JPH0819227B2 - Polyester copolymer and method for producing the same - Google Patents

Description

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

[Conventional technology]

Poly-3-hydroxybutyrate (PHB) is a thermoplastic polymer that accumulates in the cells of many microorganisms as an energy storage substance and exhibits excellent biodegradability and biocompatibility, thus preserving the environment. It has been attracting attention as a "clean" plastic, 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 for gradually releasing drugs and pesticides. 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, since PHB is a rigid polymer, it has a physical problem that it lacks impact resistance, and industrial production has been postponed because it is difficult to develop applications. Therefore, research aimed at improving the impact resistance has been earnestly conducted.

For example, JP-A-57-150393 and JP-A-59-2201
Japanese Patent Laid-Open No. 92 discloses a PHB copolymer containing 3-hydroxyvalerate (hereinafter referred to as 3HV component) as a copolymerization component. In these publications, like the conventional method for producing PHB, cells are grown in the first stage, and microorganisms are grown in the latter stage by limiting nitrogen or phosphorus to produce a copolymer. This 3HB-3HV copolymer is highly flexible and is a promising material. However, when the 3HV component increases, the melting point (Tm) drop associated with it increases significantly.
However, the Tm at 25 mol% decreases to about 120 ° C. 3 more
Since the melting point changes drastically with the change in HV content, it was extremely difficult to obtain an industrially uniform product.

[Means for solving problems]

In view of the above points, the present inventors have diligently studied to obtain a copolymer that imparts flexibility to PHB and is relatively high at the same time, and has a stable melting point, and as a result, in the subsequent culture in which nitrogen or phosphorus is restricted. And PHB in the presence of certain compounds
The present invention has been found by culturing a microorganism capable of producing and producing and accumulating a target copolymer in the cell. That is, the present invention comprises (1) 3-hydroxybutyrate units 97 to 40 mol% and 4-hydroxybutyrate units 3 to 60 mol%, and [η] measured in chloroform at 30 ° C. is 0.4 to 10.0 d.
Polyester copolymer in the l / g range.

(2) A microorganism having a poly-3-hydroxybutyrate-producing ability is proliferated in the former stage, and the latter is cultured under the restriction of nitrogen or phosphorus in the latter stage to produce poly-3 in the bacterial body.
-When producing and accumulating hydroxybutyrate,
A method for producing a polyester copolymer comprising 3-hydroxybutyrate units and 4-hydroxybutyrate units, which comprises culturing the latter stage in the presence of a compound represented by the following general formula (I) (CH ₂ XCH ₂ CH ₂ COO) _n Y (I) Exist in.

 Hereinafter, the present invention will be described in detail.

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

The microorganism used in the present invention is not particularly limited as long as it has a PHB producing ability, but in practice, for example,
Alcaligenes faecali
s), Alcaligenes r
uhlandii), Alcaligenes latus
tus), Alcaligenes
aquamarinus) and Alcaligenes eutrophs.

As representative examples of strains belonging to these strains, Alcaligenes fuecalis ATCC8750, Alcaligenes rulandii ATCC15749, Alcaligenes ratus ATCC2971
2, Alcaligenes aquamarinus ATCC14400 and Alcaligenes eutrophus H-16ATCC17699 and Alcaligenes eutrohus NCIB11597, NCIB11598, NCIB11599 and NCI which are mutants of this H-16 strain.
Examples include B11600. 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 MANUALOF DETERMINATIVE”.
BACTERIOLOGY: Eighth Edition, The Williams & Wilkin
S. Company / Baltimore "and the mycological properties of Alcaligenes eutrophus H-16 are described in J. Gen. Micl.
obiol., 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, sugar concentrate, natural products such as peptone and meat extract, sugars such as arabinose, glucose, mannose, fructose and galactose, and sorbitol, mannitol and inositol, etc., and nitrogen sources such as ammonia, ammonium salt, nitrate, etc. Inorganic nitrogen compounds and / or, for example, urea, corn steep liquor, casein, peptone, yeast extract,
Examples of organic nitrogen-containing substances such as meat extract and inorganic components include, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate salt, manganese salt, zinc salt,
Iron salt, copper salt, molybdenum salt, cobalt salt, nickel salt,
It is selected from chromium salts, boron compounds, iodine compounds 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., 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 cells of the microorganism from the culture solution obtained in the culture of the first stage, separated and recovered by a normal solid-liquid separation means such as percolation and centrifugation, or subject the cells to the culture of the second stage, or In the first-stage culture, nitrogen and / or phosphorus can be substantially depleted, and this culture solution can be transferred to the second-stage culture without separating and recovering the bacterial cells.

In the latter-stage culture, the medium or the culture medium does not substantially contain nitrogen and / or phosphorus, and the compound represented by the following general formula (I) is contained as a carbon source. Is no different from.

(CH ₂ XCH ₂ CH ₂ COO) _n Y (I) Specific examples of the compound represented by the general formula (I) 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, and aluminum. Examples thereof include salt.

The compound represented by the general formula (I) is contained in the medium or the culture solution in the subsequent culture. In the latter case, it may be at any time from the initial stage to the final stage of the culture, but the early stage of the culture is preferable.

The compound represented by the general formula (I) may be used in such an amount that it can form a copolymer and does not inhibit the growth of microorganisms. The ratio of the compound represented by the general formula (I) in the medium or the culture solution is generally increased when the ratio of the 4HB component of 4HB in the copolymer is increased.
The ratio of the ingredients increases. Usually medium or culture 1
As a compound represented by the general formula (I), 3-40
It is about g, preferably about 5 to 30 g.

In this latter-stage culture, the compound represented by the general formula (I) may be used as the sole carbon source, but other carbon sources that can be assimilated by the microorganism used-for example, glucose, fructose, methanol, ethanol, acetic acid, A small amount of propionic acid, n-butyric acid, lactic acid, and the like can be allowed to coexist. For example, when glucose is used, it is about 1.5 g / at most.

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. From the bacterial cells, by a conventional method, for example, a copolymer produced with an organic solvent such as chloroform is extracted, and to this extract, for example, a poor solvent such as hexane is added,
Precipitate the copolymer.

According to the production method of the present invention, the ratio of the 4HB component to the 3HB component in the copolymer can be arbitrarily adjusted under appropriate reaction conditions. And the copolymer obtained by the present invention, while maintaining a relatively high and stable melting point,
Since it has a low crystallinity, it is highly flexible. Therefore, the moldability such as spinning and rolling is good, and the obtained molded articles such as fibers and films are supple and tough.

〔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 9 and Comparative Example 1 A copolymer was prepared using Alcaligenes Yutrofus H-16ATCC17699. 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 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 * As a carbon source, Table 1 below. Of 4-hydroxybutyric acid and butyric acid were used.

(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.

Characteristics of Copolymer: The composition and intrinsic viscosity 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 125 MH of Example 4
The z and ¹³ C-NMR spectra are shown in FIGS. 1 and 2, respectively.

Example 10 4-Chlorobutyric acid was used as a carbon source in the second-stage culture.
Table 2 shows the results of the copolymer obtained by the same operation as in Example 1 except that 18 g was used per unit.

The chain distribution, melting temperature and heat of fusion in Table 2 were measured as follows.

Linkage distribution; the method of the present inventors and others (Y. Doi et a
l, Macromolecules, 19 , 2860 to 2864 (1986)).

Melting temperature; measured by DSC (rate of temperature increase: 10 ° C / min) Heat of fusion; measured by DSC The calculated values of C ₄ H ₆ O ₂ in elemental analysis are as follows.

C H 55.81% 7.02% Example 11 Table 2 shows the results of the copolymer obtained by the same operation as in Example 10 except that 20 g of sodium 4-hydroxybutyrate was used as a carbon source in the second-stage culture.

Example 12 Table 2 shows the results of the copolymer obtained by the same operation as in Example 11 except that Alcaligenes Yutrofus NCIB11599 was used.

[Effect of the Invention] According to the present invention, a novel polyester copolymer containing a 3HB component and a 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 to a sustained release system. It is expected to be applied to various fields such as the use of.

[Brief description of drawings]

FIG. 1 shows 500 MHz, ¹ H-NMR of the copolymer obtained in Example 4.
FIG. 2 is a 125 MHz, ¹³ C-NMR spectrum of the copolymer obtained in Example 4 as well. The numbers attached to the structural formulas in the figure correspond to the numbers of the peaks.

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Claims (2)

[Claims] 1. A composition comprising 97-40 mol% of 3-hydroxybutyrate units and 3-60 mol% of 4-hydroxybutyrate units, and [η] measured in chloroform at 30 ° C. is 0.4-1.
Polyester copolymer in the range of 0.0dl / g 2. A microorganism having the ability to produce poly-3-hydroxybutyrate is grown in the former stage, and the latter is cultured under the restriction of nitrogen or phosphorus in the latter stage to produce poly-3 in the bacterial body. -In the case of producing and accumulating hydroxybutyrate, it comprises a 3-hydroxybutyrate unit and a 4-hydroxybutyrate unit characterized in that the subsequent culture is carried out in the presence of a compound represented by the following general formula (I). Method for producing polyester copolymer. (CH ₂ XCH ₂ CH ₂ COO) _n Y (I)