JPH07113055B2 - 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), 3-hydroxyvalerate units (hereinafter referred to as 3HV
Component) and a 5-hydroxyvalerate unit (hereinafter referred to as 5HV component), and a method for producing the same, more specifically, 3HB component, 3HV component and 3HV component produced using a microorganism capable of accumulating polyester, and The present invention relates to a novel copolyester composed of 5HV components and a method for producing the same.

[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, research and development have been conducted on copolymers containing 3HB components and 3HV components and methods for producing the same.
It is described in JP-A-57-150393 and JP-A-59-220192, respectively.

The method for producing PHB in these publications is the same as in the conventional method for producing PHB, in which cells are grown in the first stage, and microorganisms are cultured in the latter stage by limiting nitrogen or phosphorus to produce a copolymer. Is.

However, in the former, by using, for example, propionic acid and isobutyric acid as substrates in the latter stage culture, the 3HB component was 99.9 to 50 mol% and, for example, 3HV.
It is described that a copolymer containing 0.1 to 50 mol% of another ester component such as a component is produced. However, in this publication, for example, in the examples, a maximum of 33 mol%
Only copolymers containing 3HV components are shown, and copolymers with more 3HV components are not specifically shown.

On the other hand, in the latter case, in the latter-stage culture, carbon from the cellular material of the waste cells after PHB extraction is used to produce a copolymer containing at least 40 mol% of 3HB component and other ester component. There is a qualitative description. However, this publication does not describe at all a copolymer in which the ratio of the 3HB component to the 3HV component is specifically shown. In addition, this method is complicated, and the components of the cellular substance are unstable because the type and amount of the substance greatly vary depending on the culture conditions and the like, which is unstable.

Furthermore, when the 3HV component of the copolymer increased from 0 to 33 mol%, the melting temperature (Tm) increased from 180 ° C to 8% with the increase.
It is known that the temperature drops rapidly to 5 ° C [TL Bluhm
et al, Macromolecules , 19, 2871-2876 (1986)], which means that it is difficult to industrially obtain a uniform product.

[Means for solving problems]

The present inventor, as a result of diligent studies to industrially advantageously and easily produce a copolymer having a relatively large proportion (molar ratio) of a copolymerization component with respect to the 3HB component, a culture for limiting nitrogen or phosphorus in the latter stage. In culturing a microorganism capable of producing PHB in the presence of the compound represented by the following general formula (I), the 3HV component and the 5H
The present invention has been achieved by obtaining new knowledge that a copolymer having a relatively large V component ratio (molar ratio) is produced and accumulated.

That is, the present invention comprises (1) 3-hydroxybutyrate unit 2 to 50 mol% 3-hydroxyvalerate unit 3 to 95 mol% 5-hydroxyvalerate unit 3 to 90 mol% and measured in chloroform at 30 ° C. [Η] is 0.4
Polyester copolymer in the range of up to 10.0 dl / g.

(2) A microorganism capable of producing poly-3-hydroxybutyrate is proliferated in the former stage, and the latter is cultured under the restriction of nitrogen or phosphorus in the latter stage to give poly-3-hydroxybutyrate in the bacterial body. In producing and accumulating the rate, the latter-stage culture is carried out in the presence of the compound represented by the following general formula (I), which is characterized by being a 3-hydroxybutyrate unit, a 3-hydroxyvalerate unit and 5-hydroxy. Method for producing polyester copolymer composed of valylate unit Exist in.

The present invention will be described in detail below.

The 3HB component, 3HV component and 5HV component contained in the copolymer in the present invention are represented by the following formulas, respectively.

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 ATCC 8750, Alcaligenes rulandii ATCC 15749, Alcaligenes ratus ATCC 29712,
Alcaligenes aquamarinus ATCC 14400 and Alcaligenes eutrophus H-16 ATCC 17699 and the mutant strains of this H-16 strain Alcaligenes eutrophus NCIB 11597, NCIB 11598, NCIB 11599, NCIB 1
1600 etc. can be mentioned. Among these, Alcaligenes eutrophus H-16 ATCC 17699 and Alcaligenes eutrophus NCIB 11599 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.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 first-stage culture, a usual 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, copalto salt, nickel salt, chromium salt, boron compound and iodine compound.

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, from the culture solution obtained in the culture of the first stage, the bacterial cells of the microorganism are separated and recovered by a usual solid-liquid separation means such as filtration and centrifugation, and the bacterial cells are subjected to the subsequent culture, or, It can also be carried out by substantially depleting nitrogen and / or phosphorus in the first-stage culture and transferring this culture medium to the second-stage culture without separating and recovering the bacterial cells.

This second-stage culture is different from the first-stage culture except that the medium or the culture solution does not substantially contain nitrogen and / or phosphorus and the compound represented by the general formula (I) is contained as a carbon source. There is no.

Specific examples of the compound represented by the general formula (I) that can be used in the present invention include 5-chlorovaleric acid, 5-hydroxyvaleric acid and their sodium salts, potassium salts,
Calcium salt etc. are mentioned.

In carrying out the present invention, 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, the culture may be performed at an early stage or a late stage at that time, but the early stage of the culture is preferable.

The compound represented by the general formula (I) used in the present invention is a strain of a microbial strain that is often used and a desired co-producer, as long as it is capable of forming a copolymer and does not inhibit the growth of the microbial organism. Although it varies depending on the polymerization ratio (molar ratio) and the like, generally about 3 to 40 g as the above-mentioned general formula (I) is suitable for the medium or culture solution 1.

In the 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, Propionic acid, n-butyric acid, lactic acid, valeric acid and the like can also be present together. For example, when glucose is used, it is about 1.5 g / l 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. 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 proportions of the 3HB component, 3HV component and 5HV component in the copolymer can be adjusted arbitrarily. In particular, when the ratio of the 3HB component is 50 mol% or less, the stability of the melting temperature becomes large, and the crystallinity becomes small, so that the strength is excellent, and molding such as spinning and rolling is easy and stable, 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 1-3 and Comparative Example 1 Copolymers were prepared using Alcaligenes Yutrofus NCIB 11599. 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 Polypebton 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 * See Table 1 as carbon source 5-chlorovaleric acid and valeric acid were used as noted. (Unit g / l medium) In Comparative Example 1, butyric acid 20 g was used.

** Mineral solution CoCl ₂ 119.0mg FeCl ₃ 9.7 g CaCl ₂ 7.8 g NiCl ₂ 118.0 mg CrCl ₂ 62.2 mg CaSO ₄ 156.4 mg dissolved in 0.1 N-HCl 1 These were dissolved in deionized water 1 to pH 7.0. It was adjusted.

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, the composition is based on the ¹ H NMR spectrum.

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

Melting temperature Tm: According to DSC measurement.

 (Raising rate 10 ° C / min) Heat of fusion ΔH: Measured by DSC.

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 a 3HB component, a 3HV component and a 5HV 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 2.
FIG. 2 is a 125 MHz, ¹³ C-NMR spectrum of the copolymer obtained in Example 2 as well. The numbers attached to the structural formulas in the figure correspond to the numbers of the peaks.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 1/20 C12R 1:05) (C12P 7/62 C12R 1:05)

Claims (2)

[Claims] 1. A 3-hydroxybutyrate unit 2-50 mol% 3-hydroxyvalerate unit 3-95 mol% 5-hydroxyvalerate unit 3-90 mol%, measured in chloroform at 30 ° C. ] Is 0.4
Polyester copolymer in the range of up to 10.0 dl / g. 2. A microbial cell capable of producing poly-3-hydroxybutyrate is proliferated in the former stage, and the microbial cell is cultured in the latter stage under the restriction of nitrogen or phosphorus to produce poly- inside the microbial cell.
When producing and accumulating 3-hydroxybutyrate, the subsequent culture is carried out in the presence of a compound represented by the following general formula (I), a 3-hydroxybutyrate unit and a 3-hydroxyvalerate unit. And a method for producing a polyester copolymer comprising 5-hydroxyvalerate units.