JP2001340078A - Method for producing polyester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a polyester by which the molar fraction of 3-hydroxyhexanoic acid (3HH) is controlled and a copolyester P[3- hydroxybutyric acid (3HB)-co-3HH] having various molar fractions of the 3HH is stably produced with high productivity. SOLUTION: This method is to produce the copolyester P(3HB-co-3HH) in which the 3HB represented by the following formula (1) is copolymerized with the 3HH represented by the following formula (2) using a microorganism and the method for producing the polyester comprises using any of a combination of oils and fats different in number of carbons, a combination of fatty acids different in the number of carbons or a combination of the oils and fats with the fatty acids different in the number of carbons as at least two kinds of carbon sources and thereby collecting the polyester different in the molar fraction of the 3HH.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a copolyester by fermentation production. More specifically, the present invention relates to a method for producing a plastic-like polymer which is decomposed by the action of microorganisms in a natural environment (in the soil, river, underwater).

[0002]

2. Description of the Related Art It has been known that polyesters are accumulated in cells as an energy storage substance in many microorganisms. A typical example is poly-3-hydroxybutyric acid (hereinafter abbreviated as P (3HB)). P (3
HB) is a thermoplastic polymer, which is biologically degraded in the natural environment, and thus attracts attention as an environmentally friendly green plastic. However, since P (3HB) has high crystallinity and has a hard and brittle property, its application range is practically limited. For this reason, research aimed at improving this property has been made.

Among them, JP-A-57-150393 and JP-A-59-220192 disclose 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3H
V) and a method for producing a copolymer P (3HB-co-3HV). This P (3HB-co-3
HV) is considered to be applicable to a wide range of applications because it is more flexible than P (3HB). The method for producing a copolymer in these publications is similar to the method for producing P (3HB), in which the cells are grown in the first stage, and the microorganisms are cultured in the second stage by limiting nitrogen or phosphorus to produce the copolymer. Was to do.

As for P (3HB-co-3HV), studies have been made to control the mole fraction of 3HV because the flexibility changes as the mole fraction of 3HV increases. For example, JP-A-57-150393 and JP-A-63-269989 use propionic acid, and JP-B-7-79705 discloses propane-1.
-Hol is used to control the 3HV molar fraction by changing the amount added to the medium, and the P (3HB-co-3H) having a 3HV molar fraction of 10 to 90 mol% is used.
V) is manufactured. However, in fact P
(3HB-co-3HV) has a small change in physical properties even when the 3HV mole fraction is increased, and the flexibility required for use in a film or the like is not improved.
It was used only in the field of hard molded products such as shampoo bottles and disposable razor handles.

In recent years, research has been conducted on a two-component copolymer polyester P (3HB-co-3HH) of 3HB and 3-hydroxyhexanoic acid (hereinafter abbreviated as 3HH) and a method for producing the same. For example, they are described in JP-A-5-93049 and JP-A-7-265065, respectively. In these publications, P (3HB-co-3H)
H) The method for producing the copolymer is described in Aeromonas cavia isolated from soil.
e) was produced by fermentation from fatty acids such as oleic acid and oils and fats such as olive oil using e).

[0006] Studies on the properties of P (3HB-co-3HH) have also been made (Y. Doi, S. Kit).
amura, H .; Abe, Macromolecule
s 28, 4822-4823 (1995)). In this report, a single type of fatty acid having a carbon number of 12 or more is used as the sole carbon source. cultivating caviae,
P (3HB-co-3H) containing 3 to 19 mol% of 3HH
H) is produced by fermentation. This P (3HB-co-3H
H) increases with increasing 3HH mole fraction, P (3H
The hard and brittle properties of B) gradually began to show soft properties, and it was revealed that they exhibited a flexibility exceeding P (3HB-co-3HV).

A. Caviae PHA synthase gene was cloned and this gene was cloned into P (3HB)
Elimination of 90% or more
It has been reported that P (3HB-co-3HH) is produced from a fatty acid as a carbon source using a recombinant strain introduced into R. ropus. (T. Fukui, Y. doi, J. Ba
cterol. vol. 179, no. 15,482
1-4830, JP-A-10-108682) Among them, sodium octanoate is used as a carbon source,
P (3HB-c having a 3HH molar fraction of 10 to 20 mol%
o-3HH) can be produced.

As described above, if a copolymer can be produced by controlling the 3HH mole fraction of the present polymer in a wide range, fermentative production from a hard copolymer to a soft copolymer can be performed, and a television housing can be produced. It can be expected to be applied to a wide range of fields, from those requiring hardness such as to those requiring flexibility such as yarns and films.
However, these methods have low productivity of the cells,
It cannot be applied as a production method for practical use of this polymer. As described above, controlling and producing the 3HH mole fraction of P (3HB-co-3HH) is indispensable for application to a wide range of fields. Therefore, there has been a demand for a production method capable of maintaining high cell productivity and polymer content and controlling the 3HH mole fraction.

[0009]

DISCLOSURE OF THE INVENTION In view of the above situation, the present invention controls the 3HH mole fraction to produce P (3HB-co-3HH) having various 3HH mole fractions with high productivity and stability. It is an object of the present invention to provide a method for producing a polyester produced by the above method.

[0010]

Means for Solving the Problems As a result of various studies, the present inventors have found that a microorganism accumulating a P (3HB-co-3HH) copolymer can be converted into a medium containing fats and oils and / or fatty acids as a carbon source. And succeeded in stably producing a copolymer having a different 3HH mole fraction while maintaining high productivity.

That is, the present invention provides a copolymerized polyester obtained by copolymerizing 3-hydroxybutyric acid represented by the following formula (1) and 3-hydroxyhexanoic acid represented by the following formula (2) using a microorganism. A method for producing P (3HB-co-3HH), wherein a combination of oils and fats having different carbon numbers, a combination of fatty acids having different carbon numbers, or an oil and fat having different carbon numbers is used as at least two carbon sources. Is a method for producing a polyester in which polyesters having different 3HH mole fractions are collected by using any one of the above combinations.

[0012]

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[0013]

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The gist of the present invention is that P (3HB-co
-3HH) copolymer accumulating at least 2
As a kind of carbon source, a combination of fats and oils with different carbon numbers,
By culturing in a medium to which either a combination of fatty acids having different carbon numbers or a combination of fats and oils having different carbon numbers and a fatty acid is added, the 3HH mole fraction is 1 to 40 mo
The present invention relates to a method for producing a copolymer polyester, which comprises accumulating a P (3HB-co-3HH) copolymer in a range of 1% in cells and collecting a polymer from the culture. The details of the present invention will be described below.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for producing a polyester of the present invention, a 3-hydroxybutyric acid represented by the above formula (1) and a 3-hydroxyhexanoic acid represented by the above formula (2) are used together with a microorganism. Copolymerized polyester P (3
HB-co-3HH). 3-hydroxybutyric acid represented by the above formula (1) and the above formula (2)
Copolymerized polyester P (3HB-co-3HH) obtained by copolymerizing with 3-hydroxyhexanoic acid represented by
For example, it is represented by the following general formula (3).

[0016]

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Wherein m and n are the same or different and
Represents an integer of 1 or more. In the method for producing the polyester of the present invention, the microorganism to be used is not particularly limited, and the genus Alcaligenes (Ralstonia) or A deposited at the depository of the strain (eg, IFO, ATCC, etc.) is used.
Bacteria such as genus eromonas and Escherichia can be used.
es eutrophus (Ralstonia eu
It is preferred to use tropha).

The microorganism used in the method for producing a polyester of the present invention is preferably a microorganism transformed with a recombinant vector containing a polyester synthase gene. When a transformant is prepared, a plasmid vector that can grow autonomously in the bacterium can be used as the vector, but may be integrated into the chromosome. The polyester polymerization gene may have an expression unit that functions in the host bacterium, such as a promoter and a terminator, in addition to the structural gene. The polyester polymerization gene used in the polyester production method of the present invention is Aeromonas caviae.
Caviae) is preferable, and for example, a gene fragment described in JP-A-10-108682 can be used.

In order to introduce a recombinant vector into a microorganism,
It can be performed by a known method. For example, the calcium method (Lederberg. EM et al.,
J. Bacteriol. 119.1072 (197
4)) and electroporation (Current
Protocols in Molecular Bi
, vol. 1, page 1.8.4, 1994).

(Method for Producing Polyester) In the method for producing polyester of the present invention, when culturing a microorganism, a combination of oils and fats having different carbon numbers and different carbon numbers are used as at least two kinds of carbon sources as described above. Using either a combination of fatty acids or a combination of fats and oils having different carbon numbers and fatty acids, a medium containing nutrients other than carbon sources, inorganic salts, and other organic nutrients can be used. In addition, the said fats and oils with different carbon numbers mean fats and oils with different carbon numbers of main fatty acids among fatty acids constituting the fats and oils. The culture temperature may be any temperature at which the bacteria can grow, but is preferably 20 ° C to 40 ° C. The culture time is not particularly limited, but may be about 1 to 7 days. Thereafter, the polyester may be recovered from the obtained cultured cells or culture. When a transformant is used, an antibiotic such as kanamycin, ampicillin, or tetracycline corresponding to the resistance gene present in the vector during the culture may be added.

In the method for producing a polyester of the present invention, a combination of oils and fats having different carbon numbers, a combination of fatty acids having different carbon numbers,
Alternatively, polyesters having different 3HH mole fractions are collected by using any one of combinations of fats and oils and fatty acids having different carbon numbers.

There are no particular restrictions on the fats and oils used as the carbon source. For example, soybean oil whose constituent fatty acids are 52% linoleic acid, 21% oleic acid, 12% palmitic acid, 11% linolenic acid and 3% stearic acid. Rapeseed oil whose fatty acid is oleic acid 59%, linoleic acid 22%, linolenic acid 11%, palmitic acid 4%, stearic acid 2%, constituent fatty acids are linoleic acid 51%, oleic acid 35%, palmitic acid 11%, stearin Corn oil with 2% acid, 75% oleic acid, 10% linoleic acid, 10% palmitic acid, 3% stearic acid, olive oil with 1% linolenic acid, 43% palmitic acid, 41% oleic acid %, Linoleic acid 10%, stearic acid 5%, myristic acid 1%, palm oil, constituent fatty acid 47% lauric acid, myristic Coconut oil, 18% acid, 9% palmitic acid, 7% oleic acid, 3% stearic acid, 2% linoleic acid, constituent fatty acids 44% lauric acid, 17% oleic acid, 14% myristic acid, 3% palmitic acid %, Linoleic acid is 3%, and at least one selected from natural fats and oils such as palm kernel oil and fatty acids having 6 to 20 carbon atoms such as hexanoic acid, octanoic acid, decanoic acid and lauric acid. Triglycerides, diglycerides, monoglycerides and the like synthesized from fatty acids and glycerol. Among oils and fats derived from natural products, coconut oil and palm kernel oil containing 40 to 50% of fatty acids having 12 or less carbon atoms as constituent fatty acids are preferable. For the synthesized triglyceride, diglyceride and monoglyceride, the constituent fatty acid is preferably hexanoic acid.

In the method for producing a polyester according to the present invention, it is preferable to use, as the fat or oil, a fat or oil selected from the group consisting of coconut oil, palm oil, palm kernel oil and hexanoic triglyceride. Examples of the fatty acids include saturated or unsaturated fatty acids such as hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linoleic acid, linolenic acid, and myristic acid, and fatty acid derivatives such as esters and fatty acid salts of these fatty acids. Is mentioned. Among them, fatty acids selected from the group consisting of saturated or unsaturated fatty acids having 6 to 10 carbon atoms, fatty acid esters and fatty acid salts thereof are preferred.

In order to obtain a polyester having a high 3HH mole fraction, it is preferable to use a fat or oil containing a fatty acid having as few carbon atoms as possible. In oils and fats, coconut oil, palm kernel oil, hexanoic triglyceride and the like containing a relatively large number of fatty acids having 12 or less carbon atoms are preferable. In fatty acids, saturated or unsaturated fatty acids having 6 to 10 carbon atoms, fatty acid esters thereof, Fatty acid salts are preferred. Among saturated or unsaturated fatty acids having 6 to 10 carbon atoms, fatty acid esters and fatty acid salts thereof, those having an even number of carbon atoms are more preferable, and hexanoic acid having 6 carbon atoms is particularly preferable.

In the method for producing a polyester of the present invention, it is preferable to control the 3HH mole fraction by changing the amount of fats or oils or fatty acids used as a carbon source. When natural oils and fats are used as carbon source, 3H
P (3HB-c) having an H mole fraction of less than 10 mol%
o-3HH) can be produced. 3HH mole fraction is 10mo
In order to produce P (3HB-co-3HH) larger than 1%, a fatty acid having 6 to 10 carbon atoms or its triglyceride, diglyceride or monoglyceride may be added.

In the method for producing a polyester of the present invention, as described above, as at least two carbon sources, a combination of oils and fats having different carbon numbers, a combination of fatty acids having different carbon numbers, or an oil and fat having different carbon numbers is used. By changing the amount of the above fats and oils or fatty acids by using any of the above combinations, 3
The HH mole fraction can be controlled between 1 and 40 mol%.

In the method for producing the polyester of the present invention, there is no particular limitation on the method for adding the fat or oil and / or fatty acid as a carbon source. However, if a large amount of fats and oils are added at one time, the concentration of dissolved oxygen in the culture solution may be reduced. Also, fatty acids are cytotoxic and may cause growth inhibition. Therefore, as a method of adding these, a method of dividing and adding an amount that does not cause growth inhibition, a method of continuously adding using a peristaltic pump or the like, and maintaining a concentration that does not cause growth inhibition are preferable. When added in portions, the concentration of the fatty acid added at one time is preferably 1 w / v% or less, and the concentration of fats and oils is preferably 5 w / v% or less, but is not particularly limited. The total amount of the fats and oils and fatty acids to be added may be such that the growth of the selected microorganism is not affected, and is preferably 20 w / v% or less, but is not particularly limited. The fats and oils and the fatty acids may be added in separate lines, but if they are compatible, it is preferable to add the fats and oils and the fatty acids in a mixed manner since the number of addition lines can be reduced.

Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate and ammonium phosphate, as well as peptone, meat extract, yeast extract and the like. Examples of the inorganic salts include potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, and sodium chloride.

Other organic nutrients include amino acids such as glycine, alanine, serine, threonine,
Vitamin such as proline, for example, vitamin B ₁ , vitamin B ₁₂ , vitamin C and the like.

In the present invention, for example, the following method can be used for recovering polyester from bacterial cells. After the culture, the cells are separated from the culture using a centrifuge or the like, and the cells are washed with distilled water, methanol and the like, and then dried. The polyester is extracted from the dried cells using an organic solvent such as chloroform. Cell components are removed from the organic solvent solution containing the polyester by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the polyester. The supernatant is removed by filtration or centrifugation, and the polyester is recovered by drying. The obtained polyester is analyzed by, for example, a gas chromatography method or a nuclear magnetic resonance method.

[0031]

The present invention will be described more specifically with reference to the following examples. However, the present invention does not limit the technical scope to these examples.

Example 1 Alcaligenes e
utrophus PHB-4 / pJRDEE32d1
3 strains (T. Fukui., Y. Doi., Appl M)
microbiol Biotechnol. , 49,33
3-336, (1998), accession number FERM P-1
5786) (hereinafter abbreviated as Aed13 strain) as follows.
Cultured. The composition of the pre-medium is 1 w / v% Meat-ext
ract, 1 w / v% Bacto-Trypton,
0.2 w / v% Yeast-extract, 0.9 w
/ V% Na_Two HPO_Four ・ 12H_Two O, 0.15 w / v%
KH_Two PO_Four , (PH 6.7). Polyester raw
The composition of the production medium is 1.1 w / v% Na_Two HPO_Four ・ 12H
_Two O, 0.19 w / v% KH_Two PO_Four , 0.6w / v%
(NH_Four )_Two SO_Four , 0.1w / v% MgSO_Four ・ 7H
_Two O, 0.5 v / v% trace metal salt solution (0.1N hydrochloric acid
1.6 w / v% FeCl_Three ・ 6H_Two O, 1w / v% Ca
Cl_Two ・ 2H_Two O, 0.02 w / v% CoCl_Two ・ 6H
_Two O, 0.016 w / v% CuSO_Four ・ 5H_Two O, 0.
012w / v% NiCl_Three ・ 6H_Two O, 0.01 w / v
% CrCl _Three ・ 6H_Two What dissolved O. ), 2w / v
% Professional extract AP-12 (Banshu seasoning), 5 × 10^-6w
/ V% kanamycin. The only carbon source is fats and oils.
Oil, palm kernel oil or coconut oil 4w / v% divided into 3 times
Was added.

The glycerol stock of the Aed13 strain was inoculated into the pre-culture medium and cultured for 20 hours, and a 10-L jar fermenter (Mulishishi Bio-Engine, M) containing 6 L of the production medium was used.
D-500) was inoculated at 1.5 v / v%. The operating conditions were a culture temperature of 30 ° C., a stirring speed of 400 rpm, an aeration rate of 1.8 L / min, and a pH of 6.6 to 6.8. For control, 5N sulfuric acid and sodium hydroxide were used. Culture was performed for up to 72 hours. The cells were collected by centrifugation, washed with methanol, freeze-dried, and the weight of the dried cells was measured.

To about 30 mg of the obtained dried cells, 2 ml of a mixed solution of sulfuric acid and methanol (15:85) and 2 ml of chloroform were added, and the mixture was sealed and heated at 100 ° C. for 140 minutes to decompose the polyester inside the cells. The methyl ester of was obtained. After cooling, 1 ml of distilled water was added thereto, and the mixture was vigorously stirred using a stirrer. After standing and separating into two layers, the lower organic solvent layer was taken out and its composition was analyzed by capillary gas chromatography. The gas chromatograph is Shimadzu GC-17A, and the capillary column is Neutra Bond-1 manufactured by GL Sciences.
(Column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 μm). Temperature condition is initial temperature 100 ℃
From 8 ° C./min. Table 1 shows the obtained results.

[0035]

[Table 1]

From these results, it was found that the productivity was improved to about 24 g / L when coconut oil was used as a carbon source.
Further, when the productivity is improved, the 3HH molar fraction is 10 m, unlike the result described in JP-A-10-108682.
ol%.

(Example 2) 1) Coconut oil 5w as a carbon source
/ V%, 2) coconut oil 4w / v% and hexanoic acid 1w / v
%, 3) coconut oil 3w / v% and hexanoic acid 2w / v%,
4) Coconut oil 2 w / v% and hexanoic acid 3 w / v%, 5) Coconut oil 1 w / v% and hexanoic acid 4 w / v%, 6) Coconut oil 0.5 w / v% and hexanoic acid 5 w / v%. Fats and fatty acids were added in various proportions. Palm oil was added at 0.5 w / v during inoculation, and the remaining palm oil and hexanoic acid were mixed and continuously added at a rate of about 10 ml / min by a peristaltic pump for 24 to 60 hours. Otherwise, culturing was performed in the same medium and under the same conditions as in Example 1, and analysis was performed. Table 2 shows the results.

[0038]

[Table 2]

From these results, it was found that the ratio of hexanoic acid was 0 to 4
It was found that the 3HH mole fraction increased from 8.1 to 32 mol% as it increased to w / v%.

Example 3 Palm oil 4 w / v as a carbon source
% And hexagonal triglyceride (HTG) 1 w / v%. In the case of only coconut oil, 5% w / v was added. The same medium as in Example 1 was used, and a 3 L jar fermenter containing 1.8 L of a production medium (M manufactured by Marubishi Bio-Engine) was used.
(DL-300 type). The operating conditions were a culture temperature of 30 ° C., a stirring speed of 550 rpm, and an aeration rate of 1.8 L /
min, and the pH was controlled between 6.6 and 6.8 with 5N sulfuric acid and sodium hydroxide. At the time of inoculation, coconut oil was added at 0.5 w / v%, and the remaining coconut oil and HTG were added.
Was mixed into three equal portions, and added 24, 36, and 48 hours after inoculation. Culture was performed for up to 72 hours. The cells were collected by centrifugation, washed with methanol, freeze-dried, and the weight of the dried cells was measured. The same analysis as in Example 1 was performed. Table 3 shows the results.

[0041]

[Table 3]

From these results, it was found that when a part of the carbon source was changed to HTG, the 3HH mole fraction was improved.

Example 4 Using the same medium and jar fermenter as in Example 1, culturing was performed under the same operating conditions. Carbon source is coconut oil 4w / v% and hexanoic acid 0.5w /
v1 or 0.5 w / v% octanoic acid
Was continuously added in the same manner as described above. 5 w / v% for coconut oil only
Was added. Table 4 shows the analysis results.

[0044]

[Table 4]

From these results, polymers having different 3HH mole fractions were obtained by changing the fatty acid to be added.

[0046]

According to the present invention, a P (3HB-co-3HH) copolymer, which is a biodegradable polymer, can be arbitrarily controlled in the range of 1 to 40 mol% of 3HH mole fraction which greatly changes physical properties. Thus, the present polymer can be stably produced with high productivity, and the present polymer having a wide range of applications can be provided.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification Symbol FI Theme Court ゛ (Reference) (C12P 7/62 C12R 1:05) C12R 1:05) C12N 15/00 A (72) Inventor Osamu Odawara (13) Inventor Keiji Matsumoto 11-33, Omoricho, Nishinomiya-shi, Hyogo (72) Inventor Yoshiharu Toi 2-1 Hirosawa, Wako-shi, Saitama F-term (Reference) 4B024 AA03 BA80 CA03 DA05 EA04 GA11 4B064 AD83 CA02 CA19 CB03 CC24 CD07 DA16 4B065 AA10Y AA12X AB01 AC14 BA02 BB08 CA12 CA60

Claims (7)

[Claims] 1. A copolymerized polyester P (3HB-) obtained by copolymerizing 3-hydroxybutyric acid represented by the following formula (1) and 3-hydroxyhexanoic acid represented by the following formula (2) using a microorganism: co-3HH), wherein as at least two carbon sources, a combination of oils and fats having different carbon numbers, a combination of fatty acids having different carbon numbers,
Alternatively, a method for producing a polyester, characterized in that polyesters having different 3HH mole fractions are collected by using any one of combinations of fats and oils and fatty acids having different carbon numbers. Embedded image Embedded image 2. The process for producing a polyester according to claim 1, wherein the 3HH mole fraction is controlled by changing the amount of the fat or oil used as a carbon source. 3. The method for producing a polyester according to claim 1, wherein the fat or oil is a fat or oil selected from the group consisting of coconut oil, palm oil, palm kernel oil and hexanoic acid triglyceride. 4. The method for producing a polyester according to claim 1, wherein the fatty acid is a fatty acid selected from the group consisting of saturated or unsaturated fatty acids having 6 to 10 carbon atoms, fatty acid esters and fatty acid salts thereof. 5. The polyester having a 3HH mole fraction of 1
The method for producing a polyester according to any one of claims 1 to 4, wherein the amount is from about 40 mol% to about 40 mol%. 6. The microorganism according to any one of claims 1 to 5, wherein the microorganism is a microorganism transformed by a recombinant vector containing a polyester synthase gene isolated from Aeromonas caviae. Method for producing polyester. 7. The method according to claim 7, wherein the microorganism is Alcaligenes.
eutrophus (Ralstonia eutro
The method for producing a polyester according to any one of claims 1 to 6, wherein the polyester is pha).