KR100462904B1 - Manufacturing process for producing Alginate lyase using Xanthomonas sp. BH-1, newly isolated marine bacterium - Google Patents

Abstract

New marine microbial xanthomonas sp. Isolated from brown algae. It relates to a high-efficiency method for producing extracellular alginic acid enzyme by culturing BHC-1677C ( Xanthomonas sp. BH-1 KCTC 10677BP) in an optimized medium. The alginic acid degrading enzyme prepared by the present invention provides alginic acid degrading enzyme which can economically mass-produce alginic acid oligosaccharides by enzymatic hydrolysis of alginic acid derived from algae such as seaweed, mussels and kelp.

Description

Novel Marine Microbial Xanthomonas sp. Manufacturing process for producing Alginate lyase using Xanthomonas sp. BH-1, newly isolated marine bacterium}

It is known that about 46 genera and 95 species of brown algae are distributed on the coast of Korea. Among them, the major edible brown algae such as seaweed, sesame and kelp have annual output of about 470,000 M / T. Carbohydrates are the major constituent of seaweeds, and alginic acid among brown algae contains about 70 ~ 80% of the building, and is composed of fucoidan and laminaran. Seaweed-derived polysaccharide alginic acid is a material extracted from brown algae such as seaweed, sesame and kelp. It is a cell wall material of brown algae, and is composed of 1,4-glycosidic bond of D-mannuronic acid and L-guluronic acid. It is a linear copolymer polysaccharide and has various properties according to these composition ratios and is widely used as a thickener, stabilizer, emulsifier, caking agent in the food, medical, paper, and dyeing industries [Biosynthesis and application of alginates, Polym. Degrad. Stab., 59: 85-91, 1998]. In general, alginic acid requires a lot of time to dissolve at room temperature and has a strong viscosity more than necessary as the concentration is increased, there is a disadvantage that loses the intrinsic characteristics of the food when more than 0.5% concentration is added to the food. Therefore, the viscosity and gelability can be properly adjusted while maintaining the properties of alginic acid. If solubility can be improved, there is no need to limit the range of addition amount as necessary, and the viscosity and gelability of food can be artificially adjusted during food production. Alginic acid can be used in a wider range. In recent years, various studies have been conducted to expand the range of alginic acid use, and one field is the study of low molecular weight or oligosaccharide of alginic acid by microorganisms. In addition, as the various functionalities of oligosaccharides have been recently revealed, researches on them are being actively conducted. There are also oligosaccharides that are produced and used industrially, and various physiological functions, anti-caries, low calorie, and selective use of bifidus bacteria have been revealed. It is of interest to many sugar-related researchers. Oligosaccharide refers to a sugar in which two to thirteen sugars are combined, and refers to a sugar having a specific physiological function. Currently, oligosaccharides are mainly produced by enzymatic methods using various sugars such as sugar and starch, and some are industrially produced and widely used in various processed foods. On the other hand, various studies on the use of seaweed-derived polysaccharides are in progress, one of them is the use in the food or pharmaceutical field by the low molecular weight or oligosaccharides of alginic acid, algae polysaccharides. The low molecular weight or oligosaccharides of seaweed polysaccharides can be obtained by enzymatic digestion or physical and chemical digestion. Alginate degrading enzymes have not been commercialized so far in the world.In Korea, acetic acid, citric acid, lactic acid, succinic acid, maleic acid, formic acid, etc. Acid hydrolysis using the same organic acid and thermal hydrolysis decomposing at high temperature and high pressure are performed in parallel. [Manufacturing method of alginic acid oligosaccharide by organic acid, Korean Patent Application No. 1999-0034911; Alginic acid oligosaccharide, which is a natural polysaccharide using alginic acid, and preparation method thereof, Korean Patent Application No. 2000-0045776; Method for preparing low molecular polyman euronate and its new use as a serum lipid improving agent and functional food and health supplement containing the same, Korean Patent Application No. 2003-0022565].

Microorganisms producing alginate degrading enzymes for oligosaccharides of seaweed polysaccharides alginic acid have been reported as Pseudomonas, Sphingomonas, Xanthomonas, Enterobacter, Bacillus . ml-Enzyme-min-Reaction time is very low and commercialization is difficult [An exotype alginate lyase in Sphingomonas sp. A1: overexpression in Escherichia coli , purification, and characterization of alginate lyase IV, Protein Expression and Purification , 29: 33-41 , 2003; Production of alginate lyase from Enterobacter cloacae M-1, US Pat. No. 5,348,875; Production of alginate lyase, Japanese Patent No. 63,039,581, Thermostable alginate lyase from Bacillus stearothermophilus , U.S. Patent No. 5,139,945] The bacteriostatic microorganism Pseudomonas sp. The alginate lyase gene of W7 was isolated and cloned into Escherichia coli to produce intracellular alginate lyase by 0.047 U / mL [Alginate lyase production of halophilic Pseudomonas sp. by recombinant Escherichia coli , J. Microbiol. Biotechnol. 5: 92-95 (1995), the soil, sea water, seaweed, abalone, snails, was navigation alginate degradation microbes by separating the microorganisms of the 176 species from a sea urchin, etc. of Enterobacter spp the alginate lyase productive 0.355 mg -Reducing sugar / mL-Enzyme showed alginate degrading enzyme activity [Isolation and Growth Characteristics of Alginate Degrading Bacteria, Journal of the Korea Industrial Microbiology , 21 (3): 207 ~ 213, 1993]. Report on the Biological Activity of Alginate Oligosaccharides and Their Use [Growth-promotion of plants with depolymerized alginates by irradiation, Rad. Phys. Chem. , 59: 97-101, 2000], and research is ongoing. As such, alginic acid, an important natural material for marine biological resources, has been shown to exhibit antimicrobial activity against some bacterial and phytopathogenic fungi strains and is known to possess antiviral and antifungal properties. [Livestock using alginic acid oligosaccharide derivatives Disease prevention and treatment, Republic of Korea Patent Registration No. 0307187; Red algae extinction agent and fish disease treatment agent using alginate oligosaccharide derivative, Republic of Korea Patent Registration No. 0322511]. In addition to the antiviral and antibacterial functions, alginic acid extracts and their derivatives are known to lower low-density cholesterol levels and stabilize immunological reactions [Medical and biotechnological applications of marine macroalgal polysaccharides, Mar. Biotechnol. , 1: 181-196, 1993], as well as biological resources, as a raw material for pharmaceuticals. The research trend on alginate and alginate oligosaccharide production is known in 1976, since the chemical method and alginate-oligosaccharide production method using alginate degrading enzyme were known in 1976. Although production of alginase from Enterobacter cloacae M-1, US Patent No. 5,348,875, Alginate oligosaccharide and method for producing the same, US Patent No. 5,516,666, produced alginate degrading enzyme There are few studies on the microorganisms that produce alginate degrading enzymes with high efficiency.

The present invention provides a novel marine microorganism for producing alginate lyase for producing alginate oligosaccharides with high separation of marine microorganisms producing alginic acid degrading enzymes and a method for producing alginate degrading enzymes using the same. will be.

The present invention relates to a novel marine microorganism isolation from seaweed and to a method for producing alginate degrading enzyme with high efficiency. In addition, the alginic acid degrading enzyme provides an alginate degrading enzyme useful for preparing alginic acid oligosaccharides by enzymatic hydrolysis of algae-derived polysaccharide alginic acid.

1 is Xanthomonas sp. Optimal medium for alginate degrading enzyme production using BH-1 (Sucrose 5 g / L, (NH ₄ ) ₂ SO ₄ 9 g / L, (NH ₄ ) ₂ HPO ₄ 3 g / L, NaCl 10 g / L, Bulk culture was performed using a 10 L fermenter containing 3 g / L KCl, 0.05 g / L CaCl ₂ ). Since it is an aerobic microorganism, air is supplied at 0.1 vmv while stirring at 400 rpm, and the result is incubated for 54 hours while automatically controlling the pH in the fermenter to 8.0 using 2 N aqueous hydrochloric acid solution and 2 N aqueous sodium hydroxide solution.

2-A shows Xanthomonas sp. In order to determine the optimal enzymatic reaction temperature of alginic acid degrading enzyme produced by BH-1, the enzymatic reaction was carried out by varying the enzyme reaction temperature up to 20 ~ 70 ℃. 2-B is a result of performing the enzyme reaction by varying the pH of the enzyme reaction from 3 to 12 pH at 55 ℃ to determine the optimum enzyme reaction pH.

3-A shows Xanthomonas sp. In order to determine the thermal stability of the alginate degrading enzyme produced by BH-1, the enzyme dilution was left for 30 minutes to 60 minutes at different temperatures up to 10-70 ° C., and the residual activity was analyzed by enzyme reaction. Figure 3-B is the result of analyzing the residual activity after leaving for 30 to 60 minutes by varying the pH to 3 to 12 to determine the pH stability of the alginic acid degrading enzyme.

In order to achieve the above object, the present inventors have conducted a continuous study to isolate strains having excellent alginate degrading enzyme activity from seaweed, and isolated new marine microorganisms having excellent alginate degrading enzyme productivity from brown algae. A method for producing alginate degrading enzyme with high efficiency by culturing under optimum conditions was developed. Hereinafter, the present invention will be described in detail, but the scope of the present invention is not limited thereto.

Example 1 Isolation and Identification of Alginate Degrading Enzyme Producing Microorganisms

A liquid medium (Na-alginate 10 g / L, Yeast extract) that was sterilized at 121 ° C for 15 minutes in order to separate microorganisms that biosynthesize alginate degrading enzymes by extracting the brown algae from the tidal flats in Sinan-gun, Jeollanam-do 2 g / L, NaCl 2 g / L, K ₂ HPO ₄ 2 g / L, MgSO ₄ 7H ₂ O 1 g / L, KCl 1 g / L, CaCl ₂ · 2H ₂ O 0.05 g / L) Only microorganisms that can metabolize alginic acid as carbon source can be survived by shaking incubator (VS-8480SF, Vision Scientific Co., Korea) maintained at 30 ° C and 200rpm by inoculating 0.5g of biomass into a 250mL Erlenmeyer flask. Incubate three times for 48 hours.

By diluting the culture medium from 10 ² to 10 ⁹ continuously (Na-alginate 10 g / L, Yeast extract 2 g / L, NaCl 2 g / L, K ₂ HPO ₄ 2 g / L) , MgSO ₄ · 7H ₂ O 1 g / L, KCl 1 g / L, CaCl ₂ · 2H ₂ O 0.05 g / L, Agar 18 g / L), 30 μl each was inoculated and dispersed well with a glass rod. After incubation for 24 hours in an incubator (IB-25G, Jeio tech Co., Korea), three colonies with alginic acid dissolving ability were isolated from solid medium inoculated with 10 ⁷ dilutions. These colonies were inoculated in the liquid medium for separating the alginic acid-decomposing microorganisms and cultured in a shaker for 24 hours to select the BH-1 strain having the best alginate-degrading enzyme activity.

For accurate phylogenetic classification of the newly isolated BH-1 strain, 16S rDNA genes were isolated and analyzed for 1,447 nucleotide sequences as shown in the following figure. ResultXanthomonasgenus, Pseudomonasgenus, Stenotrphomonasgenus BH-1 strain isolated from seaweeds showed more than 97% similar sequenceXanthomonassp. It was named BH-1, and it was deposited on August 11, 2004 at KCTC and was assigned accession number KCTC 10677BP.

Example 2 Influence of Carbon Sources on Alginate Degrading Enzyme Production

Basic medium for alginate degrading enzyme production in which alginic acid is replaced with sugar as a carbon source in the basic medium for separating alginate-degrading microorganism of Example 1 (Sucrose 10 g / L, Yeast extract 2 g / L, NaCl 2 g / L, K ₂ HPO ₄ 2 g / L, MgSO ₄ · 7H ₂ O 1 g / L, KCl 1 g / L, CaCl ₂ · 2H ₂ O 0.05 g / L) Alginic acid isolated / identified in a 250 mL Erlenmeyer flask containing 100 mL Degrading enzyme biosynthetic strain Xanthomonas sp. BH-1 was transferred to platinum and incubated for 20 hours in a shaker (KMC-8480SF, Vision Scientific Co., Korea) maintained at 30 ℃, 250rpm was used as inoculum. The initial sugar concentration as a carbon source in the alginate degrading enzyme production medium was incubated for 20 hours under the same conditions. Cell growth was measured by absorbance at 660nm using a spectrophotometer (UV / VIS 1601, Shimadzu Co., Japan) after diluting the culture so that the absorbance is less than 0.7. Alginate degrading enzyme activity was measured by adding 0.05ml of culture solution to 0.55% sodium alginate as a substrate and reacting at 50 ° C for 15 minutes, and then measuring absorbance (520nm) by DNSA method to measure the amount of D-glucuronic acid produced as uronic acid. It was. One unit of alginic acid degrading enzyme was defined as the amount of enzyme required to produce 1 μmole of uronic acid for 1 minute of enzymatic reaction. Table 1 shows the results of culturing the sugar concentration from 0 to 40 g / L to examine the effect of sugar concentration. The results were shown. Therefore, Xanthomonas sp. The optimal sugar concentration for alginate degrading enzyme production using BH-1 was found to be 5 g / L.

TABLE 1 Xanthomonas sp. Effect of Sugar Concentration as a Carbon Source on Cell Growth and Alginate Degrading Enzyme Production in BH-1 Cultures

Sugar Concentration [g / L] Cell _Growth [OD ₆₆₀ ] Alginate Enzyme Activity [U / ml] 0 3.81 0.10 5 6.66 0.38 10 4.56 0.32 15 4.17 0.36 20 4.02 0.40 25 3.87 0.41 30 3.81 0.45 35 3.45 0.27 40 3.48 0.19

Example 3 Influence of Nitrogen Sources on Alginate Degrading Enzyme Production

Replace the carbon source with 5 g / L of sugar in the basal medium of Example 2 and replace Xanthomonas sp. In order to examine the effect of the nitrogen source on the production of alginate degrading enzyme using BH-1, 10 g / L of various kinds of nitrogen sources were added to each other and cultured under the same conditions as in Example 2, and the results shown in Table 2 were obtained. When 10 g / L of ammonium sulfate was used as the nitrogen source, the alginic acid degrading enzyme activity was 0.48 U / ml.

In order to examine the effect of ammonium sulphate concentration as the optimal nitrogen source, the result of culturing with ammonium sulphate concentration of 0 ~ 21 g / L was obtained as shown in Table 3. Xanthomonas sp. The optimum nitrogen source for the preparation of alginic acid degrading enzyme using BH-1 was found to be 9 g / L ammonium sulfate.

TABLE 2 Xanthomonas sp. Effect of Nitrogen Sources on Cell Growth and Alginate Degrading Enzyme Production in BH-1 Cultures

Nitrogen source [10 g / L] Cell _Growth [OD ₆₆₀ ] Alginate Enzyme Activity [U / ml] Control 0.21 0.19 Ammonium chloride 0.30 0.20 Ammonium sulfate 4.05 0.48 Beef extract 4.35 0.38 Corn Soak 0.30 0.24 Sodium Glutamate 0.72 0.22 Malt Extract 0.63 0.02 Bactopeptone 4.35 0.20 Polypeptone 4.80 0.32 Yeast extract 5.22 0.33

TABLE 3 Xanthomonas sp. Effect of Ammonium Sulfate Concentration as an Optimal Nitrogen Source on Cell Growth and Alginate Degrading Enzyme Production in BH-1 Cultures

Ammonium Sulfate Concentration [g / L] Cell _Growth [OD ₆₆₀ ] Alginate Enzyme Activity [U / ml] 0 0.21 0.19 3 1.29 0.30 6 2.64 0.34 9 4.41 0.44 12 5.19 0.37 15 5.43 0.37 18 5.79 0.35 21 6.15 0.29

Example 4 Influence of Phosphate on Alginate Degrading Enzyme Production

In the basic medium for producing the alginate degrading enzyme of Example 2, 5 g / L of sugar was used as the carbon source and 9 g / L of ammonium sulfate as the nitrogen source, and Xanthomonas sp. In order to examine the effect of phosphate type on alginate degrading enzyme production using BH-1, 2 g / L of various kinds of phosphate were added and cultured under the same conditions as in Example 2, and the results as shown in Table 4 were obtained. The optimum phosphate types were sodium diphosphate and ammonium diphosphate, producing 0.40 U / ml in sodium diphosphate and 0.51 U / ml in ammonium diphosphate.

In order to examine the effects of sodium diphosphate and ammonium diphosphate as the optimal phosphate, the results were incubated with different concentrations of sodium diphosphate and ammonium diphosphate at 1 to 5 g / L, respectively. Xanthomonas sp. The optimal phosphate for alginate degrading enzyme production using BH-1 was found to be 3 g / L of diammonium diphosphate.

TABLE 4 Xanthomonas sp. Effect of Phosphate Types on Cell Growth and Alginate Degrading Enzyme Production in BH-1 Cultures

Phosphate [2 g / L] Cell _Growth [OD ₆₆₀ ] Alginate Enzyme Activity [U / ml] Monobasic Phosphate 0.75 0.21 Dibasic calcium phosphate 0.75 0.20 Sodium monophosphate 0.75 0.27 Dibasic Sodium Phosphate 3.75 0.40 Ammonium monophosphate 0.78 0.24 Ammonium Diphosphate 3.99 0.51

TABLE 5 Xanthomonas sp. Effect of Sodium Diphosphate and Ammonium Diphosphate As Optimal Phosphate on Cell Growth and Alginate Degrading Enzyme Production in BH-1 Cultures

Phosphate Concentration [g / L] Cell _Growth [OD ₆₆₀ ] Alginate Enzyme Activity [U / ml] Dibasic Sodium Phosphate One 3.78 0.43 2 4.26 0.52 3 3.78 0.48 4 3.81 0.49 5 4.11 0.53 Ammonium Diphosphate One 4.44 0.46 2 4.20 0.58 3 4.50 0.59 4 4.38 0.57 5 4.41 0.57

Example 5 Effect of Sodium Chloride Concentration on Alginic Acid Degrading Enzyme Production

Xanthomonas sp. In order to examine the effect of sodium chloride concentration on alginic acid degrading enzyme production using BH-1, the sodium chloride concentration was varied from 0 to 30 g / L in the optimum medium obtained from the results of Example 4, and the same as that of Example 2. Incubated under the conditions obtained as shown in Table 6, Xanthomonas sp. The optimal sodium chloride concentration for alginate degrading enzyme production using BH-1 was found to be 10 g / L.

TABLE 6 Xanthomonas sp. Effect of Sodium Chloride Concentration on Cell Growth and Alginate Degrading Enzyme Production in BH-1 Cultures

Sodium Chloride Concentration [g / L] Cell _Growth [OD ₆₆₀ ] Alginate Enzyme Activity [U / ml] 0 4.83 0.58 2 4.98 0.59 4 4.98 0.64 6 4.80 0.64 8 4.74 0.70 10 4.59 0.76 15 4.35 0.76 20 4.11 0.58 30 3.96 0.42

Example 6 Effect of Magnesium Sulfate Concentration on Alginic Acid Degrading Enzyme Production

Xanthomonas sp. In order to examine the effect of magnesium sulfate concentration on the production of alginic acid degrading enzyme using BH-1, the magnesium sulfate concentration in the optimum medium obtained from the results up to Example 5 was changed from 0 to 9 g / L, which was the same as that of Example 2. Incubated under the conditions, the results are shown in Table 7. Xanthomonas sp. For the production of alginate degrading enzyme using BH-1, it was found that magnesium sulfate was not added.

TABLE 7 Xanthomonas sp. Effect of Magnesium Sulfate Concentration on Cell Growth and Alginate Degrading Enzyme Production in BH-1 Cultures

Magnesium Sulfate Concentration [g / L] Cell _Growth [OD ₆₆₀ ] Alginate Enzyme Activity [U / ml] 0 4.11 0.95 One 4.62 0.79 2 4.59 0.69 3 4.71 0.70 4 4.77 0.71 5 4.68 0.68 6 4.68 0.65 7 4.65 0.56 8 4.50 0.54 9 4.65 0.55

Example 7: Effect of Potassium Chloride Concentration on Alginic Acid Degrading Enzyme Production

Xanthomonas sp. In order to examine the effect of potassium concentration on alginate degrading enzyme production using BH-1, the concentration of potassium concentration in the optimum medium obtained from the results of Example 6 up to 0-4.5 g / L was the same as that of Example 2. Incubated with the conditions obtained as shown in Table 8, Xanthomonas sp. The optimum potassium chloride concentration for the preparation of alginic acid degrading enzyme using BH-1 was found to be 3 g / L.

TABLE 8 Xanthomonas sp. Effect of Potassium Chloride Concentration on Cell Growth and Alginate Degrading Enzyme Production in BH-1 Cultures

Potassium chloride concentration [g / L] Cell _Growth [OD ₆₆₀ ] Alginate Enzyme Activity [U / ml] 0.0 4.17 0.82 0.5 4.20 0.85 1.0 4.26 0.85 1.5 4.35 0.87 2.0 4.29 0.90 2.5 4.26 0.90 3.0 4.29 0.91 3.5 4.29 0.91 4.0 4.29 0.91 4.5 4.32 0.91

Example 8 Effect of Calcium Chloride Concentration on Alginic Acid Degrading Enzyme Production

Xanthomonas sp. In order to examine the effect of calcium chloride concentration on the production of alginate degrading enzyme using BH-1, calcium chloride concentration was varied from 0 to 1.0 g / L in the optimum medium obtained from the results of Example 7, and the same as that of Example 2. The cultured under the conditions obtained as shown in Table 9, Xanthomonas sp. The optimal calcium chloride concentration for preparing alginic acid degrading enzyme using BH-1 was 0.05 g / L.

TABLE 9 Xanthomonas sp. Effect of Calcium Chloride Concentration on Cell Growth and Alginate Degrading Enzyme Production in BH-1 Cultures

Calcium chloride concentration [g / L] Cell _Growth [OD ₆₆₀ ] Alginate Enzyme Activity [U / ml] 0.0 3.99 0.75 0.05 4.17 0.87 0.1 4.26 0.87 0.2 4.32 0.85 0.4 4.35 0.86 0.6 4.44 0.88 0.8 4.47 0.88 1.0 4.53 0.85

Example 9 Preparation of Alginate Degrading Enzyme by Fermentor Culture

Xanthomonas sp. Obtained from the flask culture. Optimal medium for alginate degrading enzyme production using BH-1 (Sucrose 5 g / L, (NH ₄ ) ₂ SO ₄ 9 g / L, (NH ₄ ) ₂ HPO ₄ 3 g / L, NaCl 10 g / L, Bulk culture was carried out using a 10 L fermenter (KF-10L, KoBioTech Co., Korea) containing 8 g of KCl 3 g / L, CaCl ₂ 0.05 g / L). Since it is an aerobic microorganism, the air is supplied at 0.1vvm while stirring at 400rpm, and the pH in the fermenter is automatically controlled to 8.0 using 2N aqueous hydrochloride solution and 2N sodium hydroxide aqueous solution, and the incubation temperature is maintained at 30 ° C. for 54 hours with Xanthomonas sp. . As a result of culturing BH-1, as shown in FIG. 1, 4.91 U / ml (about 40,000 units) of alginic acid degrading enzyme was prepared after 48 hours of culture.

Example 10 Xanthomonas sp. Biochemical Characterization of BH-1 Alginate Degrading Enzyme

Xanthomonas sp. BH-1 culture was centrifuged at 15,000 rpm for 30 minutes (CR21G, HITACHI Co., Japan) to remove the cells, and ultrafiltration device equipped with a hollow fiber membrane cartridge with an exclusion molecular weight of 100,000 Da (U / F system, ChemiCore Inc. , Korea) and 3L (about 15,000 Unit) of the supernatant from which the cells were removed were filtered at a flow rate of 70ml / min to remove the polymer material to obtain a culture solution of 2.7L. The culture medium was concentrated to 0.75L (about 12,300 Units) by removing the low molecular weight material from the ultrafiltration device equipped with a hollow fiber membrane cartridge with an exclusion molecular weight of 10,000 Da. 2.25 L of 0.2M Potassium Phosphate Buffer (pH 7.0) was added to the concentrate in three portions, and filtered through an ultrafiltration device equipped with a hollow fiber membrane cartridge with an exclusion molecular weight of 10,000 Da, at a flow rate of 70 ml / min, followed by desalting, followed by 0.44 L. An aqueous solution of demineralized alginate degrading enzyme (about 9,200 Units) was obtained. This was lyophilized (SFDSM24, Samwon Freezing Eng. Co., Korea) for 48 hours to obtain 1.83 g (about 7,100 Units) of lyophilized alginate degrading enzyme in powder form, and the biochemical properties of the enzyme were investigated.

Xanthomonas sp. To determine the optimum enzymatic reaction temperature of alginate degrading enzyme produced by BH-1, 0.5 g of the lyophilized alginic acid degrading enzyme was dissolved in 100 ml of 0.2 M potassium phosphate buffer (pH 7.0) (2 U / ml alginate degrading enzyme standard solution). As a result of the enzymatic reaction under the same conditions as in Example 2 by varying the enzyme reaction temperature to 20 ~ 70 ℃ as shown in Figure 2-A Xanthomonas sp. The optimum enzymatic reaction temperature of alginic acid degrading enzyme produced by BH-1 was 55 ° C. Xanthomonas sp. In order to determine the optimum enzyme reaction pH of the alginic acid degrading enzyme produced by BH-1, the enzyme reaction was carried out under the same conditions as in Example 2 by varying the pH from 3 to 12 at 55 ° C. As shown, Xanthomonas sp. The optimum pH of the alginate degrading enzyme produced by BH-1 was 6-11.

Xanthomonas sp. In order to determine the thermal stability of the alginate degrading enzyme produced by BH-1, the enzyme dilution was left for 30 minutes to 60 minutes at different temperatures up to 10-70 ° C., followed by enzymatic reaction under the same conditions as in Example 2. As a result of analysis, Xanthomonas sp. Alginate degrading enzyme produced by BH-1 had thermal stability in the range of 10 ~ 55 ℃ and the optimum thermal stability was 40 ℃.

Xanthomonas sp. In order to determine the pH stability of the alginate degrading enzyme produced by BH-1, the pH was changed from 3 to 12, and left for 30 to 60 minutes, and the residual activity was analyzed by the enzyme reaction under the same conditions as in Example 2. As shown in 3-B, Xanthomonas sp. Alginate degrading enzyme produced by BH-1 had pH stability within the pH range of 7 to 10, and the optimum pH stability was pH 9.0.

Thus, Xanthomonas sp. Considering the thermal and pH stability of alginic acid degrading enzyme produced by BH-1, it was found that the optimum temperature of the enzymatic reaction process for producing alginic acid oligosaccharides by hydrolysis of alginic acid derived from algae was 40 ° C. and the optimum pH was 9.0.

As described in detail in the above embodiment, the present invention is a novel marine microorganism Xanthomonas sp. Optimized BH-1 KCTC 10677BP with medium (Sucrose 5 g / L, (NH ₄ ) ₂ SO ₄ 9 g / L, (NH ₄ ) ₂ HPO ₄ 3 g / L, NaCl 10 g / L, KCl 3 g / L , CaCl ₂ 0.05 g / L). Xanthomonas sp. After 48 hours of BH-1 incubation, 4.91 U / ml of the extracellular alginate degrading enzyme could be produced. Accordingly, the present invention provides a high-efficiency alginate degrading enzyme capable of economically mass-producing alginic acid oligosaccharides by enzymatic engineering of alginic acid, a natural polysaccharide of algae such as seaweed, mussels, and kelp.

Claims (1)

Novel Marine Microbial Xanthomonas sp. A method of producing alginate degrading enzyme, characterized by using BH-1KCTC 10677 BP ( Xanthomonas sp. BH-1 KCTC 10677BP).