JP2001247601A - Extracellular polysaccharide produced by bacteria belonging to genus rhodococcus and clarification method of marine environment using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a clarification method of the marine environment. SOLUTION: An extracellular polysaccharide produced by bacteria belonging to genus Rhodococcus can be liberated from bacterial cells by a physical impact and contains a lipid-like material. The clarification method of the marine environment utilizes the polysaccharide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to Rhodococcus (Rh).
The present invention relates to a novel extracellular polysaccharide (hereinafter referred to as “EPS”) produced by a bacterium belonging to the genus odococcus, and a method for purifying a marine environment using the same.

[0002]

2. Description of the Related Art EPS is known as one of the typical extracellular components produced by microorganisms. EPS is relatively common in many microorganisms and is considered as one of the key factors involved in the interaction between microorganisms and their surrounding environment. This EPS is a capsule polysaccharide (capsul
ar polysaccharides) and slimy polysaccha
rides). Capsule polysaccharides are said to be covalently bound to phospholipids and lipid-A on the cell surface.In contrast, slime polysaccharides are much weaker than capsule polysaccharides, as they surround the outside of cells. It is said to exist. In some cases, the two cannot be strictly distinguished.

[0003] There have been many reports on EPS in the past. In particular, E. coli K antigen and Pseudodom
Alginic acid of the genus onas) has been known for a long time, and there are many reports on its synthesis mechanism and environmental function. However, studies on EPS of bacteria belonging to the genus Rhodococcus are limited, and little is known.

EP produced by a bacterium belonging to the genus Rhodococcus
In S, E of Rhodococcus equi
PS has been studied most often, and is characterized by an acidic polymer consisting of repeating units in which multiple constituent sugars are arranged in a straight line.
Although the constituent sugars include pyruvic acid linked to acetal and lactic acid linked to ether, lipids are not mentioned.

[0005] As an example in which an extracellular component other than EPS contains lipids, mainly low molecular weight glycolipids are famous. For example, trehalose dimycolate produced by microorganisms belonging to the genus Mycobacterium, and rhamnolipid, a bacterium belonging to the genus Pseudomonas, which is a gram-negative bacterium, are shared by Escherichia coli lipid-A. LPS bound thereto, lipoteichoic acid of gram-positive bacteria, lipoglycan, and the like are also known. On the other hand, high molecular extracellular components including lipids include Acinetobacter calcoaceticas.
oaceticus) is known. This substance contains a lipid in the repeating structure of the sugar chain. However, there is no known example in which EPS contained in a bacterium belonging to the genus Rhodococcus contains lipids in a high molecular substance having a polysaccharide skeleton.

[0006]

Conventionally, in the event of a crude oil spill in the ocean, mechanical and chemical measures have been taken, but it is impossible to completely remove the spilled oil alone. And chemical treatment (administration of synthetic surfactants, etc.)
Secondary pollution, such as environmental pollution caused by water, has become a problem.
For this reason, bioremediation is being considered as an alternative to these methods, but natural purification power is slower than artificial treatment, and is not an effective solution at present. An object of the present invention is to provide a more effective bioremediation means by enhancing the natural purification ability of marine pollutants such as crude oil.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and as a result, have found that the extracellular polysaccharide produced by a bacterium belonging to the genus Rhodococcus promotes the growth of marine bacteria. The present invention has been completed based on the findings.

That is, the present invention is a polysaccharide having the following properties. (1) Extracellular polysaccharide produced by a bacterium belonging to the genus Rhodococcus (2) Can be released from cells by physical impact from bacterial cells (3) Including lipid-like substance A method for purifying a marine environment, which comprises administering a polysaccharide to an marine environment contaminated with an oil component to promote the growth of marine bacteria.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The polysaccharide of the present invention has the following properties (1) to (3). (1) Extracellular polysaccharides produced by bacteria belonging to the genus Rhodococcus. (2) It can be released from cells by physical impact from the cells. (3) Contains lipid-like substances.

[0010] Here, the "physical impact" is not particularly limited as long as the polysaccharide can be released from the cells, and the means capable of giving such impact include shaking, stirring, Means such as ultrasonic treatment and grinding treatment can be exemplified. "Lipid-like substance" means a derivative of a lipid or a substance having a long-chain fatty acid or a similar hydrocarbon chain in a molecule, and mainly comprises palmitic acid,
Refers to saturated fatty acids such as stearic acid.

Further, the polysaccharide of the present invention comprises the following (4)
More preferably, it has the property of (6). (4) As the constituent neutral sugar and uronic acid, at least one of rhamnose, fucose, mannose, glucose, galactose, and glucuronic acid is contained. (5) It has a growth promoting effect on microorganisms whose growth is inhibited under the conditions in which oil components are present. (6) It is a non-volatile hydrocarbon against microorganisms whose growth is inhibited under conditions in which oil components are present. It has a survival rate increasing effect in the presence.

Here, the term "oil component" means crude oil or petroleum which causes marine pollution, or oil produced or derived from spills thereof, and various hydrocarbons contained in the above-mentioned oils. "Microorganisms whose growth is inhibited under oil component presence conditions" refers to microorganisms whose growth is significantly inhibited by the presence of oil components,
For example, Rhodococcus rh
odochrous) R-1 strain, R-2 strain and the like are included in this microorganism. "Refractory hydrocarbon" means a high-molecular, long-chain linear or branched hydrocarbon, including hydrocarbons such as hexadecane and tetradecane.

The polysaccharide of the present invention can be obtained by culturing a microorganism belonging to the genus Rhodococcus, subjecting the microorganism to physical shock, collecting a culture supernatant, and purifying the culture supernatant by a conventional method. The microorganism to be used is not particularly limited as long as it belongs to the genus Rhodococcus and has the ability to produce the polysaccharide of the present invention, but it is preferable to use a microorganism belonging to Rhodococcus rhodochrous. Preferred strains include
Rhodococcus rhodochrous S-1 strain, Rhodococcus rhodochrous S-2 strain, Rhodococcus rhodochrous SF-3 strain
Strain, Rhodococcus rhodochrous SM-1 strain, Rhodococcus rhodochrous ATCC 53968 strain, Rhodococcus sp.PR-4
And Rhodococcus sp. PG7-2 strain.

The polysaccharide of the present invention can be used, for example, for purifying polluted marine environments. That is, the marine environment can be purified by administering the polysaccharide of the present invention to a contaminated marine environment and promoting the growth of marine bacteria.
The dose to the marine environment may be determined according to the status of contamination, etc., but usually 10 mg of EPS dry weight per 1 g of oil.
About 50 mg is appropriate. Hereinafter, the present invention will be described specifically with reference to examples. However, the present invention is not limited by this.

[0015]

EXAMPLES [Example 1] Extraction and purification of EPS 1.1 Extraction of EPS Strains belonging to Rhodococcus rhodochrous (S-1 strain, S-2 strain)
, SF-3, SM-1, ATCC53968) and Rhodococcus
Sp. strains (PR-4 strain, PG7-2 strain) were inoculated on the entire surface of an IB agar medium (square petri dish, 23 cm x 5 cm) with a sterile cotton swab.
The culture was allowed to stand at 37 ° C. for 48 hours or more.

<Composition of IB agar medium> Glucose 10 g Yeast extract 10 g MgCl ₂ .7H ₂ O 0.2 g CaCl ₂ .2H ₂ O 0.1 g NaCl 1.0 g FeCl ₂ .6H ₂ O 0.02 g (NH ₄ ) ₂ SO ₄ 0.5 g agar 15 g ultrapure water 1 L pH 7.2 A single operation was performed using a set of cells of 5 square IB agar medium. The cells were collected with a glass rod, suspended in physiological saline, and finally a cell suspension of 50 ml was obtained.

Next, a Falcon tube containing the cell suspension was fixed horizontally to REFRIGERATOR SHAKER (Takasaki Scientific Instruments), shaken at 110 rpm for 45 minutes at 25 ° C., and then at 4 ° C.
The mixture was centrifuged at 10,000 rpm for 10 minutes, and separated into a supernatant (extracellular components) and a precipitate (cells). Thereafter, 25 ml of physiological saline was added to the precipitate, mixed well with a test tube mixer, and the cells were completely suspended. Reconstitute this cell suspension with REFRIGERATOR SHA
Shaking was performed at 25 ° C. for 45 minutes at 110 rpm using KER (Takasaki Scientific Instruments). Subsequently, the mixture was centrifuged at 10,000 rpm for 10 minutes at 4 ° C., separated into a supernatant and a precipitate, and the two supernatants were combined and used for the subsequent operations. When the cells could not be completely removed, centrifugation at 10,000 rpm at 4 ° C. for 20 minutes was repeated.

The solution containing these extracellular components was added to DN
ase (1 mg / ml, final concentration) and RNase (1 mg / ml, final concentration) were added and reacted at 37 ° C. overnight. Next, proteinase K was added to these solutions to a final concentration of 10 mg / ml, and reacted at 37 ° C. for 2 hours. Subsequently, these solutions were subjected to phenol treatment and chloroform treatment. A neutral phenol solution (pH 8.0) saturated with the same amount of the sample solution as Tris-HCl was added, and the mixture was slowly inverted and stirred, and then centrifuged at 10,000 rpm at 4 ° C for 10 minutes. The upper layer was slowly drawn up and transferred to a new container. Further, an equal volume of a chloroform solution (chloroform and isoamyl alcohol in a volume ratio of 24:
(Mixed in 1), and slowly invert and stir.
The mixture was centrifuged at 10,000 rpm for 20 minutes at ℃, and the supernatant was transferred to a new container. This operation was repeated until the white intermediate layer disappeared, and the chloroform treatment was performed again.

These sample solutions were dialyzed four times each night against 5000 ml of distilled water, and the samples were freeze-dried to completely remove water. EP obtained
The weight of S was measured, and a portion thereof was taken and dissolved in sterile distilled water to prepare a 1 mg / ml EPS solution. Using a spectrophotometer, the absorbance of this solution at wavelengths of 260 nm and 280 nm was measured, and it was confirmed that the contamination of nucleic acids and proteins was small. When nucleic acids or proteins are contaminated, dry
EPS was dissolved again in sterile distilled water, subjected to an enzyme treatment, and the above-described operation was repeated. About EPS derived from all the strains extracted by the above method, the spectrophotometer 200nm ~
As a result of measuring the absorbance at 400 nm, contamination of nucleic acids and proteins was hardly confirmed.

1.2 Purification of EPS The sugar contained in EPS was fractionated by anion exchange column chromatography (25φ × 200 mm) using DEAE-Toyopearl 650 as a column carrier. A final concentration of 1 mg / EPS of 40 mg of EPS sample of each strain was added to 10 mM Tris Buffer (pH 8.0).
It was dissolved to a volume of ml and added to the column. The added sample was eluted using a continuous gradient from 0 M to 1 M NaCl. Elution of sugar was confirmed by the phenol-sulfuric acid method. The obtained fractions for each peak were collected, dialyzed against sterilized distilled water, and lyophilized to obtain an EPS purified sample.

The elution pattern by anion exchange column chromatography was about 0.3 for all seven strains used.
The elution pattern was almost the same, consisting of a peak eluted with M NaCl and two small peaks that did not adsorb to the anion exchanger. However, since most of the total amount of reducing sugars recovered was included in the peak eluted with about 0.3 M NaCl, the polysaccharide eluted with about 0.3 M NaCl used as the EPS purified sample was used. It was considered to be the main constituent polysaccharide of EPS produced by 7 strains.

Example 2 Examination of Structure and Chemical Properties of EPS 2.1 Analysis by Cellulose Acetate Membrane Electrophoresis An EPS purified sample and EPS were electrophoresed for each strain, and the mobility was compared. Place 400 ml of 0.2 M barium acetate buffer in the electrophoresis tank, and place a filter paper (bridge) in the electrophoresis tank.
It was immersed in 0.2 M barium acetate buffer and set in a migration tank. The application position of the sample was written in advance with a pencil,
The cellulose acetate membrane was gently immersed in a 0.2 M barium acetate buffer so as to prevent air bubbles from entering, thoroughly drained, and then placed on a filter paper (bridge). Air current was applied for 10 minutes at a constant current of film width × 0.5 mA. 1 μl of the sample was applied in a band shape of 1 cm, and energized for 5 hours under the same conditions as the idle energization. After the completion of the energization, the cellulose acetate membrane was taken out, immersed in a 0.5% toluidine blue solution for staining, and decolorized twice with ultrapure water.

Although the mobility in electrophoresis was slightly different between the strains used, the difference in the mobility of EPS of each strain was observed after 5 hours of electrophoresis. Did not. Therefore, since the elution pattern by anion exchange column chromatography was almost the same in all the strains used, EPS produced by the strain used in this example is an acidic polysaccharide, and EPS The electrical properties of the molecules were considered to be similar.

2.2 Analysis of Neutral Sugar and Uronic Acid The EPS purified sample of each strain was hydrolyzed, and the constituent sugars were analyzed by gas chromatography. each
The purified EPS sample was dissolved in ultrapure water to a concentration of 5 mg / ml, and a 10 mg sample per tube was dispensed and freeze-dried. At the same time, seven neutral sugars (L-rhamnose, L-fucose, L-
A standard sugar sample prepared by preparing arabinose, D-xylose, D-mannose, D-glucose, D-galactose) and two uronic acids (D-galacturonic acid, D-glucuronic acid) at a concentration of 1 mg / ml each. Prepared, dispensed 1 ml and lyophilized.
To the freeze-dried EPS sample and the standard sugar sample subjected to the hydrolysis treatment, 0.5 ml of 80% cold H ₂ SO ₄ was added in ice water, cooled on ice for 30 minutes, and left at 30 ° C. for 3 hours. Again, 6.5 ml of cold ultrapure water was added in ice water and heated at 100 ° C. for 2 hours.
Thereafter, the mixture was allowed to cool to room temperature, added with 0.8 g of calcium carbonate, and allowed to stand at 4 ° C. overnight to be sufficiently neutralized. On the other hand, a standard sugar sample without hydrolysis was also prepared. For each standard sugar samples, cold ultrapure water to 6.5 ml and 80% cold H ₂ SO ₄
After sequentially adding and mixing in 0.5 ml of ice water, 0.8 g of calcium carbonate was added, and the mixture was allowed to stand at 4 ° C. overnight to be sufficiently neutralized.
After the neutralization reaction, all the samples were subjected to suction filtration to remove calcium sulfate precipitate. These filtrates were dried under reduced pressure at 40 ° C. using a test tube concentrator.

Excess calcium ions are converted into ion-exchange resin
After removal with Amberlite IR-120B, these samples were dissolved in 1 ml of ultrapure water, 1.2 ml of 0.13 N ammonia water was added, stirred, and left for 5 minutes. Next, 1 ml of a 0.2 N acetic acid solution was added for neutralization, and then 1 ml of ultrapure water was added, followed by drying under reduced pressure at 40 ° C. After activating the solution by dissolving these vacuum-dried samples in 1 ml of 0.2 N acetic acid, the sample was added to a mini column packed with 2.5 ml of Dowexl-X8 (Acetate-type) anion exchange resin equilibrated in 0.2 N acetic acid solution. did. Neutral sugar and uronic acid were separated by the operation described below.

After the column was washed three times with 1 ml of 0.2 N acetic acid, another 10 ml of 0.2 N acetic acid was passed through the column to elute neutral sugars not adsorbed on the resin to obtain a neutral sugar fraction. In addition, uronic acid adsorbed on the resin is passed through the column three times with 1 ml of 2 N acetic acid,
It was released from the resin, and further eluted by flowing 20 ml of 2 N acetic acid (1 ml) to obtain a uronic acid fraction. The neutral sugar fraction and uronic acid fraction were dried under reduced pressure at 40 ° C. using a test tube concentrator, and 3 ml of distilled water was added thereto until the odor of acetic acid finally disappeared, and drying under reduced pressure was repeated. .

The reduction of neutral sugars and the conversion to TFA were performed in the following manner. The concentrated and dried neutral sugar fraction was dissolved in 0.5 ml of ultrapure water, and one drop of 0.13 N aqueous ammonia was added to make the mixture neutral. Then, 0.5% of 1% sodium borohydride aqueous solution
l and left at room temperature for 30 minutes. For this sample,
Activated cation exchange resin Amberlite IR-120 (H-typ
An appropriate amount of e) was added (until no foaming occurred), and the mixture was allowed to stand at 4 ° C overnight. Next, the mixed solution of the sample and the cation exchange resin was added to a mini column filled with 3 ml of activated Amberlite IR-120, together with the resin, and overlaid. This was washed three times with 8 ml of ultrapure water, and the filtrate was dried under reduced pressure at 40 ° C. using a test tube concentrator. In order to remove excess boric acid, the operation of adding 5 ml of methanol to the dried product and drying at 40 ° C. under reduced pressure was repeated three times. When a white precipitate (sodium borate) remained, the treatment with Amberlite IR-120 was performed again, and the filtrate was again dried under reduced pressure. The sample obtained in this process was dried in a vacuum desiccator containing diphosphorus pentoxide. For these dried samples, 0.2
After adding ml of trifluoroacetic anhydride (TFAA) and 0.8 ml of ethyl acetate and stirring gently, it was analyzed by gas chromatography.

The reduction of uronic acid and the conversion to TMS were performed in the following manner. The concentrated and dried uronic acid fraction was dissolved in 2 ml of ultrapure water, and 150 mg of barium carbonate was added thereto, followed by heating at 60 ° C. for 10 minutes to obtain a barium salt. This sample was subjected to suction filtration to remove the remaining barium carbonate precipitate. The filtrate was dried under reduced pressure at 40 ° C. using a test tube concentrator. 2 ml of dried matter
Dissolve in ultrapure water, add a few drops of 0.13 N ammonia water, and add
After adjusting to 10 or more, it was left at room temperature for 30 minutes. 50 mg was added to this sample to reduce the aldehyde group of uronic acid.
Was added and left at room temperature for 1 hour. To this sample was added an appropriate amount (until foaming disappeared) of the activated cation exchange resin Amberlite IR-120 (H-type), and the mixture was stirred well and left at 4 ° C. overnight. After confirming that the pH had dropped, a mixed solution of this sample and a cation exchange resin was added to a mini column filled with 3 ml of Anberlite IR-120 together with the resin, and overlaid. This was washed three times with 8 ml of ultrapure water, and the filtrate was dried under reduced pressure at 40 ° C. using a test tube concentrator. The surplus boric acid and sodium borate were removed by the same operation as the neutral sugar reduction and TFA conversion.

0.5 ml of concentrated hydrochloric acid was added to the concentrated and dried sample, and the sample was dried under reduced pressure at 80 ° C. using a rotary evaporator to lactonize aldonic acid. The concentrated and dried sample was dried in a vacuum desiccator containing phosphorous pentoxide. 0.2 ml of the dried uronic acid fraction
Anhydrous pyridine was added, 1 ml of TMS-HT was further added, and the mixture was gently stirred, left at room temperature for 5 minutes, and analyzed by gas chromatography and GC-MS.

The neutral sugar fraction and uronic acid fraction were analyzed by gas chromatography in the following manner. GC-6A and CHROMATOPAC C-R2A were used for analysis, nitrogen gas was used as carrier gas, and FID was used for detection. For the analysis of neutral sugars, a DC QF-1 column was used, and the vaporization chamber and the detector temperature were kept at 200 ° C., and the column temperature was kept at 125 ° C. for 20 minutes. Each sample
1 μl was introduced with a microsyringe. For the analysis of uronic acid, an NPGSE column was used.The temperature of the vaporization chamber and the detector was 240 ° C, and the column temperature was 160 ° C.
Up to 20 ° C., 1 μl of each sample was introduced with a microsyringe. For GC-MS analysis, GC-17A and QP5050 were used.
Helium gas was used as carrier gas, and TIC was used for detection. The column used was DB-1, and the temperature conditions followed the analysis by GC.

2.3 Analysis of Acidic Substances Contained in EPS Hydrolysis was performed on EPS purified samples of various strains, and acidic substances were extracted therefrom. Thin layer chromatography and GC-MS
Was analyzed. The purified EPS sample was dissolved in ultrapure water, dispensed so that the test amount per test tube was 3 mg, and freeze-dried. To this, add 3 ml of methanol containing 10% KOH,
For 90 minutes to hydrolyze. After cooling, 3 ml of hexane was added and stirred, and the operation of dissolving the unsaponifiable matter in the hexane layer and removing it was repeated three times. Next, an appropriate amount of HCl was added, and the pH was lowered to an acidic range (pH 2) to release acidic substances. The operation of adding 3 ml of hexane thereto and stirring, and dissolving the free acidic substance in the hexane layer and collecting the same was repeated three times. An appropriate amount of anhydrous sodium sulfate was added for dehydration. After collecting the supernatant, the sodium sulfate precipitate was washed three times with hexane, combined with the supernatant, and concentrated to dryness using a centrifugal evaporator. The dried product was dissolved in 200 μl of hexane to obtain a TLC sample. The same operation was performed for 3 mg of oleic acid to obtain a standard sample.

The detection of acidic substances by thin layer chromatography was carried out in the following manner. Hexane-ether (4: 1), chloroform-methanol-water (90: 10: 1,
Alternatively, detection was performed by TLC using 65: 25: 4) as a developing solvent. The origin at which the sample was spotted and the end point of the development were written in a thin plate of silica gel with a pencil, and after evacuation with each solvent to be used, baking was performed at 105 ° C. for 30 minutes. 20 μl of each sample was spotted on a thin layer plate and developed with each solvent. For detection, first, the sample was allowed to stand in a developing layer filled with iodine vapor, and spots were confirmed. Then, iodine was evaporated, 60% sulfuric acid was sprayed, the mixture was heated at 120 ° C. for 10 minutes, and spots were confirmed again.

The analysis of acidic substances by GC-MS was performed as follows. 20 μl of each sample was concentrated to dryness with a centrifugal evaporator, and 50 μl of methanol-benzene solution (2: 7), 1 μl
Was added and shaken for 30 minutes, and then used as a sample for GC-MS. GC-17A and QP5050 were used for GC-MS analysis, helium gas was used as carrier gas, and TIC was used for detection. The column used was DB-1, the vaporization chamber temperature and the interface temperature were both 300 ° C, the column temperature was maintained at 150 ° C for 2 minutes, and the temperature was increased by 10 ° C per minute.
Up to 300 ° C., 1 μl of each sample was introduced with a microsyringe. Table 1 shows the mass% of saccharides and lipids.

[0034]

[Table 1]

The mass% of each component is shown when the mass of the whole EPS is 100%. As shown in Table 1, from the types of constituent sugars and their abundances, groups of S-1, S-2, SM-1, AT
CC53968 group, PR-4, PG7-2 group, and
SF-3 divided into four patterns. Since all of these EPSs solubilize oil, it is considered that the type of the constituent sugars of the EPS has no effect. In addition, as a result of analysis by GC-MS, two substances having high similarity to the methyl ester of saturated fatty acid were detected in all strains. From the comparison with the retention time of the standard substance and the MS pattern, it is possible that the lipid macromolecules similar to palmitic acid and stearic acid are contained, respectively.

2.4 Analysis of Pyruvic Acid in EPS A purified EPS sample was dissolved in ultrapure water, a 10 mg sample was dispensed into a test tube with a screw cap, and freeze-dried. to this
3 ml of distilled water containing 10% KOH was added, and the mixture was heated at 100 ° C. for 90 minutes to hydrolyze. After cooling, 3 ml of hexane was added and stirred, and the operation of dissolving the unsaponifiable matter in the hexane layer and removing it was repeated three times. Next, an appropriate amount of HCl was added, and the pH was lowered to an acidic range (pH 2) to release acidic substances. 3 ml for this
Hexane was added and stirred, and the operation of transferring the free acidic substance to the hexane layer and recovering it was repeated three times. The sample was dehydrated by adding an appropriate amount of anhydrous sodium sulfate, and after collecting the supernatant, the sodium sulfate precipitate was washed three times with hexane, combined with the supernatant, and concentrated to dryness by a centrifugal evaporator. This dried product was dissolved in ultrapure water, and a 1/10 volume sample was used as a sample for pyruvate detection by an enzyme method.
For detection, a pyruvate detection kit was used, and the operation was basically performed according to the instruction manual. 1 ml of buffer, NA in cuvette
0.1 ml of a DH solution, 0.1 ml of a sample, and 1.9 ml of distilled water were added, the mixture was inverted, stirred, heated at 25 ° C. for 3 minutes, and the absorbance at 340 nm was measured. Then add 0.02 ml of L-lactate dehydrogenase solution
In addition, the mixture was inverted and stirred, and heated at 25 ° C for 5 minutes.
m was measured. The amount of pyruvate was calculated from the absorbance of each of the blank and the sample in which the same operation was performed by adding distilled water instead of the sample. EP by the above method
Pyruvic acid in S was detected, but pyruvate was not detected in EPS of any strain.

[Example 3] Investigation of the effect of EPS on other microorganisms 3-1. EPS on growth of R-1 and R-2 strains in the presence of oil
The effect of the S-2 strain was dissolved in sterile ultrapure water to a final concentration of 5 mg / ml, and this EPS was used for Rhodococcus rhodochrous R-1, R-
The effect on the two strains was examined. Rhodococcus rhodochrous R-1 and R-2 strains are colony morphological variants of S-2 strain. These form rough colonies, and S-2
EPS production is extremely low compared to stocks. In addition, its growth is extremely inhibited in the presence of oil.

An oil medium was prepared in the following manner. First, 1 g of each of the W-oil and the AF-oil was measured in a vial. W-oil is a heating oil derived from crude oil obtained by heat-treating Arabian light crude oil at 230 ° C and removing volatile components. AF-oil converts Arabian light crude oil to silica gel (C-2
00) is one of the oils derived from crude oil fractionated in
-A fraction eluted with a hexane (1: 1) solution. This includes polycyclic aromatic hydrocarbons such as naphthalene and phenanthrene. 5 ml of chloroform was added to the W-oil or AF-oil and completely dissolved. Thereafter, the liquid volume was measured and dispensed into dry heat test tubes such that the oil amount per test tube became 100 mg. These were left overnight in a fume hood to evaporate chloroform. Crude oil was measured for its specific gravity in advance, and was dispensed into dry heat test tubes immediately before culturing so that the oil amount per test tube was 100 mg.

One loopful of the R-1 and R-2 strains was inoculated into a YG liquid medium (5 ml), and cultured at 30 ° C. for 48 hours with shaking at 110 rpm (preculture). Take 50 μl of this culture and add a fresh YG liquid medium (5 m
l), and cultured again at 30 ° C. for 48 hours with shaking at 110 rpm (main culture). This main culture solution was washed three times with 5 ml of physiological saline to remove the components of the medium, and suspended again in 5 ml of physiological saline. This cell suspension was diluted 1000-fold, 100 μl of the diluted cell suspension was inoculated into 10 ml of oil medium, cultured at 30 ° C. with shaking at 110 rpm, and 100 μl was sampled over time. did. The sampling solution was appropriately diluted with physiological saline, applied to a YG agar medium, and cultured at 30 ° C.
Colonies that grew on the agar medium were counted to determine the number of bacteria. The results are shown in FIG.

EPS was added in the following manner. First 5 ml of Y
To the G liquid medium, 500 μl of a 5 mg / ml EPS solution was added and well suspended. The YG liquid medium containing the EPS was sterilized by filtration with a 0.45 μm filter, and added to a sterilized test tube or an oil test tube. Thereafter, the cells were washed as described above, ingested into an oil medium containing EPS, and cultured with shaking at 30 ° C. and 110 rpm. The measurement of the number of viable bacteria was performed in the same manner. The results are as shown in FIG.

In all the conditions examined, the growth of the R-1 and R-2 strains in the presence of oil was increased by adding EPS. Specifically, the rate of decrease in the number of viable cells in the initial stage of culture was reduced, and the total number of viable cells in the stationary phase was also increased. On the other hand, since the growth of the R-1 and R-2 strains when only EPS was added to the YG medium did not increase as compared with the case where EPS was not added, it is considered that EPS itself has no growth promoting effect. . From this, it was considered that EPS derived from the S-2 strain had an effect of promoting the growth of other microorganisms in the presence of oil.

3-2. N-Hexadecane Sensitivity Test One platinum loop of the R-1 and R-2 strains was inoculated into a YG liquid medium (5 ml), and the mixture was heated to 30 ° C.
For 48 hours at 110 rpm (preculture). 50 μl of this culture was taken into a fresh YG liquid medium (5 ml), and cultured again at 30 ° C. for 48 hours with shaking at 110 rpm (main culture). This main culture solution was washed three times with 5 ml of physiological saline to remove the components of the medium, and suspended again in 5 ml of physiological saline. The cell suspension was diluted, the suspension of the concentration of the cells 10 ⁵ cfu / ml
30 ml was prepared. These were dispensed into 6 dry heat test tubes at 5 ml each. Then, add n-hexadecane to each test tube
25, 50, 125, 250 and 500 μl were added. The remaining test tubes were used as controls. The cell suspension after the addition of n-hexadecane was mixed using a vortex mixer for 30 seconds, left for 5 seconds, and then mixed again for 30 seconds. After the aqueous layer and the organic layer were completely separated, 1 ml of the aqueous layer was sampled. After appropriately diluting this sampling solution, it was spread on a YG agar medium and cultured at 30 ° C. The grown colonies were counted, and the number of bacteria after n-hexadecane treatment was determined.

The control test tube was vortexed under the same conditions as the n-hexadecane-treated sample, diluted appropriately, spread on a YG agar medium, and cultured at 30 ° C. The colonies that grew were counted, and the number of bacteria before the treatment with n-hexadecane was determined. The survival rate was determined by the following equation. The results are shown in FIG. Survival rate = number of bacteria after n-hexadecane treatment / number of bacteria before n-hexadecane treatment x 100 As a result of the experiment, the viability of the R-1 and R-2 strains was approximately 10 to 100 times by adding EPS. Rose. In addition, Rhodococcus bacteria other than the R-1 and R-2 strains had the same effect. From this, it was considered that EPS derived from S-2 strain had a protective effect on the toxicity of non-volatile hydrocarbons such as n-hexadecane.

3-3. Effect of EPS on degradation of AF-oil by natural marine bacteria Using seawater collected from Kamaishi Bay in Iwate Prefecture as a source of marine bacteria, adding inorganic nutrients and AF-oil to this NSW-A medium was prepared. <Composition of NSW-A medium> NSW (per liter) NH ₄ NO ₃ 1.0 g FeC ₆ H ₅ O ₇・ nH ₂ O 0.02 g K ₂ HPO ₄ 0.02 g Natural seawater 800 ml Distilled water 200 ml AF-oil 10 g Prepare NSW-A medium in a volume of 10 ml per test tube,
The cells were cultured at 110 ° C. with shaking at 110 ° C. Microbial growth over time,
The analysis of oil decomposition was performed.

The growth of microorganisms was carried out by sampling each sample, diluting it appropriately with sterile seawater, and then using a direct measurement method using DAPI staining and a colony measurement method using marine agar and 1/5 marine agar. The results are shown in FIG. For the decomposition of the oil, the remaining oil was extracted from the culture solution and the culture vessel using dichloromethane and dissolved in chloroform. This sample. Analysis was performed by gas chromatography-mass spectrum. Using GC-MS (QP-5000) manufactured by Shimadzu Corporation, the aromatic compound in the residual oil was quantified by performing analysis using a selective ion detection method.

(Analysis conditions) Capillary column (DB-5, 0.25 mmφ × 30 m, manufactured by J & W Scientific) Injection volume: 1 μl (split) Injection temperature: 300 ° C. Detector temperature: 230 ° C. Heating condition: 50 ° C. (2 minutes) 50 to 300 ° C (6 ° C / min), 300 ° C (15 minutes) Carrier gas: high-purity helium The results are shown in FIG.

As a result of measuring the growth of microorganisms in seawater by the direct counting method and the colony counting method, the number of bacteria when EPS was added increased about 10 times as compared with the case where no EPS was added. Also, GC-MS
The remaining aromatic compounds were analyzed by. The remaining amount was reduced to about 50%. Naphthalene declined to the same extent with or without EPS. from these things,
By adding EPS, it became possible to promote the oil decomposition activity of microorganisms existing in seawater.

[0048]

The present invention provides a novel exopolysaccharide. Since this polysaccharide has an action of promoting the growth ability and purification ability of purification bacteria such as crude oil existing in the ocean, it can be used for purification of marine pollution.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing a time-dependent change in the number of bacteria of R-1 strain and R-2 strain in the presence or absence of EPS.

FIG. 2 is a diagram showing the results of an n-hexadecane sensitivity test on the R-1 strain and the R-2 strain.

FIG. 3 is a diagram showing a time-dependent change in the number of marine bacteria in the presence or absence of EPS.

FIG. 4 is a graph showing the resolution of marine bacteria for aromatic compounds in the presence or absence of EPS.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 1/20 (C12N 1/20 D C12R 1:01) C12R 1:01) (C12P 19 / 04 (C12P 19/04 C12R 1:01) C12R 1:01) (72) Inventor Makoto Urai 1866 Kameino, Fujisawa-shi, Kanagawa Pref. Inventor Hiroshi Ansai 1866 Kameino, Fujisawa City, Kanagawa Prefecture Nihon University Junior College, Department of Agriculture Department of Applied Biochemistry (72) Inventor Michio Sunairi 1866, Kameino, Fujisawa City, Kanagawa Prefecture Department of Applied Biological Sciences, Nihon University Laboratory of Molecular Biology and Biology (72) Inventor Mutsuyasu Nakajima 1866 Kameino, Fujisawa-shi, Kanagawa Prefecture Nihon University Faculty of Bioresource Sciences Department of Applied Biological Science Molecular Biological Biology Research indoor F-term (reference) 4B064 AF11 CA02 CD19 CD23 DA16 4B065 AA01X AA45X BB04 CA22 CA54 CA56 4C090 AA01 AA08 BA91 BB12 BB13 BB14 BB16 BB22 BC19 CA01 CA23 DA40 4D040 DD03 DD11

Claims (5)

[Claims] 1. A polysaccharide having the following properties: (1) Extracellular polysaccharide produced by bacteria belonging to the genus Rhodococcus (2) Can be released from cells by physical impact from bacterial cells (3) Contains lipid-like substances 2. The polysaccharide according to claim 1, wherein the constituent neutral sugar and uronic acid include at least one of rhamnose, fucose, mannose, glucose, galactose, and glucuronic acid. 3. The polysaccharide according to claim 1, wherein the polysaccharide has a growth promoting effect on a microorganism whose growth is inhibited under the conditions in which an oil component is present. 4. The polysaccharide according to claim 1, which has an effect of increasing the survival rate of microorganisms whose growth is inhibited in the presence of an oil component in the presence of a non-volatile hydrocarbon. 5. A method for purifying a marine environment, comprising administering the polysaccharide according to any one of claims 1 to 4 to a marine environment contaminated with an oil component to promote the growth of marine bacteria.