KR101229055B1 - Strain of methylobacterium sp. having a alginate-decomposition activity and method of producing alginate-oligomer using the same - Google Patents

Abstract

The present invention is a Methylobacterium sp. Strain having alginic acid resolution, a biocatalyst having alginic acid resolution prepared from the selected strains of Methylobacterium sp., And a method for producing an alginic acid oligomer using the biocatalyst. In particular, since the strain of the genus Methylobacterium is recognized to have a significantly superior alginic acid resolution compared to a microorganism having a previously reported alginic acid resolution, the industrial field related to food, cosmetics and medicines using alginic acid oligomer prepared from alginic acid or It can be usefully used in various industries related to bioenergy production using seaweed.

Description

Methylbacterial strain having alginate resolution and a method for preparing alginic acid oligomer using the strain {STRAIN OF METHYLOBACTERIUM SP. HAVING A ALGINATE-DECOMPOSITION ACTIVITY AND METHOD OF PRODUCING ALGINATE-OLIGOMER USING THE SAME}

The present invention relates to a methyloacterium sp. Strain having alginic acid resolution and a method for preparing an alginic acid oligomer using the strains of the methyllobacterium sp.

Alginic acid mainly forms the cell wall of brown algae and can be extracted from brown algae. Alginic acid is α-1,4 bond or β-1,4 by α-L-guluronate and β-D-manneuronic acid (1,4'-β-D-mannuronate), a C5 epimer. Heteropolysaccharide is a complex polysaccharide composed of pG block, pM block and pM / G stretch.

The alginic acid has been widely used since ancient times because of its unique physical properties. For example, it is used as a stabilizer, viscous or gelling agent in various industries including food industry, printing industry and pharmaceutical industry, as raw material of drug delivery system such as microspheres, beads, microcapsules or tablets, matrix in tissue engineering It has been used as a carrier and the like. In addition, alginic acid has been reported to have various physiological activities such as restraining obesity, healing constipation through promoting intestinal peristalsis, inhibiting anti-cholesterol, absorbing and removing heavy metals in the body, or inhibiting the toxicity of harmful substances. Research is underway to utilize it.

In addition, it is known to have a physiological activity effect such as wound dressings and hemostasis to protect the wound, as well as a variety of bioregulatory functions such as antimicrobial / anticancer activity, immune enhancement, anticholesterol effect, anticoagulant effect of alginic acid-derived oligosaccharides. Has been reported. Due to such various functionalities, research on preparing oligosaccharides using alginic acid as well as alginic acid itself is being conducted in various ways.

Existing alginic acid is a method of extracting an active ingredient such as alginic acid or alginic acid-derived oligosaccharides from seaweeds such as brown algae. For example, a method of softening and extracting algae through high pressure treatment, high temperature and high pressure treatment or radiation treatment, or a method using acid alkali treatment has been reported. However, the conventional method has a problem that the extraction efficiency of the alginic acid is somewhat difficult because most carbohydrates derived from seaweed are relatively stable to acid or alkali.

Therefore, in recent years, in order to produce functional seaweed oligosaccharides, interest in enzymatic degradation using enzymes rather than chemical degradation of chemically degrading seaweed-derived polysaccharides has been increasing.

Alginate lyase, a degrading enzyme that decomposes alginic acid, is not known for its decomposition mechanism, but is known to perform alginic acid degradation by β-elimination. Alginate degrading enzymes are classified according to their location on the pM-block (polymannuronate block) or pG-block (polyguluronate block) to break down, and one type is between mannuronate and mannuronate. Alginate degrading enzymes (EC 4.2.2.3, β-D-mannuronan lyase) that degrade β-1,4 sugar bonds, and the other type α-1 between gluuronate and guluronate And alginate degrading enzymes (EC 4.2.2.11, α-D-guluronan lyase) that break down sugar bonds.

In order to efficiently produce alginic acid oligosaccharides that can be industrially used for various purposes, alginic acid degrading enzymes are required. Therefore, research for the search for microorganisms that secrete alginic acid degrading enzymes is necessary.

Recently, due to social problems such as environmental pollution caused by fossil fuels and depletion of resources such as crude oil, interest in renewable energy is increasing, and researches related to bioenergy production using biomass of nature are attracting attention. In particular, research on the production of bioenergy using seaweed has attracted attention as an alternative to overcome the problems of bioenergy production using food resources such as corn.Alginate, which forms the cell wall of algae, is used for bioenergy production using seaweed. There is a need for development of a degrading enzyme that can decompose is excellent.

According to the necessity as described above, an object of the present invention is to provide a novel microorganism having excellent alginic acid resolution.

Another object of the present invention is to provide a method for preparing an alginic acid oligomer using a biocatalyst having alginic acid resolution according to the necessity as described above.

In order to achieve the above object, the present invention provides a methylobacterium sp. ( Methylobacterium sp.) Having an alginic acid resolution.

In another aspect, the present invention provides a biocatalyst having an alginic acid resolution including at least one selected from the group consisting of the Methylobacterium sp., A culture of the strain and a concentrate of the culture. .

In addition, the present invention provides a method for producing an alginic acid oligomer comprising the step of reacting the biocatalyst having the alginic acid resolution with seaweeds.

Hereinafter, the present invention will be described in detail.

To check that the present inventors have superior alginate resolution tumefaciens in HJM27 (Methylobacterium sp. HJM27) to the methyl novel methyl isolated from internal tumefaciens sp (Methylobacterium sp.) Of marine animals that dietary algae The present invention has been completed.

In the present invention, alginic acid (alginic acid) is a polysaccharide constituting the cell wall of algae, mainly brown algae, α-L- gluronate (guluronate) and C5 epimer (β-D-manneuronic acid (1, 4'-β-D-mannuronate) is a heteropolysaccharide consisting of α-1,4 bond or β-1,4 bond. The alginic acid is also referred to as alginate or sea acetate, and the alginic acid is polyguluronate block (pG block), polymannuronate block (pM block) and polygluronic acid-polymanuronic acid block (polyguluronate- mannuronate block, pM / G block or pG / M block).

In the present invention, alginate degrading enzyme, which is a degrading enzyme that decomposes alginic acid, is called alginase or alginate depolymerase. It is an enzyme that can decompose alginic acid through. The alginic acid can break down β-1,4-glycosidic bonds of alginic acid to produce products having 4,5-unsaturated nonreducing ends. The alginic acid degrading enzyme is a poly (M) lyase (1 → 4) -β-D-mannuronan lyase that breaks down β-1,4 sugar bonds between mannuronate and mannuronate. , EC 4.2.2.3) and poly (G) lyases (1 → 4) -α-L- that break down α-1,4 sugar bonds between guluronate and guluronate guluronan lyase, EC 4.2.2.11).

In the present invention, the biocatalyst may include a microorganism that acts as a catalyst in various chemical reactions or biochemical reactions or a substance secreted by the microorganisms, for example, a microorganism that catalyzes a chemical reaction or a biochemical reaction, It may include a microbial culture and a mixture thereof.

In the present invention, the culture medium means that the nutrients required by the culture medium for cultivating the tissues of microorganisms or animals or animals are included as main ingredients, or further include additional substances in addition to the main ingredients for special purposes. Also called culture medium, incubator or culture solution. The medium may be a natural medium, a synthetic medium or a selective medium, there are solid or liquid medium depending on the properties, but is not limited thereto.

The present invention relates to a strain of Methylbacterium genus having alginic acid resolution. Tumefaciens sp to the methyl may be methyl tumefaciens in HJM27 (Methylobacterium sp. HJM27).

In one embodiment of the present invention, the strain with alginic acid resolution according to the present invention proceeded in two steps. Specifically, the strain search is primarily for marine animals dieting algae, specifically Haliotis discus hannai , seashell ( Billillus) cornutus ), sea cucumber ( Stichopus) japonicus ), sea squirt ( Halocynthia roretzi ), and doghouse ( Urechis unicinctus ) was isolated from the sample obtained in the gut of the alginate, and the alginate resolution of the first screened strain was examined by selecting the strain having the highest resolution.

Through the above search, the strains selected as having excellent alginate resolution were identified as belonging to the genus Methylobacterium. Specifically, as a result of 16S rDNA sequencing analysis of the selected strain, the selected strain HJM27 of the present invention, as shown in Figure 2, alphaproteobacteria ( alphaproteobacteria ) Methylobacterium sp. It is showed a bacteria with the highest similarity (99%), which was designated as tumefaciens in HJM27 (Methylobacterium sp. HJM27) methyl.

The HJM27 in the methylobacterium was deposited on April 13, 2010 at the Biological Resource Center located in Yuseong-gu, Daejeon, Korea, and received the accession number KCTC 11680BP.

The MJ bacterium HJM27 (KCTC 11680BP) is an alginate degrading enzyme producing strain that has been found to have excellent alginate degrading activity, and the growth rate was sharply lowered when the concentration of NaCl in the medium was 2.5%. (halotolerant stain). According to the results confirmed, the MJ bacterium HJM27 (KCTC 11680BP), the NaCl content in the medium is expressed as a concentration relative to the weight of NaCl relative to the volume of the total composition (NaCl content (w) / total medium volume (v) NaCl content is 0.75% (w / v) to 2.75% (w / v), preferably 0.75% (w / v) to 1.25% (w / v) or 2.25% (w) / v) to 2.75% (w / v).

The MJ bacterium HJM27 (KCTC 11680BP) can be cultured in a conventional medium, for example a conventional medium containing a nitrogen source, preferably in a medium containing yeast extract (Yeast Extracct) and peptone (peptone) Can be cultured. The yeast extract is a natural additive and consists of water-soluble components such as amino acids, peptides, carbohydrates, and salts, which are components of the yeast cell. The yeast extract is made by adding edible enzymes to the edible yeast, a method of hydrolyzing the polypeptide of the edible yeast, it can be added to the salt in the manufacturing process. The peptone is an inducible protein that is an acid, an alkali, or a degradation product decomposed by a protease or peptidase, and includes a degradation product of pepsin of a protein. The peptone is a lower molecule than proteases in the proteolytic products, is water soluble and does not coagulate with heat.

In addition, the MJ bacterium HJM27 (KCTC 11680BP) can be cultured in a medium to which no alginic acid is added or a medium to which alginic acid is added.

The genus HJM27 (KCTC 11680BP) may produce alginate degrading enzymes. The optimum reaction temperature of the degrading enzyme produced by HJM27 (KCTC 11680BP) or Methylbacterium HJM27 (KCTC 11680BP) is 25 ° C., and HJM27 (KCTC 11680BP) or Methylbacterium The optimum reaction pH of the degrading enzyme produced by HJM27 (KCTC 11680BP) in Methylbacterium is pH 9.0, and can maintain high activity in the neutral and weak alkaline range. In addition, the concentration of the optimal reaction NaCl of the degrading enzyme produced by the HJM27 (KCTC 11680BP) genus Methylbacterium or HJM27 (KCTC 11680BP) genus is 1.25% (w / v) to 1.75% (w / v), preferably from 1.4% (w / v) to 1.6% (w / v).

The present invention also relates to a biocatalyst having alginic acid resolution.

The biocatalyst having the alginic acid resolution may be at least one selected from the group consisting of the strain of the genus Methylobacterium, preferably HJM27 (KCTC 11680BP) of the genus Methylbacterium, the culture of the strain and the concentrate of the culture. It may be to include.

The culture of the strain means a culture medium obtained by culturing HJM27 (KCTC 11680BP) in Methylbacterium, and may be a culture medium containing the strain or a cell-free culture medium in which the cells are removed. have. The culture of the strain may be a culture medium obtained after culturing the MJ bacterium HJM27 (KCTC 11680BP) in a liquid culture medium, but the nature of the culture is not limited to liquid or solid.

The culture medium may be a conventional medium for culturing the tissues of microorganisms or animals, preferably one or more selected from the group consisting of yeast extract (Yeast Extracct) and peptone (peptone) may be further included, More preferably, yeast extract (Yeast Extracct) and peptone (peptone) may be included.

In addition, alginic acid may be added to the culture medium, and the content of alginic acid is 0.4% (w / v) to 1.25% (w / v), preferably 0.85% (based on the content of alginic acid relative to the total medium volume). w / v) to 1.15% (w / v), more preferably 0.9% (w / v) to 1.1% (w / v).

In addition, the NaCl content of the culture medium is 0.75% (w / v) to 2.75% (w / v), preferably 0.75% (w / v) to 1.75% (w) based on the content of NaCl relative to the total medium volume. / v) or 2.25% (w / v) to 2.75% (w / v), more preferably 1.25% (w / v) to 1.75% (w / v), even more preferably 1.4% (w / v) ) To 1.6% (w / v).

The concentrate of the culture means that the culture of the strain is concentrated in a conventional manner. The concentrate of the culture may be a concentrate of a liquid culture medium in which the culture of HJM27 (KCTC 11680BP) in the methylobacterium is concentrated in a conventional manner, but the nature of the culture is not limited to liquid.

The present invention also relates to a production method for producing a biocatalyst having the alginic acid resolution.

The biocatalyst manufacturing method may include culturing the MJ bacterium HJM27 (KCTC 11680BP) in a medium.

More specifically, the biocatalyst manufacturing method may include culturing the MJ bacterium HJM27 (KCTC 11680BP) in a medium containing the yeast extract and the peptone.

NaCl content of the yeast extract and the medium containing the peptone is 0.75% (w / v) to 2.75% (w / v), preferably 0.75% (w / v) to 1.75% (w / v) or 2.25 % (w / v) to 2.75% (w / v), more preferably 1.25% (w / v) to 1.75% (w / v), even more preferably 1.4% (w / v) to 1.6% ( w / v).

The alginic acid content of the medium is 0.4% (w / v) to 1.25% (w / v), preferably 0.85% (w / v) to 1.15% (w / v), based on the weight of alginic acid relative to the total medium volume, More preferably, it may be 0.9% (w / v) to 1.1% (w / v).

The step of culturing the MJ bacterium HJM27 (KCTC 11680BP) in the culture medium may be carried out for 36 hours to 60 hours, preferably 42 hours to 54 hours, more preferably 45 hours to 51 hours.

The present invention also relates to a method for producing an alginic acid oligomer using the biocatalyst having the alginic acid resolution.

The manufacturing method may include reacting the biocatalyst having the alginic acid resolution with at least one selected from the group consisting of algae and alginic acid.

The sea algae is a word that refers to algae flying in the sea and includes a conventional algae. The algae includes at least one selected from the group consisting of green algae, brown algae, and red algae, and may be preferably brown algae. The brown algae may be, for example, 톳, wakame seaweed, kelp, rhubarb, or maban, but is not limited thereto. The alginic acid oligomer may be a common alginic acid oligomer from which alginic acid is decomposed, and for example, polygluronic acid block, polymanneuronic acid block, polygluronic acid-polymanneuronic acid block, or the polygluronic acid block, polymanneuronic acid block. And it may be a sugar polymer to which at least one selected from the group consisting of polygluronic acid-polymanneuronic acid block is bonded.

The preparation method may be performed at 23 ° C. to 27 ° C., preferably 24 ° C. to 26 ° C., more preferably at 25 ° C., and the preparation method may be pH 7.5 to pH 11, preferably pH 8 to It may be carried out at pH 10, more preferably pH 8.5 to pH 9.5.

The content of the seaweed is 0.15% (w / v) to 1.25% (w / v), preferably 0.35% (w / v) to 1.15% (based on the weight of the alginic acid included in the seaweed relative to the total volume of the reaction solution). w / v), more preferably 0.5% (w / v) to 1.0% (w / v), and the content of the alginic acid is 0.15% (w / v based on the weight of the alginic acid relative to the total volume of the reaction solution). ) To 1.25% (w / v), preferably 0.35% (w / v) to 1.15% (w / v), more preferably 0.5% (w / v) to 1.0% (w / v) have.

Methylbacterium genus HJM27 (KCTC 11680BP) of the present invention shows a remarkably superior activity compared to other microorganisms with the previously reported alginic acid resolution, it is possible to obtain the optimum reaction yield according to specific culture conditions and specific reaction conditions It is expected to be usefully used as a biocatalyst, and it is expected to contribute greatly to industries related to pharmaceuticals and functional foods.

As described above, HJM27 (KCTC 11680BP) of the genus Methylobacterium according to the present invention is remarkably excellent alginate resolution compared to other microorganisms having the previously reported alginic acid resolution, decomposing alginic acid to treat human health and disease In addition to producing a variety of alginate oligomers, which are high value-added products with various functionalities, etc., in addition to the bioenergy production using seaweeds, it is possible to increase the efficiency of bioenergy production by decomposing the main components of the cell walls of seaweeds. The value of industrial use is very high in various industries, including those related to pharmaceuticals and functional foods.

Figure 1 is a nucleotide sequence of 16S rDNA of, tumefaciens in HJM27 (Methylobacterium sp. HJM27) methyl, according to one embodiment of the present invention.
Figure 2 is a diagram showing a phylogenetic relationship with other bacteria based on the nucleotide sequence of 16S rDNA of the genus HJM27 according to an embodiment of the present invention. The number in <> next to the description about the strain species of FIG. 2 is an accession number of NCBI database.
FIG. 3 is a graph showing the degree of cell growth and relative activity of alginic acid per cell according to the nitrogen source of HJM27 in Methylbacterium according to the nitrogen source. The growth rate of the cells is represented by a straight line (line), based on the right value indicated OD ₆₀₀ , the sugar alginic acid resolution is represented by a bar graph (bar), the group showing the optimal activity (peptone) It is shown by the left figure which shows the relative activity based on the addition group).
Figure 4 is a graph showing the growth rate of the cell growth and alginate degradation per cell of the genus HJM27 according to the peptone and yeast extract according to an embodiment of the present invention. The growth rate of the cells is represented by a straight line (line), based on the right value indicated OD ₆₀₀ , the sugar alginic acid resolution is represented by a bar graph (bar), the group showing the optimum activity (peptone and yeast extract It is shown by the left figure which shows the relative activity based on the addition group, PSY medium). White bars and white straight line graphs show the group added with peptone (PS medium), black bars and black straight line graphs show the group added with peptone and yeast extract (PSY medium).
FIG. 5 is a graph showing the growth rate of cells and alginate resolution per cell of MJ bacterium HJM27 according to the amount of alginic acid added according to an embodiment of the present invention. The growth rate of the cells is represented by a straight line (line), based on the right value of OD ₆₀₀ , the sugar alginic acid resolution is represented by a bar graph (bar), the group showing the optimum activity (alginate addition amount 1.0 the relative activity based on% (w / v)).
Figure 6 is a graph showing the alginic acid resolution according to the temperature of the biocatalyst produced by the genus HJM27 in methyllobacterium according to an embodiment of the present invention.
7 is a graph showing the alginic acid resolution according to the pH of the biocatalyst produced by the genus HJM27 in Methylbacterium according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily practice the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

[Example]

Example 1 Screening and Isolation of Marine Microorganisms Having Alginic Acid Resolution

The sample for the isolation of microorganisms with alginate resolution was Haliotis , a marine animal purchased from Daebyeon Port and Gijang-eup, Gijang-gun, Busan, Korea in May 2009. discus hannai ), hermit ( Batillus) cornutus ), sea cucumber ( Stichopus japonicus ), sea urchin ( Halocynthia) roretzi ) and Gaeul ( Urechis unicinctus ) were prepared. More specifically, the sample is separated from the viscera of the marine animal, and then chopped the separated viscera sterilized diluent (NaCl 2.5 g, KH ₂ PO ₄ 0.1 g, FeSO ₄ · 7H ₂ O 0.05 g, KCl 0.05 g, NH ₄ Cl 0.1 g, distilled water 1L, pH 7) was prepared by diluting 1,000 times by a continuous dilution method.

Separation of microorganisms having alginic acid resolution using a sample prepared from the intestine of the marine animal proceeded in two steps.

More specifically, the search for the microorganism having the alginic acid resolution was carried out by a method of confirming the alginic acid resolution by first plating 0.1 mL of the sample solution diluted 1,000-fold on the multilayer plate medium. The multi-layered flat medium is composed of two layers of a lower medium and an upper medium, and the composition of the lower medium (pH 7) is 2.5% (w / v) of NaCl, 0.1% of KH ₂ PO ₄ , and the composition of the total medium composition. (w / v), FeSO ₄ .7H ₂ O 0.05% (w / v), KCl 0.05% (w / v), NH ₄ Cl 0.1% (w / v) and agar 2.0% (w / v) The composition of the upper medium (pH 7) is composed of the content of sodium alginate 1.0% (w / v) and agar 2.0% (w / v) relative to the total medium composition volume. 0.1 mL of the 1,000-fold diluted sample was plated on the multi-layered plate medium, incubated at 25 ° C. for 3 days, and then a strain having a colony size of 1 mm or more was collected using platinum teeth among the colonies formed. Cells were purified purely by the method of separating several times repeatedly by smearing onto a new multilayer plate medium.

As a result of the culture, it was confirmed that about 50,000 colonies were grown on the separation medium, and the degree of alginic acid resolution was confirmed for the strain determined to have excellent alginate resolution because the colony size was 1 mm or more.

After performing the first step of purely separating the strain, the second strain was isolated the most excellent alginate resolution by checking the alginate resolution of the purely isolated cells. Confirmation of alginic acid resolution for isolation of the strain having excellent alginic acid resolution was performed by plate assay and DNS method.

More specifically, in the first step, purely isolated strains were cultured. The culture of the pure strain isolated PYS medium (peptone 0.5% (w / v), yeast extract 0.3% (w / v), sodium alginate 1.0% (w / v), NaCl 1.5% (w / v), inoculated at pH 7) and shake cultured at 25 ° C. and 150 rpm for 24 hours.

The first confirmation of the alginic acid resolution was performed by plate assay method. The plate assay was performed in the following manner. First, 30 μL of the strain cultured on the above 24 hours shaken in PS agar medium (peptone 0.5% (w / v), sodium alginate 0.8% (w / v), agar 1.5% (w / v)) for checking alginic acid resolution. And it was incubated for 2 days at 25 ℃. Thereafter, 10% CPC (cetylpyridinium chloride monohydrate) was administered to the medium in which the strain was grown, and 10 minutes later, the activity of alginic acid degrading enzyme was confirmed. The strain isolated from about 27 colonies by this method was selected as the first excellent strain.

The second confirmation of the alginic acid resolution was performed by the DNS method for the strain selected as the first excellent strain. The DNS method is a method for confirming reducing sugar generating ability, and was performed in the following manner. Primary excellent strain in the PYS medium (peptone 0.5% (w / v), yeast extract 0.3% (w / v), sodium alginate 1.0% (w / v), NaCl 1.5% (w / v), pH 7) Inoculated with 27 strains selected as, shaking culture under conditions of 25 ℃ and 150 rpm, reducing sugar production capacity was measured by time. The reducing sugar content for measuring the reducing sugar generation capacity was used by the DNS method, the experimental method was carried out in the following manner. First, the culture broth cultured for a predetermined time was centrifuged for 5 minutes under conditions of 14,000 × g and 4 ° C., and the separated supernatant was obtained, and the obtained supernatant was used as a coenzyme solution. 500 μL of the coenzyme solution was mixed with 1.0 mL of a substrate solution containing sodium alginate (0.8%), followed by reaction at 150 rpm and 25 ° C. in a thermostat for 30 minutes, and then the reaction solution was 14,000 × g and 4 It was centrifuged for 5 minutes at the conditions of ° C. 500 mL of the supernatant separated by centrifugation was added 2 mL of DNS solution, and then heated for 10 minutes, and the absorbance was measured at 540 nm. From the measured absorbance results, the reducing sugar produced was quantified using a standard calibration curve prepared using Mannuronic acid, and it was confirmed that the reducing sugar producing ability was proportional to the alginic acid enzyme activity.

As a result of the above, HJM27, which was isolated from abalone viscera, was finally selected as a strain having the best reducing sugar production ability, that is, the alginate resolution.

< Example  2> Marine microorganisms with alginic acid resolution HJM27 The identification of

Identification of the best selected HJM27 strain showing the best resolution in the second measurement was performed by 16S rDNA sequencing. Sequencing of the HJM27 strain for 16S rDNA sequencing was performed by requesting Macrogen (Korea). 16S rDNA of the HJM27 strain confirmed as a result of the request by Macrogen Co., Ltd. as shown in Figure 1, a total of 1,411 base pair (bp) base sequence was determined.

The sequencing of the 16S rDNA of the determined HJM27 strain was performed for homology search by NCBI's Blast program (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi) based on the information registered in GenBank. After multiple alignments are performed using the Clustal program (Clustal X ver. 1.8), the phylogenetic location is determined by analyzing the neighbor-joining method and the bootstrap method (n = 1,000). The results were prepared in a phylogenetic tree, and the results are shown in FIG. 2.

As shown in FIG. 2, the isolated strain HJM27 showed 99% similarity to the strain of the genus Methylobacterium (NCBI accesision #; EU741082) belonging to alpha-proteobacteria. The strain HJM27 exhibits a tumefaciens sp with a high affinity to methyl, HJM27 was the final identified as tumefaciens sp (Methylobacterium sp.) With methyl, was named tumefaciens in HJM27 (Methylobacterium sp. HJM27) methyl .

HJM27 in the methylobacterium was deposited on April 13, 2010 at the Biological Resource Center located in Yuseong-gu, Daejeon, Korea, and received the accession number KCTC 11680BP.

< Example  3> Methyl Bacterium  genus HJM27 Characteristics of Alginate Degrading Enzymes

In order to confirm the extent of growth according to the culture conditions of the isolated and identified Methylbacterium genus HJM27, that is, the strain propagation ability and the enzyme activity according to the reaction conditions of the alginic acid degrading enzyme produced by the Methylbacterium genus HJM27, Lobacterium spp. HJM27 was cultured. The composition of the culture medium for the culture is basically 0.1% (w / v) of KH ₂ PO _4, 0.05% (w / v) of FeSO ₄ · 7H ₂ O, 0.05% (w / v) of KCl and 2.0% of agar ( w / v) and the addition of nitrogen sources (yeast extract, peptone, NH ₄ Cl, urea and (NH ₄ ) ₂ SO ₄ ), sodium alginate and NaCl depending on the culture Or the content was adjusted.

Specifically, in relation to the culture medium of the strain, first confirming the strain growth capacity and enzyme activity according to the nitrogen source, to determine the optimum medium composition, by confirming the strain growth capacity and enzyme activity according to the culture time, to determine the optimum culture time, By confirming the growth ability and enzyme activity of the strain according to the alginic acid concentration and NaCl concentration, the optimum alginic acid content and the optimum NaCl content was confirmed. In addition, by checking the enzyme activity according to the reaction temperature and the reaction pH, the optimum reaction temperature and reaction pH was confirmed. The following experiments were performed three times each, and the average value of each result was used.

3-1: Culture medium  According to the nitrogen source Methyl Bacterium  genus HJM27 Strain Proliferative capacity  And resolution of alginic acid resolution

HJM27 in the methylobacterium was added to a basic medium composition consisting of 0.1% (w / v) of KH ₂ PO _4, 0.05% (w / v) of FeSO ₄ .7H ₂ O and 0.05% (w / v) of KCl. NaCl 1.5% (w / v), sodium alginate 1.0% (w / v) and nitrogen source (pH 7) was inoculated and incubated respectively. Yeast extract (0.3% (w / v)), peptone (0.5% (w / v)), NH ₄ Cl (0.5% (w / v)), urea, 0.5% (w / v), respectively. )) And (NH ₄ ) ₂ SO ₄ (0.5% (w / v)) were added in respective amounts. The culture of HJM27 in Methylbacterium was carried out by shaking incubation at 150 rpm and 25 ° C. for 2 days using a thermostat. After the culture was carried out, the strain growth capacity was measured by measuring the OD value at 600 nm, the alginic acid resolution of each reducing sugar production capacity by measuring the DNS method of Example 1, the activity of the optimum conditions 100 As percentage, it is expressed as relative activity (%). The measurement results are shown in Fig.

As shown in FIG. 3, the growth ability of the strain was remarkably excellent in the yeast extract, and in the case of peptone, about 60% of the yeast extract was confirmed, and in the case of other nitrogen sources, the strain growth ability was found to be inadequate. . Therefore, it was confirmed from the above results that the optimum nitrogen source for culturing the genus HJM27 in the methylbacterium is a yeast extract. Also, peptone was the best in alginate degradation associated with enzymatic activity, and yeast extract was found to have a similar degree of enzymatic activity as in peptone, but was very low in other mineral nitrogen sources. Therefore, in the process of preparing the reaction solution, that is, the biocatalyst using HJM27 in Methylbacterium, it is preferable to add a yeast extract as a nitrogen source to the culture medium in order to propagate the strain of HMM27 in Methylbacterium. It was evaluated that it is preferable to add peptone as a nitrogen source to the culture medium to be used as the final biocatalyst after strain propagation.

3-2: according to the incubation period Methyl Bacterium  genus HJM27 Strain Proliferative capacity  And resolution of alginic acid resolution

HJM27 in the methyllobacterium was 0.1% (w / v) of KH ₂ PO _4, 0.05% (w / v) of FeSO ₄ .7H ₂ O, 0.05% (w / v) of KCl, 1.5% (w / v) of NaCl. ) And the composition of the content of 1.0% (w / v) sodium alginate as a nitrogen source, peptone (0.5% (w / v) and peptone (0.5% (w / v)) and yeast extract (0.3% (w / v) )) Was inoculated in each of the added medium (pH 7) and cultured.

The culture of HJM27 in Methylbacterium was carried out by shaking culture at 150 rpm and 25 ° C. for 4 days using a thermostat. While performing the incubation, strain propagation was measured by measuring the OD value at 600 nm, and alginic acid resolution was measured by the DNS method of Example 1, respectively, and then 100% of the activity under optimum conditions was measured. Relative activity (Relative activity (%)). The measurement results are shown in FIG. 4.

As shown in FIG. 4, in relation to the growth ability and alginic acid resolution of the strain, the group added with both peptone and yeast extract (PSY) was about 200% or more than the group added with only peptone and sodium alginate (PS). As a result, it was evaluated that the addition of peptone and yeast extract as a nitrogen source to the medium was preferred. These results were interpreted to be due to the smooth growth of the microorganism by the addition of the yeast extract in the growth stage of the microorganism.

In addition, in relation to the incubation period, the growth of the strain was significantly increased up to 2 days after inoculation, but decreased slightly on the third day, and the optimal incubation period was confirmed to be 2 days. In addition, with respect to the alginic acid resolution, the highest activity was shown on the second day, and the activity was significantly decreased after two days. In addition, in the group to which only peptone was added, the number of cells was decreased on the second day of culture compared to the first day of culture, but the activity was significantly increased.

From the above results, while the maximum reported alginate degradation activity of the other strains reported previously was 144 hours incubation, the MJ bacterium HJM27 improved cell growth and alginate resolution even when cultured for a short time. It is expected to be applicable to various industries.

In addition, from the above results, in terms of cell growth rate and alginic acid resolution, it was expected that culturing up to 2 days in the group to which both peptone and yeast extract were added (PSY) was performed.

3-3: according to the alginic acid content Methyl Bacterium  genus HJM27 Strain Proliferative capacity  And resolution of alginic acid resolution

HJM27 in the methyllobacterium was 0.1% (w / v) of KH ₂ PO _4, 0.05% (w / v) of FeSO ₄ .7H ₂ O, 0.05% (w / v) of KCl, 1.5% (w / v) of NaCl. ) Was inoculated in a medium (pH 7) added with a controlled content of sodium alginate in a medium composition consisting of 0.5% (w / v) of peptone and 0.3% (w / v) of yeast extract.

The culture of HJM27 in Methylbacterium was carried out by shaking incubation at 150 rpm and 25 ° C. for 2 days using a thermostat. After the culture was carried out, the strain growth capacity was measured by measuring the OD value at 600 nm, the alginic acid resolution of each reducing sugar production capacity by measuring the DNS method of Example 1, the activity of the optimum conditions 100 As%, it is expressed as Relative activity (%). The measurement results are shown in FIG. 5.

As shown in FIG. 5, the cell growth was confirmed to be smooth when the concentration of alginic acid (sodium alginate) is 0.5% (w / v) or more in relation to the growth ability and alginic acid resolution of the cells. In addition, with respect to alginic acid resolution, it was gradually increased until the alginic acid concentration was 1.0% (w / v), and it was confirmed that the alginic acid concentration was 1.0% (w / v), and the alginic acid concentration was 1.5% ( In case of w / v) or more, the activity was found to decrease. In addition, even when no alginic acid was added, it showed a certain degree of activity, and it was confirmed that a certain amount of alginic acid degrading enzyme was expressed even when the HJM27 genus Methylbacterium was cultured in a medium without adding alginic acid. In addition, it was confirmed that the enzyme activity was the best when the alginic acid concentration was 1.0% (w / v) even when comparing the enzyme activity per unit cell by dividing the enzyme activity into cell growth.

3-4: NaCl  According to the content Methyl Bacterium  genus HJM27 Strain Proliferative capacity  And resolution of alginic acid resolution

HJM27 in the methylobacterium was 0.1% (w / v) of KH ₂ PO _4, 0.05% (w / v) of FeSO ₄ .7H ₂ O, 0.05% (w / v) of KCl, 1.0% (w / v) of sodium alginate. v), NaCl content was adjusted from 0% (w / v) to 3% (w / v) in a medium composition consisting of 0.5% (w / v) peptone and 0.3% (w / v) yeast extract. The culture was inoculated with the added medium (pH 7).

The culture of HJM27 in Methylbacterium was carried out by shaking incubation at 150 rpm and 25 ° C. for 2 days using a thermostat. After the culture, strain propagation capacity was measured by measuring the OD value at 600 nm, and alginic acid resolution was measured by reducing sugar production ability (g / L) by the DNS method of Example 1. In addition, in order to confirm the alginate resolution per cell, the measured amount of reducing sugar produced was divided by the OD ₆₀₀ value. The measurement results and calculation results are shown in Table 1 below.

NaCl content (%, w / v) Biomass (OD ₆₀₀ ) Reducing sugar concentration (g / L) Reduced sugar per Biomass (g / L / OD ₆₀₀ ) 0.0 5.322 0.781 0.147 0.5 5.762 0.993 0.172 1.0 6.432 0.792 0.123 1.5 6.092 1.217 0.200 2.0 6.162 1.064 0.173 2.5 6.572 0.245 0.037 3.0 3.782 0.250 0.066

As shown in Table 1 above, the NaCl content was found to be optimal when the NaCl content was 2.5% (w / v) with respect to the proliferation ability of the cells, and was also excellent when the 1.0 (w / v). The high peak of growth was expected to be due to the formation of floc as the strain grows. On the other hand, the growth was sharply reduced when the NaCl content is 3.0% (w / v), from which it was determined that the genus HJM27 is a halolerant strain rather than basophils.

In addition, with respect to the alginic acid resolution, it was confirmed that the reduced sugar equivalent and the enzyme activity per unit cell produced (Reduced sugar per Biomass) was the best when the NaCl content is 1.5% (w / v).

Therefore, it was confirmed that the optimal NaCl content for the preparation of the biocatalyst through the culturing of HJM27 in Methylbacterium was 1.5% (w / v).

3-5: Methyl Bacterium  genus HJM27 Degradation of Alginate with Temperature of Alginate Degrading Enzymes

HJM27 in the methylobacterium was 0.1% (w / v) of KH ₂ PO _4, 0.05% (w / v) of FeSO ₄ .7H ₂ O, 0.05% (w / v) of KCl, 1.0% (w / v) of sodium alginate. v), incubated in a medium (pH 7) consisting of NaCl 0.5% (w / v), peptone 0.5% (w / v) and yeast extract 0.3% (w / v). The culture of HJM27 in Methylbacterium was carried out by shaking incubation at 150 rpm and 25 ° C. for 2 days using a thermostat. After the incubation, the reducing sugar production ability (g / L) was measured by the DNS method of Example 1 while adjusting the reaction temperature to 20 ℃ to 40 ℃. The measurement results are shown in FIG. 6.

As shown in FIG. 6, the alginic acid resolution of the biocatalyst solution showed the highest value at 25 ° C., and the alginic acid resolution represented by the relative value of the highest value at 20 ° C., that is, the relative activity was about 25%, was nearly active at 30 ° C. or higher. It was confirmed that this did not appear. Therefore, it was confirmed that the optimum reaction temperature of the biocatalyst prepared using the MJ bacterium HJM27 was 25 ° C.

3-6: Methyl Bacterium  genus HJM27 Of alginate to produce pH Resolution of alginic acid according to

HJM27 in the methylobacterium was 0.1% (w / v) of KH ₂ PO _4, 0.05% (w / v) of FeSO ₄ .7H ₂ O, 0.05% (w / v) of KCl, 1.0% (w / v) of sodium alginate. v), incubated in a medium (pH 7) consisting of NaCl 0.5% (w / v), peptone 0.5% (w / v) and yeast extract 0.3% (w / v). The culture of HJM27 in Methylbacterium was carried out by shaking incubation at 150 rpm and 25 ° C. for 2 days using a thermostat. After the incubation, each reducing sugar generation capacity (g / L) was measured by the DNS method of Example 1 while adjusting the reaction pH to pH 3 to pH 11. The measurement results are shown in FIG. 7.

Specifically, the pH is adjusted to 200 mM sodium acetate buffer (pH 3 to pH 6), 40 mM sodium phosphate buffer (pH 6 to pH 8) and 100 mM in the reaction solution glycine-NaOH buffer solution (pH 8 to pH 11) was used.

As shown in FIG. 7, the alginic acid degradability according to the pH showed the highest value in the alkaline region pH 9.0, and it was confirmed that the optimal pH of the alginic acid degrading enzyme produced by the HJM27 in the methyllobacterium was 9.0. In order to check the error according to the type of buffer solution, again, 300 mM GTA buffer solution (3,3-dimehtylglutarid acid, Tris (hydrosymethyl) aminomethane, 2-amino-2-methyl-1,3-propanediol, pH 3 to pH 10 The same result was confirmed also in the result measured using). Under acidic or alkaline conditions based on pH 9, which is the optimum pH, it was confirmed that the alginic acid resolution decreased as the acidic or alkaline properties became stronger.

Therefore, the optimum reaction pH of the biocatalyst prepared using the MJ bacterium HJM27 was found to be pH 9. In addition, by using the biocatalyst solution prepared from the culture medium of HJM27 in Methylbacterium (a supernatant removed from the culture medium), the degradation activity of polysaccharides such as starch, carrageenan, pectin, and agar was measured. It was confirmed that degrading activity was not observed with respect to alginic acid, and that the enzyme produced by HJM27 in the methyllobacterium was alginic acid degrading enzyme.

Based on the above results, the present invention can be prepared alginate degrading enzyme that can produce alginate oligomer by decomposing the alginic acid HJM27 in the present invention, the alginate degrading enzyme from the HJM27 of the methyllobacterium genus Since the optimum conditions for producing and optimum reaction conditions of the alginic acid degrading enzyme have been confirmed, the HJM27 in the methyllobacterium is expected to be applied to various industrial fields including industries related to pharmaceuticals and functional foods and bioenergy related industries. do.

Korea Biotechnology Research Institute KCTC11680 20100413

Claims (4)

Alginic acid having a resolution and the optimum alginate decomposition temperature is 25 ℃, optimum growth concentration of NaCl is 2.5% (W / V), with a salt tolerance tumefaciens sp strain methyl (Methylobacterium sp., KCTC 11680BP) . The biocatalyst having alginate resolution including at least one selected from the group consisting of the strain of claim 1, the culture solution of the strain, and the concentrate solution of the culture solution. Comprising: a yeast extract (Yeast Extract) and peptone (Peptone) is included, the I NaCl content of the medium 1.5% (w / v) of tumefaciens sp from the culture medium as methyl (Methylobacterium sp, KCTC 11680BP.) Inoculation; And culturing the strain inoculated in the culture medium for 45 hours to 51 hours.
Biocatalyst production method of claim 2 comprising a. The method of preparing an alginic acid oligomer comprising the step of reacting the biocatalyst of claim 2 at least one selected from the group consisting of seaweed and alginic acid at a temperature of 25 ° C. and a pH of pH 9.

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