KR20190033780A - enzyme-metal hybrid nanoflowers and its application in repeated batch decolorization of dyes - Google Patents

Abstract

The present invention relates to an enzyme-metal hybrid nanoflower and a repeated decolorization method of dyes using the same. The enzyme-metal hybrid nanoflower according to the present invention exhibits high stability against changes in pH and temperature, excellent storage stability and resistance to inhibitors and solvents, and an excellent repeated decolorization effect of dyes, and thus can be used in many industrial fields as an immobilization method for improving the properties of enzymes.

Description

[0001] The present invention relates to an enzyme-metal hybrid nanoflower and its application in repeated batch decolorization of dyes,

The present invention relates to an enzyme-metal fused nanoflower and a method for repeated bleaching of dyes using the same.

Enzymes are attracting attention because of their environmentally friendly properties, such as mild treatment conditions and reduced effluent production due to excellent biodegradability, and substrate specificity, which only reacts to specific substrates. However, it shows a rapid decrease in activity under the reaction conditions other than the optimal conditions of the enzyme, and it is difficult to recover and reuse after the reaction, and the production cost of the enzyme is high, so that the application to practical industry is limited. The enzyme immobilization is physically restrained in a certain place to continuously and repeatedly exhibit catalytic activity, thereby increasing the availability and stability of the enzyme. Thus, the enzyme immobilization is being watched as an alternative to overcome the limitations of the enzyme. Studies on enzyme immobilization in the textile industry have been reported on the possibility of bio-bleaching, dye bleaching and decomposition, treatment of industrial wastewater, and improvement of decontamination of laundry detergent by immobilizing lacquer, amylase, and cellulase.

Enzyme immobilization can increase the economical efficiency of the enzyme reaction process because the enzyme can easily be recovered and reused, and the reaction mode can be applied in a batch or continuous manner. The immobilized enzyme can be used repeatedly, and the product and the enzyme can be easily separated from each other, but the activity is inferior to that of the enzyme that is not immobilized. However, the immobilized enzymes can be conveniently handled above all, and thus various commercial applications are possible.

Common enzyme immobilization methods are physical adsorption methods or chemical methods. The physical adsorption method mainly uses an ion-exchange method. The ion-exchange method is advantageous in that it is non-toxic, but its bonding strength is weak. The chemical method uses a chemical reagent to fix the enzyme by forming a covalent bond by a chemical reaction. Although the method has strong cross-linking ability, due to the toxicity of the reagent used for immobilizing the enzyme, It is difficult to use it. Enzyme immobilization, in which an enzyme is immobilized by binding an enzyme to an organic or inorganic carrier to perform reuse and sequencing, is well known. The reason why organic substances (eg, cellulose, nylon, and polyacrylamide) are disadvantageous as a carrier is that the mechanical stability is poor, and corrosion due to the solvent, changes due to pH and ionic strength, and microbial infiltration, It is because. Therefore, an inorganic carrier to which an enzyme is adsorbed or covalently adsorbed has been proposed. The binding form depends on the conditions of use, the type of the enzyme, and the characteristics of the substrate. That is, if the substrate has a strong salt concentration, the adsorption method can not be applied because the adsorption of the adsorbed enzyme occurs, and the covalent bonding of the enzyme takes precedence. The surface of the carrier should include a specific functional group that induces the binding of the enzyme. Since most carriers can not cover functional groups, surface pretreatment is required. Immobilization by covalent bonding is a method of covalently bonding the surface of a carrier with an enzyme by a binder or a pier, so that the carrier should be surface-treated or a functional group should be introduced into the enzyme and the active site of the supported enzyme should not be shielded.

The inventors of the present invention have investigated a method for immobilizing an enzyme, and confirmed that the enzyme-metal fusion nanoflower is useful for immobilizing the enzyme, and its excellent dye decolorizing effect, and completed the present invention.

It is an object of the present invention to provide an enzyme-metal fusion nanoflower.

It is still another object of the present invention to provide a method for producing the nanoflower.

It is a further object of the present invention to provide a method for repeated bleaching of dyes using the nanoflower.

In order to accomplish the above object, the present invention provides an enzyme-metal fused nanoflower with an enzyme immobilized thereon.

The present invention also provides a method for producing an enzyme-metal fused nanoflower comprising the step of reacting an enzyme and a metal hydrate.

The present invention also provides a dye decolorizing method using an enzyme-metal fused nanoflower.

The enzyme-metal fused nanoflower according to the present invention exhibits high stability against changes in pH and temperature, exhibits excellent storage stability, tolerance to solvents and inhibitors, and excellent dye decolorizing effect, And can be usefully used in many industrial fields as an immobilization method.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 schematically shows the synthesis and cross-linking of a lacase-Cu ₃ (PO ₄ ) ₂ .3H ₂ O fused nanoflower.
2a to 2d are FE-SEM images of lacase-metal fused nanoflowers 2a and 2b and cross-linked lucerase fusion nanoflowers 2c and 2d.
Figures 2e and 2f are CLSM images of the lacase-metal fused nanoflowers and lacase nanoflowers labeled with FITC.
Figure 3 shows the activity of free (FE), lactase nanoflower (NF) and lactase-metal fused nanoflower (CL-NF) at various pH (a) and temperature (b) values.
Fig. 4 shows the stability (a) at 25 캜 and the storage stability at 4 캜 of glass (FE), glass (FE), lactic acid nanoflower (NF) b) and reusability (c) through relative activity.
Fig. 5 is a diagram showing the resistance to substrate specificity (a), solvent (b) and inhibitor (c) of the free lacase and the lacase-metal fused nanoflower (CL-NF) through relative activity.
6 is a graph showing a decolorization profile of (a) bromophenol blue, (b) CBBR-250 and (c) xylene cyanol using a lucerase-metal fused nanoflower, and a decolorizing effect to be.

The present invention provides an enzyme-metal fused nanoflower and a method for repeated bleaching of dyes using the same.

Hereinafter, the present invention will be described in detail.

First, the present invention provides an enzyme-metal-fused nanoflower in which an enzyme is immobilized.

The nanoflower is a nanoflower in which an enzyme is immobilized on a nanocomposite by fusion with a metal, and the enzyme is preferably immobilized on the nanoflower through cross-linking.

The cross-linking can be preferably carried out by glutaraldehyde, 3-aminopropyltriethoxysilane (APTES), carbodiimide, cyano and polyethyleneimine (PEI), more preferably with glutaraldehyde ≪ / RTI >

The nanoflower of the present invention may have an average diameter of 10 탆 or less.

The enzyme may be, but is not limited to, a laccase enzyme.

The metal may be at least one selected from the group consisting of Co, Cu, Fe, Mn, Ni, Zn, and Pd, but is not limited thereto.

The present invention also provides a method for producing an enzyme-metal fused nanoflower comprising reacting an enzyme and a metal.

The metal in the reaction in the form of Co, Cu, Fe, Mn, Ni, Zn, and one member selected from the group consisting of Pd is more than can be, but shall not limited to this, and preferably may be a Cu, more preferably from CuSO ₄ You can participate.

The preparation method may further include a crosslinking step after the reaction, and may be crosslinked preferably by glutaraldehyde.

The present invention also provides a method of decolorizing a dye using the enzyme-metal fused nanoflower.

The dye may be decolorized one to fifteen times. However, the dye is not limited to this and may exhibit high decolorization efficiency despite repeated use.

The dye may be at least one selected from the group consisting of bromophenol blue, CBBR and xylene cyanol.

The manufacturing method includes treating the nanoflower with a dye.

The enzyme-metal fused nanoflower according to the present invention exhibits high stability against changes in pH and temperature, exhibits excellent storage stability, tolerance to solvents and inhibitors, and excellent dye decolorizing effect, And can be usefully used in many industrial fields as an immobilization method.

Hereinafter, preferred examples, experimental examples, and formulation examples are provided to facilitate understanding of the present invention. However, the following examples, experimental examples and preparation examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited thereto.

Preparation Example  1. Materials and reagents

(ABTS), 2,6-dimethoxyphenol (2,6-DMP), 3,4-dihydroxy-L-phenylalanine (L -DOPA), benzoquinone, copper sulfate, ferric acid, fluorescein isothiocyanate, glutaraldehyde, from Trametes versicolor, p-phenyldiamine, pyrogallol and toluidine were purchased from Sigma-Aldrich Louis, MO, USA). PBS and ultrapure water were purchased from Life Technologies, USA. Bromophenol Blue was purchased from Shelton Scientific, Inc., USA. CBBR-250 and xylenesianol were purchased from BioShop Canada Inc., Canada. All other chemicals were analytical grade and were used without further purification.

Example  1. Enzyme-metal fusion Nano-flower  Synthetic and Crosslinking

The synthesis and cross-linking of the lacase-metal fused nanoflower is schematically shown in Fig.

As shown in FIG. 1, 5 mL of PBS (10 mM, pH 7.4) containing various concentrations of the lacquer at 0.05 to 0.5 mg mL ^-1 was mixed with CuSO ₄ (50 μL, 200 mM) for 4 hours After 24 hours of incubation, the cells were incubated at 4 ° C for 24 hours and 72 hours, respectively, using 6.0 and 0.8 mM CuSO ₄ . After incubation at 4 ° C for 4 hours with 0.1 M of glutaraldehyde in buffer (100 mM, pH, 7.0), the lacase-nanoflower was crosslinked to obtain a lacase-metal fused nanoflower (CL- .

As a control group, a synthesized laccase nanoflower (NF) was prepared after cross-linking with glutaraldehyde.

Example  2. Methods of measuring activity

The free lactase, lacase nano flower (NF) and lacase-metal fused nanoflower (CL-NF) activity were determined by measuring the absorbance at 420 nm using ABTS (1 mM) oxidation.

Example  3. Instrument analysis method

The shape of synthesized CL-NF was observed by field emission scanning electron microscopy (FE-SEM).

Absorption spectra were recorded using a UV-Vis spectrophotometer (Patel et al., 2016c).

The size of CL-NFs was measured by dynamic light scattering (DLS) analysis.

A confocal laser scanning microscope (CLSM) image of NF with FITC-labeled lacase immobilized was obtained using an FV-1000 Olympus confocal microscope.

Experimental Example  One. Lacase - metal fusion Nano-flower  Encapsulation yield ( EY ) And relative activity (RA)

As in Example 2, a fusion nanoflower was prepared using 0.25 mg mL ^-1 of lacase and incubated at a temperature (4 ° C to 25 ° C), a Cu concentration (0.8, 2.0, and 6.0 mM) The encapsulation yields (EY) and the relative activity (RA) were calculated according to the following equation (1). The encapsulation yield and relative activity were calculated using the following equation.

EY = amount of immobilized enzyme / amount of initial enzyme × 100

RA = total nonspecific activity of immobilized enzyme / total nonspecific activity of free enzyme × 100

Synthesis condition Lacase Cu
(mM) Temperature (℃) Culture time (h) EY ^a
(%) RA ^b (%) After immobilization After crosslinking 0.8 4 24 61.7 ± 5.1 137 ± 12 141 ± 12 0.8 25 72 48.3 ± 4.6 128 ± 12 132 ± 11 2.0 4 24 78.1 ± 5.1 172 ± 16 204 ± 19 2.0 25 72 80.4 ± 5.8 104 ± 10 116 ± 11 6.0 4 24 65.0 + - 5.6 89.5 ± 7.9 91.9 ± 9.0 6.0 25 72 71.3 ± 5.5 50.1 ± 4.7 50.3 ± 5.0

As shown in Table 1, EY and RA varied from 48.3% to 80.4% and from 50.1% to 172%, respectively. NF cross-linked by glutaraldehyde resulted in an 18.6% improvement in enzyme efficiency.

Of the synthetic conditions tested, a maximal immobilization efficiency of 204% was achieved after the glutaraldehyde crosslinking when using 2.0 mM of Cu with a incubation time of 24 hours at 4 ° C. The optimum conditions were set in the following experiment.

 Also, synthesis at high concentrations of Cu (6.0 mM) resulted in a reduction in RA. It was confirmed that when RA was low, the activity of lacase was inhibited by Cu at a high concentration.

The EY and RA results for the lactase immobilization indicate that the synthesis method of the present invention is more effective than the ultrasound chemical synthesis method (Batule et al., 2015).

In addition, lacase-metal fused nanoflowers were synthesized using various concentrations of lacase (0.05-0.50 mg) under the above optimal conditions, and their EY and RA are shown in Table 2 below.

Lacase
(mg mL ^-1 ) EY ^a (%) RA ^b (%) After immobilization After crosslinking 0.05 87.0 ± 4.3 140 ± 13 147 ± 13 0.10 84.5 ± 4.4 151 ± 15 168 ± 15 0.25 78.1 ± 5.1 172 ± 16 204 ± 19 0.50 41.6 ± 3.7 128 ± 12 123 ± 11

As shown in Table 2, the EA of the lactase decreased from 87.0% to 41.7% as the concentration of the lactase increased from 0.05 to 0.5 mg mL ^-1 , and the lactase-metal fused nanoflower decreased to 128-172% Of RA. ≪ / RTI >

On the other hand, in the case of the lactose nanoflower synthesized after cross-linking by glutaraldehyde, a 204% improvement of RA was observed up to 0.25 mg mL ^-1 of protein. When the protein concentration was increased to 0.50 mg mL ^-1 , RA was slightly decreased from 128% to 123%.

Experimental Example  2. Manufactured Nano-flower  Identify characteristics

FE-SEM analysis was performed to confirm the nanoflower produced, and the results are shown in Figs. 2a to 2d.

As shown in FIGS. 2A and 2B, the nanoflower morphology of the lacase-metal fused nanocomposite was confirmed and the nanoflow morphology of the cross-linked lucase fusion nanocomplex was confirmed, as shown in FIGS. 2c and 2d.

In addition, CLSM images of the lacase-metal fused nanoflowers and the lacase nanoflowers labeled with FITC were confirmed, and the results are shown in Figs. 2e and 2f.

As shown in FIGS. 2e and 2f, efficient lacase immobilization was confirmed through the high intensity FITC color of the green channel as compared to the bright channel.

In addition, the activity profile of the free lactase and the immobilized lactase nanoflower at various pH and temperature was confirmed, and the results are shown in FIG.

As shown in Fig. 3A, the optimum activity of NF and CL-NF was observed at a pH higher than 3.5, unlike the free lacase, which obtained maximum activity at pH 3.0. Specifically, NF maintained a higher residual activity over a wide pH range of 3.5-7.0 than that of the free lacase, and CL-NF showed more stable residual activity under similar conditions. NF and CL-NF were 9.0 and 17.1 times higher, respectively, than the free enzyme at pH 7.0.

Similar to the above results, as shown in FIG. 3B, NF and CL-NF retained higher residual activity in the relatively high temperature range of 40-70 DEG C compared to the free enzyme. The optimum temperature values of the free NF and CL-NF lactase were 40 ° C, 45 ° C and 45 ° C, respectively. Raccase NF and CL-NF showed 3.7 and 11.8 times higher residual activity than the free enzyme at 70 ℃, respectively.

Experimental Example  3. Convergence NF  Kinematic study

The Michaelis-Menten model was used to determine the kinetic parameters (apparent Km and Vmax) at the optimum pH of the glass, NF and CL-NF lacase at 25 ° C, and the results are shown in Table 3 below. The kinetic parameters were obtained by nonlinear regression (Prism 5, Graphpad Software, USA) using ABTS (0.005-2.0 mM) at 25 ° C (Patel et al., 2016a).

Lacase K _m (mM) V _max (μmol min ^-1 mg protein ^-1 ) k _cat K _m ^-1 (s ^-1 M ¹ ) Free 29.3 ± 2.2 1890 ± 30 71.0 ± 5.2 NF 22.9 ± 1.1 3140 ± 30 151 ± 10 CL-NF 27.0 ± 1.6 3770 ± 40 154 ± 11

As shown in Table 3, the free lactase showed Km and Vmax values of 29.3 mM and 1890 μmol min-1 mg protein-1, respectively, and the NF values were 22.9 mM and 3140 μmol min -1 mg protein-1 Respectively. After cross-linking, the values of Km and Vmax were 21.3 mM and 3770 μmol min-1 mg protein-1, respectively.

Overall, the catalytic efficiency (k _cat Km ^{- 1} ) of the lactase was improved 2.2-fold through the immobilization and cross-linking with glutaraldehyde for the free enzyme value of 71.0 s ^-1 μM ^-1 .

The improvement in k _cat Km ^-1 value is related to the cooperative effect of the immobilized enzyme over the mass transfer limit, favorable steric structure and high surface area of NF.

Experimental Example  4. Lacase - metal fusion Stability of Nanoflower  And reusability

Relative activity was measured to analyze the stability and reusability of glass, NF and CL-NF at room temperature (25 ° C) and storage temperature (4 ° C), and the results are shown in FIG.

First, equal amounts of NF and CL-NF were equally incubated in buffer solution (50 mM) under optimal conditions. The residual activity of the sample was measured at various intervals, and the stability was evaluated by considering the initial activity as 100% The results are shown in Figs. 4A and 4B.

As shown in Figure 4a, NF showed significantly higher stability compared to the free form after 10 days of incubation at 25 ° C, and surprisingly, CL-NF maintained 86.2% of the residual activity under optimal conditions, Agar was found to have completely lost its activity during 7 days of incubation.

In addition, as shown in Fig. 4B, after 60 days of incubation at 4 째 C, NF and CL-NF retained residual activity of 3.8%, 53.3%, and 91.5%, respectively, The residual activity was 14.0 and 24.1 times higher.

In addition, the reusability of NF and CL-NF laccase was evaluated under standard analytical conditions for 10 cycles. NF or CL-NF was recovered by centrifugation, washed twice with phosphate buffer, and then used in the next cycle. The initial activity of lacase was considered 100% and the reusability was evaluated. The results are shown in FIG. 4C Respectively.

As shown in FIG. 4C, after 5 cycles, NF and CL-NF showed residual activity of 85.8% and 96.8%, respectively. Residual activity was further reduced to 41.2% and 92.3% after 10 cycles of re-use, Showed a 2.2-fold higher residual activity.

For reference, the lactic acid nanoflower previously synthesized by the sonochemical method (Batule at al., 2015) retained 60% of RA after 5 cycles of reuse. This means that the lacase-metal fused nanoflower of the present invention is effective in maintaining high stability during the reuse of the enzyme.

Experimental Example  5. Lacase - metal fusion Nano-flower  Substrate specificity

In order to confirm the substrate specificity of the lacase-metal fusion nanoflower of the present invention, the activity of lacase was varied according to the type of substrate used in the assay (Addorisio et al., 2013 and Kalyani et al., 2015) , Benzoquinone, 2,3,4,4,5,6,6-DMP, L-DOPA, ferulic acid, guaiacol, p-phenyldiamine, pyrogallol and toluidine under optimal analytical conditions. And relative activity of CL-NF was measured. The results are shown in FIG. 5A.

As shown in Fig. 5 (a), it was confirmed that CL-NF exhibits a much higher oxidation potential for phenolic compounds. This means that it can be used as a biosensor for phenolic compounds.

Experimental Example  5. Lacase - metal fusion Nano-flower  Solvent and Resistance to inhibitors

Since the activity of the enzyme is greatly influenced by the solvent (Kalyani et al., 2015 and Tavares et al., 2013), the free radicals and CL-NF are reacted in several aqueous and organic solvents (25% v v-1) Was incubated at 25 DEG C for 4 hours under standard analytical conditions, and the relative activity was measured. The results are shown in FIG. 5B.

As shown in FIG. 5B, CL-NF showed higher resistance than 15.3, 12.9, 14.0, 9.1 and 10.7 for acetone, n-butane, ethanol, hexane and toluene (25%, vv-1) Respectively.

In addition, the presence of inhibitors such as L-cysteine, dithiothreitol, SDS and thiourea can variably inhibit the activity of lacquerase through active site modification (Lorenzo et al., 2005) The effect of the inhibitor (0.5 mM) containing sodium cysteine, dithiothreitol, SDS, and thiourea on the residual activity of lacucer was confirmed under standard assay conditions, and the results are shown in FIG. 5c.

As shown in Fig. 5C, the free lactase and CL-NF showed residual activity ranging from 1.2-83.5% and 27.3-97.3%, respectively. Overall, CL-NF was 6.8, 22.7, 1.2, and 2.0 times more resistant to L-cysteine, dithiothreitol, SDS, and thiourea than the free lactase, respectively. This means that CL-NF laccase can be effectively used in the presence of an inhibitor as well as an organic solvent.

Experimental Example  6. Lacase - metal fusion Nano flower  Discoloration of dyes under repeated placement conditions

Discoloration of synthetic dyes (120 μg mL-1) containing bromophenol blue (λ _max = 592 nm), CBBR-250 (λ _max = 585 nm) and xylenesianol (λ _max = 615 nm) 0.1 mM), and the results are shown in Figs. 6A to 6C. First, the decolorization was measured using a buffer solution (50 mM) at a concentration of 120 μg mL ^-1 for 48 hours (100 rpm, 25 ° C) under shaking conditions with free radicals and CL-NF. Repeated decolorization of the dye was evaluated over 10 cycles with a culture time of 12 hours. After each cycle, CL-NF was recovered by centrifugation (4 ° C) for 10 minutes and used in subsequent cycles. The initial decolorization efficiency was considered to be 100%.

As shown in FIGS. 6A to 6C, CL-NF effectively discolored these dyes to 41.2%, 73.2% and 73.0%, respectively, of the original color. The decolorization efficiencies of free radicals were low at 15.6%, 36.0% and 21.6%, respectively.

In the presence of ABTS, the maximum discoloration efficiencies of bromophenol blue, CBBR-250 and xylenecanol were 56.0%, 91.3% and 93.5%%, respectively, compared to the free enzyme values of 25.9%, 52.7% and 44.2 for CL- . The high decolorization efficiency of CL-NF is related to a significantly higher stability than the free lactase.

Repeated batch decolorization of these dyes was also evaluated during the 24 hour incubation period and the results are shown in Figure 6d.

As shown in FIG. 6D, CL-NF laccase showed 76.2%, 84.6% and 81.3% decolorization efficiency for bromophenol blue, CBBR-250 and xylene cyanol, respectively, after 10 times of reuse.

As shown in the above Experimental Example, the enzyme-metal fused nanoflower according to the present invention exhibits high stability against changes in pH and temperature, exhibits excellent storage stability, resistance to solvents and inhibitors, And thus it can be understood that the method is an immobilization method for improving the properties of the enzyme.

Claims (16)

Enzyme-immobilized enzyme-metal fusion nanoflower. The enzyme-metal fused nanofluid according to claim 1, wherein the nanoflower is immobilized to the nanocomposite through cross-linking. 3. The enzyme-metal fusion nanofluid according to claim 2, wherein the nanoflower is crosslinked by glutaraldehyde. 2. The enzyme-metal fused nanofluid according to claim 1, wherein the enzyme is laccase. The enzyme-metal fused nanofluorescent material according to claim 1, wherein the metal is at least one selected from the group consisting of Co, Cu, Fe, Mn, Ni, Zn and Pd. 6. The enzyme-metal fusion nanofluid according to claim 5, wherein the metal is Cu. A method for producing an enzyme-metal-fused nanoflower, comprising the step of reacting an enzyme and a metal. 8. The method according to claim 7, wherein the metal is at least one selected from the group consisting of Co, Cu, Fe, Mn, Ni, Zn and Pd. 9. The method according to claim 8, wherein the metal is Cu. The method of claim 7, wherein the metal is an enzyme which comprises the reaction in the form of CuSO ₄ - process for producing a metal nano-fusion flower. 8. The method of claim 7, wherein the nanoflower is immobilized to the nanocomposite through cross-linking. 12. The enzyme-metal fused nanofluid of claim 11, wherein the nanoflower is crosslinked by glutaraldehyde. A method of decolorizing a dye using the nanoflower according to any one of claims 1 to 6. 14. The method for decolorizing a dye according to claim 13, wherein the decoloring of the dye is repeated one to fifteen times. The dye decoloring method according to claim 13, wherein the dye is at least one selected from the group consisting of bromophenol blue, CBBR-250 (Coomassie Brilliant Blue R-250) and xylene cyanol. 14. The method of claim 13, further comprising treating the nanoflower with a dye.

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