JPH0714352B2 - Method for producing polyester copolymer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a 3-hydroxybutyrate unit (hereinafter referred to as 3HB component) and a 4-hydroxybutyrate unit (hereinafter referred to as 4HB component).
Component)), and more particularly to a method for producing a novel copolymerized polyester comprising 3HB and 4HB components produced by using a microorganism capable of accumulating polyester.

[Conventional technology]

Poly-3-hydroxybutyrate (PHB) accumulates in the cells of many microorganisms as an energy storage substance,
Since it is a thermoplastic polymer with excellent biodegradability and biocompatibility, it has attracted attention as a "clean" plastic that preserves the environment, and gradually releases medical materials such as surgical threads and bone fracture fixation materials as well as pharmaceuticals and agricultural chemicals. It has been expected for many years to be applied to various fields such as a sustained release system. Particularly in recent years, synthetic plastics have become a serious social problem from the viewpoint of environmental pollution and resource recycling, and PHB is attracting attention as a biopolymer that does not depend on petroleum.

[Problems to be solved by the invention]

However, PHB has been postponed for industrial production because it is inferior in impact resistance and has a problem in physical properties as well as high production cost.

Recently, a copolymer containing a 3HB component and a 3-hydroxyvalerate unit (hereinafter referred to as a 3HV component) and a method for producing the same have been studied and developed. For example, JP-A-57-150393 and JP-A- They are described in JP-A-59-220192.

However, when the 3HV component of the copolymer increased from 0 to 33 mol%, the melting temperature (Tm) increased to 180 ° C with this increase.
It is known that the temperature drops sharply from
Bluhm et al, Macromolecules, 19 , 2871 (1986)] Therefore, the copolymer with a high 3HV content was inferior in heat resistance.

On the other hand, the present inventor has conducted research and development on a copolymer containing 3HB component and 4HB component and a production method thereof,
I applied earlier (Japanese Patent Application No. Sho 62-204538). Such a copolymer is
Even if the content of the 4HB component as a copolymer component is high, it has a high melting point and thus has a high industrial value. However,
Since this method requires the use of expensive reagents as carbon sources, there has been an extremely high demand for finding a general-purpose carbon source that is easily industrially available.

[Means for solving problems]

In view of the above points, the present inventors have diligently studied to produce a copolymer consisting of a 3HB component and a 4HB component industrially advantageously and easily, as a result of culture in which nitrogen or phosphorus in the latter stage is restricted, 1. The present invention has been achieved by obtaining new knowledge that a desired copolymer is produced and accumulated in these cells when a microorganism capable of producing PHB is cultured in the presence of 4-butanediol.

That is, the present invention is that the cells of the genera Alcaligenes having poly-3-hydroxybutyrate-producing ability are grown in the first stage, and the cells are cultured in the second stage under the restriction of nitrogen or phosphorus to give poly- Produces 3-hydroxybutyrate
A method for producing a polyester copolymer comprising 3-hydroxybutyrate units and 4-hydroxybutyrate units is characterized in that the latter-stage culture is carried out in the presence of 1,4-butanediol upon accumulation.

Hereinafter, the present invention will be described in detail.

In the present invention, 3HB component and 4 contained in the copolymer
The HB component is expressed by the following equation.

The microorganism used in the present invention is not particularly limited as long as it is a microorganism capable of producing PHB, but in practice, for example, Alcaligenes faecalis,
Alcaligenes ruhlan
dii), Alcaligenes latu
s), Alcaligenes a
quamarinus) and Alcaligenes Yutrohus (Al
caligenes eutrophs) and other species of Alcaligenes.

As typical examples of strains belonging to these strains, Alcaligenes faecalis ATCC8750, Alcaligenes lulandei ATCC15749, Alcaligenes ratus ATCC29712, Alcaligenes aquamarinus ATCC14400 and Alcaligenes eutrophus H-16ATCC17699, and mutant strains of these H-16 strains Alcaligenes NC115 and Utrofus NCIB11598, NCIB11599, NCIB11600
And so on. Of these, Alcaligenes eutrophus H-16ATCC17699 and Alcaligenes eutrophus NCIB11599 are particularly preferable in practical use.

The mycological properties of these microorganisms belonging to the genus Alcaligenes include, for example, “BERGEY ′S MANUAL OF DETERMINATIVE”.
BACTERIOLOGY: Eighth Edition, The Williams & Wilkin
S. Company / Baltimore "and the mycological properties of Alcaligenes eutrophus H-16 are described in" J. Gen. Mi.
clobiol., 115 , 185-292 (1979).

Similar to the conventional method, these microorganisms are cultivated in two stages, that is, a pre-stage culture in which cells are mainly grown and a post-stage culture in which a copolymer is produced and accumulated in the cells by limiting nitrogen or phosphorus. To be done.

For the culture in the first stage, an ordinary culture method for growing a microorganism can be applied. That is, it suffices to adopt a 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, but in practice, carbon sources include, for example, synthetic carbon sources such as methanol, ethanol and acetic acid, and inorganic carbon sources such as carbon dioxide. , Yeast extract, molasses,
Natural products such as peptone and meat extract, arabinose,
Examples of the nitrogen source such as glucose, mannose, fructose and galactose, and sorbitol, mannitol and inositol include inorganic nitrogen compounds such as ammonia, ammonium salt and nitrate, and / or urea, corn steve liquor, and the like. Examples of organic nitrogen-containing substances and inorganic components such as casein, peptone, yeast extract, and meat extract include, for example, calcium salt, magnesium salt, potassium salt,
Sodium salt, phosphate, manganese salt, zinc salt, iron salt,
It is selected from copper salt, molybdenum salt, cobalt salt, nickel salt, chromium salt, boron compound, iodine compound and the like.

In addition, vitamins and the like can be used if necessary.

As the culture conditions, the temperature is, for example, about 20 to 40 ° C,
The temperature is preferably about 25 to 35 ° C, and the pH is, for example, about 6 to 10, preferably about 6.5 to 9.5. 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.

The culture method may be either batch culture or continuous culture.

The cells obtained by the culture in the first stage are further cultured under nitrogen and / or phosphorus limiting conditions.

That is, the bacterial cells of the microorganism from the culture solution obtained in the first-stage culture are separated and recovered by an ordinary solid-liquid separation means such as permeation and centrifugation, or the cells are subjected to the second-stage culture, or
Alternatively, in the culture in the first stage, nitrogen and / or phosphorus is substantially depleted, and the bacterial cells are not separated and recovered,
It is also possible to transfer this culture solution to the subsequent culture.

In the latter-stage culture, there is no difference from the former-stage culture except that the medium or the culture solution does not substantially contain nitrogen and / or phosphorus and 1,4-butanediol is contained as a carbon source.

When the culture solution contains 1,4-butanediol, it may be at any time in the early or late stages of the culture, but the early stage of the culture is preferable.

The 1,4-butanediol used in the present invention is a strain of a well-used microbial strain and a desired copolymerization ratio (mole) as long as it is capable of forming a copolymer and does not inhibit the growth of the microorganism. Ratio, etc., but generally about 3 to 40 g is appropriate for the medium or culture solution 1.

In this latter stage culture, 1,4-butanediol may be used as the sole carbon source, but other carbon sources that can be assimilated by the microorganism used, such as glucose, fructose, methanol, ethanol, acetic acid, propionic acid, n -Butyric acid, lactic acid, valeric acid, etc. can also be present together.
For example, when using glucose, at most 1.
It is said to be about 5g /.

From the culture solution obtained by culturing in this way, cells are separated and recovered by a normal solid-liquid separation means such as filtration and centrifugation, and the cells are washed and dried to obtain dried cells, which are dried. For example, a copolymer produced from an organic solvent such as chloroform is extracted from the cells by a conventional method, 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 the 3HB component and the 4HB component in the copolymer can be adjusted arbitrarily.

〔Example〕

The present invention will be described more specifically by way of examples. The present invention is not limited to these examples.

Examples 1 to 5 and Comparative Examples 1 to 3 Alkalgenes Yutrofus H16 (ATCC17699) was used to produce copolymers. That is, pre-stage culture: The above microorganism was cultured in a medium having the following composition at 30 ° C. for 24 hours, and the cells were separated from the culture solution at the end of the logarithmic growth phase by centrifugation.

Composition of preculture medium Yeast extract 10 g Polypeptone 10 g Meat extract 5 g (NH ₄ ) ₂ SO ₄ 5 g These were dissolved in deionized water 1 and adjusted to pH 7.0.

Second-stage culture: The cells obtained by the first-stage culture are suspended in a medium having the following composition at a ratio of 5 g per 1 hour, and the cells are cultured at 30 ° C for 48 hours, and the cells are centrifuged from the obtained culture solution. Separated to obtain bacterial cells.

Composition of culture medium for second-stage culture 0.5M potassium hydrogen phosphate aqueous solution 39.0ml 0.5M dipotassium hydrogen phosphate aqueous solution 53.6ml 20wt / V% magnesium sulfate aqueous solution 1.0ml Carbon source ^* mineral solution ^** 1.0ml * Various compounds were used as described in. (Unit g / media) was dissolved in ** Mineral solution _{CoCl 2 119.0mg FeCl 3 9.7g CaCl 2} 7.8g NiCl 2 118.0mg CrCl 2 62.2mg CaSO 4 dissolved 156.4mg to 0.1N-HCl1 these Deionized water 1 The pH was adjusted to 7.0.

Treatment of bacterial cells: The bacterial cells obtained in the latter-stage culture were washed with distilled water and then with acetone, and dried under reduced pressure (20 ° C, 0.1 mmHg) to obtain dried bacterial cells.

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

Properties of Copolymer: The composition, intrinsic viscosity, melting temperature and heat of fusion of the copolymer thus obtained were measured as follows. That is, according to the composition: ¹ H NMR spectrum.

Intrinsic viscosity [η]: 30 ° C in chloroform.

Table 1 shows the measurement results and the like.

The 500 MHz ¹ H-NMR spectrum and the 125 MHz ¹³ C-NMR spectrum of the copolymer obtained in Example 2 are shown in FIG. 1 and FIG. 2, respectively.

[Effect of the Invention] According to the present invention, a novel polyester copolymer containing 3HB component and 4HB component can be easily obtained.

Furthermore, since the copolymer obtained in the present invention has various excellent properties, it is extremely suitable as a raw material for medical materials such as surgical threads and materials for fixing bone fractures, and also for use in sustained release systems. It is expected to be applied to various fields such as.

[Brief description of drawings]

FIG. 1 shows 500 MHz, ¹ H-NMR of the copolymer obtained in Example 2.
2 is a 125 MHz, ¹³ C-NMR spectrum of the copolymer obtained in Example 2 as well.

Claims (1)

[Claims] 1. A bacterium of the genus Alcaligenes capable of producing poly-3-hydroxybutyrate is grown in the former stage, and the latter is cultivated under the restriction of nitrogen or phosphorus in the latter stage to give poly- in the bacterium. A polyester copolymer comprising 3-hydroxybutyrate units and 4-hydroxybutyrate units, characterized in that the subsequent culture is carried out in the presence of 1,4-butanediol in the production and accumulation of 3-hydroxybutyrate. Manufacturing method.