JP2001046094A - Separation and purification of poly-3-hydroxyalkanoic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To separate and purify the subject compound which is a biodegradable plastic by adding a surfactant to a suspension of a microbial cell of a microorganism containing a poly-3-hydroxyalkanoic acid and carrying out a physical crushing treatment of the resultant mixture liquid. SOLUTION: A surfactant is added to a suspension of a microbial cell of a microorganism (e.g. Aeromonas caviae) containing a poly-3-hydroxyalkanoic acid comprising a bipolymer of D-3-hydroxybutyrate and D-3-hydroxyhexanoate, a terpolymer, etc., of the D-3-hydroxybutyrate, D-3-hydroxyvalerate and D-3- hydroxyhexanoate and the resultant mixture liquid is then subjected to a physical crushing treatment to thereby separate and purify the objective poly-3- hydroxyalkanoic acid suitable as a raw material, etc. for a plastic product, an implant material without requiring recovery, a drug carrier, a fertilizer carrier, an agricultural mulching film, a fishing gear such as a fishing line, a bag, etc. such as a refuse bag, etc. for composts in high purity and high yield.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for separating and purifying 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 in fact, many plastic waste treatment methods cannot be dealt with simply by incineration, landfill, and recycling, and are often left in the natural world. . Therefore, biodegradable plastics that are taken into the natural material cycle after disposal and whose decomposition products are not harmful are attracting attention.
The practical application is eagerly desired.

[0003] Among these biodegradable plastics,
Poly-3-hydroxyalkanoic acid (hereinafter referred to as PHA)
Is a thermoplastic polyester that is produced and stored as an energy storage substance in the cells of many microbial species, and is expected to have little adverse effect on ecosystems because it is taken into the natural carbon cycle process. Particular attention has been paid. In the medical field, it can be used as an implant material or a drug carrier that does not require collection.

[0004] The PHA forms granules and accumulates in the microbial cells. In order to use these as plastic, it is necessary to separate and purify the PHA from the microbial cells. As a known method for separating and purifying PHA from microbial cells, broadly speaking, PH is dissolved in an organic solvent in which PHA is soluble.
There is a method of dissolving A and extracting PHA, and a method of obtaining PHA by solubilizing and removing bacterial cell components other than PHA. Among these, the latter method is preferable in that the separation of PHA is easy and the processing step is simpler.

[0005] As a method for obtaining PHA by solubilizing and removing bacterial cell components other than the PHA, for example,
J. Gen. Microbiology, 19, pp. 198-209 (1958) states:
A method is described in which a cell suspension is treated with sodium hypochlorite to solubilize cell constituents other than PHA to obtain PHA. This method is simple as a process, but is expensive due to the need to use large amounts of sodium hypochlorite. Further, it is considered that PHA is not suitable for practical use because remarkable lowering of the molecular weight of PHA is caused and chlorine which cannot be ignored in the obtained PHA is left. Japanese Patent Publication No. 4-61638 discloses that
The microbial cell suspension containing PHA is heat-treated at 100 ° C. or higher to destroy the cell structure, and then combined with proteolytic enzyme treatment and phospholipid-degrading enzyme treatment or hydrogen peroxide treatment to obtain a product other than PHA. A method for solubilizing the bacterial cell components to obtain PHA is described. This method has drawbacks such that the protein is denatured and insolubilized by the heat treatment, so that the load in the next proteolytic enzyme treatment step increases, and further, the treatment steps are many and complicated.

As another method having a step of crushing microbial cells, the nucleic acid released from the cells is treated with hydrogen peroxide, decomposed by treating with a surfactant,
A method for separating A has been proposed.
No. 415) is difficult to implement on an industrial level because it utilizes highly toxic hydrogen peroxide. Further, a method has been proposed in which PHA-containing microorganism cells are crushed with a high-pressure homogenizer to separate PHA (Japanese Patent Application Laid-Open No. 7-17789).
No. 4). However, in this method, high-purity PHA cannot be obtained unless the microbial cell suspension is repeatedly subjected to high-pressure treatment at least about three times, and the purity of the obtained PHA is as low as about 70 to 89% at the maximum. There is a disadvantage that.

[0007]

SUMMARY OF THE INVENTION The object of the present invention is to
It is an object of the present invention to provide a method for separating and purifying PHA that can obtain high-purity PHA in a small number of steps and in high yield from microbial cells containing A.

[0008]

That is, the present invention relates to a method of adding a surfactant to a suspension of microbial cells containing poly-3-hydroxyalkanoic acid (PHA) and subjecting the resulting mixture to physical suspension. The present invention relates to a method for separating and purifying PHA, which comprises crushing.

[0009]

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 (Alc
alligenes lipolytica), Alcaligenes eutrophus,
Alcaligenes such as A. latas, Pseudomonas, Bacillus (Bac)
illus), Azotobacter
er), Nocardia, and Aeromonas, and among them, Aeromonas cavier (Aeromonas c.)
aviae) or a strain into which a gene of the PHA synthase group derived from Aeromonas caviae has been introduced, for example, Alcaligenes eutrophus AC32 (Accession No. FERM P-15786) (J. Bacteriol., 17).
9, 4821-4830 (1997)).

[0010] 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 aforementioned Alcaligenes eutrophus AC32 (FERM P-15786) is used, The method described in J. Bacteriol., 179, pp. 4821-4830 (1997) is preferred.

The poly-3-hydroxyalkanoic acid (PHA) in the present invention refers to a homopolymer of D-3-hydroxybutyrate (3HB) or a copolymer of 3HB with another 3-hydroxyalkanoic acid. But especially 3HB
And D-3-hydroxyhexanoate (3HH)
Component copolymers (Macromolecules, 28, 4822-4828 (199
5)) or 3HB and D-3-hydroxyvalerate (3
HV) and a 3 component copolymer of 3HH (JP-A-8-289)
797) 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 the content of 3HB is 1 to 99 mol% and the content of 3HH unit is 1 ~ 99 mol% are preferred. 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% by mole, and the 3HV unit is Content of 1 to 96
Those having a content of 3% by mole and 3HH units of 1 to 30% by mole are preferred. The molecular weight of these PHA is 1
It is preferably at least 100,000, more preferably at least 500,000.

It is natural that the content of PHA in the microbial cells is preferably higher. For application on an industrial level, the content of PHA is preferably 20% by weight or more in the dry cells. Considering the crushing treatment, the separation operation, the purity of the separated PHA, etc., it is particularly preferably 50% by weight or more.

In the present invention, a surfactant is added to the microbial cell suspension obtained by culturing as described above.
In the present invention, the term "suspension of microbial cells" refers to a culture suspension after completion of culture or an aqueous suspension obtained by suspending cells isolated from a culture solution by centrifugation or the like in water. means.
The cell concentration in the suspension was 500 g in terms of wet cells.
/ L or less, more preferably 300 g / l or less.

The surfactant used in the present invention may be anionic, cationic, amphoteric or non-ionic. Specifically, sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium cholate, deoxychol Sodium, sodium oleate, cetyltrimethylammonium bromide, dodecylpyridinium chloride, 3-((3-cholamidopropyl) dimethylammonio) -1-propanesulfonic acid, 3-((cholamidopropyl) dimethylammonio)- 2-hydroxy-1
-Propanesulfonic acid, dodecyl-N-betaine, octylglucoside, heptylthioglucoside, polyethylethylene dodecyl ether, polyoxyethylene isooctylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene sorbitol ester, and the like. It is not limited to these. In the present invention, in particular, sodium dodecyl sulfate,
Sodium dodecyl sulfonate, sodium cholate,
Sodium deoxycholate, sodium oleate, and the like are preferred in terms of price, amount used, and effect of addition.

The amount of the surfactant added is not particularly limited, but is preferably 0.001 to 50 parts by weight, more preferably 1 to 20 parts by weight, based on 100 parts by weight of microbial cells (wet cells). preferable. The addition amount is preferably 0.001 part by weight or more from the viewpoint of a good effect of adding a surfactant,
From the viewpoint of low cost, 50 parts by weight or less is preferable.
Moreover, it is preferable to stir the obtained liquid mixture at room temperature for about 1 minute to 2 hours from the viewpoint of promoting the solubilization of bacterial cell components other than PHA.

Next, the mixture is physically crushed. In the present invention, by performing such a physical crushing treatment, there is an effect that the microbial cells are crushed and PHA is leaked out of the cells.

The physical crushing treatment in the present invention includes crushing by ultrasonic waves, crushing by a high-pressure homogenizer, a mill or the like. Examples of the high-pressure homogenizer include "Menton Gorin (trade name)" manufactured by APV Gorin of Germany, "Minilab (trade name)" of APV Runny of Denmark, and Microfluidics (U.S.A.).
dics) "Microfluidizer (trade name)" and the like. Examples of the mill include "Dino Mill (trade name)" and the like manufactured by Willy A. Bachofen of Switzerland. However, the present invention is not limited to these as long as the same crushing effect can be obtained.

The conditions of the physical crushing treatment cannot be unconditionally limited depending on the means used. For example, in the case of crushing by ultrasonic waves, the output is 5 and the output is simplified by using a sonifier manufactured by Branson of the United States. 3 with 50% missing cycles
Preferably, the sonication is performed for 0 minutes. In the case of crushing using a high-pressure homogenizer, high-pressure crushing is preferably performed at 500 kgf / cm ² for 1 hour using a mini lab manufactured by APV Lanie, Denmark. In the case of crushing by a mill or the like, crushing treatment is preferably performed for 1 hour at a flow rate of 1 l / h using a Dyno mill manufactured by Filey A. Bachoffen of Switzerland.

The physical crushing treatment is completed by placing a small amount of the crushed liquid in a centrifuge tube, centrifuging at, for example, 3000 rpm for 10 minutes, and then confirming whether a precipitate is obtained.

Next, the treatment liquid obtained by the physical crushing treatment is centrifuged to precipitate PHA. In the present invention, by performing such a centrifugation, PHA can be easily separated from bacterial cell components other than PHA. The conditions for the centrifugation are not particularly limited, and at room temperature,
It may be performed at 1000 to 15000 rpm for about 5 to 60 minutes. Centrifugation may be performed multiple times, if necessary. Further, the obtained PHA precipitate is preferably dried by a conventional method.

The purity of the obtained PHA is preferably 90% or more from the viewpoint of practical application, processability and physical properties. In addition, as a method for measuring the purity, for example, a method described in Examples described later can be mentioned.

The obtained PHA is highly pure as it is, but depending on the purpose, a known purification method, for example, a lytic enzyme such as lysozyme (Japanese Patent Publication No. 4-61638), trypsin, pronase, etc. (See JP-A-5-336982) and peroxides such as hydrogen peroxide (JP-A-8-502415) to further improve the purity.

The PHA of the present invention having the above configuration
The method for purification and separation of PHA requires less steps than conventional methods and can obtain PHA efficiently.

The PHA obtained according to the present invention has a sufficiently high purity as a practical product. Examples of the PHA include plastic products, implant materials that do not need to be collected, drug carriers, fertilizer carriers, agricultural multi-films, fishing lines and the like. Fishing gear,
It is suitably used as a raw material for compost garbage bags.

[0025]

EXAMPLES The microorganisms used in this example were Aeromonas
Alcaligenes eutrophus AC32 (Deposit No. FE) into which a PHA synthase gene derived from caviae was introduced.
RM P-15786). This is J. Bacterio
. l, 179, was cultured by the method described on pages 4821-4830 (1997) [the _{medium: Na 2 HPO 4 · 12H 2} O 11.3g, KH 2 PO 4 1.9g, (N
H ₄ ) ₂ SO ₄ 6 g, professional extract (Banshu Seasoning Co., Ltd.) 10 g,
MgSO ₄ · 7H ₂ O 1g, coconut oil 50 g, trace metal elements solution _{(composition: FeCl 3 · 6H 2 O 16.2g} , CaCl 2 · 2H 2 O 10.3g, CoCl 2
_{· 6H 2 O 0.2g, NiCl 2} · 6H 2 O 0.1g, CrCl 3 · 6H 2 O 0.1
_{g, CuSO 4 · 5H 2 O0.2g} / 1l 0.1N HCl) 5ml / 1l, pH
6.7, culture temperature 30 ° C., culture time 72 hours], poly (D-3-hydroxybutyrate-co-D-3-hydroxyhexanoate) (hereinafter referred to as poly (3HB-co-
3HH), 3HB unit: 3HH unit = 90: 1
0 (molar ratio), a molecular weight of about 1,000,000) was obtained in about 50% by weight (dry weight). Next, this was separated from the culture solution by centrifugation (5000 rpm, 10 min), and water was added to the obtained paste-form cells to obtain 100 g /
1 (wet cell equivalent) aqueous suspension. The following examples were carried out using this aqueous suspension, but the present invention is not limited to these examples.

The poly (3) obtained by separating the cells from the cells
The purity of HB-co-3HH) was determined as follows. That is, after dissolving 10 mg of the dried product of the precipitate obtained by separating the cells from the cells, 1 ml of chloroform was dissolved.
0.85 ml of methanol and 0.25 ml of concentrated sulfuric acid were added, and the mixture was treated at 100 ° C. for 140 minutes. After cooling, 0.5 ml of a saturated aqueous solution of ammonium sulfate was added, and the mixture was vigorously stirred and allowed to stand. The lower layer was analyzed by capillary gas chromatography, and the poly (3HB-co-3H) in the separated product was analyzed.
H) purity was determined.

Example 1 Sodium dodecyl sulfate was added at a concentration of 10 g / l to 1,000 ml of the above-mentioned suspension of poly (3HB-co-3HH) -containing microbial cells, and the mixture was stirred at room temperature for 1 hour. This is then called "Dynomill" (Willie A., Switzerland)
The rupture treatment was carried out at a flow rate of 1 l / h for 1 hour using a backoffen. The obtained treatment liquid is centrifuged (800
(0 rpm, 10 min) and the precipitate was collected. After drying the precipitate, the purity of poly (3HB-co-3HH) was determined to be 94%.

Example 2 Sodium dodecyl sulfate was added to a suspension of the above poly (3HB-co-3HH) -containing microbial cells at a concentration of 10 g / l and stirred at room temperature for 1 hour. Next, this was subjected to a breaking treatment at 500 kgf / cm ² for 1 hour using “Minilab” (manufactured by APV Lanie, Denmark). The processing solution obtained is centrifuged (8000 rpm, 1
0 min) and the precipitate was collected. After drying the precipitate, the purity of poly (3HB-co-3HH) was determined.
5%.

Example 3 The same operation was performed as in Example 1, except that sodium dodecyl sulfate was changed to sodium dodecyl sulfonate. After drying the precipitate, poly (3HB-co-3H)
The purity of H) was determined to be 94%.

Comparative Example 1 The same operation was performed as in Example 1 except that the breaking operation was not performed using a “Dynomill”. as a result,
No precipitate could be obtained by centrifugation, and no polymer could be separated.

Comparative Example 2 The same operation was performed as in Example 1, except that the treatment with sodium dodecyl sulfate was not performed. As a result, the purity of the precipitate obtained by centrifugation was as high as that of the poly (3H
50% same as the purity of B-co-3HH) -containing microbial cells
Met.

COMPARATIVE EXAMPLE 3 100 ml of a suspension of poly (3HB-co-3HH) -containing microbial cells was subjected to a breaking treatment at a flow rate of 1 l / h for 1 hour by using a "Dynomill" so that the suspension became 10 g / l. Sodium dodecyl sulfate was added and stirred at room temperature for 1 hour. The obtained cell suspension was very viscous, and poly (3HB-co-3HH) could not be obtained by centrifugation.

From the above results, the poly (3HB-co-3HH) obtained in Examples 1 to 3 was higher in purity than that obtained in Comparative Example 2 in which no surfactant was used. It turns out that it is.

From the results of Examples 1 to 3 and Comparative Examples 1 to 3, the method for separating and purifying poly (3HB-co-3HH) requires both physical crushing treatment and addition of a surfactant. However, a remarkable effect can be obtained by physically crushing the treatment solution containing the surfactant.

[0035]

According to the present invention, high-purity poly-3-hydroxyalkanoic acid (PHA) can be obtained efficiently and extremely easily. Therefore, the present invention improves the efficiency of industrial production of PHA and reduces cost. This greatly contributes to reduction.
The PHA obtained by the present invention has a sufficiently high purity as a practical product, for example, a plastic product, an implant material that does not need to be collected, a drug carrier, a fertilizer carrier, an agricultural multi-film, fishing gear such as a fishing line, It is suitably used as a raw material for compost garbage bags.

 ──────────────────────────────────────────────────続 き Continued on front page (72) Inventor Keiji Matsumoto 11-33 Omoricho, Nishinomiya-shi, Hyogo F-term (reference) 4B064 AD83 BA18 CA02 CA19 CC24 CE02 CE03 DA01 DA16

Claims (3)

[Claims] 1. A method for preparing a poly (3-hydroxyalkanoic acid) comprising adding a surfactant to a suspension of microbial cells containing poly-3-hydroxyalkanoic acid and subjecting the resulting mixture to physical disruption.
A method for separating and purifying 3-hydroxyalkanoic acid. 2. The method according to 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-3
The separation and purification method according to claim 1, which is a three-component copolymer of -hydroxyvalerate (3HV) and D-3-hydroxyhexanoate (3HH). 3. The method according to claim 1, wherein the microorganism containing poly-3-hydroxyalkanoic acid is poly-3 derived from Aeromonas caviae.
The method for separating and purifying according to claim 1 or 2, wherein the strain is a strain into which a gene for a hydroxyalkanoate synthase group has been introduced.