JP2012210165A - New useful deep-sea bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new bacteria producing a polymer, to provide a method for producing the polymer using the bacteria, and to provide the polymer produced by the method.SOLUTION: The new bacteria belonging to the genus Kocuria, having 16SrRNA gene including (A) a DNA comprising a specific base sequence or (B) a DNA having ≥99.5% identity with the base sequence, and having the ability to produce a polymer, especially new Kocuria sp. 4B strain or a variant thereof, is provided. A method for producing the polymer containing cystathionine as a constituent by culturing the bacteria, and the polymer thus produced, are also provided.

Description

  The present invention relates to a novel bacterium that produces a novel biopolymer, isolated from deep-sea sediment, a method for producing the polymer using the bacterium, and the biopolymer produced by the method.

  In recent years, biodegradable plastics and microorganism-derived plastics have attracted attention because of environmental considerations. In particular, microorganism-derived plastics (microbe-derived polymers) are not only expected to be biodegradable in the natural environment when discarded, but also reduce the consumption of fossil fuels during production, so that the entire life cycle of the plastic is derived. Over the years, it has been attracting attention because of its low environmental impact.

  As such a microorganism-derived polymer (polymer), for example, polyhydroxybutyrate and derivatives thereof are known. Patent Document 1 discloses a method for producing polyhydroxybutyrate using a specific bacterium that produces polyhydroxybutyrate.

  However, at present, only the above-mentioned polyhydroxybutyrate and derivatives thereof are known as main microorganism-derived polymers (polymers). Thus, there is a further need for new microorganism-derived polymers and new microorganisms that produce microorganism-derived polymers.

  Furthermore, in recent years, it has become known that plastic that has flowed into the ocean sinks by gravity and accumulates in the deep ocean, polluting the natural environment and permanently damaging the marine ecosystem. It was. Many such wastes have been confirmed in actual deep sea surveys. However, it has recently been found that even plastics that are so-called biodegradable plastics are often very slowly decomposed in the deep sea. Therefore, in order to make environmental considerations effective, there has been a further demand for microorganism-derived polymers that are expected to be decomposed well in the deep sea, and new microorganisms that produce such polymers.

JP 2009-77678 A

As described above, new microorganisms (bacteria) -derived polymers and new microorganisms (bacteria) that produce microorganisms (bacteria) -derived polymers are further demanded.
Accordingly, an object of the present invention is to provide a novel bacterium that produces a polymer (polymer), a method for producing a polymer using the bacterium, and a polymer produced by the method.

  The inventor has conducted extensive research for many years to isolate new bacteria from deep sea bottom mud. As a result, the inventors have discovered a novel bacterium belonging to the genus Kochria having polymer-producing ability, and have reached the present invention. The novel bacterium according to the present invention is a new kind of bacterium belonging to the genus Koclear and has an ability to produce a novel polymer that has not been known so far.

Therefore, the present invention includes the following [1] to [8].
[1]
A bacterium belonging to the genus Koclear that has polymer-producing ability.
[2]
Bacteria belonging to Koclear are the following (A) or (B):
(A) DNA comprising the base sequence set forth in SEQ ID NO: 1
(B) DNA having 99.5% or more identity with the base sequence set forth in SEQ ID NO: 1,
The bacterium according to claim 1, comprising a 16S rRNA gene comprising
[3]
The bacterium according to claim 1 or 2, wherein the polymer is a polymer containing cystathionine as a constituent component.
[4]
The bacterium according to [1] or [2], wherein the bacterium belonging to the genus Koclear is Kocuria sp. Strain 4B (accession number: FERM P-22056), or a variant thereof.
[5]
[1] A method for producing a polymer comprising cystathionine as a constituent by culturing the bacterium according to any one of [4].
[6]
The step of culturing the bacterium according to any one of [1] to [4] under the following conditions:
Medium containing 0.5-3.0% tryptone, 0.1-2.0% yeast extract, 0.0-4.0% sodium chloride, 1.0-5.0% monosaccharide A step of culturing at a temperature of 30 to 40 ° C. for 10 to 60 hours,
A method for producing a polymer containing cystathionine as a constituent component.
[7]
The step of culturing the bacterium according to any one of [1] to [4] under the following conditions:
Culturing in an LB medium (LBNG medium) containing 3% of glucose and sodium chloride at 37 ° C. for about 20 hours under atmospheric pressure,
A method for producing a polymer containing cystathionine as a constituent component.
[8]
Following the step of culturing, the following steps:
A step of centrifuging the obtained culture solution to obtain a supernatant;
A step of adding ethanol to the resulting supernatant to form an ethanol precipitate;
A method for producing a polymer containing cystathionine as a constituent component.
[9]
Following the step of culturing, the following steps:
A step of centrifuging the obtained culture solution to obtain a precipitate;
Washing the obtained precipitate with artificial seawater,
A step of adding a sodium hydroxide aqueous solution to the washed precipitate and dispersing it to prepare a dispersion;
A step of centrifuging the obtained dispersion to obtain a supernatant;
A step of adding ethanol to the resulting supernatant to form an ethanol precipitate;
A method for producing a polymer containing cystathionine as a constituent component.
[10]
The polymer which contains the cystathionine as a structural component manufactured by the manufacturing method in any one of [5]-[9].
[11]
The polymer according to [10], comprising cystathionine in an amount of 60 to 90% by mass among amino acid components constituting the polymer.
[12]
The following characteristics:
(I) Among amino acid components constituting the polymer, cystathionine is contained at 60 to 70% by mass,
(Ii) has biodegradability,
(Iii) has water retention and exhibits viscosity upon absorption of water,
(Iv) Sugar content (phenol-sulfuric acid method): (85 ± 5) wt%,
(V) Uronic acid content (carbazole / sulfuric acid method): (2 ± 1) wt%,
(Vi) Protein content (Bradford method): (13 ± 4) wt%,
(Vii) Molecular weight: 31 to 35 million weight average molecular weight,
(Viii) Solvent solubility: soluble in water, insoluble in ethanol,
(Ix) IR spectrum absorption band (cm @ -1): 3400-3300, 1600, 1400,
[10] The polymer according to [10].

  The bacterium of the present invention produces a novel polymer. The bacterium of the present invention is a bacterium derived from deep sea bottom mud. Therefore, the polymer (polymer) produced by the present invention is expected to have good biodegradability in the deep sea. Therefore, according to the present invention, it is possible to obtain a polymer (polymer) material that has a low environmental load and is effective in consideration of the environment.

FIG. 1 is a scanning electron micrograph of strain 4B (Kocuria sp. 4B) in which bacterial cell aggregation was visually observed. FIG. 2 shows the main amino acid content of a polymer containing cystathionine. FIG. 3 is a view showing the main sugar content of a polymer containing cystathionine. FIG. 4 is a diagram showing the measurement results of the molecular weight of a polymer containing cystathionine.

  Hereinafter, the present invention will be described in detail with reference to embodiments of the present invention. The present invention is not limited to the specific embodiments exemplified below.

[Bacteria]
The bacterium of the present invention is a bacterium belonging to the genus Koclear that has a polymer-producing ability.

In a preferred embodiment, the bacterium belonging to the genus Koclear is the following (A) or (B):
(A) DNA comprising the base sequence set forth in SEQ ID NO: 1
(B) DNA having 99.5% or more identity with the base sequence set forth in SEQ ID NO: 1,
It is a bacterium having a 16S rRNA gene containing

  In a preferred embodiment, the identity (or homology) of the base sequence is preferably 99.4% or more, more preferably 99.5% or more, further preferably 99.6% or more, and more preferably 99. 0.7% or more, more preferably 99.8% or more, and further preferably 99.9% or more.

In one embodiment of a preferred implementation, as a suitable bacterium,
DNA consisting of the base sequence described in SEQ ID NO: 1, or
DNA consisting of a base sequence in which one or several bases are added, substituted and / or deleted from the base sequence set forth in SEQ ID NO: 1,
And a bacterium belonging to the genus Koclear that has a 16S rRNA gene comprising

  In one embodiment of suitable implementation, one or several of the bases is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 9, and further Preferably 1 to 8, more preferably 1 to 7, more preferably 1 to 6, more preferably 1 to 5, further preferably 1 to 4, more preferably 1 to 3, and still more preferably One or two, more preferably one.

In a preferred embodiment, examples of the bacterium belonging to the genus Koclear having polymer-producing ability include the following strains disclosed in the present specification.
A new species of the genus Koclear 4B (Kocuria sp. 4B)
Accession number: FERM P-22056

  Kocuria sp. 4B strain is assigned to the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Central 1-1, Tsukuba City 1-1-1 Tsukuba, Japan 305-8586) : Deposited as FERM P-22056 (Reception date January 27, 2011).

  The new strain 4 of the genus Koclear is not limited to the deposited strain, and may be a strain substantially equivalent to the deposited strain. In the present specification, the strain substantially equivalent to the deposited strain is a strain belonging to the genus Koclear, which has the polymer production ability disclosed in the present specification, and the base sequence of the 16S rRNA gene is the same as that of the deposited strain. The base sequence of the 16S rRNA gene (SEQ ID NO: 1) and 99.5% or more, preferably 99.6% or more, more preferably 99.7% or more, more preferably 99.8% or more, more preferably 99.9%. As described above, a strain having 100% homology and preferably having the same mycological properties as the deposited strain is preferable.

  In a preferred embodiment, the bacterium of the present invention may be a mutant (variant) of the aforementioned strain as long as it has a polymer-producing ability.

  Mutant strains can be obtained without mutation treatment with ethyl methanesulfonic acid, a commonly used mutation agent, other chemical treatments such as nitrosoguanidine, methylmethanesulfonic acid, ultraviolet irradiation, or treatment with a mutation agent. It can also be obtained by so-called spontaneous mutation.

  For example, such a mutant strain has the ability to produce a polymer. For example, a specimen is placed in a test tube containing a liquid medium and cultured at 37 ° C. to produce and accumulate a polymer (polymer-like substance). Can be tested by visual observation. In addition, from a test tube containing a strain having a polymer-producing ability, it can be seeded on an agar medium and cultured on a plate, and the strain can be isolated and obtained as a single colony.

  Bacteriological properties indicating that it is a bacterium of the genus Koclear, for example, BERGEY'S MANUAL OF Systematic Bacteriology (Volume 1, 1984, Vol. 2, 1986, Vol. 3 1989, 4 1989).

[Culture medium]
As the medium used for the growth of the bacterium of the present invention, for example, a medium on which bacteria belonging to the genus Koclear can grow can be used. Specific examples include LB medium, LBN medium, LBNG medium, and MB medium. However, it is not limited to these. The medium used for the growth of the bacterium of the present invention contains a carbon source that can be assimilated by the bacterium of the present invention, such as glucose, and a nitrogen source that can be assimilated by the bacterium of the present invention. For example, ammonium sulfate, ammonium chloride and the like can be contained. Further, if desired, it comprises a cation such as sodium ion, potassium ion, calcium ion and magnesium ion and an anion such as sulfate ion, chlorine ion and phosphate ion. The concentration of the carbon source is about 0.01 to 10%. The concentration of inorganic salts is, for example, about 0.001 to 5%.

  The LB medium is generally a medium containing tryptone 1% (w / v), yeast extract 0.5% (w / v), and sodium chloride 1% (w / v), and the salt concentration may be changed depending on the formulation. The LBN medium is generally one in which the sodium chloride concentration of the LB medium is 3%, and 2% agar may be added according to the prescription. The LBNG medium is generally an LBN medium with 3% glucose added, and 2% agar may be added depending on the formulation. The composition of the MB medium is a medium containing 3.74% of Difco's marine broth medium powder, and 2% of agar may be added depending on the formulation.

[polymer]
The bacterium of the present invention has an ability to produce a polymer in a marine environment, particularly in a deep sea of low temperature and high pressure. Therefore, if the bacterium of the present invention is used, a polymer can be produced in an environment of ordinary temperature, ordinary pressure, ordinary temperature and high pressure, or low temperature and high pressure without causing energy degradation and thermal decomposition. In a preferred embodiment, the bacterium of the present invention can suitably produce a polymer by culturing under pressurized conditions. In culture under pressure, the bacterium of the present invention increases the growth rate of the polymer as well as the growth rate. On the other hand, general bacteria are inhibited from growing or die under pressure. For this reason, since the contamination (contamination) of the product due to contamination with other bacteria is reduced, the polymer can be suitably produced by culturing under a pressurized condition.

When the produced polymer was once separated and mixed with Kocuria sp. Strain 4B (Kocuria sp. 4B strain) and cultured under low nutrient conditions, the polymer was consumed according to the growth of the fungus. It was observed that Thus, this polymer was biodegraded by the Koclear bacterium 4B strain distributed in the deep sea floor. That is, the produced polymer is a natural material in the marine environment, particularly in the deep sea environment, and is not accumulated even in the deep sea floor like ordinary plastic waste, but is biodegraded.
Moreover, when the produced polymer was separated and immersed in river water for a certain period, the molecular weight of the polymer decreased from the above-mentioned macromolecular weight to several thousand molecular weight. Thus, this polymer is not limited to the deep seabed, but is biodegraded in a freshwater environment close to a land environment.

The polymer obtained by the present invention is a novel polymer containing cystathionine as a main constituent component (repeating unit) with respect to the amino acid component constituting the polymer.
Cystathionine is known as an intermediate of cysteine synthesis produced from homocysteine and serine by the catalyst of cystathionine β-synthase. Cystathionine is 2-amino-4-[(2-amino-2-carboxyethyl) thio] butyric acid, and has a dimer-like structure in which the side chains of amino acids are associated via S (sulfur atom). Cystathionine is a medicine, health and health drug, and a substance used as a raw material thereof, but there has never been an example in which cystathionine is contained in a polymer substance. The polymer containing cystathionine is the first polymer obtained by the present invention. Furthermore, there is no example so far from the point that such a polymer is synthesized by bacteria. The polymer according to the present invention is a completely new substance as a polymer, and is also useful as a pharmaceutical, health and health medicine and raw materials thereof as well as cystathionine, and is also useful from the viewpoint of biodegradability in the deep sea. is there.

  In a preferred embodiment, the polymer contains cystathionine among the amino acid components, preferably 30 to 90% by mass, more preferably 40 to 90% by mass, further preferably 50 to 90% by mass, and more preferably 60 to 90% by mass. 90 mass%, More preferably, it contains 60-80 mass%, More preferably, it is 60-75 mass%, More preferably, it contains in the range of 60-70 mass%.

[Production of polymer]
The step of culturing the bacterium of the present invention under the following conditions:
Medium containing 0.5-3.0% tryptone, 0.1-2.0% yeast extract, 0.0-4.0% sodium chloride, 1.0-5.0% monosaccharide A step of culturing at a temperature of 30 to 40 ° C. for 10 to 60 hours,
By performing this, a polymer containing cystathionine as a constituent component can be produced. The content (concentration) of the components of the medium is expressed as 1.0% when 10 g of the component is added per liter of the liquid medium.

  In the above culture, tryptone is generally in the range of 0.5 to 3.0%, preferably in the range of 1.0 to 2.0%, more preferably in the range of 1.3 to 1.7%, particularly preferably 1. .5% can be added and used. There is no restriction | limiting in particular as tryptone which can be used, The general tryptone used by adding to a culture medium can be used. In the above culture, yeast extract (yeast extract) is generally in the range of 0.1 to 2.0%, preferably in the range of 0.2% to 1.0%, more preferably in the range of 0.3 to 0.7. % Range, particularly preferably 0.5% can be added and used. There is no restriction | limiting in particular as yeast extract (yeast extract) which can be used, The general tryptone used by adding to a culture medium can be used. In the above culture, sodium chloride is generally in the range of 0.0 to 4.0%, preferably in the range of 0.5 to 4.0%, more preferably in the range of 2.7 to 3.3%, particularly preferably. 3.0% can be added and used. The concentration of sodium chloride can also be 0%.

  In the above culture, the monosaccharide is generally in the range of 1.0 to 5.0%, preferably in the range of 2.0 to 4.0%, more preferably in the range of 2.5 to 3.5%, particularly preferably. 3.0% can be added and used. In order to produce a sufficient amount of polymer that can be recovered, the addition of a monosaccharide is essential. As the monosaccharide, a monosaccharide used by being added to a medium can be used. For example, preferably, a C5-C6 ketose, a C5-C6 aldose, or the like can be used. Specific examples of monosaccharides include xylose, fructose, glucose, mannose, and galactose. A particularly preferred monosaccharide is glucose. As a suitable medium containing such components, an LB medium containing glucose can be exemplified.

  In a preferred embodiment, the culture can be used by adding an amino acid to the medium in addition to the components described above. Any amino acid can be used without particular limitation as long as it is an amino acid that is generally added to a medium, and generally ranges from 1.0 to 5.0%, preferably from 2.0 to 4.0%. More preferably, the range of 2.5 to 3.5%, particularly preferably 3.0%, can be added and used. In a preferred embodiment, glutamic acid can be added and used at the above concentration as an amino acid. Furthermore, in a preferred embodiment, the culture can be used by adding ethanol to the medium in addition to the components described above. Ethanol is generally in the range of 1.0 to 5.0%, preferably in the range of 2.0 to 4.0%, more preferably in the range of 2.5 to 3.5%, particularly preferably 3.0%. Can be used.

  The culture temperature is generally in the range of 30 to 40 ° C, preferably in the range of 33 to 39 ° C, more preferably in the range of 35 to 38 ° C, and particularly preferably 37 ° C. The culture time can be appropriately increased or decreased by confirming the production of the desired polymer. For example, it is generally 10 to 60 hours, preferably 15 to 50 hours, and more preferably 20 to 40 hours. For example, it can be 20 hours, 36 hours, 40 hours, or the like.

  The culture can be performed at normal pressure (atmospheric pressure), but can be performed at a high pressure equal to or higher than atmospheric pressure. In a preferred embodiment, the pressure can be in the range of atmospheric pressure (0.0101325 MPa) to 50 MPa, or in the range of atmospheric pressure (0.0101325 MPa) to 30 MPa. As high pressure conditions, it can be set as the range of 10-50 Mpa, or the range of 10-30 Mpa, for example. The high pressure condition is a condition that is advantageous for the growth of the bacterium of the present invention, and at the same time is a disadvantageous condition for the growth of normal bacteria and microorganisms. Culturing can be performed particularly advantageously from the viewpoint that contamination can be prevented.

In a preferred embodiment, the culturing can be performed, for example, by any one of steps (i) to (v) for culturing under the following conditions.
(I) Step of culturing in LBN medium containing 3% glucose at 30 ° C. for about 36 hours (ii) Step of culturing in LBN medium containing 3% glucose at 37 ° C. and 20 MPa for about 20 hours (iii) Glucose (Iv) Step of culturing at 37 ° C. for about 20 hours in LBN medium containing 3% of glutamic acid (v) Ethanol and glucose Of culturing in LBN medium containing 3% each at 37 ° C. for about 40 hours

[Polymer separation]
The polymer produced by the culture as described above can be obtained by separation from bacterial cells by the steps described below. The polymer produced by culturing is a polymer in the form of a fibrous network attached to the cells (cell-attached polymer), and is released and dispersed in the culture solution without adhering to the cells. There is a polymer in a state of being (free polymer).

  The free polymer can be obtained from the supernatant obtained by centrifuging the culture solution obtained by culturing. For example, it can be obtained by adding ethanol to the obtained supernatant to form an ethanol precipitate, and collecting this ethanol precipitate.

  The above-mentioned centrifugation can be adjusted with the condition of separation of the supernatant and the precipitate, but for example, 500 to 20000 G, preferably 1000 to 15000 G, more preferably 1500 to 10000 G, more preferably 3000 to 10000 G. For example, by centrifugation for 120 to 5 minutes, preferably 60 to 10 minutes, more preferably 45 to 15 minutes. Although general ethanol can be used as ethanol to be added, it is preferable to use high-purity ethanol, for example, 99.0 to 99.9%, preferably 99.4 to 99.9%. Concentration (purity) ethanol can be used. The amount of ethanol added for the formation of the ethanol precipitate is, for example, in the range of the same volume to 3 times the volume of the supernatant (3 times the volume), or 1. 5 times (1.5 times volume) to 2.5 times (2.5 times volume) or 2 times (2 times volume) of the supernatant volume . The ethanol precipitate can be recovered by centrifugation or decantation.

  The cell-attached polymer is prepared by centrifuging the culture solution obtained by culturing, washing the resulting precipitate with artificial seawater, adding an aqueous sodium hydroxide solution to disperse the precipitate, and preparing a dispersion. Then, this dispersion can be obtained by centrifuging, adding ethanol to the resulting supernatant to form an ethanol precipitate, and collecting this ethanol precipitate.

  The washing with artificial seawater can be performed with an aqueous solution having an approximate salt concentration, but it is preferably with artificial seawater. Washing can be performed multiple times. The precipitate washed by artificial seawater is added with an aqueous sodium hydroxide solution, and the aqueous sodium hydroxide solution is, for example, 0.05 to 0.50 M, preferably 0.10 to 0.40 M, more preferably 0.15 to 0. An aqueous sodium hydroxide solution in the range of .35M, more preferably 0.20 to 0.30M can be used. The addition amount (volume) of the aqueous sodium hydroxide solution is preferably determined according to the volume of the culture solution subjected to the initial centrifugation, and is, for example, 10 to 50%, preferably 20 with respect to the volume of the culture solution. It is preferred that a volume corresponding to -40%, more preferably 25-35%, is added to the resulting precipitate and dispersed. In order to sufficiently disperse, for example, shaking or stirring may be performed. The shaking time can be appropriately adjusted with reference to sufficient dispersion, but for example, 30 minutes to 4 hours, preferably 1 to 3 hours. The shaking for dispersion can be performed, for example, in the range of 20 to 37 ° C, for example, in the range of 25 to 35 ° C, for example, in the range of 28 to 32 ° C. Centrifugation can be performed by adjusting the conditions based on separation of the supernatant and the precipitate. For example, 500 to 20000 G, preferably 1000 to 15000 G, more preferably 1500 to 10,000 G, more preferably 3000 to 10,000 G. It can be carried out by centrifugation at an acceleration of, for example, 120 to 5 minutes, preferably 60 to 10 minutes, more preferably 45 to 15 minutes. Although general ethanol can be used as ethanol to be added, it is preferable to use high-purity ethanol, for example, 99.0 to 99.9%, preferably 99.4 to 99.9%. Concentration (purity) ethanol can be used. The amount of ethanol added for the formation of the ethanol precipitate is, for example, in the range of the same volume to 3 times the volume of the supernatant (3 times the volume), or 1. 5 times (1.5 times volume) to 2.5 times (2.5 times volume) or 2 times (2 times volume) of the supernatant volume . The ethanol precipitate can be recovered by centrifugation or decantation.

  The cell adhesion type polymer and the free type polymer had different operations for obtaining them as described above, but after collecting them as ethanol precipitates, various comparisons were made. However, the properties were the same and could not be distinguished. That is, in the polymer produced by culture, a part of the same polymer is present attached to the microbial cells, and the remaining part is present in a state of being dispersed in the culture broth free from the microbial cells. It was. The polymer attached to the cells is collected as a precipitate by centrifugation, and is observed as the formation of cell aggregates by visual observation with an optical microscope. According to this, the formation of a fibrous network was observed. That is, the polymer solubilized as a dispersion is a material capable of forming extremely small fibers.

[Polymer characteristics]
The polymer produced by cultivation has the following properties:
(I) Among amino acid components constituting the polymer, cystathionine is contained at 60 to 70% by mass,
(Ii) has biodegradability,
(Iii) has water retention and exhibits viscosity upon absorption of water,
(Iv) Sugar content (phenol-sulfuric acid method): (85 ± 5) wt%,
(V) Uronic acid content (carbazole / sulfuric acid method): (2 ± 1) wt%,
(Vi) Protein content (Bradford method): (13 ± 4) wt%,
(Vii) Molecular weight: 31 to 35 million weight average molecular weight,
(Viii) Solvent solubility: soluble in water, insoluble in ethanol,
(Ix) IR spectrum absorption band (cm @ -1): 3400-3300, 1600, 1400.
However,% by weight indicates the content of each component relative to the dry mass of the polymer.

  The content of cystathionine contained in the amino acid component constituting the polymer is as described above. The sugar content is preferably 50 to 90% by weight, more preferably 50 to 80% by weight. The uronic acid content is preferably 0.5 to 5% by weight, more preferably 1 to 3% by weight. The protein content is preferably 7 to 40% by weight, more preferably 15 to 40% by weight. The molecular weight is preferably 30 million to 40 million, more preferably 31 million to 35 million, and still more preferably 32 million to 34 million as a weight average molecular weight.

  Hereinafter, the present invention will be described more specifically with reference to examples. The present invention is not limited to these examples. Unless otherwise specified,% is% by mass. Moreover,% of the component in a culture medium is 1.0% when 10 g is contained in 1 liter of culture media.

[Separation of bacteria]
Bacteria were isolated using the following sources and media.
Source of separation:
Submarine mud collected by the core sampler from the Japan Trench Landside Slope, depth of 6181m, 40 ° 6.025 N, 144 ° 10.997 E was used as the source of separation.
Medium used for separation:
In 1 liter of solution, 15 g of tryptone, 5 g of yeast extract, 30 g of sodium chloride and 30 g of glucose were dissolved to obtain a liquid medium. This medium is expressed as containing 1.5% tryptone (Difco), 0.5% yeast extract (Difco), 3.0% sodium chloride, and 3.0% glucose. Further, 20 g of agarose was further dissolved in 1 liter of liquid medium to prepare an agar medium. This agar medium is described as containing 2.0% agarose.

Screening was performed by the following separation procedure.
Separation procedure:
1. 100 μl of a suspension obtained by suspending the bottom mud of the separation source in artificial seawater was inoculated into a liquid medium in a 2 ml plastic tube, and statically cultured at 37 MPa at 37 ° C. for 1 week.
2. After culturing, an insoluble substance formed by visual observation in the liquid medium was applied to the agar medium and cultured at 37 ° C. for 1 week at atmospheric pressure.
3. The streak culture was repeated to obtain a bacterial 4B strain producing a polymer as a single colony.

[Identification of bacteria]
Isolated single colony bacteria were identified by the following method.
Classification by 16S rRNA gene analysis Morphological observation by scanning / transmission electron microscope Analysis of biochemical characteristics by sugar utilization test Comparison with related species by analysis of fatty acid composition of cell membrane
Comparison with related species by DNA-DNA hybridization

[Identification by 16S rRNA gene sequence]
The 4B strain selected by screening was identified as follows. Physiological tests were performed according to general methods. Identification was based on Bergey's Manual of Systematic Bacteriology, Baltimore: WILLIAMS & WILKINS Co., (1984). A bacterial identification system manufactured by BIOLOG, USA was also used. The 16S rRNA gene was amplified by direct PCR, and the primers used were 27F and 1492R primer sets capable of amplifying almost the entire length of the bacterial 16S rRNA gene. Sequence analysis was performed using an Applied Biosystems Co., Model 3130xl Genetic Analyzer. From these analysis results and chromosomal DNA homology test, the 4B strain was found to be a new species of the genus Koclear.

[Morphological and physiological properties test]
The isolated 4B strain is an aerobic Gram-positive bacterium, and its morphological and physiological properties are as follows.

Form: Cocci, no flagella

Glucose utilization: Dextrin, Cellobiose, D-fructose, α-D-glucose, maltotriose, D-mannose, Palatinose, D-psicose, D-raffinose, D-ribose, Salicin, Sucrose, Turanose, L-malic acid, Pyruvic acid, Adenosine, β-cyclodextrin, amygdalin, arbutin, lactulose, maltose, 3-methyl D-galactoside, β-methyl D-glucoside, D-trehalose, Inosine, Thymidine, Uridine, DL-α-glycerol phosphate


The following Table 1 shows the sugar assimilation property in comparison with related species.

[Bacterial fatty acid composition]
When the cell membrane fatty acid composition of the strain isolated using the HPLC method was identified, the Koclear 4B strain was characterized as a strain that produces isopentadecanoic acid (iso-C _{15: 0} ) as a main component. The cell membrane composition of Koclear 4B strain is as follows.

Cell membrane composition: iso-C14: 0: 1.5%, anti iso-C15: 0: 62.5%, iso-C16: 0: 13.1%, C16: 0: 2.6%, anti iso-C17: 0: 17.5%

Table 2 below shows the fatty acid composition of cell membranes compared to related species.

[Comparison of base sequences of 16S rRNA gene]
Chromosomal DNA was prepared from the isolated strain (Kokoria genus bacterium 4B strain), and this was used as a template to amplify almost the entire length of the 16S rRNA gene of each strain by direct PCR, and its base sequence was determined. The nucleotide sequence of 1463 bases of the 16S rRNA gene of Kokria sp. 4B strain (Kocuria sp. 4B strain) (accession number: FERM P-22056) determined in this manner is shown in SEQ ID NO: 1 of the Sequence Listing. Show. Furthermore, homology search was performed based on the obtained sequence information. A comparison list of homology (%) with related species is shown in Table 3 below.

  As described above, the Koclear 4B strain showed 99.3% homology between Kocuria kristinae and the 16S rRNA gene.

[Comparison with related species by DNA-DNA hybridization]
Genomic DNA was extracted from the isolate YI # 4B strain and a quantity strain of Cochlear Christina, which was a recent relative as a result of homology search of 16S rRNA gene, and homology was examined by DNA-DNA hybridization. As a result, the homology between the two strains of genomic DNA was about 50%. In other words, the isolate 4B was a completely different species of bacteria from the recent relative, Koclear Cristina.

  From the above results, it was found that the isolate 4B strain (4B strain) is a new species of the genus Koclear. The base sequence of the 16S rRNA gene of this Koclear bacteria 4B strain (Kocuria sp. 4B strain) (Accession number: FERM P-22056) is shown in SEQ ID NO: 1 in the sequence listing.

The isolated new strain of Cochlia sp. 4B was founded on January 27, 2011, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1-1, Higashi, Tsukuba City, Ibaraki, Japan) Deposited to 6). The accession number is as follows.
New species of the genus Koclear 4B (Kocuria sp. 4B)
Accession number: FERM P-22056

[Production of polymer]
A polymer was produced by culturing Koklia sp. 4B strain isolated from deep-sea bottom mud to produce a polymer.
[Bacteria culture]
This bacterium was cultured at 37 ° C. under atmospheric pressure in a liquid or agar medium containing 1.5% tryptone (Difco) and 0.5% yeast extract (Difco).
[Polymer production by bacteria]
This bacterium is cultured at 37 ° C under atmospheric pressure in a liquid medium in which 3% glucose and 3% sodium chloride are added to the above medium. A large amount of polymer substance (polymer) was produced in the medium. Production and accumulation of the polymer starts about 11 hours after the start of the culture, and the accumulated amount increases with the passage of time, and a sufficient amount of polymer has accumulated after about 20 hours after the start of the culture. Although the accumulation amount increased, the polymer covered the entire inside of the container about 24 hours after the start of the culture, and no significant increase in accumulation was observed thereafter.

In a preliminary experiment, the sodium chloride concentration in the above medium was 0%, and the culture was carried out. The amount of polymer was slightly lower than that in the above medium having a sodium chloride concentration of 3%, but almost the same level. Recovered. Thus, the sodium chloride concentration in the culture medium and the presence or absence of sodium chloride did not significantly affect the production of the polymer by the Kocuria sp. 4B strain (Kocuria sp. 4B).
In another preliminary experiment, when the culture was performed with the glucose concentration in the above medium set to 0%, the production of polymer was quantitatively compared to the above medium with a glucose concentration of 3%. Production was so small that it could be observed by an electron microscope, but it was hardly recovered as a polymer. Thus, the addition of glucose to the medium was essentially essential for the production of the polymer by Kocuria sp. 4B.

  In a preliminary experiment, the culture was performed under pressure. When pressure culture was performed under a pressure of 20 MPa, the growth rate of the bacteria doubled compared to the culture under atmospheric pressure, and accompanying this, the rate of polymer production (production amount per unit time) ) Also doubled. Thus, growth of Kocuria sp. Strain 4B (Kocuria sp. 4B) and polymer production were increased by culture under pressure.

[Confirmation of polymer production]
It was confirmed as follows that the polymer was produced by the culture.
After culturing the cells, the culture solution was observed with an optical microscope, and the aggregation of the cells was visually confirmed. The aggregation of the cells was caused by products accumulated around the cells. Furthermore, the aggregated bacterial cells were observed with a scanning electron microscope, and it was confirmed that accumulation of filamentous products occurred.

  FIG. 1 is a scanning electron micrograph of strain 4B (Kocuria sp. 4B) in which bacterial cell aggregation was visually observed. The bar at the lower right of the photo shows 100 nm. The electron micrograph shows that a large number of polymers are accumulated in the form of fibers (threads) outside the granular cells. From Fig. 1, the electron micrograph also shows that the 4B strain produces the polymer, that the polymer is secreted and accumulated outside the cells, and that the polymer exists in the form of fibers, It has been shown. Thus, when the aggregation of the bacterial cells was visually observed, a fibrous (thread-like) polymer was produced and accumulated outside the bacterial cells also by observation with an electron microscope.

[Polymer separation]
After culturing under the above conditions, the polymer accumulated outside the cells was separated under the following conditions:
The strain 4B was cultured at 37 ° C. for about 20 hours in an LB medium containing 3% glucose, and after confirming the aggregation of the cells by visual observation, 7500 G × 30 minutes was obtained with respect to the obtained culture solution. Was centrifuged.

  An equal amount (volume) of 99.5% ethanol was added to the supernatant obtained by centrifugation, and the resulting precipitate was obtained by decantation. In this way, the product contained in the supernatant was separated. The product thus separated corresponds to the polymer (free form) dissolved or dispersed in the medium among the polymers accumulated outside the cells.

  Also, the precipitate obtained by centrifugation is washed with artificial seawater, 0.25M sodium hydroxide solution is added in 30% volume (volume) of the medium, shaken at 30 degrees for 2 hours, and then centrifuged at 7500G x 30 minutes. To the supernatant obtained by the separation, 99.5% ethanol was added in an amount (volume) twice, and the resulting precipitate was obtained by decantation. In this way, the product contained in the precipitate was separated. The product thus separated corresponds to a polymer (bacterial cell attachment type) that has been attached to the bacterial cell among the polymers accumulated outside the bacterial cell.

  As described above, as revealed by observation with an optical microscope and an electron microscope and centrifugation, the polymer produced by the culture forms a fibrous network attached to the cells. And a polymer (free polymer) in a state of being dispersed in the culture solution without adhering to the bacterial cells. These cell-attached polymer and free polymer had different operations as described above until they were recovered as ethanol precipitates, but the recovered ethanol precipitates were dissolved in distilled water, After making the polymer solution, no difference was found in the properties of any operation, and no distinction could be made. Therefore, in the following measurement, the results of measurement on a cell-attached (cell-attached) polymer solution prepared by dissolving ethanol in distilled water were used to represent the cell-attached polymer and the free polymer. Show.

[Polymer identification and component measurement]
The separated polymer was further purified by the following method for identification and component analysis.

[Polymer purification]
The product (ethanol precipitate) separated by the above method was dissolved in distilled water and subjected to molecular weight fractionation by gel filtration chromatography, and the fraction corresponding to the peak with the largest molecular weight was obtained and purified. Sepharose S-500 HR (GE Healthcare) was used as the packing material for the gel filtration chromatograph.

[Measurement of infrared absorption spectrum]
The purified polymer was measured for infrared absorption spectrum using Spectrum 65 FT-IR (PerkinElmer).

[Measurement of sugar composition]
The purified polymer was subjected to qualitative and quantitative measurement of sugar by the phenol / sulfuric acid method. For the analysis, gas chromatography / mass spectrometry (GC / MS) GCMS-QP5050A (Shimadzu Corporation) was used, and DB-5 (J & W Scientific) was used as the column. For the determination of sugar, a standard curve was prepared using glucose, galactose, mannose, inositol, rhamnose, xylose and fucose as standards.

[Measurement of acid sugar]
The purified polymer was subjected to qualitative and quantitative measurement of acidic sugars by the m-hydroxybiphenyl method. For the analysis, gas chromatography / mass spectrometry (GC / MS) GCMS-QP5050A (Shimadzu Corporation) was used, and DB-5 (J & W Scientific) was used as the column. A calibration curve was prepared using alginic acid and galacturonic acid as standards for the quantification of acidic sugars.

[Measurement of amino acid composition]
The purified polymer was hydrolyzed using 6NHCl at 110 to 125 ° C. for 20 to 24 hours, and the amino acid composition was measured using a Hitachi L-8900 amino acid analyzer. Each analytical value was quantified using a standard sample of each amino acid.

[Measurement of molecular weight]
The molecular weight of the purified polymer was determined by gel filtration chromatography using Shimadzu 10A GPC (Shimadzu Corporation). A RID-10A differential refractometer was used as a detector, and an OHPac SB-805 HQ (Shodex) was used as a column. Distilled water was used as the eluent. A calibration curve was created using 8 points (STANDARD P-82 (Shodex)) with a standard Mw = 5000 to 8000000.

[Identified properties of the polymer]
As described above, the cell-attached polymer and the free polymer were indistinguishable in nature after the collected ethanol precipitate was dissolved in distilled water to form a polymer solution. This polymer was a polymeric material containing sugar and amino products. In particular, 70% by mass of amino acids constituting the polymer was cystathionine. There have never been any reports of polymers containing cystathionine, including natural and synthetic, and this polymer was a completely new polymer. The polymer molecule was a very large polymer having a weight average molecular weight exceeding 33 million.

  Table 4 below shows the amino acid component contents obtained as a result of analysis of the produced high molecular substance (polymer). FIG. 2 shows the data of Table 4 as a pie chart. FIG. 2 shows the main amino acid content of a polymer containing cystathionine.

  Table 5 shows the sugar content (sugar composition) obtained as a result of the analysis of the polymer substance produced (polymer). FIG. 3 shows the data in Table 5 as a pie chart. FIG. 3 is a view showing the main sugar content of a polymer containing cystathionine.

  As a molecular weight measurement result obtained as a result of the analysis of the polymer substance (polymer) produced, FIG. 4 shows a differential molecular weight curve by chromatography performed for molecular weight measurement. In the graph of FIG. 4, four peaks are confirmed from the right side of the graph. The largest peak is the rightmost peak (peak number 1 in Table 6), and the small peak (peak number 2 in Table 6) on the left side thereof. There is a very small peak (peak number 3 in Table 6) on the left side, and a small peak (peak number 4 in Table 6) on the left side. The values of molecular weight and the like for these peaks with peak numbers 1 to 4 are shown in Table 6 below.

  As shown in Peak No. 1, an ultra-large macromolecule having a weight average molecular weight exceeding 33 million was the substance of the polymer containing cystathionine. The other small peaks (peak numbers 2 to 4) are small in quantity and are considered to be decomposed products of the supermacropolymer shown in peak number 1.

[Biodegradability of polymers]
When the produced polymer was once separated and mixed with Kocuria sp. Strain 4B (Kocuria sp. 4B strain) and cultured under low nutrient conditions, the polymer was consumed according to the growth of the fungus. It was observed that Thus, it was confirmed that this polymer was biodegraded.
When the produced polymer was separated and immersed in river water for a certain period of time, the molecular weight of the polymer decreased from the above-mentioned macromolecular weight to several thousand molecular weight. Thus, it was confirmed that this polymer is biodegradable in a freshwater environment close to a land environment without being limited to the deep sea bottom.

This polymer containing cystathionine has the following properties.
(1) It has biodegradability (2) It has high water retention and shows viscosity when it absorbs water.
(3) Sugar content (phenol-sulfuric acid method): (85 ± 5) wt%
(4) Uronic acid content (carbazole / sulfuric acid method): (2 ± 1) wt%
(5) Protein content (Bradford method): (13 ± 4) wt%
(6) Molecular weight: A weight average molecular weight of 33 million or more.
(7) Solvent solubility: Soluble in water, insoluble in ethanol.
(8) IR spectrum absorption band (cm @ -1): 3400-3300, 1600, 1400.

  The present invention provides novel bacteria that produce polymers. According to the present invention, the bacterium can be used to produce a novel polymer. The present invention is an industrially useful invention.

[Explanation of Sequence Listing]
Sequence number 1 of a sequence table is 1463 bases of the 16S rRNA gene of the Kokria sp. 4B strain (Kocuria.sp. 4B) (accession number: FERM P-22056) concerning this invention.

Claims (8)

Next (A) or (B):
(A) DNA comprising the base sequence set forth in SEQ ID NO: 1
(B) DNA having 99.5% or more identity with the base sequence set forth in SEQ ID NO: 1,
A bacterium belonging to the genus Koklia, which has a 16S rRNA gene containing sucrose and has a polymer-producing ability.   A bacterium belonging to the genus Cochlear having the ability to produce a polymer, which is Kocuria sp. Strain 4B (accession number: FERM P-22056).   The bacterium according to claim 1 or 2, wherein the polymer is a polymer containing cystathionine as a constituent component.   A method for producing a polymer comprising cystathionine as a constituent by culturing the bacterium according to claim 1. A step of culturing the bacterium according to any one of claims 1 to 3 under the following conditions:
Medium containing 0.5-3.0% tryptone, 0.1-2.0% yeast extract, 0.0-4.0% sodium chloride, 1.0-5.0% monosaccharide A step of culturing at a temperature of 30 to 40 ° C. for 10 to 60 hours,
A method for producing a polymer containing cystathionine as a constituent component.   The polymer which contains the cystathionine as a structural component manufactured by the manufacturing method in any one of Claims 4-5.   The polymer of Claim 6 which contains cystathionine by 60-90 mass% among the amino acid components which comprise a polymer. The following characteristics:
(I) Among amino acid components constituting the polymer, cystathionine is contained at 60 to 70% by mass,
(Ii) has biodegradability,
(Iii) has water retention and exhibits viscosity upon absorption of water,
(Iv) Sugar content (phenol-sulfuric acid method): (85 ± 5) wt%,
(V) Uronic acid content (carbazole / sulfuric acid method): (2 ± 1) wt%,
(Vi) Protein content (Bradford method): (13 ± 4) wt%,
(Vii) Molecular weight: 31 to 35 million weight average molecular weight,
(Viii) Solvent solubility: soluble in water, insoluble in ethanol,
The polymer according to claim 6, having (ix) IR spectral absorption band (cm-1): 3400-3300, 1600, 1400.