JPH0564592A - Production of bio-polyester - Google Patents

Abstract

PURPOSE:To obtain the subject polymer useful for bio-decomposable medical material, etc., in high yield by culturing a microbial strain belonging to genus Alcaligenes and capable of producing a polyester containing 3-hydroxybutyrate in a medium containing a long-chain fatty acid and collecting the product accumulated in the microbial cell. CONSTITUTION:A microbial strain belonging to genus Alcaligenes and capable of producing a polyester containing 3-hydroxybutyrate [e.g. Alcaligenes lipolytica AK201 (FERM P-12198)] is cultured in a liquid medium containing at least one kind of compound selected from long-chain fatty acid (e.g. undecanoic acid) and its derivative at 30 deg.C for 48hr under shaking at a rate of 130 strokes per minute. The culture liquid is subjected to centrifugal separation, washed with water and the collected microbial cells are freeze-dried. The freeze-dried cells are treated with hot chloroform to extract the polyester accumulated in the cell, the extracted liquid is concentrated, added with hexane and the precipitate is filtered and dried to obtain the objective bio-polyester composed of a copolymer of 3-hydroxybutyrate and 3-hydroxyvaleric acid, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an aliphatic polyester containing 3-hydroxybutyrate (3HB) as a monomer unit by utilizing the biosynthetic function of microorganisms. A group of microbial-produced polyesters represented by poly (3-hydroxybutyrate) (PHB), unlike synthetic polymers derived from petroleum, have the property of being easily decomposed in the body of microorganisms and higher animals including humans. It has (biodegradability). In recent years, enormous amounts of synthetic polymers have been used in various fields such as plastics,
It has become a serious problem that used plastic accumulates as bulky waste that does not rot and pollutes the natural environment. Since the polyester 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 and a drug carrier that are not required to be recovered.

[0002]

2. Description of the Related Art Conventionally, it has been reported that various kinds of bacteria produce PHB in cells and accumulate it as particles [H.Br.
andl et al, Adv.Biochem. Eng./Biotechnol., 41,77-93
(1990)]. Especially, Alcaligenes eutrophus (Al
caligenes eutrophus) [JP-A-57-150393],
Nocardia, Corynebacterium (C
orynebacterium), certain fungi of the genus Rhodococcus (European patent 90304267.9, April, 1990), Rhodospirillum rubrum (H.Br
andl et al, Int. J. Biol. Macromol., 11, 49-55 (1989)] et al. It has been reported to produce a copolymer as a component.
A copolymer of 3HB and 3HV (P (HB / 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, the raw materials, particularly the type of the carbon source as the main raw material, the yield based on the carbon source used, and the drying of the polyester are used. The cell content rate is an important requirement. Regarding the type of carbon source, methanol (JP-A-56-117793) and fructose (Japanese Patent Publication No.
20238), glucose (JP-A-57-15039).
3), sucrose (Japanese Patent Publication No. 63-32438) and the like have been reported to be used. However, they each have drawbacks such as low molecular weight of produced polyester, difficulty in obtaining large amount, insufficient yield based on carbon source used, and limited application to homopolymer PHB. It was

[0004]

In order to solve the above-mentioned problems, the present inventors have cultivated 3HB-containing polyester-producing bacteria with various carbon sources, and as a result, have conventionally been known to be used as carbon sources. It was found that PHB or P (HB / HV) is produced at a high yield and a high dry cell content rate by culturing a bacterium of the genus Alcaligenes using a long-chain fatty acid or its derivative as an essential carbon source.

That is, the present invention uses 3H belonging to the genus Alcaligenes using a liquid medium containing at least one compound of a long-chain fatty acid and its derivative as an essential carbon source.
The purpose of the present invention is to provide a method for producing a polyester which comprises culturing a B-containing polyester-producing bacterium, accumulating 3HB-containing polyester in the microbial cells, and collecting the polyester.

Alcaligenes used
), A.faecalis (Alcaligenes faecalis), A.denitrificans (Alcaligenes denitrificans), A. eutrophus (Alcaligenes
Known bacterial species such as Eutrophus), A. paradoxus (Alcaligenes paradoxus), and A. aquamarinus (Alcaligenes aquamarinus) can also be used, but the bacterium A. lipolytica (Alcaligenes lipolytica) AK201 isolated by the present inventor (Ministry of Microbiological Research No. 12198)
Have particularly good properties for this purpose. For the above-mentioned known bacteria, see the Berseys Manual [Be
rgey's Manual of Systematic Bacteriology, Vol.1 (198
4)] shows its mycological properties and major collected strains.

The mycological properties of A. lipolytica AK201 are as follows. (A) Morphological properties 1) Shape and size of cells: bacillus, 0.4 to 0.7 × 0.7
~ 1.5μm 2) Cell polymorphism: None 3) Motility: Yes 4) Spores: None 5) Gram stainability: Negative

(B) Culture characteristics 1) Meat broth agar plate culture: white, good growth 2) Meat broth agar slope culture: white, good growth 3) Meat broth liquid culture: cream color, good growth

(C) Physiological properties 1) Reduction of nitrate: Negative 2) Denitrification reaction: Negative 3) Formation of indole: Negative 4) Utilization of citric acid: Positive 5) Utilization of inorganic nitrogen source: Positive 6) Dye Generation: Negative 7) Urease: Positive 8) Oxidase: Positive 9) Catalase: Positive 10) Growth temperature and pH: 20-37 ° C, pH 6-8 11) Attitude toward oxygen: Aerobic 12) OF test: Negative 13) Acid production from glucose: negative 14) Gelatin decomposition: negative 15) Sugar assimilation: D-glucose, L-arabinose,
All negative for D-mannose, D-mannitol and maltose

As mentioned above, the bacterium is an aerobic, gram-negative, non-fermenting, motile bacillus, oxidase-positive,
It was identified as belonging to the genus Alcaligenes in consideration of other mycological properties such as OF test negative. The examination of the mycological properties was conducted by Kazuo Komagata [Takeharu Hasegawa (ed.), Classification and identification of microorganisms (1990); Aerobic bacteria], The Procaryotes Volume 1 [The Procaryotes; A Handbook on Ha.
bitats, Isolation and Identification of Bacteria, (E
d.) MPStarr et al (1981)]. In addition, for identification of bacteria, the Berseys Manual [Ber
gey's ManualofSystematic Bacteriology, Vol.1 (198
4)].

Comparison of the mycological properties and assimilation properties of this bacterium with those of existing strains of the genus Alcaligenes is shown in Tables 1 to 1.
Shown in 3.

[0012]

[Table 1]

[0013]

[Table 2]

[0014]

[Table 3]

As a result of the above-mentioned collation, this bacterium was determined to be a new strain belonging to the genus Alcaligenes. In addition, it is characterized by well assimilating fats and oils and long-chain fatty acids as carbon sources for growth and polyester synthesis.
I named it ca.

The long-chain fatty acid used in the present invention has 1 carbon atom.
1 to 22 straight chain fatty acids are meant. Preferably, it has 11 to 18 carbon atoms. Derivatives of long chain fatty acids include metal salts and ammonium salts of Na ⁺ , K ⁺ , Ca ²⁺ , Mg ^2+, etc., alkyl alcohols having 1 to 3 carbon atoms, alkylene glycols, mono- or polyesters of glycerol, and it includes amides (-CONH ₂₎ is. It goes without saying that fats and oils, which are triglycerides of long-chain fatty acids or mixtures thereof, are included in the derivatives.

The "essential carbon source" described in the present invention is a carbon source that induces not only the accumulation of 3HB-containing polyester, or the accumulation of the polyester but also the growth of bacterial cells itself. Means that are necessarily used in the production of the polyester of the invention. Therefore, it is not excluded that only other carbon sources effective for growth are used solely for growing the bacterial cells.

The 3HB-containing polyester produced by the present invention is a 3HB homopolymer (PHB) or 3HB and 3HB.
It is a copolymer with HV (P (HB / HV)). It has been found that which polyester is produced depends on the molecular structure of the carbon source used. For example, when the strain is cultured only with a compound in a group consisting of a long-chain fatty acid having an even number of carbon atoms and its derivative as a carbon source to induce the growth of bacterial cells and the accumulation of polyester, PHB or P (HB / H
V) is generated. On the other hand, P (HB / HV) is produced when the bacterial cells are similarly cultured with only one group of compounds consisting of a long-chain fatty acid having an odd number of carbon atoms and its derivative as a carbon source. In addition, P (HB / HV) is also produced when the compounds in each of the above groups having an even carbon number and an odd carbon number are used in combination. The type of polyester produced depends not only on the essential carbon source used, as described above, but also on the additional carbon source utilized for polyester production. That is, when the compound in the above-mentioned group 1 having an even carbon number is used as an essential carbon source and at least one kind of the compound group represented by the above chemical formula 1 is used in combination as an additional carbon source, P (HB / HV) To generate.

Cultivation of the bacterium for producing polyester is carried out in a liquid medium. The culture temperature is 20-40 ° C, preferably 24-33 ° C. The initial pH of the culture is 6.0-8.
It is 0, preferably 6.5 to 7.5. The liquid medium contains
Various materials can be used, including synthetic, semi-synthetic and natural media. When an inorganic medium is used, one or more carbon sources are added thereto and the culture is performed.

[0020] Operationally, there are roughly two types of culture methods depending on the context of the respective growth stages of bacterial cell growth and polyester accumulation. In the present invention, both culture methods should be used according to the purpose. You can Among them, in the one-stage culture method, at least one selected from the group of compounds consisting of long-chain fatty acids and their derivatives is used as a carbon source for culturing to cause microbial cell growth and polyester accumulation. At this time, an additional carbon source may be used to promote the growth of bacterial cells and / or control the properties of the produced polyester. In the other two-stage culturing method, the first stage (previous stage) causes the cells to grow exclusively or mainly, and the recovered bacterial cells are added to the second stage (second stage) medium and cultivated again. Causes the accumulation of polyester exclusively or predominantly. Therefore, the carbon source used in the two-stage culture is a carbon source advantageous for bacterial growth in the former stage, and in the latter stage, at least one selected from the group consisting of long-chain fatty acids and their derivatives alone or added. Used with a typical carbon source. Two
In the latter stage of the step culture, it is advantageous to culture at least one of the essential nutrient sources, for example, nitrogen source and phosphorus source, in a reduced amount or completely removed in order to suppress bacterial growth and promote polyester production.

Various compounds and those derived from natural products can be used as additional carbon sources effective for the growth of bacterial cells and the accumulation of polyester. As such an example, carbon number 2-10
Straight chain fatty acids up to; citric acid, gluconic acid, succinic acid,
Organic acids such as glutaric acid, adipic acid, suberic acid, azelaic acid and lactic acid; alcohols such as methanol, ethanol and glycerol; sugars such as glucose, fructose and sucrose; straight-chain aliphatic carbonization such as n-octane and n-nonane Examples thereof include hydrogen. It goes without saying that the compound of the above formula 1 is also included in the additional carbon source. Also,
Nitrogen-containing organic nutrient sources such as various amino acids, polypeptone, meat extract, casamino acid, yeast extract and molasses are suitable as carbon sources in the pre-stage of the two-stage culture.

As the nitrogen source in the medium, inorganic ammonium salts such as ammonium phosphate, ammonium sulfate,
Ammonium chloride and the like, the above-mentioned amino acids, nitrogen-containing organic nutrient sources, and organic acid ammonium and organic acid amide are used. As the inorganic ions, in addition to sodium, potassium, magnesium, calcium, chlorine, sulfuric acid, phosphoric acid, trace amounts of iron, manganese, zinc, copper, cobalt, etc. are used in whole or in part.

After completion of the culture, the cells are collected by centrifugation, filtration or the like. In the one-stage culture, after this, the bacterial cell treatment for polyester separation is performed as it is. For example, the collected bacterial cells are washed with distilled water and then freeze-dried to obtain dried bacterial cells. Next, the polyester is extracted from the bacterial 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 the polyester. In the case of two-stage culture, the collected bacterial cells are put into the latter-stage medium and the culture is performed again. After the completion of the second-stage culture, the bacteria collection, bacterial cell treatment, and polyester separation are performed according to the method described above.

[0024]

[Examples] The following are Alcaligenes repolitica A
The production of 3HB-containing polyester using K201 will be described with examples.

Examples 1 to 4 Alcaligenes lipolytica AK 201 was added to 100 ml of an inorganic medium having the composition shown in Table 4 as a carbon source, undecanoic acid (Example 1), pentadecanoic acid (Example 2), and sodium stearate (Examples). 3), 500 ml containing a liquid medium to which rapeseed oil (Example 4) was added at a rate of 3 g / l
After aseptically inoculating into a Sakaguchi flask having a volume, the culture was carried out at 30 ° C. for 48 hours with shaking at 130 strokes per minute.

[0026]

[Table 4] However, the trace element solution is obtained by dissolving the following inorganic salts in 1 liter of 1M hydrochloric acid: FeSO ₄ .7H ₂ O (2.78 g), MnCl ₂ .4
H ₂ O (1.98 g), CoSO ₄ · 7H ₂ O (2.8
1g), CaCl ₂ · 2H ₂ O (1.67g), CuC
_{l 2 · 2H 2 O (0.17g} ), ZnSO 4 · 7H 2 O
(0.29 g).

After the culture was completed, 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 hot chloroform, the solution was concentrated, and hexane was added to cause 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 the gas chromatography, dried cells were subjected to methanolysis and intracellular polyester was used as a monomer methyl ester [H. Brandl et al, Int. J. Biol. Macrom].
ol., 11, 49-55 (1989)]. In addition, the analysis of polyester is
The same was done in the following examples.

The results of the polyester biosynthesis of Examples 1 to 4 above are shown in Table 5.

[Table 5]

Examples 5 to 7 Lard (pig oil) (Example 5), olive oil (Example 6), and ethyl laurate (Example 7) were used as the sole carbon sources and cultured in the same manner as in Examples 1 to 4. After carrying out, the resulting polyester was analyzed.

The results of the polyester biosynthesis of Examples 5-7 are shown in Table 6.

[Table 6]

Example 8 A polyester produced after culturing for 72 hours under the same conditions as in Examples 1 to 4 except that 2 g / l of rapeseed oil was used as an essential carbon source and 1 g / l of sodium propionate was used as an additional carbon source. Was analyzed. As a result, the dry cell weight was 1.
The composition of the polyester was 3HB94% and 3HV6% with 9 g / l and a polyester content of 22%. Also,
The melting point was 153 ° C.

Example 9 Polypeptone 10 g, meat extract 5 g, yeast extract 10
g, and 5 g of (NH ₄ ) ₂ SO ₄ dissolved in 1 liter of water, the strain was aseptically inoculated into a Sakaguchi flask containing 100 ml of a medium, and then shake-cultured at 30 ° C. for 24 hours. After completion of the culture, the culture solution was centrifuged (8000 rpm, 10 minutes) to collect the bacterial cells, which were then collected from the latter medium (from the inorganic medium of Table 4 (NH ₄ )).
₂ Except for HPO ₄ , use a carbon source added) 100
The mixture was added to the cells and cultured with shaking at 30 ° C. for 48 hours. As a carbon source, as an essential carbon source, sodium palmitate 3 g /
1, n-nonyl acetic acid 2 g / l was used as an additional carbon source. After culturing, the produced polyester was analyzed. as a result,
Dry cell weight is 7.4 g / l, polyester content is 46
%, The composition of the polyester is 3HB 87%, 3HV1
It was 3%. The melting point was 134 ° C.

[0034]

According to the present invention, naturally rich oils and fats,
The polyester having biodegradability and thermoplasticity can be efficiently produced from long chain fatty acids.

Claims (5)

[Claims] 1. A 3-hydroxybutyrate-containing polyester-producing bacterium belonging to the genus Alcaligenes is cultivated in a liquid medium containing at least one compound of a long-chain fatty acid and its derivative as an essential carbon source, and the inside of the bacterium is cultivated. A method for producing a biopolyester, comprising collecting 3-hydroxybutyrate-containing polyester in and collecting the polyester. 2. The essential carbon source is at least one of a compound subgroup consisting of long-chain fatty acids having odd carbon numbers and derivatives thereof, and the polyester is a copolymer of 3-hydroxybutyrate and 3-hydroxyvaleric acid. The manufacturing method according to claim 1. 3. The essential carbon source is at least one of a compound subgroup consisting of long-chain fatty acids having an even number of carbon atoms and derivatives thereof, and the polyester is poly (3-hydroxybutyrate).
The method according to claim 1, which is a copolymer of 3-hydroxybutyrate and 3-hydroxyvaleric acid. 4. An essential carbon source comprising at least one compound selected from the group consisting of even-numbered long-chain fatty acids and their derivatives.
CH ₃ (CH ₂ ) _2n-1 X (wherein X represents COR or CH ₂ OR ', R is hydrogen, methoxy or ethoxy, R'is hydrogen, acetyl or propionyl, n Represents an integer of 1 to 4), a 3-hydroxybutyrate-containing polyester-producing bacterium belonging to the genus Alcaligenes is cultivated using a liquid medium containing at least one kind of compound group represented by
A method for producing a biopolyester, which comprises accumulating a copolymer of 3-hydroxybutyrate and 3-hydroxyvaleric acid in cells and collecting the copolymer. 5. The method according to claim 1, wherein the essential carbon source is triglyceride of long chain fatty acid or a mixture thereof.