KR101746398B1 - Streptomyces atrovirens WJ2, a new microbe having great activity degrading xylan - Google Patents

Abstract

The present invention relates to a novel xylan-degrading strain Streptomyces atrovirens WJ2, which is isolated from the soil of Jeju Island and deposited with the accession number of KACC92087P in the Center for Agricultural Genetic Resources , as a strain of Streptomyces atrovirens WJ2 , And a novel strain having excellent xylan degrading activity.
The novel strain of the present invention can effectively decompose xylan, and thus can be very usefully used in an industrial field that requires decomposition of xylan or an industrial field that uses various physiologically active substances produced by decomposition of xylan. In addition, the use of the strain of the present invention can very efficiently produce highly useful xylenolytic enzymes in industry.

Description

A novel xylan-degrading strain Streptomyces atrovirens WJ2, a new microbe having great activity degrading xylan,

The present invention relates to a novel xylan-degrading strain Streptomyces atrovirens WJ2, which is isolated from the soil of Jeju Island and deposited with the accession number of KACC92087P in the Center for Agricultural Genetic Resources , as a strain of Streptomyces atrovirens WJ2 , And a novel strain having excellent xylan degrading activity.

Plant biomass is the most abundant and widespread material for the production of bioproducts and biofuels. Hydrolysis of plant biomass is the most important step in the useful utilization of hemicellulosic materials present in vast amounts in nature.

Xylan is a hemicellulose, which is one of the major constituents of about 20-40% of the cell wall of the terrestrial plant, and contains acetyl, arabinosyl, and methylglucuronosyl residues Is a heteropolysaccharide consisting of a skeleton of substituted? -1,4-linked xylose units (Subramanian and PREMA 2002). Because of the complex heterogeneity of the structure, the synergistic action of many different xylenolytic enzymes is required, but mainly involves beta-1,4-endoxylanases (EC 3.2.1.8) and beta-1,4 - β-xylosidases (EC 3.2.1.37) hydrolyze the xylan skeleton. In particular, oligosaccharides derived from xylan-hydrolyzate produced by beta-1,4-endoxylanase are known to have various physiological activities such as antibacterial, antioxidant and anti-inflammation, It is expected to be used in various fields such as agriculture-livestock industry and cosmetics industry.

Xilanase is important for a variety of biotechnological and industrial applications, including pulp bleaching, food and feed, bakery industry, and bioconversion to fuel hemicellulose biomass (Beg et al ., 2001; Nath and Rao, 2001; Dhiman et al ., 2008). Recently, antiallergic, antioxidant, antimicrobial and anti-inflammatory activities and prebiotic effects of xylan-derived oligosaccharides (XOSs) have been reported (Kallel et al., 2015; Aachary and Prapulla 2009). Hydrolysis of lignocellulosic biomass by microbial enzymes has been accepted as an environmentally friendly alternative to chemical hydrolysis to produce harmful by-products (Biely 1985). Many researchers have attempted to isolate new microorganisms producing more suitable xylanase (Hwang et al., 2010; Brennan et al., 2004; Lee et al., 2006).

Streptomyces spp . Is known to produce a variety of chemicals including antineoplastic agents and antibiotics. Streptomyces is also known to be involved in the production of important proteins including proteolytic enzymes and glycosidic hydrolases such as cellulase, xylanase and agarase. it can be an industrially important strains (Shin et al 2009;. El -Sersy et al, 2010;.. Temuujin et al, 2011). In taxonomic studies, much of the work on streptomyces has focused on screening for strains producing compounds (Al-Bari et al ., 2005).

Accordingly, the present inventors have made efforts to utilize this xylanase, that is, a new strain producing xylenolytic enzyme, in a xylanase-related industry.

Al-Bari, M. A. A., M. S. A. Bhuiyan, M. E. Flores, P. Petrosyan, M. Garcia-Varela, and M. A. U. Islam. 2005. Streptomyces bangladeshensis sp. nov., isolated from soil, which produces bis- (2-ethylhexyl) phthalate. Int. J. Syst. Evol. Microbiol. 55: 1973-1977. Bajaj, B. K., and N. P. Singh. 2010. Production of xylanase from an alkalitolerant Streptomyces sp. 7b under solid-state fermentation, its purification, and characterization. Appl. Biochem. Biotechnol. 162: 1804-1818. Sasser M. 1990. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. MIDI Inc., Newark, DE. Mesbah, M., U. Premachandran, and W. B. Whitman. 1989. Precise measurement of the G + C content of deoxyribonucleic acid by high-performance liquid chromatography. Int. J. Syst. Bacteriol. 39: 159-167. Van Trappen, S., T. L. Tan, J. Yang, J. Mergaert, and J. Swings. 2004. Alteromonas stellipolaris sp. nov., a novel, budding, prosthecate bacterium from Antarctic seas, and emended description of the genus Alteromonas. Int. J. Syst. Evol. Microbiol. 54: 1157-1163.

Therefore, a main object of the present invention is to provide a novel strain useful for producing xylan degradation products by exhibiting xylan degrading activity.

It is another object of the present invention to provide a method for decomposing xylan using the strain.

It is still another object of the present invention to provide a method for producing xylanase using the strain.

According to one aspect of the present invention, the present invention provides a soil-derived Streptomyces atrovirens WJ2 (accession number KACC92087P) having xylan degrading activity.

The microorganism of the present invention has been isolated from the soil of Jeju Island and can be classified as a new sub- species of bacteria of Streptomyces artrovirans based on the results of phylogeny analysis through the 16S rRNA gene sequence.

The microorganism of the present invention exhibits Gram-positive properties and forms spores. Na ⁺ ions are required for growth, and they can grow at pH 6 ~ 10, 15 ~ 50 ℃. D-glucose, L-arabinose, D-mannose, D-mannitol, N- acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid and malic acid can be used as the carbon source. The G + C content in the genomic DNA is approximately 73.98% and is resistant to ampicillin, kanamycin, apramycin and chloramphenicol. The main fatty acids (more than 4%) _{C 15: 0 Anteiso (36.19%} ), C 15: 0 Iso (10.58%), C 16: 0 (10.20%), C 16: 0 Iso (9.84%) and C _{14: 0} Iso (4.18%).

According to another aspect of the present invention, there is provided a method for decomposing xylan using the microorganism of the present invention. According to the present invention, the microorganism of the present invention produces an endo-type xylanase which decomposes xylan into xylobiose and xylotriose. Therefore, when a method of directly inoculating the strain of the present invention or adding a culture medium to a sample for dissolving xylan, xylobiose and xylotriose can be produced by decomposing the xylan.

According to another aspect of the present invention, there is provided a method for producing xylanase, wherein the microorganism of the present invention is cultured, and the culture medium is cultured by adding xylan to the culture medium. The microorganism of the present invention exhibits the property of producing xylanase and secreting it out of the cell. Therefore, it is possible to obtain xylenolytic enzyme by simply culturing the liquid and then recovering the culture solution. Since the microorganism of the present invention has been shown to produce xylan degrading enzyme in the presence of xylan, it is necessary to include xylan in the culture medium for production of xylanase. In addition, incorporation of soytone into the nitrogen source can produce xylenolytic enzymes more efficiently. It is preferable to include xylenes in an amount of 0.2 to 0.4% (w / v) based on the volume of the culture medium and 0.1 to 0.3% (w / v) of soytone for efficient production of xylanase. It is recommended to incubate at 35 ~ 45 ℃, pH 6-8.

The novel strain of the present invention can effectively decompose xylan, and thus can be very usefully used in an industrial field that requires decomposition of xylan or an industrial field that uses various physiologically active substances produced by decomposition of xylan. In addition, the use of the strain of the present invention can very efficiently produce highly useful xylenolytic enzymes in industry.

1 is a photograph showing a WJ2 strain cultured on an LB agar medium for isolating and identifying microorganisms of the present invention.
FIG. 2 shows the Neighbor Joining system number generated using the 16S rRNA gene sequence of the microorganism of the present invention.
FIG. 3 is a graph showing the relationship between (1) WJ2; (2) S. atrovirens NRRL B-16357 ^T ; (3) S. flavoviridis NBRC12772 ^T ; (4) S. pilosus NBRC12807 ^T ; (5) S. longispororuber NBRC13488 ^T ; (6) The results of genomic DNA and DNA-DNA hybridization analysis of E. coli KCCM12119 are shown.
4 is a graph showing the activity of xylanase secreted by the microorganism WJ2 of the present invention according to the nitrogen source in the medium composition.
5 is a graph showing the activity of xylanase secreted by the microorganism WJ2 according to the carbon source in the medium composition.
6 is a graph showing the activity of xylenase secreted by the microorganism WJ2 according to the concentration of xylan in the medium composition.
FIG. 7 is a graph showing cell production and xylalanase activity in the modified medium of the present invention microorganism WJ2 ((1), (2), (3), Lanease activity; xylalanase activity from optimized media).
8 is a graph showing the effect of the pH condition on the xylanase activity of the microorganism WJ2 of the present invention (?, 20 mM MOPS buffer;?, 20 mM Tris-Cl buffer).
9 is a graph showing the effect of the temperature condition on the xylanase activity of the microorganism WJ2 of the present invention.
10 is a graph showing the thermostability against the xylanase activity of the microorganism WJ2 of the present invention.
11 is a result of TLC (Thin layer chromatography) analysis of the enzyme reaction product obtained under optimal culture conditions of the microorganism WJ2 of the present invention (X1, xylose, X2, xylobiose, X3, Xylotriose; X4, xylotetraose. Xylooligosaccharides arrow marks).

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1. Isolation of xylanase-producing microorganisms

First, strain WJ2 was isolated as a xylan-hydrolyzing bacterium from a soil sample mixed with rotten trees (see FIG. 1). Specifically, soil samples were collected from Songak Mountain in Jeju Island and diluted to 10 ^-1 ~ 10 ^-5 successively in distilled water. Then, the diluted solution was plated on LB solid medium containing 0.1% xylan azure (Sigma, USA) in an amount of 100 μl each, and then cultured at 40 ° C for 48 hours. Colonies showing xylalanase activity were selected and transferred to fresh LB medium. Of these, a colony named strain WJ2 was finally selected and used for further experiments. The 16S rRNA gene sequence of strain WJ2 was registered in GenBank as JN578482. The isolated strain was deposited under the accession number KACC92087P at the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology.

Example 2. Detection of 16S rRNA gene base sequence of strain WJ2 and analysis of phylogenetic tree

The strain WJ2 was cultivated in an LB liquid medium containing 0.2% xylenes, cultured at 40 DEG C for 2 days, and centrifuged at 10,000 rpm to recover the cells. Genomic DNA was extracted and used as a template for amplification of the 16S rRNA gene using bacterial universal primers (27F and 1492R) (Baker et al., 2003). Sequence analysis of amplified gene fragments was performed directly using Applied Biosystems 3730xl DNA Analyzer. Using the NCBI Basic Local Alignment Search Tool (BLAST) program (Altschul et al . 1990), the relevant standard strains were investigated in the GenBank database based on the 16S rRNA gene sequence of strain WJ2. The 16S rRNA gene sequences of related strain strains were collected from the ExTaxon server (Chun et al. 2007). Multiple alignments of the related streptomyces were performed using Clustal W software (Thompson et al. 1994), and the 5 'and 3' gaps were determined using the BioEdit program (Hall 1999) Respectively. The Neighbor-Joining (NJ) method (Saitou 1987) and the Maximum Parsimony (MP) method (Kluge 1969) of the PHYLIP suit program (Felsenstein 1993) were used to construct the phylogenetic tree. Bootstrap analysis was used to calculate the tree topology from the reconstructed Neighbor-Joining data of 1,000 times. The evolutionary distance matrix was calculated according to Kimura's two-parameter model (Kimura 1983).

The 16S rRNA gene (1,399 bp) from the strain WJ2 was amplified by PCR and sequenced. Table 1 shows the homology search results of 16S rRNA gene nucleotide sequences. As shown in Table 1, the result of 16S rRNA gene sequencing analysis of strain WJ2 indicates that the strain is a streptomyces .

Strain GenBank accession no. Homology Streptomyces longispororuber NBRC13488 ^T AB184440 99.425 Streptomyces atrovirens NRRL B-16357 ^T DQ026672 99.357 Streptomyces pilosus NBRC12807 ^T AB184161 99.142 Streptomyces flavoviridis NBRC12772 ^T AB184842 99.142 Streptomyces griseoflavus LMG19344 ^T AJ781322 98.928 Streptomyces speibonae PK-Blue ^T AF452714 98.928 Streptomyces viridodiastaticus NBRC13106 ^T AB184317 98.928 Streptomyces albogriseolus NRRL B-1305 ^T AJ494865 98.928 Streptomyces lusitanus NBRC13464 ^T AB184424 98.927 Streptomyces violaceochromogenes NBRC13100 ^T AB184312 98.922 Streptomyces lakyrus NBRC13401 ^T AB184877 98.856 Streptomyces coerulescens ISP5146 ^T AY999720 98.850 Streptomyces bellus ISP5185 ^T AJ399476 98.847 Streptomyces luteogriseus NBRC13402 ^T AB184379 98.782 Streptomyces djakartensis NBRC15409 ^T AB184657 98.713

As shown in Table 1, the strain WJ2 is a strain of Streptomyces longispororuber NBRC 13488 ^T , Streptomyces atrovirens NRRL B-16357 ^T , Streptomyces pilosus NBRC 12807 ^T , Streptomyces flavoviridis NBRC12772 ^T , Streptomyces griseoflavus LMG19344 ^T , Streptomyces speibonae PK- Blue ^T , Streptomyces viridodiastaticus NBRC13106 ^T , And Streptomyces albogriseolus NRRL B-1305 ^T , respectively, of the 16S rRNA gene sequence. In a Neighbor-Joining phylogenetic tree based on the 16S rRNA gene sequence, strain WJ2 formed a clade containing Streptomyces atrovirens NRRL B-16357 ^T (see FIG. 2). Based on the phylogenetic results, Streptomyces atrovirens NRRL B-16357 ^T was used as a control strain in DNA-DNA hybridization assays (Gause et al ., 1983). 16S rRNA gene sequence Since a number of studies have been reported (La Duc et al ., 2004; Satomi et al ., 2002), strains containing more than 99% of homologous homologies may not belong to the same species DNA-DNA hybridization was performed between the closest genomic control strains (see FIG. 3) .

Genomic DNA Extraction Kit (DyneBio, Korea) was prepared from the strains of WJ2 cultured in LB medium and Streptomyces atrovirens NRRL B-16357 ^{T, which} is a control strain thereof, for DNA-DNA hybridization analysis. E. coli KCCM12119 ^T was used as a negative control. DNA-DNA hybridization was performed according to the method described in the manual (Roche Applied Science, Germany) using the DIG High Prime DNA Labeling and Detection Starter Kit II and the hybridization signal was quantitatively analyzed by Quantity One Program Respectively. The value of the self-hybridization signal of strain WJ2 was converted to 100%.

As a result, the DNA-DNA hybridization value of the standard strains such as Streptomyces longispororuber NBRC13488 ^T , Streptomyces pilosus NBRC12807 ^T and Streptomyces flavoviridis NBRC12772 ^T and the strain WJ2 was about 70%. The DNA association between strain WJ2 and Streptomyces atrovirens NRRL B-16357 ^T was 87%, indicating that these strains were single species (Van Trappen et al ., 2001). However, strain WJ2 was classified as a new subspecies of S. atrovirens based on the DNA-DNA hybridization level (90% or less, but over 70%). 16S rRNA Gene Sequence Based on the results of similarity, phylogenetic analysis and DNA-DNA hybridization analysis, it was shown that the strain WJ2 could be classified as a new sub- species of bacteria of Streptomyces atrovirens , Streptomyces atrovirens WJ2. In the phylogenetic analysis based on the 16S rRNA gene sequence, the tree numbers obtained by the NJ and MP methods showed almost the same tree topology.

Example 3 Morphological-Physiological Characterization of Strain WJ2

To determine the morphological characteristics of the bacteria, the cells were stained using the Gram stain kit (BD, USA) according to the method described above and then observed with an optical microscope. As a result, the isolated strains were Gram positive bacteria and aerobic.

Of strain WJ2 Carbon utilization and enzyme production were investigated using API 20NE and API ZYM kit (Biomerieux, France) according to the proposed method. However, the bacterial suspension was prepared using AUX medium supplemented with 1.0% NaCl and trace elements. (100 占 퐂 / ml), paromomycin (100 占 퐂 / ml), thiostrepton (100 占 퐂 / ml), kanamycin (100 占 퐂 / Various antibiotics such as neomycin (100 μg / ml), ampicillin (100 μg / ml), apramycin (100 μg / ml) and chloramphenicol (100 μg / (Antibiotic susceptibility) was measured in LB medium using paper disk diffusion method. Growth at various pH (pH 4.0 ~ 11.0, pH 1.0) and various NaCl concentrations (0 ~ 10%, 1% interval) were investigated using LB solid medium.

To investigate the morphological-physiological characteristics of the strain WJ2, various agar solid media (LB, LBX, LB supplemented with 0.2% xylenes), ISP-2 (BD, USA) and ISP- agar plates for 5 days at 40 ℃ to observe morphological changes and pigment production.

As a result, diffusible pigments were not formed in agar medium, LB, ISP-2, ISP-4, and R2YE (Kieser et al ., 2000). Gray spores were formed in aerial mycelium cultured on agar plates (see Table 2).

Agar medium
(Agar medium) Spore mass
(Spore mass) Basal mycelium
(Substrate mycelium) Aerial mycelium
(Aerial mycelium) Soluble pigment
(Soluble pigments) LB none beige none none LBX * none beige White none ISP-2 Abundance, gray beige White none ISP-4 Abundance, gray beige White none R2YE Low, Gray beige White none

 * LBX is LB medium containing 0.2% xylan

Growth was also observed between 15 ° C (very weak) and 50 ° C, but not at 12 ° C and 55 ° C. Growth was observed at pH 6.0 to pH 10.0 (weak at pH 10.0) but not at pH 5.0 and pH 11.0. Cells are susceptible to thiostrepton, neomycin, ribostamycin, and paromomycin (river), while cells are susceptible to thiostrepton (about), neomycin (about), ribostamycin Resistant to ampicillin, kanamycin, apramycin, and chloramphenicol. The strain WJ2 may be selected from the group consisting of nitrate reduction, agarase, amylase, gelatinase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, phosphohydrolase), β- galactosidase (β -galactosidase), α- glucosidase (yieoteuna tested positive for the (α -mannosidase) Let α -glucosidase) and α- manno, indole production (indole production), arginine di-hydrolase dehydratase (arginine dihydrolase), urease (urease), lipase (lipase) (C14), allyl cysteine amidase (cystine arylamidase), trypsin, chymotrypsin α- (α -chymotrypsin), α- galactosidase ( α -galactosidase), it dehydratase (β -glucuronidase), N- acetyl -β- The glucosidase Sami to β- glucuronidase Kinase was negative for the (N -acetyl- β -glucosaminidase), α- Foucault let dehydratase (α -fucosidase), β- glucosidase (β -glucosidase) is a 6-bromine -2-naphythyl-β-D- A negative reaction to the 6-Br-2-naphthyl- β- D-glucopyranoside hydrolysis (API ZYM strip) was positive for the esculin hydrolysis (API 20NE strip). The use of D-glucose, L-arabinose, D-mannose, D-mannitol, N- acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid and malic acid was positive, but trisodium citrate, phenylacetic acid was negative reaction. D-Glucose fermentation was negative reaction.

Example 4. Biochemical characterization of strain WJ2

Microbial identification (MS) analysis was performed by gas chromatography (GC) using microbial identification system (MIDI). DNA G + C content in the genome was analyzed using reverse phase HPLC according to the method described (Mesbah et al ., 1989). Respiratory quinone was analyzed using reverse phase HPLC according to the method described by Komagata and Suzuki (1987) (Komagata and Suzuki, 1987).

A result of this, the major fatty acid (greater than 4%) _{C 15: 0 Anteiso (36.19%} ), C 15: 0 Iso (10.58%), C 16: 0 (10.20%), C 16: 0 Iso (9.84%) and C _{14: 0} Iso (4.18%). The total fungal fatty acid composition (≥ 1%) is shown in Table 3. The G + C content of the DNA was 73.98 mol%.

fatty acid Strain WJ2 (%) C14 _{: 0} Iso 4.18 C15 _{: 0} Iso 10.58 C _{15: 0} Anteiso 36.19 C _{15: 0} 2.96 C _{16: 1} Iso H 2.04 C _{16: 0} Iso 9.84 C _{16: 0} 10.20 C16 _{: 0} 9 Methyl 1.31 C _{17: 1} Anteiso C 1.99 C17 _{: 0} Iso 2.52 C _{17: 0} Anteiso 7.54

Example 5: Analysis of optimization characteristics of culture medium of strain WJ2

To determine the composition of the culture to improve xylanase production, the nitrogen source and carbon source of the medium were changed. The 0.2% based on the minimal medium mentioned by Hopwood et al (1985) (NH 4) 2 SO 4, 0.05% K 2 HPO 4, 0.02% MgSO 4 · 7H 2 O, 0.001% FeSO 4 · 7H 2 O , And 0.1% beechwood xylan at pH 7.2. Bacto peptone (bacto peptone), Bacto tryptone (bacto tryptone), soy tone (soytone), yeast extract (yeast extract), meat extract (meat extract), and asparagine various nitrogen source (NH ₄₎ such as (asparagines) ₂ Was added to the native medium at a final concentration of 0.2% instead of SO ₄ . Eight carbon sources such as maltose, carboxyl methyl cellulose (CMC), glucose, xylan, xylose, sucrose, starch and fructose were added to the medium containing 0.2% soytone as a sole nitrogen source at a final concentration of 0.1% Lt; / RTI >

In addition, the effect of the carbon source concentration difference in the range of 0.1 to 0.5% in the production of xylanase was measured. The liquid culture was sampled at regular intervals and centrifuged at 10,000 rpm for 20 minutes to obtain supernatant from which the cells were removed. The xylalanase activity was measured at 540 nm according to the dinitrosalicylic acid (DNS) method (Miller, 1959). 0.2% (w / v) beechwood xylan was added as a substrate to the reaction solution. One unit (U) of xylanase was defined as the amount of enzyme producing 1 μmol of xylose per minute (min) under the above analysis conditions. Xylose was used as a reference reducing sugar for the calibration curve.

To investigate the composition of the medium for fermentation, various nitrogen sources and carbon source effects on xylanase production were investigated. As a result, soytone was selected as the best nitrogen source among the tested nitrogen sources since 0.2% soytone showed xylalanine production four times higher than ammonium sulfate (see FIG. 4) ). Xylan was chosen as the best carbon source because it improves xylanase production by about 2-fold over other carbon sources (see Figure 5). In addition, the best effect of xylan concentration on xylanase production was 0.3% (see FIG. 6). Thus, an optimized medium of pH 7.2 composed of 0.2% soytone, 0.05% K ₂ HPO ₄ , 0.02% MgSO ₄ .7H ₂ O, 0.001% FeSO ₄ .7H ₂ O, and 0.3% Lt; / RTI >

The strain WJ2 was inoculated into an optimized medium and cultured at 40 DEG C for 4 days with stirring. FIG. 7 is a graph showing cell mass production and xylanase activity of WJ2, a microorganism of the present invention, in the modified medium; (1) cell growth in the specific medium; (2) cell growth from the optimized medium; Xylalanase activity; xylanase from optimized media). As shown in FIG. 7, the amount of active cells was rapidly increased to 2 days of culture, and the highest production of xylanase was slowly decreased during the days 2 and 3, followed by a decrease in production of xylanase. On the other hand, the cell growth in the native medium was relatively low, and the production of xylanase was also very low. Thus, xylan in the medium can be regarded as a limited carbon source for xylanase production, which serves to facilitate further growth by degrading xylan.

Example 6. Analysis of optimal conditions for xylanase activity of strain WJ2

In order to confirm the production characteristics of the cells and xylanase, the strain WJ2 was dissolved in 0.2% soytone, 0.05% K ₂ HPO ₄ , 0.02% MgSO ₄ .7H ₂ O, 0.001% FeSO ₄ .7H ₂ O, and 0.3 % beechwood xylan, pH 7.2, and sampled at regular intervals. The growth curve was confirmed by measuring the wet cell weight from the culture solution, and the culture solution in which the cells were removed was centrifuged at 14,000 rpm for 20 minutes and collected to measure xylanase activity. The xylanase activity of the cell lysate was measured by dinitrosalicylic acid (DNS) method (Miller, 1959) using 0.2% (w / v) xylan as a substrate. The reaction was carried out at 40 DEG C for 30 minutes. One unit (U) of xylalanase activity was defined as the amount of enzyme producing 1 μmol xylose per minute under the above analysis conditions. Xylose was used as the standard reducing sugar for the calibration curve.

To determine the optimum pH of xylalanase activity, optimal pH conditions were measured at 40 ° C under various pH conditions. 20 mM MOPS buffer solution (pH 6.0 to 7.0) and 20 mM Tris-Cl buffer solution (pH 7.0 to 9.0) were respectively used. As a result, the coenzyme of strain WJ2 showed the highest total xylanase activity when reacted at 40 < 0 > C. 8 is a graph showing the effect of the pH condition for the xylanase activity of the microorganism of the present invention (20 mM MOPS buffer, 20 mM Tris-Cl buffer). The value obtained at pH 7.0 was taken as 100%, and all the data were the mean value obtained from at least 3 repeated experiments. As shown in FIG. 8, the relative activity at pH 6.0 and 8.0 was about 70% of the maximum activity.

In order to determine the optimum temperature for the xylalanase activity, it was measured at different temperatures ranging from 45 to 70 DEG C (5 DEG C intervals) in 20 mM Tris-Cl buffer (pH 7.0). 9 is a graph showing the effect of temperature conditions for the xylanase activity of the microorganism of the present invention. The value obtained at 55 캜 was taken as 100%. Relative activity is the average of three independent experiments. As shown in FIG. 9, when the reaction was carried out at pH 7.0, it showed the maximum activity at 55 ° C.

In order to measure the thermostability, the enzyme reaction solution was sampled at regular intervals while pre-incubating the enzyme at different temperatures ranging from 55 to 65 DEG C (5 DEG C interval) for 2 hours. Residual enzyme activity was measured at 55 캜 for 30 minutes. As a result, the xylanase activity was about 80% and 50% at 60 ° C and 65 ° C, respectively. Total xylanase activity was maintained at about 50% for 20 minutes at 55 ° C and then decreased to about 30% for 120 minutes incubation. However, almost no activity was observed for 30 minutes at 60 ° C and 20 minutes at 65 ° C. 0 (zero) -the value obtained at the incubation time was converted to 100%. Relative activity is the mean value of three independent experiments (see FIG. 10).

Example 7. Analysis of Xylen hydrolyzate

To analyze hydrolysis products of xylan by xylanase in strain WJ2, crude enzyme was mixed with 80 [mu] l of crude enzyme in 20 mM Tris-Cl at pH 7.0 containing 0.5% beechwood xylan. The reaction mixture was incubated at 55 DEG C for 2 days and sampled at regular intervals. 15 μl of the reaction mixture was added to a Silica gel 60 plate (Merck, Germany), double developed in a solvent system of n-butanol: acetic acid: distilled water (2: 1: 2, v / v / v) 10% (w / v) sulfuric acid in (w / v) EtOH was sprayed and the plate was developed by heating at 120 ° C.

As a result, depolymerization of xylan caused mainly spots corresponding to xylotriose and xylotetraose (see Fig. 11). In addition, xylobiose could be detected through further incubation. TLC analysis of xylan hydrolyzate indicates that strain WJ2 produces endo-type xylanase, which degrades xylan into xylobiose and xylotriose as a major final degradation product Respectively.

The identification and characterization of microorganisms that produce xylanases suitable for industry are important prerequisites for their successful biotechnology applications. Active and stable xylianases at high temperatures are useful in a variety of industries. In the present invention, Streptomyces atrovirens WJ2 identified in soil samples was found to be an excellent producer of extracellular suitable thermotolerant xylanase. For large-scale production of industrial enzymes, the conditions and activity of the culture medium constitute one of the important factors (Bajaj et al., 2010; Gupta et al., 2001). Strain WJ2 was the most efficient source of nitrogen and used soytone. However, peptone, tryptone, yeast extract, meat extract, asparagine, ammonium sulfate and soybean meal are also known as good sources of nitrogen for the production of xylanase by various microorganisms (Bajaj et al (2001), and Sharma et al., 2005; Sharma et al., 2005; It has been found that xylans can be the best carbon source for xylanase production by strain WJ2. This means that the production of xylanase by strain WJ2 is highly dependent on the presence of xylan in the medium and that the expression of its encoding gene (s) can be regulated to be expressed according to the need for xylan degradation do. Other carbon sources including maltose, CMC, glucose, sucrose, xylose, starch, and fructose showed relatively low levels of xylanase production, which may be due to carbon catabolite repression (CCR) Bajaj et al., 2010; Virupakshi et al., 2005). The medium thus modified showed a considerable advantage for culturing microorganisms producing the mycobacterial xylanase. The bacterial mass and total xylanase activity by culturing strain WJ2 in the modified medium were about 9 times higher than those observed in LB medium. The strain WJ2 exhibited the greatest total xylanase activity at pH 7.0 and 55 ° C, indicating that for most of the other xylanases known from Streptomyces, the average of the optimum activity at pH 5.0-7.0 and 50-65 ° C (Bajaj et al., 2010; Wang et al., 2003; Techapun et al., 2002; Georis et al., 2000). This property of strains WJ2 and total xylazine are suitable for use in industrial processes, including bacterial strains for biofuel production, biological bleaching of pulp, and utilization of lignocellulosic biomass for the food and bakery industry . In the present invention, it was revealed that xylanase can be produced from a soil strain isolated from Jeju Island, Korea, by changing the composition of the medium. Although many studies have been reported on microorganisms producing xylanase, the production of xylalanase from S. atrovirens has been identified for the first time through the present invention.

Agricultural Biotechnology Research Institute KACC92087 20150916

Claims (3)

Streptomyces atrovirens WJ2 (Accession No. KACC92087P) derived from soil having xylan degrading activity. A method for degrading xylan, comprising the step of inoculating the sample containing xylan with the microorganism of claim 1 or adding a culture medium in which the microorganism of claim 1 is cultured. A method for producing xylanase according to claim 1, wherein the microorganism is cultured by adding xylan to the culture medium.

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