JP3100001B2 - Microorganisms capable of producing biopolyester - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microorganism for biosynthesizing an aliphatic polyester containing 3-hydroxybutyrate (3HB) as a monomer unit. A group of the microorganism-producing polyesters represented by poly-3-hydroxybutyrate (PHB), unlike synthetic polymers derived from petroleum, are easily degraded by microorganisms or in higher animals including humans. (Biodegradable). In recent years, an enormous amount of synthetic polymers have been used in various fields as plastics and the like, but used plastics have accumulated as bulky waste that does not rot,
Pollution of the natural environment is a serious problem. On the other hand, the microorganism-produced polyester is taken into natural material circulation by being biodegraded, and thus can be used as a plastic that enables environmental conservation. In the medical field, it can be used as an implant material or a drug carrier that does not require collection.

[0002]

2. Description of the Related Art It has been reported that various kinds of bacteria produce PHB in cells and accumulate as PHB [H.
Brandl et al, Adv. Biochem.
Eng. / Biotechnol. , 41, 77-93
(1990)]. In particular, Alcaligenes eutrophus
[JP-A-57-150393], Nocardia (No.
cardia), Corynebacterium (Coryne)
Bacterium), Rhodococcus (Rhodoc)
occus) [EP 903426
7.9, April, 1990], Rhodospirillum rubrum.
m) [H. Brandl et al, Int. J. B
iol. Macromol. , 11, 49-55 (19
89)] and the like, from a certain carbon source, a copolymer of 3HB and 3-hydroxyvaleric acid (3HV) or 3HB, 3HV
It has been reported that a copolymer containing both of the above units as components is produced. 3HB and 3HV copolymer (P (H
B / HV)) is being industrially used as a thermoplastic resin in which the physical properties of PHB having a hard and brittle defect are improved.

[0003]

When such a 3HB unit-containing polyester is industrially produced by a microorganism, the raw material, particularly the type of the carbon source which is the main raw material, the yield based on the carbon source used, and the drying of the polyester, The bacterial content is an important requirement. However, the use of the conventionally known polyester-producing bacteria has not been enough to satisfy such requirements.

[0004]

In order to solve the above-mentioned problems, as a result of searching for a 3HB-containing polyester-producing bacterium, it has been found that oils and fats or long-chain fatty acids which have not been known to be used as a carbon source, or those containing a main carbon PHB or P as source
(HB / HV) with a high yield and a high dry cell content was found. The fungus of the present invention belongs to the genus Alcaligenes and has the following bacteriological properties.

(A) Morphological properties Cell shape and size: bacilli, 0.4-0.7 ×
0.7-1.5 μm 2. Cell polymorphism: None Motility: yes 4. Flagellarity: periflagellate Spores: none Gram staining: negative (b) Culture properties 1. Gravy agar plate culture: white, good growth 2. Gravy agar slant culture: white, good growth Broth liquid culture: cream color, good growth (c) Physiological properties Nitrate reduction: negative 2. 2. Denitrification reaction: negative 3. Indole production: negative 4. Use of citric acid: positive 5. Use of inorganic nitrogen source: positive 6. Dye formation: negative Urease: positive Oxidase: positive 9. Catalase: positive10. 10. Growth temperature and pH: 20-37 ° C, pH 6-8 Attitude to oxygen: aerobic 12. OF test: negative (indicating an alkaline reaction) 13. Acid production from glucose: negative 14. Gelatin degradation: negative Sugar assimilation: D-glucose, L-arabino-
, D-mannose, D-mannitol, maltose are all negative

This strain was isolated from contaminating bacteria as described below. That is, the passaged and preserved strain Pseudomonas strain ATCC29347.
Was subjected to liquid culture. As a result, the bacteria were contaminated, and the bacteria were separated. That is, one part of the culture solution was quantitatively diluted with sterile water and plated on a Petri dish to form a colony. Bacteria were picked from five colonies of the same plate culture, and each was cultured in liquid. The growth bacteria of each liquid culture were inoculated on a slant medium and cultured for 4 days to obtain a test sample of mycological properties. In addition, as a medium for the above liquid culture and solid culture (plate, slant), the basal medium used in Reference Example (described later) was used, and 0.25 g / l of sodium n-octanoate was used as a carbon source. Was added. The culture temperature was 30 ° C., the culture time was 24 hours for liquid culture, and 4 days for solid culture.

[0007] In the results of the test of mycological properties, a strain derived from one colony designated as AK201 exhibited the properties described above in the text. AK201 showed little difference in culture characteristics compared to bacteria derived from other colonies, but the flagellation state observed by electron microscopy was different from the polar flagella common to Pseudomonas spp. Met. Furthermore, compared with bacteria from other colonies, different results were obtained in at least 5 items (reduction of nitrate, use of citric acid, urease, OF test, glucose assimilation) in the test items of physiological properties. Indicated. Therefore, as a result of examining the taxonomic position, it was found that the substance belongs to the genus Alcaligenes.

As described above, the present bacterium is an aerobic, gram-negative, non-fermentative, motile bacillus. It was identified as belonging to the genus Alcaligenes in consideration of having periflagellate and other mycological properties such as oxidase positive and OF test negative. The mycological properties were tested by Kazuo Komagata [Takeharu Hasegawa (eds.), Classification and Identification of Microorganisms (1990); Aerobic Bacteria], The Procarioates No. 1
Vol. [The Procaryotes; A Handb
books on Habitats, Isolation
and Identification of Bac
teria, (Ed.) M .; P. Starr et a
1 (1981)]. In addition, for identification of the bacterium, the Bergey's Manual
Manualflow Systematic Bacte
riology, Vol. 1 (1984)].

The mycological properties and assimilation of this bacterium are shown in Tables 1 and 2 in comparison with the mycological properties and assimilation of the existing species of the genus Alcaligenes. In the physiological tests, the oxidase test was performed by culturing with 1% peptone-containing broth and observing the color development with test paper. In the acid production test from glucose, peptone water was used as a basal medium, and in the use test of citric acid, Coser, Simmons and Christensen media were used. The morphological properties were determined by observing the flagellated state with an electron microscope and observing the other with an optical microscope according to a conventional method.

[0010]

[Table 1]

[0011]

[Table 2]

As a result of the above collation, the present bacterium was determined to be a new species belonging to the genus Alcaligenes. As described below, this bacterium is characterized by well assimilating fats and oils and long-chain fatty acids as carbon sources for growth and polyester synthesis, so that Alcaligenes lipolytica (A)
lcaligenes lypolytica) AK2
01.

The bacterium is cultured at a temperature of 20 to 40 ° C., preferably 24 to 33 ° C., like other Alcaligenes bacteria.
It is. The initial pH of the culture is 6.0 to 8.0, preferably 6.5 to 7.5. An inorganic medium can be used as the culture solution, and one or more of various carbon sources are added thereto. As the carbon source, a saturated or unsaturated linear fatty acid having 2 to 22, preferably 2 to 18 carbon atoms; fats and oils including vegetable fats and oils; citric acid, gluconic acid, succinic acid, glutaric acid, adipine Organic acids such as acid, suberic acid and azelaic acid; alcohols such as methanol, ethanol and glycerol; and linear aliphatic hydrocarbons such as n-octane and n-nonane. In addition, various amino acids may be used, and nitrogen-containing organic nutrients such as polypeptone, meat extract, casamino acid, yeast extract, and molasses can be used. As a nitrogen source, an inorganic ammonium salt,
For example, ammonium phosphate, ammonium sulfate, ammonium hydrochloride and the like, as well as the above-mentioned amino acids, nitrogen-containing organic nutrients, organic acid ammonium and organic acid amide are used. As inorganic ions, sodium, potassium,
Magnesium, calcium, chlorine, sulfuric acid, phosphoric acid,
Iron, manganese, zinc, copper, cobalt, etc. are used in combination or in all.

After completion of the culture, the cells are collected by centrifugation, filtration or the like. When a straight chain fatty acid, oil or fat, or straight chain aliphatic terminal dicarboxylic acid is used as a carbon source, the polyester is produced with a high dry cell content, and thus the cell treatment for polyester separation may be performed as it is. For example, the collected cells are washed with distilled water, and then freeze-dried to obtain dried cells.
Next, the polyester is extracted from the cells with a good solvent such as chloroform under heating, concentrated, and then reprecipitated with a poor solvent such as methanol or hexane to obtain a polyester.

When a carbon source other than straight-chain fatty acids, oils and fats, and dicarboxylic acids having both straight-chain aliphatic terminals is used, only a small amount or no polyester is formed. Incubation (referred to as second stage culture) for polyester synthesis is performed. The second stage culture is performed by culturing the collected cells in a culture solution in which at least one of certain essential nutrient elements, for example, nitrogen, phosphorus, etc. is reduced or removed, and other conditions are the same as those described above (first stage culture). culture)
Perform for a fixed time in the same manner as in. The carbon source used at this time may be the same as or different from that used in the first stage culture. Second
Bacterial collection, cell treatment, and polyester separation after the step culture are performed according to the methods described above.

The results of an expanded type of physiological test for AK201 are already shown in Table 1. Based on the results, the present bacterium was determined to be a new type of bacterium, and Alcaligenes lipolytica AK201 (Microtechnical Lab. No. 3819). Hereinafter, an example of producing a 3HB-containing polyester using the strain of the present invention will be described.

A liquid medium containing 100 g of a basic medium having the composition shown below and sodium stearate, undecanoic acid and rapeseed oil added at a ratio of 3 g / l as carbon sources was prepared.
Alkagenes lipolytica AK201 was aseptically inoculated into a 00 ml Sakaguchi flask, followed by shaking culture at 30 ° C. for 48 hours at 130 strokes per minute.

Basic medium composition (NH ₄ ) ₂ HPO ₄ 1.1 g K ₂ HPO ₄ 5.8 g KH ₂ PO ₄ 3.7 g MgSO ₄ 0.12 g Trace element solution 1 ml Water 1000 ml However, trace elements The solution was prepared by dissolving the following inorganic salts in 1 liter of 1M hydrochloric acid.

[0019] _{FeSO 4 · 7H 2 O (2.78g} ), M
_{nCl 2 · 4H 2 O (1.98g} ), CoSO 4 · 7H
₂ O (2.81 g), CaCl ₂ .2H ₂ O (1.67 g)
g), CuCl ₂ .2H ₂ O (0.17 g), ZnSO
₄ · 7H ₂ O (0.29g)

After completion of the culture, the culture was centrifuged (8000).
rpm, 15 minutes) and washed with water to collect the bacteria. The cells were freeze-dried to obtain dried cells. The polyester accumulated in the cells was extracted from the dried cells with 100 ml of hot chloroform, the solution was concentrated to about 5 ml, and precipitated by adding hexane. The precipitate was filtered and dried to obtain a polyester.

In the analysis of the polyester, gas chromatography was used to determine the composition, differential thermal analysis was used to measure the melting point, heat of fusion, and glass transition temperature, and gel filtration chromatography was used to determine the molecular weight and molecular weight distribution. In the gas chromatography, a sample obtained by subjecting dried bacterial cells to methanolysis and converting the intracellular polyester into a methyl ester of a monomer [H. Brandl et al, I
nt. J. Biol. Macromol. , 11,49
-55 (1989)]. Table 3 shows the results of the polyester biosynthesis.

[0022]

[Table 3]

[0023]

EFFECTS OF THE INVENTION The fungus of the present invention can be prepared from oils and fats which are abundant in nature, long-chain fatty acids, or P
HB and P (HB / HV) can be produced with high yield and high bacterial content. Such a 3HB-containing polyester is not only biodegradable, but also thermoplastic, and can be processed into a desired product by a molding method such as injection molding or extrusion molding or melt spinning.

Continuation of the front page (51) Int.Cl. ⁷ identification code FI C12R 1:05) (58) Investigated field (Int.Cl. ⁷ , DB name) C12N 1/20 C12P 7/62

Claims (1)

(57) [Claims] Claims: 1. A long-chain fatty acid and fats and oils assimilating, and
Alkaligenes lipolytica micromachining lab. 381 capable of producing 3-hydroxybutyrate-containing polyester
9.