KR100870561B1 - Novel cellulosimicrobium sp??hy-12 strain and xylanase produced from it - Google Patents

Abstract

Cellulosimicrobium sp. HY^-12 strain separated from intestines of Gryllotalpa orientalis and a xylanase produced from the same are provided to show an excellent xylan-analyzing activity at a wide range of temperature and pH. Xylanase is produced from Cellulosimicrobium sp. HY^-12 strain(KCTC 11155BP). The xylanase has an amino acid sequence produced from strain of a sequence number 3 and a molecular weight of 42 kDa, shows a maximum activation at a pH 5.5 ~ 6.5 and 60°C ~ 70°C. An activity of the xylanase is increased when Zn, K, Mg, Na or Mn is added. A gene ciphers the enzyme.

Description

Novel Cellulosimicrobium sp. HY-12 strain and xylanase produced from it

The present invention is cellulose microbium sp. HY-12 ( Cellulosimicrobium sp. HY-12) strain and the xylanase produced from the strain, and more specifically, Gryllotalpa micropore evacuation of sp during a novel intestinal bacterial cellulose was separated from the intestine of orientalis). HY-12 and the novel xylanase produced thereby.

Insects are the most thriving biomass on the planet, showing a variety of food habits and high biodiversity. In order to utilize insect symbiotic microorganisms in consideration of the biological characteristics of these insects as a useful biological resource, researches on intestinal microorganisms that are closely related to the growth of insects are being actively conducted (Dillon, RJ et.al). ., Annu. Rev. Entomol, 49, 71-92, 2004). In particular, many studies have been conducted to isolate cellulase, xylanase, and ligninase from the intestinal microorganisms of termites, which are the main insects affecting the woody parts of plants. Results have been reported (Varma, A. et.al., FEMS Microb Rev , 15, 9-28, 1994; Watanabe, H. et.al., Nature , 394, 330-331, 1998). In addition, in view of the eating characteristics of the herbivorous insects xylene Rana claim (xylanase) or case, remove the strain producing the cellulase (cellulase) (Teunissen, MJ et.al. , Arch. Microbiol, 156, 290-296, 1991 Watanabe, H. et.al., Nature , 394, 330-331, 1998), and the industrial application of isolated strains that produce highly efficient proteolytic enzymes from sugarless spiders that feed on animal feed (Lee, GE and HY Park., Kor. J. Microbiol . 40, 269-274, 2004), to release the strain producing the lipase with high efficiency from the top section of the long-horned beetle, and identifying cases (Park, Park DS and HY., Korean J . Appl. Entomol, 46, 1-9 , 2007) and other Lepidoptera, ecological study of intestinal microorganisms utilize molecular biological techniques on various insects including Coleoptera have been reported (Egert, M. and Friedrich, M., Appl. Environ. Microbiol, 69, 6656-6668, 2003).

Xylan is the main constituent of hemicellulose, which makes up the cell walls of plants, and after cellulose, it is abundant and renewable carbohydrates in nature. Xylan is a polymer composed of β-1,4 bonds of xylopyranose and is generally an acetyl group, L-arabinofuranose and D-glu. D-glucuronic acid is present as a substituted heterologous carbohydrate (Biely, P., Trends . Biotechnol ., 3, 286-290, 1985). Therefore, endo-1,4-β-xylanase, β-xylanase, and α-gluk are required for glycosylation and glycosylation completely. The cooperative action of several enzymes, such as ro-nicurase (α-glucuronidase), α-L-arabinofuranose and acetylesterase, is required (Bachmann, SL, et.al., Appl. Environ. Microbiol, 57, 2121-2130, 1991). Among these, xylanase is divided into three types, endo-β-xylanase, exo-β-xylanase and xylosidase, depending on the degradation mode for the substrate. It can be classified into types and plays the most important role in the hydrolysis process of xylan.

Xylanase is widely used in the bleaching process of papermaking, feed efficiency improvement, clarification of fruit drinks, high quality of baking or the use of agricultural by-products, and is produced by various microorganisms. The paper industry has been isolated from several bacteria, the alkali or heat resistance Giles Rana claim (xylanase) for application to the bleaching process (Tenkanen, M. et.al., Enzyme. Microb. Technol, 14, 566-574, 1992), Numerous xylanase-producing bacteria without cellulase activity have been reported, and studies to reduce the loss of cellulose for papermaking have been reported (Khashin, A. et.al., Appl. .. Environ Microbiol, 59, 1725-1730 , 1993; Kosugi, A. et.al., J. Bacteriol, 183, 7037-7043, 2001). In addition, xylanase treatment can be used to improve bread quality (Courtin, CM et.al., J. Agric . Food . Chem , 47, 1870-1877, 1999), or β-xylosidase ( As a yeast in which the genes of β-xylosidase) and xylanase have been introduced, studies have been reported on glycosylation of agroforestry by-products for use by microorganisms (La Grange, DC et.al., Appl . Environ . Microbiol , 67). , 5512-5519, 2001). In the feed industry, xylanase is marketed and used as an enzyme for feed addition. The enzyme acts to lower the intestinal viscosity of livestock due to hemicellulose when eating grain feed. It is known to prevent disease and improve feed efficiency (McCracken, KJ et.al., Br . Poult. Sci , 42, 638-642, 2001).

Currently known domestic xylanase-related patents include a patent for a novel Streptomyces genus JL-2 strain that produces xylanase (Korean Patent Laid-Open Patent 2001-0111986), a signal sequence gene of Bacillus subtilis endozylanase. Patent for a recombinant plasmid containing the method and a method for producing a foreign protein using the same (Korean Patent Publication No. 2000-0034279), a novel xyl encoding the xylanase of Bacillus sp. AMX-4 strain Lanase gene (Korean Patent Laid-Open Publication No. 2003-0085679) and the novel Fanibacilli sp. Xylanase (HK-8 2007-0082329), etc. of the HV-8 strain, but is not actually used in many fields in Korea. Therefore, the study of xylanase with new properties was required.

  Accordingly, the present inventors completed the present invention by selecting a highly efficient xylanase producing strain that produces novel xylanase from various insects, separating and purifying the xylanase isolated therefrom, and confirming its characteristics.

An object of the present invention is a land dog ( Gryllotalpa) orientalis ) microbial cellulose microbiium sp. It is to provide a Cellulosimicrobium sp. HY-12 (HY-12) strain and a novel xylanase produced therefrom.

In order to achieve the above object, the present invention is a novel cellulose microbiium sp. HY-12 ( Cellulosimicrobium sp. HY-12) strain is provided.

The present invention also provides a novel xylanase enzyme produced from the strain and a gene encoding the same.

In addition, the present invention is cellulose microbium sp. Provided are methods for producing xylanase using the HY-12 strain.

Cellulose microbium sp. HY-12 ( Cellulosimicrobium sp. HY-12) and xylanases produced therefrom exhibited excellent xylan degradation activity over a relatively wide range of temperature and pH, and because of its novelty, many research and industrial fields using xylanase It can be usefully used in.

Hereinafter, the present invention will be described in detail.

The present invention is a novel cellulose microbiium sp. HY-12 ( Cellulosimicrobium sp. HY-12) strain is provided.

The microorganism of the present invention is ground dog ( Gryllotalpa) Orientalis ) Inoculated with bacterial colonies formed from the intestinal separation medium from the intestine and inoculated in a restriction medium containing Birchwood xylan and cultured supernatant using the culture supernatant as coenzyme solution The microorganisms producing xylanase were selected by screening. As a result of analyzing the characteristics of the selected strains, Gram-positive bacilli did not have flagella (see Fig. 1), and the partial sequence of 16S rDNA (SEQ ID NO: 1) was determined. 97% homology with the cellulose strain was confirmed.

The microorganisms were examined for physiological and biochemical properties using API 20E (BioMerieux) microorganism identification kit. As a result, it produces beta-galactosidase and acetone and is the carbon source of glucose, mannitol, rhamnose, sucrose, amygdalin and arabinose. Positive for (see Table 1). In addition, the cell membrane fatty acid composition was analyzed, and anteiso-C _{15 : 0} 55.6% _, iso-C _{16 : 0} 13% and anteiso-C _{17: 0} 12% were present as main fatty acids (see Table 2).

In view of the above characteristics, this strain was identified as a strain belonging to cellulose microbium sp. And named "cellulose microbium sp. HY-12", and the strain was July 12, 2007 It was deposited in the Gene Bank of Korea Research Institute of Bioscience and Biotechnology (Accession Number; KCTC 11155BP).

The present invention also provides a novel xylanase enzyme produced from the strain and a gene encoding the same.

The inventors have isolated and identified cellulose microbium sp. In order to purify the xylanase produced by the HY-12 strain, the strain was incubated in a liquid medium containing oat xylan, and then a precipitant was added to precipitate the water-soluble protein, and the precipitate was recovered. After dissolving the precipitate, the dialysis membrane was dialyzed to purify DEAE-anion exchange resin column chromatography and Mono-Q anion exchange resin column chromatography step by step (see FIG. 2A).

As described above, the molecular weight of the purely isolated xylanase was measured by polyacrylamide gel electrophoresis (see FIG. 2B), the purified protein was treated with trypsin, and the digested fragments were separated by reverse phase HPLC. The degenerate primer was prepared based on the sequence, and the total nucleotide sequence of the xylanase (SEQ ID NO: 2) and amino acid sequence (SEQ ID NO: 3) were determined using inverse-PCR and DNA walking techniques. . As a result, a protein having a molecular weight of 42 kDa consisting of 274 amino acid residues was identified.

Cellulose microbium sp. Xylanase produced by the HY-12 strain is acidothermus known as bacterial xylanase. cellulolyticus ) 52% amino acid sequence homology with xylanase , and a novel xylase with 47% homology with xylanase from Thermobifida alba and Streptomyces avermitilis Turned out to be lanase.

In the present invention, as described above, cellulose microbium sp. Since the nucleotide sequence of the gene encoding the xylanase isolated from the HY-12 strain has been revealed, a recombinant vector containing the gene and a transformant into which the recombinant vector is introduced can be prepared using conventional methods known in the art. .

In the present invention, the optimum reaction conditions of xylanase were examined, and the temperature conditions showed the maximum activity at 60 ° C. to 70 ° C., preferably 70 ° C., and the acidity showed the maximum activity at pH 5.5 to 6.5. It was confirmed to be xylanase (see FIGS. 3A and 3B). In order to investigate the stability to heat, after the treatment of purified enzyme solution for 30 minutes at each temperature, the remaining enzyme activity was measured, and it was confirmed that the enzyme activity remained stable at a temperature lower than 50 ° C (see FIG. 3C). ). Further, the addition of a total of nine kinds of metal ions, the activity of the xylene Rana agent is Fe ^{2 +} ions, Cu ^{2 +} ions, Ca and was reduced the addition of the activity of Co, Mn, Zn, K, Mg, Na, etc. In addition, it was confirmed that the activity was increased substantially (see Fig. 3D).

In addition, the present invention

1) cellulose microbium sp. In a medium containing xylan. Culturing the HY-12 strain or the transformant and then centrifuging to obtain a supernatant;

2) adding a precipitant to the supernatant of step 1) to induce aqueous protein precipitation;

3) removing the insoluble precipitate from the precipitate of step 2) and dialysis to obtain a crude enzyme solution; And

4) It provides a xylanase production method comprising the step of purifying the crude enzyme solution of step 3) by column chromatography.

In the above method, as the precipitant of step 2), any one selected from the group consisting of ammonium sulfate, acetone, isopropanol, methanol, ethanol and polyethylene glycol can be used. The precipitation may be performed by concentrating by replacing the ultrafiltration method using a membrane having various pore sizes.

In the above method, the column chromatography of step 4) may be purified by performing column chromatography using a filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide, Toyopearl, and XAD resin. Column chromatography can be carried out several times by selecting the appropriate filler as required.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the examples.

< Example  1> Strain Isolation and Screening

The present inventors collected land puppies used in the search for microorganisms having xylanase production activity near Daejeon, transported them to the laboratory in a live state, and used them. To isolate the xylanase-producing bacteria, the target insects were immersed in 70% (w / w) ethanol for 1 to 2 minutes to remove the contaminants from the surface of the carcass, and then sterilized distilled water to remove the ethanol from the carcass. Washed twice. The washed carcasses were dissected to separate the viscera and then pulverized with buffer (Phospate buffered saline; 0.8% NaCl, 0.02% KCl, 0.144% Na ₂ HPO ₄ , 0.024% KH ₂ PO ₄ ). After dilution, smear on a solid medium containing 0.5% birch wood xylan and incubate at 25 ° C. for 2 days, followed by Congo-red staining (Skiper, N. et.al., Science , 230, 958-960, 1985). Strains forming transparent rings around colonies grown as) were selected first. Strains selected by the above method were limited to medium containing 0.5% birch wood xylan (K ₂ HPO ₄ 7g / L, KH ₂ PO ₄ 2g / L, (NH ₄ ) ₂ SO ₄ 1g / L, Inoculated with 3 g of MgSO ₄ ㆍ 7H ₂ O 1.1g / L, yeast extract 0.6g / L) and incubated for 48 hours in a shaking incubator at 25 ℃ and centrifuged to recover the supernatant to measure the activity of xylanase, Among them, strains excellent in xylanase activity were finally selected. The enzyme activity was determined by DNS (dinitrosalicylic acid) assay (Miller, GL, Anal . Chem ., 55, 952-959, 1959), specifically 100 μl of substrate solution (1% oat in 100 μl enzyme solution). Add 50mM sodium phosphate (pH 6.0) containing xylan, react for 10 minutes at 50 ℃, add 700μL of DNS (3,5-Dinitrosalicylic acid) solution, treat at 100 ℃ for 5 minutes and absorb at 540 nm. Measured. One unit of enzyme was defined as the amount of enzyme that releases 1 μmol of reducing sugar in one minute.

< Example  2> Identification of Isolated Strains

The inventors of the present invention identified the strains isolated from the intestine of the ground dog in Example 1 were examined morphological, physiological biochemical, fatty acid irradiation and the nucleotide sequence of 16S rDNA. In order to observe the morphological characteristics of the isolated strain, the appearance was examined using Gram staining and Transmission Electron Microscope (TEM). As shown in FIG. 1, the cell size of 0.7 μm and the cells of 1.75 μm were observed. It was rod-shaped with length and appeared to be nonmotile bacilli without flagella. In addition, the genomic DNA of the strain was isolated to determine the 16S rDNA sequence of the microorganism, and the reaction for PCR was 50 μL of the reaction with the following composition. 10-fold Tag DNA polymerase buffer (MgCl ₂ ) in 1 μl genomic DNA (50-100 ng / μl) 2 μl, 2.5 μm dNTPs, 2 μl, 10 pmol of forward primer (27F: 5′-agagtttgatcmtggctcag-3 ′, SEQ ID NO: 4) and reverse primer (1492R: 5′-ggttaccttgttacgactt-3 ′, SEQ ID NO: 5) 1 μl and 1 to 2 units of tag DNA polymerase (Promega, USA) were added thereto, and distilled water was added to the final 50 μl reaction solution. Primers were synthesized for nucleotide amplification of 1373 bp corresponding to the 16S rDNA site of eukaryotic bacteria. PCR reaction was performed after 5 minutes of denaturation at 94 ° C, followed by 30 seconds of denaturation at 94 ° C, 30 seconds of annealing at 50 ° C, and 3 minutes of extension at 72 ° C. Finally it was finished by extension at 72 ° C. for 7 minutes and kept at 4 ° C.

As a result, the nucleotide sequence (SEQ ID NO: 1) of the obtained amplified fragment was determined, and the result of searching the Genebank database showed a homology of 97% with the cellulose microbiium cellulose strain.

Physiological and biochemical investigation of the strain was carried out at 25 ℃ and used a commercial kit (API) of API 20E (BioMerieux). As a result, beta-galactosidase and acetone were produced as shown in Table 1 below, and glucose, mannitol, rhamnose, sucrose, amygdalin, arabin It was positive for the carbon source of arabinose (see Table 1). In addition, the cell membrane fatty acid composition was further analyzed for accurate identification of isolates. Component analysis and quantification of cell fatty acids were performed using MIS library generation software (Microbial ID), and the results were compared with the MIDI Aerobe Library (ver. 3.8). As a result, anteiso-C _{15 : 0} 55.6% _, iso-C _{16 : 0} 13% and anteiso-C _{17: 0} 12% were present as main fatty acids (see Table 2).

In view of the above characteristics, the isolate was identified as belonging to Cellulosimicrobium sp. Named HY-12. The cellulose microbium sp. The HY-12 strain was deposited on July 12, 2007 at the Gene Bank of Korea Biotechnology Research Institute, an international depository institution (Accession Number; KCTC 11155BP).

Cellulosic mibium sp . HY Morphological and Physiological Characteristics of -12 Strains  characteristic Responsive  Gram staining +  Mortility -  Cell form rod  β-galactosidase +  Ornithine decarboxylase -  Citrate utilization -  H2S production -  Urease -  Tryptophane deaminase -  Indole production -  Acetone Production +  Proteolysis of gelatin -  Arginine dihydrolase -  Lysine decarboxylase -  Glucose +  Mannitol +  Inositol -  Sorbitol -  Rhamnose +  Sucrose +  Melibiose -  Amygdaline +  Arabinose +

Cellulosic mibium sp . HY Major Cell Fatty Acids of -12 Strains fatty acid content(%)  14: 0 iso 1.1 ± 0.7  14: 0 1.3 ± 0.4  15: 0 iso 9.5 ± 2.1  15: 0 anteiso 55.6 ± 7.7  16: 0 iso 13.0 ± 5.4  16: 0 4.8 ± 2.7  17: 0 anteiso 12.5 ± 1.7  18: 1 w7c 0.6 ± 0.6

< Example  3> Xylanase  Characterization

<3-1> Xylanase  Separation and Purification

The present inventors have isolated and identified cellulose microbium sp. Xylanase produced by the HY-12 strain was purified as follows. Specifically, K ₂ HPO ₄ 7g / L, KH ₂ PO ₄ 2g / L, (NH ₄ ) ₂ SO ₄ 1g / L, MgSO ₄ 7H ₂ O 1.1g / L, yeast extract 0.6g / L, oat xylan After incubation for 48 hours in a liquid medium consisting of 3g / L (Sigma Chem. Co.) and added to the supernatant recovered by centrifugation 70% saturated ammonium sulfate powder and then centrifuged to recover the precipitate. 50 mM sodium phosphate (pH 6.0) buffer was added to the precipitate to dissolve the precipitate, followed by centrifugation at 12,000 rpm for 10 minutes to remove insoluble precipitate, followed by dialysis at 4 ° C. with 20 mM phosphate buffer (pH 7.0) for 12 hours. PIERCE, MW cutoff 10 kDa) to obtain a crude enzyme solution. An FPLC system (Amersham-Pharmacia Biotech) was used for column chromatography of the enzyme. First, samples were loaded on a Hiprep 16/10 DEAE (Amersham Pharmacia Biotech Inc) column equilibrated with 20 mM phosphate buffer and eluted at a flow rate of 5 ml / min as a linear gradient of 0 to 0.5 M NaCl. At this time, the enzyme activity fraction eluted at NaCl concentration of about 0.35 to 0.4 M was taken and the mono Q HR 5/5 ion exchange resin (Amersham Pharmacia Biotech Inc) column equilibrated with 20 mM bis-Tris buffer was used at a flow rate of 1 ml / min. The concentration of NaCl was eluted with a concentration gradient from 0 to 0.5M (see FIG. 2A), and only the enzyme activity fraction eluted at about 0.35 M NaCl concentration was taken and purified by SDS-PAGE. The active fractions were lyophilized and used at -20 ° C.

<3-2> Xylanase  Molecular Weight and Amino Acid Sequencing

In Example <3-1>, the purely isolated xylanase was confirmed molecular weight by SDS-PAGE. In FIG. 3B, lane 1 is a marker protein of known size, lane 2 is a culture supernatant, and lane 3 is a finally purified xylanase protein via a Mono Q column. It is shown. As a result of measuring the molecular weight by SDS-PAGE, the xylanase of the present invention was found to have a molecular weight of about 42 kDa (see FIG. 2B).

In addition, in order to determine the partial sequencing of the separated xylanase, 200 μg of the purified protein was vacuum dried, and then dissolved in 25 μl of 8M urea and 25 μl 0.4M ammonium bicarbonate. 45 μl of DTT was added and reacted at 50 ° C. for 15 minutes. After cooling at room temperature, 5 µl of 100 mM ioacetamide was added thereto, followed by normalization for 15 minutes, followed by 60 µl of distilled water, and 5 µg of trypsin (Sigma Chem. Co.). It was. The resulting peptide fragment was separated on a C18 reversed phase chromatography column (YMC Pack Protein RP, 250 x 4.6 mm) equilibrated with 0.5% TFA in RP-HPLC with a 0 to 60% acetonitrile linear gradient and the resulting fraction results in Institute using Applied Biosystems Procise 491 sequencer was analyzed and some amino acid sequence of the protein to know Dorsal mousse cellulose utility kusu (Acidothermus cellulolyticus ) trypsin digestion fragment homologous to xylanase. Based on this result, the xylanase sequence provided by NCBI was downloaded, and the sequences of several highly homologous xylanases were compared to prepare degenerate primers based on high amino acid sequences (forward primer: 5'-atytggcaatgggacgt-). '3, SEQ ID NO: 6; reverse primer: 5'-tggtcngcttcgtgrgccca-'3 (SEQ ID NO: 7), cellulose cymbium sp. Through PCR and DNA walking techniques (DNA walking speedup kit, Seegene). The total nucleotide sequence and amino acid sequence of HY-12 xylanase were determined. PCR amplification conditions were performed using a Hot Start PCR kit (Roche, USA) to denature the degenerate primer pairs and template described above at 94 ° C. for 5 minutes, 30 seconds at 95 ° C., 30 seconds at 40 ° C., and 72 ° C. The reaction was carried out for 30 seconds and extended at 72 ° C. for 7 minutes to terminate the reaction.

As a result, the xylene Rana of the present invention is composed of the 274 amino acid residues (SEQ ID NO: 2) The gene bank data source as a protein base (Genebank database) search results, Syracuse know Dorsal mousse utility Cellulofine (Acidothermus cellulolyticus ) showed 52% amino acid sequence homology with xylanase and thermobifida alba ) and Streptomyces avermitilis have been identified as new xylanase with 47% homology to xylanase.

<3-3> Xylanase  Biochemical Characterization

In order to investigate the optimal reaction conditions of xylanase, the effects of reaction temperature, pH of reaction solution and metal ions were investigated.

The optimum temperature of enzyme activity was measured at 10 ℃ intervals in the range of 30 ~ 90 ℃, and after 30 minutes of treatment at 10 ℃ ~ 90 ℃ temperature, the enzyme activity was measured by measuring the residual activity of enzyme. Thermal stability was measured. The optimum pH for enzyme activity was 50 mM Sodium Citrate Buffer (pH 3.0-6.0), 50 mM Sodium Phosphate Buffer (pH 6.0-7.0), 50 mM Tris-HCl Buffer (pH 7.0-8.0) And 50 mM boric acid buffer (pH 8.0-10.0). First, the effect of reaction temperature and pH on the activity of xylanase was examined. As a result, the maximum activity was observed at pH 5.5 to 6.5 and 70 ° C., indicating that xylanase was highly active at neutral pH (see FIGS. 3A and 3B). . In addition, in order to investigate the stability to heat, the residual activity was measured after leaving the purified enzyme solution at different temperatures at different temperatures and times. When the treatment was performed at 60 ° C. for 30 minutes, the activity decreased to about 35%. After 30 minutes of treatment, the residual activity was about 84%, but it was confirmed that the enzyme activity remained stable at a temperature lower than 50 ° C (see FIG. 3C). In addition, as a result of investigating the influence of metal ions on xylanase, the activity of xylanase was Fe when 9 kinds of metal ions (Sigma Chem. Co.) including cobalt ions were added and reacted at a concentration of 10 mM ^{2 +} , When the Cu ^{2 +} , Ca, Co ions were added, the activity was decreased, but when the K, Mn, Na ions were added, the activity was generally increased (see FIG. 3D).

1 is cellulose microbium sp. Electron micrograph of the strain of Cellulosimicrobium sp.

FIG. 2 is a diagram showing the separation and purification of xylanase from HY-12 strain as column chromatography (FIG. 2A) and electrophoresis with acrylamide gel (FIG. 2B).

3 is a graph showing the biochemical properties of xylanase isolated from HY-12 strains:

    A: optimum reaction pH of xylanase;

    B: optimum reaction temperature of xylanase;

    C: stability to heat of xylanase; And

    D: effect of metal ions on xylanase,

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel Cellulosimicrobium sp. HY-12 strain and xylanase produced          from it <130> 7p-08-12 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 1373 <212> DNA <213> 16S rDNA of Cellulosimicrobium sp. HY-12 <400> 1 tgaagcccgc ttgctgggtg gatcagtggc gaacgggtga gtaacacgtg agtaacctgc 60 ccctgacttt gggataactc cgggaaaccg gggctaatac cggatatgag ctcttgctgc 120 atggtgaggg ttggaaagat ttatcggttg gggatgggct cgcggcctat cagcttgttg 180 gtggggtgat ggcctaccaa ggcgacgacg ggtagccggc ctgagagggc gaccggccac 240 actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa tattgcacaa 300 tgggcgcaag cctgatgcag cgacgccgcg tgagggatga cggccttcgg gttgtaaacc 360 tctttcagca gggaagaagc gcaagtgacg gtacctgcag aagaagcgcc ggctaactac 420 gtgccagcag ccgcggtaat acgtagggcg caagcgttgt ccggaattat tgggcgtaaa 480 gagctcgtag gcggtttgtc gcgtctgctg tgaaatcccg aggctcaacc tcgggcttgc 540 agtgggtacg ggcagactag agtgcggtag gggagactgg aattcctggt gtagcggtgg 600 aatgcgcaga tatcaggagg aacaccgatg gcgaaggcag gtctctgggc cgcaactgac 660 gctgaggagc gaaagcatgg ggagcgaaca ggattagata ccctggtagt ccatgccgta 720 aacgttgggc actaggtgtg gggctcattc cacgagttcc gtgccgcagc taacgcatta 780 agtgccccgc ctggggagta cggccgcaag gctaaaactc aaaggaattg acgggggccc 840 gcacaagcgg cggagcatgc ggattaattc gatgcaacgc gaagaacctt accaaggctt 900 gacatatacg agaatccgct ggagacagcg gcctctttgg acactcgtat acaggtggtg 960 catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1020 ctcgtcccat gttgccagcg ggttatgccg gggactcatg ggagactgcc ggggtcaact 1080 cggaggaagg tggggatgac gtcaaatcat catgcccctt atgtcttggg cttcacgcat 1140 gctacaatgg ccggtacaaa gggctgcgat accgtaaggt ggagcgaatc ccaaaaagcc 1200 ggtctcagtt cggattgggg tctgcaactc gaccccatga agtcggagtc gctagtaatc 1260 gcagatcagc aacgctgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcaagt 1320 cacgaaagtc ggtaacaccc gaagcccatg gcccaaccct tgtgggggga gtg 1373 <210> 2 <211> 1188 <212> DNA <213> nucleotide sequence of xylanase <400> 2 atggccactg cacctgtcac caccacctct cccaccccgc gacggcaccg ccgagccatc 60 gtgctcggag ccggcatcgc cgtcgtcggc ctggtcgccg cgcccctcgc ggcatccgcc 120 cacggcggct ctccgtgggg ccaccacgcc gtccccggcg cccccagcca cggccacaag 180 ccgcccaccc agcccaccac cggcgacgac accctgcgga gcctcgcggc gccgtcgggc 240 ctgcggatcg gtgcagccat caacaccgac aagctcggca ccgacgacgc ctacacgacg 300 atcgccggcg agcagttctc caccgtcagc ccggagaacg tcatgaagtg ggacacgatc 360 gagcccacgc agggcacgta caacttcgcg ccggccgaca agctcgtggc cttcgcgcag 420 cagcacggcc agctcgtccg cggccacacc ctggtctggc acaaccagct cccgtcctgg 480 ctcaccgccg aagcggacag cctcaccgcc gaccagctcc gggcgatcct caagaagcac 540 atccagaccg aggtcaagca cttcaagggc aagatctggc agtgggacgt cgtcaacgag 600 gccttcgccg acgacggcac gctccgcgac gacatctggt cgcagaagct cggcgacagc 660 tacatcgccg acgcgttccg ctgggcccac gaggccgacc cgaaggccaa gctcttctac 720 aacgactaca acatcgagta cacgggcgcg aagagcgaag ccgtctacgc gatggtcaag 780 aagctgcagg cccagggcgt gcccatcgac ggcgtcggtt tccaggacca cctcgacacg 840 cagtacggca cgccgaacct ccaggagacg atgcagaagt tcgccgacct gggcctcgac 900 accgccgtga ccgaggccga cgtccgcacc acgctccccg tcaccaccgt ggagcagacc 960 gcccagaaca gcatgtggtc gcagtcgctg tccgcgtgcc tgctggtcaa gcgctgcatc 1020 tcgttcaccg tctggggcat cgacgacggc tcgtcctggg tgccgagcac gttcgagggc 1080 gagggcgcgg ccctgctctg ggacgacgac ttccagccga aggcccagtt cggcgtcctc 1140 cagcagacgc tcgagctcgc cgccggggca ccgcgccgga cgcgttga 1188 <210> 3 <211> 395 <212> PRT <213> amino acid sequence of xylanase <400> 3 Met Ala Thr Ala Pro Val Thr Thr Thr Ser Pro Thr Pro Arg Arg His   1 5 10 15 Arg Arg Ala Ile Val Leu Gly Ala Gly Ile Ala Val Val Gly Leu Val              20 25 30 Ala Ala Pro Leu Ala Ala Ser Ala His Gly Gly Ser Pro Trp Gly His          35 40 45 His Ala Val Pro Gly Ala Pro Ser His Gly His Lys Pro Pro Thr Gln      50 55 60 Pro Thr Thr Gly Asp Asp Thr Leu Arg Ser Leu Ala Ala Pro Ser Gly  65 70 75 80 Leu Arg Ile Gly Ala Ala Ile Asn Thr Asp Lys Leu Gly Thr Asp Asp                  85 90 95 Ala Tyr Thr Thr Ile Ala Gly Glu Gln Phe Ser Thr Val Ser Pro Glu             100 105 110 Asn Val Met Lys Trp Asp Thr Ile Glu Pro Thr Gln Gly Thr Tyr Asn         115 120 125 Phe Ala Pro Ala Asp Lys Leu Val Ala Phe Ala Gln Gln His Gly Gln     130 135 140 Leu Val Arg Gly His Thr Leu Val Trp His Asn Gln Leu Pro Ser Trp 145 150 155 160 Leu Thr Ala Glu Ala Asp Ser Leu Thr Ala Asp Gln Leu Arg Ala Ile                 165 170 175 Leu Lys Lys His Ile Gln Thr Glu Val Lys His Phe Lys Gly Lys Ile             180 185 190 Trp Gln Trp Asp Val Val Asn Glu Ala Phe Ala Asp Asp Gly Thr Leu         195 200 205 Arg Asp Asp Ile Trp Ser Gln Lys Leu Gly Asp Ser Tyr Ile Ala Asp     210 215 220 Ala Phe Arg Trp Ala His Glu Ala Asp Pro Lys Ala Lys Leu Phe Tyr 225 230 235 240 Asn Asp Tyr Asn Ile Glu Tyr Thr Gly Ala Lys Ser Glu Ala Val Tyr                 245 250 255 Ala Met Val Lys Lys Leu Gln Ala Gln Gly Val Pro Ile Asp Gly Val             260 265 270 Gly Phe Gln Asp His Leu Asp Thr Gln Tyr Gly Thr Pro Asn Leu Gln         275 280 285 Glu Thr Met Gln Lys Phe Ala Asp Leu Gly Leu Asp Thr Ala Val Thr     290 295 300 Glu Ala Asp Val Arg Thr Thr Leu Pro Val Thr Thr Val Glu Gln Thr 305 310 315 320 Ala Gln Asn Ser Met Trp Ser Gln Ser Leu Ser Ala Cys Leu Leu Val                 325 330 335 Lys Arg Cys Ile Ser Phe Thr Val Trp Gly Ile Asp Asp Gly Ser Ser             340 345 350 Trp Val Pro Ser Thr Phe Glu Gly Glu Gly Ala Ala Leu Leu Trp Asp         355 360 365 Asp Asp Phe Gln Pro Lys Ala Gln Phe Gly Val Leu Gln Gln Thr Leu     370 375 380 Glu Leu Ala Ala Gly Ala Pro Arg Arg Thr Arg 385 390 395 <210> 4 <211> 20 <212> DNA <213> Sense primer for 16S rDNA of Cellulosimicrobium sp. HY-12 <400> 4 agagtttgat cmtggctcag 20 <210> 5 <211> 19 <212> DNA <213> Antisense primer for 16S rDNA of Cellulosimicrobium sp. HY-12 <400> 5 ggttaccttg ttacgactt 19 <210> 6 <211> 17 <212> DNA <213> Sense primer for xylanase <400> 6 atytggcaat gggacgt 17 <210> 7 <211> 20 <212> DNA <213> Antisense primer for xylanase <400> 7 tggtcngctt cgtgrgccca 20

Claims (13)

Cellulose microbium sp. To produce xylanase. HY-12 ( Cellulosimicrobium sp. HY-12) strain (KCTC 11155BP). A xylanase having the amino acid sequence of SEQ ID NO: 3 produced from the strain of claim 1. delete 3. The xylanase according to claim 2, wherein the xylanase has a molecular weight of 42 kDa. The xylanase according to claim 2, wherein the xylanase exhibits maximum activity at 60 ° C to 70 ° C. The xylanase of claim 2, wherein the xylanase exhibits maximum activity at pH 5.5 to 6.5. The xylanase according to claim 2, wherein the xylanase increases enzymatic activity upon addition of Zn, K, Mg, Na or Mn. Gene encoding the enzyme of claim 2. 9. The gene of claim 8, wherein the gene is set forth in SEQ ID NO: 2. Recombinant vector comprising the gene of claim 8. A transformant into which the recombinant vector of claim 10 is introduced. 1) Cellulose microbium sp. Of claim 1 in a medium containing xylan. Culturing the HY-12 strain or the transformant of claim 11 and then centrifuging to obtain a supernatant; 2) adding a precipitant to the supernatant of step 1) to induce aqueous protein precipitation; 3) removing the insoluble precipitate from the precipitate of step 2) and dialysis to obtain a crude enzyme solution; And 4) xylanase production method comprising the step of purifying the crude enzyme solution of step 3) by column chromatography. The method of claim 12, wherein the precipitant of step 2) is selected from the group consisting of ammonium sulfate, acetone, isopropanol, methanol, ethanol and polyethylene glycol.

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