KR100457546B1 - A microsphere and process for producting thereof using polyfructose and its derivatives - Google Patents

Abstract

The present invention relates to microspheres using levan, a polyfructose, and derivatives thereof, and more particularly, to leban, a polymer [(2-6) -beta-di-fructan] of fructose, which is a strong hydrophilic And a method for producing polyfructose copolymer microspheres by dissolving the derivatives in a basic solution and then adding them to an oil-based reverse suspension polymerization method and a crosslinking agent, and the use of the prepared microspheres.

The polyfructose copolymer microspheres of the present invention are useful as gel filtration, affinity, ion exchange, hydrophobic chromatography fillers, enzyme and cell immobilization carriers, drug delivery carriers, other functional cosmetic raw materials, skin supplements for protein purification. There is an excellent effect that can be used.

Description

Microspheres using polyfructose or derivatives thereof and a method for producing the same {A MICROSPHERE AND PROCESS FOR PRODUCTING THEREOF USING POLYFRUCTOSE AND ITS DERIVATIVES}

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to microspheres using polyfructose and derivatives thereof, and to a method for producing the same, and more particularly, to Levan [(2,6) -beta-di, which is a hydrophilic polyprotose -Fructan] is dissolved in a basic solution, and then it is related to polyprotose copolymer microspheres and a method for producing the same by reverse suspension polymerization and the addition of a crosslinking agent and the use of the prepared microspheres.

(2,6) -beta-di-fructan, Levan, was a by-product produced by the microorganisms that first contaminated the sugar juice, an undesirable by-product of increasing the viscosity of the preparation during sugar production. Levan is produced by microorganisms, but is also reported to be produced by the plant itself. In 1881, when Lippman reported, it was called Levulan, but it is now simply called Levan. The chemical structure is a polymer of fructose. The main bond consists of beta 2,6 bonds, and branches are formed by beta 2,1 bonds. The molecular weight produced by plants is not high in molecular weight, but the microorganisms produce high molecular weights of about 2 × 10 ⁷ .

Lebanese-producing microorganisms have been reported by Acetobacter pasteurianus and 27 species (Han, YW, Advances in Applied Microbiology, 35, 171 (1992)). For the production of levane , fermentation and enzymatic synthesis have been studied, and in the present invention, Zymmonas mobilis and Bacillus subtilis , which are obtained by expressing the Levanschkrase genes of E. coli, respectively. What was obtained using a genetically recombined Levanschkrase was used.

Levan, a homopolymer of fructose, is a plasma substitute (Dodender et al., Bull. Soc. Chim. Biol., 39, 438 (1957); Schechter et al., J. Lab. Clin. Med., 61, 962 (1963). ), As a colloidal stabilizer, immunological agent, pharmacological effect enhancer (Leibovici et al., Anticancer Res., 5, 553 (1985); Stark et al., Br. J. Exp. Path, 67, 141 ( 1986), as a quality improver, stabilizer, or additive in health foods (Hatcher et al., Bioprocess, Technol., Ll, 1 (1989)), which can be used in the food field and in the printing and cosmetic fields. Whiting et al., J. Inst. Brew., 73, 422 (1961); Han, Bioprecess Technol., 12, 1 (1990).

The inventors of the present invention have shown that the chemically bonded type is an inorganic polymer with low mechanical strength and inorganic polymers such as dextran gel (European Patent No. 974,054 and US Patent No. 4,794,177), agarose gel, polyacrylamide gel, etc. In order to solve the problem of silica gel vulnerable to alkali, the research resulted in the development of biodegradable, inexpensive and easily obtainable Levan polymer microspheres. Microspheres prepared by lowering the molecular weight of Levan, a biopolymer, have excellent affinity for peptides or proteins, and have strong mechanical strength due to strong covalent bonds between polymers, and unlike synthetic polymers, they are harmless to the natural environment. The present invention can be accomplished by using the same as gel filtration, affinity, ion exchange, filler for hydrophobic chromatography, enzyme and cell fixation carrier, drug delivery medium, functional cosmetic raw material and skin supplement. It came to the following.

Accordingly, an object of the present invention is to provide a porous polyfructose copolymer microsphere and a method for preparing the same by reacting polyfructose with an agent which is a crosslinking reagent in an oil emulsion to have cross-linkage. To provide.

Another object of the present invention is to provide a polyfructose copolymer microsphere, wherein the polyfructose copolymer obtained by the above method is used as a filler for chromatography and a carrier for enzyme immobilization.

The above object of the present invention was achieved by preparing polyfructose microspheres using Levan or its derivatives, and identifying the use of the microspheres of the present invention as a filler for chromatography and a carrier for enzyme immobilization.

Hereinafter, the configuration of the present invention will be described in detail.

1 is a scanning electron micrograph of polyfructose microspheres prepared by the method of Example 1 of the present invention.

2 is a polyfructose microsphere of the present invention showing a microsphere size of 45-150 μm (dry sample), an expansion ratio of 4 mL / g, and a pressure difference of 7.0 psi at a linear velocity of 130 cm / h. Chromatogram of bovine serum albumin (BSA) and sodium chloride.

Figure 3 is a thyroid gland using the present invention polyfructose microspheres showing a pressure difference of 7.0 psi at a linear velocity of 45-150 μm (dry sample), an expansion rate of 11 mL / g, and a linear velocity of 80 cm / h. It is a chromatogram fractionated from globulin (Thyroglobulin), bovine serum albumin (cytochrome C).

The present invention is a process for preparing polyfructose into hydrophilic and porous copolymers in the form of microspheres. In particular, the present invention is characterized in that under a basic solution, a hydroxyl group of polyfructose and a bifunctional organic substance used as a crosslinking agent react to form an ether bridge between polymers.

The method for preparing a copolymer, in which a bifunctional substance reacts with polyfructose containing a hydroxy group in the presence of an alkaline substance, sufficiently stirs a dispersion medium capable of forming a two phase with the polyfructose solution. The polyfructose solution is converted into a suspension point in the dispersion medium, and the polyfructose and the crosslinking agent react until the gel spherical particles are formed.

Polyfructose microspheres used in the present invention refers to microspheres prepared using Levan or derivatives thereof (hereinafter, abbreviated as 'polyfructose microspheres'). The term copolymer encompasses a product by chemical bonding of numerous similar units forming a single molecule.

In order to form a continuous phase (dispersion medium) in two phases, when polyfructose having a hydroxyl group is dissolved in water, it is an organic solvent having no solubility in water, and is aliphatic and aromatic hydrocarbon, halogenated aliphatic and aromatic. Hydrocarbons include dichloromethane, 1,2-dichloroethane, 1,2-dibromoethane, o -dichlorobenzene, toluene, mineral oil octane, heptane, cyclohexane, hexadecane, and soybean oil. .

In preparing the microspheres of the present invention, the concentration of polyfructose is very important because it determines the swelling capacity (water content) of the final copolymer. Low concentrations of polyfructose result in higher swelling of the final product as compared to those with high concentrations. Lebanese polymer concentrations can range from 5 to 70 percent (weight ratios), with positive results in the range of 10 to 50 percent. Examples of polyfructose having a hydroxy group suitable for such a reaction and derivatives thereof include leban, methyllevan, ethyllevan, hydroxy propyllevane, and aryl levan.

In addition, the Levan complex microspheres are mixed with one or more selected from the group consisting of biopolymers such as agarose, dextran, cellulose, chitin and chitosan, derivatives thereof, monomers of synthetic polymers, and ceramic materials, in an appropriate ratio. Can be synthesized.

In the present invention, the bifunctional organic substance used as a crosslinking agent has an X-R-Z form. R is an aliphatic residue having 3-10 carbon atoms, X is a halogen or methoxy group Z is an epoxy group. Suitable amphoteric substances in this reaction include epichlorohydrin, dichlorohydrin, 1,2-3,4 diepoxybutane, bis-epoxypropyl-ether, ethylene glycol-bis-epoxy propyl ether, 1 , 4-butane-diol-bis-epoxy-propyl ether, gamma-glycidoxypropyltrimethoxysilane, and the like can be used. Aliphatic residues that form crosslinks of the copolymer are blocked by oxygen atoms or cause substitution reactions with hydroxyl groups. The molecular ratio of the polyfunctional organics having a bifunctional organic substance and a hydroxy group should be at least 1:10, and in order to increase the effect of the reaction, stirring to obtain the desired size of the suspension point. Under conditions, it is preferable to stir a mixed solution of polyfructose and an alkali substance with a liquid serving as a continuous phase (dispersion medium) in an ideal system. Thereafter, a crosslinking agent is added to the ideal system. However, the crosslinking agent may be first dissolved in a liquid serving as a continuous phase and then reacted.

According to this invention, in order to stabilize the dispersion | distribution of the solution in which polyfructose is melt | dissolved, a stabilizer is added to the liquid which forms a continuous phase (dispersion medium). As the stabilizer, polymers having high molecular weight, such as polyvinylacetate, polystyrene, polyisobutylene, cellulose-acetate-butylate, and ethyl cellulose, which are insoluble in water, are suitable. On average, stabilizers having a relatively higher molecular weight than low molecular weight stabilizers have proven to be highly effective in the stability of dispersion under any of the conditions. A suitable amount of stabilizer may be about 0.1-15 grams, based on 100 ml of the continuous phase, and positive results may be obtained in the range of about 0.5-10 grams. Under certain conditions, the reactants are sorbitan monorate, polyoxyethylene sorbitan monooleate, sorbitan sesquiolit, sorbitan trioleate, polyoxyethylene sorbitan monostyrene, polyoxyethylene isooctyl in the form of a surfactant It is also desirable to add surface active substances such as phenyl ether. Such materials do not act as suspension stabilizers but may be used if one wishes to obtain a smaller size copolymer. During the initial period of the polymerization process, the size of the suspension point at which the conditions of stirring and stabilizer are dispersed is determined. Therefore, when experimenting at different stirring speeds, the stirring speed should be determined to obtain the desired result.

Alkali substances required for this reaction are mostly contained in the polyfructose solution. However, it is also possible to use an alkaline substance in the form dissolved in a dispersion medium forming a continuous phase in an ideal system. In theory, any substance that exhibits alkalinity in solution can be used in this reaction. However, alkali metal oxides such as sodium hydroxide are mainly used as alkaline materials. Materials such as quaternary ammonium materials, alkali metal / alkaline earth metal carbonates and alkaline earth oxides are also used.

In the present invention, the stabilizer can be removed from the gel formed by working up with a suitable solvent. Regarding the ability of high molecular weight polymers to be hydrolyzed under relatively mild conditions, the gel is first treated with a hydrolyzable substance, such as an alkali metal oxide solution, followed by washing the gel with a solvent for these substances. Remove the degraded material. In other words, high molecular weight esters such as polyvinylacetate and cellulose acetate butyrate remaining in the formed gel are treated with a dilute aqueous alkali metal oxide solution which causes its saponification (hydrolysis). High molecular weight alcohols formed after saponification (hydrolysis) can be removed by washing with a suitable solvent.

On the other hand, the time for the gel to form depends on the content of polyfructose, the amount of bifunctional substance, the temperature and the like in the solution to be dispersed. However, this reaction will proceed until the reaction is stopped or until all of the bifunctional material is consumed. After the gel is formed, stirring no longer affects the final particle size of the copolymerization. The reaction temperature will determine the rate at which copolymerization occurs. The possible reaction temperature is about 90 ° C at room temperature, and positive results can be obtained in the range of 30-60 ° C.

As described above, the gel obtained by the present invention has excellent mechanical strength and high hydrophilicity, which is very effective as a filler such as gel filtration chromatography used to separate and purify water-soluble proteins, enzymes, polysaccharides, amino acids and antibiotics. It can be used as ion exchange chromatography, affinity chromatography and enzyme fixation carrier by introducing ion exchange groups such as sulfone groups, amine groups and carboxyl groups using hydroxyl groups present on the surface.

In addition, although the present invention exemplifies polyfructose microspheres for protein separation and enzyme immobilization, the application of the microspheres as drug delivery carriers, functional cosmetic raw materials, and skin supplements has the common knowledge to which the present invention belongs. Will be self-evident.

Hereinafter, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited thereto, and it is obvious that those skilled in the art can make changes to technical values and equivalents.

Example 1 Preparation of Polyfructose Microspheres Using Levan

194 g of Levan was dissolved in a 2N sodium hydroxide solution, and then 0.9 g of sodium borohydride was added to the solution and allowed to stand for 2 hours. A 3-phase paddle-shaped blade agitator (2 L) with a heat jacket was installed, and 21 g of cellulose acetate butyrate as a stabilizer was added thereto, and about 2-fold 1,2-dichloroethane was slowly added to the Levan solution as a dispersion medium. It was added and then dissolved homogeneously. The water layer, which is a Levan's solution, was added to the organic layer, which was a 1,2-dichloroethane solution, with agitation and mixed to make a uniform suspension colloidal solution. Within 1 hour, the temperature was raised to 50 ° C. and 1-chloro-2,3-epoxypropane, a crosslinking agent, was added. The reaction was carried out for 16 to 20 hours while stirring at this temperature. After completion of the reaction, the reactor was cooled slowly. Acetone was added to the product, and only the solvent was separated from the formed gel into the supernatant, followed by the removal of most of the cellulose acetate butyrate by the addition of acetone and the supernatant only again. To remove the stabilizer film on the gel surface, 95% ethanol and aqueous sodium hydroxide solution were dispersed in a 50:50 solution for about 15 minutes. The gel was then neutralized with diluted aqueous hydrochloric acid solution and filtered. The gel obtained after the filtration was washed with distilled water, dehydrated using ethanol and dried in vacuo at 70 ℃ to obtain a polyfructose copolymer microspheres. 1 is a scanning electron micrograph of the present invention polyfructose microspheres prepared by the method of Example 1. FIG.

Example 2 Swellability of Polyfructose Microspheres According to Levan Content

Determining the properties of the copolymer as a molecular sieve is a fundamental property that affects the pore size, which is cross-link density, which is an exclusion limit expressed in terms of molecular weight that cannot pass into the gel spherical particles. exclusion limit). Related to the crosslink density is the swelling capacity (water content). This can be defined as calculating the volume of water recovered by centrifuging 1 g of dry sample after swelling in water, and defining the bed volume, which is the volume it expands after swelling 1 g of dry sample in water. Can be.

The swelling properties of the polyfructose microspheres prepared in Example 1 were investigated as follows. A sample of 1 g was filled into a glass column of 0.9 cm in diameter and 90 cm in length with a glass filter, and distilled water was added thereto. The glass column was swollen up and down for 30 minutes while shaking up and down, and then left still to obtain a swelling volume (bed volume) from the scale of the column.

As a result, as can be seen in Table 1, the polyfructose microspheres of the present invention was found to increase rapidly as the content of the Levan is lowered.

Swellability of Polyfructose Microspheres According to Levan Content density(%) Swellability (ml / g) 35 4-5 30 6-7 25 10-12 20 15-17 15 20-25 10 50-60

Example 3 Swellability of Polyfructose Microspheres According to Crosslinking Agent Content

Among the polyfructose microspheres prepared in Example 1, polyfructose microspheres having different contents using 1-chloro-2,3-epoxypropane as a crosslinking agent based on the microspheres having a Leban content of 30% Was prepared and the swelling property was measured by the method similar to Example 2.

Experimental Results As can be seen in Table 2, the polyfructose microspheres of the present invention was found to increase the swelling property as the content of the crosslinking agent is lowered.

Swelling property of polyfructose microspheres according to the amount of crosslinking agent (leban amount) content(%) Swellability (ml / g) 10 19.3 15 13.8 25 9.35 35 5.6 50 4.5 60 4

Example 4 Formation of Polyfructose Microspheres According to Stabilizer Type

Based on the microspheres having a Levan content of 30% among the polyfructose microspheres prepared in Example 1, the production of polyfructose microspheres according to the type of stabilizer was investigated.

As can be seen in Table 3, when cellulose-acetate-butylate was used as a stabilizer, polyfructose microsphere formation state was the best.

Formation of Polyfructose Microspheres According to Stabilizers Type of stabilizer Microsphere Creation Status Cellulose-acetate-butyrate ++++ Polyisobutylene ++ Polyvinylacetate +++ Polystyrene ++ Sorbitan monorate +++ Polyoxyethylene Sorbitan Monoolet + Polyoxyethylene Isooctylphenyl Ether +

Example 5 Formation of Polyfructose Microspheres According to the Type of Dispersant (organic Phase)

Based on the microspheres having a Levan content of 30% among the polyfructose microspheres prepared in Example 1, the production of polyfructose microspheres according to the type of dispersion medium was investigated.

As can be seen in Table 4, when 1,2-dichloroethane was used as the dispersion medium (organic phase), polyfructose microsphere formation state was the best.

Formation of Polyfructose Microspheres According to the Type of Dispersant Type of dispersion medium Microsphere production aspect 1,2-dichloroethane ++++ 1,2-Dibromoethane +++ 1,2-dichloromethane ++ o -dichlorobenzene ++ toluene ++ Cyclohexane +

Example 6 Preparation of Polyfructose Microspheres Using Levan Derivatives

As a levan derivative, a polyfructose derivative copolymer microsphere was obtained by the same method as Example 1 using methyllevan, ethyllevan, hydropropyl propylban, and allylban, respectively, and the physicochemical properties of the microspheres were It was confirmed that there was no polyfructose microspheres and caudal difference.

Example 7 Application of Polyfructose Microspheres as Gel Filtration Chromatography Filler

Chromatography was performed using polyfructose microspheres of the present invention having different expansion rates as chromatographic fillers.

Experimental Example 1 Gel Filtration Chromatography Using Microspheres with an Expansion Rate of 4 mL / g

Using the polyfructose microspheres prepared in Example 1, desalting chromatography experiments for the separation of proteins and inorganic salts, which are applications of gel filtration chromatography, were carried out. The polyfructose microspheres used in this experiment had a size of 45-150 μcm (dry sample), an expansion rate of 4 mL / g, and a very low pressure difference (7.0 psi) even at a high linear velocity of 130 cm / h. Has characteristics. The column used in the experiment (Amicon, USA) was 1.1 cm (D) x 25 cm (H) and the amount of microspheres used was 15.7 ml. In order to examine the separation performance of the protein and the inorganic salt sodium chloride (NaCl), three proteins of various sizes, hirudin, lysozyme, and bovine serum albumin were tested. 400 μL (5 mg-protein / mL-buffer + 0.5 N NaCl aqueous solution) was injected into the column of each protein, and the flow rate of the buffer solution (pH 7.0, 50 mM Tris-HCl buffer) was 1.0 mL / min. While maintaining, the behavior of protein and sodium chloride was examined using an ultraviolet detector (UV @ 280) and a conductivity detector. As can be seen in FIG. 2, the protein BSA was preferentially fractionated at about 10.5 minutes, and elution did not begin until about 19 minutes for sodium chloride having a very small molecular size. In the case of hirudin, which has a relatively small protein size, tailing was observed at the end of the protein band, and the larger the molecular size of the protein, the more clearly it could be separated from the inorganic salt. Therefore, by selecting the microspheres having a suitable pore size according to the size of the protein it can be applied to the separation and purification of the target protein from impurities other than inorganic salts.

Experimental Example 2 gel filtration chromatography using microspheres with an expansion rate of 11 mL / g

In the same manner as in Example 1, polyfructose microspheres prepared by controlling the concentration of the leban and the content of the crosslinking agent were subjected to gel filtration chromatography experiments for protein separation.

The polyfructose microspheres used in this experiment had a size of 45-150 μm (dry sample), an expansion rate of 11 mL / g, and a very low pressure difference (7.0 psi) even at a high linear velocity of 80 cm / h. Have The column used in the experiment (Sigma, USA) was 1.0 cm (D) x 60 cm (H) and the amount of microspheres used was 45.5 mL. To examine the separation of proteins from proteins, three proteins of various sizes, thyroglobulin, bovine serum albumin, and cytochrome C were tested. Inject 600 μL (5 mg-protein / mL-buffer) of each protein into the cluster and keep the flow rate of buffer solution (pH 7.0, 50 mM Tris-HCl / 10 mM NaCl buffer) at 0.13 mL / min. An ultraviolet detector (UV @ 280) was used to examine the behavior of the proteins. As can be seen in FIG. 3, Thyloglobulin (molecular weight 669000) was preferentially fractionated at about 1.7 hours, and then BSA, a molecular size, was separated at about 2.05 hours. And after about 3.8 hours Cytochrome C was isolated. Protein molecules could be separated in order. Therefore, by selecting the microspheres having a suitable pore size according to the size of the protein it can be applied to the separation and purification of the target protein from impurities other than the protein.

Example 9 Application of Polyfructose Microspheres as Carrier for Enzyme Immobilization

Polyfructose microspheres (10 g) were washed with distilled water and filtered using distilled water. Suspend the washed microspheres in 0.6 M sodium hydroxide (10 mL) and 20 mg sodium borohydride, add 1,4-butane-diol-bis-epoxy-propylether (10 mL), and then slowly at room temperature for 8 hours. Was stirred. Then, the resultant was washed with 5 L of distilled water using a glass filter. In this way, the enzyme and polyfructose microspheres were covalently linked to introduce an epoxy group (epoxy group) capable of stably binding the enzyme to the surface of the microspheres. In this connection, very stable C-N bonds are formed between the microspheres and the enzyme, and thus there is no room for the formation of nonionic residues on the surface of the microspheres, and the possibility of non-selective physisorption is reduced. Polyfructose microspheres having an epoxy group were added with 1,4-butane-diol-bis-epoxy-propyl ether so that one epoxy group reacts with hydroxyl groups on the surface of the microspheres, and the other epoxy group is combined with an immobilizing enzyme. It was prepared by allowing the reaction to bind. The enzymes used in this experiment were Lipase-OF (Meito Sangyo Co., Japan) and Lipase VII (Sigma Chemical Co., USA), two commercially available lipase enzymes. 2 g of two lipases were dissolved in 40 mL of buffer solution (pH 6.0, pH 8.5, 1 M potassium phosphate buffer) and 1 g of microspheres with epoxy groups was added. The immobilization reaction was carried out at room temperature for 3 days for sufficient reaction between the enzyme and the microspheres, which were immobilized carriers, and then washed five times with a buffer solution to remove unreacted enzymes on the surface of the microspheres. (R, S) -ketoprofen ethyl ester was used as a substrate to investigate the activity of the enzymes used in the experiment. After immobilization, the enzyme activity was maintained over 80% of the initial activity and the optical selectivity of the two types of lipase was also maintained.

Although the present embodiment exemplifies only an epoxy group as a functional group, it is obvious to those skilled in the art to introduce various functional groups such as an amino group, a bromocyan group, a carboxyl group, an aldehyde group, and a thiol group according to a suitable use. It will be true.

As is apparent from the above Examples and Experimental Examples, the polyfructose and derivative microspheres of the present invention can adjust the content of polyfructose and derivatives and the swelling ability as a crosslinking agent, and modify the properties of the microspheres according to the stabilizer. Dehydration, gel filtration, affinity and ion exchange chromatography, fillers for enzyme and cell immobilization, carrier for drug delivery, functional cosmetic raw materials by introduction of various functional groups such as amino group, bromocyanic group, carboxyl group, aldehyde group, thiol group It can be used very effectively as a skin supplement. In addition, due to the excellent affinity for peptides and strong intrinsic bonds between polymers, they have a relatively strong strength and do not contaminate the natural environment unlike synthetic polymers.

Claims (4)

delete (1) a carrier for the isolation of a protein, peptide or bioactive material comprising the following steps; (2) a carrier for immobilizing enzymes or cells; (3) drug delivery media; Or (4) a method for producing polyfructose or derivatives thereof having the use of a skin supplement: (a) dissolving polyfructose or a derivative thereof in an alkaline solution at a concentration of 10-50%; (b) stabilizers selected from the group consisting of cellulose-acetate-butylate, polyisobutylene, polyvinylacetate, polystyrene and sorbitan monorate and 1,2-dichloroethane, 1,2-dibromoethane, 1 Preparing a uniform suspended colloidal solution by adding and mixing a dispersion medium selected from the group consisting of 2-2-chlorochloromethane, o -dichlorobenzene and toluene; (c) heating the colloidal solution, reacting with addition of a crosslinking agent, and cooling to prepare a gel; And (d) removing the stabilizer from the surface of the gel to obtain microspheres. The method of claim 2, wherein at least one selected from the group consisting of agarose, dextran, cellulose, chitin, chitosan and derivatives thereof, monomers of synthetic polymers, and ceramic materials is added to step (a). The method according to claim 2, wherein a functional group selected from the group consisting of an epoxy group, an amino group, a bromocyan group, a carboxyl group, an aldehyde group and a thiol group is introduced to the surface of the microspheres obtained in step (d).