ENZYME IMMOBILIZATION USING IRON OXIDE YOLK-SHELL NANOSTRUCTURE

Abstract

This invention relates to a carrier for immobilizing a biocatalyst including a Fe.sub.2O.sub.3 yolk-shell structure, to an immobilized enzyme using the carrier, and to realizing an increase in the stability of the enzyme and stability in organic solvents by cross-linking the enzyme. According to this invention, the carrier for immobilizing a biocatalyst and the enzyme immobilized thereon can be reused, have increased stability, facilitate the control of reactivity, pH, and temperature, and can be widely useful in various biochemical engineering industries.

Claims

1. A carrier composition for immobilizing a biocatalyst, comprising a Fe.sub.2O.sub.3 yolk-shell structure.

2. The carrier composition of claim 1, wherein the Fe.sub.2O.sub.3 yolk-shell structure has one or more pores having an average diameter of 10 to 50 nm on a surface thereof.

3. A method of immobilizing an enzyme using the carrier composition of claim 1.

4. The method of claim 3, comprising immobilizing an enzyme on the Fe.sub.2O.sub.3 yolk-shell structure and cross-linking the immobilized enzyme to form a crosslink.

5. The method of claim 3, wherein the enzyme is a laccase enzyme.

6. The method of claim 4, wherein the cross-linking is performed using glutaraldehyde.

7. A Fe.sub.2O.sub.3 yolk-shell structure-enzyme complex composition comprising a Fe.sub.2O.sub.3 yolk-shell structure on which an enzyme is immobilized.

8. The Fe.sub.2O.sub.3 yolk-shell structure-enzyme complex composition of claim 7, wherein the enzyme is a laccase enzyme.

9. A method of decolorizing a dye, comprising treating a dye wastewater with the Fe.sub.2O.sub.3 yolk-shell structure-enzyme complex composition of claim 7.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0023] FIGS. 1A and B show electron microscope images of the surface of a Fe.sub.2O.sub.3 yolk-shell structure before and after immobilization of laccase on the Fe.sub.2O.sub.3 yolk-shell structure, and FIG. 1C shows an electron microscope image of the surface of the Fe.sub.2O.sub.3 yolk-shell structure;

[0024] FIG. 2 is a graph showing the FTIR absorbance when cross-linking laccase immobilized on the Fe.sub.2O.sub.3 yolk-shell structure;

[0025] FIG. 3 is a graph showing the optimal reaction temperature of laccase immobilized and cross-linked by the Fe.sub.2O.sub.3 yolk-shell structure, wherein: =pure laccase enzyme, ◯=laccase enzyme immobilized on the Fe.sub.2O.sub.3 yolk-shell structure, and .Math.=laccase enzyme immobilized and then cross-linked on the Fe.sub.2O.sub.3 yolk-shell structure;

[0026] FIG. 4 is a graph showing the optimal reaction pH of laccase immobilized and cross-linked by the Fe.sub.2O.sub.3 yolk-shell structure, wherein: =pure laccase enzyme, ◯=laccase enzyme immobilized on the Fe.sub.2O.sub.3 yolk-shell structure, and .Math.=laccase enzyme immobilized and then cross-linked on the Fe.sub.2O.sub.3 yolk-shell structure;

[0027] FIG. 5 is a graph showing the stability of the enzyme depending on the number of cycles of reuse of laccase immobilized on the Fe.sub.2O.sub.3 yolk-shell structure, wherein: grey square=laccase enzyme immobilized on the Fe.sub.2O.sub.3 yolk-shell structure and .square-solid.=laccase enzyme immobilized and then cross-linked on the Fe.sub.2O.sub.3 yolk-shell structure; and

[0028] FIG. 6 is a graph showing the stability of the enzyme depending on the number of cycles of reuse of laccase immobilized on the Fe.sub.2O.sub.3 yolk-shell structure, regarding resistance of the cross-linked immobilized enzyme to the organic solvent, wherein: .square-solid.=pure laccase enzyme and grey square=laccase enzyme immobilized and then cross-linked on the Fe.sub.2O.sub.3 yolk-shell structure.

MODE FOR INVENTION

[0029] A better understanding of the present invention may be obtained via the following non-limiting examples, which are set forth to illustrate, but are not to be construed as limiting the scope of the present invention.

Example 1: Synthesis of Fe.SUB.2.O.SUB.3 .Yolk-Shell Structure Using Spray Pyrolysis

[0030] The corresponding Fe.sub.2O.sub.3 yolk-shell structure was synthesized using a spray pyrolysis process as follows. A metal salt and dextrin as a drying aid are dissolved to give a transparent spray solution, which is then dried using a spray drying process, thereby synthesizing a metal oxide-carbon complex powder. The metal oxide-carbon complex is mass produced and then subjected to simple post-heat treatment at 300° C. or more, thus synthesizing a yolk-shell structure through stepwise combustion of the carbon complex. The detailed synthesis conditions are described below. [0031] Preparation of solution: 0.15 M Fe nitrate is added to distilled water and completely dissolved. 10 g of dextrin is dissolved in 200 ml of an aqueous solution. [0032] The prepared solution is sprayed into a spray-drying reactor using a nozzle, thus recovering particles. [0033] Preparation conditions (spray-drying device operating conditions): an inlet temperature of 300° C., an outlet temperature of 120° C., and a nozzle pressure of 2.4 bar. [0034] Reagents: iron nitrate (Junsei), dextrin (Samchun)

[0035] Using a transmission electron microscope, the Fe.sub.2O.sub.3 yolk-shell structure was observed before and after immobilization with laccase (FIG. 1: A-before immobilization, B-after immobilization). As shown in C of FIG. 1, the Fe.sub.2O.sub.3 yolk-shell structure is configured to have a predetermined sphere in which a movable small sphere is included, with porous particles having a size of 21 nm. Based on the results of analysis with a transmission electron microscope, multiple shells of the Fe.sub.2O.sub.3 yolk-shell structure are produced due to the stepwise combustion of dextrin. Conventional micrometer-sized particles are able to immobilize an enzyme only on the outermost portion thereof, whereas the yolk-shell Fe.sub.2O.sub.3 structure enables the immobilization of the enzyme up to the inside of the particles, thus making it possible to immobilize an enzyme in a large amount per unit volume and mass, namely in an amount at least three to four times the amount of conventional micrometer-sized particles. In the present invention, as the enzyme support, a Fe.sub.2O.sub.3 yolk-shell structure having superior performance was synthesized.

Example 2: Immobilization of Laccase Enzyme

[0036] The Fe.sub.2O.sub.3 yolk-shell nanostructure is activated through treatment with glutaraldehyde as follows. Specifically, the Fe.sub.2O.sub.3 yolk-shell nanostructure is washed two times with distilled water. Thereafter, the Fe.sub.2O.sub.3 yolk-shell nanostructure is treated with 1 M glutaraldehyde. Then, in order to aid activation, reaction is carried out in a shaking incubator at 25° C. and 250 rpm for 4 hr. The activated Fe.sub.2O.sub.3 yolk-shell nanostructure is washed with 30 ml of distilled water and then washed once with a 100 mM phosphate buffer (pH 7).

[0037] 10 mg of the activated carrier and 1 mg of a purified enzyme are mixed with a 50 mM phosphate buffer (pH 7) and then reacted in a shaking incubator at 4° C. and 150 rpm for 24 hr. The protein not coupled with the activated carrier is washed with distilled water and a 100 mM phosphate buffer (pH 7).

Example 3: Cross-Linking of Enzyme Immobilized on Fe.SUB.2.O.SUB.3 .Yolk-Shell Nanostructure

[0038] Cross-linking was performed to maximize the stability of immobilized laccase. The enzyme immobilized on the Fe.sub.2O.sub.3 yolk-shell nanostructure was treated with glutaraldehyde in various concentrations ranging from 0.01 to 1.00 M in the presence of a phosphate buffer at pH 7.0 (50 mM) under conditions of 4° C. 150 rpm and 2 to 8 hr.

Example 4: Results of Cross-Linking of Laccase Immobilized on Fe.SUB.2.O.SUB.3 .Yolk-Shell Structure

[0039] FIG. 2 is a graph showing the FTIR absorbance when cross-linking laccase immobilized on the Fe.sub.2O.sub.3 yolk-shell structure. As is apparent from the absorbance of 1600 to 1800 cm.sup.−1 in the FTIR spectrum of FIG. 2, an amide bond (N═C═O) can be found to be formed due to the cross-linking.

Example 5: Immobilization Efficiency of Laccase Enzyme Immobilized on Various Nano-Carriers

[0040] Using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, available from Sigma-Aldrich) as a substrate, the immobilized enzyme prepared as above and the enzyme immobilized on various carriers were measured for activity (Table 1). 1 mM ABTS and 0.05 μg of the immobilized enzyme were added to 1 ml of a reaction medium (50 mM sodium citrate buffer, pH 3.0), after which the oxidation of ABTS was carried out at a reaction temperature of 25° C. for 5 min. After the completion of the reaction for 5 min, the immobilized enzyme was separated from the reaction mixture using a magnet, and the product obtained through the oxidation of ABTS was analyzed by observing the absorbance at 420 nm.

[0041] Laccase was immobilized on each of commercial carriers and synthesized carriers, after which the immobilization yield (IY) and immobilization efficiency (IE) thereof were compared, whereby the immobilization yield was determined to range from 18.7 to 90.6% and the immobilization efficiency was determined to range from 18.4 to 87.5%. Under similar conditions among various carriers, the Fe.sub.2O.sub.3 yolk-shell structure exhibited the greatest immobilization yield of 90.6% and immobilization efficiency of 87.5%.

TABLE-US-00001 TABLE 1 Immobilization Nano-particles Immobilization yield (IY) % Efficiency (IE) % Commercial particles Al.sub.2O.sub.3 45.5 ± 3.7 37.8 ± 3.5 SnO.sub.2 18.7 ± 1.5 24.5 ± 2.1 Fe.sub.2O.sub.3 64.2 ± 5.1 30.8 ± 2.6 Fe.sub.3O.sub.4 37.4 ± 3.2 55.6 ± 5.1 SiO.sub.2 (15 nm) 35.6 ± 3.0 48.4 ± 4.1 SiO.sub.2 (20 nm) 48.2 ± 4.2 34.8 ± 3.0 SiO.sub.2 (80 mn) 63.5 ± 5.3 69.0 ± 6.1 SrFe.sub.12O.sub.19 42.5 ± 3.6 30.5 ± 2.5 TiO.sub.2 53.0 ± 4.1 40.1 ± 3.2 Y.sub.3Fe.sub.5O.sub.12 45.7 ± 3.8 23.2 ± 2.0 ZrO.sub.2 26.4 ± 2.1 18.4 ± 1.4 Synthesized particles Fe.sub.2O.sub.3 yolk-shell 90.6 ± 6.5 87.5 ± 7.1 Fe.sub.2O.sub.3anti-cave 44.5 ± 4.8 58.2 ± 4.6 NiO@void@SiO.sub.2 47.5 ± 4.2 52.1 ± 4.4 Co.sub.3O.sub.4 (nanotube) 42.4 ± 4.0 46.1 ± 4.0 SnO.sub.2 (Tube-in-Tube) 48.6 ± 4.1 48.2 ± 4.2 NiO@void@SiO.sub.2 10% 53.8 ± 4.0 64.5 ± 5.1 NiO@void@SiO.sub.2 40% 59.1 ± 4.3 48.5 ± 3.8

[0042] Table 1 shows the immobilization efficiency of laccase on various nano-carriers.

Example 6: Properties of Laccase Immobilized on Fe.SUB.2.O.SUB.3 .Yolk-Shell Structure Depending on Changes in Temperature

[0043] FIG. 3 shows the optimal temperatures of pure laccase, laccase immobilized on the Fe.sub.2O.sub.3 yolk-shell structure (YS-IM) and laccase obtained by cross-linking the immobilized enzyme (YS-IMC). Measurement was performed in the temperature range from 25 to 70° C. The optimal temperatures of the YS-IM and YS-IMC enzymes were 5° C. higher than that of the free laccase enzyme (FLac). Also, in the temperature range from 50 to 70° C., YS-IMC exhibited residual activity higher than those of FLac and YS-IM.

Example 7: Properties of Laccase Immobilized on Fe.SUB.2.O.SUB.3 .Yolk-Shell Structure Depending on Changes in pH

[0044] FIG. 4 shows the residual activity of laccase depending on changes in pH. The optimal pH was 3 for FLac, 4 for YS-IM, and 4 for YS-IMC. In the pH range of 5 to 7, the residual activity of YS-IMC was higher than those of FLac and YS-IM. That is, residual activity of YS-IMC was increased 2.7-, 4.5-, and 8.3-fold under the same conditions compared to FLac.

Example 8: Stability of Laccase Upon Reaction Using Immobilized Enzyme

[0045] Changes in relative activity depending on the number of cycles of reuse of the immobilized enzyme were measured to determine the stability of the enzyme. The reaction was carried out at 25° C. using 1 mM ABTS and 0.05 μg of the immobilized enzyme. As shown in FIG. 5, .square-solid. and the grey square show changes in the relative activity depending on the number of cycles of reuse of YS-IMC and YS-IM, respectively. In FIG. 5, when the number of cycles of reuse reached 5 and 10, the relative activity of YS-IMC was 94.1 and 87.5% or more, and the relative activity of YS-IM was 88.6 and about 70.6%. Thus, the enzyme immobilized on YS-IMC was determined to be more stable.

Example 9: Stability of Immobilized Laccase in Organic Solvent

[0046] The resistance of FLac to 12 organic solvents (25% v/v) was evaluated through reaction at 25° C. for 4 hr. YS-IMC exhibited the residual activity of 15.8 to 84.7%, whereas the residual activity of FLac was only 8%. The organic solvent having the lowest toxicity to YS-IMC was acetone, and upon reaction for 4 hr and 12 hr, the residual activity was increased 13-fold and 32-fold respectively compared to FLac (FIG. 6).