KR100494566B1 - Ester type perpolymer and preparation method thereof - Google Patents

Abstract

The present invention is a compound of formula 1, 2 or 2 characterized in that a compound having a double bond in the sock end is obtained by reacting a hydroxyl group having an unsaturated bond with a hydroxyl group in the sock end of the polyol skeleton as a starting material. Obtaining a novel microorganism immobilized ester photocurable prepolymer represented by the formula (3), by adding and adding a photopolymerization initiator to the mixture, the immobilization material is added to water or a buffer solution to make a suspension, and then mixed with the prepolymer, It is a method of making a hydrogel containing enzymes or cells by dropping silicone oil into droplets through a nozzle device of an appropriate diameter or by directly adding a mixed solution while stirring the mixed organic solvent. Yeast, methane bacteria and environmental purification for useful enzymes or alcohol fermentation By fixing the bacteria such as nitrifying bacteria, denitrification bacteria, phenol decomposition bacteria and activated sludge at a high concentration was able to solve the problems caused in the conventional photocurable resin for microorganism immobilization.

[Formula 1]

(Wherein Y is H or CH ₃ and n is an integer from 1 to 150).

[Formula 2]

(Wherein Y is H or CH ₃ , m is an integer from 1 to 50, and n is an integer from 1 to 150).

[Formula 3]

(Wherein Y is H or CH ₃ , m is an integer from 1 to 50, and n is an integer from 1 to 150).

Description

Ester-type photocurable prepolymer for immobilization of microorganism and preparation method thereof and method for immobilization of microorganism using same {ESTER TYPE PERPOLYMER AND PREPARATION METHOD THEREOF}

The present invention relates to an ester-based photocurable prepolymer for immobilizing microorganisms for comprehensively immobilizing cells such as enzymes, microorganisms and activated sludge, and more particularly, to a hydroxyl group in a sock end of a polyol skeleton as a starting material. The present invention relates to an ester-based photocurable prepolymer for immobilization of microorganisms prepared from a novel material having a double bond at a sock end by reacting an acryl-based compound, and a method for preparing the same and a method for immobilizing microorganisms using the same.

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Conventionally, alginate, a natural polymer, has been mainly used as a material for immobilizing a high concentration of useful enzymes or bacteria such as yeast for methanol, methane, environmental nitrifying bacteria, denitrifying bacteria, phenolic bacteria and activated sludge. . Alginic acid, K-carrageenan, etc., which are compositions of alginate, have excellent affinity with microorganisms, and thus are easy to immobilize microorganisms. .

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In order to solve this problem, the use of polyacrylamide gel, which is a synthetic polymer, has been proposed, but polyacrylamide is known to have an inhibitory effect on a biocatalyst, and an initiator, a crosslinking agent, and a polymerization accelerator must be used and polymerized. Due to the complexity of the polymerization method, such as the cells have a sensitive effect on the heat of reaction, it is not used well in the actual scale process.

Recently, urethane-based photocurable resin prepolymers based on polyethylene glycol or polypropylene glycol, which are cured by ultraviolet (UV) irradiation and can be used as a material for immobilizing microorganisms without the addition of a crosslinking agent or a polymerization accelerator, are developed as a photocurable resin prepolymer. It became.

That is, a polyol skeleton containing polyethylene glycol (PEG) and polypropylene glycol (PPG) (molecular weight: 400 to 10,000) is reacted with a diisocyanate such as toluene diisocyanate, xylene diisocyanate, hexamethylene diisocyanate or isophorone diisocyanate. The resultant was prepared by reacting an unsaturated monohydroxy compound such as 2-hydroxyethyl acrylate or 2-hydroxyethyl methacrylate, and a urethane-based photocurable resin having a double bond, which is an unsaturated group, was synthesized at the end. It has been.

The conventional photocurable resin is a prepolymer in a solution state, and these resins are mixed with a photopolymerization initiator and a molding agent, and then the enzymes and microorganisms are suspended in water or a buffer solution, mixed with the resin solution, and then the mixed solution is molded and exposed to ultraviolet rays. Enzymes or microorganisms were immobilized.

However, conventional photocurable resins are hydrolyzed or deteriorated in mechanical strength over long periods of use, and must be passed through nozzles (Ψ: 3 to 5 mm) of suitable diameter to pellet or gel resin suspensions containing enzymes or microorganisms. In addition, since the extruded pellets are formed as long sticks by nozzles, they must be cut to a suitable length, so that the fine parts of the cut portion and the outer surface are recessed when cutting. In addition, since polyatoms are formed by dropping an inorganic salt solution into drops, there is a problem in that a uniform and large amount of gel is limited.

Accordingly, the present invention has been made to solve the above problems, has a three-dimensional cross-linked structure having a permeability and breathability of the substrate and a compressive strength of 1 kg / ㎠ or more, and can be used to immobilize useful enzymes or cells at high concentrations. An object of the present invention is to provide an ester-based photocurable prepolymer for preparing microorganisms and a method for preparing the same.

Another object of the present invention is a novel ester-based photocurable prepolymer produced for the above purpose by mixing the photopolymerization initiator and the cells, respectively, and UV-cured, so that a three-dimensional crosslinked structure having a permeability and breathability and a compressive strength of more than 1kg / cm The present invention provides a method for immobilizing microorganisms to immobilize enzymes and cells at high concentration by preparing in a gel form.

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In the present invention for achieving the above object as well as another object that can be easily expressed, by reacting an acrylic compound having an unsaturated bond in the hydroxyl group in the sock end of the polyol skeleton as a starting material to the sock end A novel microorganism-immobilized ester-based photocurable prepolymer, characterized in that a compound having a double bond is obtained, is added to a photopolymerization initiator and mixed therein, and then the immobilization target is added to water or a buffer solution to form a suspension. Next, the mixture is mixed with the prepolymer and dropped into the silicone oil through a nozzle device of an appropriate diameter, or the mixed solution is directly added while stirring the mixed organic solvent to form a gel. How to make a hydrogel containing cells, useful enzymes and By immobilizing the cells, such as yeast for fermentation call, methane bacteria, and environmental purification of nitrifying bacteria, denitrifying bacteria, phenol-decomposing bacteria and activated sludge in a high concentration, it was able to solve the problems generated in the conventional photo-curable resin immobilized microorganisms.

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

The microorganism-immobilized ester photocurable prepolymer according to the present invention reacts an acrylic compound having an unsaturated bond to a polyol skeleton such as polyethylene glycol (PEG), polypropylene glycol (PPG) and polytetramethylene glycol (PTMG). In more detail, an acrylic or acrylic compound (V) having an unsaturated bond is reacted with a hydroxyl group (-OH) at the end of the polyol (IV) skeleton as shown in Scheme 1 below. It is obtained by adding a promoter such as benzene or triethylamine to promote the reaction, and according to the substance introduced into the polyol (IV) skeleton, a double bond in the sock end as shown in the following formula (1), (2) or (3) Ester based copolymers are produced.

(IV) (V), where X is -H, -CH ₃ or -CH ₂ CH ₂ , and Y is H or CH ₃

[Formula 1] when X = -H ,

When X = -CH ₃

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When X = -CH ₂ CH ₂

(In the above formulas 1,2,3, Y is H or CH ₃ , m is an integer of 1 to 50, n is an integer of 1 to 150, the ratio of a and b is 0.3: 0.7 to 0, 5: 0.5 to 0.7 : 0.3)

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The polyol (IV) is polyethylene glycol (PEG), polypropylene glycol (PPG) or polytetramethylene glycol (PTMG) as described above, the polyethylene oligomer of the polyethylene glycol (PEG) structure is hydrophilic, polypropylene glycol The resin oligomers of (PPG) and polytetramethylene glycol (PTMG) structures have relatively hydrophobic properties. Therefore, the hydrophilicity of the photocured product and the aqueous medium are determined according to the mixing ratio (molar ratio) of the polyethylene glycol (PEG) and the polypropylene glycol (PPG), the polyethylene glycol (PEG) and the polytetramethylene glycol (PTMG), and the chain length of the polyol. The swelling property and mechanical properties at the time of absorption can be changed arbitrarily. In addition, it has the elasticity, such as rubber, but is given the function of a soft hydrogel, there is an advantage that can be easily immobilized microbial cells and friendly and non-toxic with biological tissues.

In addition, accelerators such as benzene and triethylamine are used to promote the reaction of the polyol (IV) and the acrylic compound (V) having an unsaturated bond, and are not limited thereto, and adversely affect the reaction as well as benzene and triethylamine. Anything can be used as long as it promotes the reaction.

In the ester-based photocurable prepolymer prepared by the above-described method, the molecular structure of the ester-based prepolymer is differently formed by a substance introduced into the polyol skeleton, as in Scheme 1, that is, X, and when X = -H (hydrogen group) An ester-based prepolymer having a molecular structure as in Formula 1 is obtained, and when X = -CH 3 (methyl group), an ester-based prepolymer having a molecular structure as in Formula 2 is obtained. And, if X = -CH ₂ CH ₂ (acetylene group) to obtain an ester-based prepolymer having a molecular structure as in the formula (3). After mixing the photocurable prepolymer prepared by adding a photoinitiator to the mixture, the immobilized microorganism is added to water or a buffer solution to make a suspension, and the photocurable prepolymer is added to the suspension to obtain a mixed solution. The gel is formed by dropping the silicone oil into the droplet through the nozzle apparatus or by directly adding the mixed liquid while stirring the mixed organic solvent. In the semi-anaerobic state, ultraviolet rays were irradiated to fix enzymes or cells.

That is, the synthesized prepolymer, the photopolymerization initiator, the molding aid, and the cells were added to the organic solvent, followed by injection molding with stirring in an organic solvent, followed by UV curing to obtain a bead-type hydrogel as a target product.

In this case, as the photopolymerization initiator, azobisisobutyronitrile, 2,2-dimethoxy-2-phenylacetophenone, benzoin methyl ether, benzoyl peroxide, and the like are used, and the photocurable resin is 100% (W / V). The photoinitiator is mixed with 1wt% of the photocurable resin by weight, and the immobilization object is made of photocurable resin at a ratio of 1: 1 (W: W).

Then, beads are formed by dropping the mixed solution into the silicone oil through a nozzle (Ψ: 3 to 5 mm) or a normal nozzle (Ψ: 3 to 5 mm) mounted in a constant temperature bath (20 ° C), and the formed beads are 300 Using a UV lamp with a wavelength of ˜400nm to obtain a hydrogel by light for 5 to 10 minutes, it is washed with sterile distilled water or buffer to complete the immobilization.

As a result of measuring and evaluating the compressive strength and the hydrolyzability, which are physical properties of the prepared hydrogel, the physical properties were superior to those of the conventional photocurable hydrogel, and the transparent and high swelling property with respect to water (swelling rate content: 72-96%).

The following examples illustrate the invention in more detail, but do not limit the scope of the invention.

Example 1

3.4 g (3.4 mmol) of polyethylene glycol having a molecular weight of 1,000 were placed in a well-dried 500 mL round bottom flask, and 50 mL of benzene was added thereto, and then refluxed at a temperature of 60 ° C. in an oil bath. Completely dissolved.

Then, 0.68 ml (7 mmol) of methacryloyl chloride was added and 1 ml (7.2 mmol) of triethylamine were added and reacted for 3 hours.

The reaction was cooled to room temperature, filtered to remove triethylamine hydrochloride from the reaction, the reaction was added dropwise to excess n-hexane, and then dried in a vacuum oven at 40 ° C. according to the present invention. An ester photocurable prepolymer was obtained.

After NMR analysis of the prepared prepolymer, an analysis graph is shown in FIG. 1, and the analysis values are shown in Table 1 below.

NMR Functional group δ (ppm) peak 1.9 a 5.56.1 b 4.25 c 3.6 d

Example 2

An ester-based photocurable prepolymer was prepared in the same manner as in Example 1, but the prepolymer was prepared by changing the type of polyol and changing the compounding ratio to 0.7: 0.3, 0.5: 0.5, 0.3: 0.7, and the like.

For example, polyethyleneglycol (n having a molecular weight of 1,000 to 8,000 mol) sock ends are unsaturated monocarboxylic acids (n × 2.2 mol) having unsaturated double bonds such as acrylic acid and methacrylic acid or acid chlorides (n × 2.2 mol) such as acryloyl chloride and methacryloyl chloride. Subsequently, the ester-based photocurable prepolymer of the present invention was obtained.

Thus, 10 parts of a photocurable resin prepolymer obtained by various mixing ratios such as PEG / PPG and PEG / PTMG (weight molar), and 0.1 part of benzoin methyl ether, a photopolymerization initiator, and 10 parts of a suspension of enzyme or microbial cells are mixed. The mixture was added dropwise to the silicone oil through a 5 mm nozzle mounted in a thermostat (20 ° C.). The gel thus formed was exposed to light for 10 minutes using an ultraviolet lamp having a wavelength of 300 to 400 nm to obtain a hydrogel, which was washed with sterile distilled water to complete the immobilization.

The pore size of the molded hydrogel was 10-200㎛, and it is a soft rubbery elastomer with high swellability (72% to 96%) with respect to water. The strength was 1 kg / cm ³ or more and had a three-dimensional crosslinked structure.

As described above, in the present invention, a compound having a double bond in the sock end is obtained by reacting an acrylic compound having an unsaturated bond with a hydroxyl group in the sock end of the polyol skeleton as a starting material. , To obtain a novel ester-based prepolymer represented by the formula (2) or (3), mixed with the addition of a photopolymerization initiator, and then the immobilized object is mixed with a suspension suspended in water or a buffer solution, and then mixed with a mixed organic solvent while stirring Formed in the form of a gel, and fixed in a high concentration by exposing the ultraviolet light in a semi-anaerobic state was able to solve the problems occurring in the conventional photocurable resin for microbial immobilization.

1 is an NMR analysis graph of the ester photocurable prepolymer synthesized in Example 1. FIG.

Claims (9)

A microorganism immobilized ester photocurable prepolymer represented by the following formula having a double bond in a sock end by reacting a hydroxyl group in a sock end of a polyol skeleton with an unsaturated bond. (Wherein Y is H or CH ₃ and n is an integer from 1 to 150) According to claim 1, wherein the prepolymer has an average molecular weight of 1,000 ~ 8,000, characterized in that the microorganism immobilized ester-based photocurable prepolymer. Novel compound represented by the formula (1) by mixing an acrylic compound (V) having an unsaturated bond with a hydroxyl group in the sock end of the polyol (IV) skeleton and reacting the hydroxyl group with the acrylic compound by adding an accelerator A method for producing an ester photocurable prepolymer for immobilizing microorganisms to prepare an ester photocurable prepolymer.      (Ⅳ) (Ⅴ) [Formula 1] (Where X is -H, Y is H or CH ₃ and n is an integer from 1 to 150). A novel ester-based photocurable prepolymer and a photopolymerization initiator represented by Chemical Formula 1 are respectively mixed in a suspension in which a microorganism to be immobilized with water or a buffer is suspended, and then molded into a gel, followed by curing with ultraviolet rays to fix the microorganism. Microorganism immobilization method using ester photocurable prepolymer for microorganism immobilization. The microorganism of claim 4, wherein the photoinitiator uses any one or more of azobisisobutyronitrile, 2,2-dimethoxy-2-phenolacetophenone, benzoin methylether or benzoyl peroxide. Microorganism Immobilization Method Using Immobilized Ester-based Photocurable Prepolymer.  The method of claim 4 or 5, wherein the photoinitiator is a microorganism immobilization method using a microorganism immobilized ester-based photocurable prepolymer, characterized in that containing 1% by weight relative to the solid content of the photocurable prepolymer. The method of claim 4, wherein the curing is performed by irradiating light with an ultraviolet lamp having a wavelength of 300 to 400 nm for 5 to 10 minutes. The method of claim 4, wherein the gel is A method for immobilizing microorganisms using an ester-based photocurable prepolymer for immobilizing microorganisms, wherein the compressive strength is 1 kg / cm 3 to 3.1 kg / cm 3 and molded to have swellability (swelling rate content: 72 to 96%). delete