JP2001238668A - Microorganism producing poly-3-hydroxy-4-phenoxy-n- butyric acid and preparation method for poly-3- hydroxy-4-phenoxy-nbutyric acid using the microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microoganism having productivity of poly-3-hydroxy-4- phenoxy-n-butyric acid which is useful for a device material, a medical material and the like, and a preparation method for poly-hydroxy-3-4-n-butyric acid using the microorganism. SOLUTION: This method comprises the following process: Pseudomonas fluorescens 127 stain (Pseudomonas fluorescens N127, FERM P-17745) is cultured in a culture medium containing 4-phenoxy-n-butyric acid represented by formula (2) (n represents an integer number <=10.), and poly-3-hydorxy-n-butyric acid produced by culturing the above 127 strain and represented by formula (1) is extracted.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the production of poly-3-hydroxy-4-phenoxy-n-butyric acid (PHPxB) among microorganism-produced polyhydroxyalkanoates (PHA) in comparison with conventional microorganisms. And a novel microorganism that accumulates a larger amount of PHPxB in the cells. Further, the present invention relates to a method for producing PHPxB using the microorganism.

[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, published by NTS Co., Ltd., P178-
197, 1995). These polymers can be used for production of various products by melt processing or the like, similarly to conventional plastics. Further, since it is biodegradable, it has the advantage of being completely degraded by microorganisms in nature, and does not remain in the natural environment and cause contamination unlike many conventional synthetic polymer compounds. Also,
It is also excellent in biocompatibility and is expected to be applied as a soft medical member.

[0003] Such PHA produced by microorganisms depends on the type of microorganism used for the production, the composition of the medium, the culture conditions, and the like.
It is known that various compositions and structures can be obtained, and studies on such composition and structure control have been made mainly from the viewpoint of improving the physical properties of PHA.

[0004] For example, Alcaligenes eutropus H16 (ATCC No. 1769)
9) and its mutants are capable of producing 3-hydroxybutyric acid (3HB) and 3-hydroxybutyric acid (3HB) by changing the carbon source during the culture.
It has been reported that copolymers with hydroxyvaleric acid (3HV) are produced in various composition ratios (Japanese Patent Application Laid-Open No. Hei 6-156).
Japanese Patent Publication No. 04, Japanese Patent Publication No. Hei 7-14352, Japanese Patent Publication No. Hei 8-
No. 19227).

[0005] Also, in Japanese Patent Publication No. 2642937,
Pseudomonas oleovorans ATCC29347 (Pseudom
ona oleovorans ATCC29347), by giving acyclic aliphatic hydrocarbons as carbon sources, the number of carbon atoms is 6 to 1;
It is disclosed that PHAs having up to two 3-hydroxyalkanoate monomer units are produced.

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-hydroxyhexane are obtained by culturing Aeromonas caviae using oleic acid or olive oil as a carbon source. It is disclosed that a two-component copolymer of acid (3HHx) is produced.

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

Furthermore, in certain microorganisms, various substituents, for example, groups obtained from unsaturated hydrocarbons, ester groups, allyl groups, cyano groups, groups obtained from halogenated hydrocarbons, epoxides and the like are introduced. It has been reported that PHA is produced, and attempts to improve the physical properties of microorganism-produced PHA by such techniques have begun.
For example, Makromol. Chem., 191, 1957-1965, 1990, Macr.
omolecules, 24, 5256-5260, 1991; Chirality, 3, 492
-494, 1991, etc., reported that Pseudomonas oleovorans produces PHA containing 3HPV monomer units obtained from 3-hydroxy-5-phenylvaleric acid (3HPV). A change in the physical properties of the polymer, which is considered to be caused, has been observed.

[0010]

As described above, the type of PHA in which a substituent is introduced into the side chain is selected by selecting the introduced substituent in accordance with desired characteristics and the like. Due to the properties and the like, it can also be expected to develop as a `` functional polymer '' having extremely useful functions and properties, and an excellent polymer having such functionality and biodegradability, and a method for producing the same, and The development of a method for efficiently producing a desired polymer using such a microorganism was considered to be extremely useful and important.

In general, when an attempt is made by a microorganism to produce a PHA containing a monomer unit having a special functional group introduced therein, compared to a case where a microorganism produces a PHA consisting of only a general monomer unit such as 3HB, The amount of accumulated PHA in the cells tends to decrease significantly. Therefore, PHA having such a special substituent introduced into the side chain
Has been an important technical issue in developing the polymer.

Examples of a method for increasing the amount of PHA accumulated in a microorganism include a method of culturing a microorganism in a medium containing a fatty acid containing a desired functional group and an auxiliary substrate such as n-nonanoic acid. However, in the above-mentioned method, it is inevitable that a large amount of monomer units derived from the auxiliary substrate besides the monomer units containing the desired functional group are mixed in the produced PHA. It cannot be used when it is desired to obtain a homopolymer composed only of a monomer unit containing.

An object of the present invention is to provide poly-3-hydroxy-
An object of the present invention is to provide a microorganism having a high ability to produce 4-phenoxy-n-butyric acid and a method for producing poly-3-hydroxy-4-phenoxy-n-butyric acid using the microorganism.

[0014]

Means for Solving the Problems The present inventors have prepared poly-3.
-Hydroxy-4-phenoxy-n-butyric acid (PHPx
Aiming to develop B) as a functional polymer,
Search for new microorganisms that accumulate more high-purity PHPxB in cells, and PHPx using such microorganisms
We have intensively studied a method for producing B at a high yield. As a result, the following equation (2):

[0015]

Embedded image

4-phenoxy-n-butyric acid (P
xBA) as a raw material and the following formula (1):

[0017]

Embedded image

[0018] PHPxB having a repeating unit represented by the formula (n is an integer of 10 or more) was obtained, for example, by using Pseudomonas putida P9 strain.
1, Pseudomonas fluorescens N127 strain (Pseudomona), which is a novel microorganism having the ability to accumulate in larger amounts as compared with FERM P-17409, Japanese Patent Application No. 11-371867.
fluorescens N127, FERM P-17745), and further developed a method for producing PHPxB in high yield using this microorganism.

That is, the method for producing PHPxB of the present invention provides a method for producing a PHxB having a repeating unit represented by the above formula (1).
A method for producing PxB, comprising the step of:
Culturing in a medium containing PxBA represented by the above formula (2), and PHPx produced by culturing the N127 strain.
And extracting B.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The details of the present invention will be described below.

First, the present inventors searched for a microorganism having the ability to produce PHPxB using PxBA as a substrate. As a result, microorganisms producing more PHPxB and accumulating in the cells were successfully separated from the soil, and this was designated as strain N127.

The bacteriological properties of strain N127 according to the present invention are as follows.

<Bacteriological properties of strain N127> (a) Morphological properties Cell shape: Bacillus Cell size: 0.5 μm × 1.5-2.0 μm Presence or absence of cell polymorphism: − Motility : + Flagella formation state: polar hair, 2-3 spores:-Gram stainability:-(b) Cultural properties Colony shape: round, whole edge smooth, low convex, surface smooth, creamy gravy Liquid medium: cloudy Litmus milk:-(c) Physiological properties Reduction of nitrate:-Denitrification reaction:-MR test:-VP test:-Formation of indole:-Formation of hydrogen sulfide:-Formation of pigment King's B agar Medium: + starch hydrolysis:-urease:-oxidase: + catalase: + arginine dihydrolase: + growth temperature 25 ° C: + 30 ° C: + 37 ° C: + 42 ° C:-attitude to oxygen: aerobic O- F-Tess : Oxidized glucose acidification:-Esculin hydrolysis:-Gelatin hydrolysis: + Levan production from sucrose:-Ornithine decarbonization:-Lysine decarbonization:-β-galactosidase:-Lecithinase: + Lipase:- Acid production from saccharides / gas production L-arabinose:-/-D-sorbitol:-/-inositol:-/-D-xylose:-/-D-galactose:-/-sucrose:-/-glycerin: -/-D-fructose:-/-maltose:-/-lactose:-/-D-mannose:-/-trehanose:-/-D-mannitol:-/-assimilation of carbon source glucose: + L- Arabinose: + D-mannose: + D-mannitol: + N-acetyl-D-glucosamine: + Maltose:-gluconic acid Um: + n-capric acid: + adipic acid: + dl-malic acid: + sodium citrate: + phenyl acetate:-From the above mycological properties, the Burgers Manual of Systematic Bacteriology, Volume 1 (Be
rgey's Manual of Systematic Bacteriology, Volume
1) (1984) and Bergey's Manual of Defective Bacteriology (Bergey's M
anual of Determinative Bacteriology) 9th edition (1994
), The N127 strain was found to be Pseudomonas fluorescen.
s). Therefore, this strain was named Pseudomonas fluorescens N127 strain.

The strain was deposited under the accession number "FERM P-17745".
It has been deposited with the Ministry of International Trade and Industry, the National Institute of Advanced Industrial Science and Technology, the Institute of Biotechnology and Industrial Technology, and the Patent Microorganisms Depositary.

[0025] Ordinary culture of microorganisms used in the method for producing PHPxB of the present invention, for example, preparation of a preserved strain, PHPxB
For cultivation to ensure the number of bacteria and the active state required for the production of B, a general natural medium (such as a broth medium) or a nutrient source should be used unless it adversely affects the growth and survival of the microorganism. Any type of medium, such as an added synthetic medium, can be used. Culture conditions such as temperature, aeration and stirring are appropriately selected depending on the microorganism used.

On the other hand, PHPxB is produced using microorganisms.
In the case of accumulation, an inorganic medium containing at least PxBA is used.

The inorganic medium used in the above culture method includes a phosphorus source (for example, phosphate), a nitrogen source (for example,
Any material may be used as long as it contains a component that allows microorganisms to grow, such as ammonium salts and nitrates. Examples of the inorganic salt medium include an MSB medium and an M9 medium.

The M9 used in the embodiment of the present invention
The composition of the medium 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 (pH 7.0 in 1 liter of medium) Culture conditions are, for example, 15 to 40. ° C, preferably 2
Shaking culture and stirring culture under aerobic conditions at 0 to 35 ° C are mentioned.

In the culturing step, any method used for culturing ordinary microorganisms such as a batch type, a flow batch type, a semi-continuous cultivation, a continuous cultivation, a reactor type and the like can be used.
A multi-stage system in which these steps are connected in a plurality of stages may be adopted.

For example, an inorganic substance containing about 0.1% to 1.0% by weight of a growth substrate such as yeast extract or glucose and about 0.01% to 0.5% by weight of PxBA. After culturing from the late logarithmic phase to the stationary phase in a medium or the like, and the cells are collected by centrifugation or the like, PxBA
Was further cultured in an inorganic medium containing no nitrogen source containing 0.01% to 0.5% by weight of
There is a method of extracting PxB. 0.1-1% by weight
A growth substrate such as yeast extract or glucose,
There is also a method in which PxBA of about 0.01% by weight to 0.5% by weight is fed and cultured, and cells are collected from late logarithmic growth phase to stationary phase to extract PHPxB.

[0032] PHPx from cells in the method of the present invention
For the recovery of B, the usual extraction with an organic solvent such as chloroform is the easiest, but in an environment where an organic solvent is difficult to use, treatment with a surfactant such as SDS, treatment with an enzyme such as lysozyme, etc. , EDTA, sodium hypochlorite, ammonia and other chemicals to remove bacterial components other than PHPxB.
Can be used.

In addition, culture of microorganisms, PHP by microorganisms
xB production and accumulation in cells, and PH from cells
The recovery of PxB is not limited to the above method.

[0034]

EXAMPLES Examples will be shown below, but the present invention is not limited by the following examples.

Example 1 N127 strain was inoculated into 200 ml of M9 medium containing 0.5% by weight of yeast extract and 0.1% by weight of PxBA,
Shaking culture was performed at 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 20 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. Obtained PH
A: After performing methanolysis according to a conventional method, a gas chromatography-mass spectrometer (GC-MS, Shimadzu Q
(P-5050, EI method), and the methyl ester of the monomer unit constituting PHA was identified.

As a result, as shown in Table 1, the PHA was almost 3-hydroxy-4-phenoxy-n-butyric acid (3H
PHPxB containing only PxB) as a monomer unit
The productivity is 1.8% by weight per dry cell weight.
Met. When PHPxB was produced by the same method using the P91 strain, the productivity was 0.1% per dry cell weight.
It was 5% by weight.

[0038]

[Table 1]

Example 2 D-glucose 0.5% by weight, PxBA 0.1% by weight
N127 strain was inoculated into 200 ml of M9 medium containing
Shaking culture was performed at 125 ° C. and 125 strokes / min. After 48 hours, the cells were collected by centrifugation, and resuspended in 200 ml of M9 medium containing 0.5% by weight of D-glucose and 0.1% by weight of PxBA and not containing a nitrogen source (NH ₄ Cl). And shaking culture at 30 ° C. and 125 strokes / min. After 48 hours, the cells were collected by centrifugation, washed once with cold methanol and freeze-dried.

The lyophilized pellet was suspended in 20 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. Obtained PH
A: After performing methanolysis according to a conventional method, a gas chromatography-mass spectrometer (GC-MS, Shimadzu Q
(P-5050, EI method), and the methyl esterified product of the monomer unit constituting the PHA was identified.

As a result, as shown in Table 2, the PHA contained almost only 3HPxB as a monomer unit.
PxB, and its productivity is 7.1 per dried cell weight.
%Met.

[0042]

[Table 2]

[0043]

Industrial Applicability According to the present invention, a novel microorganism capable of accumulating more highly poly-3-hydroxy-4-phenoxy-n-butyric acid in cells, and a poly-3 using the microorganism. Hydroxy-4-phenoxy-n-
A method for high yield production of butyric acid is provided. Thereby, poly-3-hydroxy-4-phenoxy-n-butyric acid useful as a functional polymer can be efficiently produced, and application to various fields can be expected.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:39) C12R 1:39) (72) Inventor Tetsuya Yano 3-30-2 Shimomaruko, Ota-ku, Tokyo No. Within Canon Inc. (72) Inventor Kei Takashi 3-30-2 Shimomaruko, Ota-ku, Tokyo F-term within Canon Inc. (reference) 4B064 AD83 CA02 CB12 CB24 CC03 CD07 CE08 CE17 DA01 DA20 4B065 AA43X AC14 BA23 BB08 BD11 BD16 CA12 CA44 CA60

Claims (2)

[Claims] 1. A poly-3-hydroxy-4-phenoxy-n-butyric acid having a repeating unit represented by the following formula (1) and a 4-phenoxy-n-butyric acid represented by the following formula (2): Pseudomonas fluorescens N127 strain (Pseudomo
nas fluorescens N127, FERM P-17745). Embedded image 2. A method for producing poly-3-hydroxy-4-phenoxy-n-butyric acid having a repeating unit represented by the following formula (1), comprising the step of producing Pseudomonas fluorescens strain N127 (Pseu:
domonas fluorescens N127, FERM P-17745) in a medium containing 4-phenoxy-n-butyric acid represented by the following formula (2), and poly-3-hydroxy-produced by culturing the N127 strain. Extracting 4-phenoxy-n-butyric acid. A method for producing poly-3-hydroxy-4-phenoxy-n-butyric acid. Embedded image