KR20120000856A - A paenibacillus sp. hpl-003 strain for producing xylanase and an thermostable, wide ph spectrum, and highly active xylanase produced thereby and a transgenic mass-producing method thereof - Google Patents

Abstract

PURPOSE: A novel protein isolated from novel Penibacillus sp. is provided to develop biopolymers, foods, and feed. CONSTITUTION: A xylanase has an amino acid sequence of sequence number 4. A strain producing xylanase is Paenibacillus sp. HPL-003(deposit number KCTC 18179P). A polynucleotide encoding the xylanase has a base sequence of sequence number 3. A recombinant expression vector contains the polynucleotide encoding xylanase. A method for producing the xylanase comprises: a step of introducing the recombinant expression vector to a host cell to prepare a tranformant; a step of culturing the transformant; and a step of centrifuging to collect crude enzyme liquid.

Description

Novel phenavacillus sp.HPL-003 strain producing heat resistance, broad pH, high activity xylanase, new xylanase enzyme isolated from it and its mass production method using transformants thereof {A Paenibacillus sp.HPL- 1003 strain for producing xylanase and an thermostable ， wide pH spectrum ， and highly active xylanase produced thereby and a transgenic mass-producing method

The present invention relates to a novel Phenibacillus sp. Strain that produces novel xylanases.

To cope with international environmental regulations such as reducing CO2 emissions, human beings living in the 21st century are faced with the task of developing alternative resources for fossil fuels.In preparation for global warming and depletion of petroleum resources, solar power, wind and hydro, nuclear power, biomass, etc. The main subject is research. Among these, in the biorefinery process of saccharifying wood fiber cellulose biomass raw materials to produce biofuels and chemical raw materials, it is inevitable that 15-30% of xylans contained in wood materials are generated as by-products. Since this is impossible, most of them are treated as waste resources.

To date, hydrolysis of xylan has been mainly performed by chemical method. It is converted into the form of decomposition products xylose and xyloligosaccharide by adding sulfuric acid to fibrin-based biomass and steam pressurization at 130 ° C, Various impurities are generated additionally, and thus, purification technology thereof is required. In addition, a large amount of energy is consumed, and an expensive device capable of withstanding acid and high temperature is required, and the production cost is increased due to the disposal cost of waste generated at this time. On the other hand, the decomposition of xylan by a biological method using xylanase (an enzyme that decomposes and glycosylates xylan, the main component of hemicellulose into xylose, is generally referred to as xylanase enzyme) is compared with chemical decomposition. Waste is low and the waste generated is not only a small amount, but also easy to process, it is very economically advantageous.

Xylanase is not only required in the biorefinery (biochemical industry) process, but is also used for various purposes such as bleaching process of paper, improving feed efficiency, cleaning of fruit drinks, high-quality baking or using agricultural by-products. Can be. To date, it has been produced by a variety of microorganisms, especially in the paper industry, alkali or heat resistant xylanase has been isolated from various bacteria for application in the bleaching process (Tenkanen, M. e t . Al ., Enzyme.Microb). (Technol, 14, 566-574, 1992), many xylanase-producing bacteria without cellulase activity have been reported, and the results of research to reduce the loss of cellulase for papermaking have been reported. (Khashin, A. e t . Al ., Appl. Environ. Microbiol, 59, 1725-1730, 1993; Kosugi, A. e t . Al ., J. Bacteriol, 183, 7037-7043, 2001). In addition, improving the quality of bread by Giles Rana handle dehydratase (xylanase), or let a (Courtin, CM e t. Al ., J. Agric. Food. Chem, 47, 1870-1877, 1999), β- claim Giles (β-xylosidase) and xylene Lana's study of glycosylation were reported to the perch forestry by-products as the gene is introduced into yeast kinase (xylanase) microorganism can utilize (La Grange, DC e t. 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 ingesting grain feed. are known to prevent disease and improve feed efficiency (McCracken, KJ e t. al ., Br. Poult. Sci, 42, 638-642, 2001).

Was of the xylene Rana dehydratase producing microorganism reported are Trichoderma (Trichoderma) in fungal strains are generally used, their enzyme productivity represents the maximum activity at substantially dominant but mainly acidic conditions compared to the bacteria that produce xylene Rana dehydratase (Tenkanen et al . , Enzyme and Microbial Technology 14 (7): 566-574, 1992). Bacteria producing xylene Rana dehydratase may include difficulties Pseudomonas (Aeromonas) genus Bacillus (Bacillus) genus, Clostridium (Clostridium) genus Streptomyces (Streptomyces), An asbestos spread loose (Aspergillus), A bacterial Depending on the characteristics of the xylanase produced, various genes encoding these enzymes have been reported.

The domestic and foreign technical status of xylanase enzyme is the trichoderm a sp. C-4 strains were selected but higher activity is required for industrialization (Sul et al . , Appl Microbiol Biotechnol . 66 (1): 63-70, 2004). Dr. Kang Myung-kyu's team at Donghae University produces Cephalosporium sp. RYM-202 strains have been selected and studied for their use in pulp processing (Kang, Myung-kyu et al., Korean Journal of Environmental Biology, 17 (2): 191-198, 1999). Professor Kim's team at KAIST constructed Bacillus subtilis DB104 / pJHKJ4, a recombinant strain that produces Bacillus- derived endoxylanase extracellularly (Kim JH et al . , J. Microbiol . Biotechnol . , 10 (4): 551-553, 2000). In Taiwan, anaerobic fungi Neocallimastix Xylanase , a gene cloned from patriciarum , has increased base resistance through directional enzyme evolution (Yew-Loom Chen et. al . , Can . J. Microbiol . 47 (12): 10881094, 2001) Recently, Bacillus , a basic strain resistant from wastewater generated after the pulp process, firmus has been isolated and identified (Pochih Chang, Biochemical and Biophysical Research Communications 319: 1017-1025, 2004). This xylanase has a high activity in a wide range from pH 4 to pH 11, and has a high heat resistance to show a residual activity of about 70% of the initial activity even after 16 hours incubation at 62 ℃. As such, various strains have been discovered in many countries around the world, and their functions have been improved through directional evolution.

Looking at the existing patents related to xylanase, after identifying strains that produce xylanase, using wild strains or making recombinant strains using E. coli, etc., a method of producing xylanase is used (International Patent Publication) 93/08275, International Publication No. 92/01793, International Publication No. 92/17573, Republic of Korea Publication No. 10-0072225, Republic of Korea Publication No. 10-02211204, Republic of Korea Publication No. 10-0411771 ). In addition, patents related to xylanase as a feed additive include a patent for a novel Streptomyces genus JL-2 strain producing xylanase (Korean Patent Publication No. 2001-0111986), a signal sequence of Bacillus subtilis endo xylanase. Patent for a recombinant plasmid containing a gene and a method for producing a foreign protein using the same (Korean Patent Publication No. 2000-0034279), encoding xylanase of Bacillus sp. AMX-4 strain Patents related to the novel xylanase gene (Korean Patent Publication No. 2003-0085679) and the like.

Currently known domestic xylanase-related patents include a patent for a novel Streptomyces genus JL-2 strain producing xylanase (Korean Patent Laid-Open Patent 2001-0111986), and the 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 novel Fanibacilli sp. Xylanase (Korean Patent Publication No. 2007-0082329) of the HY-8 strain, but there is no mention of a clear enzyme activity or actually has not been used in many fields in Korea. However, since the development and polymerization technology of biomass-based various value-added compounds, especially polymer compounds (plastics, etc.), the development of xylanase with new characteristics is more urgently needed than ever before.

In order to effectively utilize the biomass, the saccharification process of cellulose using cellulase should be developed as well as the saccharification process of hemicellulose using xylanase. The enzymatic glycosylation process using xylanase is characterized by the enzyme properties required according to the biomass pretreatment method.For biomass pretreatment using acid, acid-resistant glycosylation is required, but the current pulp and paper process or bio using alkali Alkali-resistant glycosylase is required in the saccharification process after mass pretreatment. In addition, heat resistance may be required for simultaneous processes of saccharification and fermentation. However, since commercialized xylanase is mostly suitable for acidic conditions, development of xylanase that is active under alkaline conditions is required. More preferably, the development of xylanase having high activity in both acidic and alkaline conditions is required. Therefore, in order to use the industry directly, it is necessary to secure a xylanase gene that maintains activity even under high temperature and pH conditions by searching for a new microorganism and enzyme system instead of existing xylanase. You will be able to solve the problem.

Accordingly, the inventors of the present invention, in order to be able to expand the world as well as domestic development in order to avoid or break the intellectual property rights that are all oligopolized by the developed countries of the existing xylanase and the strains producing them. In the development of a novel xylanase, a novel strain of the genus Penibacillus HPL-003 ( Paenibacillus sp. HPL-003) and the xylanases produced therefrom have been reported so far compared to other xylanases. Unit = mM product / mg protein / min), heat resistance, the best in all of the optimum pH range was confirmed by the solid culture measurement method and / or liquid culture enzyme activity measurement method was completed.

It is an object of the present invention to provide xylanase with high activity at high temperatures and a wide range of pH.

In addition, another object of the present invention to provide a strain for producing the xylanase.

It is another object of the present invention to provide a polynucleotide encoding xylanase.

It is another object of the present invention to provide a recombinant expression vector operably linked to the polynucleotide.

Another object of the present invention is to provide a transformant in which the recombinant expression vector is introduced into a host cell.

In addition, another object of the present invention is to provide a xylanase production method comprising the step of obtaining a crude enzyme solution by culturing the strain or the transformant in the medium and centrifugation.

Another object of the present invention is to provide a xylan degrading agent comprising the xylanase, the strain or the transformant.

In addition, another object of the present invention to provide a composition for processing xylan in food comprising the xylanase.

In addition, another object of the present invention to provide a feed additive comprising the xylanase.

In addition, another object of the present invention is to provide a composition for a papermaking process comprising the xylanase.

In addition, another object of the present invention is to provide a xylan decomposition method comprising the step of adding the xylanase, the strain or the transformant to a fibrin-based biomass or xylan-containing solution.

In addition, another object of the present invention is to provide a feed manufacturing method comprising the step of adding the xylanase, the strain or the transformant to the feed material of the animal.

In order to achieve the above object, the present invention provides a xylanase having the following characteristics (a) to (d):

(a) the molecular weight on SDS-PAGE is about 65 kDa;

(b) maximum activity at pH 5 to pH 11;

(c) maximum activity at 50-60 ° C .; And,

(d) 95 unit / min mg at 50 ° C., pH 5 to pH 11 and at least 100 unit / min mg xylose at 60 ° C., pH 6 to pH 9;

The present invention also provides a strain for producing the xylanase.

The present invention also provides polynucleotides encoding xylanase.

The present invention also provides a recombinant expression vector operably linked to the polynucleotide.

The present invention also provides a transformant in which the recombinant expression vector is introduced into a host cell.

The present invention also provides a method for producing xylanase comprising culturing the strain or the transformant in a medium and then centrifuging to obtain a crude enzyme solution.

The present invention also provides a xylan degrading agent comprising the xylanase, the strain or the transformant.

The present invention also provides a composition for processing xylan in a food comprising the xylanase.

The present invention also provides a feed additive comprising the xylanase.

In addition, the present invention provides a composition for a papermaking process comprising the xylanase.

The present invention also provides a method for decomposing xylan comprising the step of adding the xylanase, the strain or the transformant to a fibrin-based biomass or xylan-containing solution.

In addition, the present invention provides a feed preparation method comprising the step of adding the xylanase, the strain or the transformant to the feed material of the animal.

The novel strain of the present invention, Penibacillus ( Paenibacillus ) sp. HPL-003 and xylanase isolated from it show excellent xylan decomposition activity at 60 ℃ heat resistance and wide pH condition (4-11), so it is used in glycosylation process of fibrous biomass as well as in feed industry, paper and detergent industry. It can be usefully used to produce raw materials, such as petroleum substitute raw materials, special functional substances, biopolymers.

1 is an electron micrograph of the selection strain.
Figure 2 shows the results of screening for active clones from 1,248 gDNA libraries.
3 is a diagram showing an ORF analysis and the results of each ORF amino acid homology analysis.
Figure 4 shows the activity after separation and purification of xylanase from the transformant.
5 is an optimal pH graph of xylanase activity.
6 is an optimal temperature graph of xylanase activity.
7 is enzyme properties (kinetics) after xylanase isolation and purification.

Hereinafter, the present invention will be described in detail.

The present invention provides xylanases having the following characteristics (a) to (d):

(a) the molecular weight on SDS-PAGE is about 65 kDa;

(b) maximum activity at pH 5 to pH 11;

(c) maximum activity at 50-60 ° C .; And,

(d) 95 unit / min mg at 50 ° C., pH 5 to pH 11 and at least 100 unit / min mg xylose at 60 ° C., pH 6 to pH 9;

The amino acid sequence of the xylanase of the present invention preferably includes any one of the following sequences, but is not limited thereto:

a) the amino acid sequence set forth in SEQ ID NO: 4;

b) an amino acid sequence having at least 70%, preferably at least 80%, most preferably at least 90% homology with the amino acid sequence set forth in SEQ ID NO: 4;

c) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 3;

d) an amino acid of a protein that is functionally identical to a protein comprising one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 4, wherein said amino acid sequence is substituted, deleted, inserted, and / or added, and comprises the amino acid sequence set forth in SEQ ID NO: 4; order; And,

e) an amino acid sequence of a protein functionally identical to a protein which is encoded by a DNA hybridized under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3, and which comprises the amino acid sequence set forth in SEQ ID NO: 4.

Such stringent conditions are determined upon cleaning after hybridization. One of the stringent conditions is 15 minutes with 6 ± SSC, 0.5% SDS at room temperature, 30 minutes with 2 ± SSC, 0.5% SDS at 45 ° C, 30 minutes with 0.2 ± SSC, 0.5% SDS at 50 ° C. Repeat the wash twice. More preferred stringent conditions are to use higher temperatures than above. The other part of the stringent conditions are carried out in the same manner, but the last two 30 minutes are washed with 0.2 ± SSC, 0.5% SDS at 60 ° C. Another stringent condition is to wash the last two times at 65 ° C. with 0.1 ± SSC, 0.1% SDS under these stringent conditions. Those skilled in the art can clearly set these conditions in order to obtain the stringent conditions required.

The xylanase of the present invention exhibits maximum activity at pH 5 to pH 11, preferably pH 6 to pH 10, more preferably pH 6 to pH 9, and preferably at 50 to 60 ° C. It is not limited to this.

In a specific embodiment of the present invention, after the mushroom cultivation collected from the middle of Garasan located in Dadae-ri, Namje-myeon, Geoje-si, Gyeongnam, strains with excellent xylan degradability were selected from the soil containing waste wood residues.

In addition, in a specific embodiment of the present invention, the inventors prepared a gDNA library to isolate a gene encoding an enzyme protein having xylanase activity from the strain, and then tested xylanase activity. One clone was selected through the above experiment (see FIG. 2), and the nucleotide sequence of the inserted fragment DNA was analyzed. The analysis showed that the size of the DNA fragment was 6,956 bp (SEQ ID NO: 2) and included 11 ORFs in the range of 100 or more amino acids (see FIG. 3). The inventors found homology of the protein encoded by the gene in the ORF with 48% homology with endo-1,4-beta-xylanase (GenBank Accession Number: YP_001817989). Eggplant ORF9 (1620 bp, 539 aa) was confirmed by the transformation of E. coli in the xylanase activity was confirmed that the xylanase activity is excellent. In addition, the nucleotide sequence of the target DNA fragment of the transformant was confirmed, and the ORF of the novel xylanase gene of the nucleotide sequence described in SEQ ID NO: 3 was reconfirmed.

In a specific embodiment of the present invention, a transformant overexpressing a large amount of novel xylanase of the amino acid sequence set forth in SEQ ID NO: 4 was produced. Examination of the enzyme activity of about 65 kD of novel xylanase (see FIG. 4) produced and isolated from the transformed Escherichia coli confirmed that it exhibited maximum activity at a temperature of 50 ° C. to 60 ° C. at pH 4-11 (FIG. 5 to 6). In addition, 74%, 28%, 12%, 46% were inhibited by 1 mM Cu + 2, Zn + 2, Fe + 2, EDTA, etc. among the elements added as a heavy metal source, but they were not affected under 100 μM It was shown to slightly increase enzyme activity (see Table 1). In addition, as a result of analyzing the reaction characteristics (Enzyme Kinetics), the Km value indicating the substrate affinity was 0.2 (see Fig. 7), and most of the hydrolysis products were xyloligomers.

Therefore, as a result of sequencing and activity analysis, it was confirmed that the xylanase produced from the strain identified in the present invention is a novel xylanase different from the existing xylanase.

The present invention also provides a strain for producing the xylanase.

The strain may be, but is not limited to, prokaryotic cells including Escherichia coli, or eukaryotic cells including yeast, animal cells, and insect cells, and the strain producing xylanase is HPP genus HPL deposited with accession number KCTC18179P. -003 (Paenibacillus sp. HPL-003) is preferred, but any strain producing xylanase of the present invention is included in the scope of the present invention.

In a specific embodiment of the present invention, after the mushroom cultivation collected from the middle of Garasan located in Dadae-ri, Namje-myeon, Geoje-si, Gyeongnam, strains with excellent xylan degradability were selected from the soil containing waste wood residues. The selected strains were identified as gram positive bacilli, rod-shaped cells having a cell size of 1.1 µm and cell lengths of 2.5 µm to 4 µm, and lacking flagellar mobility (see FIG. 1). The strain has a 16S rRNA as set forth in SEQ ID NO: 1, and as a result of homology analysis on the rRNA, 95.0% of the genus Penisbacillus sp. CSH12-5 (Paenibacillus sp. (T) [Paenibacillus daejeonensis (T) (GenBank Accession No. AM141; AF391124)] and 95.7% homology, but no further homology was found, so the strain of the present invention HPL- It was named 003 (Paenibacillus sp. HPL-003), and it was deposited with the Korea Research Institute of Bioscience and Biotechnology on September 21, 2009. The accession number is KCTC18179P.

The present invention also provides polynucleotides encoding xylanase.

The polynucleotide encoding the xylanase of the present invention preferably includes any one of the following sequences, but is not limited thereto:

a) the nucleotide sequence set forth in SEQ ID NO: 3;

b) a nucleotide sequence having at least 70%, preferably at least 80%, most preferably at least 90% homology with the nucleotide sequence set forth in SEQ ID NO: 3;

c) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 4;

d) an amino acid of a protein that is functionally identical to a protein comprising one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 4, wherein said amino acid sequence is substituted, deleted, inserted, and / or added, and comprises the amino acid sequence set forth in SEQ ID NO: 4; Base sequences encoding sequences; And,

e) The base sequence of the protein which is a DNA base sequence hybridized under stringent conditions with the DNA comprising the base sequence of SEQ ID NO: 3, and functionally identical to the protein comprising the amino acid sequence of SEQ ID NO: 4.

The present invention also provides a recombinant expression vector operably linked to the polynucleotide.

Since the present invention has revealed a nucleotide sequence of a novel gene encoding xylanase isolated from the genus HPL-003 strain, it is possible to prepare a recombinant vector containing the gene using conventional methods known in the art. The recombinant vector of the present invention may use a commercially available vector, but is not limited thereto, and those skilled in the art may prepare and use a suitable recombinant vector.

The present invention also provides a transformant in which the recombinant expression vector is introduced into a host cell.

The host cell that can be used in the present invention is not limited, but is preferably selected from the group consisting of E. coli, prokaryotic cells including bacteria, yeast, animal cells, and prokaryotic cells including insect cells, more preferably using E. coli. One is not limited thereto.

The present invention also provides a method for producing xylanase comprising culturing the strain or the transformant in a medium and then centrifuging to obtain a crude enzyme solution.

The production method of the present invention may further comprise the step of purifying xylanase from the obtained crude enzyme solution.

In the above method, the medium is preferably selected from among the commercially known media known to those skilled in the art, such as HPL-003 strain (KCTC18179P) or the transformant of the present invention.

The present inventors use a specific resin binding vector for column chromatography when preparing a transformant as a method for increasing the purification efficiency while simplifying the purification process, and in the purification process, it is preferable to separate and separate only the enzyme bound to the resin. . As strict conditions, glutathione binding vectors, calmodulin binding vectors, maltose binding vectors, and the like can be used, and the resin for column filling is determined according to the use vector. In the present invention, but the present results using the glutathione binding vector and the resin is not limited thereto.

The present invention also provides a xylan degrading agent comprising the xylanase, the strain or the transformant.

The xylan degrading agent of the present invention can be used as the xylan degrading agent as well as the xylanase produced by the strain or the transformant.

The present invention also provides a composition for processing xylan in a food comprising the xylanase.

The present invention also provides a feed additive comprising the xylanase.

In addition, the present invention provides a composition for a papermaking process comprising the xylanase.

The composition or feed additive of the present invention may include the same or similar ingredients in addition to the xylanase of the present invention, and preferably contains 1 to 90% of the xylanase of the present invention as a whole composition or feed additive. It doesn't happen.

Xylanase of the present invention, unlike the existing xylanase shows a high activity in a wide range of conditions (pH 5-11) and heat resistance temperature range (50 ~ 60 ℃) (see Figs. 5 to 6), It can be used for the purpose of heat resistance, a wide range of pH and high activity xylanase.

The applications currently occupying the xylanase enzyme market can be broadly categorized into food, feed and technology (Bedford and Morgana, World's Poultry Science Journal 52: 61-68, 1996). In the food (fruit and vegetable production, brewing and liquor production, bakery and confectionery) market, it is used for quality improvement through softening and refining efficiency of materials, viscosity reduction, and extraction and filtration efficiency. In the feed market, it is used to reduce non-starch carbohydrates, improve intestinal viscosity, and increase digestibility of protein and starch in feeds such as pigs, poultry and ruminants (Kuhad and Singh, Crit. Rev. Biotechnol. 13, 151-172 , 1993). In addition, it is used in the papermaking process, biological whitening process, reduction of chlorine consumption, energy reduction through reduction of mechanical papermaking process, deinking effect, separation of starch and gluten, renewable fuel (bioethanol) and chemical raw material production. .

Therefore, the novel xylanase of the present invention can be usefully used for paper production and waste paper regeneration, feed addition and food quality improvement or decomposition of xylanase for industrial use, which is obvious to those skilled in the art. The compositions can be formulated and formulated in a manner known to those skilled in the art.

The present invention also provides a method for decomposing xylan comprising the step of adding the xylanase, the strain or the transformant to a fibrin-based biomass or xylan-containing solution.

In this method, the amount of the strain, the transformant or the xylanase produced by the strain or the transformant can be adjusted by those skilled in the art.

In addition, the present invention provides a feed preparation method comprising the step of adding the xylanase, the strain or the transformant to the feed material of the animal.

In this method, the amount of the strain, the transformant or the xylanase produced by the strain or the transformant can be adjusted by those skilled in the art.

[Example]

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

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example  1> Strain Isolation and Screening

<1-1> Strain Isolation

The strain of the present invention was isolated from the soil containing the waste wood residue after mushroom cultivation collected from the middle of Garasan located in Dadae-ri, Namdae-myeon, Geoje-si, Gyeongsangnam-do. Soil samples were collected from 2 to 5 cm layers from the surface of the soil, and the soil was selected by passing through 2 mm after air drying. 30 g of selected soil is placed in 270 ml of sterile saline solution (8.0 g / l of NaCl) and shaken with shaking incubator (37 ℃, 200 rpm) for about 20 minutes and then left at room temperature for 30 minutes to coarse soil particles and impurities. Was precipitated to the bottom, and the supernatant was transferred to a sterile container as a first diluent. After stirring this well, 10 ml of the solution was added to 90 ml of physiological saline to prepare 100 ml of the second dilution. 10 ml of the same solution was taken in the same manner as above while stirring the second dilution sufficiently. 100 ml was prepared. After the 6th dilution was prepared in the same manner. 3, 4, 5, and 6th dilutions were dispensed in 0.25 ml portions three times in TSA (Tryptic Soy Agar, Difco Co.) medium for strain isolation, and then spread evenly and cultured in a 37 ° C. incubator for 2 days. Colonies of microorganisms were selected. At this time, the colony was separated in consideration of various factors such as shape, size, and color. The separated colonies were subcultured again in TSA medium to separate pure strains, and were stored at -70 ° C for use as parent strains.

<1-2> Strain Selection

Selection of active strains having xylanase activity among purely isolated strains was performed by making soft agar double medium containing 0.5-1.0% of birch xylan (Fluka Bio Chemika. Co.) in TSA medium and incubating overnight by inoculating strains. Congo-red staining method (Theater RM, PJ . Wood . Appl) Environ Microbiol 43, 777-780, 1982; Beguin P. Analytical Biochemistry , 131 (2): 333-336, 1983), strains and active clones forming halo around cultured colonies were selected. Reproducibility was confirmed by measuring the xylan resolution of the selected strains once again, and among them, the best strains were selected as microorganisms producing xylanase.

< Example  2> Strain Identification

The present inventors incubated the strain producing the highest activity xylanase isolated in Example 1 at 30 ° C., followed by Gram staining and Spore Staining, to generate spores. It was identified as Gram-positive bacilli. As a result of observing the morphology with an electron microscope, as shown in Figure 1, it was a rod having a cell size of 1.1 μm and a cell length of 2.5 μm to 4 μm, and showed no mobility without flagella. In addition, after obtaining the 1,234 bp rDNA described in SEQ ID NO: 1 by 16S rRNA sequencing of the strain, and searching the GenBank database, Pantoae agglomerans ZFJ-15 (Pantoea agglomerans ZFJ- 15; GenBank Accession No. EU931554) had 96.4% homology and 96.3% matched with Penicillus genus WPCB158 (Paenibacillus sp. WPCB158; GenBank Accession No. FJ006910), but no further homology was found. The strain was named as HPL-003 strain in the genus Penivacillus, and was deposited on September 21, 2009 by the Korea Research Institute of Bioscience and Biotechnology, accession number is KCTC18179P.

< Example  3> new Xylanase  detach

<3-1> Penny Bacillus  Gene library production and assay of strain

The present inventors extracted the genomic DNA from the genus HPL-003 strain identified in Example 1 and Example 2 to extract the gene encoding the enzyme protein having xylanase activity, the gene having a size of 5 kb or less A gDNA library with fragments was produced. In the preparation of the library, DNA fragments of 1-6 kb size were made by randomly cutting the DNA extracted from the strain, and the size of the DNA fragments around 5 kb was secured by selecting the size by electrophoresis on an agarose gel. It was inserted into pCB31 plasmid vector and transformed into E. coli DH10B cells. The thus prepared 1,248 clones of the library were tested for xylanase activity in solid or liquid conditions.

<3-2> Xylanase  Activity test

The determination of xylanase activity (xylan degradability) of isolated strains, active clones, transformants, and isolated purified enzymes was performed using one or both of the following two methods. The first method is a solid culture measuring method, which is made of soft agar double medium containing 0.5-1.0% of birch xylan (Fluka Bio Chemika. Co.) in LB medium, inoculated with the strain and incubated overnight, and then cultured by Congo red staining the next day. Strains and active clones forming halo around the colonies were selected. The second method is a liquid culture enzyme activity measuring method (DN, 3,5-dinitrosalicylic acid) assay (Miller GL Anal Chem 31, 426-428, 1959), specifically, 50 μl of enzyme solution, 50 μl of substrate solution (50 mM Tris-HCl with 2% birch xylan, pH 7.0) was added at 50 ° C. After reacting for 20 minutes, 200 μl of DNS solution was added thereto, and then treated at 90 ° C. for 5 minutes, and then absorbed at 540 nm. One unit of the enzyme was defined as the enzyme activity in which 1 mg of xylanase produced 1 μmol of reducing sugar (xylose) in 1 minute. Top 1 clones GM3-SLX1 were selected through liquid and solid culture activity tests (FIG. 2).

<3-3> Xylanase  Active clone selection and genetic analysis

The base sequence of the fragment DNA inserted into the clone selected in Example 3-2 was analyzed. Sequence analysis showed that the size of the DNA fragment inserted into the plasmid was 6,956 bp (SEQ ID NO: 2), which was encoded by NCBI's Blast P or Blast N program (//www.ncbi.nlm.nih.gov/). An open reading frame (ORF) was analyzed. Of the ORFs analyzed, 11 ORFs with a size of 100 or more amino acids were selected as SLX-O1, SLX-O2, SLX-O3, SLX-O4, SLX-O5, SLX-O6, SLX-O7, SLX-O8, SLX-O9 , SLX-O10, SLX-O11. Homology search results of the protein encoded by the gene in the ORF SLX-O9 (SEQ ID NO: 1,4-beta-xylanase (AJ006646)) and 48% homology A primer was prepared by adding Xho I and Bam HI restriction enzyme recognition sites based on the nucleotide sequence, and then amplified by PCR and inserted into a pGEM-T-Easy vector (Promega) vector to prepare a recombinant plasmid. It was.

Specifically, 1 ng of GM3-SLX1 plasmid template is composed of a forward primer described by SEQ ID NO: 5 (5'-CTCGAGATGGATACATTGAAGTTGTATGTG-3 ') and a primer pair consisting of a reverse primer described by SEQ ID NO: 6 (5'-GGATCCCTATTCGTTGCTCCCC-3'). After mixing with (10 mmol), PCR amplification conditions were performed for 5 minutes denaturation at 94 ° C using PCR Premix (GenetBio), followed by 30 seconds of denaturation at 94 ° C and 30 seconds of binding at 55 ° C. Annealing), the extension of 1 minute at 72 ° C was repeated 30 times, and finally, the reaction was terminated after finishing the extension for 7 minutes at 72 ° C and maintained at 4 ° C. The product amplified by PCR was purified using GENCLEAN II Kit (Q-Biogene), and recombinant DNA was generated using T4 ligase (RBC) in the pGEM-T-Easy vector. The recombinant plasmid was transformed into E. coli JM109 to prepare a transgenic E. coli . After culturing the transformant in LB liquid medium, plasmid DNA was extracted using HiYield ™ Plasmid Mini Kit (RBC) and digested with restriction enzymes Xho I (NEB) and Bam HI (NEB). It was confirmed that the DNA fragment is inserted. In addition, the transformant was tested for xylanase activity under liquid conditions as in Example 3-2, and it was confirmed that xylanase activity was shown.

The nucleotide sequence of the target DNA fragment of the transformant was confirmed to reconfirm the ORF of the novel xylanase gene of the nucleotide sequence set forth in SEQ ID NO: 4.

<3-4> Xylanase Overexpression  making( pIVEX - GST - PX3 )

In order to prepare a transformant that overexpresses the novel xylanase, PCR was performed using a primer pair of SEQ ID NO: 5 and SEQ ID NO: 6 using GM3-SLX1 plasmid as a template. PCR reaction solution and reaction conditions were the same as in Example 3-2. The amplified product was purified and digested with Xho I (NEB) and Bam HI (NEB), and then inserted into the protein overexpression vector pIVEX-GST (Roche) to prepare a recombinant overexpression plasmid, which was obtained in E. coli BL21 (RBC). The transformed E. coli overexpressed xylanase was produced. After culturing the overexpressed transformed E. coli, the plasmid DNA was extracted and digested with the restriction enzyme, and then electrophoresis confirmed that the vector and the target DNA were successfully recombined, and the identified cells were transformed into LB liquid medium (100 ampicillin). / Ml addition) After 18 hours incubation (37 ℃, 250 rpm, A600 = 1.0) and re-inoculated in a new LB liquid medium treated with 1 mM IPTG when the A600 absorbance value is 0.4 ~ 0.6 and incubated for 3 hours Was harvested. The harvested cells were suspended, sonicated, and centrifuged at 10,000 g to separate the supernatant and the precipitate, and the SDS PAGE confirmed that the target protein was overexpressed in the supernatant and had a molecular weight of about 65 kD (FIG. 4).

<3-5> Xylanase  Active expression condition analysis

The activity of xylanase overexpressed and isolated from the novel xylanase overexpressing transformed Escherichia coli prepared in Example 3-4 was investigated by the method of Example 3-2 for each pH, temperature and metal ion. The pH of the reaction solution was adjusted to pH 4-5 with citric acid buffer, pH 6-8 with Phosphate buffer, pH 7-9 with Tris / HCl buffer, glycine / Sodium hydroxide (Glycine / NaOH) buffer solution was performed at pH 9-11. As metal ions, 1 mM CaCl ₂ , MgCl ₂ , MgCl ₂ , CuCl ₂ , ZnCl ₂ , FeCl _3, etc. were added to investigate the effect on xylanase activity. Other NaCl, LiCl, KCl, NH ₄ Cl, EDTA The effects of CsCl ₂ , 2-ME (2-Mercaptoethanol), DTT (Dithiothreitol), PMSF (Penylmethylsulfonyl fluoride), acetate, and furfural were added to investigate the effects on xylanase activity.

As a result, as shown in FIG. 5, the novel xylanase exhibited maximum activity at 50 ° C. to 60 ° C. as shown in FIG. 6 at pH 4 to 11. In addition, as shown in Table 1, xylanase activity was inhibited to 74, 28, 12, 46% by 1 mM of heavy metal or additives such as Cu ⁺² , Zn ⁺² , Fe ⁺² , EDTA and the like.

additive Relative activity (%) depending on additive concentration 1 mM Negative control group 100 NaCl 105 LiCl 101 KCl 101 NH4Cl 99 CaCl ₂ 102 MgCl ₂ 93 MnCl ₂ 98 CsCl ₂ 97 CuSO ₄ 26 ZnSO ₄ 72 FeCl ₃ 88 EDTA 54 2-ME 89 DTT 95 PMSF 95 Acetate 101 Furfural 98

< Example  4> new Xylanase  massive production

PIVEX GST-xylanase recombinant vector (Bioprogen Co., Ltd., Korea) comprising a gene encoding a novel xylanase as set forth in SEQ ID NO: 4 was isolated from Escherichia coli BL21-Gold (DE) (Stratagene, USA). ) And inoculated in liquid medium (LB 25 g / L) to which ampicillin was added (50 ㎍ / ml) to OD ₆₀₀ Stirring was incubated at 37 rpm 150 rpm until the value is 0.4 ~ 0.6. In order to induce E. coli expression of the target protein, IPTG (isopropyl-D-thiogalactoside) was added to the suspension to a final concentration of 1 mM, followed by further incubation for 3 hours. The precipitate recovered by centrifugation of the culture solution at 10,000 rpm for 10 minutes was washed twice with PBS. The washed precipitate was resuspended in PBS again, and the cells were crushed using an ultrasonic crusher (Cosmo Bio Co., LTD), and then the supernatant was recovered by centrifugation (12,000 rpm, 10 minutes). A glutathione-transferase column (GST binding resin column, Novagen) was used to purely separate the xylanase in the recovered supernatant. Xylase pure separation is attached to the prepared supernatant in a glutathione-transferase column (GST binding resin column, Novagen) equilibrated with Washing buffer solution (50 mM Tris-HCl, 100 mM NaCl; pH 7.0) After the treatment, factor Xa protease (NEB) was treated and purified by using a buffer (Washing buffer solution; 50 mM Tris-HCl, 100 mM NaCl; pH 7.0). Xylanase enzyme activity was measured from each sample recovered in the purification step, and purification of the enzyme active fraction was confirmed by SDS-PAGE. Protein content was measured using the Bradford method (Bradford, Sigma Aldrich), BSA (bovine serum albumin) was used as a standard protein.

As a result, glutathione resin column chromatography was able to produce a large amount of xylanase simply. In addition, the xylanase activity bound to the resin showed that the xylanase activity was not changed, and thus it can be seen that it can be applied to a high efficiency conversion process through the enzyme immobilization method. The enzyme purified by this method showed more than five times more activity than the enzyme before purification. Enzyme activity was shown to be 97.37, 124.19, 122.21, 124.61, 122.95, 103.27, and 96.08 units at pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, pH 10.0, and pH 11.0 at 50 ° C. for very wide pH applications. Sex. Also at 60 ° C, 26.93, 105.19, 123.56, 120.95, 112.25, 29.89, and 22.76 units (1 mg of enzyme per minute) at pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, pH 10.0, and pH 11.0, respectively. Both the heat resistance and high activity of the produced xylose μM amount were shown.

< Example  5> Mass-produced new Xylanase  Enzyme properties

To characterize the enzyme, purified xylanase was added to the test tube and 50 mM Tris-Hcl (pH 7.0) buffer containing various amounts of birch xylan was added. The reaction mixture was reacted at 50 ° C. for 20 minutes to determine the enzyme reaction rate (Lineweaver-Burk).

As a result, as shown in FIG. 7, the affinity K _m value for the xylan substrate was 0.2, and most of the hydrolysis products were xylose and xyloligomer. The xylanase activity against xylans (beech, oats, etc.) other than birch xylan was similar.

Korea Research Institute of Bioscience and Biotechnology KCTC18179 20090921

<110> Korea Research Institute of Chemical Technology <120> A Paenibacillus sp. HPL-003 strain for producing xylanase and an          thermostable, wide pH spectrum, and highly active xylanase          produced thereby and a transgenic mass-producing method <130> 9p-11-42 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 1393 <212> DNA <213> Paenibacillus sp. HPL-003 rDNA <400> 1 ggctcaggac gaacgctggc ggcgtgccta atacatgcaa gtcgagcggg gttatgttaa 60 aagcttgctt ttaacataac ctagcggcgg acgggtgagt aacacgtagg caacctgccc 120 atcagactgg gataactacc ggaaacggta gctaataccg gatacatcct ttccctgcat 180 ggggagagga ggaaagacgg agcaatctgt cactgatgga tgggcctgcg gcgcattagc 240 tagttggtgg ggtgaaggcc taccaaggcg acgatgcgta gccgacctga gagggtgatc 300 ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt agggaatctt 360 ccgcaatggg cgaaagcctg acggagcaac gccgcgtgag tgatgaaggt tttcggatcg 420 taaagctctg ttgccaggga agaacgtctt gtagagtaac tgctacaaga gtgacggtac 480 ctgagaagaa agccccggct aactacgtgc cagcagccgc ggtaatacgt agggggcaag 540 cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg ctctttaagt ctggtgttta 600 atcccgaggc tcaacttcgg gtcgcactgg aaactgggga gcttgagtgc agaagaggag 660 agtggaattc cacgtgtagc ggtgaaatgc gtagatatgt ggaggaacac cagtggcgaa 720 ggcgactctc tgggctgtaa ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt 780 agataccctg gtagtccacg ccgtaaacga tgaatgctag gtgttagggg tttcgatacc 840 cttggtgccg aagttaacac attaagcatt ccgcctgggg agtacggtcg caagactgaa 900 actcaaagga attgacgggg acccgcacaa gcagtggagt atgtggttta attcgaagca 960 acgcgaagaa ccttaccagg tcttgacatc cctctgatcg gtctagagat agatctttcc 1020 ttcgggacag aggagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080 gttaagtccc gcaacgagcg caacccttat gcttagttgc cagcaggtca agctgggcac 1140 tctaagcaga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa atcatcatgc 1200 cccttatgac ctgggctaca cacgtactac aatggccggt acaacgggaa gcgaaagagc 1260 gatctggagc gaatcctaga aaagccggtc tcagttcgga ttgcaggctg caactcgcct 1320 gcatgaagtc ggaattgcta gtaatcgcgg atcagcatgc ccgcggtgaa tacgttcccg 1380 ggtcttgtac aca 1393 <210> 2 <211> 6956 <212> DNA <213> Paenibacillus sp. <400> 2 catcctctct ataagcttaa cagaagccta gcccaataga caaaagacgg gtataccgtc 60 cccggtcccg tcttttctta tcatgaatcg ctgctgtata tgccaggcga gctggaagtg 120 aattgcgctt caaccttatt tcttggttgt tgttactact ttagctgctg cttttggagc 180 tgttttcact gctgtttttg gagctgtttt taccactgtt tttgctggag tcactttttt 240 aacaggagct ttgtgagccg cttttttagc tacaacatgg tgtgcacgag ttttgtgagc 300 tgttgcttta tgttttactg cataatgtct tttagcatga tgtttaacgg tacggtgctt 360 cgcttttaca gcttttgctt tatgtgccgt ttttttggca acagcttttt tggcaggagc 420 ttttttagct gttgttactt tatgagctgt tgcagctttt ttaacaggtg ctttcaccac 480 tggtgttttc gtcgcaggtg cttttactgc tggtgctttg gtagccgggg tcgtaaccgc 540 cggtgctttc gttgctggtg cagttgtagc tggcgttgtt gtcgctgtag cggcgaatgc 600 actgcttgct cctcccaata cgcttactac ggtcaatacg gctgctgcat ttttagtcca 660 ttttttcatg attgtatcac ctctccttta aagtatcttt agcttatcag gagaattcgg 720 gataattgtg gaacaaatgt gtgaaaagta tatgaaaaag cgtgcagccc cgtattcaaa 780 aaatacgtga gtctgcacgc tgtacgtttt acacaattac attcactaac cgatggcaat 840 ccaggaaaca ataccgtagt tgtgcgcggt ttcgttccat ttggttacta caatcaccgc 900 tccattcggc gtctgcgatt tcagcgtcgc atgaaagaga ggatggttcg tcatcgctac 960 cagcgtatag tccgggttca taaaaggctc tgcgaatata atgatcactt ccgtctctcc 1020 ttcgctgccc ttcaaaagaa acggcgctct gccaaactgc tgcaatgcgg actgattggc 1080 acaagtacga accggggtga tgctcagatg ctccggcttc actgcgttca gctccagctc 1140 acgcgagcct acggagcctt ccaccagatg atgcccttca atgctttgct ctgccaagtg 1200 atggctttgt accgcagcgg tctgtagatg ctccgatcct acagcttccg ggctcagcac 1260 ctcgctgtcg acggcctgag ctttcagatg gctgcgagat acactatctg cctggagtct 1320 gtcgccactg ataccttcct ccggcaaaag ccccgagaga tgaatgtccg tggacaactg 1380 agccagcgtc actgcgcccg gcttcaggtg gtgcgcatca atggcttctg ctccaatgtg 1440 tcggccttca atggcttcct cagccacatg ctgggactgt accgctccat ttgcaagctt 1500 gccggacgta accgcttctt cttgcaatgt gtcttccgac accacctgcc agcctatatg 1560 ctgcggctgg atggcacctg cttgcagatg ctccgactgg acggaatcca aggcgatatg 1620 ccttgattgc accgcgcctt ctgccagcgc cgattcctgt accgccccat cggccagatg 1680 aaggtggtgt accgcttgct ccaccagatg tacggagctt acgctggctt ccgccaaatt 1740 cgaagcacgc accgcccgaa gcgcaagctt ctcagcattc acgctcccgg cctggagcgc 1800 gtgaacagat acacttcccg gcgcaatatg ccgggtcatc actgctgaag cctgcaaatg 1860 gtcagaggtg acggcttccg gggccaaatg gctcgattgg atcgcctctt ctgccaaatg 1920 gttcccttgc accgcttcgt ccgtaatgtg ccatggctgt accgagtggg cggacagctt 1980 ggccggccgt cacgctctca tccgccagtt tgccggacgt aacagactca tcctgcaagg 2040 catcggtcga tatcgcctgt gatgcgatat gctcgctctg aatggttccg atgtgcagat 2100 gatgtggctg gatggactcc ggtgcgatat gcacggactt cactgccccc tctgccagcg 2160 ctctgtcctg taccgctcca tctgccagat gccgggggtg tacggcctgc gcagacaaat 2220 gtgaagggcc gacgatgcct tccgcgaggt tggtcgcttg cacggcccga gccgccagct 2280 tctccgaggt gacgctttcc gcttgaagcg cacgaccgga tacgctatcc ggggccagat 2340 gtctcgtaag caccgccgaa gcctgcaaat ggtcagaggt gacggcttcc ggggccaaat 2400 ggctcgattg gatcgcttct tctgccaaat ggttcccttg caccgcttcg tccgtaatgt 2460 gccatggctg taccgagtga gcggacaact tggcggccgt cacactctca tccgccagtt 2520 tgtcagacgt aacagactca tcctgcaagg catcggtcga tatcgcctgt gatgcgatat 2580 gctcgccctg aatggttccg atgtgcagat gatgtggctg gatggactcc ggtgcgatat 2640 gcacggactt tactgcccct tctgccagcg ctctgtcttg taccgctcca gctgccaaat 2700 gccgggggtg tacggcctgc gcagacaaat gtgaagggcc gacgctgcct tccgcgagat 2760 tcgtcgcttg caccgagcgt gctgccagct tttccgaggt gatgctttcc gcttgaagcg 2820 cacgatcgga tacactatcc ggggccagat gtctcgtatg caccgacgaa gactgtaaat 2880 gaaccgaggt gaccgcttcg gctgccagat gtgtcgaatc cacagccgcc acagccaggt 2940 gactgctttg tacagcttca tctgtaatat gctcggactg cactgcatgg gcagatagct 3000 tgatggaggt tacacttcct ccgccagttt gtcggacgta acagactcat tctgcaaagc 3060 atcggtcgat atcgcctgtg atgtgatatg ctcgccctga atggttccga tgtgcagatg 3120 gtgcggctgg atagactccg gtgcgatgtg cacggacttc actgccccct ctgccagcgc 3180 tctttcctgt accgctccaa ctgctagatg ccgggggtgt acggcctgcg cagataaatg 3240 cggaggaccc acgctgcctt ccgcgaggtt cattccttgt accgagcgcg ccgccagctt 3300 ttccgaggta atgctttctg cttgaagcgc acgaccggat acgctatccg gggccagatg 3360 tctcgtaagc accgtggagg cttgtagatg taccgaggtg accgctccgg ccgccagatg 3420 cgccgaatcg acagccgcca cagccagatg gttgcttctc ctgattcgtg agctggggat 3480 gcttggaagc ttctgggtgt atatcgtgcc tgggctgatc ggggtgttca atgtgattgt 3540 aattcgctcc tttattgagg gcttaccgga agggatcttg gagtcggcgc gtatagatgg 3600 ggcgggggag tttgcgactt ttatgcgtat cgttttgccg ctatgtgtcc cggtgctggc 3660 aacggtgtcg ctgttcacgg cagtagcgca atggaactcc tggttcgatg tatttttgta 3720 caattcgtcg tatgagcagt ggagtaccct ccagtatgag ctgatgaaaa tattgcaaaa 3780 ttccaacacg tccgtgaacg ctcaggatta tgccagccaa ttcgcaggct cggaaaatct 3840 ggcgaaggcg gttactccta cctccattcg tgccacgatg acaattgtgg cgtctgtgcc 3900 tattattttg gtctatccat ttttgcagaa atattttgtg aagggtatga ctttgggcgg 3960 tgtcaaggga taagcaagga ccggaaatgt gtgaaactcg ctttcttata ttttgaaaat 4020 atgggcgtag gagggattgc gttgaggtca tttttgtata aatctttggg tctggtgctg 4080 gtcggggtgt tgttattgcc agtggggtgg tgggggctgt ctgtagcgga ggcgactcca 4140 actgtggaac attcccagtc agttgcggat acggttctgt cgacggggag cgtgaataag 4200 accatcggga cgtatggatt tgagcaaggc aacgttgagg gctggaagcc tcgggggact 4260 tatacccaaa tcgcaaccgt ctcagaagct gcatatggcg gcgtacacag cctgaaggta 4320 accgctcgca cggaggtttg gaacggtgcg gagctggatg tgaagtcgct gttgcagccg 4380 ggtgtggaat atgagattag cggctatgtg aagcaggacg gtaattctac gacgccgagc 4440 gtgattaagt ttacggtgga gcagcagccg acaggcgggg ccacgacatg gaagacggtt 4500 gcgcagacag agacgacgga tacatcgtgg gccaaacttc aagggacata tacattcacc 4560 ggggggatgg atacattgaa gttgtatgtg gaaagctcga atcctgcaca ggcctattat 4620 ctggatgagg tggaaatcag gcaggtgtcg gagacaccaa ccactccccc aacggagccg 4680 acaagtggta tcgaatccag atttgaagat ggtacggctc aaggctgggt atcccgtatg 4740 ggcaccgaga cggtgcaggt gtcgaatgcc gatgcacgaa ccggatcgta cagtctgttg 4800 acgacaggca gacaacaaac atatgccggg ccaaagctgg acgtgactgc tacagtgcaa 4860 aaaggcagcc gttacacggt cagcgcttgg gtaaagctgg caccgggcga gcagcagcct 4920 gccaaggtgc gcctcagcgt acagcgcgat catcaaggtg aaagtacgta tgaaactgtg 4980 gtaggcaaca cggccatcac gactggggga tggacgcatt tgtacggaac gtatactctc 5040 gcgcatgagg cagacaccgt ctctatgtat ttggaaacgc cagaaggtac ggcttccttt 5100 tacatggatg atttcgagct gtcgcttgtg ccgccgttgg ctattgagaa ggacattccc 5160 tctttgcatg gattgtatca gggacagttc agcatcggta cggctattga agctttccag 5220 acagaagggg cttacgggga gctggtgcag aagcatttta acagcgtcgt cgccggaaat 5280 gcgatgaagc cgatctcctt gcagccgtcc gaaggacagt tccattggga agaagcagac 5340 cggattgtgc aatttgccca gcagcatggg attgctatcc gtttccatac actggtgtgg 5400 cataaccaga ccggcgattg gatgtttaag gataagaatg gacagccgat gacgccgacc 5460 gcggaaaata aaaagctttt gctggatcgg ctggagacgc atattcgtgc tgttgcagcc 5520 cgttataaaa atgtaatcac cgattgggat gtggtgaatg aagtcattga tcccgaccag 5580 ccggacggta tgcgccgcag caagtggtat cagattaccg gcacggatta tattgacaaa 5640 gcattccgtg tcacgaggga agcggcgggt ccgaacgccc ggctgtacat taacgattac 5700 aacacgcatg aaccgaagaa acgggatttt ctgtacaatt tggtgcgtga tttactcgct 5760 aaaggcgtac cgatcgatgg cgtaggtcac caatcgcata tccgcctgga gttccctgct 5820 attgacgaga tggagcagtc tattgaaaag tttgcttcgc tcggcctgga taatcagatc 5880 acggagctgg atatgggcct gtattctaat gatacagacc actacgaaac gatacctgaa 5940 gccatgctga tccggcaggc tcaccgctat cgggcgttgt tcgatatgtt ttcccgacag 6000 caggagcata tcagtaatgt gacgatttgg ggtacggatg atggaaatac gtggctcagt 6060 atgttcccga ttgcccggct ggataagccg ctgctgttcg acgaacggtt aaaggccaaa 6120 tatgcctatt gggcgcttgt tgacccgtcc aaagtaccgc cgctgccagc ggggagcaac 6180 gaatagcaac ataggtatgg attacacgtg gaaatcattt ctatacacca aaaaggcagg 6240 ccgcctctga catgccacaa ggacatgcga aggacgatct gccttttact gtaaaataat 6300 acgatctcta ctccagattg gcgtgttgac caaacataga ttcagaagtc agcttgcgta 6360 gctccatcgt tctgagcacc aatgcatcgc catacagcag caaggtttgc tcaaatagtg 6420 aacccatggg ctgaatggtt tggtaatcct tgttggatgg atccttgggt gcgcccggaa 6480 gcctgacgat aatatcagcc agcttgccaa tggtcgatcc aggggaggtg gtcagaagtg 6540 ccagagaagc ccccagcttt ttcgtctttt ctgccatcga ggtcaaactt ttggtttcac 6600 cagagcctga accgataatc agcaagtcac cttcgcccaa acctggagtt actgtttccc 6660 cgaccacata agcctgaaca cccatatgca tcagccgcat cgccagcgaa cggatcatga 6720 atccagaacg gcctgcgcct gcgacgaaca ccttattcgc tgaggtaatg gactggatca 6780 gttgctctga ttcctcatca ttgattagct gtggaaccaa ttgcagttcc ttgagcacct 6840 cggataaata ttgagaggtt tccattggaa ttaaccttgt ttaatgagct gctgcatttc 6900 ggaagcaacg gcctttttgt cgtcctcacc agtgatcccg ccgccaacaa taacca 6956 <210> 3 <211> 1620 <212> DNA <213> SLX-O9 <400> 3 atggatacat tgaagttgta tgtggaaagc tcgaatcctg cacaggccta ttatctggat 60 gaggtggaaa tcaggcaggt gtcggagaca ccaaccactc ccccaacgga gccgacaagt 120 ggtatcgaat ccagatttga agatggtacg gctcaaggct gggtatcccg tatgggcacc 180 gagacggtgc aggtgtcgaa tgccgatgca cgaaccggat cgtacagtct gttgacgaca 240 ggcagacaac aaacatatgc cgggccaaag ctggacgtga ctgctacagt gcaaaaaggc 300 agccgttaca cggtcagcgc ttgggtaaag ctggcaccgg gcgagcagca gcctgccaag 360 gtgcgcctca gcgtacagcg cgatcatcaa ggtgaaagta cgtatgaaac tgtggtaggc 420 aacacggcca tcacgactgg gggatggacg catttgtacg gaacgtatac tctcgcgcat 480 gaggcagaca ccgtctctat gtatttggaa acgccagaag gtacggcttc cttttacatg 540 gatgatttcg agctgtcgct tgtgccgccg ttggctattg agaaggacat tccctctttg 600 catggattgt atcagggaca gttcagcatc ggtacggcta ttgaagcttt ccagacagaa 660 ggggcttacg gggagctggt gcagaagcat tttaacagcg tcgtcgccgg aaatgcgatg 720 aagccgatct ccttgcagcc gtccgaagga cagttccatt gggaagaagc agaccggatt 780 gtgcaatttg cccagcagca tgggattgct atccgtttcc atacactggt gtggcataac 840 cagaccggcg attggatgtt taaggataag aatggacagc cgatgacgcc gaccgcggaa 900 aataaaaagc ttttgctgga tcggctggag acgcatattc gtgctgttgc agcccgttat 960 aaaaatgtaa tcaccgattg ggatgtggtg aatgaagtca ttgatcccga ccagccggac 1020 ggtatgcgcc gcagcaagtg gtatcagatt accggcacgg attatattga caaagcattc 1080 cgtgtcacga gggaagcggc gggtccgaac gcccggctgt acattaacga ttacaacacg 1140 catgaaccga agaaacggga ttttctgtac aatttggtgc gtgatttact cgctaaaggc 1200 gtaccgatcg atggcgtagg tcaccaatcg catatccgcc tggagttccc tgctattgac 1260 gagatggagc agtctattga aaagtttgct tcgctcggcc tggataatca gatcacggag 1320 ctggatatgg gcctgtattc taatgataca gaccactacg aaacgatacc tgaagccatg 1380 ctgatccggc aggctcaccg ctatcgggcg ttgttcgata tgttttcccg acagcaggag 1440 catatcagta atgtgacgat ttggggtacg gatgatggaa atacgtggct cagtatgttc 1500 ccgattgccc ggctggataa gccgctgctg ttcgacgaac ggttaaaggc caaatatgcc 1560 tattgggcgc ttgttgaccc gtccaaagta ccgccgctgc cagcggggag caacgaatag 1620                                                                         1620 <210> 4 <211> 539 <212> PRT <213> SLX-O9 <400> 4 Met Asp Thr Leu Lys Leu Tyr Val Glu Ser Ser Asn Pro Ala Gln Ala   1 5 10 15 Tyr Tyr Leu Asp Glu Val Glu Ile Arg Gln Val Ser Glu Thr Pro Thr              20 25 30 Thr Pro Pro Thr Glu Pro Thr Ser Gly Ile Glu Ser Arg Phe Glu Asp          35 40 45 Gly Thr Ala Gln Gly Trp Val Ser Arg Met Gly Thr Glu Thr Val Gln      50 55 60 Val Ser Asn Ala Asp Ala Arg Thr Gly Ser Tyr Ser Leu Leu Thr Thr  65 70 75 80 Gly Arg Gln Gln Thr Tyr Ala Gly Pro Lys Leu Asp Val Thr Ala Thr                  85 90 95 Val Gln Lys Gly Ser Arg Tyr Thr Val Ser Ala Trp Val Lys Leu Ala             100 105 110 Pro Gly Glu Gln Gln Pro Ala Lys Val Arg Leu Ser Val Gln Arg Asp         115 120 125 His Gln Gly Glu Ser Thr Tyr Glu Thr Val Val Gly Asn Thr Ala Ile     130 135 140 Thr Thr Gly Gly Trp Thr His Leu Tyr Gly Thr Tyr Thr Leu Ala His 145 150 155 160 Glu Ala Asp Thr Val Ser Met Tyr Leu Glu Thr Pro Glu Gly Thr Ala                 165 170 175 Ser Phe Tyr Met Asp Asp Phe Glu Leu Ser Leu Val Pro Pro Leu Ala             180 185 190 Ile Glu Lys Asp Ile Pro Ser Leu His Gly Leu Tyr Gln Gly Gln Phe         195 200 205 Ser Ile Gly Thr Ala Ile Glu Ala Phe Gln Thr Glu Gly Ala Tyr Gly     210 215 220 Glu Leu Val Gln Lys His Phe Asn Ser Val Val Ala Gly Asn Ala Met 225 230 235 240 Lys Pro Ile Ser Leu Gln Pro Ser Glu Gly Gln Phe His Trp Glu Glu                 245 250 255 Ala Asp Arg Ile Val Gln Phe Ala Gln Gln His Gly Ile Ala Ile Arg             260 265 270 Phe His Thr Leu Val Trp His Asn Gln Thr Gly Asp Trp Met Phe Lys         275 280 285 Asp Lys Asn Gly Gln Pro Met Thr Pro Thr Ala Glu Asn Lys Lys Leu     290 295 300 Leu Leu Asp Arg Leu Glu Thr His Ile Arg Ala Val Ala Ala Arg Tyr 305 310 315 320 Lys Asn Val Ile Thr Asp Trp Asp Val Val Asn Glu Val Ile Asp Pro                 325 330 335 Asp Gln Pro Asp Gly Met Arg Arg Ser Lys Trp Tyr Gln Ile Thr Gly             340 345 350 Thr Asp Tyr Ile Asp Lys Ala Phe Arg Val Thr Arg Glu Ala Ala Gly         355 360 365 Pro Asn Ala Arg Leu Tyr Ile Asn Asp Tyr Asn Thr His Glu Pro Lys     370 375 380 Lys Arg Asp Phe Leu Tyr Asn Leu Val Arg Asp Leu Leu Ala Lys Gly 385 390 395 400 Val Pro Ile Asp Gly Val Gly His Gln Ser His Ile Arg Leu Glu Phe                 405 410 415 Pro Ala Ile Asp Glu Met Glu Gln Ser Ile Glu Lys Phe Ala Ser Leu             420 425 430 Gly Leu Asp Asn Gln Ile Thr Glu Leu Asp Met Gly Leu Tyr Ser Asn         435 440 445 Asp Thr Asp His Tyr Glu Thr Ile Pro Glu Ala Met Leu Ile Arg Gln     450 455 460 Ala His Arg Tyr Arg Ala Leu Phe Asp Met Phe Ser Arg Gln Gln Glu 465 470 475 480 His Ile Ser Asn Val Thr Ile Trp Gly Thr Asp Asp Gly Asn Thr Trp                 485 490 495 Leu Ser Met Phe Pro Ile Ala Arg Leu Asp Lys Pro Leu Leu Phe Asp             500 505 510 Glu Arg Leu Lys Ala Lys Tyr Ala Tyr Trp Ala Leu Val Asp Pro Ser         515 520 525 Lys Val Pro Pro Leu Pro Ala Gly Ser Asn Glu     530 535 <210> 5 <211> 30 <212> DNA <213> SLX-O9 sense primer <400> 5 ctcgagatgg atacattgaa gttgtatgtg 30 <210> 6 <211> 22 <212> DNA <213> SLX-O9 antisense primer <400> 6 ggatccctat tcgttgctcc cc 22

Claims (18)

Xylanase having the characteristics of (a)-(d):
(a) the molecular weight on SDS-PAGE is about 65 kDa;
(b) maximum activity at pH 5 to pH 11;
(c) maximum activity at 50-60 ° C .; And,
(d) 95 unit / min mg at 50 ° C., pH 5 to pH 11, at least 100 unit / min mg xylose at 60 ° C., pH 6 to pH 9;
The xylanase of claim 1, wherein the xylanase has any one of the following sequences:
a) the amino acid sequence set forth in SEQ ID NO: 4;
b) an amino acid sequence having at least 70% homology with the amino acid sequence set forth in SEQ ID NO: 4;
c) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 3;
d) an amino acid of a protein that is functionally identical to a protein comprising one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 4, wherein said amino acid sequence is substituted, deleted, inserted, and / or added, and comprises the amino acid sequence set forth in SEQ ID NO: 4; order; And,
e) an amino acid sequence of a protein functionally identical to a protein which is encoded by a DNA hybridized under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3, and which comprises the amino acid sequence set forth in SEQ ID NO: 4.
The strain producing the xylanase of claim 1.
4. The strain according to claim 3, wherein the strain is Paenibacillus sp. HPL-003 ( Penibacillus sp. HPL-003) deposited with accession number KCTC18179P.
A polynucleotide encoding the xylanase of claim 1.
The gene of claim 5, wherein the polynucleotide has any one of the following sequences:
a) the nucleotide sequence set forth in SEQ ID NO: 3;
b) a nucleotide sequence having 95% homology with the nucleotide sequence set forth in SEQ ID NO: 3;
c) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 4;
d) an amino acid of a protein functionally identical to a protein comprising one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 4, wherein said amino acid sequence is substituted, deleted, inserted and / or added, and comprises the amino acid sequence set forth in SEQ ID NO: 4; Base sequences encoding sequences;
e) The base sequence of the protein which is a DNA base sequence hybridized under stringent conditions with the DNA comprising the base sequence of SEQ ID NO: 3, and functionally identical to the protein comprising the amino acid sequence of SEQ ID NO: 4.
A recombinant expression vector operably linked to the polynucleotide of claim 5.
A transformant comprising the recombinant expression vector of claim 7 introduced into a host cell.
The transformant according to claim 8, wherein the host cell is a prokaryotic cell including E. coli or a eukaryotic cell including yeast, animal cells and insect cells.
A method for producing xylanase comprising culturing the strain of claim 3 or the transformant of claim 8 in a medium and then centrifuging to obtain a crude enzyme solution.
The production method according to claim 10, further comprising the step of purifying xylanase from the obtained crude enzyme solution.
A xylan degrading agent comprising the xylanase of claim 1, the strain of claim 3, or the transformant of claim 8.
A composition for processing xylan in a food comprising the xylanase of claim 1.
Feed additive comprising a xylanase of claim 1.
Composition for a papermaking process comprising the xylanase of claim 1.
A method for decomposing xylan comprising adding a xylanase of claim 1, a strain of claim 3, or a transformant of claim 8 to a fibrin-based biomass or xylan-containing solution.
17. The method for decomposing xylan according to claim 16, which is used in the production process of renewable fuels or chemical raw materials.
A feed preparation method comprising the step of adding the xylanase of claim 1, the strain of claim 3, or the transformant of claim 8 to an animal feed material.

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