KR101771960B1 - Paenibacillus jamilae BRC15-1 strain producing cellulase and use thereof - Google Patents

Abstract

The present invention relates to a strain of Paenibacillus jamilae BRC15-1 having cellulase producing ability and a cellulase produced using the same. Since the cellulase produced by the strain of the present invention has excellent fibrinolytic activity, it can be used for saccharification of fibrous biomass for bioethanol production. If it is used as an enzyme for feed addition, the effect of increasing the nutrient utilization efficiency of livestock .

Description

Staphylococcus aureus strain BRC151 and its use {Paenibacillus jamilae BRC15-1 strain producing cellulase and use thereof}

The present invention relates to a strain of Paenibacillus jamilae BRC15-1 having a cellulase producing ability and a cellulase produced using the same.

In recent years, not only global warming but also limited oil resources and soaring oil prices have demanded the development of new alternative energy globally. Bioethanol and biofuels, which can be produced from renewable plant resources, have been attracting much attention (Lynd LR et al., 2008. Nature Biotechnology 26 (2): 169-172). Bioethanol as transport fuel is currently produced from sugar-based sources such as sugarcane in Brazil and fermented from yeast using glucose from starchy sources such as corn (Cassman KG et al., 2007. Biofuels Bioproducts & Biorefining-Biofpr 1 (1): 18-23; Mabee WE. 2007. Biofuels 108: 329-357). However, these carbohydrate and starchy resources have been linked to food resources and have long been a problem in raw material supply and demand, and research has been conducted to utilize lignocellulose (ie, agricultural and forestry byproducts) rather than food resources (Bak JS et al. , 2009. Bioresource Technology 100 (3): 1285-1290; Purwadi R, Brandberg T, Taherzadeh MJ 2007. International Journal of Molecular Sciences 8: 920-932; Sakai S et al., 2007. Applied and Environmental Microbiology 73 7): 2349-2353; Zaldivar J et al., 2001. Applied Microbiology and Biotechnology 56 (1-2): 17-34).

Generally, lignocellulose is the most abundant renewable biomass on the planet and is the main constituent of plants. Lignocellulose is composed mainly of carbohydrate cellulose, hemicellulose, and lignin, a polymer of aromatic compounds (Reddy N et al., 2005. Trends in Biotechnology 23 (1): 22 -27; Stocker M. 2008. Angewandte Chemie-International Edition 47 (48): 9200-9211; Zhang YHP 2008. Journal of Industrial Microbiology & Biotechnology 35 (5): 367-375). Hydrolysis of cellulose into celluloses can result in glucose that can be fermented by microorganisms, so lignocellulose can be used as a raw material to produce glucose and produce biofuels such as ethanol (McKendry P. 2002. Bioresource Technology 83 (1): PII S0960-8524 (01) 00118-3; Stocker M. 2008. Angewandte Chemie-International Edition 47 (48): 9200-9211; Zhang YHP 2008. Journal of Industrial Microbiology & Biotechnology 35 (5): 367-375). Another carbohydrate, hemicellulose, is a polysaccharide composed of various sugars such as xylose, arabinose, galactose, mannose, and glycosides (Krull LH et al., 1980. Journal of Agricultural and Food Chemistry 28 (5): 917-919; Ren JL, Peng F, Sun RC. 2008.).

Cellulase is an enzyme that hydrolyzes cellulose, and is distributed throughout the biota such as fungi, bacteria, higher plants, and molluscs, but the main origins are fungi and bacteria. Recently, the utility of cellulase as an enzyme for feed addition has been increasing. Feed grains, which supply mainly carbohydrates and small amounts of protein for the energy metabolism of animals, contain fiber that most animal species can not use. The fiber in the grains is composed of non-electrolytic polysaccharides and lignin. The cell wall of the plant is composed of the non-electrolytic polysaccharide composed of hemicellulose and beta-glucan and lignin, which surround the cellulosic layer on the outermost side of the cell and the whole inside of the cell. This nonspecific polysaccharide is not only decomposed by the digestive enzymes of the livestock but also contains the starch and protein in the cereal, which lowers the efficiency of using the nutrients. In the small intestine, the non-electrolytic polysaccharide is sticky and melted in a thick gel state, preventing digestive enzymes from accessing nutrients and inhibiting the flow of digestive enzymes. Therefore, there is a problem that microorganisms adhere to the digestive tract and cause abnormal fermentation, resulting in loss of nutrients. Cellulase is added to the grain feed to hydrolyze the cellulosic layer surrounding the algae to improve the availability of nutrients in the algae as well as improve the condition of intestinal digestion.

Accordingly, the present inventors have completed the present invention by selecting a strain of Penicillium jamilee BRC15-1 which is a novel microorganism producing a cellulase, and confirming the activity of degrading the cellulose of the cellulase separated therefrom.

 Lynd LR et al., 2008. Nature Biotechnology 26 (2): 169-172.  Cassman KG et al., 2007. Biofuels Bioproducts & Biorefining-Biofpr 1 (1): 18-23; Mabee WE. 2007. Biofuels 108: 329-357.  See JS et al., 2009. Bioresource Technology 100 (3): 1285-1290.  Purwadi R, Brandberg T, Taherzadeh MJ. 2007. International Journal of Molecular Sciences 8: 920-932.  Sakai S et al., 2007. Applied and Environmental Microbiology 73 (7): 2349-2353.  Zaldivar J et al., 2001. Applied Microbiology and Biotechnology 56 (1-2): 17-34.  Reddy N et al., 2005. Trends in Biotechnology 23 (1): 22-27.  Stocker M. 2008. Angewandte Chemie-International Edition 47 (48): 9200-9211.  Zhang YHP. 2008. Journal of Industrial Microbiology & Biotechnology 35 (5): 367-375.  McKendry P. 2002. Bioresource Technology 83 (1): PII S0960-8524 (01) 00118-3.  Krull LH et al., 1980. Journal of Agricultural and Food Chemistry 28 (5): 917-919; Ren JL, Peng F, Sun RC. 2008.

It is an object of the present invention to provide a strain of Paenibacillus jamilae BRC15-1 (accession number: KACC92074P) having cellulase producing ability.

It is still another object of the present invention to provide a saccharifying composition comprising the cellulase obtained from the strain.

It is still another object of the present invention to provide a feed additive composition comprising the cellulase obtained from the strain.

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

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

The present invention provides a strain of Paenibacillus jamilae BRC15-1 (accession number: KACC92074P) having cellulase producing ability.

The above-mentioned Paenibacillus jamilae ) BRC15-1 strain may be isolated from, but not limited to, soil collected from reclaimed land.

In one embodiment of the present invention, the morphological characteristics of the strain Paenibacillus jamilae BRC15-1 were observed. As a result, the shape was circular, the surface was smooth, It was moist, the height was raised, the edges were undulate, opaque, and white in color. Further, as a result of Gram staining, Gram positive (+) was observed (see Table 1 and Fig. 2).

In another embodiment of the present invention, the strain Paenibacillus jamilae BRC15-1 has a high cellulose decomposing activity of 0.356 IU (International Unit) / mL for decomposing cellulose into glucose. This is because the comparative group of P. pomymyxa (KACC10098) and P. jamilae (KCTC13919) 0.330 IU / mL and 0.275 IU / mL, respectively (see FIG. 4).

As used herein, the term "cellulase" is a generic term for enzymes that hydrolyze cellulose.

The cellulase produced by the strain of the present invention is preferably endo-beta-1,4-glucanase, but is not limited thereto. The endo-beta-1,4-glucanase is an enzyme that cleaves the beta-1,4-glucosidic bond of cellulose internally. It is also called CMCase because it uses the normally soluble CMC (carboxymethyl cellulose) as a substrate.

The present invention also provides a saccharifying composition comprising a cellulase obtained from a strain of Paenibacillus jamilae BRC15-1 according to the present invention.

As used herein, the term "saccharification" refers to the conversion of a high molecular weight carbohydrate, such as starch, fibrin, etc., into a saccharide that is hydrolyzed by the action of an enzyme or acid to produce a low molecular weight (monosaccharide or disaccharide) Lt; / RTI > Therefore, when the saccharifying composition of the present invention is used, saccharification capable of producing a monosaccharide or a disaccharide from fibrin is possible.

The present invention also provides a composition for feed addition comprising a cellulase obtained from a strain of Paenibacillus jamilae BRC15-1 according to the present invention.

The feed composition may help digest fibrils in the body of an animal, preferably a livestock animal.

The cellulose may be cellulose, preferably carboxymethyl cellulose (CMC), but is not limited thereto.

The composition for feed addition may additionally include, but is not limited to, an additive or a nutrient that can aid digestion or provide nutrients of an animal, preferably a livestock animal.

The present invention also relates to a pharmaceutical composition comprising Paenibacillus < RTI ID = 0.0 > The present invention provides a method for producing a cellulase using a BRC15-1 strain, more specifically,

1) Preparation of Paenibacillus < RTI ID = 0.0 > jamilae ) BRC15-1 strain; And

2) recovering the cellulase from the culture broth of the strain.

The culture of step 1) is preferably carried out at a temperature of 34 to 44 DEG C, and most preferably, it is carried out at a temperature of 40 DEG C, but is not limited thereto. In one embodiment of the invention, the Paenibacillus < RTI ID = 0.0 > As a result of confirming the optimal culture temperature condition of the jamilae BRC15-1 strain, it was confirmed that culturing at 40 ° C is the condition in which the cellulase activity is the highest (see FIG. 5).

The culture of step 1) is preferably carried out at pH 6-8, most preferably at pH 7, but is not limited thereto. In one embodiment of the invention, the Paenibacillus < RTI ID = 0.0 > As a result of confirming the optimum culture pH of the strain BRC15-1 , it was confirmed that culturing at pH 7 is the condition that exhibits the highest cellulase activity (see FIG. 6).

The culture of step 1) may be carried out by adding a carbon source such as carboxymethyl cellulose (CMC), glucose, xylose, mannose, arabinose, sucrose, starch, , And it is most preferable to add carboxymethyl cellulose (CMC), but the present invention is not limited thereto. In one embodiment of the invention, the Paenibacillus < RTI ID = 0.0 > As a result of confirming the optimum substrate carbon source of BRC15-1 strain, it was confirmed that the cellulase activity was high in medium containing CMC (see FIG. 7).

The culture of step 1) is carried out by adding to the culture medium a nitrogen source such as peptone, skim milk, beef extract, ammonium sulfate, malt extract, urea and nitrate Ammonium nitrate, and it is most preferred to add peptone, but it is not limited thereto. In one embodiment of the invention, the Paenibacillus < RTI ID = 0.0 > As a result of confirming the optimum substrate nitrogen source of the jamilae BRC15-1 strain, it was confirmed that the cellulase activity was high in the medium containing the peptone (see FIG. 8).

The culture of step 1) is preferably performed for 10-80 hours, more preferably 12-72 hours, most preferably 24 hours, but not limited thereto. In one embodiment of the present invention, the strain Paenibacillus jamilae BRC15-1 showed a logarithmic growth until 12 hours, and the activity of the enzyme produced by the strain was increased during the logarithmic growth period . Enzyme activities were maintained constant for 12 hours after incubation. The activity values were higher in 24 - hour culture and 48 - hour culture. Thus, it was found that 24-hour culture was optimal for enzyme production (see FIG. 9).

The present invention also relates to a pharmaceutical composition comprising Paenibacillus < RTI ID = 0.0 > The present invention provides a glycation method using a BRC15-1 strain, more specifically,

1) Preparation of Paenibacillus < RTI ID = 0.0 > jamilae ) BRC15-1 strain;

2) centrifuging the culture of step 1) to obtain a supernatant;

3) concentrating the supernatant of step 2) to recover the cellulase; And

4) treating the cellulose with the cellulose in step 3) to decompose the cellulose.

The cellulase treatment of step 4) is preferably performed at pH 5-7 and 30-50 ° C, and most preferably, it is performed at pH 6 and 40 ° C, but is not limited thereto. In one embodiment of the invention, the Paenibacillus < RTI ID = 0.0 > jamilae) results confirm the optimal reaction temperature and pH of the enzyme produced by the strain BRC15-1, were the pH of the substrate into the reaction when the enzyme and substrate appeared the cellulase activity the highest when 6 days at pH 10 or more 55% (See Fig. 10), and showed the highest enzyme activity at a reaction temperature of 40 캜, 78% of high enzyme activity even at a low temperature of 30 캜, and more than 30% of enzyme activity even at a high temperature of 80 캜 and 90 캜 Reference).

In one embodiment of the present invention, an experiment was conducted to verify the glycation ability of the strain Paenibacillus jamilae BRC15-1. Paenibacillus jamilae strain BRC15-1 was cultured and centrifuged, and only the supernatant was concentrated 10 times by using an ultrafiltration device. Then, the fibrous biomass, singe water gas, was pretreated with alkali to be used as a substrate As a result, it was confirmed that the single-stranded sugar gas was decomposed into monosaccharides such as glucose, xylose, and arabinose. Thus, it was confirmed that this strain could be used for saccharification of fibrin or feed additives.

The cellulase produced by the strain Pennibacillus jamilee BRC15-1 of the present invention is suitable for saccharification of fibrous biomass for the production of bioethanol because of its excellent saccharifying activity and has excellent fibrinolytic activity, And can be usefully used as a feed additive.

Brief Description of the Drawings Fig. 1 is a view showing a BRC15-1 strain in which a clear zone is formed in a blue plate of a trypan blue:
a. Colonies of BRC15-1; And
b. Enzyme active colony of BRC15-1.
FIG. 2 is a graph showing the results of Gram staining of strain BRC15-1. FIG.
3 is a diagram showing a phylogenetic tree of BRC15-1 by 16S rRNA sequencing.
Fig. 4 is a graph showing the distribution of P. jamilae BRC15-1 and Paenibacillus sp. 2 is a graph comparing the cellulase activities of the two strains.
5 is a graph showing the activity of cellulase according to the culture temperature of P. jamilae BRC15-1 strain.
6 is a graph showing cellulase activity according to pH of the initial medium of P. jamilae BRC15-1 strain.
7 is a graph showing cellulase activity according to the carbon source of P. jamilae BRC15-1 strain.
8 is a graph showing cellulase activity of the P. jamilae BRC15-1 strain according to the nitrogen source.
FIG. 9 is a graph showing the growth curve and cellulase activity (CMCase activity) of P. jamilae BRC15-1 according to the culture time.
10 is a graph showing cellulase activity of P. jamilae BRC15-1 according to pH of the enzyme reaction.
11 is a graph showing cellulase activity of P. jamilae BRC15-1 according to the enzyme reaction temperature.
12 is a graph showing the results of performing enzymatic saccharification using an enzyme produced by a strain of P. jamilae BRC15-1 against a single-stranded sugar gas pretreated with an alkali.

Hereinafter, the present invention will be described in more detail in the following Examples. It should be noted, however, that the following examples are illustrative only and do not limit or limit the scope of the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

< Example  1> Strain search and selection

&Lt; 1-1 > Isolation of fibrinolytic enzyme producing strains

The present invention was carried out in order to search for a saccharification strain having excellent fibrin degradation ability from soil and plant samples collected from reclaimed land such as Incheon, Gyeonggi-do, Seosan, Jeonbuk, Gunsan, Buan, Jeollanam-do and Haenam.

Specifically, 1 g of each sample was mixed with 9 mL of sterilized water and stirred. Then, a cellulose-decomposing activity strain was separated by a plate dilution culture method. The medium used was CMC (0.008% tryphan blue) supplemented with trypan blue (1% carboxymethyl cellulose, 0.5% bacto-peptone, 0.25% yeast extract, 1.5% agar) and the incubation temperature and incubation time were 37 ° C and 24 hr. After cultivation, 39 strains showing clear enzyme activity such as BRC15-1 were firstly selected (Fig. 1).

<1-2> Fibrinolytic activity Outgrowth  Selection

The present inventors measured the activity of the enzyme using the DNS method to select strains having excellent cellulolytic activity among the primary selection strains forming a clear zone.

Specifically, each of the active strains selected in Example 1-1 was inoculated into 100 mL of CMC liquid medium (1% carboxymethyl cellulose, 0.5% bacto-peptone, 0.25% yeast extract) , For 24 hours. The culture broth was centrifuged at 15,000 rpm at 4 ° C for 10 min, and the supernatant was used as a coenzyme to confirm the enzyme activity. Enzyme activity was determined by modifying an experiment such as TK GHOSE ("Measurement of carboxymethyl cellulase activity", Anal. Biochem . 2: 127-132 (1960)). 0.5 mL of supernatant (coenzyme) centrifuged in the above was added to 0.5 mL of 2% CMC, and enzyme and substrate were reacted at 50 ° C. for 30 min. Immediately after the reaction, 3 mL of dinitrosalicylic acid (DNS) was added and reacted at 100 ° C for 5 min. After the reaction was completed, it was immediately cooled in ice water, and then 20 mL of distilled water was added and the absorbance was measured at 540 nm. For the determination of reducing sugar, 0.1-2.0 mg / mL of glucose was prepared as a standard sugar and the absorbance was measured at 540 nm to prepare a quantitative curve. The enzyme activity was expressed as IU (International Unit), which was defined as the amount of enzyme that produced 1 μmol of glucose for 1 minute.

As a result, cellulase degrading activity of cellulose was found to be high as 0.356 IU / mL of BRC 15-1 enzyme, and this strain was selected as an excellent strain for the degradation of cellulose, and this strain was identified.

< Example  2> Morphological, biochemical and 16S rRNA sequencing of selected strains

The present inventors examined the morphological and biochemical characteristics of BRC15-1 strains with excellent fibrinolytic activity and analyzed 16S rRNA nucleotide sequences.

Specifically, the morphological characteristics of the BRC15-1 strain having excellent fibrinolytic activity selected in Example 1-2 were inoculated on a PCA (plate count agar) medium and observed. Then, Gram staining was carried out and the cells were inoculated with an API kit (BioMeriex, France) and cultured at 37 ° C for 24 hours. Biochemical properties were confirmed (Table 2, Fig. 2). Then, gene sequence analysis and phylogenetic tree of this strain were submitted to Daejeon, Korea (Fig. 3).

Strain shape surface Texture Height edge Opacity Color Gram stain BRC15-1 circle
(circular) lubricity
(smooth) With viscosity
(moist) Bulged
(raised) Wavy
(undulate) opacity
(opaque) White
(white) G (+)

Through 16S rRNA sequencing, BRC15-1 strain was identified as Paenibacillus The strain which showed 99% homology with jamilae was named Paenibacillus jamilae ) BRC15-1, and optimization of enzyme production and enzyme activity of this strain was performed.

< Example  3> Paenibacillus jamilae BRC15 Cellulase activity of strain-1

The present inventors tried to verify the superiority of the strain BRC15-1 of Paenibacillus jamilae selected as an excellent strain for the degradation of cellulose.

Specifically, in Example 2, Paenibacillus jamilae ) Paenibacillus polymyxa (KACC10098) and Paenibacillus jamilae (KCTC13919), two strains of which were most homologous with BRC15-1, were deposited with KACC (National Institute of Agricultural Science and Technology) And the microbial resource center (KCTC), respectively. Paenibacillus sp. Strains were inoculated into 100 mL of CMC medium and cultured at 37 ° C and 150 rpm for 24 hours to confirm the enzyme activity. 2 mL of each culture was centrifuged at 4 ° C and 15,000 rpm for 10 min, and the supernatant was used as the coenzyme. The enzymatic activity of these cellulases was determined by the DNS method described in Example 1-2. 0.5 mL of crude enzyme solution was added to 0.5 mL of 2% CMC, and enzyme and substrate were reacted at 40 ° C for 30 min. Immediately after the reaction, 3 mL of dinitrosalicylic acid (DNS) was added and reacted at 100 ° C for 5 min. After the reaction was completed, it was immediately cooled in ice water, and then 20 mL of distilled water was added and the absorbance was measured at 540 nm.

As a result, Paenibacillus jamilae BRC15-1 strain showed the highest activity (0.357 IU / mL) as compared with the comparative group (FIG. 4). P.polymyxa (KACC10098) and P. jamilae (KCTC13919), respectively, 0.330 IU / mL, and 0.275 IU / mL activity, respectively.

< Example  4> Optimization of culture conditions of selected strain

<4-1> Optimization of culture temperature

The present inventors optimized the culture temperature condition of the strain BRC15-1 of Paenibacillus jamilae selected as an excellent strain for decomposing cellulose.

Specifically, Paenibacillus < RTI ID = 0.0 > jamilae ) strain BRC15-1 was inoculated into 100 mL of CMC medium and cultured at 25 ° C to 50 ° C at 5 ° C intervals. After culturing, the culture was incubated at 4 ° C and 15,000 rpm for 10 min, and the supernatant was used as the coenzyme. The enzymatic activity of these cellulases was determined by the DNS method described in Example 1-2.

0.5 mL of crude enzyme solution was added to 0.5 mL of 2% CMC, and the enzyme and substrate were reacted at 50 ° C. for 30 min. Immediately after the reaction, 3 mL of dinitrosalicylic acid (DNS) was added and reacted at 100 ° C for 5 min. After the reaction was completed, it was immediately cooled in ice water, and then 20 mL of distilled water was added and the absorbance was measured at 540 nm.

As a result, Paenibacillus It was confirmed that culturing the strain of BRC15-1 at 40 DEG C for the jamilae strain is a condition in which cellulase activity is highest (FIG. 5).

<4-2> Optimization of initial medium pH

The present inventors optimized the initial pH conditions of the strain BRC15-1 of Paenibacillus jamilae , which was selected as an excellent strain for decomposing cellulose.

Specifically, Paenibacillus < RTI ID = 0.0 > jamilae ) BRC15-1 strain was inoculated in 100 mL liquid medium after adjusting the pH of CMC liquid medium with NaOH and HCl to pH 4 ~ 10, and cultured at 40 ° C and 150 rpm for 24 hr. 2 mL of each culture was centrifuged at 15,000 rpm at 4 ° C for 10 min, and only the supernatant was used as the coenzyme. The enzymatic activity of these cellulases was determined by the DNS method described in Example 1-2. 0.5 mL of crude enzyme solution was added to 0.5 mL of 2% CMC, and the enzyme and substrate were reacted at 50 ° C. for 30 min. Immediately after the reaction, 3 mL of dinitrosalicylic acid (DNS) was added and reacted at 100 ° C for 5 min. After the reaction was completed, it was immediately cooled in ice water, and then 20 mL of distilled water was added and the absorbance was measured at 540 nm.

As a result, Paenibacillus Jamilae strain BRC15-1 was found to have the highest cellulase activity when cultured at pH 7 (Fig. 6).

<4-3> Optimization of Substrate Substrate

The present inventors have optimized the substrate conditions for the cultivation of the strain BRC15-1 of Paenibacillus jamilae selected as an excellent strain for the degradation of cellulose.

Specifically, Paenibacillus < RTI ID = 0.0 > Jamilae BRC15-1 strain was inoculated in 100 mL of 1% CMC medium supplemented with 7 carbon sources and inoculated in 100 mL of CMC medium supplemented with 7 kinds of nitrogen sources at 40 ° C and 150 rpm for 24 hours Lt; / RTI &gt; The substrate sources used were glucose, xylose, mannose, arabinose, sucrose, starch, carboxymethyl cellulose (CMC), nitrogen-free skim milk skim milk, beef extract, ammonium sulfate, malt extract, peptone, urea, and ammonium nitrate were used. After culturing, 2 mL of each culture medium in each substrate medium was centrifuged at 15,000 rpm for 10 min at 4 ° C, and the supernatant was used as the coenzyme. The enzymatic activity of these cellulases was determined by the DNS method described in Example 1-2. 0.5 mL of crude enzyme solution was added to 0.5 mL of 2% CMC, and the enzyme and substrate were reacted at 50 ° C. for 30 min. Immediately after the reaction, 3 mL of dinitrosalicylic acid (DNS) was added and reacted at 100 ° C for 5 min. After the reaction was completed, it was immediately cooled in ice water, and then 20 mL of distilled water was added and the absorbance was measured at 540 nm.

As a result, Paenibacillus Jamilae ) BRC15-1 strain was found to have high cellulase activity under medium conditions of carboxymethyl cellulose (CMC) as a carbon source and peptone as a nitrogen source (FIGS. 7 and 8).

<4-4> Optimization of incubation time

The present inventors have optimized the culture time of the strain BRC15-1 of Paenibacillus jamilae selected as an excellent strain for decomposing cellulose.

Specifically, Paenibacillus < RTI ID = 0.0 > jamilae ) strain BRC15-1 was inoculated into 100 mL of CMC medium and cultured at 40 ° C and 150 rpm for 72 hours. In order to measure the growth curve of the strain, the absorbance was measured at intervals of 2 hr from 0 hr to 12 hr, 3 hr from 12 hr to 24 hr, 6 hr from 24 hr to 48 hr, and 12 hr from 48 hr to 72 hr 600 nm. Cellulase activity was measured at 6 hr interval from 0 hr to 30 hr in order to optimize cellulase activity according to incubation time, and enzyme activity of 48 hr and 72 hr was measured. The enzyme activity was determined by taking 2 mL of the culture solution, centrifuging at 15,000 rpm at 4 ° C for 10 min, and using the supernatant as the coenzyme. The enzymatic activity of these cellulases was determined by the DNS method described in Example 1-2. 0.5 mL of crude enzyme solution was added to 0.5 mL of 2% CMC, and the enzyme and substrate were reacted at 50 ° C. for 30 min. Immediately after the reaction, 3 mL of dinitrosalicylic acid (DNS) was added and reacted at 100 ° C for 5 min. After the reaction was completed, it was immediately cooled in ice water, and then 20 mL of distilled water was added and the absorbance was measured at 540 nm.

As a result, Paenibacillus Jamilae ) BRC15-1 strain showed logarithmic growth until 12 hr. Cellulase activity increased during the logarithmic growth period, but showed constant activity after 12 hr (FIG. 9). These results suggest that Tricoderma ressi [Wen Z et al., 2005. Bioresour Technol. 96 (4): 491-9.], Aspergillus niger [PB Acharya et al., 2008. Arfican Joumal of Biotechnology. 7 (22): 4147-4152] The incubation time for enzyme production of bacteria Compared with 4 to 8 days, the short incubation time of this bacterium suggests that it is advantageous for economic production of enzyme production.

< Example  5> Optimization of enzymatic reaction conditions of the selected strain

<5-1> Enzyme reaction pH optimization

The present inventors have optimized the enzyme reaction pH conditions of the strain Paenibacillus jamilae BRC15-1 selected as an excellent strain for the degradation of cellulose.

Specifically, Paenibacillus < RTI ID = 0.0 > jamilae ) strain BRC15-1 was inoculated into 100 mL of CMC medium and cultured at 40 ° C and 150 rpm for 24 hours. 10 mL of the culture was taken and centrifuged at 4 ° C and 15,000 rpm for 10 min. The supernatant was used as the coenzyme. The enzymatic activity of these cellulases was determined by the DNS method described in Example 1-2. 0.5 mL of crude enzyme solution was added to 0.5 mL of 2% CMC adjusted to pH 4 to 10, and enzyme and substrate were reacted at 50 ° C. for 30 min. The buffer solution used was 0.2 M sodium citrate buffer (pH 4, 5), 0.2 M sodium phosphate buffer (pH 6, 7), 0.2 M Tris-HCL buffer , 10). Immediately after the reaction, 3 mL of dinitrosalicylic acid (DNS) was added and reacted at 100 ° C for 5 min. After the reaction was completed, it was immediately cooled in ice water, and then 20 mL of distilled water was added and the absorbance was measured at 540 nm.

As a result, Paenibacillus Jamilae BRC15-1 showed the highest cellulase activity when the substrate pH was 6 when reacting the enzyme with the substrate in the DNS measurement, and maintained the enzyme activity over 55% even at pH 10 (FIG. 10).

<5-2> Optimization of enzyme reaction temperature

The present inventors optimized the enzyme reaction temperature condition of strain BRC15-1 of Paenibacillus jamilae selected as an excellent strain for decomposing fibrinolytic activity.

Specifically, Paenibacillus < RTI ID = 0.0 > jamilae ) strain BRC15-1 was inoculated into 100 mL of CMC medium and cultured at 40 ° C and 150 rpm for 24 hours. 2 mL of the culture was taken, centrifuged at 15,000 rpm at 4 ° C for 10 min, and the supernatant was used as the coenzyme. The enzymatic activity of these cellulases was determined by the DNS method described in Example 1-2. 0.5 mL of crude enzyme solution was added to 0.5 mL of 2% CMC, and the enzyme and substrate were reacted for 30 min at 30 ° C to 90 ° C at 10 ° C intervals. Immediately after the reaction, 3 mL of dinitrosalicylic acid (DNS) was added and reacted at 100 ° C for 5 min. After the reaction was completed, it was immediately cooled in ice water, and then 20 mL of distilled water was added and the absorbance was measured at 540 nm.

As a result, Paenibacillus Jamilae ) BRC15-1 showed the highest enzyme activity at 40 ℃ when the enzyme and substrate were reacted with DNS at 78 ℃ and 78% at 30 ℃. At 80 ° C and 90 ° C, the enzyme activity was 30% or more (Fig. 11).

< Example  6> Penny Bacillus Jamilé ( Paenibacillus jamilae ) BRC15 -1 strain Glycation ability  black

The present inventors examined the glycation ability of BRC15-1 strain of Paenibacillus jamilae selected as an excellent strain for the degradation of cellulose.

More specifically, the number of organs such as Orghum bicolor var. Dulciusculum Ohwi bagasse; Sediment of the fiber which separated the sucrose by squeezing the stem of a single water sample) was cultivated and harvested as a pilot project in Muan region in 2013, and the sugar was completely removed and pretreated with alkali (1M NaOH). The saccharification experiment was carried out by modifying the enzymatic saccharification experiment of ligno fiber biomass of NREL LAP (Laboratory Analytical Procedure) proposed by National Renewable Energy Laboratory (NREL) (Seling, M. et al., Enzymatic saccharification of lignocellulosic biomass in The Laboratory Anltical Procedure (LAP). Technical report NREL (National Renewable Energy Laboratory) / TP-510-42629 (2008)). Enzymes were prepared by centrifuging Paenibacillus jamilae BRC15-1 (initial pH of medium 7, culture temperature 40 ° C, 150 rpm, 1 day) at optimal conditions and centrifuging the supernatant only in an ultrafiltration device Sartorius Slice 200, 10 kD MWCO). Enzymatic saccharification was carried out at 50 ° C and 200 rpm for 3 days by adding 10 ml of concentrated enzyme, and a single water bath pretreated with 1.0 M NaOH to contain 0.1 g of glucan. HPLC analysis was performed using a Waters 2414 pump, an Aminex HPX-87H column (column temperature 65 ° C), a Waters RI detector (detector, Temperature: 40 ° C), the mobile phase was 0.5 mM H ₂ SO ₄ , and the flow rate was 0.5 mL / min.

As a result of the analysis, it was confirmed that the singly water-absorbent fiber biomass pretreated with alkali was decomposed into glucose (2.75 ± 0.03 mg), xylose (0.79 ± 0.08 mg) and arabinose (1.12 ± 0.05 mg) (Fig. 12).

Agricultural Biotechnology Research Institute KACC92074P 20150514

Claims (12)

Paenibacillus jamilae BRC15-1 strain (accession number: KACC92074P), which is capable of producing cellulase, isolated from soil and plant in reclaimed land.
The method of claim 1, wherein the cellulase is the endo -β-1,4- glucanase penny Bacillus chair millet (Paenibacillus jamilae) BRC15-1 strain, characterized in that (endo-β-1,4-glucanase ) is.
The term &quot; Paenibacillus &quot; Jamilae ) BRC15-1 strain.
The term &quot; Paenibacillus &quot; A composition for feed addition comprising a cellulase obtained from a jamilae BRC15-1 strain.
1) Inoculation of strain Paenibacillus jamilae BRC15-1 (accession number: KACC92074P) isolated from the soil and plant of the reclaimed land on a culture medium containing carboxymethyl cellulose (CMC) and peptone, &Lt; / RTI &gt; and a pH of 6 to 8 for 10 to 36 hours; And
2) recovering the cellulase from the culture broth of the strain.
delete delete delete delete delete 1) Inoculation of strain Paenibacillus jamilae BRC15-1 (accession number: KACC92074P) isolated from the soil and plant of the reclaimed land on a culture medium containing carboxymethyl cellulose (CMC) and peptone, &Lt; / RTI &gt; and a pH of 6 to 8 for 10 to 36 hours;
2) centrifuging the culture of step 1) to obtain a supernatant;
3) concentrating the supernatant of step 2) to recover the cellulase; And
4) treating the concentrated cellulase of step 3) with cellulose pretreated with alkali to decompose the cellulose.
The saccharifying method according to claim 11, wherein the cellulase treatment of step 4) is carried out at pH 5-7 and 30-50 ° C.

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