KR101982618B1 - Antimicrobial hybrid materials combined with titanium dioxide and glucose oxidase and method for fabricating the same - Google Patents

Abstract

In view of the fact that glucose is decomposed by glucose oxidase to generate hydrogen peroxide (H ₂ O ₂ ), the present invention is based on the fact that by coating glucose oxidase on the surface of titanium dioxide particles, the photocatalytic reaction of titanium dioxide (O ₂ ^- , · OH) and hydrogen peroxide (H ₂ O ₂ ) to induce glucose degradation and hydrogen peroxide generation by glucose oxidase as well as the antibacterial effect of microorganisms The present invention relates to a composite antimicrobial substance combined with a titanium dioxide and a glucose oxidase, which can reduce glucose as a carbon source, and a preparation method thereof. Characterized in that the titanium dioxide particle is provided with a glucose oxidase, Generates free radicals through the photocatalytic reaction in the environment, the glucose oxidase is to produce hydrogen peroxide with a decomposition of glucose also in an environment where the glucose is present.

Description

TECHNICAL FIELD The present invention relates to a composite antimicrobial substance comprising titanium dioxide and a glucose oxidase,

More particularly, the present invention relates to a complex antimicrobial substance comprising titanium dioxide and a glucose oxidase, and more particularly, to a method for producing a complex antimicrobial material comprising hydrogen peroxide (H ₂ O ₂ ) when glucose is degraded by glucose oxidase, Noting in that it generate, to thereby coat the glucose oxidase on the titanium dioxide particle surface as well as the antimicrobial effect by the photocatalytic reaction of titanium dioxide leads to glucose degradation and hydrogen peroxide generated by the glucose oxidase, the active oxygen (O ₂ ^- (OH) and hydrogen peroxide (H ₂ O ₂ ), as well as titanium dioxide and glucose oxidase, which can reduce glucose, which is an organic carbon source of microorganisms, and a method for producing the same will be.

Titanium dioxide (TiO ₂ ) has been used to decompose pollutants or to microbialize microorganisms due to the photocatalytic effect after reported photocatalytic action in the 1970s. Titanium dioxide, which is exposed to ultraviolet rays in a water environment, loses electrons and generates holes and generates active oxygen such as superoxide (O ₂ ^- ) and hydroxyl radical (· OH) while being excited in an unstable state. Active oxygen generated by the photocatalytic reaction of titanium dioxide is involved in antibacterial activity by decomposing contaminants or altering the structure of organic materials or by oxidizing action which interferes with the growth and division of microorganisms. Due to such strong oxidizing power, titanium dioxide is bound to various supports to decompose pollutants or to use them for antibacterial use.

On the other hand, the presence of microorganisms in the water environment or the possibility of propagation means that there is a high possibility that there is an energy source available for microorganism growth in the water environment, and the energy source that microorganisms can use is mainly carbon-based organic sources. Among the organic carbon used as an energy source, glucose is the most commonly used carbon source.

If an organic carbon source such as glucose is present in an excessive amount in the water environment, it can be used as an energy source to multiply microorganisms. Therefore, an antibacterial method using only titanium dioxide is not effective in an environment where an excessive amount of organic carbon energy source such as glucose is present. In addition, there have been frequent cases of adverse health effects by using chemicals which are not known to be toxic for contact with microorganisms or for microbial antibiosis which are present in household appliances such as humidifiers as well as water environments. Therefore, it is increasingly necessary to safely and effectively antibacterial microorganisms present in the surrounding environment.

Korean Patent Registration No. 935512

Disclosure of the Invention In view of the above problems, the present invention has been made in view of the fact that when glucose is decomposed by glucose oxidase, hydrogen peroxide (H ₂ O ₂ ) is produced, (O ₂ ^- , · OH) and hydrogen peroxide (H ₂ O ₂ ) as well as antibiotic effect by photocatalytic reaction of titanium dioxide, as well as glucose degradation and hydrogen peroxide generation by glucose oxidase, Which is capable of effectively decomposing microorganisms and reducing glucose as an organic carbon source of microorganisms, and a method for producing the same.

In order to accomplish the above object, the present invention provides a composite antimicrobial substance comprising titanium dioxide and glucose oxidase, wherein the surface of the titanium dioxide particle is provided with glucose oxidase.

The titanium dioxide particles generate active oxygen through a photocatalytic reaction in an environment irradiated with ultraviolet rays, and the glucose oxidase decomposes glucose and generates hydrogen peroxide in the presence of glucose.

And the surface of the titanium dioxide particles may be adsorbed by glucose oxidase. In addition, the titanium dioxide particles and the glucose oxidase can be bound to each other in the form of a covalent bond, an ionic bond, a weak van der Waals bond, a hydrophilic-hydrophilic bond, a hydrophobic-hydrophobic bond, or a mixed bond thereof.

On the other hand, titanium dioxide particles and glucose oxidase can be bound through a crosslinking agent. The surface of the titanium dioxide particles is provided with polyacrylic acid (PAA), and the carboxyl group of PAA and the amine group of the glucose oxidase can be bound by a peptide bond. The crosslinking agent may be EDC / NHS (N-ethyl-N '- (3- (dimethylamino) propyl) carbodiimide / N-hydroxysuccinimide.

A method for preparing a complex antimicrobial material comprising titanium dioxide and a glucose oxidase according to the present invention comprises the steps of: preparing a first solution in which titanium dioxide particles are dispersed and a second solution in which glucose oxidase is dissolved; And mixing and stirring the first solution and the second solution to adsorb the glucose oxidase on the surface of the titanium dioxide particles.

The present invention also provides a method for preparing a composite antimicrobial material comprising a titanium dioxide and a glucose oxidase conjugate, comprising the steps of: binding polyacrylic acid (PAA) to the surface of titanium dioxide particles; And binding the carboxyl group of the PAA and the amine group of the glucose oxidase through a crosslinking agent to coat the surface of the titanium dioxide particle with the glucose oxidase.

The composite antimicrobial substance combined with the titanium dioxide and the glucose oxidase according to the present invention and the preparation method thereof have the following effects.

Through the photocatalytic reaction of titanium dioxide and glucose degradation by glucose oxidase, glucose, which is an organic carbon source of microorganisms, can be reduced and active oxygen and hydrogen peroxide can be effectively used to remove microorganisms and organic pollutants. Thus, titanium dioxide alone The antimicrobial effect can be doubled.

FIG. 1 is a reference view showing a complex antimicrobial substance combined with a titanium dioxide and a glucose oxidase according to an embodiment of the present invention, and a reaction mechanism therefor.
FIG. 2 shows FT-IR analysis results of the compound antibacterial substance prepared in Experimental Example 1. FIG.
FIG. 3 shows the results of the antimicrobial properties of the titanium dioxide particles (TiO ₂ ) not coated with glucose oxidase and the complex antimicrobial material (TiO ₂ -GO _x ) prepared in Experimental Example 1 with or without glucose.
Fig. 4 shows results of antibacterial tests against Escherichia coli and Bacillus sp.

The present invention provides a complex antimicrobial substance in which titanium dioxide (TiO ₂ ) and glucose oxidase are combined.

As is well known, titanium dioxide (TiO ₂ ) generates active oxygen such as superoxide (O ₂ ^- ) and hydroxyl radical (OH) through photocatalytic reaction when ultraviolet rays are irradiated in the presence of water (H ₂ O) The active oxygen (O ₂ ^- , · OH) generated by the photocatalytic reaction of titanium dioxide (TiO ₂ ) oxidizes and decomposes organic contaminants and microorganisms.

Glucose oxidase degrades glucose and converts it to gluconic acid. Hydrogen peroxide (H ₂ O ₂ ) is produced as a by-product in the process of converting glucose to gluconic acid See equation 1 below). Glucose is a major organic carbon source of microbial growth as described in the Background of the Invention, and is present in a large amount in a polluted environment.

(Equation 1)

In view of the above points, the present invention has been made to solve the above problems and to provide a method for removing glucose from a reaction between glucose oxidase and glucose in addition to the photocatalytic reaction of titanium dioxide to generate hydrogen peroxide, To inhibit the growth of microorganisms and to induce the action of decomposing microorganisms and organic pollutants through the generated hydrogen peroxide.

That is, according to the present invention, both of the photocatalytic reaction of titanium dioxide and the decomposition reaction of glucose by the glucose oxidase can be performed, and the glucose, which is an organic carbon source of the microorganism, can be effectively removed through glucose decomposition reaction by glucose oxidase, (O ₂ ^- , · OH) produced by the photocatalytic reaction of glucose and oxidized and decomposed microorganisms and organic pollutants by using hydrogen peroxide (H ₂ O ₂ ) generated through glucose degradation reaction by glucose oxidase .

In other words, according to the present invention, microorganisms can be removed by active oxygen (O ₂ ^- , OH) and hydrogen peroxide (H ₂ O ₂ ) while inhibiting the growth and proliferation of microorganisms by glucose decomposition.

Hereinafter, a composite antimicrobial substance combined with titanium dioxide and glucose oxidase according to an embodiment of the present invention and a method for producing the same will be described in detail with reference to the drawings.

Referring to FIG. 1, a composite antimicrobial substance having titanium oxide (TiO ₂ ) and glucose oxidase bonded thereto according to an embodiment of the present invention is formed by a core-shell (core-shell) structure.

The titanium dioxide particles may have any one of an anatase crystal phase and a rutile crystal phase, or may be composed of a mixture thereof. As the glucose oxidase, glucose oxidase derived from various sources may be used. In one embodiment, fungus Aspergillus niger may be used.

The titanium dioxide particles are not limited in size. That is, titanium dioxide particles can be designed in nanometer (nm) to millimeter (mm) size. In addition, the amount of the glucose oxidase contained on the surface of the titanium dioxide particles can be set in the range of 0.1 to 99.9 wt% based on the weight of the titanium dioxide particles.

FIG. 1 shows a photocatalytic reaction and a glucose degradation reaction by a complex antimicrobial substance combined with titanium dioxide and glucose oxidase according to an embodiment of the present invention.

As shown in FIG. 1, when ultraviolet rays are irradiated in the state where glucose oxidase 2 is provided on the surface of titanium dioxide particle 1, superoxide (O ₂ ^- ), hydroxyl radical (OH) and the like are generated (refer to 'I' in FIG. 1). In addition to the photocatalytic reaction of titanium dioxide, D-glucose is decomposed by glucose oxidase (GO _x ) in the presence of glucose (D-glucose) do. D-glucose is decomposed by glucose oxidase (GO _x ) and converted to gluconic acid, and hydrogen peroxide (H ₂ O ₂ ) is produced as a by-product ('II' in FIG. 1) Reference).

As described above, it can be seen that both the photocatalytic reaction of titanium dioxide and the degradation reaction of glucose with glucose oxidase are both possible. Hydrogen peroxide (H ₂ O ₂ ) is produced by glucose decomposition reaction by glucose oxidase, and active oxygen (O ₂ ^- , · OH) is produced by photocatalytic reaction of titanium dioxide. Therefore, it is possible to inhibit the growth and proliferation of microorganisms by removing glucose, and to effectively and efficiently control microorganisms and organic pollutants by using active oxygen (O ₂ ^- , · OH) and hydrogen peroxide (H ₂ O ₂ ) Can be removed.

The complex antimicrobial substance combined with titanium dioxide and glucose oxidase according to the present invention can be used for various purposes such as an antibacterial filter, an antibacterial paint, an antibacterial paste, and an antibacterial cosmetic.

Meanwhile, the complex antimicrobial substance in which titanium dioxide and glucose oxidase are combined according to an embodiment of the present invention can be produced through various methods. In one embodiment, it can be prepared via adsorption or crosslinking methods.

When the adsorption method is used, a solution in which titanium dioxide particles are dispersed and a solution in which glucose oxidase is dissolved are prepared, and these two solutions are mixed and stirred to adsorb glucose oxidase on the surface of the titanium dioxide particles, Materials can be prepared. The titanium dioxide particles and the glucose oxidase may be bound in any combination of covalent bond, ionic bond, weak van der Waals bond, hydrophilic-hydrophilic bond, hydrophobic-hydrophobic bond, or a combination thereof.

In the case of using the crosslinking method, the carboxyl group of PAA and the amine group of glucose oxidase are reacted with EDC / NHS (N-ethyl-N '- (3- (dimethylamino) propyl) carbodiimide / N-hydroxysuccinimide) can be used to coat glucose oxidase on the surface of titanium dioxide particles.

The complex antimicrobial substance combined with the titanium dioxide and the glucose oxidase according to one embodiment of the present invention and the preparation method thereof have been described above. Hereinafter, the present invention will be described in more detail with reference to experimental examples.

≪ Experimental Example 1: Preparation of a complex antibacterial substance &

The titanium dioxide particles are dispersed at 20 mg / ml in a PBS (phosphate buffered saline) solution (pH 7). Similarly, dissolve in PBS solution at pH 7 so that glucose oxidase is 10 mg / ml. Take 1 mL of each of the two prepared solutions, mix and mix at 200 rpm for 3 hours at room temperature. After mixing for 3 hours, the mixture is centrifuged at 13,500 rpm for 20 minutes, and the supernatant is discarded. Add 1 mL PBS (pH 7) solution to disperse the particles, and centrifuge at 13,500 rpm for 20 minutes. This procedure was repeated two more times. Finally, the particles were dispersed with 2 mL of PBS (pH 7.0) and stored at 4 ° C for refrigeration.

In another method, PAA (polyacrylic acid) is bonded to titanium dioxide particles to convert the carboxyl group of PAA and the amine group of glucose oxidase into EDC / NHS (N-ethyl-N '- (3- (dimethylamino) propyl) carbodiimide / N-hydroxysuccinimide ) May be used to bond the glucose oxidase to the titanium dioxide through peptide bonds, precisely by covalent bonding. That is, although the embodiment employs adsorption bonding, the manner in which titanium dioxide and glucose oxidase are bonded may be any of covalent bonding, ionic bonding, weak van der Waals bonding, hydrophilic-hydrophilic bonding, hydrophobic-hydrophobic bonding and hydrogen bonding .

<Experimental Example 2: Surface analysis of composite antimicrobial substance>

The complex antimicrobial material prepared in Experimental Example 1 was subjected to Fourier Transform Infrared Spectroscopy (FT-IR) to analyze its surface. In the complex antimicrobial substance prepared through Experimental Example 1, the glucose oxidase was bound to about 15 wt% of the titanium dioxide particle weight. FIG. 2 shows FT-IR analysis results of the complex antimicrobial substance prepared in Experimental Example 1. FIG.

Referring to FIG. 2, in the region of 1655 cm ^-1 and 1528 cm ^-1 of the FT-IR signals of the complex antimicrobial substance prepared in Experimental Example 1, signals more noticeable than titanium dioxide can be observed. Significant signals at 1650 cm ^-1 signify C═O bonds and prominent signals at 1528 cm ^-1 appear by NH. Titanium dioxide itself is an inorganic substance of TiO ₂ and can not be found in bonds such as C═O or NH, and C═O or NH bonds can be found in proteins such as glucose oxidase, which consists of bonds of amino acids. Thus, the remarkable 1655 cm ^-1 and 1528 cm ^-1 signals of the complex antimicrobial substance prepared in Experimental Example 1 indicate that glucose oxidase is bound to the titanium dioxide particle surface.

&Lt; Experimental Example 3: Antimicrobial properties of a complex antimicrobial substance &

Antimicrobial properties of the titanium dioxide particles not coated with glucose oxidase and the composite antimicrobial substance prepared in Experimental Example 1 were tested with and without glucose.

Add 20 mL of PBS (pH 7.0) solution to a 50 mL quartz chamber, insert the magnetic bar into the chamber, and mount the quartz chamber on the magnetic stirrer. At this time, the quartz chamber and the magnetic stirrer are mounted in a black box where the light is blocked. On the left and right sides of the quartz chamber, there is an ultraviolet lamp, which can be adjusted to turn on or off the ultraviolet lamp. Ultraviolet lamps of 4W intensity having a wavelength of 352 nm were used but are not necessarily limited thereto. 20 mL of PBS (pH 7.0) solution may or may not contain glucose. Escherichia coli is cultured at an appropriate concentration, and the culture is washed with PBS and suspended in 20 mL of PBS to 1 × 10 ⁷ CFU / mL. Titanium dioxide or a titanium dioxide-glucose oxidase complex is added to a quartz chamber in which E. coli is suspended to a concentration of 0.2 mg / mL. For the antimicrobial effect, 0.1 mL of the sample is taken at a fixed time interval after the UV lamp is turned on, the sample is diluted to a range of 10 ^-5 to 10 ^-7 , plated on a nutrient solid medium, and cultured in a 37 ° C thermostat for about 17 hours . Count colonies of Escherichia coli grown in each nutrient solid medium and calculate the change in the number of Escherichia coli and the antibacterial efficiency.

FIG. 3 shows experimental results of antimicrobial properties of titanium dioxide particles (TiO ₂ ) not coated with glucose oxidase and the composite antimicrobial material (TiO ₂ -GO _x ) prepared in Experimental Example 1 according to presence or absence of glucose.

As shown in FIG. 3, the titanium dioxide particles (TiO ₂ ) not coated with glucose oxidase after 45 minutes as shown in FIG. 3 showed about 90% (log (N / N ₀ ) value), but in the case of the composite antimicrobial material (TiO ₂ -GO _x ) prepared in Experimental Example 1, about 70% of the cells were killed. That is, the antibacterial activity of the complex antimicrobial material (TiO ₂ -GO _x ) prepared in Experimental Example 1 was lower than that of titanium dioxide particles (TiO ₂ ) not coated with glucose oxidase in the absence of glucose. This can be interpreted as follows. Since the titanium dioxide surface of the complex antimicrobial substance (TiO ₂ -GO _x ) prepared in Experimental Example 1 is covered with glucose oxidase, the amount of active oxygen formed on the surface thereof is lower than that in the case where only titanium dioxide is present under ultraviolet light . That is, the formation of some glucose oxidase may act like a membrane, which may interfere slightly with the active oxygen production of titanium dioxide by ultraviolet light.

The results of the antimicrobial properties test in the presence of glucose are as follows. 3, the antimicrobial effect of titanium dioxide particles (TiO ₂ ) not coated with glucose oxidase in the ultraviolet light condition when glucose of 0.5, 2.5 and 5.0 mg / ml was gradually increased in PBS (pH 7.0) It can be seen that it is considerably weakened from 90% to about 50%. That is, the antimicrobial effect was rather inhibited. This suggests that glucose can be used as an energy source for E. coli, and therefore, the glucose component acts as a resistance to the antimicrobial effect of titanium dioxide. On the contrary, in the complex antimicrobial material (TiO ₂ -GO _x ) prepared in Experimental Example 1, the antimicrobial effect was gradually increased in 45 minutes as glucose concentration increased. It is confirmed that 99.9% antimicrobial effect is shown in the case of containing mg / ml of glucose. It can be seen that glucose was used for the growth of microorganisms under the condition that only titanium dioxide was present. However, when the complex antimicrobial material (TiO ₂ -GO _x ) prepared in Experimental Example 1 exists, glucose is oxidized by glucose oxidase And hydrogen peroxide water was formed to increase the antimicrobial effect.

<Experimental Example 4: Antibacterial properties against Gram-positive bacteria and Gram-negative bacteria>

Titanium dioxide particles not coated with glucose oxidase and the complex antimicrobial substance prepared in Experimental Example 1 were tested for antimicrobial activity against Gram-positive Bacillus subtilis and Gram- The experiment was carried out. The conditions in which Gram-positive bacteria and Gram-negative bacteria exist are a PBS solution (pH 7.0) in which 5.0 mg / ml of glucose is dissolved, and FIG. 4 shows the results of antibacterial tests against Escherichia coli and Bacillus bacteria.

4, it can be seen that the antibacterial effect of the composite antimicrobial substance prepared in Experimental Example 1 is more effective than that of titanium dioxide particles not coated with glucose oxidase at all time points, for Gram-positive bacillus and Gram-negative bacteria, Escherichia coli .

1: titanium dioxide particle 2: glucose oxidase

Claims (10)

A composite antimicrobial substance comprising titanium dioxide and glucose oxidase, characterized in that the surface of the titanium dioxide particle is provided with a glucose oxidase.
The method according to claim 1, wherein the titanium dioxide particles generate active oxygen through a photocatalytic reaction in an environment irradiated with ultraviolet rays, wherein the glucose oxidase decomposes glucose in the presence of glucose and generates hydrogen peroxide Is a complex antimicrobial substance in which titanium dioxide and glucose oxidase are combined.
The complex antimicrobial substance according to claim 1, wherein the surface of the titanium dioxide particle is adsorbed with glucose oxidase.
[Claim 2] The method according to claim 1, wherein the titanium dioxide particle and the glucose oxidase are bound to each other in the form of a covalent bond, an ionic bond, a weak van der valence bond, a hydrophilic-hydrophilic bond, or a hydrophobic-hydrophobic bond, Composite antimicrobial substance combined with titanium dioxide and glucose oxidase.
The complex antimicrobial substance according to claim 1, wherein the titanium dioxide particles and the glucose oxidase are bound through a crosslinking agent.
6. The method of claim 5, wherein the surface of the titanium dioxide particle is provided with polyacrylic acid (PAA), and the carboxyl group of the PAA and the amine group of the glucose oxidase are bound through a crosslinking agent. Complex antimicrobial substance.
7. The method of claim 6, wherein the crosslinking agent is EDC / NHS (N-ethyl-N '- (3- (dimethylamino) propyl) carbodiimide / N-hydroxysuccinimide) Antimicrobial substance.
Preparing a first solution in which titanium dioxide particles are dispersed and a second solution in which glucose oxidase is dissolved; And
And mixing and stirring the first solution and the second solution to adsorb the glucose oxidase on the surface of the titanium dioxide particles.
Coupling polyacrylic acid (PAA) to the surface of the titanium dioxide particle; And
A step of binding the carboxyl group of PAA with the amine group of the glucose oxidase through a cross-linking agent to coat the surface of the titanium dioxide particle with a glucose oxidase, and then the titanium dioxide and glucose oxidase-conjugated antimicrobial &Lt; / RTI &gt;
The method of claim 9, wherein the crosslinking agent is EDC / NHS (N-ethyl-N '- (3- (dimethylamino) propyl) carbodiimide / N-hydroxysuccinimide) A method for producing an antimicrobial substance.

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