JPH06181784A - Production of copolyester - Google Patents

Abstract

PURPOSE:To obtain a copolyester having adequate mechanical strength, biocompatibility and biodegradability, useful as e.g. a medical material, by culture of a kind of Alcaligenes bacteria using, as carbon source, a combination of sucrose, gamma-butyrolactone, 4-hydroxybutyric acid and a specific glycol. CONSTITUTION:A medium comprising (A) as carbon source, a combination of sucrose, gamma-butyrolactone, 4-hydroxybutyric acid and an alkylene glycol of formula HO(CH2)nOH (n is an even number of 4-12) and (B) as nitrogen source, an inorganic nitrogen compound such as ammonia, ammonium salt or nitrate, or an organic nitrogen compound such as urea, casein or peptone, is inoculated with a kind of Alcaligenes bacteria [e.g. Alcaligenes latas (ATCC 29713)] to conduct shaking culture at 30 deg.C for 48hr, and the resultant microbes are separated and dried, and then subjected to extraction with hot chloroform, thus affording the objective copolyester made up of 97-80mol% of 3-hydroxybutyrate unit and 3-20mol% of 4-hydroxybutyrate unit.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing a polyester copolymer comprising 3-hydroxybutyrate units (hereinafter referred to as 3HB units) and 4-hydroxybutyrate units (hereinafter referred to as 4HB units). More specifically, it relates to a method for producing a biodegradable polyester copolymer having improved productivity.

[0002]

2. Description of the Related Art Poly-3-hydroxybutyrate (hereinafter referred to as PHB) is a thermoplastic polymer that accumulates in the cells of many microorganisms as an energy storage substance and exhibits excellent biodegradability and biocompatibility. Therefore, it has attracted attention as a clean plastic that preserves the environment, and it has been expected for many years to be applied to various fields such as medical materials such as surgical threads and materials for fixing bone fractures, and sustained-release systems that gradually release drugs and pesticides. It was Particularly in recent years, synthetic plastics have become a serious social problem from the viewpoint of environmental pollution and resource recycling,
PHB and its copolymers are attracting attention as petroleum-independent biopolymers. However, PHB has a problem in physical properties that it is inferior in impact resistance.

Recently, a copolymer containing 3HB units and 3-hydroxyvalerate units (hereinafter referred to as 3HV units) and a method for producing the same have been researched and developed, for example, JP-A-57-150393. Sho 59-22
No. 0192. However, the copolymer having a high 3HV unit content was inferior in heat resistance. That is, it was known that when the 3HV unit of the copolymer increased from 0 to 33 mol%, the melting temperature (Tm) rapidly decreased from 185 ° C. to 85 ° C. with this increase (T.
L. Bluhm et al Macromolecule
les, 19, 2871 (1986).

On the other hand, research and development have also been carried out on copolymers containing 3HB units and 4HB units and methods for producing the same, for example, Japanese Patent Laid-Open No. 48821, 1-22227.
88, 1-304891, 279292. Such a copolymer has a high melting point even when the content of 4HB units is high, and since the impact resistance is improved, it has a high industrial value.

However, the method for producing a copolymer containing the above 3HB units and 4HB units is a method using fructose as a carbon source, or γ-butyrolactone, 4
- hydroxybutyric acid, or _{HO (CH 2) n OH (} n is 4
Only a method using a compound represented by (even number of -12) has been reported. Therefore, there is a problem in productivity and cost of raw materials for production on an industrial scale.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing a copolymer comprising 3HB units and 4HB units, which is mainly composed of inexpensive raw materials and has excellent productivity.

[0007]

Means for Solving the Problems The inventors of the present invention have made earnest studies on a method capable of industrially producing a copolymer consisting of 3HB units and 4HB units. As a result, in the presence of Alcaligenes spp., Particularly Alcaligenes rattus. , A copolymer comprising a specific 3HB unit and a specific 4HB unit can be efficiently produced by using a specific carbon source in combination with sucrose, and an expensive carbon source such as γ-butyrolactone is highly efficiently used in a 4HB unit. As a result, they have found that they can be introduced into a copolymer, and have reached the present invention.

That is, the gist of the present invention is to produce a polyester copolymer comprising 97 to 80 mol% of 3-hydroxybutyrate units and 3 to 20 mol% of 4-hydroxybutyrate units in the presence of Alcaligenes. In the method, sucrose and γ-butyrolactone as carbon sources,
4-hydroxybutyric acid, HO (CH ₂ ) _n OH (n is 4 to
The present invention also relates to a method for producing a polyester copolymer, characterized in that it is used in combination with at least one selected from compounds represented by the even number 12).

The present invention will be described in detail below. The polyester copolymer of the present invention contains 3 to 80 mol% of HB units.
And 4HB units 3-20%. Where 3HB
The unit and the 4HB unit are constituent units represented by the following formulas, respectively.

[0010]

## STR1 ## 3HB _{units; -OCH (CH 3) CH 2} CO- 4HB units; -OCH ₂ CH ₂ CH ₂ CO-

Regarding the microorganisms used in the present invention, P
There is no particular limitation as long as it is an Alcaligenes genus capable of producing HB. For example, Alcaligenes faecalis (A
lcaligenes faecalic, Alcaligenes r
uhlandii), Alcaligenes ratus (Alc
aligenes ratus, Alcaligenes eutroph
s) etc., and representative examples of strains belonging to these strains are Alcaligenes faecalis ATCC 8750, Alcaligenes rulandi ATCC15749, Alcaligenes ratus ATCC 29712, ATCC29.
713: Alcaligenes Yutrofus H-16ATC
C17699 etc. are mentioned. Of these, Alcaligenes ratus ATCC29712, ATCC2971
3 is particularly preferable in terms of productivity.

The mycological properties of these microorganisms belonging to the genus Alcaligenes are described, for example, in "BERGEY'S MAN".
UAL OF DETERMINATIVE BACT
ERILOGY: Eight Edition,
The mycological properties of Alcaligenes eutrophus H-16 are described, for example, in “J. Gen. Microbiol., 1
15 , 185-192 (1979).

As the culture method of the present invention, a usual culture method for growing a microorganism is applied. For example, a two-stage batch culture consisting of a pre-stage culture (proliferation of cells) and a post-stage culture (production of a copolymer under the restriction of a nitrogen source), or a single stage in which cell growth and copolymer production are performed simultaneously Batch culture is possible. Furthermore, continuous culture can be carried out in which a bacterial cell is grown and a copolymer is produced while dropping a nitrogen source or a carbon source.

The carbon sources used are sucrose and γ-
Bularolactone, 4-hydroxybutyric acid, and HO (CH
₂ ) Used in combination with at least one selected from compounds represented by _n OH (n is an even number of 4 to 12). Of these, it is most preferable to use sucrose and γ-butyrolactone together. The concentration of the above carbon source is preferably in the range of 3 to 100 g / l in terms of the productivity of the copolymer, and 5 to 20 g / l.
Is particularly preferred. Also, the above γ used in combination with sucrose
-The amount ratio of butyrolactone and the like (hereinafter referred to as γ-BLs) is preferably 0.5 to 6% by weight, and particularly preferably 1 to 5% by weight based on the total carbon source in order to efficiently introduce 4HB units. ~ 3% by weight. When the amount of γ-BLs is less than the above range, the introduction rate of the 4HB component into the copolymer is low, and when it is more than the above range, the production amount of the copolymer itself and the γ-BLs to the 4HB component are changed. Conversion efficiency is reduced.

Incidentally, within the scope of the object of the present invention,
It is also possible to use other known carbon sources in combination. Examples of such other carbon sources include synthetic carbon sources such as methanol, ethanol and acetic acid, inorganic carbon sources such as carbon dioxide, natural products such as yeast extract, molasses, peptone and meat extract, arabinose, fructose, glucose and mannose. And the like.

Examples of the nitrogen source include inorganic nitrogen compounds such as ammonia, ammonium salts and nitrates and / or nitrogen-containing substances such as urea, corn steve liquor, casein, peptone, yeast extract and meat extract. Particularly preferred is ammonium sulfate. As the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt,
Nickel salts, chromium salts, boron compounds and iodine compounds, preferably potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and the like can be mentioned.

If necessary, vitamins and the like can also be used. As the culture conditions, the temperature is, for example, about 20 to 45 ° C., preferably about 30 to 42 ° C., and the pH is, for example, about 6 to 10 and preferably about 6.5 to 9.5. It It cultures aerobically under such conditions. When the culture is carried out under these conditions, the growth of microorganisms becomes relatively poor, but it does not prevent the culture under these conditions.

From the culture solution obtained by culturing in this way,
The cells are separated and recovered by a usual solid-liquid separation means such as filtration and centrifugation, and the cells are washed and dried to obtain dried cells. From the dried cells, for example, chloroform is used. A copolymer produced with a different organic solvent is extracted, and a poor solvent such as hexane is added to the extract to precipitate the copolymer. According to the production method of the present invention, the ratio of 3HB units and 4HB units in the copolymer can be arbitrarily adjusted by the ratio of the added carbon source γ-BLs.

[0019]

EXAMPLES The present invention will be described more specifically by way of examples. The present invention is not limited to these examples. <Examples 1-4 and Comparative Examples 1-2> Alcaligenes
The copolymer was prepared using ratus (ATCC 29713).

Cultivation of bacterial cells 100 ml of a medium in Kolben having the following composition was inoculated with the above-mentioned microorganism and shake-cultured at 30 ° C. for 48 hours. The cells were separated from the obtained culture solution by centrifugation to obtain the cells.

[0021]

[Table 1]

* Trace-element solu
composition (in 1 liter) is H₃BO₃ 0.
3g, CoCl₂・ 6H₂O 0.2g, ZnSO_Four・
7H ₂O 0.1g, MnCl₂・ 4H₂O 30m
g, NaMoO_Four・ 2H₂O30mg, NiCl / 6H
₂O 20mg and CuSO_Four・ 5H₂O 10 mg
is there. ** Sucrose and γ-as described in Table 1 below as carbon sources
Butyrolactone or 4-hydroxybutyric acid was used.
(Unit: g / l medium) The cells were washed with distilled water and then with acetone,
This is dried under reduced pressure (20 ° C, 0.1 mmHg) and dried.
Got the body

Separation and recovery of the copolymer: The copolymer was extracted from the dried cells thus obtained with hot chloroform, hexane was added to the extract to precipitate the copolymer, and the precipitate was filtered. It was taken and dried to obtain a copolymer.

Characteristics of Copolymer The composition of the copolymer thus obtained and other properties were measured as follows, and the results are shown in Table 1. Composition: 3HB unit, 4HB unit molar ratio H-NMR
It was determined from the (270 MHz) spectrum.

Dry cell weight: The cells collected by centrifugation were freeze-dried. Polyester content: Polyester content in dry cells (mol%) 4HB conversion: Ratio of γ-BLs introduced into the copolymer as 4HB with respect to γ-BLs used.

[0026]

[Table 2]

[0027]

According to the present invention, a copolymer comprising 3HB units and 4HB units can be efficiently obtained in the presence of a small amount of γ-BLs. Since the copolymer obtained by the method of the present invention has various excellent mechanical properties and biocompatibility or biodegradability, it is suitable as a raw material for medical materials such as surgical threads and fracture fixation materials. In addition, it is expected to be applied to various fields such as use in sustained release systems.

Claims (4)

[Claims] 1. 3-hydroxybutyrate units 97-8
0 mol% and 4-hydroxybutyrate units 3-20
In the method for producing a polyester copolymer comprising mol% in the presence of a bacterium of the genus Alcaligenes, sucrose and γ-butyrolactone as a carbon source, 4-hydroxybutyric acid and HO (CH ₂ ) _n OH (n is 4 to 12). And at least one selected from the compounds represented by the formula (1), the method for producing a polyester copolymer. 2. The Alcaligenes genus is Alcaligenes
The method for producing a polyester copolymer according to claim 1, which is Latus. 3. The amount of γ-butyrolactone in the total carbon source is 0.5.
The method for producing a polyester copolymer according to claim 1 or 2, wherein the content is from 6 to 6% by weight. 4. The process for producing a polyester copolymer according to claim 1, wherein the concentration of the total carbon source is 3 to 100 g / liter.