Construct and method for making it

专利摘要:
Having a substrate and a polyhydroxyalkanoate, at least a portion of the substrate is covered with the polyhydroxyalkanoate, and the polyhydroxyalkanoate is a 3-hydroxypropionic acid unit, 3-hydroxy-n- A structure containing a 3-hydroxyalkanoic acid unit except a butyric acid unit and a 3-hydroxy-n-yl acetic acid unit.
公开号:KR20020083519A
申请号:KR1020020023282
申请日:2002-04-27
公开日:2002-11-02
发明作者:혼마쓰토무；야노데쓰야；노모토쓰요시；고자키신야
申请人:캐논 가부시끼가이샤；
IPC主号:

专利说明:

Structure and its manufacturing method {CONSTRUCT AND METHOD FOR MAKING IT}
[5] The present invention relates to a structure comprising a polyhydroxyalkanoate and a substrate, and wherein the polyhydroxyalkanoate has a structure covering at least a part of the substrate, and a method for producing the same. More specifically, a polyhydride containing at least 3-hydroxyalkanoic acid unit other than 3-hydroxypropionic acid unit, 3-hydroxy-n-butyric acid unit or 3-hydroxy-n-yl acetic acid unit as a monomer unit. A hydroxyalkanoate and a base material are provided, and this polyhydroxyalkanoate coat | covers at least one part of this base material, It is related with the structure characterized by the above-mentioned. In addition, the present invention immobilizes the polyhydroxyalkanoate synthase involved in the medium-chain polyhydroxyalkanoate biosynthesis reaction to the substrate, polymerizes 3-hydroxyacyl coenzyme A by the enzyme, and polyhydroxyal At least a part of this base material is covered with polyhydroxyalkanoate by synthesizing a canoate, to thereby provide a structural method. In addition, the structure of the present invention includes a capsule structure in which a polyhydroxyalkanoate is coated on a granular substrate, and a flat or film-shaped substrate in which a polyhydroxyalkanoate is coated on a flat or film-like substrate. Laminated structures are included.
[6] The structure of the present invention can be used for a wide range of applications as a functional structure. For example, the capsule structure can be used as a capsule toner for electrophotography, and the laminated structure can be used as a recording medium such as an OHP film or ink jet recording paper. have.
[7] Polymer materials are indispensable for the modern industry and life, and have been used in various fields such as housing materials for home appliances, packaging materials, cushioning materials, and textile materials due to their low cost and lightness and good moldability. On the other hand, various functional materials such as liquid crystal materials and coating materials have also been obtained by introducing substituents that express various functions in the molecular chain of the polymer by utilizing the stable properties of these polymer materials. Since these functional materials have higher added value than polymers as structural materials, even a small amount of production can expect a large market need. Such a polymer functional material has been obtained by an organic synthetic chemical method by modifying the synthesized polymer with a substituent in the synthesis process of the polymer or until now. In most cases, the polymer serving as the basic skeleton of the polymer functional material is obtained from petroleum-based raw materials by an organic synthetic chemical method. Polyethylene, polyethylene terephthalate, polyester, polystyrene, polyvinyl chloride, polyacrylamide and the like are typical examples.
[8] Therefore, the inventors of the present invention have focused on a weight structure coated with a substrate by a polymer compound as one element technology to give a large added value to the polymer compound. Thus, by coating a specific substrate with a high molecular compound, a composite structure having extremely useful functionality can be obtained. Specific applications of such a structure include, for example, an electrophotographic capsule toner consisting of a microcapsule structure containing a toner component in a polymer compound, an inkjet recording method for coating a sheet-like substrate with a polymer compound, and the like. Can be mentioned.
[9] In general, the electrophotographic method uses a photoconductive material, forms an electrical latent image on the photoconductor by various means, and then develops the latent image using toner, and toner image on a transfer material such as paper as necessary. After the transfer, the film is fixed by heating pressure, solvent vapor, or the like to obtain a radiant image. As a toner used for this purpose, "pulverized toner" having a desired particle size has been used by melt-mixing and uniformly dispersing a colorant consisting of salts and pigments in a thermoplastic resin and then using a fine grinding device and a classifier. Although this toner has excellent ability, there is a problem such that there is a certain limitation on the selection range of the material because brittleness is required in the manufacturing process. In order to overcome such a problem, the manufacture of "polymerization toner" by suspension polymerization method is proposed by Japanese Unexamined Patent Publication No. 36-10231. In the suspension polymerization method, a polymerizable monomer, a colorant, a polymerization initiator, a crosslinking agent, a charge control agent and the like are uniformly dissolved or dispersed as necessary, and then a continuous phase (for example, an aqueous phase) containing a dispersion stabilizer is mixed with a stirrer. It disperse | distributes and a polymerization reaction is performed simultaneously and a desired toner is obtained. This method does not involve a pulverization step, so that the toner does not need brittleness, and there is an advantage that a soft material can be used. However, the polymerization toner of such a small particle size tends to expose the colorant and the charge control agent to the toner surface layer, so that the influence of the colorant is likely to occur, and the uniformity of charging is feared. In order to solve these problems, a so-called "capsule toner" has been proposed in which the surface of the polymer particles is coated with a single layer or a plurality of outer layers of a polymer compound.
[10] As such a capsule toner, for example, Japanese Unexamined Patent Application Publication No. 8-286416 discloses a capsule toner for developing electrostatic charge coated with polymer particles in an outer shell containing a polar resin and a method of manufacturing the same. In this method, an outer skin is formed by an organic synthetic chemical method around a core made of polymerized particles containing a toner component. This method improves the above-mentioned problems, and improves image durability, realizes uniformity and stabilization of charging. Capsule developing toner can be supplied. In addition, Japanese Laid-Open Patent Publication No. 9-292735 discloses an image forming capsule toner coated with a core made of a release agent material and a material having a large thermal expansion by a firm coat resin. The toner is a functional microcapsule configured to extrude the release agent material contained by the thermally expandable material in the core due to overheating at the time of fusing, thereby destroying the outer shell, and to prevent offset when using a film heating type fixing device. In addition, in the case of using a roller electrodeposition machine, an effect of enabling fixing at low pressure and reducing paper wrinkles can be expected. Similarly, capsule toners having a high molecular compound and a method of manufacturing the same are disclosed in Japanese Patent Application Laid-Open Nos. 5-119531, 5-249725, 6-332225, and 9-43896. , Japanese Patent Application Laid-Open No. 10-78676, Japanese Patent Laid-Open No. 11-7163, Japanese Patent Laid-Open No. 2000-66444, Japanese Patent Laid-Open No. 2000-112174 and Japanese Patent Laid-Open No. 2000-330321 are disclosed. All of them produce desired capsule structures by organic synthetic chemical methods such as suspension polymerization method, emulsion polymerization method, precipitation polymerization method, dispersion polymerization method, Soupfree emulsion polymerization method and sheet polymerization method.
[11] However, the manufacturing method of these capsule toners also had the problem of using a large amount of solvents and surfactants in the manufacturing process which makes the manufacturing process extremely complicated.
[12] On the other hand, as a recording medium which consists of a laminated structure which covered the sheet-like base material with the high molecular compound, the recording medium in inkjet recording system is mentioned, for example. The ink jet recording method is to record images, characters, and the like by attaching small droplets of ink to a recording medium such as paper by flying the ink droplets by various operating principles. In this recording method, since the ink contains a large amount of solvent such as water, a mixture of water and an organic solvent, it is necessary to use a large amount of ink to obtain a high color density. In addition, since the droplets are continuously ejected, when the first droplet is ejected, the droplets fuse and the beads of the ink are bonded to each other, resulting in a disturbing image. For this reason, the ink jet recording medium is required to have a large ink absorption amount and a high absorption speed.
[13] To this end, a recording medium has been proposed in which an ink receiving layer is formed on a substrate to improve ink absorption. For example, Japanese Patent Laid-Open No. 55-146786 and the like have proposed a recording medium in which a substrate is coated with a water-soluble resin such as polyvinyl alcohol or polyvinyl pyrrolidone. In addition, Japanese Patent Laid-Open No. 5-221112 or the like has proposed a recording medium using a water-resistant resin. In addition, recording media using ionic resins as ink receiving layers are proposed (e.g., Japanese Patent Application Laid-Open No. 11-78221, Japanese Patent Laid-Open No. 2000-190631, etc.), and water wettability, water resistance, dye fixability, and the like. A recording medium excellent in this absorption and excellent in ink absorption dryness and image sharpness has been obtained.
[14] As a method of forming an ink absorbing layer on a substrate, conventionally, a method by smearing is generally used. For example, a blade coating method, an air knife coating method, a roll coating method, a flash coating method, a gravure coating method, a key coating method It has been carried out by a method using a method, a die coating method, an extrusion coating method, a slide hopper coating method, a curtain coating method, a spray coating method and the like.
[15] In any of the methods exemplified above, the polymer compound used for coating the substrate is synthesized and structured by an organic synthetic method, and various functions are added thereto.
[16] On the other hand, in recent years, the research which manufactures a high molecular compound by the biotechnological method has been actively performed, and has been put to practical use in part. For example, a microbial-derived high molecular compound is a copolymer of poly-3-hydroxy-n-butyric acid (PHB), 3-hydroxy-n- butyric acid and 3-hydroxy-n-gilic acid (PHB / V Polyhydroxyalkanoate (PHA), polysaccharides, such as bacterial cellulose and pullulan, and polyamino acids, such as poly- (gamma) -glutamic acid and polylysine, are known. In particular, PHA can be used in various products by melt processing and the like as conventional plastics, and is also excellent in biocompatibility, and is also expected to be applied as a medical soft member.
[17] Many microorganisms have been reported to produce PHA and accumulate in cells. For example, Alcaligenes eutrophus H16, ATCC No. 17699, Methylobacterium sp., Paracoccus sp., Alcaligenes sp. .), Production of PHB / V by microorganisms of Pseudomonas sp. Has been reported (Japanese Patent Application Laid-Open No. 5-74492, Japanese Patent Application Laid-Open No. 6-15604, Japanese Patent Application Laid-Open No. 7-14352). 8-19227, etc.), and Comamonas acidovorans IFO 13852 contains 3-hydroxy-n-butyric acid and 4-hydroxy-n-butyric acid in monomer units. Production of PHA is disclosed (Japanese Patent Laid-Open No. 9-191893). It is also disclosed to produce a copolymer of 3-hydroxy-n-butyric acid and 3-hydroxyhexanoic acid by Aeromonas caviae (Japanese Patent Application Laid-Open No. 5-93049, Copper). Japanese Patent Laid-Open No. 7-265065).
[18] The biosynthesis of these PHBs and PHB / Vs is based on (R) -3-hydroxybutyryl CoA or (R) -3-hydroxyvaleryl CoA, which are produced through various metabolic pathways in vivo from various carbon sources. It is carried out by a polymerization reaction by an enzyme. The enzyme catalyzing this polymerization reaction is PHB synthase (also called PHB polymerase or PHB synthase). CoA is an abbreviation of coenzyme A, and its chemical structure is shown in the following chemical formula.
[19]
[20] In recent years, the research on the polyhydroxyalkanoate (mcl-PHA) consisting of 3-hydroxyalkanoic acid units having a medium chain length of 3 to 12 carbon atoms has been vigorously conducted. .
[21] For example, Japanese Patent Laid-Open No. 2642937 has a monomer unit of 3-hydroxyalkanoic acid having 6 to 12 carbon atoms by assigning acyclic aliphatic hydrocarbons to Pseudomonas oleovorans ATCC 29347. It is disclosed that PHA is produced. In addition, Appl. Environ. Microbiol., 58, 746 (1992) found that Pseudomonas resinovorans has 3-hydroxy-n-butyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3, with octanoic acid as a single carbon source. Produces PHA with hydroxydecaic acid as monomer unit, 3-hydroxy-n-butyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxy with hexanoic acid as single carbon source It has been reported to produce PHA in units of decaic acid. Here, it is considered that the introduction of the 3-hydroxyalkanoic acid monomer unit having a longer chain length than the fatty acid of the raw material is via the fatty acid synthesis route described later.
[22] Int. J. Biol. In Macromol., 16 (3), 119 (1994), Pseudomonas sp. Strain 61-3 has 3-hydroxy-n-butyric acid, 3-hydroxyhexane as a single carbon source using sodium gluconate. 3-hydroxyalkanoic acid, such as acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid, and 3-hydroxy-5-cis-decenoic acid, 3-hydroxy-5 It has been reported to produce PHAs in units of 3-hydroxyalgenic acid, such as -cis-dodecenoic acid.
[23] The PHA is PHA (usual-PHA) consisting of a monomer unit having an alkyl group in the side chain. However, when considering a wider range of applications, for example as functional polymers, side chains of substituents other than alkyl groups (e.g., phenyl groups, unsaturated hydrocarbons, ester groups, allyl groups, cyano groups, halogenated hydrocarbons, epoxides, etc.) PHA (unusual-PHA) introduced in is extremely useful.
[24] As an example of the biosynthesis of unusual-PHAs with phenyl groups, it has been reported, for example, that Pseudomonas oleoboranth produces PHAs comprising 3-hydroxy-5-phenylglyacetic acid units from 5-phenylglyceic acid (Polymers, 24, 5256-5260 (1991), Macromol, Chem., 191, 1957-1965 (1990): Chirality, 3, 492-494 (1991)). In addition, Pseudomonas Oleoboranth is a 3-hydroxy-5- (4-tolyl) yl group from 5- (4-tolyl) gylic acid (5- (4-methylphenyl) gylic acid) in Polymers, 29 1762-1766 (1996). It has been reported to produce PHA containing acetic acid units. Polymers, 32, 2889-2895 (1999) also show that Pseudomonas oleoboranth is 3-hydroxy-5- (2,4-dinitrophenyl) glymic acid from 5- (2,4-dinitrophenyl) glymic acid. It is reported to produce PHAs comprising units and 3-hydroxy-5- (4-nitrophenyl) ylacetic acid units.
[25] Moreover, as an example of unusual-PHA which has a phenoxy group, Macromol. Chem. phys., 195, 1665-1672 (1994) comprising 3-hydroxy-5-phenoxyglyoxyacetic acid units and 3-hydroxy-9-phenoxynonanoic acid units from 11-phenoxyundecanoic acid in Pseudomonas oleoborane It is reported to produce PHA. Polymers, 29, 3423-3435 (1996) also show that Pseudomonas oleoboranth is a 3-hydroxy-4-phenoxy butyric acid unit and a 3-hydroxy-6-phenoxyhexanoic acid unit from 6-phenoxyhexanoic acid. PHA containing 3-hydroxy-4-phenoxy butyric acid unit, 3-hydroxy-6-phenoxyhexanoic acid unit and 3-hydroxy-8-phenoxyoctanoic acid unit from 8-phenoxyoctanoic acid. It has been reported to produce PHAs comprising Phydroxy comprising 3-hydroxy-5-phenoxyylacetic acid units and 3-hydroxy-7-phenoxyheptanoic acid units from 11-phenoxyundecanoic acid. Also, Can. J. Microbiol., 41, 32-43) 1995) Pseudomonas puteoborans ATCC 29347 strain and Pseudomonas putida KT 2442 strains were used as p-cyanophenoxyhexanoic acid or p-nitrophenoxyhexane. It is reported to produce PHAs comprising 3-hydroxy-p-cyanophenoxyhexanoic acid units or 3-hydroxy-p-nitrophenoxyhexanoic acid units from acids. Japanese Patent No. 2989175 discloses a homopolymer consisting of 3-hydroxy-5- (monofluorophenoxy) yl acetate unit or 3-hydroxy-5- (difluorophenoxy) yl acetate unit, at least 3- Copolymers containing hydroxy-5- (monofluorophenoxy) pentanoate units or 3-hydroxy-5- (difluorophenoxy) pentanoate units and methods for preparing the same are described. It is said that, as an effect, stereoregularity and water repellency can be imparted while maintaining a high melting point and good workability.
[26] In addition, as an example of an unusual-PHA having a cyclohexyl group, polymers, 30, 1611-1615 (1997), have been reported to produce this PHA from Pseudomonas oleoboranes cyclohexyl butyric acid or cyclohexyl oxyacetic acid.
[27] The biosynthesis of these mcl-PHA and unusual-PHA is (R) -3-hydroxyacyl CoA produced through various metabolic pathways (eg, β-oxidation system or fatty acid synthesis pathway) in vivo from various alkanoic acid raw materials. It is carried out by a polymerization reaction by an enzyme using as a substrate. The enzyme that catalyzes this polymerization reaction is PHA synthase (called PHA polymerase or PHA polymerase). Below, the reaction until the alkanoic acid becomes PHA through polymerization by β-oxidation system and PHA synthase is shown.
[28]
[29] On the other hand, in the case of the fatty acid synthesis route, (R) -3-hydroxyacyl CoA converted from (R) -3-hydroxyacyl-ACP (ACP refers to acyl carrier protein) generated in this pathway is used as a substrate. Similarly, it is thought that PHA is synthesized by PHA synthase.
[30] Recently, attempts have been made to synthesize PHA in a virto by taking the PHB synthase or PHA synthase out of cells.
[31] For example, Proc. Natl. Acad. Sci. USA, 92, 6279-6283 (1995) succeeded in synthesizing PHB consisting of 3-hydroxy-n-butyric acid units by acting 3-hydroxybutyryl CoA on PHB synthase derived from alkali genes eutropus. Doing. Int. J. Biol. Macromol., 25, 55-60 (1999) discloses 3-hydroxy-n-butyric acid by acting 3-hydroxybutyryl CoA or 3-hydroxyvaleryl CoA on PHB synthase derived from alkaline genes eutropus. The synthesis of PHA consisting of a unit or a 3-hydroxy-n-yl acetate unit has been successful. In addition, in this report, racemic 3-hydroxybutyryl CoA was reacted. According to the stereoselectivity of the enzyme, PHB composed of only the 3-hydroxy-n-butyric acid unit of R was synthesized. Macromol. Also in Rapid Commun., 21, 77-84 (2000), PHB synthesis in the cell using a PHB synthetase derived from alkaline genes eutropus has been reported.
[32] FEMS Microbiol. Lett., 168, 319-324 (19998) synthesized PHB consisting of 3-hydroxy-n-butyric acid units by acting 3-hydroxybutyryl CoA on PHB synthase derived from Chromatium vinosum. It is successful in doing.
[33] Appl. In Microbiol, Biotechnol., 54, 37-43 (2000), PHA consisting of 3-hydroxydecanoic acid units was formed by acting 3-hydroxydecanoyl CoA on PHA synthase of Pseudomonas aeruginosa. Synthesizing
[34] As described above, by applying the biotechnological method to the synthesis of the polymer compound, it is expected that the synthesis of the new polymer compound and the provision of a new function and structure, which were difficult to realize in the conventional organic synthesis method, are possible. In addition, in the conventional organic synthetic chemical technique, a manufacturing process requiring multiple reactions can be realized in only one step, and the effect of simplifying the manufacturing process, reducing the cost, and reducing the time required is also expected. It is becoming. In addition, it is possible to reduce the use of organic solvents, acids, alkalis, surfactants, etc., to set mild reaction conditions, to synthesize from non-petroleum-based materials and low-purity raw materials, and to realize a more environmentally-friendly and resource-cycling synthesis process. This becomes possible. In addition, the synthesis from low-purity raw materials will be described in more detail. In the biotechnological synthesis process, since the substrate specificity of enzymes, which are generally catalysts, is high, a low-purity raw material can be used to selectively advance a desired reaction. Therefore, it can be expected to use waste or recycled materials.
[35] On the other hand, as described above, the inventors of the present application have focused on a structure coated with a base material with a polymer compound as an element technology for imparting a large added value to the polymer compound. Thus, by coating a specific substrate with a high molecular compound, a composite structure having extremely useful functionality can be obtained. In order to manufacture the structure as described above, a lot of conventional organic synthesis techniques have been used, but this technique has certain limitations.
[36] If such a structure can be produced by the above-described biotechnological method, it can be expected that the use of a new polymer compound, or the provision of a new function and structure, which has been difficult to realize by the conventional organic synthetic method. In addition, it is thought that the environmental load and the resource recycling type manufacturing process can be realized with a low cost. For example, it is covered with new functional polymer compounds or extremely high chirality polymer compounds, which are difficult to realize in conventional organic synthetic chemical methods by using extremely precise molecular recognition ability and stereoselectivity peculiar to biological catalysis. The encapsulated capsule structure and the laminated structure can be manufactured by a very simple and low environmental load process.
[37] Accordingly, an object of the present invention is to provide a high-performance polymer compound structure that can be produced by a biotechnological method. The present invention also provides an efficient method for producing a structure coated with a substrate by a high molecular compound widely available as a functional composite structure.
[1] BRIEF DESCRIPTION OF THE DRAWINGS The figure which shows the GC-MS analysis result of the shell of the capsule structure in Example 1;
[2] Fig. 2 shows the results of GC-MS analysis of the shell of the capsule structure in Example 4;
[3] Fig. 3 shows the results of GC-MS analysis of the shell of the capsule structure in Example 5;
[4] Fig. 4 is a diagram showing the results of GC-MS analysis of the coating layer of the laminated structure in Example 11;
[38] As a result of earnestly examining by the present inventors in order to achieve the said objective, the PHA synthase was immobilized on the surface of a base material, 3-hydroxyacyl CoA was added to it, and it reacted, synthesize | combining desired PHA on the surface of a base material, and covering a base material with PHA. It has been found that a structure can be obtained and the present invention has been completed. Moreover, it discovered that the structure which improved various characteristics etc. by applying chemical formula to this PHA was obtained. More specifically, it has been found that, for example, by introducing a graft chain into the PHA, a structure having a PHA having various characteristics attributable to the graft chain can be coated with at least a part of the substrate. In addition, it has been found that by crosslinking this PHA, a PHA having desired physicochemical properties (eg, mechanical strength, chemical resistance, heat resistance, etc.) can obtain a structure in which at least a part of the substrate is coated. In addition, the chemical modification in this invention means changing the molecular structure of this polymer material by making a chemical reaction in the molecule | numerator or between molecules of a polymer material, or between a polymer material and another chemical substance. . In addition, crosslinking refers to making a network structure by chemically or physicochemically bonding intramolecular or intermolecular molecules of a polymer material, and a crosslinking agent refers to the polymer material added to perform the crosslinking reaction. It refers to a substance that has a certain reactivity with.
[39] That is, the present invention is a polyhydroxyalkane containing 3-hydroxyalkanoic acid unit (except 3-hydroxypropionic acid unit, 3-hydroxy-n-butyric acid unit or 3-hydroxy-n-yl acetic acid unit). It relates to a structure wherein the canoate covers at least a portion of the substrate.
[40] In addition, the present invention is a medium-chain polyhydroxyalkanoate synthase is immobilized on the surface of the substrate, by polymerizing 3-hydroxyacyl coenzyme A by the enzyme to synthesize at least a part of the substrate It relates to a method for producing a structure, characterized in that the coating with polyhydroxyalkanoate.
[41] In addition, the present invention relates to a capsule structure having a base as a core (core material) and having an outer surface of mcl-PHA or unusual-PHA, and more specifically, a capsule comprising at least a colorant in the core. A structure, a capsule structure in which the colorant contains at least a pigment, or a capsule structure in which the core is a pigment. The present invention also relates to a laminated structure in which at least part of a flat plate or film-like substrate is coated with mcl-PHA or unusual-PHA.
[42] The present invention also relates to an electrophotographic capsule toner comprising the capsule structure or a recording medium comprising the laminated structure.
[43] The present invention also relates to an image forming method and an image forming apparatus using the toner.
[44] (Description of Preferred Embodiments)
[45] The structure of the present invention is a structure having a form coated with a substrate by PHA containing monomer units of various structures having substituents in the side chain, and is extremely useful as a high functional structure such as an electrophotographic capsule toner or a recording medium. The present invention will be described in more detail below.
[46] <PHA>
[47] PHA usable in the present invention is not particularly limited as long as PHA can be synthesized by PHA synthase involved in the synthesis of mcl-PHA (ie, various mcl-PHAs and unusual-PHAs). As described above, the PHA synthetase is an enzyme that catalyzes the final step in the PHA synthesis reaction system in living organisms, so that all PHAs known to be synthesized in living organisms are catalyzed by this enzyme and synthesized. It becomes. Therefore, a structure coated with a substrate of various types of PHAs known to be synthesized in living organisms by acting on this enzyme immobilized on the substrate of the present invention corresponding to the desired PHA is 3-hydroxyacyl CoA. It is possible to write.
[48] As such PHA, PHA containing at least the monomer unit represented by following General formula [1]-[10] can be illustrated specifically ,.
[49]
[50] Wherein the monomer unit is at least one selected from the group consisting of monomer units wherein the combination of R1 and a is any one of the following.
[51] (1) a monomer unit wherein R1 is a hydrogen atom (H) and a is any of an integer of 3 to 10;
[52] (2) a monomer unit wherein R1 is a halogen atom (H) and a is any of integers from 1 to 10;
[53] (3) a monomer unit wherein R1 is a chromophore and a is any one of integers from 1 to 10;
[54] (4) a monomer unit wherein R1 is a carboxy group or a salt thereof and a is any one of integers from 1 to 10;
[55] (5) R1 is
[56]
[57] And a is any one of integers of 1-7.
[58]
[59] B represents any one of integers from 0 to 7, and R2 is composed of a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F ₇ Any one selected from the group is displayed.
[60]
[61] Where c represents any one of integers from 1 to 8, and R3 is composed of a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F ₇ Any one selected from the group is displayed.
[62]
[63] Where d represents any one of integers from 0 to 7, and R4 is hydrogen atom (H), halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F ₇ Any one selected from the group is displayed.
[64]
[65] Where e represents any one of integers from 1 to 8, R5 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F ₇ ,- CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ Any one selected from the group consisting of.
[66]
[67] F represents one of integers of 0-7 here.
[68]
[69] G represents any one of the integers of 0-7 here.
[70]
[71] Where h represents any one of integers from 1 to 7, and R6 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -COOR ', -SO ₂ R ", -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ , -CH (CH ₃ ) ₂ , -C (CH ₃ ) ₃ represents any one selected from the group consisting of R 'is a hydrogen atom (H), Na, K,- CH ₃ , and -C ₂ H ₅ , and R "is -OH, -ONa, -OK, a halogen atom, -OCH ₃ , -OC ₂ H ₅ .
[72]
[73] I represents any one of integers of 1 to 7, and R7 represents any one selected from the group consisting of a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -COOR ', -SO ₂ R " Where R 'is one of hydrogen atom (H), Na, K, -CH ₃ , and -C ₂ H ₅ , and R "is -OH, -ONa, -OK, halogen atom, -OCH ₃ , -OC ₂ H ₅
[74]
[75] Here, j represents any one of the integers of 1-9.
[76] Specific examples of the halogen atom include fluorine, chlorine and cancellation. In addition, the chromophore is not particularly limited as long as the 3-hydroxyacyl CoA body can be catalyzed by the PHA synthetase, but considering the steric hindrance during polymer synthesis, the 3-hydroxyacyl CoA molecule It is preferable to have a methylene chain having 1 to 5 carbon atoms between the carboxyl group to which CoA is bonded and the chromophore in the compound. In addition, when the light absorption wavelength of the chromophore is in the visible range, a colored structure can be obtained, and even if there is a light absorption wavelength in addition to the visible range, it can be used as various electronic materials. Examples of such chromophores are nitroso, nitro, azo, diarylmethane, triarylmethane, chianthene, acridine, quinoline, methine, thiazole, indamine, indophenol, lactones, aminoketones, hydroxyketones, Stilbene, azine, oxazine, thiazine, anthraquinone, phthalocyanine, indigoid, and the like.
[77] As the PHA used in the present invention, it is also possible to use a random copolymer or a block copolymer containing a plurality of the above monomer units, and to control the physical properties of the PHA using the properties of each monomer unit or the functional group included and to impart a plurality of functions. In addition, the expression of new functions using interactions between functional groups is possible.
[78] Further, by changing the composition, such as the type and concentration of 3-hydroxyacyl CoA as a substrate over time, the monomer unit of the PHA in the direction from the inside to the outside when the structure is granular, or in the vertical direction when the structure is planar. It is also possible to change the composition.
[79] Thus, for example, in the case of capsule toner, PHA having a high glass transition temperature is formed on the toner surface layer, and PHA having a low glass transition temperature is formed on the inner layer, thereby providing excellent blocking resistance during storage and low temperature fixing during fixing. It is possible to hold a plurality of functions such as excellent performance.
[80] For example, when it is necessary to form a coating structure with PHA having a low affinity with a substrate, the substrate is first coated with PHA having a high affinity with the substrate, and the monomer unit composition of the PHA having a high affinity with the substrate, By changing the monomer unit composition of the desired PHA in the direction from the inside to the outside or in the vertical direction, for example, the multilayer structure or the gradient structure, it is possible to form a PHA film having a strong bond with the substrate. Done.
[81] For example, when it is necessary to form a coating structure with PHA having a low affinity with a substrate, the substrate is first coated with PHA having a high affinity with the substrate, and the monomer unit composition of the PHA having a high affinity with the substrate, By changing the monomer unit composition of the desired PHA in the direction from the inside to the outside or in the vertical direction, for example, the multilayer structure or the gradient structure, it is possible to form a PHA film having a strong bond with the substrate. .
[82] In addition, 3-hydroxypropionic acid unit, 3-hydroxy-n-butyric acid unit, 3-hydroxy-n-acetic acid unit, 4-hydroxy-n-butyric acid unit, and the like, mcl-PHA or Although not applicable to unusual-PHA, PHAs in which these monomer units are mixed in the monomer units as exemplified above can be used in the present invention. Moreover, you may perform chemical formula etc. further after synthesize | combining PHA as needed or during synthesis. It is preferable that the molecular weight of PHA is about 10 to 10 million in number average molecular weight, for example, when the structure is used as an electrophotographic capsule toner, about 30 to 1 million.
[83] In addition, PHA synthesize | combined with the PHA synthetase used for the structure of this invention is an isotactic polymer generally comprised only by R form.
[84] <3-hydroxyacyl CoA>
[85] As 3-hydroxyacyl CoA used as a substrate of the PHA synthase of the present invention, specifically, 3-hydroxyacyl CoA represented by the following formulas [11] to [20] can be exemplified.
[86]
[87] (Wherein -SCoA represents at least one coenzyme A bound to an alkanoic acid, wherein the combination of R1 and a is at least one selected from the group below, and is a monomer represented by the above formula [1] Corresponds to R1 and a in the unit, where R1 is a hydrogen atom (H) and a is a monomer unit of any of 3 to 10, and R1 is a halogen atom and a is a monomer of any of 1 to 10. A monomer unit wherein R1 is a chromophore and a is any one of integers from 1 to 10, a monomer unit wherein R1 is a carboxyl group or a salt thereof and a is any of integers from 1 to 10, R1 and a is an integer from 1 to 7 Any one of the monomer units.)
[88]
[89] (Wherein -SCoA represents coenzyme A bound to alkanoic acid,
[90] b represents one of the integers of 0-7 corresponding to b in the monomer unit represented by the said Formula [2], and R2 corresponds to R2 in the monomer unit represented by the said Formula [2] , Hydrogen atom (H), halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F _{7 It} represents any one selected from the group consisting of.
[91]
[92] (Wherein -SCoA represents coenzyme A bound to alkanoic acid,
[93] c represents any one of integers 1 to 8 corresponding to c in the monomer unit represented by the formula [3], and R3 corresponds to R3 in the monomer unit represented by the formula [3]. , Hydrogen atom (H), halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F _{7 It} represents any one selected from the group consisting of.
[94]
[95] (Wherein -SCoA represents coenzyme A bound to alkanoic acid,
[96] d represents any one of the integers of 0-7 corresponding to in the monomer unit represented by the said Formula [4], and R4 corresponds to R4 in the monomer unit represented by the said Formula [4], Any one selected from the group consisting of a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F ₇ is indicated.
[97]
[98] (Wherein -SCoA represents coenzyme A bound to alkanoic acid,
[99] e represents any of the integers 1-8 corresponding to e in the monomer unit represented by the said Formula [5], and R5 corresponds to R5 in the monomer unit represented by the said Formula [5] , Hydrogen atom (H), halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F _7, -CH ₃ , -C ₂ H _5, -C ₃ H ₇ Any one selected from the group is displayed.
[100]
[101] (Wherein -SCoA represents coenzyme A bound to alkanoic acid,
[102] f represents any one of the integers of 0-7 corresponding to f in the monomer unit shown by the said Formula [6].
[103]
[104] (Wherein -SCoA represents coenzyme A bound to alkanoic acid,
[105] g represents any one of the integers of 1-8 corresponding to g in the monomer unit represented by the said Formula [7]. ₂
[106]
[107] (Wherein -SCoA represents coenzyme A bound to alkanoic acid,
[108] h represents any of the integers 1-7 corresponding to h in the monomer unit represented by the said Formula [8], and R6 corresponds to R6 in the monomer unit represented by the said Formula [8] , Hydrogen atom (H), halogen atom, -CN, -NO ₂ , -COOR ', -SO ₂ R ”, -CH ₃ , -C ₂ H _5, -C ₃ H _7, -CH (CH ₃ ) ₂ , -C (CH ₃ ) ₃ represents any one selected from the group consisting of
[109] Where R 'is one of hydrogen atom (H), Na, K, -CH ₃ , -C ₂ H ₅ , and R "is -OH, -ONa, -OK, halogen atom, -OCH ₃ , -OC ₂ Is any one of H _5. )
[110]
[111] (Wherein -SCoA represents coenzyme A bound to alkanoic acid,
[112] i represents any of the integers 1-7 corresponding to i in the monomer unit represented by the said Formula [9], and R7 corresponds to R7 in the monomer unit represented by the said Formula [9] , Hydrogen atom (H), halogen atom, -CN, -NO ₂ , -COOR ', -SO ₂ R "represents any one selected from the group consisting of,
[113] Where R 'is one of hydrogen atom (H), Na, K, -CH ₃ , -C ₂ H ₅ , and R "is -OH, -ONa, -OK, halogen atom, -OCH ₃ , -OC ₂ Is any one of H _5. )
[114]
[115] (Wherein -SCoA represents coenzyme A bound to alkanoic acid,
[116] j represents any one of integers 1 to 9 corresponding to j in the monomer unit represented by the above formula [10].)
[117] These 3-hydroxyacyl CoA can be synthesize | combined and used by the method suitably chosen from the in vitro synthesis method using an enzyme, the in vivo synthesis method using organisms, such as microorganisms and plants, and a chemical synthesis method, for example. In particular, the enzyme synthesis method is a method generally used for the synthesis of the substrate, the following reaction using a commercially available acyl CoA synthase (acyl CoA ligase E, C, 6, 2, 1, 3),
[118] Acyl CoA Synthetase
[119] A method using 3-hydroxyalkanoic acid = CoA-> 3-hydroxyacyl CoA, etc. is known (Eur. J. Biochem., 250, 432-439 (1997), Appl. Microbiol. Biotechnol., 54, 37- 43 (2000) et al. In a synthetic step using an enzyme or a living organism, a batch synthetic method may be used, or may be continuously produced using immobilized enzymes or immobilized cells.
[120] <PHA synthase and its producing bacteria>
[121] The PHA synthetase used in the present invention can be one produced by a microorganism suitably selected from the microorganisms producing the enzyme or by a transformant into which the PHA synthase gene of these microorganisms is introduced.
[122] As microorganisms producing PHA synthetase, for example, mcl-PHA or unusual-PHA-producing bacteria can be used, and as such microorganisms, Pseudomonas oleoboranth, Pseudomonas redinoborane, Pseudomonas genus 61 -3 weeks, Pseudomonas putida P91 strain (Pseudomonas putida P91), Pseudomonas chiteria, H45 strain separated by the present inventors other than Pseudomonas putida KT2442 strain, Pseudomonas aelginosa, etc. Pseudomonas cichorii H45), Pseudomonas cichorii YN2 strain (Pseudomonas cichorii YN2), Pseudomonas jeseni. Microorganisms of the genus Burkholderia, such as the genus Burkholderia sp.OK3, FERM 9-17370, and the Burkholderia sp.OK4 strain (Burkholderia sp.OK4, FERM 9-17371) described in Japanese Patent Application Laid-Open No. 2001-69968. Can be used. Subjected to biological, ah in Monastir (Aeromonas sp.), It is possible that in such a coma in Monastir (Comamonas sp.), And the use of microorganisms to produce mcl-PHA and unusual-PHA in the.
[123] In addition, P91 shares the accession number FERM BP-7373, H45 shares the accession number FERM BP-7374, YN2 the accession number FERM BP-7375, P161 the accession number FERM BP-7376, and the international approval of the deposit of microorganisms in the patent procedure In accordance with the Butafest Treaty, the Institute has been deposited internationally at the Institute of Patented Biotechnology of the Institute of Industrial Technology (Industrial Industrial Technology Research Institute (formerly Tongsan Industrial Technology Research Institute)). .
[124] In addition, the above-mentioned bacteriological properties of the P91, H45, YN2 and P161 strains are as follows. For the P161 strain, the nucleotide sequence of 16S rRNA is shown in SEQ ID NO: 1.
[125] (Bacterial Properties of Pseudomonas, Putida and P91)
[126] (1) morphological properties
[127] Shape and size of cells: rod fungus, 0.1㎛ × 1.5㎛
[128] Cell Polymorphism: None
[129] Mobility: Yes
[130] Spore Formation: None
[131] Gram Dyeing: Negative
[132] Colony geometry
[133] : Round, smooth all edges, low convex shape,
[134] Surface is smooth, glossy, creamy.
[135] (2) physiological properties
[136] Catalase: positive
[137] Oxidase: Positive
[138] O / F test: Oxidation type
[139] Reduction of Nitrate: Negative
[140] Generation of indole: voice
[141] D-glucoacidification: negative
[142] Arginine dihydrolase: positive
[143] Urease: negative
[144] Esculin Hydrolysis: Negative
[145] Gelatin Hydrolysis: Negative
[146] β-galactosidase: negative
[147] Fluorescent Pigment Production in King's B Agar: Positive
[148] (3) substrate fairy tale capacity
[149] D-glucose: positive
[150] L-Arabinose: Voice
[151] D-Mannose: Voice
[152] D-mannitol: negative
[153] N-acetyl-D-glucosamine: negative
[154] Maltose: voice
[155] Potassium Gluconate: Positive
[156] n-capric acid: positive
[157] Adipic acid: negative
[158] DL-malic acid: positive
[159] Sodium citrate: positive
[160] Phenyl Acetate: Positive
[161] (Pseudomonas CHI KOREA-H45 strains)
[162] (1) morphological properties
[163] Shape and size of cells: rods, 0.8 μm × 1.0 to 1.2 μm
[164] Cell Polymorphism: None
[165] Mobility: Yes
[166] Sporulation: None
[167] Gram Dyeing: Negative
[168] Colony shape: round, smooth all edges, low convex shape,
[169] Smooth, glossy, creamy surface
[170] (2) physiological properties
[171] Catalase: positive
[172] Oxidase: Positive
[173] O / F test: Oxidation type
[174] Reduction of Nitrate: Negative
[175] Generation of indole: voice
[176] D-glucoacidification: negative
[177] Arginine dihydrolase: negative
[178] Urease: negative
[179] Esculin Hydrolysis: Negative
[180] Gelatin Hydrolysis: Negative
[181] β-galactosidase: negative
[182] Fluorescent Pigment Production in King's B Agar: Positive
[183] Growth in 4% NaCl: negative
[184] Accumulation of poly-β-hydroxybutyric acid: negative
[185] (3) substrate fairy tale capacity
[186] D-glucose: positive
[187] L-Arabinose: Voice
[188] D-Mannose: Positive
[189] D-mannitol: positive
[190] N-acetyl-D-glucosamine: positive
[191] Maltose: Voice
[192] Potassium Gluconate: Positive
[193] n-capric acid: positive
[194] Adipic acid: negative
[195] dl-malic acid: positive
[196] Sodium citrate: positive
[197] Phenyl Acetate: Positive
[198] (Pseudomonas chi korea has YN2 strains)
[199] (1) morphological properties
[200] Shape and size of cells: rods, 0.8 μm × 1.5 to 2.0 μm
[201] Cell Polymorphism: None
[202] Mobility: Yes
[203] Sporulation: None
[204] Gram Dyeing: Negative
[205] Colony shape: round, smooth all edges, low convex shape,
[206] Surfaces are smooth, glossy, and translucent
[207] (2) physiological properties
[208] Catalase: positive
[209] Oxidase: Positive
[210] O / F test: Oxidation type
[211] Reduction of Nitrate: Negative
[212] Generation of indole: positive
[213] D-glucoacidification: negative
[214] Arginine dihydrolase: negative
[215] Gelatin Hydrolysis: Negative
[216] β-galactosidase: negative
[217] Fluorescent Pigment Production in King's B Agar: Positive
[218] Growth at 4% NaCl: Positive (weak growth)
[219] Accumulation of poly-β-hydroxybutyric acid: negative
[220] Hydrolysis of Tween 80: Positive
[221] (3) substrate fairy tale capacity
[222] D-glucose: positive
[223] L-arabinose: positive
[224] D-Mannose: Voice
[225] D-mannitol: negative
[226] N-acetyl-D-glucosamine: negative
[227] Maltose: Voice
[228] Potassium Gluconate: Positive
[229] n-capric acid: positive
[230] Idipic acid: voice
[231] dl-malic acid: positive
[232] Sodium citrate: positive
[233] Phenyl acetate; positivity
[234] (Bacterial properties of Pseudomonas, cessini, P161 strain)
[235] (1) morphological properties
[236] Cell shape and size: spherical, 0.6 μm in diameter,
[237] Normal balance, 0.6 μm × 1.5 to 2.0 μm
[238] Cell polymorphism: Yes (kidney type)
[239] Mobility: Yes
[240] Sporulation: None
[241] Gram Dyeing: Negative
[242] Colony shape: round, smooth all edges, low convex shape,
[243] Smooth surface, pale yellow
[244] (2) physiological properties
[245] Catalase: positive
[246] Oxidase: Positive
[247] O / F test: Oxidation type
[248] Reduction of nitrates: positive
[249] Generation of indole: voice
[250] Arginine dihydrolase: positive
[251] Urease: negative
[252] Esculin Hydrolysis: Negative
[253] Gelatin Hydrolysis: Negative
[254] β-galactosidase: negative
[255] Fluorescent Pigment Production in King's B Agar: Positive
[256] (3) substrate fairy tale capacity
[257] D-glucose: positive
[258] L-arabinose: positive
[259] D-Mannose: Positive
[260] D-mannitol: positive
[261] N-acetyl-D-glucosamine: positive
[262] Maltose: Voice
[263] Potassium Gluconate: Positive
[264] n-capric acid: positive
[265] Adipic acid: negative
[266] dl-malic acid: positive
[267] Sodium citrate: positive
[268] Phenyl Acetate: Positive
[269] For normal culture of microorganisms used in the production of PHA synthase according to the present invention, for example, preparation of a conserved strain, propagation for securing the number of bacteria required for the production of PHA synthase and the active state, etc. A medium containing a component necessary for the growth of microorganisms is appropriately selected and used. For example, any kind of medium may be used, such as general natural medium (exclamation medium, yeast extract, etc.), or synthetic medium containing nutrient sources, as long as it does not adversely affect the growth or survival of microorganisms.
[270] The culture may be any method as long as the microorganism is propagated, such as liquid culture or solid culture. In addition, batch cultures, feed batch cultures, semi-continuous cultures, continuous cultures, and the like can also be used. As a form of liquid batch culture, there are a method of supplying oxygen by shaking with a shaking flask and a method of supplying oxygen in a stirred aeration system by a japper. Moreover, you may employ | adopt the multistage system which connected these processes in multiple stages.
[271] When PHA synthase is produced using the above-described PHA producing microorganisms, for example, the microorganism is propagated in an inorganic medium containing alkanoic acid such as octanoic acid or nonanoic acid, and from the logarithmic growth phase to the beginning of the normal phase. The microorganisms can be recovered by centrifugation or the like to extract a desired enzyme. In addition, when cultured under the above conditions, mcl-PHA derived from the added alkanoic acid is synthesized in the cells, but in this case, in general, PHA synthase binds to the fine particles of PHA formed in the cells. It is supposed to be. However, studies by the present inventors have found that a considerable amount of enzymatic activity also exists in the supernatant obtained by centrifugation of the lysate of cells cultured by the above method. This is presumably because the PHA synthase in the free state is also present in a relatively early stage of culture from the logarithmic growth phase as described above, since the enzyme is actively produced in the cells.
[272] The inorganic medium to be used in the above cultivation method may be any material as long as it contains components capable of proliferating microorganisms such as human body (for example, phosphate) and nitrogen source (for example, ammonium salt and nitrate). Examples of the inorganic salt medium include MSB medium, E medium (J. Biol, chem, 218, 97-106 (1956), M9 medium, etc.). The composition of M9 medium is as follows.
[273] Na ₂ HPO ₄ : 6.2 g
[274] KH ₂ PO ₄ : 3.0g
[275] NaCl: 0.5g
[276] NH ₄ Cl: 1.0 g
[277] (PH 7.0 in 1L medium)
[278] In addition, for good growth and production of PHA synthase, it is preferable to add about 0.3% (v / v) of the small amount component solution shown below to the inorganic salt medium.
[279] (Small amount component solution)
[280] Nitrile to acetic acid: 1.5 g
[281] MgSO ₄ : 3.0g
[282] MnSO ₄ : 0.5g
[283] NaCl: 1.0g
[284] FeSO ₄ : 0.1g
[285] CaCl ₂ : 0.1g
[286] CoCl ₂ : 0.1g
[287] ZnSO ₄ : 0.1g
[288] CuSO ₄ : 0.1g
[289] AIK (SO ₄ ) ₂ : 0.1 g
[290] H ₃ BO ₃ : 0.1g
[291] Na ₂ MoO ₄ : 0.1g
[292] NiCl ₂ : 0.1g
[293] (Out of 1ℓ)
[294] The culture temperature may be any temperature at which the strain can be satisfactorily propagated, for example, 14 to 40 ° C, preferably about 20 to 35 ° C.
[295] It is also possible to produce a desired PHA synthetase by using a transformant having a PHA synthase gene introduced into the PHA-producing bacterium. Cloning of the PHA synthase gene, preparation of the expression vector, and preparation of the transformant can be performed according to a conventional method. In transformants obtained by using bacteria such as Escherichia coli as a host, natural or synthetic media such as LB medium and M9 medium may be used as a culture medium. In addition, the culture temperature is in the range of 25 to 37 ° C., and microbial growth is achieved by incubating aerobicly for 8 to 27 hours. Thereafter, the cells are collected and the PHA synthase accumulated in the cells can be recovered. If necessary, antibiotics such as kanamycin, ampicillin, tetracycline, chloramphenicol and streptomycin may be added to the medium. In the case of using an inducible promoter in an expression vector, when culturing the transformant, the promoter may be added to the medium by inducing a corresponding inducer, thereby prompting expression. For example, isopropyl- (beta) -D- thiogalacnopyranoside (IPTG), tetracycline, indole acrylic acid (IAA), etc. are mentioned as an inducer.
[296] As PHA synthetase, a crude enzyme such as microbial cell lysate or sulphate salts in which protein components are precipitated and recovered by ammonium sulfate or the like may be used, or purified enzymes purified by various methods may be used. If necessary, stabilizers and activators such as metal salts, glycerin, dithiositol, and EDTA bovine serum albumin (BSA) can be appropriately added to the enzyme.
[297] As a method for isolating and purifying the PHA synthetase, any method can be used as long as the enzyme activity of the PHA synthase is maintained. For example, after breaking up the obtained microbial cells using a French press, an ultrasonic crusher, a lysozyme or various surfactants, etc., to the defect enzyme solution obtained by centrifugation or the sulphate prepared from here, affinity Purified enzyme can be obtained by combining single or appropriate means, such as chromatography, cation or anion exchange chromatography, and gel filtration. In particular, the recombinant protein can be more easily purified by expressing it in the form of a fusion protein in which the [tag] such as histigin residues are bound to the N- and C-terminals, and binding to the affinity resin via this tag. In order to separate the target protein from the fusion protein, a method such as adding a high concentration of imidazole as a binding competing agent that lowers the pH by cutting with proteases such as thronbin and blood coagulation factor Xa may be used. Alternatively, when the tag contains intein as in the case of using pTYBI (manufactured by New Englan Biolab) as an expression vector, the tag is cleaved with dithiothreitol or the like as reducing conditions. Fusion proteins that allow purification by affinity chromatography include glutathione S-transferase (GST), kitchen binding domain (CBD), martose binding protein (MBP), or thioredoxin (TRX) in addition to histigene tags. ) Is also known. GST fusion protein can be refine | purified by GST affinity resin.
[298] For the activity measurement of PHA synthase, various methods of notation can be used. For example, CoA released in the process of 3-hydroxy acyl CoA polymerized by PHA synthase and becomes PHA, 5, It can measure according to the method shown below which makes it a coloring principle and color-measures with 5'-dithiobis (2-nitro benzoic acid), and measures it. Reagent 1: Dissolve 3.0 mg / ml in bovine serum albumin (manufactured by Sigma) in 0.1 M Tris hydrochloric acid buffer (pH 8. 0), and Reagent 2: 3 -Hydroxyoctanoyl CoA in 0.1 M Tris hydrochloric acid buffer Dissolve 3.0 mM in pH 8.0, Dissolve 3 mg trichloroacetic acid in 10 mg / ml in 0.1 M Tris hydrochloric acid buffer, pH 8. 0, Reagent 4: 5,5'-dithiobis (2- / Nitrobenzoic acid) was dissolved in 0.1 M Tris hydrochloric acid buffer (pH 8. 0), and the first reaction (PHA synthesis reaction): 100 μl of reagent 1 was added to 100 μl of the sample (enzyme) solution, and mixed at 30 ° C. Plane incubate for 1 min. 100 μl of reagent 2 is added thereto, mixed, incubated at 30 ° C. for 1 to 30 minutes, and then reagent 3 is added to stop the reaction. Second reaction (color reaction of free CoA): The reaction mixture was centrifuged (15,000 x g, 10 minutes), and 500 µl of reagent 4 was added to 500 µl of this supernatant, and incubated at 30 ° C for 10 minutes. Then, the absorbance of 412 nm is measured. Calculation of enzyme activity: The amount of enzyme that releases 1 μmol of CoA in 1 minute is 1 unit (U).
[299] In addition, PHA synthesize | combined by the said enzyme is an isotactic polymer generally comprised only by R body.
[300] <Base material>
[301] As a base material used by the method of this invention, if a PHA synthetase can be immobilized, it can select suitably from a general high molecular compound, an inorganic solid substance, for example, resin, glass, a metal, etc., and can use. Moreover, the kind and structure of a base material can be suitably selected and used according to the immobilization method of PHA synthase, the form of application of the produced structure, etc.
[302] For example, styrene, α-methylstyrene, β-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4 dimethyl styrene, as a base material (core) in the capsule structure of the present invention, styrenic polymerizable monomers such as pn-butyl styrene, p-tert butyl styrene, pn-hexyl styrene, pn-octyl styrene, pn-tesy styrene, pn-dodecyl styrene, p-methoxy styrene, p-phenyl styrene, Methyl acrylate, ethyl acrylate, n-propyl acrylate, iso-propyl acrylate, n-butyl acrylate, iso-butyl acrylate, tert-butyl acrylate, n-amyl acrylate, n-hexyl acrylate, 2-ethyl hexyl acrylate, n-octyl acrylate, n-nonyl acrylate,
[303] Acrylic polymerizable monomers such as cyclohexyl acrylate, benzyl acrylate, dimethyl phosphate ethyl acrylate, diethyl phosphate ethyl acrylate, dibutyl phosphate ethyl acrylate, 2-benzoyloxyethyl acrylate, methyl methacrylate, Ethyl methacrylate, n-propyl methacrylate, iso-propyl methacrylate, n-butyl remethacrylate, iso-butyl methacrylate, tert-butyl methacrylate, n-amyl methacrylate, n-hexyl Methacrylic polymerizable monomers, such as methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, n-nonyl methacrylate, diethyl phosphate ethyl acrylate, and dibutyl phosphate ethyl methacrylate, and methylene Aliphatic monocarboxylic acid esters, vinyl acetate, vinyl propionate, vinyl benzoate, vinyl butyrate, benzo Vinyl polymerization such as vinyl esters such as vinyl acid and vinyl formate, vinyl ethers such as vinyl methyl ether, vinyl ethyl ether and vinyl isobutyl ether, and vinyl ketones such as vinyl methyl ketone, vinyl hexyl ketone and vinyl isopropyl ketone Resin fine particles prepared by polymerizing a polymerizable monomer selected from the group consisting of a monomer, or resin fine particles prepared by adding various additives such as a polar group polymer or a coloring agent to the monomer system, paraffin wax, polyolefin wax, or fitschatte Fine particles containing fischer wax, amide wax, higher fatty acid, ester wax and derivatives thereof or graft or block compounds thereof, clay minerals such as kaolinite, bentonite, talc, mica, metal oxides such as alumina and titanium dioxide Insoluble inorganic salts, such as silica gel, hydroxyapatite and calcium phosphate gel Black pigments such as rack, copper oxide, manganese dioxide, aniline black, activated carbon, nonmagnetic ferrite, magnetite, sulfur lead, zinc sulfur, yellow iron oxide, cadmium yellow, mineral first yellow, nickel titanium yellow, naive yellow, naphthol yellow S, Chinese characters Yellow G, Kanji Yellow 10G, Benjijin Yellow G, Benjijin Yellow GR, Quinoline Yellow Lake, Permanent Yellow NCG, Tatrazin Lake, etc. Orange pigments such as Benzine Orange G, Indus Ren Brilliant Orange RK, Indus Ren Brilliant Orange GK,
[304] Ben Color, Cadmium Red Podium, Mercury Sulfide, Cadmium, Permanent Red 4R, Resolred, Pyrazorone Red, Watching Red, Calcium Salt, Lake Red C, Lake Red D, Brilliant Carmine 6B, Brilliant Carmine 3B, Eokisin Lake, Roda Red pigments such as Minlake B and Alizarin Lake, blue pigments such as wire blue, cobalt blue, alkali blue lake, Victoria blue lake, phthalocyanine blue, metal-free phthalocyanine mlulu, phthalocyanine blue, partial chlorine compounds, first sky blue, and indusren blue BC Purple pigments such as manganese purple, first violet B, methyl violet ake, green pigments such as chromium oxide green, pigment green B, marachite green latex, final yellow green G, zinc oxide, titanium oxide, antimony white, zinc sulfide, etc. White pigment, barita powder, barium carbonate, mud, silica, white carbon, talc, alumina But they not used, and extender pigments including, of course, not limited to these. Although the shape of a core can be selected suitably according to the use, For example, what is necessary is just to use the particle | grains which have a particle diameter in the range of 0.1 micrometer-1.0 mm of particle diameters. In the case where the structure is used as a capsule toner for electrophotography, the particle diameter may be selected within a range of 3.0 µm to 10 µm.
[305] Moreover, as a base material of the laminated structure of this invention, polyethylene terephthalate (PET), diacetate, tri acetate, cellophane, celluloid, polycarbonate, polyimide, polyvinyl chloride, polyvinylidene chloride, polyacrylate , Film made of plastic such as polyethylene, polypropylene, polyester, polyvinyl chloride, polyvinyl alcohol, acetyl cellulose, polycarbonate, nylon, polypropylene, polyethylene, Teflon, etc. Paper such as cotton, rayon, acrylic, silk, polyester, fine paper, solid paper, art paper, bond paper, recycled paper, variety paper, cast coated paper, corrugated paper, resin coated paper, etc. can be used, of course, these It is not limited to. Moreover, the surface of the said base material may be smooth, a thing with unevenness | corrugation may be sufficient, and any of transparent, translucent, and opaque may be sufficient. Moreover, what bonded together two or more types among these base materials may be sufficient.
[306] <Production of structure>
[307] The manufacturing method of the structure of this invention includes the process of synthesize | combining PHA by making 3-hydroxy acyl CoA react with PHA synthetic enzyme to a base material.
[308] As a method of immobilizing the PHA synthetase on the substrate, the activity of the enzyme can be maintained and, if applicable to the desired substrate, it can be arbitrarily selected and used among the enzyme immobilization methods which are usually performed. For example, a covalent bonding method, an ion adsorption method, a hydrophobic adsorption method, a physical adsorption method, an affinity adsorption method, a crosslinking method, a lattice encapsulation method, etc. can be illustrated, but the immobilization method using ion adsorption or hydrophobic adsorption is especially easy.
[309] Enzyme proteins such as PHA synthetase are polypeptides in which a large number of amino acids are bound and exhibit properties as an ion adsorbent by amino acids having free ionic groups such as lysine, histidine, arginine, aspartic acid, glutamic acid, and alanine, varin, It is based on amino acids having free hydrophobic groups such as leucine, isoleucine, methionine, tryptophan, phenylalanine, and proin, and also has properties as a hydrophobic adsorbent in terms of organic polymers. Therefore, although there is a difference in degree, it is possible to adsorb to a solid surface having ionic, hydrophobic, or both ionic and hydrophobic properties.
[310] In the method of immobilizing PHA synthase mainly by ion adsorption, a core expressing an ionic functional group may be used. For example, clay minerals such as kaolinite, bentite, talc, mica, alumina, and dioxide Insoluble inorganic salts, such as metal oxides, such as titanium, a silica gel, hydroxy apatite, and a calcium phosphate gel, etc. can be used as a core. In addition, polymers having ionic functional groups, such as inorganic pigments, ion exchange resins, chitosan, and polyaminophosphesterene, which are the main components thereof, can also be used as the ion-adsorbing core.
[311] Moreover, in the method of immobilizing PHA synthase mainly by hydrophobic adsorption, what is necessary is just to use a nonpolar core, for example, a styrene polymer, an acryl polymer, a methacryl polymer, vinyl esters, a vinyl polymer, etc. Many polymers which do not express an ionic functional group on the surface or express a hydrophobic functional group on the surface can be used as the core. Organic pigments such as an azo pigment having a plurality of aromatic rings, a phthalocyanine pigment of a condensed polycyclic ring, an anthraquinone pigment, carbon black and the like are hydrophobic adsorptive.
[312] Immobilization of the PHA synthase to the core by the ion adsorption method or the hydrophobic adsorption method is achieved by mixing the enzyme and the core in a predetermined reaction solution.
[313] At this time, it is preferable to shake or stir the reaction vessel to an appropriate strength so that the enzyme is evenly adsorbed on the surface of the core.
[314] As the pH, salt concentration, and temperature of the reaction solution change the charge, the amount of charge, and the hydrophobicity of the surface charges of the core and PHA synthetase, the adjustment of the solution within the allowable range of enzyme activity depends on the nature of the core used. It is preferable to carry out. For example, if the core is predominantly ion adsorbent, by lowering the salt concentration. The amount of charge that contributes to the adsorption of the core with the PHA synthase can be increased. In addition, by changing the pH, the opposite charges of both can be increased.
[315] If the core is mainly hydrophobic, the hydrophobicity of both can be increased by increasing the salt concentration. In addition, it is also possible to set the solution conditions suitable for adsorption by measuring electrophoresis, small angles, etc. in advance, and examining the charged state and hydrophobicity of the core and the PHA synthase. In addition, the adsorption amount between the core and the PHA synthase may be directly measured to request the conditions. The measurement of the adsorption amount is
[316] For example,
[317] After adding a concentration known PHA synthase solution to the solution in which the core is dispersed, and performing the adsorption treatment, a method of measuring the concentration of the PHA synthetase in the solution and determining the amount of adsorption enzyme by the difference method may be used.
[318] In the case of the core material which is difficult to immobilize the enzyme by the ion adsorption method or the hydrophobic adsorption method, the covalent bonding method may be used in consideration of the complicated operation and the possibility of deactivation of the enzyme. For example, the method which diazotizes the solid particle which has an aromatic amino group, and diazo couples an enzyme to this, the method of forming a peptide bond between the solid particle which has a carboxyl group and an amino group, and an enzyme, the solid which has a halogen group Alkylation between the particle and the amino group of the enzyme, method of reacting the polysaccharide particles activated with cyanide bromide with the amino group of the enzyme, crosslinking between the amino group of the solid particles and the amino group of the enzyme, aldehyde group or ketone And a method of reacting a solid particle having a carboxyl group and an amino group with an enzyme in the presence of a compound having a group and an isocyanide compound, and a method of exchanging reaction between a solid particle having a disulfide group and a thiol group of the enzyme.
[319] Moreover, you may adsorb | suck to solid particle by affinity adsorption. Affinity adsorption is biological adsorption between a biopolymer and a specific substance called a ligand, which has a specific affinity for it, for example, information substances such as enzymes and substrates, antibodies and hinges, receptors and acetylcholine, mRNA and tRNA. In general, as a method of immobilizing enzymes using affinity adsorption, an enzyme substrate, a reaction product, an antagonist, a coenzyme, an allosteric effector, and the like are bound to a solid as a ligand, and the enzyme is added to the solid to form affinity. A method of adsorption is taken. However, for PHA synthetase, for example, when 3-hydroxyacylCoA, which is a substrate, is used as a ligand, PHA is blocked because the active site catalyzing the synthesis of PHA of the enzyme is blocked by the binding to the ligand. The problem of not being able to synthesize occurs. However, other biopolymers can be fused to PHA synthetase and the ligand of the biopolymer can be used for affinity adsorption to maintain the activity of the PHA synthetase even after immobilization. The fusion of the PHA synthetase with the biopolymer may be performed by a genetic engineering technique or may be chemically bound to the PHA synthetase. As the biopolymer to be used, any ligand can be used as long as the corresponding ligand is readily available and the ligand can be easily bound to the core. However, when the fusion is expressed by gene recombination, the protein is preferably a protein. Specifically, using a Escherichia coli linking the gene sequence of the PHA synthetase to the right electron sequence expressing GST by transformation, a fusion protein of GST and PHA synthetase is produced, and this is Sepharose which binds glutathione which is a ligand of GST. By the addition, PHA synthase can be affinity adsorbed onto Sepharose.
[320] In addition, a peptide containing an amino acid sequence having a binding ability to a substrate is fused to a PHA synthetase, and PHA synthesis is performed on the surface of the substrate based on the binding property between the peptide portion of the amino acid sequence having a binding ability to the substrate and the substrate. The enzyme can also be immobilized.
[321] The amino acid sequence having the binding ability to the substrate can be determined by screening a random peptide library, for example. In particular, a phage display peptide library prepared by linking a random synthetic gene to the N-terminal gene of the surface protein (for example, gene III protein) of M13 phage can be suitably used. To determine the amino acid sequence, follow these steps: In other words, the substrate or at least one component constituting the substrate is brought into contact with the addition of phage dispeptide dry brush to separate the bound phage and the unbound phage by washing. The substrate-binding phage is eluted with an acid or the like, neutralized with buffer, infected with E. coli, and amplified. Repeating this selection several times concentrates a plurality of clones having binding capacity to the target substrate. Here, colonies are made on the medium plate with E. coli again to obtain a single clone. After culturing each single colony in a liquid medium, the phages present in the medium supernatant are precipitated and purified by polyitylene glycol, etc., and the structure of the peptide can be known by analyzing the nucleotide sequence.
[322] The amino acid sequence of the peptide which has the binding ability with respect to the base material obtained by the said method is used by fuse | conjugating to PHA synthase using a conventional genetic engineering technique. Peptides having a binding ability to the substrate can be expressed by being linked to the N terminus or C terminus of the PHA synthetase. Moreover, an appropriate spacer array can also be inserted and expressed. As the spacer arrangement, about 3 to about 400 amino acids are preferable, and the spacer arrangement may contain any amino acid.
[323] Most preferably, the spacer arrangement does not prevent the PHA synthetase from functioning and does not interfere with the binding of the PHA synthetase to the substrate.
[324] The immobilized enzyme produced by the above method can be used as it is, but can also be used after giving lyophilization or the like.
[325] In the reaction where PHA is synthesized by the polymerization of 3-hydroxy acyl CoA, when the amount of PHA synthesized enzyme is 1 unit (U), the amount of CoA released is 1 μmol in 1 minute. For example, when a base material is a core of a capsule structure, it is good to set it in the range of 10 unit (U) to 1,000 unit (U), preferably 50 unit (U) to 500 unit (U) per 1g of base materials.
[326] The immobilized enzyme is introduced into an aqueous reaction solution containing 3-hydroxy acyl CoA, which is a raw material of the desired PHA, and PHA is synthesized by PHA synthase on the surface of the substrate, thereby forming a structure in which the substrate is coated with PHA. . The aqueous reaction solution should be configured as a reaction system adjusted to the conditions capable of exerting the activity of the PHA synthetase, for example, usually from pH 5.5 to pH 9.0, preferably from pH 7.0 to pH 8.5, Prepared by However, depending on the intellectual pH and pH stability of the PHA synthetase to be used, setting conditions other than the above range is not excluded. The type of buffer can be appropriately selected and used according to the pH range to be set as long as it can exert the activity of the PHA synthetase to be used. For example, the buffer used for general biochemical reactions, specifically acetic acid buffer, Phosphate buffer, potassium phosphate buffer, 3- (N-morpholino) propane sulfonic acid (MOPS) buffer, N-tris (hydroxymethyl3-amino propane sulfonic acid (TAPS) buffer, tris hydrochloric acid buffer, glycine buffer, 2- ( Cyclohexyl amino) ethane sulfonic acid (CHES) buffer, etc. The concentration of the buffer is not particularly limited as long as it can exert the activity of the PHA synthetase to be used, but is usually 1.0 M at 1.0 M, preferably What is necessary is just to use the thing of the density | concentration of 0.1 M to 0.2 M. Although reaction temperature is suitably set according to the characteristic of the PHA synthetase to be used, it is 4 degreeC-50 degreeC normally, Preferably it is 20 degreeC It is sufficient to set the temperature to 40 ° C. However, depending on the intellectual temperature and the heat resistance of the PHA synthetase to be used, the setting of conditions other than the above range is not excluded.The reaction time depends on the stability of the PHA synthetase to be used, The concentration is suitably selected within the range of 1 minute to 24 hours, preferably 30 minutes to 3 hours, and the concentration of 3-hydroxyacyl CoA in the reaction solution can exert the activity of the PHA synthetase to be used. Although it sets suitably within a range, it is usual to set in the range of 0.1mM to 1.0M, Preferably it is 0.2mM to 0.2M. Moreover, when 3-hydroxyacyl CoA concentration in a reaction liquid is high, it is general Therefore, since pH of a reaction system tends to fall, when setting 3-hydroxy acyl CoA density | concentration high, setting the said buffer concentration also seems to be a little high. Is recommended.
[327] In the above step, the composition of the 3-hydroxyacyl CoA in the aqueous reaction liquid is changed over time, so that the structure is formed in a direction from the inside to the outside when the structure is particulate. If it is a planar shape, the monomer unit composition of PHA which coat | covers a base material can be changed in a vertical direction.
[328] As the form of the structure in which the monomer unit composition is changed, for example, the composition in which the composition change of the PHA film is continuously coated with a base material coated with a base material in which a gradient of composition is formed in a direction from the inside to the outside or in a vertical direction Can be mentioned. As a manufacturing method, it is good by the method of adding 3-hydroxyacyl CoA of a separate composition to reaction liquid, for example, synthesize | combining PHA.
[329] As another form, the composition change of a PHA film is stepwise, and the form in which PHA from which a composition differs coat | covered the base material on the multilayer. As this manufacturing method, after synthesize | combining PHA by the composition of any 3-hydroxy acyl CoA, the structure in preparation is once collect | recovered from the reaction liquid by centrifugation, etc., and this consists of the composition of another 3-hydroxy acyl CoA. The reaction liquid may be added again.
[330] The structure obtained by the said reaction is provided to a washing | cleaning process as needed. The method for cleaning the structure is not particularly limited as long as the structure is not subjected to undesirable changes to the structure for the purpose of manufacturing the structure. In the case where the structure is a capsule structure in which the substrate is used as the core and the PHA is the outer shell, for example, the structure can be precipitated by centrifugation and the supernatant can be removed to remove unnecessary components contained in the reaction solution. . Further washing may be performed by adding a washing agent insoluble in PHA such as water, a buffer solution, methol and the like to perform centrifugation. Instead of centrifugation, a method such as filtration may be used. On the other hand, when a structure is a structure which coat | covered the flat base material with PHA, it can wash | clean by immersing in the said detergent, for example. In addition, the above-mentioned can be provided to a drying process as needed. Moreover, it can also be used by giving various structures, such as secondary processing and chemical modification, to the said structure.
[331] For example, a chemical modification is performed on PHA coated with a base material, whereby a structure having useful functions and characteristics can be obtained.
[332] For example, by introducing a graft chain into the PHA, a structure in which PHA having various characteristics due to the graft chain is coated with at least a part of the substrate can be obtained. In addition, by crosslinking the PHA, it is possible to control the mechanical strength, chemical resistance, heat resistance and the like of the structure.
[333] The method of chemical modification is not particularly limited as long as it satisfies the purpose of obtaining a desired function and structure, but, for example, a PHA having a reactive functional group in the side chain is synthesized, and the chemical formula of the functional group is used to form a chemical formula. How to do it can be used as a suitable method.
[334] The kind of the reactive functional group is not particularly limited as long as it satisfies the purpose of obtaining a desired function and structure. For example, the epoxy group described above can be exemplified. PHA which has an epoxy group in a side chain can perform chemical conversion similar to the polymer which has a normal epoxy group. Specifically, for example, it is possible to convert to a hydroxyl group or to introduce a sulfone group. Moreover, the graft chain of a polymer is formed by adding and reacting the compound which has a thiol and an amine, specifically, the compound which has an amino group with high reactivity with an epoxy group at the terminal, etc.
[335] As a compound which has an amino group at the terminal, amino modified polymers, such as polyvinyl amine, polyethyleneimine, and an amino modified polysiloxane (amino modified silicone oil), can be illustrated. Among these, as amino modified polysiloxane, a commercially available modified silicone oil may be used, and J. Amer. It can also synthesize | combine and use it by the method of Chem Soc., 78,2278 (1956) etc., and can enjoy effects, such as the improvement of heat resistance by addition of the graft chain of the said polymer.
[336] Moreover, as another example of the chemical conversion of the polymer which has an epoxy group, the crosslinking reaction by diamine compounds, such as nucleated methylenediamine, succinic anhydride, 2-ethyl-methylimidazole, electron beam irradiation, etc. is mentioned. Among these, reaction of PHA which has an epoxy group in a side chain, and hexamethylenediamine advances in the form as shown in the following scheme, and a crosslinked polymer produces | generates.
[337]
[338] As a method of confirming that the base material is coated with PHA about the obtained structure, generally, the method which combined the compositional analysis by gas chromatography and the form observation by an electron microscope etc., and the flight time, for example By using a type secondary ion mass spectrometer (TOP-SIMS) and ion sputtering techniques, a method of determining the structure from the mass spectra of the respective constituent layers can be used. However, a combination of Nile blue A staining and fluorescence microscopy newly developed by the present inventors can also be used as a direct and simple identification method. MEANS TO SOLVE THE PROBLEM As a result of continuing earnest examination about the method which can easily determine the synthesis | combination of PHA in vitro using PHA synthetase, it is a chemical | medical agent which has the property which binds specifically to PHA and fluoresces. , APP1, Environ. Microbio., 44,238-241 (1982) reported that Nile Blue A, which is reported to be available for simple determination of pha production in microbial cells (in vivo), is acellular based by setting appropriate use methods and conditions of use. The above method was completed by finding that it can also be used for the determination of PHA summation in. That is, in this method, the Nile Blue A solution of a predetermined concentration is filtered, and then mixed with a reaction solution containing PHA and irradiated with a constant wavelength of excitation light under a fluorescence microscope to emit fluorescence only from the synthesized PHA. By observing this, it is possible to easily and easily determine PHA synthesis in the cell-free system. As long as the substrate used does not have the property to fluoresce under the above conditions, the above-described method can be applied to the production of the structural CPD of the present invention, whereby the PHA covering the surface of the substrate can be directly observed and evaluated.
[339] In addition, the composition distribution in the direction from the inside to the outside of the PHA covering the base material or in the vertical direction can be evaluated by combining ion sputtering technology and time-of-flight secondary ion mass spectrometer (TOF-SIMS).
[340] <Use of structure>
[341] One of the features of the present invention is to enable the production of a structure that was difficult to manufacture in a conventional organic synthetic chemical method, and thus to provide excellent characteristics not present in a capsule structure or a laminated structure produced by a conventional organic synthetic chemical method. A structure can be obtained, for example, the use of a new high molecular compound and the provision of a new function and structure which were difficult to realize by the conventional organic synthetic method become possible. More specifically, by using a very precise molecular recognition ability and stereoselectivity peculiar to the catalysis of living organisms, a new functional high molecular compound or an extremely high chirality high molecular compound that was difficult to realize in conventional organic synthetic chemical techniques It is possible to manufacture a coated capsule structure, a laminated structure and the like in an extremely simple process.
[342] An example of the application of such a structure is an electrophotographic high performance capsule toner. As mentioned above, in the electrophotographic capsule toner, the manufacturing process is extremely complicated. There existed problems, such as using a large amount of solvents and surfactant in a manufacturing process. According to the method of the present invention, the above-mentioned problem is solved, and the method which can manufacture a capsule toner easily is provided. In addition, the thickness of the shell, the composition of the monomer unit, and the like can be controlled relatively easily. In addition, according to Japanese Patent Application Laid-Open No. 8-286416, it is believed that by containing a polar resin such as polyester in the outer surface of the capsule toner, it is possible to obtain an effect of improving image quality durability, uniformizing and stabilizing charging. The same effect as described above can be expected in the outer shell made of PHA in the capsule toner obtained by the method of the present invention. Further, in the method of the present invention, PHA having various functional groups can be used as the outer shell. In addition, it is possible to control the surface physical properties of the toner by these functional groups, to impart new SRLSMD properties, and the like. Except for the manufacturing process of the core part, since little or no organic solvent, no surface activity, etc. are used in the production, and the reaction conditions are extremely mild, the environmental load on the manufacturing can be greatly reduced. Do.
[343] As another example of the application of the structure of the present invention, for example, a recording medium in the inkjet recording method is mentioned. As described above, as a method of forming an ink absorbing layer on a substrate in the recording medium, a method by smearing has conventionally been common. According to the method of the present invention, a recording medium can be newly manufactured without using the method. That is, the substrate on which the enzyme is immobilized. For example, the following general formula [21],
[344]
[345] By reacting 3-hydroxypimeryl CoA represented by
[346] [22]
[347]
[348] It is possible to manufacture a recording medium in which PHA, i.e., PHA having a carboxyl group, which is an anionic functional group, in the side chain is represented as an ink receiving layer. With the method of the present invention, it is possible to manufacture such a novel functional recording medium, which is difficult to manufacture by the conventional method, but there is no report that the recording medium was manufactured by such a method.
[349] In addition, the structure of this invention, its usage method, and its manufacturing method are not limited to said method.
[350] EXAMPLE
[351] Hereinafter, the present invention will be described more specifically by way of example. However, although the Example described below is an example of the best embodiment of this invention, the technical scope of this invention is not limited to these Examples. In addition, "%" in the following is a basis of weight except having specifically abandoned.
[352] (Reference Example 1) Preparation of a transformant having a PHA synthase production ability
[353] After cultivating YN2 strain in 30 ml of overnight in 100 ml of LB medium (1% polypeptone (product made in Japan Pharmaceutical Co., Ltd.), 0.5% yeast extract (product made in Difco), 0.5% sodium chloride, pH7.4), horses The chromosomal DNA was separated and recovered by the method of. The obtained chromosomal DNA was linked to a restriction enzyme HindIII onnalysis fragment. E. coli (Escherichia coli) HB101 strain was transformed using the plasmid vector incorporating the chromosomal DNA fragment, thereby producing a DNA library of YN2 strain. Next, to select a DNA fragment containing the PHA synthase gene of the Y N strain, a probe for colony hybridization was prepared. An oligonucleotide consisting of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 was synthesized (Amature Siamese Bacteria Biotech Co., Ltd.), using this oligonucleotide as a primer, and PCR was carried out using chromosomal DNA as a template. Carried out. The PCR amplified DNA fragment was used as a probe. Labeling of the probe was performed using a commercially available labeling enzyme-based AlkPhosDirect (manufactured by Siamese Biosma Biotech Co., Ltd.). Using the labeling probe obtained,
[354] E. coli strains having a recombinant plasmid containing a PHA synthase gene were selected from the chromosomal DNA library of the YN2 strain by colony hybridization. From the selected strain, the DNA fragment containing the PHA synthase gene was obtained by recovering the plasmid by the alkali method. The gene DNA fragment obtained here was reassembled into the vector pBBR122 (Mo Bi Tec) which contains the wide-range replication region which does not belong to either incompatible group IncP, IncQ, or IncW. When this reassorted plasmid was transformed into the PHA synthetic defect strain by the electropotassium method, the PHA synthesis ability of the YN2 ml strain was restored, and showed complementarity. Therefore, it is confirmed that the selected DNA DNA fragment contains a PHA synthase gene region which can be translated by PHA synthetase within YN2 ml of Sudmonas te korea.
[355] The base sequence was determined by the acid value method for this DNA fragment. As a result, it was confirmed that base sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5, which encode peptide chains, exist in the determined nucleotide sequences, respectively. For these PHA synthase genes, PCR was performed using chromosomal DNA as a template, and the full length of the PHA synthase gene was re-prepared. That is, the PHA synthetase of the nucleotide sequence represented by the upstream primer (SEQ ID NO: 6) and the downstream primer (SEQ ID NO: 7), SEQ ID NO: 5 to the PHA synthase gene of the nucleotide sequence represented by SEQ ID NO: 4 The upstream primer (array number; 8) and the downstream primer (array number: 9) for the gene were synthesized respectively (Amateur Siamese Biomasia Biotech Co., Ltd.).
[356] Using these primers, PCR was performed for each of the nucleotide sequences represented by SEQ ID NO: 4 and SEQ ID NO: 5 to amplify the full length of the PHA synthase gene (LA-PCR kit; manufactured by Takara Co., Ltd.). In addition, the obtained PCR amplification fragment and the expression vector pTrc99A were cleaved with the restriction enzyme HindIII, and dephosphorylated (Molecular Cloning, Vol. 1, p. 572, 1989; published by Cold Spring Harbor Laboratory), followed by the expression vector pTrc99A. At the cleavage site, a DNA fragment containing the full length of the PHA synthetase gene except for the useless base sequence of the sock end was prepared by DNA ligation kit Ver. It connected using II (manufactured by Takara Casting Co., Ltd.).
[357] E. coli (Escherichia coli) HB101 (Takara casting) was transformed with the obtained recombinant plasmid by the calcium chloride method. The obtained recombinants were cultured, the recombinant plasmids were amplified, and the recombinant plasmids were recovered. Gene of SEQ ID NO; 4
[358] The recombinant plasmid holding the gene DNA of pYN2-C1 and the SEQ ID NO: 5 recombinant plasmid holding the gene DNA were set to pYN2-C2. Recombinant Escherichia coli, pYN2-C1 recombinant strain, pYN2-C2 recombinant strain which transform Escherichia coli (Escher ichia coli HB101fB fadB defective strain) into pYN2-C1, pYN2-C2 by calcium chloride method, and maintain each recombinant plasmid Got.
[359] Reference Example 2 Production of PHA Synthetase 1
[360] For pYN2-C1, oligonucleotides (SEQ ID NO: 10) serving as upstream primers and oligonucleotides (SEQ ID NO: 11) serving as downstream primers were designed and synthesized respectively (Amasham Palmasha Biotech Co., Ltd.). . PCR was performed using this oligonucleotide as a primer and pYN2-C1 as a ten plate to amplify the full length of the pha synthase gene having a BamHI restriction site upstream and an Xhol restriction site downstream (LA-PCR kit; Takarazu Co., Ltd.). ) My).
[361] Similarly, for pYN2-C2, an oligonucleotide (SEQ ID NO: 12) and an oligonucleotide (SEQ ID NO: 13), which are downstream primers, were designed and synthesized (Amasham Palmasha Biotech ( week)). PCR was carried out using this oligonucleotide as a primer and pYN2-C2 as a ten plate to amplify the full length of the PHA synthetase gene having a BamHI restriction site upstream and a Xhol restriction site downstream (LA-PCR kit: Takara Co., Ltd.) ) My).
[362] Each purified PCR amplification product was digested with BamHI and Xhol, and inserted into the corresponding site of plasmid pGEX-6P-1 (manufactured by Amasham Palmasha Biotech Co., Ltd.). E. coli (JM109) was transformed using these vectors to obtain an expression strain. The strain was identified by DNA fragments obtained by treating plasmid DNA prepared in large quantities using Miniprep (Wizard Minipreps DNA Purification Systems, manufactured by PROMEGA) with BamHI and Xhol. After the obtained strain was pre-calculated with 10 ml of LB-Amp medium overnight, the 0.1 ml was added to 10 ml of LB-Amp medium, followed by shaking culture at 37 ° C and 170 rpm for 3 hours. Thereafter, IPTG was added (final concentration 1 mM), and the culture was continued at 37 ° C for 4-12 hours.
[363] IPTG-induced E. coli was collected (8,000 × g, 2 minutes, 4 ° C.), and 1/10 amount of 4 ° C. phosphate buffered saline (PBS; 8 g NaC 1, 1.44 g Na ₂ HPO 4, 0.24 g KH ₂ PO _4, 0.2 g KC1, 1,000 ml purified water). The cells were crushed by freezing thawing and sonication, and centrifuged (8,000 × g, 10 minutes, 4 ° C.) to remove solid contaminants. After confirming that the expressed protein of interest exists in the supernatant by SDS-PAGE, the induced and expressed GST fusion protein was purified by glutathione Sepharose 4B (made by Amarsham Palmasha Biotech Co., Ltd.). The glutathione sepharose to be used previously performed the process which suppresses nonspecific adsorption. That is, after glutathione sepharose was washed three times with the same amount of PBS (8,000 × g, 1 min, 4 ° C.), 4% bovine serum albumin-containing PBS was added and treated at 4 ° C. for 1 hour. After the treatment, the resultant was washed twice with the same amount of PBS and suspended again in 1/2 amount of PBS. 40 µl of pretreated treated glutathione sepharose was added to 1 ml of the cell-free extract, and the mixture was stirred at 4 ° C silently. As a result, the fusion proteins GST-YN2-C1 and GST-YN2-C2 were adsorbed to the glutathione sepharose. After adsorption, centrifugation (8,000 g, 1 minute, 4 ° C) was performed, and the glutathione sepharose was collected and washed three times with 400 µl of PBS. Thereafter, 40 µ1 of 10 mM glutathione was added, the mixture was stirred at 4 ° C for 1 hour, and the adsorbed fusion protein was eluted. The supernatant was collected by centrifugation (8,000 g, 2 minutes, 4 ° C), and then dialyzed against PBS to purify the GST fusion protein. Single band was confirmed by SDS-PAGE.
[364] 500 g of each GST fusion protein was digested with PreScisson protease (manufactured by Amarsham Palmasha Biotech Co., Ltd., 5U), and then the protease and GST were removed by glutathione sepharose. The final purification of the expression proteins YN2-C1 and YN 2-C2 was obtained over a Sephadex G200 column where the flowthrough fraction was also equilibrated with PBS. Single bands of 60.8 kDa and 61.5 kDa were identified by SDS-PAGE, respectively.
[365] The enzyme was concentrated using a biological solution sample thickener (Mizutori Group AB-1100, manufactured by Ato Corporation) to obtain a purified enzyme solution of 10 U / ml.
[366] Each purified enzyme activity was measured by the method described above. In addition, the protein concentration in the sample was measured by the micro BCA protein quantitative reagent kit (made by Paas Chemical). Table 1 shows the results of the activity measurement of each purified enzyme.
[367] Disabled pYN2-C14.1U / mg Protein pYN2-C23.6U / mg Protein
[368] Reference Example 3 Production of PHA Synthetase 2
[369] p91 strains, H45 strains, YN2 strains or P161 strains were cultured in 200 ml of M9 medium containing 0.5% yeast extract (manufactured by Difco Co., Ltd.) and 0.1% octanoic acid, and shaken at 30 ° C for 125 strokes / minute. After 24 hours, the cells were collected by centrifugation (10,000 × g, 4 ° C. for 10 minutes), resuspended in 200 ml of 0.1 M tris hydrochloric acid buffer (pH8.0), and washed again by centrifugation. After resuspending in 2.0 ml of M-tris hydrochloric acid buffer (pH 8.0), crushed with an ultrasonic crusher, the supernatant was collected by centrifugation (12,000 xg, 4 degreeC 10 minutes), and the coarse enzyme was obtained.
[370] Each purified enzyme activity was measured by the method described above, and the results are shown in Table 2.
[371] activation P91 Week0.1U / ml H45 Note0.2U / ml YN 2 weeks0.4U / ml P161 Weeks0.2U / ml
[372] Example 1 Preparation of Capsule Structure 1
[373] 1 part by mass of alumina particles (particle diameter 0.12 µm to 135 µm) and 39 parts by mass of PBS were added to 10 parts by mass of the PHA synthetase solution derived from the pYN2-C1 recombinant strain, and shaken gently at 30 ° C. for 30 minutes. PHA synthase was then adsorbed onto the alumina surface. This was centrifuged (10,000 × g, 4 ° C. for 10 minutes), the precipitate was suspended in PBS solution, and again centrifuged (10,000 × g, 4 ° C. for 10 minutes) to obtain an immobilized enzyme.
[374] The immobilized enzyme was suspended in 48 parts by mass of 0.1 M phosphate buffer (pH 7.0) and described in (R) -3-hydrokisioctanoyl CoA (Eur. J. Biochem., 250, 432-439 (1997)). 1 mass part of preparation) and 0.1 mass part of bovine serum albumin (made by Si gma) were added by the method, and it stirred gently at 30 degreeC for 2 hours.
[375] 10 μl of the reaction solution was collected on a slide glass, 10 μl of a 1% Nile Blue A aqueous solution was added, mixed on a slide glass, and then covered with a cover glass and mounted on a fluorescence microscope (330 to 380 nm excitation filter, 420 nm long pass absorption filter). , Nikon Corporation) observation was performed. As a result, it was confirmed that the surface of the alumina particles emits fluorescence. Therefore, it turned out that the said alumina particle has coat | covered the surface by PHA. As a control, 1 part by mass of alumina particles was added to 49 parts by mass of 0.1 M phosphate buffer (pH 7.0), and gently shaken at 30 ° C. for 2.5 hours, followed by staining with Nile Blue A in the same manner, followed by fluorescence microscopy. As a result, the surface of the alumina particles did not fluoresce at all.
[376] In addition, a part of the dissolved particles was collected by centrifugation (10,000 × g, 4 ° C. for 10 minutes), dried in vacuo, suspended in chloroform, and stirred at 60 ° C. for 20 hours to extract PHA forming an envelope. The extract was filtered with a Methylene filter with a pore diameter of 0.45 μm, concentrated under reduced pressure with a rotary evaporator, and then subjected to metanosis according to the conventional method, followed by a gas chromatography-mass spectrometer (GS-MS, Shimadzu QP-5050, EI). Method), and the methyl ester cargo of the PHA monomer unit was classified. As a result, as shown in FIG. 1, it was confirmed that the solution PHA is PHA which consists of 3-hydroxy octanoic acid units.
[377] In addition, the molecular weight of the PHA was evaluated by Germination Chromatography (GPC: Tosoh HLC-8020, column; polymer laboratory PLgel MIXED-C (5 μm), solvent; chloroform, column temperature: 40 ° C., polystyrene conversion) , Mn = 21,000 and Mw = 40,000.
[378] Example 2 Preparation of Capsule Structure 2
[379] 1 mass part of alumina particles (particle diameter 0.12 micrometers-135 micrometers), PBS 39 mass part are added to 10 mass parts of PHA synthase solutions (10U / ml) derived from a pYN2-C2 recombinant strain, and shaken gently at 30 degreeC for 30 minutes. PHA synthase was adsorbed onto the alumina surface. This was centrifuged (10,000 × g, 4 ° C. for 10 minutes), and the precipitate was suspended in PBS solution, followed by re-dimensional separation (10,000 × g, 4 ° C., 10 minutes) to obtain an immobilized enzyme.
[380] The immobilized enzyme was suspended in 48 parts by mass of 0.1 M phosphate buffer (pH 7. 0), and (R) -3-hydroxyoctanoyl CoA (Eur. J. Biochem., 250, 432-439 (1997). 1 mass part of preparation) and 0.1 mass part of bovine serum albumin (made by Sigma) were added to (), and it stirred gently at 30 degreeC for 2 hours.
[381] 10 μl of the reaction solution was collected on a slide glass, 10 μl of a 1% Nile Blue A aqueous solution was added, and mixed on the slide glass. Then, the cover glass was loaded with a fluorescence microscope (330-380 nm excitation filter, 420 nm long pass absorption filter). , Nikon Corporation) observation was performed. As a result, it was confirmed that the surface of the alumina particles emits fluorescence. Therefore, it turned out that the said alumina particle has coat | covered the surface by PHA.
[382] In addition, some of the particles were collected by centrifugation (10,000 × g, 4 ° C. for 10 minutes), dried in vacuo, suspended in chloroform, and stirred at 60 ° C. for 20 hours to extract PHA, which forms an envelope. The extract was filtered with a membrane filter with a pore diameter of 0.45 μm, concentrated under reduced pressure with a rotary evaporator, and then subjected to metanosis according to the conventional method, followed by a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method) was used to classify the methyl ester of the PHA monomer unit. As a result, as in the result of Example 1, it was confirmed that the PHA was PHA composed of 3-hydroxy octanoic acid units.
[383] Example 3 Preparation of Capsule Structure 3
[384] 1 part by mass of alumina particles (particle diameter 0.12 μm to 135 μm) is added to 99 parts by mass of the PHA synthetase derived from YN2, H45, P91, or P161, and shaken gently at 30 ° C. for 30 minutes to synthesize PHA. The enzyme was adsorbed onto the alumina surface. This was centrifuged (10,000 × g, 4 ° C. for 10 minutes), and the precipitate was suspended in a PBS solution, followed by re-dimensional separation (10,000 × g, 4 ° C. for 10 minutes) to obtain an immobilized enzyme.
[385] Each immobilized enzyme was suspended in 48 parts by mass of 0.1 M phosphate buffer (pH 7. 0), and (R) -3-hydroxyoctanyl CoA (Eur. J. Biochem. 250, 432-439 ( 1 part by mass) and 0.1 part by mass of bovine serum albumin (manufactured by Sigma) were added by the method described in 1997), and the mixture was shaken gently at 30 ° C for 2 hours.
[386] 10 μl of the reaction solution was collected on a slide glass, 10 μl of a 1% Nile Blue A aqueous solution was added, mixed on a slide glass, and then covered with a cover glass, followed by a fluorescence microscope (330-380 nm excitation filter, 420 nm long pass absorption filter). , Nikon Corporation) observation was performed. As a result, it was confirmed that the surface of the alumina particles fluoresced with respect to the reaction solution of any immobilization enzyme. Therefore, it turned out that all the said alumina particle has coat | covered the surface by PHA.
[387] In addition, some of the particles were collected by centrifugation (10,000 × g, 4 ° C. for 10 minutes), dried in vacuo, suspended in chloroform, and stirred at 60 ° C. for 20 hours to extract PHA having an envelope. The extract was filtered with a membrane filter having a pore diameter of 0.45 μm, concentrated under reduced pressure with a rotary evaporator, and then subjected to metanosis according to the conventional method, followed by a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method) was used to classify methyl esters of PHA monomer units. As a result, as in the result of Example 1, it was confirmed that the PHA was PHA composed of 3-hydroxy octanoic acid units.
[388] Example 4 Preparation of Capsule Structure 4
[389] 1 part by mass of alumina particles (particle size 0.12 μm to 135 μm) and 39 parts by mass of PBS are added to 10 parts by mass of the PHA synthetase solution (10 U / ml) derived from the pYN2-C1 recombinant strain, and shaken gently at 30 ° C. for 30 minutes. The PHA synthase was adsorbed onto the alumina surface. This was centrifuged (10,000 × g, 4 ° C. for 10 minutes), and the precipitate was suspended in PBS solution, again centrifuged (10,000 × g, 4 ° C. for 10 minutes) to obtain an immobilized enzyme.
[390] The immobilized enzyme was suspended in 48 parts by mass of 0.1 M phosphate buffer (pH 7. 0), and (R) -3-hydroxy-5-phenylbareryl CoA (3-hydroxyphenyl obtained by Reformatsky reaction). After hydrolyzing valeric acid ester to obtain 3-hydroxy-5-phenylvaleric acid, 1 part by mass (prepared by the method described in Eur. J. Biochem. 250, 432-439 (1997)), 1 part by mass of bovine serum albumin 0.1 mass part (made by Sigma) was added, and it stirred gently at 30 degreeC for 2 hours.
[391] 10 μl of the reaction solution was collected on a slide glass, 10 μl of a 1% Nile Blue A aqueous solution was added, mixed on a slide glass, and then covered with a cover glass, followed by a fluorescence microscope (330-380 nm excitation filter, 420 nm long pass absorption filter). , Nikon Corporation) observation was performed. As a result, it was confirmed that the surface of the alumina particles emits fluorescence. Therefore, it turned out that the said alumina particle has coat | covered the surface by PHA.
[392] In addition, some of the particles were collected by centrifugation (10,000 × g, 4 ° C. for 10 minutes), dried in vacuo, suspended in chloroform, and stirred at 60 ° C. for 20 hours to extract PHA that forms an envelope. The extract was filtered with a membrane filter with a pore diameter of 0.45 μm, concentrated under reduced pressure with a rotary evaporator, and then subjected to metanosis according to the conventional method, followed by a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method) and classify | categorized the methyl ester cargo of the PHA monomer unit. As a result, as shown in Fig. 2, the PHA was confirmed to be PHA composed of 3-hydroxy-5-phenylvaleric acid unit.
[393] In addition, the molecular weight of the PHA was evaluated by gel permeation chromatography (GPC: Tosoh HLC-8020, column; polymer laboratory PLgel MIXED-C (5 μm), solvent; chloroform, column temperature; 40 ° C., polystyrene conversion). , Mn = 16, 000 and Mw = 36, 000.
[394] Example 5 Preparation of Capsule Structure 5
[395] To 1 part by mass of alumina particles (particle diameter 0.12 μm to 135 μm) and 39 parts by mass of PBS are added to 10 parts by mass of a PHA synthetase solution (10 u / ml) derived from a pYN2-C1 recombinant strain, and shaken gently at 30 ° C. for 30 minutes. PHA synthase was adsorbed onto the alumina surface. This was centrifuged (10,000 × g, 4 ° C. for 10 minutes), and the precipitate was suspended in PBS solution, and again centrifuged (10,000 × g, 4 ° C. for 10 minutes) to obtain an immobilized enzyme.
[396] The immobilized enzyme was suspended in 48 parts by mass of 0.1 M phosphate buffer (pH 7. 0) and obtained by (R) -3-hydroxy-5- (4-fluorophenyl) vareryl CoA (ReformatskY reaction). Hydrolysis of 3-hydroxy phenylvaleric acid ester to give 3-hydro 5- (4-fluorophenyl) valeric acid, followed by the method described in (Eur. J. Biochem. 250, 432-439 (1997)). Preparation) 1 mass part and 0.1 mass part of bovine serum albumin (made by Sigma) were added, and it stirred gently at 30 degreeC for 2 hours.
[397] 10 μl of the reaction solution was collected on a slide glass, 10 μl of a 1% Nile Blue A aqueous solution was added, mixed on a slide glass, and then covered with a cover glass, followed by a fluorescence microscope (330-380 nm excitation filter, 420 nm long pass absorption). A filter, manufactured by Nikon Corporation, was observed. As a result, it was confirmed that the surface of the alumina particles emits fluorescence. Therefore, it turned out that the said alumina particle has coat | covered the surface by PHA.
[398] In addition, some of the particles were collected by centrifugation (10,000 × g, 4 ° C. for 10 minutes), dried in vacuo, suspended in chloroform, and stirred at 60 ° C. for 20 hours to extract PHA that forms an envelope. The extract was filtered with a membrane filter with a pore diameter of 0.45 μm, concentrated under reduced pressure with a rotary evaporator, and then subjected to metanosis according to the conventional method, followed by a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method) and classify | categorized the methyl ester cargo of the PHA monomer unit. As a result, as shown in FIG. 3, it was confirmed that the said PHA is PHA which uses 3-hydroxy-5- (4-fluorophenyl) valeric acid as a monomer unit.
[399] Example 6 Preparation of Capsule Structure 6
[400] 1 part by mass of alumina particles having a particle diameter (volume average particle diameter of 1.45 μm) and 39 parts by mass of PBS were added to 10 parts by mass of PHA synthetase solution (10 u / ml) derived from the pYN2-C1 recombinant strain. The PHA synthase was adsorbed onto the alumina surface by gentle shaking for 30 minutes at &lt; RTI ID = 0.0 &gt; This was centrifuged (10,000 × g, 4 ° C. for 10 minutes), and the precipitate was suspended in PBS solution, again centrifuged (10,000 × g, 4 ° C. for 10 minutes) to obtain an immobilized enzyme.
[401] The immobilized enzyme was suspended in 48 parts by mass of 0.1 M phosphate buffer (pH 7. 0), and (R) -3-hydrokixyoctanoyl CoA (Eur. J. Biochem., 250, 432-439 (1997) 1 mass part of preparation) and 0.1 mass part of bovine serum albumin (made by Sigma) were added, and it stirred gently at 30 degreeC, respectively for 2 hours.
[402] After the reaction, 10 μl of the reaction solution was collected on a slide glass, 10 μl of a 1% Nile Blue A aqueous solution was added, mixed on the slide glass, and then covered with a cover glass, followed by a fluorescence microscope (330-380 nm excitation filter, 420 nm). Long-pass absorption filter, Nikon Corporation make observation. As a result, it was confirmed that the surface of the alumina particles emits fluorescence. Therefore, it turned out that the said alumina particle has coat | covered the surface by PHA.
[403] In addition, the capsule structure was collected by centrifugation (10,000 × g, 4 ° C. for 10 minutes), and after drying, the mass of the polymer formed on the surface of the particulate matter was measured using a time-of-flight type secondary ion mass spectrometer (TOF -SIMS IV, CAMECA). From the obtained mass spectrum, it was found that the surface of the capsule structure was composed of a homopolymer of polyhydroxy octanoate. The mass spectra were similarly measured by TOF-SIMS while the surface of the capsule structure was little by little by ion spattering, but they were all composed of a homopolymer of polyhydroxy octanoate. Thereby, it turned out that the capsule structure of this comparative example was a capsule structure which directly coated hydrophilic inorganic particle with the homopolymer of hydrophobic polyhydroxy octanoate.
[404] Here, the molecular weight of the PHA was evaluated by gel permeation chromatography (GPC: Tosoh HLC-8020, column; polymer laboratory PLgel MIXED-C (5 μm), solvent; chloroform, column temperature; 40 ° C., polystyrene conversion). , Mn = 23, 000 Mw = 42, 000.
[405] Example 7 Preparation of Capsule Structure 7
[406] 1 part by mass of alumina particles having a particle diameter (volume average particle diameter of 1.45 μm) and 39 parts by mass of PBS were added to 10 parts by mass of PHA synthetase solution (10 u / ml) derived from the pYN2-C1 recombinant strain. The PHA synthase was adsorbed onto the alumina surface by gentle shaking for 30 minutes at &lt; RTI ID = 0.0 &gt; This was centrifuged (10,000 × g, 4 ° C. for 10 minutes), and the precipitate was suspended in PBS solution, again centrifuged (10,000 × g, 4 ° C. for 10 minutes) to obtain an immobilized enzyme.
[407] The immobilized enzyme was suspended in 48 parts by mass of 0.1 M phosphate buffer (pH 7. 0), and described in (R) -3-hydroxypimeryl CoA (J. Bacteriol., 182, 2753-2760 (2000). 1 part by mass of the prepared) and 0.1 parts by mass of bovine serum albumin (manufactured by Sigma) were added and shaken gently at 30 ° C for 10 minutes. Subsequently, (R) -3-hydroxyoctanoyl CoA (prepared by the method described in Eur. J. Biochem., 250, 432-439 (1997)) was added to this reaction solution with gentle shaking at 30 ° C. 0.1 mass part of 0.1 M phosphate buffer (pH 7.0) containing 1 mass part and 0.1 mass part of bovine serum albumin (made by Sigma) ratio using a micro tube pump (MP-3N by Tokyo Rika Chemical Co., Ltd.) Was added. Furthermore, after 1 hour and 30 minutes, the produced particulate body was recovered by centrifugation (10,000 × g, 4 ° C. for 10 minutes), the supernatant was removed, and then (R) -3-hydroxyoctanoyl CoA was added to the particulate matter. (Prepared by the method described in Eur. J. Biochem., 250, 432-439 (1997)) 0.1 M phosphate buffer (pH 7. 0) containing 1 part by mass and 0.1 part by mass of bovine serum albumin (manufactured by Sigma). Was added 25 parts by mass and gently shaken at 30 ° C for 20 minutes.
[408] After the reaction, 10 μl of the reaction solution was collected on the slide glass, 10 μl of 1% Nile Blue A aqueous solution was added, mixed on the slide glass, and then covered with a cover glass, followed by a fluorescent microscope (330-380 nm excitation filter, 420 nm). Long-pass absorption filter, Nikon Corporation make observation. As a result, it was confirmed that the surface of the alumina particles emits fluorescence. Therefore, it turned out that the said alumina particle has coat | covered the surface by PHA.
[409] In addition, the capsule structure was recovered by centrifugation (10,000 × g, 4 ° C. for 10 minutes), and after drying, the mass of the polymer formed on the surface of the particulate body was measured using a time-of-flight type secondary ion mass spectrometer (TOF -SIMS IV, made by CAMECA). From the obtained mass spectrum, it was found that the surface of the capsule structure was composed of a homopolymer of polyhydroxy octanoate. In addition, the mass spectra were measured by TOF-SIMS in the same manner as the surface of the capsule structure was little by little by ion spattering. A copolymer of 3-hydroxyoctanoic acid and 3-hydroxypimeric acid (molar ratio 21: 1) Appears, the composition ratio of 3-hydroxyoctanoic acid in the copolymer gradually decreases as it goes to the granular body, on the contrary, the composition ratio of 3-hydroxypimeric acid increases, and finally polyhydroxypi It was confirmed to change to homopolymer of methacrylate. Thereby, the capsule structure of this Example coat | covers a hydrophilic particulate base material with polyhydroxy pimerate which has a hydrophilic functional group, and 3-hydroxy pimeric acid which has a hydrophilic functional group and 3 which has a hydrophobic functional group on it The capsule which coated the outermost layer with the homopolymer of polyhydroxy octanoate by covering with the copolymer of -hydroxy octanoic acid, increasing the composition ratio of 3-hydroxy octanoic acid as it reached the surface layer. It turns out that it is a structure.
[410] Here, the average molecular weight of the PHA was determined by gel permeation chromatography (Gp C: Tosoh HLC-8020, column: polymer laboratory PLgel MIXED-C (5 μm), solvent: chloroform, column temperature: 40 ° C., polyester conversion). As a result of evaluation, Mn = 21, 000 and Mw = 40,000.
[411] (Example 8) Evaluation of Example 6 and Example 7
[412] When the capsule structure of Example 6 and Example 7 and 1 mass part of alumina particles (volume average particle diameter 1.45 micrometers) with a particle diameter were added according to the sedimentation method as a comparative example, 10 mass parts of pure water was added, and it was left still as it is. In the case of vigorous stirring for 5 minutes with a vortex mixer, the volume average particle diameter of the capsule structure and the volume average particle diameter after storage at room temperature for 30 days were measured. The measurement was performed using a laser toppler system particle size distribution analyzer (UPA-150: Nikkiso Co., Ltd.), and the result at the time of standing still without stirring was shown in Table 3, and the result at the time of stirring was shown in Table 4.
[413] Example 6Example 7Comparative example Volume average particle diameter (before storage)1.581.681.45 Volume average particle diameter (after storage)1.601.713.42
[414] (Μm)
[415] Example 6Example 7Comparative example Volume average particle diameter (before storage) Volume average particle diameter (after storage1.472.051.651.171.413.56
[416] (Μm)
[417] As a result, when stirring was not performed, it turned out that the volume average particle diameter of the capsule structure of Example 6 and Example 7 showed the substantially equivalent value before and after storage, and was excellent in storage stability. On the other hand, the volume average particle diameter after storage of the capsule structure of a comparative example increased compared with before storage.
[418] Moreover, when stirring was performed, the volume average particle diameter of the capsule structure of Example 7 showed the substantially equivalent value before and after storage, but the volume average particle diameter after the storage of the capsule structure of Example 6 compared with before storage. Slightly increased. The comparative example showed the same result as the case of not stirring.
[419] In addition, when the capsule structure after storage at the time of stirring of Example 6 and Example 7 was observed with the optical microscope, in Example 7, it was seen that each capsule structure was disperse | distributing well, but Example 6 In the above, aggregation of the capsule structure was observed, and the capsule structure in which PHA coated was further peeled was observed.
[420] The inorganic base material is coated with PHA having an inorganic base and a high affinity hydrophilic functional group, and further, by a copolymer of a hydrophilic PHA monomer unit and a hydrophobic PHA monomer unit. By reaching the surface layer and coating while increasing the composition ratio of the hydrophobic PHA monomer unit, it was found that a hydrophobic PHA capsule capable of containing the inorganic base material more stably can be produced.
[421] Example 9 Preparation of Capsule Structure 8
[422] 1 part by mass of alumina particles (particle diameters from 0 .12 μm to 135 μm) and 39 parts by mass of PBS were added to 10 parts by mass of a PHA synthetase solution (10 u / ml) derived from a pYN2-C1 recombinant strain, and shaken gently at 30 ° C. for 30 minutes. PHA synthase was adsorbed onto the alumina surface. This was centrifuged (10,000 × g, 4 ° C. for 10 minutes), and the precipitate was suspended in PBS solution and centrifuged again (10,000 × g, 4 ° C. for 10 minutes) to obtain an immobilized enzyme.
[423] The immobilized enzyme is suspended in 48 parts by mass of 0.1 M phosphate buffer (pH 7. 0), and (R, S) -3-hydroxy-5-phenoxybareryl CoA (3-phenoxy propanol and bromine After hydrolysis of the 3-hydroxy-5-phenoxyvaleric acid ester obtained by the Reformatsky reaction with ethyl acetate to obtain 3-hydroxy-5-phenoxyvaleric acid, Eur. J. Biochem., 250 , 432-439 (1997)) 0.8 parts by mass, (R, S) -3-hydroxy-7, 8-epoxyoctanoyl CoA (Int. J. Biol, Macromol., 12, 85 The unsaturated portion of 3-hydroxy 7-octenic acid synthesized by the method described in -91 (1990) was epoxidized with 3-chlorobenzoic acid, and then described in Eur. J. Biochem. 250, 432-439 (1997). 0.2 mass part of preparation) and 0.1 mass part of bovine serum albumin (made by SiGma) were added, and sample 1 was shaken gently at 30 degreeC for 2 hours.
[424] As a comparative control, Sample 2 was obtained in the same manner as described above, except that (R, S) -3-hydroxy-7,8-epoxyoctanoyl CoA was changed to 3-hydroxyoctanoyl CoA.
[425] 10 μl of the sample was taken on the slide glass, 10 μl of a 1% Nile Blue A aqueous solution was added, mixed on the slide glass, and then the cover glass was loaded and a fluorescence microscope (330-380 nm excitation filter, 420 nm long pass absorption). A filter, manufactured by Nikon Corporation, was observed. As a result, it was confirmed that the alumina particle surface fluoresced also in any sample. Therefore, it turned out that the said alumina particle has coat | covered the surface by PHA.
[426] As a control, 1 part by mass of alumina particles was added to 49 parts by mass of 0.1 M phosphate buffer (pH 7.0), and gently shaken at 30 ° C. for 2.5 hours, followed by dyeing with Nile Blue A in the same manner, followed by fluorescence microscopy. As a result, the surface of the alumina particles did not fluoresce at all.
[427] A part of the sample was collected by centrifugation (10,000 × g, 4 ° C., 10 minutes), dried in vacuo, suspended in chloroform, and stirred at 60 ° C. for 20 hours to extract PHA that forms an envelope. This extract was subjected to ¹ H-NMR analysis (device used: FT-NMR: B ruker DPX400, measured nuclide: ¹ H, solvent used: heavy chloroform (with TMS)). Table 5 shows the unit% of each side chain unit calculated therefrom.
[428] Composition of shell PHA of capsule structure ( ¹ H-NMR, unit%) Monomer UnitSample 1Sample 2 3-hydroxy-5-phenoxyvaleric83%77% 3-hydroxy-7,8-epoxy octanoic acid17%- 3-hydroxy octanoic acid-23%
[429] The sample 1 was centrifuged at 50 parts by mass (10,000 × g, 4 ° C. for 10 minutes) to recover the capsule structure, and the suspension was suspended three times in 50 parts by mass of purified water, followed by hexamethylenediamine as a crosslinking agent in the suspension. 0.5 parts by mass were dissolved. After confirming the dissolution, water was removed by lyophilization (this is referred to as Sample 3). In addition, sample 3 was allowed to react at 70 ° C for 12 hours (this is referred to as sample 4).
[430] The sample 3 and the sample 4 were suspended in chloroform, stirred at 60 ° C. for 20 hours to extract PHA constituting the skin, and chloroform was removed by vacuum drying, followed by differential scanning calorimetry (DSC; manufactured by Parkinerma, PYris 1, Temperature rise: 10 degree-C / min) The measurement was performed. As a result, in Sample 3, a clear exothermic peak was observed around 90 ° C, and the reaction between the epoxy group in the polymer and hexamethylenediamine occurred, indicating that the crosslinking between the polymers was progressing. On the other hand, in Sample 4, no clear heat flow was observed, and the crosslinking reaction was almost completed.
[431] Moreover, infrared absorption was measured about the same sample (FT-IR; Parkinerma, make, 1720X). As a result, the peaks of the amine (near 3340 cm ⁻¹ ) and the epoxy (near 822 cm ⁻¹ ) seen in Sample 3 disappeared in Sample 4.
[432] In the above result, it became clear that a crosslinked polymer can be obtained by making PHA which has an epoxy unit in a side chain, and hesamethylenediamine react.
[433] On the other hand, although the same evaluation was performed about sample 2 as a comparative control, the evaluation result which clearly indicated the crosslinking of the above polymers was not obtained.
[434] Example 10 Preparation of Capsule Structure 9
[435] PHA synthetase solution (10 u / ml) derived from pYN2-C1 recombinant strain was added to 10 parts by mass of alumina particles (particle size from 0.12 μm to 135 μm) and 39 parts by mass of PBS, followed by gentle shaking at 30 ° C. for 30 minutes. Synthetic enzymes were adsorbed onto the alumina surface. This was centrifuged (10,000 × g, 4 ° C for 10 minutes), and the precipitate was suspended in PBS solution, and again centrifuged (10,000 × g, 4 ° C for 10 minutes) to obtain an immobilized enzyme.
[436] The immobilized enzyme is suspended in 48 parts by mass of 0.1 M phosphate buffer (pH 7. 0), and (R, S) -3-hydroxy-5-phenoxybareryl CoA (3-phenoxy propanol and bromine After hydrolysis of the 3-hydroxy-5-phenoxyvaleric acid ester obtained by the Reformatsky reaction with ethyl acetate to obtain 3-hydroxy-5-phenoxyvaleric acid, Eur. J. Biochem., 250 , 432-439 (1997)) 0.8 parts by mass, (R, S) -3-hydroxy-7, 8-epoxyoctanoyl CoA (Int. J. Biol, Macromol., 12, 85 The unsaturated portion of 3-hydroxy 7-octenic acid synthesized by the method described in -91 (1990) was epoxidized with 3-chlorobenzoic acid, and then described in Eur. J. Biochem. 250, 432-439 (1997). 0.2 mass part of preparation) and 0.1 mass part of bovine serum albumin (made by SiGma) were added, and it stirred gently at 30 degreeC for 2 hours, and obtained sample 5.
[437] 10 microliters of said sample 5 were extract | collected on the slide glass, 10 microliters of 1% Nile Blue A aqueous solution was added, and it mixed on the slide glass, and the cover glass was loaded, and a fluorescence microscope (330-380 nm excitation filter, 420 nm long) was carried out. A pass absorption filter, manufactured by Nikon Corporation, was observed. As a result, it was confirmed that the alumina particle surface fluoresced also in any sample. Therefore, it turned out that the said alumina particle has coat | covered the surface by PHA.
[438] As a control, 1 part by mass of alumina particles was added to 49 parts by mass of 0.1 M phosphate buffer (pH 7.0), and gently shaken at 30 ° C. for 2.5 hours, followed by dyeing with Nile Blue A in the same manner, followed by fluorescence microscopy. As a result, the surface of the alumina particles did not fluoresce at all.
[439] A part of Sample 5 was recovered by centrifugation (10,000 × g, 4 ° C., 10 minutes), dried in vacuo, suspended in chloroform, and stirred at 60 ° C. for 20 hours to extract PHA, which forms an envelope. This extract was subjected to ¹ H-NMR analysis (device used: FT-NMR: B ruker DPX400, measured nuclide: ¹ H, solvent used: heavy chloroform (with TMS)). The unit% of each side chain calculated here was 83% of 3-hydroxy-5-phenoxy valeric acid and 17% of 3-hydroxy-7,8-epoxyoctanoic acid.
[440] 50 mass parts of said sample 5 was centrifuged (10,000 * g, 4 degreeC, 10 minutes), the capsule structure was collect | recovered, and the procedure which suspended in 50 mass parts of purified water was repeated 3 times, and water is removed by freeze drying. did. 10 mass parts of terminal amino modified polysiloxane (modified silicone oil TSF4700, GE Toshiba Silicone Co., Ltd. product) were added, and it was made to react at 70 degreeC for 2 hours. This was suspended in methanol and centrifuged (10,000 × g, 4 ° C. for 20 minutes), and washed and dried to obtain a capsule structure having a graft chain of polysiloxane.
[441] Example 11 Preparation of Laminated Structure
[442] The porous sheet of 10 mm in length × 10 mm in width × 1 mm in thickness was immersed in 1% glutaraldehyde for 1 hour and washed with pure water, and then washed in pure water and then 30 ° C. in a PHA synthase solution derived from pYN2-C1 recombinant strain (10 u / ml). I soaked for 30 minutes and immobilized the enzyme. Unreacted PHA synthase was washed with PBS to remove the immobilized enzyme.
[443] 30 mM (R) -3-hydroxyoctanoyl CoA Eur. J. Biochem. Prepared by the method described in 250, 432-439 (1997)) The immobilized enzyme was immersed in a 0.1 M phosphate buffer (pH 7.0) containing 0.1% bovine serum albumin (manufactured by Sigma), and gently stirred at 30 ° C for 2 hours. Shaken. After the reaction was completed, the resultant was washed with 0.1 M phosphate buffer (DH 7.0) to remove unreacted substances.
[444] The glass sheet after reaction was dyed with 1% Nile blue A aqueous solution, and fluorescence microscope (330-380 nm excitation filter, 420 nm long pass absorption filter, the Nikon Corporation make) observation was performed. As a result, since the fluorescence was recognized by the surface of the glass sheet, it turned out that the said particle | grain is a laminated structure by which the base material which consists of a glass sheet was coat | covered with the film | membrane which consists of PHA.
[445] Moreover, after vacuum drying the said laminated structure, it immersed in chloroform, and it stirred for 20 hours at 60 degreeC, and extracted PHA which forms a coating layer. Filter the extract with a Methylene filter with a hole diameter of 0.45 μm. After concentrating under reduced pressure with a rotary evaporator, metanosis was carried out according to a conventional method, and analyzed by a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method) to classify the methyl ester cargo of the PHA monomer unit. Carried out. As a result, as shown in FIG. 4, it was confirmed that this PHA is PHA which uses 3-hydroxy octanoic acid as a monomer unit.
[446] Example 12 Preparation of Capsule Toner
[447] First, polymer microparticles were manufactured as a core as follows. 450 mass parts of 0.1-M Na3PO4 aqueous solution was thrown into 710 mass parts of ion-exchange water, and it heated at 60 degreeC, and then crossed at 12,000 rpm using the TK-type homomixer (made by Tokushuki Chemical Co., Ltd.). Thereto, 1. OM CaCl ₂ aqueous solution was gradually added to 68 parts by weight, Ca ₃ (PO ₄₎ to obtain an aqueous medium containing the _two. Next, 165 parts by mass of styrene monomer, 35 parts by mass of n-butyl acrylate, 12 parts by mass of copper phthalocyanine pigment, 10 parts by mass of unsaturated polyester (fumaric acid-propylene oxide modified bisphenol A), 60 parts by mass of ester wax, and polymerization to this 10 mass parts of initiators 2, 2'- azobis (2, 4- dimethyl vareronitrile) are melt | dissolved, the polymerizable monomer composition is prepared, this is heated at 60 degreeC, and TK-type homomixer (Tokushukigako Co., Ltd. )), And it melt | dissolved and disperse | distributed uniformly at 12,000 rpm. The polymerizable monomer composition was introduced into the aqueous medium, and stirred at 10,000 rpm for 10 minutes in a TK-type homo mixer under 60 ° C and N ₂ atmosphere to form particles. Then, temperature was raised to 80 degreeC, stirring by paddle stirring blades, and it was made to react for 10 hours. After the completion of the polymerization reaction, the suspension of the polymer was cooled, 3.6 parts by mass of Na ₂ CO ₃ was added to pH 11. Next, 0.3 parts by mass of potassium persulfate as a polymerization initiator was added to the polymerizable monomer system consisting of 82 parts by mass of styrene monomer, 12 parts by mass of n-butyl acrylate, unsaturated polyester, and 0.05 parts by mass of (fumaric acid-propylene oxide modified bisphenol A). After the addition-dissolved solution was added dropwise, the temperature was raised to 80 ° C and polymerized again for 6 hours. Next, this was cooled to room temperature, hydrochloric acid was added to dissolve and remove calcium phosphate, and the core component fine particles of the toner were obtained by filtration and drying.
[448] To 100 parts by mass of a PHA synthetase solution (10 u / ml) derived from a pYN2-C1 recombinant strain, 10 parts by mass of the fine particles and 390 parts by mass of PBS are added, and the mixture is shaken gently at 30 ° C. for 30 minutes, and the PHA synthesizing enzyme is dispersed. Adsorbed on the surface. This was centrifuged (10,000 × g, 4 ° C. for 10 minutes), and the precipitate was suspended in PBS solution, again centrifuged (10,000 × g, 4 ° C. for 10 minutes) to obtain an immobilized enzyme.
[449] 10 parts by mass of the immobilized enzyme was suspended in 480 parts by mass of 0.1 M phosphate buffer (pH 7. 0) and subjected to (R) -3-hydroxy-5- (4-fluorophenyl) bareryl CoA (Refor matsky reaction). After hydrolysis of the 3-hydroxy phenylvaleric acid ester obtained by to obtain 3-hydroxy-5- (4-fluorophenyl) valeric acid, Eur. J. Biochem., 250, 432-439 (1997) 10 mass parts of preparation) and 1 mass part of bovine serum albumin (made by Sigma) were added, and it stirred gently at 30 degreeC for 2 hours.
[450] After the completion of the reaction, the mixture was centrifuged at 10,000 x g for 10 minutes, the precipitate was suspended in 1000 parts by mass of 0, 1 M phosphate buffer (pH 7. 0), and the centrifugation was repeated three times to recover the precipitate. . From this, the capsule structure was obtained through each process of filtration and drying. To 10 mass parts of the obtained capsule structure, 0.12 mass part of hydrophobic titanium oxide fine powder was added outside, and the capsule toner A which has a titanium oxide fine powder on the capsule structure surface was obtained.
[451] On the other hand, 0.12 parts by mass of hydrophobic titanium oxide fine powder was added to 10 parts by mass of the above-mentioned core component fine particles, and toner B having titanium oxide fine powder on the surface of the toner was obtained.
[452] To 6 parts by mass of each capsule toner, 144 parts by mass of ferrite carrier coated with an acrylic resin was mixed to obtain a two-component developer.
[453] Using the above developer, images were copied in a commercial copier NP 6000 (manufactured by Canon Corporation) at 23 ° C and 60% RH environment, and evaluated for image durability, toner scattering, and fading. As a result, as shown in Table 6, in the developer prepared using the capsule toner A, the occurrence of image defects such as image density drop, toner scattering, and fading was not recognized even for the durability of 100,000 sheets, and showed good image quality durability. Moreover, although the Tribo value was measured about charging property, it was stabilized at -33mC / kg initial stage and -31mC / kg after durability. Moreover, no problem was recognized regarding settlement. On the other hand, in the developer prepared using the capsule toner B, the occurrence of image defects such as image density drop, toner scattering, and fading was recognized for the durability of 200 sheets, and high-definition image quality faithful to the original document could not be obtained. , Tribo value was measured, but it was uneven at initial -24mC / kg and -18mC / kg after endurance, and showed unstable charging property. Moreover, in the fixability test, it was inferior to high temperature offset resistance.
[454] ManufacturabilityBurn densityQualityDaejeonFixability Capsule Toner A○○○○○ Capsule Toner B○△△○△
[455] Example 13 Preparation of Capsule Toner
[456] To 100 parts by mass of the PHA synthetase solution (10 u / ml) derived from the pYN2-C1 recombinant strain, 10 parts by mass of the core component fine particles of the toner produced in the same manner as in Example 12 and 390 parts by mass of PBS were added thereto, and 30 ° C. The mixture was shaken gently for 30 minutes at, and the PHA synthase was adsorbed onto the surface of the particulates. This was centrifuged (10,000 × g, 4 ° C. for 10 minutes), and the precipitate was suspended in a PBS solution and again centrifuged (10,000 × g, 4 ° C. for 10 minutes) to obtain an immobilized enzyme.
[457] 10 parts by mass of the immobilized enzyme was suspended in 480 parts by mass of 0.1 M phosphate buffer (pH 7. 0) and subjected to (R) -3-hydroxy-5- (4-fluorophenyl) bareryl CoA (Refor matsky reaction). After hydrolysis of the 3-hydroxy phenylvaleric acid ester obtained by to obtain 3-hydroxy-5- (4-fluorophenyl) valeric acid, Eur. J. Bio chem., 250, 432-439 (1997 10 mass parts of preparation) and 1 mass part of bovine serum albumin (made by Sigma) were added, and it stirred gently at 30 degreeC for 2 hours. After the completion of the reaction, the mixture was centrifuged at 10,000 x g for 10 minutes to recover the precipitate.
[458] This precipitate is then suspended in 480 parts by mass of 0.1 M phosphate buffer (pH 7. 0) and (R) -3-hydroxy-5-phenoxybareryl CoA (J. Org. Chem., 3-hydroxy-5 phenoxy obtained by Reformatsky reaction with zinc as a raw material using 3-phenoxy propanel and bromoethyl acetate synthesized by the method described in 55, 1490-1, 492 (1990). Hydrolysis of the civaleric acid ester to obtain 3-hydroxy-5-phenoxyvaleric acid, and then prepared by the method described in Eur. J. Bio chem., 250, 432-439 (1997)) 5 parts by mass, 1 mass part of bovine serum albumin (made by Sigma) was added, and it stirred gently at 30 degreeC for 30 minutes.
[459] After the completion of the reaction, the mixture was centrifuged at 10,000 x g for 10 minutes, the precipitate was suspended in 1000 parts by mass of 0, 1 M phosphate buffer (pH 7. 0), and the centrifugation was repeated three times to recover the precipitate. . From this, the capsule structure was obtained through each process of filtration and drying.
[460] The mass of the polymer formed on the surface of this capsule structure was measured by a time-of-flight secondary ion mass spectrometer (TOF-SIMS IV, manufactured by CAMECA). From the obtained mass spectrum, it was confirmed that the surface of the capsule structure was composed of poly-3-hydroxy-5-phenoxyvaleric acid. In addition, mass spectra were measured by TOF-SIMS while cutting the surface of the capsule structure little by little by ion spattering. At some point, the polymer constituting the capsule structure was poly-3-hydroxy 5- (4- It was confirmed that the fluorine phenyl) valeric acid is substituted. As a result, the capsule structure of the present embodiment was prepared by coating the core fine particles on a low poly-3-hydroxy-5- (4-fluorophenyl) valeric acid having a low glass transition temperature. It was found that it is a desired capsule structure coated with -hydroxy-5-phenoxy valeric acid.
[461] To 10 parts by mass of this capsule structure, 0.12 parts by mass of hydrophobic titanium oxide fine powder was added to obtain a capsule toner C having titanium oxide fine powder on the surface of the capsule structure.
[462] 144 mass parts of ferrite carriers coated with acrylic resin were mixed with 6 mass parts of this capsule toner to make a two-component developer.
[463] Using the developer described above, images are copied in a commercial copier NP6000 (manufactured by Canon Corporation) in the same manner as in Example 7, at 23 ° C and 60% RH, and subjected to image durability, toner scattering, and fading. It evaluated and showed good performance similarly to the capsule toner A of Example 7.
[464] In addition, in order to evaluate low-temperature fixability of the capsule toner A and the capsule toner C, a fixing test was performed by an external fixing unit having a fixing unit configuration such as NP6000. As a method of the fixing test, a sheet having a width of 2 cm and a length of 10 cm is made, and an unfixed image is fixed thereon by passing a roller along the length direction of the sheet while monitoring the temperature of the upper roller of the external fixing unit, For the fixed image obtained, fixability was judged whether or not an offset could be seen at the rear of the unit. As a result, it was found that the fixing start temperature was as low as 95 ° C and excellent in low temperature fixability, as in the capsule toner A and the capsule toner C.
[465] In addition, in order to evaluate the blocking resistance of the capsule toner A and the capsule toner C, the cohesion after leaving the capsule toner for 3 days at a temperature set at 1 ° C. to 50 to 70 ° C. was observed for each capsule toner. It developed as mentioned above and image evaluation was performed. And the blocking point of the change of the roughness state of a highlight area | region was made into. As a result, the blocking temperature of the capsule toner C was 61 ° C, the blocking temperature of the capsule toner A was 58 ° C, and the capsule toner C was excellent in blocking resistance.
[466] From the above, low temperature fixability can be realized by encapsulating the toner by PHA having a low glass transition temperature, and furthermore, a capsule coated with PHA having a low glass transition temperature is coated with PHA having a high glass transition temperature, thereby achieving low temperature fixability. It turned out that blocking resistance can be realized simultaneously.
[467] Example 14 Preparation of Capsule Toner
[468] Into a three-liter four-necked separabur fresco, a reflux condenser, a thermometer, a nitrogen inlet tube, a stirrer, 1200 parts of ion-exchanged water, 15 parts of polyvinyl alcohol, 0.1 part of sodium dodecyl sulfate, styrene monomer A mixture consisting of 75 parts, 25 parts of 1 n-butyl acrylate, 5 parts of di-tert-butyl chromium salt salt, 5 parts of copper phthalocyanine, and 6 parts of 2,2-azobis (2,4-zimethylvareronitrile) was added. Then, after stirring at 10,000 rpm for 10 minutes with the high speed stirring apparatus TK-homo mixer, and making particle composition, the rotation speed was decelerated at 1,000 rpm, and the ringing by nitrogen gas was fully performed. Next, the stirring apparatus was changed into the crescent-shaped stirring blade, and it superposed | polymerized for 16 hours in the oil bath heated at 80 degreeC with gentle stirring.
[469] After completion | finish of a polymerization reaction, after cooling a reaction container to room temperature, the dispersion liquid was wash | cleaned by 5 times of decantation, and it filtered, washed with water and dried, and obtained core particle which is blue powder. The PHA synthetase derived from pYN2-C1 recombinant strain was immobilized to the core particles in the same manner as in Example 12 to obtain an immobilized enzyme.
[470] 10 parts by mass of the immobilized enzyme was suspended in 480 parts by mass of 0.1 M phosphate buffer (pH 7. 0), and (R, S) -3-hydroxy-5-phenoxybareryl CoA (3-phenoxy propane) Hydrolysis of the 3-hydroxy-5-phenoxyvaleric acid ester obtained by Reformatsky reaction with ethyl bromoacetate to obtain 3-hydroxy-5-phenoxyvaleric acid, followed by Eur. J. Bio chem. , 250, 432-439 (1997)) 8 parts by mass, (R, S) -3-hydroxy-7, 8-epoxyoctanoyl CoA (Int J. Biol. Macromol., 12, 85-91 (1990), after the unsaturated moiety of 3-hydroxy-7-octenic acid synthesized by the method described in Ep. J. Biochem., 250, 432-439 ( 2 parts by mass) and 1 part by mass of bovine serum albumin (manufactured by Sigma) were added by the method described in 1997), and the mixture was shaken gently at 30 ° C for 2 hours.
[471] After completion of the reaction, the reaction solution was centrifuged (10,000 × g, 4 ° C. for 10 minutes) to recover the capsule structure, and the operation of suspending 100 parts by mass of purified water was repeated three times to recover the precipitate. From here, the blue capsule structure D was obtained through each process of filtration and drying. To 10 parts by mass of the capsule water tank D, 0.2 parts by mass of hydrophobic silica having a disintegrated pulverized BET value of 360 m ² / g was mixed with a Henschel mixer and added outside to give Toner D.
[472] In addition, 10 parts by mass of the capsule structure D was suspended in purified water, and 5 parts by mass of hexamethenediamine was dissolved as a crosslinking agent. After confirming dissolution, water was removed by lyophilization and reacted at 70 ° C for 12 hours. After completion of the reaction, the resultant was centrifuged at 10.000 × g for 10 minutes, and the precipitate was suspended in 1000 parts by mass of 0.1 M phosphate buffer (pH 7.0) and centrifuged again to recover the precipitate. From here, the blue capsule structure E was obtained through each process of filtration and drying. 0.2 mass parts of hydrophobic silica having a BET value of 360 m ² / g subjected to dismantling treatment was mixed with a Henschel mixer with respect to 10 mass parts of the capsule structure E10, and added outside to obtain Toner E.
[473] In order to perform the image evaluation of the produced toner, six parts of the toner obtained above were mixed and stirred by a tubular mixer with 94 parts of a carrier coated with a silicone resin on a ferrite cord having an average particle diameter of 35 μm. Made my Then, this developer is mounted on the color laser copier CLC-500 (manufactured by Canon Co., Ltd.), and observes the image after the initial and 10,000 copies under 23 ° C, 60% RH environment by SEM, Evaluation and the deterioration state of the developer were evaluated.
[474] As evaluation of the image quality, the reproducibility of the microscopic spots was evaluated by microscopic observation by recording a large number of values by pulse width modulation (PWM) of the laser in one pixel. Moreover, the developer after 10,000 copies was observed with the scanning electron microscope.
[475] In order to evaluate the blocking resistance of the toner, the cohesiveness after leaving the toner obtained above for 3 days under various temperature conditions was observed, and each toner composition was developed in the same manner using the carrier described above to perform image evaluation. did.
[476] Moreover, as evaluation of fixability, the fixing test by the external fuser which has a fuser structure like CLC-500 was performed. As a method of the fixing test, a sheet having a width of 2 cm and a length of 10 cm is made, and the unfixed image thereon is fixed by passing a roller along the length direction of the sheet while monitoring the temperature of the upper roller of the external fixing unit, With respect to the obtained fixed image, fixability was judged whether or not an offset could be seen at the rear of the unit.
[477] In addition, the above evaluation was performed using the toner F used as it was, without encapsulating the core particles instead of the capsule structure. The results are shown in Table 7.
[478] QualityBlocking resistanceaccuracy Toner D◎○◎ Toner E◎◎◎ Toner D○○△
[479] ※ Remarks: Symbols in the table ◎: Very good, ○: Good, △: Slightly bad, ×: Bad
[480] (However, the above 0, Δ, × does not correspond to 0, △, × in the twelfth embodiment.)
[481] Example 15 Preparation of Capsule Toner 3
[482] Core particles were prepared in the same manner as in Example 14, and PHA synthetase derived from pYN2-C1 recombinant strain was immobilized to obtain an immobilized enzyme. 10 parts by mass of this immobilized enzyme is suspended in 480 parts by mass of 0.1 M phosphate buffer (pH 7. 0), and (R, S) -3-hydroxy-5-phenoxybareryl CoA (the same method as in Example 14). Prepared) 8 parts by mass, (R, S) -3-hydroxy-7,8-epoxy octanoyl CoA (prepared in the same manner as in Example 14), 2 parts by mass, and 1 part by mass of bovine serum albumin (manufactured by Sigma) It was added and shaken gently at 30 degreeC for 2 hours.
[483] After the completion of the reaction, the reaction solution was centrifuged (10.000 x g for 10 minutes), the capsule structure was recovered, and the operation of suspending 100 parts by mass of purified water was repeated three times to recover the precipitate. From here, the blue capsule structure G was obtained through each process of filtration and drying. To 10 parts by mass of the capsule structure G, 0.2 parts by mass of hydrophobic silica with a disintegrated BET value of 360 m ² / g was mixed with a Henschel mixer and added outside to give Toner G.
[484] Moreover, 100 mass parts of terminal amino modified polysiloxane (modified silicone oil TSF 4700, GE Toshiba Silicone Co., Ltd. product) were added to 10 mass parts of capsule structures G, and it was made to react at 70 degreeC for 2 hours. This was suspended in methanol and centrifuged (10.000 × g at 4 ° C. for 20 minutes), followed by washing and drying to obtain a blue capsule structure H having a graft chain of polysiloxane. 0.2 part by mass of hydrophobic silica having a disintegrated and ground BET value of 360 m ² / g was mixed with a Henschel mixer with respect to H10 parts by mass of the capsule structure, and added outside to give Toner H.
[485] The toner was evaluated in the same manner as in Example 14 to evaluate the image quality and the deteriorated state of the developer.
[486] Instead of the capsule structure, the same evaluation was performed using Toner 1, which was used as it is without encapsulating the core particles. The results are shown in Table 8.
[487] QualityBlocking resistanceaccuracy Toner G◎○◎ Toner H◎◎◎ Toner I○○△
[488] ※ Remarks: Symbols in the table ◎: Very good, ○: Good, △: Slightly bad, ×: Bad
[489] (However, the above 0, Δ, × does not correspond to 0, △, × in the twelfth embodiment.)
[490] Example 16 Production of a Recording Medium
[491] PET film (100Q80D, product made by Toray Co., Ltd.) was immersed in 1% glutaraldehyde solution for 1 hour, and after washing with pure water, in PHA synthase solution derived from pYN2-C1 recombinant strain (10U / ml) at 30 ° C. It was immersed for 30 minutes and the said enzyme was immobilized. Unreacted PHA synthase was washed with PBS to remove the immobilized enzyme.
[492] 30 mM (R) -3-hydroxypimeryl CoA (prepared by the method described in J. Bacteriol., 1982, 2753-2760 (2000)), 0. 0% containing bovine serum albumin (manufactured by Sigma). The PET film was immersed in 1 M phosphate buffer (pH 7.0) and gently shaken at 30 ° C for 30 minutes. After the completion of the reaction, the resultant was washed with 0.1 M phosphate buffer (pH 7. 0), and unreacted substances were removed, followed by air drying, whereby poly- (R) -3-hydroxypimeric acid, an ionic polymer, was used as the ink receiving layer. A desired recording medium was obtained. As a comparative control, a PET film subjected to the same treatment in a system containing only PHA synthetase derived from the pYN2-C1 recombinant strain was produced.
[493] For these recording media, black and color images were printed using an inkjet printer (BJC-4301, manufactured by Canon Corporation), and ink bleeding and beading were confirmed, respectively. As a result, the smearing and the beading were hardly recognized in the recording medium of the present invention because the smearing and the beading were recognized very much in comparison and contrast, and an excellent image was obtained.
[494] Example 17 Production of a Recording Medium
[495] As in Example 16, PET film (100Q80D, manufactured by Toray Co., Ltd.) was immersed in 1% glutaraldehyde solution for 1 hour, and after washing with pure water, PHA synthase solution derived from pYN2-C1 recombinant strain (10U / ml ) Was immersed at 30 ° C. for 30 minutes to fix the enzyme. Unreacted PHA synthase was washed with PBS to remove the immobilized enzyme.
[496] PET film immobilized with this enzyme was prepared by 30 mM (R) -3-hydroxyoctanoyl CoA (prepared by the method described in Eur. J. Biochem., 250, 432-439 (1997)), 0.1% bovine serum albumin ( Sigma Co., Ltd.) was immersed in 100 parts by mass of 0.1 M phosphate buffer (pH 7. 0). Then, 30 mM (R) -3-hydroxypimeryl CoA (prepared by the method described in J. Bacteriol, 182, 2753-2760 (2000)), 0.1% 0.1 M phosphate buffer (pH 7.0) containing serum albumin (Sigma) was added at a rate of 25 parts by mass using a microtube pump (MP-3N manufactured by Tokyo Rikagi Co., Ltd.) in 1 minute.
[497] After shaking for 30 minutes, it was washed with 0.1M phosphate buffer (pH 7.0), and unreacted materials were removed, followed by air drying to prepare a recording medium.
[498] The mass of the polymer formed on the surface of the recording medium was measured by a time-of-flight secondary ion mass spectrometer (TOF-SIMS IV, manufactured by CAMECA). From the obtained mask vector, it was found that the recording medium surface is composed of a copolymer of 3-hydroxypimelic acid and 3-hydroxyoctanoic acid (molar ratio 17: 1). Moreover, the mask vector was measured by TOFR-SIMS similarly by scraping off the glass sheet surface little by little by ion sputtering, and the composition ratio of 3-hydroxypimelic acid in the said copolymer gradually decreased, and 3- The composition ratio of hydroxyoctanoic acid increased. From this, the recording medium of this embodiment is coated with a polyhydroxy pimelate having a hydrophilic functional group, and the surface thereof is 3-hydroxy pimelic acid having a hydrophilic functional group and a 3-hydroxyoctanoic acid having a hydrophobic functional group. The copolymer was found to be a desired recording medium in which the coated layer was used as the ink receiving layer while increasing the composition ratio of 3-hydroxyoctanoic acid as it reached the lower layer.
[499] This recording medium was subjected to an inkjet printer (BJC-4301, manufactured by Canon Corporation) in the same manner as in Example 16, to print black and color images, and to check for ink bleeding and beading, respectively. Comparison was made with the recording medium produced in Example 16. As a result, it was found that the recording medium of this embodiment is less bleeding in the ink receiving layer than in the recording medium of Example 16, and a clearer image can be obtained. This is considered to be because in the recording medium of this embodiment, since the PHA film becomes hydrophobic as it reaches the lower layer, diffusion of ink absorbed from the surface into the ink receiving layer is gradually suppressed.
[500] If the structure of the present invention is a particulate structure, for example, when used for an electrophotographic toner, it is possible to obtain a high quality image with little blurring and good fixability. On the other hand, when a flat or film-like structure is used as the recording medium, a good image without blurring can be obtained.

权利要求:
Claims (46)
[1" claim-type="Currently amended] In a structure having a substrate and a polyhydroxyalkanoate, at least a portion of the substrate is covered with the polyhydroxyalkanoate, and the polyhydroxyalkanoate is a 3-hydroxypropionic acid unit, 3-hydroxy A structure characterized by containing a 3-hydroxyalkanoic acid unit excluding an -n-butyric acid unit and a 3-hydroxy-n-yl acetic acid unit.
[2" claim-type="Currently amended] The structure according to claim 1, wherein the polyhydroxyalkanoate is a polyhydroxyalkanoate containing at least one selected from the group consisting of monomer units represented by the following formulas [1] to [10].

(However, this monomer unit is at least one selected from the group consisting of monomer units wherein the combination of R1 and a is any one of the following.
R 1 is a hydrogen atom (H) and a is a monomer unit in which any one of an integer of 3 to 10;
A monomer unit wherein R 1 is a halogen atom and a is any one of integers from 1 to 10;
A monomer unit in which R 1 is a chromophore and a is any integer of 1 to 10;
A monomer unit wherein R1 is a carboxy group or a salt thereof and a is any integer of 1 to 10;
R1 is

Wherein a is any one of integers from 1 to 7.

(Wherein b represents any one of integers from 0 to 7, and R2 represents a hydrogen atom (H), a halogen atom, -CN, NO ₂ , -CF ₃ -C ₂ F _5, -C ₃ F ₇ ) Any one selected from the group consisting of :)

(Wherein C represents any one of integers from 1 to 8, and R3 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F Any one selected from the group consisting of ₇ ).

(Wherein d represents any one of integers from 0 to 7, and R4 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F Any one selected from the group consisting of ₇ ).

(Wherein e represents any one of integers of 1 to 8, and R5 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F ₇ , -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ Any one selected from the group consisting of:

(Wherein f represents one of integers of 0 to 7).

(Wherein g represents any one of integers of 1 to 8.)

(Wherein h represents any one of integers from 1 to 7, R6 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -COOR ', -SO ₂ R ", -CH ₃ , Any one selected from the group consisting of -C ₂ H ₅ , -C ₃ H ₇ , -CH (CH ₃ ) ₂ , -C (CH ₃ ) ₃ ,
Wherein R 'is one of hydrogen atom (H), Na, K, -CH ₃ , -C ₂ H ₅ , and R' is -OH, -ONa, -OK, halogen atom, -OCH ₃ , -OC ₂ Is any one of H _5. )

(Wherein i represents any one of integers from 1 to 7, and R7 is selected from the group consisting of hydrogen atom (H), halogen atom, -CN, -NO ₂ , -COOR ', -SO ₂ R ") Which one to display,
Where R 'is one of hydrogen atom (H), Na, K, -CH ₃ , -C ₂ H ₅ , and R' is -OH, -ONa, -OK, halogen atom, -OCH ₃ , -OC ₂ Is any one of H _5. )

(Wherein j represents any one of integers from 1 to 9.)
[3" claim-type="Currently amended] The structure of claim 1, wherein at least a portion of the polyhydroxyalkanoate is a chemically modified polyhydroxyalkanoate.
[4" claim-type="Currently amended] The structure according to claim 3, wherein the chemical formula is a graft chain.
[5" claim-type="Currently amended] The structure according to claim 4, wherein the graft chain is a graft chain formed by a chemical formula of polyhydroxyalkanoate containing at least a monomer unit having an epoxy group of polyhydroxyalkanoate.
[6" claim-type="Currently amended] The structure according to claim 4, wherein the graft chain is a graft chain of a compound having an amino group.
[7" claim-type="Currently amended] The structure according to claim 6, wherein the compound having an amino group is a terminal amino modified compound.
[8" claim-type="Currently amended] 8. The structure of claim 7, wherein said terminal amino modified compound is selected from the group consisting of polyvinylamine, polyethyleneimine, and terminal amino modified polysiloxane.
[9" claim-type="Currently amended] 4. The structure of claim 3 wherein at least a portion of the polyhydroxyalkanoate is crosslinked polyhydroxyalkanoate.
[10" claim-type="Currently amended] 10. The structure according to claim 9, wherein the polyhydroxyalkanoate is crosslinked between monomer units having an epoxy group.
[11" claim-type="Currently amended] The structure according to claim 1, wherein the substrate is a granular body.
[12" claim-type="Currently amended] The structure according to claim 11, wherein the substrate contains a colorant.
[13" claim-type="Currently amended] 13. The structure according to claim 12, wherein the colorant contains a pigment.
[14" claim-type="Currently amended] The structure according to claim 12, wherein the colorant contains a dye.
[15" claim-type="Currently amended] 12. The structure of claim 11, wherein said substrate is a pigment.
[16" claim-type="Currently amended] The structure of claim 1, wherein the substrate is in the form of a plate or a film.
[17" claim-type="Currently amended] 12. The structure according to claim 11, wherein the monomer unit composition of the polyhydroxyalkanoate is changed in a direction from the inside to the outside of the structure.
[18" claim-type="Currently amended] The structure according to claim 16, wherein the monomer unit structure of the polyhydroxyalkanoate is changed in a direction perpendicular to the surface of the structure.
[19" claim-type="Currently amended] The structure according to claim 1, wherein the polyhydroxyalkanoate has a molecular weight of 1,000 to 10,000,000.
[20" claim-type="Currently amended] 20. The structure of claim 19, wherein the polyhydroxyalkanoate has a molecular weight of 3,000 to 1,000,000.
[21" claim-type="Currently amended] The structure according to claim 1, wherein a polyhydroxyalkanoate synthase is immobilized on the substrate.
[22" claim-type="Currently amended] Fixing a medium chain polyhydroxyalkanoate synthase to the substrate;
Coating at least a part of the substrate with polyhydroxyalkanoate by polymerizing 3-hydroxyacylboenzyme A with the enzyme to synthesize polyhydroxyalkanoate;
Method for producing a structure comprising a.
[23" claim-type="Currently amended] 23. The polyhydroxyalkanoate according to claim 22, wherein said polyhydroxyalkanoate is a polyhydroxyalkanoate containing at least one selected from the group consisting of monomer units represented by formula [1] to formula [10]. A method for producing a construct, wherein the corresponding 3-hydroxyacyl coenzyme A is 3-hydroxyacyl coenzyme A represented by the formulas [11] to [20] in order.

(However, this monomer unit is at least one selected from the group consisting of monomer units in which the combination of R1 and a is any one of the following.
R 1 is a hydrogen atom (H), and a is a monomer unit in which any one of an integer of 3 to 10;
A monomer unit wherein R 1 is a halogen atom and a is any of 1 to 10 integers;
R 1 is a chromophore, and a is any one monomer unit having an integer of 1 to 10;
R 1 is a carboxyl group or a salt thereof, and a is a monomer unit wherein a is an integer of 1 to 10;
R1 is

Wherein a is any one of integers of 1 to 7.

(Wherein b represents any one of integers from 0 to 7, and R2 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ H ₅ , -C ₃ F Any one selected from the group consisting of ₇ ).

(Wherein C represents any one of integers of 1 to 8, and R3 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F Any one selected from the group consisting of ₇ ).

(Wherein d represents any one of integers from 0 to 7, and R4 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F Any one selected from the group consisting of ₇ ).

(Wherein e represents any one of integers of 1 to 8, and R5 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -CF ₃ , -C ₂ F ₅ , -C ₃ F) ₇ , -CH ₃ , -C ₂ H ₅ , -C ₃ H ₇ is selected from the group consisting of.

(Where f denotes any one of 0 to 7 integers.)

(Wherein g represents any one of integers of 1 to 8.)

(Wherein h represents any one of integers of 1 to 7, R6 represents a hydrogen atom (H), a halogen atom, -CN, -NO ₂ , -COOR ', -SO ₂ R ", -CH ₃ , Any one selected from the group consisting of -C ₂ H ₅ , -C ₃ H ₇ , -CH (CH ₃ ) ₂ , -C (CH ₃ ) ₃ ,
Where R 'is one of hydrogen atom (H), Na, K, -CH ₃ , -C ₂ H ₅ , and R' is -OH, -ONa, -OK, halogen atom, -OCH ₃ , -OC ₂ Is any one of H _5. )

(Wherein i represents any one of integers of 1 to 7, and R7 is selected from the group consisting of hydrogen atom (H), halogen atom, -CN, -NO ₂ , -COOR ', -SO ₂ R ") Which one to display,
Where R 'is one of hydrogen atom (H), Na, K, -CH ₃ , -C ₂ H ₅ , and R' is -OH, -ONa, -OK, halogen atom, -OCH ₃ , -OC ₂ Is any one of H _5. )

(Wherein j represents any one of 1 to 9)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and R1 and a are the same as in formula [1].)

(Wherein, -SCoA represents coenzyme A bound to alkanoic acid, and R2 and b are as shown in the formula [2].)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and R3 and c are as defined in formula [3].)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and R4 and d are the same as those in the monomer unit represented by formula [4].)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and R5 and e are the same as those in the monomer unit represented by formula [5].)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and f is the same as that in the monomer unit represented by formula [6].)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and g is the same as that in the monomer unit represented by formula [7].)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and R6 and h are the same as those in the monomer unit represented by formula [8].)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and R7 and i are the same as those in the monomer unit represented by formula [9].)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and j is the same as that in the monomer unit represented by formula [10].)
[24" claim-type="Currently amended] 23. The method of manufacturing a structure according to claim 22, further comprising a step of chemically modifying at least a portion of the polyhydroxyalkanoate covering the substrate.
[25" claim-type="Currently amended] The method for producing a structure according to claim 24, wherein the step of performing chemical modification is a step of adding a graft chain to at least a part of the polyhydroxyalkanoate.
[26" claim-type="Currently amended] The method for producing a structure according to claim 25, wherein the step of adding the graft chain is a step of reacting at least a part of the polyhydroxyalkanoate with a compound having a reactive functional group at its terminal.
[27" claim-type="Currently amended] The method for producing a structure according to claim 24, wherein the step of carrying out the chemical modification is a step of crosslinking at least a part of the polyhydroxyalkanoate.
[28" claim-type="Currently amended] 28. The method of claim 27, wherein the crosslinking step is a step of reacting at least a portion of the polyhydroxyalkanoate with a crosslinking agent.
[29" claim-type="Currently amended] 29. The method of claim 28, wherein the crosslinking agent is selected from the group consisting of diamine compounds, succinic anhydride, 2-methyl-4-methylimidazole.
[30" claim-type="Currently amended] 30. The method of claim 29, wherein the diamine compound is hexamethylenediamine.
[31" claim-type="Currently amended] The method for producing a structure according to claim 27, wherein the crosslinking step is a step of irradiating an electron beam to the polyhydroxyalkanoate.
[32" claim-type="Currently amended] 23. The structure according to claim 22, wherein the monomer unit composition of the polyhydroxyalkanoate is changed in a direction from the inside to the outside of the structure by changing the composition of the 3-hydroxyacylcoenzyme A over time. Manufacturing method.
[33" claim-type="Currently amended] The method for producing a construct according to claim 22, wherein said polyhydroxyalkanoate synthase is produced using a microorganism having a production capacity of this enzyme.
[34" claim-type="Currently amended] 23. The method for producing a construct according to claim 22, wherein said polyhydroxyalkanoate synthase is produced by a transformant having a gene involved in the production capacity of the enzyme.
[35" claim-type="Currently amended] 35. The method of claim 34, wherein the gene is a gene obtained from a microorganism having a production capacity of a polyhydroxyalkanoate synthase.
[36" claim-type="Currently amended] The method of claim 33, wherein the microorganism is Pseudomonas Sp.
[37" claim-type="Currently amended] The method of claim 36, wherein the microorganism belonging to the Pseudomonas Sp.Pseudomonas putida P91, FERM BP-7373, Pseudomonas cichorii H45 strain (Pseudomonas cichorii H45, FERM BP-7374), Pseudomonas chi Korea is at least one microorganism selected from the group consisting of Pseudomonas cichorii YN2, FERM BP-7375, Pseudomonas jessenii P161, FERM BP-7376 .
[38" claim-type="Currently amended] 34. The method of claim 33, wherein the microorganism is a microorganism belonging to the genus Burkholdreia sp.
[39" claim-type="Currently amended] 39. The microorganism of claim 38, wherein the microorganism of the genus Berkholderia is composed of the OK3 strain of the Berkholdereria (Burkholdreia sp. At least one microorganism selected from the group.
[40" claim-type="Currently amended] 35. The method of claim 34, wherein the host microorganism of the transformant having the ability to produce the polyhydroxyalkanoate synthase is Escherichia coli.
[41" claim-type="Currently amended] A substrate containing a pigment; And
Having a polyhydroxyalkanoate containing 3-hydroxyalkanoic acid except 3-hydroxypropionic acid unit, 3-hydroxy-n-butyric acid unit, and 3-hydroxy-n-yl acetic acid unit,
At least partially coated with a polyhydroxyalkanoate.
[42" claim-type="Currently amended] A substrate containing a pigment; And
Having a polyhydroxyalkanoate containing 3-hydroxyalkanoic acid except 3-hydroxypropionic acid unit, 3-hydroxy-n-butyric acid unit, and 3-hydroxy-n-yl acetic acid unit,
And the substrate is at least partially coated with polyhydroxyalkanoate.
[43" claim-type="Currently amended] Preparing a particulate substrate;
Coating at least a portion of the substrate with polyhydroxyalkanoate to obtain a particulate construct; And
Manufacturing a toner using the granular structure;
Toner manufacturing method comprising a.
[44" claim-type="Currently amended] Preparing a base material which is a flat plate or a film; And
Coating at least a portion of the substrate with polyhydroxyalkanoate to produce a plate structure;
Method of manufacturing a recording medium comprising a.
[45" claim-type="Currently amended] An image forming method, comprising applying the toner according to claim 41 to a recording medium.
[46" claim-type="Currently amended] An image forming apparatus comprising means for applying the toner according to claim 41 to a recording medium to form an image.

类似技术:

---

公开号 | 公开日 | 专利标题

---

CN104423185B|2018-07-06|Toner

---

Zhuang et al.2013|Multi-stimuli responsive macromolecules and their assemblies

---

Cao et al.2016|Preparation and characterization of immobilized lipase from Pseudomonas cepacia onto magnetic cellulose nanocrystals

---

Cai et al.2013|MUC-1 aptamer-conjugated dye-doped silica nanoparticles for MCF-7 cells detection

---

Lomas et al.2011|Polymersome‐loaded capsules for controlled release of DNA

---

Jelinek et al.2013|Polydiacetylenes–recent molecular advances and applications

---

KR101566070B1|2015-11-04|Toner

---

Lutz2007|1, 3‐Dipolar cycloadditions of azides and alkynes: a universal ligation tool in polymer and materials science

---

Wu et al.2015|Surface-initiated controlled radical polymerizations from silica nanoparticles, gold nanocrystals, and bionanoparticles

---

US8008379B2|2011-08-30|Fluorescent polymer fine particle, method for forming thereof, fluorescence detection kit, and method for detecting fluorescence

---

Herdt et al.2007|Encapsulated magnetic nanoparticles as supports for proteins and recyclable biocatalysts

---

Landfester et al.2010|Encapsulation by miniemulsion polymerization

---

CN101004563B|2011-03-30|Electrophotographic toner and electrophotographic developer and image forming method

---

Nayak et al.2005|Soft nanotechnology with soft nanoparticles

---

JP2536995B2|1996-09-25|Enzyme immobilization method

---

CN101727030B|2013-09-25|Toner compositions and processes

---

Park et al.2007|Preparation and characterization of polyion complex micelles with a novel thermosensitive poly | shell via the complexation of oppositely charged block ionomers

---

US5834121A|1998-11-10|Composite magnetic beads

---

US7615233B2|2009-11-10|Particulate construct comprising polyhydroxyalkanoate and method for producing it

---

Cao et al.2006|Preparation of silica encapsulated quantum dot encoded beads for multiplex assay and its properties

---

Numata et al.2009|Bioengineered silk protein-based gene delivery systems

---

CN1614520B|2010-04-14|Color toner and two-component developer

---

Heyes et al.2007|Synthesis, patterning and applications of star-shaped poly | biofunctionalized surfaces

---

Blasco et al.2014|A novel photoresponsive azobenzene-containing miktoarm star polymer: Self-assembly and photoresponse properties

---

US7354995B2|2008-04-08|Magnetic substance-biosubstance complex structure, peptide fragment capable of linking to magnetic substance and gene therefor, and process for producing the complex structure

同族专利:

---

公开号 | 公开日

---

JP3684175B2|2005-08-17|

---

US20030104302A1|2003-06-05|

---

EP1253160A2|2002-10-30|

---

KR100555993B1|2006-03-03|

---

DE60239244D1|2011-04-07|

---

EP1253160B1|2011-02-23|

---

JP2003011312A|2003-01-15|

---

EP1253160A3|2003-10-22|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

法律状态:
2001-04-27|Priority to JP2001131694

---

2001-04-27|Priority to JPJP-P-2001-00131694

---

2001-07-10|Priority to JP2001208704A

---

2001-07-10|Priority to JPJP-P-2001-00208704

---

2002-04-27|Application filed by 캐논 가부시끼가이샤

---

2002-11-02|Publication of KR20020083519A

---

2006-03-03|Application granted

---

2006-03-03|Publication of KR100555993B1

优先权:

---

申请号 | 申请日 | 专利标题

---

JP2001131694|2001-04-27|

---

JPJP-P-2001-00131694|2001-04-27|

---

JP2001208704A|JP3684175B2|2001-04-27|2001-07-10|Structure and manufacturing method thereof|

---

JPJP-P-2001-00208704|2001-07-10|