KR20130046205A - Enzyme immobilized on au-doped magnetic silica nanoparticle, method for producing the same, and hydrolytic degradation method of biomass thereby - Google Patents

Abstract

PURPOSE: A customized enzyme-fixed gold-magnetic silica nanoparticle, a method for preparing the same, and a continuous hydrolytic method using the particles are provided to enhance mounting efficiency of an enzyme by fixing the enzyme through chemical bond. CONSTITUTION: An enzyme-fixed gold-magnetic silica nanoparticle contains a magnetic silica nanoparticles, a plurality of -SH groups, one or more gold nanopaticles, and one or more enzymes. The plurality of -SH groups is introduced on the surface of the magnetic silica nanoparticles. The one or more gold nanoparticles bind to the -SH group and are conjugated on the surface of the magnetic silica nanoparticles. The enzymes are conjugated to the gold nanoparticles. A method for preparing the enzyme-fixed gold-magnetic silica nanoparticles comprises: a step of preparing the magnetic nanoparticles; a step of preparing the magnetic silica nanoparticles using the magnetic nanoparticles; a step of introducing the -SH groups onto the surface of the magnetic silica nanoparticles; a step of conjugating the gold nanoparticles to the magnetic silica nanoparticles and preparing the gold-magnetic silica nanoparticles; and a step of fixing the enzyme to the gold-magnetic silica nanoparticles.

Description

Customized immobilized on Au-doped Magnetic silica nanoparticles, method for producing the same, and hydrolytic degradation method of biomass hence}

The present invention relates to an immobilized enzyme and an enzyme immobilization method. More specifically, the enzyme-enhanced gold-magnetic silica nanoparticles having a new structure, which enhances the enzyme mounting efficiency and easily separates from a substrate and is easily regenerated when the enzyme is immobilized, It relates to a production method and a continuous biomass hydrolysis method using the particles.

Enzyme is widely used to convert cellulose processed from biomass to glucose, but despite the price drop of enzyme, it has disadvantages such as high price and low processing speed to apply to domestic process. Development of hydrolase is required.

Therefore, enzyme immobilization technology is a particularly noteworthy flow among the three directions of research and development for securing the economics of biofuel production. If the development of hydrolysing enzymes, which are biofuel production catalysts, and fermentation catalysts for ethanolization are mainly focused on the redesign of metabolic pathways, another direction for R & D is to optimize the transformation of biomass into new or optimal new biomass. The last method is enzyme fixation technology.

Fundamental concepts of enzyme immobilization are related to the technology of using enzymes in biochemical processes using enzymes, which can be divided into technology fields for improving the biological activity of enzymes and technology fields for stabilizing and economically utilizing the enzymes. The latter has been developed around the immobilization method, whereas the research has been conducted in the direction of the research.

An important purpose of immobilizing the enzyme is to increase the economics of the enzymatic reaction process because the enzyme can be easily recovered and reused, and the reaction mode can be variously applied batchwise or continuously. In other words, since the enzyme is water soluble in nature, it is difficult to repeatedly use the enzyme, because if the water soluble enzyme can be removed, problems such as mixing with by-products, difficulty in separation and purification, and weak stability can be easily solved. For this reason, hydrophilicity and hydrophobicity may appear depending on which protein raw material is used, and this characteristic changes the efficiency of enzymatic action. In addition, most enzyme fixation techniques or entrapment techniques bring about changes in the enzyme structure, which ultimately affects yield.

Therefore, in order to effectively use natural enzymes in a biochemical process, the enzymes must be immobilized. A general enzyme immobilization method is a physical adsorption method or a chemical method. The physical adsorption method mainly uses an ion-exchange method, but the ion-exchange method has the advantage of being non-toxic, but has a problem in that its binding strength is weak. In addition, the chemical method is to use a reagent to fix the enzyme by forming a covalent bond by a chemical reaction, the method has a strong cross-linking force, but because the reagent used for immobilization of the enzyme is toxic Another disadvantage is that it is difficult to use in the pharmaceutical industry.

Enzyme immobilization, which involves the enzymatic immobilization by binding an enzyme to an organic or inorganic carrier, reuses and performs continuous processing is well known. Organics (e.g. cellulose, nylon, polyacrylamide) are disadvantageous as carriers because they have poor mechanical stability and can break down the bonds with enzymes due to corrosion by solvents, changes in pH and ionic strength and invasion by microorganisms. Because it can. Therefore, an inorganic carrier to which an enzyme is adsorbed or covalently adsorbed has been proposed. The binding type depends on the conditions of use of the enzyme and the characteristics of the substrate. In other words, if the substrate has a strong salt concentration, the adsorption method cannot be applied because the adsorption of the adsorbed enzyme occurs. Therefore, covalent bonds of enzymes take precedence. The surface of the carrier should encompass specific functional groups that induce the binding of enzymes. Most carriers cannot cover functional groups and require surface pretreatment.

In most cases, silica was used to fix the enzyme and have a diameter of 3-6 nanometers, and silica was widely used. The small size was 2 nanometers, the medium size was 2-50 nanometers, and the large size was over 50 nanometers. Production is possible. (Lignocellulosic cells have a size of 0.2-0.5 nanometers in diameter). The advantages of silica are low cost, rapid immobilization in seconds, environment for particle formation, room temperature and neutral acidity, high diffusivity, matrix design, and physical properties suitable for flow. In addition, the stability of the enzyme has been proven, and by changing the silica deposition, it is possible to control the diameter. In particular, silica sol-gel encapsulation was used to fix biomolecules, which is mainly applied to biosensors. However, when silica gel-sol is used, there is a problem that the efficiency of encapsulating the enzyme or the enzyme is very low.

Therefore, there is a need for the development of a new structure of immobilized enzyme that can improve the efficiency of loading, maintain the activity for a long time, can be more stable and use for a long time, can be easily separated from the substrate, and can be easily regenerated. It became.

Furthermore, in order to convert cellulose processed from biomass to glucose, so far, enzymes have been used a lot. However, despite the price drop of enzymes, it has disadvantages such as high price and low processing speed to apply to domestic processes. There is a need for a new artificial hydrolase development.

The present inventors have completed the present invention by developing a new structure of immobilized enzyme, which is easy to be separated from a substrate, and is easily regenerated.

Therefore, an object of the present invention is to enhance the mounting efficiency of the enzyme by immobilizing the enzyme through the chemical bonding to the magnetic silica nanoparticles coated with gold nanoparticles, it is possible to maintain the long-term activity by reducing the sensitivity of the enzyme to changes in external conditions, It is to provide an enzyme-fixed gold-magnetic silica nanoparticles of a structure that is more stable and can be used for a long time and a method for producing the same.

Another object of the present invention can be easily separated from the substrate after the end of the enzyme reaction using a magnet, enzyme-free gold-magnetic silica of a structure that is easy to regenerate by simply removing the inactivated enzyme and recombining the new active enzyme It is to provide a nanoparticle and a method of manufacturing the same.

Another object of the present invention can be used in a continuous reactor, if a plurality of enzymes are required for the reaction of a specific substrate enzyme fixed gold-magnetic silica nano structure of the structure to increase the enzyme reaction efficiency by fixing a plurality of enzymes required It is to provide a particle and a method for producing the same.

Another object of the present invention is enzyme-fixed gold-magnetic silica nano, which can be more usefully used in the case where the amount of processing of the substrate is very large and the enzyme required for processing the substrate is expensive, such as hydrolysis of pretreated cellulose-based biomass. It is to provide a particle and a method for producing the same.

It is still another object of the present invention to realize the possibility of a continuous biomass hydrolysis method which can drastically reduce the unit production cost according to the use of a catalyst, which accounts for about 40% of the total cost of ethanol production from biomass by recycling a plurality of times. to provide.

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention described above, first, the present invention is a magnetic silica nanoparticle; A plurality of -SH groups introduced to the surface of the magnetic silica nanoparticles; One or more gold nanoparticles bonded to the -SH group and bonded to the surface of the magnetic silica nanoparticles; And it provides an enzyme-fixed gold-magnetic silica nanoparticles comprising one or more enzymes coupled to the gold nanoparticles.

In a preferred embodiment, the cysteine bond is formed by the -SH group.

In a preferred embodiment, the magnetic silica nanoparticles are synthesized using Fe ₃ O ₄ as a precursor.

In a preferred embodiment, the magnetic silica nanoparticles have a diameter of 5 to 50 nm.

In a preferred embodiment, the gold nanoparticles have a diameter of 2 to 10nm.

In a preferred embodiment, the enzyme can be reused more than 10 times while maintaining the activity of 90% or more.

In a preferred embodiment, the enzyme can be reused more than 20 times while maintaining the activity of 50% or more.

In a preferred embodiment, if the activity of the enzyme is less than 50%, after removing the enzyme bound to the gold nanoparticles can be recycled by binding other enzymes required to the gold nanoparticles.

In addition, the present invention comprises the steps of preparing a magnetic nanoparticle; Preparing magnetic silica nanoparticles using the magnetic nanoparticles; Introducing a plurality of -SH groups onto the surface of the prepared magnetic silica nanoparticles; Preparing gold-magnetic silica nanoparticles by combining gold nanoparticles with the magnetic silica nanoparticles having the —SH group introduced therein; And fixing the enzyme to the gold-magnetic silica nanoparticles.

In a preferred embodiment, the step of preparing the magnetic nanoparticles is a step of mixing the iron dichloride aqueous solution and the iron trichloride solution; Reacting the mixed solution by adding ammonium hydroxide solution while stirring; And separating the black product obtained by the reaction. At this time, the step of separating the black product is preferably centrifuged and washed with water and ethanol and then vacuum drying.

In a preferred embodiment, the preparing of the magnetic silica nanoparticles may include: ultrasonically dispersing the prepared magnetic nanoparticles in a solvent; Adding TEOS (tetrathyl orthosilicate) to the dispersed solution and then adding an ammonium hydroxide solution while stirring to maintain a pH of 10 or more; And washing and vacuum drying the refluxed product. Here, the solvent used during the ultrasonic dispersion may be an alcohol solvent, the pH is preferably maintained at 12 or more, reflux may be made at 90 ℃ or more.

In a preferred embodiment, the step of introducing a plurality of -SH group is the step of refluxing after adding the magnetic silica nanoparticles to a solution containing -SH group; And separating, washing and vacuum drying the product passed through the above steps. In this case, the solution containing the -SH group is not limited as long as the compound containing the -SH group is dissolved in an organic solvent. Preferably, Mercaptopropyltriethoxysilane is dissolved in an organic solvent, particularly toluene, under a nitrogen stream.

In a preferred embodiment, the step of preparing the gold-magnetic silica nanoparticles comprises the steps of preparing a gold nanoparticle solution; Adding and reacting the magnetic silica nanoparticles having the -SH group introduced thereinto the gold nanoparticle solution; And washing and vacuum drying the reaction product.

In a preferred embodiment, the step of immobilizing the enzyme on the gold-magnetic silica nanoparticles is a step of ultrasonic dispersion after adding a buffer to the gold-magnetic silica nanoparticles; Reacting by adding an enzyme to the dispersed product; And washing the enzyme-fixed gold-magnetic silica nanoparticles obtained by the reaction and separating the same using a magnet.

In addition, the present invention provides a continuous hydrolysis method characterized in that the use of the enzyme-fixed gold-magnetic silica nanoparticles as a hydrolase.

In a preferred embodiment, after the enzymatic reaction for hydrolysis is completed, the enzyme-fixed gold-magnetic silica nanoparticles are separated by a magnet and then washed and reused.

In a preferred embodiment, when the substrate of the hydrolysis is biomass, the enzyme-fixed gold-magnetic silica nanoparticles are combined with endo-glucosidase, exo-glucosidase, β-glucanase.

In addition, the present invention comprises the steps of removing the immobilized enzyme from the enzyme-locked gold-magnetic silica nanoparticles of any one of claims 1 to 8, wherein the immobilized enzyme activity is reduced in number; And fixing the new enzyme to the gold-magnetic silica nanoparticles from which the enzyme has been removed.

In a preferred embodiment, removing the immobilized enzyme comprises sonicating at 90 to 110 ° C.

The present invention has the following excellent effects.

First, according to the present invention, immobilization of enzymes through chemical bonding to magnetic silica nanoparticles coated with gold nanoparticles increases the mounting efficiency of enzymes, and reduces the sensitivity of enzymes to changes in external conditions, thereby maintaining long-term activity. It is more stable and can be used for a long time.

In addition, according to the present invention, the magnet can be easily separated from the substrate after the enzymatic reaction is completed, and the inactivated enzyme is easily removed and the new active enzyme is recombined to facilitate regeneration.

In addition, according to the present invention can be used in a continuous reactor, if a plurality of enzymes are required for the reaction of a specific substrate can be fixed by fixing a plurality of enzymes required to increase the enzyme reaction efficiency.

In addition, according to the present invention, such as the hydrolysis of the pre-treated cellulose-based biomass can be more useful when the amount of substrate treatment is very large and the enzyme required for the treatment of the substrate is expensive.

In addition, according to the present invention, by recycling a plurality of times, it is possible to drastically reduce the unit production cost according to the use of a catalyst, which accounts for about 40% of the total cost of ethanol production from biomass.

In FIG. 1, (a) is a schematic diagram of simultaneously immobilizing three cellulase enzymes on gold nanoparticles, and (b) is a schematic diagram of simultaneously immobilizing three cellulase enzymes on magnetic silica nanoparticles coated with gold nanoparticles.
Figure 2 is a schematic diagram of a continuous hydrolysis method using the enzyme-fixed gold-magnetic silica nanoparticles according to an embodiment of the present invention as a hydrolase.
Figure 3 is a graph showing the result of measuring the immobilization rate when fixing the cellulase enzyme in gold nanoparticles and gold-magnetic silica nanoparticles.
Figure 4 is a graph of pH stabilization test results of the hydrolase immobilized in the enzyme-fixed gold-magnetic silica nanoparticles according to an embodiment of the present invention using the pNPG activity test method.
Figure 5 is a graph of the pH stabilization test results of the hydrolase immobilized in the enzyme-fixed gold-magnetic silica nanoparticles according to an embodiment of the present invention using the CMC activity test method.
Figure 6 is a graph showing the results of testing the pH effect of the immobilized cellulase enzyme using the filter paper activity test in the enzyme-fixed gold-magnetic silica nanoparticles according to an embodiment of the present invention.
Figure 7 is a graph showing the thermal stability of the cellulase enzyme immobilized in the enzyme-fixed gold-magnetic silica nanoparticles according to an embodiment of the present invention.
Figure 8 is a graph of the reusability test results when using the enzyme enzyme gold-magnetic silica nanoparticles hydrolase according to another embodiment of the present invention.
9 is a graph showing the results of recyclability test after removing the inactivated enzyme and recombining new active enzyme according to another embodiment of the present invention.
FIG. 10 shows the results of before and after removal of cellulase from the surface of gold-magnetic silica nanoparticles by TEM (transmission electron microscopy) and EDX (Energy Dispersive X-ray Spectroscopy).
FIG. 11 is an electron micrograph showing a change in filter paper form after hydrolysis is performed according to another embodiment of the present invention.
12 is a graph showing the results of activity test of xylanase immobilized on enzyme-fixed gold-magnetic silica nanoparticles immobilized with xylanase according to another embodiment of the present invention.
13 is a graph showing the reuse test results of enzyme-fixed gold-magnetic silica nanoparticles with xylanase immobilized according to another embodiment of the present invention.

Although the terms used in the present invention have been selected as general terms that are widely used at present, there are some terms selected arbitrarily by the applicant in a specific case. In this case, the meaning described or used in the detailed description part of the invention The meaning must be grasped.

Hereinafter, the technical structure of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Like reference numerals used to describe the present invention throughout the specification denote like elements.

The technical feature of the present invention is to enhance the enzyme loading efficiency when the enzyme is fixed, it is easily separated from the substrate, and the new structure of the immobilized enzyme is easy to regenerate.

In other words, by immobilizing the enzyme through the chemical bonding to the gold-magnetic silica nanoparticles to which the gold nanoparticles are bonded by introducing -SH group to the magnetic silica particles, the enzyme mounting efficiency is increased, and the sensitivity of the enzyme to changes in external conditions is reduced. This is because the activity can be maintained for a long time, can be easily separated from the substrate after the enzymatic reaction by using a magnet, and it is easy to regenerate by simply removing the inactivated enzyme and recombining the new active enzyme.

Therefore, the enzyme-fixed gold-magnetic silica nanoparticles of the present invention are magnetic silica nanoparticles as shown in the schematic diagram shown in Figure 1 (b); A plurality of -SH groups introduced to the surface of the magnetic silica nanoparticles; One or more gold nanoparticles bonded to the -SH group and bonded to the surface of the magnetic silica nanoparticles; And one or more enzymes bound to the gold nanoparticles.

The magnetic silica nanoparticles are preferably synthesized using Fe ₃ O ₄ as a precursor, and preferably have a diameter of 5 to 50 nm. If the size of the magnetic silica nanoparticles is smaller than 5 nm, the magnetic silica nanoparticles are difficult to form and have small magnetic properties. This may be difficult to separate, and if the size is larger than 50 nm, the contact area with the substrate may be small.

Cysteine bonds are formed by a plurality of -SH groups introduced to the surface of the magnetic silica nanoparticles, and the -SH group increases not only the enzyme mounting efficiency but also the activity of the enzyme.

Gold nanoparticles are preferably one having a diameter of 2 to 10nm because one or more of the gold nanoparticles are bonded to the surface of the magnetic silica nanoparticles to bind the enzyme.

Due to the structure described above, the enzyme-fixed gold-magnetic silica nanoparticles of the present invention can be reused 10 times or more in a state where the activity of the fixed enzyme is maintained at 90% or more, and the state of the enzyme is maintained at 50% or more. Can be reused more than 20 times.

In addition, when the activity of the immobilized enzyme falls below 50%, the enzyme bound to the gold nanoparticles is easily removed by heat treatment or sonication, and then other enzymes necessary for binding to the gold nanoparticles can be easily regenerated.

When the enzyme-enhanced gold-magnetic silica nanoparticles of the present invention is used as a hydrolase, continuous hydrolysis can be easily performed. After the enzymatic reaction is completed, the enzyme-enriched gold-magnetic silica nanoparticles are separated by a magnet. This is because it is possible to increase the efficiency of enzyme reaction by fixing a plurality of enzymes necessary when a plurality of enzymes are required for the reaction of a specific substrate.

Therefore, if the substrate of the hydrolysis is biomass, as shown in Figure 2 when using the enzyme-enhanced gold-magnetic silica nanoparticles of the present invention is combined endo-glucosidase, exo-glucosidase, β-glucanase, Since it is possible to recycle enzyme-fixed gold-magnetic silica nanoparticles at once, it is possible to drastically reduce the unit production cost due to the use of catalysts, which account for about 40% of the total cost of ethanol production from biomass.

Example 1

Cellulase Fixed Gold-Magnetic Silica Nanoparticles Preparation

(1) Magnetic Nanoparticle Preparation

Mix 80 mL of 2M FeCl ₂ -4H ₂ O (iron dichloride) with 320 mL of 1M FeCl ₃ -6H ₂ O (iron trichloride). 4 L of 0.7 M NH 4 OH (ammonium hydroxide) solution is dropped dropwise while stirring the mixed solution rapidly. The black product after reaction is centrifuged. After washing with water and ethanol and vacuum drying to obtain magnetic nanoparticles.

(2) Magnetic Silica Nanoparticles Preparation

The prepared magnetic nanoparticles are added to ethanol and dispersed for about 30 minutes using an ultrasonic disperser. 20 mM tetraethylolsosilicate (TEOS) was added thereto and stirred, while 0.7 M ammonium hydroxide solution was slowly added to maintain pH at 12. The mixture was refluxed at 100 ° C. for 24 hours, washed three times with ethanol, and dried in vacuo to obtain magnetic silica nanoparticles.

(3) Preparation of magnetic silica nanoparticles having -SH group introduced to the surface

In order to introduce the surface of the gold nanoparticles, -SH group was introduced to the magnetic silica nanoparticles using Mercapto propyltriethoxysilane. First, 125 mg of mercapto propyltriethoxysilane is dissolved in 5 mL of toluene under a stream of nitrogen. 40 mg of prepared magnetic silica nanoparticles are added and refluxed at 100 ° C. for 24 hours. The product was separated using a vacuum filter, washed 2-3 times with THF, and then vacuum dried to obtain magnetic silica nanoparticles into which -SH group was introduced.

(4) Preparation of Gold Nanoparticle Solution

First we make 2.5 ㅧ 10 ^-4 M HAuCl ₄ Prepare 20 mL of 2.5 x 10 ^-4 M trisodium citrate solution each. To this slowly add 0.6 mL of 0.1 M NaBH ₄ . Gold nanoparticles that turn reddish brown can be obtained. Measure the average size of the particles using TEM. Add 9.0 mL of 2.5 ㅧ 10 ^-4 M HAuCl ₄ and 0.10M CTAB solution to the resulting seed gold nanoparticles. Here a 0.10 M ascorbic acid was added to 50 μ L, and begin stirring. Centrifugation gave a dark gold nanoparticle solution.

(5) Preparation of Gold-Magnetic Silica Nanoparticles

To 0.5 g of the prepared magnetic silica nanoparticles, 10 mL of the gold nanoparticle solution is added and stirred well at room temperature for 24 hours. The product was washed three times with distilled water and dried in vacuo to prepare gold-magnetic silica nanoparticles.

(6) Preparation of enzyme-fixed gold-magnetic silica nanoparticles

Gold - then followed by the addition of 10 mg magnetic silica nanoparticles in phosphate buffer solution (PBS / NaCl, KCl, Na 2 HPO 4, KH 2 PO 4, 200 μ L) dispersion for 30 minutes by an ultrasonic dispersing machine. Add 50 mg of three kinds of cellulase, endo-glucanase (EGIVCBDII), exo-glucanase (CBHII), and BglB (β-glucosidase) and stir well at 4 ℃ for 1-2 days. Cellulase-fixed gold-magnetic nanoparticles were washed with DW (distilled water) and magnetically separated to prepare cellulase-fixed gold-magnetic silica nanoparticles. In the figure, cellulase-fixed gold-magnetic silica nanoparticles are represented as MSNP @ Au @ Cellulases, Cellulases @ Au-MSNP or Au-MSNP @ Cellulases.

Example 2

Filter paper, pNPG, and CMC solutions were prepared with cellulose substrates, and the prepared cellulose substrates were added to buffer solutions of pH 4.5-5.0, respectively. Then, the cellulase-fixed gold-magnetic silica nanoparticles prepared in Example 1 was added to the buffer solution to which the substrate was added, followed by reaction for 30 minutes to 1 hour at 50 ° C. or 80 ° C., which is an optimum reaction temperature according to each substrate. Continuous hydrolysis was performed as shown in 2.

After the hydrolysis reaction, the cellulase-fixed gold-magnetic silica nanoparticles were separated using magnetic and the amount of cellobiose and glucose was measured. At this time, the cellulase fixed gold-magnetic silica nanoparticles separated by the magnet may be washed and reused using DW (distilled water).

Example 3

Xylanase-fixed gold-magnetic silica nanoparticles were prepared in the same manner as in Example 1 except that xylanase was added instead of cellulase. In the figure, xylanase-fixed gold-magnetic silica nanoparticles are represented as Au-MSNP @ xylanase.

Example 4

Recycling Method of Enzyme Fixed Gold-Magnetic Silica Nanoparticles

In order to remove from the surface of the cellulase-immobilized gold-magnetic silica nanoparticles, the immobilized cellulase which was reused in the continuous hydrolysis reaction performed in Example 2 and had little activity was sonicated at 100 ° C. for about 1 hour.

Thereafter, the step (6) enzyme fixation of gold-magnetic silica nanoparticles of Example 1 was repeated to prepare recycled cellulase-fixed gold-magnetic silica nanoparticles.

Comparative Example 1

Example 1 phosphate buffer solution on the gold nanoparticles prepared in solution and then the mixture (PBS / NaCl, KCl, Na 2 HPO 4, KH 2 PO 4, 200 μ L) is dispersed for 30 minutes by an ultrasonic dispersing machine. Add 50 mg of three kinds of cellulase and stir well at 4 ℃ for 1-2 days. Cellulase-fixed gold nanoparticles were washed and separated using DW (distilled water) to prepare cellulase-fixed gold nanoparticles. In the figure, cellulase-fixed gold nanoparticles are represented as Au @ Cellulases, Cellulases @ AuNP or AuNP @ Cellulases.

Comparative Example 2

Except for the use of enzyme-fixed gold nanoparticles in the same manner as in Example 2, a large number of reusable immobilized cellulase in continuous hydrolysis reactions were removed at the surface of the cellulase-fixed gold nanoparticles at 100 ° C. for 1 hour. Sonication was enough. Thereafter, recycled cellulase-fixed gold nanoparticles were prepared in the same manner as in Comparative Example 1.

Comparative Example 3

Except for using xylanase but not cellulase, xylanase-fixed gold nanoparticles were prepared in the same manner as in Comparative Example 1. In the figure, xylanase immobilized gold nanoparticles are represented by AuNP @ xylanase.

Experimental Example 1

In order to measure the ratio of enzyme immobilization when cellulase enzyme is immobilized to gold-magnetic silica nanoparticles as in Example 1 and the immobilization ratio when cellulase enzyme is immobilized to gold nanoparticles as in Comparative Example 1, that is, the binding efficiency of cellulase The amount of cellulase was immobilized by increasing the amount of cellulase to 2mg of nanoparticles and 2ml of gold nanoparticles, respectively. The amount of cellulase remaining without binding to the nanoparticles was measured using the brad-ford method and the respective immobilization ratios were measured. The result is shown in FIG.

From FIG. 3, it can be seen that the gold-magnetic silica nanoparticles have better enzyme immobilization efficiency than the gold nanoparticles.

Experimental Example 2

Three main cellulase enzymes, endo-glucanase (EGIVCBDII), exo-glucanase (CBHII), and BglB, respectively, immobilized on the cellulase-fixed gold-magnetic silica nanoparticles according to Example 1 and the cellulase-fixed gold nanoparticles according to Comparative Example 1 In order to determine the pH effect of β-glucosidase) was investigated using pNPG activity at each pH (3.0-7.0), the results are shown in FIG. At this time, the temperature conditions were fixed at 80 degreeC.

As a result, as shown in FIG. 4, the activity was optimal at both pH 4.5 and cellulase-immobilized gold-magnetic silica nanoparticles over unimmobilized cellulase and cellulase-immobilized gold nanoparticles (AuNP @ cellulases). (Au-MSNP @ Cellulases) was found to be more stable at each pH.

Experimental Example 3

The pH effect of the cellulase enzyme immobilized was examined in the same manner as in Experiment 2 except that the CMC activity test was used and the temperature condition was fixed at 50 ° C., and the results are shown in FIG. 5.

It was optimal at pH 5.0 from FIG. 5, and the cellulase immobilized gold-magnetic silica nanoparticles (Au-MSNP @ Cellulases) and the cellulase immobilized gold nanoparticles (AuNP @ more stable at each pH than cellulases).

Experimental Example 4

The pH effect of the cellulase enzyme immobilized in the same manner as in Experimental Example 2 was investigated except that the filter paper activity test was used and the temperature condition was fixed at 50 ° C., and the results are shown in FIG. 6.

As a result, as shown in Figure 6 was optimal at pH 4.0, as shown in Figures 4 and 5, it can be seen that the cellulase fixed gold-magnetic silica nanoparticles (Au-MSNP @ Cellulases) is most stable at each pH there was.

Experimental Example 5

Thermal stability of the cellulase immobilized gold-magnetic silica nanoparticles according to Example 1 and the cellulase enzyme immobilized on the cellulase immobilized gold nanoparticles according to Comparative Example 1 was investigated and the results are shown in FIG. 7. Thermal stability was investigated up to 48 hours at pH 4.5 and 80 degrees.

As shown in FIG. 7, the cellulase-fixed gold-magnetic silica nanoparticles (Au-MSNP @ Cellulases) remained active even for more than 36 hours, but the cellulase mixture (free cellulase) and the cellulase-fixed gold nanoparticles (AuNP @ cellulases) began to fall from 36 hours. From this result, it was found that the cellulase immobilized on the gold-magnetic silica nanoparticles was more thermally stable than the other cases.

Experimental Example 6

The reusability of the cellulase-fixed gold-magnetic silica nanoparticles according to Example 1 and the cellulase immobilized on the cellulase-fixed gold nanoparticles according to Comparative Example 1 was tested through repeated hydrolysis experiments as in Example 2 and the The results are shown in FIG.

As shown in FIG. 8, the cellulase-fixed gold nanoparticles began to drop activity to 80% after the third reuse, and the activity decreased by about 60% than the first when the seventh use. On the other hand, the cellulase-fixed gold-magnetic silica nanoparticles were found to maintain about 90% activity even after the seventh reuse. From this result, it can be seen that when the enzyme is immobilized on the gold-magnetic silica nanoparticles, reusability is much better than that of the enzyme immobilized on the gold nanoparticles.

Experimental Example 7

The reusability of the cellulase-fixed gold-magnetic silica nanoparticles recycled according to Example 4 and the cellulase immobilized on the cellulase-fixed gold nanoparticles recycled according to Comparative Example 2 was repeatedly performed as in Example 2 Through experiments and the results are shown in Figure 9 (a) and (b), respectively. In addition, the reusability of repurified cellulase was also investigated. (1st: 1st recycle, 2nd: 2nd recycle, 3rd: 3rd recycle, 4th: 4th recycle)

As shown in Figure 9, when immobilized in gold nanoparticles can be seen that the activity of the cellulase is reduced from the second recycle. However, in the case of immobilization on gold-magnetic silica nanoparticles, it was found that the cellulase enzyme activity was well maintained even after recycling 4 times. In addition, recrystallized cellulase was found to maintain well over 90% in the gold-magnetic silica nanoparticles until the seventh.

Experimental Example 8

In the case where the activity of the enzyme immobilized by using a large number of cellulase-fixed gold-magnetic silica nanoparticles obtained in Example 1 is almost lost, before removal of cellulase which is almost inactive on the surface of the nanoparticles (Before Rinsing) and described in Example 4 After removing the cellulase almost no activity in the manner (After Rinsing) the results were confirmed by TEM (transmission electron microscopy) and EDX (Energy Dispersive X-ray Spectroscopy), it is shown in FIG.

As shown in FIG. 10, the transmission electron microscopic observation showed that the cellulase was well removed from the surface of the gold-magnetic silica nanoparticles after rinsing, and the same result was obtained in the EDX result. Before rinsing, carbon, oxygen, and nitrogen elements obtained from cellulase were seen, but after removal, it was confirmed that cellulase was well removed from the surface of gold-magnetic silica nanoparticles.

Experimental Example 9

After freezing the cellulase immobilized on the free cellulase mixture and the gold-magnetic silica nanoparticles prepared in Example 1 using filter paper, the surface of the filter paper was subjected to SEM (scanning electron microscope). It confirmed and observed and the result is shown in FIG. (a) is before the cellulase treatment, (b) is the case of a mixture of unimmobilized cellulase and (c) is the result of treatment with cellulase immobilized on gold-magnetic silica nanoparticles.

11, it can be seen that when treated with cellulase immobilized on the enzyme-fixed gold-magnetic silica nanoparticles, that is, the gold-magnetic silica nanoparticles, the diameters of the celluloses were found to be more effectively degraded than when not immobilized.

Experimental Example 10

The activity of the xylanase immobilized on the xylanase-fixed gold-magnetic silica nanoparticles (Au-MSNP @ xylanase) obtained in Example 3 using a known Xylanase activity test method is shown in FIG. 12.

12 shows that xylanase immobilized on gold-magnetic silica nanoparticles has better activity than other cases.

Experimental Example 11

The reusability of the cellulase-fixed gold-magnetic silica nanoparticles obtained in Example 3 and the xylanase immobilized on the xylanase-fixed gold nanoparticles obtained in Comparative Example 3 was tested through the repeated performance of a known Xylanase activity test method. The results are shown in FIG.

13 shows that the xylanase-fixed gold nanoparticles began to drop to 60% of activity at the time of the second reuse, and decreased at least 60% of activity from the first at the third use. On the other hand, the xylanase-fixed gold-magnetic silica nanoparticles were found to maintain about 50% or more of activity after the fifth reuse. From this result, it can be seen that even if the xylanase is fixed to the gold-magnetic silica nanoparticles, the reusability is much higher than that of the xylanase is fixed to the gold nanoparticles.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, Various changes and modifications will be possible.

Claims (19)

Magnetic silica nanoparticles;
A plurality of -SH groups introduced to the surface of the magnetic silica nanoparticles;
One or more gold nanoparticles bonded to the -SH group and bonded to the surface of the magnetic silica nanoparticles; And
Enzyme-fixed gold-magnetic silica nanoparticles comprising one or more enzymes bonded to the gold nanoparticles.
The method of claim 1,
Enzyme-fixed gold-magnetic silica nanoparticles, characterized in that the cysteine bond is formed by the -SH group.
The method of claim 1,
The magnetic silica nanoparticles are immobilized gold-magnetic silica nanoparticles, characterized in that synthesized as a precursor to Fe ₃ O ₄ .
The method of claim 3, wherein
The magnetic silica nanoparticles are fixed gold-magnetic silica nanoparticles, characterized in that having a diameter of 5 to 50 nm.
The method of claim 1,
The gold nanoparticles are fixed in gold-magnetic silica nanoparticles, characterized in that having a diameter of 2 to 10nm.
The method of claim 1,
Enzyme-fixed gold-magnetic silica nanoparticles, characterized in that the enzyme activity can be reused more than 10 times to maintain more than 90%.
The method of claim 1,
Enzyme-fixed gold-magnetic silica nanoparticles, characterized in that the enzyme activity can be reused more than 20 times to maintain more than 50%.
The method of claim 1,
When the activity of the enzyme falls below 50%, the enzyme-fixed gold-magnetic silica nanoparticles, characterized in that after removing the enzyme bound to the gold nanoparticles and other enzymes required to be bonded to the gold nanoparticles and recycled.
Preparing magnetic nanoparticles;
Preparing magnetic silica nanoparticles using the magnetic nanoparticles;
Introducing a plurality of -SH groups onto the surface of the prepared magnetic silica nanoparticles;
Preparing gold-magnetic silica nanoparticles by combining gold nanoparticles with the magnetic silica nanoparticles having the —SH group introduced therein; And
Enzyme-fixed gold-magnetic silica nanoparticles manufacturing method comprising the step of fixing the enzyme to the gold-magnetic silica nanoparticles.
The method of claim 9, wherein preparing the magnetic nanoparticles
Mixing the aqueous iron dichloride solution with an iron trichloride solution;
Reacting the mixed solution by adding ammonium hydroxide solution while stirring; And
Enzyme-fixed gold-magnetic silica nanoparticles manufacturing method comprising the step of separating the black product obtained by the reaction.
The method of claim 9, wherein preparing the magnetic silica nanoparticles
Ultrasonically dispersing the prepared magnetic nanoparticles in a solvent;
Adding TEOS (tetrathyl orthosilicate) to the dispersed solution and then adding an ammonium hydroxide solution while stirring to maintain a pH of 10 or more; And
Enzyme-fixed gold-magnetic silica nanoparticles manufacturing method comprising the step of refluxing the product passed through the above step and washed with vacuum drying.
The method of claim 9, wherein the step of introducing a plurality of -SH groups
Adding the magnetic silica nanoparticles to a solution including a -SH group and then refluxing it; And
Enzymatic fixed gold-magnetic silica nanoparticles manufacturing method comprising the step of separating and washing the product passed through the step and vacuum drying.
The method of claim 9, wherein the step of preparing the gold-magnetic silica nanoparticles is
Preparing a gold nanoparticle solution;
Adding and reacting the magnetic silica nanoparticles having the -SH group introduced thereinto the gold nanoparticle solution; And
Enzyme-fixed gold-magnetic silica nanoparticles manufacturing method comprising the step of washing the reaction product and vacuum drying.
The method of claim 9, wherein fixing the enzyme to the gold-magnetic silica nanoparticles comprises
Ultrasonically dispersing after adding a buffer to the gold-magnetic silica nanoparticles;
Reacting by adding an enzyme to the dispersed product; And
Enzyme-fixed gold-magnetic silica nanoparticles prepared by the reaction comprising washing and separating using a magnet, characterized in that it comprises a step of separating using a magnet.
9. The continuous hydrolysis method according to any one of claims 1 to 8, wherein the enzyme-fixed gold-magnetic silica nanoparticles are used as a hydrolase.
The method of claim 15,
After the enzymatic reaction for hydrolysis is terminated, the enzyme-fixed gold-magnetic silica nanoparticles are separated by a magnet, and then washed and reused.
The method of claim 15,
If the substrate of the hydrolysis is biomass, the enzyme-fixed gold-magnetic silica nanoparticles are continuous hydrolysis method characterized in that the endo-glucosidase, exo-glucosidase, β-glucanase is combined.
Removing the immobilized enzyme from the enzyme-fixed gold-magnetic silica nanoparticles of any one of claims 1 to 8, wherein the immobilized enzyme activity is reduced in number; And
Enzyme-fixed gold-magnetic silica nanoparticles recycling method comprising the step of fixing the new enzyme to the enzyme-free gold-magnetic silica nanoparticles.
The method of claim 18,
Removing the fixed enzyme is an enzyme-fixed gold-magnetic silica nanoparticles recycling method comprising the step of ultrasonication at 90 to 110 ℃.

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