 Ala Phe Ala Glu Ala Asn His Gln Gln Val Arg Gly His Thr Leu Leu         115 120 125 Trp His Ser Gln Asn Pro Ala Trp Val Thr Ala Ala Ser Ala Thr Trp     130 135 140 Thr Cys Asp Asp Ala Arg Ala Val Leu Glu Asp His Ile Arg Thr Val 145 150 155 160 Val Gly His Phe Lys Gly Lys Ile Tyr Glu Trp Asp Val Ala Asn Glu                 165 170 175 Ile Phe Gln Asp Glu Trp Asp Asn Gly Gly Val Lys Leu Arg Thr Thr             180 185 190 Ala Asn Pro Phe Leu Lys Ala Cys Ala Ala Asp Pro Val Gly Leu Leu         195 200 205 Ala Asp Ala Phe Arg Trp Ala His Glu Ala Asp Pro Asp Ala Val Leu     210 215 220 Phe Leu Asn Asp Tyr Asn Ala Glu Gly Ile Asn Ala Lys Thr Asp Ala 225 230 235 240 Tyr Tyr Ala Leu Ala Gln Gln Leu Leu Ala Ala Gly Ala Pro Leu Gly                 245 250 255 Gly Phe Gly Ala Gln Gly His Leu Ser Leu Leu Tyr Gly Phe Asp Thr             260 265 270 Ser Ile Gln Ala Asn Phe Glu Arg Ser Ala Ala Leu Gly Leu Lys Val         275 280 285 Ala Val Thr Glu Ala Asp Val Arg Ile Pro Leu Gln Glu Gly Glu Thr     290 295 300 Gly Pro Thr Pro Glu Gln Val Ala Val Gln Ala Glu Arg Tyr Asp Ala 305 310 315 320 Met Leu Gln Ala Cys Leu Asn Val Thr Ala Cys Ser Ser Phe Thr Val                 325 330 335 Trp Gly Phe Ser Asp Ala Tyr Ser Trp Val Pro Gly Val Phe Pro Gly             340 345 350 Glu Gly Trp Ala Thr Ile Thr Asp Glu Lys Phe Thr Pro Lys Pro Ala         355 360 365 Phe Tyr Ala Leu Leu Gly Ser Leu Arg Asp Ala Thr Pro Gly Thr Ser     370 375 380 Pro Arg Arg Gly 385 <210> 8 <211> 1167 <212> DNA <213> Mycobacterium <400> 8 atgcgccgat cgatcagatc gctgctggtg gccacggctg ccgccaccgc tctcgtcctt 60 cccctctgcg ccgcgaccgc ggccagcgcc gcgcctcccg gattcagcaa cgccgacaag 120 gatgcgctgc gcaaccaagc gccccgcgac ctcgccatcg gcagcgcagt ctgggcgcag 180 cagcacctcg tcggctacga cgcctctgcg ccgaccgagt tccagcaggt gctcgccggc 240 cagttctcgt cgctgacgcc cgagaacgac atgaaatggg atgccgtcca ccccgcgccg 300 ggcgtgtacg acttcacgtc ggcggacgcg ctgatcgcat tcgccgaggc gaaccaccag 360 caggtgcgcg gacacacgct gctgtggcac agccagaacc cggcatgggt gacggcggcc 420 agcgccacct ggacgtgcga cgacgcacgg gccgtgctgg aggaccacat ccgcaccgtc 480 gtcggtcact tcaaaggcaa gatctacgag tgggacgtgg ccaacgagat cttccaggac 540 gagtgggaca acgggggcgt gaagctgcgc accacggcga acccgttcct caaggcctgc 600 gccgcggatc cggtcgggct cctggcggac gcgttccgct gggcccatga ggccgacccc 660 gacgcggtgc tgttcctgaa cgactacaac gccgagggca tcaacgcgaa gaccgacgcc 720 tactacgcgc tcgcgcagca gctgctggcg gcgggtgcac cgctcggcgg cttcggcgcg 780 cagggccacc tcagcctgct gtacggcttc gacacgtcca tccaggccaa cttcgagcgc 840 tccgccgccc tcggcctcaa ggtggcggtg acggaggccg acgtgcgcat cccgctgcag 900 gagggcgaga cgggcccgac gcccgagcag gtcgcggtgc aggccgagcg ctacgacgcg 960 atgctgcagg cctgcctcaa cgtcacggca tgctcctcct tcacggtgtg gggcttctcg 1020 gacgcctact cgtgggtccc gggcgtcttc cccggcgagg gctgggcgac gatcaccgac 1080 gagaagttca cgccgaagcc cgccttctac gcgctgctcg gctcgctgcg cgacgcgaca 1140 ccgggcacct cgccccgacg cggctga 1167

Claims (13)

Microbacterium sp. HY-17 ( Microbacterium sp. HY-17) strain (KCTC 12338BP) producing xylanase.
7. An xylanase having an amino acid sequence represented by SEQ ID NO: 7 produced from the strain of claim 1.
3. The xylanase according to claim 2, wherein the xylanase has a molecular weight of 42 kda.
3. The xylanase according to claim 2, wherein the xylanase exhibits maximum activity at pH 5.5 to pH 10.0.
3. The xylanase according to claim 2, wherein the xylanase exhibits maximum activity at 60 &lt; 0 &gt; C.
3. The xylanase according to claim 2, wherein the xylanase exhibits endo-beta-1,4-xylanase activity.
3. The xylanase according to claim 2, wherein the xylanase is a glycoside hydrolase-10 domain (GH-10 domain).
A method for producing a recombinant vector comprising the steps of: 1) culturing a transformant into which a recombinant expression vector comprising a polynucleotide having the nucleotide sequence of SEQ ID NO: 8 encoding xylanase has been introduced into a host cell and then centrifuging to obtain a crude enzyme solution step; And
2) purifying xylanase in the crude enzyme solution obtained in the step 1).
9. The method according to claim 8, wherein the host cell is any one selected from the group consisting of prokaryotes including E. coli, yeast, animal cells, and insect cells.
9. The method of claim 8, wherein step (2)
1) inducing precipitation of a soluble protein by adding a precipitant to the supernatant obtained by centrifuging the culture solution of the transformant of claim 8;
2) removing the insoluble precipitate from the precipitate of step 1) and then dialyzing to obtain a crude enzyme solution; And
3) purifying the crude enzyme solution of step 2) by column chromatography.
A feed additive comprising the xylanase of claim 2 as an active ingredient
A food additive comprising the xylanase of claim 2 as an active ingredient.
A composition for papermaking process comprising the xylanase of claim 2 as an active ingredient.

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