Process for production of a random copolymer comprising D-(-)-3-hydroxybutyrate units and D-(-)-3-hydroxyvalerate.

0001This invention relates to a random copolymer and a process for its production. More specifically, it relates to a novel random copolymer comprising D-(-)-3-hydroxybutyrate units (to be sometimes referred to as units B) and D-(-)-3-hydroxyvalerate units (to be sometimes referred to as units V) and having a units V/units B mole ratio of more than 1, and a process for producing a random copolymer comprising the units B and units V.

0002Poly-3-hydroxybutyrate (to be referred to sometimes as PHB) is a thermoplastic polymer which is accumulated as an energy storage substance within the cells of many microorganisms and shows excellent bio­degradability and biocompatibility. Hence, it has aroused interest as "clean" plastics for preserving environments and has long been expected to find appli­cation in many fields of utility, for example, as a medical material such as a surgical suture and a fixing material for treatment of bone fracture, and as a material for use in slow-releasing systems for slowly releasing pharmaceuticals and agricultural chemicals. Particularly, as synthetic plastics have recently raised a serious social problem from the standpoint of environ­mental pollution and recycling of resources, PHB has attracted attention as a biopolymer not dependent on petroleum.

0003However, since PHB has low impact strength and the cost of its production is high, it has not yet gone into commercial production.

0004Japanese Laid-Open Patent Publications Nos. 150393/1982 and 220192/1984 propose copolymers comprising units B and units V, and processed for production thereof. The processes for producing PHB disclosed in these patent documents, like conventional processes for produc­ing PHB, involve growing the cells of a microorganism in a first step and cultivating the microorganism while limiting the amount of nitrogen or phosphorus, thereby to produce a copolymer.

0005Japanese Laid-Open Patent Publication No. 150393/1982 states that by using propionic acid and isobutyric acid as a substrate in the second step, a copolymer comprising 99.9 to 50 mole % of B units and 0.1 to 50 mole % of other ester units such as V units. However, this patent document describes a working example in which a copolymer containing at most 33 mole % of V units was obtained, and does not specifically describe a copolymer containing the V units in a larger proportion.

0006Japanese Laid-Open Patent Publication No. 220192/1984, on the other hand, has a qualitative de­scription that a copolymer comprising at least 40 mole % of B units and units of another ester can be produced when carbon from a cellular substance of the waste cells left after extraction of PHB is used in the second step. This patent document quite fails to describe copolymers indicating specific proportions of the units B and the units V. Furthermore, the process of this patent document is complex, and the type, amount, etc. of the cellular substance formed vary greatly depending upon the cultivation conditions. Hence, the process is unstable and not practical.

0007It is known that as the proportion of the V units in the copolymer increases from 0 to 33 mole %, the melting temperature (Tm) abruptly decreases from 180 to 85 °C [T. L. Bluhm et al., Macromolecules, <u style="single">19</u>, 2871-2876 (1986)]. This means that industrially, it is difficult to obtain a uniform product.

0008It is an object of this invention to provide a novel random copolymer comprising D-(-)-3-hydroxybutyrate units and D-(-)-3-hydroxyvalerate in which the pro­portion. of the D-(-)-3-hydroxyvalerate units is prefer­ably more than 50 mole % based on the total amount of these units.

0009Another object of this invention is to provide a process which can produce the random copolymer of this invention containing the D-(-)-3-hydroxyvalerate units in a proportion of more than 50 mole % based on the total amount of the two units in a yield sufficient for practi­cal application, and in which the content of the D-(-)-3-hydroxyvalerate units can be easily controlled to a range of more than 50 mole % to 95 mole %.

0010Further objects and advantages of this inven­tion will become apparent from the following description.

0011According to this invention, these objects and advantages of the invention are achieved by a random copolymer comprising less than 50 mole % of D-(-)-3-­hydroxybutyrate units and more than 50 mole % of D-(-)-3-hydroxyvalerate units, the percenrages being based on the total amount of the two units.

0012In the present invention, the units B and the units V in the copolymer are represented by the following formulae.     units B: -OCH(CH₃)CH₂CO-     units V: -OCH(C₂H₅)CH₂CO-

0013Preferably, the copolymer of this invention comprises 93 to 5 mole % of the B units and 7 to 95 mole % of the V units based on the total amount of the two units.

0014Preferably, the copolymer of this invention consists essentially of the units B and the units V.

0015The copolymer of this invention has an intrinsic viscosity [η], measured in a chloroform solution at 30 °C, of 0.1 to 10 dl/g, more preferably 1 to 9 dl/g.

0016According to this invention, the copolymer can be produced by a process which comprises <ul id="ul0001" list-style="none"><li>(1) cultivating a microorganism having the ability to produce poly-3-hydroxybutyrate to grow the cells of the microorganism,</li><li>(2) cultivating the cells in the presence of a valeric acid while limiting the amount of nitrogen or phosphorus to produce and accumulate a random copolymer comprising D-(-)-3-hydroxybutyrate units and D-(-)-3-­hydroxyvalerate units within the cells, and thereafter,</li><li>(3) recovering the copolymer from the cells.</li></ul>

0017Any microorganism which has the ability to produce PHB may be used in the process of this invention. In industrial practice, <u style="single">Alcaligenes</u><u style="single">faecalis</u>, <u style="single">Alcaligenes</u><u style="single">ruhlandii</u>, <u style="single">Alcaligenes</u><u style="single">latus</u>, <u style="single">Alcaligenes</u><u style="single">aquamarinus</u> and <u style="single">Alcaligenes</u><u style="single">eutrophus</u>, for example, are advantageously used.

0018Specific examples of strains belonging to these microorganism species include <u style="single">Alcaligenes</u><u style="single">faecalis</u> ATCC 8750 <u style="single">Alcaligenes</u><u style="single">ruhlandii</u> ATCC 15749, <u style="single">Alcaligenes</u><u style="single">latus</u> ATCC 29712, <u style="single">Alcaligenes</u><u style="single">aquamarinus</u> ATCC 14400 <u style="single">Alcaligenes</u><u style="single">eutrophus</u> H-16 ATCC 17699, and <u style="single">Alcaligenes</u><u style="single">eutrophus</u> NCIB 11597, NCIB 11598, NCIB 11599 and NCIB 11600 which are mutants of <u style="single">Alcaligenes</u><u style="single">eutrophus</u> H-16. In industrial practice, <u style="single">Alcaligenes</u><u style="single">eutrophus</u> H-16 ATCC 17699 and <u style="single">Alcaligenes</u><u style="single">eutrophus</u> NCIB 11599 are especially preferred.

0019The microbiological properties of these micro­organisms of the genus <u style="single">Alcaligenes</u> are described, for example, in "BERGEY'S MANUAL OF DETERMINATIVE BACTERI­OLOGY: English Edition, The Williams &amp; Wilkins Company, Baltimore". The microbiological properties of <u style="single">Alcaligenes</u><u style="single">eutrophus</u> H-16 are described, for example, in "J. Gen. Microbiol., <u style="single">115</u>, 185-192 (1979)".

0020According to this invention, these micro­organisms are cultivated in two steps as in conventional processes. In a first step, the microorganisms is cultivated to grow and proliferate the cells mainly, and in a second step, the microorganism cells are cultivated while limiting the amount of nitrogen or phosphorus in the cultivation system to form and accumulate the copoly­mer in the cells.

0021The first-step cultivation may be carried out by an ordinary cultivation method for growing and pro­liferating microorganisms using a medium and cultivation conditions suitable for growing and proliferating the microorganisms.

0022The components of the culture medium may be any substances which the microorganism used can utilize. In industrial practice, suitable carbon sources include synthetic carbon sources such as methanol, ethanol and acetic acid, inorganic carbon sources such as carbon dioxide, naturally occurring materials such as yeast extract, molasses, peptone and meat extract, carbo­hydrates such as arabinose, glucose, mannose, fructose and galactose, and sugar alcohols such as sorbitol, mannitol and inositol. Suitable nitrogen sources include inorganic nitrogen compounds such as ammonia, ammonium salts and nitrates and organic nitrogen compounds such as urea, corn steep liquor, casein, peptone, yeast extract and meat extract. Suitable inorganic materials include 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, boron compounds and iodine compounds. As required, vitamins may be used.

0023The cultivation temperature may be, for ex­ample, 20 to 40 °C, preferably about 25 to 35 °C. The pH of the culture medium may be, for example, about 6 to 10, preferably about 6.5 to 9.5. Under these conditions, the microorganism is cultivated aerobically.

0024When cultivation is carried out under con­ditions outside the above-mentioned conditions, pro­liferation of the microorganism becomes relatively poor. But since the proliferation does not stop under the other conditions, it is permissible to cultivate the micro­organism under the other conditions.

0025The cultivation may be carried out batchwise or continuously.

0026According to the process of this invention, the cells obtained by the cultivation in the first step are cultivated further while restricting the amount of nitrogen and/or phosphorus.

0027For example, the microorganism cells are sepa­rated and recovered by ordinary solid-liquid separation means from the culture broth obtained in the first-step cultivation, and the recovered cells are cultivated in the second step. Alternatively, in the first-step culti­vation, nitrogen and/or phosphorus is substantially exhausted, and without separating and recovering the cells from the culture broth, the culture broth <u style="single">per</u><u style="single">se</u> is subjected to cultivation in the second step.

0028The second-step cultivation is the same as the first-step cultivation except that nitrogen and/or phosphorus is substantially exhausted in the culture medium or the culture broth and a valeric acid is in­cluded as a carbon source.

0029The valeric acid is preferably a compound which is an acid represented by the following formula     CH₂XCHYCH₂CH₂COOH wherein X represents a hydrogen atom, a halogen atom or a hydroxyl group, and Y represents a hydrogen atom, a halogen atom, a hydroxyl group or an alkyl group, preferably a methyl group, or its salt.

0030Example of the valeric acid are valeric acid, 4-chlorovaleric acid, 4-hydroxyvaleric acid, 4-methyl­valeric acid, 4-ethylvaleric acid, 5-hydroxyvaleric acid and 5-chlorovaleric acid. Examples of its salt are sodium and potassium salts of the above acids.

0031The valeric acid is included in the culture medium used in the second-step cultivation or the culture broth in the second-step cultivation. In the latter case, it may be added at any stage of cultivation from its start to end. Preferably, it is included at the initial stage of the cultivation.

0032The amount of the valeric acid used may be one which is sufficient to produce the copolymer without inhibiting the growth and proliferation of the micro­organism, and may be varied with the type of the micro­organism strain used and the ratio of the units V to the units B in the copolymer. Generally, as the concen­tration of the valeric acid in the culture medium or the culture broth is increased, the proportion of the units V in the copolymer increases. For example, to adjust the proportion of the units V in the copolymer to more than 50 mole %, the concentration of the valeric acid in the culture medium and the culture broth is usually about 5 to 40 g, preferably 10 to 30 g, as valeric acid per liter of the culture medium or the culture broth.

0033In the second-step cultivation, the valeric acid may be used as a sole carbon source. It is possible however to use together another carbon source which the microorganism used can utilize, such as glucose, fructose, methanol, ethanol, acetic acid, propionic acid, n-butyric acid or lactic acid. If glucose is used as the other carbon source, its concentration should be adjusted to not more than about 1.5 g/ℓ.

0034According to this invention, the second-step cultivation is preferably carried out in the copresence of a valeric acid and an n-butyric acid. The valeric acid is preferably the compound represented by the above formula or its salt.

0035The n-butyric acid is preferably a compound which is an acid of the following formula     CH₂X¹CH₂CH₂COOH wherein X¹ represents a hydrogen atom, a halogen atom or a hydroxyl group, or its salt.

0036Examples of the n-butyric acid are n-butyric acid, 4-chloro-n-butyric acid and 4-hydroxy-n-butyric acid. Examples of its salt are sodium and potassium salts of these acids.

0037The valeric acid and the n-butyric acid are included in the culture medium used in the second-step cultivation or the culture broth in the second-step cultivation. In the latter case, they may be included at any stage of the cultivation from its start to end. Preferably, they are added at the initial stage of the cultivation.

0038The amounts of the valeric acid and the n-butyric acid may be one sufficient to form the copoly­mer without inhibiting the growth and proliferation of the microorganism, and may vary depending upon the type of the microorganism strain used, and the desired mole ratio of the units V to the units B. The total concen­tration of the valeric acid and the n-butyric acid in the culture medium and the culture broth is usually to 5 to 40 g, preferably to 10 to 30 g, calculated as valeric acid or n-butyric acid per liter of the culture medium or the culture broth.

0039The molar proportion of the units V in the copolymer can be higher as the proportion of the valeric acid in the culture medium or the culture broth is made higher or the proportion of the n-butyric acid is made lower.

0040Usually, it is advantageous to limit the n-butyric acid/valeric acid mole ratio to more than 0 to 10, preferably more than 0 to 5.

0041In the second-step cultivation, the valeric acid and the n-butyric acid may be used as sole carbon sources. They may, however, be used in combination with a small amount of another carbon source which the micro­organism can utilize, for example glucose, fructose, methanol, ethanol, acetic acid, propionic acid and lactic acid. However, if glucose is used as the other carbon source, its concentration should be adjusted to not more than about 1.5 g/liter.

0042According to this invention, the cells are separated and recovered from the resulting culture broth by ordinary solid-liquid separating means such as filt­ration and centrifugation. The cells are then washed and dried to obtain dry cells. The resulting copolymer is extracted from the dry cells in a customary manner, for example by using an organic solvent such as chloroform.

0043By the process of this invention, the ratio of the units V to the units B can be freely adjusted, and a copolymer having a major proportion of the units V and a minor proportion the units B can be obtained.

0044Because of these excellent properties, the copolymers obtained by this invention are expected to find many applications, for example as medical materials such as surgical sutures and fixing materials in the treatment of bone fracture, and in slow-releasing systems.

0045Those copolymers of this invention which contain a major proportion of the units V have a lowered melting temperature, high stability at the melting temperature and low crystallinity. Hence, they can be spun, rolled, or otherwise molded easily and stably to give fibers or films having high strength, pliability and toughness.

0046The following examples illustrate the present invention more specifically.

EXAMPLE 1

0047A copolymer was produced using <u style="single">Alcaligenes</u><u style="single">eutrophus</u> NCIB 11599.

First-step cultivation

0048The above microorganism was cultivated at 30°C for 24 hours in a culture medium having the following composition. The cells were separated from the culture broth in the final phase of the logarithmic growth stage.

Composition of the culture medium:

0049Yeast extract      10 g Polypeptone      10 g Meat extract      5 g (NH₄)₂SO₄      5 g

0050These ingredients were dissolved in 1 liter of deionized water and its pH was adjusted to 7.0.

Second-step cultivation

0051The cells obtained by the first-step culti­vation were suspended in an amount of 5 g per liter in a culture medium having the following composition, and cultivated at 30°C for 48 hours. The cells were sepa­rated by centrifugation from the resulting culture broth.

Composition of the culture medium:

00520.5M aqueous potassium hydrogen phosphate solution      39.0 ml 0.5M aqueous dipotassium hydrogen phosphate solution      53.6 ml 20 wt/v % aqueous magnesium sulfate solution      1.0 ml Carbon source (*) Mineral solution (**)      1.0 ml

0053The above ingredients were dissolved in 1 liter of deionized water and its pH was adjusted to 7.0. (*) Valeic acid and/or glucose (g/liter-­medium) was used in the following proportions as the carbon source. <tables id="tabl0001" num="0001"><img file="EP0288908A2_D0001.tif" /></tables> (**) Composition of the mineral solution CoCl₂      119.0 mg FeCl₃      9.7 g CaCl₂      7.8 g NiCl₂      118.0 mg CrCl₂      62.2 mg CaSO₄      156.4 mg

0054The ingredients were dissolved in 1 liter of 0.1N-HCl.

Treatment of the Cells

0055The cells obtained by the second-step culti­vation were washed with distilled water and subsequently washed with acetone. The washed cells were dried at 20°C under a pressure of 0.1 mm Hg. There was no substantial difference in the weight of dry cells among the uses of (1), (2), (3) and (4).

Separation and recovery of the copolymer

0056The copolymer was extracted with hot chloro­form from the resulting dry cells. Hexane was added to the extract to precipitates the copolymer. The precipi­tate was collected by filtration and dried to recover the copolymer.

Characteristics of the copolymer

0057The composition, intrinsic viscosity, melting temperature, and the heat of fusion of the resulting copolymer were measured by the following methods. Composition: 500 MHz, ¹H-NMR spectrum Intrinsic viscosity [η]: in chloroform at 30°C Melting temperature Tm: DSC measurement (temperature elevation rate 10°C/min.) Heat of fusion ΔH: DSC measurement

0058The results are shown in Table 1.

0059The sequence distribution of the copolymer obtained when (1) was used was determined from 125 MHz ¹³C NMR spectrum.

0060Specifically, by the method of the present inventor and others [Y. Doi et al., "Macromolecules, <u style="single">19</u>, 2860-2864 (1986)], the dyad sequence distribution of the units B and units V was determined from the multiplet resornance structure of carbonyl carbon. This copolymer was found to have the following sequence distribution. BB (butyrate-butyrate) dyad fraction      3 % BV (butyrate-valerate) and VB (valerate-butyrate) dyad fractions      12 % VV (valerate-valerate) dyad fraction      85 %

0061This sequence distribution shows that the co­polymer has a random sequence distribution of monomeric units B and V.

EXAMPLE 2

0062Example 1 was repeated except that <u style="single">Alcaligenes</u><u style="single">eurotophus</u> H-16 ATCC 17699 was used, and valeric acid was used as the carbon source in an amount of 20 g/liter-­culture medium. The results are shown in Table 1. <tables id="tabl0002" num="0002"><img file="EP0288908A2_D0002.tif" /></tables>

EXAMPLE 3

0063<u style="single">Alcaligenes</u><u style="single">europhus</u> NCIB 11599 was cultivated in the same way as in Example 1 except that valeric acid and n-butyric acid in the following proportions were used in combination as the carbon source. <tables id="tabl0003" num="0003"><img file="EP0288908A2_D0003.tif" /></tables>

0064In the same way as in Example 1, the resulting cells were treated and the copolymer was separated and recovered from the dry cells. The properties of the copolymers were examined. The weight of the dry cells and the properties of the copolymers are shown in Table 2.

0065The copolymer obtained when (3) was used as the carbon source was found to have the following sequence distribution. BB (butyrate-butyrate) dyad fraction      32 % BV (butyrate-valerate) and VB (valerate-butyrate) dyad fractions      48 % VV (valerate-valerate) dyad fraction      20 %

0066This sequence distribution shows that the copolymer has a random copolymer sequence distribution. <tables id="tabl0004" num="0004"><img file="EP0288908A2_D0004.tif" /></tables>