KR20070082329A - Novel paenibacillus sp. hy-8 strain and xylanase isolated from it - Google Patents

Abstract

A novel Paenibacillus sp. HY-8 strain and xylanase isolated from the same strain are provided to increase efficiency of fodder by decomposing cellulose in various plant fodder raw materials, so that the microorganism is useful as a fodder additive. The Paenibacillus sp. HY-8 strain(KCTC 10896BP) producing xylanase is isolated from insects. The xylanase is isolated from Paenibacillus sp. HY-8 strain(KCTC 10896BP), has the amino acid sequence of SEQ ID NO:5, and shows optimum activity at 45-60 deg.C and pH 6.0-6.5, and increased activity by addition of Ca, Zn, K, Mg, Na or Co. A gene encoding the xylanase has the nucleotide sequence of SEQ ID NO:4. A method for producing the xylanase comprises the steps of: (1) culturing Paenibacillus sp. HY-8 strain(KCTC 10896BP) or a microorganism transformed with a recombinant vector containing the xylanase gene, and centrifuging the cultured medium to obtain the supernatant containing coenzyme solution; (2) adding a precipitating agent into the coenzyme solution to induce precipitation of water-soluble proteins; and (3) centrifuging and purifying the precipitation-induced coenzyme solution, wherein the precipitating agent is ammonium sulfate, acetone, isopropanol, methanol, ethanol or polyethyleneglycol.

Description

New Fanibacilli sp. HV-8 strain and xylanase isolated therefrom [Novel Paenibacillus sp. HY-8 strain and xylanase isolated from it}

1 is a pannibacillus sp. Electron micrograph of the strain HY-8 ( Paenibacillus sp. HY-8),

2 is a graph showing the enzyme activity of xylanase isolated from HY-8 strain,

FIG. 3 is a diagram showing the results of the separation and purification of xylanase from HY-8 strain as column chromatography and electrophoresis with acrylamide gel. FIG.

4 is a graph showing the biochemical properties of xylanase isolated from HY-8 strain,

Figure 5 is a graph showing the result of xylose generated by xylan decomposition when xylanase isolated from HY-8 strain is added to the plant feed.

The present invention is a novel F. ni. Sp. HY-8 ( Paenibacillus sp. HY-8) strain and xylanase (xylanase) isolated from the strain, and more specifically, isolated from insects, and excellent fibrinolytic ability of F. nirvana sp. HY-8 strain and xylanase isolated from said strain.

Xylanase is one of a variety of enzymes produced by microorganisms. To completely decompose and glycosylate xylan, the main component of hemicellulose, three types of enzymes, namely, endozylanase (endo-β-xylanase), exo-β-xylanase and xylosidase (β-xylosidase) are known to work together, these are collectively referred to as xylanase enzymes (Biely, P ., Trends. Biotechnol., 3 , 286-290, 1985). Production and Waste Reclamation of High-Quality Paper (Beg, Q. and GS Hoondal., Appl. Micribiol. Biotechnol . 56, 326-338, 2001), Destroying Fruit Beverages and Beer, Extracting Coffee and Vegetable Oil and Starch And for a variety of applications, such as increasing feed efficiency (Bedford, M. R and Classen, HL, Elsevier Amsterdam , 361-370, 1992; Wong, KK Y and Saddler, JN, Crit. Rev. Biotechnol . 12, 413- 435, 1992). Recently, the availability of xylanase as a feed additive is increasing, which means that feed grains that provide carbohydrates and small amounts of protein for animal energy metabolism contain fiber that most animals do not use. This is because the fiber can be broken down.

The fiber in the grain is composed of nonstarch polysaccharides and lignin, and the cell wall of the plant covers the outermost layer of cellulose and the whole grain inside the cell, with xylan being the main ingredient. A combination of lignin and non-starchy polysaccharides consisting of phosphorus hemicellulose and beta-glucan. The non-starch polysaccharide is not only degraded by the digestive enzymes of the livestock, but also encapsulates the starch or protein in the grain, thereby reducing the efficiency of the livestock using nutrients. In particular, the water-soluble xylan contained in the cell walls of wheat and rye absorbs moisture in the digestive tract and becomes a sticky gel, which prevents digestive enzymes from accessing nutrients and inhibits the flow of digestive products in the digestive tract. Adherence to the digest leads to abnormal fermentation resulting in loss of nutrients (Kuhad, RC and Singh, A., Crit. Rev. Biotechnol . 13, 151-172, 1993).

Therefore, the addition of xylanase to the grain feed can hydrolyze the hemicellulose layer surrounding the grains, thereby increasing the availability of nutrients in the grains and improving the condition of the intestinal digest of livestock. In particular, when wheat products with high hemicellulose content are used for feed, it is easy to cause stool or redness in livestock. Xylanase can prevent this phenomenon and increase the value as a feed and also improve the quality variation of wheat products. As it enables wheat products to be used as a stable feedstock, its value is high as a feed additive enzyme. In addition, as the consumption of meat is increasing not only in developed countries but also in many countries with improved living conditions and incomes, xylanase is considered to be a very important enzyme as a feed additive for unit animals such as pigs and chickens. (Bedford. MR, Animal Feed Science and Technology . 53, 145-155, 1995).

The microorganisms used to produce xylanase for feed addition have been reported to be fungi. Among the fungi, strains of Trichoderma genus have been mainly used, and their enzyme productivity is the enzyme productivity of bacteria that produce xylanase. It is generally superior to, and exhibits maximum activity mainly in acidic conditions (Tenkanen, H. and Poutanen, K., Enzyme. Microb. Technol . 14, 566-574, 1992). However, although the physiological conditions of the digestive tract of pigs and chickens vary from site to site, the post-intestine pH condition at which xylanase should act is around 6.5. Therefore, xylanase having a high activity in acidic conditions near neutral than xylanase derived from mold having high activity near pH 5.0 is required as an enzyme for feed addition. Bacteria producing xylene Rana claim having a high activity pH near neutral is difficulty Pseudomonas (Aeromonas) genus Bacillus (Bacillus) genus, Clostridium (Clostridium) genus Streptomyces (Streptomyces) variety belonging like in There are bacteria, and the reaction characteristics of xylanase produced by the bacteria also vary. In addition, the above-mentioned xylan is composed of 30% sugar in grass hemicellulose and 20% sugar in legume hemicellulose. Since hemicellulose in legume feed consists of more complex glycocomplex than herb hemicellulose, more complicated enzymatic digestion process is required to decompose it, so the development of feed additives such as xylanase is urgently needed. do.

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 Laid-Open Publication No. 2000-0034279), a novel coding for xylanase of Bacillus sp. AMX-4 strain There is a xylanase gene (Korean Patent Publication No. 2003-0085679), but the situation is not actually used as feed additives in Korea.

Insects, on the other hand, are the most flourishing biomes on the planet, showing a variety of food habits and high biodiversity. Recently, researches on utilizing the symbiotic microorganisms of insects in consideration of the biological characteristics of these insects as useful biological resources have been increasing, and researches on the intestinal microorganisms, which are closely related to the growth of insects, have been actively conducted. For example, studies have shown that intestinal microorganisms are involved in the digestion and nutrition of termites (Breznak, JA and A. Brune., Ann. Rev. Entomoi . 39, 453-457, 1994) and high efficiency from sugarless spiders that feed on animal food. Isolation of Protease-producing Strains for Industrial Application (Lee, GE and HY Park., Kor. J. Microbiol. 40, 269-274, 2004), and various insects including Lepidoptera and Coleoptera Ecological studies of intestinal microorganisms using molecular biological techniques have been published (Markus, E and Friedrich, W., Appl. Environ. Microbiol . 69, 6659-6668. 2003; Nichole, A. and Handelsman, J., appl. Environ. Microbiol. 70, 293-300, 2004).

Accordingly, the present inventors screened high-efficiency xylanase producing strains from a number of insects that feed on xylan-rich plants such as leaves and bark of plants, and treated the xylanase isolated therefrom to the plant feed material to reduce the sugar content. The present invention was completed by confirming the increase.

An object of the present invention is selected from insects that feed on plants rich in xylans. It is to increase the feed efficiency by separating and purifying xylanase produced from the HY-8 strain and treating it with vegetable feed and low grade feed.

In order to achieve the above object, the present invention is a novel Fanibacillus sp. HY-8 ( Paenibacillus sp. HY-8) 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 Penicillus sp. Provided are methods for producing xylanase using the HY-8 strain.

In addition, the present invention is Penicillus sp. Provided is a feed additive for improving xylan degradation including the HY-8 strain.

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, the present invention is a novel Fanibacillus sp. HY-8 ( Paenibacillus sp. HY-8) strain is provided.

The microorganism of the present invention was inoculated with bacterial colonies formed by smearing a medium for separating bacteria from about 60 kinds of insect guts into a culture medium containing oat xylan, followed by culturing, and the culture supernatant was used as a coenzyme solution. Excellent strains were selected to select microorganisms that produce xylanase. Analysis of the characteristics of the selected strains, Gram-positive bacilli (Gram-positive bacilli) do not have flagella (see Fig. 1), the result of determining the partial sequence of the 16S rDNA (SEQ ID NO: 1), the Fanibacillus Pabuli, It was confirmed that it exhibits a high homology of 98-99% or more with strains such as Fanibacilli militicus. Accordingly, this strain was identified as a strain belonging to the F. ni. Sp. HY-8 ”and the strain was deposited on January 17, 2006 to the Gene Bank of Korea Biotechnology Research Institute, an international depository institution (accession number; KCTC 10896BP).

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 Fanibacilli sp. After culturing the HY-8 strain in a liquid medium containing oat xylan, a precipitant (eg, ammonium sulfate) was added to precipitate a water-soluble protein, which was dialyzed with a dialysis membrane and purified by a column to obtain xylanase (FIG. 3). b, c). The specific activity of purely isolated xylanase against oat xylan was 147.8 U / mg, with a recovery of 19.5% and 26.9 times pure separation (see Table 1).

As described above, the molecular weight of the purely isolated xylanase was measured by polyacrylamide gel electrophoresis (see FIG. 3A), and the purely isolated protein was treated with trypsin, followed by high homology through MS / MS analysis and database search. Degenerate primers were prepared based on high homology by searching for other bacterial xylanases and comparing these bacterial xylanases, and using 633 bases of xylana using PCR and DNA walking techniques. Proteins having a molecular weight of 22.9 kDa (SEQ ID NOs: 4 and 5) consisting of 211 amino acid residues having a transcriptional transcription framework were identified.

Fanibacilli sp. Xylanase produced by the HY-8 strain showed the highest amino acid sequence homology of 88% with Aeromonas punctata xylanase, known as bacterial xylanase, and Geobacillus stearothermophillus . And novel xylanases showing 88% and 87% homology with endo-beta-1,4-xylanase from Paenibacilus polymyxa , respectively.

In the present invention, as described above, Fanibacillus sp. Since the nucleotide sequence of the gene encoding the xylanase isolated from the HY-8 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. .

Subsequently, as a result of investigating the optimum reaction conditions of xylanase, the temperature condition showed the maximum activity at 46 ℃ to 60 ℃ preferably 50 ℃ and the acidity showed the maximum activity at pH 6.0-6.5 so that the high activity even at neutral pH It was confirmed that it is lanase (see FIGS. 4A and 4B). In order to investigate the stability to heat, the enzyme activity was measured after varying the temperature and time and the purified enzyme solution was treated for 30 minutes at each temperature. It was confirmed that the activity remained stable (see FIG. 4C). In addition, when 9 kinds of metal ions were added, the activity of xylanase decreased when Fe ²⁺ and Cu ²⁺ ions were added, but when Ca, Zn, K, Mg, Na, etc. were added The activity was generally increased, especially in the presence of cobalt ions, the enzyme activity was confirmed to increase more than two times (see Figure 4d).

In addition, the present invention

1) In the medium containing xylan, claim 1 of the Pannibacillus sp. Culturing the HY-8 strain or the transformant followed by centrifugation to obtain a supernatant crude enzyme solution;

2) adding a precipitant to the coenzyme liquid obtained in step 1 to induce water-soluble protein precipitation; And

3) It provides a xylanase production method comprising the step of centrifugation and purification of the crude enzyme solution induced precipitation in step 2.

As the precipitant of step 2, ammonium sulfate, acetone, isopropanol, methanol, ethanol, polyethylene glycol and the like may be used.

In addition, the present invention is Penicillus sp. Provided is a feed additive for improving xylan degradation including the HY-8 strain.

In order to investigate whether the xylanase of the present invention is suitable as an additive for vegetable feed, the present invention is applied to soybean meal, cottonseed meal, rapeseed gourd, embryo gourd, soybean meal, soybean, palm gourd and lupine seed which are rich in xylan After xylanase treatment, the amount of reducing sugar produced by enzymatic activity was analyzed. As a result, it was confirmed that most of the vegetable feed is effective in the production of reducing sugar by decomposition of xylan. As a negative control, the reducing sugar produced by pure xylanase was measured by irradiating the reducing sugar with respect to the test group without addition of the enzyme, and then subtracting the amount from the treated group. As a comparative control, xyl derived from Trichoderma Lanase was added in the same unit to measure the amount of reducing sugar produced by the same method as the treatment group. As a result, as shown in Figure 5, the Fanibacillus sp. Xylanase derived from HY-8 showed better decomposition of xylan than the control for vegetable feed components such as cottonseed, wheat buckwheat, soybean, soybean meal, palm and lupine. In the case of chaejongbak, the comparative control showed a slightly better degradation effect (see Figure 5). From the above results, it was confirmed that the xylanase of the present invention can be used as an additive of feed containing plant feed by promoting xylan decomposition.

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 inventors of the present invention immersed for about 1 minute in 70% (w / w) ethanol for 1 minute to 70% (w / w) ethanol to remove the contaminant surface of the filling surface for the separation of bacteria producing xylanase (xylanase) In order to remove the ethanol from the carcass, it was washed twice with sterile distilled water. After washing, the carcasses were dissected and the intestines were taken out with tweezers and pulverized in physiological saline (Phospate-buffered saline; 0.8% NaCl, 0.02% KCl, 0.144% Na ₂ HPO ₄ , 0.024% KH ₂ PO ₄ ). The extract was spread on a solid medium (5 g / L yeast extract, 10 g / L tryptone, 5 g / L sodium chloride. 20 g / L agar) to which 0.5% oat xylan (Sigma Chem. Co.) was added and incubated at 25 ° C for 2 days. Strains that form plaque around colonies grown after congo-red staining (Theater, RM and Wood, PJ, Appl . Enviro n. Microbiol . 43, 777-780, 1982) It was.

Restriction medium containing 0.5% oat xylan was first selected by the above method (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) inoculated in 3 ml and incubated for 48 hours in a shaking incubator at 25 ℃ and centrifuged to recover the supernatant to measure the xylanase activity, of which xylana The strain with the highest activity was finally selected. Enzyme activity was determined by DNS (dinitrosalicylic acid) assay (Miller, GL, Anal . Chem ., 55, 952-959, 1959), specifically 100 μl of enzyme solution in 100 μl of enzyme solution (1% oat xylan). Add 50 mM sodium phosphate solution, pH 6.0, and react for 10 minutes at 50 ° C., add 700 μl of DNS (3,5-Dinitrosalicylic acid) solution, and then treat the solution at 100 ° C. for 5 minutes, and then absorb 540 nm. Measured at One unit of enzyme was defined as the amount of enzyme that releases 1 μmol of reducing sugar in one minute.

Example 2 Strain Identification and Characterization

The present inventors incubated the strain producing the highest activity xylanase isolated in Example 1 at 25 ° C., followed by gram staining and spore staining (Cappuccino and Sherman, Microbiology a laboratory manual, 2nd edition, The Benjamin / Cummings Publishing Co.) produced spores and was identified as Gram-positive bacteria. 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. In addition, the genomic DNA of the strain was isolated to determine the 16S rDNA nucleotide sequence of the strain (DNeasy Tissue kit, Qiagen) and the reaction for PCR was 50 ul of the reaction with the following composition. 5 μl of 10-fold Taq DNA polymerase buffer (MgCl ₂ added) to 1 μl of total DNA (50-200 ng / ul), 5 μl of 2.5 mM dNTPs (dATP, dTTP, dCTP, dGTP), forward of 3.2 p mol 5 μl of primers (SEQ ID NOs: 6 and 7) and 1-2 units of Taq DNA polymerase (Promega, USA) were added, and distilled water was added to the final 50ul reaction solution. Primers were synthesized for nucleotide amplification of 1465 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 65 ° 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 partial base sequence of the obtained amplified fragment was determined, and the genBank database search result showed that it showed a high homology of 98-99% or more with the strains of Fanibacillus Pavli and Fanibacillus amiliticus. Was identified as belonging to Bacillus sp. Named HY-8. Said Fanibacillus sp. The HY-8 strain was deposited on January 17, 2006 to the Gene Bank of Korea Research Institute of Bioscience and Biotechnology (Accession No .; KCTC 10896BP).

Example 3 Characterization of Xylanase

<3-1> Isolation and Purification of Xylanase

The present inventors have identified and identified the Pannibacillus sp. Xylanase produced by the HY-8 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 for 24 hours with 50 mM sodium phosphate buffer (pH 6.0). (Spectrum, MW cutoff 12 kDa) to obtain a crude enzyme solution. The FPLC system (Amersham-Pharmacia Biotech) was used for column chromatography of the enzyme. First, the coenzyme solution was eluted at a flow rate of 2.5 ml / mim according to the size of the molecule using a Hiload superdex 200 gel chromatography (Amersham Pharmacia Biotech Inc) column (see FIG. 3A). The concentration of NaCl was reduced to 0-0.5M at a flow rate of 1 ml / min using Mono S HR 5/5 ion exchange resin (Amersham Pharmacia Biotech Inc) column equilibrated with 20 mM MES buffer, pH 7.6 After eluting as a concentration gradient (see FIG. 3B), only the enzyme active fractions were taken and purified by SDS-PAGE (see FIG. 3C). The active fractions were lyophilized and used at -20 ° C.

<3-2> Determination of enzyme activity of xylanase

Xylanase enzyme activity was measured from each sample recovered in the purification step of Example <3-1>. Protein content was measured using the Bradford method (Bradford, MM, Anal . Biochem , 86, 142-146, 1997), and BSA (bovine serum albumin) was used as a standard protein. Finally, the specific activity for oat xylan of pure xylase isolated from 1 L culture was 147.8 U / mg, and the yield was purified to 19.5%, 26.9 times pure separation, and total enzymatic activity and specific enzyme activity of each purification step. Purity and recovery are shown in Table 1.

<3-3> Molecular weight and amino acid sequence determination of xylanase

The present inventors measured the molecular weight of xylanase purely separated in Example <3-1> by polyacrylamide gel electrophoresis. In FIG. 3C, 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 S column. It is shown. The molecular weight measured by SDS-PAGE showed that the xylanase of the present invention had a molecular weight of about 22.5 kDa (FIG. 3C).

In addition, the cloning of the isolated xylanase was treated with trypsin, a protease, and then commissioned by the Korea Basic Science Research Institute for MS / MS analysis and (Q-TOF, Micromass, UK) database search. Trypsin digestion fragments showing high homology with Aeromonas punctata xylanase were identified. Based on this result, the bacterial xylanase sequence provided by NCBI was downloaded, and the sequences of various bacterial xylanases with high homology were compared to prepare degenerate primers based on the high amino acid conserved sequence (sequence). Nos. 2 and 3) through the PCR and DNA walking technique (DNA walking speedup kit, Seegene ver 2.1). The total nucleotide sequence and amino acid sequence of HY-8 xylanase were determined. PCR amplification conditions using the PCR Premix kit (Bioneer) to denature the primer pair and template described above for 5 minutes at 94 ℃, 30 seconds at 94 ℃, 40 seconds at 52 ℃ and 30 times for 1 minute at 72 ℃ The reaction was terminated by extending for 7 minutes at 72 ° C.

As a result, Fanibacilli sp. HY-8 is a 22.9 kDa molecular weight protein consisting of 211 amino acid residues with an open reading frame of 633 base xylanase transcription.As a GenBank database search, it is known that Eromonas puntata, known as bacterial xylanase ( Aeromonas punctata ) showed the highest amino acid sequence homology of 88% with xylanase and endo-beta-1,4-xylanase from GeoBacillus stearothermophillus and Paenibacilus polymyxa And 88% and 87% homology, respectively.

<3-4> Biochemical Characterization of Xylanase

In order to investigate the optimal reaction conditions of xylanase, the effects of reaction temperature, pH of the 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 a temperature of 10 ℃ to 80 ℃, the enzyme activity was measured by measuring the residual activity of the 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, and the maximum activity was confirmed at 50 ° C. and pH 6.0-6.5 to confirm that xylanase had high activity even at neutral pH (FIGS. 4A and 4B). 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 was reduced to about 50%. After 30 minutes of treatment, the residual activity was about 80%, but it was confirmed that the enzyme activity remained stable at a temperature lower than 50 ° C (FIG. 4C).

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.) such as cobalt ions were added and reacted at a concentration of 10 mM. ^The activity decreased when ²⁺ ions and Cu ²⁺ ions were added, but when Ca, Zn, K, Mg, and Na were added, the activity was generally increased, especially in the presence of cobalt ions. It was confirmed (Fig. 4d)

Example 4 Validation of Xylanase as a Feed Additive

In order to investigate whether the xylanase of the present invention is suitable as an additive for vegetable feed, the present invention is applied to soybean meal, cottonseed meal, rapeseed gourd, embryo gourd, soybean meal, soybean, palm gourd and lupine seed which are rich in xylan After xylanase treatment, the amount of reducing sugar produced by enzymatic activity was analyzed.

Specifically, 1 gram of vegetable feed was suspended in 10 ml of 50 mM sodium phosphate (pH 6.0) buffer and xylanase was added to 100 units / ml and treated at 37 ° C. for 24 hours, as shown in FIG. 5. It was found that most of the plant feeds were effective in the production of reducing sugars by decomposition of xylan. As a negative control, the reducing sugar produced by pure xylanase was measured by irradiating the reducing sugar with respect to the test group without addition of the enzyme, and then subtracting the amount from the treated group. As a comparative control, xyl derived from Trichoderma Lanase was added in the same unit to measure the amount of reducing sugar produced by the same method as the treatment group. As a result, as shown in Figure 5, the Fanibacillus sp. Xylanase derived from HY-8 showed better decomposition of xylan than the control for vegetable feed components such as cottonseed, wheat buckwheat, soybean, soybean meal, palm and lupine. In the case of chaejongbak, the comparative control showed a slightly better decomposition effect.

In view of the above results, it was confirmed that xylanase derived from the strain of the present invention is suitable as an additive of a feed comprising a vegetable feed by promoting the decomposition of xylan of the vegetable feed.

The novel strain of the present invention, Fanibacillus sp. HY-8 ( Paenibacillus sp. HY-8) and xylanase isolated therefrom showed excellent xylan degrading activity over a relatively wide range of temperature and pH conditions, as well as excellent activity at neutral pH compared to acid-based xylase derived from mold. Compared to Trichoderma-derived xylanase, which is widely used as a feed additive, it is judged to be an excellent enzyme showing better or similar xylan decomposition efficiency in most plant feed materials, and it is effective in decomposing xylan, which accounts for 20-40% of feed material. It is possible to increase the feed efficiency through the use of the enzyme as a feed additive is considered to be high.

<110> Korea Research Institute of Bioscience & Biotechnoloy <120> Novel Paenibacillus pabuli genus HY-8 strain producing xylanase          and its application for effective feeds <130> 6p-01-22 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 568 <212> DNA <213> 16S rDNA of Paenibacillus pabuli HY-8 <400> 1 gggtgagtaa cacgtaggca acctgccctc aagcttggga caactaccgg aaacggtagc 60 taataccgaa tagttgtttt cttctcctga agagaactgg aaagacggag caatctgtca 120 cttggggatg ggcctgcggc gcattagcta gttggtgggg taacggctca ccaaggcgac 180 gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact 240 cctacgggag gcagcagtag ggaatcttcc gcaatgggcg aaagcctgac ggagcaatgc 300 cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gccagggaag aacgcttggg 360 agagtaactg ctctcaaggt gacggtacct ganaagaaag ccccggctaa ctacgtgcca 420 gcagccgcgg taatacgtag ggggcaagcg ttgtccggaa ttattgggcg taaagcgcgc 480 gcaggcggtc atttaagtct ggtgtttaat cccggggctc aaccccggat cgcactggaa 540 actgggtgac ttgagtgcag aaaaagag 568 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of gene coding xylanase <400> 2 tattggcaga attggacc 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of gene coding xylanase <400> 3 tggtcggtat gtgcccca 18 <210> 4 <211> 633 <212> DNA <213> open reading frame (ORF) of Paenibacillus pabuli HY-8 xylanase <400> 4 atgtttaaat ttagcaaaaa aatgttgacg gtcattcttg cggcttccat gagttttggc 60 gtgtttgcag caacctcaag tgcagcgaca gattattggc agaattggac cgatggcggc 120 ggtacggtta atgctgtgaa tggatcgggc ggaaattaca gtgtgacatg gcaaaatacg 180 gggaactttg ttgttggtaa aggctggaat gttggatctc caaaccggac aattaactat 240 aatgctgggg tttggtctcc atccggcaat ggctatttga cactctatgg gtggacgaga 300 aatgcactta ttgaatatta cgttgtagat agttggggca cataccgacc aaccggaacg 360 tttaaaggta ctgtcaacag tgatggagga acctatgaca tctatacaac gatgcggtat 420 aatgcgcctt ccattgacgg cacacaaact tttgcccagt actggagcgt aagacagtcg 480 aagagagcga ccggggttaa ctccgcaatt gcgttcagca accatgttaa cgcatgggca 540 agcaaaggaa tgaatctggg tagcagttgg tcttaccagg tgttagcgac agaaggatat 600 caaagtagtg gaagttccaa cgtaacggtg tgg 633 <210> 5 <211> 211 <212> PRT <213> Amino acid of Paenibacillus pabuli HY-8 xylanase <400> 5 Met Phe Lys Phe Ser Lys Lys Met Leu Thr Val Ile Leu Ala Ala Ser   1 5 10 15 Met Ser Phe Gly Val Phe Ala Ala Thr Ser Ser Ala Ala Thr Asp Tyr              20 25 30 Trp Gln Asn Trp Thr Asp Gly Gly Gly Thr Val Asn Ala Val Asn Gly          35 40 45 Ser Gly Gly Asn Tyr Ser Val Thr Trp Gln Asn Thr Gly Asn Phe Val      50 55 60 Val Gly Lys Gly Trp Asn Val Gly Ser Pro Asn Arg Thr Ile Asn Tyr  65 70 75 80 Asn Ala Gly Val Trp Ser Pro Ser Gly Asn Gly Tyr Leu Thr Leu Tyr                  85 90 95 Gly Trp Thr Arg Asn Ala Leu Ile Glu Tyr Tyr Val Val Asp Ser Trp             100 105 110 Gly Thr Tyr Arg Pro Thr Gly Thr Phe Lys Gly Thr Val Asn Ser Asp         115 120 125 Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Met Arg Tyr Asn Ala Pro Ser     130 135 140 Ile Asp Gly Thr Gln Thr Phe Ala Gln Tyr Trp Ser Val Arg Gln Ser 145 150 155 160 Lys Arg Ala Thr Gly Val Asn Ser Ala Ile Ala Phe Ser Asn His Val                 165 170 175 Asn Ala Trp Ala Ser Lys Gly Met Asn Leu Gly Ser Ser Trp Ser Tyr             180 185 190 Gln Val Leu Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser Asn Val         195 200 205 Thr Val Trp     210 <210> 6 <211> 20 <212> DNA <213> 27F primer <400> 6 agagtttgat cmtggctcag 20 <210> 7 <211> 19 <212> DNA <213> 1492R primer <400> 7 ggttaccttg ttacgactt 19

Claims (13)

Paenibacillus sp. HY-8 strain (KCTC 10896BP) producing xylanase. Xylanase produced from the strain of claim 1. The xylanase of claim 2, wherein the xylanase is set forth in SEQ ID NO: 5. 4. The xylanase of claim 2, wherein the xylanase exhibits maximum activity at 45 ° C to 60 ° C. The xylanase of claim 2, wherein the xylanase exhibits maximum activity at pH 6.0-6.5. The xylanase according to claim 2, wherein the xylanase increases enzymatic activity when Ca, Zn, K, Mg, Na or Co is added. Gene encoding the enzyme of claim 2. 8. The gene of claim 7, wherein said gene is set forth in SEQ ID NO: 4. Recombinant vector comprising the gene of claim 7. A transformant having a recombinant vector of claim 9 introduced therein. 1) In the medium containing xylan, claim 1 of the Pannibacillus sp. Culturing the HY-8 strain or the transformant of claim 10 and then centrifuging to obtain a supernatant crude enzyme solution; 2) adding a precipitant to the coenzyme liquid obtained in step 1 to induce water-soluble protein precipitation; And 3) Xylanase production method comprising the step of centrifugation and purification of the crude enzyme solution induced in precipitation in step 2. 12. The method of claim 11 wherein the precipitant of step 2 is selected from the group consisting of ammonium sulfate, acetone, isopropanol, methanol, ethanol and polyethylene glycol. A feed additive for improving xylan decomposition, comprising the xylase of claim 2.

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