JP4510842B2 - Polyhydroxyalkanoate-coated liposome - Google Patents

Description

  The present invention relates to a polyhydroxyalkanoate-coated liposome having excellent biocompatibility, high membrane stability, and controlled sustained release of a supported substance, and a method for producing the same.

  Liposomes formed when lipids are dispersed in water have a very close approximation as a model of cell membrane structure, and are used as pharmaceutical materials such as tissue-oriented drug carriers, artificial red blood cells, cell repair agents, and enzyme-immobilized bases. Is expected to be actively used. Further, not only in the medical and pharmaceutical fields, but also in the cosmetics field, it is expected as a material that allows an active ingredient having a problem in stability to be selectively transferred to an affected area to provide sustained release. Liposomes have such a wide range of application fields, but as a problem, the vulnerability of the membrane structure has been pointed out. That is, the membrane is broken by the chemical or physical change of the lipid, which is a film-forming substance, and the retention substance is likely to leak, which is still insufficient in terms of improving the drug reachability to the target tissue. Therefore, as a method for strengthening the liposome membrane, there are conventionally known methods such as adding sphingomyelin to the membrane to impart and strengthen hydrogen bonding, and adding tocopherol or the like to prevent oxidation of unsaturated lipids. .

  There is also a need for liposomes with high liposome membrane stability and for producing liposomes that are excellent in stabilizing the liposome membrane, being excellent in drug retention inside, and achieving drug release to the target tissue.

  Conventionally, in order to improve the mechanical strength of liposomes under physiological conditions and to provide sustained release, JP-A 06-178930 discloses that the surface of the membrane is a homopolymer of 2-methacryloyloxyethyl phosphorylcholine or 2 A liposome membrane coated with a copolymer of methacryloyloxyethyl phosphorylcholine and a monomer has been proposed.

  JP-A 06-298638 proposes a liposome characterized in that it is coated with a sterol and / or sterol glucoside such as sitosterol, campesterol, stigmasterol, or brassicasterol.

On the other hand, as an example of a drug-encapsulated capsule using polyhydroxyalkanoate as a biodegradable / biocompatible material other than phospholipid, USP614665 includes a porous granule made of polyhydroxyalkanoate. A method for producing a pharmaceutical composition is disclosed in which fine particles encapsulating a hydrophilic drug and oil droplets in which a lipophilic drug is dissolved as a core material are encapsulated in a shell made of polyhydroxyalkanoate.
Japanese Patent Laid-Open No. 06-178930 JP 06-298638 A U.S. Pat.No. 614665

  A liposome coated with a homopolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) or a copolymer of 2-methacryloyloxyethyl phosphorylcholine and a monomer disclosed in Japanese Patent Application Laid-Open No. 06-178930 does not Although the mechanical strength is improved, it is not always sufficient and is not excellent in biocompatibility.

  USP614665 discloses a fine particle in which a hydrophilic drug is captured in a porous granule made of polyhydroxyalkanoate, which is non-toxic, biodegradable, and capable of capturing the drug in situ, but has a porous structure. For this reason, the hydrophilic drug is quickly released by diffusion, and it is difficult to control the sustained release.

  That is, the problem to be solved by the present invention is to combine the drug retention ability / sustained release properties of liposomes with the mechanical strength of polyhydroxyalkanoates, and to hydrophilic drugs, other water-soluble substances, lipophilic drugs, and other hydrophobic substances. However, it is to provide drug holding particles that are excellent in holding properties and can control sustained release properties.

  A means for solving the above-mentioned problems is to provide a polyhydroxyalkanoate-coated liposome characterized by having a structure in which at least a part of the outer wall is coated with polyhydroxyalkanoate.

  As described above, the PHA-coated liposome of the present invention is excellent in biocompatibility, has high membrane stability, and can control the sustained release property of the supported substance. The PHA-coated liposome of the present invention has various uses such as pigment-encapsulated liposome, dye-encapsulated liposome, agricultural chemical-encapsulated liposome, hemoglobin-encapsulated liposome, cosmetic component-encapsulated liposome, fertilizer component-encapsulated liposome, and drug component-encapsulated liposome. Available to:

  In the present invention, liposomes are monolayer liposomes and multilamellar liposomes composed of lipids, particularly phospholipids alone, or composed of phospholipids and sterols, and conventionally known methods can be used as production methods thereof.

  The substance carried on the liposome may be either a water-soluble substance or a fat-soluble substance. However, the water-soluble substance is accommodated in the liposome lumen, and the fat-soluble substance is accommodated in the liposome membrane. In addition, these substances may be adsorbed chemically and physically on the surface of the liposome membrane. In the present invention, the above three cases, that is, “intraluminal retention”, “containment in membrane” and “membrane surface” “Holding” is also referred to as “carrying”. In addition to drugs that are unstable in vitro, in vivo, gradually released in the body, or desired to be quickly distributed to specific organs, such substances include markers, plasmids, DNA, RNA There is no particular limitation as long as it is effective when administered in vivo.

  The present inventors immobilized polyhydroxyalkanoate synthase on the liposome membrane, added 3-hydroxyacyl coenzyme A to the reaction, and thereby allowed to coat the liposome with polyhydroxyalkanoate. By selecting an appropriate type of 3-hydroxyacyl coenzyme A, it is possible to coat the liposome envelope with a non-toxic and biodegradable polyhydroxyalkanoate, and at least one of the membranes with polyhydroxyalkanoate. It has been found that the liposome coated with a part has enhanced physical and mechanical strength, and can impart the characteristics that the sustained release property of the encapsulated substance can be controlled, thereby completing the present invention.

That is , the polyhydroxyalkanoate-coated liposome of the present invention has
Having polyhydroxyalkanoate synthase on the surface of at least a part of the outer wall of the liposome ;
A polyhydroxyalkanoate-coated liposome characterized by being coated with a plurality of layers of the polyhydroxyalkanoate having different compositions in the direction from the inside to the outside of the liposome.

Polyhydroxyalkanoate-coated liposome of the present invention is a polyhydroxyalkanoate-coated liposome obtained by coating at least part of an outer wall by polyhydroxyalkanoate, a step of dispersing the liposome in an aqueous medium, is dispersed in an aqueous medium A step of immobilizing polyhydroxyalkanoate synthase on the surface of the liposome, a step of adding 3-hydroxyacyl CoA as a substrate, and a polyhydroxyalkanoate synthesis reaction cause at least a part of the liposome surface to be polymerized. a step of coating a hydroxy alkanoate, may be produced by a production method characterized by having a.

  The liposome in the present invention has a constitution in which at least a part of the surface is coated with polyhydroxyalkanoate, and the entire surface does not necessarily have to be coated within a range in which the desired properties of the liposome can be obtained. In a state where the entire surface is coated, a polyhydroxyalkanoate-coated microcapsule having a liquid as a core and a polyhydroxyalkanoate coating layer as a shell can be obtained.

  Hereinafter, the present invention will be described in more detail.

  Prior liposomes coated with polyhydroxyalkanoate to be used in the polyhydroxyalkanoate-coated liposome of the present invention can be prepared by a conventional production method generally known as a method for producing liposomes. The phospholipid used here is phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), sphingomyelin (SPM), etc. Several types can be used in combination. The phospholipid can also include hydrogenated phospholipids or enzyme-treated phospholipids. The phospholipid may be synthesized, may be obtained from natural products such as eggs and soybeans, or may be commercially available.

  Cholesterol and phospholipid can also be combined for the purpose of adjusting the hardness and permeability of a lipid bilayer made of phospholipid. The ratio of cholesterol and phospholipid used can vary depending on the lipophilicity of the encapsulated substance, but the cholesterol: phospholipid molar ratio can be exemplified as 1: 3 to 1: 1. Liposomes having a lipid bilayer membrane composed of the phospholipid as a shell can be prepared by a conventional production method generally known as a method for producing liposomes. That is, as a known technique, the method of Bangham (J. Mol. Biol., 13, 238 (1965)), etc., and its modified method (Japanese Patent Laid-Open No. 52-1114013). , JP-A-59-173133, JP-A-2-13929, JP-A-7-241487, ultrasonic treatment (Biochemical and Biophysical Research Communication (Biochem. Biophys. Res. Commun., 94, 1367 (1980)), ethanol injection method (J. Cell. Biol., 66, 621 (1975)), French press method (FEBS Lett .), 99, 210 (1979)), ether injection method (New York Academy Science (NY Acad. Sci., 308, 250 (1978))), cholic acid (surfactant) method (Biokimika et Biophysica)・ Actor (Biochim. Biophys. Acta, 455, 322 (1976)), calcium fusion method (Biochim. Biophys. Acta, 394, 483 (1978)), freeze-thaw method (Archives of biochemistry, And Biophysics (Arch. Biochem. Biophys.), 212, 186 (1981)), reverse phase evaporation (Proceeding of the National Academy of Sciences USA) (Pro. NAS USA, 75, 4194 (1978)), a method using a glass filter (ThirdUS-Japan Symposium on Drug Delivery System (1995)), a method using a commercially available kit such as coatsome, etc. These known methods Any of the liposomes (liposomes) prepared by the above can be used in the present invention.

  The liposome preparation method reported by Bangham et al. Is that phospholipid is dissolved in an organic solvent such as chloroform, placed in an eggplant type flask, and the solvent is removed under reduced pressure using a rotary evaporator. A thin film is formed. When an aqueous buffer solution containing a substance to be supported is added to this and swollen with shaking, and then the film is mechanically peeled off using vortex or the like, multilamellar liposomes are formed. The swelling operation needs to be performed at a temperature about 10 ° C. higher than the phase transition temperature of the phospholipid. Multilamellar liposomes produced by this method have a particle size distribution of 0.2 to 5 μm, but if it is necessary to make the distribution as small as possible, shake more intensely, or 5 to 10 W What is necessary is just to irradiate a low output ultrasonic wave for a short time.

  The liposome preparation method by ultrasonic treatment is to irradiate a suspension of multilamellar liposomes prepared by the method of Bangham et al. With higher output ultrasonic waves. Ultrasonic irradiation gradually reduces the size and finally yields small unilamellar liposomes with a particle size of 20 to 30 nm. Ultrasonic irradiation tends to cause chemical decomposition of phospholipid molecules and oxidation of lipids by dissolved oxygen. Therefore, it is necessary to operate in an inert gas stream such as nitrogen or argon so that the temperature does not increase too much.

  The liposome production method by the ethanol injection method is a method in which lipid is dissolved in ethanol and injected with a microsyringe into an aqueous buffer solution containing a substance to be supported at a phase inversion temperature or higher. The liposomes produced are small unilamellar liposomes whose size depends on the lipid concentration, with a particle size of about 30 nm at low lipid concentration (3 mM) and about 110 nm at high lipid concentration (36 mM). Are produced respectively. Since this method does not involve sonication, it is suitable for carrying unstable substances. However, since the liposome is diluted, a concentration operation such as ultrafiltration may be required. There are also disadvantages such as complete removal of ethanol from liposomes is difficult.

  The liposome production method by the French press method is a method in which a multilamellar liposome produced by the method of Bangham et al. Is put into a French press cell and extruded at a pressure of about 20,000 psi to form a single membrane liposome, and this operation is repeated. In other words, small unilamellar liposomes having a particle size of about 30 to 50 nm are formed. Since this method does not involve sonication, it is suitable for carrying unstable substances. When a low pressure is applied, single-membrane liposomes having a particle size of about 50 to 150 nm are formed.

  The liposome production method by the ether injection method is to dissolve phospholipids in diethyl ether or a mixed solvent of diethyl ether and methanol, and inject the solution into an aqueous buffer solution containing the substance to be supported, and remove the solvent. . The organic solvent is removed by heating the aqueous solution to 55 to 65 ° C. or heating to 30 ° C. under reduced pressure. Large unilamellar liposomes can be prepared by the ether injection method.

  The liposome preparation method by the cholic acid (surfactant) method is a lipid membrane membrane or multilamellar liposome prepared by the method of Bangham et al., Or a small unilamellar liposome prepared by other methods. A relatively large unilamellar liposome (30 to 180 nm) is produced by mixing with a surfactant such as an acid and removing the surfactant from the mixture. In order to remove the surfactant, a dialysis method or a gel filtration method can be used. The size of the liposome produced depends on the ratio of lipid to surfactant, the amount of cholesterol contained, the dialysis rate, and the like. This method has a problem of how to reduce the removal rate of the surfactant in the liposome bilayer. For this reason, it is necessary to repeat dialysis for a long time and several times of gel filtration.

  Liposome preparation by the calcium fusion method is to induce membrane fusion by adding calcium ions to liposomes containing acidic phospholipids and other methods, and then adding a chelating agent such as EDTA to remove calcium To produce large unilamellar liposomes (200 to 1000 nm). This method is limited to acidic phospholipids such as phosphatidylserine (PS) and phosphatidic acid (PA) and mixed liposomes thereof.

  The liposome preparation method by freeze-thawing is a method in which the sonicated liposome solution is frozen in liquid nitrogen, then allowed to thaw at room temperature, and then the resulting milky white suspension is held for a short period of sonication. Is to do. Large unilamellar liposomes having a particle size of 50 to 500 nm are prepared. There is an advantage that retention efficiency is improved due to the concentration effect during freezing.

  The liposome preparation method by the reverse phase evaporation method is to add water to a phospholipid ether solution and sonicate to form a w / o emulsion, then distill off the ether, and shake using a vortex. The phase is then reversed, and ether is removed under reduced pressure to produce large unilamellar liposomes (200 to 1000 nm). The size of the liposome is affected by the lipid composition and the ionic strength of the aqueous solution, and the addition of cholesterol increases the size, and the increase in ionic strength decreases the size. The substance carried on the liposome is appropriately selected depending on the use of the polyhydroxyalkanoate-coated liposome of the present invention.

  When the polyhydroxyalkanoate-coated liposome of the present invention is used as, for example, a liposome for an agrochemical composition, the substance carried by the liposome may be any substance described in the Agricultural Handbook (published by the Japan Plant Protection Association). Anything can be used. For example, carbamate insecticides, organophosphorus insecticides, pyrethroid insecticides, urea insecticides, anilide fungicides, azole fungicides, dicarboximide fungicides and the like can be exemplified. These can be used in combination of two or more as required. Liposomes carrying active ingredients are treated with ultrasonic treatment, ethanol injection, French press method, ether injection method, cholic acid (surfactant) method, calcium fusion method, freeze-thaw method, reverse phase evaporation method, glass filter. It can adjust by the above-mentioned method, such as the method used, the method using commercially available kits, such as coatsome.

  When the polyhydroxyalkanoate-coated liposome of the present invention is used as, for example, a liposome for a fertilizer composition, examples of substances supported on the liposome include ammonium sulfate, ammonium sulfate, ammonium nitrate, urea, acetaldehyde condensed urea, isobutyraldehyde condensed urea, and the like. Aqueous solution of nitrogenous fertilizer, aqueous solution of phosphate fertilizer such as superphosphate lime, heavy superphosphate, molten fertilizer, aqueous solution of fertilizer such as potassium sulfate, potassium chloride, fish cake, bone meal, soybean oil Water suspension of organic fertilizer such as dregs, rapeseed oil dregs, etc., aqueous solution of ternary complex fertilizers such as phosphorus ammonium and potassium phosphate, aqueous solution of trace element compound fertilizer, etc. Can be used in combination. Liposomes carrying fertilizer components use ultrasonic treatment, ethanol injection, French press method, ether injection method, cholic acid (surfactant) method, calcium fusion method, freeze-thaw method, reverse phase evaporation method, glass filter Or a method using a commercially available kit such as coatsome.

  When the polyhydroxyalkanoate-coated liposome of the present invention is used as, for example, a cosmetic liposome, the substances supported on the liposome include moisturizing ingredients, herbal extracts, tyrosinase, superoxide dismutase, lipase enzymes, retinol, ascorbine Moisture such as acids, vitamins such as tocopherol, pyridoxal, riboflavin, organic pigments such as β-carotene, chlorophyll, glycerin, sorbitol, urea, lactic acid, propylene glycol, polyethylene glycol and copolymers, glucose derivatives, etc. Ingredients, paraffin, stearyl alcohol, cetyl alcohol, squalane, silicone oil, stearis and other emollient ingredients, treatment ingredients, dandruff inhibiting ingredients, hair nourishing ingredients, hair restoration ingredients, External absorber, an antioxidant, can be mentioned perfumes, it may be used in combination of two or more as necessary. Liposomes carrying cosmetic ingredients are treated with ultrasonic treatment, ethanol injection, French press, ether injection, cholic acid (surfactant) method, calcium fusion method, freeze-thaw method, reverse phase evaporation method, glass filter. It can adjust by the above-mentioned method, such as the method used, the method using commercially available kits, such as coatsome.

  When the polyhydroxyalkanoate-coated liposome of the present invention is used as, for example, an artificial erythrocyte liposome, examples of the substance carried on the liposome include hemoglobin, hemocyanin, and the like. Can be used in combination. Liposomes carrying hemoglobin use ultrasonic treatment, ethanol injection, French press method, ether injection method, cholic acid (surfactant) method, calcium fusion method, freeze-thaw method, reverse phase evaporation method, glass filter Or a method using a commercially available kit such as coatsome.

  When the polyhydroxyalkanoate-coated liposome of the present invention is used as, for example, a liposome for ink or paint, examples of the substance supported on the liposome include aqueous dye solutions and pigment dispersions. I. Acid Red 52, C.I. I. Acid Blue 1, C.I. I. Acid Black 2, Acid Dye 123, etc., C.I. I. Basic Blue 7, C.I. I. Basic dyes such as basic red 1, C.I. I. Direct Black 19, C.I. I. Direct dyes such as direct blue 86 and the like, CI Solvent Black 7, 123, CI Solvent Red 8, 49, 100, CI Solvent Blue 2, 25, 55 70, CI Solvent Green 3, CI Solvent Yellow 21, 61, CI Solvent Orange 37, CI Solvent Violet 8, 21 and the like, oil-soluble dyes such as C.I. I. Reactive Yellow 15, 42; C.I. I. Reactive Red 24, 218; C.I. I. Reactive dyes such as reactive blue 38 and 220, carbon black, copper oxide, manganese dioxide, aniline black, activated carbon, non-magnetic ferrite, magnetite and other black pigments, yellow lead, zinc yellow, yellow iron oxide, cadmium yellow, mineral Yellow pigments such as Fast Yellow, Nickel Titanium Yellow, Navels Yellow, Naphthol Yellow S, Hanser Yellow G, Hansa Yellow 10G, Benzidine Yellow G, Benzidine Yellow GR, Quinoline Yellow Lake, Permanent Yellow NCG, Tartrazine Lake, Red Yellow Orange pigments such as lead, molybdenum orange, permanent orange GTR, pyrazolone orange, vulcan orange, benzidine orange G, indanthrene brilliant orange RK, indanthrene brilliant orange GK, Nkara, Cadmium Red Panda, Mercury Sulfide, Cadmium, Permanent Red 4R, Risor Red, Pyrazolone Red, Watching Red, Calcium Salt, Lake Red C, Lake Red D, Brilliant Carmine 6B, Brilliant Carmine 3B, Eoxin Lake, Rhodamine Lake B, red pigments such as alizarin lake, bitumen, cobalt blue, alkali blue lake, Victoria blue lake, phthalocyanine blue, metal-free phthalocyanine blue, phthalocyanine blue partially chlorinated compounds, blue pigments such as first sky blue, indanthrene blue BC, manganese Purple pigments such as purple, fast violet B, methyl violet lake, chromium oxide, chrome green, pigment green B, malachite green lake, finale -Green pigments such as green G, white pigments such as zinc white, titanium oxide, antimony white, zinc sulfide, and extender pigments such as barite powder, barium carbonate, clay, silica, white carbon, talc, and alumina white. Of course, it is not limited to these. These can be used in combination of two or more as required. Liposomes carrying ink components use ultrasonic treatment, ethanol injection, French press method, ether injection method, cholic acid (surfactant) method, calcium fusion method, freeze-thaw method, reverse phase evaporation method, glass filter Or a method using a commercially available kit such as coatsome.

  When the polyhydroxyalkanoate-coated liposome of the present invention is used as, for example, a drug sustained-release capsule, as a drug for a pharmaceutical preparation carried on the liposome, an easily water-soluble drug and a poorly water-soluble (lipid-soluble) drug are used. Both drugs can be mentioned. Examples of such drugs include sterols (eg cholesterol, sitosterol), estrogens (eg estrone, estradiol and esters thereof, ethinyl estradiol etc.), corticoids and esters thereof, peptide hormones such as calcitonin, antibiotics (eg gentamicin, vancomycin, Amikacin, kanamycin, streptomycin, minocycline, tetracycline, etc., chloramphenicol, macrolide antibiotics (eg erythromycin and its derivatives, especially its palmitate and stearate, or spiramycin), antiparasitic and dermatological agents ( Clotrimazole, miconazole, dithranol, etc.), anti-inflammatory analgesics (eg indomethacin, dithranol) Rofenakku, flurbiprofen, ketoprofen, and 4-biphenyl acetic acid and its ethyl ester), vitamins such as cyanocobalamin, enzymes, such as urokinase, fluorouracil, and the like carcinostatic system as Arashichijin. These can be used in combination of two or more as required. Liposomes carrying these drugs use ultrasonic treatment, ethanol injection, French press method, ether injection method, cholic acid (surfactant) method, calcium fusion method, freeze-thaw method, reverse phase evaporation method, glass filter Or a method using a commercially available kit such as coatsome. Liposomes prepared by the above method have physical and mechanical strength enhanced by covering at least a part of the outer wall with polyhydroxyalkanoate, biocompatibility and biodegradability are also imparted, and the liposomes are released slowly. If it has the characteristics, controlled release can be controlled. The method for coating the liposome with polyhydroxyalkanoate will be described below.

  <PHA> The PHA usable in the present invention is not particularly limited as long as it is a PHA that can be synthesized by a PHA synthase involved in the biosynthesis reaction of PHA.

  Here, the biosynthesis of PHA uses (R) -3-hydroxyacyl CoA produced from various alkanoic acids as raw materials through various metabolic pathways in the living body (for example, β-oxidation system and fatty acid synthesis pathway). It is carried out by a polymerization reaction using an enzyme as a substrate. The enzyme that catalyzes this polymerization reaction is PHA synthase (also called PHA polymerase or PHA synthase). CoA is an abbreviation for coenzyme A, and its chemical structure is as shown below.

  The following shows the reaction until the alkanoic acid becomes PHA through the polymerization reaction by the β-oxidation system and PHA synthase.

  On the other hand, when passing through the fatty acid synthesis pathway, (R) -3-hydroxyacyl converted from (R) -3-hydroxyacyl-ACP (ACP is an acyl carrier protein) produced in the pathway Similarly, PHA is thought to be synthesized by PHA synthase using CoA as a substrate.

  Furthermore, it is also known that the above-mentioned PHB synthase and PHA synthase can be taken out of the microbial cells and PHA can be synthesized in a cell-free system (in vitro), and there are the following examples. For example, in Proc.Natl.Acad.Sci.USA, 92, 6279-6283 (1995), 3-hydroxybutyryl CoA is allowed to act on PHB synthase derived from Alcaligenes eutrophus. We have succeeded in synthesizing PHB consisting of -n-butyric acid units. In Int. J. Biol. Macromol., 25, 55-60 (1999), by causing 3-hydroxybutyryl CoA or 3-hydroxyvaleryl CoA to act on the PHB synthase derived from Alcaligenes eutrophas, We have successfully synthesized PHA consisting of 3-hydroxy-n-butyric acid units and 3-hydroxy-n-valeric acid units. Furthermore, in this report, when racemic 3-hydroxybutyryl-CoA was allowed to act, PHB consisting only of R-form 3-hydroxy-n-butyric acid unit was synthesized by the stereoselectivity of the enzyme. Macromol. Rapid Commun., 21, 77-84 (2000) also reports extracellular PHB synthesis using a PHB synthase derived from Alkaligenes eutrophas. In FEMS Microbiol. Lett., 168, 319-324 (1998), 3-hydroxy-n-butyric acid is obtained by allowing 3-hydroxybutyryl CoA to act on a PHB synthase derived from Chromatium vinosum. It has succeeded in synthesizing PHB consisting of units. In Appl. Microbiol. Biotechnol., 54, 37-43 (2000), PHA consisting of 3-hydroxydecanoic acid units is allowed to act on PHA synthase of Pseudomonas aeruginosa by acting 3-hydroxydecanoyl CoA. Is synthesized.

As described above, the PHA synthase is an enzyme that catalyzes the final stage in the PHA synthesis reaction system in the living body, and therefore any PHA that is known to be synthesized in the living body can be used. It is synthesized under the catalytic action of. Therefore, any kind of PHA known to be synthesized in an organism by allowing 3-hydroxyacyl-CoA corresponding to the desired PHA to act on the enzyme immobilized on the substrate in the present invention. It is possible to make polyhydroxyalkanoate-coated liposomes.

Specific examples of PHA used in the present invention include PHA containing at least monomer units represented by the following formulas [1] to [10].

  (However, the monomer unit is at least one selected from the group consisting of monomer units in which the combination of R1 and a is any of the following:

  A monomer unit in which R1 is a hydrogen atom (H), a is any integer from 0 to 10, R1 is a halogen atom, a monomer unit in which a is any integer from 1 to 10, and R1 is colored A monomer unit in which a is an integer from 1 to 10, R1 is a carboxyl group or a salt thereof, and a monomer unit in which a is an integer from 1 to 10, R1 is

A monomer unit in which a is any integer from 1 to 7. )

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

(However, c in the formula represents any of integers from 1 to 8, R3 is hydrogen atom (H), a halogen _{atom, -CN, -NO 2, -CF 3} , -C 2 F 5 and -C ₃ F ₍ Represents any one selected from the group consisting of ₇ )

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

(Wherein in e represents any of integers from 1 to 8, R5 is a hydrogen atom (H), a halogen _{atom, -CN, -NO 2, -CF 3} , -C 2 F 5, -C 3 F ₇ represents any one selected from the group consisting of —CH ₃ , —C ₂ H ₅ and —C ₃ H _7. )

(In the formula, f represents any integer of 0 to 7.)

(In the formula, g represents an integer of 1 to 8.)

(In the formula, h represents an integer of 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, and —C (CH ₃ ) ₃ , wherein R ′ represents a hydrogen atom ( H), Na, K, —CH ₃ and —C ₂ H ₅ , and R ″ is any of —OH, —ONa, —OK, a halogen atom, —OCH ₃ and —OC ₂ H ₅ . .)

(Wherein i represents an integer of 1 to 7, and R7 is selected from the group consisting of a hydrogen atom (H), a halogen atom, —CN, —NO ₂ , —COOR ′, and —SO ₂ R ″). Any one selected, wherein R ′ is a hydrogen atom (H), Na, K, —CH ₃ , —C ₂ H ₅ , R ″ is —OH, —ONa, -OK, it is either a halogen atom, -OCH ₃ and -OC ₂ H _5.)

(In the formula, j represents an integer from 1 to 9.) Specific examples of the halogen atom include fluorine, chlorine, bromine and the like.

As 3-hydroxyacyl CoA used as a substrate for synthesizing the above PHA, specifically, there can be mentioned 3-hydroxyacyl CoA expressed by the formula [21] from the following formula [12].

(However, the formula -SCoA represents coenzyme A bound to an alkanoic acid, R1 and a in the formula is defined as in the formula [1].)

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

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

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and d and R4 are defined in the same manner as in the above formula [4].)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and e and R5 are defined in the same manner as in the above formula [5] .)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and f is defined in the same manner as in the above formula [6].)

(Wherein —SCoA represents coenzyme A bound to alkanoic acid, and g is defined in the same manner as in the above formula [7].)

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, and h and R6 are defined in the same manner as in the above formula [8].)

(Wherein, -SCoA represents coenzyme A bound to alkanoic acid, and i and R7 are defined in the same manner as in the above formula [9].)

(Wherein —SCoA represents coenzyme A bound to alkanoic acid, and j is defined in the same manner as in the above formula [10].)
In addition, when the polyhydroxyalkanoate-coated liposome of the present invention is used suspended in an aqueous medium, those having a hydrophilic functional group are used as PHA constituting the polyhydroxyalkanoate-coated liposome. Any hydrophilic functional group may be used, but an anionic functional group may be used, and any anionic functional group may be used, and in particular, a carboxyl group may be used. Examples of PHA having a carboxyl group include PHA containing at least one selected from monomer units represented by the following formula [11].

(Where k is an integer from 1 to 10)
Further, among the above PHA, more specifically, a PHA containing 3-hydroxypimelic acid represented by the following formula [23]

Can be illustrated.

  Examples of 3-hydroxyacyl CoA used as a substrate for synthesizing PHA represented by the above formula [11] include 3-hydroxyacyl CoA represented by the following formula [22].

(Wherein -SCoA represents coenzyme A bound to alkanoic acid, wherein k is at least one selected from the group consisting of the following, and in the formula [11]: Corresponds to k in the monomer unit represented, where k is an integer from 1 to 10.)
Further, as 3-hydroxyacyl CoA used as a substrate for synthesizing PHA containing 3-hydroxypimelic acid represented by the above formula [23], 3-hydroxypimeryl CoA represented by the following formula [24]

Can be shown.

  Specific examples of the halogen atom include fluorine, chlorine, bromine and the like. The chromophore is not particularly limited as long as the 3-hydroxyacyl CoA form can be catalyzed by PHA synthase. However, in consideration of steric hindrance during polymer synthesis, 3- In the hydroxyacyl CoA molecule, it is desirable that a methylene chain having 1 to 5 carbon atoms is present between the carboxyl group to which CoA is bonded and the chromophore. Moreover, if the light absorption wavelength of the chromophore is in the visible range, colored polyhydroxyalkanoate-coated liposomes can be obtained. Examples of such chromophores include nitroso, nitro, azo, diarylmethane, triarylmethane, xanthene, acridine, quinoline, methine, thiazole, indamine, indophenol, lactone, aminoketone, hydroxyketone, stilbene, azine, ocasazine, Examples include thiazine, anthraquinone, phthalocyanine, and indigoid.

  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 control the physical properties of the PHA using the characteristics of each monomer unit and the functional group contained therein. It is possible to give a plurality of functions, develop new functions using interactions between functional groups, and the like. Furthermore, a block copolymer having an arbitrary order and composition ratio can be synthesized on the liposome surface by appropriately controlling the amount and order of addition of 3-hydroxyacyl CoA as a substrate. If necessary, chemical modification or the like may be further performed after the synthesis of PHA or during the synthesis.

  For example, it is possible to change the monomer unit composition of PHA in the direction from the inside to the outside of the polyhydroxyalkanoate-coated liposome by changing the composition such as the type and concentration of 3-hydroxyacyl CoA as a substrate over time. Is possible. Thus, for example, when it is necessary to form a polyhydroxyalkanoate-coated liposome with PHA having a low affinity with the liposome, the liposome is first coated with PHA having a high affinity with the liposome, and the PHA having a high affinity with the liposome is then coated. By changing the monomer unit composition to the target PHA monomer unit composition in the stacking direction, for example, a multilayer structure or a gradient structure, it is possible to form a PHA film having a strong bond with liposomes.

  In addition, by subjecting the PHA to chemical modification, polyhydroxyalkanoate-coated liposomes with improved various characteristics can be obtained. For example, by introducing a graft chain into the PHA, a polyhydroxyalkanoate-containing structure in which at least a part of the liposome is coated with PHA having various characteristics resulting from the graft chain can be obtained. In addition, polyhydroxyalkanoate in which at least a part of the liposome is coated with PHA having desired physicochemical properties (for example, mechanical strength, chemical resistance, heat resistance, etc.) by crosslinking the PHA. A containing structure can be obtained. The chemical modification in the present invention refers to a chemical reaction of the polymer material by causing a chemical reaction within or between the molecules of the polymer material or between the polymer material and another chemical substance. This refers to modifying the molecular structure. Crosslinking refers to the formation of a network structure by chemically or physicochemically bonding within or between molecules of a polymer material, and the crosslinking agent refers to the crosslinking reaction. A substance having a certain reactivity with the polymer material, which is added in order to carry out the process.

  The PHA synthesized by the PHA synthase used in the structure of the present invention is generally an isotactic polymer composed only of the R isomer.

  3-hydroxyacyl-CoA, which is a synthetic substrate for PHA, is selected from, for example, in vitro synthesis methods using enzymes, in vivo synthesis methods using organisms such as microorganisms and plants, chemical synthesis methods, etc. Can be synthesized and used. In particular, the enzyme synthesis method is a method generally used for the synthesis of the substrate, and the following reaction using a commercially available acyl CoA synthetase (acyl CoA ligase, E.C.6.2.1.3),

(Eur. J. Biochem., 250, 432-439 (1997), Appl. Microbiol. Biotechnol., 54, 37-43 (2000), etc.) are known. In the synthesis step using an enzyme or an organism, a batch-type synthesis method may be used, or continuous production may be performed using an immobilized enzyme or an immobilized cell.

  <PHA synthase and its producing bacteria> The PHA synthase used in the present invention is produced by a microorganism appropriately selected from microorganisms producing the enzyme, or by a transformant in which the PHA synthase gene of these microorganisms is introduced into a host. Can be used.

  As microorganisms that produce PHA synthase, PHB and PHB / V producing bacteria can be used. Examples of such microorganisms include Aeromonas sp., Alcaligenes sp., Chromatium sp. .), Comamonas sp., Methylobacterium sp., Paracoccus sp., Pseudomonas sp., And the like. In addition, Burkholderia cepacia KK01 strain (Burkholderia cepacia KK01), Ralstonia eutropha TB64 strain (Ralstonia eutropha TB64), Alcaligenes sp. TL2 strain (Alcaligenes sp. TL2) and the like can be used. The KK01 strain is deposited under the deposit number FERM BP-4235, the TB64 strain is deposited under the deposit number FERM BP-6933, and the TL2 strain is deposited under the deposit number FERM BP-6913. It has been deposited. In addition, mcl-PHA and unusual-PHA-producing microorganisms can be used as microorganisms that produce PHA synthase. Examples of such microorganisms include Pseudomonas oleovorans, Pseudomonas renonovans, Pseudomonas genus strain 61-3, Pseudomonas sp. In addition to Peptida KT2442 strain, Pseudomonas aeruginosa, etc., Pseudomonas putida P91 strain (Pseudomonas putida P91), Pseudomonas chicoryii H45 strain (Pseudomonas cichorii H45), Pseudomonas chicoryii. YN2 strain (Pseudomonas cichorii YN2), Pseudomonas jessenii P161 (Pseudomonas jessenii P161) and other Pseudomonas microorganisms, and Burkholderia sp. OK3, FERM described in JP-A-2001-78753 P-17370), and Burkholderia genus described in JP-A-2001-69968 / OK Burkholderia microorganisms such as 4 strains (Burkholderia sp. OK4, FERM P-17371) can be used. In addition to these microorganisms, microorganisms belonging to the genus Aeromonas (Composition), Comamonas sp., Etc. and producing mcl-PHA and unusual-PHA can also be used.

  In addition, the P91 strain has the deposit number FERM BP-7373, the H45 strain has the deposit number FERM BP-7374, the YN2 strain has the deposit number FERM BP-7375, and the P161 strain has the deposit number FERM BP-7376. Based on the Budapest Treaty on the international recognition of the deposit of microorganisms, the depository is internationally deposited at the Institute of Industrial Technology, Ministry of Economy, Trade and Industry (former Institute of Industrial Technology, Ministry of International Trade and Industry) and the Patent Microbiology Deposit Center of the Institute of Biotechnology.

  It is used for normal culture of microorganisms used for production of the PHA synthase according to the present invention, for example, preparation of conserved strains, growth for ensuring the number of bacteria and activity required for production of PHA synthase, etc. A medium containing components necessary for the growth of microorganisms is appropriately selected and used. For example, as long as it does not adversely affect the growth and survival of microorganisms, any kind of medium such as a general natural medium (meat medium, yeast extract, etc.) or a synthetic medium to which a nutrient source is added can be used.

  Any method can be used for culturing as long as the microorganism grows, such as liquid culture or solid culture. Furthermore, the types such as batch culture, fed-batch culture, semi-continuous culture, and continuous culture are not limited. As a form of liquid batch culture, there are a method of supplying oxygen by shaking with a shake flask, and a method of supplying oxygen by stirring aeration using a jar fermenter. Further, a multistage system in which these steps are connected in a plurality of stages may be adopted.

  When producing a PHA synthase using a PHA-producing microorganism as described above, for example, the microorganism is grown on an inorganic medium containing an alkanoic acid such as octanoic acid or nonanoic acid, and from the logarithmic growth phase to the early stationary phase. A method of extracting a desired enzyme by collecting the microorganisms by centrifugation or the like can be used. In addition, when cultured under the above conditions, mcl-PHA derived from the added alkanoic acid is synthesized in the microbial cells. In this case, generally, PHA synthase is formed in the microbial cells. It is said that it is bound to PHA fine particles. However, according to the study by the present inventors, it has been found that a considerable degree of enzyme activity is also present in a supernatant obtained by centrifuging a lysate of cells cultured by the above method. This is because the enzyme continues to be actively produced in the cells relatively early in the culture from the logarithmic growth phase to the early stationary phase as described above, and thus there is a considerable amount of free PHA synthase. Presumed.

  Any inorganic medium can be used for the above culture method as long as it contains a component capable of growing microorganisms, such as a phosphorus source (for example, phosphate) and a nitrogen source (for example, ammonium salt, nitrate, etc.). Examples of the inorganic salt medium include MSB medium, E medium (J. Biol. Chem., 218, 97-106 (1956)), M9 medium, and the like. The composition of the M9 medium used in the examples of the present invention is as follows.

Na ₂ HPO ₄ : 6.2 gKH ₂ PO ₄ : 3.0 gNaCl: 0.5 g NH ₄ Cl: 1.0 g (pH 7.0 in 1 liter of medium)
Furthermore, for good growth and production of PHA synthase, it is preferable to add about 0.3% (v / v) of the following trace component solution to the inorganic salt medium.

(Trace component solution)
Nitrilotriacetic acid: 1.5 g MgSO ₄ : 3.0 gMnSO ₄ : 0.5 g NaCl: 1.0 g FeSO ₄ : 0.1 g CaCl ₂ : 0.1 g CoCl ₂ : 0.1 gZnSO ₄ : 0.1 g CuSO ₄ : 0.1 g AlK (SO ₄ ) ₂ : 0.1 gH ₃ BO ₃ : 0.1 gNa ₂ MoO ₄ : 0.1 g NiCl ₂ : 0.1 g (in 1 liter)
The culture temperature may be any temperature at which the above-mentioned strain can grow well, for example, 14 to 40 ° C, preferably about 20 to 35 ° C.

  It is also possible to produce a desired PHA synthase using a transformant introduced with the PHA synthase gene possessed by the aforementioned PHA producing bacterium. Cloning of the PHA synthase gene, preparation of an expression vector, and preparation of a transformant can be performed according to a conventional method. In a transformant obtained using a bacterium such as E. coli as a host, examples of the medium used for the culture include a natural medium or a synthetic medium, such as an LB medium or an M9 medium. The culture temperature is in the range of 25 to 37 ° C., and aerobic culture is performed for 8 to 27 hours to promote the growth of microorganisms. Thereafter, the cells can be collected and the PHA synthase accumulated in the cells can be collected. If necessary, an antibiotic such as kanamycin, ampicillin, tetracycline, chloramphenicol, or streptomycin may be added to the medium. In the case where an inducible promoter is used in the expression vector, when the transformant is cultured, an inducing substance corresponding to the promoter may be added to the medium to promote expression. Examples of the inducer include isopropyl-β-D-thiogalactopyranoside (IPTG), tetracycline, indoleacrylic acid (IAA) and the like.

  PHA synthase may be a crude enzyme such as microbial cell disruption solution or ammonium sulfate salted-out precipitate from which protein components are precipitated and recovered with ammonium sulfate, etc., and purified enzymes purified by various methods. Also good. The enzyme can be used by appropriately adding stabilizers and activators such as metal salts, glycerin, dithiothreitol, EDTA, bovine serum albumin (BSA), as necessary.

  As a method for separating and purifying PHA synthase, any method can be used as long as the enzyme activity of PHA synthase is maintained. For example, the obtained microbial cells are crushed using a French press, an ultrasonic crusher, lysozyme, various surfactants, etc., and then centrifuged to obtain a crude enzyme solution, or ammonium sulfate salt prepared therefrom. With respect to the precipitate, a purified enzyme can be obtained by alone or suitably combining means such as affinity chromatography, cation or anion exchange chromatography, and gel filtration. In particular, genetically engineered proteins can be expressed more easily by expressing them in the form of a fusion protein in which a “tag” such as a histidine residue is bound to the N-terminus or C-terminus, and binding to an affinity resin via this tag. Can be purified. In order to separate the target protein from the fusion protein, it is preferable to use a method such as cleaving with a protease such as thrombin or blood coagulation factor Xa, lowering the pH, or adding a high concentration of imidazole as a binding competitor. Alternatively, when the tag contains intein as in the case of using pTYB1 (manufactured by New Englan Biolab) as an expression vector, it is cleaved as a reducing condition with dithiothreitol or the like. Known fusion proteins that can be purified by affinity chromatography include glutathione S-transferase (GST), chitin-binding domain (CBD), maltose-binding protein (MBP), and thioredoxin (TRX) in addition to the histidine tag. is there. The GST fusion protein can be purified by a GST affinity resin.

  For measuring the activity of PHA synthase, various methods already reported can be used. For example, CoA released in the process of polymerization of 3-hydroxyacyl CoA by the catalytic action of PHA synthase to become PHA, The measurement can be carried out by the following method based on the principle of measurement by coloring with 5′-dithiobis- (2-nitrobenzoic acid). Reagent 1: Bovine serum albumin (Sigma) dissolved in 3.0 M / mL in 0.1 M Tris-HCl buffer (pH 8.0), Reagent 2: 3-hydroxyoctanoyl CoA in 0.1 M Tris-HCl buffer (pH 8.0) 3.0 mM dissolution, Reagent 3: Trichloroacetic acid dissolved in 0.1 M Tris-HCl buffer (pH 8.0) 10 mg / mL, Reagent 4: 5, 5'-dithiobis- (2-nitrobenzoic acid) in 0.1 M Tris-HCl buffer Dissolve 2.0 mM in (pH 8.0). First reaction (PHA synthesis reaction): Add 100 μl of reagent 1 to 100 μl of sample (enzyme) solution, mix, and preincubate at 30 ° C. for 1 minute. Here, 100 μl of reagent 2 is added and mixed, and after incubation at 30 ° C. for 1 to 30 minutes, reagent 3 is added to stop the reaction. Second reaction (coloring reaction of free CoA): Centrifugation (15,000 × g, 10 minutes) of the first reaction solution that has stopped the reaction, 500 μl of reagent 4 is added to 500 μl of this supernatant, and 10 minutes at 30 ° C. After incubation, measure the absorbance at 412 nm. Calculation of enzyme activity: The amount of enzyme that releases 1 μmol of CoA per minute is defined as 1 unit (U).

<Method for Producing PHA-Coated Liposomes> As an example of the method for producing PHA-coated liposomes of the present invention, as illustrated in FIG. 1, (1) a step of dispersing liposomes produced by the above-described method in an aqueous medium (FIG. 1 [ A]), (2) Step of immobilizing PHA synthase on dispersed liposomes (Fig. 1 [B]), (3) Step of adding substrate 3-hydroxyacyl CoA, (4) PHA synthesis reaction (5) Collecting the PHA-coated liposomes, drying them as needed, or dispersing them in a medium appropriately selected according to their use and processing them as a dispersion system The method which has a process at least can be mentioned.

  The step of dispersing the liposome in the aqueous medium is performed by adding the selected one or more liposomes to the aqueous medium, performing the dispersion treatment, and then classifying the liposome into a desired particle size range if necessary.

  The particle size of the dispersed liposome varies depending on the use, but it is desirable to disperse the particle size in a monodisperse state up to about 100 nm to 100 μm. When the particle size of the dispersed liposome is not within a desired range, it can be adjusted by performing classification by filtration or sedimentation.

  The particle size of the dispersed liposome can be measured by a known method such as an absorbance method, a static light scattering method, a dynamic light scattering method, or a centrifugal sedimentation method. For example, a particle size measuring device such as Coulter Counter Multisizer is used. be able to.

  The composition of the aqueous medium for PHA synthesis in this step can disperse the liposomes in the desired state, and does not interfere with the subsequent steps of immobilizing the enzyme on the liposome and the step of performing the PHA synthesis reaction. However, in order to omit the subsequent steps, a composition that can exhibit the activity of PHA synthase can be used. Here, as a composition capable of exerting the activity of PHA synthase, for example, a buffer solution can be used. As the buffer, common buffers used in biochemical reactions, such as acetate buffer, phosphate buffer, potassium phosphate buffer, 3- (N-morpholino) propane sulfonic acid (MOPS) buffer, N-Tris. (Hydroxymethyl) methyl-3-aminopropane sulfonic acid (TAPS) buffer, Tris-HCl buffer, glycine buffer, 2- (cyclohexylamino) ethane sulfonic acid (CHES) buffer, etc. are preferably used. The concentration of the buffer capable of exhibiting the activity of PHA synthase can be used at a general concentration, that is, in the range of 5 mM to 1.0 M, but preferably 10 to 200 mM. Further, the pH is adjusted to 5.5 to 9.0, preferably 7.0 to 8.5. However, depending on the optimum pH and pH stability of the PHA synthase used, setting conditions other than the above ranges is not excluded.

  In addition, in order to maintain the dispersion state of the liposome in the aqueous medium, a suitable surface activity can be used as long as it is of a type and concentration that do not interfere with the subsequent steps, and further, a type and concentration that does not hinder the purpose of the PHA-coated liposome of the present invention. An agent may be added. Examples of such surfactants include anions such as sodium oleate, sodium dodecyl sulfonate, sodium dodecyl sulfate, sodium dodecyl-N-sarcosinate, sodium cholate, sodium deoxycholate, sodium taurodeoxycholate, etc. Surfactant, cationic surfactant such as cetyltrimethylammonium bromide, dodecylpyridinium chloride, 3-[(coleamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS), 3-[(3-coleamide Propyl) dimethylammonio] -2-hydroxy-1-propanesulfonic acid (CHAPSO), palmitoyl lysolecithin, dodecyl-β-alanine and other zwitterionic surfactants, octyl glucoside, octyl thioglucosi , Heptylthioglucoside, decanoyl-N-methylglucamide (MEGA-10), polyoxyethylene dodecyl ether (Brij, Lubrol), polyoxyethylene-i-octylphenyl ether (Triton X) polyoxyethylene nonylphenyl ether (Nonidet) Nonionic surfactants such as P-40, Triton N), polyoxyethylene fatty acid ester (Span), and polyoxyethylene soliitol ester (Tween) can be exemplified.

  Further, in order to maintain the dispersion state of the liposome in the aqueous medium, the type and concentration that do not interfere with the subsequent steps, and further, the type and concentration that do not interfere with the characteristics necessary for the use of the PHA-coated liposome of the present invention, A suitable co-solvent may be added. As the auxiliary solvent, for example, one kind selected from linear aliphatic hydrocarbons such as hexane, monohydric alcohols such as methanol and ethanol, polyhydric alcohols such as glycerol, fatty acid ethers, and derivatives such as carboxylic acid esters Alternatively, two or more types can be selected and used.

  The step of immobilizing the PHA synthase on the liposome can be performed by adding the PHA synthase to the previous liposome dispersion and subjecting it to an immobilization treatment. The immobilization treatment can be carried out by arbitrarily selecting from the usual enzyme immobilization methods as long as the activity of the enzyme can be maintained and can be applied to the desired liposome. Can do. For example, a covalent bond method, an ion adsorption method, a hydrophobic adsorption method, a physical adsorption method, an affinity adsorption method, a cross-linking method, a lattice-type inclusion method, etc. can be exemplified, but in particular immobilization using ion adsorption or hydrophobic adsorption The method is simple.

  Enzyme proteins such as PHA synthesizing enzymes are polypeptides in which a large number of amino acids are linked, exhibiting properties as ion adsorbents by amino acids having free ionic groups such as lysine, histidine, arginine, aspartic acid, and glutamic acid. It has the properties of a hydrophobic adsorbent in that it is an organic polymer due to an amino acid having a free hydrophobic group such as alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, and proline. Therefore, it can be adsorbed to liposomes having ionicity, hydrophobicity, or both ionic and hydrophobic properties, to varying degrees.

  In the method of immobilizing PHA synthesizing enzyme mainly by ion adsorption, liposomes expressing ionic functional groups may be used. For example, phosphatidylserine, phosphatidic acid, dicetyl phosphate, phosphati Liposomes may be prepared using a lipid that imparts a negative charge, such as diluinositol, or a lipid that imparts a positive charge, such as stearylamine.

  Immobilization of the PHA synthase to the liposome by the ion adsorption method or the hydrophobic adsorption method is achieved by mixing the liposome and the PHA synthase so as to have a predetermined concentration in a predetermined aqueous medium. At this time, it is desirable that the reaction vessel is shaken or stirred with an appropriate strength so that the enzyme is evenly adsorbed on the surface of the liposome. In the above-described immobilization treatment, the composition of the aqueous medium in which the liposome and the enzyme are mixed varies depending on the pH and salt concentration of the aqueous medium, because the positive and negative surface charges, the amount of charge, and the hydrophobicity of the liposome and PHA synthase change. It is desirable that the composition take into consideration. For example, when the liposome is mainly ion-adsorptive, the amount of charge contributing to adsorption between the liposome and PHA synthase can be increased by lowering the salt concentration. Moreover, the opposite charge of both can be increased by changing pH. The composition can also be obtained by directly measuring the amount of adsorption between the liposome and the PHA synthase. The amount of adsorption can be measured, for example, by adding a PHA synthase solution with a known concentration to a solution in which liposomes are dispersed and performing an adsorption treatment, then measuring the concentration of PHA synthase in the solution, and subtracting the amount of adsorbed enzyme. Or the like.

  In the case of liposomes where it is difficult to immobilize enzymes by ion adsorption method or hydrophobic adsorption method, they can be immobilized by covalent bonding method by taking into consideration the complexity of operation and the possibility of enzyme deactivation as needed. Absent. For example, there is a method of performing an exchange reaction between a liposome having a thiol reactive group (a reactive group capable of reacting with a thiol group such as a dithiopyridyl group and a maleimide group) and a thiol group of an enzyme. Also, the hydroxyl group, amine or carboxyl group of the enzyme is activated by monofunctional activation reagents such as N-hydroxysuccinimide, ethyl chloroformate, dicyclohexylcarbodiimide (DCC), Woodward reagent K, cyanuric acid and trifluoromethanesulfonyl chloride. The enzyme can be activated by a number of bifunctional cross-linking reagents that contain different reactive groups such as diisocyanates and coupled to liposomes containing amino group-containing phospholipids such as phosphatidylethanolamine.

  Alternatively, the enzyme may be immobilized on a liposome into which a ligand has been introduced by affinity adsorption. In this case, any ligand can be selected as long as it can perform affinity adsorption while maintaining the enzyme activity of PHA synthase as a ligand. Alternatively, the enzyme may be immobilized by binding other biopolymer such as protein to PHA synthase and affinity-adsorbing the bound biopolymer. The binding of PHA synthase and biopolymer may be performed by genetic recombination or the like, or may be performed chemically.

  In addition, a peptide containing an amino acid sequence capable of binding to liposomes is fused with polyhydroxyalkanoate synthase, and the peptide portion of the amino acid sequence capable of binding to liposomes is bound to liposomes. Based on the above, it is also possible to immobilize polyhydroxyalkanoate synthase on the liposome surface.

  The amino acid sequence capable of binding to the liposome can be determined, for example, by screening a random peptide library. In particular, for example, a phage display peptide library prepared by linking a random synthetic gene to the N-terminal gene of the surface protein of M13 phage (for example, geneIII protein) can be preferably used. In order to determine the amino acid sequence possessed, the following procedure is taken. That is, the phage display peptide library is brought into contact with the liposome or the phospholipid constituting the liposome, and then the bound phage and the unbound phage are separated by washing. The liposome-bound phage is eluted with acid or the like, neutralized with a buffer, and then infected with E. coli to amplify the phage. When this selection is repeated a plurality of times, a plurality of clones capable of binding to the target liposome are concentrated. Here, in order to obtain a single clone, colonies are made on the medium plate in a state of being again infected with E. coli. After culturing each single colony in a liquid medium, the phage present in the medium supernatant is precipitated and purified with polyethylene glycol or the like, and the base sequence is analyzed to know the peptide structure.

  The amino acid sequence of the peptide having the binding ability to the liposome obtained by the above method is used by fusing to a polyhydroxyalkanoate synthase using an ordinary genetic engineering technique. A peptide capable of binding to liposomes can be expressed by linking to the N-terminus or C-terminus of polyhydroxyalkanoate synthase. It can also be expressed by inserting an appropriate spacer sequence. The spacer sequence is preferably about 3 to about 400 amino acids, and the spacer sequence may contain any amino acid. Most preferably, the spacer sequence is one that does not interfere with the functioning of the PHA synthase and does not prevent the PHA synthase from binding to the liposomes.

  The liposomes immobilized with the enzyme prepared by the above method can be used as they are, but can also be used after lyophilization or the like.

  When the amount of CoA released in the reaction of synthesizing PHA by polymerization of 3-hydroxyacyl CoA is 1 μmol per minute and the amount of PHA synthase is 1 unit (U), the amount of enzyme immobilized on the liposome is: It may be set within the range of 10 units (U) to 1,000 units (U), preferably 50 units (U) to 500 units (U) per gram of phospholipid.

  The time for performing the enzyme immobilization treatment is preferably 1 minute to 24 hours, and more preferably 10 minutes to 1 hour. Excessive standing or standing is not preferable because it causes liposome aggregation and a decrease in enzyme activity.

  Alternatively, the step of dispersing the liposome in the previous step may be omitted, and the liposome before being dispersed in the aqueous medium may be directly added to the enzyme solution, and the enzyme may be immobilized on the liposome while being dispersed in the enzyme solution. . In this case, the liposome is easily dispersed in the aqueous medium due to electrical repulsion or steric hindrance by the ionic functional group possessed by the enzyme immobilized on the liposome, and the addition of a surfactant to the aqueous medium is unnecessary. It becomes possible to make it small or to make it small.

  The step of adding 3-hydroxyacyl-CoA as a substrate is performed by adding a separately prepared 3-hydroxyacyl-CoA stock solution to the aqueous dispersion of liposomes to which the enzyme in the previous step is immobilized so as to reach the target concentration. Is achieved by doing The substrate 3-hydroxyacyl CoA is generally added at a final concentration of 0.1 mM to 1.0 M, preferably 0.2 mM to 0.2 M, more preferably 0.2 mM to 1.0 mM.

  In addition, in the above process, the monomer unit composition of PHA that coats the liposome in the direction from the inside to the outside of the liposome by changing the composition such as the type and concentration of 3-hydroxyacyl CoA in the aqueous reaction solution over time Can be changed.

  Examples of the form of the liposome with the changed monomer unit composition include a form in which the composition change of the PHA coating is continuous and a layer of PHA having a composition gradient in the direction from the inside to the outside coats the liposome. Can do. As a production method, for example, a method of adding 3-hydroxyacyl CoA having another composition to the reaction solution while synthesizing PHA may be used.

  As another form, there can be mentioned a form in which the composition change of the PHA coating is gradual and PHA having different compositions coats the liposome in multiple layers. As this production method, after synthesizing PHA with a certain composition of 3-hydroxyacyl CoA, the liposomes being prepared are once recovered from the reaction solution by centrifugation, gel filtration, etc. What is necessary is just to add the reaction liquid which consists of a composition again.

The PHA synthesizing step can exert the activity of PHA synthase when the composition of the reaction solution is not prepared by the previous step so that the PHA-coated liposome of the desired shape can be obtained by the synthesized PHA. Preparation is carried out so as to obtain a composition, and the reaction temperature and reaction time are adjusted.

  The concentration of the buffer of the reaction solution capable of exhibiting the activity of PHA synthase can be used at a general concentration, that is, in the range of 5 mM to 1.0 M, but preferably 10 to 200 mM. Further, the pH is adjusted to 5.5 to 9.0, preferably 7.0 to 8.5. However, depending on the optimum pH and pH stability of the PHA synthase used, setting conditions other than the above ranges is not excluded.

  The reaction temperature is appropriately set according to the characteristics of the PHA synthase to be used, and is usually set to 4 ° C to 50 ° C, preferably 20 ° C to 40 ° C. However, depending on the optimum temperature and thermostability of the PHA synthase used, setting conditions other than the above ranges is not excluded. The reaction time depends on the stability of the PHA synthase used, but is usually selected and set appropriately within the range of 1 minute to 24 hours, preferably 30 minutes to 3 hours.

  PHA-coated liposomes can be obtained by this process. The monomer unit structure of PHA constituting the liposomes is extracted from PHA-coated liposomes with chloroform, analyzed by gas chromatography, etc., and time-of-flight secondary ions. It can be determined using a mass spectrometer (TOF-SIMS) and ion sputtering technology.

  The molecular weight of PHA is not particularly limited, but in order to maintain the strength of the PHA-coated liposome, the number average molecular weight is in the range of 1,000 to 10,000,000, more preferably in the range of 3,000 to 1,000,000. It is desirable to do. The molecular weight of PHA may be measured by GPC (gel permeation chromatography) after extracting PHA from chloroform from PHA-coated liposomes.

  The coating amount of PHA depends on the use of the PHA-coated liposome, but is, for example, a weight composition ratio in the range of 1 to 30% by mass, preferably in the range of 1 to 20% by mass, with respect to the liposome dry mass. Preferably it is set as the range of 1-15 mass%.

  The particle diameter of the PHA-coated liposome obtained by the above process is usually 50 μm or less, preferably 10 μm or less, more preferably 0.01 to 10 μm, although it depends on the use of the PHA-coated liposome. The particle size of PHA-coated liposomes can be measured by known methods such as absorbance method, static light scattering method, dynamic light scattering method, and centrifugal sedimentation method. For example, use a particle size measuring device such as Coulter Counter Multisizer. Can do.

  In addition, the PHA-coated liposome obtained in this step can be used after being subjected to various secondary processing and chemical modification.

  For example, PHA-coated liposomes having more useful functions and characteristics can be obtained by chemically modifying PHA on the surface layer of PHA-coated liposomes. For example, by introducing a graft chain, PHA-coated liposomes having various characteristics resulting from the graft chain can be obtained. For example, if polysiloxane described later is introduced as a graft chain, PHA-coated liposomes having further improved mechanical strength, dispersibility, weather resistance, water repellency (water resistance), heat resistance and the like can be obtained. Moreover, it is possible to further improve the mechanical strength, chemical resistance, heat resistance, etc. of the PHA-coated liposome by crosslinking the PHA of the PHA-coated liposome.

  The method of chemical modification is not particularly limited as long as it satisfies the purpose of obtaining a desired function / structure. For example, PHA having a reactive functional group in the side chain is synthesized and the chemical reaction of the functional group is used. Thus, a method of chemically modifying can be used as a suitable method.

  Although the kind of said reactive functional group will not be specifically limited if the objective of obtaining a desired function and structure is satisfy | filled, For example, an above-described epoxy group can be illustrated. PHA having an epoxy group in the side chain can be chemically converted in the same manner as a polymer having an ordinary epoxy group. Specifically, for example, it can be converted into a hydroxyl group or a sulfone group can be introduced. It is also possible to add a compound having a thiol or an amine. For example, a compound having a reactive functional group at the terminal, specifically, a compound having an amino group having a high reactivity with an epoxy group at the terminal is added. Thus, a polymer graft chain is formed.

  Examples of the compound having an amino group at the terminal include amino-modified polymers such as polyvinylamine, polyethyleneimine, and amino-modified polysiloxane (amino-modified silicone oil). Among these, as the amino-modified polysiloxane, commercially available modified silicone oil may be used, and it is synthesized by the method described in J. Amer. Chem. Soc., 78, 2278 (1956), etc. It is also possible to expect effects such as improvement in mechanical strength, dispersibility, light resistance, weather resistance, water repellency (water resistance) and heat resistance due to the addition of the graft chain of the polymer.

  In addition, as another example of chemical conversion of a polymer having an epoxy group, a crosslinking reaction with a diamine compound such as hexamethylene diamine, succinic anhydride, 2-ethyl-4-methylimidazole, or the like is an example of physicochemical conversion. Crosslinking reaction by electron beam irradiation etc. is mentioned. Among these, the reaction between PHA having an epoxy group in the side chain and hexamethylenediamine proceeds in a form as shown in the following scheme to produce a crosslinked polymer.

  The step of recovering the liposomes coated with the PHA of the present invention, drying as necessary, or dispersing in a medium and processing as a dispersion system is replaced with a dispersion medium appropriately selected according to the use of the PHA-coated liposome. Is achieved.

  When the PHA-coated liposome of the present invention is used as, for example, a liposome for an agricultural chemical composition, the slurry obtained in the previous step can be used as it is as an agricultural chemical composition, but an aqueous suspension, wettable powder, powder It is used as a preparation in a form that is easier to use, such as granules, and is preferably used as an aqueous suspension. The aqueous suspension is prepared by adding a stabilizer such as a thickening agent, an antifreezing agent, a specific gravity adjusting agent, and an antiseptic to the liposome slurry obtained as described above. As thickeners used, carboxymethylcellulose, xanthan gum, lambzan gum, locust bean gum, carrageenan, welan gum and other synthetic polymers such as sodium polyacrylate, aluminum magnesium silicate, smectite, bentonite, hectorite, dry process Examples include mineral fine powders such as silica and alumina sol. Examples of the antifreezing agent include alcohols such as propylene glycol. Examples of the specific gravity regulator include water-soluble salts such as sodium sulfate and urea.

  When the PHA-coated liposome of the present invention is used as, for example, a liposome for a fertilizer composition, the slurry obtained in the previous step can be used as it is as a fertilizer composition, but an aqueous suspension, wettable powder, powder It is used as a preparation in a form that is easier to use, such as granules, and is preferably used as an aqueous suspension. The aqueous suspension is prepared by adding a stabilizer such as a thickening agent, an antifreezing agent, a specific gravity adjusting agent, and an antiseptic to the liposome slurry obtained as described above. As thickeners used, carboxymethylcellulose, xanthan gum, lambzan gum, locust bean gum, carrageenan, welan gum and other synthetic polymers such as sodium polyacrylate, aluminum magnesium silicate, smectite, bentonite, hectorite, dry process Examples include mineral fine powders such as silica and alumina sol. Examples of the antifreezing agent include alcohols such as propylene glycol. Examples of the specific gravity regulator include water-soluble salts such as sodium sulfate and urea.

  When the PHA-coated liposome of the present invention is used as, for example, a cosmetic liposome, the dispersion medium may be a hydrocarbon such as solid or liquid paraffin, crystal oil, ceresin, ozokerite, or montan wax; olive, ground wax, carnauba wax, lanolin Or plant or animal fats and waxes such as whale wax; stearic acid, palmitic acid, oleic acid, glycerin monostearate, glyceryl distearate, glycerin monooleate, isopropyl myristate, isopropyl stearate or Fatty acids such as butyl stearate and esters thereof; methyl polysiloxane, methyl polycyclosiloxane, methyl phenyl polysiloxane, silicone polyether copoly Silicones such as ethanol; alcohols such as ethanol, isopropyl alcohol, cetyl alcohol, stearyl alcohol, palmityl alcohol or hexyldecyl alcohol; known cosmetic groups such as polyhydric alcohols having a moisturizing action such as glycol, glycerin or sorbitol Prepared by dispersing in an agent. When the PHA-coated liposome of the present invention is used as, for example, an artificial erythrocyte liposome, the slurry obtained in the previous step is suspended in physiological saline, and coarse particles are removed by a known means such as gel filtration or centrifugation. .

  When the PHA-coated liposome of the present invention is used as, for example, a liposome for ink, it is dispersed in an aqueous medium. For the purpose of assisting dispersion in an aqueous medium, a surfactant, a protective colloid, a water-soluble organic solvent, and the like can be added within a range that does not significantly reduce the water resistance of the coating film. Moreover, antiseptic | preservative, a viscosity modifier, a pH adjuster, a chelating agent, etc. can also be added. Specific examples of protective colloids that may be added to ink liposomes include natural proteins such as glue, gelatin, casein, albumin, gum arabic, and fish mulberry, alginic acid, methylcellulose, carboxymethylcellulose, polyethylene oxide, hydroxyethylcellulose, and polyvinyl alcohol. , Synthetic polymers such as polyacrylamide, aromatic amide, polyacrylic acid, polyvinyl ether, polyvinyl pyrrolidone, acrylic and polyester. The protective colloid is used as necessary for the purpose of improving the fixing property, viscosity adjustment, and quick drying, and the content of the protective colloid in the ink is preferably 30% by mass or less, and preferably 20% by mass or less. Particularly preferred.

  As the surfactant that may be added to the liposome for ink, any of anionic, cationic, zwitterionic, and nonionic active agents may be used. Examples of anionic surfactants include fatty acid salts such as sodium stearate, potassium oleate, and semi-cured tallow fatty acid sodium; alkyls such as sodium dodecyl sulfate, tri (2-hydroxyethyl) ammonium dodecyl sulfate, and sodium octadecyl sulfate. Sulfate esters; benzenesulfonates such as sodium nonylbenzenesulfonate, sodium dodecylbenzenesulfonate, sodium octadecylbenzenesulfonate, sodium dodecyldiphenyl ether disulfonate; naphthalenesulfone such as sodium dodecylnaphthalenesulfonate, formalin condensate of naphthalenesulfonate Acid salts; sulfosuccinic acid ester salts such as sodium dododecyl sulfosuccinate and dioctadecyl sodium sulfosuccinate; Sodium polyoxyethylene sulfate, polyoxyethylene dodecyl ether sulfate tri (2-hydroxyethyl) ammonium sulfate, polyoxyethylene octadecyl ether sodium sulfate, polyoxyethylene dodecyl phenyl ether sodium sulfate polyoxyethylene sulfate ester salts; potassium dodecyl phosphate And phosphate ester salts such as sodium octadecylphosphate. Examples of cationic surfactants include alkylamine salts such as octadecyl ammonium acetate and coconut oil amine acetate; fourth amines such as dodecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, dioctadecyldimethylammonium chloride, and dodecylbenzyldimethylammonium chloride. Examples include quaternary ammonium salts. Examples of zwitterionic activators include alkylbetaines such as dodecylbetaine and octadecylbetaine; amine oxides such as dodecyldimethylamine oxide and the like. Examples of nonionic surfactants include polyoxyethylene dodecyl ether, polyoxyethylene hexadecyl ether, polyoxyethylene octadecyl ether, polyoxyethylene alkyl ethers such as polyoxyethylene (9-octadecenyl) ether; polyoxy Polyoxyethylene phenyl ethers such as ethylene octyl phenyl ether and polyoxyethylene nonyl phenyl ether; oxirane polymers such as polyethylene oxide and co-polyethylene oxide propylene oxide; sorbitan dodecanoate, sorbitan hexadecanoate, sorbitan octadecane Acid ester, sorbitan (9-octadecenoic acid) ester, sorbitan (9-octadecenoic acid) triester, polyoxyethylene sorbitandodecanoic acid Steal, polyoxyethylene sorbitan hexadecanoic acid ester, polyoxyethylene sorbitan octadecanoic acid ester, polyoxyethylene sorbitan octadecanoic acid triester, polyoxyethylene sorbitan (9-octadecenoic acid) ester, polyoxyethylene sorbitan (9-octadecenoic acid) tri Examples include sorbitan fatty acid esters such as esters; sorbitol fatty acid esters such as polyoxyethylene sorbitol (9-octadecenoic acid) tetraester; and glycerin fatty acid esters such as glycerin octadecanoic acid ester and glycerin (9-octadecenoic acid) ester. Among these nonionic active agents, those having an HLB of 14 or more are particularly preferred. The amount of the surfactant used in the present invention varies depending on whether it is used alone or in combination of two or more, but is 0 to 10% by weight, preferably based on the total amount of the aqueous ink composition. Is 0-5 mass%. The aqueous pigment ink composition of the present invention preferably contains 20 to 95% by weight of water and 1 to 60% by volume of pigment based on the total amount of the composition.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the examples described below are examples of the best mode of the present invention, but the technical scope of the present invention is not limited to these examples.

(Reference Example 1)
Production of transformant having ability to produce PHA synthase and production of PHA synthase Transformant having ability to produce PHA synthase was produced by the following method. That is, after culturing YN2 strain in 100 mL of LB medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, pH 7.4) at 30 ° C. overnight, chromosomal DNA was separated and recovered by the method of Marmer et al. The obtained chromosomal DNA was completely digested with the restriction enzyme HindIII. PUC18 was used as a vector and digested with the restriction enzyme HindIII. After dephosphorylation of the ends (Molecular Cloning, 1,572, (1989); published by Cold Spring Harbor Laboratory), DNA ligation kit Ver.II (Takara Shuzo) was used to cleave the site of the vector (cloning site) and chromosome The DNA was ligated with a HindIII fully degraded fragment of DNA. Using a plasmid vector incorporating this chromosomal DNA fragment, Escherichia coli HB101 strain was transformed to prepare a DNA library of YN2 strain.

  Next, in order to select a DNA fragment containing the PHA synthase gene of the YN2 strain, a probe for colony hybridization was prepared. Oligonucleotides comprising the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 were synthesized (Amersham Pharmacia Biotech), and PCR was performed using this oligonucleotide as a primer and chromosomal DNA as a template. A DNA fragment that had been PCR amplified was used as a probe. The labeling of the probe was performed using a commercially available labeling enzyme system AlkPhosDirect (Amersham Pharmacia Biotech). Using the resulting labeled probe, an E. coli strain having a recombinant plasmid containing the PHA synthase gene was selected from the chromosomal DNA library of the YN2 strain by colony hybridization. By recovering the plasmid from the selected strain by the alkaline method, a DNA fragment containing the PHA synthase gene could be obtained.

  The gene DNA fragment obtained here was recombined into a vector pBBR122 (Mo Bi Tec) containing a broad host range replication region that does not belong to any of the incompatibility groups IncP, IncQ, or IncW. When this recombinant plasmid was transformed into Pseudomonas chicoryai YN2ml strain (strain lacking PHA synthesis ability) by electroporation, the PHA synthesis ability of the YN2ml strain was restored and showed complementarity. Therefore, it is confirmed that the selected gene DNA fragment contains a PHA synthase gene region that can be translated into PHA synthase in Pseudomonas chicory eye YN2ml strain.

  The nucleotide sequence of the DNA fragment containing this PHA synthase gene was determined by the Sanger method. As a result, it was confirmed that the determined nucleotide sequences contained the nucleotide sequences represented by SEQ ID NO: 2 and SEQ ID NO: 4, which respectively encode peptide chains. As described below, it is confirmed that each protein consisting of individual peptide chains has enzyme activity, and that the base sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 are PHA synthase genes, respectively. I was able to. That is, the amino acid sequence shown in SEQ ID NO: 1 encodes the base sequence of SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 3 encodes the base sequence of SEQ ID NO: 4. It was confirmed that the ability to synthesize PHA was exhibited only with a protein having the amino acid sequence of

  The PHA synthase gene having the nucleotide sequence represented by SEQ ID NO: 2 was subjected to PCR using chromosomal DNA as a template, and the full length of the PHA synthase gene was re-prepared.

  An oligonucleotide having a base sequence upstream of its start codon (SEQ ID NO: 7), which serves as an upstream primer, and a downstream primer, downstream of a stop codon, with respect to the base sequence represented by SEQ ID NO: 2. Were designed and synthesized (Amersham Pharmacia Biotech). PCR was performed using this oligonucleotide as a primer and chromosomal DNA as a template to amplify the full length of the PHA synthase gene (LA-PCR kit; Takara Shuzo).

  Similarly, the PHA synthase gene having the nucleotide sequence represented by SEQ ID NO: 4 was also subjected to PCR using chromosomal DNA as a template, and the full length of the PHA synthase gene was re-prepared. An oligonucleotide having a base sequence upstream of its start codon (SEQ ID NO: 9), which serves as an upstream primer, and a downstream primer, downstream of a stop codon, with respect to the base sequence represented by SEQ ID NO: 4 Were designed and synthesized (Amersham Pharmacia Biotech), respectively. Using this oligonucleotide as a primer, PCR was performed to amplify the full length of the PHA synthase gene (LA-PCR kit; Takara Shuzo).

  Next, the PCR amplified fragments containing the full length of the obtained PHA synthase gene were completely digested with the restriction enzyme HindIII for each. The expression vector pTrc99A was also cleaved with the restriction enzyme HindIII and dephosphorylated (Molecular Cloning, Vol. 1, page 572, 1989; Cold Spring Harbor Laboratory published). A DNA fragment containing the full length of the PHA synthase gene excluding unnecessary nucleotide sequences at both ends was ligated to the cleavage site of this expression vector pTrc99A using a DNA ligation kit Ver.II (Takara Shuzo).

  Escherichia coli HB101 (Takara Shuzo) was transformed with the obtained recombinant plasmid by the calcium chloride method. The obtained recombinant was cultured, the recombinant plasmid was amplified, and each recombinant plasmid was recovered. The recombinant plasmid retaining the gene DNA of SEQ ID NO: 2 was designated as pYN2-C1 (derived from SEQ ID NO: 2), and the recombinant plasmid retaining the gene DNA of SEQ ID NO: 4 was designated as pYN2-C2 (derived from SEQ ID NO: 4).

  E. coli (Escherichia coli HB101fB fadB deficient strain) is transformed with the pYN2-C1 and pYN2-C2 by the calcium chloride method, and the recombinant Escherichia coli strain, pYN2-C1 recombinant strain, pYN2-C2 pair that holds the respective recombinant plasmids Obtained a replacement stock.

  Each of the pYN2-C1 recombinant strain and the pYN2-C2 recombinant strain was inoculated into 200 ml of M9 medium containing 0.5% yeast extract and 0.1% octanoic acid, and cultured with shaking at 37 ° C. at 125 strokes / minute. After 24 hours, the cells were collected by centrifugation, and plasmid DNA was collected by a conventional method.

  For pYN2-C1, an oligonucleotide (SEQ ID NO: 11) serving as an upstream primer and an oligonucleotide (SEQ ID NO: 12) serving as a downstream primer were respectively designed and synthesized (Amersham Pharmacia Biotech). PCR was performed using this oligonucleotide as a primer and pYN2-C1 as a template to amplify the full length of the PHA synthase gene having a BamHI restriction site upstream and a XhoI restriction site downstream (LA-PCR kit; Takara Shuzo).

  Similarly, for pYN2-C2, the oligonucleotide (SEQ ID NO: 13) that serves as the upstream primer and the oligonucleotide (SEQ ID NO: 14) that serves as the downstream primer were each designed and synthesized (Amersham Pharmacia Biotech) . PCR was performed using this oligonucleotide as a primer and pYN2-C2 as a template to amplify the full length of the PHA synthase gene having a BamHI restriction site upstream and a XhoI restriction site downstream (LA-PCR kit; Takara Shuzo).

  Each purified PCR amplification product was digested with BamHI and XhoI and inserted into the corresponding site of plasmid pGEX-6P-1 (Amersham Pharmacia Biotech). Escherichia coli (JM109) was transformed with these vectors to obtain an expression strain. The strain was confirmed by DNA fragments obtained by treating plasmid DNA prepared in large quantities using Miniprep (Wizard Minipreps DNA Purification Systems, PROMEGA) with BamHI and XhoI. The obtained strain was precultured overnight with 10 mL of LB-Amp medium, and 0.1 mL thereof was added to 10 mL of LB-Amp medium and cultured with shaking 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 to 12 hours.

  E. coli induced by IPTG was collected (8000 × g, 2 minutes, 4 ° C.) and resuspended in 1/10 volume of 4 ° C. PBS. The cells were disrupted by freeze-thawing and sonication, and centrifuged (8000 × g, 10 minutes, 4 ° C.) to remove solid contaminants. After confirming that the target expressed protein was present in the supernatant by SDS-PAGE, the induced and expressed GST fusion protein was purified with Glutathion Sepharose 4B beads (manufactured by Amersham Pharmacia Biotech).

  The glutathione sepharose used was previously treated to suppress nonspecific adsorption. That is, glutathione sepharose was washed three times with the same amount of PBS (8000 × g, 1 minute, 4 ° C.), and then the same amount of 4% BSA-containing PBS was added and treated at 4 ° C. for 1 hour. After the treatment, it was washed twice with the same amount of PBS and resuspended in 1/2 amount of PBS. 40 μL of pretreated glutathione sepharose was added to 1 mL of cell-free extract and gently stirred at 4 ° C. Thereby, the fusion proteins GST-YN2-C1 and GST-YN2-C2 were adsorbed to glutathione sepharose.

  After adsorption, the glutathione sepharose was collected by centrifugation (8000 × g, 1 minute, 4 ° C.) and washed 3 times with 400 μL of PBS. Thereafter, 40 μL of 10 mM glutathione was added and stirred for 1 hour at 4 ° C. to elute the adsorbed fusion protein. The supernatant was collected by centrifugation (8000 × g, 2 minutes, 4 ° C.) and then dialyzed against PBS to purify the GST fusion protein. It was confirmed by SDS-PAGE that a single band was shown.

  Each GST fusion protein (500 μg) was digested with PreScission protease (Amersham Pharmacia Biotech, 5 U), and then passed through glutathione sepharose to remove the protease and GST. The flow-through fraction was further applied to a Sephadex G200 column equilibrated with PBS to obtain final purified products of the expressed proteins YN2-C1 and YN2-C2. It was confirmed by SDS-PAGE that single bands of 60.8 kDa and 61.5 kDa were exhibited, respectively.

  Each purified enzyme activity was measured by the method described above. The protein concentration in the sample was measured with a micro BCA protein quantitative reagent kit (Pierce Chemical Co., Ltd.). The results of measuring the activity of each purified enzyme are shown in Table 1.

  The enzyme was concentrated using a biological solution sample concentrating agent (Mizubutori-kun AB-1100, manufactured by Ato Co., Ltd.) to obtain a 10 U / ml purified enzyme solution.

(Reference Example 2)
Production of PHA synthase 2 Inoculate P91 strain, H45 strain, YN2 strain or P161 strain into 200 ml of M9 medium containing 0.5% yeast extract (manufactured by Difco) and 0.1% octanoic acid. Cultured with shaking for minutes. After 24 hours, the cells are collected by centrifugation (10,000 xg, 4 ° C, 10 minutes), washed by resuspending in 200 ml of 0.1M Tris-HCl buffer (pH 8.0) and centrifuging again. did. The cells are resuspended in 2.0 ml of 0.1M Tris-HCl buffer (pH 8.0), crushed with an ultrasonic crusher, and centrifuged (12,000 × g, 4 ° C, 10 minutes) to obtain the supernatant. Recovery gave a crude enzyme. The results of measuring the activity of each crude enzyme are shown in Table 2.

Each enzyme was concentrated using a biological solution sample concentrating agent (Mizubutori-kun AB-1100, manufactured by Ato Co., Ltd.) to obtain a purified enzyme solution of 10 U / ml.

(Reference Example 3)
Synthesis of 3-hydroxyacyl CoA (R) -3-Hydroxyoctanoyl-CoA is based on Rehm BHA, Kruger N, Steinbuchel A (1998) Journal of Biological Chemistry 273 pp24044-24051 went. Acyl-CoA synthetase (manufactured by Sigma) is dissolved in Tris-HCl buffer (50 mM, pH 7.5) containing 2 mM ATP, 5 mM MgCl ₂ , 2 mM coenzyme A, 2 mM (R) -3-hydroxyoctanoate, 0.1 milliunit / microliter. The mixture was kept warm in a 37 ° C. bath, sampled in a timely manner, and the reaction progress was analyzed by HPLC. After stopping the enzymatic reaction by adding sulfuric acid to the sampled reaction solution to 0.02 N, (R) -3-hydroxyoctanoate which is an unreacted substrate was extracted and removed with n-heptane. For HPLC analysis, an RP18 column (nucleosil C18, 7 mm, Knauser) was used and eluted with a linear gradient of acetonitrile using 25 mM phosphate buffer (pH 5.3) as the mobile phase, and a diode array detector. The thioester compound produced by the enzyme reaction was detected by monitoring the absorption spectrum from 200 to 500 nm. Similarly, (R) -3-hydroxy-5-phenylvaleryl CoA and (R) -3-hydroxy-5- (4-fluorophenyl) valeryl CoA were prepared.

Example 1
Ink-supported PHA-coated liposomal dipalmitoylphosphatidylcholine, distearyl phosphatidylcholine and cholesterol, respectively, 159.7mg, 172.0mg, 168.3mg (1: 1: 2 molar ratio total 500mg) in a 1 liter beaker It was dissolved in 70 mL of a 1: 1 mixed solution of chloroform and isopropyl ether. To this was added 10 mL of a solution of water-soluble dye direct special black AXN (manufactured by Nippon Kayaku Co., Ltd.) and emulsified with a probe type ultrasonic oscillator (Ohtake) to prepare a w / o type emulsion. Ultrasonic irradiation was repeated 10 times for 30 seconds under the condition of 50 watts. The emulsion thus prepared was subjected to a rotary evaporator, and the organic solvent was distilled off under reduced pressure at 60 ° C. to obtain a liposome carrying a dye. The degree of vacuum of the evaporator was high at the beginning, and the degree of vacuum was adjusted to prevent bumping as the evaporation of the organic solvent progressed. Thereafter, a small amount of the organic solvent remaining in the liposome carrying the dye was distilled off by wiping with nitrogen gas. Add the appropriate amount of 10 mM phosphate buffer (pH 7.0) to the liposome carrying the dye to make 30 mL, filter through a 1.2 micron filter (Acrodisc, Gelman), and use a dialysis membrane (Spectrapor, SpectrumMedical). The unsupported dye was removed by dialysis in 10 mM phosphate buffer for 24 hours to obtain a liposome carrying the dye. According to the dynamic light scattering method, the average particle size was 650 nm.

  PHA synthase YN2-C1 derived from Pseudomonas cichorii YN2 prepared in Reference Example 1 was added to the liposome carrying the dye at 100 U / mL and allowed to stand at 20 ° C. for 30 minutes. Next, (R) -3-hydroxyoctanoyl CoA prepared in Reference Example 3 was added to a final concentration of 5 mM. The synthesis reaction was performed by incubating at 37 ° C. for 30 minutes. The reaction solution was subjected to size fractionation by gel filtration (Sephadex G-50 column) to obtain PHA-coated liposomes. According to the dynamic light scattering method, the average particle size of the PHA-coated liposome was in a monodispersed state of 750 nm.

  A portion of the prepared PHA-coated liposome was vacuum-dried, suspended in 20 ml of chloroform, and stirred at 60 ° C. for 20 hours to extract PHA forming the outer coat. The extract is filtered through a membrane filter with a pore size of 0.45μm, concentrated under reduced pressure on a rotary evaporator, then methanolyzed according to a conventional method, and analyzed with a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method). Then, the methyl esterified product of the PHA monomer unit was identified. As a result, as shown in FIG. 2, the PHA was confirmed to be PHA having 3-hydroxyoctanoic acid as a monomer unit. Furthermore, 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.

(Example 2)
Liposomes carrying vancomycin as a PHA-coated liposome antibiotic carrying antibiotics were prepared as follows. Purified egg yolk lecithin 2.1 g and cholesterol 0.9 g were weighed and dissolved in 20 ml of chloroform in an eggplant-shaped flask. Next, chloroform was removed by a rotary evaporator, and a membrane component mixture completely dried by a vacuum dryer was obtained. To this mixture, 20 ml of a 5% glucose solution and 0.4 g of vancomycin were added, and the mixture was sonicated to disperse the mixture and then freeze-thawed to obtain a multilamellar vesicle containing vancomycin. Gel filtration using a Sephadex G-50 column was performed to remove vancomycin not supported on the liposomes, and a size-fractionated liposome fraction was obtained. According to the dynamic light scattering method, the average particle size was 750 nm.

  PHA synthase YN2-C2 derived from Pseudomonas cichorii YN2 prepared in Reference Example 1 was added to 100 U / mL and allowed to stand at 20 ° C. for 30 minutes. Next, (R) -3-hydroxy-5-phenylvaleryl CoA prepared in Reference Example 3 was added to a final concentration of 5 mM. The synthesis reaction was performed by incubating at 37 ° C. for 30 minutes.

  The reaction solution was subjected to size fractionation by gel filtration (Sephadex G-50 column) to obtain PHA-coated liposomes. According to the dynamic light scattering method, the average particle size of the PHA-coated liposome was in a monodispersed state of 820 nm.

  A portion of the prepared PHA-coated liposome was vacuum-dried, suspended in 20 ml of chloroform, and stirred at 60 ° C. for 20 hours to extract PHA forming the outer coat. The extract is filtered through a membrane filter with a pore size of 0.45μm, concentrated under reduced pressure on a rotary evaporator, then methanolyzed according to a conventional method, and analyzed with a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method). Then, the methyl esterified product of the PHA monomer unit was identified. As a result, as shown in FIG. 3, the PHA was confirmed to be PHA having 3-hydroxy-5-phenylvaleric acid as a monomer unit. Furthermore, 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 = 18,000 and Mw = 38,000.

(Example 3)
PHA-coated liposome carrying a pesticide A liposome carrying O, O-dimethyl O- (3-methyl-4-nitrophenyl) phosphorothioate as an agrochemical active ingredient compound was prepared as follows. Purified egg yolk lecithin 2.1 g and cholesterol 0.9 g were weighed and dissolved in 20 ml of chloroform in an eggplant-shaped flask. Next, chloroform was removed by a rotary evaporator, and a membrane component mixture completely dried by a vacuum dryer was obtained. To this mixture was added 20 ml of 5% O- (3-methyl-4-nitrophenyl) phosphorothioate aqueous solution, and the mixture was sonicated to disperse the mixture, and then freeze-thawed to obtain O- (3-methyl-4-nitrophenyl). Multilamellar vesicles containing phosphorothioate were obtained. Gel filtration using a Sephadex G-50 column was performed to remove O- (3-methyl-4-nitrophenyl) phosphorothioate not supported on the liposomes, and a size-fractionated liposome fraction was obtained. According to the dynamic light scattering method, the average particle size was 730 nm.

  PHA synthase derived from the P161 strain prepared in Reference Example 2 was added to 100 U / mL, and the mixture was allowed to stand at 20 ° C for 30 minutes. Next, (R) -3-hydroxy-5- (4-fluorophenyl) valeryl CoA prepared in Reference Example 3 was added to a final concentration of 5 mM. The synthesis reaction was performed by incubating at 37 ° C. for 30 minutes.

  The reaction solution was subjected to size fractionation by gel filtration (Sephadex G-50 column) to obtain PHA-coated liposomes. According to the dynamic light scattering method, the average particle size of the PHA-coated liposome was a monodispersed state of 790 nm.

  A portion of the prepared PHA-coated liposome was vacuum-dried, suspended in 20 ml of chloroform, and stirred at 60 ° C. for 20 hours to extract PHA forming the outer coat. The extract is filtered through a membrane filter with a pore size of 0.45μm, concentrated under reduced pressure on a rotary evaporator, then methanolyzed according to a conventional method, and analyzed with a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method). Then, the methyl esterified product of the PHA monomer unit was identified. As a result, as shown in FIG. 4, the PHA was confirmed to be PHA having (R) -3-hydroxy-5- (4-fluorophenyl) valeric acid as a monomer unit. Furthermore, 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 = 15,000 and Mw = 35,000.

Example 4
Liposomes carrying 2,4-dihydroxybenzophenone, which is an example of a UV absorber as a PHA-coated liposome cosmetic carrying a cosmetic, were prepared as follows. Dipalmitoylphosphatidylcholine, distearyl phosphatidylcholine and cholesterol, respectively, 159.7 mg, 172.0 mg, 168.3 mg (500 mg total in a 1: 1: 2 molar ratio), 1: 1 of chloroform and isopropyl ether in a 1 liter beaker. 1 was dissolved in 70 mL of the mixed solution. To this was added 10 mL of a 5 mass% aqueous solution of 2,4-dihydroxybenzophenone and emulsified with a probe type ultrasonic oscillator (Ohtake) to prepare a w / o type emulsion. Ultrasonic irradiation was repeated 10 times for 30 seconds under the condition of 50 watts. The emulsion thus prepared was subjected to a rotary evaporator, and the organic solvent was distilled off under reduced pressure at 60 ° C. to obtain liposomes carrying 2,4-dihydroxybenzophenone. The degree of vacuum of the evaporator was high at the beginning, and the degree of vacuum was adjusted to prevent bumping as the evaporation of the organic solvent progressed. Thereafter, a small amount of the organic solvent remaining in the liposome carrying 2,4-dihydroxybenzophenone was distilled off by wiping with nitrogen gas. In addition, an appropriate amount of 10 mM phosphate buffer (pH 7.0) was added to the obtained liposomes supporting 2,4-dihydroxybenzophenone to make 30 mL, filtered through a 1.2 micron filter (Acrodisc, Gelman), and a dialysis membrane (Spectrapor And 2,4-dihydroxybenzophenone were removed by dialysis for 24 hours in 10 mM phosphate buffer using Spectrum Medical, to obtain liposomes carrying 2,4-dihydroxybenzophenone. According to the dynamic light scattering method, the average particle size was 660 nm.

  To the liposomes carrying 2,4-dihydroxybenzophenone, the PHA synthase derived from the H45 strain prepared in Reference Example 2 was added to 100 U / mL and allowed to stand at 20 ° C. for 30 minutes. Next, (R) -3-hydroxyoctanoyl CoA prepared in Reference Example 3 was added to a final concentration of 5 mM. The synthesis reaction was performed by incubating at 37 ° C. for 30 minutes.

  The reaction solution was subjected to size fractionation by gel filtration (Sephadex G-50 column) to obtain PHA-coated liposomes. According to the dynamic light scattering method, the average particle size of the PHA-coated liposome was in a monodispersed state of 730 nm.

  A portion of the prepared PHA-coated liposome was vacuum-dried, suspended in 20 ml of chloroform, and stirred at 60 ° C. for 20 hours to extract PHA forming the outer coat. The extract is filtered through a membrane filter with a pore size of 0.45μm, concentrated under reduced pressure on a rotary evaporator, then methanolyzed according to a conventional method, and analyzed with a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method). Then, the methyl esterified product of the PHA monomer unit was identified. As a result, it was confirmed that the PHA was PHA having 3-hydroxyoctanoic acid as a monomer unit. Furthermore, 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 = 17,000 and Mw = 37,000.

(Example 5)
Using a blood collection bag containing a liposome anticoagulant for artificial red blood cells, 1.5 L was collected from the vein of a cow. The collected blood was aseptically transported and stored in a sealed container at 4 ° C. The following steps were all aseptically performed at a low temperature of 4 ° C. Centrifugal washing was performed with physiological saline using a continuous centrifuge to obtain 500 mL of coarsely washed red blood cells from which platelets, white blood cells, and plasma were removed. Further washing was performed with physiological saline using a plasma separator having a pore size of 0.45 μm. 1 L of pyrogen-free distilled water was added to 500 mL of washed erythrocytes to cause hemolysis. Using a plasma separator with a pore size of 0.45μ and a plasma component separator with a pore size of 0.1μ, the erythrocyte membrane was removed and the filter was sterilized. About 1.2 L of erythrocyte membrane-removed hemoglobin having a hemoglobin concentration of 8% (w / w) was obtained. About 180 mL of erythrocyte membrane-removed hemoglobin having a hemoglobin concentration of 50% (w / w) was obtained by concentrating by ultrafiltration using a dialyzer TAF10W (a cellulosic hollow dialyzer manufactured by Terumo).

  27.76 g of purified phosphatidylcholine with a hydrogenation rate of 80%, 6.96 g of cholesterol, and 3.75 g of purified phosphatidic acid with a hydrogenation rate of 80% were dissolved in chloroform. The lipid solution was placed in an eggplant flask, evaporated to remove chloroform, and a lipid film was formed on the bottom of the eggplant flask. Further, vacuum drying was performed for 16 hours to completely remove chloroform.

  Erythrocyte membrane-removed hemoglobin (180 mL) was added to the lipid membrane prepared in the preparation of the liposome-forming lipid, and a suspension was prepared using a portex mixer, which was used as a raw material solution. The raw material solution was placed in a pressurized container with a slit nozzle and a pearl cell crusher (manufactured by PAR, USA), and nitrogen gas was introduced to pressurize to 130 kg / cm2. Let stand for 30 minutes to allow nitrogen gas to fully penetrate into the raw material solution. Next, the nozzle valve was gradually opened, and the raw material solution was discharged while maintaining a pressure of 130 kg / cm @ 2.

  The liquid after pressure discharge was fractionated by a gel filtration method (Sephadex G-50 column) to remove hemoglobin not supported on the liposomes, thereby obtaining liposomes supporting hemoglobin.

  PHA synthase YN2-C1 derived from Pseudomonas cichorii YN2 prepared in Reference Example 1 was added to the hemoglobin-supported liposomes at 100 U / mL and allowed to stand at 20 ° C. for 30 minutes. Next, (R) -3-hydroxyoctanoyl CoA prepared in Reference Example 3 was added to a final concentration of 5 mM. The synthesis reaction was performed by incubating at 37 ° C. for 30 minutes.

  The reaction solution was subjected to size fractionation by gel filtration (Sephadex G-50 column) to obtain PHA-coated liposomes. According to the dynamic light scattering method, the average particle size of the PHA-coated liposome was a monodispersed state of 720 nm.

  A portion of the prepared PHA-coated liposome was vacuum-dried, suspended in 20 ml of chloroform, and stirred at 60 ° C. for 20 hours to extract PHA forming the outer coat. The extract is filtered through a membrane filter with a pore size of 0.45μm, concentrated under reduced pressure on a rotary evaporator, then methanolyzed according to a conventional method, and analyzed with a gas chromatography-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method). Then, the methyl esterified product of the PHA monomer unit was identified. As a result, it was confirmed that the PHA was PHA having 3-hydroxyoctanoic acid as a monomer unit. Furthermore, 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 = 17,000 and Mw = 37,000.

(Example 6)
500 mg of sustained-release dipalmitoylphosphatidylcholine from PHA-coated liposomes carrying calcein in the lumen was dissolved in 70 mL of a 1: 1 mixture of chloroform and isopropyl ether in a 1-liter beaker. To this was added 10 mL of an aqueous solution of a water-soluble fluorescent dye calcein and emulsified with a probe type ultrasonic oscillator (Ohtake) to prepare a w / o type emulsion. Ultrasonic irradiation was repeated 10 times for 30 seconds under the condition of 50 watts. The emulsion thus prepared was subjected to a rotary evaporator, and the organic solvent was distilled off under reduced pressure at 60 ° C. to obtain a liposome carrying a dye. The degree of vacuum of the evaporator was high at the beginning, and the degree of vacuum was adjusted to prevent bumping as the evaporation of the organic solvent progressed. Thereafter, a small amount of the organic solvent remaining in the liposome carrying the dye was distilled off by wiping with nitrogen gas. Add the appropriate amount of 10 mM phosphate buffer (pH 7.0) to the resulting liposome carrying the dye to make 30 mL, filter through a 1.2 micron filter (Acrodisc, Gelman), and add the calcein-loaded liposome in the lumen. Obtained.

  PHA synthase YN2-C1 derived from Pseudomonas cichorii YN2 prepared in Reference Example 1 was added to a part of the liposome supporting calcein at 100 U / mL and allowed to stand at 20 ° C. for 30 minutes. Next, (R) -3-hydroxyoctanoyl CoA prepared in Reference Example 3 was added to a final concentration of 5 mM. The synthesis reaction was carried out by incubating at 20 ° C. for 90 minutes. The reaction solution was subjected to size fractionation by gel filtration (Sephadex G-50 column) to obtain PHA-coated liposomes.

  The time-dependent release behavior of calcein supported in the lumen of this PHA-coated liposome and liposome not coated with PHA was examined by measuring the fluorescence intensity of calcein. The results are shown in FIG.

  Regarding sustained release at a temperature lower than the phase transition temperature of the phospholipid membrane (25 ° C.), the PHA-coated liposome was superior in retention ability compared to the liposome not coated with PHA. Regarding the sustained release at the phase transition temperature (about 42 ° C.) of the phospholipid membrane, the PHA-coated liposome showed a quicker release than the uncoated liposome. When the temperature was returned to 25 ° C again, the release of PHA-coated liposomes was suppressed.

  Therefore, it was revealed that the PHA-coated liposome of this example had improved sustained-release temperature sensitivity compared with the liposome not coated with PHA. The improvement in retention at room temperature (25 ° C.) is thought to be due to the suppression of leakage of the contents from the liposomes due to the difference in osmotic pressure inside and outside the liposomes due to the high mechanical strength of the coated PHA. On the other hand, the improvement in releasability at the phase transition temperature is that the difference in osmotic pressure of liposomes coated with PHA is kept larger than the difference in osmotic pressure of liposomes not coated with PHA. This is thought to be because the leakage of the material was accelerated, and the PHA in the outer shell was porous so that the contents permeated quickly.

(Example 7)
A gradient polyhydroxyalkanoate-coated liposome dipalmitoylphosphatidylcholine (500 mg) was dissolved in a 1: 1 mixture of chloroform and isopropyl ether in 70 mL in a 1-liter beaker. To this was added 10 mL of an aqueous solution of a water-soluble fluorescent dye calcein and emulsified with a probe type ultrasonic oscillator (Ohtake) to prepare a w / o type emulsion. Ultrasonic irradiation was repeated 10 times for 30 seconds under the condition of 50 watts. The emulsion thus prepared was subjected to a rotary evaporator, and the organic solvent was distilled off under reduced pressure at 60 ° C. to obtain a liposome carrying a dye. The degree of vacuum of the evaporator was high at the beginning, and the degree of vacuum was adjusted to prevent bumping as the evaporation of the organic solvent progressed. Thereafter, a small amount of the organic solvent remaining in the liposome carrying the dye was distilled off by wiping with nitrogen gas. Add the appropriate amount of 10 mM phosphate buffer (pH 7.0) to the resulting liposome carrying the dye to make 30 mL, filter through a 1.2 micron filter (Acrodisc, Gelman), and add the calcein-loaded liposome in the lumen. Obtained.

  PSE synthase YN2-C1 derived from Pseudomonas cichorii YN2 prepared in Reference Example 1 was added to a part of the liposome loaded with calcein to 100 U / mL, and gently shaken at 20 ° C for 30 minutes to add PHA synthase. Immobilized on the liposome surface.

  Size fractionation was performed by gel filtration (Sephadex G-50 column) to obtain a synthetic enzyme-immobilized liposome fraction. 30 mM (R) -3-hydroxyoctanoyl CoA (prepared by the method described in Eur. J. Biochem., 250, 432-439 (1997)), 0.1% bovine serum albumin 100 parts by mass of 0.1 M phosphate buffer (pH 7.0) containing (manufactured by Sigma) was added. Subsequently, 30 mM (R) -3-hydroxypimeryl CoA (prepared by the method described in J. Bacteriol., 182, 2753-2760 (2000)) was added to the reaction solution while gently shaking at 30 ° C. 0.1 M phosphate buffer (pH 7.0) containing 1% bovine serum albumin (manufactured by Sigma) at a rate of 25 parts by mass per minute using a micro tube pump (MP-3N, manufactured by Tokyo Rika Kikai Co., Ltd.) Added.

  After shaking for 30 minutes, it was washed with 0.1 M phosphate buffer (pH 7.0) to remove unreacted substances and air-dried to obtain polyhydroxyalkanoate-coated liposomes.

  The polyhydroxyalkanoate-coated liposome was freeze-dried, and the mass of the polymer formed on the surface was measured with a time-of-flight secondary ion mass spectrometer (TOF-SIMS IV, manufactured by CAMECA). From the obtained mass spectrum, it was found that the surface of the polyhydroxyalkanoate-coated liposome was composed of a copolymer of 3-hydroxypimelic acid and 3-hydroxyoctanoic acid (molar ratio 17: 1). In addition, when the mass spectrum was measured by TOF-SIMS while the surface of the polyhydroxyalkanoate-coated liposome was gradually scraped by ion sputtering, the composition ratio of 3-hydroxypimelic acid in the copolymer gradually decreased. However, the composition ratio of 3-hydroxyoctanoic acid increased. As a result, the polyhydroxyalkanoate-coated liposome of this example was coated with polyhydroxypimelate having a hydrophilic functional group on the surface, and 3-hydroxypimelic acid having a hydrophilic functional group and a hydrophobic surface under the polyhydroxypimelate. It was found that a 3-hydroxyoctanoic acid copolymer having a functional group had a gradient structure coated while increasing the composition ratio of 3-hydroxyoctanoic acid toward the lower layer.

  The average 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 = 21,000, Mw = 40,000.

(Example 8)
Preparation of polyhydroxyalkanoate-coated liposome (chemical modification)
In the same manner as in Example 7, an enzyme-immobilized liposome in which calcein was encapsulated and PHA synthase YN2-C1 derived from Pseudomonas cichorii YN2 prepared in Reference Example 1 was immobilized on the surface was prepared.

  1 part by mass of the enzyme-immobilized liposome is suspended in 48 parts by mass of 0.1 M phosphate buffer (pH 7.0), and (R, S) -3-hydroxy-5-phenoxyvaleryl CoA (3-phenoxypropanal) is suspended. After hydrolyzing 3-hydroxy-5-phenoxyvaleric acid ester obtained by Reformatsky reaction of bromoacetate with ethyl bromoacetate to give 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) After epoxidizing the unsaturated portion of 3-hydroxy-7-octenoic acid synthesized by the method described in US Pat. No. 91 (1990) with 3-chlorobenzoic acid, Eur. J. Biochem., 250, 432-439 (1997) )) 0.2 parts by mass, bovine serum albumin (manufactured by Sigma) 0.1 part by mass, In 2 hours gentle shaking to obtain a sample 1.

  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.

  Collect 10 μl of the above sample on a slide glass, add 10 μl of 1% Nile Blue A aqueous solution, mix on the slide glass, place the cover glass, and place a fluorescence microscope (330-380 nm excitation filter, 420 nm long-pass absorption) (Filter, manufactured by Nikon Corporation) was observed. As a result, it was confirmed that the liposome surface emitted fluorescence in any sample. Therefore, it was found that the liposome was coated on the surface with PHA.

  As a control, 1 part by mass of a polyhydroxyalkanoate-coated liposome was added to 49 parts by mass of 0.1 M phosphate buffer (pH 7.0), gently shaken at 30 ° C. for 2.5 hours, and then similarly. The sample was stained with Nile Blue A and observed with a fluorescence microscope. As a result, the liposome surface did not fluoresce at all.

Further, a part of the sample was collected by centrifugation (10,000 xg, 4 ° C, 10 minutes), vacuum-dried, suspended in chloroform, stirred at 60 ° C for 20 hours, and PHA forming the outer cover was removed. Extracted. This extract was subjected to ¹ H-NMR analysis (device used: FT-NMR: Bruker DPX400, measurement nuclide: ¹ H, solvent used: deuterated chloroform (with TMS)). The unit% of each side chain unit calculated from this is shown in Table 3.

  The above sample 1 was centrifuged at 50 parts by mass (10,000 × g, 4 ° C., 10 minutes) to recover polyhydroxyalkanoate-coated liposomes and suspended in 50 parts by mass of purified water three times. Thereafter, 0.5 part by mass of hexamethylenediamine was dissolved in the suspension as a crosslinking agent. After confirming dissolution, water was removed by freeze-drying (this is designated as sample 3). Furthermore, sample 3 was reacted at 70 ° C. for 12 hours (this is referred to as sample 4).

  Sample 3 and sample 4 were suspended in chloroform, stirred at 60 ° C. for 20 hours to extract PHA forming the outer coat, chloroform was removed by vacuum drying, and a differential scanning calorimeter (DSC; manufactured by Perkin Elmer, Pyris) (1, temperature increase: 10 ° C./min). As a result, in Sample 3, a clear exothermic peak was observed at around 90 ° C., indicating that a reaction between the epoxy group in the polymer and hexamethylenediamine occurred, and the cross-linking of the polymers proceeded. On the other hand, Sample 4 shows no clear heat flow, indicating that the crosslinking reaction is almost complete.

Furthermore, infrared absorption was measured for the same sample (FT-IR; manufactured by PerkinElmer, 1720X). As a result, the peaks of amine (near 3340 cm ⁻¹ ) and epoxy (near 822 cm ⁻¹ ) seen in sample 3 disappear in sample 4.

  From the above results, it was clarified that a crosslinked polymer can be obtained by reacting PHA having an epoxy unit in the side chain with hexamethylenediamine.

  On the other hand, the same evaluation was performed for Sample 2 as a comparative control. However, as described above, an evaluation result clearly showing crosslinking between polymers was not obtained.

It is a flowchart showing an example of a method for producing a PHA-coated liposome of the present invention. FIG. 3 is a diagram showing the results of GC-MS analysis of the outer shell of the PHA-coated liposome of Example 1. FIG. 4 is a diagram showing the results of GC-MS analysis of the outer shell of the PHA-coated liposome of Example 2. Example 3 is a diagram showing the results of GC-MS analysis of the PHA-coated liposome outer shell. It is a figure which shows the release | release behavior of calcein from the PHA coating liposome in Example 6 at 25 degreeC and 42 degreeC, and the liposome which is not coat | covered with PHA with time.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Liposomes 2 Water-soluble substance carried in liposome lumen 3 Fat-soluble substance carried in liposome membrane 4 Polyhydroxyalkanoate synthase immobilized on liposome 5 Polyhydroxyalkanoate 6 Polyhydroxyalkanoate coated from liposome Release behavior of calcein 7 Release behavior of calcein from liposomes not coated with polyhydroxyalkanoate

Claims (9)

Having polyhydroxyalkanoate synthase on the surface of at least a part of the outer wall of the liposome ;
A polyhydroxyalkanoate-coated liposome, which is coated with a plurality of layers of the polyhydroxyalkanoate having different compositions in the direction from the inside to the outside of the liposome. The polyhydroxyalkanoate-coated liposome according to claim 1, wherein the polyhydroxyalkanoate contains at least one selected from the group consisting of monomer units represented by the formulas [1] to [10].

(However, the monomer unit is at least one selected from the group consisting of monomer units in which the combination of R1 and a is any of the following:
A monomer unit in which R1 is a hydrogen atom (H), and a is any integer of 0 to 10,
A monomer unit in which R1 is a halogen atom and a is any integer of 1 to 10,
A monomer unit wherein R1 is a chromophore and a is any integer from 1 to 10;
A monomer unit in which R1 is a carboxyl group or a salt thereof, and a is an integer of 1 to 10,
R1 is

A monomer unit in which a is any integer from 1 to 7. )

(However, b in the formula represents either 0-7 integer, R2 is a hydrogen atom (H), a halogen _{atom, -CN, -NO 2, -CF 3} , -C 2 F 5 and -C ₃ F ₍ Represents any one selected from the group consisting of ₇ )

(However, c in the formula represents any of integers from 1 to 8, R3 is hydrogen atom (H), a halogen _{atom, -CN, -NO 2, -CF 3} , -C 2 F 5 and -C ₃ F ₍ Represents any one selected from the group consisting of ₇ )

(However, d in the formula represents either 0-7 integer, R4 is a hydrogen atom (H), a halogen _{atom, -CN, -NO 2, -CF 3} , -C 2 F and -C ₃ F ₇ Represents any one selected from the group consisting of

(Wherein in e represents any of integers from 1 to 8, R5 is a hydrogen atom (H), a halogen _{atom, -CN, -NO 2, -CF 3} , -C 2 F 5, -C 3 F ₇ represents any one selected from the group consisting of —CH ₃ , —C ₂ H ₅ and —C ₃ H _7. )

(In the formula, f represents any integer of 0 to 7.)

(In the formula, g represents an integer of 1 to 8.)

(In the formula, h represents an integer of 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, and —C (CH ₃ ) ₃ , wherein R ′ represents a hydrogen atom ( H), Na, K, —CH ₃ , —C ₂ H ₅ , and R ″ is —OH, —ONa, —OK, a halogen atom, —OCH ₃ , or —OC ₂ H ₅ . .)

(Wherein i represents an integer of 1 to 7, and R7 is selected from the group consisting of a hydrogen atom (H), a halogen atom, —CN, —NO ₂ , —COOR ′, and —SO ₂ R ″). Any one selected, wherein R ′ is any one of a hydrogen atom (H), Na, K, —CH _3, and —C ₂ H ₅ , and R ″ is —OH, —ONa, -OK, it is either a halogen atom, -OCH ₃ and -OC ₂ H _5.)

(Where j represents any integer from 1 to 9) The polyhydroxyalkanoate-coated liposome according to claim 1 or 2 , wherein the polyhydroxyalkanoate has a hydrophilic functional group. The polyhydroxyalkanoate-coated liposome according to any one of claims 1 to 3 , wherein at least a part of the polyhydroxyalkanoate is a polyhydroxyalkanoate chemically modified to have a specific function. The polyhydroxyalkanoate-coated liposome according to claim 4 , wherein the chemically modified polyhydroxyalkanoate has at least a graft chain. 6. The polyhydroxyalkanoate-coated liposome according to claim 5 , wherein the graft chain is a graft chain obtained by chemical modification of polyhydroxyalkanoate containing at least a monomer unit having an epoxy group. The polyhydroxyalkanoate-coated liposome according to claim 5 or 6 , wherein the graft chain is a graft chain of a compound having an amino group. The polyhydroxyalkanoate-coated liposome according to claim 4 , wherein at least a part of the polyhydroxyalkanoate is a crosslinked polyhydroxyalkanoate. In said liposome, pigment suspension, a dye, agricultural chemical ingredients, hemoglobin, cosmetic component, polyhydroxyalkanoate according to any one of claims 1 to nutrients or pharmaceutical active ingredients are the encapsulating 8 Coated liposome.

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