JPH0714354B2 - 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 inventor has diligently studied to produce a copolymer consisting of 3HB component and 4HB component industrially advantageously and easily, and γ-in culture in which nitrogen or phosphorus in the latter stage is restricted. 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 butyrolactone.

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 subsequent culture is carried out in the presence of γ-butyrolactone for accumulation.

Hereinafter, the present invention will be described in detail.

In the present invention, 3HB component and 4 contained in the copolymer
Each 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 latus,
Alcaligenes aquama
rinus) and Alcaligenes Yutrohus (Alcalig
enes eutrophs) and other Alcaligenes spp.

Representative examples of strains belonging to these strains are Alcaligenes faecalis ATCC8750, Alcaligenes lulandei ATCC15749, Alcaligenes ratus ATCC29712, Alcaligenes aquamarinus ATCC14400 and Alcaligenes eutrophus H-16 ATCC17699, and mutant strains of these H-16 strains Alcaligenes NC115 Yutrofus. , Same NCIB11598, same NCIB11599, same NCIB11
600 can be mentioned. Of these, Alcaligenes Yutrohus H-16 ATCC17699
And Alcaligenes eutrophus NCIB 11599 are particularly preferred.

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 salts and nitrates and / or, for example, urea, corn steep liquor, 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, microbial cells of the microorganism obtained from the culture solution obtained in the first-stage culture are separated and recovered by a usual solid-liquid separation means such as filtration and centrifugation, and 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 γ-butyrolactone is contained as a carbon source.

When the culture solution contains γ-butyrolactone,
It may be at any point in the early or late period of the culture, but the early stage of the culture is preferred.

The γ-butyrolactone used in the present invention is a strain of a microorganism often used and a desired copolymerization ratio (molar ratio), etc., as long as it is capable of producing a copolymer and does not inhibit the growth of the microorganism. Although it varies depending on the type, about 3 to 100 g is generally suitable for the medium or culture solution 1.

In this second-stage culture, γ-butyrolactone may be the only carbon source, but other carbon sources that can be assimilated by the microorganism used, for example, glucose, fructose, methanol, ethanol, acetic acid, propionic acid, n-butyric acid,
Lactic acid and valeric acid, 4-hydroxybutyric acid and salts thereof, and diols such as 1,4-butanediol can also be present together. For example, when glucose is used, it is about 1.5 g / at most.

From the culture solution obtained by culturing in this manner, 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, which are dried. By a conventional method, a copolymer produced with an organic solvent such as chloroform is extracted from the cells, 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 2 Copolymers were produced using Alcaligenes Yutrofus H16 (ATCC17699). 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 bacterial cells obtained by the above-mentioned culture are suspended in a medium having the following composition at a ratio of 5 g per 1 hour and cultured at 30 ° C. for 48 hours, and the bacterial cells are centrifuged from the obtained culture solution. Separated to obtain bacterial cells. Composition of culture medium for post-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 as listed in the table were used. (Unit g / Medium) ** Mineral solution _{CoCl 2 119.0mg FeCl 3 9.7 g CaCl} 2 7.8g NiCl 2 118.0mg CrCl 2 62.2mg CaSO 4 dissolved in 0.1 N-HCl 1 dissolved them 156.4mg deionized water 1 And adjusted to pH 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 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 collected by filtration 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 of 500 MHz.

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

Melting temperature and heat of fusion: According to DSC measurement (10 ° C / min).

Table 1 shows the measurement results and the like.

[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.

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. In producing and accumulating 3-hydroxybutyrate, the latter-stage culture is carried out in the presence of γ-butyrolactone, which is a polyester copolymer comprising 3-hydroxybutyrate units and 4-hydroxybutyrate units. Production method.