JPH05276934A - Microorganism capable of producing bio-polyester - Google Patents

Abstract

PURPOSE:To provide a new microorganism capable of producing an aliphatic polyester containing 3-hydroxybutyrate (3HB) as a monomer unit in a high yield and high content in dry microbial cell. CONSTITUTION:The new microorganisms capable of producing the polyester was separated and identified and it has been clarified that the microorganism is a bacterial strain belonging to the genus Alcaligenes. The strain showed microbiological characteristics different from those of any known strain of the genus Alcaligenes and exhibited the characteristic nature to be grown with oil and fat or long-chain fatty acid to produce the above polyester. Accordingly, the bacteria was named as Alcaligenes lipolytica AK201. A bio-decomposable thermoplastic polyester can be produced from an oil and fat or a long-chain fatty acid abundantly available from nature in high efficiency by the use of the subject microorganism.

Description

Detailed Description of the Invention

[0001]

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 above-mentioned microorganism-produced polyesters represented by poly-3-hydroxybutyrate (PHB), unlike synthetic polymers derived from petroleum, is easily decomposed by microorganisms or in the body of higher animals including humans. It has (biodegradability). In recent years, enormous amounts of synthetic polymers have been used in various fields such as plastics, but used plastics accumulate as bulky waste that does not decay,
The pollution of the natural environment has become a serious problem. On the other hand, since the polyester produced by microorganisms is biodegraded and taken into the natural material cycle, it can be used as a plastic that enables environmental protection. Further, in the medical field, it can be used as an implant material or a drug carrier that does not require recovery.

[0002]

2. Description of the Related Art Heretofore, it has been reported that various kinds of bacteria produce PHB in cells and accumulate it as particles [H.
Brandl et al, Adv. Biochem.
Eng. / Biotechnol. , 41, 77-93
(1990)]. In particular, Alcaligenes eutrophus
[JP-A-57-150393], genus Nocardia (No.
cardia), genus Corynebacterium (Coryne)
Bacterium), Rhodococcus (Rhodoc)
occus) of certain fungi [European Patent 9030426
7.9, April, 1990], Rhodospirillum rubrum
m) [H. Brandl et al, Int. J. B
iol. Macromol. , 11, 49-55 (19
89)] is a copolymer of 3HB and 3-hydroxyvaleric acid (3HV) or 3HB, 3HV from a certain carbon source.
It is reported that a copolymer containing each of the units as a component is produced. Copolymer of 3HB and 3HV (P (H
B / HV)) is industrially utilized as a thermoplastic resin having improved physical properties of PHB, which has the drawback of being hard and brittle.

[0003]

When such a 3HB unit-containing polyester is industrially produced by a microorganism, raw materials, particularly the type of carbon source which is the main raw material, the yield based on the carbon source used, and the drying of the polyester. The cell content rate is an important requirement. However, the use of the conventionally known polyester-producing bacteria has been insufficient 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 long-chain fatty acids, which have not been known to be used as a carbon source, or the main carbon As a source, PHB or P
We have found a novel bacterial species that produces (HB / HV) with high yield and high dry cell content. The bacterium of the present invention belongs to the genus Alcaligenes and the mycological properties are as follows.

(A) Morphological properties 1. Cell shape and size: bacilli, 0.4-0.7x
0.7-1.5 μm 2. Cell polymorphism: None 3. Motility: Yes 4. Flagella: Periflagellates 5. Spore: None 6. Gram stainability: Negative (b) Culture property 1. Meat broth agar plate culture: white, good growth 2. Broth agar slope culture: white, good growth 3. Broth liquid culture: cream color, good growth (c) Physiological properties 1. Nitrate reduction: negative 2. Denitrification reaction: Negative 3. Generation of indole: negative 4. Use of citric acid: positive 5. Use of inorganic nitrogen source: positive 6. Dye formation: negative 7. Urease: positive 8. Oxidase: Positive 9. Catalase: Positive 10. Growth temperature and pH: 20 to 37 ° C., pH 6 to 8 11. Attitude toward oxygen: Aerobic 12. OF test: negative (indicated alkaline reaction) 13. Acid production from glucose: negative 14. Degradation of gelatin: negative 15. Utilization of sugar: D-glucose, L-arabino-
Negative for glucose, D-mannose, D-mannitol and maltose

This strain was isolated from contaminating bacteria as shown below. That is, the strain ATCC29347 of the genus Pseudomonas that has been passage-preserved
Was liquid-cultured. As a result, it was contaminated with miscellaneous bacteria, so the bacteria were separated. That is, one part of the culture solution was quantitatively diluted with sterilized water and plated on a Petri dish to form colonies. Bacteria were picked from 5 colonies of the same plate culture, and each was liquid-cultured. A slant medium was inoculated with the growing bacteria of each liquid culture and cultured for 4 days to obtain a test sample of mycological properties. The basal medium used in the reference example (described later) is used as the medium for the above liquid culture and solid culture (plate, slant), and is equivalent to 0.25 g / l of sodium n-octanoate as a carbon source. Was added. The culture temperature was 30 ° C., the culture time was 24 hours in liquid culture, and the culture was in solid culture for 4 days.

As a result of the test of mycological properties, a strain derived from one colony named AK201 exhibited the properties described above in the text. AK201 was less likely to have a significant difference in culture properties as compared with bacteria derived from other colonies, but the flagellation state of the flagella by electron microscopic observation was different from the polar flagella common to Pseudomonas spp. Met. Furthermore, compared with bacteria derived from other colonies, different results were obtained in at least 5 items (reduction of nitrate, utilization of citric acid, urease, OF test, glucose assimilation) among the implementation test items of physiological properties. Indicated. Therefore, as a result of examining the taxonomic position, it was found to belong to the genus Alcaligenes.

As described above, this bacterium is an aerobic, gram-negative, non-fermenting, motile bacillus. In addition, it is identified as belonging to the genus Alcaligenes in consideration of having periflagellates and other mycological properties such as oxidase positive and OF test negative. The test of mycological properties was conducted by Kazuo Komagata [Takeharu Hasegawa (ed.), Classification and Identification of Microorganisms (1990); Aerobic Bacteria], The Procalioats No. 1
Volume [The Procariotes; A Handb
look on Habitats, Isolation
and Identification of Bac
teria, (Ed.) M. P. Starr et a
1 (1981)]. In addition, for identification of bacteria, the Berseys Manual [Bergey's
Manualal System Bacte
riology, Vol. 1 (1984)].

The mycological properties and assimilation properties of this bacterium are shown in Tables 1 and 2 in comparison with those of existing strains of the genus Alcaligenes. Among the physiological tests, the oxidase test was performed by culturing in 1% peptone-containing broth and observing the color development on a test paper. In the acid production test from glucose, peptone water was used as the basal medium, and in the utilization test of citric acid, a coser, Simmons and Christensen's media were used. In addition, the morphological characteristics were determined by observing the state of flagella attachment with an electron microscope, and otherwise observing with an optical microscope according to a conventional method.

[0010]

[Table 1]

[0011]

[Table 2]

As a result of the above collation, it was determined that this bacterium is a new species of bacterium 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. Therefore, Alcaligenes lipolytica (A
lcaligenes lypolytica) AK2
It was named 01.

The bacterium, like other bacterium of the genus Alcaligenes, has a culture temperature of 20 to 40 ° C., preferably 24 to 33 ° C.
Is. The initial pH of the culture is 6.0 to 8.0, preferably 6.5 to 7.5. The culture medium may be an inorganic medium, to which one or more of various carbon sources are added. As the carbon source, saturated or unsaturated linear fatty acids having 2 to 22 carbon atoms, preferably 2 to 18 carbon atoms; oils and fats including vegetable oils and animal fats; citric acid, gluconic acid, succinic acid, glutaric acid, adipine Examples thereof include acids, organic acids such as suberic acid and azelaic acid; alcohols such as methanol, ethanol and glycerol; and linear aliphatic hydrocarbons such as n-octane and n-nonane. Further, various amino acids may be used, and nitrogen-containing organic nutrient sources such as polypeptone, meat extract, casamino acid, yeast extract, and sugar-tight can be used. Inorganic ammonium salt as nitrogen source,
For example, ammonium phosphate, ammonium sulfate, ammonium chloride, etc., and the above-mentioned amino acids, nitrogen-containing organic nutrient sources and organic acid ammonium, organic acid amide are used. As inorganic ions, sodium, potassium,
In addition to magnesium, calcium, chlorine, sulfuric acid, phosphoric acid,
Iron, manganese, zinc, copper, cobalt and the like are used in whole or in part.

After completion of the culture, the bacterial cells are collected by centrifugation, filtration or the like. When linear fatty acids, oils and fats, and linear aliphatic both-end-dicarboxylic acids are used as carbon sources, the polyester is produced at a high dry cell content, and thus the cell treatment for polyester separation may be performed as it is. For example, the collected bacterial cells are washed with distilled water and then freeze-dried to obtain dried bacterial cells.
Then, the polyester is extracted from the bacterial cells with a good solvent such as chloroform under heating, concentrated, and reprecipitated with a poor solvent such as methanol or hexane to obtain the polyester.

When a carbon source other than linear fatty acids, oils and fats, and dicarboxylic acids having both linear and aliphatic terminals is used, only a small amount of polyester is produced or no polyester is produced at all. Incubation for polyester synthesis (referred to as second stage culture) is performed. The second-stage culture is carried out by culturing the collected cells in a culture medium in which at least one of certain essential nutrient elements such as nitrogen and phosphorus is reduced or removed. culture)
Same as above for a certain period of time. The carbon source used at this time may be the same as or different from that used in the first stage culture. Second
After the stage culture, the bacteria collection, bacterial cell treatment, and polyester separation are performed according to the methods described above.

The results of the tests conducted by expanding the types of physiological tests for AK201 are already shown in Table 1. From this result, this bacterium was judged to be a new strain, and Alcaligenes lipolytica AK201 3819). Below, the example which manufactured 3HB containing polyester is shown using the strain of this invention.

A liquid medium was prepared by adding 100 g of a basic medium having the composition shown below with sodium stearate, undecanoic acid and rapeseed oil as carbon sources at a rate of 3 g / l.
The Sakaguchi flask with a volume of 00 ml was inoculated with Alcaligenes lipolytica AK201 aseptically, and then shake-cultured at 30 strokes / min for 48 hours at 130 strokes / min.

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 The solution is prepared by dissolving the following inorganic salts in 1 liter of 1M hydrochloric acid.

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

After completion of the culture, the culture solution 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 bacterial cells was extracted from the dried bacterial cells with 100 ml of hot chloroform, the solution was concentrated to about 5 ml, and hexane was added for precipitation. The precipitate was filtered and dried to obtain polyester.

For the analysis of 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 addition, for gas chromatography, a sample was used in which dried bacterial cells were subjected to methanolysis to use intracellular polyester as a methyl ester of a monomer [H. Brandl et al, I
nt. J. Biol. Macromol. , 11, 49
-55 (1989)]. The results of polyester biosynthesis are shown in Table 3.

[0022]

[Table 3]

[0023]

INDUSTRIAL APPLICABILITY The bacterium of the present invention is derived from naturally occurring abundant fats and oils and long-chain fatty acids, or using it as a main carbon source
It has become possible to produce HB and P (HB / HV) with high yield and high cell content. Such 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 front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1:05)

Claims (1)

[Claims] 1. A long-chain fatty acid and a fat and oil assimilation, and
Alkagenes lipolytica microscopic research institute Kojiro 381 capable of producing 3-hydroxybutyrate-containing polyester
9.