JPH01156320A - Polyester copolymer and production thereof - Google Patents

Abstract

PURPOSE:To produce the title copolymer advantageously and easily by multiplying an alcaligenes which can produce poly-3-hydroxybutyrate, and then cultivating it in the presence of two specific compounds. CONSTITUTION:An alcaligenes which can produce poly-3-hydroxybutyrate (e.g., Alcaligenes faecalis ATCC8750) is inoculated in an ordinary medium containing sources of carbon and nitrogen, inorganic components, etc., and is cultivated aerobically at a pH of 6-10 and a temperature of 20-40 deg.C to multiply the fungus. Then, the cells are cultivated in a culture broth which is free from N2 and P and contains a compound of formula I (wherein X is H, halogen, or hydroxy; Y is alkyl; n is 1-4; and Z is H or a monovalent-tetravalent metal element) and a compound of formula II (wherein M is hydroxy or halogen; and N is Z) in an amount of 3-40g/l to produce a polyester copolymer comprising 10-90mol.% 3-hydroxybutyrate unit, 3-60mol.% 4-hydroxybutyrate unit, and 5-87mol.% 3-hydroxyvalerate unit and having an intrinsic viscosity of 0.4-10.0dl/g (30 deg.C, in chloroform).

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a 3-hydroxybutyrate unit (hereinafter referred to as 3HB component), a gu-hydroxybutyrate unit (hereinafter ≠H
3HB component produced using a microorganism capable of accumulating polyester, The present invention relates to a new copolyester comprising a ≠HB component and a 3H■ component, 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 helping to preserve the environment. It has been attracting attention as a "Vaclean" 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 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 carried out on copolymers containing 3HB and 3HV components and methods for producing the same. has been done.

The PHB manufacturing method described in these publications is similar to the conventional PHB manufacturing method, in which microbial cells are grown in the first stage, and microorganisms are cultivated in the second stage with limited nitrogen or nitrogen to produce a copolymer. It is something.

However, in the former case, by using, for example, propionic acid and isobutyric acid as substrates in the subsequent culture, the 3HB component Tata, Ri~SO morti and other ester components such as the 3H■ component O0/~ 5
There is a description that a copolymer containing 0 mol of rice is produced. However, in this publication, for example, the examples only show copolymers containing a maximum of 33 moles of 3HV components, and do not specifically show copolymers containing more 3HV components. .

On the other hand, in the latter case, carbon from the cell material of waste bacterial cells after PHB extraction is used in the subsequent culture to at least ≠
There is a qualitative description that a copolymer containing the 3HB component of θmolti and other ester components is produced. However, this publication does not describe any copolymer that specifically indicates the ratio of the 3HB component to the 3HV component.

In addition, this method is complicated, and the components of the cell material are unstable due to large variations in the type and amount of substances depending on the culture conditions, etc., and thus are not practical.

Furthermore, as the 3Hv component of the copolymer increases from O to 33M, the melting temperature (Tm) increases by 1
It is known that the temperature decreases rapidly from 10℃ to ♂j℃ (T, L, Bluhm etal, Macrom
olecules, /ri, 2za7/-217t(
This means that it is difficult to obtain a uniform product industrially.

[Means for solving problems]

The present inventor has discovered that the copolymerization component for the 3HB component, that is, ≠H
As a result of intensive studies to industrially advantageously and easily produce a copolymer with a relatively large proportion (molar ratio) of B and 3HV components, we found that (a) the following general When a microorganism capable of producing PHB is cultured in the presence of a compound represented by formula (1) and (b) a compound represented by the following general formula (II), ≠ HB component and 3HV component relative to 3HB component in the microbial cell. The present invention was achieved based on the new finding that copolymers with a relatively large proportion (molar ratio) of .

(CH2XCHYCH2CH2COO) Z
(1) (wherein, Indicates an atom.) (CH2M CH2CH2COO) N (
II) (In the formula, M is a hydroxy group or a halogen atom, n is an integer from / to ≠, and N is a hydrogen atom or a metal atom with a valence of /-1d.) That is, the present invention is based on the 3HB:IO-taO molar relationship. ,ψHB:
3-O mole section, 3Hy: consisting of t-g7 mole candy [η
] is 0. Hiro ~ / 0. It consists in a polyester copolymer in the range of Odl/it' and a method for producing the same.

The present invention will be explained in detail below.

In the present invention, the 3HB component contained in the copolymer, FR
The B component and the 3HV component are each expressed by the following formula.

3HB component: OCR (CH3) CH2C - The microorganism used in the present invention is not particularly limited as long as it has the ability to produce PHB, but in practical use, for example, Alcaligenes faecalis
ecalis), Alcaligenes leulandii (
Alca]igenes ruhlandii).

Alcaligenes ratus (Alcaligenes 1)
atus).

Alcaligenes aquamarinus (Alcali-g)
enes aquamarinus) and Alcaligenes eutophus (Alcaligenes eut
rophs) and other members of the genus Alcaligenes.

A typical example of a strain belonging to these bacterial species is Alcaligenes faecalis ATCCIqso.

Alcaligenes Roulandii ATCC/! ;7≠ri.

Alcaligenes latus ATCC2ri7/2,7 Lucarigenes aquamarinus ATCC/≠ti-o.

and Alcaligenes eutrophus H-/4AT
CC/7 Otata and this H-//; mutant strain of Alcaligenes eutrophus NCIB//3ri7
．． Same NCIB //! ; Rig, same NCIB//3 Tata,
Examples include NCIB i/Otsuoo. Of these, in practice, Alcaligenes eutrophus H.
Particularly preferred are ATCC/74 Tata and Alcaligenes eutrophus NCIB//j Tata.

The mycological properties of these microorganisms belonging to the genus Alcaligenes are, for example, "BERGEY'SMANUAL"
OF DETERMINATIVE BACTE
RI-0LOGY: Eighth Editio
n, The Williams & Wilkins
Company/Baltimore", and the mycological properties of Alcaligenes eutrophus H-/&, for example, "J, Gen, Microbio.
l.

//j /doj~/ri2 (/ri7ri) respectively.

Similar to conventional methods, these microorganisms are cultivated through a combination of a first stage culture in which the bacterial cells are mainly grown, and a second stage culture in which nitrogen or phosphorus is limited to produce and accumulate copolymers within the bacterial bodies. 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 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 and galactose and nrubitol, mannitol and inositol, as nitrogen sources, for example inorganic nitrogen compounds such as ammonia, ammonium salts, nitrates and/or for example urea, corn steep liquor, Examples of organic nitrogen-containing substances such as casein, peptone, yeast extract, and meat extract as well as inorganic components include calcium salts, magnesium salts, potassium salts,
Sodium salts, sinate, manganese salts, zinc salts, iron salts,
Each is selected from copper salts, molybdenum salts, cobalt salts, nickel salts, chromium salts, boromine compounds, iodine compounds, and the like.

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

As for the culture conditions, the temperature is, for example, about λO to ψO℃, preferably about 25 to 30℃, and the pH is
For example, about 6 to IO, preferably B, 5 to RI,
It is said to be around evening. 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.

In this latter stage of culture, the medium or culture solution is not substantially free from nitrogen and/or phosphorus (a) the compound represented by the general formula (I) and (
b) There is no difference from the previous culture except that the compound represented by the general formula (If) is contained as a carbon source.

Specifically, the compounds represented by the general formula (I) that can be used in the present invention include valeric acid, ≠-chlorovaleric acid, ≠-hydroxyvaleric acid, ≠-methylvaleric acid, ψ-ethylvaleric acid, j Examples include -hydroxyvaleric acid, j-chlorovaleric acid, and their respective sodium and potassium salts.

Specifically, the compounds represented by the general formula (II) that can be used in the present invention include butyric acid derivatives such as ψ-hydroxybutyric acid, ≠-chlorobutyric acid, ≠-bromobutyric acid, and their sodium and potassium salts, Examples include calcium salts.

In carrying out the present invention, the compounds represented by the general formula (1) and general formula (I[) are contained in the medium or culture solution in the subsequent culture. In the latter case,
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.

The general formula (I) and -general formula (I) used in the present invention
The compound represented by f) may be used in an amount that can produce a copolymer and does not inhibit the growth of microorganisms, and may be used in combination with the strain of microorganism used and the desired copolymerization ratio (
-For boat fishing, the above general formula (1) and general formula (If) are added to the medium or culture solution/l.
Is the total 3~≠O? The degree is appropriate.

In this latter stage of cultivation, the compounds represented by the general formulas (1) and (II) may be used as the only carbon sources, but other carbon sources that can be assimilated by the microorganisms used, such as glucose, fructose, Methanol, ethanol, acetic acid, propionic acid, n-butyric acid, lactic acid, valeric acid, and the like can also be present. For example, when glucose is used, the amount is at most /, sr/.

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 ratio of the HB component and the JHV component can be adjusted as desired. The ratio of each component is 3HB: 10 to 0 mole, ≠H
B: 3 to 60 moles, JHV:, t to lr7 moles can be selected, for example, 3HB:
/ 0-do0 mole section, ≠HB: 3 ~ jO mole section, 3H■
: 10 to 70 molar ratio is selected.

〔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/~Hiroshi and Comparative Examples/~2 Copolymers were produced using Alcaligenes eutrophus NCIB //j Tata. First-stage culture: The above microorganisms were cultured at 30°C in a medium with 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 medium for first stage culture Yeast extract 10g Polypeptone 10g Meat extract g (N'H4)2 S04 g These were dissolved in deionized water/l and adjusted to pH 7.0.

Post-stage culture: The microbial cells obtained in the first-stage culture were suspended in a medium having the following composition at a ratio of jg/l and cultured at 30°C for ttg hours. were separated to obtain bacterial cells.

Composition of medium for subsequent culture 0.5M potassium hydrogen phosphate aqueous solution 3,0
m1o, s M dipotassium hydrogen phosphate aqueous solution
33. Otsuvt1.20 Wj/v% magnesium sulfate aqueous solution /, 0.1 Carbon source 1 Mineral solution ** /, 0@18
≠-Hydroxybutyric acid and valeric acid were used as carbon sources as described in Table/. (Unit g/medium) *8 Mineral solution CoCl2 / / ta, 0rtq
FeC13, 7g CaCl27.1 g NiCh / / fOmg CrC12 20.2 7ng CaSO4i s A4 mg 0. Dissolved in IN-HCl/t These were dissolved in deionized water/l and adjusted to I) H7.0.

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, Oolwn).
Hg) 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, 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 thus obtained copolymer were measured as follows. That is, Composition: Based on 'HNMR spectrum.

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

Melting temperature Tm: Based on DSC measurement.

(Temperature increase rate 70℃/min) Heat of fusion ΔH: Based on DSC measurement.

The measurement results are shown in Table 1.

In addition, r o OMHz' of the copolymer obtained in Example/
The H-NMR spectrum is shown in Figure 1, /2! ; M
The Hz13C-NMR spectra are shown in FIG. 2.

〔Effect of the invention〕

According to the present invention, 3HB component, 4tHB component and 3H■
Novel polyester copolymers containing the components can be easily obtained.

Furthermore, the copolymer obtained by 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, and can also be used for sustained release systems. It is expected that it will be applied in many fields.

[Brief explanation of the drawing]

Figure/ is the ro of the copolymer obtained in Example/. MHz, 'H-NMR spectrum, Figure 2 shows the i,zsMHz of the copolymer obtained in the same Example. 13C-NMR spectrum is shown. Applicant: Mitsubishi Chemical Industries, Ltd. Agent: Patent attorney: Yo Hase - and others/names

Claims (2)

[Claims] (1) 3-hydroxybutyrate units 10 to 90 mol%
Consisting of 3 to 60 mol% of 4-hydroxybutyrate units and 5 to 87 mol% of 3-hydroxyvalyrate units, at 30°C
[η] measured in chloroform is 0.4-10.0d
Polyester copolymers in the range of l/g. (2) In the first stage, the cells of Alcaligenes bacteria capable of producing poly-3-hydroxybutyrate are grown, and in the second step, the cells are cultured under nitrogen or phosphorus limitation, so that poly-3- When producing and accumulating hydroxybutyrate, the subsequent culture is carried out in the presence of (a) a compound represented by the following general formula (I) and (b) a compound represented by the following general formula (II). A method for producing a polyester copolymer comprising 3-hydroxybutyrate units, 4-hydroxybutyrate units and 3-hydroxyvalerate units. (CH_2XCHYCH_2CH_2COO)_nZ(
I) (However, in the formula, (represents a metal atom) (CH_2MCH_2CH_2COO)_nN(II) (However, in the formula, M represents a hydroxyl group or a halogen atom, n represents an integer of 1 to 4, and N represents a hydrogen atom or a monovalent to tetravalent metal atom)