JPH0412713B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to D-(-)-3-hydroxybutyrate (hereinafter referred to as component B) and D-(-)-3
- Concerning a method for producing a copolymer containing hydroxyvalerate (hereinafter referred to as component V), more specifically, a method for producing a copolymer containing hydroxyvalerate (hereinafter referred to as component V), in which the ratio of component B and component V can be arbitrarily controlled within a wide range, and in a high yield. It relates to a manufacturing method for obtaining this copolymer. [Prior art and problems to be solved by the invention] Poly-3-hydroxybutyrate (hereinafter referred to as
PHB (sometimes written as PHB) is a thermoplastic polymer that is produced and accumulated in the cells of many microorganisms as an energy storage substance, and exhibits excellent biodegradability and biocompatibility. It has attracted 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 conservation, and PHB is attracting attention as a biopolymer that does not depend on petroleum. 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 component B and component V, and methods for producing the same. There is. The method for producing copolymers in these publications is similar to the conventional method for producing PHB, in which bacterial cells are grown in the first step, and microorganisms are cultured in the second step with limited nitrogen or phosphorus to produce the copolymer. It is manufactured. However, in the former, by using, for example, propionic acid and isoacetic acid as substrates in the subsequent culture, 99.9 to 50 mol% of the B component and 0.1 to 50 mol% of other ester components such as the V component, for example. There is a description that a copolymer containing the following is produced. However, in this publication, for example, in the examples, only copolymers containing up to 33 mol% of V component as other ester components are shown, and copolymers containing more V component than this are not specifically described. is not shown. On the other hand, in the latter case, PHB
There is a qualitative description that a copolymer containing at least 40 mol% of component B and other ester components is produced using carbon from the cell material of waste bacterial cells after extraction. However, this publication does not disclose any copolymer that specifically indicates the proportions of component B and component V. In addition, this method is complicated, and the components of the cell material are unstable as the type and amount of the material vary widely depending on the culture conditions, etc., and thus is not practical. On the other hand, the present inventor previously invented a copolymer containing component B and component V, in which the V component is 50 mol% or more, and a method for producing the same, and filed a Japanese patent application (Japanese Patent Application Laid-Open No. 63-269989). However, in this invention, the V component is 50 to 95
Although it can be controlled arbitrarily within the range of mol%, the yield of the copolymer produced and accumulated within the bacterial cells is insufficient and is not suitable for practical use. [Means and effects for solving the problems] The present inventors can save the amount of valeric acid used and arbitrarily control the ratio of component B and component V within a wide range,
Furthermore, in order to improve the yield of the copolymer, as a result of intensive research, we have been able to control the nitrogen or phosphorus in the latter stage to produce and accumulate a copolymer containing component B and component V mainly within the bacterial cells. By replacing a portion of valeric acid with n-acetic acid during culture, a copolymer with the molar ratio of component B and component V arbitrarily controlled over a wide range can be produced in high yield within the bacterial cells. , we have obtained new knowledge that it is accumulated, and have arrived at the present invention based on this new knowledge. That is, the present invention involves culturing bacteria that belong to the genus Alcaligenes and have the ability to produce poly-3-hydroxybutyrate, by mainly growing the bacterial cells in the first stage and under nitrogen or phosphorus limitation in the second stage. When producing and accumulating a copolymer containing D-(-)-3-hydroxybutyrate and other ester components in the bacterial cells, (a) valeric acid, a derivative thereof, or a salt thereof; (b) Cultivating the bacteria in the coexistence of n-acetic acid or its derivatives or salts thereof, and injecting D-(-)-3-hydroxybutyrate and D-(-)-3-hydroxybutyrate into the bacterial cells. This is a method for producing a copolymer, which is characterized by producing and accumulating a copolymer containing a compound. In the present invention, component B and component V contained in the copolymer are represented by the following formulas. That is, _component B: -OCH( _CH3 ) _CH2CO- component V: -OCH( _C2H5 ) _CH2CO- The bacteria used in the present invention belong to the genus Alcaligenes and have the ability to produce PHB. Good to have,
There are no particular restrictions, but in practice, for example, Alcaligenes facalis,
Alcaligenes denitrificans, Alcaligenes denitrificans
Rattus (Alcaligenes latus), Alcaligenes
Aquamarinus (Alcaligenes aquamarinus) and Alcaligenes eutrophus (Alcaligenes eutrophus) are used. Representative examples of strains belonging to these bacterial species include Alcaligenes fuecalis ATCC 8750, Alcaligenes denitrificans subspecies xylose oxidans ATCC 15749,
Alcaligenes latus ATCC 29712, Alcaligenes aquamarinus ATCC 14400 and Alcaligenes eutrophus H-16
ATCC 17699 and Alcaligenes eutrophus, a mutant strain of this H-16 strain.
NCIB 11597, NCIB 11598, NCIB
11599, NCIB 11600, etc. Among these, Alcaligenes eutrophus H-16 ATCC 17699 and Alcaligenes eutrophus NCIB 11599 are particularly preferred for practical purposes. The mycological properties of these bacteria belonging to the genus Alcaligenes are described, for example, in “BERGEY'S MANUAL
OF DETERMINATIVE BACTERIOLOGY:
Eighth Edition, The Williams & Wilkins
Company/Baltimore”, and the mycological properties of Alcaligenes eutrophus H-16.
For example, “J.Gen.Microbiol., 115, 185-192
(1979)". These bacteria are produced in the same manner as in conventional production methods, mainly through the initial cultivation of bacterial cells, and
It is cultured in two stages, with nitrogen or phosphorus being restricted and the latter stage of culture mainly producing and accumulating copolymers within the bacterial cells. For the first-stage culture, a normal culture method for growing bacteria can be applied. In other words, selecting a medium and culture conditions that allow the bacteria to grow,
Just adopt it. There are no particular restrictions on the medium components as long as they can be assimilated by the bacteria 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. Carbon sources, natural products such as yeast extract, molasses, pepotone and meat extract, sugars such as arabinose, glucose, mannose, fructose and galactose and sugar alcohols such as sorbitol, mannitol and inositol, etc.
Nitrogen sources include, for example, inorganic nitrogen compounds such as ammonia, ammonium salts, nitrates and/or
Or, for example, urea, corn stapler
Organic nitrogen-containing substances such as liquor, casein, peptone, yeast extract, and meat extract; and inorganic components such as calcium salts, magnesium salts, potassium salts, sodium salts, phosphates, manganese salts, zinc salts, and iron. salts, copper salts, molybdenum salts, cobalt salts, nickel salts, chromium salts, boron 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, 20~40℃.
The temperature is about ℃, preferably about 25 to 35℃, and the PH
is, for example, about 6 to 10, preferably 6.5 to 9.5
It is considered to be a degree. It is cultivated aerobically under these conditions. When cultured outside of these conditions, bacterial growth becomes relatively poor, but this does not prevent cultivation outside of these conditions. The culture method may be batch culture, continuous culture or semi-continuous culture. The bacterial cells obtained in the first stage of culture were further cultured.
Cultivate under nitrogen and/or phosphorus-limited conditions. That is, from the culture solution obtained in the previous stage of culture,
Sterile bacterial cells are separated and recovered by ordinary solid-liquid separation means such as filtration and centrifugation, and these cells are subjected to subsequent culture, or nitrogen and/or phosphorus are substantially removed during the initial culture. This can also be done by depleting the bacterial cells, or by leaving a small amount of nitrogen and/or phosphorus remaining, and using this culture solution to transfer to the subsequent culture without separating and recovering the bacterial cells. In this latter stage of cultivation, the medium or culture solution does not substantially contain nitrogen and/or phosphorus, or contains a small amount of nitrogen and/or phosphorus, and (a) valeric acid, its derivatives, or salts thereof (including (sometimes collectively referred to as valeric acids) and (b)n-acetic acid,
There is no difference from the previous culture except for the inclusion of derivatives thereof or salts thereof (sometimes collectively referred to as n-butyric acids). Valeric acid and its derivatives have the general formula CH ₂ XCHYCH ₂ CH ₂ COOH (wherein, ), and as a representative example of this derivative, 4
-chlorovaleric acid, 4-hydroxyvaleric acid, 4-methylvaleric acid, 4-ethylvaleric acid, 5-hydroxyvaleric acid, and 5-chlorovaleric acid. Representative examples of the respective salts of valeric acid and its derivatives include sodium salt and potassium salt. n-Butyric acid and its derivatives are represented by the general formula CH ₂ XCH ₂ CH ₂ COOH (wherein, Examples include butyric acid and 4-hydroxy n-butyric acid. Representative examples of the respective salts of n-acetic acid and its derivatives include sodium salt and potassium salt. Valeric acids and n-butyric acids 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 final stage of the culture, but the early stage of the culture is preferable. The amounts of valeric acids and n-butyric acids to be used may be such that the copolymer can be produced and the growth and proliferation of bacteria is not inhibited, depending on the strain of bacteria used and the copolymer. Although it varies depending on the desired molar ratio of component V to component B of the polymer, the total concentration of valeric acids and n-butyric acids in the medium and culture solution is usually determined as a free acid in the medium. Alternatively, the amount is about 5 to 40 g per culture solution, preferably about 10 to 30 g. In addition, the molar ratio of the V component in the copolymer can be increased as the ratio of valeric acids is increased and the ratio of n-butyric acid is decreased in the medium or culture solution. In this latter stage of cultivation, the carbon sources may be both valeric acids and n-butyric acid, but other carbon sources that can be assimilated by the microorganisms used, such as glucose, fructose, methanol, ethanol, acetic acid, A small amount of propionic acid, lactic acid, etc. may also be present. For example, when glucose is present, the concentration of glucose is about 1.5 g/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, or the bacterial cells are produced from the wet bacterial cells by a conventional method. The copolymer is extracted with an organic solvent such as chloroform, and a poor solvent such as hexane is added to the extract to precipitate the copolymer. [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. Example 1 Alcaligenes eutrophus NCIB 11599
A copolymer was produced using That is, first-stage culture: Next, the above-mentioned microorganisms are cultured at 30% in a medium having the following composition.
The cells were cultured at ℃ for 24 hours, and the bacterial cells were separated and recovered from the culture solution in the winter logarithmic growth phase by centrifugation. Composition of medium for first stage culture Yeast extract 10g Polypeptone 10g Meat extract 5g (NH ₄ ) ₂ SO ₄ 5g These were dissolved in 1 part of deionized water and adjusted to pH 7.0. Post-stage culture: The bacterial cells obtained in the first-stage culture were suspended in a medium having the following group name at a rate of 3.5 g per 30
The cells were cultured at ℃ for 48 hours, and the bacterial cells were separated and recovered from the resulting culture solution by centrifugation. 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 ^* The following amounts as carbonate source of valeric acid and n-butyric acid were used, respectively. Valeric acid (g/) n-Butyric acid (g/) 18 2 16 4 14 6 12 8 10 10 5 15 ^** Mineral solution CoCl ₂ 119.0mg FeCl ₃ 9.7g CaCl ₂ 7.8g NiCl ₂ 118.0mg CrCl ₂ 62.2mg CaSO 156.4 mg of ₄ was dissolved in 1 part of 0.1N HCl. These were dissolved in 1 part of deionized water and the pH was adjusted to 7.0. 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℃,
0.1 mmHg) to obtain dried bacterial cells. Table 1 shows the dry bacterial body weights in each culture solution when the above-mentioned ~ were used as carbon sources. Separation and recovery of copolymer: From the dried bacterial cells thus obtained, extract the copolymer with hot chloroform, add hexane to this extract to precipitate the copolymer, and collect this precipitate by filtration. A copolymer was obtained by drying. 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: According to ¹ H NMR spectrum. Intrinsic viscosity [η]: 30℃, in chloroform. Melting temperature Tm: Based on DSC measurement. (heating rate
10℃/min) Heat of fusion: Based on DSC measurement. The measurement results are shown in Table 1. In addition, in this example, as a carbon source ~
Regarding the copolymers obtained using each of
The chain distribution of the copolymer was determined using 125NH ₂ ¹³ C NMR spectrum. That is, the method of the present inventor and others [Y. Doi et al. Maclomolecules, 19, 2860-
2864, (1986)], the dyad chain distribution of the B and V components was determined from the multiplet resonance structure of carbonyl carbon. For example, it was found that the case has the following linkage distribution. Namely, BB (butyrate-butyrate) chains 32% BV (butyrate-valerate) and VB (valerate-butyrate) chains 48% VV (valerate-valerate) chains 20% This chain distribution indicates that the copolymer has a random copolymerization chain distribution. It shows that it has. Comparative Example The same procedure as in Example 1 was carried out except that valeric acid was used alone or valeric acid and glucose were used together as a carbon source. That is, Valeric acid (g/) Glucose (g/) 20.0 0 19.5 0.5 19.0 1.0 18.0 2.0 The results are shown in Table 1.

〔Effect of the invention〕

By the production of the present invention, the molar ratio of the V component to the B component of the copolymer can be arbitrarily adjusted within a wide range, and therefore, a copolymer having a large molar ratio of the V component to the B component can also be produced. Furthermore, the copolymer can be obtained in high yield. The copolymer obtained in the present invention is D-(-)-3-
Compared to poly-3-hydroxybutyrate consisting only of hydroxybutyrate, the melting temperature is lower,
The melting temperature stability is high and the crystallinity is low, so it has excellent strength, is easy to form by spinning and rolling, and is stable.
The resulting finished products, such as fibers and films, are flexible and strong. In addition, the copolymer obtained by the present invention has various excellent properties as mentioned above, and is therefore extremely suitable as a raw material for medical materials such as surgical threads and fracture fixing materials. It has the potential to be applied in a variety of fields, including use in radioactive systems.

Claims (1)

[Claims] 1. Bacteria belonging to the genus Alcaligenes and having the ability to produce poly-3-hydroxybutyrate are mainly grown in the first stage, and in the second stage, the bacteria are cultured under nitrogen or phosphorus limitation, so that D. −
When producing and accumulating a copolymer containing (-)-3-hydroxybutyrate and other ester components, (a) valeric acid or a derivative thereof or a salt thereof and (b) n-acetic acid are added in a later stage. or a derivative thereof, or a salt thereof, and culturing the bacterium in the coexistence with
A method for producing a copolymer, which comprises producing and accumulating a copolymer containing D-(-)-3-hydroxybutyrate and D-(-)-3-hydroxyvalerate in the bacterial cells.