NOVEL XYLAN DECOMPOSITION STRAIN, STREPTOMYCES ATROVIRENS SUBSPECIES WJ2

Or less, more detailed embodiment which utilizes the present invention to less than 1000. For example since only these embodiments of the invention, the scope of this invention interpreted not limited by these embodiments.

Embodiments 1. Separation of xylose it recovers, sacrifice producing microorganism

First, a soil sample from the dead trees mixed bacteria strain WJ2 Xyl (xylan-a hydrolyzing bacterium) separating as his (also reference 1). Specifically, collecting samples of soil have jeju-do[song[song] evil acid, 10 continuously distilled water^-1 - 10^-5 His diluent. Then, the dilute solution each 100 micro l by 0. (Sigma, USA) containing 1% xylan azure LB solid medium after 48 40 °C him as time in human herpesvirus. Selected colonies comprising xylose it recovers, sacrifice activity was new LB medium passaging. Among these, as well as additional experiments finally selected WJ2 strain named as the colonies. GenBank JN578482 16S rRNA gene sequence of strain WJ2 registered to him. The isolated strains of dielectric resource center consignee number been strain brocadcast FM with agriculture KACC92087P agricultural Institute.

2 embodiments. 16S rRNA gene sequence of strain WJ2 decryption and channel possibility analysis

A strain WJ2 0. 2 culture in a liquid medium containing 2% commercial LB xylose by culturing is 10,000 rpm 40 °C after cyclone bacterial cells that were unsafe. Dielectric by extracting DNA, bacterial universal primer (on 27F 1492R) (template) was used for mold using 16S rRNA gene amplification in (Baker et al. , 2003). The nucleotide sequence of genes that are amplified fragments using Applied Biosystems 3730xl DNA Analyzer analysis directly conducting. Of NCBI Basic Local Alignment Search Tool (BLAST) program (Altschul Et al. 1990) WJ2 16S rRNA gene sequence based on the GenBank database using strain within an associated standard strains of a visit from the police. Related type strains of 16S rRNA gene nucleotide sequence was collecting in the ExTaxon server (Chun et al. 2007). A multiple sequence alignment (Multiple alignment) associated e two three [su[su] molded Clustal W software (Thompson et al. 1994) have performed using, 5 'and 3' (Hall 1999) BioEdit program using his editing the gap (gap). (Felsenstein 1993) PHYLIP suit program (NJ) method (Saitou 1987) and (Kluge 1969) (MP) of Neighbour provided Joining Maximum Parsimony method was used in the production of a channel possibility (Phylogenetic tree). Bootstrap analysis has been employed to compute from an 1,000 Conference reconstructed Neighbor provided Joining tree topology. (Evolutionary distance matrix) Kimura's two a-parameter model (Kimura 1983) ancestor distance has been the total value.

From strain WJ2 16S rRNA gene (1, 399bp) PCR was a into a base sequence analysis. Table 1 indicating the 16S rRNA gene sequence homology search results are disclosed. Such as in table 1, that the strains of Streptomyces strain WJ2 16S rRNA gene sequence analysis ofExhibits belonging to the genus Department.

1 the table such as, strain WJ2 is Streptomyces longispororuber NBRC 13488^T , Streptomyces atrovirens NRRL B provided 16357^T , Streptomyces pilosus NBRC12807^T , Streptomyces flavoviridis NBRC12772^T , Streptomyces griseoflavus LMG19344^T , Streptomyces speibonae PK-a Blue^T , Streptomyces viridodiastaticus NBRC13106^T , And Streptomyces albogriseolus NRRL B provided 1305^T Of each of the 99. 42%, 99. 35%, 99. 14%, 99. 14%, 98. 92%, 98. 92%, 98. 92% and 98. 16S rRNA gene sequence homology of 92% and viscoelasticity. In the 16S rRNA gene sequence based on Neighbor provided Joining channel possibility (phylogenetic tree), strain WJ2 is Streptomyces atrovirens NRRL B provided 16357^T (Clade) comprising clay for use in rear (also reference 2). Based on the results of channel possibilityStreptomyces atrovirens NRRL B provided 16357^T Compared to DNA-a DNA hybridization analysis was used persimmons (Gause Et al. , 1983). 16S rRNA gene sequence comprising at least 99% homologous strains such as not belonging to the plurality study that reported is shown twisted (La Duc Et al. , 2004; Satomi Et al. , 2002), strain WJ2 genetically closest contrast in the strain on DNA-a DNA hybridization between conducting (3 also reference).

The DNA-a DNA hybridization analysis, strain cultured in LB WJ2 machine is added with verification Streptomyces atrovirens NRRL B provided 16357^T Genomic DNA Extraction Kit (DyneBio, Korea) was performing firing processes for preparing DNA from cells. E. Coli KCCM12119^T As well as the voice matching group. DNA-a DNA hybridization experiments using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Science, Germany) that have been performed in accordance with the proposed method to the personal computer, the hybridization signal of a Quantity One Program (Bio non-rad, USA) were measured. A self-a hybridization signal data corresponding to values of 100% in terms of strain WJ2 to him.

Utilization, Streptomyces longispororuber NBRC13488^T , Streptomyces pilosus NBRC12807^T , Streptomyces flavoviridis NBRC12772^T Up to about 70% of strains such as strain WJ2 DNA-a DNA hybridization value is standard. Strain WJ2 and Streptomyces atrovirens NRRL B provided 16357^T DNA association identifies a pulse between 87%, exhibits a single being a bell, these strains (Van Trappen Et al. , 2001). However, strain WJ2 is DNA-a DNA hybridization levels (or 90% or less, 70% or more) based on the S. AtrovirensBeen classified into new subspecies. 16S rRNA gene sequence homology (similarity) search, based on strain WJ2 determinants analysis (phylogenetic analysis) and DNA-a DNA hybridization analysis result is Streptomyces atrovirensClassified in the novel bacterial subspecies appears to be a starting point, the Streptomyces atrovirens WJ2 was referred to. In reference to the 16S rRNA gene sequence determinants analysis, NJ and MP channel possibility obtained by almost identical purges a tree topology.

3 embodiments. Strain - WJ2 physiological characteristic analysis of morphological

The recombinant expression vector of morphological characteristics separate, Gram stain kit (BD, USA) in accordance with the proposed method using the optical microscope after dyeing process observed. Utilization, and aerobic gram-positive strains isolated hypercholesterolemia.

Of WJ2 strainAPI ZYM kit (Biomerieux, France) on carbon using and enzyme production is API 20NE using with the proposed method according to visit from the police. Stage, the bacterial suspension (bacterial suspension) 1. 0% NaCl and trace element added AUX Royal and adhesive. Ribo-star feed (ribostamycin) (100 micro g/Ml), yeast (paromomycin) (100 micro g/Ml), macrocyclic (thiostrepton) (100 micro g/Ml), Kanamycin (kanamycin) (100 micro g/Ml), Neo (neomycin) erythromycin (100 micro g/Ml), cancerous blood thread phosphorus (ampicillin) (100 micro g/Ml), as antiprotozoal oh [phu[phu] lama (apramycin) (100 micro g/Ml) and chloramphenicol (chloramphenicol) (100 micro g/Ml) antibiotics using paper disk diffusion method for antibiotic susceptibility (antibiotics susceptibility) various areas such as using LB medium (paper disk diffusion method) were measured. Various pH (pH4. 0 - 11. 0, pH 1. 0 interval) and various NaCl concentration (0 - 10%, 1% interval) growth in LB was irradiated using a solid growth medium.

In order to physiological properties of strain WJ2 morphological - extracting, LB, LBX (0. 2% added Xyl LB), ISP provided 2 (BD, USA), ISP provided 4 (BD, USA) in 5 such as solid growth medium (agar plates) in liver by culturing various cap 40 °C observed morphological change and dye production.

Utilization, diffusing dye (Diffusible pigment) is cap, LB, ISP provided 2, ISP provided 4, and R2YE indications to be formed (Kieser Et al. , 2000). The cap formation in cultured hypha gray spores (agar plates) to (aerial mycelium) (table 2 reference).

The * LBX 0. By LB medium containing 2% xylan

Also, 15 °C (by very weak) on growth is observed between 50 °C but, in 12 °C and 55 °C was not observed. PH6. 0 to pH10. 0 (pH10. 0 weak in by) observed but grown in, pH5. 0 and pH11. 0 not observed in her. The macrocyclic (thiostrepton) (Cells) cells (about), Neo (neomycin) erythromycin (about), ribo-star feed (ribostamycin) (steel), and susceptible to yeast (paromomycin) while (steel) exhibit, cancerous blood thread phosphorus (ampicillin), Kanamycin (kanamycin), resistant to chloramphenicol (chloramphenicol) leucine (apramycin) and oh [phu[phu] lama mistletoe. Strain WJ2 is nitrate reduction, agarase (agarase), amylase (amylase), gelatinase (gelatinase), alkali phosphatase (alkaline phosphatase), esterase (esterase) (C4), esterase lipase (esterase lipase) (C8), leucine will be biting and Oh the sacrifice which gets torn (leucine arylamidase), valine will be biting and Oh the sacrifice which gets torn (valine arylamidase), acid phosphatase (acid phosphatase), naphthol - AS-a BI - pos id roll under gun Oh sacrifice (naphtol provided AS-a BI provided phosphohydrolase), β - galactosidase (β- Galactosidase), α - glucosidase (α- Glucosidase) and α --mannosidase (α- Mannosidase) but a positive reaction, indole production (indole production), arginine id roll under D Oh sacrifice (arginine dihydrolase), urease (urease), lipase (lipase) (C14), cystine it will inform and Oh the sacrifice which gets torn (cystine arylamidase), trypsin, α - chymotrypsin (α- Chymotrypsin), α - galactosidase (α- Galactosidase), β - it is a [ni[ni] in writing base [khwu[khwu], Oh sacrifice (β- Glucuronidase), it is a writing base nose company mini, Oh sacrifice N - acetyl - β - (N- Acetyl -β- Glucosaminidase) voice reaction for fluorescence, α - fucose it is sour, Oh sacrifice (α- Fucosidase), β - glucosidase (β- Glucosidase) is 6 - bromine - 2 a-naphythyl β-a D - on hair (6 a-Br-a 2 provided naphythyl -β- D non-glucopyranoside) voice reaction for hydrolysis (API ZYM strip) but, s [khwul[khwul] Phosphorus (esculin) hydrolysis (API 20NE strip) has been a positive reaction. D non-glucose, L-a arabinose, D non-mannose, D-a mannitol, N- Acetyl-a glucosamine, D-a maltose, potassium gluconate, malic acid and adipic acid (about) but the use of a positive reaction, trisodium citrate, capric acid and phenylacetic acid has been voice reaction. Voice been D provided Glucose fermentation reaction.

4 embodiments. Strain WJ2 biochemical characteristic analysis

Process gas chromatography (GC) using Methyl esters by Microbial Identification system fatty acids (MIDI) was analyzed. According to the method referred to in a Genome DNA G + C content (Mesbah Et al. , 1989) reverse phase HPLC was analyzed. Respiratory quinone (Respiratory quinone) on the Komagata Suzuki (1987) was analyzed by reverse phase HPLC according to the method referred (Komagata and Suzuki, 1987).

Utilization, major fatty acid (4% or more) is 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%) been. The tables shown to total process fatty acid (≥ 1%) 3. G + C content of the DNA is 73. 98mol % min.

5 embodiments. Production of strain WJ2 optimization of characterizing

Extracting it recovers, sacrifice xylose culture medium composition in order to enhance production, nitrogen-free medium was changing won and a carbon source. Hopwood et al (1985) based on the minimum medium referred by 0. 2% (NH₄ )₂ SO₄ , 0. 05% K₂ HPO₄ , 0. 02% MgSO₄ , 7H₂ O, 0. 001% FeSO₄ , 7H₂ O, and 0. A unique medium containing 1% beechwood xylan pH7. 2 was prepared. (Bacto peptone) peptone sphingosine, sphingosine trip tone (bacto tryptone), SOI tone (soytone), (yeast extract) yeast extract, meat extract (meat extract), and various nitrogen source such as asparagine (asparagines) (NH₄ )₂ SO₄ 0 instead. Contains 2% final concentration was unique. Won 0 as nitrogen alone. To media containing 2% SOI tone (soytone) maltose, carboxyl methyl cellulose (CMC), glucose, xylan, xylose, sucrose, starch, and fructose 0 8 each species such as carbon source. Was added to a final concentration of 1%.

Also, in the production it recovers, sacrifice xylose 0. 1 to 0. In accordance with a difference effect concentration range of carbon won 5% were measured. Liquid culture in 20 minutes at constant intervals to 10,000 rpm sampling centrifuging removing bacterial cells obtained dissolves. Dinitrosalicylic acid (DNS) method (Miller, 1959) xylose it recovers, sacrifice activity in the 540 nm were measured. 0. 2% (w/v) was added as a substrate beechwood xylan of reaction solution. 1 unit (U) (min) 1 μmol of xylose it recovers, proposal the analysis conditions (xylose) per amount of enzyme producing powdered food component was defined. For the manufacture as well as the reducing sugar xylose with five [su[su] reference calibration curves.

To replace the animal-separate composition, various xylose yield production it recovers, sacrifice nitrogen won and carbon cancels the effects of a visit from the police. Utilization, 0. 2% ammonium sulfate (ammonium sulfate) 4 times than the SOI tone (soytone) indicating high it recovers, sacrificewith [u[u] pigment production, SOI tone (soytone) was selected for testing during nitrogen won best nitrogen won (reference 4 also). Other carbon won about 2 times (xylan) Xyl xylose it recovers, sacrifice cause increased production, best carbon won was selected (reference 5 also). Also, for best effect xylose production it recovers, sacrifice Xyl concentration of 0. Up to 3% (reference 6 also). The, 0. 2% soytone, 0. 05% K₂ HPO₄ , 0. 02% MgSO₄ , 7H₂ O, 0. 001% FeSO₄ , 7H₂ O, and 0. Xyl pH7 consisting of 3%. 2 WJ2 optimization of fermentation on an advanced strain was used.

Strain WJ2 optimised perpendicularly installed in 40 °C 4 him as during stirring. Figure 7 improved medium WJ2 microorganism of the present invention in cells production and xylose represents a it recovers, sacrifice activity are disclosed (■, unique media process growth ;●, optimized medium from cells growth; □, xylose media it recovers, sacrifice unique active; ○, optimized from it recovers, sacrifice xylose medium). Such as in Figure 7, the rapid rise in pigment production was highest it recovers, sacrifice culture to active contact portions 2 shown in 2 and 3 have slowly during culture, then a reduction in the inhibin it recovers, sacrifice xylose production. On the other hand, relatively low level pulse cells growth is unique media, also been it recovers, sacrifice xylose production is very low. The xylose medium serving to facilitate growth of xylose by decomposing column far infrared ray for the production of xylose it recovers, sacrifice more limited carbon won can be considered.

6 embodiments. Analyzing characteristic optimum condition for xylose WJ2 it recovers, sacrifice activity of strain

Process and to make sure that the xylose it recovers, sacrifice production characteristics, a strain WJ2 0. 2% SOI tone (soytone), 0. 05% K₂ HPO₄ , 0. 02% MgSO₄ , 7H₂ O, 0. 001% FeSO₄ , 7H₂ O, and 0. 3% beechwood xylan, pH 7. 2 consisting of optimized cultured in regular intervals have his sampling. Measuring cells harvested from the contact portions (wet cell weight) grown in culture has been removed 20 minutes 14,000 rpm formate curve centrifugal separating and collecting a xylose to it recovers, sacrifice activity were measured. 0 xylose it recovers, sacrifice activity of mesenchymal cells has been removed. 2% (w/v) dinitrosalicylic acid (DNS) method (Miller, 1959) by using far infrared ray xylose as a substrate were measured. 30 minutes in 40 °C reaction is conducting. The xylose it recovers, sacrifice activity (U) of enzyme producing 1 μmol 1unit with five [su[su] amount of xylose sugar under conditions analysis was defined. For the manufacture as well as the reducing sugar xylose with five [su[su] standard calibration curves.

Measuring it recovers, sacrifice optimum pH for activity of xylose, optimal pH conditions 40 °C were measured in various pH conditions. 20 mm MOPS buffer (pH 6. 0 - 7. 0) and 20 mm Tris a-Cl buffer (pH 7. 0 - 9. 0) was used respectively. Utilization, as a coenzyme in the highest xylose it recovers, sacrifice activity can be constructed 40 °C WJ2 strains react mistletoe. Figure 8 represents a pH for activity of the microorganisms in a xylose it recovers, sacrifice condition effect are disclosed (●, 20 mm MOPS buffer ;■, 20 mm Tris a-Cl buffer). PH 7. 0 to 100% when the value obtained, at least 3 times all data obtained from experiments alert disclosed. As in Figure 8 the, pH 6. 0 and 8. 0 maximum activity relative activity was about 70%.

Measuring a temperature optimum for xylose it recovers, sacrifice activity, 20 mm Tris a-Cl buffer (pH 7. 0) in 45 to 70 °C (5 °C interval) were measured in a different temperature range. Figure 9 shows a xylose of microorganisms are also active for on condition represents a it recovers, sacrifice effect are disclosed. Obtained 55 °C value is set to 100%. Average of 3 independent experiments relative sialidase Conference are disclosed. As in Figure 9 the, pH 7. 0 react as shown in 55 °C in mistletoe.

For measuring the thermostable (Thermostability), enzyme 2 time 55 to 65 °C (5 °C interval) culturing the pre - a different temperature range while the liquid reaction mixture was sampled at regular intervals enzyme. 55 °C remaining enzymatic activity in 30 minutes were measured. Utilization, about 80% and 50% respectively been 60 °C and 65 °C xylose it recovers, sacrifice activity. About 50% xylose it recovers, sacrifice activity was maintained in 20 minutes total 55 °C, 120 minutes then been reduced to about 30% culture. However, 60 °C in 30 minutes, 20 minutes as in the picomolar activity in 65 °C substantially lost. 0 (zero) at the time the value was obtained by culturing - 100% in terms of value. The average value of 3 independent experiments relative activity are disclosed (10 also reference) Conference.

7 embodiments. Xyl hydrolysates analysis

Strain WJ2 it recovers, in sacrificecolumn xylose by hydrolysis of xylose product analyze, 0. PH 7 containing 5% beechwood xylan. 0 to 80 micro l to final volume of 20 mm Tris a-Cl(crude enzyme) coenzyme solution is mixed. The reaction mixture was sampled at a 2 in at regular intervals by means 55 °C during culturing. 15 Silica gel 60 plate (Merck, Germany) added to the reaction mixture a micro l, n - butanol: acetic acid: distilled water (2:1:2, v/v/v) dual electrophoresis when the solvent system, then 100% (w/v) during 10% EtOH (w/v) sulfuric acid spray and plate 120 °C to color have been heated.

Utilization, depolymerization (depolymerization) xylose column (xylotriose) and corresponding method for the rotating coating comprising xylose lot five [su[su] xylose (xylotetraose) spot mainly appeared (reference 11 also). Also, the deterioration detecting capable of xylose (xylobiose) through additional culture lobby. TLC analysis strain WJ2 Xyl hydrolysates of xylose (xylobiose) and far infrared ray method for the final mainly comprises xylose (xylotriose) into a decomposition product lot are resolved into xylose it recovers, sacrificeto typefive [su[su] lobby xylose (endo-a type xylanase) that produce has been confirmed.

Industry xylose it recovers, sacrifice suitable microorganism producing successful are those compositions and giant applications precondition Alzheimer's disease are disclosed. Active in the high temperature stable it recovers, sacrifice xylose variety of industry a dilutant. In the present invention, identifying a soil in a sample Streptomyces atrovirens WJ2 cells (extracellular) has excellent heat resistance (thermotolerant) it recovers, proposal turnstile xylose production out proper found. Industrial enzyme produced billet, of cell culture media is an important active condition and one of the JPO (Bajaj et al. , 2010; Gupta et al. , 2001). The most efficient to SOI tone (soytone) nitrogen won WJ2 strain is by water. However, peptone, trip tone, (yeast extract) yeast extract, meat extract (meat extract), asparagine, ammonium sulfate and soy foil for improving various by such microorganisms producing xylose it recovers, proposal good nitrogen won also be known (Bajaj et al. , 2010; Sharma et al. , 2005; Virupakshi et al. , 2005; Sa Pereria et al. , 2002; Bakri et al. , 2003; Ding et al. , 2004; Subramanian et al. , 2001). For the best carbon circle dayit recovers, proposal xylose WJ2 xylose by commercial strains can be found. This strain is very dependent columnit recovers, proposal WJ2 xylose production medium by the presence of xylose, (encoding gene (s)) of the expression of a gene encoding an (are) who expressed the need arises Xyl may be big. Maltose, CMC, glucose, sucrose, xylose, starch, fructose and different applications including activated carbon source it recovers, sacrifice representing a relatively low level of xylose production, this carbon (carbon catabolite repression, CCR) can be founded on the use of metabolism inhibitor (Bajaj et al. , 2010; Virupakshi et al. , 2005). The xylose medium contact portions change out 45,000 it recovers, sacrifice incubating the purges significant advantages. Cultivation of strain change media contact portions and total xylose WJ2 LB medium observed in about 9 times higher it recovers, sacrifice activity yet. The pH7 WJ2 strain. 0 xylose it recovers, sacrifice activity of maximum total 55 °C and activated, this known other Streptomyces produce xylose it recovers, proposal pH 5. 0 - 7. 0 and 50 - 65 °C average activity within the range are disclosed (Bajaj et al. , 2010; Wang et al. , 2003; Techapun et al. , 2002; Georis et al. , 2000). Strain WJ2 it recovers, sacrifice bacterial strains of these attributes and total xylose is biofuel production, biological bleaching of the pulp, and food and baking industrial lignocellulose biomass (lignocellulosic biomass) utilization may be suitable for use in industrial processes comprising the mistletoe. A Korean jeju-do isolated from soil that are capable of producing xylose it recovers, sacrifice through change of media composition acid are described. Producing xylose it recovers, sacrifice against microorganisms should forward more reporting, S. AtrovirensThis is therefore at a producing xylose from it recovers, sacrifice has been identified.