JP2001057895A - Extraction of 3-hydroxyalkanoic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently extract and separate the subject compound by adding a divalent or polyvalent metal salt and a surfactant to a suspension of a microbial cell of a poly-3-hydroxyalkanoic acid-containing microorganism in an extraction solvent and flocculating and removing an undissolved cell residue. SOLUTION: A divalent or polyvalent metal salt (e.g. calcium chloride, etc.), and/or a surfactant (e.g. benzyltrimethlammonium chloride, etc.), is added to a suspension of a microbial cell of poly-3-hydroxyalkanoic acid (PHA)-containing microorganism [e.g. Aicaligenes eutrophus A32C(FERM P-15786) strain into which a PHA synthase gene derived from Aeromonas caviae is transferred, etc.], and an extraction solvent (e.g. chloroform, etc.), and undissolved cell residue is flocculated and removed from the PHA-containing solution to readily obtain a high-purity poly-3-hydroxyalkanoic acid useful as a biodegradable plastic, etc., in an improved efficiency of industrial production at a low cost.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for extracting and separating poly-3-hydroxyalkanoic acid from microbial cells.

[0002]

2. Description of the Related Art At present, plastic waste is treated by incineration, landfill, and the like. However, these treatment methods have problems such as global warming and ground loosening of a landfill. For this reason, with the increasing public awareness of plastic recycling, recycling systems are being promoted.
However, recyclable applications are limited, and as a practical matter, many plastic waste treatment methods cannot be dealt with only by incineration, landfill, and recycling, and are often left in the natural world. Therefore, biodegradable plastics which are taken into the material cycle in the natural world after disposal and whose decomposition products do not become harmful have been attracting attention, and their practical use has been eagerly sought.

[0003] Among these biodegradable plastics, poly-3-hydroxyalkanoic acid (hereinafter referred to as PHA) is a biodegradable thermoplastic polyester that is produced and accumulated as an energy storage substance in the cells of many microbial species. It is of particular interest because it is expected to have little adverse effect on ecosystems due to its inclusion in the natural carbon cycle process. Also, in the medical field, it is considered that it can be used as an implant material or a drug carrier that does not require collection.

[0004] PHA produced by microorganisms forms granules and accumulates in the cells, and in order to utilize them as plastic, it is necessary to separate them from the cells of the microorganisms. Known methods for separating and purifying PHA from microbial cells are roughly classified into a method in which PHA is dissolved and extracted in an organic solvent in which PHA is soluble, and a method in which cell components other than PHA are solubilized and removed. To obtain a PHA.

As a method of extracting and separating PHA using an organic solvent, a method using a hydrophobic halogen-containing hydrocarbon such as 1,2-dichloroethane or chloroform as an extraction solvent (JP-A-55-118394, JP-A-55-118394, 5
7-65193) and dioxane (Japanese Unexamined Patent Publication No.
198991) or propanediol (Japanese Unexamined Patent Publication No.
-69187) or tetrahydrofuran (Japanese Unexamined Patent Publication No.
No. 7,797,788), a method using a hydrophilic extraction solvent has been proposed. However, in these methods, when attempting to dissolve PHA to a practically usable concentration, the extract becomes extremely viscous, and it is very difficult to separate the bacterial residue remaining in the extraction solvent from the solvent layer containing PHA. It is disadvantageous in that it is difficult.

On the other hand, some methods have been proposed to obtain PHA by solubilizing and removing bacterial components other than PHA (J. Gen. Microbiolo).
gy, 19, pp. 198-209 (1958), Tokuho 0
4-61638, JP-T-08-502415, JP-A-07-177894), there are problems such as the remarkable reduction of the molecular weight of PHA, and the purity of the obtained PHA is low. At present, any of these methods are not suitable for practical use, for example, they are complicated, require highly toxic chemicals, or are extremely expensive.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for efficiently separating a PHA-containing solvent layer from a bacterial residue that has not been dissolved in an extraction solvent in the extraction of PHA from microbial cells. To provide.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies on a method capable of industrially producing PHA, and as a result, have found that a suspension of microbial cells containing PHA and an extraction solvent has a bivalent or higher valence. It has been found that by adding a metal salt or a surfactant, cell residues not dissolved in the extract can be aggregated and efficiently separated and removed, and the present invention has been achieved.

That is, the present invention relates to a method for preparing a suspension of a microbial cell containing PHA and an extraction solvent in a suspension of a divalent or higher valent metal salt.
Alternatively, a surfactant is added to aggregate and remove undissolved cell residues from the solution containing PHA.
The present invention relates to a method for extracting and separating HA.

[0010] In a preferred embodiment, the present invention relates to the above extraction separation method wherein the surfactant is cationic.

[0011] In another preferred embodiment, the microorganism containing PHA is a microorganism derived from Aeromonas caviae.
The present invention relates to the above-mentioned extraction / separation method, which is a strain into which the A synthase group gene has been introduced.

In still another preferred embodiment, PH
A is a two-component copolymer of 3HB and 3HH, or 3
The present invention relates to the above extraction and separation method, which is a ternary copolymer of HB, 3HV and 3HH.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism used in the present invention is not particularly limited as long as it is a microorganism accumulating PHA in cells. For example, Alcaligenes lipolytica (Aical
geneneslipolytica), Alcaligenes eutrophus (Aicaligenes eutr)
opus), Alcaligenes latus (Aicali)
genus latas, etc. (Al)
caligenes), Pseudomonas (Pseud)
omonas), the genus Bacillus, the genus Azotobacter, the genus Nocardia, the genus Aeromonas (Aer)
(Amonas caviae), or a strain into which a gene of the PHA synthase group derived from Aeromonas caviae has been introduced, such as Alcaligenes yutrophus A32C (Accession No. FERM).
P-15786) and the like are more preferred.

The method of culturing these microorganisms is not particularly limited as long as PHA can be efficiently accumulated in a large amount in the cells. For example, when the above-mentioned Alcaligenes eutrophus A32C (FERM P-15786) is used, J. Bacteriol. , 179, 4821-4
The method described on page 880 (1997) is preferred.

[0015] The poly-3-hydroxyalkanoic acid (PHA) in the present invention is not particularly limited.
Hydroxybutyrate (3HB) homopolymer and 3H
A copolymer of B and another 3-hydroxyalkanoic acid is preferable, and a two-component copolymer of 3HB and D-3-hydroxyhexanoate (3HH) (Macromol
ecules, 28, 4822-4828 (199
5)) or a ternary copolymer of 3HB, D-3-hydroxyvalerate (3HV) and 3HH (Japanese Unexamined Patent Publication No.
No. 289797) is more preferable from the viewpoint of physical properties.
Here, the composition ratio of each monomer unit constituting the two-component copolymer of 3HB and 3HH is not particularly limited, but a composition ratio in which the content of the 3HB unit is 1 to 99 mol% is preferable. It is. Further, the composition ratio of each monomer unit constituting the three-component copolymer of 3HB, 3HV and 3HH is not particularly limited. For example, the content of the 3HV unit is 1 to 95 mol%, and the content of the 3HV unit is 3%. Those having a composition ratio of 1 to 96 mol% and a 3HH unit content of 1 to 30 mol% are preferred. The molecular weight of these PHA is preferably 100,000 or more, more preferably 500,000 or more.

It is natural that the content of PHA in the microbial cells is preferably higher, and in the application on an industrial level, it is preferably 20% by weight or more in the dry cells. Considering the polymer purity etc., 50
% By weight or more is particularly preferred. In the present invention, the microbial cells obtained by culturing as described above may be used directly as wet cells separated from the culture solution, or the wet cells may be subjected to a drying treatment with a freeze dryer or the like. And may be used as dried cells. Furthermore, a part of the cells is destroyed by a physical crushing treatment such as a mill or a high-pressure homogenizer, or a chemical treatment such as a surfactant, sodium hypochlorite or an organic solvent, or a part of the cells is removed. A material having an increased PHA content may be used.

The extraction solvent of PHA used in the present invention is not particularly limited as long as PHA can be dissolved. For example, chloroform, methylene chloride, 1,2-dichloroethane, pyridine, and 1,2-propylene carbonate can be used. Such cyclic carbonates, tetrahydrofuran, ethyl lactate, acetonitrile and the like, and mixtures of these solvents, for example, a mixed solvent system such as a mixture of chloroform and methanol and a mixture of chloroform and tetrahydrofuran are exemplified.

The metal salt used in the present invention is not particularly limited as long as it is a metal salt composed of a divalent or higher valent metal ion and a general counter ion.
Calcium, magnesium, iron, zinc, aluminum,
Barium, manganese, copper, cobalt, and the like, and counter ions include chloride ion, sulfate ion, phosphate ion, nitrate ion, carbonate ion, and the like. Specific examples of the metal salt include calcium chloride. , Magnesium chloride, ferrous chloride, ferric chloride, zinc chloride, barium chloride, cobalt chloride, copper chloride, manganese chloride, aluminum chloride, magnesium sulfate, zinc sulfate, calcium carbonate, magnesium carbonate and the like. The surfactant used in the present invention may be anionic, cationic, amphoteric or nonionic, but is preferably a cationic surfactant, and specifically, cetyltrimethylammonium. Bromide, dodecylpyridinium chloride, tetradecylammonium bromide, cetylpyridinium chloride, triethylhexylammonium bromide, 4,4-trimethylenebis (1-methylpiperidin), trimethylphenylammonium bromide, benzyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, acetamine 86 (manufactured by Kao Corporation) Coatamine 24P (manufactured by Kao Corporation).

The amount of the metal salt or surfactant used in the present invention is not particularly limited, but it is preferable to add the metal salt or the surfactant in a concentration within the range of 0.001 to 10% by weight per 1 L of the microbial cell suspension. More preferably, a concentration in the range of 0.01 to 5% by weight is more preferable. When the concentration is less than 0.001% by weight, the effect is low, and when the concentration exceeds 10% by weight, it is not preferable in terms of cost.

In the present invention, the metal salt and the surfactant may be used alone or in combination. The metal salt or the surfactant may be charged into the cell suspension as a liquid or a solid and dissolved therein, or may be prepared as a solution and then charged into the cell suspension. When charging the metal salt or the surfactant, it is preferable to stir the cell suspension in order to promote the dispersion of the metal salt or the surfactant in the cell suspension. The stirring time and the stirring temperature for aggregating the undissolved cell residue of the microbial cells can be appropriately set.

In the present invention, a suspension of a microbial cell containing PHA and an extraction solvent is treated with a metal salt or a surfactant as described above, so that undissolved cells in the suspension are treated. Since the residue aggregates, the PHA solution can be easily separated. The separation operation that can be used here is not particularly limited. For example, generally known methods such as filtration, decantation, centrifugal separation, and membrane separation can be used. For the separation operation by filtration, generally used filter media, specifically, filter paper, filter cloth, mesh (mesh), porous ceramic, porous metal plate, porous film, and the like can be used. As a separation operation by decantation, for example, after stirring the microorganism suspension, the suspension is allowed to stand for 5 minutes to 2 hours, preferably 10 minutes to 1 hour, and the clear solution at the top of the suspension is removed by an appropriate method such as a suction device. You only have to collect it. For the separation operation using a centrifuge, generally known conditions can be used, and the centrifuge can be used in either a batch type or a continuous type.

The polymer purity of the PHA solution obtained by separating it from the undissolved cell residue in this way is very high, and high-purity PHA can be obtained by removing the solvent by a known method. Of course, the purity can be further improved by crystallization or other purification methods according to the purpose.

[0023]

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

(Example 1) Alkagenes gene into which a PHA synthase group gene derived from Aeromonas caviae was introduced.
Eutrofus AC32 (Deposit No. FERM P-1
5786). Bacteriol. , 179,
Were cultured by the method described in Section 4821-4830 (1997) _{(Medium: Na 2 HPO 4 · 12H 2} O 11.3g, KH 2 PO 4 1.9g, (NH 4) 2
SO ₄ 6g, professional extract (Banshu Seasoning Co., Ltd. 10g, MgSO ₄・
7H ₂ O 1g, coconut oil 50g, trace metal element solution (composition: FeCl _{2
· 6H 2 O 16.2g, CaCl 2} · 2H 2 O 10.3g, CoCl 2 · 6H 2 O 0.2g, Ni
_{Cl 3 · 6H 2 O 0.1g,} CrCl 3 · 6H 2 O 16.2g, CuSO 4 · 5H 2 O 0.2g /
1L 0.1N-HCl) 5ml / 1L, pH 6.7, culture temperature 30 ℃,
A bacterium containing about 50% by weight of a two-component copolymer of 3HB and 3HH (3HB unit: 3HH unit = 90: 10 (molar ratio), molecular weight about 1,000,000) was obtained. This is centrifuged (5000 rpm, 10
min) and separated from the culture solution to obtain wet cells. The wet cells were freeze-dried to obtain dried cells, suspended in chloroform at 50 g / l with the dried cells, and stirred at room temperature for 5 hours to obtain a two-component copolymer of 3HB and 3HH. The union was extracted. To the extract containing the microbial cells, benzyltrimethylammonium chloride as a cationic surfactant was added at a concentration of 10 g / l, and the mixture was further stirred for 1 hour to coagulate the undissolved cell residue. Suction filtration with a Kiriyama funnel using No. 4)
Aggregated cell residue was separated and removed. At this time, filtration could be performed without clogging. Methanol was added to the obtained filtrate with stirring to precipitate crystals of a binary copolymer of 3HB and 3HH, and the crystals were collected by filtration and dried under reduced pressure. The calculated recovery of the two-component copolymer of 3HB and 3HH was 98%.

Example 2 The same operation was performed as in Example 1, except that benzyltrimethylammonium chloride, which was a cationic surfactant, was changed to hexadecyltrimethylammonium bromide. 3HB obtained
The recovery of the two-component copolymer of and 3HH was 96%.

Example 3 The same operation as in Example 1 was performed except that the extraction solvent was changed from chloroform to tetrahydrofuran. 2 of the obtained 3HB and 3HH
The recovery of the component copolymer was 87%.

Example 4 The wet cells obtained in Example 1 were suspended in tetrahydrofuran at 50 g / l without drying, and the suspension was stirred for 5 hours under reflux with heating to obtain 3HB and 3HH. Was extracted. 10 g of calcium chloride was added to the extract containing the microbial cells.
/ L and further stirred for 1 hour to aggregate the undissolved cell residue, which is then filtered with filter paper (Kiriyama Seisakusho, No. 4).
The mixture was subjected to suction filtration using a Kiriyama funnel to separate and remove aggregated cell residue. At this time, filtration could be performed without clogging. The obtained filtrate was cooled to room temperature with stirring to precipitate crystals of a binary copolymer of 3HB and 3HH. The crystals were collected by filtration and dried under reduced pressure. The recovery of the obtained two-component copolymer of 3HB and 3HH is 80.
%Met.

Example 5 The same operation as in Example 4 was performed except that calcium chloride was changed to benzyltrimethylammonium chloride. 3HB and 3 obtained
The recovery of the two-component copolymer with HH was 83%.

Example 6 Alcaligenes eutrophus (ATCC 17699) strain was cultured using glucose as a carbon source (medium: glucose 20 g, Na ₂ HPO ₄ .12H ₂ O).
_{9g, KH 2 PO 4 1.5g,} (NH 4) 2 SO 4 6g, MgSO 4 · 7H 2 O 0.2g,
Trace metal element solution (Composition: 16.2 g of FeCl ₂ .6H ₂ O, CaCl ₂ .2H
₂ O 10.3 g, CoCl ₂ .6H ₂ O 0.2 g, NiCl ₃ .6H ₂ O 0.1 g, CrCl _{3.
6H 2 O 16.2g, CuSO 4 ·} 5H 2 O 0.2g / 1L 0.1N-HCl) 5ml / 1
L, pH 6.8, culture temperature 30 ° C, culture time 48 hours),
Cells containing about 60% by weight of 3HB homopolymer (3HB unit 100%) in the cells were obtained. This was centrifuged (5000 rpm, 10 min) and separated from the culture solution to obtain wet cells. The wet cells were freeze-dried to obtain dried cells, suspended in chloroform at 50 g / l with the dried cells, and stirred at room temperature for 5 hours to give 3H.
Extraction of B homopolymer was performed. To the extract containing the microbial cells, benzyltrimethylammonium chloride, a cationic surfactant, was added to a concentration of 10 g / l, and the mixture was further stirred for 1 hour to aggregate cell residues that were not dissolved in chloroform. Filter paper (Kiriyama Seisakusho, No. 4)
The mixture was subjected to suction filtration using a Kiriyama funnel to separate and remove aggregated cell residue. At this time, filtration could be performed without clogging. Methanol was added to the obtained filtrate while stirring to precipitate 3HB homopolymer crystals, and the crystals were collected by filtration and dried under reduced pressure. 3HB obtained
The calculated recovery of the homopolymer was 95%.

Example 7 A similar operation was performed as in Example 6, except that benzyltrimethylammonium chloride, which was a cationic surfactant, was changed to hexadecyltrimethylammonium bromide. 3HB obtained
The recovery of the homopolymer was 94%.

Example 8 The same operation as in Example 6 was performed except that the extraction solvent was changed from chloroform to tetrahydrofuran. The recovery of the obtained 3HB homopolymer was 85%.

Example 9 The wet cells obtained in Example 6 were suspended in tetrahydrofuran at 50 g / l without drying and stirred for 5 hours under reflux with heating to obtain a 3HB homopolymer. An extraction was performed. Calcium chloride was added to the extract containing the microbial cells at a concentration of 10 g / l, and the mixture was further stirred for 1 hour to aggregate undissolved cell residues.
This was subjected to suction filtration using a filter paper (manufactured by Kiriyama Seisakusho, No. 4) with a Kiriyama funnel to separate and remove flocculent cell residue. At this time, filtration could be performed without clogging. The obtained filtrate was cooled to room temperature with stirring to precipitate crystals of 3HB homopolymer, and the crystals were collected by filtration and dried under reduced pressure. The recovery of the obtained 3HB homopolymer was 81%.

Example 10 The same operation as in Example 9 was carried out except that calcium chloride was changed to benzyltrimethylammonium chloride as a cationic surfactant. The recovery of the obtained 3HB homopolymer was 83
%Met.

(Example 11) In Example 1, Alcaligenes eutrophus AC32 (FERMP-1) was used.
5786) was changed to Aeromonas caviae FA440 (Accession No. FERMBP-3432), and cultured under the same conditions, and a two-component copolymer of 3HB and 3HH (3HB unit: 3HH unit = 10: 90 (molar ratio)) ) Was obtained at about 30% by weight. This was centrifuged (5000 rpm, 10 min) and separated from the culture solution to obtain wet cells. The wet cells were freeze-dried to obtain dried cells, suspended in chloroform at 50 g / l with the dried cells, and stirred at room temperature for 5 hours.
Extraction of a two-component copolymer of HB and 3HH was performed. 10 g / l of benzyltrimethylammonium chloride, a cationic surfactant, was added to the extract containing the microbial cells.
The mixture was further stirred for 1 hour to aggregate the undissolved cell residue, and this was filtered with a Kiriyama funnel using a filter paper (manufactured by Kiriyama Seisakusho, No. 4) to separate the aggregated cell residue. Removed. At this time, filtration could be performed without clogging. Methanol was added to the obtained filtrate with stirring to precipitate crystals of a binary copolymer of 3HB and 3HH, and the crystals were collected by filtration and dried under reduced pressure. The calculated recovery of the obtained two-component copolymer of 3HB and 3HH was 96%.

Comparative Example 1 The same operation as in Example 1 was performed except that benzyltrimethylammonium chloride was not added. At the stage of filtration, clogging was so severe that cell residue could not be separated.

Comparative Example 2 The same operation as in Example 6 was carried out except that benzyltrimethylammonium chloride was not added. As a result, clogging was so severe at the filtration stage that bacterial cell residues could not be separated.

[0037]

According to the present invention, a very simple operation of adding a divalent or higher-valent metal salt or a surfactant to a suspension of a microbial cell containing PHA and an extraction solvent is achieved.
The present invention makes it possible to aggregate and remove undissolved cell residues and easily obtain high-purity PHA.
It greatly contributes to improving the efficiency of industrial production of PHA by microorganisms and reducing costs.

Claims (4)

[Claims] 1. A poly-3-hydroxyalkane obtained by adding a divalent or higher valent metal salt and / or a surfactant to a suspension of microbial cells containing poly-3-hydroxyalkanoic acid and an extraction solvent. A method for extracting and separating poly-3-hydroxyalkanoic acid, comprising agglutinating and removing undissolved cell residues from a solution containing an acid. 2. The method according to claim 1, wherein the surfactant is cationic.
The method for extracting and separating poly-3-hydroxyalkanoic acid according to the above. 3. The method according to claim 1, wherein the microorganism containing poly-3-hydroxyalkanoic acid is poly-3 derived from Aeromonas caviae.
The method according to claim 1 or 2, wherein the strain is a strain into which a gene for a hydroxyalkanoate synthase group has been introduced. 4. The method of claim 1, wherein the poly-3-hydroxyalkanoic acid is D-
Binary copolymer of 3-hydroxybutyrate (3HB) and D-3-hydroxyhexanoate (3HH), or D-3-hydroxybutyrate (3HB) and D-
4. The extraction separation method according to claim 1, which is a three-component copolymer of 3-hydroxyvalerate (3HV) and D-3-hydroxyhexanoate (3HH).