JP2023165504A - Homopolymer of 3-mercapto-2-methyl propionic acid, and polyhydroxyalkanoic acid copolymer composed of 3-hydroxybutanoic acid unit and 3-mercapto-2-methyl propionic acid unit - Google Patents

Abstract

An object of the present invention is to provide a novel polythioester having better mechanical properties.
[Solution] A polyhydroxyalkanoic acid copolymer consisting of 3-hydroxybutanoic acid units and 3-mercapto-2-methylpropionic acid units, a homopolymer of 3-mercapto-2-methylpropionic acid, and the polymer A composition comprising.
[Selection diagram] Figure 6

Description

The present invention relates to polyhydroxyalkanoic acid copolymers comprising 3-hydroxybutanoic acid units and 3-mercapto-2-methylpropionic acid units, and homopolymers having 3-mercapto-2-methylpropionic acid as monomer units. , and compositions containing the polymer.

Polyhydroxyalkanoic acid (PHA), a homopolymer or copolymer of 3-hydroxyalkanoic acid, is a biopolyester that microorganisms accumulate intracellularly. In recent years, it has attracted attention not only as a biodegradable plastic material but also as a biomass-derived plastic material.

In the polyester processing process, a high crystallization rate of polyester is very important because it leads to a reduction in processing time.
The most common PHA, a homopolymer containing (R)-3-hydroxybutanoic acid (3HB) as a constituent unit (hereinafter referred to as "P(3HB)"), is hard and brittle due to its high crystallinity. Poor practicality. Furthermore, since the melting temperature (180°C) and the thermal decomposition temperature (approximately 200°C) are close to each other, deterioration during molding, such as the polymer having a low molecular weight during melting, becomes a problem, making it unsuitable for industrial production. As one means of improving these physical properties, various studies have been made on copolymerization of 3HB and other monomers.

Recently, the present inventors have developed a polyhydroxyalkanoic acid copolymer (hereinafter referred to as P(3HB-co- 3H2MB)) (Patent Document 1). The P(3HB-co-3H2MB) contains a low fraction of 3H2MB, which is the second component, and has excellent melt processability because it melts at a low temperature while having a high crystallization rate.

On the other hand, although polythioester (PTE) was chemically synthesized about 70 years ago, it has never been produced on an industrial scale due to high production costs, low yields, and the use of toxic substances. However, PTE has a property of having a lower crystal order than PHA, and PTE is expected to be highly useful from the viewpoint of processing.

In recent years, it has been reported that, as a biosynthetic polythioester, for example, a copolymer of 3HB and 3-mercaptopropionic acid (3MP) can be produced using Ralstonia eutropha (Non-Patent Documents 1 and 2). It has also been reported that a copolymer of 3HB and 3-mercaptobutanoic acid (3MB) can be biosynthesized in Ralstonia eutropha (Non-Patent Document 3).

Furthermore, it has been reported that PTE homopolymers such as 3MP homopolymer, 3MB homopolymer, and 3-mercaptovaleric acid (3MV) homopolymer were obtained from recombinant strains of Escherichia coli (non-patent References 4, 5). Specifically, in Non-Patent Document 1, various PTE homopolymers are obtained by a non-natural pathway involving butyrate kinase using a recombinant strain of Escherichia coli (Fig. 1b). Homopolymers of PTE have lower glass transition temperatures (Tg) and/or higher melting temperatures (Tm) than the corresponding polyoxyesters, and may even have higher elongations to break. This is believed to be because, for example, a copolymer of 3HB and 3-mercaptopropionic acid (3MP) has higher elasticity than the corresponding polyoxyester copolymer.

However, the mechanical properties of the above-mentioned PTE are not known, and there is a demand for the development of PTE having better mechanical properties.

Japanese Patent Application Publication No. 2016-155936

Lutke-Eversloh, et al. , Microbiology 2001, 147, 11-19 Lutke-Eversloh, et al. , Biomacromolecules 2001, 2, 1061-1065 Lutke-Eversloh, et al. , Nat Mater 2002, 1, 236-40 Yu F. , et al. , Macromol Biosci 2007, 7, 810-819 J. Kawada, et al. , Biomacromolecules 2003, 4, 1698-1702

Therefore, the present invention aims to provide a new PTE with better mechanical properties.

As a result of intensive studies in view of the above circumstances, the present inventors discovered that polyhydroxyalkanoic acid polymerase in the presence of a carbon source and 3-mercapto-2-methylpropionic acid (3M2MP) having a methyl group at the 2-position. By using a transformant in which a gene is introduced into a β-oxidation-deficient strain of a specific microorganism, polyhydroxyalkanoic acid copolymers containing a high percentage of 3M2MP units, and polyhydroxyl copolymers consisting essentially only of 3M2MP units, can be produced. It was discovered that an alkanoic acid copolymer (ie, a 3M2MP homopolymer) can be obtained efficiently, and the present invention was completed. The inventors also discovered that these copolymers have higher elasticity than conventional PHA and PTE, leading to the completion of the present invention.

That is, the present invention provides the following.
(1) A polyhydroxyalkanoic acid copolymer consisting of 3-hydroxybutanoic acid units and 3-mercapto-2-methylpropionic acid units.
(2) The polyhydroxyalkanoic acid copolymer according to (1), wherein the mole fraction of 3-mercapto-2-methylpropionic acid units is 6 mol% or more.
(3) The polyhydroxyalkanoic acid copolymer according to (1), wherein the molar fraction of 3-mercapto-2-methylpropionic acid units is 10 mol% or more.
(4) The polyhydroxyalkanoic acid copolymer according to (1), wherein the mole fraction of 3-mercapto-2-methylpropionic acid units is substantially 100 mol%.
(5) Homopolymer of 3-mercapto-2-methylpropionic acid.
(6) A composition comprising the polyhydroxyalkanoic acid copolymer according to any one of (1) to (4) and/or the homopolymer according to (5).
(7) A film or sheet comprising the composition described in (6).

The polyhydroxyalkanoic acid copolymer containing 3M2MP units and 3HB units and the 3M2MP homopolymer of the present invention have an abnormally large elongation at break, and can be effectively used as plastic materials such as films.

The left side of FIG. 1 is a schematic diagram of the biosynthetic pathway of P(3HB-co-3M2MP). The right side of FIG. 1 is a schematic diagram of the biosynthetic pathway of P(3M2MP). n and m are the repeating numbers, each representing an integer from 1 to 20,000. FIG. 2(a) is a ¹ H NMR spectrum of P(3M2MP) (sample number 7 in Table 1). Symbols A to D in structural formula P (3M2MP) represent protons, and each is observed as a corresponding signal in the ¹ H NMR spectrum. FIG. 2(b) is a ¹³ C NMR spectrum of P(3M2MP) (sample number 7 in Table 1). Symbols E to H in structural formula P (3M2MP) represent carbon, and each is observed as a corresponding signal in the ¹³ C NMR spectrum. FIG. 3 shows a comparison of ¹ H NMR spectra of copolymers of sample numbers 1-7. Sample number 1 (3M2MP 5.5 mol%); Sample number 2 (10.1 mol%); Sample number 3 (34.2 mol%); Sample number 4 (53.9 mol%); Sample number 5 (54.8 mol%) ; Sample number 6 (10.7 mol%): Sample number 7 (100 mol%). The samples represented by sample numbers 1 to 7 in FIG. 3 refer to the samples listed in Table 1, respectively. The same applies to the following drawings. FIG. 4 shows DSC thermograms of copolymers of sample numbers 1-7. (a) shows the change in heat capacity when the temperature is raised for the first time, and (b) shows the change in heat capacity when the temperature is raised for the second time. FIG. 5 shows photographs of films obtained from the polymers of sample numbers 1 to 5 and 7 by the solvent cast film method. FIG. 6 shows the stress-strain curves of the polymers of sample numbers 1-7. FIG. 7 shows the elongation at break and instantaneous recovery after deformation of the copolymer of sample number 7. (a) Before stretching; (b) When stretched; (c): During recovery.

The novel polyhydroxyalkanoic acid copolymer of the present invention is a polyhydroxyalkanoic acid copolymer consisting of 3-hydroxybutanoic acid (3HB) units and 3-mercapto-2-methylpropionic acid (3M2MP) units, represented by formula (I). It is a hydroxyalkanoic acid copolymer (sometimes referred to as P(3HB-co-3M2MP)). Further, the novel polyhydroxyalkanoic acid copolymer of the present invention is a 3M2MP homopolymer (sometimes referred to as P(3M2MP)) represented by formula (II). In this specification, these may be collectively referred to as PHA or polythioester (PTE).

(In the formula, n and m are the number of repetitions, each representing an integer from 1 to 20,000.)

(In the formula, m is the number of repetitions and represents an integer from 1 to 20,000.)

The proportion of 3M2MP units in the copolymer (P(3HB-co-3M2MP)) of formula (I) of the present invention is at least 6 mol%, preferably at least 10 mol%, more preferably at least 10 mol%. Preferably at least 20 mol%, more preferably at least 30 mol%, more preferably at least 40 mol%, more preferably at least 50 mol%, more preferably at least 60 mol%, more preferably at least 70 mol%, more preferably At least 80 mol%, more preferably at least 90 mol%, more preferably at least 95 mol%, more preferably at least 98 mol%, even more preferably at least 99 mol%.

P(3HB-co-3M2MP) and P(3M2MP) of the present invention, especially P(3M2MP), exhibit unusually high elongations at break, and are typically found in natural or synthetic rubbers (typically with elongations in the range of 100-800%). ), or copolymers and/or homopolymers, the elongation of which is approximately 1000% higher than that of well-known PHAs such as poly(4-hydroxybutyrate) (P(4HB)). It has elasticity. Since P(3HB-co-3MP) also has significant elastic properties (Non-Patent Document 4), it is thought that the excellent elasticity of PTE is due to the sulfur atoms in the polymer skeleton.

The Pauling electronegativity of sulfur in PTE (2.58) is significantly lower than that of oxygen (3.44) in the polyoxyester PHA, and is much lower than that of the carbon atom (2.55). ) (Non-Patent Document 3), PTE has a lower crystalline order than PHA and may be able to more easily mobilize the polymer structure upon stretching and contraction (Non-Patent Document 4).

The alpha carbon methyl group of the 3M2MP unit may also affect its mechanical properties by interfering with the packing of the polymer structure.

P (3HB-co-3M2MP) of the present invention is produced using a β-oxidation-deficient strain of a specific microorganism as a host.
Specifically, in the presence of a carbon source and 3-mercapto-2-methylpropionic acid (3M2MP), a polyhydroxyalkanoic acid polymerase gene is added to a broad host range vector for expressing the target gene in the host. , and a propionyl CoA transferase (pct) gene and a known monomer supply gene to increase the supply of 3M2MP-CoA transferase by promoting the conversion reaction of 3M2MP to 3M2MP-CoA, and obtain a plasmid, It can be obtained by introducing the plasmid into host cells.

FIG. 1 shows an example of a typical biosynthetic pathway for P(3HB-co-3M2MP) of the present invention (left panel). Glucose, an example of a carbon source, is converted to pyruvate and then to acetyl-CoA, which is then dimerized (PhaA) by β-ketothiolase (PhaA) and reduced by acetoacetyl-CoA reductase (PhaB). and converted to 3HB-CoA. On the other hand, 3M2MP is converted to 3M2MP-CoA by propionyl-CoA transferase. The monomers supplied through each route are used as substrates for polyhydroxyalkanoic acid polymerase (PhaC), and P(3HB-co-3M2MP) is synthesized.

Further, FIG. 1 shows an example of a typical biosynthetic pathway for P(3M2MP) of the present invention (right panel). 3M2MP is converted to 3M2MP-CoA by propionyl-CoA transferase in a host that does not express phaA and phaB. 3M2MP-CoA is used as a substrate for polyhydroxyalkanoic acid polymerase (PhaC), and P(3M2MP) is synthesized.

Furthermore, a gene encoding a protein called phasin is introduced upstream of the polyhydroxyalkanoic acid polymerase gene (for example, see JP-A No. 2013-42697). Fasin is known to colocalize with PHA granules within bacterial cells, and is thought to be involved in the formation and stabilization of PHA granules.

As a wide host range vector for expressing a gene of interest in a host, known vectors having a promoter, ribosome binding site, gene cloning site, terminator, etc. can be used.

Examples of the polyhydroxyalkanoic acid polymerase gene include Pseudomonas sp. 61-3, Pseudomonas stutzeri, Pseudomonas sp. A33, and Allochromatium vinosum. ), Bacillus Bacillus megaterium, Bacillus cereus, Bacillus sp. INT005, Lamprocystis roseopersicina, Nocardia Nocardia corallina, Rhodobacter shaeroides , Ralstonia eutropha, Rhodococcus sp. NCIMB 40126, Thiocapsa pfennigii, Aeromonas caviae ), and microorganisms selected from Aeromonas hydrophila Examples include those derived from

Furthermore, the polyhydroxyalkanoic acid polymerase gene may be a gene encoding a variant of the above polyhydroxyalkanoic acid polymerase. Such mutants are proteins that have an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence of wild-type polyhydroxyalkanoic acid polymerase, and that have polyhydroxyalkanoic acid polymerization activity. It is. By using such variants, the production amount of polyhydroxyalkanoic acid polymers is increased. For example, the PHA polymerase mutant used in the present invention includes substitution of asparagine at position 149 from the N-terminus of microorganism-derived PHA polymerase (PhaC) with serine, and/or substitution of aspartic acid at position 171 of PhaC with glycine. Examples include variants containing substitutions.
As phasin (PhaP), a variant having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the 20th amino acid region from the N-terminus is also preferably used. For example, there is a mutant in which the fourth aspartic acid from the N-terminus of phasin is replaced with asparagine (for example, see JP-A No. 2013-42697). Variants of phasin similarly improve productivity of polyhydroxyalkanoic acid polymers.
Variants containing all of the above-mentioned substitutions of the 149th amino acid of PhaC, the 171st amino acid substitution of the same enzyme, and the 4th amino acid substitution of phasin are also preferred.
The microorganism from which PHA polymerase and phasin are derived is, for example, a microorganism belonging to the genus Aeromonas, and specifically includes Aeromonas caviae.
Hereinafter, the mutant in which the 4th aspartic acid of PhaP is replaced with asparagine is referred to as "PhaP _Ac D4N", the 149th asparagine of PhaC is replaced with serine, and the 171st aspartic acid of PhaC is replaced with glycine. The double mutant is sometimes referred to as "PhaC _Ac NSDG".
A method for producing PhaP _Ac D4N is disclosed, for example, in JP-A No. 2013-42697, and a method for producing PhaC _Ac NSDG is disclosed, for example, in Tsuge T, et al. , FEMS Microbial Lett 277 (2007) 217-222.

Known monomer-supplying genes include, for example, phbA, phbB, phaA, and phaB derived from Ralstonia eutropha (Peoples, O.P. and Sinskey, A.J., J. Biol. Chem. 264:15293-15297 (1989) ), phaJ derived from Aeromonas caviae (Fukui, T. and Doi, Y., J. Bacteriol. 179:4821-4830 (1997)), and the like.

The host microorganism used in the present invention has good growth properties when using sugars and fats and oils as a carbon source, has a highly stable strain, and is relatively easy to separate from the culture medium. Examples include, but are not limited to, Escherichia genus, Cupriavidus genus, Pseudomonas genus, Aeromonas genus, Alcaligenes genus, Bacillus genus, Corynebacterium spp. Rium Microorganisms of the genus Corynebacterium, Brevibacterium, Serratia, or Vibrio are preferred. More preferred is the genus Escherichia.

In the present invention, for the synthesis of P(3HB-co-3M2MP), for example E. E. coli LS5218 fadB gene and fadJ gene knockout mutant strain. Two plasmids: pTTQ19-PCT and pBBR1phaP(D4N)JC _Ac (NSDG)AB _Re can be introduced into E. coli LSBJ strain (β-oxidation deficient strain) (J. Biosci Bioeng 2012, 113, 480-486) as a host. can.
pTTQ19-PCT contains the propionyl-CoA transferase (PCT) gene from Megasphaera elsdenii for the supply of 3M2MP-CoA. pBR1phaP(D4N)JC _Ac (NSDG)AB _Re is a clone of the phasin gene from Aeromonas caviae (phaP _Ac ) [J. Gen Appl NIcrobial 2015, 61, 63-66], A. caviae-derived PHA synthase gene (phaC _Ac ) [FEMS Microbial Let 2007, 277, 217-222], (R) specific enoyl-CoA hydratase gene (phaJ _Ac ), and supply of 3HB precursor for PhaC _Ac polymerization. for R. It contains genes for 3-ketothiolase (PhaA _Re ) and acetoacetyl-CoA reductase (PhaB _Re ), which are enzymes derived from eutropha H16.
In addition, for the biosynthesis of P(3M2MP), for example, E. Two plasmids: pTTQ19-PCT and pBBR1phaP(D4N)C _Ac (NSDG) can be introduced into E. coli LSBJ strain (β-oxidation deficient strain) as a host. pBBR1phaP(D4N)C _Ac (NSDG) is a plasmid obtained by deleting the phaAB _Re gene and phaJ _Ac gene from pBBR1phaP(D4N)JC _Ac (NSDG)AB _Re .

The presence of the point mutation D4N of the PhaJ _Ac gene and the double point mutation NSDG of the PhaC _Ac , described above, results in high polymer accumulation and high incorporation of the second monomer (3M2MP) unit into the 3HB unit. In fact, in the present invention, due to the presence of the point mutation D4N and the double point mutation NSDG of PhaC _Ac , the production of P(3HB-co-3-mercaptoalkanoate) significantly exceeds that reported so far. (Non-Patent Document 1; Lutke-Eversloh, et al., Biomacromolecules 2001, 2, 1061-1065; Lutke-Eversloh, et al., FEMS Microbiology Letters 2003, 221 , 191-196).

As the carbon source, for example, saccharides, carboxylic acids, fats and oils, etc. can be used. Examples of sugars include glucose, fructose, gactose, xylose, arabinose, sucrose, maltose, starch, and starch hydrolysates. Examples of carboxylic acids include acetic acid and lactic acid. The oils and fats are preferably vegetable oils, such as soybean oil, corn oil, cottonseed oil, peanut oil, coconut oil, palm oil, palm kernel oil, or fractionated oils thereof, such as palm W oleic oil (palm oil is added twice). palm kernel oil olein (low boiling point fraction obtained by solvent-free fractionation of palm kernel oil), or synthetic oils obtained by chemically or biochemically processing these oils and fats and their fractions. or a mixture of these oils.

The culture temperature is a temperature at which bacteria can grow, preferably 15 to 40°C, particularly preferably 20 to 40°C, and even more preferably 28 to 34°C. The culture time is not particularly limited, but for example, batch culture is preferably 1 to 7 days, and continuous culture is also possible. The culture medium is not particularly limited as long as it can be used by the host of the present invention. A medium containing, in addition to a carbon source, a nitrogen source, inorganic salts, other organic nutrient sources, etc. can be used.
Examples of the nitrogen source include ammonia, ammonium salts such as ammonium chloride, ammonium sulfate, and diammonium hydrogen phosphate, peptone, meat extract, yeast extract, and the like.
Examples of the inorganic salts include primary potassium phosphate, secondary potassium phosphate, magnesium hydrogen phosphate, magnesium sulfate, and sodium chloride.
Examples of other organic nutritional sources include amino acids such as glycine, alanine, serine, threonine, and proline; vitamins such as vitamin B1, vitamin B12, biotin, nicotinamide, pantothenic acid, and vitamin C.

The PHA of the present invention can be recovered from bacterial cells, for example, by the following method. After the culture is completed, the bacterial cells are separated from the culture solution using a centrifuge, etc., and the bacterial cells are washed with distilled water, methanol, etc., and then dried. to extract the copolymer. Next, bacterial cell components are removed from the organic solvent solution containing this copolymer by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the copolymer. The supernatant liquid can be removed from the precipitated copolymer by filtration or centrifugation, and the copolymer can be recovered by drying. The obtained copolymer can be analyzed by, for example, gas chromatography, nuclear magnetic resonance, or the like.

The P(3HB-co-3M2MP) and P(3M2MP) of the present invention, especially P(3M2MP), have an unusually large elongation at break and recover their shape almost instantaneously after deformation. Such characteristics are a great advantage when processing as a plastic material. Therefore, compositions containing these copolymers can be used for films, sheets, containers, bottles, packaging materials, and the like.

EXAMPLES Next, the present invention will be explained in detail with reference to Examples, but the present invention is not limited to these Examples in any way.

[Materials and preparation method]
(1) Microorganism Escherichia coli E. E. coli LSBJ strain (β-oxidation deficient strain) was used as a host. LSBJ stock is E. This is a mutant strain of E. coli LS5218 (Spratt, S. et al., J. Bacteriol. 1981, 146, 1166) in which fadB and fadJ genes, which are responsible for part of β-oxidation, are deleted (Tappel et al., J. Biosci.Bioeng.2012, 113, 480).

(2) Medium (a) Lysogeny Broth (LB) medium Prepared by dissolving 10 g of Bacto trypton, 5 g of yeast extract, and 10 g of NaCl in 1 L of deionized water and autoclaving at 121° C. for 20 minutes. Further, an agar medium was prepared by adding agar to the above components at a concentration of 1.5 to 2% (vol/vol) and autoclaving the mixture. Antibiotics (kanamycin: final concentration 50 μg/mL, carbenicillin: final concentration 50 μg/mL) were added to both the liquid medium and the agar medium after autoclaving.
(b) M9 improved medium _{Na2HPO4.12H2O 17.1g} , _{KH2PO4 3g} , _{NH4Cl 0.5g} , bacto-yeast extract 2.5g, NaCl 0.5g, 1M _MgSO4 2mL, 1M It was prepared by dissolving 0.1 mL of CaCl ₂ in 1 L of deionized water and autoclaving at 121° C. for 20 minutes. Kanamycin 50 mg and carbenicillin 50 mg were added after autoclaving.

(3) Plasmid (a) pBBR1phaP(D4N)CJ _Ac AB _Rc NSDG
pBBR1phaP(D4N)CJ _Ac AB _Rc NSDG was produced according to the literature (Saika et al., J. Biosci. Bioeng. 2014, 117, 670). pBBR1phaP(D4N)CJ _Ac AB _Rc NSDG has the PHA operon of Aeromonas caviae, and in phaP, aspartic acid (D) at position 4 is converted to asparagine (N), and in phaC, asparagine (N) at position 149 is converted to serine. (S), and aspartic acid (D) at position 171 is mutated to glycine (G) (Ushimaru et al., J. Gen. Appl. Microbiol. 2015, 61, 63).
(b) pBBR1phaP(D4N)C _Ac NSDG
The region not containing phaJ and phaAB of pBBR1phaP(D4N)CJ _Ac AB _Rc NSDG (Saika et al., J. Biosci. Bioeng. 2014, 117, 670) was amplified by PCR method, and the 7 kb amplified fragment was made blunt-ended. pBBR1phaP(D4N)C _Ac NSDG was produced by phosphorylating and self-circularizing.
(c) pTTQ19-PCT
pTTQ19-PCT was created by amplifying the Nhe I-Mun I fragment containing the pct gene from pTVpctC1STQKAB _Re by PCR, and then inserting the fragment into the Xba I-EcoR I site of pTTQ19 (Furutate et al. ., J. Polym. Res. 2017, 24:221). The pTTQ19-PCT has a propionate CoA transferase gene (pct) derived from Megasphaera elsdenii (same document).

Example 1 Production of polymer 1. Biosynthesis of P(3HB-co-3M2MP) The above Escherichia coli E. Plasmid pBBR1phaP(D4N)CJ _Ac NSDG was added to the E. coli LSBJ (β-oxidation-deficient strain) strain, and pTTQ19-PCT was introduced.
Recombinant E. coli bacteria formed on LB agar medium. A single colony of E. coli LSBJ was inoculated into 20 mL of Lysogeny Broth (LB) medium in a 50 mL shake flask and cultured overnight at 37° C. and 160 strokes/min. 5 mL of this culture was mixed with 95 mL of M9 modified medium (17.1 g/L _{Na2HPO4.12H2O} , 3 g/L _KH2PO4 , 0.5 _g /L NaCl, 2 mL of 1M ₎ in _a 500 mL shake flask. MgSO _{4.7H 2} O, 0.1 mL 1M CaCl ₂ , 2.5 g/L Bacto-yeast extract, 20 g/L glucose, 0.25-2.5 g/L (RS)-3-mercapto-2-methyl Propionic acid (3M2MP), 50 mg/L kanamycin, 50 mg/L carbenicillin, 1 mM inducer IPTG) were inoculated and cultured at 30° C. and 130 strokes/min for 72 hours. After culturing, cells were collected by centrifugation (5960×g) for 10 minutes, washed with water, and then freeze-dried.

2. Biosynthesis of P(3M2MP) To produce P(3M2MP), E. Plasmid pBBR1phaP(D4N)C _Ac NSDG was added to the E. coli LSBJ (β-oxidation deficient strain) strain, and pTTQ19-PCT was introduced. First, recombinant E. coli LSBJ was inoculated into 1.7 mL of LB medium (containing 50 mg/L kanamycin and 50 mg/L carbenicillin) in a 5 mL test tube, and cultured with shaking at 160 rpm at 37° C. for 4 hours. This culture solution (0.2 mL) was inoculated into 20 mL LB medium (containing 50 mg/L kanamycin and 50 mg/L carbenicillin) in a 50 mL flask, and cultured at 30°C for 18 hours with shaking at 160 rpm. It was used as a culture solution. Next, 5 mL of the preculture was added to 100 mL of M9 modified medium (2 mL/L of 1 M MgSO ₄ 7H ₂ O, 0.1 mL of 1 M CaCl ₂ , 50 mg/L of kanamycin, and 50 mg/L of kanamycin in a 500 mL shake flask). containing carbenicillin) and cultured with shaking at 130 rpm for 4 hours at 30°C. Thereafter, 3.75 g/L glucose, 1.2 g/L 3-mercapto-2-methylpropionic acid (pH neutralized), and 1 mM IPTG were added to the medium, and culture was continued for 76 hours. After culturing, cells were collected by centrifugation (5960×g) for 10 minutes, washed with water, and then freeze-dried.

3. Collection of P(3HB-co-3M2MP), P(3M2MP) After culturing, the bacterial cells were collected by centrifugation, washed twice with distilled water, and lyophilized for 72 hours in a pre-weighed tube to determine the dry bacterial weight. was calculated. Polymer content was determined by ultrasonic extraction with a modification of the method described in the literature (Arikawa et al., J Biosci Bioeng 2017, 124, 250-254). That is, about 100 mg of freeze-dried bacterial cells were added to a 50 mL plastic tube weighed in advance and resuspended in 20 mL of distilled water. Subsequently, 13 mL of a 10% solution of sodium dodecyl sulfate (SDS) was added to give a final concentration of approximately 4% SDS. The solution was then continuously sonicated for 4 min at power 10, followed by 3 rinsing steps (20 mL distilled water, then 5 mL methanol, then 20 mL distilled water, collected by centrifugation), and lyophilized for 24 hours. The amount of polymer was measured.

4. Purification of P(3HB-co-3M2MP), P(3M2MP) PHA When purifying a large amount of polymer, transfer the dried bacterial cells that have accumulated PHA to a 100 mL glass bottle with a lid, add 100 mL of chloroform, and stir with a stirrer. Stirred for 72 hours. The solution after stirring was transferred to No. 1 filter paper (manufactured by Advantech) and transferred to an eggplant-shaped flask. Thereafter, the filtrate was completely evaporated using a rotary evaporator to precipitate the polymer. Next, the precipitated polymer was washed with a small amount of methanol, completely dissolved in about 20 mL of chloroform, and then added dropwise little by little to 400 mL of methanol while stirring for purification. The purified copolymer was transferred to No. 1 filter paper and dried in a fume hood for 48 hours. The results are shown in Table 1.

Example 2 Analysis of structure and physical properties of P(3HB-co-3M2MP) and P(3M2MP) (1) ¹ H-NMR and ¹³ C-NMR
Measurements were performed using a nuclear magnetic resonance spectrometer (Biospin AVANCE III 400A, Bruker or AVANCE III HD, Bruker). For one-dimensional (1D) ¹ H-NMR, two-dimensional (2D) ¹ H- ¹ H COSY (correlation spectroscopy), and ¹ H- ¹³ C HSQC (heteronuclide single quantum coherence) NMR, purified and filtered 10-20 mg of polymer was dissolved in ₁ ml CDCl and filtered through a 0.45 μm polyvinylidene fluoride (PVDF) filter membrane; for ^13C -NMR, 40 mg was dissolved in ₁ ml CDCl and filtered through a 0.45 μm It was filtered through a PVDF filter membrane and used as a measurement sample. The ¹ H-NMR and ¹³ C-NMR spectra of sample number 7 P (3M2MP) are shown in FIG. FIG. 3 shows a comparison of ¹ H-NMR spectra of sample numbers 1 to 6 of P (3HB-co-3M2MP) and sample number 7 of P (3M2MP).

From the ¹ H-NMR spectrum of P(3M2MP), four peaks A (3.14 ppm), B (3.01 ppm), C (2.86 ppm) and D (1.25 ppm) were identified. The peak integral values were 0.99 to 1 for peaks A to C and 3.16 for peak D. From ^{the 13} C-NMR spectrum, peaks E, F, G, and H were 201.3, 48.4, 31.6, and 17.6 ppm, respectively.
The content of 3M2MP units of P(3HB-co-3M2MP) was determined by integration of the methine (>CH-) peak in the ¹ H-NMR spectrum. Methine (3HB-CH) of 3HB appeared at 5.25 ppm, methylene (3HB-CH ₂ ) of 3HB appeared at around 2.5 ppm, and methyl (3HB-CH ₃ ) of 3HB appeared at 1.27 ppm.
Furthermore, as a copolymer having two monomer units, there are four possible combinations of copolymers (3HB-3M2MP, 3HB-3HB, 3M2MP-3M2MP, and 3M2MP-3HB); Sequence distribution was predicted by calculating the parameter D (Macromolecules 1989, 22, 1676-1682).

(In the formula, 3HB is O, 3M2MP is S, and, for example, Fos is the molar fraction of carbonyl groups in the 3HB-3M2MP sequence in the ¹³ C-NMR spectrum.)
For example, if D is 1, the copolymer is significantly random, but if D is significantly greater than 1, the copolymer is blocky. When D is significantly less than 1, the copolymer is an alternating copolymer. Attribution of peaks for the four possible copolymer combinations was performed by ¹ H- ¹³ C-HMBC (heteronuclear multiple bond correlation) NMR. The results are shown in Table 2.

Table 2 shows that the D values for all samples are significantly greater than 1. This result shows that all samples can have a block-like order degree distribution. When the mole fraction of 3M2MP units was low, the degree of block arrangement tended to be high.

Finally, the polymer molecules were analyzed using Attenuated Total Reflection (ATR), Fourier Transform Infrared (FTIR) spectroscopy with a FT/IR-4600 spectrometer (Jasco, Japan) with model ATR PRO400-S (Jasco, Japan). The occurrence of thioester chemical groups in the sample was confirmed (not shown).

(2) GPC (gel permeation chromatography)
The number average molecular weight (Mn) and molecular weight distribution (Mw/Mn) (Mw: weight average molecular weight) of the polymer purified from the bacterial cells were determined by GPC measurement. Since polystyrene is used as a standard substance, the molecular weight determined in the present invention is a relative molecular weight in terms of polystyrene. The molecular weights of the five types of polystyrene used are 3,790, 30,300, 219,000, 756,000 and 4,230,000.
A GPC sample was prepared by dissolving the purified polymer in chloroform to a concentration of about 1 mg/mL, and filtering the solution using a syringe equipped with a Millex-FH PVDF filter (manufactured by Millipore) with a pore size of 0.45 μm.
For the GPC measurement, Nexera 40 182 GPC system (detector: Shodex RI-504 refractive index detector) manufactured by Shimadzu Corporation was used, and K-806M and K-802 manufactured by Shodex were used as columns. Chloroform was used as the moving phase, the total liquid flow rate was set at 0.3 mL/min, and the column temperature was set at 40°C. A calibration curve was drawn from a polystyrene standard, and the molecular weight and molecular weight distribution in terms of polystyrene were calculated by comparing this with sample data.

(3) Differential scanning calorimetry (DSC)
For the DSC sample, approximately 10 mg of the purified polymer was weighed out, placed in a 5 mL sample vial, and completely dissolved in approximately 1 to 2 mL of chloroform. A cast film was produced by drying this in a draft for about 3 weeks. Approximately 3 mg of this cast film was weighed and placed in a special aluminum pan, which was used as a sample for DSC measurement.
For the measurement, DSC 8500 manufactured by PerkinElmer was used. The measurement was performed under a nitrogen atmosphere at 20 mL/min. An aluminum pan containing no sample was used as a control. The measurement conditions were as follows: First, the sample was heated from -50°C to 200°C at a temperature increase rate of 20°C/min, held at 200°C for 1 minute, and then rapidly cooled to -50°C at -500°C/min. Next, after holding at -50°C for 1 minute, it was heated to 200°C at a temperature increase rate of 20°C/min.
The cast film used was one that had been kept at room temperature for three weeks or more to allow crystal formation to proceed. The melting point Tm of the measured sample was determined from the thermogram when the temperature was first raised. The glass transition temperature Tg was taken as the midpoint of the change in heat capacity during the second temperature rise.
The results are shown in Table 3 and Figure 4.
_Tcc : Cold crystallization point. Cold crystallization is a phenomenon in which crystals are formed from a supercooled liquid state above the glass transition temperature when the temperature is increased.

From Table 3, sample 7 exhibited clear amorphous behavior, negative Tg, and no melting point and low temperature crystallization peaks were detected. Also, in samples 1 to 3, Tg and ΔH _m decreased as the molar fraction of 3M2MP units increased, and sample 4 resulted in amorphousness.

Example 3 Mechanical properties of P(3HB-co-3M2MP) and P(3M2MP) Cast films of the copolymers of sample numbers 1 to 6 produced in Example 1 and cast films of the polymer of sample number 7 were The yield strength, tensile strength, elongation at break, and Young's modulus were determined.
<Device>
Tensile tester: AGS-H/EZTest (Shimadzu Corporation)
Analysis software: TRAPEZIUM 2 (Shimadzu Corporation)
<Sample preparation>
(a) Preparation of cast film The purified polymer was dissolved in an appropriate amount of chloroform and spread on a flat petri dish. The weight of the polymer used here was calculated in consideration of the area of the Petri dish so that the thickness would be 150 μm. After development, the petri dish was covered with aluminum foil with holes and left on a flat surface at room temperature until the chloroform was completely vaporized. After drying, the film was carefully peeled off from the petri dish and left at room temperature for one week. Polymers that were difficult to peel off from the petri dish were removed by dropping ultrapure water between the petri dish and the polymer.
(b) Preparation of tensile test piece The produced cast film was cut into 20 mm long and 3 mm wide pieces to be used as samples. A razor was used for cutting, and the polymer was cut by pressing down from directly above it.
<Measurement conditions>
The distance between the grips was 10 mm, the tensile speed was 10 mm/min, and the measurement was performed at room temperature.
<Data analysis>
In the tensile test, the stress at which plasticity of the polymer begins was defined as yield strength, the stress at which the polymer broke was defined as tensile strength, and the elongation rate was defined as elongation at break. Young's modulus was defined as the slope of the starting point in the stress-strain curve.
The results are shown in Table 4 and FIGS. 5 to 7. FIG. 5 shows photographs of each cast film, FIG. 6 shows stress-strain curves, and FIG. 7 shows manual elongation (b) and instantaneous recovery after deformation (c) of sample 7.

From FIG. 5, it can be seen that as the mole fraction of 3M2MP units increases, the cast film becomes more transparent and more flexible. Moreover, from Table 4, as the molar fraction of 3M2MP units increased, yield strength, tensile strength, and Young's modulus decreased, while elongation at break increased. In particular, sample 4 showed an elongation at break of 1500% or more. Sample 5, which was composed of 3M2MP units with approximately the same molar fraction as sample 4, showed a lower elongation at break than sample 4, which was attributed to the lower molecular weight and difference in microstructure compared to sample 4. It will be done.
Sample 7 exhibited an abnormal elongation at break of 2605%, and most of the deformed film instantly recovered its shape. This means that P(3M2MP) has exceptional elasticity, which is particularly advantageous in processing.

The polyhydroxyalkanoic acid copolymer containing 3M2MP units and 3HB units and the 3M2MP homopolymer of the present invention can be effectively used as plastic materials such as films.

Claims (7)

A polyhydroxyalkanoic acid copolymer consisting of 3-hydroxybutanoic acid units and 3-mercapto-2-methylpropionic acid units. The polyhydroxyalkanoic acid copolymer according to claim 1, wherein the mole fraction of 3-mercapto-2-methylpropionic acid units is 6 mol% or more. The polyhydroxyalkanoic acid copolymer according to claim 1, wherein the mole fraction of 3-mercapto-2-methylpropionic acid units is 10 mol% or more. The polyhydroxyalkanoic acid copolymer according to claim 1, wherein the mole fraction of 3-mercapto-2-methylpropionic acid units is substantially 100 mol%. Homopolymer of 3-mercapto-2-methylpropionic acid. A composition comprising the polyhydroxyalkanoic acid copolymer according to any one of claims 1 to 4 and/or the homopolymer according to claim 5. A film or sheet comprising the composition according to claim 6.