JPH0731490A - Method for production copolymer and microorganism production the same - Google Patents

Abstract

PURPOSE:To efficiently obtain a copolymer useful for medicines, foods, sanitary articles, etc., by culturing a microorganism belonging to the genus Pseudomonas and producing a copolymer having specific physicochemical properties, specified constituents, etc. CONSTITUTION:The production method of the objective copolymer comprises culturing a microorganism belonging to the genus Pseudomanas, such as Pseudomonas S.P. 31-1 strain (FERM P-13280) and producing a copolymer in an amount of 80-95X in the dried cells, the copolymer comprising (A) (i) 1-40% of 3-hydroxyhexanoate units, (ii) 10-95% of 3-hydroxyoctanoate units, (iii) 0-70% of 3-hydroxydecanoate units, and (iv) 0-40% of 3-hydroxydodecanoate units, and having physicochemical properties comprising (B) a number-average mol.wt. of 50000-1000000, (C) a melting point of 35-70 deg.C or no clear melting point, (D) a glass transition point of -20 to -50 deg.C. and (E) solubility: soluble in chloroform, dichloromethane, 1,2-dichloroethane, acetone; and in soluble in water.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for producing a biodegradable copolymer applicable to a wide range of fields such as pharmaceuticals, foods, hygiene products, agricultural and horticultural products, and packaging materials, and a method for producing the copolymer. Regarding microorganisms.

[0002]

2. Description of the Related Art It is known that many microorganisms produce various copolymers. These copolymers range from a thermoplastic so-called plastic to a viscoelastic rubber-like copolymer, and all of them are biodegradable.

[0003] For example, poly-3-hydroxybutyrate [P
(3HB)], JP-A-56-117793,
No. 57-74084, No. 57-150393, 3
Regarding the copolymer of hydroxybutyrate and 3-hydroxyvalerate [P (3HB-co-3HV)], JP-A Nos. 57-150393, 58-69224, 58-212792 and 59-220192 are disclosed. Issue 5
9-205992, 61-293385, 63
-2699989, 3-hydroxybutyrate and 4-
Hydroxybutyrate [P- (3HB-co-4H
B)], JP-A-1-48821 and 1-15.
6320, 1-222788, 1-30489.
No. 1, No. 2-279992, No. 2-234683, No. 3-216193, etc. disclose copolymers and methods for producing the same, respectively.

Furthermore, A. Steinbuchel et al.
Appl. Environ. Microbiol. Vo
l. 56, No. 11, p3360-3367 (199
0) is a copolymer of 3-hydroxyhexanoate and 3-hydroxyoctanoate [P (3HHx-co-
It has been reported that a copolymer having no 3HB unit such as 3HO)] is produced by a microorganism. That is,
A nutrient medium supplemented with octanoic acid as a carbon source, such as Pseudomonas aureofascin DSM500
When 82 is cultivated, 18.0% [P (3HH
x-co-3HO)] [3HHx20.8 mol%, 3H
O89.2 mol%] was produced and Pseudomonas citronellolis DSM50332 was cultured to produce 7 out of 7 dry cells.
5.3% [P (3HO-co-3HD)] [3HHx
93.8 mol%, 3HD6.2 mol%], and Pseudomonas oleovorans ATCC293
When 47 was cultured, 44.0% [P (3HH
x-co-3HO, 3HD)] [3HHx 5.4 mol%, 3HO 92.0 mol%, 3HD 2.6 mol%]. Furthermore, in a nutrient medium supplemented with gluconic acid as a carbon source, for example, Pseudomonas mendocina DSM
When 50017 was cultured, 50.7% P (3
HHx-co-3HO, 3HD, 3HDD) [3HHx
4.3 mol%, 3HO29.8 mol%, 3HD61.9
Mol%, 3HDD 4.2 mol%].

These biodegradable copolymers are hydrolyzable, and are decomposed into carbon dioxide and water by the action of microorganisms in soil, river water, seawater, and living bodies to return to the natural environment. In recent years, attention has been paid to the increased awareness of global environment conservation, research and development have been made, and practical application is being considered.

[0006]

However, the copolymers produced by microorganisms have a wide variety of physicochemical properties, and it is necessary to search for a copolymer that is suitable for each purpose of use.

On the other hand, in the production of the copolymer by the microorganism, the medium components are more expensive, the polymer production rate of the microorganism is slower, and the polymer content in the bacterial cell is lower than that produced by the ordinary chemical synthesis method. Therefore, there is a drawback that the production cost becomes high, and it has been desired to solve this problem.

[0008]

[Means for Solving the Problems] As a result of intensive studies in view of such circumstances, the present inventors have found that if bacteria belonging to the genus Pseudomonas isolated from soil in Sakura City, Chiba Prefecture are cultivated, 3
It was found that a copolymer having no HB unit can be obtained in an extremely high yield, and the present invention has been completed.

That is, the present invention belongs to the genus Pseudomonas and has 80 to 95% of the following physicochemical properties in dry cells.
The present invention provides a method for producing a copolymer, which comprises culturing a microorganism producing a copolymer having (A) to (E) and collecting the copolymer.

(A) Constituent Components and Compositions (a), (b), (c) and (d): (a) Represented by the following formulas (1) and / or (2) and / or (3). 3-hydroxyhexanoate unit (3HHx) 1-40 mol%,

[0011]

[Chemical 5]

(B) 3-hydroxyoctanoate unit (3HO) represented by the following formula (4) and / or (5) and / or (6): 10 to 95 mol%,

[0013]

[Chemical 6]

(C) 0 to 70 mol% of 3-hydroxydecanoate unit (3HD) represented by the following formula (7) and / or (8) and / or (9):

[0015]

[Chemical 7]

(D) 3-hydroxydodecanoate unit (3HDD) represented by the following formula (10) and / or (11) and / or (12) 0 to 40 mol%,

[0017]

[Chemical 8]

A copolymer consisting of: (B) Number average molecular weight of 50,000 to 1,000,000. (C) Melting point 35-70 ° C or no clear melting point. (D) Glass transition point -20 to -50 ° C. (E) Solubility Soluble in chloroform, dichloromethane, 1,2-dichloroethane and acetone, insoluble in water.

The present invention further provides Pseudomonas s which produces the above copolymer.
p. ) 31-1 strain (Ministry of Microbiological Research No. 13280) is provided.

The mycological properties of the microorganism producing the above copolymer are as follows.

(1) Morphological properties 0.6 to 0.8 μm x after overnight culture on agar medium at 30 ° C.
It is a straight bacillus of 1.7 to 2.3 μm. Liquid medium, 30
0.8-1.1μm x 2.5-5.7μ in overnight culture at ℃
It is a straight rod of m. Polar hair is observed by flagella staining, and motility is recognized. Polymorphism, spores, Gram stain and acid resistance are not observed.

(2) Growth state in various media (a) Meat broth agar plate culture The colonies are smooth and the margins are slightly rough. No characteristic colony dye or diffusible dye is produced. (B) Meat broth agar slope medium The moss is smooth and the periphery is rather rough. No characteristic colony dye or diffusible dye is produced. (C) Meat juice liquid medium Growth is observed throughout the medium, but no surface film is observed. (D) Meat broth gelatin stab culture Growth is observed at the top of the medium, but no liquefaction is observed. (E) Litmus milk Alkali is produced but coagulation is not observed.

(3) Physiological properties (a) Reduction of nitrate: Negative (b) Denitrification reaction: Negative (c) MR test: Negative (d) VP test: Negative (e) Indole formation: Negative (f) Production of hydrogen sulfide TSI agar: Negative Lead acetate agar: Negative (G) Starch hydrolysis: Negative (H) Use of citrate Koser's medium: Positive Christensen's medium: Positive (R) Use of inorganic nitrogen source Nitrate: Positive Ammonium salt: Positive (nu) Pigment formation Colony: Negative Water solubility: Negative (L) Urease: Negative (wo) Oxidase: Positive (wa) Catalase: Positive (mosquito) Growth range pH: 5.5-9. 0 Temperature: −3 to 36 ° C. (Yo) Attitude toward oxygen: Aerobic (ta) OF test (Hugh Leifson method):
Generation of Acid and Gas from O (R) Sugars: Described in Table 1

[0024]

[Table 1]

From the above-mentioned mycological properties, this microorganism belongs to the genus Pseudomonas, and the same strain does not exist as compared with known strains. Therefore, it is judged as a new strain, and Pseudomonas sp.
p. ) 31-1 and named to the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology, as a micromachine researcher No. 13280 (FERM P-
13280).

The microorganisms of the present invention are natural or ultraviolet, X-ray,
Mutation is caused by chemical agents. Therefore, these mutants can also be used in the method for producing the copolymer of the present invention.

The method for producing the copolymer of the present invention using the strain of the present invention is as follows. It is a method of collecting coalescence. An appropriate amount of assimilable carbon source, nitrogen source and other nutrient sources are contained in this medium. These are not particularly limited, but specific examples of the carbon source include sodium octanoate, sodium gluconate, valeric acid, broth, sucrose, starch, molasses, arabinose, sorbitol, methanol, carbon dioxide, and the like. Ammonium chloride as a nitrogen source,
Ammonium sulfate, other inorganic nitrogen sources such as sodium nitrate, yeast extract, meat extract, peptone, soybean flour, organic nitrogen sources such as oil cake can also be mentioned, as other additives, magnesium, sodium, if necessary, Potassium, calcium, manganese, copper, molybdenum, zinc,
Metal salts such as iron, phosphates, nitrates, chlorides, carbonates, etc.
Organic acid salts such as sodium citrate may be mentioned.

The culture is preferably carried out under aerobic conditions,
Any of stationary culture, shaking, and aeration and agitation culture are possible,
Shaking or aeration-agitation culture is advantageous. The culture temperature is preferably about 10 to 36 ° C, particularly about 20 to 32 ° C. Further, the pH of the medium is suitably about 5.5 to 9.0, and particularly 6.5 to 8.0 is most suitable.

The culture period varies depending on the culture conditions such as medium composition and temperature, but is usually about 0.5 to 6 days, preferably about 1 to 3 days.

Further, at this time, it is preferable that the culture is carried out in two stages, that is, a pre-stage culture in which the cells are mainly grown and a post-stage culture in which the copolymer is produced and accumulated in the cells by limiting nitrogen and / or phosphorus. Usually, it is preferable because the amount of the copolymer produced increases. That is, the first-stage culture is carried out under the above-mentioned culture conditions, and the cells are separated and recovered from the obtained culture solution by means such as filtration or centrifugation, and the cells are restricted to the latter-stage nitrogen and / or phosphorus-containing medium. To culture in
Alternatively, nitrogen and / or phosphorus may be depleted in the first-stage culture, and the culture solution may be transferred to the second-stage culture without separating and recovering the bacterial cells. The latter-stage culture differs from the former-stage culture only in that the culture medium does not substantially contain nitrogen and / or phosphorus.

The amount of the carbon source such as sodium octoate in the medium in the above culture may be any amount as long as it can produce the copolymer and does not inhibit the growth of microorganisms. It is preferable to change it depending on the kind of the monomer unit or the ratio of the number of monomer units. Usually, it is about 1 to 100 g, preferably about 5 to 30 g per liter of the culture solution.

From the culture cultivated as described above, cells are separated and recovered by a usual means such as filtration or centrifugation, and the cells are washed and dried to obtain dried cells. The yield of bacterial cells is about 1 to 10 g / l. In order to collect the desired copolymer from this microbial cell, the copolymer produced by an organic solvent such as chloroform is extracted by a conventional method, and the poor copolymer such as methanol or hexane is difficult to dissolve the copolymer. Is added to precipitate the copolymer.

The copolymer thus obtained is a copolymer having the above-mentioned physicochemical properties in which monomer units of 3HHx and 3HO, and optionally 3HD and 3HDD are ester-bonded. The ratio and molecular weight of each monomer unit can be controlled by changing the type and concentration of carbon sources such as sodium octanoate and sodium gluconate.

[0034]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

Example 1 Restoration and Preculture of Preserved Strain Pseudomonas sp 31-1 strain (Microtechnology Research Institute, No. 13280) thawed and frozen was thawed at room temperature and inoculated into a broth medium containing 1% glucose. It was restored by culturing at 30 ° C. for 24 hours. This reconstituted strain was inoculated into 5 ml of a broth liquid medium containing 1% glucose and cultured at 30 ° C. for 24 hours with reciprocal shaking. Pre- stage culture The above-mentioned Pseudomonas sp.
The strain -1 was aerobically cultured at 30 ° C for 20 hours.

[0036]

[Table 2] (Media composition) NH ₄ Cl 0.4 g KH ₂ PO ₄ 2.7 g NaH ₂ PO ₄ 3.2 g MgSO ₄ 0.2 g Mineral solution ^* 1.0 ml Distilled water 1,000 ml pH 7.0

[0037]

[Table 3] *: CoCl 119.0 mg CaCl ₂ 7.8 mg CrCl ₂ 62.2 mg FeCl ₃ .6H ₂ O 9.7 mg NiCl ₃ 118.0 mg CuSO ₄ 156.0 mg 0.1N-HCl aqueous solution 1 .01

Second- stage culturing Next, aerobically culturing was carried out at 30 ° C. for 30 hours in a medium in which 5 g / l of sodium octanoate as a carbon source was added to the same medium composition as that of the first-stage culturing. pH is unadjusted 7.
It was 0 to 7.5. Separation of bacterial cells The bacterial cells were separated from the obtained culture by centrifugation (10,000 rpm). Next, the obtained cells were freeze-dried to obtain 8.0 g / l of dried cells. Separation and Purification of Copolymer Coated with chloroform (water bath 90
℃) was added to extract and concentrate the copolymer, methanol was added to this to precipitate the copolymer, the supernatant methanol was removed, and the residue was dried in a vacuum dryer to obtain 88% of the dried cells. A copolymer (1) was obtained. Physicochemical Properties of Copolymer (1) The composition, number average molecular weight, melting point, glass transition point and solubility of copolymer (1) obtained as described above were measured by the following.

[0039]

Table 4 Composition: ¹ H-NMR spectrum, ¹³ C-NMR spectrum Molecular weight: Gel permeation chromatography (GP
C) Measurement (Hitachi L-6200, Data processing device D-2520) Melting point: Differential scanning calorimeter (DSC) measurement (10 ° C / min)
) (Shimadzu DSC-50) Glass transition point: Differential scanning calorimeter (DSC) measurement (20 ° C)
/ Min) Solubility: Solubility in various solvents

The measurement results are also shown in Table 5. The copolymer (1) in the dried cells had a high content rate of 88%. The solubility of the copolymer (1) is chloroform, dichloromethane,
It was soluble in 1,2-dichloroethane and acetone and insoluble in water. The copolymer (1) was rubbery. Also, 1
The 00 MHz ¹³ C-NMR spectrum is shown in FIG.
The MHz ¹ H-NMR spectrum is shown in FIG. 2, the GPC measurement chart is shown in FIG. 3, and the DSC measurement chart is shown in FIG.

Example 2 Sodium octanoate 1 was used as a carbon source in the latter culture.
The same procedure as in Example 1 was repeated except that the medium was 2 g / l.

The measurement results are also shown in Table 5. The copolymer (2) in the dried cells had a high content of 93%. Also,
The solubility of the copolymer (2) was soluble in chloroform, dichloromethane, 1,2-dichloroethane and acetone, but insoluble in water. The copolymer (2) was rubbery.

Example 3 Example 1 was carried out without dividing the culture into two stages, a first stage and a second stage.
The same procedure as in Example 1 was performed except that 5 g / l of sodium octanoate as a carbon source was added to the pre-stage culture and the culture was performed for 72 hours, and the post-stage culture was not performed.

The measurement results are also shown in Table 5. The copolymer (3) in the dried cells had a high content rate of 83%. The solubility of the copolymer (3) is chloroform, dichloromethane,
It was soluble in 1,2-dichloroethane and acetone and insoluble in water. The copolymer (3) was rubbery.

Example 4 0.5 g of NH ₄ Cl in the composition of the medium for the pre-stage culture and the post-stage culture
/ L medium, and sodium gluconate 15 g /
The same procedure as in Example 1 was carried out except that 1 medium was added.

The measurement results are also shown in Table 5. The copolymer (4) in the dried cells had a high content rate of 80%. The solubility of the copolymer (4) is chloroform, dichloromethane,
It was soluble in 1,2-dichloroethane and acetone and insoluble in water. The copolymer (4) was a muddy oil. The 100 MHz ¹³ C-NMR spectrum is shown in FIG. 5, the 500 MHz ¹ H-NMR spectrum is shown in FIG.
A GPC measurement chart is shown in FIG. 7, and a DSC measurement chart is shown in FIG.

[0047]

[Table 5]

[0048]

EFFECT OF THE INVENTION By the production method of the present invention, 8
High content monomer unit 3HH occupying 0 to 95%
A copolymer of x, 3HO, 3HD and 3HDD can be obtained. As a result, compared with the conventional manufacturing method,
It is possible to significantly reduce the production cost, and the industrial merit is great. Furthermore, the copolymer obtained by the present invention is expected to be applied in a wide range of fields such as pharmaceuticals, foods, hygiene products, agricultural and horticultural products, and packaging materials.

[Brief description of drawings]

1 is a drawing showing a ¹³ C-NMR spectrum at 100 MHz of the copolymer obtained in Example 1. FIG.

FIG. 2 is a drawing showing the ¹ H-NMR spectrum of the copolymer obtained in Example 1 at 500 MHz.

3 is a drawing showing a GPC measurement chart of the copolymer obtained in Example 1. FIG.

4 is a drawing showing a DSC measurement chart of the copolymer obtained in Example 1. FIG.

FIG. 5 is a drawing showing a ¹³ C-NMR spectrum at 100 MHz of the copolymer obtained in Example 4.

FIG. 6 is a drawing showing the ¹ H-NMR spectrum of the copolymer obtained in Example 4 at 400 MHz.

7 is a drawing showing a GPC measurement chart of the copolymer obtained in Example 4. FIG.

8 is a drawing showing a DSC measurement chart of the copolymer obtained in Example 4. FIG.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technology display location (C12N 1/20 C12R 1:38) (72) Inventor Genzo Hirano 1-chome Takanodai, Yotsukaido, Chiba No. 3 Stock Company, Japan Steel Works

Claims (2)

[Claims] 1. A microorganism belonging to the genus Pseudomonas and producing a copolymer having 80 to 95% of the following physicochemical properties (A) to (E) in a dry cell is cultured, and the copolymer is collected. A method for producing a copolymer, comprising: (A) Constituent Components and Compositions (a), (b), (c) and (d) below: (a) 3-Hydride represented by the following formula (1) and / or (2) and / or (3) Roxyhexanoate unit 1-40 mol%, (B) 3-hydroxyoctanoate unit represented by the following formula (4) and / or (5) and / or (6): 10 to 95 mol%; (C) 3-hydroxydecanoate unit represented by the following formula (7) and / or (8) and / or (9): 0 to 70 mol%; (D) 3-hydroxydodecanoate unit represented by the following formula (10) and / or (11) and / or (12): 0 to 40 mol%, A copolymer consisting of. (B) Number average molecular weight of 50,000 to 1,000,000. (C) Melting point 35-70 ° C or no clear melting point. (D) Glass transition point -20 to -50 ° C. (E) Solubility Soluble in chloroform, dichloromethane, 1,2-dichloroethane and acetone, insoluble in water. 2. Pseudomonas s which produces the copolymer of claim 1.
p. ) 31-1 strain (Ministry of Microbiological Research, No. 13280).