JPH0532768A - Novel polyester copolymer and production thereof - Google Patents

Abstract

PURPOSE:To produce a biodegradable and thermoplastic polyester copolymer by culturing a microorganism having a poly-3-hydroxybutyrate-producing ability in a medium containing lebulinic acid. CONSTITUTION:A microorganism having a poly-3-hydroxybutyrate-producing ability such as Alcaligenes.hydrogenophilus is cultured in a medium containing lebulinic acid or a salt thereof such as sodium salt, and the cultured solution is subjected to a centrifugal separation etc., to recover the cells of the microorganisms, which are washed to provide the objective copolymer having repeating units of-OCH(CH2)CH3CO-,-OCH(CH2CH3)CH2-, and-OCH(CH3)CH2 CH2 CO-.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel polyester copolymer having biodegradability and thermoplasticity and a method for producing the same. The polyester copolymer is produced from general-purpose plastics (fishing nets, various films, etc.). It can be widely used for special purpose plastics (polymers for medical use, implant materials, etc.).

[0002]

2. Description of the Related Art 3-Hydroxybutyrate (hereinafter referred to as 3HB
Since it was discovered in 1925 that poly-3-hydroxybutyrate (hereinafter abbreviated as PHB) having a structural unit of () is accumulated in microorganisms, many studies have been conducted on polyesters produced by microorganisms. Came. Since the polyester is a thermoplastic polymer, it can be melt processed to produce various products such as fibers and films. In addition, since it is stored in microorganisms as an energy storage substance and shows biodegradability, it has the advantage that it can be completely assimilated by microorganisms in the natural world, and like many synthetic polymer compounds currently in widespread use. It does not remain in the natural environment and cause no adverse effects. Also, because it has excellent biocompatibility, it is expected to be applied to medical materials such as base materials for drug delivery systems that gradually release drugs, etc., as surgical threads and implant members, or directly to the affected area. Has been done.

However, since PHB has high crystallinity, it is hard and brittle, and has a physical property defect of lacking impact resistance, and industrial production has been postponed. Therefore, research aimed at improving this physical property has been earnestly conducted.

For example, in JP-A-57-150393 and JP-A-59-220192, 3-hydroxyvaleate (hereinafter abbreviated as 3HV) is used as a copolymerization component.
Copolymers containing units, and JP-A-64-48821 discloses copolymers each containing 4-hydroxybutyrate (hereinafter abbreviated as 4HB) units as a copolymer component. These copolymers are more flexible than PHB and are excellent in various properties as a polymer compound, but their physical properties have not been sufficiently improved, and their use was limited. . Therefore, from the need for further improvement, a copolyester having a novel component has been desired.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems and provides a novel polyester copolymer to provide a biodegradable polyester copolymer having unprecedented properties and physical properties. It is an attempt to provide coalescence.

[0006]

The novel polyester copolymer of the present invention is characterized by having the following repeating units a, b and c. _{-OCH (CH 3) CH 2 CO-} ... a -OCH (CH 2 CH 3) CH 2 CO- ... b -OCH (CH 3) CH 2 CH 2 CO- ... c also the polyester copolymer poly -3 -Hydroxybutyrate-producing microorganisms, levulinic acid and /
Alternatively, it can be produced by culturing in a medium containing the salt.

[0007]

The novel polyester of the present invention has the above-mentioned a, b, c
Of the above, that is, 3HB units, 3HV units and 4-hydroxyvaleate (hereinafter abbreviated as 4HV) units, and a polyester copolymer containing the above 4HV units was first found by the present inventors. It is a thing.
As repeating units other than the above structural units, 3-hydroxyalkanoate, 4-hydroxybutyrate, 5-
Hydroxyvaleate or the like may be contained to the extent that physical properties are not impaired.

In producing the above polyester copolymer, it may be possible to synthesize it by chemical synthesis, but at the present time, it is considered preferable to produce it by a microorganism.

The microorganism used in the present invention is not particularly limited as long as it is a microorganism capable of producing PHB.
Alcaligenes de
nitrificans), Alcaligenes eutrophus (Alca
ligenes eutrophus), Alcaligenes fuecalis (Alc
aligenes faecalis), Alcaligenes ratus (Alcalig
enes latus), Alcaligen Paradukas (Alcaligen)
es paradoxus) and Alcaligenes hydrogenophilus, and other genus Alcaligenes, Bacillus cereus, Bacillus megaterium, and other genus Bacillus, Pseudomonas cepacia
, Pseudomonas oleo
vorans) and other Pseudomonas species.

As a typical example of the microorganism belonging to the genus Alcaligenes, Alcaligenes denitrificans JCM
(Japan Collection of Microorganisms) 5491, Alcaligenes eutrophus ATCC 17697, 17699, Alcaligenes fuecalis JCM 1474, Alcaligenes ratus ATCC 17713 and Alcaligenes hydrogenophilus ATCC 33178.

The mycological properties of these microorganisms are
Y'S MANUAL OF SYSTEMATIC BACTERIOLOGY; The Williams
& Wilkins Publishing ”, and the mycological properties of Alcaligenes hydrogenophilus ATCC 33178 are described in J.Gen.App.
L. Microbial., 25 , 53-58 (1979) and the like, and these strains are easily available from public microbial preservation organizations.

In culturing these microorganisms,
As in the conventional method, there are mainly pre-culture (proliferation culture) for growing microbial cells and post-culture (polyester production culture) for restricting nitrogen or phosphorus to produce and accumulate polyester copolymer in the cells. It is preferable to culture in two stages.

Proliferation culture is carried out merely to increase the number of microorganisms, and ordinary culture methods can be applied. That is, it may be appropriately set according to the culture medium and culture conditions in which the microorganism used can grow.

The medium component is not particularly limited as long as it is a substance that can be assimilated by the microorganism to be used, and examples of the carbon source include synthetic carbon sources such as methanol, ethanol, acetic acid and gluconic acid, molasses, and starches of cereals. , Natural products such as fibrous fibers,
Glucose, sugars such as fructose, and nitrogen sources include
For example, ammonia, ammonium salts, inorganic nitrogen compounds such as nitrates, or urea, peptone, tryptone, organic nitrogen compounds such as yeast extract, meat extract, inorganic components,
For example, calcium salts, magnesium salts, potassium salts, sodium salts, phosphates and the like, and if necessary, growth factors such as vitamins can be used. Bacteria that can utilize carbon dioxide, such as those belonging to the genus Alcaligenes (autotrophic bacteria)
In this case, the culture can be performed without adding the above-mentioned carbon source.

The culture conditions are appropriately determined according to the type of microorganisms used, but it is generally preferable to culture under the following conditions. The temperature is the temperature at which the microorganism grows, that is, 20 to 40 ° C.
It is preferably about 30 ° C., and the pH is about pH at which the microorganism grows, that is, about 6 to 10, preferably about 6.5 to 8.5. It is preferable to culture aerobically under such culture conditions. The culture method may be either batch culture or continuous culture.

The cells obtained by the growth culture are further subjected to polyester production culture under nitrogen or phosphorus limitation. That is, bacterial cells of microorganisms are separated and recovered from a culture solution obtained by growth culture by a usual solid-liquid separation method such as filtration and centrifugation, and the bacterial cells are subjected to polyester production culture, or substantially nitrogen or Phosphorus is depleted and cells are transferred to the polyester production culture without recovery.

In the polyester production culture, the medium or culture solution used is substantially free of nitrogen or phosphorus, and contains lebronic acid and / or a salt thereof as a copolymer constituent precursor. , Proliferation culture is performed.

As the salt of levulinic acid, sodium,
Examples thereof include salts such as potassium, magnesium and calcium. These compounds may be added during growth culture or during polyester production culture. In the latter case, it may be at any point in the early and final stages of the culture, but the early stage is preferable.

From the culture broth obtained by culturing in this manner, cells are collected and washed by a solid-liquid separation method such as filtration or centrifugation. Thereafter, the obtained bacterial cells are treated by a conventional method to recover the polyester copolymer. The polyester copolymer produced from the bacterial cells is extracted with an organic solvent such as chloroform, and the extract is extracted with n-
The copolymer is precipitated and recovered by adding it to a solvent such as hexane.

The present invention will be described in more detail with reference to the following examples, but the following examples do not limit the present invention.
All modifications and implementations that do not depart from the spirit of the description below are included in the technical scope of the present invention.

[0021]

【Example】

Example 1 <Proliferation culture and production culture> A polyester copolymer was produced using Alcaligenes Hydrogenophilus ATCC33178. First, as growth culture, 200 ml of a medium having the composition shown below was placed in a 500 ml Erlenmeyer flask and sterilized, and then the above microorganism was inoculated and shake-cultured at 30 ° C. for 24 hours.
The cells were recovered from the culture solution at the end of the logarithmic growth phase by aseptic centrifugation.

[0022]

Next, as a polyester production culture, 200 ml of a medium having the following composition was placed in a 500 ml Erlenmeyer flask and sterilized, and the cells obtained by the growth culture were added to 1 ml per 200 ml.
It was suspended at a rate of g and cultured at 30 ° C. for 3 days. The cells were recovered from the obtained culture solution by centrifugation.

[0024]                     [Polyester culture medium composition]                                           (Addition amount)                 ・ Potassium dihydrogen phosphate 2.66 g                 ・ Dipotassium hydrogen phosphate 4.67 g                 ・ Magnesium sulfate heptahydrate 0.2 g                 ・ Levulinic acid *                 ・ Mineral solution * * 1.0 ml                 ・ Distilled water 1000 ml                                     (Adjusted to pH 7)            * Levulinic acid was added by changing the ratio shown in Table 1 below.           ** Composition of mineral solution                                           (Addition amount)                 ・ Cobalt chloride 11.90 mg                 ・ Nickel chloride 11.80 mg                 ・ Chromium chloride 6.22 mg                 ・ Calcium sulfate 15.64 mg                 ・ Iron chloride 970.00 mg                 ・ Calcium chloride 780.00 mg                 ・ 0.1N hydrochloric acid solution 100 ml

<Treatment of bacterial cells> The viable cells (5.24 g / l) obtained by the polyester production culture were washed with distilled water, washed with acetone, left to dry in a desiccator containing silica gel, and dried. Cells (1.58 g / l) were obtained. <Recovery of Copolymer> A polyester copolymer is extracted from the dried bacterial cells obtained as described above with hot chloroform using a Soxhlet extractor, and this extract is mixed with about 10 to 20 volumes of n-hexane. In addition to the above, a polyester copolymer was precipitated, and this precipitate was recovered and dried.

The composition of the polyester copolymer thus obtained was measured by gas chromatography, the average molecular weight by gel permeation chromatography (GPC), and the structure by nuclear magnetic resonance (NMR). The measurement results and the like are shown in Table 1 and the NMR results are shown in FIGS.

[0027]

[Table 1]

Example 2 Alcaligenes eutrophus ATCC 1769 as bacterial cells
Polyester production culture was performed according to the conditions of Experiment No. 5 of Example 1 except that 7 was used. As a result, the dry cell yield was 1.773 g / l, the polyester accumulation was 0.858 g / l, the polyester accumulation was 48.4% by weight, and the ratio of 4HV in polyester was 4HV.
It was 3.6 mol%.

[0029]

Industrial Applicability According to the present invention, it is possible to obtain a novel polyester copolymer containing 3HB units, 3HV units and 4HV units. Furthermore, the copolymer obtained in the present invention is biodegradable and is considered to have unprecedented performance, and is expected to be applied to various fields. Since it is a compound similar to PHB, it is expected to be a biocompatible material, and it is also expected to be applied to raw materials for medical materials such as surgical threads and materials for fixing bone fractures, as well as materials for drug delivery systems.

[Brief description of drawings]

FIG. 1 shows 75 MHz, ¹³ C of the copolymer obtained by the present invention.
It is an NMR spectrum, and the numbers shown in the structural formulas in the figure correspond to the numbers of the respective peaks.

FIG. 2: 300 MHz, ^{1 of the} copolymer obtained by the present invention
It is an H-NMR spectrum, and the numbers shown in the structural formulas in the figure correspond to the numbers of the respective peaks.

Claims (2)

[Claims] 1. A novel polyester copolymer having the following repeating units a, b and c. _{-OCH (CH 3) CH 2 CO-} ... a -OCH (CH 2 CH 3) CH 2 CO- ... b -OCH (CH 3) CH 2 CH 2 CO- ... c 2. The novel polyester copolymer according to claim 1, which comprises a step of culturing a microorganism having a poly-3-hydroxybutyrate-producing ability in a medium containing levulinic acid and / or a salt thereof. Method for producing polymer.