Crystallization accelerator

Abstract

To provide a new technique for accelerating crystallization of a polylactic acid.SOLUTION: A crystallization accelerator for accelerating crystallization of a polylactic acid contains copolyester of a lactic acid and other hydroxy carboxylic acid. The other hydroxy carboxylic acid is preferably a 3-hydroxyalkanoic acid. The copolyester is preferably a random copolymer of a lactic acid and other hydroxy carboxylic acid. A resin composition contains a polylactic acid and the crystallization accelerator.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a crystallization accelerator for promoting the crystallization of polylactic acid and its use.

Background Art

[0002] Plastic is difficult to decompose in the natural environment, so environmental pollution caused by a large amount of waste has been taken up as a serious problem. To address such problems, the practical application of biodegradable plastics that can be decomposed into water and carbon dioxide by the action of microorganisms has been promoted.

[0003] Polylactic acid, which is one of the typical biodegradable plastics, is a type of biopolymer that can be synthesized from biomass-derived raw materials. Because it has relatively high rigidity, strength, and transparency, its use in a wide range of applications as an alternative to petroleum-derived plastics has been promoted.

[0004] Thermoplastic resins such as polylactic acid are desired to be heated and melted during molding and then solidified in a short time from the viewpoint of industrial productivity. As a method for improving such solidification properties, it is known to blend various additives with thermoplastic resins.

[0005] For example, Patent Document 1 discloses that adding basic zinc cyanurate particles to polylactic acid increases the crystallization rate of polylactic acid. Also, Patent Document 2 discloses that adding inorganic particles composed of talc and / or boron nitride having a specific particle size or less to polylactic acid increases the crystallization rate of polylactic acid.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] Although Patent Documents 1 and 2 disclose techniques for promoting the crystallization of polylactic acid, the effects are not sufficient, and there is a concern that mixing a substance having a significantly different chemical structure from polylactic acid with polylactic acid may impair the transparency and mechanical properties of polylactic acid.

[0008] An object of the present invention is to provide a novel technique for promoting the crystallization of polylactic acid.

Means for Solving the Problems

[0009] The present inventor has found that a copolyester of lactic acid and another hydroxycarboxylic acid can promote the crystallization of polylactic acid, and has thus arrived at the present invention.

[0010] That is, the present invention relates to a crystallization accelerator for promoting the crystallization of polylactic acid, which contains a copolyester of lactic acid and another hydroxycarboxylic acid. The present invention also relates to a resin composition containing polylactic acid and the above-mentioned crystallization accelerator. Furthermore, the present invention also relates to a molded article formed by molding the above-mentioned resin composition. Still further, the present invention also relates to a method for promoting the crystallization of polylactic acid, which is characterized by mixing a copolyester of lactic acid and another hydroxycarboxylic acid with polylactic acid. Still further, the present invention also relates to the use of a copolyester of lactic acid and another hydroxycarboxylic acid as a crystallization accelerator for promoting the crystallization of polylactic acid.

Effects of the Invention

[0011] The present invention can provide a novel technique for promoting the crystallization of polylactic acid. According to a preferred embodiment, crystallization can be promoted without reducing the biomass degree of polylactic acid. According to a preferred embodiment of the present invention, it is possible to promote the crystallization of polylactic acid without substantially reducing the transparency of polylactic acid. Furthermore, it is also possible to plasticize polylactic acid and improve elongation.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0013] Hereinafter, embodiments of the present invention will be described in detail. The crystallization accelerator according to the present embodiment is an additive for promoting the crystallization of polylactic acid by being mixed with polylactic acid and used. This crystallization accelerator contains at least a copolymer polyester of lactic acid and another hydroxycarboxylic acid.

[0014] (Polylactic acid) Polylactic acid is a polyester having lactic acid as a constituent monomer. As described above, it is desired that polylactic acid undergoes solidification after heat melting in a short time during molding processing. By using the crystallization accelerator according to the present embodiment, the crystallization of polylactic acid is promoted, and the solidification after heat melting proceeds in a shorter time, thereby improving the productivity of the molded body containing polylactic acid.

[0015] Polylactic acid may be a conventionally known polylactic acid and may be either crystalline or amorphous. Polylactic acid is preferably a homopolymer of lactic acid, but may contain a trace amount of other monomers in addition to lactic acid.

[0016] The lactic acid that constitutes polylactic acid may be either the L-form or the D-form, or may contain both. In the latter case, the ratio of the L-form to the D-form is not particularly limited. The polylactic acid may be any of a poly(L-lactic acid) resin, a poly(D-lactic acid) resin, and a poly(DL-lactic acid) resin. A blend of these may also be used.

[0017] Examples of the other monomers that may be contained in the polylactic acid include aliphatic hydroxycarboxylic acids other than lactic acid, aliphatic polyhydric alcohols, aliphatic polycarboxylic acids, and polyfunctional polysaccharides. When the polylactic acid is a copolymer of lactic acid and other monomers, from the viewpoint of the crystallinity of the polylactic acid, the content ratio of the other monomers is preferably about 0 to 3 mol%, more preferably 0 to 2 mol%, based on the total monomers contained in the polylactic acid.

[0018] The lactic acid raw material for producing polylactic acid is not particularly limited, and L-lactic acid, D-lactic acid, DL-lactic acid, or a mixture thereof, L-lactide, D-lactide, meso-lactide, or a mixture thereof, etc. can be used. Lactic acid obtained by microbial fermentation from renewable raw materials such as starch can be preferably utilized. As a method for producing polylactic acid, known methods such as a dehydration polycondensation method or a ring-opening polymerization method can be applied and are not particularly limited.

[0019] The molecular weight of the polylactic acid is not particularly limited and may be appropriately set according to its use, but the weight average molecular weight is preferably 1,000 to 700,000, more preferably 10,000 to 300,000.

[0020] (Copolyester) The crystallization accelerator according to this embodiment contains, as an active ingredient for promoting crystallization, a copolymer polyester of lactic acid and other hydroxycarboxylic acids. By mixing the copolymer polyester with polylactic acid, the crystallization of polylactic acid can be promoted.

[0021] In addition, since the copolyester contains lactic acid as one of the constituent monomers, it has good compatibility with polylactic acid and can form a uniform mixture with polylactic acid. As a result, a substantial decrease in the transparency of polylactic acid can be avoided. In addition, polylactic acid can be plasticized, and the elongation of polylactic acid can also be improved.

[0022] In addition, since the copolyester contains a hydroxycarboxylic acid other than lactic acid as one of the constituent monomers, it phase-separates from polylactic acid, but also has the compatibility with polylactic acid as described above. As a result, the copolyester can be extremely finely dispersed in the polylactic acid matrix to form a sea-island structure in which the hydroxycarboxylic acid portion other than lactic acid is the island and polylactic acid is the sea. It is considered that the crystallization of polylactic acid is promoted by the fine dispersion of the hydroxycarboxylic acid portion other than lactic acid and the formation of fine crystals.

[0023] The copolyester itself is a polymeric material showing biodegradability. Since bacteria that decompose the copolymer of lactic acid and 3-hydroxybutyric acid have been isolated from the environment (see Polymer Degradation and Stability, 2014, 110, 44, and Applied Microbiology and Biotechnology, 2015, 99, 9555), it is expected that the copolyester shows high biodegradability in the environment. In addition, when an enzyme secreted by the isolated copolyester-degrading bacteria was isolated, it was revealed that it decomposes poly-D-lactic acid of oligomers of 31-mer or less.

[0024] The copolyester may be any of a random copolymer, an alternating copolymer, a block copolymer, and a graft copolymer. However, from the viewpoints of crystallization promoting effect, transparency, mechanical properties, and further availability, the copolyester is preferably a random copolymer. A random copolymer refers to a copolymer in which two or more types of monomer units are arranged in a non-regular order. When the copolyester is produced by a microorganism, it is usually a random copolymer.

[0025] The lactic acid monomer unit in the copolyester-polyester may be either an L-lactic acid monomer unit or a D-lactic acid monomer unit. It may contain either one or both.

[0026] When the copolyester is produced by a microorganism, the lactic acid monomer unit in the copolyester is substantially composed of only D-lactic acid monomer units. Substantially composed of only D-lactic acid monomer units means that the proportion of D-lactic acid monomer units in the total amount of lactic acid monomer units is usually 90% or more, preferably 95% or more, more preferably 99% or more.

[0027] When the lactic acid monomer unit in the copolyester is substantially composed of only D-lactic acid monomer units, it is preferable from the viewpoint of forming a stereocomplex crystal with the L-form lactic acid monomer unit contained in the polylactic acid. It is considered that the network of the formed stereocomplex crystal deforms in a flow field to form a structure in which the molecular chains are highly stretched, significantly accelerating the crystallization rate of the entire resin composition.

[0028] The hydroxycarboxylic acid other than lactic acid contained in the copolymer polyester is not particularly limited as long as it is a hydroxyalkanoic acid capable of copolymerizing with lactic acid. The number of carbon atoms of the hydroxyalkanoic acid is preferably 3 or more. The upper limit of the number of carbon atoms is preferably 15 or less, more preferably 10 or less, still more preferably 8 or less, even more preferably 6 or less, and particularly preferably 5 or less.

[0029] Examples of the hydroxyalkanoic acid include 2-hydroxyalkanoic acid, 3-hydroxyalkanoic acid, and 4-hydroxyalkanoic acid. Among them, 3-hydroxyalkanoic acid is particularly preferred. Specific examples of the 3-hydroxyalkanoic acid include 3-hydroxybutanoic acid (hereinafter may be abbreviated as 3HB), 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxypentadecanoic acid, 3-hydroxyhexadecanoic acid, and the like. Only one of these may be contained in the copolyester, or two or more may be contained. Among them, it is preferable that at least 3HB is contained. In particular, P(LA-co-3HB) (hereinafter may be abbreviated as LAHB), which is a copolyester of lactic acid and 3HB, is most preferable as the copolyester.

[0030] The ratio of the lactic acid unit contained in the copolyester is not particularly limited. However, since the effect of promoting the crystallization of polylactic acid is high, the molar fraction of the lactic acid unit with respect to all the monomer units constituting the copolyester is preferably 5 to 70 mol%, more preferably 10 to 60 mol%, and still more preferably 15 to 50 mol%. The value of the molar fraction of the lactic acid unit can be determined using HPLC. In addition, it can also be determined using NMR or GC.

[0031] The molecular weight of the copolymerized polyester is not particularly limited, but the weight average molecular weight Mw may be, for example, from 10,000 to 1,000,000, preferably from 50,000 to 700,000. The upper limit of the weight average molecular weight is more preferably 300,000 or less, even more preferably 200,000 or less, and particularly preferably 100,000 or less. In the present application, the value of the weight average molecular weight can be determined based on standard polystyrene using gel permeation chromatography (GPC) (manufactured by Shimadzu Corporation) equipped with tandem TSKgel Super HZM-H columns (manufactured by Tosoh Corporation).

[0032] The copolymerized polyester of lactic acid and other hydroxycarboxylic acids can be produced using biomass, an organic resource derived from organisms other than fossil fuels, and can be manufactured from 100% biomass-derived raw materials.

[0033] The method for producing the copolymerized polyester of lactic acid and other hydroxycarboxylic acids is not particularly limited and may be a conventionally known method. It may be biosynthesized by microorganisms or produced by chemical synthesis. Among them, as an example of the method for producing P(LA-co-3HB), a production method using recombinant microorganisms as described in International Publication No. 2009 / 131186 and International Publication No. 2006 / 126796 can be mentioned.

[0034] The crystallization accelerator according to the present embodiment may be composed only of the copolymerized polyester of lactic acid and other hydroxycarboxylic acids, or may contain components other than the copolymerized polyester. Examples of such other components include known resin additives, specifically, plasticizers, hydrolysis inhibitors, compatibilizers, antioxidants, ultraviolet absorbers, processing aids, antistatic agents, colorants, crystal nucleating agents, inorganic or organic particles, lubricants, mold release agents, water repellents, inorganic fillers, antifungal agents, antibacterial agents, foaming agents, flame retardants, and the like.

[0035] The content of the copolymerized polyester in the crystallization accelerator is not particularly limited, and for example, it may be 10 to 100% by weight. The lower limit may be 30% by weight or more, 50% by weight or more, 70% by weight or more, 90% by weight or more, or 99% by weight or more.

[0036] (Usage mode) By mixing the crystallization accelerator according to this embodiment with polylactic acid, an effect of promoting the crystallization of polylactic acid can be obtained. It is preferable to uniformly mix the crystallization accelerator and polylactic acid. For example, it is particularly preferable to uniformly mix the two components by melt-kneading, mixing in an organic solvent, and subsequent solvent removal.

[0037] (Usage amount) The usage amount of the crystallization accelerator according to this embodiment may be any amount that can promote the crystallization of polylactic acid by using the crystallization accelerator. From the perspective of the balance between the crystallization promoting effect and the transparency and mechanical properties of polylactic acid, it is preferable to use the crystallization accelerator in such an amount that the amount of the copolymerized polyester, which is the active ingredient of the crystallization accelerator, is 1 part by weight or more and 100 parts by weight or less with respect to 100 parts by weight of polylactic acid. The lower limit is preferably 5 parts by weight or more, more preferably 10 parts by weight or more, further preferably 15 parts by weight or more, and particularly preferably 20 parts by weight or more. The upper limit is preferably 80 parts by weight or less, more preferably 60 parts by weight or less, further preferably 50 parts by weight or less, still more preferably 40 parts by weight or less, and particularly preferably 30 parts by weight or less.

[0038] (Resin composition) One aspect of the present invention may be a resin composition containing polylactic acid and the crystallization accelerator. Since the crystallization of polylactic acid is promoted by the blending of the crystallization accelerator, the solidification after heating and melting of the resin composition can proceed in a short time. Therefore, the productivity of the molded body composed of the resin composition can be improved.

[0039] In the resin composition, the copolymerized polyester of the lactic acid and another hydroxycarboxylic acid is preferably finely dispersed because it has an excellent effect of promoting crystallization of polylactic acid. Specifically, the average particle diameter of the copolymerized polyester in the resin composition is preferably 50 nm or more and 10 μm or less, more preferably 100 nm or more and 1 μm or less. Such an average particle diameter can be achieved by uniformly mixing polylactic acid and a crystallization accelerator by the method as described above. The average particle diameter can be determined by analyzing a photograph of a cross-section of the resin composition observed with a scanning electron microscope using image analysis software.

[0040] (Other components) The resin composition may contain a thermoplastic resin other than polylactic acid and the copolymerized polyester. Such other thermoplastic resins are not particularly limited, and conventionally known resins can be used. Specifically, biodegradable aliphatic polyesters other than polylactic acid and the copolymerized polyester, aromatic polyesters, etc. can be mentioned.

[0041] The blending amount of the other thermoplastic resin is not particularly limited, but it may be, for example, 0 parts by weight or more and 200 parts by weight or less with respect to 100 parts by weight of polylactic acid. The upper limit may be 100 parts by weight or less, 50 parts by weight or less, 30 parts by weight or less, or 10 parts by weight or less.

[0042] In addition, a resin having low compatibility with polylactic acid may deteriorate the transparency of polylactic acid. Therefore, it is preferably not blended, or, if blended, it is preferably blended in a small amount. Although not particularly limited, for example, the blending amount of polyhydroxyalkanoate resin, which is one of the resins having low compatibility with polylactic acid, is preferably about 0 to 100 parts by weight, more preferably about 0 to 50 parts by weight with respect to 100 parts by weight of polylactic acid.

[0043] The resin composition may appropriately contain other additives as long as the effects of the invention are not inhibited. Such additives are not particularly limited, and examples thereof include plasticizers, hydrolysis inhibitors, compatibilizers, antioxidants, ultraviolet absorbers, processing aids, antistatic agents, colorants, crystal nucleating agents, inorganic or organic particles, lubricants, mold release agents, water repellents, inorganic fillers, fungicides, antibacterial agents, foaming agents, flame retardants, and the like. The content of each additive can be appropriately determined according to the purpose. Also, only one type of additive may be blended, or two or more types may be blended.

[0044] As the plasticizer, plasticizers generally used as plasticizers for polymers can be used. Specifically, polyester-based plasticizers, glycerin-based plasticizers, polyvalent carboxylic acid ester-based plasticizers, polyalkylene glycol-based plasticizers, epoxy-based plasticizers, and the like can be mentioned.

[0045] (Use) After melt-kneading each component, the resin composition can be extruded into strands and then cut into pellets. After drying the obtained pellets to remove moisture, any molded article can be obtained by performing molding processing by a known molding processing method. Such a molded article also constitutes one aspect of the present invention. Examples of the molding processing method include film molding, sheet molding, injection molding, blow molding, fiber spinning, extrusion foaming, bead foaming, and the like.

[0046] The method for manufacturing a film molded article is not particularly limited, and examples thereof include T-die extrusion molding, calender molding, roll molding, and inflation molding. Further, the obtained film can be thermoformed, vacuum formed, or press formed by heating.

[0047] As a method for manufacturing an injection molded article, for example, an injection molding method such as an injection molding method, a gas assist molding method, or an injection compression molding method generally employed when molding a thermoplastic resin can be adopted. Further, according to other purposes, in addition to the above methods, an in-mold molding method, a gas press molding method, a two-color molding method, a sandwich molding method, PUSH-PULL, SCORIM, etc. can also be adopted. However, the injection molding method is not limited to these.

[0048] The resin composition may be processed into pellets or molded articles such as films, sheets, or fibers using an extrusion molding machine, or may also be processed into a molded article of a predetermined shape by injection molding.

[0049] When the resin composition contains a foaming agent, the molded article may be a foamable molded article, or may be a molded foam obtained by foaming the foamable molded article.

[0050] The resin composition can be processed into molded articles of various shapes. Examples of the molded article include paper, film, sheet, tube, plate, rod, container, bag, parts, etc. Further, the molded article can also be combined with other molded articles (for example, fibers, yarns, ropes, woven fabrics, knitted fabrics, non-woven fabrics, paper, film, sheet, tube, plate, rod, container, bag, parts, foams, etc.) made of materials different from the resin composition according to the present embodiment.

[0051] The use of the molded article is not particularly limited, and it can be suitably used in agriculture, fishery, forestry, horticulture, medicine, sanitary products, clothing, non-clothing, packaging, automobiles, building materials, and other fields.

[0052] In the following items, preferred embodiments in the present disclosure are listed, but the present invention is not limited to the following items. [Item 1] A crystallization accelerator that promotes the crystallization of polylactic acid, the crystallization accelerator containing a copolymer polyester of lactic acid and another hydroxycarboxylic acid. [Item 2] The crystallization accelerator according to item 1, wherein the other hydroxycarboxylic acid is 3-hydroxyalkanoic acid. [Item 3] The crystallization accelerator according to item 1 or 2, wherein the other hydroxycarboxylic acid is at least one selected from the group consisting of 3-hydroxybutanoic acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxypentadecanoic acid, and 3-hydroxyhexadecanoic acid. [Item 4] The crystallization accelerator according to any one of items 1 to 3, wherein the other hydroxycarboxylic acid is 3-hydroxybutanoic acid. [Item 5] The crystallization accelerator according to any one of items 1 to 4, wherein the copolyester is a random copolymer of lactic acid and another hydroxycarboxylic acid. [Item 6] The crystallization accelerator according to any one of items 1 to 5, wherein the lactic acid monomer in the copolyester is a D-lactic acid monomer. [Item 7] The crystallization accelerator according to any one of items 1 to 6, wherein the molar fraction of the lactic acid units in the copolyester is 5 to 70 mol%. [Item 8] The crystallization accelerator according to any one of items 1 to 7, wherein the weight average molecular weight of the copolyester is 10,000 to 1,000,000. [Item 9] A resin composition containing polylactic acid and the crystallization accelerator according to any one of items 1 to 8. [Item 10] The resin composition according to item 9, wherein the amount of the copolyester relative to 100 parts by weight of the polylactic acid is 0.1 to 8 parts by weight. [Item 11] The resin composition according to item 9 or 10, wherein the average particle diameter of the copolyester is 50 nm or more and 10 μm or less. [Item 12] A molded article obtained by molding the resin composition according to any one of Items 9 to 11. [Item 13] A method for promoting crystallization of polylactic acid, characterized by bringing a copolymer polyester of lactic acid and another hydroxycarboxylic acid into contact with polylactic acid. [Item 14] Use of a copolymer polyester of lactic acid and another hydroxycarboxylic acid as a crystallization promoter for promoting crystallization of polylactic acid.

Examples

[0053] Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited to these examples. Overall genetic manipulation can be carried out as described, for example, in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Also, enzymes, cloning hosts, etc. used for genetic manipulation can be purchased from market suppliers and used according to their instructions. The enzyme is not particularly limited as long as it can be used for genetic manipulation.

[0054] (Synthesis Example 1) [Synthesis of P54LAHB] According to the description in PNAS 105(45)17323 - 17327(2008), a copolymer polyester of lactic acid and 3-hydroxybutanoic acid was biosynthesized using recombinant Escherichia coli. Cultivation was carried out with an aeration rate of 0.5 vvm and stirring at 500 rpm, and the biosynthesized copolymer polyester was extracted from the cells with chloroform. Of the obtained copolymer polyester 1The 1H-NMR spectrum was measured using a nuclear magnetic resonance apparatus (Avance III 600 MHz manufactured by Bruker), with deuterated chloroform as the solvent, at room temperature, and under the condition of 8 accumulations. Tetramethylsilane (0 ppm) was used as the internal standard substance. The signals appearing in the regions of 4.9 - 5.2 ppm and 5.2 - 5.4 ppm were attributed to the protons of the methine groups of the lactic acid unit and the 3-hydroxybutyric acid unit, respectively. When the areas of these regions were measured and designated as X and Y respectively, the molar fraction of lactic acid in the copolyester was calculated by the formula: X / (X + Y)×100. The molar fraction of lactic acid in the copolyester calculated by this method was 54 mol%, and the weight average molecular weight Mw was 73,000. This was designated as P54LAHB.

[0055] (Synthesis Example 2) [Synthesis of P40LAHB] Using the same method as above, culturing was carried out with an aeration rate of 0.5 vvm and stirring at 500 rpm. The obtained copolyester had a lactic acid molar fraction of 40 mol% and a weight average molecular weight Mw of 99,000. This was designated as P40LAHB.

[0056] (Synthesis Example 3) [Synthesis of P25LAHB] Using the same method as above, culturing was carried out with an aeration rate of 1 vvm and stirring at 500 rpm. The obtained copolyester had a lactic acid molar fraction of 25 mol% and a weight average molecular weight Mw of 66,000. This was designated as P25LAHB.

[0057] (Synthesis Example 4) [Synthesis of P22LAHB] Using the same method as above, culturing was carried out with an aeration rate of 2 vvm and stirring at 500 rpm. The obtained copolyester had a lactic acid molar fraction of 22 mol% and a weight average molecular weight Mw of 76,000. This was designated as P22LAHB.

[0058] (Synthesis Example 5) [Synthesis of P16LAHB] Using the same method as above, culturing was carried out with an aeration rate of 2 vvm and stirring at 500 rpm. The obtained copolyester had a lactic acid molar fraction of 16 mol% and a weight average molecular weight Mw of 75,000. This was designated as P16LAHB.

[0059] [Breeding of Cupriavidus necator H16 strain for the production of copolyester (LAHB) hydrogen bacteria] The Cupriavidus necator H16 strain was genetically recombined, and the PHA polymerase gene phaC on the genome 1Re was replaced with another PHA polymerase gene, and the PHA degrading enzyme gene phaZ 1,2,6 was disrupted. To enhance glucose assimilation ability, the 793rd base G of the nagE gene, which is the N-acetylglucosamine uptake gene, was replaced with C, and the gene nagR, which encodes a transcriptional regulator, was disrupted to obtain the KNK005ΔphaZ 1,2,6 / nagE G793C.dR strain (see International Publication No. 2017 / 104722) was prepared.

[0060] Furthermore, the PHA polymerase gene phaC on the genome of the KNK005ΔphaZ 1,2,6 / nagE G793C.dR strain 1Re was replaced with the STQK mutant (PHA polymerase in which the 325th serine of the polymerase PhaC1 derived from Pseudomonas sp. 61-3 Ps was converted to threonine and the 481st glutamine was converted to lysine) to obtain H16 phaC 1Re ::STQK ΔphaZ 1,2,6 / nagE G793C.dR was prepared. This strain was used as host (1).

[0061] [Preparation of plasmid for gene disruption] Using the genomic DNA of C. necator H16 strain as a template, PCR was performed with the oligonucleotide DNAs shown in SEQ ID NO: 1 and SEQ ID NO: 2 as primers. PrimeSTAR GXL DNA polymerase (manufactured by Takara Bio Inc.) was used as the DNA polymerase. Similarly, PCR was performed using the DNAs shown in SEQ ID NO: 3 and SEQ ID NO: 4 as primers. Using the two DNA fragments obtained by the above PCR as templates, overlap PCR was performed using the DNAs shown in SEQ ID NO: 3 and SEQ ID NO: 4 as primers. The obtained DNA fragment is a fragment in which approximately 500 base pairs upstream and approximately 500 base pairs downstream of the ORF of D-lactate dehydrogenase (Locus tag: H16_A3091) are ligated. This DNA fragment was treated with the restriction enzyme SmiI and ligated using the vector pNS2X-sacB (described in JP 2007-259708) which was also treated with SmiI and DNA ligase. The gene disruption plasmid containing the nucleotide sequence shown in SEQ ID NO: 5 thus obtained was named pNS2X-sacB-Δdld. This gene disruption plasmid (1) is a plasmid used to disrupt the gene dld: A3091 encoding D-lactate dehydrogenase.

[0062] Similarly, the gene disruption plasmid pNS2X-sacB-ΔphaA was prepared. This gene disruption plasmid (2) is a plasmid used to disrupt the gene phaA: A1438 encoding acetyl-CoA acetyltransferase. Table 1 summarizes the genes to be disrupted by each plasmid, the SEQ ID NOs showing the base sequences of the primers used in the preparation of each plasmid, and the SEQ ID NOs showing the base sequences inserted by each plasmid.

[0063] [Table 1]

[0064] (Preparation of plasmid for gene introduction) To introduce the gene group necessary for LAHB production onto the genome of C. necator, a plasmid for gene transfer was constructed. In a form of replacing the ORF of phaJ4b (Locus tag: H16_B0397) on the genome of the host (1), a gene sequence expressing lactic acid dehydrogenase LDH derived from Leuconostoc mesenteroides under the REP promoter Lm pNS2X-sacB-phaJ4b::REP-LDH into which the gene sequence can be introduced Lm was constructed. This plasmid is a plasmid obtained by inserting the DNA fragment represented by SEQ ID NO: 11 into the pNS-sacB vector by ligation. By using this plasmid, it becomes possible to endow C. necator with the ability to produce D-lactic acid from glucose. This plasmid is used as the gene transfer plasmid (1).

[0065] Similarly, in a form of replacing the ORF of phaJ4a (Locus tag: H16_A1070) on the genome of the host (1), propionyl-CoA transferase PCT derived from Epulopiscium sp. under the lacN17 promoter Es pNS2X-sacB-phaJ4a::lacN17-PCT into which the gene sequence can be introduced Es was constructed. This plasmid is a plasmid obtained by inserting the DNA fragment represented by SEQ ID NO: 12 into the pNS2X-sacB vector by ligation. By using this plasmid, it becomes possible to add CoA to the lactic acid produced from glucose to supply the substrate for the copolyester polymerization enzyme. This plasmid is used as the gene transfer plasmid (2).

[0066] (Genetic modification of C. necator by homologous recombination) The gene disruption plasmid or the gene transfer plasmid was introduced into Escherichia coli S17-1 strain (ATCC47055) by electroporation, and co-cultured with the target C. necator recombinant strain on Nutrient Agar medium (manufactured by Difco) to perform conjugative transfer.

[0067] From the microbial community after co-culture, strains with plasmids inserted into their genomes were selected on Simmons agar medium (sodium citrate 2 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, diammonium hydrogen phosphate 1 g / L, agar 15 g / L, pH 6.8) containing 250 mg / L of kanamycin sulfate and isolated. After further purifying the strains isolated on Nutrient Agar medium containing 250 mg / L of kanamycin sulfate, they were inoculated onto Nutrient Agar medium containing 15% sucrose to obtain strains in which the plasmid had been excised. By homologous recombination, at the stage of plasmid excision, two types of strains occurred: those that reverted to the original genomic sequence and those with the desired genetic modification. The latter was isolated and obtained by colony PCR. The obtained genetically modified strains were purified again on Nutrient Agar medium containing sucrose to obtain homologous recombinant strains.

[0068] Using plasmid for gene transfer (1) and plasmid for gene transfer (2), gene modification was performed on host (1) by the above method to obtain H16 phaC 1Re ::STQK ΔphaZ 1,2,6 / nagE G793C.dR phaJ4a::lacN17-PCT Es phaJ4b::REP-LDH Lm This was designated as host (2). Furthermore, gene modification was performed on this strain using plasmid for gene disruption (1): pNS2X-sacB-Δdld by the above method to obtain H16 phaC 1Re ::STQK ΔphaZ 1,2,6 / nagE G793C.dR phaJ4a::lacN17-PCT Es phaJ4b::REP-LDH Lm Δdld. This was designated as host (3).

[0069] Furthermore, gene modification was performed on host (2) using plasmid for gene disruption (2): pNS2X-sacB-ΔphaA by the above method to obtain H16 phaC 1Re ::STQK ΔphaZ 1,2,6 / nagE G793C.dR phaJ4a::lacN17-PCT Es phaJ4b::REP-LDH Lm ΔphaA was prepared. This was used as the host (4).

[0070] A DNA fragment encoding STQK was amplified by PCR and ligated with a DNA fragment obtained by treating the pCUP2 vector (see International Publication No. 2007 / 049716) with MunI and SpeI using DNA ligase to obtain a vector for expression under a strong lacUV5 promoter (SEQ ID NO: 13). Strains obtained by introducing this expression vector into hosts (3) and (4) by electroporation were designated as hosts (3') and (4'). Kanamycin was added as appropriate to retain the plasmid.

[0071] (Synthesis Example 6) [Synthesis of HP15LAHB] Using host (3'), a copolymer polyester (LAHB) was produced using a jar fermenter with glucose as the carbon source.

[0072] First, as a preculture, a meat medium (composition: 1% (w / v) meat extract, 1% (w / v) bactotryptone, 0.2% (w / v) yeast extract, 0.9% (w / v) disodium hydrogen phosphate dodecahydrate, 0.15% (w / v) potassium dihydrogen phosphate, 50 μg / L kanamycin) was used for overnight culture at 30°C.

[0073] This preculture solution was added to 500 ml of Sakaguchi flasks containing 100 ml of meat medium and cultured with shaking at 30°C for 6 hours.

[0074] Subsequently, the above culture solution was inoculated into a 5 L jar fermenter (Bioneer Neo type manufactured by Marubishi Bioengine) containing 1.8 L of PHA production medium. The operating conditions were a culture temperature of 30°C, a stirring speed of 500 rpm, an aeration rate of 1.8 L / min, and the pH was controlled between 6.7 and 6.8 for 48 hours of culture. A 7% aqueous ammonium hydroxide solution was used for pH control.

[0075] The composition of the PHA production medium was 0.578% (w / v) disodium hydrogen phosphate dodecahydrate, 0.101% (w / v) potassium dihydrogen phosphate, 0.437% (w / v) ammonium sulfate, 0.15% (w / v) magnesium sulfate heptahydrate, 0.75% (v / v) trace metal salt solution (dissolved 1.6% (w / v) iron(II) chloride hexahydrate, 1% (w / v) calcium chloride dihydrate, 0.02% (w / v) cobalt(II) chloride hexahydrate, 0.016% (w / v) copper(II) sulfate pentahydrate, 0.012% (w / v) nickel(II) chloride hexahydrate in 0.1N hydrochloric acid). The carbon source was glucose, with an initial concentration of 20 g / L. When the glucose was consumed to 10 g / L, it was then maintained at 10 g / L thereafter.

[0076] The cells were recovered from the culture broth by centrifugation, purified with pure water and ethanol, and then dried to a solid state by vacuum drying. The polymer was extracted from the dried cells with chloroform, and the chloroform was completely removed using an evaporator and a vacuum dryer to obtain a copolymer polyester (LAHB) of lactic acid and 3-hydroxybutanoic acid.

[0077] The copolymer polyester obtained from the host (3’) 1 had a lactic acid molar fraction of 15 mol% and a weight average molecular weight of 540,000 as measured by 1H NMR. This was designated as HP15LAHB.

[0078] (Synthesis Example 7) [Synthesis of HP20LAHB] The copolymer polyester obtained in the same manner as in Synthesis Example 6 except that the host (4’) was used instead of the host (3’) 1 had a lactic acid molar fraction of 20 mol% and a weight average molecular weight of 370,000 as measured by 1H NMR. This was designated as HP20LAHB.

[0079] (Example 1) [Measurement of crystallization peak time] 10 g of a mixture composed of 80 wt% of polylactic acid (PDLA: D-form ratio 99.8%, weight average molecular weight 220,000, manufactured by BMG Corporation) and 20 wt% of copolymerized polyester P54LAHB was dissolved in chloroform, and the solvent was removed and dried to obtain a polymer blend sample. The obtained sample was molded into a film with a thickness of 0.2 mm at 180 °C using a vacuum heating press, punched out, and subjected to DSC measurement. The DSC measurement was performed using DSC7000X manufactured by Hitachi High-Tech Corporation. Under a nitrogen stream, after dissolving the sample at 200 °C for 1 minute, it was rapidly cooled to 90 °C at a cooling rate of -70 °C per minute or more, and the DSC signal under isothermal conditions was measured. Typical data are shown in Figure 1. Taking the time point when the temperature became constant as 0 minute, the time until the crystallization signal reached its peak was defined as the crystallization peak time and used as an index of the crystallization rate. The obtained crystallization peak time was 4.90 minutes.

[0080] (Example 2) The experiment was conducted in the same manner as in Example 1 except that P40LAHB was used instead of copolymerized polyester P54LAHB. The obtained crystallization peak time was 3.90 minutes.

[0081] (Example 3) The experiment was conducted in the same manner as in Example 1 except that P25LAHB was used instead of copolymerized polyester P54LAHB. The obtained crystallization peak time was 1.29 minutes.

[0082] (Example 4) The experiment was conducted in the same manner as in Example 1 except that P22LAHB was used instead of copolymerized polyester P54LAHB. The obtained crystallization peak time was 2.60 minutes.

[0083] (Example 5) The experiment was conducted in the same manner as in Example 1 except that 99 wt% of polylactic acid (PDLA) and 1 wt% of copolymerized polyester P16LAHB were used. The obtained crystallization peak time was 6.86 minutes.

[0084] (Example 6) The experiment was conducted in the same manner as in Example 1, except that 95% by weight of polylactic acid (PDLA) and 5% by weight of copolymer polyester P16LAHB were used. The obtained crystallization peak time was 4.15 minutes.

[0085] (Example 7) The experiment was conducted in the same manner as in Example 1, except that 80% by weight of polylactic acid (PDLA) and 20% by weight of copolymer polyester P16LAHB were used. The obtained crystallization peak time was 0.80 minutes.

[0086] (Example 8) The experiment was conducted in the same manner as in Example 1, except that 70% by weight of polylactic acid (PDLA) and 30% by weight of copolymer polyester P16LAHB were used. The obtained crystallization peak time was 3.29 minutes.

[0087] (Example 9) The experiment was conducted in the same manner as in Example 1, except that 50% by weight of polylactic acid (PDLA) and 50% by weight of copolymer polyester P16LAHB were used. The obtained crystallization peak time was 3.75 minutes.

[0088] (Example 10) The experiment was conducted in the same manner as in Example 1, except that 70% by weight of polylactic acid (PDLA) and 30% by weight of copolymer polyester HP15LAHB were used. The obtained crystallization peak time was 3.88 minutes.

[0089] (Example 11) The experiment was conducted in the same manner as in Example 1, except that 70% by weight of polylactic acid (PDLA) and 30% by weight of copolymer polyester HP20LAHB were used. The obtained crystallization peak time was 3.48 minutes.

[0090] (Comparative Example 1) The experiment was conducted in the same manner as in Example 1 using only polylactic acid (PDLA). The obtained crystallization peak time was 10.79 minutes. The results of Examples 1 to 11 and Comparative Example 1 are summarized in Table 2.

[0091]

Table 2

[0092] (Example 12) 10 g of a mixture consisting of 80% by weight of polylactic acid (PLLA: D-form ratio 0.0%, weight-average molecular weight 230,000, manufactured by BMG Corporation) and 20% by weight of copolymerized polyester P54LAHB was dissolved in chloroform, and the solvent was removed and dried to obtain a polymer blend sample. The crystallization time measured by the same DSC method as in Example 1 for the obtained sample was 3.10 minutes.

[0093] (Example 13) The experiment was conducted in the same manner as in Example 12 except that P40LAHB was used instead of copolymerized polyester P54LAHB. The obtained crystallization peak time was 2.98 minutes.

[0094] (Example 14) The experiment was conducted in the same manner as in Example 12 except that P25LAHB was used instead of copolymerized polyester P54LAHB. The obtained crystallization peak time was 3.37 minutes.

[0095] (Example 15) The experiment was conducted in the same manner as in Example 12 except that 95% by weight of polylactic acid (PLLA) and 5% by weight of copolymerized polyester P22LAHB were used. The obtained crystallization peak time was 5.94 minutes.

[0096] (Example 16) The experiment was conducted in the same manner as in Example 12 except that 80% by weight of polylactic acid (PLLA) and 20% by weight of copolymerized polyester P22LAHB were used. The obtained crystallization peak time was 2.31 minutes.

[0097] (Example 17) The experiment was conducted in the same manner as in Example 12 except that 70% by weight of polylactic acid (PLLA) and 30% by weight of copolymerized polyester P16LAHB were used. The obtained crystallization peak time was 6.63 minutes.

[0098] (Example 18) The experiment was conducted in the same manner as in Example 12, except that 80% by weight of polylactic acid (PLLA) and 20% by weight of copolymerized polyester P16LAHB were used. The obtained crystallization peak time was 3.02 minutes.

[0099] (Example 19) The experiment was conducted in the same manner as in Example 12, except that 70% by weight of polylactic acid (PLLA) and 30% by weight of copolymerized polyester HP15LAHB were used. The obtained crystallization peak time was 4.78 minutes.

[0100] (Example 20) The experiment was conducted in the same manner as in Example 12, except that 50% by weight of polylactic acid (PLLA) and 50% by weight of copolymerized polyester HP15LAHB were used. The obtained crystallization peak time was 4.11 minutes.

[0101] (Example 21) The experiment was conducted in the same manner as in Example 12, except that 80% by weight of polylactic acid (PLLA) and 20% by weight of copolymerized polyester HP15LAHB were used. The obtained crystallization peak time was 3.63 minutes.

[0102] (Example 22) The experiment was conducted in the same manner as in Example 12, except that 80% by weight of polylactic acid (PLLA) and 20% by weight of copolymerized polyester HP20LAHB were used. The obtained crystallization peak time was 4.62 minutes.

[0103] (Comparative Example 2) The experiment was conducted in the same manner as in Example 12 using only polylactic acid (PLLA). The obtained crystallization peak time was 9.79 minutes. The results of Examples 12 to 22 and Comparative Example 2 were summarized in Table 3.

[0104]

Table 3

[0105] (Example 23) 10 g of a mixture consisting of 80 wt% of polylactic acid (D-isomer ratio 4%, weight-average molecular weight 180,000, Ingeo Biopolymer 2003D manufactured by NatureWorks) and 20 wt% of copolymerized polyester P16LAHB was dissolved in chloroform, and the solvent was removed and dried to obtain a polymer blend sample. The crystallization time of the obtained sample measured by the same DSC method as in Example 1 was 19.9 minutes.

[0106] (Example 24) 10 g of a mixture consisting of 80 wt% of polylactic acid (D-isomer ratio 4%, Luminy LX175 manufactured by TotalEnergies Corbion) and 20 wt% of copolymerized polyester P16LAHB was dissolved in chloroform, and the solvent was removed and dried to obtain a polymer blend sample. The crystallization time of the obtained sample measured by the same DSC method as in Example 1 was 13.8 minutes.

[0107] (Comparative Example 3) Using only polylactic acid (D-isomer ratio 4%, weight-average molecular weight 180,000, Ingeo Biopolymer 2003D manufactured by NatureWorks), the experiment was conducted in the same manner as in Example 23, but no crystallization peak was detected even after 40 minutes.

[0108] (Comparative Example 4) Using only polylactic acid (D-isomer ratio 4%, Luminy LX175 manufactured by TotalEnergies Corbion), the experiment was conducted in the same manner as in Example 24, but no crystallization peak was detected even after 40 minutes. The results of Examples 23 and 24 and Comparative Examples 3 and 4 were summarized in Table 4.

[0109]

Table 4

[0110] From Tables 2 to 4, it can be seen that by adding a copolymerized polyester of lactic acid and another hydroxycarboxylic acid to polylactic acid, the crystallization peak time becomes shorter compared to the case of polylactic acid alone, and the crystallization of polylactic acid is promoted.

[0111] (Example 25) [Preparation of Compound Pellets] After drying 10 g of a mixture consisting of 97% by weight of polylactic acid (Ingeo 2003D, manufactured by NatureWorks) and 3% by weight of HP20LAHB at 50°C for 12 hours, the temperature of the barrel and die was set to 190°C and melt-kneaded and extruded using a twin-screw extruder (ULT Nano05 manufactured by Techno bel, screw diameter 1.5 cm, L / D = 13.33). The molten strand extruded from a die with a diameter of 2.5 mm was taken up, air-cooled in the cooling process, and then cut into lengths of about 4 mm with a pelletizer to obtain compound pellets.

[0112] (Comparative Example 5) An experiment was conducted in the same manner as in Example 25 except that only 10 g of polylactic acid (Ingeo 2003D, manufactured by NatureWorks) was used to obtain polylactic acid pellets.

[0113] The compound pellets and pellets obtained in Example 25 or Comparative Example 5 were dried in a vacuum heating dryer (DP300 manufactured by Yamato Scientific) under vacuum at 80°C for 4 hours, and then a press plate with a thickness of 4 mm was produced using a hydraulic vacuum heating press (IMC-11FD type manufactured by Imoto Seisakusho) under vacuum at 170°C and a forming pressure of 1.4 MPa.

[0114] The obtained press plate was frozen with liquid nitrogen and broken, and photographs of the fracture surface observed with a scanning electron microscope (SEM) (JSM-IT300HR manufactured by JEOL Ltd.) are shown in FIGS. 2 and 3. In the test piece of Example 25 shown in FIG. 2, a large number of circular depression structures formed by the extremely fine dispersion of the copolymer polyester of 3-hydroxybutanoic acid and lactic acid in polylactic acid were observed. On the other hand, such a structure was not confirmed in the test piece of Comparative Example 5 shown in FIG. 3.

[0115] Regarding the SEM observation results in FIG. 2, the diameters of 200 circular depression structures were measured using image analysis software ("ImageJ"), and the arithmetic mean value was calculated as the average particle size. The average particle size was 185 nm.

Claims

1. A crystallization accelerator for promoting the crystallization of polylactic acid, the crystallization accelerator containing a copolymer polyester of lactic acid and another hydroxycarboxylic acid.

2. The crystallization accelerator according to claim 1, wherein the other hydroxycarboxylic acid is 3-hydroxyalkanoic acid.

3. The crystallization accelerator according to claim 1, wherein the other hydroxycarboxylic acid is at least one selected from the group consisting of 3-hydroxybutanoic acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3-hydroxypentadecanoic acid, and 3-hydroxyhexadecanoic acid.

4. The crystallization accelerator according to claim 1, wherein the other hydroxycarboxylic acid is 3-hydroxybutanoic acid.

5. The crystallization accelerator according to any one of claims 1 to 4, wherein the copolymer polyester is a random copolymer of lactic acid and another hydroxycarboxylic acid.

6. The crystallization accelerator according to any one of claims 1 to 4, wherein the lactic acid monomer in the copolymer polyester is a D-lactic acid monomer.

7. The crystallization accelerator according to any one of claims 1 to 4, wherein the molar fraction of lactic acid units in the copolymer polyester is 5 to 70 mol%.

8. The crystallization accelerator according to any one of claims 1 to 4, wherein the weight average molecular weight of the copolymer polyester is 10,000 to 1,000,000.

9. A resin composition containing polylactic acid and the crystallization accelerator according to any one of claims 1 to 4.

10. The resin composition according to claim 9, wherein the amount of the copolymer polyester relative to 100 parts by weight of the polylactic acid is 1 to 100 parts by weight.

11. The resin composition according to claim 9, wherein the average particle diameter of the copolymer polyester is 50 nm or more and 10 μm or less.

12. A molded article formed by molding the resin composition according to claim 9.

13. A method for promoting the crystallization of polylactic acid, characterized by mixing a copolymer polyester of lactic acid and another hydroxycarboxylic acid with polylactic acid.

14. Use of a copolymer polyester of lactic acid and another hydroxycarboxylic acid as a crystallization accelerator for promoting the crystallization of polylactic acid.

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