KR20130064909A - Branched polymer microspheres with silica shell - Google Patents

Abstract

PURPOSE: A branched polymer microsphere is provided to be easily collected and reused in case of being used as an enzyme immobilized carrier because the branched polymer microsphere is simply and easily separated by a magnet by including a magnetic particle. CONSTITUTION: A branched polymer microsphere includes one or more magnetic particles. The magnetic particle is made of a magnetic material or a magnetic alloy. A composite includes a silica layer which surrounds the microsphere and has a diameter of 500-8000μm. [Reference numerals] (AA) Branched polymer microsphere : electric spray + complexation; (BB) Branched polymer microsphere with a silica layer : deformed sol-gel reaction; (CC) Ferment stabilizing system : CE fixing

Description

Branched polymer microspheres with silica shell

The present invention relates to a branched polymer microsphere having a silica layer which can be used in the field using an enzyme technique and can be separated by a magnet.

Enzymes belong to a specific class of biocatalysts with a variety of uses in the food and pharmaceutical industry, chemical modification processes, biological environmental purification, biofuel cells, and the like. Recently, enzymes have been found to have many advantages in developing green energy and sustainable energy or chemicals for catalytic conversion techniques derived from biomass. Enzymes usually function at mild conditions, such as at room temperature, pressure, physiological pH, water, or even organic solvents, and retain chemoselectivity, site selectivity and enantioselectivity. Despite these advantages, their application is limited due to the lack of implementation and storage stability associated with recovery, reuse and product contamination. However, these obstacles can generally be overcome by immobilization techniques. Numerous methods of immobilization, such as binding, encapsulation or capture using solid carriers, and crosslinking of specific substances with enzymes have been reported. As a result, enzymes immobilized on certain carriers can improve storage and enforcement stability. In addition, the separation for reuse is easy, recoverable, and can be applied to enzyme-based industrial processes by maintaining activity and selectivity.

Organic carrier solid carriers are particularly advantageous in terms of recovery and reuse as carriers for enzymes. Microbead types of microspheres or polymeric materials modified with amines are both attractive both academically and industrially. This is because the amine group can be provided as a moiety for binding to amino acids in the enzyme and can be immobilized. Modification of the surface of the carrier with amine groups can be synthesized by various polymerization techniques such as suspension, emulsification and precipitation polymerization. However, the low enzymatic activity resulting from mechanical stability and limited content delivery prevents the use of these polymers as carriers. Thus, many researchers have recently paid more attention to mixed species. Previous studies have shown that organic-inorganic hybrid structures can improve unique features, such as advantages between inorganic materials and organic polymers, such as strength, elasticity, plasticity, and chemical bonding in a suitable microenvironment. Among them, alginate-silica composites have been noted for their uniform size, spherical shape and ease of manufacture. Practically, alginate microspheres or beads formed with a silica layer by sol-gel chemistry seem to be promising because they allow the coupling of a rough, non-expanding component of silica with a flexible biocompatible component of alginate. Sol-gel silica is formed on the surface of the alginate as a silica shield, so-called diatom, which can be considered as a reserve room for bonding as well as protecting denaturation from the environment. Moreover, due to their porous nature, silica coatings can not only block enzyme release but also act as permeable membranes for small molecules. Although the alginate microspheres in which these silica layers are formed can overcome some of the salt-induced swelling, they have significant mechanical drawbacks and are not suitable for applying them while maintaining enzymatic activity under normal industrial enzymatic processes such as very stringent shaking conditions. impossible.

1. International Publication No. 2010/078202, 2010.07.08

J. Biomater. Sci. Polymer Edn, vol. 18 (1), pp. 71-80, 2007 Drug Delivery, vol. 14, pp. 129-138, 2007 3. Journal of colloid and interface science, vol. 333, pp. 329-334, 2009

Disclosure of Invention An object of the present invention is to provide a microcomposite having a structure in which a silica layer surrounds branched polymer microspheres having improved mechanical strength and a method of manufacturing the same.

Another object of the present invention is to provide a use of the microcomplex as an enzyme immobilization carrier.

Still another object of the present invention is to provide a method for fixing an enzyme using the microcomplex.

In order to achieve the above object,

Branched polymer microspheres containing one or more magnetic particles; And

It provides a microcomposite comprising a silica layer surrounding the microspheres.

The present invention also

Preparing magnetic-polymer blend microspheres by mixing magnetic particles, branched polymers, alginates and crosslinking agents under divalent metal ions;

Preparing magnetic-polymer microspheres from which the alginate-divalent metal ion complex has been removed by complexing the magnetic-polymer blend microspheres; And

It provides a method for producing a microcomposite comprising the step of preparing a microcomposite having a silica layer formed on the outer surface of the microspheres through a sol-gel reaction of the magnetic-polymer microspheres and the silica precursor.

The preparing of the magnetic-polymer blend microspheres may include performing first sonication on a mixed solution of magnetic particles, branched polymers, and alginates;

Second sonicating the mixed solution and a crosslinking agent; And

The magnetic-polymer solution obtained in the above step may be added dropwise to the divalent metal ion solution to prepare a magnetic-polymer blend microsphere.

According to one embodiment, the method may further comprise reacting the magnetic-polymer blend microspheres with a crosslinking agent.

The present invention also provides an enzyme immobilization carrier comprising the microcomplex.

The present invention also provides an enzyme immobilization method comprising the step of reacting the microcomplex and the enzyme.

Since the microcomposite of the present invention includes magnetic particles, it can be easily and easily separated by a magnet, and thus, when used as an enzyme immobilization carrier, recovery and reuse are advantageous.

In addition, the microcomposite has an amine group having a (+) charge coupled to the surface silica layer, so that the microcomposite can act as a moiety for the immobilization of the enzyme, so that the enzyme can be stable for a long time while maintaining excellent activity.

Since the microcomplex of the present invention is effective for enzymatic immobilization, it can be applied to various fields of application of enzyme technology, such as applied biological preparation, biological environment purification, and microenzyme reactors.

FIG. 1 briefly illustrates a process of preparing a microcomposite of the present invention having a structure in which a branched polymer microsphere is surrounded by a silica layer, and fixing the enzyme to the microsphere.
Figure 2 shows a SEM photograph (a) and a cross-sectional SEM photograph (b) of the microcomposite of the present invention.
Figure 3 shows the FT-IR spectrum of the microcomposite of the present invention.
Figure 4 shows the EDX spectrum of the microcomposite of the present invention.
5 shows a digital image of the microcomposite of the present invention.
6 shows the effect of the used number of microcomposites of the present invention on the initial activity of immobilized carboxyl esterases.
Figure 7 shows the reuse (a) and storage stability (b) of the carboxyl esterase immobilized on the microcomposite of the present invention.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

In order to solve the above-mentioned disadvantages of alginate-silica-based materials used as enzyme immobilization carriers, the present invention has developed a microcomposite having a structure in which a silica layer surrounds branched polymer microspheres as a new type of organic-inorganic hybrid material. The outer shell of the microcomposite can improve mechanical strength by forming a strong silica protective layer through a strong chemical double bond by a silica precursor and a sieve base bond.

Therefore, the present invention is a branched polymer microsphere containing one or more magnetic particles (branched polymer microsphere); And

It provides a microcomposite comprising a silica layer surrounding the microspheres.

In the microcomposite of the present invention, one or more magnetic particles are dispersed in a branched polymer microsphere, and the surface of the microsphere may have a structure coated by a silica layer.

The magnetic particles may be dispersed through physicochemical bonds in the branched polymer microspheres. For example, the magnetic particles may be modified to contain functional groups such as amine groups, and may be dispersed in the branched polymer matrix through an amine, which is a crosslinking agent, or an imine bond (or a sieve base bond).

In addition, the microcomposites of the present invention have improved mechanical stability through strong chemical double bonds (eg, imine bonds, etc.) of the branched polymer and the silica layer, and the silica layer has a functional group capable of providing a moiety for the enzyme binding site. It can be used as a carrier for enzyme immobilization. In addition, the magnetic particles are dispersed on the branched polymer microspheres and can be easily and quickly recovered by a magnet, which is reusable.

As used herein, "branched polymer" refers to the graft copolymer by substitution of a hydrogen atom on a monomer subunit in polymer chemistry, by another covalently bonded chain of the polymer, or by another type of chain. In this case, it means a polymer having branches. For example, it may include a graft polymer, a star polymer, a comb polymer, a brush polymer, a polymer network, or a dentrimer. In the present specification, in particular, it may include a biocompatible branched polymer having a terminal functional group that may be involved in chemical bonding in the microspheres. For example, -NHS, -NHNH ₂ , carbonyl imidazole, nitrophenyl, isocyanate, sulfonyl chloride, aldehyde, glyoxal, epoxide polyethylene having one or more terminal functional groups selected from the group consisting of epoxides, carbonates, cyanuric halides, dithiocarbonates, tosylates and maleimides glycol, PEG), polypropylene glycol (PPG), polyoxyethylene (POE), polytrimethylene glycol, polylactic acid and its derivatives, polyacrylic acid and its derivatives, poly (Amino acids), poly (vinyl alcohol), polyurethane, polyphosphazenes, poly (L-lysine) [poly (L-lysine)], polyalkylene oxides (polyalky) branches such as lene oxide (PAO), polysaccharide, dextran, polyvinyl pyrrolidone, polyvinyl alcohol (PVA), or polyacryl amide It may include a polymer form. More specifically, examples of the branched polymer having a terminal functional group may be 3 to 8 arms of polyalkylene glycol containing an amine group. The weight average molecular weight of the branched polymer may be 200 to 100,000, specifically, 1,000 to 40,000, and more specifically 1,000 to 10,000. According to an embodiment of the present invention, when the branched polymer has an amine group as the terminal functional group, the amine group may react with an aldehyde which is a crosslinking agent to form an imine bond. In addition, the amine group that does not participate in the reaction can provide a moiety for the immobilization of the enzyme can be fixed to the enzyme chemically on the microcomposite.

As used herein, the term "magnetic-polymer blend microsphere" means a spherical bead formed by dropwise addition of magnetic particles, branched polymer and alginate to a divalent metal ion solution. The alginate has a feature of forming a gel through ion exchange with divalent metal ions. Thus, the magnetic-polymer blend microspheres have magnetic particles dispersed in a branched polymer matrix in an alginate-2 divalent metal ion gel. The microspheres may be embedded.

As used herein, "magnetic-polymer microspheres" refers to alginate-divalent metals in magnetic-polymer blend microspheres by introducing a complexing agent into the magnetic-polymer blend microspheres to dissolve the alginate-2 divalent metal ion gel. It means a state in which the ion complex is removed.

As used herein, “branched polymer microspheres containing one or more magnetic particles” refers to magnetic-polymer microspheres that have been surface-modified through treatment with the magnetic-polymer microspheres or crosslinking agents.

In the present specification, the "branched high molecular microspheres having a silica layer" means a microcomposite of the present invention having a structure in which the surface of the magnetic-polymer microspheres is surrounded by a silica layer.

The magnetic particles are magnetic so that the microcomposite of the present invention can be easily and quickly separated by a magnet. Therefore, the cost of recovery or separation of the microcomposite can be reduced.

The magnetic particles may be magnetic, but particles having a diameter of 1 nm to 1000 nm may be used, but are not particularly limited thereto. For example, it may be a metal, magnetic material, or magnetic alloy having the diameter.

The metal is not particularly limited, but Pt, Pd, Ag, Cu, Au, etc. may be used alone or in combination of two or more.

The magnetic material is not particularly limited, but Co, Mn, Fe, Ni, Gd, Mo, MM ' ₂ O ₄ , Or M _x O _y Etc. may be used alone or in combination of two or more, wherein M and M 'each independently represent Co, Fe, Ni, Mn, Zn, Gd, or Cr, and x and y each represent the formula “0 <x ≦ 3”. And "0 <y ≤ 5".

The magnetic alloy is not particularly limited, but CoCu, CoPt, FePt, CoSm, NiFe, or NiFeCo may be used alone or in combination of two or more.

In addition, the magnetic particles may be surface modified to have a functional group. For example, when having the amine group as the functional group can form an imine bond with the aldehyde crosslinking agent can be dispersed through the chemical bond in the microspheres.

The branched polymer may increase the structural strength of the microspheres through the branched form, and may include terminal functional groups to participate in chemical bonding with the silica layer or provide a moiety upon enzymatic fixation.

The silica layer surrounding the branched polymeric microspheres not only increases the structural strength but also has a positively charged amine group, thereby increasing the mechanical stability and the opportunity for binding to biomolecules such as enzymes.

The branched polymer microspheres and the silica layer may be chemically bonded (eg, imine bond) to form an outer shield to improve mechanical stability, and the inside of the microcomposite has pores of uniform size so that chemical diffusion may be achieved. Can happen.

The microcomposite is not particularly limited, but may have a uniform sphere size, and may have a diameter of 500 to 8000 μm, more specifically 500 to 2000 μm, and most specifically, 500 to 600 μm.

The present invention also

Preparing magnetic-polymer blend microspheres by mixing magnetic particles, branched polymers, alginates and crosslinking agents under divalent metal ions;

Preparing magnetic-polymer microspheres from which the alginate-divalent metal ion complex has been removed by complexing the magnetic-polymer blend microspheres; And

 It relates to a method for producing a microcomposite comprising the step of preparing a microcomposite having a silica layer formed on the outer surface of the microspheres through a sol-gel reaction of the magnetic-polymer microspheres and the silica precursor.

The method for preparing a microcomposite of the present invention forms an alginate-based gel including branched polymer, removes alginate through chemical complexation, and then performs a modified sol-gel method sequentially to disperse the magnetic particles in the branched polymer matrix. It is characterized by producing a microcomposite having a structure in which the silica sphere is surrounded by the microspheres. A detailed description with reference to FIG. 1 is as follows.

The first step is to prepare a magnetic-polymer blend microspheres,

First sonicating the mixed solution of the magnetic particles, the branched polymer, and the alginate;

Second sonicating the mixed solution and a crosslinking agent; And

The magnetic-polymer solution obtained in the above step may be added dropwise to the divalent metal ion solution to prepare a magnetic-polymer blend microsphere.

More specifically, the solution of the magnetic particles, the branched polymer and the alginate is first sonicated at 20 to 30 ° C. for 20 to 40 minutes, and a crosslinking agent is added to the solution at 50 to 2 minutes at 20 to 30 ° C. After the second sonication for a period of time, it is added dropwise to a divalent metal ion solution to prepare a magnetic-polymer blend microsphere in which a magnetic-branched polymer microsphere is embedded in an alginate-divalent metal ion gel.

The magnetic particles may be prepared in the form of particles using a metal, magnetic material, magnetic alloy, or the like of the above-mentioned type through a conventional manufacturing process, or magnetic beads may be used, and the present invention is not particularly limited thereto.

It can also be surface modified with functional groups for dispersion by chemical bonds in branched polymeric microspheres. The functional group may be appropriately adopted depending on the type of crosslinking agent, terminal functional group of the branched polymer, but is not particularly limited, but may be, for example, an amine group.

The branched polymer may be a biocompatible branched polymer having a terminal functional group, but the kind is as described above.

The alginate is not particularly limited as long as it can react with divalent metal ions to form a gel, for example, Na ⁺ -alginate, K ⁺ -alginate, PEG-alginate (polyethylene glycol-alginate), NH ₄ -alginate, or mixtures thereof can be used.

The two divalent metal ions with ^{2 +} Ca, Zn ^{2 +,} Cu ^{2 +,} Fe ^{2 +,} Ba ^{+ 2} or Sr ^{+ 2,} but can use, but is not particularly limited to this.

The crosslinking agent may increase the mechanical strength of the microspheres or the microcomposites by inducing chemical bonding with terminal functional groups such as magnetic particles, branched polymers, and silica described below. As the kind of the crosslinking agent, polyaldehydes such as glutaraldehyde, dialdehyde starch, succinate aldehyde and the like can be used, and more specifically, glutaraldehyde is preferably used.

According to one embodiment, the aldehyde, which is a crosslinking agent, may react with an amine group, which is a terminal functional group such as magnetic particles, a branched polymer, and silica described below, to induce a chemical bond through an imine bond.

The crosslinking agent may be included in an amount of 0.4 to 0.6 parts by weight based on 100 parts by weight of the mixed solution of the magnetic particles, the branched polymer, and the alginate in order to prepare a microcomposite having excellent mechanical strength.

The magnetic-polymer blend microspheres may be obtained by dropwise addition of a magnetic-polymer solution subjected to secondary sonication to a divalent metal ion solution.

The dropping method may be electric spray, syringe pumping, and the like, but is not particularly limited thereto.

According to one embodiment of the invention, an electric spray can be used.

When the electrospray is performed, the size of the microspheres can be controlled by changing the distance between the capillary tip and the electrode collector, the electric force, the viscosity of the polymer solution, and the like.

Therefore, the electric spray conditions can be appropriately applied at the level of those skilled in the art in consideration of the size of the microspheres are not particularly limited. For example, according to one embodiment, the distance between the tips 3 to 7 cm for a mixed solution of calcium compound and crosslinking agent in a 3-4 kV Gamma high voltage Research (ES series), and 30 gauge syringe needle It can be done by fixing it vertically and setting the flow rate to 2-3 mL / h.

The magnetic-polymer blend microspheres formed above may be further reacted with a crosslinking agent to harden crosslinks in the microspheres.

The reaction may be carried out by mixing the magnetic-polymer blend microspheres and the crosslinking agent overnight at room temperature, but is not particularly limited thereto.

The type and content of the crosslinking agent are as described above.

The second step is to prepare a magnetic-polymer microsphere from which the alginate-divalent metal ion complex is removed through the complexing reaction of the magnetic-polymer blend microsphere.

The step is to dissolve the alginate divalent metal ion gel from the magnetic-polymer blend microspheres using a metal complexing agent to separate the magnetic-polymer microspheres.

The complexing agent may be used without particular limitation as long as it is a material capable of forming a complex with a divalent metal ion. For example, sodium citrate, sodium phosphate, or the like may be used.

The third step is to prepare a microcomposite having a silica layer formed on the outer surface of the microspheres through a modified sol-gel reaction of the magnetic-polymer microspheres and the silica precursor.

The silica layer may contain an amine group to improve the mechanical strength of the microspheres and provide a moiety for fixing the enzyme on the surface. To this end, after the branched polymer microspheres and the aminoalkylsilane derivatives are reacted, a silanol group-containing silica precursor is added thereto to form a silica layer on which the amine group is introduced through the sol-gel process on the surface of the branched polymer microspheres.

The aminoalkylsilane derivatives include β-aminoethyl-γ-aminopropylmethyldimethoxysilane, aminopropylmethyldiethoxysilane, γ-aminopropyltrimethoxysilane, or γ-aminopropyltriethoxysilane alone or two. More than one species can be used.

The silanol group-containing silica precursor may be tetraethyl orthosilicate, tetramethyl orthosilicate, methyl triethoxysilane, phenyl triethoxysilane, dimethyl dimethoxy silane, ethyl triethoxysilane, or mixtures thereof. There is no restriction in particular.

The aminoalkylsilane derivative and the silanol group-containing silica precursor may be mixed in a volume ratio of 1: 0.5 to 2: 1, but may be appropriately adopted within the understanding of those skilled in the art.

According to one embodiment of the present invention, the silica coating may be reacted by mixing the polymer microspheres with an organic solvent, such as hexane, stirring, shaking with addition of an aminoalkylsilane derivative, and adding a silanol group-containing silica precursor thereto. have. After the silica coating may be washed with distilled water to remove the silica residue.

The present invention also relates to an enzyme immobilization carrier comprising the microcomplex.

The microcomposite of the present invention improves mechanical stability, and the terminal functional group or the silica layer of the branched polymer in the branched polymer microsphere has an amine group capable of providing a moiety to the enzyme binding site, and thus has excellent enzyme fixation ability. It is stable and can be reused repeatedly while maintaining initial activity even under shaking conditions. In addition, it contains magnetic particles and can be easily and quickly separated by a magnet, so that it can be easily reused repeatedly and applied to fields where enzyme technology is applied, such as applied biological preparation, biological environment purification, and micro-scale enzyme reactor. .

According to one embodiment, when immobilized on the microcomposite of the present invention using a carboxyl esterase as a model enzyme, the amine group of the enzyme may be immobilized through a cip base bond with silica or a branched polymer by an aldehyde which is a crosslinking agent. .

According to one embodiment, when immobilized to the microcomplex of the present invention using a carboxyl esterase as a model enzyme, it is stable while maintaining at least 90% of the initial activity for 100 days or more, and repeatedly 15 even under very strict shaking conditions. Can be reused more than once. On the other hand, free enzymes deactivate enzyme activity rapidly due to denaturation and autolysis. Existing alginate-based beads swell in phosphate buffer due to exchange with Na ⁺ ions, followed by enzymes. Leakage and suppression of reuse of the immobilized enzyme in the reaction medium occurs, in the case of the microcomposites of the present invention, the silica layer inhibits the expansion of the microspheres, provides a functional group for the enzyme immobilization, and provides a strong mechanical stability to provide strong aggregation of the enzyme. By inhibiting the enzyme leaching and denaturation, enzyme activity can be maintained for a long time and enzyme leakage can be suppressed.

In addition, in the case of alginate-based microspheres prepared by a conventional method, because the holder is used to recover, it is difficult to separate or collect small molecules and cause mechanical damage of the microspheres. On the other hand, the microcomposite of the present invention can be separated by a magnet, there is an advantage that can be separated from the microcomposite without mechanical damage and leakage of the fixed enzyme. This is advantageous in that the microcomplex can be easily separated from the reaction medium for reuse in the reaction process using an enzyme.

The enzyme may be used without limitation so long as it can react with the amine group in the silica layer of the microcomposite of the present invention. For example, carboxyl esterase may be used.

The present invention also relates to an enzyme immobilization method comprising the step of reacting the microcomplex and the enzyme.

The enzyme immobilization method of the present invention is a method of immobilizing an enzyme on a microcomposite through a covalent bond between an amine group of an enzyme and a functional group of a microcomposite by a crosslinking agent. Induces a reaction with the terminal functional groups of the enzyme allows the enzyme to be fixed to the microcomposite through covalent bonds.

Conditions of the enzyme immobilization reaction may vary depending on the type of enzyme is not particularly limited.

In one embodiment of the present invention, a carboxyl esterase may be used as a kind of enzyme, and the enzyme may be immobilized through the following steps. In other words,

Mixing and shaking the enzyme and microcomplex; And

The addition of a solution containing a crosslinking agent may include shaking and incubating.

The step of mixing and leaving the enzyme and the microcomposite may be carried out by vortexing for 20 minutes to 40 minutes at 100 to 300 rpm at room temperature, followed by shaking for 1 to 3 hours at 50 to 100 rpm at 4 ° C. .

Next, a solution containing a crosslinking agent is added to the mixed solution of the enzyme and the microcomposite, shaken for 1 to 3 hours at 100 to 300 rpm at room temperature, and then incubated at 20 to 40 rpm at 4 ° C. overnight. The enzyme can be immobilized on the microcomplex of.

The type and content of the crosslinking agent are as described above.

The fixation capacity of the enzyme can be increased in a concentration-dependent manner of the microcomplex of the present invention.

After the crosslinking reaction, the microcomplex in which the enzyme is immobilized is sufficiently washed with a suitable buffer to remove the easily detachable enzyme and excess crosslinking agent having insufficient binding.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention, but the scope of the present invention is not limited by the following Examples.

Example 1 Preparation of a Microcomposite with a Silica Layer on the Outer Shell of Branched Polymer Microspheres

10,000, Jenkem technology), 알긴산 소듐 염(alginic acid sodium salt, Sigma), 염화칼슘(calcium chloride, Aldrich), 소듐 시트레이트(sodium citrate, Sigma), 테트라메틸 오르토실리케이트(tetramethyl orthosilicate, TMOS) (Aldrich), 테트라에틸 오르토실리케이트(tetraethyl orthosilicate, TEOS) (Aldridh), 3-아미노프로필트리에톡시실란(3-aminopropyltriethoxysilane, APTES) (Sigma-Aldrich), 글루타알데히드(glutaraldehyde, Aldrich), N,N,-디메틸포름아마이드(N,N-dimethylformamide, DMF) (99 %, Sigma-Aldrich), p-니트로페닐 부티레이트(p-nitrophenyl butyrate, Aldrich), Dynabeads M-270 Amine (Amine-magnetic beads) (Invitrogen)을 구입하여 추가 정제 없이 사용하였다. 효소 고정화를 위해 사용하는 리조퍼스 오리자(Rhyzopus oryzae) 유래의 카복실 에스터라아제(Carboxyl esterase (E.C 3.1.1.1)는 Biochemik Chemicals (Aldrich)에서 구입하였다. To produce a highly stable branched polymer / silica-shell hybrid microsphere that can be separated by a magnet, a microscopic polymer is subjected to an electrospray, complexation process, silica sol-gel coating with addition of TMOS and APTES. Microspheres were prepared. The experimental procedure is shown in FIG. 8 arm polyethylene glycol amine (hexaglycerol), Amine-PEG) (Mw) used to prepare microcomposites with silica layers formed on the outer shell of branched polymer microspheres

10,000, Jenkem technology), alginic acid sodium salt (Sigma), calcium chloride (Aldrich), sodium citrate (Sigma), tetramethyl orthosilicate (TMOS) (Aldrich), Tetraethyl orthosilicate (TEOS) (Aldridh), 3-aminopropyltriethoxysilane (APTES) (Sigma-Aldrich), glutaraldehyde (glutaraldehyde, Aldrich), N, N, -dimethyl buy-nitrophenyl butyrate (p -nitrophenyl butyrate, Aldrich), Dynabeads M-270 Amine (Amine-magnetic beads) (Invitrogen) - formamide (N, N-dimethylformamide, DMF ) (99%, Sigma-Aldrich), p It was used without further purification. Rhyzopus used for enzyme immobilization Carboxyl esterase (EC 3.1.1.1) from oryzae ) was purchased from Biochemik Chemicals (Aldrich).

In order to prepare a microcomposite having a silica layer formed on the outer shell of the branched polymer microspheres, 30 parts by weight of amine-PEG, 1 part by weight of alginic acid salt and 5 parts by weight of amine-magnetic beads were dissolved in distilled water, and ultrasonically at 25 ° C. for 30 minutes. Treated. The polymer-magnetic bead solution was mixed with 0.5 parts by weight of glutaaldehyde and further sonicated at 25 ° C. for 60 minutes. Aliquots of the polymer-magnetic bead solution prepared in the above step were loaded into a 10 mL-syringe. For a mixed solution of 100 mM calcium chloride solution and 0.5 parts by weight of glutaaldehyde in a 3-4 kV Gamma high voltage research (ES series), the distance between the tips is 5 cm, the 30 gauge syringe needle is fixed vertically and the flow rate The electrospray was performed with the speed set at 2.5 mL / h. The electrospun crosslinked alginate-PEG microspheres obtained therefrom were collected for a short time in a mixed solution of calcium chloride and glutaraldehyde, and the microspheres were stirred overnight at room temperature to harden crosslinks. Alginate-PEG microspheres are separated using a magnet, washed three times or more with distilled water, and finally, sodium citrate is used as a calcium complexing agent to dissolve the Ca-alginate gel embedded with crosslinked branched polymer. Branched polymeric microspheres were prepared. In fact, the polymer blend microspheres obtained after the electrospray were incubated for 15 minutes at room temperature in 100mM sodium citrate solution, washed several times with distilled water and stored at 4 ° C.

Branched polymeric microspheres that can be separated by magnets are silica coated by a modified sol-gel process of 3-aminopropyltriethoxysilane (APTES) and tetramethyl orthosilicate (TMOS). Organic-inorganic hybrid microspheres were obtained.

That is, the microspheres were mixed with n-hexane and stirred for 2 minutes. After addition of 3-aminopropyltriethoxysilane, shaking for another 5 minutes, tetraethyl orthosilicate was added to the mixed solution. The volume ratio of microspheres: n-hexane: APTES: TMOS was 25: 50: 4: 3. For the branched polymer / silica-shell structure, shaking for 2 hours was followed by removing uncoated silica residue from the microspheres, washing with distilled water for 5 minutes, and storing at 4 ° C.

(Characterization of Microcomposites)

Morphology and elemental analysis of the microspheres were investigated by scanning electron microscopy (SEM; S-2360N equiped with Energy-dispersive X-ray spectroscopy (EDAX), Hitachi Co. Ltd., JAPAN). Fourier transform infrared (FT-IR) spectra of microspheres were measured on an Infinity gold FT-IR spectrometer (Thermomattson. Co. Ltd., USA) using an ATR accessory.

Experimental Example 1 Physical and Chemical Properties of the Microcomposite

As a result of analyzing the shape of the microcomposite prepared in Example 1 by SEM, as shown in Figure 2A, the average size of the microcomposite is 500 to 600 ㎛, spherical, uniform.

Although homogeneous spherical polymeric microspheres can be easily synthesized, their mechanical stability is not stable in buffered system containing salts due to ion exchange. Thus, the present inventors anticipated that the sieve base double bonds derived from chemical biofunctional agents through branched polymers and magnetic beads could be an alternative to inhibit the expansion of microspheres. Indeed, yellow changes that occur during imine reactions were observed during the strengthening and crosslinking processes. In conclusion, the enhanced mechanical properties are demonstrated by the complexation method from sodium citrate treatment.

As shown in the cross-sectional photograph shown in FIG. 2B, the inorganic silica coating induces morphological changes outside of the microspheres. As shown in the enlarged view of the hybrid microspheres, there are pores of uniform size inside. Therefore, the microcomposite can not only form an outer shield capable of improving mechanical stability but also obtain pores which are advantageous in chemical diffusion.

As shown in FIG. 3, the FT-IR spectra of the microcomposites are 3274 cm ⁻¹ (NH stretching), 2869 cm ⁻¹ (aldehyde stretching), 950 cm ⁻¹ (ethyl alkane bending), 1541 cm ⁻¹ (NH ( 2 ° -amide) bending), and 1648 cm ⁻¹ (C = N stretch: imine). On the other hand, polymer microspheres for a silica coating on the (matrix) from TMOS and APTES mixing technique due to the Si-O-Si and CH ₃ 1040 cm ^-1 and 1457 cm ^- a very distinct intensity of the band at ¹ was observed .

In addition, as a result of EDX analysis of the microcomposite, as shown in FIG.

Due to the presence of magnetic beads, the overall color of the microspheres is light brown, and they can be easily observed with the naked eye during the microencapsulation process. However, the microspheres turn yellow when the crosslinking agent glutaaldehyde (GA) is treated. As shown in FIG. 5, the microcomposites showed yellow, which means seed base bonds.

Another advantage is the possibility of magnetic separation of the microcomposites from the liquid medium. As shown in FIG. 5, the microcomposite can be separated by a magnet in 2 seconds. This feature, that is, the magnetoscopic resolution of the microcomposite, allows the application of biomolecule-microsphere hybrid molecules to reduce the cost of recovery or separation from the mixture.

Example 2 Preparation of Enzyme-immobilized Microcomposites

For enzyme immobilization in the microcomplex prepared in Example 1, a carboxyl esterase (CE) solution was prepared at 10 mg / mL in 100 mM phosphate buffer (pH 8.0). In a glass vial, the microcomplex of Example 1 and 1 mL of the enzyme solution were mixed and vortexed at 200 rpm for 30 minutes at room temperature. The glass vial was then placed in a rocker and shaken at 4 ° C. at 60 rpm for 2 hours. For enzyme agglomeration, first, 2 mL of a solution containing 1.0 parts by weight of glutaraldehyde and 0.5 g ammonium sulfate was added to the vial to aggregate the enzyme, allowed to stand on a shaker for 2 hours at 200 rpm at room temperature, and Incubated overnight on a rocker (30 rpm). The next day, discard all solutions, transfer the remaining enzyme immobilized on the microcomposite to a new vial, wash briefly with 100 mM phosphate buffer, and leave the unbound functional groups on glutaaldehyde 100 mM Tris-HCl (pH). 7.8). After capping, the fixed CE was washed three times with 100 mM phosphate buffer (pH 6.5). The prepared enzyme-microcomposite was dispersed in 2 mL of 100 mM phosphate buffer (pH 6.5) and stored at 4 ° C.

Experimental Example 2 Enzyme Activity and Stability Measurement

Life of the enzyme fixed on the micro-composite material prepared in Example 2 Castle N, N- dimethyl formamide was dissolved in p-it was checked by monitoring the production of nitrophenol-p from the hydrolysis of nitrophenyl butyrate.

That is, 4.95 mL of 100 mM phosphate buffer (pH 6.5) containing 50 μl of 50 mM p -nitrophenyl butyrate dissolved in N, N-dimethylformamide was prepared as a substrate. Fixed CE was dispersed in the substrate solution and shaken at 200 rpm. After a short reaction, initial activity was calculated from the absorbance at 400 nm for each time period, and the absorbance was averaged by the concentration of p -nitrophenol.

For enzyme stability experiments, the CE immobilized on the microcomposites after activity measurement was washed three times with 100 mM phosphate buffer (pH 6.5) and stored at shaking conditions at 200 rpm at room temperature. All measurements were performed in three replicates for the standard deviation. One unit (U) of carboxyl esterase activity was defined as the amount of enzyme that releases 1 μmol p -nitrophenol per minute under assay conditions. All samples were performed in three replicates for error analysis. Standard deviations for the results are shown as error intervals on the graph.

As shown in FIG. 5, after enzyme immobilization, color change occurred in the microcomposite. That is, ammonium sulphate and glutaaldehyde with beige color can induce a change from beige to dark brown. This demonstrates that the enzymes are immobilized in the microspheres through the sieve base binding, which can increase the stability between the microcomplex and the enzyme.

Since the number of microcomplexes may affect the enzyme loading efficiency due to the amine moiety, the concentration of the carboxyl esterase for immobilization was fixed and only the content of the microcomposites was changed.

As shown in Figure 6, as the number of microcomposites increases, the initial activity of immobilized CE also increases. Of course, natural changes from crosslinked CE can affect the structural arrangement in the enzyme. In addition, the silica shell can interfere with the free migration of the substrate to the active site of CE so that the initial rate of the hydrolysis reaction will be delayed compared to the free enzyme.

Nevertheless, by controlling the number of microcomposites, some more effective immobilization systems with much higher activity can be performed. Therefore, the loss of CE from the microcomposite was inhibited by the maintenance of enzyme activity and measured by examining these effects. The stability of free CE and immobilized CE was measured by incubating them in aqueous buffer under shaking conditions (200 rpm) in order to mimic the natural environment by standing in actual enzyme reactor conditions. In typical industrial enzymatic processes, the enzymatic reaction is carried out under very stringent conditions and it is desirable to maintain the enzyme system and / or the enzyme system immobilized under such conditions.

CE immobilized on the microcomposite showed good reuse and long term storage stability under stringent shaking conditions as shown in FIG. The branched polymer / silica shell may allow the CE to be stored under strict conditions, rather than being maintained without shear stress.

Initial activity of the immobilized CE in the microcomposite was set to 100%, and the relative activity of the immobilized CE was determined as the ratio of activity to the initial enzyme activity.

As shown in FIG. 7A, the relative activity of immobilized CE was well maintained, while free CE showed rapid inactivation of enzyme activity due to denaturation and autolysis of CE. After 15 repeated uses, at least 90% of the relative activity was preserved. Typically, homogeneous spherical alginate based beads expand in phosphate buffer due to exchange with Na ⁺ ions, followed by enzymatic leakage and inhibition of the reuse of the immobilized enzyme in the reaction medium. Silica-shells, however, not only inhibit the expansion of microspheres but also provide functional groups that can be used for enzyme immobilization. Therefore, enzyme leakage was almost suppressed with improved stability during the enzymatic reaction.

In addition, as a result of measuring the storage stability of the immobilized CE, as shown in FIG. 7B, the relative activity of the free enzyme decreased from 100% to 10% within 3 days due to its autolysis. However, the relative activity of CE immobilized in the microcomposites decreased only by 100% to 90% for 100 days. This may be due to the mechanical stability from the outer silica-shell of the branched polymer microspheres, which inhibits the strong aggregation of enzymes in the microcomposites and enzyme leaching and denaturation.

In addition, the CE fixed to the microcomposite could be separated by a magnet. Electrospray can be used to produce microspheres of fine size, but it is difficult to separate or collect small molecules because of the use of common holders, and can cause mechanical damage and leakage of enzymes in hybrid microspheres. However, the CE fixed to the microcomposite that can be separated by the magnet can be separated from the reaction solution within 2 seconds using a strong magnet without any loss. Above all, the pulverization is achieved through the magnet so that it can be easily separated from the reaction medium for subsequent reuse. This is the first report of the possibility of a microcomplex based enzyme stabilization system as a very stable carrier for enzyme reuse under very stringent storage conditions.

In conclusion, the present invention successfully provides a highly active, stable and magnetically separable enzyme system comprising microcomposites. The microcomplex is usually applicable to many different enzymes, and can be applied to various fields of enzyme technology, such as applied biological preparation, biological environmental purification, microscale enzyme reactors.

Claims (20)

Branched polymer microspheres containing one or more magnetic particles; And
Microcomposite comprising a silica layer surrounding the microspheres.
The method of claim 1,
Magnetic particles are metal, magnetic material or magnetic alloy fine composite.
The method of claim 2,
Magnetic materials include Co, Mn, Fe, Ni, Gd, Mo, MM ' ₂ O ₄ , And at least one selected from the group consisting of M _x O _y , wherein M and M 'each independently represent Co, Fe, Ni, Mn, Zn, Gd, or Cr, and x and y are each represented by the formula “0 <x Microcomposites satisfying ≤3 "and" 0 <y ≤5 ".
The method of claim 1,
The branched polymeric microspheres are -NHS, -NHNH ₂ , carbonyl imidazole, nitrophenyl, isocyanate, sulfonyl chloride, aldehyde, glyoxal , One or more terminal functional groups selected from the group consisting of epoxides, carbonates, cyanuric halides, dithiocarbonates, tosylates and maleimides Microcomposites made from compatible branched polymers.
The method of claim 1,
The silica layer is a microcomposite containing an amine group.
The method of claim 1,
Microcomposites are microcomposites having a diameter of 500 to 8000 μm.
Preparing magnetic-polymer blend microspheres by mixing magnetic particles, branched polymers, alginates and crosslinking agents under divalent metal ions;
Preparing magnetic-polymer microspheres from which the alginate-divalent metal ion complex has been removed by complexing the magnetic-polymer blend microspheres; And
A method for producing a microcomposite comprising the step of preparing a microcomposite having a silica layer formed on an outer surface of the microspheres through a sol-gel reaction of the magnetic-polymer microspheres and the silica precursor.
The method of claim 7, wherein preparing the magnetic-polymer blend microspheres
First sonicating the mixed solution of the magnetic particles, the branched polymer, and the alginate;
Second sonicating the mixed solution and a crosslinking agent; And
A method of producing a microcomposite comprising the step of dropwise adding a magnetic-polymer solution obtained in the step to a divalent metal ion solution to prepare a magnetic-polymer blend microsphere.
The method of claim 7, wherein
Branch polymers include -NHS, -NHNH ₂ , carbonyl imidazole, nitrophenyl, isocyanate, sulfonyl chloride, aldehyde, glyoxal, epoxy Biocompatible branches having one or more terminal functional groups selected from the group consisting of epoxides, carbonates, cyanuric halides, dithiocarbonates, tosylates and maleimides Method for producing a microcomposite that is a polymer.
9. The method of claim 8,
The first ultrasonic treatment step is a method for producing a microcomposite that is carried out at 20 to 30 ℃ for 20 to 40 minutes.
9. The method of claim 8,
The crosslinking agent is at least one selected from the group consisting of glutaraldehyde, dialdehyde starch and succinate aldehyde.
9. The method of claim 8,
Crosslinking agent is a method for producing a microcomposite containing 0.4 to 0.6 parts by weight based on 100 parts by weight of the mixed solution.
9. The method of claim 8,
Second ultrasonic treatment step is a method for producing a microcomposite is carried out at 20 to 30 ℃ 50 minutes to 2 hours.
9. The method of claim 8,
The divalent metal ion is Ca ^{2 +} , Zn ^{2 +} , Cu ^{2 +} , Fe ^{2 +} , Ba ^{2 +} or Sr ^{2 + A} method for producing a microcomposite.
9. The method of claim 8,
A method of producing a microcomposite further comprising the step of reacting the magnetic-polymer blend microspheres with a crosslinking agent.
The method of claim 7, wherein
The silica layer is a method for producing a microcomposite formed by reacting an aminoalkylsilane derivative with a silanol group-containing silica precursor.
17. The method of claim 16,
The aminoalkylsilane derivative is at least one selected from the group consisting of β-aminoethyl-γ-aminopropylmethyldimethoxysilane, aminopropylmethyldiethoxysilane, γ-aminopropyltrimethoxysilane and γ-aminopropyltriethoxysilane. Method for producing a microcomposite.
17. The method of claim 16,
The silanol group-containing silica precursor is at least one fine selected from the group consisting of tetraethyl orthosilicate, tetramethyl orthosilicate, methyl triethoxysilane, phenyl triethoxysilane, dimethyl dimethoxy silane, ethyl triethoxysilane and mixtures thereof Method for preparing a composite.
An enzyme immobilization carrier comprising the microcomplex of claim 1.
An enzyme immobilization method comprising the step of reacting the microcomplex of claim 1 with an enzyme.

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