JPH0412712B2 - - Google Patents

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention provides D-(-)-3-hydroxybutyrate (hereinafter referred to as component B) and D-(-)-3
-Regarding a method for producing a copolymer containing hydroxyvalerate (hereinafter referred to as component V). [Prior art and problems to be solved by the invention] Poly-3-hydroxybutyrate (PHB) is
It is a thermoplastic polymer that is accumulated in the bodies of many microorganisms as an energy storage substance and exhibits excellent biodegradability and biocompatibility, so it has attracted attention as a "clean" plastic that protects the environment.
It has been expected for many years to find applications in a wide range of fields, including medical materials such as surgical threads and fracture fixation materials, and sustained-release systems that gradually release pharmaceuticals and agricultural chemicals. Especially in recent years, synthetic plastics have become a serious social problem from the perspective of environmental pollution and resource recycling, 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 PHB production methods in these publications are different from conventional PHB.
As in the production method described above, in the first stage, the microbial cells are grown, and in the second stage, the microorganisms are cultured with limited nitrogen or phosphorus to produce the copolymer. However, in the former, by using, for example, propionic acid and isobutyric 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, only copolymers containing up to 33 mol% of V component are shown in the examples, and copolymers containing more V component than this are not specifically shown. . On the other hand, in the latter case, PHB
There are qualitative descriptions of using carbon from the cell material of waste bacterial cells after extraction to produce a copolymer containing at least 40 mol % of component B and other ester components. However, this publication does not describe any copolymer that specifically indicates the ratio of component B to 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 considerably depending on the culture conditions, etc., and thus it is not practical. Therefore, there has been a desire for an industrially advantageous method that can arbitrarily control the ratio of component V to component B in a copolymer. [Means and effects for solving the problem] The present inventors have made extensive efforts to develop an industrially advantageous production method that can arbitrarily control the ratio (molar ratio) of component V to component B. As a result, a bacterium belonging to the genus Alcaligenes and capable of producing poly-3-hydroxybutyrate was grown in the first stage, and in the second stage, the bacterium was cultured under nitrogen or phosphorus limitation to produce D-( When producing and accumulating a copolymer containing -)-3-hydroxybutyrate and D-(-)-3-hydroxyvalerate, the bacteria are grown using a carbon compound other than glucose and fructose as a carbon source in the first step. We have discovered an efficient method that allows us to arbitrarily control the ratio of component V to component B in a copolymer by growing it and culturing it in the presence of valeric acid, its derivatives, or salts thereof in the latter stage, and have arrived at the present invention. did. In the present invention, component B and component V contained in the copolymer are each represented by the following formulas. That is, _component B: -OCH( _CH3 ) _CH2CO- component V: -OCH( _C2H5 ) _CH2CO- The microorganism used in the present invention belongs to the genus Alcaligenes and has the ability to produce PHB. There is no particular restriction if it is, but for practical purposes, for example, Alcaligenes faecalis
ruhlandii), Alcaligenes latus, Alcaligenes aquamarinus and Alcaligenes eutrophus
eutrophus). Representative examples of strains belonging to these bacterial species include Alcaligenes fuecalis ATCC8750, Alcaligenes roulandii ATCC15749, Alcaligenes latus ATCC29712, Alcaligenes aquamarinus ATCC14400, and Alcaligenes eutrophus H-
16ATCC17699 and the mutant strain of this H-16 strain, Alcaligenes eutrophus.
NCIB11597, NCIB11598, NCIB11599,
Examples include NCIB11600. Among these, Alcaligenes eutrophus H-16 ATCC17699 and Alcaligenes
Eutrophus NCIB11599 is particularly preferred. The mycological properties of these microorganisms belonging to the genus Alcaligenes are, for example, “BERGEY′S
MANUAL OF DETERMINATIVE
BACTERIOLOGY: Eighth Edition, The
Williams & Wilkins Company/Baltimore”
In addition, the mycological properties of Alcaligenes eutrophus H-16 are described in, for example, “J.Gen.Microbiol .
115, 185-192 (1979), respectively. 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. The medium components may be any substances that can be assimilated by the microorganisms used. Practically speaking, carbon sources include synthetic carbon sources such as methanol, ethanol and acetic acid, inorganic carbon sources such as carbon dioxide, yeast extract, Natural products such as peptone and meat extract are used. However, glucose and fructose cannot be used. Nitrogen sources include, for example, inorganic nitrogen compounds such as ammonia, ammonium salts, nitrates and/or e.g. urea, corn staple, liquor, casein, peptone, yeast extract,
Examples of organic nitrogen-containing substances and inorganic components such as meat extract include calcium salts, magnesium salts, potassium salts, sodium salts, phosphates, manganese salts, zinc salts, iron salts, copper salts, molybdenum salts,
Each is selected from 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℃.
It is about ℃, preferably about 25 to 35℃, and
PH is, for example, about 6 to 10, preferably 6.5 to 10.
It is said to be around 9.5. 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, 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 This can also be achieved by substantially depleting nitrogen and/or phosphorus in the first-stage culture and transferring this culture solution to the second-stage culture without separating and recovering the bacterial cells. In this latter stage of cultivation, the medium or culture solution should be substantially free of nitrogen and/or phosphorus, and valeric acid, its derivatives, or salts thereof (these may also be collectively referred to as valeric acids). There is no difference from the previous culture except for the inclusion of a certain amount of carbon dioxide as a carbon source. 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. Valeric acids are contained in the culture 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 amount of valeric acids to be used may be such that the copolymer can be produced and the growth of microorganisms is not inhibited. Although it varies depending on the desired ratio, in general, as the concentration of valeric acids in the medium or culture solution increases, the ratio of component V in the copolymer increases. For example, in order to make the V component of the copolymer 50 mol% or more, the concentration of valeric acids in the medium or culture solution is usually 5 to 40% as valeric acid per medium or culture solution.
It is about 10 to 30 g, preferably about 10 to 30 g. In this latter stage of culture, valerates may be used as the sole carbon source, 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, etc. , lactic acid, etc., may also be present in small amounts. For example, when glucose is used, the amount is about 1.5 g/at most. From the culture solution obtained by culturing in this way, 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, From this dried bacterial body, by the usual method,
For example, the produced 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. By the production method of the present invention, the ratio of the V component to the B component of the copolymer can be arbitrarily adjusted, and furthermore, a copolymer with a large ratio of the V component to the B component can be obtained, and the B component is 50% Even new copolymers with a V component of less than 50 mol % can be obtained. The copolymer obtained in the present invention has a relatively large ratio of component V to component B, which lowers the melting temperature, increases the stability of the melting temperature, and decreases the degree of crystallinity. In addition, it has excellent strength and can be easily and stably formed by spinning, rolling, etc., and the obtained molded products such as fibers and films are flexible and strong. [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 In other words, first-stage culture: microorganisms in the first stage are grown at 30°C in a medium with the following composition.
The cells were cultured for 24 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 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 composition at a ratio of 5 g per 1 cell at 30°C.
The cells were cultured for 48 hours, and the cells were separated from the resulting culture solution by centrifugation to obtain cells. Composition of secondary culture medium 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 proportions as carbon source valeric acid and/or glucose (g/medium) were used. Valeric acid Glucose 20 0 20 0.5 20 1.0 0 20 *Mineral solution CoCl ₂ 119.0mg FeCl ₃ 9.7g CaCl ₂ 7.8g NiCl ₂ 118.0mg CrCl ₂ 62.2mg CaSO ₄ Dissolve 156.4mg in 0.1N-HCl 1 Deionize these It was dissolved in water 1 and adjusted to pH 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°C,
0.1 mmHg) to obtain dried bacterial cells. There was virtually no difference in the dry bacterial weight when using the above-mentioned ~. 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. Namely, Composition: According to ^1HNMR spectrum. Intrinsic viscosity [η]: 30℃, in chloroform. Melting temperature Tm: Based on DSC measurement. (Heating rate 10
℃/min) Heat of fusion ΔH: Based on DSC measurement. The measurement results are shown in Table 1. Incidentally, the chain distribution of the copolymer was determined using 125MH ₂ ¹³ C NMR spectrum. That is, the method of the present inventor and others [Y. Doi et al, Macromolecules, 19 , 2860-
2864 (1986)], the dyad chain distribution of B and V units was determined from the multiplet resonance structure of carbonyl carbon. This copolymer was found to have the following chain distribution. BB (butyrate-butyrate) chains 3% BV (butyrate-valerate) and VB (valerate-butyrate) chains 12% VV (valerate-valerate) chains 85% This chain distribution indicates that the copolymer has a random copolymerization chain distribution. It shows that it has. Example 2 Alcaligenes eutrophus H-16
Using ATCC17699, valeric acid 20 as carbon source
The procedure was the same as in Example 1 except that g/medium was used. The results are shown in Table 1. [Table] [Effects of the Invention] By the production method of the present invention, a copolymer having a large ratio of component V to component B can be obtained easily and efficiently, and even a novel copolymer can be obtained. I can do it. 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 is also suitable for use in sustained release systems. It is expected that it will be applied in many fields, such as the use of

Claims (1)

[Claims] 1 Bacteria belonging to the genus Alcaligenes and capable of producing poly-3-hydroxybutyrate are grown in the first stage, and in the second stage, the bacteria are cultured under nitrogen or phosphorus limitation to produce D-(- )-3-Hydroxybutyrate and D-(-)-3-hydroxyvalerate-containing copolymer, in the first step, the bacteria are grown using a carbon compound other than glucose and fructose as a carbon source. In the latter stage, D-(-) is cultured in the presence of valeric acid or its derivatives or salts thereof.
-3-hydroxybutyrate and D-(-)-3
- A method for producing a copolymer, which comprises producing and accumulating a copolymer containing hydroxyvalerate.