JP2989175B1 - Polyester and method for producing the same - Google Patents

Abstract

Abstract: 3-Hydroxy, 5- (monofluorophenoxy) pentanoate (3H5 (MFP) P) unit or 3-hydroxy, 5- (difluorophenoxy)
Homopolymers composed of pentanoate (3H5 (DFP) P) units, copolymers containing at least 3H5 (MFP) P units or 3H5 (DFP) P units; Pseudomonas.
Putida; a method for producing the polymer using Pseudomonas sp. [Effect] By utilizing a long-chain fatty acid having a substituent, it is possible to synthesize a polymer having a phenoxy group substituted with one or two fluorine atoms at the side chain terminals, and has a high melting point and good processability. While providing stereoregularity and water repellency.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel polyester, a microorganism for fermenting and synthesizing the same, and a method for producing the same. More specifically, the present invention relates to a plastic-like polymer which decomposes under the action of microorganisms in a natural environment (in the soil, river, underwater) and a method for producing the same.

[0002]

2. Description of the Related Art It has been known that a large number of microorganisms accumulate polyester as an energy storage substance in cells. A typical example is poly-3-hydroxybutyrate (hereinafter referred to as P
(Abbreviated as (3HB)), and is a homopolymer comprising a monomer unit (3HB) represented by the following formula.

[0003]

Embedded image 3HB

[0004] P (3HB) is a polymer that can be decomposed in the natural environment, but when it is viewed as a polymer material, it has high crystallinity, is hard and brittle, and is not practically sufficient. Met. To solve this, Japanese Patent Laid-Open No.
-150393, JP-A-58-69225, JP-A-63-269989, JP-A-64-4
According to 8821, JP-A-1-156320 and JP-A-5-93049, this is achieved by incorporating a monomer unit having 3 to 6 carbon atoms structurally different from 3HB as a monomer unit for synthesizing a polyester. It has been proposed to overcome such disadvantages.

According to JP-A-63-229291, a hydrocarbon-utilizing bacterium, Pseudomonas oleovorans ATCC 29347, is provided with a 3-hydroxyalkanoate having 6 to 12 carbon atoms (abbreviated as 3HA) in a monomer unit. It has been reported that the copolymer P (3HA) can be synthesized by fermentation. This type of copolymer has a large number of methylene groups in the side chain and is a sticky polymer.

[0006]

Embedded image 3HA

As described above, at present, copolymers of a type in which the side chain length is changed have been proposed. That is, physical properties are controlled by the number of methylene groups in the side chain.
However, it has been said that fermentation synthesis using microorganisms is inefficient compared to chemical mass synthesis, and it is difficult to counter the cost of general-purpose plastics. For this reason, the search for a strain capable of synthesizing a high value-added polymer having both functions has been an issue.

[0006]

Means for Solving the Problems The present inventors have been searching for microorganisms that biosynthesize and accumulate polyester in cells by assimilating fatty acids that do not exist in nature and that have been chemically synthesized. The present inventors have discovered microorganisms and conducted further research to complete the present invention.

That is, the microorganism found by the present inventors is a 27N01 strain which grows using phenoxyalkanoic acid having one or two fluorine atoms substituted on a phenoxy group as a sole carbon source to synthesize polyester. Analysis of the monomer units of the polymer fermented and synthesized by this microorganism revealed that the structure was substituted with a fluorine atom such as 3-hydroxy, 5- (monofluorophenoxy) pentanoate (abbreviated as 3H5 (MFP) P), 3-hydroxy, −
(Difluorophenoxy) pentanoate (3H5 (D
FP) P), 3-hydroxy, 7- (monofluorophenoxy) heptanoate (3H7 (MFP) Hp
NMR) confirmed that 3-hydroxy, 7- (difluorophenoxy) heptanoate (abbreviated as 3H7 (DFP) Hp) was completely a polymer. When this microorganism was identified, the 27N01 strain was found to be Pseudomonas putida.

[0008]

Embedded image 3H5 (MFP) P

Embedded image 3H5 (DFP) P

Embedded image 3H7 (MFP) Hp

Embedded image 3H7 (DFP) Hp

The present invention is based on the finding of this microorganism. That is, the gist of the present invention is to provide (1) a polyester consisting of only 3-hydroxy, 5- (monofluorophenoxy) pentanoate (3H5 (MFP) P) unit, and (2) 3-hydroxy, 5- (difluorophenoxy) pentanoate. Polyester consisting only of (3H5 (DFP) P) units, (3) 3-hydroxy-5- (monofluorophenoxy) pentanoate (3H5 (MFP) P) units from 70 mol% to 99 mol%
Mole%, a copolymer polyester containing 30 to 1 mole% of 3-hydroxy, 7- (monofluorophenoxy) heptanoate (3H7 (MFP) Hp) unit,
(4) 3-hydroxy, 5- (difluorophenoxy)
70 pentanoate (3H5 (DFP) P) units
Mol% to 99 mol%, 3-hydroxy, 7- (difluorophenoxy) heptanoate (3H7 (DFP) H
p) a copolymer polyester containing 30 to 1 mol% of units, (5) at least 3-hydroxy, 5-
(Monofluorophenoxy) pentanoate (3H5
(MFP) Copolymer polyester comprising three-component monomer units containing (P) units, (6) three-component containing at least 3-hydroxy, 5- (difluorophenoxy) pentanoate (3H5 (DFP) P) units (7) As the second and third components, 3-hydroxyhexanoate (3HHx) unit, 3-hydroxyheptanoate (3HHp) unit, and 3-hydroxyoctanoate A copolymerized polyester with (3H5 (MFP) P) having two units selected from the group consisting of a (3HO) unit, a 3-hydroxynonanoate (3HN) unit and a 3-hydroxydecanoate (3HD) unit; 8) As the second and third components, 3-hydroxy It consists of a hexanoate (3HHx) unit, a 3-hydroxyheptanoate (3HHp) unit, a 3-hydroxyoctanoate (3HO) unit, a 3-hydroxynonanoate (3HN) unit and a 3-hydroxydecanoate (3HD) unit. 3H5 having two units selected from the group
(DFP) a copolymerized polyester with P, (9) Pseudomonas putida for synthesizing the polyester described in the above (1) to (8), and

(10) The present invention relates to the method for producing a polyester according to the above (1) to (9) using a microorganism of the genus Pseudomonas. Specifically, 1) a microorganism of the genus Pseudomonas is used as a carbon source under the restriction of nutrient sources other than the carbon source, using a fatty acid having one aromatic atom and a phenoxy group bonded to the aromatic ring in the molecule. 3-hydroxy, 5-characterized by culturing
(Monofluorophenoxy) pentanoate (3H5
(MFP) A method for producing a polyester having a (P) unit. 2) A microorganism of the genus Pseudomonas is carbonized by using, as a carbon source, a fatty acid having in its molecule a phenoxy group having two fluorine atoms bonded to an aromatic ring. Characterized by culturing under the restriction of nutrients other than the source, 3-hydroxy, 5-
(Difluorophenoxy) pentanoate (3H5 (D
The present invention relates to a method for producing a polyester having FP) and P) units.

A method for producing the polyester of the present invention using a microorganism of the genus Pseudomonas has not been reported so far.

The microbiological properties of the microorganism of the present invention, Pseudomonas putida, are as shown in Table 1 shown for 27N01. The 27N01 strain found as such a microorganism of the present invention was isolated from the soil of Horikoshi-cho, Nishi-ku, Nagoya-shi, and the 27N01 strain has been deposited under the Patent Microbial Center; Accession No. FERM P-16953.

[Table 1]

The Pseudomonas putida 27N01 strain of the present invention differs from Pseudomonas oleovorans, a known representative P (3HA) -producing bacterium, in the polyester biosynthesis ability. That is, the specificity of the polymerase for 3-hydroxyalkanyl-CoA, and this 27N01 strain has a wider range of substrates to act on.

The present invention discloses a microorganism of Pseudomonas having the above-mentioned properties, a microorganism-producing polyester fermented and synthesized by the microorganism, and a method for producing the same, and a technique for producing a polyester into which a fluorine group has been introduced. It provides a means of strategic.

Specifically, a microorganism of the genus Pseudomonas is given as a carbon source a fatty acid in which a methylene group having 5 or more carbon atoms is substituted with a fluorophenoxy group at the terminal, and the microorganism is usually subjected to the restriction of nutrient sources other than the carbon source. The target polyester can be obtained only by aerobically culturing under nitrogen limitation. When it is desired to increase the composition of a unit having only a methylene group, a fatty acid having 6 or more carbon atoms may be provided as a carbon source at the end of culture.

As described above, in the present invention, various polyesters having a phenoxy group substituted by fluorine can be fermented and synthesized utilizing the characteristics of microorganisms of the genus Pseudomonas. At present, Pseudomonas oleovorans has been reported as a microorganism capable of synthesizing a polyester having a functional group, that is, Macromolecule.
s, 1996, pp. 4572-4581, reported the results of fermentative synthesis of a polyester using a carboxylic acid in which hydrogen was replaced by fluorine on a methyl group as a carbon source. According to this, polyester was a copolymer. So,
It does not have the ability to synthesize a homopolymer like this microorganism.

In the fermentative synthesis of polyester using the microorganism of the present invention, usually, under the limitation of nutrient sources other than carbon source,
Although it can be easily obtained by culturing under conventionally known nitrogen-limited conditions, polyester can be derived even when essential nutrients other than carbon sources, such as phosphorus, minerals and vitamins, are restricted. In this case, since the growth of the cells is limited, the fermentation synthesis of the polyester is usually performed in a two-stage system.

The first stage is intended for the growth of cells, and is cultured under conditions rich in nutrient sources. At this time, since the cells hardly perform polyester synthesis, the carbon source is not limited to fatty acids, and can be freely selected as long as it can be assimilated. The cells obtained in the first step are washed and collected, and in the second step, a new carbon source is added to induce and culture the polyester. Therefore, the culture conditions in the second stage are important,
The carbon source provided in the second stage is a raw material for polyester synthesis, and it can be said that the chemical structure of this carbon source determines the structure of the polyester. Therefore, in the present invention, the carbon source means the carbon source provided in the second stage,
By variously adjusting the carbon source, the polyester containing a fluorine atom can be fermented and synthesized by utilizing the characteristics of microorganisms of the genus Pseudomonas. In addition, the culture conditions in the second stage are usually pH 6 to 8, temperature 25 to 35 ° C,
The aeration amount is 0.5 to 2 vvm, and the culture time is 48 to 96 hr.

The polyester synthesized by fermentation can be recovered from the cells by a conventional method. For example, after completion of the culture, the cells are washed with distilled water, methanol, and the like,
The dried cells obtained by drying under reduced pressure are subjected to an extraction treatment using chloroform or the like, and the cells are removed by centrifugation, filtration, etc., and then methanol is added to the extract to precipitate and recover the polyester.

[0020]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to the following Examples. Example 1 Pseudomonas putida 27N01 strain (Patent Microorganisms Biological Center; Accession No. FERM P-16953) was cultured with shaking at 30 ° C. for 24 hours using the following medium.
That is, water is added to the following composition and the total amount is 1
Liter (pH 7.0) to prepare a medium. Citric acid _{4g Na 2 HPO 4 2g KH 2} PO 4 2g MgSO 4 · 7H 2 O 0.2g yeast extract 0.3g

After the completion of the culture, the culture broth was centrifuged to collect the cells, and the whole was added to the following medium for 3 hours.
Shaking culture was performed at 0 ° C. for 96 hours. That is, water is added to the following medium composition to make the total volume 1 liter (pH 7.
0), a medium was prepared. Difluorophenoxy undecanoic acid _{Na 2 HPO 4 2g KH 2 PO} 4 2g NaHCO 3 1.5g MgSO 4 · 7H 2 O 0.2g FeSO 4 · 7H 2 O 0.02
g After completion of the culture, the cells are washed with distilled water and methanol,
The dried cells were dried under reduced pressure. The dried cells thus obtained were extracted at 30 ° C. for 5 hours. After removing the cells, a 10-fold amount of methanol was added to the chloroform extract to precipitate and recover the polyester. The obtained polyester is
The mixture was subjected to methanolysis at 90 ° C. for 90 minutes, and subjected to temperature rise analysis by a capillary gas chromatograph equipped with a light scattering molecular weight measuring device using a monomer as a methyl ester. The capillary gas chromatograph is HP5890 (Hewl
ett Packard) and a light scattering molecular weight measuring apparatus were performed using miniDAWN (Wyatt Technology). The column used was a fused silica capillary column DB-5 manufactured by J & W (column inner diameter 0.25 mm, liquid layer thickness 0.25 μm, column length 30).
m). Initial temperature 90 ° C, 5 minutes, heating rate 5 ° C /
And a final temperature of 250 ° C. for 2 minutes. FIG. 1 shows the results of gas chromatographic analysis of the methyl esterified product of the obtained polymer. FIG. 2 shows an analysis result of ¹³ C-NMR (100 MHz) of the polyester.
From this result, it was confirmed that this polyester was a homopolymer composed of one component of 3H5 (DFP) P unit.

Example 2 A similar experiment was carried out in the first culture of Example 1 except that octanoic acid was used instead of citric acid as a carbon source. As a result, a three-component copolymer composed of 3HHx, 3HO, and 3H5 (DFP) P units was obtained.

Example 3 A similar experiment was conducted in the second culture of Example 1 except that monofluorophenoxyundecanoic acid was used instead of difluorophenoxyundecanoic acid as a carbon source. as a result,
It was confirmed that this was a homopolymer composed of one component of the 3H5 (MFP) P unit.

Example 4 A similar experiment was carried out in the first culture of Example 3 except that octanoic acid was used instead of citric acid as a carbon source. As a result, a three-component copolymer composed of 3HHx, 3HO, and 3H5 (MFP) P units was obtained.

Example 5 A similar experiment was conducted in the first stage of culture in Example 1 except that nonanoic acid was used instead of citric acid as a carbon source. as a result,
A ternary copolymer comprising 3HHp, 3HN, and 3H5 (DFP) P units was obtained.

Example 6 A similar experiment was carried out in the first stage culture of Example 3 except that nonanoic acid was used instead of citric acid as a carbon source. as a result,
A ternary copolymer composed of 3HHp, 3HN, and 3H5 (MFP) P units was obtained.

Example 7 When the melting points of a polymer having no fluorinated phenoxy group and a polymer having the same structure having two fluorine groups were examined, there was a difference of about 40 ° C. The polymer with groups had a melting point above 100 ° C.

[0028]

The plastics produced by fermentation of microorganisms have been well studied as biodegradable plastics. Those having a fluorine group introduced into the side chain have existed conventionally, but have not been used as a homopolymer but as a copolymer unit.
% Or less. In the present invention, a homopolymer containing 100% of a unit having a fluorine group can be synthesized by using Pseudomonas putida having a wide range of assimilation properties and by introducing a fluorine group on the aromatic ring of a phenoxy group. This polymer can have a melting point of at least 100 ° C., which cannot be achieved by conventional polymers containing a substituent, and can be expected to improve physical properties. Further, by controlling the amount of these units contained in the polymer, desirable physical properties can be obtained. In addition, optical resolution can be expected due to water repellency and stereoregularity unique to in vivo synthesis.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ identification code FI (C12P 7/62 C12R 1:40) (58) Investigated field (Int.Cl. ⁶ , DB name) C08G 63/00-63 / 91 C12N 1/20-1/21 C12P 7/62 CA (STN) REGISTRRY (STN)

Claims (11)

(57) [Claims] 1. A polyester comprising only 3-hydroxy, 5- (monofluorophenoxy) pentanoate (3H5 (MFP) P) units. Embedded image 2. A polyester comprising only 3-hydroxy, 5- (difluorophenoxy) pentanoate (3H5 (DFP) P) units. Embedded image 3. A compound comprising 3-hydroxy-5- (monofluorophenoxy) pentanoate (3H5 (MFP) P) units in an amount of 70 mol% to 99 mol%, 3-hydroxy 7-
(Monofluorophenoxy) heptanoate (3H7
(MFP) Copolymer polyester containing 30 mol% to 1 mol% of Hp) units. Embedded image 4. A compound comprising 3-hydroxy-5- (difluorophenoxy) pentanoate (3H5 (DFP) P) units in an amount of 70 to 99 mol%, 3-hydroxy, 7-
(Difluorophenoxy) heptanoate (3H7 (D
FP) A copolymer polyester containing 30 mol% to 1 mol% of Hp) units. Embedded image 5. At least 3-hydroxy, 5- (monofluorophenoxy) pentanoate (3H5 (MFP)
P) Copolymer polyester comprising three-component monomer units containing units. 6. At least 3-hydroxy, 5- (difluorophenoxy) pentanoate (3H5 (DFP)
P) Copolymer polyester comprising three-component monomer units containing units. 7. The second and third components are 3-hydroxyhexanoate (3HHx) unit, 3-hydroxyheptanoate (3HHp) unit, 3-hydroxyoctanoate (3HO) unit, and 3-hydroxynonanoate. The copolyester according to claim 5, comprising two units selected from the group consisting of an ate (3HN) unit and a 3-hydroxydecanoate (3HD) unit. Embedded image Embedded image Embedded image Embedded image Embedded image 8. The second and third components include a 3-hydroxyhexanoate (3HHx) unit, a 3-hydroxyheptanoate (3HHp) unit, a 3-hydroxyoctanoate (3HO) unit, and a 3-hydroxynonano. 7. The copolymerized polyester according to claim 6, comprising two units selected from the group consisting of an acrylate (3HN) unit and a 3-hydroxydecanoate (3HD) unit. 9. Pseudomonas putida for synthesizing the polyester according to claim 1, 2, 3 or 4. 10. A microorganism of the genus Pseudomonas cultivated under the restriction of nutrient sources other than a carbon source, using a fatty acid having one fluorine atom in an aromatic ring and a phenoxy group bonded to the molecule in the molecule as a carbon source. For producing polyester having 3-hydroxy, 5- (monofluorophenoxy) pentanoate (3H5 (MFP) P) unit 11. A microorganism of the genus Pseudomonas cultivated under the restriction of nutrients other than the carbon source, using a fatty acid having two fluorine atoms in the aromatic ring and a phenoxy group bonded to the molecule in the molecule as a carbon source. 3-hydroxy, 5- (difluorophenoxy) pentanoate (3
Method for producing polyester having H5 (DFP) P) unit