KR101844951B1 - Novel gluconacetobacter strain producing cellulose with high yield - Google Patents

Abstract

The present invention relates to novel strains belonging to the genus Gluconacetobacter having a cellulose production activity. More specifically, the present invention relates to a novel strains belonging to the genus Gluconacetobacter, capable of producing cellulose having a nanostructure at a very high efficiency. The strain of the present invention is excellent in cellulose production activity and the cellulose produced by the strain of the present invention is a nanostructured bacterial cellulose having excellent thermal characteristics and can be effectively used as a bio-nanofiber. Particularly, , And the usual cellulose based resin can be effectively used for a substrate of a liquid crystal display device.

Description

[0001] The present invention relates to a novel glucone acetobacterium strain having high yield of cellulose production activity,

The present invention relates to novel strains belonging to the genus Gluconacetobacter, capable of producing cellulose. More specifically, the present invention relates to novel strains belonging to the genus Gluconacetobacter, which can produce cellulose having a nanostructure at a very high yield.

Cellulose is one of the most abundant natural polymers on the planet. It is estimated that approximately 10 ¹¹ tonnes of cellulose are biosynthesized. Most of them synthesize cellulosic fibrous components of higher plants. The toughness of cellulose is due to the entire structure, whose molecules are chains or microfibers of up to 14,000 units, forming a twisted rope-like bundle, which is retained by hydrogen bonding. Due to the abundant amount of cellulose and different physical properties from other natural polymers, many researches have been conducted for various applications as recycled resources.

Meanwhile, since the fact that acetic acid bacteria produce cellulose by AJ Brown in 1886 has been reported, bacterial cellulose (hereinafter referred to as BC) produced by microorganisms has been continuously studied as a new material . Particularly, cellulose (BC) derived from microorganisms is composed of ribbon-like bundles, while cellulose derived from plants is composed of bundles of microfibrils.

Cellulose (BC) derived from microorganisms composed of the ribbine-like bundles is free from lignin or hemicellulose, unlike cellulose derived from plants, which is composed of bundles of microfibrils And is being developed for various applications in various industrial fields due to the unique advantages of only high bacterial cellulose (BC) such as high mechanical strength, high moisture resistance, high crystallinity and biodegradability.

In particular, in order to be used as a substrate material of a conventional liquid crystal display device (LCD), the coefficient of thermal expansion is low, and the transmittance is high and must have a small thickness. At present, expensive s-glass is used. However, it has been reported that the nanostructured cellulose improves thermal properties over s-glass, and the nanostructured cellulose is known to be produced by microorganisms (Schraamm, M. and S. Hestrin, 1054 Factors affecting production of cellulose in the air / liquid interface of a culture of Acetobacter xylinum.J.Gen. Microbiol. 11: 123-129). Therefore, when the microorganism having the cellulosic production activity is developed, the economic effect is very high.

However, acetobacter xylinum is known as a typical microorganism for producing bacterial cellulosic (BC), but it is highly required to develop a new microorganism that can replace the acetobacter xylinum because of low efficiency of cellulosic production.

Accordingly, the inventors of the present invention have developed a novel strain belonging to the genus Glacial acetoobacter in the study to develop a new microorganism having a very high efficiency of producing cellulose, and confirmed that the strain can produce cellulose effectively, thereby completing the present invention .

Accordingly, the present invention is a gluconacetobacter strain having cellulase production activity, wherein the strain is selected from the group consisting of gluconacetobacter sp.) strain D44 (accession number KCCM11078P) or Gluconacetobacter intermedius) and an object thereof is to provide a gluconate acetonitrile bakteo spp characterized in that the D47 strain (Accession No. KCCM11079P).

It is another object of the present invention to provide a culture of the strain of the genus Gluconacetabacter.

The present invention also provides a composition for producing cellulose comprising at least one member selected from the group consisting of the strain of the genus Gluconacetobacter, the culture of the strain, the concentrate of the strain or the culture, and the dried product thereof do.

Also, the present invention provides a method for producing a recombinant vector comprising the steps of: And

Obtaining cellulose from the culture

The present invention also provides a method for producing cellulose.

Also, the present invention provides a method for producing a cellulosic material, comprising the steps of: preparing cellulose produced by the strain of the genus Gluconacetobacter;

And impregnating the cellulose with a resin

And a method for producing the cellulose-based resin.

In order to solve the above problems, the present invention provides a gluconacetobacter strain having cellulase production activity, wherein the strain is selected from the group consisting of gluconacetobacter sp.) strain D44 (accession number KCCM11078P) or Gluconacetobacter intermedius D47 strain (Accession No. KCCM11079P).

The present invention also provides a culture of the strain of the above-mentioned Gluconacetobacter.

The present invention also provides a composition for producing cellulose comprising at least one selected from the group consisting of the strain of the genus Gluconacetobacter, the culture of the strain, the concentrate of the strain or the culture, and the dried product thereof.

Also, the present invention provides a method for producing a recombinant vector comprising the steps of: And

Obtaining cellulose from the culture

Wherein the cellulose is a cellulose acetate.

Also, the present invention provides a method for producing a cellulosic material, comprising the steps of: preparing cellulose produced by the strain of the genus Gluconacetobacter; And

Impregnating (impregnating) the cellulose with the resin

Based on the total weight of the cellulose-based resin.

Hereinafter, the present invention will be described in more detail.

The gluconacetobacter subspecies of the present invention, specifically gluconacetobacter sp. D44 strain (Accession No. KCCM11078P) or Gluconacetobacter intermedius intermedius D47 strain (Accession No. KCCM11079P) is a novel strain having excellent ability to produce cellulose.

The strain of the present invention is a kind of acetobacteraceae and may be isolated from vinegar, but not limited thereto, and preferably can be isolated from persimmon vinegar, more preferably persimmon vinegar.

In one embodiment of the present invention, it is possible to isolate the strain of the present invention having a high yield of cellulose production activity from the persimmon vine membrane, and to have the cellulase production activity can be confirmed according to a known method. For example, whether the strain of the present invention is inoculated onto HS medium (see Table 1 below) to form a tabular membrane on the medium or whether it causes color change by inoculation on a Carr medium (see Table 1 below) , But is not limited thereto (see Example 1).

In order to identify the strain of the present invention, the morphological and biochemical characteristics of the strain of the present invention were analyzed. As a result, the strain of the present invention has the morphological characteristics shown in the following Table 2, and is shown in Table 3 (D44) and Table 4 It is a strain of the genus Gluconacetobacter having the described biochemical characteristics (see Example 2).

In addition, whether the strain of the present invention is a strain belonging to the genus Gluconacetobacter can be determined by obtaining a 16s rDNA sequence (D44) represented by the nucleotide sequence of SEQ ID NO: 1 and a 16s rDNA sequence (D47) represented by the nucleotide sequence of SEQ ID NO: 2 As a result, the D44 strain was most similar to the 16s rDNA sequence of gluconacetobacter rhaeticus , but the similarity was only 87.215%. The strain of the present invention was found to be a novel strain belonging to the genus Gluconacetobacter, and D47 The strain is Gluconacetobacter intermedicus , and the similarity was found to be 99.776%, indicating that the strain of the present invention is a strain belonging to the genus Gluconacetobacter (see Example 2)

The present inventors isolated the strains of the present invention isolated and identified as described above, respectively, from Gluconacetobacter sp.) strain D44 and Gluconacetobacter < RTI ID = 0.0 > intermedius) called D47, and was deposited with the above strain on November 12, 2009, the Korea Culture Center of Microorganisms (each KFCC11470P deposited in the KCCM) depository number (D44) and KFCC11471P accession number (D47), since March 2010, (D44) and KCCM11079P deposit number (D47), respectively, to the depository of the Korean Microorganism Conservation Center (KCCM) on the 26th.

In addition, the culture of the strain of the genus Gluconacetobacter of the present invention can be produced using a culture medium and a known culture medium used for culturing the microorganism of the strain of the present invention.

In order to cultivate the strain of the genus Gluconacetobacter of the present invention, a medium used for culturing the microorganism, preferably a medium used for culture of acetic acid bacteria, more preferably a medium described in the following Table 1 or Table 7, More specifically, if the medium for cellulosic production, specifically the HS medium described in Table 1 below, or the coconut mixed medium (2) described in Table 7 below is used, it is preferable to use cellulose for the production of cellulose, Can be produced at a higher yield (see Example 4).

The D44 strain is preferably glucose 20g / L, yeast extract 5g / L, peptone _{5g / L, Na 2 HPO 4} 2.7g / L and 1.15g of sodium citrate / L HS medium or coconut juice 100% (w containing / v), and the yeast of the yeast extract (CAS No.: 8013-01-02) (registered name of European existing chemical: 232-387-9) contains less than 40% protein And has a specific odor physico-chemical characteristic, which is a yellowish white platelet-like powder with no fermentability of not more than 0.12 mg of diamine hydrochloride, not more than 0.04 mg of riboflamine and not more than 0.25 mg of nicotinic acid per g.

Preferably, the D47 strain comprises 85 to 94.9 parts by weight of the coconut stock solution, 5 to 15 parts by weight of sugar, 0.1 to 20 parts by weight of oily solution, To 1 part by weight of a coconut mixed medium.

The yuan is an abbreviation of ammonium sulfate and refers to a white crystal prepared by reacting sulfuric acid with ammonia, preferably a purity of 10 to 99.9%, more preferably a purity of 10 to 30%, and most preferably a purity of 15 to 25% Ammonium sulfate may be used.

The culture of the strain of the present invention can be produced by inoculating the strain of the present invention into the medium and culturing the strain according to a microorganism culture method (for example, stationary culture or agitation culture) known in the art, , More preferably 1 wt% (based on the total weight of the culture medium), and culturing at 28 to 30 DEG C for 4 to 7 days. More preferably, the strain of the present invention can be prepared by inoculating the culture medium with 1% by weight (based on the total weight of the culture medium) and culturing at 30 캜.

The culture of the present invention may include, but is not limited to, cellulose produced by the strain of the present invention.

Meanwhile, the composition for producing cellulose of the present invention comprises at least one member selected from the group consisting of the strain of the present invention, the culture of the strain, the concentrate of the strain or the culture, and the dried product thereof.

Since the strain of the present invention can produce cellulose very efficiently, the strain, the culture of the strain, the concentrate of the strain or the culture, and the dried product thereof can be usefully used as a composition for producing cellulose.

The method for producing the culture of the above strain is as described above, and preferably, the strain of the present invention is used for the cellulose production medium, the HS medium described in Table 1 below, or the coconut mixed medium 2 , And the culture can be produced by, but not limited to, stationary culture or agitation culture, preferably stationary culture. The concentrate of the strain or culture and the dried product thereof can be easily produced by a method of concentrating or drying a microorganism or a culture known in the art.

The strain of the genus Gluconacetobacter of the present invention, the culture of the strain, the strain or the concentrate of the strain or the culture and the dried product thereof are not limited thereto, but they are preferably contained in the composition for producing cellulose of the present invention in an amount of 1% .

Meanwhile, the method for producing cellulose according to the present invention comprises the steps of: culturing the strain of the genus Glacial Acetobacter; And obtaining cellulose from the culture.

The method for culturing the strain is the same as described above. In order to produce the cellulose of the present invention, the strain of the present invention is not limited to this method. However, the cellulose formed by the method of the present invention is immersed in a sodium hydroxide solution and washed with distilled water Pure cellulose can be produced. The cellulose thus produced may be in the form of a membrane, although not limited thereto (see Example 3).

The cellulose produced by the strain of the present invention is smooth, has excellent rigidity and elasticity, has a thickness of about 0.3 mm to about 12 mm, and has a remarkably improved thickness compared to conventional bacterial cellulose, (See Examples 3 and 4). &Lt; tb > < TABLE >

Furthermore, the strain of the present invention is excellent in cellulase production activity, and is remarkably superior to acetobacter xylinum , which is a typical microorganism producing bacterial cellulose (BC), by about three times as much as about 97.5 times as much as acetobacter xylinum It is possible to produce cellulose with high efficiency (see Example 3 and Example 4).

Therefore, the cellulose produced by the strain of the present invention is a nanostructured bacterial cellulose having excellent thermal characteristics, and thus can be usefully used as a bio-nanofiber.

The method for producing a cellulose-based resin according to the present invention comprises the steps of: preparing cellulose produced by the glucone acetobacterium strain of the present invention; And impregnating the cellulose with the resin.

The cellulose-based resin may be produced according to a production method known in the art, as long as it uses cellulose produced by the strain of the glucone acetobacterium of the present invention. For example, the cellulose produced by the glucone acetobacterium strain of the present invention can be produced by mixing a thermoplastic resin and a binder and extruding the mixture.

The thermoplastic resin is preferably selected from the group consisting of polypropylene, polyethylene, polystyrene and polyvinyl chloride, and the binder is preferably at least one selected from the group consisting of maleated ethylene, maleated propylene and silane. The mixture composed of cellulose, a thermoplastic resin and a binder may further be mixed with at least one additive selected from stabilizers, pigments, dyes, inorganic or organic additives, electromagnetic wave shielding agents or mixtures thereof.

The cellulose resin thus prepared contains a nanostructured bacterial cellulose having excellent thermal characteristics and can be used for a substrate of a liquid crystal display device.

Accordingly, the present invention provides a method for producing a cellulose resin, comprising the steps of: preparing a cellulose resin; And producing a substrate of a liquid crystal display device using the cellulose-based resin.

The present invention also provides a liquid crystal display device substrate manufactured using the cellulose resin.

Celluloses in cellulosic produce are known to defend cells from the lethal effects of UV light, to form colonies of bacteria, to provide protection from competitors using the same substrate as a nutrient source and to provide moisture to cells to prevent drying There is a bar. In addition, since the cellulose produced by the microorganism according to the present invention has high crystallinity and hygroscopicity of the fiber, it is highly likely to have industrial applications such as a speaker diaphragm and the like. The cellulose membrane has good skin feel to the skin in the gel state and is easily fused to the body surface, The maintenance ability of the material component is high and the surface of the skin can be maintained in a humidified state, so that it can be applied to medical pads, cosmetic pads (e.g., mask packs), artificial skins, and the like. Currently, mask packs using cellulose are being commercialized. Furthermore, it has been found that the cellulose membrane is treated with 5% NaOH or 0.5% NaCl to remove materials other than the cellulose component, and then pressed to form a high elastic membrane. In the Japanese company Sony, the high- have.

Also, when paper is made using cellulose produced by microorganisms as in the present invention, the strength and elastic modulus of the paper are increased 2.5 times as compared with the control, and 5-20% of the synthetic polymer fiber, aromatic polyamide or polyamide short fiber is added When a sheet is formed, a sheet excellent in strength, thermal stability, and shape stability can be obtained as compared with synthetic fibers. If cellulose fibers are mixed with glass fibers as well as synthetic fibers to form a sheet, they can be applied to filters for air filtration, printed wiring boards and floor materials.

As described above, the cellulose derived from the microorganism according to the present invention can be widely used for industrial materials and food materials, and in particular, it is an eco-friendly material and has unlimited development potential and diversity (Jeong, YJ, Lee, IS view of utilizing cellulose produced by acetobacter bacteria . Food Industry and Nutrition ., Vol. 5, 25-29, 2000. Reference).

The strain of the present invention is excellent in cellulose production activity and the cellulose produced by the strain of the present invention is a nanostructured bacterial cellulose having excellent thermal characteristics and can be effectively used as a bio-nanofiber. Particularly, And the cellulose-based resin can be effectively used for a substrate of a liquid crystal display device.

Fig. 1 shows morphological observation results of the strain D44 of the present invention. Fig. 1 (a) shows the result of observation with an optical microscope, and Fig. 1 (b) shows a result of observation with a scanning electron microscope (SEM).
FIGS. 2A to 2C show the results of comparing the 16s rDNA sequence of the D44 strain of the present invention with the 16s rDNA sequence of Gluconacetobacter rhaeticus (*: the nucleotide sequence matches).
FIG. 3 shows the phylogenetic tree for the gulconacetobacter genus, the genus Gluconobacter, and the genus Acetobacter belonging to the acetic acid bacteria of the strain D44 of the present invention.
Fig. 4 shows the results of confirming whether or not cellulose of the strain D44 of the present invention was produced.
Fig. 5 shows morphological observation results of the strain D47 of the present invention. Fig. 5 (a) shows the result of observation with an optical microscope, and Fig. 5 (b) shows a result of observation with a scanning electron microscope (SEM).
FIGS. 6A to 6C show the results of comparing the 16s rDNA sequence of the D47 strain of the present invention with the 16s rDNA sequence of Gluconacetobacter intermedius (*: the nucleotide sequence matches).
Fig. 7 shows the phylogenetic numbers of gulconacetobacter, gluconobacter and acetoobacter belonging to the acetic acid bacteria of the D47 strain of the present invention.
Fig. 8 shows the results of confirming whether or not the D47 strain of the present invention produced cellulose.
FIGS. 9A and 9B show cellulose produced by culturing the D47 strain of the present invention in HS medium (FIG. 9A) and coconut mixed medium (FIG. 9B), respectively.

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

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Reference example  1>

The medium and culture conditions used in the experiment

In order to isolate and identify the novel strain of the present invention, the medium described in the following Table 1 was used. Unless otherwise specified in the following examples, the strain producing the cellulose of the present invention was cultured at 30 ° C for 7 days The experiment was carried out.

Types and composition of medium Types of badges Furtherance Related literature H.S badge 5 g / L of glucose 20 g / L yeast extract, 5 g / L of peptone, 2.7 g / L of Na ₂ HPO ₄ , 1.15 g / L of sodium citrate, Schraham, M. and S. Hestrin. 1054. Factors affecting production of cellulose in the air / liquid interface of a culture of Acetobacter xylinum.J.Gen. Microbiol. 11: 123-129. GYC badge 50 g / L of glucose, 10 g / L of yeast extract, 30 g / L of calcium carbonate, and 25 g / L of agar Jang, OY, Joo, KH, Lee, JH, Baik, CG,
Growth Characteristics and Production of Celulose of Microorganisms in Static Culture Vinegar.Journal J.Food Sci. Technol.Vol. 35, No. 6, pp. 1150 to 1154 (2003) Frateur medium L of ethanol, 10 g / L of yeast extract, 10 g / L of peptone, 10 g / L of calcium carbonate and 20 g / L of agar Krig, N.R., J.G. Holt. 1984. Bergey ' s Manual of Systematic Bacteriology Vol. 1. The William and Wilkins Co.U.S.A. Carr badge 30 g / L of yeast extract, 20 ml / L of ethanol, 0.022 g / L of bromocresol purple, 20 g / L of agar, pH 6.0 Krig, N.R., J.G. Holt. 1984. Bergey ' s Manual of Systematic Bacteriology Vol. 1. The William and Wilkins Co.U.S.A. YEG solid medium 10 g / L of yeast extract, 30 g / L of glycerol, 20 g / L of agar Jang. O.Y., Joo. K. H., Lee. J.H., Baik. C.G. 2003. Growth characteristics and production of cellulose in microorganisms in static culture vinegar. Korean J. Food SCI. tech. vol. 35, No. 6, pp. 1150 ~ 1154. SM badge 5 g / L of yeast extract, 50 g / L of glucose Jang. O.Y., Joo. K. H., Lee. J.H., Baik. C.G. 2003. Growth characteristics and production of cellulose in microorganisms in static culture vinegar. Korean J. Food SCI. tech. vol. 35, No. 6, pp. 1150 ~ 1154. YPC badge 5 g / L of yeast extract, 5 g / L of peptone, 5 g / L of calcium carbonate Jang. O.Y., Joo. K. H., Lee. J.H., Baik. C.G. 2003. Growth characteristics and production of cellulose in microorganisms in static culture vinegar. Korean J. Food SCI. tech. vol. 35, No. 6, pp. 1150 ~ 1154. GYP badge 30 g / L of glucose, 5 g / L of yeast extract, 2 g / L of peptone Jang. O.Y., Joo. K. H., Lee. J.H., Baik. C.G. 2003. Growth characteristics and production of cellulose in microorganisms in static culture vinegar. Korean J. Food SCI. tech. vol. 35, No. 6, pp. 1150 ~ 1154.

< Example  1>

Isolation of novel strains producing cellulose in high yield

< Example  1-1>

Glucone acetobromide  genus D44  Isolation of strain

The strains used in this experiment were isolated from the membranes of conventional persimmon vinegar (Yongsoo Lee, Bongdong - eup, Wonju - gun, Jeonbuk). Specifically, various parts of persimmon vinegar were directly sampled and classified into A, B, C, and D according to the collected parts. Among them, D is a sample taken from the vinegar vinegar.

The persimmon vinegar A, B, C and D stock solutions were inoculated in 100 ml of HS medium at 1% each and cultured at 30 ° C for 7 days. After culturing, a single strain was diluted to 10 ^-7 in a D sample which produces cellulose in the HS medium membrane and plated on a solid HS medium. Then, the colonies were taken at random and inoculated into a liquid HS medium again. The strain forming the membrane was isolated.

The strains isolated from the persimmon D portion were inoculated onto a Carr medium containing a bromocresol purple having a color change range of pH 5.2 to 6.8 and an acidity of yellow, basic to purple, and a color change from yellow to purple to purple to yellow It was once again confirmed that the isolated strain had an oxidation pathway for oxidizing ethanol to acetic acid and reoxidizing it with carbon dioxide and water. As a result, it was found that the strain had the characteristics of a cellulosic producing strain. (Jang, O., Joo, KH, Lee, JH, Baik, CG, 2003. Growth characteristics and production of cellulose in microorganisms in static culture vinegar J. Food Sci. ~ 1154)

The present inventors named the strain having the cellulase-producing activity as 'D44'.

< Example  1-2>

Glucone acetobromide Intermedius ( Gluconacetobacter intermedius ) D47  Isolation of strain

The strains used in this experiment were isolated from the membranes of conventional persimmon vinegar (Dongsan - ri, Gosan - myeon, Wonju - gun, Jeonbuk). Specifically, the film from the preparation of the conventional persimmon vinegar was obtained from the NACF and thoroughly cut with sterilized scissors for dissection, and then a small amount was inoculated into the HS medium and cultured at 30 ° C for 7 days. After the incubation, HS to the medium pyomak cellulose is a single strain 10 in the production of the sample ^- the smeared on solid HS culture medium was diluted to ^7, and then randomly taking the colony again inoculated into the liquid HS medium and Table film on of which the medium The strains were separated.

The strains isolated from the persimmon vinegar portion were inoculated into a Carr medium containing a pH range of 5.2 to 6.8 and an acidic to yellow, basic to purple, bromocresol purple, and yellow to purple to purple to yellow Confirming that the isolated strain had cellulase-producing activity.

The present inventors named the strain having the cellulase-producing activity as 'D47'.

< Example  2>

Identification and Deposit of New Strains Producing Highly Cellulose

<2-1> Morphological characteristics

The size, shape, Gram stain and colony shape and color of each of D44 and D47 isolated in Example 1 were observed. Specifically, colonies of D44 and D47 were observed in GYC medium and D44 The strains produced by the strain and D47 strain were observed under an optical microscope and a scanning electron microscope (SEM) in a state in which the bacteria were not removed.

More specifically, the shape and Gram staining of strain D44 and strain D47 were examined using a scanning electron microscope (SEM) using the optical microscope (Nikon ECLIPSE E600, Japan), and the sizes of D44 and D47 bacteria and the cellulosic fibers produced by the bacteria Respectively. In particular, the electron microscope was modified using Chae's method (Chae, CM 1977. Maceration methods for SEM obserbation 'A legal medicine lecture room', Kyungpook National University, p 40-58) (v / v) glutaraldehyde for 2 hours, fixed with 2% (w / v) OsO ₄ for 2 hours, dehydrated sequentially with ethanol, treated with isoamyl acetate Dried at 55 ° C and gold coated. The above observation results are shown in Table 2 and Figs. 1 and 5.

Morphological characteristics of strain D44 and strain D47 Morphological feature D44 D47 Gram stain voice voice Cell morphology Bacillus Bacillus Cell size (um) 0.5 × 1.8 μm 0.6 to 0.8 × 1.6 to 2.0 μm Spore formation voice voice Colony shape Convex Convex Colony color White Light yellow (pale yellow) Colony surface Smooth and rough Smooth and rough Whether colony is transparent opacity opacity

As shown in Table 2 and FIG. 1 and FIG. 5, the D44 and D47 strains are gram-negative and exist in single or pairs without spores as bacilli. In particular, as in Figures 1 and 5, the strains are internal to the cellulosic fibers produced by strain D44 and D47.

<2-2> Biochemical characteristics

To examine the biochemical characteristics of the D44 and D47 strains, D44 and D47 strains were cultured in H.S liquid medium, respectively, and the collected strains were subcultured in a fresh medium.

Specifically, the novel D44 strain and the D47 strain of the present invention were inoculated into the Frateur medium, respectively, and the ethanol oxidation pathway of the D44 strain and the D47 strain was confirmed by confirming whether the CaCO ₃ in the medium was neutralized.

In order to determine whether ketone formation occurs in glycerol, each of the above strains was streaked on a YEG solid medium and cultured at 30 ° C. for 5 days. Then, a few drops of Fehlings solution were dropped on the colony, Respectively.

To determine the growth and cellulogenesis of ethanol and sodium chloride, medium supplemented with sodium chloride 0.5, 1.0, 1.5, 2.0% (w / v) and ethanol 10% (v / v) D44 strain and D47 strain were inoculated, respectively, and cultured for 7 days at 30 ° C.

In addition, D44 and D47 were streaked on GYC solid medium and cultured for 7 days at 30 ° C to confirm the formation of brown pigment. To confirm the presence of γ-pyrone formation, glucose 50 g / L, fructose The bacteria were plated on a YPC medium containing 50 g / L each and cultured for 7 days at 30 ° C, followed by dropping 5% (w / v) solution of ferric chloride. When it turned purple, it was determined that the strain could produce γ-pyrone using glucose and fructose (Jang, Y., Joo, KH, Lee, JH, Baik, CG 2003. Growth characteristics and production of cellulose Korean J. Food Sci., vol. 35, No. 6, pp. 1150-1154).

In order to examine the resistance of D44 and D47 to acetic acid, D44 and D47 were inoculated in GYP medium with acetic acid concentration adjusted from 2% (v / v) to 10% (v / v) And cultured for 7 days at 30 ° C.

Biochemical experiments were performed using the API 20NE kit (Bio-Merieux, France), and the results were identified through Bergy's manual of systematic bacteriology (Krig, NR, JG Holt 1984. Bergey's Manual of Systematic Bacteriology Vol. The William and Wilkins Co.USA).

The results of the experiment are shown in Tables 3 and 4, and the results are compared with that of acetobacter spp., Gluconobacter spp., And Gluconacetobacter spp. The Gluconacetobacter spp. OS, Jang, SY, Jeong, YJ 2002. Effect of Ethanol on the Production of Cellulose and Acetic Acid by Gluconacetobacter persimmonensis KJ145 J. Korean Soc. Food Sci. Nutr. 31 (4) .572-577 ). &Lt; / RTI &gt;

Biochemical characteristics of strain D44 Characteristic D44 Acetobacter
xylinum KCCM40274 Gluconobacter
oxidance KCCM21183 Gluconacetobacter genus Overoxidation of ethanol + ¹⁾ + - ²⁾ + Ketogenesis from glycerol + + + + Oxidation of acetate to CO ₂ and H ₂ O + + - + Oxidation of lactate to CO ₂ and H ₂ O - + - + Oxidation of Fructose to CO ₂ and H ₂ O + ND Brown pigmentation on GYC agar - + - - Cellulose formation + + - + Oxidase - - - - Indole production - - - - Urease - - - - γ-pyrone from 5% glucose - - + - γ-pyrone from 5% fructose - - + - ß-galactocidase + + - + Nitrate to nitritc + + + - Gelatin lizuefaction - - - - Acid produced from Arbionose - - - + Manose - ± + + Manitol + + + + Glucose + + + + Growth in the presence of 10% ethanol - - - - 0.5% NaCl + ND ³⁾ 1.0% NaCl + ND ³⁾ 1.5% NaCl + ND ³⁾ 2.0% NaCl + ND ³⁾ 2-10% acetate - ND ³⁾

+ ¹⁾ : positive, - ²⁾ : negative, ND ³⁾ : undetermined

As described in Table 3, it is possible to observe that is a novel D44 strain After a culture was inoculated in Frateur medium of the present invention, the medium in the CaCO ₃ neutralized formed transparent ring, CaCO ₃ the material at the 12 day culture It can be confirmed that it is settled. As a result, it is known that D44 strain oxidizes ethanol to acetic acid and acetic acid to CO ₂ and H ₂ O (Son. HJ, Kim, KK, Kim, HS 1999. Isolation and identification of cellulose-producing bacteria J. Agri. Tech & Dev. Inst.).

In addition, the D44 strain of the present invention can not produce γ-pyrone using 5% (w / v) glucose and fructose, and is positive in the ketone formation reaction from glycerol. In addition, the D44 strain is β-galactocidase-positive, reducing nitrate to nitrite and oxidizing acetate, fructose, glucose and mannitol but not lactate.

The strain D44 of the present invention is negative in the production of indole, uricase and gelatin, and is capable of growth in 0.5, 1.0, 1.5, 2.0% (w / v) sodium chloride, and particularly in 0.5% (w / v) Can also be generated. However, at a concentration of 2% (v / v) to 10% (v / v) acetate, D44 was not viable and was resistant to tolerance, producing no brown pigment and forming a clear white colony.

Based on the above results, it can be seen that the strain D44 of the present invention is a strain belonging to the genus Gluconacetobacter .

Biochemical characteristics of strain D47 Characteristic D47 Acetobacter
xylinum KCCM40274 Gluconobacter
oxidans KCCM21183 Gluconacetobacter genus Overoxidation of ethanol + ¹⁾ + - ²⁾ + Ketogenesis from glycerol - + + + Oxidation of acetate to CO ₂ and H ₂ O + + - + Oxidation of lactate to CO ₂ and H ₂ O - + - + Oxidation of Fructose to CO ₂ and H ₂ O + ND Brown pigmentation on GYC agar ± + - - Cellulose formation + + - + Oxidase - - - - Indole production - - - - Urease - - - - γ-pyrone from 5% glucose - - + - γ-pyrone from 5% fructose - - + - ß-galactocidase - + - + Nitrate to nitritc - + + - Gelatin lizuefaction - - - - Acid produced from Arbionose - - - + Manose - ± + + Manitol + + + + Glucose + + + + Growth in the presence of 10% ethanol - - - - 0.5% NaCl + ND ³⁾ 1.0% NaCl + ND ³⁾ 1.5% NaCl + ND ³⁾ 2.0% NaCl + ND ³⁾ 2-10% acetate - ND ³⁾

+ ¹⁾ : positive, - ²⁾ : negative, ND ³⁾ : undetermined

As shown in Table 4, when the D47 strain of the present invention was inoculated into the Frateur medium, it was observed that the CaCO ₃ was neutralized in the medium to form a transparent ring, and CaCO ₃ was reprecipitated after 8 days of culture Can be confirmed. Finally, the strain D47 is found to oxidize ethanol to acetic acid and acetic acid to CO ₂ and H ₂ O (Son. HJ, Kim, KK, Kim, HS 1999. Isolation and identification of cellulose-producing bacteria J. Agri. Tech & Dev. Inst.).

In addition, the D47 strain of the present invention can not produce γ-pyrone by using 5% glucose (w / v) and fructose. Unlike the D44 strain, D47 is negative in the ketone formation reaction from glycerol. The D47 strain was β-galactocidase negative, unlike the D44 strain, and could not be reduced to nitrite from nitrate. However, like the D44 strain, acetate, fructose, glucose and mannitol can be oxidized, but lactate can not be oxidized.

In addition, the D47 strain of the present invention is negative in the production of indole, urease and gelatin, and can grow on 0.5, 1.0, 1.5, 2.0% (w / v) sodium chloride, and in particular can also produce cellulose in 0.5% sodium chloride . However, at the concentration of 2% (v / v) to 10% (v / v) acetate, D47 was not viable and was not resistant, and unlike the D44 strain, pale brown colonies were formed.

Based on the above results, it can be seen that the strain D47 of the present invention is a strain of Gluconacetobacter .

<2-3> 16s rDNA  analysis

In order to remove the gDNA of strain D44 and D47 isolated in Example 1, cellulase (non-cellulase) was added to 1% (v / v) And incubated for 1 hour with stirring. The 16s rDNA was extracted and amplified using DNeasy Blood & Tissue kit (QIAGEN, Germany). Through the 16s rDNA sequencing, the whole sequence of the 16s rDNA of the D44 strain of SEQ ID NO: 1, 2 &lt; / RTI &gt; of the D47 strain, respectively. Genomic DNA was isolated from candidate strains using the DNeasy® Blood & Tissue kit (QIAGEN, Germany) for strain identification. Purification of the DNA followed the manufacturer's instructions.

For the cloning of 16S rDNA of strain D44 and D47, 5'-AGAGTTTGATCMT GGCTCAG-3 '(Forward) of SEQ ID NO: 3 and 5'-ACGGGCGGGTGTGTRC-3' (Reverse) of SEQ ID NO: 4 were used as primers Amplification was performed. Specifically, PCR amplification was performed at 95 ° C for 30 seconds using 100 ng template DNA, 0.5 μM primer DNA, 0.2 mM dNTPs, 10 × Ex Taq buffer and 0.025 U / μl of Taq polymerase (TaKaRa bio Inc., Japan) 35 cycles of binding at 58 ° C for 30 seconds and elongation at 72 ° C for 1 minute 30 seconds were amplified with a Biometra thermocycler (Tampa, USA). PCR products were confirmed by 1% agarose gel electrophoresis. The identified PCR products were purified using a PCR purification kit (Bioneer, Korea) and ligated to pGEM T-easy vector (Promega, USA) using T4 ligase (Promega, USA) Sequencing was performed to obtain the nucleotide sequence information of each of the above strains.

From the 16s rDNA sequence of the D44 and D47 strains obtained, the homology of the DNA from the ribosomal gene bank was examined and referenced for identification. The Bioedit program (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) , Clustal X program, and Mega program (http://www.megasoftware.net/) were used to draw a phylogenetic tree with similar microorganisms to observe the relationship between the species.

First, the 16s rDNA sequence of the D44 strain and the Gluconacetobacter As a result of comparing the similarities of the 16s rDNA sequence (AY180961) of rhaeticus, the similarity was only 87.215%. Usually, the similarity of the strain identification should be 98 ~ 99%, but the D44 and Gluconacetobacter rhaeticus degree of similarity can be seen that the D44 strain of the present invention with only a 87.215% are novel.

The similarity of D44 and Gluconacetobacter rhaeticus of the present invention was again confirmed using the Clustal X program, and the results are shown in Figs. 2a to 2c. As shown in Figs. 2A to 2C, the similarity between D44 and Gluconacetobacter rhaeticus is only 87.215%.

The Bioedit program, the Clustal X program, and the Mega program were used to study the system with D44 based on the microorganisms belonging to the same section with reference to the above similarity result. A gluconate acetonitrile bakteo (Gluconacetobacter) in the glucono bakteo (Gluconobacter), A phylogenetic tree created by the Neighbor-joining method for similar gyundeul in the acetonitrile bakteo (acetobacter) belonging to the particular chosangyun (acetobacteraceae) are shown in Fig.

Next, the 16s rDNA sequence of the D47 strain and the Gluconacetobacter As a result of comparing the similarity of intermedius 16s rDNA sequence (Y14694), it showed 99.774% similarity. Thus, the D47 strain is Gluconacetobacter intermedius was identified as Gluconacetobacter intermedius D47.

Also, the similarity of D47 of the present invention and Gluconacetobacter intermedius was confirmed again using the Clustal X program, and the results are shown in FIGS. 6A to 6C. As shown in FIGS. 6A to 6C, D47 and Gluconacetobacter intermedius were 99% similar.

However, unlike the conventional strain of the genus Gluconacetobacter , as shown in Table 4 of <Example 2-2>, the D47 strain has ketogenesis which forms dihydroxyacetone in glycerol, (W / v) NaCl was added to the HS culture medium to form cellulose on the culture medium. Thus, the biochemical properties of the strain were completely different from those of the conventional strain of Gluconacetobacter It was confirmed that the strain was a novel strain and that the cellulose production ability was significantly higher than that of the conventional strain of the genus Gluconacetobacter as described later.

The Bioedit program, the Clustal X program, and the Mega program were used to study the D47 lineage based on the microorganisms belonging to the same category. Specifically, the phylogenetic trees prepared by the Neighbor-joining method on the bacteria belonging to the acetobacteraceae family and those belonging to the genus Gluconacetobacter belonging to the genus Acucobacteraceae and those belonging to the genus Gluconobacter and acetobacter are shown in FIG.

&Lt; 2-4 > The deposit of the strain of the present invention

The inventors of the present invention carried out the above-mentioned isolated and identified strains belonging to the gluconeacetobacter strain of the present invention to KCCM International Preservation Center (KCCM) on March 26, 2010, respectively, as KCCM 11078P Deposit No. (D44) and KCCM11079P Deposit No. (D47)

< Example  3>

Cellulose production activity of the strain of the present invention

&Lt; 3-1 > Cellulose production activity of the strain of the present invention

The strain D44 and the strain D47 were each inoculated into the H.S medium and the results of confirming whether or not cellulose was produced are shown in FIGS. 4 and 8. FIG.

As shown in FIG. 4 and FIG. 8, the D44 strain and the D47 strain of the present invention form cellulose on the skin after 2 to 3 days of culture, and as the culture period passes, the cellulose film formed by D47 becomes thicker A layer is formed.

The D44 and D47 strains were inoculated at 1% by weight (relative to the total weight of the culture medium) in the HS medium, respectively, and cultured at 30 DEG C for 7 days. Cellulose produced was measured for dry weight and the results are shown in Tables 5 and 6 9a. Specifically, the celluloses formed on the surface of the H.S medium were collected, washed with tap water, and then boiled in a small amount of 0.1N NaOH solution for 1 hour to allow lysis. The recovered cellulose was further washed with tap water and neutralized by putting in a small amount of distilled water for one day. The neutralized cellulose was filtered and dried at 90 ° C until it became a constant amount (anti-weight), and after the dried cellulose was cooled in a desiccator, the dry weight was measured.

The dry weight (BC: Bacterial Cellulose) of cellulose produced by strain D44 and strain D47 BC production (Dry weight, g / L) D44 1.70 + 0.9072 D47 2.17 ± 0.3741

The cellulosic membrane produced using the strain D44 of the present invention has a smooth surface, firmness, elasticity, and thickness of about 0.3 mm. The cellulose membrane produced using the D47 strain of the present invention has a smooth surface, firmness, And the cellulosic activity was much higher in the thickness of about 4 mm. Furthermore, as described later, the thickness of the cellulose membrane produced using the D47 strain in the coconut mixed medium 2 showed a remarkably high cellulose activity of about 12 mm.

<3-2> Comparison of Cellulose Production Activity of the Strain of the Present Invention

The cellulose production activity of the D44 strain and the D47 strain of the present invention was compared with the known strain used for producing cellulose, Acetobacter xylinum strains (KCCM 40274).

The D44 strain and the D47 strain of the present invention, Acetobacter xylinum KCCM 40274 strain was inoculated 1% by weight into the HS medium (relative to the total weight of the culture medium), followed by incubation at 30 ° C for 7 days. Thereafter, the dry weight of the cellulose produced according to Example <3-1> was measured The results are shown in Table 6 below.

The dry weight (BC: Bacterial Cellulose) of cellulose produced by strain D44 and strain D47 Acetobacter xylinum
(KCCM 40274) Gluconacetobacter
sp . D44 Gluconacetobacter
intermedius D47 BC production (g / 1L) 0.50 0.2828 1.70 + 0.9072 2.17 ± 0.3741

As shown in Table 6, the D44 strain and the D47 strain of the present invention can produce a cellulose of about 3 times in the case of the strain D44 and about 4.34 times in the case of the strain D47 in comparison with Acetobacter xylinum . Among the strains, the D47 strain can produce about 1.27 times more cellulose than the D44 strain, indicating that cellulose can be produced at a very high yield.

< Reference example  2>

The medium and the culture conditions used for evaluating the cellulosic yield according to the medium composition

The strains of the present invention were one of the factors for further enhancing the amount of cellulose produced in the H.S medium. In order to confirm the effect of the medium composition on the medium, bacteria were cultured in the medium prepared as shown in Table 7 below.

The culture medium was adjusted to 1% (v / v), and the temperature was maintained at 30 ° C. After 7 days of culture, the dry weight was measured and used as a mean value through repeated experiments.

Medium composition for culture of strain D44 and strain D47 Composition badge H.S badge coconut
Liquid medium coconut
Mixed medium ① coconut
Mixed medium ② Glucose 20g / L - - - Yeast extract
(Difco, USA) 5g / L - - - peptone 5g / L - - - Na ₂ HPO ₄ 2.7 g / L - - - Sodium citrate 1.15 g / L - - - Coconut stock solution ^{1 )} - 1000g / L 895 g / L 895 g / L Sugar - - 100 g / L 100 g / L Yuan ^{2 )} - - 5g / L - Yuan ^{3 )} - - - 5g / L pH, temperature (캜) 5, 30 5, 30 5, 30 5, 30

1) Coconut stock solution: 100% raw stock from www.onedrinks.com (One world enterproses, LLC, USA. Mede in brazil)

2) Yuan: 99% purity ammonium sulphate manufactured by Sigma (USA)

3) Yuan: Chia Tai Co., Ltd. (21% purity ammonium sulphate

< Example  4>

Test for promoting cellulase production activity of the strain of the present invention according to the composition of the medium

As shown in Table 7, the dry weight and thickness of the cellulose produced by strain D44 and strain D47 of the present invention were measured by varying the composition of the medium, and the results are shown in Table 8 below. The measurement results are shown in Table 1 below. Acetobacter xylinum strains (KCCM 40274).

Comparison of Cellulose Production Dry Weight of Strain by Media Composition Strain badge Dry weight (g / L) Acetobacter xylinum
(KCCM 40274) H.S badge 0.10 Coconut liquid medium 0.02 Coconut mixed medium ① 0.20 Coconut mixed medium ② 0.20 Gluconactetobacter sp . D44 H.S badge 1.90 Coconut liquid medium 1.00 Coconut mixed medium ① 0.20 Coconut mixed medium ② 0.20 Gluconacetobacter intermedius D47 H.S badge 2.20 Coconut liquid medium 1.95 Coconut mixed medium ① 0.80 Coconut mixed medium ② 5.10

As shown in Table 8, the D44 strain and the D47 strain of the present invention are the representative strains producing cellulose, Acetobacter xylinum Cellulose production was generally higher than that of strain D47. In particular, strain D47 produced the largest amount of cellulose in all of the strains.

Specifically, the dry weight of the cellulose produced by strain D44 in HS medium and coconut liquid medium was determined as Acetobacter xylinum strains were 19 times and 50 times, respectively, and D47 strains were 22 and 97.5 times, respectively. Thus, it was confirmed that the strains of the present invention produced cellulose at a much higher yield.

On the other hand, when the amount of cellulose produced in the coconut mixed media (1) and (2) is examined, the representative strain Acetobacter xylinum strain of the present invention and the D44 strain of the present invention did not show a difference in the amount of cellulose produced. However, in the case of D47 strain of the present invention, the dry weight of the produced cellulose was 0.80 g / L in the coconut mixed medium 1, L, respectively, showing cellulosic production of 4 times and 25.5 times, respectively. Acetobacter xylinum strain of the present invention, as well as the D44 strain of the present invention.

In conclusion, the strains of the present invention are selected from the group consisting of Acetobacter xylinum Cellulose can be produced at a much higher yield than that of the strain, and such cellulose production efficiency can be varied depending on the composition of the medium.

That is, the D44 strain produced more cellulose than the coconut mixed medium at 9.5 times in the HS synthetic medium and the D47 strain at about 2.32 times in the coconut mixed medium prepared by adding the sugar and the yuan to the coconut stock solution than the HS synthetic medium And it produced about 6.4 times more cellulose than the coconut mixed medium (1). Also, the thickness of the cellulose produced by the D47 strain in the coconut mixed medium (2) was found to be significantly increased by about 12 mm, which was about three times higher than that measured from the HS synthetic medium (FIG. 9 9b).

Korea Microorganism Conservation Center (overseas) KCCM11078 20100326 Korea Microorganism Conservation Center (overseas) KCCM11079 20100326

<110> SAMSUNG ELECTRONICS CO., LTD. <120> Novel gluconacetobacter strain producing cellulose with high          yield <130> dpp20106168kr <150> KR10-2010-0018886 <151> 2010-03-03 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 1328 <212> DNA <213> Artificial Sequence <220> <223> 16s rDNA of D44 <400> 1 gggggcaagc gttgctcgga atgactgggc gtaaagggcg cgtaggcggt tgacacagtc 60 agatgtgaaa ttcctgggct taacctgggg gctgcatttg atacgtggcg actagagtgt 120 gagagagggt tgtgaaattc ccagtgtaga ggtgaaattc gtagatattg ggaagaacac 180 cggtggcgaa ggcggcaacc tggctcatga ctgacgctga ggcgcgaaag cgtggggagc 240 aaacaggatt agataccctg gtagtccacg ctgtaaacga tgtgtgcttg gatgttgggt 300 gactttgtca ttcagtgtcg tagttaacgc gataagcaca ccgcctgggg gagtacggcc 360 mccaaggttg aaactcaang ctttcagcgg ggacgatgat gacggtaccc gcagaagaag 420 ccccggctaa cttcgtgcca gcagccgcgg taatacgaan gggggcaagc gttgctcgga 480 atgactgggc gtaaagggcg cgtaggcggt tgacacagtc agatgtgaaa ttcctgggct 540 taacctgggg gctgcatttg atacgtggcg actagagtgt gagagagggt tgtgaaattc 600 ccagtgtaga ggtgaaattc gtagatattg ggaagaacac cggtggcgaa ggcggcaacc 660 tggctcatga ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt agataccctg 720 gtagtccacg ctgtaaacga tgtgtgctgg atgttgggtg actttgtcat tcagtgtcgt 780 agttaacgcg ataagcacac cgcctgggga gtacggccgc aaggttgaaa ctcaaaggaa 840 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa ccgcagaacc 900 ttaccagggc ttgacatgcg gaggccgtgt ccagagatgg gcatttctcg caagagacct 960 ccagcacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1020 gcaacgagcg caaccctcgc ctttagttgc cagcacgtct gggtgggcac tctaaaggaa 1080 ctgccggtga caagccggag gaaggtgggg atgacgtcaa gtcctcatgg cccttatgtc 1140 ctgggctaca cacgtgctac aatggcggtg acagtgggaa gccaggtagc gataccgagc 1200 cgatctcaaa aagccgtctc agttcggatt gcactctgca actcgagtgc atgaaggtgg 1260 aatcgctagt aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca 1320 ccgcccgt 1328 <210> 2 <211> 1347 <212> DNA <213> Artificial Sequence <220> <223> 16s rDNA of D47 <400> 2 agagtttgat catggctcag agcgaacgct ggcggcatgc ttaacacatg caagtcgcac 60 gaacctttcg gggttagtgg cggacgggtg agtaacgcgt agggatctgt ccacgggtgg 120 gggataactt tgggaaactg aagctaatac cgcatgacac ctgagggtca aaggcgcaag 180 tcgcctgtgg aggaacctgc gttcgattag ctagttggtg gggtaaaggc ctaccaaggc 240 gatgatcgat agctggtctg agaggatgat cagccacact gggactgaga cacggcccag 300 actcctacgg gaggcagcag tggggaatat tggacaatgg gcgcaagcct gatccagcaa 360 tgccgcgtgt gtgaagaagg ttttcggatt gtaaagcact ttcagcgggg acgatgatga 420 cgcgtacccg cagaagaagc cccggntnan ttngtgccag cagccgcggt aatangaagg 480 ggncaagcgt tgctcggaat gactgggcgt aaagggcgcg taggcggttg acacagtcag 540 atgtgaaatt cctgggctta acctgggggc tgcatttgat acgtggcgac tagagtgtga 600 gagagggttg tggaattccc agtgtagagg tgaaattcgt agatattggg aagaacaccg 660 gtggcgaagg cggcaacctg gctcatgact gacgctgagg cgcgaaagcg tggggagcaa 720 acaggattag ataccctggt agtccacgct gtaaacgatg tgtgctggat gttgggtgac 780 tttgtcattc agtgtcgtag ttaacgcgat aagcacaccg cctggggagt acggccgcaa 840 ggttgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 900 cgaagcaacg cgcagaacct taccagggct tgacatgcgg aggccgtgtc cagagatggg 960 catttctcgc aagagacctc cagcacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg 1020 agatgttggg ttaagtcccg caacgagcgc aaccctcgcc tttagttgcc atcacgtctg 1080 ggtgggcact ctaaaggaac tgccggtgac aagccggagg aaggtgggga tgacgtcaag 1140 tcctcatggc ccttatgtcc tgggctacac acgtgctaca atggcggtga cagtgggaag 1200 ccaggtggtg acaccgagcc gatctcaaaa agccgtctca gttcggattg cactctgcaa 1260 ctcgagtgca tgaaggtgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg 1320 ttcccgggcc ttgtacacac cgcccgt 1347 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for cloning 16s rDNA <400> 3 agagtttgat cmtggctcag 20 <210> 4 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for cloning 16s rDNA <400> 4 acgggcgggt gtgtrc 16

Claims (11)

delete delete delete delete delete In acetonitrile gluconate bakteo with cellulose production activity (gluconacetobacter sp.) D44 strain (Accession No. KCCM11078P) glucose 20g / L, yeast extract 5g / L, peptone _{5g / L, Na 2 HPO 4} 2.7g / L sodium citrate, and 1.15 g / L in a culture medium containing HS. L of a gluconacetobacter sp. D44 strain (accession number KCCM11078P) having cellulosic activity, glucose, 20 g / L, yeast extract 5 g / L, peptone 5 g / L, Na ₂ HPO ₄ 2.7 g / 1. &lt; / RTI &gt; 15 g / L; And
Obtaining cellulose from the culture obtained in the culturing step
&Lt; / RTI &gt; delete delete In acetonitrile bakteo gluconate (gluconacetobacter sp.) D44 strain (Accession No. KCCM11078P) the glucose 20g / L, yeast extract 5g / L, peptone _{5g / L, Na 2 HPO 4} 2.7g / L sodium citrate and 1.15g / L &Lt; / RTI &gt; to obtain cellulose from the culture cultured in the HS medium containing; And
Impregnating the cellulose with the resin
Based on the weight of the cellulose-based resin. The method for producing a cellulose resin according to claim 10, wherein the cellulose resin is used for a substrate of a liquid crystal display.

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