Polyesters containing 4-hydroxybutyrate units

Abstract

To provide a high molecular weight polyhydroxyalkanoate containing 4-hydroxybutyrate units, which has high breaking strength and high breaking elongation. A polyester having a weight average molecular weight of 1,250,000 or more as measured by gel permeation chromatography relative to polystyrene standards, containing at least 3-hydroxybutyrate units and 4-hydroxybutyrate units as polymerized units, and the proportion of 4-hydroxybutyrate units to all monomer units is 14 mol % to 40 mol %.

Description

[Technical field]

[0001] The present invention relates to polyesters containing 3-hydroxybutyrate units and 4-hydroxybutyrate units and having high molecular weights. [Background technology]

[0002] Many microorganisms accumulate polyhydroxyalkanoates (PHAs) in their living bodies as energy and carbon source storage substances. It is well known that accumulation of PHA occurs when there is a sufficient carbon source and nutrients such as nitrogen, phosphorus, sulfur, oxygen, and magnesium are limited. PHA is a thermoplastic polyester that has attracted attention as a biodegradable and biocompatible plastic, and has been the subject of much research (Non-Patent Document 1). More than 100 types of monomer units that constitute PHA are known, and the most representative is poly-3-hydroxybutyrate (hereinafter abbreviated as P(3HB)) consisting of (R)-3-hydroxybutyrate (hereinafter abbreviated as 3HB) (Non-Patent Document 1).

[0003] P(3HB) has a high melting point similar to that of polypropylene (hereafter referred to as PP), and its breaking strength is similar to that of PP, but its breaking elongation is less than 5%, and it is a highly crystalline, hard, and brittle material.When trying to use PHA industrially, there are two ways to improve its physical properties: by introducing a second component monomer unit to make it a copolymer, or by increasing the molecular weight. An example of a method for introducing a second component monomer unit to form a copolymer is a copolymer incorporating 4-hydroxybutyrate (hereinafter, 4HB) (hereinafter, abbreviated as P(3HB-co-4HB)) (Non-Patent Document 2).

[0004] In addition, as a method for increasing the molecular weight, the P(3HB) biosynthesis gene (phaCAB) extracted from the P(3HB)-synthesizing bacterium Cupriavidus necator is introduced into Escherichia coli XL1-Blue, which does not have a PHA synthesis or degradation system, and the genetically modified bacteria is cultured at pH 6 to produce ultra-high molecular weight P(3HB) (Non-Patent Document 3).

[0005] The weight-average molecular weight Mw of P(3HB) in wild-type strains that produce P(3HB) is generally said to be about 500,000 to 1.5 million, about 200,000 to 2 million, or even about 10,000 to 3 million. Wild-type microorganisms have many decomposition enzymes in their cells, making it difficult to synthesize ultra-high molecular weight P(3HB) with Mw of 3 million or more. In addition, since P(3HB) accumulates in microorganisms as an energy source and carbon source storage substance, it has been investigated in many microorganisms that it is decomposed and used when the carbon source is depleted. However, there are also examples showing that PHA synthesis and decomposition occur simultaneously. The physiological significance of PHA synthesis and decomposition occurring simultaneously has not yet been elucidated. In addition, the simultaneous synthesis and decomposition of PHA in PHA-producing wild-type strains is one of the factors that make it difficult to synthesize ultra-high molecular weight PHA.

[0006] Many studies on the production of P(3HB-co-4HB) have also been conducted, and P(3HB-co-4HB) is produced by culturing the P(3HB)-producing wild strain Cupriavidus necator while feeding it carbon sources such as 4-hydroxybutyric acid (4HB), γ-butyrolactone, 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, and 1,12-dodecanediol.

[0007] Methods for producing P(3HB-co-4HB) and P(4HB) by genetically modifying Escherichia coli, a P(3HB) non-producing bacterium, instead of a P(3HB)-producing wild-type strain, have also been reported. Initially, in addition to the phaA, phaB, and phaC genes of β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA polymerase (PhaC) derived from Cupriavidus necator, which are necessary for producing P(3HB) from acetyl-CoA, the genes of the succinic acid decomposition pathway (sucD, 4hbD, and orfZ) derived from Clostridium kluyveri were introduced to supply 4HB-CoA from succinic acid, and P(3HB-co-4HB) with a molecular weight Mw of approximately 1.8 million was produced in Escherichia coli using glucose as a carbon source, but the 4HB ratio in PHA was low at 1.3 to 1.5% (Non-Patent Document 4).

[0008] There are also reports of the use of ε-caprolactone or its saponification product 6-hydroxyhexanoate (or its salt) in the production of P(3HB-co-4HB). It has been reported that when Cupriavidus necator was cultured using ε-caprolactone as a carbon source, P(3HB-co-4HB) with a PHA content of 26 to 38% and a 4HB ratio of 30 to 36% was accumulated (Non-Patent Document 5), but there is no description of the molecular weight or mechanical tests. Furthermore, although the production of P(3HB-co-4HB) with high molecular weight and high elasticity has been reported (Non-Patent Document 6), the breaking strength was insufficient.

[0009] Patent Document 1 also describes an aliphatic polyester resin composition containing polyhydroxyalkanoate (A), a copolymer (B) containing vinyl acetate, and pentaerythritol (C), in which the polyhydroxyalkanoate (A) and the copolymer (B) containing vinyl acetate are incompatible. The aliphatic polyester resin composition described in Patent Document 1 aims to improve the slow crystallization, which is a drawback of polyhydroxyalkanoate, to improve processability in molding such as injection molding, increase the processing speed, and suppress blooming from the obtained molded body. Patent Document 1 achieves the above object by blending the copolymer (B) containing vinyl acetate and pentaerythritol (C) and further making the polyhydroxyalkanoate (A) and the copolymer (B) containing vinyl acetate incompatible. Patent Document 1 does not discuss the conditions for providing a 4HB-containing PHA that can produce a film with high breaking strength and high breaking elongation.

[0010] Patent Document 2 describes a medical material for soft tissues, which is composed of a fiber assembly and a polyester copolymer having 3-hydroxybutyrate units and 4-hydroxybutyrate units. Patent Document 2 describes that the 4HB unit content of the 3HB-4HB polyester copolymer is usually 30-99 mol%, preferably 40-98 mol%, and particularly preferably 60-95 mol%. Patent Document 3 describes a method for producing a polyester copolymer consisting of 97-80 mol% of 3-hydroxybutyrate units and 3-20 mol% of 4-hydroxybutyrate units in the presence of Alcaligenes bacteria. Patent Documents 2 and 3 also do not discuss the conditions for providing a 4HB-containing PHA that can produce a film having high breaking strength and high breaking elongation. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. WO2015 / 098001

Patent Document 2

Patent Document 3

Non-Patent Document

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0013] Copolymerization and high molecular weight of PHA are expected to improve the physical properties of PHA. P(3HB) has hard and brittle physical properties, and copolymerization with 3-hydroxyvalerate (3HV) units does not improve the physical properties much because of co-crystallization. However, copolymerized PHAs consisting of second component units that do not co-crystallize with 3HB units, such as 4HB units and 3-hydroxyhexanoate (3HH) units, are expected to have significant improvements in physical properties by changing the ratio of the second unit components. In particular, PHAs consisting of 3HB units with side chains and other 3-hydroxyalkanoates do not show lipase degradability, whereas P(3HB-co-4HB), which is a copolymer of 4HB units that do not have side chains compared to 3HB, is known to be degraded not only by PHA degrading enzymes but also by lipases, and is expected to have improved degradability in the body and is expected to be used as a medical material. However, in the manufacturing method using PHA-producing wild strains using 1,4-butanediol, γ-butyrolactone, or 4HB, which are the 4HB unit precursors that have been commonly used in the past, there is no known method for obtaining a P(3HB-co-4HB) copolymer with a 4HB ratio of 14 mol% to 40 mol% and a weight average molecular weight Mw of 1.25 million or more. When using a genetically modified bacterium (Delftia acidovorans), there are reports of the production of P(4HB) with a weight average molecular weight Mw of 2.47 million and a number average molecular weight Mn of 480,000. There are also reports of the production of P(3HB-co-4HB) with a molecular weight of 1.8 million using genetically modified Escherichia coli, but the 4HB ratio is only 1.3 to 1.5%.

[0014] As described above, there is no known method for producing a P(3HB-co-4HB) copolymer that has a 4HB ratio of 14 mol% to 40 mol% and a weight average molecular weight Mw of 1.25 million or more, which is expected to significantly improve physical properties.

[0015] PHA-producing wild-type strains have PHA-degrading enzymes within their cells that allow them to degrade and utilize accumulated PHA as needed, making it difficult for them to synthesize ultra-high molecular weight PHA. It is also understood that the gradual decrease in molecular weight of PHA throughout the cultivation period is a common phenomenon.

[0016] When PHA is used as a medical material, advanced purification techniques, including endotoxin removal, are used, but generally, the more advanced the purification, the more PHA decomposes and the lower its molecular weight tends to be. In addition, the molecular weight reduction also progresses when heat treatment such as heat melting is applied, so if the molecular weight of PHA needs to be high after purification or commercialization, it is required that the molecular weight is sufficiently high in the culture stage before purification. Even if it is intended for general industrial products rather than medical materials, a certain degree of purification process is always necessary, and a higher molecular weight is required to improve the physical properties of the purified PHA. Therefore, a method for obtaining higher molecular weight PHA than before at the culture stage has been required.

[0017] An object of the present invention is to provide a high molecular weight 4HB-containing PHA having high breaking strength and high breaking elongation. [Means for solving the problem]

[0018] As a result of intensive research conducted by the present inventors to solve the above problems, it was found that P(3HB-co-4HB) with various molecular weights can be obtained even from PHA-producing wild strains that do not use genetic recombination, and that P(3HB-co-4HB) with a weight-average molecular weight of 1,250,000 or more and a ratio of 4-hydroxybutyrate units to all monomer units of 14 mol% to 40 mol% in particular exhibits excellent physical properties. The present invention was completed based on the above findings.

[0019] That is, according to the present invention, the following inventions are provided. (1) A polyester having a weight average molecular weight of 1,250,000 or more as measured by gel permeation chromatography relative to polystyrene standards, containing at least 3-hydroxybutyrate units and 4-hydroxybutyrate units as polymerization units, and the proportion of 4-hydroxybutyrate units to all monomer units is 14 mol % to 40 mol %. (2) The polyester according to (1), which has a weight average molecular weight of 1,380,000 or more as measured by gel permeation chromatography relative to polystyrene standards. (3) The polyester according to (1) or (2), which has a weight average molecular weight of 1,800,000 or more as measured by gel permeation chromatography relative to polystyrene standards. (4) The polyester according to any one of (1) to (3), which has a number average molecular weight of 300,000 or more as measured by gel permeation chromatography relative to polystyrene standards. (5) The polyester according to any one of (1) to (4), wherein the polymerized units consist only of 3-hydroxybutyrate units and 4-hydroxybutyrate units. (6) The polyester according to any one of (1) to (5), which is a random polymer. (7) A film comprising the polyester according to any one of (1) to (6). Effect of the Invention

[0020] P(3HB-co-4HB) provided by the present invention has a weight-average molecular weight of 1,250,000 or more and a ratio of 4-hydroxybutyrate units to all monomer units of 14 mol % to 40 mol % and exhibits excellent physical properties. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 shows the 1H-NMR spectrum of the PHA of Example 8. [Diagram 2] FIG. 2 shows the 13C-NMR spectrum of the PHA of Example 8. [Diagram 3] FIG. 3 shows the 1H-NMR spectrum of the PHA of Example 9. [Figure 4] FIG. 4 shows the 13C-NMR spectrum of the PHA of Example 9. [Diagram 5] FIG. 5 shows the PHA composition analysis (GC-FID chromatogram) of the PHA-containing cells of Example 8. [Figure 6] FIG. 6 shows the identification results (GC-MS spectrum) of each peak shown in the chromatogram of FIG. [Figure 7] FIG. 7 shows the identification results (GC-MS spectrum) of each peak shown in the chromatogram of FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Hereinafter, an embodiment of the present invention will be described. [polyester] The polyester of the present invention has a weight average molecular weight of 1,250,000 or more as measured by gel permeation chromatography in terms of polystyrene, contains at least 3-hydroxybutyrate units and 4-hydroxybutyrate units as polymerization units, and the ratio of 4-hydroxybutyrate units to all monomer units is 14 mol % to 40 mol %.

[0023] As described above, one of the features of the polyester of the present invention is that the weight average molecular weight (Mw) measured by gel permeation chromatography in terms of polystyrene is a high molecular weight of 1.25 million or more. The weight average molecular weight measured by gel permeation chromatography in terms of polystyrene is preferably 1.38 million or more, more preferably 1.8 million or more, and even more preferably 1.9 million or more. The weight average molecular weight measured by gel permeation chromatography in terms of polystyrene may be 2 million or more, 2.1 million or more, 2.2 million or more, 2.3 million or more, 2.4 million or more, 2.5 million or more, 2.6 million or more, 2.7 million or more, 2.8 million or more, 2.9 million or more, 3 million or more, 3.1 million or more, 3.2 million or more, 3.3 million or more, 3.4 million or more, 3.5 million or more, 3.6 million or more, 3.7 million or more, 3.8 million or more, 3.9 million or more, or 4 million or more. The upper limit of the weight average molecular weight as measured by gel permeation chromatography in terms of polystyrene is not particularly limited, but is generally 20 million or less, and may be 10 million or less, 8 million or less, 7 million or less, 6 million or less, or 5 million or less. By making the weight average molecular weight of the polyester 1.25 million or more, it becomes possible to produce a film having high breaking strength.

[0024] P(3HB-co-4HB) is known to be extensible and flexible, but it has been discovered in the present invention that when the weight average molecular weight is 1.25 million or more, preferably when it is an ultrahigh molecular weight material having a weight average molecular weight of more than 3 million, it has both extensibility and improved breaking strength.

[0025] The polyester of the present invention preferably has a number average molecular weight (Mn) of 300,000 or more as measured by gel permeation chromatography in terms of polystyrene. The number average molecular weight as measured by gel permeation chromatography in terms of polystyrene may be 350,000 or more, 400,000 or more, 450,000 or more, 500,000 or more, 550,000 or more, 600,000 or more, 650,000 or more, 700,000 or more, 750,000 or more, 800,000 or more, 850,000 or more, 900,000 or more, 950,000 or more, or 1 million or more. The upper limit of the number average molecular weight as measured by gel permeation chromatography in terms of polystyrene is not particularly limited, but is generally 10 million or less, and may be 5 million or less, 400 or less, 3 million or less, or 2 million or less.

[0026] The ratio of Mw to Mn (Mw / Mn) is not particularly limited, but is preferably 1.0 to 10.0, more preferably 1.0 to 8.0, further preferably 1.0 to 7.0, preferably 1.0 to 6.0, 1.0 to 5.0, and further preferably 1.0 to 4.0.

[0027] The weight average molecular weight can be measured by gel permeation chromatography in terms of polystyrene standards in the same manner as described in the Examples below.

[0028] The polyester of the present invention contains at least 3-hydroxybutyrate units and 4-hydroxybutyrate units as polymerization units. That is, the polyester of the present invention may be a polyester containing only 3-hydroxybutyrate units and 4-hydroxybutyrate units as polymerization units (that is, the polymerization units consist of only 3-hydroxybutyrate units and 4-hydroxybutyrate units), or may contain 3-hydroxybutyrate units and 4-hydroxybutyrate units as polymerization units, and may further contain other polymerization units other than those mentioned above. Examples of the other polymerization units mentioned above include lactate (LA), glycolate (GA), 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), 5-hydroxyvalerate (5HV), 5-hydroxyhexanoate (5HH), 6-hydroxyhexanoate (6HH), or 3-hydroxyhexanoate (3HH), or hydroxyalkanoates having 7 or more carbon atoms.

[0029] In the present invention, the 3-hydroxybutyrate unit and the 4-hydroxybutyrate unit are represented by the following formulas, respectively. 3-Hydroxybutyrate unit: -OCH(CH3)CH2C(=O)- 4-Hydroxybutyrate unit: -OCH2CH2CH2C(=O)-

[0030] One of the characteristics of the polyester of the present invention is that the ratio of 4-hydroxybutyrate units to all monomer units is 14 mol % to 40 mol %. The ratio of 4-hydroxybutyrate units to all monomer units may be 15 mol% or more, 16 mol% or more, 17 mol% or more, 18 mol% or more, 19 mol% or more, or 20 mol% or more, or may be 20.2 mol% or more, 20.6 mol% or more, 21 mol% or more, 22 mol% or more, 23 mol% or more, 24 mol% or more, 25 mol% or more, 26 mol% or more, 27 mol% or more, or 28 mol% or more. The ratio of 4-hydroxybutyrate units to all monomer units may be 35 mol% or less, 34 mol% or less, 33 mol% or less, 32 mol% or less, 31 mol% or less, 30 mol% or less, 29.9 mol% or less, 29.8 mol% or less, 29 mol% or less, 28 mol% or less, 27 mol% or less, 26 mol% or less, or 25 mol% or less.

[0031] The ratio of 4-hydroxybutyrate units to all monomer units is preferably 20.2 mol% or more, 20.6 mol% or more, 21 mol% or more, 22 mol% or more, 23 mol% or more, 24 mol% or more, or 25 mol% or more, and 29.9 mol% or less, 29.8 mol% or less, 29 mol% or less, 28 mol% or less, or 27 mol% or less. An example of the ratio of 4-hydroxybutyrate units to all monomer units is 21 mol% or more and 29 mol% or less.

[0032] When the weight-average molecular weight of the polyester is 1,250,000 or more, and the ratio of 4-hydroxybutyrate units to all monomer units is in the range of 14 mol% to 40 mol%, the breaking elongation of the film produced tends to increase as the ratio of 4-hydroxybutyrate units to all monomer units increases. In the present invention, by combining the feature of a weight-average molecular weight of the polyester being 1,250,000 or more and the feature of a ratio of 4-hydroxybutyrate units to all monomer units being 14 mol% to 40 mol%, it is possible to achieve both high breaking strength and high breaking elongation.

[0033] The ratio of 4-hydroxybutyrate units to the total monomer units can be measured according to the method described in the Examples below.

[0034] The polyesters of the present invention may be random, block, alternating or graft polymers, but are preferably random polymers.

[0035] [Manufacturing method of polyester] The polyester of the present invention can be produced by culturing a microorganism capable of producing P(3HB) in the presence of ε-caprolactone (also known as 6-hexanolactone) as a carbon source, or its saponification product, 6-hydroxyhexanoate, or a salt thereof.

[0036] Microorganisms capable of producing P(3HB) include those of the genera Cupriavidus, Alcaligenes, Ralstonia, Delftia, Comamonas, Hydrogenophaga, Microorganisms belonging to the genus Burkholderia, Escherichia, Azotobacter, Methylobacterium, or Paracoccos, Acinetobacter, Aeromonas, Allochromatium, Azorhizobium, Bacillus, Caulobacter, Chromobacterium, Ectothiorhodospira, Klebsiella, Nocardia, Pseudomonas, Rhodobacter, Rhodococcus, Rhodospirillum, Rickettsia, Sinorhizobium, Sphingomonas, Synechocystis, Thiococcus, Thiocystis, Vibrio, or Wautersia can be used. Among the above, the genus Cupriavidus is preferred, and Cupriavidus necator is more preferred. As an example, Cupriavidus necator strain H16 (ATCC17699) can be used.

[0037] In addition, Cupriavidus necator H16 wild type strain can fully incorporate 3HB, 3HV, 4HB, 5HV, etc. into PHA, but if a genetically modified strain into which a PHA polymerizing enzyme gene with different substrate specificity is introduced is used, other hydroxy acids can also be polymerized into PHA. Therefore, not only Cupriavidus necator H16 strain, but also its genetically modified strains and, as mentioned above, other hydroxy acids of the genera Cupriavidus, Alcaligenes, Ralstonia, Delftia, Comamonas, Hydrogenophaga, It is also possible to use microorganisms that have the ability to polymerize PHA or that have been imparted with the ability to polymerize PHA, such as the genera Burkholderia, Escherichia, Azotobacter, Methylobacterium, Paracoccos, Acinetobacter, Aeromonas, Allochromatium, Azorhizobium, Bacillus, Caulobacter, Chromobacterium, Ectothiorhodospira, Klebsiella, Nocardia, Pseudomonas, Rhodobacter, Rhodococcus, Rhodospirillum, Rickettsia, Sinorhizobium, Sphingomonas, Synechocystis, Thiococcus, Thiocystis, Vibrio, and Wautersia.

[0038] The pH of the culture medium is generally about 4 to about 10, preferably about 5 to about 8, and more preferably about 5.8 to about 7.5. The culture temperature is generally 15°C to 45°C, preferably 20°C to 40°C, and more preferably 25°C to 38°C. The culture method may be any of batch culture, fed-batch culture, and continuous culture.

[0039] There are no particular limitations on the medium components, so long as they are substances that can be assimilated by the microorganisms used. Examples of carbon sources that can be used include organic carbon sources such as methanol, ethanol, butanol, acetic acid, or butyric acid; inorganic carbon sources such as carbon dioxide; natural products such as yeast extract, molasses, peptone, and meat extract; sugars such as arabinose, glucose, mannose, fructose, and galactose; and sorbitol, mannitol, and isocitol.

[0040] Nitrogen sources that can be used include inorganic nitrogen compounds such as ammonia, ammonium salts (ammonium chloride, ammonium sulfate, ammonium phosphate), nitrates, etc., and / or organic nitrogen-containing substances such as urea, corn steep liquor, casein, peptone, yeast extract, meat extract, etc.

[0041] Examples of the inorganic component are selected from 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, and more specific examples include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, and the like.

[0042] Other organic nutrient sources include, for example, amino acids such as glycine, alanine, serine, threonine, and proline; vitamins such as vitamin B1, vitamin B12, and vitamin C; and the like.

[0043] Methods for producing 4HB-containing PHA include culturing a PHA-producing wild strain with a precursor that can become 4HB-CoA, and introducing a supply pathway for 4HB-CoA by genetic recombination. In the present invention, when culturing a PHA-producing wild strain with a precursor that can become 4HB-CoA, it is preferable to use ε-caprolactone or 6-hydroxyhexanoate or a salt thereof (e.g., 6-hydroxyhexanoate Na, 6-hydroxyhexanoate K) rather than the conventionally well-known 4HB, γ-butyrolactone, or an even-numbered diol having 4 or more carbon atoms.

[0044] 3HB-CoA is converted from two acetyl-CoAs to acetoacetyl-CoA by β-ketothiolase (PhaA), which then converts it to 3HB-CoA by acetoacetyl-CoA reductase (PhaB), and 3HB-CoA is polymerized to P(3HB) by PHA polymerase (PhaC). Many bacteria that synthesize PHA consisting of short-chain units (3-5 carbon atoms) have the genes phaA, phaB, and phaC for these three enzymes, and have the ability to synthesize P(3HB) from acetyl-CoA produced by the metabolism of carbon sources such as sugars, fats, and alcohols. In such bacteria, when propionic acid is present, 3HV-CoA is supplied by condensation reduction of acetyl-CoA and propionyl-CoA, and both 3HB-CoA and 3HV-CoA are present, resulting in the accumulation of P(3HB-co-3HV). 3HV-CoA is also supplied from valerate and pentanol, and is incorporated into PHA as a 3HV unit.

[0045] In addition, when 4HB itself, which is a precursor of 4HB-CoA, or γ-butyrolactone, 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, etc. are present, 4HB-CoA will be supplied, and both 3HB-CoA and 4HB-CoA will be present, resulting in the accumulation of P(3HB-co-4HB). In even-numbered diols with six or more carbon atoms, acetyl-CoA is produced by the β-oxidation pathway, and 4HB-CoA is also produced at the same time.

[0046] In the conventional synthesis of P(3HB-co-4HB) using 4HB, γ-butyrolactone, 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, etc. (Appl. Microbiol. Biotechnol. (1989), 30, 569-573, and Polymer International Volume 39, Issue 3, pages 169-174, March 1996), it was not possible to obtain high molecular weight substances, and there were problems in industrializing the process while maintaining good physical properties.

[0047] When ε-caprolactone is added to the culture, it is ring-opened to become 6-hydroxyhexanoate (6HH), CoA is added to become 6HH-CoA, and acetyl-CoA is removed in the β-oxidation system, leaving 4HB-CoA, which becomes a 4HB unit when incorporated into PHA. Due to the substrate specificity of PHA polymerase, 6HH-CoA is not easily incorporated into PHA, and P(3HB-co-4HB) accumulates. If 4HB-CoA also undergoes β-oxidation, acetyl-CoA is also produced.

[0048] A compound with a hydroxyl group enters the enzyme-S-PHA complex of the elongating PHA polymerizing enzyme, cleaves the thioester between the enzyme and the PHA polymer chain, and the PHA polymer chain moves from the enzyme to the chain transfer agent, terminating the PHA polymerization. This reaction is called a chain transfer reaction, following radical polymerization. 4HB and diols are compounds with hydroxyl groups, and γ-butyrolactone also opens its ring to become 4HB, so these compounds with hydroxyl groups act as chain transfer agents during PHA polymerization and may terminate the PHA polymerization. Diols in particular are particularly prone to causing termination of PHA polymerization, as either of the two hydroxyl groups at the terminals can be involved in chain transfer, making it difficult to obtain high molecular weight PHA.

[0049] When ε-caprolactone is ring-opened, 6HH is produced, which also has a hydroxyl group. Just as 4HB-CoA can be a substrate for PHA synthase but 6HH-CoA is not, 6HH probably acts less as a chain transfer agent than 4HB, and this is why it is thought that higher molecular weight PHA can be obtained when ε-caprolactone is used.

[0050] Substance production by microorganisms can be classified into growth-coupled production and growth-uncoupled production. In growth-coupled PHA production, PHA accumulates at the same time that other bacterial components grow. In growth-coupled PHA production, acetyl-CoA is competed for by both PHA synthesis and bacterial growth, so acetyl-CoA is unlikely to become surplus. During growth-coupled PHA production, the generation of free hydroxy acids due to PHA decomposition is suppressed, and chain transfer reactions are unlikely to occur, which is presumably why the molecular weight becomes relatively high.

[0051] In non-growth-coupled PHA production, PHA accumulation occurs after the growth of the bacterial components stops, leading to an increase in the PHA content. Because bacterial growth has stopped, excess acetyl-CoA is used for PHA production. During non-growth-coupled PHA production, the components that were once incorporated into the form of PHA appear to be redifferentiated and the free hydroxy acid is excreted from the bacterial body to the outside. If the nitrogen source becomes depleted in the latter half of the culture, the production will shift from growth-coupled to growth-uncoupled production, and chain transfer reactions will occur frequently, creating a state in which synthesis and decomposition are likely to occur simultaneously (a state in which the molecular weight is likely to decrease).

[0052] In the present invention, it was discovered that when PHA is accumulated in a growth-linked manner, a higher molecular weight PHA is accumulated than when PHA is accumulated in a growth-independent manner under nutrient-limited conditions. That is, in the production of the polyester of the present invention, it is preferable to use growth-linked PHA production in which the growth of the bacterial cell and the accumulation of PHA occur simultaneously, rather than growth-independent PHA production by nutrient limitation in which the growth of the bacterial cell and the accumulation of PHA are separated.

[0053] From the culture solution obtained by culturing according to the method of the present invention, the cells are separated and collected by a conventional solid-liquid separation means such as filtration and centrifugation, and the cells are washed and dried to obtain dry cells. The polyester produced can be extracted from the dried cells by a conventional method, for example, with an organic solvent such as chloroform, and the polyester of the present invention can be precipitated and collected by adding a poor solvent such as hexane to the extract.

[0054] [film] The present invention further provides a film comprising the above-described polyester of the present invention. The method for producing the film of the present invention is not particularly limited, and the film can be produced by a conventional method such as a solvent casting method or a melt extrusion molding method.

[0055] The breaking strength of the film of the present invention is preferably 18 N / mm 2 Above 19N / mm 2 More than 20N / mm 2 Above, 21N / mm 2 Above, 22N / mm 2 Above, 23N / mm 2 Above, 24N / mm 2 More than 25N / mm 2 Above, 26N / mm 2 Above, 27N / mm 2 Above, 28N / mm 2 Above, 29N / mm 2 or more than 30N / mm 2 The upper limit of the breaking strength is not particularly limited, but is generally 100 N / mm 2 Less than 50N / mm 2 The following is also fine. The breaking strength was measured in accordance with ISO 527-1 and JIS K7161.

[0056] The elongation at break of the film of the present invention is preferably 300% or more, and may be 400% or more, 460% or more, 470% or more, 480% or more, 490% or more, or 500% or more. The upper limit of the elongation at break is not particularly limited, but is generally 1000% or less, and may be 900% or less, 800% or less, or 700% or less. The breaking elongation was measured in accordance with ISO 527-1 and JIS K7161.

[0057] The elastic modulus of the film of the present invention is preferably 240 N / mm 2 Above 250N / mm 2 More than 270N / mm 2 More than 300N / mm 2 More than 350N / mm2 More than 400N / mm 2 More than 450N / mm 2 or more than 500N / mm 2 The upper limit of the elastic modulus is not particularly limited, but is generally 1000 N / mm 2 Less than 900N / mm 2 or less than 800N / mm 2 The following is also fine. The elastic modulus was measured in accordance with ISO 527-1 and JIS K7161.

[0058] When the weight-average molecular weight of the polyester is 1.25 million or more, the elastic modulus tends to be easily affected by the ratio of 4-hydroxybutyrate units to all monomer units. That is, when the ratio of 4-hydroxybutyrate units to all monomer units is in the range of 14 mol% to 40 mol%, the elastic modulus of the film produced tends to decrease as the ratio of 4-hydroxybutyrate units to all monomer units increases.

[0059] A film having the above-mentioned physical properties can be obtained by forming a polyester provided by the present invention, which has a weight average molecular weight of 1,250,000 or more as measured by gel permeation chromatography relative to polystyrene standards, contains at least 3-hydroxybutyrate units and 4-hydroxybutyrate units as polymerization units, and has a ratio of 4-hydroxybutyrate units to all monomer units of 14 mol% to 40 mol%.

[0060] When producing the film of the present invention, it is sufficient to form the polyester provided by the present invention, and it is not necessary to blend a copolymer containing vinyl acetate and / or pentaerythritol. That is, one embodiment of the film of the present invention is a film that contains the polyester provided by the present invention, does not contain a copolymer containing vinyl acetate, and does not contain pentaerythritol.

[0061] The film of the present invention has the above-mentioned excellent physical properties and is made of P(3HB-co-4HB), which has excellent biodegradability and biocompatibility, and is useful for medical instruments, packaging materials for food and other products, agricultural vinyl sheets, seedling pots, construction and civil engineering sheets, etc.

[0062] The present invention will be described in more detail with reference to the following examples, but the present invention is not particularly limited to the following examples. EXAMPLES

[0063] [Polymer production (jar culture)] <Example 1> Cupriavidus necator strain H16 (ATCC17699) was used to produce PHA. Sterilized medium 1 consisting of KH2PO4 2.72g / L, Na2HPO4 4.26g / L, NaHCO3 0.3g / L, (NH4)2SO4 2g / L, MgSO4 7H2O 0.2g / L, yeast extract 0.2g / L, and 3.5mL of the mineral solution listed below was supplemented with fructose at 14.24g / L. Test tube shaking culture was carried out at 30℃ for 24 hours to obtain a pre-preculture solution.

[0064] Mineral solution: FeC6H5O7·xH2O 6g / L, ZnSO4·7H2O 2g / L, CuSO4·5H2O 0.1g / L, MnCl2·4H2O 1g / L, KI 0.1g / L, (NH4)6Mo7O 24 0.1g / L ·4H2O, 0.1g / L CoCl2·6H2O, 0.2g / L H3BO3, 5g / L NaCl, 4g / L CaCl2·2H2O dissolved in water.

[0065] 1 mL of the pre-preculture solution was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of the above medium 1 supplemented with 14.24 g / L of fructose or 8.86 g / L of fructose and 5.38 g / L of ε-caprolactone, and cultured at 30°C and 150 rpm for 48 to 96 hours to prepare the main culture medium (preculture solution).

[0066] 2 L of the above medium 1, with (NH4)2SO4 changed to 12.5 g / L, was prepared in a 3 L jar fermenter, sterilized, and 100 mL of the culture medium was inoculated. 42% by mass fructose and ε-caprolactone were aseptically fed through a sterilizing filter (PTFE 0.2 μm pores). The feed rate and feeding ratio of the carbon source can be set arbitrarily, but in order to avoid the bacterial growth being stopped due to the carbon source not being consumed by the bacterial cells and remaining in excess in the culture tank, the culture was started at a low flow rate of about 1 to 2 g / h (0.5 to 1 g / h·L) for 42% by mass fructose and about 0.2 to 0.5 g / h (0.1 to 0.25 g / h·L) for ε-caprolactone, and the feed rates were increased stepwise or continuously according to the growth of the bacterial cells. The aeration rate was controlled at 0.2-0.3 L / min, the stirring speed at 500-700 rpm, the culture temperature at 36°C, the lower limit of culture pH at 6.0, and 12.5% ​​ammonia water was used as an alkali for adjusting pH. The ratio of ε-caprolactone:fructose was about 0.5. The culture was completed 100 hours after the start of the culture.

[0067] After the cultivation, the cells were collected by centrifugation, frozen at -20°C, and then freeze-dried. The method for extracting and purifying PHA from the cells was as follows. In a glass Erlenmeyer flask with a screw cap, about 4 to 10 g of freeze-dried cells were suspended in 400 mL of chloroform, and extraction was carried out at 30°C for 24 to 48 hours. The resulting viscous solution was filtered through filter paper to remove the cell residue. The resulting clear liquid was concentrated to about 100 to 200 mL using an evaporator, and PHA was precipitated using 5 times the amount of hexane, a poor solvent. The resulting white precipitate was washed with ethanol and then vacuum dried to obtain purified PHA.

[0068] <Example 2> The same procedure as in Example 1 was carried out except that the culture temperature in the jar culture was 34° C. and the culture time was 112.5 hours.

[0069] <Example 3> The same procedure as in Example 1 was repeated except that the culture temperature in the jar culture was changed to 30° C., the (NH4)2SO4 concentration was changed to 15 g / L, and the culture time was changed to 149 hours.

[0070] <Example 4> The same procedure as in Example 1 was carried out, except that the (NH4)2SO4 concentration in the jar culture medium was changed to 10 g / L.

[0071] <Example 5> The same procedure as in Example 1 was repeated except that the (NH4)2SO4 concentration in the jar culture was changed to 15 g / L and the culture time was changed to 184.8 hours.

[0072] <Example 6> The same procedure as in Example 1 was carried out except that the ratio of ε-caprolactone:fructose in the jar culture was 0.7 and the culture time was 113.5 hours.

[0073] <Example 7> The same procedure as in Example 1 was carried out, except that the culture time in the jar culture was 112 hours.

[0074] <Example 8> The same procedure as in Example 1 was repeated except that the (NH4)2SO4 in the jar culture was changed to 7.5 g / L, a 4N NaOH solution was used as the alkali for adjusting the pH, and the culture time was 122.5 hours.

[0075] <Example 9> The same procedure as in Example 1 was repeated except that the culture temperature in the jar culture was 37°C, the medium used had a (NH4)2SO4 concentration of 7.5 g / L, 4N NaOH solution was used as the alkali for adjusting the pH, and the culture time was 102.5 hours.

[0076] <Example 10> The same procedure as in Example 1 was carried out except that the ratio of ε-caprolactone:fructose in the jar culture was 0.4 and the culture time was 94.5 hours.

[0077] <Example 11> The same procedure as in Example 1 was carried out except that the culture temperature in the jar culture was 37°C.

[0078] <Example 12> The same procedure as in Example 1 was carried out except that the culture temperature in the jar culture was 38°C.

[0079] <Example 13> The same procedure as in Example 1 was repeated except that the culture temperature in the jar culture was 38° C., the culture medium had a (NH4)2SO4 concentration of 7.5 g / L, and the culture time was 114.3 hours.

[0080] <Example 14> The same procedure as in Example 1 was repeated except that the culture temperature in the jar culture was 38°C, the medium used had a (NH4)2SO4 concentration of 7.5 g / L, 4N NaOH solution was used as the alkali for adjusting the pH, and the culture time was 114.3 hours.

[0081] <Example 15> The same procedure as in Example 1 was repeated, except that the (NH4)2SO4 concentration in the jar culture medium was changed to 7.5 g / L, 25 g / L of sodium acetate was added to the 42% by mass fructose solution, and the culture time was 122.5 hours.

[0082] <Example 16> The same procedure as in Example 1 was repeated except that the (NH4)2SO4 in the jar culture was changed to 17.5 g / L, γ-butyrolactone was used instead of ε-caprolactone, and the culture time was 185.8 hours.

[0083] <Example 17> The (NH4)2SO4 in Medium 1 was changed to 2.5 g / L, and NaCl was added to the medium at 3 g / L, and fructose was added at 8.86 g / L and ε-caprolactone was added at 5.38 g / L. 1 mL of the test tube preculture solution was inoculated into a 500 mL Erlenmeyer flask containing 125 mL of the medium, and cultured at 30°C and 150 rpm for 96 hours. The recovery of the bacterial cells and the extraction and purification of PHA were performed in the same manner as in Example 1.

[0084] <Comparative Example 1> The same procedure as in Example 1 was repeated except that the culture temperature in the jar culture was 30° C., a medium containing 7.5 g / L (NH4)2SO4 was used, and the culture time was 136.5 hours.

[0085] <Comparative Example 2> The same procedures as in Example 1 were carried out except that the culture temperature in the jar culture was 36°C, a medium containing 7.5 g / L (NH4)2SO4 was used, γ-butyrolactone was used instead of ε-caprolactone, 2N NaOH solution was used as the alkali for adjusting the pH, and the culture time was 108 hours. The extracted PHA was stored in a dehumidified and deoxygenated state (RP pack manufactured by Mitsubishi Gas Chemical Company) at 25°C for 6 months.

[0086] <Comparative Example 3> The PHA sample prepared by culture extraction in Comparative Example 2 was stored at 100% humidity and 40° C. for one month.

[0087] <Comparative Example 4> The PHA sample prepared by culture extraction in Comparative Example 2 was stored at 25° C. and 100% humidity for 6 months.

[0088] <Comparative Example 5> The PHA sample prepared by culture extraction in Comparative Example 2 was stored in a dehumidified and deoxygenated state (RP pack manufactured by Mitsubishi Gas Chemical Company) at 60° C. for 3 months.

[0089] <Comparative Example 6> The PHA sample prepared by culture extraction in Comparative Example 2 was stored in a dehumidified and oxygen-free state (RP pack manufactured by Mitsubishi Gas Chemical Company) at 60° C. for 6 months.

[0090] <Comparative Example 7> The PHA sample prepared by culture extraction in Comparative Example 2 was stored at 100% humidity and 40° C. for 6 months.

[0091] <Comparative Example 8> The PHA sample prepared by culture extraction in Comparative Example 2 was stored at 60° C. and 100% humidity for one month.

[0092] [Polymer analysis] <1H-NMR and 13C-NMR> The composition analysis and chain analysis of the purified PHA produced in each example and comparative example were determined using a nuclear magnetic resonance spectrometer (JASCO ECA500). The purified PHA was dissolved in CDCl3 at a concentration of 1.5% by mass to obtain a measurement sample. 1 The 1H-NMR spectrum was measured at 500 MHz, 13 and the 13C-NMR spectrum was measured at 125 MHz at room temperature.

[0093] The 4HB ratios measured by NMR for the PHA of each example and comparative example are shown in Table 3 below.

[0094] For the PHA of Example 8, 1 the 1H-NMR and 13 13C-NMR are shown in FIGS. 1 and 2. For the PHA of Example 9, 1 the 1H-NMR and 13 13C-NMR are shown in FIGS. 3 and 4.

[0095] 1 The results of the composition analysis of PHA based on the 1H-NMR analysis results are shown in Table 1 below. 13 The results of the chain analysis based on the 13C-NMR analysis results are shown in Table 2 below. The polymers produced in the examples were random polymers.

[0096]

Table 1

[0097]

Table 2

[0098] <PHA Composition Analysis (GC Method)> The composition analysis of the PHA contained in the cells was carried out by GC as follows. Approximately 10 mg of the obtained dried cells was weighed into a test tube with a screw cap, mixed with 2 mL of chloroform and 2 mL of a methanol-sulfuric acid mixture containing an internal standard (internal standard: benzoic acid 0.5 g / L, sulfuric acid 3.7% by mass), heated at 121°C for 90 minutes, and cooled to room temperature to methylate the PHA. After the reaction was completed, 1 mL of pure water was added, vigorously stirred, and centrifuged to obtain an organic solvent layer. This organic solvent layer was dehydrated with sodium sulfate and then analyzed by gas chromatography to calculate the PHA content. The GC conditions were as follows.

[0099] Gas chromatographic analysis conditions Equipment: Shimadzu GC-2025 Capillary column: DB-1 (0.25 mm (id) x 60 m, film thickness 1 μm) Carrier gas: He (3.23 ml / min) Column temperature: 125℃ 6.5min - rate25℃ / min-260℃ Make-up flow rate: 30mL / min H2 flow rate: 40ml / min Air flow rate: 400ml / min Injection: 250℃ Detector: FID (260℃) Split: 1:20 Injection volume: 1μL Analysis time: 21.5 minutes

[0100] A typical chromatogram in GC-FID (PHA produced in Example 8) is shown in FIG. When PHA containing 4HB or dried cells containing the PHA are methyl-esterified and analyzed by GC-FID using a high-resolution capillary column, multiple peaks originating from 3HB and 4HB are generated. Therefore, each peak was identified using GC-MS (Shimadzu GCMS-QP2010 Plus). The GC conditions were the same as for GC-FID. The results of identifying each peak are shown in Figures 6 and 7.

[0101] <Measurement of PHA Molecular Weight (Gel Permeation Chromatography (GPC) Method)> The measurement of the PHA molecular weight was performed by the gel permeation chromatography method as follows. The measurement results of the molecular weight of the PHA in each example and comparative example are shown in Table 3 below. Chloroform was added to the purified PHA to a concentration of about 0.5 mg / ml, and the mixture was dissolved at 60 °C for 4 hours. After returning to room temperature, it was filtered through a PTFE filter with a pore size of 0.2 μm to remove insoluble matter, and used as the measurement sample. The GPC conditions are as follows. Apparatus: HPLC Prominence system manufactured by Shimadzu Corporation Column: Shodex K-806L (two columns in series) manufactured by Showa Denko Column temperature: 40 °C Mobile phase: Chloroform (1 ml / min) Detector: RI (40 °C) Standard: Shodex polystyrene molecular weight standard (6.87 million - 12.7 million) Injection volume: 60 μl Analysis time: 30 minutes

[0102] [Preparation of PHA Film (Solvent Casting Method)] 5 g of PHA (PHA of each example and each comparative example) was dissolved in 200 ml of chloroform, poured into a flat container with a 18 cm square glass bottom, and the chloroform was slowly evaporated to prepare a solvent cast film. A film with a thickness of about 0.1 - 0.2 mm was prepared. After the evaporation of chloroform, the film was peeled off from the container, vacuum dried for more than two weeks, and then used for the tensile test.

[0103] [Tensile Test Method] The solvent cast film was punched into a test dumbbell in the shape of JIS K7127 Type 5, and using an Autograph AGS-X (manufactured by Shimadzu Corporation), the breaking strength, elongation at break, and elastic modulus of the film were measured under the conditions of a temperature of 23 °C and a test speed of 100 mm / min. The measurement results are shown in Table 3 below.

[0104]

Table 3

[0105] [Production of Polymer (Flask Culture)] [Examples 18 to 31] PHA was produced using Cupriavidus necator H16 strain (ATCC17699). A sterilized medium 1 consisting of KH2PO4 2.72 g / L, Na2HPO4 4.26 g / L, NaHCO3 0.3 g / L, (NH4)2SO4 2 g / L, MgSO4·7H2O 0.2 g / L, yeast extract 0.2 g / L, and 3.5 mL of the same mineral solution as in Example 1 was added with fructose at 14.24 g / L, and test tube culture was carried out at 30 °C for 24 hours to obtain a preculture solution.

[0106] 1 mL of the above preculture medium was inoculated into 500 mL Erlenmeyer flasks each containing 100 mL of a medium added with fructose at 8.86 g / L and ε-caprolactone at 5.38 g / L (Examples 21 to 33, but the ammonium sulfate concentration is as described in the table), or a medium added with fructose at 8.86 g / L and ε-caprolactone at 6.46 g / L (Example 34, but the ammonium sulfate concentration is as described in the table), and cultured at 30 °C and 150 rpm for the time described in the table. The pH before the start of culture was about 6.8 to 7.5. After the culture was completed, the cells were collected by centrifugation, freeze-dried, and the dry cell weight was measured. Also, the results of PHA content and composition by GC analysis after methyl esterification and molecular weight measurement by gel permeation chromatography (GPC method) are shown in Table 4 below. The PHA composition analysis by GC method was carried out in the same manner as in Examples 1 to 17 and Comparative Examples 1 to 8.

[0107] [Measurement of PHA Molecular Weight (GPC Method)] The measurement of PHA molecular weight was carried out by gel permeation chromatography in the same manner as in Examples 1 to 17 and Comparative Examples 1 to 8, except that freeze-dried cells were used instead of purified PHA.

[0108]

Table 4

Claims

1. A film containing a polyester having a weight average molecular weight of 2.6 million or more as measured by gel permeation chromatography relative to polystyrene standards, containing at least 3-hydroxybutyrate units and 4-hydroxybutyrate units as polymerization units, and a ratio of 4-hydroxybutyrate units to all monomer units of 14 mol % to 25 mol %, a breaking strength of 23.0 N / mm 2 to 31.6 N / mm 2 ; An elongation at break of 410% to 637%, and Elastic modulus of 297 N / mm 2 to 977 N / mm 2 ; A film having

2. The film described in claim 1, wherein the number average molecular weight of the polyester measured by gel permeation chromatography in polystyrene equivalent is 300,000 or more.

3. A film described in claim 1 or 2, wherein the polyester polymerization units consist only of 3-hydroxybutyrate units and 4-hydroxybutyrate units.

4. A film described in any one of claims 1 to 3, wherein the polyester is a random polymer.

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