JPH082297B2 - Alginic acid-degrading enzyme, its production method, bacteria used, and alginic acid-degrading method - Google Patents

Description

TECHNICAL FIELD The present invention relates to an alginate-degrading enzyme produced by an Enterobacter bacterium, a method for producing the same, a bacterium to be used, and an alginate-decomposing method.

Since the alginic acid-degrading enzyme of the present invention is produced in a large amount by Enterobacter cloacae M-1 bacterium, this enzyme is used to partially decompose and decompose alginic acid to bring about a change in physical properties, which is likely to be new. The present invention is extremely useful for manufacturing alginic acid products and for studying the structure of alginic acid.

(Prior Art and Problems) Conventionally, alginate-degrading enzymes are flavobacterium, Pseudomonas sp. (Japanese Patent Laid-Open No. 59-143597) and Vibrio sp. (Journal of the Fisheries Society of Japan 55 (4), 709-713 (1989). It is known to be produced by

However, in recent years, the demand for alginate-degrading enzymes has increased in the processing and treatment of alginate, and there has been a demand for superior mass production of alginate-degrading enzymes.

(Means for Solving the Problems) The inventors of the present invention have made an intensive search for a strain that produces a large amount of alginic acid degrading enzyme, and found that a bacterium that produces a large amount of alginic acid degrading enzyme from soil in which the alginic acid extraction residue was discarded. It came to separate one strain. This bacterium was designated as M-1 bacterium, and when classified and searched, it was recognized as belonging to Enterobacter chloecae. In addition, M-1 bacterium has been deposited as FERM BP-2577 at the Micro Incorporated Research Institute.

The mycological properties of Enterobacter chloecae M-1 are as follows.

Morphology Bacillus Grac stain − Spores − Motility + Oxidase − Catalase + OF F Gas production from glucose + Indole production − Methyl red − VP + citrate utilization + Hydrogen sulfide production − Urea decomposition − Phenylalanine deamino − Lysine decarboxylation-Arginine dihydrolase + Ornithine decarbonation + Gelatin liquefaction-Nitrate reduction + ONPG + Yellow pigment production-Acid production Adonitol-Arabinose + Inositol-Sucrose + Dulucitol-Sorbitol + Mannitol + Mellibiose + Lactose enol The growth of Bacterus chloejeae M-1 bacterium is extremely fast, and the activity of alginate-degrading enzyme increases with the growth of the bacterium, and reaches its maximum 12 hours after starting the main culture.

As a culture medium for the bacterium, a medium containing an appropriate amount of sodium alginate, a nitrogen source, and inorganic salts is used. Here, as the nitrogen source, there are peptone, yeast extract, ammonium sulfate, ammonium nitrate and the like, and as inorganic salts, phosphoric acid 1
Potassium, 2 potassium phosphate, magnesium sulfate and the like are used as appropriate.

The culture temperature is 30 to 40 ° C, and about 12
Incubate for about an hour until the enzyme production reaches the maximum.

Since alginate-degrading enzyme is produced outside the cells, the obtained culture solution is centrifuged at about 10,000 G to obtain the supernatant as the enzyme solution.

The enzyme was subjected to ion fractionation chromatography such as acetone fractionation, ammonium sulfate fractionation, DEAE-Sephadex A-50, CM-Sephadex C-50, etc. from the obtained supernatant, gel filtration by Bio-Gel P-100, Hydroxyapatite. Can be purified by adsorption chromatography or the like.

Acetone fractionation was performed by slowly adding the crude enzyme solution 1 to 2 parts of cold acetone. However, precipitation of residual alginic acid in the crude enzyme solution also occurred at the same time, and the recovery rate was considerably low at less than 10%.

Ammonium sulfate fractionation was performed with 20, 40, 60, 80, 90% saturated ammonium sulfate. As a result, the best recovery was obtained in the precipitation fraction with 90% saturated ammonium sulfate, which was about 70%. However, in this case as well, about 25% of the activity remained in the supernatant after the ammonium sulfate fractionation.

Ion exchange chromatography shows 0.02M phosphoric acid 1
Equilibrate the column with potassium buffer (pH 7.8),
It was carried out by supplying a crude enzyme solution dialyzed with the same buffer thereto. Elution was performed with a NaCl gradient in the same buffer. As a result, this enzyme was eluted as a non-adsorbed fraction in DEAE-Sephadex A-50, the recovery rate was 65%, and the purification rate was 15 times. In the case of CM-Sephadex C-50, it was eluted as an adsorption fraction, the recovery rate was 25%, and the purification rate was
It was 35 times. Since the enzyme is auxotrophic for metal ions, 1 mM calcium was added to the same buffer to give C
M-Sephadex C-50 was performed. As a result, the recovery rate is 45
%, The purification rate increased to 90 times. . Gelo filtration with Bio-Gel P-100 is performed with 0.02M monopotassium phosphate buffer (pH
After equilibrating the column in 7.8), the crude enzyme solution was supplied. As a result, the recovery rate was 42% and the purification rate was 34 times.

The adsorption chromatography was carried out by supplying a crude enzyme solution dialyzed with the same buffer to a column equilibrated with 0.02 M monopotassium phosphate buffer (pH 7.8). The elution was performed with 0.2 M potassium phosphate buffer (pH 7.8).
As a result, the recovery rate was 80% and the purification rate was 3.3 times.

In the present invention, a culture supernatant enzyme solution, a concentrated solution, a crude enzyme such as ascent fractionation, a purified enzyme obtained by various purification means, etc. all become the alginate-degrading enzyme of the present invention, and can be used according to each application. It is a thing.

Next, the physicochemical properties of the alginate-degrading enzyme of the present invention measured using the supernatant alginate-degrading enzyme solution obtained in Example 1 are shown.

1. Action It acts on alginic acid, and desorbs and decomposes alginic acid like lyase.

2. Substrate specificity Alginate is cleaved and decomposed by lyase to produce unsaturated uronic acid or oligosaccharide having unsaturated uronic acid residue.

1% sodium alginate (M / G = 0.93) solution (pH 7.
8) and the crude enzyme solution which had been inactivated by heating at 100 ° C. for 5 minutes were mixed at a ratio of 1: 1 to give a blank. The reaction solution was sampled by 200 μl at regular intervals and subjected to the same treatment. Subsequently, each sample was measured by TBA reaction. That is, 200 μl of sample
It was added with 0.125NH ₂ S0 ₄ solution 0.25ml containing HI0 ₄ of 0.025N
The sample was allowed to stand for 20 minutes to perform periodate oxidation of the sample. The reaction was stopped by adding 0.5N HCl solution containing 2% sodium arsenite and letting it stand for 2 minutes, and then 2 ml of 0.3% thiobarbituric acid solution was added to 100 ° C.
This was heated in a hot water bath for 10 minutes to carry out the condensation reaction. The reaction liquid showing red color was allowed to cool, and then the absorbance at 548 nm was measured with an absorptiometer. However, if the absorbance at 548 nm exceeds 1.0, dilute the enzyme reaction solution with distilled water and add TB
It was measured so that the absorbance in the A reaction was 1.0 or less.

 The results are shown in Figure 1.

As is clear from FIG. 1, the production of unsaturated uronic acid or oligosaccharide having an unsaturated uronic acid residue by this enzyme is almost completed in 6 hours.

The system for producing a red compound from alginic acid is as follows.

3. Action pH and stable pH Fig. 2A shows that the optimum pH of the enzyme reaction is 7.8, and Fig. 2B shows the stable pH. In FIG. 2B,
The residual activity of the enzyme after treatment at 30 ℃ for 3 hours at various pHs was stable around pH8, but below 6.5 and pH9.
Above, it fell to below 50%.

4. Action temperature and temperature stability As shown in Fig. 2C, the optimum temperature of the enzyme reaction is 35 ° C, and the residual activity of the enzyme after treatment for 3 hours at pH 7.8 at various temperatures is up to 30 ° C. Was stable, but the residual activity decreased with increasing temperature, and it was completely deactivated at 60 ° C (Fig. 2D).

5. Effect of metal salts Figure 3 shows the effect of metal salts on enzyme activity. The activity of this enzyme was strongly inhibited by the addition of 1 mM EDTA.
The activity was remarkably activated by the addition of 2 mM cadmium or calcium.

6. Decrease in viscosity of sodium alginate due to enzymatic action 5 ml of supernatant enzyme solution in 5 ml of 1% sodium alginate solution (pH 7.8)
Was added and the change with time in viscosity was measured by an Ostwald viscometer at 35 ° C.

The results are shown in FIG. The specific viscosity (α) of the reaction solution after 5 minutes reached 0.83 and after 60 minutes reached 0.97, and finally the viscosity decreased to almost the same value as pure water.

In FIG. t _o = falling time (second) of the reaction mixture at the time of 0 t _t = falling time (second) of the reaction mixture at the time t t _w = falling time of water (second)

7. Degradation product of sodium alginate 5 ml of 1% sodium alginate solution (pH 7.8) was added with 5 ml of enzyme solution, and the enzyme reaction was carried out at 35 ° C. The reaction solution was sampled with time, and the change of the product was traced by TLC. (The developing solvent is n-butanol: acetic acid: water = 5: 2: 3). The result is shown in FIG.

8. Thiobarbituric acid reaction of sodium alginate decomposition product Add 5 ml of enzyme solution to 5 ml of 1% sodium alginate solution (pH 7.8), carry out enzyme reaction at 35 ° C, and sample the reaction solution over time.
548 nm of red compound produced by thiobarbituric acid reaction
It was measured at. The results are shown in FIG. Since the absorbance at 548 nm increased rapidly with the progress of the reaction, this enzyme is considered to be a lyase that cleaves alginate desorbingly.

9. Molecular weight The molecular weight of alginate degrading enzyme is 38,000 (SDS-PAGE
Law).

 Next, examples of the present invention will be described.

Example 1 B medium composition: Na alginate 1.0%. , Peptone 0.6%,
0.3% yeast extract, KH ₂ P0 ₄ 0.5% and MgS0 ₄ · 7H ₂ 0.05
%.

Enterobacter chloecae M-1 bacterium, FERM BP-2
577 was inoculated into the medium having the above-mentioned medium B composition and shake-cultured at 35 ° C. for 24 hours to obtain a seed culture solution.

Prepare 6.4 liters of the above medium B composition in a 10 liter culture tank and
Sterilize by autoclaving at ℃ for 20 minutes, then cool to 35 ℃.

This culture medium was inoculated with 600 ml of the above inoculum solution in a culture tank,
Culturing was carried out at a temperature of 350 to 450 rpm and an aeration rate of 1.5 l / min.

 Figure 6 shows the time course of enzyme production.

Enzyme production reached a maximum 12 hours after the start of culture. After completion of the culture, the supernatant obtained by removing the bacteria by centrifugation at 10,000 G for 20 minutes was used as an alginate-degrading enzyme solution.

The enzyme activity in the obtained alginate-degrading enzyme solution was 20 × 10
^-3 u / ml.

The method for measuring the activity of alginate-degrading enzyme is as follows. 0.5 ml solution of Na alginate dissolved in Tris-HCl buffer containing 1 mM CaCl ₂ to a concentration of 1%
The enzyme solution (0.5 ml) was added to the mixture, the mixture was reacted at 35 ° C for 30 minutes, and the activity was measured by the thiobarbituric acid method. The enzyme activity was defined as 1 unit of the amount of enzyme that produced 1 μmol of uronic acid equivalent per minute under the same conditions.

Example 2 of D-mannuronic acid (M) and L-guluronic acid (G)
1% aqueous solution of sodium alginate with M / G ratio of 0.93 (p
H7.8) 5 ml of the supernatant alginate-degrading enzyme solution 5 obtained in Example 1
ml was added, and the enzyme reaction was performed at 35 ° C.

The reaction solution was sampled with time, and the red compound produced by the thiobarbituric acid reaction was measured at 548 nm to obtain the results shown in FIG.

[Brief description of drawings]

Fig. 1 is a graph showing the time course of the measurement of the red compound produced by the thiobarbituric acid reaction by allowing this supernatant alginate-degrading enzyme solution to act on sodium alginate with an M / G ratio of 0.93.
Fig. 2 shows the action pH (A), stable pH (B), action temperature (C) and temperature stability (D) of this enzyme. Fig. 3 shows the inhibition of this enzyme by EDTA and various metal salts. Fig. 4 is a diagram showing activation by addition, Fig. 4 is a diagram showing a decrease in viscosity of sodium alginate by the action of this enzyme, and Fig. 5 is an alginate by this enzyme.
FIG. 6 is a diagram showing changes over time in the decomposition products of Na by thin layer chromatography, and FIG. 6 is a diagram showing changes over time in culture and enzyme production in Example 1.

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Claims (4)

[Claims] 1. An alginate-degrading enzyme which is produced by an alginate-degrading enzyme-producing bacterium belonging to the genus Enterobacter and has the following properties and which decomposes alginate in a lyase-like manner: (a) Substrate specificity Alginate is detached and decomposed in a lyase-like manner. Then, an unsaturated uronic acid or an oligosaccharide having an unsaturated uronic acid residue is produced. (B) Optimum pH and stable pH range The optimum pH is 7.8 and the stable pH range is 5.5 to 9.5. (C) Optimum temperature and working temperature range The optimum temperature is 35 ° C and the working temperature range is 20 to 40 ° C. (D) Molecular weight 38,000 (by SDS-PAGE method) 2. A method for producing an alginate-degrading enzyme, which comprises culturing an alginate-degrading enzyme-producing bacterium belonging to the genus Enterobacter and collecting the alginate-degrading enzyme from the culture. 3. An alginate-degrading enzyme-producing bacterium M-1 bacterium belonging to the genus Enterobacter. 4. A culture of a microorganism which belongs to the genus Enterobacter and produces an alginate-degrading enzyme having the following properties:
An alginic acid decomposition method characterized by adding the treated product, a crude enzyme or a purified enzyme to a sodium alginate-containing solution and allowing them to act: (a) Substrate specificity Alginic acid is lyase-eliminating and decomposed to give unsaturated uronic acid or It produces oligosaccharides with unsaturated uronic acid residues. (B) Optimum pH and stable pH range The optimum pH is 7.8 and the stable pH range is 5.5 to 9.5. (C) Optimum temperature and working temperature range The optimum temperature is 35 ° C and the working temperature range is 20 to 40 ° C. (D) Molecular weight 38,000 (by SDS-PAGE method)