JP2925385B2 - Method for producing polyhydroxy organic acid ester - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microbiological process for producing polyhydroxy organic acid esters using a microorganism of the genus Rhodobacter.

[0002]

2. Description of the Related Art Conventionally, various microorganisms have their own nutrient sources,
It is known to produce and accumulate polyhydroxybutyric acid as an energy source. Polyhydroxybutyric acid produced by microorganisms has biodegradability and has been expected to be used in various fields as a material for molding resins, pharmaceutical / agrochemical formulations, medical equipment and the like.

Recently, from the viewpoint of environmental protection, there has been a demand for a polymer having natural degradability in the treatment of industrial waste such as plastics, and production of polymers utilizing various microorganisms has been proposed. For example,
High, in British Patent No. 1370892 follower micro bi um-Variapire, certain strains of the Pseudomonas Rosea species, also, Journal of General Microbiology (J.of GeneralMicrobiology) 1977, 98, 265
Pp. 272, Methylobacterium, Organophilum, Pseudomonas AM-1 and the like; JP-A-56-117793 discloses Methylobacterium organophilum species;
JP-A-57-74084 discloses a genus Alcaligenes (A. faecalis, A. roullandii, A. lass, A. aquamarinus, A. eutrophus).
No. 3 discloses A.I. Utrofus, JP 59-220192
No., Nocardia, Azotobacter, Bacillus, Micrococcus, Rhizobium, Rhodospirillum, Methylobacterium, Pseudomonas, Hydrogenomonas, JP-A-60-199392 discloses Alcaligenes reitas,
Japanese Patent Application Laid-Open No. 60-214888 discloses Alcaligenes, Japanese Patent Application Laid-Open No. 60-251889 discloses Azotobacter binenlandi or a mutant strain, and Japanese Patent Application Laid-Open No. 61-293385 discloses Alcaligenes Eutrophus. JP-A-62-55094 discloses the genus Protomonas, and JP-A-63-198991 discloses Azotobacter binenlandi, Alcaligenes eutrophus, Zugrea ramigera, and Bacillus megaterium. Pseudomonas oleovorans species are each described. In addition, poly-β-hydroxybutyrate has been found along with polysaccharides and polyphosphates as microbial cell accumulations of microorganisms belonging to the red sulfur-free bacteria (Bergey's Manual of Systemic Bacteriology (Bergey's Manual of Systemic Bacteriology)).
ematic Bacteriology), p. 1658), but there is no proposal to produce a polyhydroxy organic acid ester using a microorganism belonging to the genus Rhodobacter.

[0004] Accordingly, the effect of pH on the yield of polyhydroxy organic acids when using microorganisms belonging to the genus Rhodobacter, better carbon sources and effective additives have not been studied at all, and are therefore completely unknown. Not.

[0005] Further, no study has been made on the addition of yeast to the microbiological production of polymers.

[0006]

SUMMARY OF THE INVENTION In view of the above circumstances, the present invention provides a method for efficiently producing a biodegradable polymer by using microorganisms which have a high production rate, a large amount of cells accumulated in cells, and are safe and easy to handle. The purpose is to provide.

[0007]

SUMMARY OF THE INVENTION The present invention is characterized in that a microorganism belonging to the genus Rhodobacter is cultured under aerobic conditions, and a polymer comprising a polyhydroxy organic acid ester is accumulated in the cells. The present invention relates to a method for producing a polyhydroxy organic acid ester.

[0008]

The present inventors have conducted intensive studies to achieve the above-mentioned object, and as a result, have found that a polyhydroxy organic acid ester can be effectively produced by using a microorganism belonging to the genus Rhodoctor which has no pathogenicity and is therefore safe and easy to handle. Surprisingly, the rate of growth of organisms and the rate of accumulation of polyhydroxy organic acid esters in the microbial cells are extremely high under anaerobic conditions (conditions in the absence of oxygen), which has been conventionally accepted as common knowledge. It has been found that accumulation is very effective under aerobic conditions (oxygen-containing conditions) which are low and have not been adopted at all, and that the accumulation is effectively performed by one-stage culture or two-stage culture. .

Further, as a result of further studies, it has been found that, in a microbiological production method of a polyhydroxy organic acid ester using a microorganism belonging to the genus Rhodobacter, the pH of the culture solution is 4.
When it is less than 5, the consumption of the polyhydroxy organic acid ester accumulated in the cells progresses, and the addition of yeast to the culture medium increases the accumulation of the polyhydroxy organic acid ester,
Further, it has been found that the production of polyhydroxy organic acid esters can be effectively performed by mainly using mannitol as a carbon source of the medium components. The present invention has been completed based on many of the above findings.

Next, a method for producing the polyhydroxy organic acid ester of the present invention will be described.

As the microorganism belonging to the genus Rhodobacter used in the present invention, any microorganism belonging to the genus Rhodobacter which can accumulate the polymer in the cells by aerobic cultivation is used. For example Rhodobacter cap thread Itasu (R.capsulatus), Rhodobacter sphaeroides (R.sphaeroides), Rhodobacter sulfide Filth (R.sulfidophilus), Rhodobacter Adria Genetics (R.adriaticus) and Rhodobacter Berudokanpyi (R.veldkampii), and their the polymer Mutant strains and the like.

As a method for culturing a microorganism belonging to the genus Rhodobacter according to the present invention, as described above, the cultivation under anaerobic conditions has a very low growth rate of organisms and a very low rate of accumulation of polyhydroxy organic acid esters in the cells. Culture under aerobic conditions is very effective. The aerobic condition usually means culturing in the presence of oxygen. Under conditions in the absence of oxygen (anaerobic conditions), the method of the invention is very inefficient.

[0013] As a condition for producing the polymer by a microorganism belonging to the genus Rhodobacter, culture under photosynthetic conditions is not particularly necessary.

The culturing temperature is usually 45 ° C. or lower, preferably 30 to
40 ° C. pH is usually 4.5 or more, preferably 6-9,
More preferably, it is 6.5-8.

The nitrogen source is essential for the growth of the microorganism, but not for the accumulation of the polymer. Therefore,
After the cells are grown (pre-cultured) in a medium containing a nitrogen source and harvested, nitrogen starvation culture is performed, and the polymer can be accumulated in the cells by a two-step method. The term "nitrogen starvation" as used herein means that the nitrogen content in the medium is 1 g / l or less, preferably 0.5 g / l or less, more preferably 0.2 g / l or less. If pre-culture is performed, under aerobic conditions, without nutrient restrictions, until the logarithmic growth phase, i.e., the concentration of the number of bacteria is at least 10 ⁷ / ml
It is performed until it becomes.

Alternatively, as described above, a two-stage method of growing the bacteria and then producing the polymer of the present invention by nitrogen-starving culture is also possible, but the medium is mixed with a nitrogen source from the beginning to grow. At the same time, it is also possible to accumulate the polymer by a one-stage method of accumulating the polymer. By being able to culture in one stage in this way,
There is no need to collect the cells grown in the preculture (in the presence of nitrogen) and transfer them to a new nitrogen-starved medium for cultivation. Since the production process is not performed, the loss of bacterial cells is extremely small, which is extremely industrially advantageous.

The nitrogen source can be selected from inorganic substances such as ammonium sulfate and ammonium chloride, and the organic substances can be amino acids, urea and the like.

As a nutrient source of the microorganism, any carbon source which does not inhibit the culture of the microorganism can be used. For example, inexpensive products such as alcohols such as methanol, ethanol and propanol, organic acid salts such as sodium acetate and sodium propionate, and saccharides such as glucose can be used. Preferred are mannitol, acetic acid, propionic acid, fructose, and glucose.

However, when mannitol is used as a carbon source, organic acids do not accumulate in the culture solution.
H does not become 7 or less. In addition, a small amount of NH ₄
Even in the presence of salt, there is no aminocarbinol reaction associated with the autoclave treatment, and therefore, the product of this reaction does not inhibit the growth of cells, which is very advantageous. One of the production methods of the present invention is a method for producing a polyhydroxy organic acid ester by culturing a microorganism belonging to the genus Rhodobacter using a carbon source containing mannitol as a main component.

The concentration of the carbon source in the culture solution is usually 5% or less (% by weight, hereinafter referred to as%), preferably 0.5 to 5%.
3%, more preferably 1-2%. In addition, other components essential for culturing microorganisms, for example, inorganic salts such as magnesium sulfate and potassium phosphate, required trace elements such as thiamine and biotin, and other naturally necessary components are required. These components may use a reagent, and may be a material obtained as a natural product, for example, a waste itself or a synthetic medium partially using the same.

[0021] One of the production methods of the present invention is a method for producing a polyhydroxy organic acid ester, which further comprises adding yeast to a medium. The addition of yeast significantly increases the amount of polyhydroxy organic acid esters that accumulate in the cells. Here, "yeast" means yeast cells, yeast dead cells, or yeast extract. The yeast to be added is preferably a yeast extract. The amount of yeast added is usually 0.1 to 3 g / l, more preferably 0.5 to 2 g / l, and the amount of yeast extract added is usually 0.1 to 2 g / l, preferably 0.2 to 1.5 g / l.

A nutrient source such as a carbon source may be added at the beginning of the cultivation, but may be added at any time depending on the normal consumption. For example, they can be added continuously or intermittently.

The pH during cultivation increases with the consumption of an acid sodium salt or the like as a carbon source, but decreases with the consumption of a saccharide as a carbon source. I do. On the other hand, when the pH of the culture solution becomes less than 4.5, the consumption of the polyhydroxy organic acid ester accumulated in the cells progresses rapidly. One of the production methods of the present invention is to adjust the pH of a culture solution to 4.
5 or more, preferably 6.0 to 9.0, more preferably 6.
This is a production method characterized by maintaining the value at 5 to 8.0. p
The method for retaining H is not particularly limited. For example,
A sterilized diluted solution of sodium hydroxide, potassium hydroxide, or hydrochloric acid can be dropped by operating a pump in conjunction with the pH to maintain an appropriate pH value. By maintaining the pH above 4.5, the yield of polyhydroxy organic acid ester is significantly increased.

The accumulation of the polymer in bacteria can be monitored with an optical microscope, a phase contrast microscope, an electron microscope, or the like. The accumulation rate of the polymer in the bacterium in the present invention varies depending on conditions, but is usually at least 20%, preferably at least 20%.
It is 40%, more preferably at least 60%. One of the features of the present invention is that the accumulation rate is higher than previously reported, the polymer production per cell weight is large, and the extraction efficiency is excellent.

When the accumulation of the polymer reaches at least about 20%, preferably at least 40%, bacteria are separated from the culture solution. As a method for separating and recovering the produced polymer from the cells, any of conventionally known methods may be used.

For example, if the polymer is eluted by adding, for example, chloroform as a non-polar solvent to the freeze-dried cells, hot-air-dried cells, or dehydrated cells with acetone or methanol, and the solvent is distilled off, a residue mainly composed of the polymer is obtained. available. Pre-processing is also effective to increase extraction efficiency. For example, if the cell suspension is lysed by adding an organic solvent such as a lytic enzyme or xylene to the cell suspension, and then extracted with a solvent such as chloroform, the extraction time is shortened and purification is facilitated.

Alternatively, when the content of the produced polymer inside the cells is high, the cells may be used together with the cells without extracting the polymer from the cells, or the cells may be softened with a solvent, an enzyme or the like. May be used. The extracted polymer can be purified or fractionated, if necessary, and reconstituted to remove impurities.

As the polyhydroxy organic acid ester obtained by the production method of the present invention, for example, a polymer composed of a poly-3-hydroxybutyrate (hereinafter referred to as PHB) represented by the following formula or a polymer composed of PHB as a main component: Polymers.

[0029]

Embedded image

As other components, an organic acid ester component having 5 or more carbon atoms may be contained, but the amount is usually 10% by weight.
It is as follows.

The qualitative analysis of the structure can be performed by IR, NMR and the like. Also, the property to heat is
Possible by DSC, TMA, etc. The molecular weight of the polymer can be measured by a viscosity method, a GPC method, or the like. Biodegradability is
This is possible by molding the polymer into a film or the like, embedding it in soil, and measuring changes in physical properties (strength, elongation), morphology (surface, cross section), molecular weight, and the like over a certain period of time.

The polymers according to the invention are soluble in organic solvents, such as, for example, chloroform, dichloromethane, 1,2-dichloroethane and 1,2-dichloropropane. Can be extracted. In addition, when a film of the polymer is buried in general garden soil, cracks start to be generated on the film surface from about one week, followed by a decrease in strength and elongation, and it is apparent that the film is clearly decomposed. Show.

The polymer obtained by the present invention is useful for applications utilizing biodegradability, for example, plastic bottles, fishing lines, wrapping paper, surgical lines, medical coating agents and the like.

Next, the method for producing the polyhydroxy organic acid ester of the present invention will be described based on examples, but the present invention is not limited to these examples.

Example 1 Rhodobacter capsulatus ATCC 11166 was transplanted from a holding medium into an L-shaped tube containing 10 ml of nutrient broth and cultured at 37 ° C. for 24 hours with shaking (amplitude 6 cm, 92 times /
And the same in Examples 2 and 3) for 3 passages. Next, 100ml of nutrient broth of the same composition
Was prepared, and the subcultured solution was transplanted to a ratio of 2%, cultured with shaking at 37 ° C. for 48 hours, and then centrifuged at 3000 G for 15 minutes. The bacteria were collected.
This was suspended in 200 ml of physiological saline, and again centrifuged in the same manner to collect cells. The same operation was repeated three times to obtain washed cells.

Next, nitrogen starvation culture was performed under aerobic conditions. That is, sodium acetate 10 g, potassium phosphate 1 g, potassium diphosphate 1 g, magnesium sulfate 7
0.2 g of water salt, 0.05 g of calcium chloride monohydrate, 0.005 g of thiamine hydrochloride, 1 g of yeast extract, 1 liter of water, pH 7.
A starvation culture medium of 0 was made. The washed cells were added to one liter of the starvation culture medium, mixed, and suspended. 500ml
100 ml each was dispensed into 10 Nosakaguchi flasks, and shaking culture was carried out at 37 ° C. for 8 days. After culturing, the cells were collected by centrifugation under the same conditions as above, suspended in 100 ml of acetone, allowed to stand for 10 minutes, and then centrifuged at 3000 G for 5 minutes to collect the cells. This was repeated four times to dehydrate the cells. Acetone adhering to the cells was volatilized by air drying.

Next, 200 ml of chloroform was added thereto to form a suspension, and the mixture was transferred to a 500-ml eggplant-shaped flask. The mixture was kept in a 70 ° C. water bath with a reflux condenser for 3 hours. After cooling to room temperature, Toyo Filter Paper No. .2 The solution was filtered through a filter paper to separate the chloroform solution from the cells. The cells were washed with chloroform and combined with the chloroform solution. Next, the filtrate was transferred to a 500 ml eggplant-shaped flask and distilled under reduced pressure at 35 ° C. to remove chloroform. The film-form polymer remaining in the flask was washed with methanol, acetone, hexane and ether in this order, dried, dissolved again in chloroform, and once filtered to obtain a chloroform solution. Chloroform is distilled off, and the polymer is left in the flask to obtain 6 g.
Could be obtained. The same purification operation was repeated three times,
A polymer containing poly-3-hydroxybutyrate as a main component was obtained.

As a comparative example, after culturing under photosynthetic conditions under anaerobic illumination in a medium having the same composition, nitrogen-starved cultivation was continued under anaerobic illumination for exactly the same time. The weight was only 0.2 g per gram of cells.

The results clearly show that under aerobic conditions, the production of a polymer containing polyhydroxybutyrate as a main component is high.

Example 2 Cultivation was carried out in exactly the same manner as in Example 1 except that the nitrogen source and its content were changed. Ammonium sulfate was used as the nitrogen source, and the cells were cultured at a ratio of the nitrogen source to the carbon source that is the nutrient of the microorganism, that is, C / N of about 1.8 to ∞. Table 1 shows the amount of the polymer accumulated in the cells per dried cell of the obtained cells. The fact that the culture is not nitrogen-starved is apparent from the measurement of the nitrogen source after the culture, which indicates the residual amount.

As shown in Table 1, the production of the polymer is possible without complete nitrogen starvation, and when the nitrogen content is 2 g / l or less, the production of the polymer in the nitrogen starvation state and the production of the polymer do not increase. It can be seen that there is no change in productivity. This is a great feature using the Rhodobacter bacterium of the present invention.

[0042]

[Table 1]

Example 3 In the same manner as described in Example 1, the culture conditions in starvation culture were changed, the carbon dioxide source was glucose, and the culture temperature was
At 27 ° C., the culture time was 4 days. The initial glucose concentration is shown in Table 2.

The number of viable cells after culturing was measured by a plate dilution culture method (using a YPS agar medium).
In all cases, the number of viable bacteria showed an approximate viable bacterial concentration, and was (2.7 × 7.9) × 10 ⁷ / ml. After 2 days of starvation culture, when observed with a phase contrast microscope with live cells, polymer granules were accumulated in the cells, and 3 to 3 cells per cell were observed.
There were 5 particles. Further culturing was continued, and on the fourth day after the start of the culturing, the morphology of the cells changed to a shape close to a sphere (1 μ in diameter), and all the cells were filled with the polymer granules. Table 2 shows the results.

The following results show that polyhydroxybutyrate is produced even at a culture temperature of 27 ° C., and that the polymer is produced even when glucose, a typical sugar, is used as a carbon source. After culturing for 4 days, the cells were filled with a large amount of the polymer, and the accumulation process could be monitored using an optical microscope. Therefore, it was possible to determine an appropriate time for ending the culture without extracting and analyzing the polymer.
The polymer was confirmed by a conventional method in which cells were fixed on an object glass with a flame, stained with Sudan black, and then examined under a microscope.

[0046]

[Table 2]

Example 4 A culture medium containing Rhodobacter capsulatus ATCC 11166 containing 10 ml of YPS medium (the composition is shown below) was added.
Transfer to a 0ml Sakaguchi flask and shake culture at 37 ° C for 24 to 48 hours (amplitude 3.5cm, 100 times / min, all the same below)
Then, the cells were collected by centrifugation at 3000 G for 15 minutes. This was suspended in 100 ml of physiological saline, and again centrifuged similarly to collect cells. The same operation was repeated three times to obtain washed cells.

The cells were added to a nitrogen-starved medium with 1.0 g / l of yeast extract (manufactured by Nacalai Tesque) and 1.0 g / l of yeast extract (manufactured by Difco), respectively.
It was transplanted into a 500 ml Sakaguchi flask containing 0 ml.

[0049] 0.3% yeast composition of YPS medium extract polypeptone _{0.3% MgSO 4 · 7H 2 O} 0.05% CaCl 2 0.03% (pH 7.40) the composition of nitrogen starvation medium _{KH 2 PO 4 1.0 g / l} K 2 HPO 4 1.0 g _{/ l MgSO 4 · 7H 2 O} 0.2 g / l CaCl 2 · 2H 2 O 0.05g / l FeSO 4 · 6H 2 O 0.01g / l thiamine hydrochloride 5 mg / l nicotinic acid 2 mg / l biotin 2 mg / l glucose 10mg / l (pH 7.40) After shaking culture at 37 ° C for 86 hours, the cells were collected by centrifugation under the same conditions as above, suspended in 100 ml of acetone, allowed to stand for 10 minutes, and then centrifuged. 3000G,
The reaction was performed for 15 minutes to collect the cells. This was repeated three times to dehydrate the cells. Acetone adhering to the cells was volatilized by air drying.

Next, 20 ml of chloroform was added thereto to form a suspension, and the mixture was transferred to a 500-ml eggplant-shaped flask, kept in a hot water bath at 65 ° C. for 3 hours with a reflux condenser, and cooled to room temperature.
The solution was filtered through Toyo No. 2 filter paper to separate the chloroform solution from the bacterial cells. The cells were washed with chloroform and combined with the chloroform solution. Next, the filtrate was transferred to a 500 ml eggplant-shaped flask and distilled under reduced pressure at 35 ° C. to remove chloroform. The polymer in the form of a film remaining in the flask was washed with methanol, acetone, hexane and ether in this order, dried, dissolved again in chloroform and filtered once to obtain a chloroform solution. Chloroform was distilled off to obtain an ester. The same purification operation was repeated three times,
PHB-based polymer was obtained.

The amount of the polymer thus obtained is determined by the former
0.0953 g / l and the latter at 0.0925 g / l. The intracellular content of the polymer (that is, the ratio of the obtained polymer weight to the weight of the bacterial cells) was 36.0% and 41.0%, respectively.

From the above results, it can be seen that there is no difference in the obtained polymer production amount and in the intracellular content depending on the type of the yeast extract to be added.

Example 5 Rhodobacter Capsulatus ATCC 11166 was added with and without yeast extract at 1 g / l.
Starvation was performed in the same manner as described above. The yeast extract used was manufactured by Nakarai Tesque. After culturing at 32 ° C. for 86 hours, a polymer was obtained in the same manner as in Example 1.

When the yeast extract was added, the obtained polymer was 109.9 mg / 100 ml medium, and when no yeast extract was added, the polymer was 14.1 mg / 100 ml medium. The intracellular content of the polymer was 38.6% and 32.9%, respectively.

From these results, it is found that the amount of polymer per medium is significantly increased by adding yeast extract to the medium.

However, since the starvation medium contains a sufficient amount of thiamine, biotin, and nicotinic acid, which are essential for the growth of Rhodobacter capsulatus ATCC 11166, the addition of yeast extract is generally known for growth. is not. As can be seen from the increase in the intracellular content of the polymer, the addition of the yeast extract performed here has the effect of increasing the amount of polymer accumulated, not growth.

Example 6 In exactly the same manner as in Example 5, the polymer was obtained by culturing while changing the ratio of the yeast extract (manufactured by Nacalai Tesque) to be added to the nitrogen-starved medium. The results are shown below.

[0058]

[Table 3]

Table 3 shows the following.

(1) When the yeast extract was not added, the amount of polymer produced was 0.01 g per liter of the medium, and the amount of the yeast extract was 0.1 g.
It is 1/10 or less when 5 g / l is added.

(2) When 2.0 g / l of yeast extract was added, the polymer content was lower than when 1.0 g / l was added, and the intracellular content was also reduced. Therefore, the larger the amount added, the more the polymer production does not increase.

Example 7 Cultivation was carried out in the same manner as in Example 3, except that instead of yeast extract, Ebios tablets (trade name, Tanabe Seiyaku Co., Ltd.) powdered with milk stick were used. . Table 4 shows the results.

[0063]

[Table 4]

It can be seen that good results can be obtained by using dried cells such as Evios as yeast. In the case of shrimp, the polymer content is high when 1.0 g is added per liter of medium.

Example 8 Rhodocobacter capsulatus ATCC 11166 was transferred to 300 ml of YPS medium and cultured in the same manner as in Example 1 to obtain washed cells. To this, 7 ml of physiological saline was added to prepare a suspension, and 2 ml of each suspension was taken and transplanted to a nitrogen starvation medium described below. A nitrogen-starved medium was prepared by adding 1 g / l of yeast extract (manufactured by Difco) to the composition described in Example 1. p
H makes three types of 4.7, 6.0 and 7.0.
ml was placed in a 500 ml Sakaguchi flask. After culturing for 15 hours, each pH is adjusted to 4.4, 4.6 to 1 using 1N hydrochloric acid.
The culture was 4.7 and 6.0 to 6.3, and the culture was continued for another 50 hours. After the culture, the cells were collected, the polymer was extracted by the method described in Example 4, and the content in the cells was measured. The results are summarized in Table 5.

That is, it is shown that the accumulation of the polymer (1) is remarkably reduced at pH 4.4 and hardly accumulated. In the case of culturing at pH 5.9 to 6.1 for 15 hours in (2), the polymer was accumulated, but it was 24% because it was kept at pH 4.6 to 4.7 for 50 hours. (3) 15 hours pH 6.6 ~
If you set the time to 6.0 and 6.3 after 50 hours, 42
% Capacity accumulated. The cell yield after culturing was not much different.

[0067]

[Table 5]

Example 9 Rhodobacter Capsulatus ATCC 11166 and Rhodobacter Sphaeroide
s) IFO 12203 was treated with YSP medium 20 in the same manner as in Example 4.
After transplanting to 0 ml and culturing for 48 hours at 27 ° C., cells were collected and washed cells were obtained.

The washed cells were transplanted into 200 ml of the medium described in Example 4 in which the carbon source for starvation culture was adjusted to 10 g / l of mannitol and 1 g / l of yeast extract (manufactured by Nacalai Tesque, Inc.).
The cells were cultured with shaking for a time. The cells were collected and the polymer was extracted in the same manner as in Example 4, and the content in the cells was measured. Table 6 shows the results
It was shown to.

[0070]

[Table 6]

From the results, it was found that the pH did not decrease,
It is clear that mannitol, which does not have to worry about control of cell growth by the aminocarbinol reaction, can be effectively used as a carbon source for PHB production.

Example 10 In order to confirm the properties of the polymer produced by the aerobic culture described in Example 1, the molecular weight distribution, thermal analysis (DSC), infrared absorption spectrum, and nuclear magnetic resonance spectrum were measured.
As a result of NMR analysis, the polymer was found to contain poly-3-hydroxylbutyric acid as a main component and a trace amount (5 mol
l%) of poly-3-hydroxyvaleric acid. Further, as a result of GPC measurement, it was found that the oligomer portion having several hundred Mw and the high molecular weight portion having several hundred thousand Mw had peaks. As a result of DSC measurement, it has a melting point at 163-165 ° C. The crystallinity was found to be significantly higher than the heat of fusion.

Example 11 In order to confirm the properties of the polymer produced in Example 4, thermal analysis (DSC), measurement of molecular weight distribution (GPC) and measurement of nuclear magnetic resonance spectrum (NMR) were performed. The conditions and results are shown below.

[0074] (1) GPC (test solution) after the m- cresol sample of about 30mg was completely dissolved at 10ml was added 40 ° C., measured up to 50ml with CHCl _3, 0.
The solution filtered with a 45 μm membrane filter was used as a test solution.

(Measurement conditions) Pump: CCPD (Tosoh) Column: GMHXL (Tosoh) Eluent: m-cresol / CHCl ₃ (1: 4) Flow rate: 1.0 ml / min Temperature: 40 ° (thermal bath CO-) 8011 Tosoh) Det .: RI-8010 (Tosoh) Injection amount: 50 μl (Molecular weight calibration curve) Prepared from standard polystyrene, and the molecular weight is represented by the converted value of the standard polystyrene molecular weight.

(Data) The molecular weight distribution curve is shown in FIG.

(Results) From the data, it was found that the polymer of Example 4 had Mw of 1.74 million and Mn of 800,000. Mw / Mn
Was 2.2.

(2) FIG. 2 shows the DSC measurement results.

From these results, the Tm of the polymer of Example 1 was
It was found to be 163.7 ° C.

(3) NMR NMR was performed with a magnetic field of 300 MHz and deuterated chloroform as a solvent.

[0093] ¹ for the polymer obtained in Example 4
FIG. 3 shows the results of H-NMR measurement, and FIG. 4 shows the results of ¹³ C-NMR measurement.
It was shown to.

From the NMR results, it was found that the polymer obtained in Example 4 was hydroxybutyric acid (PHB) and hydroxyvaleric acid (PHV)
It was found to contain units and the ratio was about 95: 5.

Example 12 The polymer recovered in Exp. No. 4 of Example 2 and the polymer produced and recovered in Example 4 according to the present invention were dissolved in chloroform, cast on a glass plate, and air-dried to form a film. This film was transferred to our laboratory (4-1, Kanebocho, Hofu City).
No.) and observed the strength, elongation, and morphological changes over time. The results are shown in Table 7, and it can be seen that the composition was decomposed until the strength was completely lost in about 60 days.

[0084]

[Table 7]

[0085]

According to the method for producing a polyhydroxy organic acid of the present invention, the production efficiency of the polymer, the rate of accumulation in cells and the stability of the produced polymer are higher than those of the conventionally proposed method. It has a major feature that has not been found. Further, since the intracellular accumulation rate can be extremely high, it is very advantageous for polymer extraction and purification. Further, the production process and conditions are very industrially advantageous as compared with conventional methods, such that the polymer can be produced without using light energy and without depending on nitrogen starvation. Therefore, its economic value is also very large.

[Brief description of the drawings]

FIG. 1 is a molecular weight distribution curve of a polymer obtained in Example 4.

FIG. 2 is a graph showing the measurement results of thermal analysis of the polymer obtained in Example 4.

FIG. 3 is a chart showing the result of ¹ H-NMR spectrum analysis of a polymer obtained in Example 4.

FIG. 4 is a chart showing the results of ¹³ C-NMR spectrum analysis of a polymer obtained in Example 4.

──────────────────────────────────────────────────続 き Continued on the front page (56) References NATO ASI Series. Applied Sciences, Vol. 186, p. 427-430 (1990) Archives of Microbiology, Vol. 155, No. 4, p. 337-340 (1991) Prog. Biomed. Eng. (Polymers in Medicine III), p. 19-26 (1988) Prikladaya Bioh imiyai Mikrobiolo giya, Vol. 25, No. 6, p. 785-789 (1989) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12P 7/62

Claims (10)

(57) [Claims] 1. A method for producing a polyhydroxy organic acid ester, comprising culturing a microorganism belonging to the genus Rhodobacter under aerobic conditions to produce a polymer comprising the polyhydroxy organic acid ester. 2. The method for producing a polyhydroxy organic acid ester according to claim 1, wherein the culturing of a microorganism belonging to the genus Rhodobacter is performed in one step in a medium containing a nitrogen source. 3. The method for producing a polyhydroxy organic acid ester according to claim 1, wherein the cultivation of the microorganism belonging to the genus Rhodobacter is performed in two stages: a preculture for growth and a subsequent nitrogen starvation culture. 4. The polyhydroxy organic acid ester according to claim 1, 2 or 3, wherein the culture is maintained while maintaining the pH of the culture solution from the start of polymer accumulation until the cell collection to 4.5 or more, and the polymer is accumulated in the cells. Manufacturing method. 5. The pH of the culture solution from the start of accumulation to the collection of bacteria is adjusted to 4.
5. The method for producing a polyhydroxy organic acid ester according to claim 4, wherein said ester is kept at 5 to 8.5. 6. The method for producing a polyhydroxy organic acid ester according to claim 1, wherein yeast is added to a medium for accumulating the polymer in cells. 7. The method for producing a polyhydroxy organic acid ester according to claim 6, wherein the yeast added to the medium is a yeast extract. 8. The method for producing a polyhydroxy organic acid ester according to claim 1, wherein a medium in which the main component of the carbon source is mannitol is used. 9. The polyhydroxy organic acid ester is poly-3
The method according to claim 1, which is a polymer containing hydroxybutyric acid as a main component. 10. The method according to claim 1, wherein the polyhydroxy organic acid ester is a copolymer containing an organic acid ester unit having 5 or more carbon atoms.