JPH07303490A - Method of extracting polyhydroxyalkanoate - Google Patents

Abstract

PURPOSE: To efficiently extract a polyhydroxyalkanoate in a halophilic bacterium by carrying out the lysis, concentration and centrifugation of a halophilic cell growing in a high-concentration salt medium, then diluting and resuspending the resultant substance with a low-concentration salt medium and further centrifuging, sedimenting or filtering the resultant suspension.

CONSTITUTION: A halophilic cell growing in a high-concentration salt medium such as a halobacterium type (e.g. Haloferax Mediterranei strain ATCC33500) is cultured in a culture medium and the resultant culture solution is then centrifuged, concentrated, strongly stirred in a crusher, pulverized, lysed using 0-2% of sodium dodecylsulfonate and 0-6 mM of ethylenediaminetetraacetic acid, 0-6 mM of taurocholic acid or 0-6 mM of sodium deoxycholate in order to promote the lysis and centrifuged to afford a dense white sediment. The supernatant thereof is then decanted and resuspended in a low-concentration salt medium, e.g. soft water or distilled water and the produced suspension is centrifuged to carry out the sedimentation or filtration and thereby efficiently extract the objective polyhydroxyalkanoate.

COPYRIGHT: (C)1995,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to bacterias halof.
The present invention relates to a method for extracting polyhydroxyalkanoate (hereinafter referred to as PHA) produced from cells of ilas; halophile bacterium).

[0002]

PHA is accumulated intracellularly in the form of granules by many bacteria. In order to use these polymers as thermoplastics, the residual cellular material must be separated to a sufficient purity. Therefore, PHA from a complex mixture represented by cell biomass by lysis / precipitation method.
Numerous methods based on the use of selective solvents and precipitants to extract polymers have been described in the literature.

In US Pat. No. 3,107,172, it is proposed to dry the cells by spreading and use the resulting material directly for casting. In some other patented methods, extraction and purification of the polymer is performed by utilizing a solvent that dissolves the PHA and thus can leach the PHA from the residual cellular material.
For example, in British Patent No. 7,906,076, an aqueous suspension of bacteria is dried in a stream of hot gas,
It has been proposed to use a mixture of solvents (dichloroethane, chloroform, etc.) and non-solvents (acetone and methanol) for recovery of the purified polymer. Another method consists of centrifuging, concentrating the suspension, treating with acetone, drying and disrupting the cells. The polymer is then extracted with a suitable solvent (pyridine in U.S. Pat. No. 3,036,959, dichloromethane-ethane mixture in U.S. Pat. No. 3,044,942). It is then necessary to recover the polymer, and finally the solvent, usually by rectification (eg EP 84302508.1). US Pat. No. 3,27
No. 5,610 describes a method in which cells are suspended in water and subjected to the action of a vibration field to leach PHA. Once the cells are disrupted, the product is centrifuged and dried. Subsequently, PHA is purified by treatment with a solvent (chloroform). This operation usually consumes a lot of energy. U.S. Pat. No. 4,101,5
No. 33 proposes to avoid the solvent recovery step by using the carboxylic acid cyclic ester hot and allowing it to cool and then precipitate the polymer. Similarly, PHA can be extracted directly from aqueous cell suspensions using some solvents (chloroform, dichloromethane, dichloroethane) directly without the need to remove water by centrifugation or drying with gas. However, sometimes if the cell membrane is stiff, a prior milling step is required to break the cells. In this method, the solvent acts on the lipids and pigments to create a relatively stable emulsion, which complicates the purification of PHA and controls the extraction conditions to avoid leaving it impure. Need.

In either case, the process for producing PHA is expensive and involves the use of large amounts of organic solvent. These solvents must be recovered and reused to make PHA production economically feasible.

[0005]

The object of the present invention is to provide a method which does not have the drawbacks of the conventional manufacturing methods as described above.

[0006]

The method of the present invention can be applied to the extraction of PHA granules produced by halobacteria and other halophilic bacteria. The present invention discloses that when exposed to low concentrations of salts, such as soft water, halophilic cells lyse (destroy) cells under these conditions, releasing all of their cellular components into the medium. It is based on the weakness of the cell membrane. Since the PHA granules have a considerably large particle size and density, they can be recovered from a suspension of once damaged cells by low speed centrifugation, sedimentation, filtration or the like. The above properties of halophilic bacteria, such as Haloferax Mediterranei (Patent No. 890347), are used in both continuous and discontinuous processes in order to obtain polymers in high yield and purity, It makes it possible to carry out the method of the invention using a concentration of detergent.

It is thus possible to obtain a sediment of PHA granules with a minimum content of impurities consisting of lipids and proteins. In order to remove these residual impurities, the precipitate can be washed once or several times with a detergent that carries away proteins, for example, sodium dodecyl sulfate (SDS) (the detergent is anionic, cationic, It can be non-ionic or amphoteric). After various washings with detergent and finally with water, the material is recovered to give a fine powder of PHA of sufficient purity for direct use in polymer processing machines.

The use of the method of the present invention obviates the need for drying aqueous bacterial suspensions and the use of organic solvents,
The handling is considerably simplified, the use of solvents and the need for recycling are eliminated, thus making the extraction process of the product considerably cheaper and the possibility of industrial scale production being significantly advantageous and attractive.

The first step of the method of the invention is the elimination of the salt-containing medium in which the bacteria grow. For that purpose, it is necessary to maximize the concentration of the bacterial suspension. For halobacterium, for example, 20 to 25% by weight of NaCl and various magnesia salts are used. To cause cell lysis, the concentration of these salts in the cell-disrupting medium is less than 0.5% for NaCl and 0.1 for magnesium.
Must be reduced to less than%. Otherwise, the dissolution will not be effective. To do this, the medium must be removed and the cells resuspended in sufficient water to keep the salt concentration at these levels. In order to lyse all cells in the shortest possible time, a detergent that already facilitates cell membrane lysis at this stage, a bile acid such as taurocholate or deoxycholate, which is extremely effective in causing lysis, is already present. Salts and cation complexers may be added. On the other hand, using an excessive amount of water makes the extraction expensive by increasing the consumption of water and / or detergent and also increases the energy consumption and time in centrifugation and / or sedimentation, filtration. Let

The suspension can also be heated, for example at 50 to 60 ° C. for 20 to 30 minutes in order to activate the cell lysis. Similarly, cells can be subjected to other mechanical agitation systems for accelerated lysis, including vigorous agitation, sudden vacuum, freeze-thaw and the like.

Once a granule suspension of significant particle size is obtained in which complete cells and cell debris have been removed, sedimentation or low speed centrifugation is performed to collect highly pure PHA granules. At this stage, it is important that the medium to be separated is as clean as possible, free of specific substances that bind the granules and become impurities in the final product.

Impurities consisting of lipids and proteins are usually present in the sediment containing the granules. These impurities can be reduced by washing once or several times with water and a detergent such as SDS that dissolves proteins. The purity of the product is characteristic of the sediment in the early stages and can be followed visually by the disappearance of the pink color resulting from the presence of impurities in the microbial cell membrane and intact cells which have not yet been lysed.

As the detergent to be used, anionic detergents (sodium and potassium salts of linear fatty acid, linear alkylbenzene sulfonate (LAS), paraffin sulfonate, α-olefin sulfonate, dialkyl sulfosuccinate). , Alkylsulfates, alkylpolyestersulfates, alkylphosphates, long-chain alkylbenzenesulfonates such as SAS, LAS, sodium cholate, sodium laurylsulfate, etc .; cationic detergents (aliphatic amines and their salts, Quaternary ammonium salt,
Polyethoxylated aliphatic amine), nonionic detergent (polyethoxylated alkylphenol, polyethoxylated fatty alcohol, polyethoxylated fatty acid, alkanolamide or alkanolamide condensate, alfol,
Nonaphenol) and amphoteric detergents (N-alkyl betaines, N-alkyl sulfobetaines, alkyl imidazolines, N-alkyl-β-aminopropionic acids, etc.)
Can be mentioned.

[0014]

The present invention will be further described by the following examples.

Example 1 Haloferrax Mediterranei Strain ATCC335
00 was used as the starting material [ATCC number is American Type Culture Collection, Bethesda City, Maryland, USA]
It is the strain number stored in. The properties of this microorganism as a PHA producer are described in patent 890347. 500m culture containing 10g / l of biomass (6g / l of which is PHA)
1 was centrifuged at 600 times / min for 15 minutes to obtain a concentrated precipitate, and the supernatant was removed by decantation.

Next, distilled water containing 0.1% of SDS 5
The pellet was resuspended in 00 ml. The sediment was stirred vigorously in the suspension medium for resuspension, a homogenous suspension was obtained and then left until the turbidity disappeared. This took 1 to 20 minutes.

The suspension was centrifuged at 2000 rpm for 5 minutes to obtain a dense whiteish precipitate. Decant the supernatant and again 500 ml of water containing SDS (0.1%)
Suspended in water, centrifuged as before, and distilled water (5
00 ml) and re-centrifuged.

Water was removed by decantation and the resulting paste was mixed with 7
About 3 g of PHA having a purity of 98.99% was obtained by drying in one step by spraying in a fluidized bed in an air stream at 0 ° C. for about 2 hours.

Example 2 The cells were separated by centrifugation in the same manner as above, resuspended in distilled water with vigorous stirring in a laboratory disruptor. The suspension was heated to 65 ° C. for 20 minutes. After this step, 2
The whole procedure of Example 1 was repeated, which consisted of centrifugation at 00 rpm for 5 minutes, followed by a previous treatment with water-detergent and a subsequent centrifugation. Purity 98.9
9% product was obtained with a yield of 97-99%.

Example 3 This example demonstrates a clear relationship between PHA and water and detergent in a novel process in which each step is repeatedly contacted with a mixture of water and detergent (single step). Washing in can require very large amounts of water and cleaning agents). About 10 g / l of biomass (5-6 g / l of which is PH
Cell disruption step in which the concentrate is treated with a solution consisting of salt-free water and a detergent, for example 1 liter containing medium (which is A) (this treatment is optional and the yield will be reduced if not carried out) Was subjected to similar process steps to Example 1. Once centrifuged at 8000 rpm for 20 minutes, the resulting suspension was divided into 3 parts. One of them is 0.
250 ml of a 2% SDS aqueous solution was used for treatment three times, another one was treated with 125 ml of the same solution three times, and the last was treated three times with 75 ml of the solution. As a final treatment, after washing with water to remove the detergent, a dryer or 70 ° C
Was dried in a fluidized bed with a stream of air for 2 hours. In all cases the yields are above 95% and the purity measured by dissolving a sample of PHA and weighing the insoluble residue is 98-99% in the first case and 90-91 in the second case. % And 87-90% in the third case.

Example 4 In order to improve the yield, a cell lysis step was performed before countercurrent washing. In order to increase the purity in the dissolution step, in addition to the detergent, a cation sequestering agent, that is, a complex former (for example, ethylenediaminetetraacetic acid, EDTA) can be used. For example, the concentrate obtained in Example 1 is treated with 500 ml of water containing 0.2% SDS three times or SD.
S and ethylenediaminetetraacetic acid (EDTA) 0.6 mM
The purity obtained after three treatments with 500 ml of water containing 78.8% was 78.8%, compared with 92% of the second case.

Example 5 Another example of an agent that favors bacterial lysis prior to the onset of countercurrent washing is a bile salt such as taurocholic acid, which, without causing any apparent loss of purity, can be washed with water and after. It is possible to reduce the consumption of the cleaning agent. For example, processing 1 l of medium, 800
Centrifuge at 0 times / minute for 20 minutes, and precipitate the resulting sediment with S
Solution 12 containing DS 0.2%, Taurochol 6 mM
125 ml of a solution containing 5 ml, then 0.2% SDS
The final product with a purity of 96-97% was obtained by washing with 1.

Example 6 3 l of medium containing 3 g / l PHA was treated. Of this, 1 liter was 3 times with 500 ml of 0.2% SDS, the second was 1 time with 500 ml of 0.2% SDS and 0.1% SD.
S 2 times with 500 ml, and the third with 0.1% SD
S was treated with 500 ml three times. In all cases, pre-washing (lysis step) was performed with SDS and 0.6 mM EDTA. Final purities are 91.9% and 76.9, respectively.
% And 59.3%.

Example 7 Washing was carried out in countercurrent. Tests were carried out on sediments similar to those described in Example 1, but resulting from the treatment with EDTA of Example 5. It was treated with 150 ml of 0.2% SDS aqueous solution in a continuous / countercurrent type three-stage washing device.
The purity obtained was 97%.

Claims (7)

[Claims] 1. When extracting polyhydroxyalkanoate contained in halophilic bacteria, lysis or destruction of halophilic cells (for example, those of halobacterium type) growing in a high-concentration salt medium, concentration, centrifugation. Method for extracting polyhydroxyalkanoates, characterized in that a separation is carried out, followed by a dilution-resuspension with a low-concentration salt medium such as soft or distilled water and centrifugation, sedimentation or filtration of the suspension thus formed. . 2. Anionic, cationic, nonionic or amphoteric detergents such as sodium dodecyl sulfate, linear alkylbenzene sulfonate, sodium cholate, alfol, nonaphenol and low concentrations of 0 to 1.5%. The process according to claim 1, wherein any of the cation complex products of the ethylenediaminetetraacetic acid type is used for promoting lysis of bacteria and improving yield and purity of the resulting polyhydroxyalkanoate. 3. The method according to claim 1, wherein crushing, stirring, shaking, depressurizing or heating is performed in order to promote cell lysis. 4. A process according to claim 1 or 2 in which a detergent of the above type is used for washing the polyhydroxyalkanoates obtained and for removing lipids and proteins. 5. Washing may be continuous or discontinuous, in one step or in several steps with fresh solvent each time,
Or the method of Claim 4 performed in countercurrent. 6. The method according to claim 5, wherein the final washing step is carried out with water to remove the detergent. 7. To accelerate dissolution, sodium dodecyl sulfonate 0 to 2% and ethylenediaminetetraacetic acid 0 to 6 mM or taurocholic acid 0 to 6 mM or sodium deoxycholate 0 to 6 mM are used in one step or several steps. For countercurrent washing, a wash water to polyhydroxyalkanoate ratio of 200: 1 and sodium dodecyl sulphonate 0 to 3% were used to obtain a purity of more than 98% and a yield of more than 98%. A method according to claim 1 obtained.