KR101207584B1 - Strain of sanguibacter keddieii having an alginate-decomposition activity and method of producing alginate-oligomer using the same - Google Patents

Abstract

PURPOSE: A Sanguibacter keddieii NC9 with an ability of decomposing alginic acid is provided to be used in various industries such as foods, cosmetic products, pharmaceutical products, and bioenergy production. CONSTITUTION: A Sanguibacter keddieii NC9(KCTC 11916BP) has an ability of decomposing alginic acid. A biocatalyst with an ability of decomposing alginic acid contains the strain, a culture medium thereof, or a concentrate of the culture medium. A method for preparing the biocatalyst comprises a step of culturing the strain in a culture medium containing peptone and alginic acid. A method for preparing the alginic acid oligomer comprises a step of reacting the biocatalyst with marine algae or alginic acid.

Description

Sangobacter Cadiai strain having alginate resolution and alginate oligomer production method using the strain

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

Alginic acid (alginate) constitutes 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 used extensively since ancient times for its unique physical properties such as gel forming ability, high viscosity, and film forming ability. 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 addition, recently, due to social problems such as environmental pollution caused by fossil fuels and resource depletion 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.

Studies related to alginic acid degrading enzymes, in particular, studies on microorganisms having alginic acid degrading ability include Bacillus sp. N7151-B ( Bacillus sp. N7151-B, KACC 91428P, Korean Patent Publication No. 2010-0087959), Pseudomonas in strains of Pseudomonas species N7151-6 (Pseudomonas sp. N7151-6, KACC 91353P, Republic of Korea Patent Application Publication No. 2009-0092613 call) or in the Vibrio Vibrio spp S21 (Vibrio sp. S21, KCTC 18103P, Republic of Korea Patent Application Publication No. 2008-0006931) or Vibrio sp. AEBL-211 ( Vibrio sp. AEBL-211, Korean Journal of Life Science Vol. 13, No. 5, 718-722 (2003)) and the like. Since the activity was focused on the microorganisms studied, the efficient production of alginic acid oligosaccharides that can be industrially used for various purposes, especially for the efficient production of alginic acid oligosaccharides from seaweeds, abundant natural resources Accordingly, the necessity of research for searching for microorganisms having alginic acid degradability among various marine microorganisms or microorganisms derived from marine resources and alginic acid degrading enzymes produced by the microorganisms continues to increase.

According to the necessity as described above, an object of the present invention is to provide a novel microorganism having 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 Sanguibacter sp. Strain having 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 Sanguibacter sp., Algaic acid degrading strain ( Sanguibacter sp.), The culture medium of the strain and the concentrate of the culture medium. do.

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.

The present inventors have found that a new strain isolated from a mud flat in sanggwo bakteo South Coast of the Republic of Korea (Sanguibacter sp.) Of sanggwo bakteo Kane dieyi NC9 strain (Sanguibacter keddieii It was confirmed that NC9) has an excellent alginic acid resolution, and further confirmed the optimum growth conditions and alginic acid degradation conditions of the Sangobacter Cadiai NC9 strain to complete the present invention.

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, alginic acid degrading enzymes that decompose alginic acid are referred to as alginase or alginate depolymerase, and the complete mechanism for the degradation is not yet known. It is an enzyme that has the ability to 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 genus S. aureus having alginic acid resolution. The strain of genus S. may be Sanguibacter keddieii NC9.

In one embodiment of the present invention, the strain search with alginic acid resolution of the present invention proceeded in two steps. In detail, the search for strains having alginic acid resolution was carried out by diluting step by step using a sterilized dilution by taking a sample from a tidal flat in Namhae-gun, Gyeongsangnam-do, Korea (Longitude 128: 1: 38 and Latitude 34:53:57). A sample dilution liquid was prepared by the method described above. The strain having alginate resolution was isolated from the sample dilution solution first, and the alginate resolution of the first screened strain was examined to select the strain having the highest resolution.

Through the above screening process, NC9 strain selected as having superior alginate resolution was identified as genus Sanguibacter. sp.). Specifically, as a result of 16S rDNA sequencing analysis of the selected strain shown in Figure 1, the selected NC9 strain of the present invention was identified as belonging to Sanguibacter keddieii , the strain is Sanggubacter keddie NC9 ( Sanguibacter keddieii NC9).

Sanguibacter Cadiay NC9 ( Sanguibacter) keddieii NC9) was deposited on April 13, 2011 to the Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon, Korea, and was assigned the deposit number KCTC 11916BP.

Sangguibacter keddieii NC9 (KCTC 11916BP), which has been granted the accession number KCTC 11916BP, may be cultured in a conventional medium, for example, a conventional medium containing a nitrogen source, and specifically, peptone and yeast extract. (Yeast Extract) and mixtures thereof may be cultured in a medium containing any one selected from the group consisting of, more specifically, a medium containing peptone. The peptone is an induced protein which 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 Sangobacter Kedei NC9 can be cultured in a medium to which no alginic acid is added or a medium to which alginic acid is added, preferably in a medium to which alginic acid is added. As an example, the Sangobacter cadiai NC9 has an alginate of 0.02% (w / v) to 0.15% (w / v) or 0.05% (w / v) to 0.125% (w / v) in terms of cell growth and culture period. ) Or 0.09% (w / v) to 0.11% (w / v) in a medium containing 2 days.

In addition, the Sangyobacter cardiac NC9 has alginic acid degradability, and in this aspect, the Sangyobacter cardiac NC9 can produce alginic acid degrading enzyme. The degrading enzyme produced by the Sangobacter Cadiai NC9 or Sangobacter Cadiai NC9 exhibits excellent degradation activity at pH 9 to pH 7, preferably pH 9, showing high activity in the neutral and weak alkaline range. Confirmed. In addition, the degrading enzyme produced by the Sangobacter Cadiai NC9 or Sangobacter Cadiai NC9 exhibited excellent degradation activity up to 100 minutes even at a temperature of 40 ℃, the optimum activity was maintained up to 60 minutes, it was confirmed to have heat resistance .

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

The biocatalyst having the alginic acid resolution may include one or more selected from the group consisting of the Sangguibacter keddieii NC9 (KCTC 11916BP), the culture of the strain, and the concentrate of the culture.

The culture of the strain refers to a culture medium obtained by culturing Sangobacter caddie NC9, may be a culture medium containing the strain or a cell-free culture medium (cell-free). The culture of the strain may be a culture medium obtained after culturing Sangobacter Cadiai NC9 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 a tissue of a microorganism or a plant or animal, and any one selected from a nitrogen source, for example, a yeast extract, peptone, and a mixture thereof may be further added.

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 strains of the genus S. guobacter are concentrated in a conventional manner, but the shape of the culture is not limited to a 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 strain of the genus S. guobacter in a medium. More specifically, the biocatalyst manufacturing method includes culturing the Sanguobacter cadiai NC9 in a medium containing alginic acid and any one selected from the nitrogen source, preferably the yeast extract, peptone, and mixtures thereof. It may be.

The content of alginic acid in the medium is 0.02% (w / v) to 0.15% (w / v) or 0.05% (w / v) to 0.125% (w / v) based on the weight of alginic acid relative to the total medium volume or 0.09% (w / v) to 0.11% (w / v).

The step of culturing the Sangguibacter keddieii NC9 (KCTC 11916BP) in the culture medium is 20 to 50 hours or 24 to 40 hours or 30 to 38 hours or 35 to 37 hours in terms of alginic acid resolution. Can be done for 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 pH 6.0 to pH 9.5 or pH 7.0 to pH 9.5 or pH 8.5 to pH 9.5. In addition, the production method may be carried out at a temperature condition of 30 ℃ to 40 ℃ or 35 ℃ to 40 ℃.

Sangubacter caddie NC9 strain of the present invention ( Sanguibacter keddieii NC9, KCTC 11916BP) has alginic acid resolution unlike other S. gubacterium strains. In addition, it has different activity and reaction conditions from different microorganisms and genus different from other microorganisms with alginic acid degrading ability of different genera, and it is useful as a new biocatalyst because the optimum reaction yield can be obtained according to specific culture conditions and specific reaction conditions. It is expected to be a major contributor to the industries related to pharmaceuticals and functional foods.

As described above, the strains of Sangobacterium spp. According to the present invention have alginic acid resolution unlike other Sangobacter spp. Strains, which are remarkably superior to alginic acid degrading ability compared to other microorganisms having previously reported alginic acid degradation, thereby decomposing alginic acid. In addition to producing a variety of alginate oligomers, which are high value-added products with various functionalities related to human health and disease treatment, as well as bioenergy production using algae, bioalgae production efficiency is decomposed by decomposing the main components of the algal cell wall. It is possible to increase the industrial use value in various industries, including those related to pharmaceuticals and functional foods.

1 is a nucleotide sequence of 16S rDNA of Sangobacter Cadiai NC9 according to an embodiment of the present invention.
Figure 2 is a phylogenetic diagram showing the phylogenetic relationship with other bacteria based on the nucleotide sequence of Sangobacter Cadiai NC9 according to an embodiment of the present invention.
Figure 3 is a photograph showing the results of confirming the alginic acid resolution using the Sanggubacter Cadiai NC9 according to an embodiment of the present invention.
Figure 4 is a graph showing the results of measuring the cell growth of Sangobacter Cadiai NC9 according to the concentration of alginic acid in the medium according to an embodiment of the present invention.
Figure 5 is a graph showing the results of measuring the alginic acid resolution of Sangobacter Cadiai NC9 according to the alginic acid concentration in the medium according to an embodiment of the present invention.
Figure 6 is a graph showing the results of measuring the alginic acid resolution of Sangobacter Cadiai NC9 according to the culture medium pH according to an embodiment of the present invention.
7 is a graph showing the results of confirming the heat resistance of the alginic acid resolution of Sangobacter Cadiai NC9 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  1. Screening and Isolation of Marine Microorganisms with Alginate Resolution

Samples for the isolation of microorganisms with alginic acid resolution were collected from cold spring tidal flats (Longitude 128: 1: 38 latitude 34:53:57) in Namhae-gun, Gyeongsangnam-do, Korea. More specifically, the sample used for microbial separation of the present invention is a sterilized dilution solution (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 tidal flat was carried out in two steps.

More specifically, the screening of the microorganism having the alginate resolution was performed by first plating 100 μl of the diluted sample solution in alginate medium (sodium alginate 8.0 g / L, peptone 5.0 g / L, agar 15.0 g / L). It was carried out by the method of confirming. The medium to which the diluted sample solution was spread was incubated at 30 ° C. for 3 days using an incubator. After transferring the strain using a sterilized velvet cloth from the cultured medium for 3 days, the strain was purely separated by culturing the strain again for 3 days under the same conditions.

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 method.

More specifically, the purely isolated strain in step 1 was plated in a solid medium containing sodium alginate 10.0 g / L, peptone 5.0 g / L, agar 15.0 g / L and incubated in a 30 ℃ incubator for 24 hours. Thereafter, 10% CPC (cetylpyridinium chloride) was administered to the medium in which the strain was grown, and after 10 minutes, the CPC added to the medium with distilled water was removed, and then the alginate resolution was confirmed according to whether clear zones were generated. The confirmed result is shown in FIG. 3. As shown in FIG. 3, it was confirmed that a transparent ring was formed around the colony, and among the strains purely separated in step 1, NC9, which was found to have the highest alginate resolution, was selected as the final isolated strain, and then the experiment was performed.

Example  2. Microorganisms with Alginic Acid Resolution NC9 The identification of

Identification of the NC9 strain isolated from the secondary measurement was performed by 16S rDNA sequencing. Sequencing of the NC9 strain for 16S rDNA sequencing was performed by requesting Macrogen (Korea). As shown in FIG. 1, 16S rDNA of NC9 strain confirmed as a result of request by Macrogen Co., Ltd. was determined in total of 1,449 base pair (bp) base sequences. The determined base sequence is shown in FIG.

The NCBI Blast program (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi) is used for information registered in GenBank using the nucleotide sequence of 16S rDNA of the NC9 strain shown in FIG. 1. Homology screening, multiple alignment using Clustal program (Clustal X ver. 1.8), and then phylogenetic analysis by analyzing neighbor-joining method and bootstrap method (n = 1,000). The location was determined, and the result was prepared as a phylogenetic tree, and the result is shown in FIG. 2.

As shown in Figure 2, the isolated strain is Sanguibacter keddieii ST74 and Sanguibacter Showing high similarity to keddieii DSM 10542, the NC9 strain was finally identified as Sangobacter keddie, named Sanguibacter keddieii NC9.

Sanguibacter Cadiay NC9 ( Sanguibacter) keddieii NC9) was deposited on April 13, 2011 at the Korea Institute of Bioscience and Biotechnology, located in Yuseong-gu, Daejeon, Korea, and was assigned accession number KCTC 11916BP.

Example  3. Sanggubacter Keddie NC9  Growth Characteristics and Alginate Resolution of Strains

The growth degree according to the culture conditions of Sanguibacter keddieii NC9 KCTC 11916BP isolated and identified in Example 2, that is, the growth of the strain and the reaction conditions of the alginic acid degrading enzyme produced by Sanggubacter keddie NC9 In order to confirm the enzymatic activity according to the above, the Sangobacter caddie NC9 was incubated. The composition of the culture medium for the culture was basically configured to include sodium alginate 8.0 g / L, peptone 5.0 g / L and agar 15.0 g / L. The addition of nitrogen source (peptone), sodium alginate or the amount of the content was adjusted according to the confirmation through the culture.

Specifically, first check the cell growth and enzyme activity of the strain according to the alginic acid concentration of the strain, to determine the optimal alginic acid content. In addition, by checking the enzyme activity according to the reaction temperature and the reaction pH, it was confirmed the optimum reaction temperature and reaction pH. The following experiments were performed three times each, and the average value of each result was used.

3-1. According to the alginic acid content Sanggubacter Keddie NC9 Cell growth ability  Confirm

The Sangobacter caddie NC9 was inoculated into a medium containing 0.2 g to 1 g of alginic acid in the medium composition based on 1 L of the medium, followed by shaking culture at 30 ° C. and 150 rpm.

After the culture was carried out, the strain growth capacity was measured by measuring the O.D. value at 600 nm, the results are shown in FIG.

As shown in FIG. 4, the optimum culture medium condition of the Sangobacter cadydia NC9 was found to be 1 g, that is, 0.1% (w / v) of alginic acid based on 1 L of medium. Since the cell population increased by day, that is, 48 hours, 48 hours were found to be the most suitable.

3-2. According to the alginic acid content Sanggubacter Keddie NC9 Of Alginate Degrading Enzyme Produced by

Alginate resolution according to the culture conditions and culture time of the medium incubated under the same conditions as in Example 3-1 was measured every 12 hours for each medium, the coenzyme for confirming the resolution is the culture solution 4 ℃ and 14,000 rpm After centrifugation for 5 minutes under the conditions of, the supernatant was obtained by separating. Determination of the alginic acid degradability of the coenzyme is a mixture of the crude enzyme solution and 1.0 g / L of the alginic acid solution 1: 1 (coenzyme solution: alginic acid solution) on the basis of the volume ratio, using a thermostat at pH 7 and 30 ℃ The reaction was carried out for 30 minutes, and was performed by measuring the reducing sugar produced using the DNS method. The DNS method was performed by adding 2 ml of the DNS solution to 500 µl of the reaction solution in which the enzyme reaction was carried out, heating for 10 minutes, and measuring the absorbance at 540 nm. Enzyme activity of the alginic acid degrading enzyme produced by the above enzyme concentration according to the alginic acid concentration, that is, Sangobacter Cadiai NC9 is shown in FIG. 5. The enzyme activity was expressed as relative activity (Relative activity (%)) with the activity of the optimum conditions as 100%.

As shown in FIG. 5, in terms of the resolution of the alginic acid degrading enzyme produced by the Sangobacter cadiai NC9, as the optimal culture medium condition, 1 g of 0.1 g (0.1% (w / v)) of alginic acid is based on 1 L of the medium. It was confirmed that the resolution was increased up to 36 hours under the above medium conditions, and then decreased, so that the 36 hours was found to be most suitable.

3-3. Sanggubacter Keddie NC9 Of Alginate Degrading Enzyme Produced by pH  Check condition

Using the method of Example 3-2 to change the activity according to pH using a medium in which alginic acid, the medium condition showing the maximum enzymatic activity identified in Example 3-2, was added at a concentration of 1.0 g / L. Confirmed. The pH effect on the enzyme activity was confirmed in the pH condition of pH 5 to pH 9, and the pH was GTA buffer (3.3-dimethylglutaric acid 16.02 g / L, Tris (hydroxymethyl) aminomethane 12.11 g / L, 2-amino-2 -methyl-1,3-propanediol 10.51 g / L) was adjusted, and the alginate (alginate) was added to each buffer at a concentration of 1.0 g / L to measure the activity, the results are shown in Figure 6 Shown in The enzyme activity was expressed as Relative activity (%) with a resolution of 100% in the condition of pH 7 shown as 100% activity in Example 3-2.

As shown in FIG. 6, the enzyme activity was found to be the best at pH 9.0. More specifically, it showed similar activity in the range of pH 5.0 to pH 8.0, a slight increase in enzyme activity was confirmed as the pH was increased, but the enzyme activity was slightly lowered at pH 8.0, significantly higher than other pH at pH 9.0 Excellent resolution was confirmed. From the above results, alginic acid degrading enzyme produced by Sangobacter kdeii NC9 shows excellent activity in all of weak acidity, neutrality and weak alkalinity, but especially in weak alkali condition.

3-4. Sanggubacter Keddie NC9 Heat resistance of alginic acid dehydrogenase

The heat resistance of the alginic acid degrading enzyme produced by the Sanguobacter Cadiei NC9 was confirmed using the alginate 1% (w / v) medium, which is the medium condition showing the maximum enzymatic activity identified in Example 3-2.

As a preliminary experiment, the enzyme activity according to the temperature is mixed by the crude enzyme solution and alginic acid solution of 1.0 g / L concentration in a ratio of 1: 2 (coenzyme solution: alginic acid solution) using the method of Example 3-2. Then, the reaction was carried out for 30 minutes using a thermostat for each 10 ℃ at pH conditions of pH 7.0 and temperature conditions of 20 ℃ to 60 ℃.

After the reaction, enzyme activity was measured by DNS method as in Example 3-2. As a result of the measurement, the enzyme activity was found to be the highest at 40 ?.

In the case of 40 ℃ excellent in the enzyme activity, since the temperature conditions higher than the growth conditions of the room temperature and marine microorganisms, the stability of the enzyme at the temperature conditions was confirmed, and the heat resistance was confirmed.

In order to confirm the heat resistance, the enzyme activity with time at 40 ℃ and pH 9 was measured at 30 minutes intervals using the DNS method, the results are shown in FIG. The enzyme activity was expressed as Relative activity (%) with a resolution of 100% in the condition of pH 7 shown as 100% activity in Example 3-2.

As shown in FIG. 7, the optimum enzyme activity was maintained until 60 minutes of activity at 40 ° C., and the excellent enzyme activity was maintained up to 100 minutes, resulting in the degradation of alginic acid produced by Sanggubacter cadiai NC9. The enzyme was confirmed to have heat resistance for a certain time even at 40 ℃.

Based on the results as described above, the present invention Sanggubacter keddie NC9 can produce alginic acid degrading enzyme that can produce alginic acid oligomer by decomposing alginic acid, the optimum production of alginic acid degrading enzyme from the Sanggubacter keddie NC9 Since conditions and optimum conditions for the reaction of the alginic acid degrading enzymes have been confirmed, it is expected that the Sangoobacter caddie NC9 can be applied to various industrial fields including industries related to pharmaceuticals and functional foods, and bioenergy related industries.

Korea Biotechnology Research Institute KCTC11916BP 20110413

Claims (4)

Sanguibacter keddieii NC9, KCTC 11916BP with alginic acid resolution. 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. The biocatalyst preparation method of claim 2, comprising culturing the strain of claim 1 in a culture medium containing peptone and alginic acid. The alginic acid oligomer manufacturing method comprising the step of reacting the biocatalyst of claim 2 with at least one selected from the group consisting of algae and alginic acid.

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