JPH0716092A - Porphyran degrading enzyme, neo-agaro oligosaccharide and methods for producing them - Google Patents

Abstract

(57) [Summary] (Modified) [Object] To provide an enzyme capable of decomposing porphyran, which is a polysaccharide of the seaweed of the genus Amari, a method for producing the same, and a novel neo-agarooligosaccharide using the enzyme and a method for producing the same. [Structure] A porphyran degrading enzyme having the following physicochemical properties. (1) Enzyme action: Porphyran is hydrolyzed to give a degree of polymerization of 6
And catalyzes the reaction of forming neo-agarooligosaccharides containing 8 as a main component. (2) Substrate specificity: High specificity for porphyran, and low specificity for polysaccharides contained in red algae and brown algae other than the genus Amanori. Further, the present invention relates to a method for producing the above-mentioned enzyme, in which a microorganism belonging to the genus Pseudomonas and capable of decomposing porphyran is cultured, and a porphyran-degrading enzyme is collected from the culture. Furthermore, a neo-agarooligosaccharide mainly having a degree of polymerization of 6 or 8 obtained by acting the above enzyme on porphyran, and a method for producing the same.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a porphyran-degrading enzyme which acts on porphyran to produce neo-agaro-oligosaccharide and agaromegalo-sugar and a method for producing the porphyran-degrading enzyme, which is obtained by decomposing porphyran using the above-mentioned degrading enzyme. Novel neo agarooligosaccharides and a method for producing the same.

[0002]

BACKGROUND ART In recent years, oligosaccharides have been used not only as low-calorie sweeteners having low digestibility but also as excellent physical properties such as moisturizing property, water activity regulating property, protein protection / stabilization, and starch aging suppression. It is known to have the following properties, and is attracting attention as a useful material in the food industry. In addition, oligosaccharides are difficult to caries, anti-cariogenic, bacteriostatic,
It also has useful physiological effects such as an intestinal regulating effect and an inhibitory effect on blood sugar elevation, and it has come to be noticed as a functional food material.

The oligosaccharide is a general term for saccharides formed by chemically bonding 2 to 10 monosaccharides. In particular, recently, starch, by using various hydrolases and glycosyltransferases of microbial origin,
New oligosaccharides related to sucrose, lactose, xylose, agarose, etc. are being developed.

As a method for producing an oligosaccharide, there are a method of synthesizing using a glycosyltransferase and a method of decomposing a polysaccharide by using a glycolytic enzyme as described above, but the former method is often used. . On the other hand, seaweeds have recently been used as raw materials for emulsifiers, gelling agents, thickeners, etc. in the food industry, but they are also attracting attention as a polysaccharide raw material for producing oligosaccharides. Research on the degrading enzyme has been made. Pseudomonas spp., Vibrio spp.
genus brio), genus Flavobacterium,
Cytophaga spp., Achromobacter (Ac
hromobacter), Corynebacteriu
m) Microorganisms belonging to genus etc. have been reported.

By the way, there are polysaccharides such as mannan, xylan, and porphyran in the dietary fiber of the genus Amanori.
Of these, Porphyran is the most abundant and is the main component. Porphyrans include galactose, 3,6-anhydrogalactose, 6-O-methylgalactose and 6
It is a sulfated polysaccharide composed of -O-galactose sulfate and structurally similar to agar, but having no gelling ability. There have been some studies on porphyrans related to structure, but few studies on utilization. In addition, oligosaccharides derived from porphyran are completely unknown fields. It is known that an enzyme capable of decomposing agar decomposes not only agar but also porphyran, but an enzyme that decomposes only porphyran without decomposing agar is not known at present.

[0006]

The present invention has been made in view of the above circumstances, and an object thereof is to decompose porphyran, which is a polysaccharide of seaweed, to produce a novel and useful oligosaccharide derived from porphyran. It is an object of the present invention to provide an enzyme having such porphyran decomposing ability, and further to provide a method for producing such an enzyme.

[0007]

Means for Solving the Problems That is, the present invention has the following physicochemical properties (1) Enzyme action: Porphyran is hydrolyzed to give a polymerization degree of 6
And catalyzes the reaction of forming neo-agarooligosaccharides containing 8 as a main component. (2) Substrate specificity: High specificity for porphyran, and low specificity for polysaccharides contained in red algae and brown algae other than the genus Amanori. And a porphyran degrading enzyme having

The present invention also relates to Pseudomonas (Pseudo)
The present invention relates to a method for producing a porphyran-degrading enzyme, which comprises culturing a porphyran-degrading microorganism belonging to the genus monas) and collecting the porphyran-degrading enzyme from the culture.

The present invention also relates to a novel neo-agarooligosaccharide mainly having a degree of polymerization of 6 and 8 obtained by hydrolyzing porphyran contained in the seaweed of the genus Porphyra using the above enzyme, and a method for producing the same. Regarding

The neo-agarooligosaccharide of the present invention exhibits an activity of inhibiting angiogenesis in the hen egg chorioallantoic membrane by the hen egg chorioallantoic membrane method. Further, the method for producing a neo-agarooligosaccharide of the present invention is characterized in that porphyran or a porphyran-containing substance contained in the seaweed of the genus Amanori is hydrolyzed under acidic or weakly acidic conditions using the enzyme. .

Examples of microorganisms belonging to the genus Pseudomonas and capable of decomposing porphyrans used in the present invention include Pseudomonas B-2411 (Deposit No. P-13637 of the Institute of Industrial Science). A transmission electron micrograph of the strain is shown in FIG. Staining was performed with uranium acetate.

Next, the properties of the above strains will be shown. (a) Morphology (1) Cell shape and size Bacteria, width 0.5-0.8 μm, length 0.8-3 μm (2) Cell polymorphism Presence / absence of flagella (3) Motility presence / absence, Flagella sessile state Almost no motility, polar flagella (4) Presence or absence of spores (5) Gram stain negative (6) No acid resistance

(B) Growth state in each medium (1) Meat broth agar plate size: diameter 4 to 4.5 mm degree of growth: medium shape: circular or elliptical ridge: hill shape: regular shape surface shape : Smooth color: Milky white Gloss: Yes Property: Viscous (2) Slope culture of broth agar Shape: Filament Growth: Medium Uplift: Hilly odor: None Surface shape: Smooth

(3) Liquid culture of broth Presence or absence of surface growth: Formation of bacterial ring Turbidity: White turbidity from the second day of culture Precipitation: None Gas production: None Change in color tone of indicator: Neutral (green-blue in BTB) Odor: None (4) Meat broth gelatin stab culture Growth condition: Grows only on the top 5 mm, ciliated in the medium Liquefaction of gelatin: None (5) Litmus milk medium reaction: None (blue purple) Coagulation: None

(C) Physiological properties (1) Reduction of nitrate: None (2) Denitrification reaction: None (3) MR test: Negative (4) VP test: Negative (5) Indole formation: None (6) Production of hydrogen sulfide: None (7) Hydrolysis of starch: None (8) Utilization of citric acid: None (9) Utilization of inorganic nitrogen source (nitrate): Yes (ammonium salt): Yes (10) Pigment formation: None (11) Urease: None (12) Oxidase: Negative (13) Catalase: Positive (14) Growth range (pH): Initial pH 4.5 to 7.5, not pH 4 (Temperature): Growth above 35 ° C No (15) Attitude toward oxygen: Aerobic (16) OF test: No decomposition

(17) Presence or absence of acid and gas generation from sugars L-arabinose: None D-xylose: None D-glucose: None D-mannose: None D-fructose: None D-galactose: None Maltose: None Sucrose : None Lactose: None Trehalose: None D-Sorbit: None Inosit: None Glycerin: None Starch: None

(D) Properties showing characteristics of the new species (1) Decomposition products of sugars: Produce oligosaccharides mainly composed of hexasaccharides and octasaccharides from porphyran (2) Decomposition of arginine: None (3) Temperature Resistance: 85 ° C, 10 minutes or 80 ° C, 3
Kills in 0 minutes (4) Sodium chloride resistance: Grows at 0-3% (peptone water) (5) Lipase: Tween 80 is degraded (1% Tween 80-peptone water) (6) G + C of DNA in cells Content: 62.7 mol% (high performance liquid chromatography method)

From the above characteristics, it is considered that the strain belongs to the genus Pseudomonas. Although the physiological properties are close to those of Pseudomonas marina, they show different properties such as not requiring NaCl and degrading Tween 80, so that it is appropriate to be a new strain of Pseudomonas. It should be noted that some strains belonging to the genus Pseudomonas that have agar-decomposing ability have been reported so far, but the properties such as sugar fermentation ability, oxidase generation, and nitrate reduction ability of these strains are the same as those of the present invention. It is different from the above-mentioned strains, and the strain of the present invention is different from these. In addition, the determination of the taxonomic status of the above-mentioned strains is performed by "Burger's Manual of Systematic Biology", Vol. 1 (Williams and Wilkins,
USA, 1984).

Next, the method for producing the enzyme of the present invention will be described. The medium for culturing the strain may be liquid or solid.
Usually, it is advantageous to use a liquid medium. Aerial agitation culture is industrially preferable. It is necessary to include in the medium a carbon source, a nitrogen source, and other components necessary for the growth of the strain and enzyme production that can be used by the strain.

As the carbon source, starch, dextrin, sucrose, lactose, maltose, glucose, galactose, fructose, molasses, etc. can be used for the growth of the strain, but porphyran or a raw material containing porphyran is added for enzyme production. Must. As the nitrogen source, inorganic or organic nitrogen-containing substances such as ammonium salts, nitrates, corn steep liquor, peptone, meat extract, casamino acid, soybean flour, wheat bran and urea are used. In addition, yeast extract, dry yeast, etc. are effective in increasing the amount of enzyme production. As the inorganic salts, magnesium salts, calcium salts, sodium salts, phosphates, iron salts, manganese salts, zinc salts and the like are used, and magnesium salts are particularly important. In addition, nutrients such as vitamins and growth promoting substances may be appropriately added.

Examples of preferable medium composition include a combination of peptone, yeast extract, galactose, porphyran, dipotassium hydrogen phosphate, sodium chloride, magnesium sulfate and calcium chloride.

The culture temperature is preferably about 20 to 30 ° C.,
The culture pH is preferably 4-8. Incubate for 2-5 days under aerobic conditions. Since the above enzyme is produced in the medium as a result of the culture, the bacterial cells are removed from the culture solution by filtration or centrifugation to obtain an enzyme solution. The enzyme solution can be further purified and purified by further treating it with an organic solvent precipitation, salting out, concentration under reduced pressure, adsorption / desorption with an ion exchanger, gel fractionation and the like according to a conventional method for enzyme purification. it can.

[0023]

[Function] The enzyme of the present invention specifically acts on and hydrolyzes porphyran, which is a component of seaweed of the genus Amari,
Produces neo-agarooligosaccharides.

Various properties of the enzyme of the present invention are shown in Table 1 and FIGS. That is, although these tables and drawings will be further referred to in Examples, when these are explained here, FIG. 2 shows the influence of pH on the enzyme activity of the present enzyme, and the solid line shows the enzyme by measuring the reducing sugar amount. The activity (mU / ml) is shown, and the broken line shows the enzyme activity (U / ml) by the viscosity reduction rate measurement. In the figure, pH 3-6
Area is 100 mM sodium acetate buffer, pH 5.5-8
Area is 60 mM phosphate buffer, pH 7.5-9 is 50 m
M Tris-HCl buffer, pH 8.5-10 is 100 mM
With glycine-sodium hydroxide buffer.

FIG. 3 shows the effect of temperature on the enzyme activity of the present enzyme. As with FIG. 2, the solid line shows the enzyme activity measured by the reducing sugar amount, and the broken line shows the enzyme activity measured by the viscosity reduction rate (hereinafter , FIG. 4 and FIG. 5). Figure 4
FIG. 5 is a diagram showing the pH stability of the enzyme activity of this enzyme.
FIG. 3 is a diagram showing the thermostability of this enzyme.

Further, FIG. 7 shows a diagram in which oligosaccharides and megalosaccharides produced by reacting the porphyran with the enzyme of the present invention were detected by TLC and arranged sequentially. In FIG.
(A) is the enzyme of CM-IIbaa fraction, (B) is C
This is the case of the enzyme of the M-II Idaa fraction. In the figure, S ₁ is D-galactose, S ₂ is neoagarobiose (DP
2) + neoagarotetraose (DP4) + neoagarohexaose (DP6), S ₃ is a porphyran degradation product by an enzyme derived from Pseudomonas Atlantica.

The neo-agarooligosaccharide obtained in the present invention is
Porphyran has further enhanced activities such as serum cholesterol lowering, blood pressure lowering, immunostimulation, anti-mutagenicity, and angiogenesis inhibition, and the angiogenesis inhibitory effect is particularly remarkable. Also, since it is water-soluble and has low viscosity, it is easier to handle than porphyran as a food material. As described above, the enzyme activity was determined by measuring both an increase in reducing sugar and a decrease in viscosity caused by the decomposition of porphyran, and the measuring method thereof will be described below.

That is, the reducing sugar was measured according to the Somogyi-Nelson method, and a reaction solution obtained by decomposing a porphyran solution with the enzyme was used as a sample solution, and 1 ml of this was mixed with solution A (copper sulfate pentahydrate 2 g, potassium sodium tartrate 6 g). , 12 g of anhydrous sodium carbonate and 10 g of anhydrous sodium sulfate were dissolved in distilled water and adjusted to a constant volume of 500 ml), 1 ml was added, and the mixture was boiled for 20 minutes and cooled with water for 5 minutes. Solution B (ammonium molybdate 25 g, sulfuric acid 21 ml, arsenic acid) Dissolve 3 g of disodium in distilled water and add 1 ml of 500 ml) and mix well, then add 4 ml of distilled water and measure the absorbance at 660 nm. The obtained numerical value is
The amount of reducing sugar was determined as a galactose conversion value by reading from a standard line prepared using D-galactose as a standard product.

The measurement by decreasing the viscosity was conducted with a sample solution of 5.5 m.
Using an Oswald viscometer (No. 4), the flow velocity at 37 ° C. was measured, and the viscosity decrease rate (A%) was calculated from the following equation.
Sought as. A% = (Ta-Tt) × 100 / Ta-To Ta: Flow-down time (second) immediately after mixing of porphyran solution and enzyme solution Tt: Flow-down time (second) after reaction time t of the mixed solution To: Water Flow time (second)

[0030]

【Example】

Example 1. A medium consisting of porphyran 0.1% peptone 0.1% K ₂ HPO ₄ 1.0% NaCl 1.0% MgSO ₄ .7H ₂ O 0.05% was adjusted to an initial pH of 7 and added to a 300 ml Erlenmeyer flask for 100 ml. Pour and sterilize by heating under pressure at 121 ° C for 20 minutes. One platinum loop of the strain B-2411 was inoculated into this medium, and cultivated at 25 ° C. for 4 days with shaking and shaking (200 rp).
m)

On the other hand, a medium containing porphyran 0.5% peptone 0.42% yeast extract 0.01% NaCl 0.1% MgSO ₄ .7H ₂ O 0.25% CaCl ₂ .2H ₂ O 0.01% was used. The initial pH is adjusted to 7, and 20 ml is put in a 100 ml Erlenmeyer flask and sterilized at 121 ° C. for 20 minutes. 200 μl of the above culture solution was inoculated into this, and the temperature was 25 ° C.
The cells were cultivated with shaking at 200 rpm for 24 hours. The enzyme activity of the supernatant obtained by centrifuging the culture broth at 20,000 xg for 10 minutes was 3.24 U / ml by the viscosity reduction method and 96 mU / ml by the reducing sugar measurement method.

Example 2. Nori powder (dried nori crushed with a crusher to a size of about 0.25 mm) 2% D-glucose 0.1% peptone 0.1% yeast extract 0.05% NaCl 0.1% MgSO ₄ · A medium consisting of 7H ₂ O 0.25% CaCl ₂ · 2H ₂ O 0.01% was adjusted to an initial pH of 6, and 300 ml of this was adjusted.
Dispense 30 ml into an Erlenmeyer flask and sterilize by heating under pressure at 121 ° C for 20 minutes. 200 μl of the culture solution prepared in the same manner as in Example 1 was inoculated into this, and 2 days at 28 ° C. for 2 days.
Culture was performed with orbital shaking at 00 rpm. Culture medium 20,000
The enzyme activity of the supernatant obtained by centrifugation at 0 × g for 10 minutes was
It was 199 mU / ml as determined by reducing sugar.

Example 3. The same medium as in Example 2 was added to 30
Dispense 15 l into a l jar fermentor and sterilize with steam. 150 ml of the culture solution obtained in the same manner as in Example 2 was inoculated into this, 28 ° C., 400 rpm, aeration rate.
The medium pH was adjusted to 7 with 10% hydrochloric acid under the condition of 15 l / min.
The culture was performed for 60 hours while adjusting to 5. The culture solution was filtered using a filter aid (Celite), and a filtrate (crude enzyme solution) having an activity of 322 mU / ml according to the reducing sugar assay was 11,880.
ml was obtained.

The crude enzyme solution was concentrated to 800 ml with a Zaltocon module membrane (limit molecular weight 10,000), and 30-70% saturated fraction was fractionated using ammonium sulfate. The fraction was passed through a cellulose dialysis membrane to 40 mM acetate buffer (pH 5.5).
Was dialyzed for 12 hours. The dialysate was concentrated to 66 ml with polyethylene glycol 2000. The enzymatic properties at this stage are shown in FIGS. The concentrated enzyme solution was further added to CM-Sephadex C-50 (pH 5.
5) After passing through the column, a column non-adsorbed fraction (CM-II) and a 0.2 M sodium chloride elution fraction (CM-III) were obtained as active fractions (FIG. 6).

CM-II is further dialyzed, concentrated, DEAE-S
ephadex A-50 column treatment, Buty1-T
C. by treatment with yopearl 650M column
M-III fraction is dialyzed, concentrated, Toyopearl HW
-55F column treatment, DEAE-Sephadex A
By performing -50 column processing, both are SDS-PE
It could be purified by AGE electrophoresis to a single band. The enzymatic properties of both fractions are shown in Table 1.

[0036]

[Table 1]

A characteristic of both fractions is the substrate specificity, which is capable of decomposing porphyrans and partially desulfurized porphyrans but not acting on polysaccharides such as agar, carrageenan, alginic acid, dextran and pectin. The TLC of the reaction solution obtained by decomposing porphyran in each of the two fractions is shown (Fig. 7). The oligosaccharide estimated to have a degree of polymerization of 8 in CM-II ((A) in Fig. 7) and the degree of polymerization in CM-III are shown. It is understood that a large amount of oligosaccharides considered to be 6 ((B) in FIG. 7) are produced, and oligosaccharides having fewer sugars and megalosaccharides having a larger number of sugars increase with the passage of time.
The mechanism of action of both fractions is clearly different, and the effect of using both in combination is greater than the action of each alone.

Next, an example in which the enzyme of the present invention is allowed to act on porphyran to produce the neo-agarooligosaccharide of the present invention will be described.

Example 4 10 mM acetate buffer (pH
5.5) To 500 ml, add 15 g of crude porphyran,
Melt by heating under pressure at 121 ° C. for 20 minutes. After allowing it to cool, add 75 units of enzyme solution to this solution and stir at 37 ° C for 48 hours.
Reacted for hours.

After the reaction was completed, the insoluble substance was added to 20,000 ×.
Removed by centrifugation at 20 g for 20 minutes. (Fraction A). Ethanol was gradually added to the obtained supernatant to a final concentration of 70% to precipitate a polymer substance, and the formed precipitate was removed by centrifugation again under the above conditions (fraction B). . When the total amount of sugar in the supernatant was quantified by the phenol-sulfuric acid method, 5.9 g of a porphyran degradation product in terms of galactose could be obtained.

300 ml of the supernatant obtained by removing the fraction B was concentrated to 43 ml with an evaporator, and then Su was added with water as an eluent.
It was passed through a column (2.6 × 40 cm) packed with per-Q-Toyopearl 650M. Fraction C is the non-adsorbed fraction
The adsorbed fraction was eluted with 0.5 M sodium chloride and then concentrated to 50 ml. The concentrate is Sephade
Gel filtration was performed using a x G-10 column, and fractions D to F were separated.

The sugar composition of the obtained 6 fractions (A to F) was determined by TL.
Analyzed in C. The developing reagent was n-butanol: acetic acid: water (2: 1: 1), the mixture was developed at room temperature for 80 minutes, and then the diphenylamine-aniline reagent was sprayed on it at 105 ° C for 1 minute.
Color was developed by heating for 0 minutes. The results are shown in Fig. 8. Fraction A
There are no low molecular weight substances in B and B, Fraction C is a megalo-sugar fraction with 8 or more sugars, Fraction D is a sugar segment with a high degree of polymerization, and Fractions E and F have 6 and 8 sugars. Individual oligosaccharides are found. In the figure, S ₁ is neo agarobiose + neo agaro tetraose + neo agaro hexaose, and S ₂ is D-galactose. The sugar composition of each fraction can be determined by comparison with the agarooligosaccharide sample used as a standard.

The total sugar amount, 3,6-anhydrogalactose amount, sulfate group amount, and yield of the fractions A to F are summarized in Table 2. The yield from the total amount of sugar was highest in Fraction F, and the sulfate group content was also high in Fraction F.

[0044]

[Table 2]

Example 2 Fractions C, D, E and F obtained in Example 1 and the substrate porphyran were freeze-dried and the angiogenesis-inhibiting effect was measured by the chorioallantoic membrane (CAM) method of chicken eggs. The test is DHA usprunk et
al., J. Dev. Biol., 38: 237 (1974).

A fresh fertilized egg is obtained, and the eggshell on the air chamber side on the fourth day of the egg is peeled off by about 3 cm in diameter and the chorioallantoic membrane is exposed. On the 2mm diameter chorioallantoic membrane in the center of the bare part,
A silicon ring having an inner diameter of 3 mm is placed, and a test sample solution dissolved with 1% methylcellulose physiological saline is put therein (1 mg of sample per egg). The treated eggs were kept warm at 37.8 ° C, and 40 to 46 hours later, the degree of neovascularization of the chorioallantoic membrane was observed. The diameter of the site where the angiogenesis was inhibited was measured and scored as follows.

Grade 1; 6 mm or more Grade 2; 4 mm or more and less than 6 mm Grade 3; 2 mm or more and less than 4 mm Grade 4; less than 2 mm

[Equation 1]

The results are shown in FIG. 9. The activity of oligosaccharide fraction F is considerably stronger than that of the substrate porphyran. Weak activity was observed in the megalo sugar fraction C and the highly polymerized sugar fraction D.

Angiogenesis is a phenomenon deeply related to hyperplasia of capillaries at the site of injury and inflammation, hypercapillary formation of Kaposi's sarcoma and hemangiomas, and extension of blood vessels that support the growth of solid tumors. It is also found in various ophthalmic diseases that cause blindness, such as sexual retinopathy. In such various diseases, its inhibitor is expected to be effectively used for eliminating these pathological phenomena.

[0050]

INDUSTRIAL APPLICABILITY The enzyme of the present invention specifically acts on porphyran, which is a component of seaweed of the genus Amari, and hydrolyzes it to form neo-agarooligosaccharide. Neo agarooligosaccharide can be obtained from The neo-agarooligosaccharide obtained in the present invention has an angiogenesis-inhibiting activity and is therefore effective against various diseases caused by the angiogenic action.

[Brief description of drawings]

FIG. 1 Strain B-2411 obtained by culturing in broth liquid medium
The figure which shows the transmission electron micrograph of this.

FIG. 2 is a graph showing the influence of pH on the enzyme activity of the enzyme of the present invention.

FIG. 3 is a graph showing the influence of temperature on the enzyme activity of the enzyme of the present invention.

FIG. 4 is a diagram showing pH stability of enzyme activity of the enzyme of the present invention.

FIG. 5 shows the thermostability of the enzyme of the present invention.

FIG. 6 shows the major active fractions CM-II and CM-II when the enzyme solution was column-treated with CM-Sephadex C-50.
The figure showing the separation pattern of I.

FIG. 7 shows the oligosaccharides and megalosaccharides produced by the action of CM-II and CM-III on porphyran, detected by TLC, and sequenced over time, (A).
Shows CM-II and (B) shows CM-III.

FIG. 8 is a view showing a TLC development pattern of each section obtained by enzymatically decomposing porphyran.

FIG. 9 is a view showing an angiogenesis-inhibiting effect on each porcine chorioallantoic membrane of each porphyran-degraded fraction.

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[Procedure amendment]

[Submission date] May 19, 1994

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[0007]

Means for Solving the Problems That is, the present invention has the following physicochemical properties (1) Enzyme action: Porphyran is hydrolyzed to have a polymerization degree of 2
It catalyzes the reaction of forming neo-agarooligosaccharides composed mainly of 4 and 4 . (2) Substrate specificity: High specificity for porphyran, and low specificity for polysaccharides contained in red algae and brown algae other than the genus Amanori. And a porphyran degrading enzyme having

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The present invention also relates to a novel neo-agarooligosaccharide mainly having a degree of polymerization of 2 and 4 , which is obtained by hydrolyzing porphyran contained in seaweed of the genus Porphyra using the above enzyme, and a method for producing the same. Regarding

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The (d) The new properties indicating characteristics of (1) sugars decomposition products: disaccharide from Porphyran, 4 sugar to produce oligosaccharides that mainly (2) decomposition of arginine: None (3) Temperature Resistance: 85 ° C, 10 minutes or 80 ° C, 3
Kills in 0 minutes (4) Sodium chloride resistance: Grows at 0-3% (peptone water) (5) Lipase: Tween 80 is degraded (1% Tween 80-peptone water) (6) G + C of DNA in cells Content: 62.7 mol% (high performance liquid chromatography method)

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A characteristic of both fractions is the substrate specificity, which is capable of decomposing porphyrans and partially desulfurized porphyrans but not acting on polysaccharides such as agar, carrageenan, alginic acid, dextran and pectin. The TLC of the reaction liquid which decomposed each porphyran in both fractions is shown (FIG. 7). The explanation of FIG. 7 is as described in the section of the above-mentioned action. As shown in this figure, it can be seen that oligosaccharides having a smaller number of sugars and megalosaccharides having a larger number of sugars increase with the passage of time. The mechanism of action of both fractions is clearly different, and the effect of using both in combination is greater than the action of each alone.

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The sugar composition of the obtained 6 fractions (A to F) was determined by TL.
Analyzed in C. The developing reagent was n-butanol: acetic acid: water (2: 1: 1), the mixture was developed at room temperature for 80 minutes, and then the diphenylamine-aniline reagent was sprayed on it at 105 ° C for 1 minute.
Color was developed by heating for 0 minutes. The results are shown in Fig. 8. Fraction A
There are no low molecular weight substances in B and B, the fraction C is a megalosaccharide fraction having 6 or more sugars, the fraction D is a saccharide segment with a high degree of polymerization, and oligosaccharides are found in fractions E and F. In the figure, S ₁ is neo agarobiose + neo agaro tetraose + neo agaro hexaose, and S ₂ is D-galactose. The sugar composition of each fraction can be determined by comparison with the agarooligosaccharide sample used as a standard. Next, each of the above-mentioned fractions E and F was purified using a Toyopear 1 HW-40S column to obtain two spot components of oligosaccharide in FIG. 7. These components were desulfated and reduced under an alkaline condition using sodium borohydride to obtain corresponding neutral sugar alcohols. This was analyzed by TLC after removing unreacted materials by anion exchange chromatography. Furthermore, S ₁ in FIG. 7 was also reduced to the corresponding sugar alcohol, and simultaneously analyzed by TLC. The results are shown in Fig. 10. In FIG. 10, is a standard neutral sugar with a known degree of polymerization,
Is a sugar alcoholized by reducing, is a fraction having a low degree of polymerization in the E fraction and the F fraction of the present invention, and is a neutral sugar alcohol, is the E fraction and F of the present invention. Among the fractions, the fraction with a high degree of polymerization is a neutral sugar alcohol. According to this figure, it was found that the sugar alcohol coincided with the top spot of the standard sugar alcohol (sugar alcohol having a degree of polymerization of 2) and had a degree of polymerization of 2. Similarly, the sugar alcohol was found to have a degree of polymerization of 4 in agreement with the second spot of the standard sugar alcohol (a sugar alcohol having a degree of polymerization of 4). From the above experiments, it was found that the oligosaccharides obtained in the present invention mainly had a degree of polymerization of 2 and a degree of polymerization of 4.

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The results are shown in FIG. 9. The activity of oligosaccharide fraction F is considerably stronger than that of the substrate porphyran. Oligo <br/> Toga fraction C, and weak activity in the high polymerization sugar fraction D was observed.

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[Brief description of drawings]

FIG. 1 Strain B-2411 obtained by culturing in broth liquid medium
The figure which shows the transmission electron micrograph of this.

FIG. 2 is a graph showing the influence of pH on the enzyme activity of the enzyme of the present invention.

FIG. 3 is a graph showing the influence of temperature on the enzyme activity of the enzyme of the present invention.

FIG. 4 is a diagram showing pH stability of enzyme activity of the enzyme of the present invention.

FIG. 5 shows the thermostability of the enzyme of the present invention.

FIG. 6 shows the major active fractions CM-II and CM-II when the enzyme solution was column-treated with CM-Sephadex C-50.
The figure showing the separation pattern of I.

FIG. 7 shows the oligosaccharides and megalosaccharides produced by the action of CM-II and CM-III on porphyran, detected by TLC, and sequenced over time, (A).
Shows CM-II and (B) shows CM-III.

FIG. 8 is a view showing a TLC development pattern of each section obtained by enzymatically decomposing porphyran.

FIG. 9 is a view showing an angiogenesis-inhibiting effect on each porcine chorioallantoic membrane of each porphyran-degraded fraction.

FIG. 10: E compartment obtained by enzymatically decomposing porphyran
And the F compartment were purified and desulfated and reduced
Development pattern of neutral sugar alcohol obtained by liquefaction
FIG.

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[Figure 10]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display location (C12N 9/14 C12R 1:01) (72) Inventor Hiromi Ashino 19-1 Hommachi, Funabashi, Chiba Ebigawa Mansion 609

Claims (8)

[Claims] 1. A porphyran degrading enzyme having the following physicochemical properties. (1) Enzyme action: Porphyran is hydrolyzed to give a degree of polymerization of 6
And catalyzes the reaction of forming neo-agarooligosaccharides containing 8 as a main component. (2) Substrate specificity: High specificity for porphyran, and low specificity for polysaccharides contained in red algae and brown algae other than the genus Amanori. 2. The optimum pH for porphyran decomposition is 6.0, the optimum temperature is 60 ° C., and the pH stable range is 6.0 to 8.
0, the molecular weight is about 28,000, and the isoelectric point is pH 5.
2. The porphyran degrading enzyme according to claim 1, which has a Km value of 0.02% with respect to porphyran. 3. The optimum pH for porphyran decomposition is 5.0, the optimum temperature is 50 ° C., and the pH stable range is 5.0-9.
5, the molecular weight is about 42,000, and the isoelectric point is pH 6.
8. The porphyran degrading enzyme according to claim 1, which has a Km value of 0.098% with respect to porphyran. 4. A method for producing a porphyran-degrading enzyme, which comprises culturing a porphyran-degrading microorganism belonging to the genus Pseudomonas and collecting the porphyran-degrading enzyme from the culture. 5. A microorganism belonging to the genus Pseudomonas and having a porphyran degrading property is Pseudomonas B-2411 (Deposit P-13 of the Institute of Industrial Science and Technology).
637) and mutants thereof, the method for producing a porphyran-degrading enzyme according to claim 4. 6. Obtained by hydrolyzing porphyran contained in seaweed of the genus Amanori with the enzyme according to claim 1.
A neo-agarooligosaccharide characterized by mainly having a degree of polymerization of 6 and 8. 7. The neo-agarooligosaccharide according to claim 6, which exhibits an activity of inhibiting angiogenesis in the hen egg chorioallantoic membrane by the hen egg chorioallantoic membrane method. 8. A neo-agarooligosaccharide characterized by hydrolyzing a porphyran or a porphyran-containing substance contained in a seaweed belonging to the genus Amanori, with the enzyme according to claim 1 under acidic or weakly acidic conditions. Production method.