JP2008237182A - Method for separating and purifying polyhydroxy butyric acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To inexpensively extract PHB (polyhydroxy butyric acid) at an ordinary temperature and reduce lord to the environment. <P>SOLUTION: PHB from which impurities are certainly removed is produced by preparing a culture solution for microbial cells containing PHB, obtaining the microbial cells with accumulated PHB from the culture solution, subsequently freezing the wet cells by a freezing means, physically crushing the PHB-containing frozen cells by a crushing means, subsequently separating the crushed cells (crushed pieces or crushed powder) by suspending the same in a solvent. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for separating and purifying polyhydroxybutyric acid capable of extracting and purifying polyhydroxybutyric acid from microbial cells.

  Some microorganisms existing in nature accumulate polyhydroxybutyric acid (hereinafter referred to as PHB), which is a kind of polyester resin, as a hibernating substance when given appropriate nutrients. This PHB is made of a thermoplastic polyester having complete biodegradability and biocompatibility, which is produced and stored as an energy storage material in microbial cells, and is required to be industrially used in the industry.

In this regard, Patent Document 1 discloses, as a known general method for separating and producing PHB from microbial cells, PHB is extracted from the cells by a Soxhlet extraction method using a solvent in which PHB is soluble, for example, chloroform. How to do is described.
JP-A-7-177894 (see paragraph 0003)

  However, in an extraction method using chloroform as a solvent in which PHB is soluble, for example, about 100 ml of chloroform is required to elute to recover 3 g of PHB. When the amount of chloroform is 100 ml or less, it becomes a highly viscous solution, which makes it difficult to handle. Further, in order to precipitate PHB eluted in chloroform, a PHB-insoluble solvent such as methanol, ethanol, and acetone having a volume of 10 times that amount is further required. For this reason, the manufacturing cost increases and harmfulness is pointed out.

  Examples of the method for extracting PHB using chemicals other than chloroform include, for example, a method of obtaining only PHB by solubilizing unnecessary proteins in water using various enzymes such as lysozyme for decomposing bacterial cell walls. And extraction using another organic solvent such as tetrahydrofuran.

  However, in the former extraction method using various enzymes, the enzyme itself is not mass-produced and is expensive, and it is difficult to reduce the manufacturing cost. In the latter extraction method using an organic solvent, Although the toxicity is lower than that of chloroform, there is a problem that a process of heating to a predetermined temperature is required to increase the reactivity, which is complicated. Furthermore, there is a concern that a high load is applied to the surrounding environment by using such chemicals.

  The present invention has been made in view of the above-mentioned problems and the like, and a general object of the present invention is to extract PHB at a low temperature in a normal temperature range and to reduce the burden on the environment. The object is to provide a method for separating and purifying butyric acid.

  Another object of the present invention is to separate polyhydroxybutyric acid that can reliably remove unnecessary components other than PHB, and can extract only PHB by a very simple method and with a higher purity than conventional methods. It is to provide a purification method.

  In the present invention, the wet cells in which polyhydroxybutyric acid is accumulated are frozen by a freezing means, and then the frozen body containing the polyhydroxybutyric acid is physically crushed by a crushing means. In order to extract only polyhydroxybutyric acid from microbial cells containing polyhydroxybutyric acid, it is necessary to remove cell walls and nucleic acids in the cells, and by crushing the frozen cells of the wet cells, the cell walls in the cells As a result, the cell wall, nucleic acid, and the like can be suitably removed from the microbial cells. Subsequently, the crushed crushed body (crushed pieces or crushed powder or the like) is suspended in a solvent and separated, so that impurities are reliably removed and polyhydroxybutyric acid having high purity can be obtained.

  In this case, a liquefied gas such as liquid nitrogen or liquid helium may be used as the freezing means. The crushing means includes, for example, a ball mill, but is not limited thereto, and other mechanical devices may be used. Furthermore, as said solvent, although distilled water etc. are mentioned, if it is other than an organic solvent, it will not be limited to the said distilled water. This is because when an organic solvent is used, a recovery step, a disposal step, and the like are required, and the purification step becomes complicated.

  Further, in the present invention, the wet microbial cell in which the polyhydroxybutyric acid is accumulated is obtained by centrifuging the liquid in which the microbial cell containing polyhydroxybutyric acid is cultured before freezing by the freezing means. . By centrifuging a liquid in which a predetermined microbial cell is cultured with a centrifugal separator or the like and removing the supernatant, a wet cell in which polyhydroxybutyric acid is accumulated can be easily obtained.

  Furthermore, in the present invention, when the wet cells in which polyhydroxybutyric acid is accumulated are frozen at a predetermined temperature and physically crushed, for example, physical impact force is exerted on the cell walls, cytoplasm, and nucleic acids of the microbial cells. The cell wall, cytoplasm, nucleic acid, and the like are easily separated out of the microbial cell, and the cell wall, cytoplasm, nucleic acid, and the like can be efficiently and well removed from the microbial cell. In other words, by making the wet cell body into a frozen body at a predetermined temperature, the impact force is imparted over substantially the entire frozen body, and the separation of the cell wall, cytoplasm, nucleic acid, and the like outside the cell body is preferably promoted. Because.

  As described above, in the present invention, the cell wall, cytoplasm, and nucleic acid of the microbial cell can be easily removed by subjecting the wet cell in which polyhydroxybutyric acid is accumulated to freeze-fracturing, and further centrifugation. By removing the removed cell wall, cytoplasm and nucleic acid by the above-mentioned method, polyhydroxyl can be used in a low environmental load state at a low temperature, industrially at low cost and without giving a high load to the surrounding environment. Butyric acid itself can be extracted by a simple method.

  In the normal temperature range, only polyhydroxybutyric acid is extracted at a low cost, and a recovery process and a disposal process when using various existing organic chemicals are not required, and the burden on the environment can be reduced. Moreover, unnecessary components other than polyhydroxybutyric acid can be reliably removed, and only polyhydroxybutyric acid can be extracted with a very simple method and with high purity.

  The microbial cell used in the present invention is not particularly limited as long as it is a microbial cell having polyhydroxybutyric acid producing ability. Practically, for example, alkaline genus Eutropha and alkaline generatus included in the genus Alcaligenes, and other examples include Sinorizobium fredi and Bacillus megaterium. As the freezing means, for example, a liquefied gas such as liquid nitrogen or liquid helium may be used.

  Further, examples of the crushing means include a ball mill, but the invention is not limited to this. For example, various homogenizers may be used in a low temperature atmosphere. Furthermore, examples of the solvent include distilled water, but are not limited to the distilled water as long as it is other than an organic solvent. Furthermore, the freezing temperature of the wet cells may be lower than −20 ° C.

  Next, in order to confirm the effect of the present invention, the following experiment was performed based on various processes, which will be described in detail. Note that the present invention is not limited to the following experimental examples.

1. Bacterial culturing process As a microorganism of a microorganism containing polyhydroxybutyric acid (hereinafter referred to as PHB), alkaline genus Eutropha, which is one of the bacteria included in the genus Alcaligenes, was used in the experiment. Pre-cultivate this alkaline genus eutrophic fungus in NB medium (concentration of 8 g per liter) in the order of 5 ml, then 100 ml, respectively, for 24 hours at 30.degree. To obtain 100 ml × 3 sets of preculture liquid.

  Subsequently, the pre-culture solution consisting of 100 ml × 3 sets was put into a culture apparatus (fermenter) (not shown), and main culture was performed with the culture apparatus. The adjustment of the liquid medium in the main culture is as follows.

  That is, about 1.7 L of MSM (Mineral Salts Media) culture solution composed of the components shown in FIG. 1 and FIG. 2 is prepared, and 2.0 L together with the pre-culture solution (100 ml × 3 sets). It was made to become.

  As the conditions for the main culture, the culture was kept at 32 ° C. for 48 hours. The stirring speed was appropriately set so that dissolved oxygen was sufficiently distributed in the liquid within a range of 200 to 350 rpm. The main culture is based on semi-batch culture (fed-batch culture). The main nutrient carbon source is a 40% Na-gluconate aqueous solution so as to maintain a concentration of about 5% in the solution, and the nitrogen source is in the solution. A 20% NH 4 Cl aqueous solution was used so that the concentration was maintained at about 0.05%. In addition, acid and a base, respectively 4N hydrochloric acid and 4N NaOH aqueous solution were used so that pH might be maintained at 7.0.

2. Crushing process of bacterial cells Only 50 ml of the liquid after completion of the main culture in the above process is collected, and this liquid is centrifuged at a rotational speed of 15000 rpm for 3 minutes to separate it from the supernatant liquid. Accumulated wet cells (1) were obtained. Note that the wet cells accumulated on the lower side could be easily obtained by removing the supernatant liquid separated by the centrifugal separation action. Subsequently, the wet cells (1) that had been cultured were frozen with liquid nitrogen at a liquid nitrogen temperature, and then the frozen cells were freeze-ground with a ball mill. In the experiment, wet cells are frozen at a liquid nitrogen temperature, but this freezing temperature may be lower than −20 ° C., for example.

  Specifically, a small amount of the wet cells (1) after centrifugation was collected in a dedicated tube attached to the ball mill, a dedicated crusher was set, and the wet cells were immersed in liquid nitrogen for 1 minute. The frozen body thus frozen was set on a ball mill and oscillated along the vertical direction to crush the frozen body into crushed pieces or crushed powder.

  Further, the crushed pieces or crushed powder containing PHB was washed several times by suspending it with distilled water and centrifuging it. And the microbial cell (2) which concerns on this Example which the crushing and washing | cleaning were completed, and the wet microbial cell which concerns on the comparative example which is the state which main culture has completed as mentioned above, and has not been subjected to the freezing crushing process (1) was placed in an oven at about 60 ° C. and dried by heating, and then samples for measurement of gas chromatography (Gas Chromatography, GC) were obtained.

3. Purity measurement step Two measurement samples obtained in the previous step, that is, wet cells (1) according to a comparative example in which freeze-fracture treatment was not performed and the state where main culture was completed was maintained The purity of PHB contained in each of the dried sample and the dried sample of the bacterial cell (2) according to the present example that had been subjected to freeze-fracture treatment and washing treatment was measured by the GC method. The GC method is described in Brauegg, G. et al. , Et al. , Eur. J. et al. Appl. Microbiol. 6, 29 (1978). The measurement conditions are as shown in FIG.

  As a result, as shown in FIG. 4, in the wet cells (1) according to the comparative example which was not subjected to freeze-fracture treatment and was maintained in the state where the main culture was completed, PHB was measured by GC measurement. While the purity is 59%, the fungus body (2) according to the present example subjected to the freeze crushing treatment and the washing treatment has a PHB purity of 85% as measured by GC, thereby achieving high purity. I was able to.

  From the above, in the experiment, the bacterial cell (2) according to the present example can reduce the burden on the environment without using various existing organic chemicals, and only polyhydroxybutyric acid can be reduced by a very simple method. In addition, it was confirmed that the effect of the present invention that it can be extracted with high purity was achieved.

It is a figure which shows the component structure of the MSM culture solution used for adjustment of a liquid culture medium. It is a figure which shows the component structure of TES contained in the said MSM culture solution. It is a figure which shows the measurement conditions of the sample by a gas chromatography. It is the measurement result by the said gas chromatography, Comprising: It is the figure which compared the purity of a present Example and a comparative example.

Claims (2)

A method for separating and purifying the polyhydroxybutyric acid from microbial cells containing polyhydroxybutyric acid,
Freezing the wet cells in which the polyhydroxybutyric acid is accumulated by a freezing means;
Physically crushing the frozen body containing the polyhydroxybutyric acid by crushing means;
A step of obtaining polyhydroxybutyric acid by suspending and separating the crushed crushed material in a solvent;
Separation and purification method of polyhydroxybutyric acid having The method for separating and purifying polyhydroxybutyric acid according to claim 1,
Before the step of freezing by the freezing means, a step of obtaining a wet microbial cell in which the polyhydroxybutyric acid is accumulated is obtained by centrifuging the liquid in which the microbial cell containing polyhydroxybutyric acid is cultured. A method for separating and purifying polyhydroxybutyric acid.

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