KR101316085B1 - Microbacterium oxydans Having Property for Degrading Laminarin and Alginic acid - Google Patents

Abstract

The present invention relates to microorganisms having laminarin and alginic acid resolution, and more particularly, to microbacterium oxydans having laminarin and alginic acid resolution, which are by-products generated during processing of brown algae that are difficult to process or during extraction of brown algae. will be.
The microorganisms having laminarin and alginic acid resolution are characterized in that the microbacterium oxydans FS4 ( Microbacterium oxydans FS4) deposited with the accession number KACC91657P in the National Institute of Agricultural Science.
According to the present invention, it is possible to produce reducing sugars, such as glucose, from laminarin and alginic acid, which are by-products generated during the processing of brown algae, which are difficult to process, or during the extraction of brown algae, and thus have an environmental protection effect. It has the effect of recycling.

Description

Microbacterium oxydans Having Property for Degrading Laminarin and Alginic acid}

The present invention relates to microorganisms having laminarin and alginic acid resolution, and more particularly, to microbacterium oxydans having laminarin and alginic acid resolution, which are by-products generated during processing of brown algae that are difficult to process or during extraction of brown algae. will be.

As the climate change convention and environmental regulations are strengthened around the world, researches on renewable energy such as bio, hydrogen, and solar to replace petroleum and coal, and to develop stable and environmentally friendly energy are being actively conducted.

Leading countries in renewable energy production, such as the United States, Brazil, and Germany, are conducting research and production using raw materials such as sugarcane and corn as raw materials for bioenergy production, and wood-based resources as forestry and agricultural by-products. The limitation of biofuel production using grains and wood-based resources has been revealed due to problems such as limitation of governing area and difficulty in supplying nutrients according to the types of crops, and economic efficiency by additional pretreatment processes such as hemicellulose and lignin other than cellulose.

In order to solve these problems, marine algae, in which cell walls are composed of many fibers and various polysaccharides, have recently attracted attention as a new bioenergy source. The main producers of seaweed are Asian countries such as Korea, Japan, China, and Indonesia, and they have a larger cultivable ocean area than land. In addition, algae grows faster than wood and plant-based cellulose, and absorbs carbon dioxide in the air through photosynthetic reactions, thereby reducing greenhouse gases, and simplifying pretreatment processes because of less lignin content in seaweeds. It has many potentials as a raw material. Among the seaweeds accounting for about 25% of Korea's aquatic production, most of the brown algae that are suitable for the climate of Korea are farmed. About 60-95% of these large seaweeds are water, and more than 50% of the rest are carbohydrates. Large algae have various types and binding forms of polysaccharides according to species. Especially, brown algae such as kelp and seaweed have many similarities to green plants, and 5.7% to 14.3% of cellulose is composed of structural polysaccharides. It is composed of alginic acid, fucoidan, which is a sex polysaccharide, and laminarin, which is a hypotonic polysaccharide.

Laminarine, a by-product of brown algae processing and extraction, is composed mainly of β-1,3-glucose bonds and partially composed of β-1,6-glucose bonds. L-Gluronic acid is composed of β-1,4 bonds, polymanneuronic acid (MM), polygluronic acid (GG) form bonded in the homopolymer form or heteropolymer (MG) form a mixture of the two components It is composed of mixed irregularly. Therefore, using microorganisms that decompose laminarin and alginic acid, which are purely separated from soil, mud flats, and intestines of seabed animals, not only glucose acid required for ethanol production, but also peat sugar, samtan sugar, oligosaccharide, etc. The same degradation metabolites can be useful bioactives. To date, algae, alginic acid, and fucoidan, which are algae polysaccharides, are extracted from brown algae through processing such as alkali, acid, or enzyme treatment, and are widely used as food additives and health food resources. However, it is difficult to use as a substrate for biofuel production due to the complicated structure of brown algae itself, and the treatment of by-products and wastes generated in addition to the materials currently used remains a problem. As a member of the OECD, Korea is obliged to follow the '96 Protocol, adopted by the London Convention on Waste Dumping, which cannot be ruled out as a result of the disposal of seaweeds discarded after 2012.

In this regard, Korean Unexamined Patent Publication No. 2010-0087959 discloses a new microorganism producing alginate degrading enzyme, Bacillus sp. N7151-Z, and Korean Patent No. 0985841 discloses a novel microorganism Pseudomonas sp. N7151-producing alginate degrading enzyme. 6, Korean Patent Laid-Open No. 2008-0006931 discloses marine microorganisms Vibrio sp. Although a method for preparing alginic acid degrading enzyme using S21 strain has been disclosed, it is urgent to develop microorganisms having not only alginic acid but also laminarin degrading ability and excellent enzymatic activity.

Accordingly, the present inventors have made intensive efforts to develop a method for recycling laminarin and alginic acid, which are by-products generated in the brown algae processing process, which are difficult to process, or generated during the brown algae extraction process, and thus purely isolated microbacterium oxydans FS4 No. KACC91657P) was used to decompose laminarin and alginic acid to produce bioethanol and bioactive substances, thus completing the present invention.

It is an object of the present invention to provide a microorganism capable of effectively decomposing laminin and alginic acid, which are storage polysaccharides, among brown algae constituent polysaccharides.

Another object of the present invention is to provide a method for preparing laminarin or alginic acid degrading enzyme and a method for degrading laminarin or alginic acid.

Still another object of the present invention is to provide a method for producing oligosaccharides, which are glucose and bioactive substances used in bioethanol production, from laminarin.

In order to achieve the above object, the present invention provides a micro tumefaciens oxy thiooxidans FS4 (Microbacterium oxydans FS4) deposited as accession No. KACC91657P the National Genetic Resources Center, Agricultural Research agriculture.

In the present invention, the microbacterium oxidans FS4 is characterized by having a laminarin degrading enzyme ability and alginic acid degrading enzyme ability.

In the present invention, the microbacterium oxidans FS4 is characterized by having the 16S -rDNA of SEQ ID NO: 3.

The present invention also provides a method for producing laminarin or alginic acid degrading enzyme, characterized in that the culture of the microbacterium oxidans FS4.

The present invention also provides a method for decomposing laminarin, which comprises using a laminarin degrading enzyme prepared by the above method or a microbacterium oxydans FS4 cell containing the laminarin degrading enzyme.

The present invention also provides a method for decomposing alginic acid, which comprises using an alginic acid degrading enzyme prepared by the above method or a microbacterium oxydans FS4 cell containing the alginic acid degrading enzyme.

The present invention has an effect of providing a microorganism capable of effectively dissolving laminin and alginic acid which are the major polysaccharide components of brown algae to produce useful substances. According to the present invention, it is possible to produce reducing sugars such as glucose from laminarin and alginic acid, which are by-products generated during the processing of brown algae, which are difficult to process, or during the extraction of brown algae, and thus have an environmental protection effect. The effect can be recycled into.

1 is inoculated with the six strains (A) FS1, (B) FS2, (C) FS3, (D) FS4, (E) FS5, (F) FS6 separated in a laminarin liquid medium in a tube unit This is the result showing naryn resolution [pH (▲); Cell density (●); Toatl reducing sugars (■)].
Figure 2 shows the results of laminin degradation when incubated in a 100 ml flask for 15 days to isolate strains [pH (▲); Cell density (●); Total reducing sugars (■), Glucose (◆)].
Figure 3 is a graph showing the results of laminarin decomposition of (a) 1 g / L, (b) 5 g / L, (c) 10 g / L Microbacterium oxydans FS4 for each concentration of laminarin [pH ( ▲); Cell density (●); Total reducing sugars (■), Glucose (◆)].
Figure 4 Laminarin 5 g / L Laminarin digested cultures with microbacterium oxydans FS4 for (a) 0 days, (b) 3 days, (c) 6 days, (d) 9 days, (e) 12 days, and (f) 15 days at concentration. HPLC shows the result of HPLC analysis [1: Laminarin, 2: Oligo-saccharides 3: Glucose].
5 laminarin 10 g / L Laminarin digested cultures with microbacterium oxydans FS4 for (a) 0 days, (b) 3 days, (c) 6 days, (d) 9 days, (e) 12 days, and (f) 15 days at concentration. HPLC shows the result of HPLC analysis [1: Laminarin, 2: Oligo-saccharides, 3: Glucose].
Figure 6 is a clear ring by laminarin degradation when the isolated strain in a laminarin solid plate medium (a) 0 days, (b) 2 days, (c) 4 days, (d) 6 days at 37 ℃ ( This shows the appearance of forming a clear zone.
7 is a clear ring by alginate degradation when microbacterium oxydans FS4 is incubated at 37 ° C. for (a) 0 days, (b) 2 days, (c) 4 days, and (d) 6 days in an alginate solid plate medium. This is a result showing the appearance of forming a (clear zone).
Figure 8 shows the large amount of β-1,3-glucanase gene obtained from the pyrococcus puriosus strain using E. coli, and then isolated and purified β-1,3-glucanase for 7 days (a) 37 ℃, (b) Laminarin decomposition reaction was carried out at 60 ℃ [pH (▲); Total reducing sugars (■)].
9 shows a large amount of β-1,3-glucanase gene obtained from Pycococcus puriosus strain using Escherichia coli, followed by reaction of isolated and purified β-1,3-glucanase at 37 ° C. for 15 days. Narine decomposition result [pH (▲); Total reducing sugars (■), Glucose (◆)].

In the present invention, microorganisms capable of efficiently decomposing laminin and alginic acid, which are waste algae polysaccharides generated in brown algae processing and extraction processes, are separated from samples collected in various environments such as soil, seawater, and tidal flats. It was confirmed that laminarin and alginic acid had a resolution.

That is, in one embodiment of the present invention, FS1 to FS6 strains that decompose laminarin from the intestines of soil, mud flats and marine animals are separated, and double FS4 has the highest laminarine resolution, and then 16S of FS4 strain -rDNA was identified and identified as a microbacterium oxydans strain, which was deposited with the accession number KACC91657P at the National Institute of Agricultural Science.

In addition, in another embodiment of the present invention it was confirmed that the FS4 strain has an alginate resolution as well as laminarin.

Accordingly, the present invention relates to In one aspect, the National Academy of Agricultural Sciences agricultural genetic accession No. KACC91657P micro tumefaciens oxy thiooxidans FS4 (Microbacterium oxydans FS4) deposited in the center.

The microbacterium oxidans FS4 is characterized by having the ability to laminarin degrading enzyme and alginic acid degrading enzyme.

In addition, the microbacterium oxidans FS4 is characterized by having the 16S-rDNA of SEQ ID NO: 3.

On the other hand, the present inventors cultivate the microbacterium oxydans FS4 having the laminarin degrading enzyme ability and the alginic acid degrading enzyme ability to produce a laminarin degrading enzyme or alginic acid degrading enzyme, so when separated from the laminarin degrading enzyme or alginic acid degrading It was predicted that the enzyme could be prepared.

Accordingly, the present invention relates to a method for producing laminarin or alginic acid degrading enzyme, characterized in that the microbacterium oxidans FS4 is cultured in another aspect.

The medium for culturing the microbacterium oxydans FS4 is not limited as long as the growth of the strain is smooth, but includes the laminarin or alginic acid in the medium for the smooth growth of the strain and mass production of laminarin or alginic acid degrading enzyme It is desirable to.

The laminarin degrading enzyme or alginic acid degrading enzyme can be recovered from the culture by the following conventional method.

The supernatant is recovered from the culture by centrifugation, filtered at 0.45 um, and then concentrated using ammonium sulfate. In addition, the collection of laminarin or alginic acid degrading enzyme from the culture medium is separated using methods known in the art, such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, FPLC (Fast performance liquidchromatography). Can be purified.

On the other hand, by decomposing laminarin using the microbacterium oxydans FS4, it was predicted that oligosaccharides, which are glucose and bioactive substances used for bioethanol production, could be prepared.

In another embodiment of the present invention, the microbacterium oxydans FS4 (Accession Number: KACC91657P) was cultured in a medium containing laminarin, it was confirmed that glucose and oligosaccharides are produced.

Therefore, in another aspect, the present invention relates to a method for decomposing laminarin, which comprises using a laminarin degrading enzyme prepared by the above method or a microbacterium oxydans FS4 cell containing the laminarin degrading enzyme. .

Laminarin is broken down into glucose and oligosaccharides by the microbacterium oxydans FS4 cells containing the laminarin degrading enzyme or the laminarin degrading enzyme.

Examples of the oligosaccharides include lamina Lindaose, laminarine triose, laminarineheptaose, and the like.

In another aspect, the present invention relates to a method for decomposing alginic acid, which comprises using alginate degrading enzyme prepared by the above method or microbacterium oxydans FS4 cells containing the alginic acid degrading enzyme.

In the present invention, the microbacterium oxidans bacteria FS4 is a term that includes the microbacterium oxydans FS4 culture and the microbacterium oxydans FS4 lysate.

[Example]

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

Example 1 Pure Separation of Laminarin Degrading Microorganisms

Bacteria that effectively degrade laminarin were isolated from samples taken from the soil and wetlands around the Nakdong River, and from the guts of sea cucumbers and small fish.

Samples were collected and inoculated in a 250-ml flask of kelp addition medium (NH ₄ Cl 0.1g / L, kelp pieces 1 cm × 1 cm or less, pH 6.8), followed by shaking culture at 37 ° C. and 180 rpm for 8 days, respectively. It was plated on a solid medium containing 1.5% agar in 0.8% nutrient medium (Nutrient broth, pH 6.8). Each of the cultured microorganisms was continuously plated on a new solid medium until pure colonies were separated, and each strain was finally purified. The isolated pure strains were subcultured every 2 weeks with storage at 4 ℃.

In order to screen useful microorganisms in the isolated pure strain, laminarin medium (Laminarin 1 g / L, MgSO ₄ 0.1 g / L, NaCl 0.1 g / L, CaCl ₂ 0.1 g / L, (NH ₄ ) ₂ SO ₄ 2 g / L, 0.5 g / L of KH ₂ PO ₄ , 0.5 ml of Mineral, 0.5 ml of Vitamin, pH 6.8) and the pure strain was added to a solid medium to which 1.5% agar was added.

As a result, six strains forming pure colonies in laminarin agar medium were identified, and the six isolates were named FS1 to FS6.

Example 2 Screening of Laminarin Degrading Microorganisms

In order to confirm whether or not to produce an antagonist against each other among the six pure strains isolated in Example 1 was plated to cross each other in laminarin solid medium and incubated for 2 days at 37 ℃, each strain in a 15 ml tube Inoculated in 8 ml laminarin liquid medium was shaken for 8 days at 37 ℃, 180 rpm, the growth curve, total reducing sugar and pH of the strain was measured to test the laminarin resolution.

As a result, it was confirmed that the six isolates are strains that do not produce antagonists to each other, and that the strain showing the highest resolution in laminarin liquid medium is FS4 (FIG. 1).

Example 3. Identification and Deposit of Laminarin Degrading Microorganisms

FS4 strain showing the highest laminarin resolution in Example 2 was identified primarily through Gram staining, catalase test, colony morphology and microscopic examination.

The FS4 strain is a gram-positive bacillus having a vigorous motility in a nutrient state, having a width of 0.5-1 μm and a length of 0.1-1.5 μm, and is clustered with each other. And when cultured in 0.8% nutrient solid medium, 1% laminarin solid medium at 37 ℃, 2 days, yellow colonies and white colonies were formed, respectively, catalase test results, it was shown to be positive.

Secondary identification was performed by molecular sifting based on 16S rDNA (16S rRNA coding gene) sequence. Chromosomal DNA of each strain was isolated using AccuPrep chromosome DNA extraction kit (Bionia, Korea), PCR was performed using the primers of SEQ ID NO: 1 and SEQ ID NO: 2 with the isolated DNA as a template.

PCR was performed under the following conditions. Initial modification was performed at 95 ° C. for 5 minutes, 30 cycle amplification was performed at 95 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds, and the final stretching step was performed at 72 ° C. for 7 minutes.

As a result of PCR, approximately 1500 bp fragment of 16S-rDNA gene was amplified in each strain.

[SEQ ID NO 1]: 5'-CCAGCAGCCGCGGTAATACG-3 '

[SEQ ID NO 2]: 5'-TACCAGGGTATCTAATCC-3 '

The amplified PCR product was commissioned by Macrogen Co., Ltd. for 16S-rDNA sequencing analysis, and then confirmed the 16S-rDNA subsequence (SEQ ID NO: 3) of the FS4 strain, and the BioEdit Sequence Alignment Editor (ver. 5.0.9, Hall, Homology searches were performed in GenBank's Advanced BLAST (http://www.ncbi.nlm.nih.gov) using T., Nucleic Acids Symposium Series , 41: 95, 1999 (Altschul et al ., Nucleic) . Acids Res. , 25: 3389, 1997).

As a result, the FS4 strain was identified as Microbacterium oxidans species (see Table 1).

microbe 16S-rDNA
size GenBank
Accession No. Strain name Homology FS4 1429 bp HQ113206.1 Microbacterium oxydans 98%

The FS4 strain identified as microbacterium oxydans was entrusted to the National Institute of Agricultural Science, Agricultural and Genetic Resources Center on August 3, 2010 (Accession Number: KACC91657P).

Example 4 Determination of Laminarin Resolution of Microbacterium Oxydans FS4

Laminarin of the microbacterium oxydans FS4 strain was confirmed the resolution and the possibility of producing useful substances.

Microbacterium oxydans FS4 strain was used after harvesting by centrifugation at the end of the logarithmic growth phase in 0.1% laminarin liquid medium.

A 100 ml flask containing 40 ml laminarin liquid medium was inoculated with 80 µg (wet cell) of the strain, and the degradation reaction was performed at 37 ° C. and 180 rpm for 15 days. During the biodegradation reaction, samples in the flask were taken at three day intervals to characterize the degradation reaction. Changes in pH in the culture medium during the degradation reaction were measured using a pH meter (ISTECH, Korea), and the concentration of the strain was measured by using a spectrophotometer (Hanbit Nano Biotech, Korea), after diluting the sample diluted three-fold in cuvettes. Measurement was made at a specific wavelength (375 nm). The total reducing sugars produced during the decomposition reaction were uniformly mixed with 100 μl of the supernatant obtained by centrifugation (7000 rpm, 10 minutes) and 1 ml DNS (3.5-dinitrosalicylic acid) solution using vortex. Then, after reacting for 10 minutes in boiling water, and then cooled in cold water for 5 minutes, the value was measured at a wavelength of 570 nm using a spectrophotometer.

In addition, the concentration of glucose and other oligosaccharides produced by laminarin digestion was analyzed by HPLC / RID (Agilent Technologies 1200 series, USA) using Sugar-pak ^TM 1 (Waters, 6.5 × 300 mm) column. . As the solvent for analysis, water of 0.8 ml / min flow rate was used, and the detector and oven temperatures were set to 35 ° C. and 80 ° C., respectively.

In the result of decomposition of laminarin, the pH of 6.8 was gradually decreased, and the final pH value of 15 days of cultivation was 3.98. I could see that. The total reducing sugar and glucose concentration produced were 4.23 g / L, respectively. And 2.23 g / L, stoichiometrically, it was found that when the laminarin is decomposed, an intermediate metabolite such as oligosaccharide can be produced in addition to glucose as a reducing sugar (FIG. 2).

Example 5 Decomposition of Microbacterium Oxydans FS4 by Laminarin Concentration

The resolution of the microbacterium oxydans FS4 strains at various laminarin concentrations (1, 5 and 10 g / L) was confirmed.

The culture was inoculated with 0.2% (w / v) of the strain in a liquid medium containing different concentrations of laminarin and incubated at 37 ° C. and 180 rpm for 15 days, and the laminarin resolution was measured in the same manner as in Example 4. Confirmed.

In the results of the decomposition experiments for each laminarin concentration, the decomposition reaction did not occur well at the concentration of laminarin of 1 g / L, the total reducing sugar and glucose concentration was not measured, 5 g / L and 10 g / L The laminarin concentration was confirmed to be reduced to 4.18 and 4.07 after 6 days of culture at the initial pH value of 6.8 as the laminarin was decomposed. 5 g / L At laminarin concentration, the total reducing sugar and glucose concentration after 15 days of culture were 2.04 g / L, respectively. And 0.85 g / L, 10 g / L At laminarin concentrations, the total reducing sugar and glucose concentrations were 5.54 g / L, respectively. And 3.36 g / L were generated. Compared with the results obtained using the 1 g / L laminarin concentration it was clearly seen the difference in the resolution (Fig. 3).

5 g / L and 10 g / L As a result of analyzing the microbial cultures collected at the laminarin concentration by HPLC, it was found that as the reaction time elapsed, the concentration of the laminarin decreased and the concentration decreased.In addition to glucose, peat sugar and samtan are reduced sugars. It was confirmed that intermediate metabolites such as sugar were produced (FIGS. 4 and 5).

In the results of the decomposition experiment for each concentration of laminarin, it was found that the best decomposition reaction occurred at the concentration of laminarin of 10 g / L.

Example 6 Determination of Laminarin and Alginic Acid Resolution of Microbacterium Oxydans FS4

It was confirmed that the microbacterium oxydans FS4 strain is capable of degrading alginic acid, which is a representative polysaccharide of brown algae, in addition to laminarin.

In order to observe the ability of the strain to degrade laminarin and alginic acid, laminarin medium (Laminarin 0.1 g / L, MgSO ₄ 0.1 g / L, NaCl 0.1 g / L, CaCl ₂ 0.1 g / L, (NH ₄ ) ₂ SO ₄ 2 g / L, 0.5 g / L KH ₂ PO ₄ , 0.5 ml of Mineral, 0.5 ml of Vitamin, pH 6.8) and a solid flat medium containing 1.5% agar in Alginate medium (Peptone 0.5 g / L, Sodium alginate 0.1 g / L, pH 6.8) 10 μl of the microbacterium oxydans FS4 strain culture medium incubated in nutrient liquid medium (0.8% Nutrient broth) for 2 days was dropped and dried, followed by incubation in a 37 ° C. incubator. After the strain formed colonies on the medium, the laminarin plate medium was stained for 15 minutes by administering 10 ml of 0.5% (w / v) Congo red solution, and then decolorized twice with 1 M NaCl, followed by Congo red solution. The presence of yellow ring formation around colonies on red stained plate medium and the change in size of transparent ring with time (Lee, DS, et al., Cloning and expression of a β-1,3-glucanase gene from Bacillus circulans KCTC3004 in Escherichia coli.Biotechnology letters 17 (4): 355-360, 1995). 10 ml of 10% cetylpyridinium chloride monohydrate (CPC) was applied to the alginic acid plate medium for 10 minutes.Then, the solution was removed and the surface of the medium was washed with distilled water to make the color opaque by 10% CPC. Ring formation and changes in size were observed (Peter, G., et al., Plate assay for simultaneous detection of alginate lyases and determination of substrate specificity. Amerian Society of Microbiology 56 (7): 2265-2267, 1990).

As a result of the experiment, as a result of the 2, 4, and 6 days of colonies cultured in laminarin plate medium, yellow rings having a radius of 1 cm, 1.5 cm, and 1.9 cm were formed, and micro It was confirmed that the ring size gradually increased by the degradation of laminarin of the Bacterium oxydans FS4 strain (FIG. 6), and in the alginate plate medium, the transparent ring around the colonies was changed to 2, 4 and 6 days. It was confirmed that the radius of the ring gradually increased to 1 cm, 1.7 cm, and 1.9 cm by decomposition of alginic acid of the Leeum oxydans FS4 strain (FIG. 7).

That is, it was confirmed that the microbacterium oxidans FS4 strain has the ability to decompose alginic acid other than laminarin among the polysaccharides contained in brown algae.

Comparative Example 1. Comparison of laminin resolution of β-1,3-glucanase isolated and purified from pyrococcus puriosus strains

In order to compare the laminarin resolution of the microbacterium oxydans FS4 strain, β-1,3-glucanase gene obtained from the pyrococcus puriosus strain (Accession No .: KCCM 41005), which is a thermophilic bacterium, was isolated from E. coli. (Kim, DG, et al., Applicaiton of β-1,3-glucanase from pyrococcus furisous for ethanol production using laminarin.Journal of Life Science 21 (1): 6, 69-73) 2011) An enzyme was used for the experiment with the consent and cooperation of the deposit depositor.

Β-1,3-glucanase enzyme at 5 ㎍ / ml concentration was added to 50 mM Tris-HCl buffer (pH 7.5) and 1% laminarin was reacted at 180 rpm at 37 ℃ and 60 ℃ for 7 days to decompose laminarin. Was analyzed. The pH change in the solution during the laminarin digestion reaction was measured using a pH meter (ISTECH, Korea). The total reducing sugar concentration produced was 100 μl of enzyme digestion solution and 1 ml of DNS (3.5-dinitrosalicylic acid) solution. The mixture was uniformly mixed, reacted in boiling water for 10 minutes, cooled in cold water for 5 minutes, and the sample was put in a cuvette and measured at 570 nm using a spectrophotometer (Hanbit Nano Biotech, Korea). .

Laminarin decomposition of β-1,3-glucanase enzyme showed no pH change in solution during each degradation reaction at 37 ° C and 60 ° C, but the total amount of reducing sugars produced at the decomposition reaction at 60 ° C was increased at 37 ° C. More than the amount produced (FIG. 8). Comparing this result with the result of Example 4 (FIG. 2), the maximum total reduction sugar concentration produced in the result of laminarin degradation by the microbacterium oxydans FS4 strain for 15 days was 4.84 g / L, β-1 The total reducing sugar concentrations produced at 37 ° C. and 60 ° C. by, 3-glucanase enzyme were 5.88 g / L and 7.13 g / L, respectively.

The difference may be that the amount of laminin degrading enzyme in the microbacterium oxydans FS4 strain used in the experiment may be less than that of β-1,3-glucanase enzyme at a concentration of 5 ㎍ / ml, It is estimated that low concentrations of reducing sugars can be produced because the laminarin decomposition reaction was not performed under optimal conditions. However, if the decomposition reaction of laminarin using microorganisms does not fall significantly compared to the reaction of the purified and purified enzymes, the microorganism oxidase FS4 strain is used to decompose the laminarin compared to the isolated and purified enzymes. It is obvious that is more economic.

Β-1,3-glucanase enzyme at a concentration of 5 μg / ml was treated with basal medium (MgSO ₄ 0.1 g / L, NaCl 0.1 g / L, CaCl ₂ 0.1 g / L, (NH ₄ ) ₂ SO ₄ 2 g / L, KH ₂ PO ₄ 0.5 g / L, Mineral 0.5 ml, Vitamin 0.5 ml, pH 6.8) in a solution of 1% laminarin was added to the laminarin decomposition reaction for 15 days at 37 ℃, 180 rpm. The pH change occurring in the solution during the laminarin decomposition reaction was measured in the same manner as the above method. Production concentrations of glucose and other oligopolysaccharides were analyzed in the same manner as in Example 4 above using a Sugar-pak ^™ 1 (Waters, 6.5 × 300 mm) column.

As a result, the pH value did not change significantly during the laminarin decomposition reaction, but the final concentrations of reducing sugar and glucose were 6.24 g / L and 1.13 g / L, respectively. Comparing the results of laminarin digestion using the microbacterium oxydans FS4 strain of Example 4 itself (FIG. 2) and laminarin digestion by the isolated and purified β-1,3-glucanase enzyme, microbacterium The concentration of total reduced sugars produced by the oxydans FS4 strain was relatively low, but the production rate of glucose in the total reduced sugars was much higher. As a result, the degradation of laminarin by the microbacterium oxydans FS4 strain resulted in bioethanol. It can be seen that it can be used more useful for production.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Agricultural Biotechnology Research Institute KACC91657P 20110803

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Claims (8)

Microbacterium oxydans FS4 deposited with KACC91657P with accession to the National Institute of Agricultural Science.
The method of claim 1, wherein the microbacterium oxydans FS4 ( microbacterium oxydans FS4) characterized in that it has the ability of laminin degrading enzyme and alginic acid degrading enzyme.
The method of claim 1, wherein the microbacterium oxydans FS4 ( Microbacterium oxydans FS4), characterized in that having a 16S-rDNA of SEQ ID NO: 3.
A method for producing laminarin or alginic acid degrading enzyme, comprising culturing the microbacterium oxydans FS4 of claim 1.
The method according to claim 4, wherein the microbacterium oxydans FS4 is cultured in a medium containing laminarine or alginic acid.
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