JPH04325094A - Production of copolyester - Google Patents

Abstract

PURPOSE:To produce a copolyester comprising a 3-hydroxybutylate unit (3HB) and a 4-hydroxybutyrate(4HB) unit, having a higher ratio (molecular ratio) of 4HB composition to 3HB component industrially and readily. CONSTITUTION:When a bacterium capable of producing a poly 3- hydroxybutylate is incorporated with citric acid and/or a citrate in the presence of a substrate and cultured a copolyester having a higher ratio of 4HB component to 3HB component is formed to readily produce a copolyester having a high ratio of 4HB component.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention is directed to the production of polyester copolymers containing 3-hydroxybutyrate units (hereinafter referred to as 3HB component) and 4-hydroxybutyrate units (hereinafter referred to as 4HB component) using microorganisms. The present invention relates to a manufacturing method for producing the product. More specifically, the present invention relates to a novel production method in which a microorganism capable of producing poly-3-hydroxybutyrate is cultured in the presence of a substrate to easily produce a copolymerized polyester with a high content of 4HB component.

0002

[Prior Art] Poly-3-hydroxybutyrate (hereinafter referred to as PHB) is known as an energy storage substance that is accumulated in the cells of many microorganisms, and is a thermoplastic material that exhibits excellent biodegradability and biocompatibility. Since it is a resin, it is expected to be applied in a wide range of fields, including medical materials such as surgical threads and fracture fixation materials, and system materials for sustained release of pharmaceuticals and agricultural chemicals.

[0003] In recent years, expectations have increased for PHB as a substitute for synthetic plastics that are associated with environmental pollution, but industrial application has been postponed due to physical property problems such as poor impact resistance.

From this point of view, 3HB components and 4H
Research and development has been carried out on copolymers containing component B and methods for producing the same, including, for example, JP-A-64-48821, JP-A-1-222788, and JP-A-1-30.
4891 and JP-A-2-27992, respectively.

[0005] The method for producing the copolymer disclosed in these publications is similar to the conventional method for producing PHB, in which microorganisms are grown in the first step, microorganisms are cultured under limited nitrogen or phosphorus, and
This is to produce polyester copolymers.

In the subsequent culture, substrates such as 4-hydroxybutyric acid, γ-butyrolactone and 1,
There is a description that a copolymer containing 40 to 97 mol% of 3HB component and 3 to 60 mol% of 4HB component is produced by using 4-butanediol (see above, JP-A-64
-48821).

The 4HB component content of the copolymers shown in the examples of this specification is at most 49%, and in particular, no polyester copolymers with a 4HB component content of more than 60 mol% have been shown. do not have.

[0008]

[Problems to be Solved by the Invention] The present inventors have determined the ratio (molar ratio) of the 4HB component to the 3HB component in the copolymer.
As a result of extensive research in order to industrially easily produce polyester copolymers with relatively large PHB, it was found that by culturing microorganisms capable of producing PHB in the presence of a substrate with the addition of citric acid and/or citrate. , 4H for the 3HB component
It was discovered that a polyester copolymer having a large proportion of component B can be produced, and the present invention was completed.

[0009]

[Means for Solving the Problems] That is, the present invention involves culturing a microorganism capable of producing poly-3-hydroxybutyrate in the presence of a substrate, and producing 3-hydroxybutyrate units and 4-hydroxybutyrate units. The present invention provides a method for producing a polyester copolymer, which comprises culturing in the presence of citric acid and/or citrate.

The present invention will be explained in detail below.

In the present invention, 3H contained in the copolymer
The B component and 4HB component are each expressed by the following equations. 3HB -OCH(CH3)CH2CO-4
HB -OCH2CH2CH2CO-

00
12] The microorganisms used in the present invention are not particularly limited as long as they have the ability to produce PHB;
For example, Alcaligenes faeca has the ability to be produced and accumulated within bacteria.
lis), Alcaligenes roulandii (Alca
ligenes ruhlandii), Alcaligenes latus
), Alcaligenes aquamarinus (Alcali
Genes aquamarinus) and Alcaligenes eutrophus
Microorganisms of the genus Alcaligenes such as P. trophus are used.

Representative examples of strains belonging to these microorganisms include Alcaligenes faecalis ATCC8750,
Alcaligenes roulandii ATCC15749,
Alcaligenes leitus ATCC29712, Alcaligenes aquamarinus ATCC14400, and Alcaligenes eutrophus ATCC17
699, NCIB11599, NCIB11600
etc. can be mentioned.

Among these strains, Alcaligenes eutrophus ATCC 17699 and Alcaligenes eutrophus NCIB 11599 are particularly preferred for practical purposes.

[0015] In the present invention, for example, the above-mentioned microorganism, in the presence of a carbon source serving as a substrate for 4HB and citric acid,
It is cultivated by restricting nitrogen and/or phosphorus in the medium or culture solution to produce and accumulate the copolymer within the bacterial cells. If necessary, a culture method (preliminary culture) for mainly propagating the bacterial cells may be combined before culturing for polymer production, and the culture conditions adopt the culture medium and culture conditions that allow the bacterial cells to proliferate. Just do it.

Substrates used in the present invention include butyric acid derivatives such as 4-hydroxybutyric acid, 4-chlorobutyric acid, and 4-bromobutyric acid, and their respective sodium salts, potassium salts, magnesium salts, calcium salts, aluminum salts, etc. γ
-Butyrolactone and diols of the following general formula are preferred. HO(CH2)nOH (n=2,4,6,8,
10,12) The amount of these substrates is between 3 and 100 g/l, preferably 3
It is preferable to use it at ~50 g/l, and by changing the amount of substrate, the 4HB component content can be changed. Generally, as the amount of substrate increases, the content of the 4HB component tends to increase.

[0017] Regarding the culture conditions used in the present invention, the pH is 6 to 10, preferably 7 to 8, and the dissolved oxygen concentration is 0.5 to 40 ppm, preferably 5 to 20 ppm. If culture is carried out under these conditions, the polyester content produced and accumulated in the dried cells becomes extremely low, making it ineffective for industrial production. The culture temperature is about 20 to 40°C, preferably about 25 to 35°C.

The amount of citric acid or citrate used in the present invention may be such as to increase the content of 4HB component in the copolymer and not inhibit the growth of microorganisms. Although it varies depending on the bacterial strain and desired copolymer composition, 0.3 to 1 liter of culture medium or culture solution is used.
It is about 40g, preferably about 1 to 30g. Citric acid and citrate salts may be used alone or in combination, but if the amount is too large, the amount of polymer obtained will decrease. Examples of the citrate used in the present invention include Na salt, K salt, and ammonium salt.

[0019] From the culture solution obtained by culturing in this way,
For example, bacterial cells are separated and recovered by ordinary solid-liquid separation means such as filtration and centrifugation, washed and dried to obtain dry bacterial cells, and from these dried bacterial cells, chloroform or The produced copolymer is extracted with an organic solvent such as acetone, and a poor solvent such as hexane is added to this extract to precipitate the copolymer.

By the production method of the present invention, 3 in the copolymer
The ratio of 4HB component to HB component is increased, and 4HB
The component is 60 mol% or more, preferably 65 mol% or more,
More preferably, a polyester copolymer of 70 mol% or more can be easily obtained.

The copolymer obtained in the present invention has 3HB
Due to the high ratio of the 4HB component to the components, the melting temperature is lower and the stability of the melting temperature is greater. In addition, it has excellent strength, is easy to form by spinning and rolling, and is stable, and the obtained molded products such as fibers and films are flexible, exhibit rubber elasticity, and are strong.

[0022]

EXAMPLES The present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Examples 1, 2, 3, 4 Alcaligenes eutrophus (ATCC1)
7699) to first culture the cells (hereinafter referred to as the first stage culture), put the cells in 200 ml of a medium having the following composition into a 2 liter Sakaguchi flask and incubate at 28°C for 24 hours. Incubate for an hour and centrifuge (800
0r. p. m. 15 minutes) to separate the bacterial cells.

[0024] First-stage culture medium composition (in 1 liter) Yeast extract 10g Polypeptone 10g
Meat extract 5g (NH4)2SO4 5g Dissolve these in 1 liter of deionized water and adjust the pH to 7.0.

[0025] The bacterial cells obtained in the above-mentioned first stage culture were added to a copolymer production medium having the following composition at a concentration of 4 per liter.
It was suspended in a proportion of g. 500ml of this suspension
The cells were placed in a Sakaguchi flask and cultured at 28° C. for 48 hours, and the cells were separated from the resulting culture solution by centrifugation to obtain cells.

Composition of copolymer production medium (in 1 liter)
Na2HPO4 4.4g KH2PO4 1.2g MgSO4 0.2g Sodium 4-hydroxybutyrate 15.0g Sodium citrate was used in the amounts (unit: g/l) shown in Table 1 in Examples 1 to 4. Dissolve the ingredients in 1 liter of deionized water and adjust the pH to 7.0.

[0027] Next, as a bacterial cell treatment, the obtained bacterial cells were
The cells were washed with distilled water and dried under reduced pressure to obtain dry bacterial cells.

The copolymer is extracted from the dried bacterial cells thus obtained with hot chloroform, and this extract is concentrated and precipitated dropwise into a large amount of hexane.The precipitate is collected by filtration and dried to obtain the copolymer. was separated to obtain the desired polyester copolymer. The composition of the copolymer thus obtained was determined as 1H-N
Determined by MR spectrum (solvent CDCl3).
The results of Examples 1 to 4 are shown in Table 1. Tg, crystallization temperature, and Tm were determined by DSC measurement (heating rate 10°C/m
in).

[0029]

[Table 1] Na citrate Dry bacterial weight Polymer amount Composition ratio (mol%) Example (g/l) (g/l)
(g/l) 3HB:4HB------
−−−−−−−−−−−−−−−−−−−−−−−−−
-- 1 1.0
5.6 1.2
30:70---------------
−−−−−−−−−−−−−−− 2
5.0 5.4
1.1 19:81------
−−−−−−−−−−−−−−−−−−−−−−−−−
--- 3 15.0
5.3 0.7
17:83---------------
−−−−−−−−−−−−−−− 4
30.0 4.3
0.1 6:94------
−−−−−−−−−−−−−−−−−−−−−−−−−
--- Comparative example 0
5.7 1.2
54:46---------------
−−−−−−−−−−−−−−−−

The copolymer obtained in Example 2 had a Tg of -41.9°C, a crystallization temperature of 9.4°C, a Tm of 52.4°C, and a weight average molecular weight determined by GPC of 645,000.

[0031]

Effects of the Invention As can be seen from the above examples, according to the production method of the present invention, the 4HB component is reduced to 6% compared to the 3HB component.
A polyester copolymer containing 0 mol% or more can be easily produced with good yield.

Furthermore, the polyester copolymer with a high 4HB content obtained by the present invention has various excellent properties and is therefore extremely suitable as a raw material for medical materials such as surgical threads and bone fracture fixation. It is also expected to be applied in many fields such as controlled release systems and synthetic plastic substitutes in response to environmental issues.

Claims (1)

[Claims] Claim 1: Cultivating a microorganism capable of producing poly-3-hydroxybutyrate in the presence of a substrate,
When producing a polyester copolymer having 3-hydroxybutyrate units and 4-hydroxybutyrate units, the cultivation is carried out in the presence of citric acid and/or citrate. Production method.