JPH02234683A - Method for producing polyester copolymer - Google Patents

Abstract

PURPOSE:To readily obtain a polyester copolymer consisting of 3- and 4- hydroxybutylate units in a bacterial cell by culturing a microoganism having poly-3-hydroxybutylate producing ability in the presence of 1,6-hexanediol in culture restricting N2 or P. CONSTITUTION:An Alcaligenes bacterium (e.g. Alcaligenes faecalis) having poly-3-hydroxybutylate (PHB) producing ability is cultured and proliferated as well as hitherto. As the culture conditions, e.g. conditions such as about 20-40 deg.C and about pH 6-10 are adapted. Then the resultant bacterium cell is cultured in a culture medium containing 1,6-hexanediol as a carbon source without substantially containing N2 and/or P. Then the bacterial cell is separated and recovered from the culture medium by an ordinary solid-liquid separation means such as filtration and centrifugation and cleaned and dried. Then the above-mentioned polyester copolymer produced from the dried bacterium cell by an organic solvent according to an ordinary method is extracted and recovered.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a 3-hydroxybutyrate unit (hereinafter referred to as 3 I
Regarding the method for producing a copolymer containing 4-hydroxybutyrate units (hereinafter referred to as the 98 B component) and 4-hydroxybutyrate units (hereinafter referred to as the 98 B component), for more details, see / Produced using a microorganism capable of accumulating polyester. 3
This invention relates to a method for producing a novel copolyester comprising an HB component and a +HB component.

[Conventional technology]

Poly-3-hydroxybutyrate (PH8) is a thermoplastic polymer that accumulates in the cells of many microorganisms as an energy storage substance and exhibits excellent biodegradability and biocompatibility, helping to preserve the environment. It has been attracting attention as a "clean" plastic, and has long been expected to be used in a variety of fields, including medical materials such as surgical threads and fracture fixation materials, and sustained-release systems that gradually release pharmaceuticals and pesticides. .

Particularly in recent years, synthetic plastics have become a serious social problem in terms of environmental pollution and resource recycling, and PH8 is attracting attention as a biopolymer that does not depend on petroleum.

[Problem that the invention seeks to solve]

However, industrial production of PH8 has been postponed due to physical property problems such as poor impact resistance and high production costs.

Recently, research and development have been carried out on copolymers containing a 3HB component and 3-hydroxyvalerate units (hereinafter referred to as 3HV component) and methods for producing the same. Publication r0393 and JP-A-Sho! ? -2
Each of these is described in Publication No. 20/9J.

It is known that the melting temperature (Tm) decreases rapidly from igo℃ to g3℃9 (T.L.Bluh
metal, macromolecules, /
9, 2 and 7/(/l'4)) Therefore, copolymers with high 3HV component content were inferior in heat resistance.

On the other hand, the present inventor has conducted research and development on a copolymer containing a 3HB component and a QHB component and a method for producing the same, and has previously filed an application (Japanese Patent Application No. 42-204'53g; -1367 Hiro 8, 43-i
7ggu hong). Even when the copolymer content of the +HB component is high, the darkening copolymer has a high melting point and therefore has high industrial value. However, since this method requires the use of expensive reagents as a carbon source, there has been an extremely high demand for finding a general-purpose carbon source that can be easily obtained industrially.

[Means for solving problems]

In view of the above points, the present inventor has determined that the 3HB component and the QHB component
As a result of intensive studies aimed at industrially advantageous and easy production of copolymers consisting of these components, we found that microorganisms capable of producing PH8 were grown in the presence of l,6-hexanediol in the subsequent nitrogen- or phosphorus-limiting culture. The present invention was achieved based on the new finding that a desired copolymer is produced and accumulated in the bacterial cells when cultured.

That is, in the present invention, cells of the genus Alcaligenes having the ability to produce poly-3-hydroxybuterate are grown in the first stage, and in the second stage, the cells are cultured under nitrogen or phosphorus limitation to inject poly-3-hydroxybuterate into the cells. From 3-hydroxybuterate units and gu-hydroxybuterate units, which is characterized in that when producing and accumulating 3-hydroxybuterate, the subsequent cultivation is carried out in the presence of /,A-hexanediol. The present invention relates to a method for producing a polyester copolymer.

The present invention will be explained in detail below.

In the present invention, the 3HB component and +HB component contained in the copolymer are each represented by the following formula.

3HB component; -OCH(CH3)CH2ClIHB
Ingredients: -OCH2CH2CH2C The microorganism used in the present invention is not particularly limited as long as it has the ability to produce PH8, but for practical purposes, for example, Alcaligenes faecalis
), Alcaligene Lulandei 4 (Alcalig
enes ruhlandii), alcaligenes
Alcaligenes latus, Alcaligenes aquamarinus
s aquamarinus ) and Alcaligenes eutrophus ( Alcaligenes eutropus ).
hs) and other members of the genus Alcaligenes.

Representative examples of strains belonging to these bacterial species include Alcaligenes faecalis kTccgqzo, Alcaligenes faecalis
roulandii ATCC /z Kug9, Alcaligenes latus ATCC 2wa7/2, Alcaligenes aquamarinus ATCCllIdaoo and Alcaligenes eutrophus H-/6ATCC/7699 and the mutant strain of this H-/A strain Alcaligenes eutrophus NCIB//! 97, same NCIB//J9g,
Examples include the same NCIB/l1 spirit and the same NCIBllAOθ. Of these, in practice, Alcaligenes Yut I:l;'s H-/AATCC/749? and Alcaligenes eutrophus NCIB/Ik9ta are particularly preferred.

The mycological properties of these microorganisms belonging to the genus Alcaligenes are, for example, "BERGEY'SMANUAL"
OF DETERMINATIVE BACTERIO
-LOGY; Ei. ghth Edition,T
he Will liams & Wj. Iki. ns
Company/8altimore”, and the mycological notes of Alcaligenes eutrophus H-/O are as follows:
For example, “J. Gen. Microbiol, ,/
/! ;, Igta~/9U (/979), respectively.

These microorganisms are developed in the same way as in conventional methods, with the first stage of culturing mainly in which bacterial cells are grown, and the second stage of culturing in which nitrogen or oxygen is limited to produce and accumulate copolymers within the bacterial cells. Cultivated.

For the first-stage culture, a normal culture method for propagating microorganisms can be applied. That is, it is sufficient to adopt a medium and culture conditions that allow the microorganisms used to proliferate.

There are no particular restrictions on the medium components as long as they can be assimilated by the microorganisms used, but in practical terms carbon sources include 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,
Sugars such as glucose, mannose, fructose, oyohygalactose, and solpitol, mannitol and inositol. As nitrogen sources, inorganic nitrogen compounds such as ammonia, ammonium salts, nitrates and/or e.g. urea, corn stizo liquor, etc. Examples of organic nitrogen-containing substances and inorganic components such as casein, beptone, yeast extract, and meat extract include calcium salts, magnesium salts, potassium salts,
Sodium salts, phosphates, manganese salts, zinc salts, iron salts,
Each is selected from copper salts, molybdenum salts, cobalt salts, nickel salts, chromium salts, boron compounds, iodine compounds, and the like.

However, vitamins can also be used if necessary.

As for the culture conditions, the temperature is, for example, about 20°C to 10°C, preferably about 25 to 35°C, and the pH is, for example, about 6 to 10°C, preferably about 6° to 10°C. S
It is considered to be a degree. Like this -1? − Cultivate aerobically under suitable conditions.

If culture is performed under these conditions, the growth of microorganisms will be relatively poor, but this does not preclude cultivation under these conditions.

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

The bacterial cells obtained in the first stage of culture are further cultured under nitrogen and/or phosphorus-limited conditions.

That is, either the microbial cells are separated and recovered from the culture solution obtained in the first-stage culture by ordinary solid-liquid separation means such as F filtration and centrifugation, and the microbial cells are subjected to the second-stage culture, or
Alternatively, in the first stage of culture, nitrogen and/or phosphorus are substantially depleted, without separating and collecting the bacterial cells.
This can also be done by transferring this culture solution to the subsequent stage of culture.

There is no difference between this second stage cultivation and the first stage cultivation except that the medium or culture solution does not substantially contain nitrogen and/or nitrogen and contains /,A-hexanediol as a carbon source.

When /,A-hexanediol is added to the culture solution, it may be added at any time from early to late stages of culture, but preferably at the early stages of culture.

/,A-hexanediol used in the present invention can be used in an amount that can produce a copolymer and does not inhibit the growth of microorganisms. ratio) etc., but
Generally, a ratio of about 3 to 1/001 per liter of medium or culture solution is appropriate.

In this latter stage of cultivation, /,6-hexanediol may be used as the sole carbon source, but other carbon sources that can be assimilated by the microorganisms used may be used, such as glucose, fructose, methanol, ethanol, acetic acid, propionic acid, n Monobutyric acid, lactic acid, valeric acid, etc. can also be present.

For example, when using glucose, at most Ha! It is said to be about rf/l.

From the culture solution obtained by culturing in this way, the bacterial cells are separated and recovered by ordinary solid-liquid separation means such as filtration and centrifugation, and the bacterial cells are washed and dried to obtain dry bacterial cells. The entire copolymer produced from the dried bacterial cells is extracted using an organic solvent such as chloroform using a conventional method, and a poor solvent such as hexane is added to this extract to extract the copolymer. precipitate.

If the production method of the present invention is used, the 3HB component in the copolymer, the 4
'The ratio of HB components can be adjusted as desired.

〔Example〕

The present invention will be explained in more detail with reference to Examples. Note that the present invention is not limited to these embodiments.

Examples 1-3 and Comparative Examples/- Coal Caligenes eutrophus H/A (ATCCtqb
qq) was used to produce a copolymer. That is, first stage culture: 2q of the above microorganisms at 30°C in a medium having the following composition.
After culturing for several hours, the bacterial cells were separated from the culture solution at the end of the logarithmic growth phase by centrifugation.

Composition of medium for first stage culture Yeast extract /Og Polypeptone /09 Meat extract 3,! i' (NH4)2SO4 k
，! i'il rinse these with deionized water! Dissolved in pH 7
．． Adjust fc to 0.

Post-stage culture: The bacterial cells obtained in the first-stage culture were suspended in a medium having the following composition at a ratio of 9/0 to 20jj/73 and cultured at 30°C for yg hours to obtain f'Lfc culture solution. The bacterial cells were separated by centrifugation to obtain bacterial cells.

Composition of secondary culture medium OJM Potassium hydrogen phosphate aqueous solution 2
2.7mlOJM dihydrogen phosphate IJium aqueous solution //0.7yd20wt/V% magnesium sulfate aqueous solution /. A compound of 8-11 carbon sources was used. (Unit 11/13 medium) ** Mineral solution CoCl2 / / 9. OttqFeCl
3 9.79 CaCl2 h. t
gNiCl2//t. 0■CrCl2
62.2mgCaSO4/j 6.1
1 mf to 0. Dissolved in IN-HC 1/II These were dissolved in deionized water/It and adjusted to pH 74.

Treatment of bacterial cells: The bacterial cells obtained in the post-culture were washed with distilled water, followed by acetone, and dried under reduced pressure (20°C,
) to obtain dried bacterial cells.

Separation and recovery of copolymer: Extract the copolymer from the dried bacterial cells thus obtained with hot chloroform, add hexane to this extract to precipitate the copolymer, take 1 inch of this precipitate, and dry. A copolymer was obtained.

The composition of the copolymer obtained in this way was determined by 'H-NMR.
It was measured by spectrum.

The measurement results are shown in Table 1.

In addition, the copolymer obtained in Example/! ;00MHz
"H-NMR spectrum f /JjMHZ"C
-NMR spectra are shown in FIG.

[Effect of the invention] According to the present invention, . ? A new polyester copolymer containing the I-{B component and the HB component can be easily obtained.

Furthermore, the copolymer obtained in the present invention has various excellent properties and is therefore extremely suitable as a raw material for medical materials such as surgical threads and materials for fixing bone fractures. It is expected that it will be used in a wide range of fields, including use in radioactive systems.

[Brief explanation of the drawing]

Claims (1)

[Claims] (1) In the first stage, cells of the Alcaligenes genus having the ability to produce poly-3-hydroxybutyrate are grown, and in the second stage, the cells are cultured under nitrogen or phosphorus limitation, and poly-3- Production of a polyester copolymer consisting of 3-hydroxybutyrate units and 4-hydroxybutyrate units, characterized in that the latter stage of cultivation is carried out in the presence of 1,6-hexanediol when producing and accumulating hydroxybutyrate. Method.