JPH0998793A - Production of polyester copolymer containing 4-hydroxybutyrate unit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject copolymer having a biodegradability and a bio-compatibility, and used for a medical material, etc., by culturing microorganisms belonging to Comamonas genus having an ability of producing poly-3-hydroxybutyrate in a medium containing gluconic acid and 1,4-butanediol. SOLUTION: This objective polyester copolymer excellent in biodegradability and bio-compatibility is obtained by inoculating microorganisms belonging to Comamonas genus and having an ability to produce poly-3-hydroxybutyrate (e.g.; Comamonas acidovorans, IFO13852) to a medium containing gluconic acid and 1,4-butanediol, performing an aerobic culture at 30 deg.C for 72hr to grow cells while aerating, producing and storing a polyester copolymer consisting of 3-hydroxybutyrate units expressed by the formula -OCH(CH3 )CH2 CO- and 4-hydroxybutyrate units expressed by -O(CH2 )3 CO- in the cells, and then collecting the product with an extraction, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to 3-hydroxybutyrate unit (hereinafter referred to as 3HB component) and 4-hydroxybutyrate, which are thermoplastic polymers which can be decomposed by microorganisms and have excellent biocompatibility. Unit (hereinafter 4HB
Ingredients. ) And a method for producing a copolyester consisting of

[0002]

2. Description of the Related Art Polyhydroxyalkanoate, which accumulates in cells of various kinds of microorganisms as an energy storage substance, has been attracting attention as a plastic material that can be decomposed by microorganisms. Among such polyhydroxyalkanoates, poly-3-hydroxybutyrate, which is composed of only 3HB component, is a functional material which is excellent in degradability by microorganisms and is also hydrolyzable, biocompatible, and optically active. Is evaluated as. However, since this poly-3-hydroxybutyrate is brittle because of its high crystallinity, it has not yet been put to practical use.

On the other hand, in recent years, the hydrogen bacterium Alcaligenes eutrophus is
It has been confirmed that a polyester copolymer composed of 3HB component and 4HB component is produced by culturing using 4-hydroxybutyric acid or 1,4-butanediol as a carbon source. , JP-A-64-488
21 and Japanese Patent Laid-Open No. 1-156320.

The copolymerized polyester composed of such 3HB component and 4HB component is the same as 4H in the copolymer.
It has been confirmed that depending on the amount of the component B present, it can be a polymeric material having a wide range of properties from highly crystalline plastics to highly elastic rubber. Therefore, by controlling the amount of the 4HB component present in the copolymer so as to have properties according to the intended use, it is expected to meet various uses. In particular, it has been clarified that a copolyester having a high abundance ratio of 4HB component exhibits a tough property of having a tensile strength of 100 MPa or more and is rapidly decomposed by enzymes such as PHB depolymerase and lipase ( Y. Saito and Y. Doi, Int. J. Biol. M
acromol., 16 , 99 (1994)).

However, when manufactured by the method using Alcaligenes eutrophus described in the above-mentioned JP-A-64-48821 and JP-A-1-156320,
Since only 4 mol% or less of the 4HB component in the copolymer can be obtained, it can be applied to applications requiring extremely flexible and tough properties such as structural materials, fishing line, and surgical line. No copolyester can be obtained.

Further, since Alcaligenes eutrophus is difficult to produce the above-mentioned copolyester in the presence of nitrogen or phosphorus, it is necessary to produce the copolyester by the method described in the above-mentioned publication. , The culture of the cells of Alcaligenes eutrophus is divided into the front and rear stages, and after the proliferation of the cells in the first stage, the culture of the cells in a medium containing a carbon source and limited in phosphorus and / or nitrogen in the second stage. It is necessary to culture, and the manufacturing process is complicated.

[0007]

The present invention has been made in order to solve the above-mentioned problems of the prior art, and the object of the present invention is to provide a copolyester comprising 3HB components and 4HB components. It is an object of the present invention to provide a method capable of producing a product having a high content ratio of 4HB component and further producing such a copolyester by one-step culture of a microorganism.

[0008]

The present invention relates to a comamonas (C
a microorganism belonging to the genus omamonas) and capable of producing poly-3-hydroxybutyrate is aerobically cultured in a medium containing gluconic acid and 1,4-butanediol to grow the cells, A 3-hydroxybutyrate unit represented by the following formula (1): —OCH (CH ₃ ) CH ₂ CO— (1) and the following formula (2)
: Generating copolyesters consisting of _{-OCH 2 CH 2 CH 2 CO- (} 2) represented by 4-hydroxybutyrate units, characterized in that to accumulate,
A method for producing the copolyester is provided.

The microorganism used in the present invention is Comamonas
(Comamonas) is not particularly limited as long as it is a microorganism having poly-3-hydroxybutyrate-producing ability, for example, Comamonas acidovor
ans) and the like, and among such strains, a strain easily available is Comamonas acidoboranes IFO13852.

In the present invention, these microorganisms are aerobically cultivated in a liquid medium containing gluconic acid and 1,4-butanediol as carbon sources to grow microbial cells. The culture is carried out aerobically, but the aeration rate per 1 liter of the medium per minute is preferably 0.1 to 1.7 liter, more preferably 0.2 to 1.0 liter.

The above-mentioned microorganism grows cells by aerobically culturing in a medium containing gluconic acid and 1,4-butanediol. In order to obtain a copolyester having a high proportion of 4HB component, The microorganisms are grown aerobically in a medium containing gluconic acid, and then, in the stationary phase of growth, 1,4-butanediol is fed to the medium to aerobically culture the cells to grow the cells. preferable. In this case, after the addition of 1,4-butanediol, it is preferable to culture microaerobically for the reason that the growth of cells is suppressed and polyester is accumulated in the cells as an energy storage substance. When performing microaerobic culture, the aeration amount per 1 liter of medium per minute is preferably 0.1 to 1.0 liter,
More preferably, it is 0.1 to 0.3 liter.

Gluconic acid and 1,4-butanediol may be used in such amounts that a copolyester composed of 3HB component and 4HB component can be produced and does not inhibit the growth of microorganisms. Depending on the strain of the microorganism used and the desired copolyester, etc., the total of gluconic acid and 1,4-butanediol is usually
It is about 10 to 40 g per liter of medium.

Further, first, the above-mentioned microorganism is aerobically cultured in a medium containing gluconic acid and aspartic acid or glutamic acid, and then 1,4-butanediol is fed to the medium at a stationary phase of growth to aerobically culture. When the bacterial cells are grown by doing so, the content of the 4HB component is higher than that of the copolymer polyester.
90 mol% or more of copolyester can be produced. In this case, the amount of aspartic acid or glutamic acid used is usually about 3 to 5 g per liter of the medium.

In addition to the above-mentioned gluconic acid, 1,4-butanediol, aspartic acid and glutamic acid, the medium contains ammonium sulfate, magnesium sulfate, potassium dihydrogen phosphate, disodium hydrogen phosphate, cobalt chloride, nickel chloride, Contains iron chloride, chromium chloride, calcium chloride, copper sulfate, etc. Culture is usually carried out in a medium as described above.
26-37 ° C, preferably 26-30 ° C, usually pH 6.0-
It is carried out at 8.0, preferably 6.5 to 7.0 for about 2 to 4 days.

After culturing as described above, the bacterial cells are separated and recovered from the culture solution by a usual method such as filtration and centrifugation, the bacterial cells are washed and dried, and the obtained dried bacterial cells are obtained. Is mixed with chloroform or methylene chloride, and the copolyester can be extracted by heating. Furthermore, the copolyester can be precipitated and isolated by adding the extract to hexane or water which is a poor solvent. According to the manufacturing method of the present invention,
The ratio of the 3HB component and the 4HB component in the copolyester can be adjusted arbitrarily, and the ratio of the 4HB component is 90%.
It is also possible to produce copolymerized polyesters in an amount of more than mol%.

[0016]

EXAMPLES The present invention will be described more specifically with reference to examples. The present invention is not limited to these examples. [Examples 1 to 6] In each example, a copolymer was produced using Comamonas acid bordans IFO13852 as follows. Medium with the following composition (pH 7.0)
Then, after inoculating Comamonas acidoborans IFO13852, the amount of aeration (liter) per minute to 1 liter of the medium was set to the amount shown in Table 1, and the cells were cultured at 30 ° C. for 72 hours.

Medium composition Deionized water 1.00 L Carbon source Sodium gluconate 15.14 g 1,4-Butanediol 5.44 g Ammonium sulfate 4.72 g Magnesium sulfate heptahydrate 0.30 g Potassium dihydrogen phosphate 2.65 g Disodium hydrogen phosphate 12 water 7.16g Mineral solution (with following composition) 3.00ml Composition of mineral solution 0.1N HCl 1.00L CoCl ₂ 119.0 mg NiCl ₂ 118.0 mg FeCl ₂ 9.7 mg CrCl ₂ 62.2 mg CaCl ₂ 7.8 mg CuSO ₄ 156.4 mg

After the completion of the culture, the culture solution was centrifuged at 9000 rpm for 5 minutes to separate and collect the bacterial cells, which were then freeze-dried to obtain dried bacterial cells. The dry cell weight per liter of medium is shown in Table 1. The dried bacterial cells obtained were treated with hot chloroform to extract the copolyester produced and accumulated in the bacterial cells, the obtained extract was concentrated, and then hexane was added to precipitate the copolyester. , Recovered. The weight of the dried cells and the weight of the dried copolyester were measured to calculate the content of the copolyester in the cells. Table 1 shows the results.

Further, the extracted copolyester was analyzed by gas chromatography to examine the ratio of 3HB and 4HB components. Table 1 shows the results. In addition, “PHA” in Table 1 represents a copolyester, and “3HB” represents
3-hydroxybutyrate unit, "4HB" represents 4-hydroxybutyrate unit, respectively.

[0020]

[Table 1]

As shown in Table 1, the cells of Comamonas acidoboranes (IFO13582) were active under aerobic conditions with an aeration rate of 1.667 liters per 1 liter of medium per minute, and the PHA content was dry. It was 1% based on the cell weight. On the other hand, under the conditions of airflow of 0.333 liters and 0.667 liters, a copolymerized polyester having a ratio of 4HB component of 50 mol% or more was synthesized at a content of 19% based on the dry cell weight.

Example 7 3 L of the following medium containing sodium gluconate and L-aspartic acid as carbon sources
Was used. After inoculating Comamonas acidoborans (IFO13582) cells cultured in 100 ml of this medium, the culture was started at a culture temperature of 30 ° C. with an aeration rate of 1.667 liter per 1 liter of the medium per minute. The turbidity of the culture solution was continuously monitored (see Fig. 1), and when it became steady (after 20 hours), 1,4-butanediol was adjusted to 5.44 g per liter of the culture medium. Fed-batch. From this point, the aeration amount per 1 liter of the medium per minute was reduced to 0.333 liter, and the culture was continued for further 52 hours.

Medium composition Deionized water 1.00 L Carbon source Sodium gluconate 15.14 g L-Aspartic acid 5.00 g 1,4-Butanediol 5.44 g Ammonium sulfate 4.72 g Magnesium sulfate heptahydrate 0.30 g Potassium dihydrogen phosphate 2.65 g Phosphorus Disodium hydrogen hydrate 12 hydrate 7.16 g Mineral solution (composition similar to Examples 1 to 6) 3.00 ml

The culture broth was collected over time, and the dry cell weight per liter of the medium (g / L), the PHA in the dry cell
The content (wt%), and the respective content ratios (mol%) of the 3HB component and the 4HB component were measured with time by the same method as in Examples 1 to 6 above. The content (g / L) of carbon source (gluconic acid, 1,4-butanediol, L-aspartic acid) and nitrogen source (ammonium sulfate) per liter of medium in the supernatant was measured by high performance liquid chromatography. It was quantified over time. FIG. 1 is a graph showing changes with time of the components contained in the medium, turbidity, cell weight, PHA content, and composition of the copolymerized polyester when L-aspartic acid was added to the medium.

FIG. 2 shows the same results as above when L-aspartic acid was not added to the medium. From FIG. 1, under the condition that L-aspartic acid was added to the medium, 1,
The proportion of 4HB component increased after feeding 4-butanediol, and after 72 hours, a copolymerized polyester having a proportion of 4HB of 93 mol% was synthesized at a content of about 30% based on the dry cell weight. Recognize. On the other hand, under the condition that L-aspartic acid was not added to the medium (FIG. 2), poly-3-hydroxybutyrate was accumulated at a content of 15% or more based on the dry cell weight from the start of culture to the stationary phase. Furthermore, the proportion of the 4HB component increased after the feeding of 1,4-butanediol, but the proportion after 72 hours was about 80 mol%, and the content was about 20% based on the dry cell weight. there were.

FIG. 3 is a summary of the productivity calculated from the above results. The fraction of 4HB component in the copolyester with and without the addition of L-aspartic acid in the medium ( Mol%), 4 in 1 liter of medium
3 is a graph showing changes over time in the amount (g / L) of HB component and the yield (% by weight) of 4HB component from 1,4-butanediol. Under the condition that L-aspartic acid was added to the initial medium, the concentration of 4HB component per unit volume after 72 hours was
It was 1.5 g / L, which was about twice the result of the case where L-aspartic acid was not added. Furthermore, looking at the yield of 4HB component from 1,4-butanediol when L-asparaic acid was added, it reached nearly 30% after 72 hours, which was more than twice the value when not added. ing. in this way,
By adding L-aspartic acid to the initial medium, the productivity of the 4HB component after adding 1,4-butanediol can be further improved.

Example 8 3 L of the following medium containing sodium gluconate and L-glutamic acid as carbon sources was used. After inoculating Comamonas acidoborans (IFO13582) cells cultured in 100 ml of this medium, the culture was started at a culture temperature of 30 ° C. with an aeration rate of 1.667 liter per 1 liter of the medium per minute. The turbidity of the culture solution was continuously monitored (see Fig. 4), and when it reached a steady state (after 20 hours), 1,4-butanediol was adjusted to 5.44 g per liter of the culture medium. Fed-batch.
From this point, the aeration amount per 1 liter of the medium per minute was reduced to 0.333 liter, and the culture was continued for further 52 hours.

Medium composition Deionized water 1.00 L Carbon source Sodium gluconate 15.14 g L-Glutamic acid 5.00 g 1,4-Butanediol 5.44 g Ammonium sulfate 4.72 g Magnesium sulfate heptahydrate 0.30 g Potassium dihydrogen phosphate 2.65 g Phosphoric acid Disodium hydrogen dodecahydrate 7.16 g Mineral solution (same composition as Examples 1 to 6) 3.00 ml

The culture broth was collected over time, and the dry cell weight (g / L) per liter of the medium, the PHA in the dry cell
The content (wt%), and the respective content ratios (mol%) of the 3HB component and the 4HB component were measured with time by the same method as in Examples 1 to 6 above. The content (g / L) of the carbon source (gluconic acid, 1,4-butanediol, L-glutamic acid) and the nitrogen source (ammonium sulfate) in the supernatant per liter of medium was measured by high performance liquid chromatography. Quantitatively. FIG. 4 is a graph showing changes with time of the components contained in the medium, turbidity, cell weight, PHA content, and composition of the copolymerized polyester when L-aspartic acid was added to the medium.

From FIG. 4, the 4HB fraction increased after feeding 1,4-butanediol, and the 4HB fraction 90 after 72 hours of culture.
It can be seen that% of the copolyester is accumulated at a content of 26% based on the dry cell weight. Further, the yield of 1,4-butanediol to 4HB component was 23%.

[0031]

According to the present invention, a polyester copolymer having a high abundance ratio of 3HB component and 4HB component, especially 4HB component, can be produced in good yield. Since the microorganism used in the present invention can grow even in the presence of phosphorus or nitrogen, it is not necessary to divide the culture of the bacterium into front and rear stages, and the microbial growth and the copolymer synthesis are performed in one step. It is possible,
It is efficient. Further, the copolymer obtained in the present invention,
Since it has excellent various properties, it is extremely suitable as a raw material for medical materials such as surgical systems and materials for fixing bone fractures, and is expected to be applied to various fields such as use in a controlled release system.

[Brief description of drawings]

FIG. 1 is a diagram showing a graph showing changes with time in composition of medium, turbidity, cell weight, PHA content, and composition of copolymerized polyester when L-aspartic acid was added to the medium in Example 7. is there.

FIG. 2 shows the medium components, turbidity, cell weight, and P when the medium was not mixed with L-aspartic acid in Example 7.
It is the figure showing the graph which shows the time-dependent change of HA content and the composition of copolyester.

FIG. 3 shows the proportion of 4HB component in the copolyester with and without L-aspartic acid in the medium in Example 7, 4HB in 1 liter of the medium.
It is the figure showing the graph which shows the change over time of the amount of a component, and the yield from the 1,4-butanediol of 4HB component.

FIG. 4 is a diagram showing a graph showing time-dependent changes in medium components, turbidity, cell weight, PHA content, and composition of copolyester in the case where L-glutamic acid was added to the medium in Example 8. .

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takashi Tomozawa 1-25-1 Nishishinjuku, Shinjuku-ku, Tokyo Taisei Corporation

Claims (4)

[Claims] 1. A polymycin which belongs to the genus Comamonas
A microorganism capable of producing -3-hydroxybutyrate is aerobically cultured in a medium containing gluconic acid and 1,4-butanediol to grow the microbial cells, whereby the following formula (1): _{-OCH (CH 3) CH 2 CO-} (1) and 3-hydroxybutyrate units represented by the following formula (2)
: Generating copolyesters consisting of _{-OCH 2 CH 2 CH 2 CO- (} 2) represented by 4-hydroxybutyrate units, characterized in that to accumulate,
A method for producing the copolyester. 2. The growth of the bacterial cells is preferably carried out by first aerobically culturing the microorganism in a medium containing gluconic acid and then feeding 1,4-butanediol to the medium at a stationary phase of the growth. The method for producing a copolyester according to claim 1, which is carried out by air culture. 3. The method for producing a copolymerized polyester according to claim 2, wherein the aerobic culture after feeding the 1,4-butanediol to the medium is a microaerobic culture. 4. A copolymerized polyester containing 90 mol% or more of the unit represented by the above formula (2) is produced and accumulated in the cells by using a medium further containing aspartic acid or glutamic acid as the medium. The method for producing a copolyester according to claim 3, characterized in that.