JPH0227992A - Production of polyester copolymer - Google Patents

Abstract

PURPOSE:To produce a polyester copolymer by multiplying a cell of a bacterium belonging to the genus Alcaligenes capable of producing poly-3-hydroxybutylate in a first step and cultivating the cell under limitation of nitrogen or phosphorus. CONSTITUTION:A cell of a bacterium belonging to the genus Alcaligenes capable of producing poly-3-hydroxybutylate is multiplied in a first step, the cell is cultivated under limitation of nitrogen or phosphorus in the presence of gamma- butyrolactone and poly-3-hydroxybutylate is formed and accumulated in the cell. The cell is separated, from the cultivated culture solution by centrifuging, etc., recovered, washed and dried to form the dried cell, which is extracted with an organic solvent to collect a polyester copolymer comprising a 3- hydroxybutylate unit and 4-hydroxybutylate unit.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a 3-hydroxybutyrate unit (hereinafter referred to as 3HB
component) and 4-hydroxybutyrate unit (hereinafter referred to as 4
Regarding the method for producing a copolymer containing HB component),
More specifically, the present invention relates to a method for producing a novel copolyester comprising a 3HB component and a 48B component, which is produced using a microorganism capable of accumulating polyester.

[Conventional technology]

Poly-3-hydroxybutyrate (PHB) is a thermoplastic polymer that accumulates in the bodies of many microorganisms as an energy storage substance and exhibits excellent biodegradability and biocompatibility, making it environmentally friendly. It has been attracting attention as a "clean" plastic for conservation, 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 agricultural chemicals.

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.

[Problem that the invention seeks to solve]

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

Recently, research and development have been conducted on copolymers containing a 3HB component and 3-hydroxyvalerate units (hereinafter referred to as 3HV component) and methods for producing the same. Japanese Patent Publication No. 59-22
Each of these is described in Publication No. 0192.

However, it is known that when the 3HV component of the copolymer increases from O to 33 mol%, the melting temperature (Tm) decreases rapidly from 180°C to 85°C (T, L, Bluhmet al, Macrom
olecules.

2, 2871 (1986)) Therefore, copolymers with a 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 4HB component and a method for producing the same, and has previously filed a patent application (Japanese Patent Application No. 62-204538). Such a copolymer has a high melting point even when the copolymerization component content of the 4HB component is high, 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 4HB component
As a result of intensive studies to industrially advantageously and easily produce a copolymer consisting of these components, we found that in the presence of T-butyrolactone in the subsequent nitrogen or phosphorus limiting culture,
The present invention was achieved based on the new finding that when a microorganism capable of producing B is cultured, a desired copolymer is produced and accumulated in the microorganism.

That is, in the present invention, cells of alkaline earth metal bacteria capable of producing poly-3-hydroxybutyrate are grown in the first step, and in the second step, the cells are cultured under limited nitrogen or phosphorus to inject into the cells. Produces poly-3-hydroxybutyrate.
The present invention provides a method for producing a polyester copolymer comprising 3-hydroxybutyrate units and 4-hydroxybutyrate units, characterized in that during accumulation, the latter stage of cultivation is carried out in the presence of T-butyrolactone.

The present invention will be explained in detail below.

In the present invention, the 3HB component and 4HB component contained in the copolymer are expressed by the following formulas.

3HB component; -0CH(CH3)CH, C-4HB component; OCH! CHt CHt C - The microorganism used in the present invention is not particularly limited as long as it has the ability to produce PHB, but in practice, for example, AIcaligenes fae
calts), Alcaligenes roulandii (A
lcaligenes ruhlandti),
Alcaligenes latus (Alcaligenes I)
atuS).

Alcaligenes aquamarinus (Alcaligen)
es aquamarinus) and Alcaligenes eutophus (Alcaligenes eut
rophs) and other alkaline earth metals.

Representative examples of strains belonging to these bacterial species include Alcaligenes faecalis ATCC8750 and Alcaligenes faecalis ATCC8750.
Rulandii ATCC15749, Alcaligenes
Rattus ATCC29712゜Alcaligenes aquamarinus ATCC14400 and Alcaligenes
Eutrophus H-16ATCC17699 and this H
-16 strain, Alcaligenes eutrophus NClB11597. Same NClB11598゜ Same N
ClB11599. Examples include the same NClB11600. Among these, Alcaligenes eutrophus H-16ATCC17699 and Alcaligenes eutrophus NClB11599 are particularly preferred for practical purposes.

The mycological properties of these microorganisms belonging to alkaline earth metals are, for example, “BERGEY” SMANUAL
OF DETERMINATIVE BACTE
RIOLOGY: Eighth Edition, The
Williams & Wilkins Com
Pany/Baltimore", and the mycological properties of Alcaligenes eutrophus H-16 are described, for example, in "J.

Qen, Microbiol, 115, 185-1
92 (1979).

Similar to conventional methods, these microorganisms are cultured in two stages: a first stage culture in which the bacterial cells are mainly grown, and a second stage culture in which nitrogen or phosphorus is restricted to produce and accumulate copolymers within the bacterial cells. be done.

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 used 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,
Nitrogen sources include, for example, ties such as glucose, mannose, fructose and galactose, and sorbitol, mannitol and inositol.
Inorganic nitrogen compounds such as ammonia, ammonium salts, nitrates and/or organic nitrogen-containing substances and inorganic components such as, for example, urea, corn steep liquor, casein, peptone, yeast extract, meat extract, etc.
For example, calcium salts, magnesium salts, potassium salts, sodium salts, phosphates, manganese salts, zinc salts, iron salts, copper salts, molybdenum salts, cobalt salts, nickel salts, chromium salts, boromine and iodine compounds. Each is selected from the following.

Additionally, vitamins and the like can be used as needed.

As for 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 6.5 to 9.5.
It is considered to be a degree. Cultivate aerobically under these 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 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.

This second-stage culture is similar to the first-stage culture except that the medium or culture solution does not substantially contain nitrogen and/or phosphorus and contains γ-butyrolactone as a carbon source.

In addition, when containing γ-butyrolactone in the culture solution,
It may be carried out at any time from the early stage to the late stage of the culture, but the early stage of the culture is preferable.

γ-Butyrolactone used in the present invention may be used in an amount that can produce a copolymer and does not inhibit the growth of microorganisms, such as the strain of the microorganism used and the desired copolymerization ratio (molar ratio). Although it varies depending on the situation, in general, about 3 to 100 g is appropriate for the medium or culture solution Il.

Although T-butyrolactone may be used as the sole carbon source in this subsequent culture, other carbon sources that can be used by the microorganisms used may be used, such as glucose, fructose, methanol, ethanol, acetic acid, propionic acid, n-acetic acid,
Diols such as lactic acid and valeric acid, 4-hydroxyacetic acid and salts thereof, and 1,4-butanediol can also be present. For example, when glucose is used, the amount is about 1.5 g/l at most.

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. A copolymer produced from the bacterial cells is extracted using an organic solvent such as chloroform 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 3HB component in the copolymer, the 4
The proportion of the HB component 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 examples.

Examples 1 to 5 and Comparative Examples 1 to 2 Alcaligenes eutrophus H16 (ATCC176
99) was used to produce the copolymer.

That is, preliminary culture: The above microorganisms were incubated at 30°C for 24 hours in a medium having the following composition.
The cells were cultured for several hours, and the bacterial cells were separated from the culture solution at the end of the logarithmic growth phase by centrifugation.

Composition of culture medium for first stage: Yeast extract 10g Polypeptone 10g Meat extract 5g (Ntla)zs045g These were dissolved in 1z of demineralized water and adjusted to pH 7.0.

Post-stage culture: The bacterial cells obtained in the above culture were suspended in a medium having the following composition at a ratio of 5 g per 11 cells, cultured at 30°C for 48 hours, and the bacterial cells were removed from the resulting culture solution by centrifugation. The cells were separated to obtain bacterial cells. Composition of medium for second stage culture 0,5 M Potassium hydrogen phosphate aqueous solution 39.0 0,5 M Dipotassium hydrogen phosphate aqueous solution 53.6 1 20wt/V% Magnesium sulfate aqueous solution 1.0++1
1 Carbon source 0 Mineral solution 1.0s+t! * Various compounds as listed in Table 1 below were used as carbon sources. (Unit: g/l medium) ** Mineral solution co c J z 119. O
wzFeCls 9.7gCa
CIt z 7.8 gN
Amount ct, 11B,
0 small ■-ran [Cr C41z
62.2111Ca S Oa 1
56.4 w to 0. Dissolved in 1N HCl II1 These were dissolved in deionized water 11 and adjusted to pH 7.0.

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

0.1 mmHg)/L/dry cells were obtained.

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, collect this precipitate by filtration, and dry. A copolymer was obtained.

Properties of copolymer: The composition, intrinsic viscosity, melting temperature and heat of fusion of the copolymer thus obtained were measured as follows. That is, composition: 'H-NM of 500MHz
According to R spectrum.

Intrinsic viscosity [η] = 30°C, in chloroform.

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

The measurement results are shown in Table 1.

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

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- A method for producing a polyester copolymer consisting of 3-hydroxybutyrate units and 4-hydroxybutyrate units, which comprises performing the subsequent cultivation in the presence of γ-butyrolactone when producing and accumulating hydroxybutyrate. .