JP2001178486A - Method for producing polyhydroxyalkanoate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new polyhydroxyalkanoate having phenyl group as a substituent group on the side chain and a method for producing the polyhydroxyalkanoate by using a bacterium. SOLUTION: A bacterium is cultured in a culture medium containing 6- phenylhexanoic acid and polyhydroxyalkanoate accumulated in the microbial cells of a microorganism is extracted to produce the objective new polyhydroxyalkanoate containing a monomer unit derived from 3-hydroxy-6- phenylhexanoic acid represented by the chemical formula (I).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel polyhydroxyalkanoate (PHA) and a method for producing the same. More specifically, the present invention relates to 3-hydroxy-6
The present invention relates to a method for producing such a microbial polyhydroxyalkanoate with high selectivity using a microorganism capable of producing and accumulating polyhydroxyalkanoate (PHA) having phenylhexanoic acid as a monomer unit.

[0002]

2. Description of the Related Art It has been reported that many microorganisms produce poly-3-hydroxybutyric acid (PHB) or other PHA and accumulate in cells ("Biodegradable Plastic Handbook", Edible Plastics Study Group, NTT Corp., P178-19
7). These microorganism-producing polymers can be used for production of various products by melt processing or the like, similarly to conventional plastics. Furthermore, because it is biodegradable,
It has the advantage of being completely degraded by microorganisms in nature. Therefore, unlike conventional synthetic polymer compounds, they do not remain in the natural environment and cause pollution, and there is no need to perform combustion treatment. , A material without any concerns. It is also excellent in biocompatibility and is expected to be applied as a soft medical member.

[0003] It is known that microorganism-produced PHA can have various compositions and structures depending on the kind of microorganism used for its production, the composition of the medium, the culture conditions, and the like. Until now, mainly from the viewpoint of improving the physical properties of PHA, studies on methods for controlling the composition and structure of PHA produced by microorganisms have been made.

[0004] For example, Alcaligenes eutropus H16 (ATCC No.17699)
In addition, it has been reported that the mutants produce copolymers of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) at various composition ratios by changing the carbon source during culture. (Table 6-15604)
Gazette, Japanese Patent Publication No. Hei 7-14352, Japanese Patent Publication No. Hei 8-19
227, etc.).

Also, Japanese Patent No. 2642937 discloses that
Pseudomonas oreobolans ATCC 29347
It is disclosed that a strain (Pseudomonas oleovorans ATCC29347) is provided with an acyclic aliphatic hydrocarbon as a carbon source to produce PHA having a monomer unit of 3-hydroxyalkanoate having 6 to 12 carbon atoms. .

Japanese Patent Application Laid-Open No. 5-74492 discloses a genus Methylobacterium sp., A genus Paracoccus sp., And a genus Alcalige.
nessp.) and a method of producing a copolymer of 3HB and 3HV by contacting a microorganism of the genus Pseudomonas sp. with a primary alcohol having 3 to 7 carbon atoms.

[0007] JP-A-5-93049 and JP-A-7-265065 disclose that 3HB and 3-hydroxyhexanoic acid (Aeromonas caviae) are cultured by using oleic acid or olive oil as a carbon source. 3HHx) is disclosed to produce a two-component copolymer.

Japanese Patent Application Laid-Open No. 9-191893 discloses that Comamonas acidovorans IFO13852 strain (Comamo
nas acidovorans IFO13852), when cultured using gluconic acid and 1,4-butanediol as carbon sources, 3
It is disclosed to produce a polyester having HB and 4-hydroxybutyric acid as monomer units.

Further, some microorganisms produce PHA into which various substituents, for example, unsaturated hydrocarbon groups, ester groups, allyl groups, cyano groups, halogenated hydrocarbon groups, epoxides, etc. are introduced. And attempts have been made to improve the physical properties of microorganism-produced PHA by such techniques.

For example, Makromol. Chem., 191, 1957-196
5,1990, Macromolecules, 24, 5256-5260,1991, Chiral
, 3, 492-494, 1991, etc., Pseudomonas oleovorans dissolves 3-hydroxy-5-phenylvaleric acid (3
It has been reported to produce PHA containing (HPV) as a monomer unit, and a change in polymer physical properties, which may be caused by the inclusion of 3HPV, has been observed.

[0011]

As described above, as described above,
In microbial PHA, several types of compositions and structures have been obtained by changing the type of microorganism used, the medium composition, the culture conditions, etc., but the main purpose of these attempts is exclusively The purpose was to change the ratio of a plurality of hydroxyalkanoic acids (HA) contained in the PHA to improve the physical properties as a plastic.

On the other hand, PHAs having various substituents introduced are
The development as a “functional polymer” having extremely useful functions and characteristics due to the characteristics of the introduced substituents can be expected. A new polymer that has such functionality in addition to biodegradability, a method for producing it using microorganisms, and the development and selection of microorganisms that can produce the polymer and accumulate in the cells Separation is considered extremely useful and important.

The present invention solves the above-mentioned problems, and
An object of the present invention is to provide a novel PHA which is biodegradable and has a phenyl group as a substituent, and a method for producing the above-mentioned PHA having a phenyl group as a substituent using a microorganism. To provide new microorganisms. In particular, a method for producing a novel PHA using 3-hydroxy-6-phenyl-hexanoic acid (3HPHx) as a single monomer unit and a PHA having the phenyl group as a substituent using a microorganism, An object of the present invention is to provide a novel microorganism to be used.

[0014]

In order to solve the above-mentioned problems, the present inventors have developed a novel PHA having a phenyl group as a substituent on a side chain, which is particularly useful as a device material or a medical material. In search of microorganisms capable of producing new PHA and accumulating in the cells,
Using the searched microorganisms, the intense research was conducted on a method for producing a desired PHA. As a result, using 6-phenylhexanoic acid (PHxA) as a raw material,
The present inventors have found that there is a microorganism capable of producing and accumulating a novel PHA containing 6-phenyl-hexanoic acid (3HPHx) as a monomer unit and accumulating the same in a microbial cell, and completed the present invention.

That is, the novel polyhydroxyalkanoate provided by the present invention has a chemical formula (I):

[0016]

Embedded image

A polyhydroxyalkanoate containing a monomer unit represented by the formula:

Further, the method for producing the polyhydroxyalkanoate of the present invention comprises a process represented by the following chemical formula (II):

[0019]

Embedded image

6-phenylhexanoic acid (PH
A method for producing polyhydroxyalkanoate, which comprises culturing a microorganism in a medium containing xA). Usually, after culturing a microorganism, 3-hydroxy-6-phenylhexanoic acid (3HPH) represented by the above chemical formula (I)
A step of extracting a polyhydroxyalkanoate containing a monomer unit derived from x) may be provided.

The microorganism having the ability to produce and accumulate PHA to be used is Pseudomonas sp.
domonas sp.). For example, Pseudomonas jessenii P161 strain (Ps
eudomonas jessenii P161, FERM P-17445)
Is more preferable.

[0022]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS First, the production method of the present invention comprises the steps of:
Using phenylhexanoic acid (PHxA) as a raw material (substrate), 3-hydroxy-6-phenylhexanoic acid (3
This is a method that utilizes a microorganism that has the ability to produce PHA containing HPHx) as a monomer unit and accumulate it in cells. A microorganism exhibiting such a novel enzyme reactivity has been found by the present inventors' search. One example of the preferred strain is P161 strain, which is a microorganism newly isolated from the soil by the present inventors.

<Mycological properties of strain P161> Morphological properties Shape and size of cells: spherical, φ0.6 μm rod, 0.6 μm × 1.5-2.0 μm Cell polymorphism: yes (elongation) Motility: Yes Sporulation: No Gram stain: Negative Colony shape: Circular, whole edge smooth, low convex, surface smooth, pale yellow physiological properties Catalase: Positive oxidase: Positive O / F test: Oxidized nitrate Reduction: Positive Indole formation: Negative Glucose acidification: Negative Arginine dihydrolase: Positive urease: Negative Esculin hydrolysis: Negative Gelatin hydrolysis: Negative β-galactosidase: Negative Fluorescent dye production on King's B agar: Positive substrate Chemoactive glucose: positive L-arabinose: positive D-mannose: positive D-mannitol: positive N-acetyl-D-glucose Samin: Positive Maltose: Negative Potassium gluconate: Positive n-Capric acid: Positive Adipic acid: Negative dl-Malic acid: Positive Sodium citrate: Positive Phenyl acetate: Positive Systematic Bacteriology Volume 1 (Be
rgey's Manual of Systematic Bacteriology, Volume1)
(1984) and the Burgess Manual of
Deterministic bacteriology (Bergey's Ma
nual of Determinative Bacteriology) 9th edition (199
The P161 strain was found to belong to the genus Pseudomonas (Pseudomonas sp.), However, the taxonomic position could not be determined from its mycological properties.

Therefore, in order to try classification based on genetic properties, the nucleotide sequence of 16S rRNA of the P161 strain was determined (SEQ ID NO: 1, cDNA to rRNA), and the nucleotide sequence of 16S rRNA of the known microorganism of the genus Pseudomonas was determined. Were examined for homology. It has been found that the nucleotide sequence homology between the P161 strain and Pseudomonas jessenii is extremely high. In addition, System.Appl.
Microbiol., 20, 137-149 (1997) and System.App
High similarity was also observed between the mycological properties of Pseudomonas gessenii described in l. Microbiol., 22, 45-58 (1999) and the mycological properties of strain P161. Based on the above results, it was determined that the P161 strain belonged to Pseudomonas jessenii. Therefore, the P161 strain was named Pseudomonas jessenii P161.
The Pseudomonas jessenii P161 strain has been deposited with the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Biotechnology and Industrial Technology Research Institute (Patent Microorganisms Depositary Center) under the deposit number "FERM P-17445".

In the method for producing a polyhydroxyalkanoate of the present invention, 3-hydroxy-6-phenylhexanoic acid belonging to the genus Pseudomonas, such as the above-mentioned strain P161, is prepared by using 6-phenylhexanoic acid (PHxA) as a raw material (substrate). It is preferable to use a microorganism that converts to (3HPHx) and produces PHA. In addition to microorganisms belonging to the genus Pseudomonas, Aeromonas (Aeromonas sp.)
Genus, Comamonas sp., Burkholderia sp., Etc., and using 6-phenylhexanoic acid (PHxA) as a raw material (substrate).
-Hydroxy-6-phenylhexanoic acid (3HPHx)
It is also possible to use a microorganism that produces PHA containing as a monomer unit.

When the microorganism used in the present invention is usually grown, any natural medium (such as a broth medium) or a synthetic medium to which nutrients are added, as long as it does not adversely affect the growth and survival of the microorganism, may be used. Different types of media can also be used.

The microorganism used in the present invention has a chemical formula (I) derived from 3HPHx:

[0028]

Embedded image

PHA containing a monomer unit represented by the following formula:
If it has the ability to produce and accumulate, PHA containing other monomer units may be mixed, medium-chain fatty acids such as octanoic acid and nonanoic acid as a carbon source for growth,
After culturing from the late logarithmic growth phase to the stationary phase in an inorganic medium or the like containing PHxA and collecting cells by centrifugation or the like, an inorganic medium containing a medium-chain fatty acid and PVA and containing no nitrogen source is used. And a method of further culturing. Alternatively, the concentration of the nitrogen source in the inorganic salt medium is restricted to about 1/10, and medium-chain fatty acids and PHxA are fed and cultured. Can be used. In the method of adding a medium-chain fatty acid as a carbon source for growth to a medium, the obtained PHA becomes PHA in which monomer units derived from the medium-chain fatty acid added as a carbon source for growth are mixed. I have.

In the method of producing PHPHx of the present invention, when selectively producing and accumulating only PHPHx using a microorganism, it is preferable to culture the microorganism in a medium containing a yeast extract and PHxA. For example, about 0.1% to 1.0% of yeast extract and 0.01% of PHxA are added to a medium.
The microorganism is cultured from the late logarithmic growth phase to the stationary phase using an inorganic medium or the like containing about 0.5% to 0.5%, and the cells are collected by centrifugation or the like.
A two-stage culturing method can be employed in which culturing is further performed in an inorganic medium containing no nitrogen source and containing about 5%. Also,
About 0.1% to 1.0% of yeast extract and PHx
A is cultivated in an inorganic medium containing about 0.01% to 0.5% of A, and the cells are collected from the late logarithmic phase to the stationary phase,
It is also possible to use a one-stage culture method for extracting a desired PHA from the cells.

In the above-mentioned culturing method, the inorganic medium used for preparing the medium contains components such as a phosphorus source (for example, phosphate) and a nitrogen source (for example, ammonium salt, nitrate, etc.) that can be proliferated by microorganisms. Any medium may be used as long as it is suitable for use, and examples thereof include an MSB medium and an M9 medium. The composition of the M9 medium used in Examples, which is a specific example of the present invention described below, is as follows. Na ₂ HPO ₄ : 6.2 g, KH ₂ PO ₄ : 3.0
g, NaCl: 0.5 g, NH ₄ Cl: 1.0 g (in 1 liter of medium, pH 7.0).

The concentration of PHxA as a raw material to be added to the medium is appropriately selected depending on the genus of the microorganism, the cell density, or the culturing method. % To about 0.5%. In addition, it is preferable to add yeast extract to the medium at the same time.At this time, the concentration of the yeast extract to be added is appropriately selected depending on the genus of the microorganism, the cell density, or the culture method. Is selected from about 0.1% to about 1.0% and added.

As the above-mentioned culturing method, any method used for ordinary culturing of microorganisms, such as a batch type, a flow batch type, a continuous culturing, a reactor type, etc., can be used.
At this time, the culture temperature is preferably maintained in a temperature range in which the microorganism to be used can grow and proliferate, for example, about 20 ° C to 30 ° C.

In the production method of the present invention, the simplest method of recovering PHA from cultured cells is the usual extraction with an organic solvent such as chloroform. Other than the extraction method using this organic solvent, without using an organic solvent, for example,
Surfactant treatment such as SDS, enzyme treatment such as lysozyme,
It is also possible to adopt a method of recovering only PHA by removing intracellular components other than PHA by treatment with a chemical such as EDTA, sodium hypochlorite, and ammonia.

In the present invention, the cultivation of microorganisms, production of PHA by microorganisms, accumulation in microorganisms, and recovery of PHA from microorganisms are not limited to the specific methods exemplified above. Absent.

[0036]

The present invention will be described in more detail with reference to specific examples. These specific examples are examples of the best mode of the present invention, but the present invention is not limited by the following specific examples.

Example 1 0.1% yeast extract, PHx
The P161 strain was inoculated into 200 ml of M9 medium containing 0.1% of A, and cultured with shaking at 30 ° C. and 125 strokes / min.
After 24 hours, the cells were collected by centrifugation, and PHxA
M9 without 0.1% nitrogen source (NH ₄ Cl)
The cells were resuspended in 200 ml of the medium, and cultured with shaking at 30 ° C. and 125 strokes / min. After 24 hours, the cells were collected by centrifugation, washed once with cold methanol and freeze-dried.

The lyophilized pellet was suspended in 100 ml of chloroform and stirred at 60 ° C. for 20 hours to extract PHA. After the extract was filtered with a membrane filter having a pore size of 0.45 μm, the extract was concentrated with a rotary evaporator, the concentrate was reprecipitated in cold methanol, and only the precipitate was recovered and dried in vacuo to obtain PHA.

The above PHA (100 mg) was mixed with chloroform (1).
and then added normal hexane until cloudy. This was centrifuged to collect a precipitate, and dried under vacuum. The operation of dissolving this in 1 ml of chloroform again, adding normal hexane, and collecting the precipitated portion was repeated three times.

The obtained precipitate was subjected to methanolysis according to a conventional method, and then analyzed by a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method) to obtain a methyl ester of a PHA monomer unit. Was identified. The molecular weight of the PHA was determined by gel permeation chromatography (GPC; Tosoh Corporation).
HLC-8020, column; Polymer Laboratory P
Lgel MIXED-C 5 μm, solvent; chloroform, molecular weight in terms of polystyrene). Table 1
Table 2 shows the identification results, the average molecular weight, and the yields and yields of the lyophilized pellets and the recovered polymer. As a result, Table 1
As shown in the figure, it was confirmed that the PHA was a monomer unit containing only 3HPHx as a monomer unit.

[0041]

[Table 1]

This PHA is prepared by a nuclear magnetic resonance apparatus (F
T-NMR: using Bruker DPX400)
The analysis was performed under the following conditions. Measurement nuclide: ¹ H, Solvent used: deuterated chloroform (containing TMS). FIG. 1 shows the ¹ H-NMR spectrum measurement results and Table 2 shows the assignment.

[0043]

[Table 2]

As in this example, the yeast extract and the raw material 6
It is also confirmed from the measurement results shown in Table 2 that when cultured in an inorganic medium supplemented with -phenylhexanoic acid, the PHA accumulated in the cells is a PHA containing only 3HPHx as a monomer unit.

[0045]

Industrial Applicability According to the production method of the present invention, it is possible to selectively produce a novel polyhydroxyalkanoate containing 3-hydroxy-6-phenylhexanoic acid as a monomer unit, which is a kind of polyhydroxyalkanoate produced by microorganisms. Become. In addition, the novel polyhydroxyalkanoate containing 3-hydroxy-6-phenylhexanoic acid as a monomer unit can be efficiently produced using, for example, strain P161. It is also useful as a functional polymer, and in particular, its homopolymer can be expected to be applied to various fields such as device materials and medical materials.

[0046]

[Sequence List] SEQUENCE LISTING <110> CANON INC. <120> Preparation of Poly-hidroxyalkanoic Acid <130> 4052004 <160> 1 <170> Microsoft Word <210> 1 <211> 1501 <212> DNA <213> Pseudomonas jessenii P161; FERM P-17445 <400> 1 tgaacgctgg cggcaggcct aacacatgca agtcgagcgg 40 atgacgggag cttgctcctg aattcagcgg cggacgggtg 80 agtaatgcct aggaatctgc ctggtagtgg gggacaacgt 120 ctcgaaaggg acgctaatac cgcatacgtc ctacgggaga 160 aagcagggga ccttcgggcc ttgcgctatc agatgagcct 200 aggtcggatt agctagttgg tgaggtaatg gctcaccaag 240 gcgacgatcc gtaactggtc tgagaggatg atcagtcaca 280 ctggaactga gacacggtcc agactcctac gggaggcagc 320 agtggggaat attggacaat gggcgaaagc ctgatccagc 360 catgccgcgt gtgtgaagaa ggtcttcgga ttgtaaagca 400 ctttaagttg ggaggaaggg cattaaccta atacgttagt 440 gttttgacgt taccgacaga ataagcaccg gctaactctg 480 tgccagcagc cgcggtaata cagagggtgc aagcgttaat 520 cggaattact gggcgtaaag cgcgcgtagg tggtttgtta 560 agttggatgt gaaagccccg ggctcaacct gggaactgca 600 ttcaaaactg acaagctaga gtatggtaga gggtggtgga 640 atttcctgtg tagcggtgaa atgcgtagat ataggaagga 680 acaccagtgg cgaaggcgac cacctggact gatactgaca 720 ctgaggtgcg aaagcgtggg gagcaaacag gattagatac 760 cctggtagtc cacgccgtaa acgatgtcaa ctagccgttg 800 ggagccttga gctcttagtg gcgcagctaa cgcattaagt 840 tgaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa 880 tgaattgacg ggggcccgca caagcggtgg agcatgtggt 920 ttaattcgaa gcaacgcgaa gaaccttacc aggccttgac 960 atccaatgaa ctttccagag atggatgggt gccttcggga 1000 acattgagac aggtgctgca tggctgtcgt cagctcgtgt 1040 cgtgagatgt tgggttaagt cccgtaacga gcgcaaccct 1080 tgtccttagt taccagcacg taatggtggg cactctaagg 1120 agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1160 caagtcatca tggcccttac ggcctgggct acacacgtgc 1200 tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg 1240 agctaatccc acaaaaccga tcgtagtccg gatcgcagtc 1280 tgcaactcga ctgcgtgaag tcggaatcgc tagtaatcgc 1320 gaatcagaat gtcgcggtga atacgttccc gggccttgta 1360 cacaccgccc gtcacaccat gggagtgggt tgcaccagaa 1400 gtagctagtc taaccttcgg gaggacggtt accacggtgt 1440 gattcatgac tggggtgaag tcgtaccaag gtagc cgtag 1480 gggaacctgc ggctggatca c 1501

[Brief description of the drawings]

FIG. 1 shows the ¹ H-NMR spectrum measurement results of poly-3-hydroxy-6-phenylhexanoic acid produced by strain P161 by the production method of the present invention.

FIG. 2: Pseudomonas jesseny P161 strain (Ps
eudomonas jessenii P161; FERM P-17445)
1 shows the nucleotide sequence of 16S rRNA.

Continued on the front page (72) Inventor Tetsuya Yano 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc. (72) Inventor Sakae 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc. (72) Inventor Tatsu Kobayashi F-term (reference) 4B064 AD83 CA02 CC03 CD07 DA16 4J029 AA02 AB04 AC01 AE06 EB01, 3-30-2 Shimomaruko, Ota-ku, Tokyo

Claims (1)

[Claims] [Claim 1] Chemical formula (I): A method for producing a polyhydroxyalkanoate containing a monomer unit represented by the following formula: In a medium containing 6-phenylhexanoic acid represented by the following formula, Pseudomonas jessenii P161 strain (Pseudomonas je
ssenii P161, FERM P-17445), which comprises culturing the polyhydroxyalkanoate.