CN110628755A - Prussian blue structure-enzyme complex and preparation method thereof - Google Patents
Prussian blue structure-enzyme complex and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological catalytic materials, and discloses a Prussian blue structure-enzyme complex and a preparation method thereof. Respectively preparing metal ion solution and K4[Fe(CN)6]Solutions, surfactant solutions and enzyme solutions; mixing metal ion solution, surfactant solution and enzyme solution, stirring and preheating, and then dropwise adding K4[Fe(CN)6]Reacting the solution to generate precipitate particles, and then cooling to 4-20 ℃ for continuous reaction; and carrying out centrifugal separation, washing and drying on the obtained precipitate particles to obtain the Prussian blue structure-enzyme complex. The prussian blue structure-enzyme compound is prepared by a one-step in-situ embedding method, the method is simple, convenient and efficient, the catalytic activity of the enzyme is obviously improved, and the stability of the enzyme under industrial catalytic conditions is improved.
Description
Technical Field
The invention belongs to the field of biological catalytic materials, and particularly relates to a Prussian blue structure-enzyme complex and a preparation method thereof.
Background
The enzyme is used as a natural high-efficiency catalyst and has wide application in the fields of biological medicine, fine chemicals, food fine processing and the like. However, under industrial catalysis conditions, natural enzyme molecules have the characteristics of poor stability, non-recoverability and the like, and the expansion of the natural enzyme molecules in industrial application is greatly limited. Immobilized enzyme technology provides one of the effective solutions to the above challenges.
The immobilized enzyme technology has already realized partial industrialization and has achieved certain success. However, the existing immobilized enzyme method still has certain defects, such as complicated preparation method, use of toxic reagents in the preparation process, low enzyme retention activity after immobilization and the like, so that further exploration of the preparation method of the immobilized enzyme with simple, convenient and efficient preparation and high enzyme retention activity after immobilization is very important.
Prussian blue can be used as an antidote for heavy metal thallium poisoning in medicine, and has good biological safety and biocompatibility. Prussian blue synthetic raw materials are low in price, the preparation method is simple, the reaction conditions are mild, and the Prussian blue synthetic raw materials have been reported to have pseudo-enzyme catalytic activity.
Disclosure of Invention
In view of the above disadvantages and shortcomings of the prior art, the present invention is primarily directed to a method for preparing prussian blue structure-enzyme complex.
Another object of the present invention is to provide a prussian blue structure-enzyme complex prepared by the above method.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a Prussian blue structure-enzyme complex comprises the following preparation steps:
(1) respectively preparing metal ion solution and K4[Fe(CN)6](yellow blood salt) solution, surfactant solution and enzyme solution;
(2) mixing a metal ion solution, a surfactant solution and an enzyme solution, stirring and preheating;
(3) adding K dropwise into the mixed solution obtained in the step (2)4[Fe(CN)6]Reacting the solution to generate precipitate particles, then cooling to 4-20 ℃, and thenContinuing the reaction;
(4) and (4) carrying out centrifugal separation, washing and drying on the precipitate particles obtained in the step (3) to obtain the Prussian blue structure-enzyme complex.
Preferably, the metal ions in step (1) include at least one of iron ions, copper ions, nickel ions, zinc ions, cobalt ions, calcium ions and magnesium ions.
Preferably, the concentration of the metal ion solution in the step (1) is 2-50 mM.
Preferably, said K in step (1)4[Fe(CN)6]The concentration of the solution is 2-200 mM.
Preferably, the surfactant in step (1) comprises at least one of polyvinylpyrrolidone, sodium dodecylbenzenesulfonate and pluronic.
Preferably, the enzyme in step (1) comprises at least one of cytochrome C, horseradish peroxidase, lipase, carbonic anhydrase, amylase, sucrase, tyrosinase, superoxide dismutase, glucose oxidase, alcohol dehydrogenase, laccase, aldo-ketone reductase and trypsin.
Preferably, the concentration of the enzyme solution in the step (1) is 0.05-10 mg/ml.
Preferably, the temperature of said preheating in step (2) is 30 deg.C～Preheating time is 1-30 min at 60 ℃.
Preferably, the metal ions in step (2) and K in step (3)4[Fe(CN)6]The amount ratio of the substances (A) is 1:4 to 4: 1.
Preferably, the cooling to 4-20 ℃ in the step (3) is carried out for 0.5-24 h.
Preferably, the drying in step (4) is freeze drying, vacuum drying or a combination thereof.
Preferably, the solutions in the above preparation methods are all prepared with deionized water.
A Prussian blue structure-enzyme complex is prepared by the method.
The principle of the invention is as follows: the metal ion solution and the xanthate solution are mixed to generate the nano-particles with the Prussian blue structure, the formation of the Prussian blue crystal structure is further induced by adding the enzyme protein molecules and the surfactant molecules, and the enzyme protein molecules are embedded into the Prussian blue structure at the same time, so that the Prussian blue structure-enzyme compound of the Prussian blue structure embedded enzyme molecules is formed.
The preparation method and the obtained product have the following advantages and beneficial effects:
(1) the prussian blue structure-enzyme compound is prepared by a one-step in-situ embedding method, and the method is simple, convenient and efficient;
(2) the method for immobilizing the enzyme molecules by using the prussian blue and the analogues can obviously improve the catalytic activity of the enzyme or improve the stability of the enzyme under the industrial catalytic condition.
Drawings
Fig. 1 is a scanning electron micrograph of the prussian blue material obtained in example 1.
Fig. 2 is a scanning electron micrograph of the prussian blue copper-based analogue material obtained in example 2.
FIG. 3 is a scanning electron micrograph of the Prussian blue-cytochrome C complex obtained in example 3.
Fig. 4 is an XRD pattern of the prussian blue-cytochrome C complex (PB @ Cyt C) and Prussian Blue (PB) materials obtained in example 3.
FIG. 5 is a graph showing the comparison of the activity of the Prussian blue-cytochrome C complex (PB @ Cyt C) obtained in example 3 with that of free cytochrome C (Cyt C) and Prussian Blue (PB).
FIG. 6 is a graph comparing the activity retention of the Prussian blue-cytochrome C complex (PB @ Cyt C) and free cytochrome C (Cyt C) of example 3 after treatment in organic solvent.
FIG. 7 is a scanning electron micrograph of a Prussian blue copper-based analogue-tyrosinase complex obtained in example 5.
FIG. 8 is a graph comparing the activity retention of the Prussian blue copper-based analogue-tyrosinase complex (CuFe @ TYR) and native Tyrosinase (TYR) in example 5 after treatment under high temperature conditions.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
(1) FeCl was prepared at a concentration of 5mM each3Solution, 20mM K4[Fe(CN)6]Solution, 250mM PVP solution.
(2) Taking FeCl in the step (1)31mL of the solution, 2mL of the PVP solution and 1mL of deionized water are mixed, and the mixture is preheated for 5min by water bath magnetic stirring at 40 ℃.
(3) Slowly dropwise adding 1mL of K into the mixed solution obtained in the step (2)4[Fe(CN)6]The solution reacts for 5min after the dropwise addition is finished, and a large amount of precipitated particles are obtained; then the reaction system is transferred to a water bath with the temperature of 20 ℃ to continue stirring and react for 30 min.
(4) And (4) taking out the reaction solution obtained in the step (3), centrifuging to obtain precipitate particles, washing, and freeze-drying to obtain powder, namely the Prussian blue material.
The scanning electron micrograph of the prussian blue material obtained in this example is shown in fig. 1. It can be seen that the prussian blue particles obtained by the aqueous phase in-situ growth method are of a cubic lamellar structure with the particle size of 50-200 nm.
Example 2
(1) CuCl with the concentration of 5mM is prepared respectively2Solution, 5mM K4[Fe(CN)6]Solution, 250mM PVP solution.
(2) Taking CuCl in the step (1)21mL of the solution, 2mL of the PVP solution and 1mL of deionized water are mixed, and the mixture is preheated for 5min by water bath magnetic stirring at 40 ℃.
(3) Slowly dropwise adding 1mL of K into the mixed solution obtained in the step (2)4[Fe(CN)6]The solution reacts for 5min after the dropwise addition is finished, and a large amount of precipitated particles are obtained; then the reaction system is transferred to a water bath with the temperature of 20 ℃ to continue stirring and react for 30 min.
(4) And (4) taking out the reaction solution in the step (3), centrifuging to obtain precipitate particles, washing, and freeze-drying to obtain powder, namely the Prussian blue copper-based analogue material.
The scanning electron micrograph of the prussian blue copper-based analogue material obtained in this example is shown in fig. 2. It can be seen that the Prussian blue copper-based analogue material obtained by the aqueous phase in-situ growth method is of a cubic lamellar structure with the particle size of 50-200 nm.
Example 3
(1) FeCl was prepared at a concentration of 5mM each3Solution, 20mM K4[Fe(CN)6]Solution, 250mM PVP solution, 2mg/mL cytochrome C solution.
(2) Taking FeCl in the step (1)31mL of the solution, 2mL of the PVP solution, 0.5mL of the cytochrome C solution and 0.5mL of deionized water are mixed, and the mixture is preheated for 5min by water bath magnetic stirring at 40 ℃.
(3) Slowly dropwise adding 1mL of K into the mixed solution obtained in the step (2)4[Fe(CN)6]The solution reacts for 5min after the dropwise addition is finished, and a large amount of precipitated particles are obtained; then the reaction system is transferred to a water bath with the temperature of 20 ℃ to continue stirring and react for 30 min.
(4) And (4) taking out the reaction solution obtained in the step (3), centrifuging to obtain precipitate particles, washing, and freeze-drying to obtain powder, namely the Prussian blue-cytochrome C compound.
The scanning electron microscope image of the prussian blue-cytochrome C complex obtained in this example is shown in fig. 3, and is a cohesive cubic lamellar structure, and it can be seen that in the aqueous phase in-situ embedding process, the enzyme protein participates in the growth process of prussian blue crystal, and affects the morphology of the finally formed prussian blue-cytochrome C complex. The XRD pattern of the obtained prussian blue-cytochrome C complex is shown in fig. 4, and it can be seen that the crystal structure of prussian blue itself is not affected by the doping of protein.
The activity of the prussian blue-cytochrome C complex prepared in this example was measured. The specific operation steps are as follows: 50 μ L of Prussian blue-cytochrome C complex (PB @ Cyt C), equivalent amount of free cytochrome C (Cyt C), and equivalent amount of Prussian Blue (PB) were added to 850 μ L of ABTS (0.28mg/mL) and 100 μ L H, respectively2O2(10mM) acetic acid buffer (50mM, pH 4.5) was rapidly mixed and the dynamic change of the 415nm absorbance within 1min was measured with an ultraviolet spectrophotometer. The enzyme activity is compared by the slope of the light absorption value and the time. The results of the experiment are shown in FIG. 5. As can be seen from FIG. 5, Prussian blue itself has catalytic activity, and reacts with cellsThe activity of the Prussian blue-cytochrome C compound obtained by compounding the pigment C is higher than the sum of the activities (PB + Cyt C) of the same amount of Prussian blue and the same amount of cytochrome C, and the activity is improved by 2.6 times. The improvement of the activity of the Prussian blue-cytochrome C compound is probably related to that heme groups of an active center of the cytochrome C are more exposed after the Prussian blue in-situ embedding, and the cytochrome C is more easily contacted with substrate molecules.
The prussian blue-cytochrome C complex prepared in this example and an equal amount of free cytochrome C were dispersed in 50 μ L of an aqueous solution, 450 μ L of an organic solvent (dimethyl sulfoxide, acetonitrile, acetone) was added thereto and mixed well, and heated in a water bath at 30 ℃ for 30 min. 50 μ L of Prussian blue-cytochrome C complex solution treated by organic solvent and equivalent amount of free cytochrome C solution treated by organic solvent are respectively added with 850 μ L of ABTS (0.28mg/mL) and 100 μ of L H2O2(10mM) acetic acid buffer (50mM, pH 4.5) was rapidly mixed and the dynamic change of the 415nm absorbance within 1min was measured with an ultraviolet spectrophotometer. The enzyme activity is compared by the slope of the light absorption value and the time. The activity retention of the prussian blue-cytochrome C complex (PB @ Cyt C) and free cytochrome C (cytc) after treatment with organic solvents is shown in fig. 6. The result shows that the apparent activity of free cytochrome C is improved by more than 1 time due to the damage of protein structure and the exposure of heme group after the treatment of dimethyl sulfoxide, acetonitrile and acetone, and the activity of Prussian blue immobilized cytochrome C is not obviously changed after the treatment of organic solvent. The organic solvent tolerance of the prussian blue-cytochrome C compound is obviously improved compared with that of the free cytochrome C.
Example 4
(1) FeCl was prepared at a concentration of 5mM each3Solution, 5mM K4[Fe(CN)6]Solution, 250mM PVP solution, 0.5mg/mL horseradish peroxidase solution.
(2) Taking FeCl in the step (1)3Mixing 1mL of solution, 2mL of PVP solution, 0.5mL of horseradish peroxidase solution and 0.5mL of deionized water, and carrying out magnetic stirring and preheating for 5min in a water bath at 60 ℃.
(3) Slowly dropwise adding 1mL of K into the mixed solution obtained in the step (2)4[Fe(CN)6]Solution, reacting after completion of dropwise addition5min, obtaining a large amount of precipitate particles; then the reaction system is transferred to a water bath with the temperature of 4 ℃ to continue stirring and react for 30 min.
(4) And (4) taking out the reaction solution in the step (3), centrifuging to obtain precipitate particles, washing, and freeze-drying to obtain powder, namely the prussian blue-horseradish peroxidase compound.
The activity of the prussian blue-horseradish peroxidase complex prepared in this example was measured. The measurement method was the same as in example 3. The activity measurement shows that the catalytic activity of the prussian blue-horseradish peroxidase is 60 percent of that of equivalent free enzyme after the prussian blue in-situ growth. Compared with cytochrome C, the catalytic activity of horseradish peroxidase is more than 10 times that of Prussian blue, after the Prussian blue is embedded in situ, the loss of the horseradish peroxidase activity can be caused by the increase of mass transfer resistance, and the catalytic activity of the Prussian blue is lower than that of the lost enzyme activity, so that the activity of the compound is not as good as that of free enzyme.
Example 5
(1) CuCl with the concentration of 10mM is prepared respectively2Solution, 40mM K4[Fe(CN)6]Solution, 250mM PVP solution, 0.5mg/mL tyrosinase solution.
(2) Taking CuCl in the step (1)21mL of the solution, 2mL of the PVP solution, 0.5mL of the tyrosinase solution and 0.5mL of deionized water are mixed, and the mixture is preheated for 5min by magnetic stirring in a water bath at 60 ℃.
(3) Slowly dropwise adding 1mL of K into the mixed solution obtained in the step (2)4[Fe(CN)6]The solution reacts for 5min after the dropwise addition is finished, and a large amount of precipitated particles are obtained; the reaction system is transferred to a water bath with the temperature of 4 ℃ to continue stirring and react for 30 min.
(4) And (4) taking out the reaction solution in the step (3), centrifuging to obtain precipitate particles, washing, and freeze-drying. The obtained powder is the Prussian blue copper-based analogue-tyrosinase compound.
The scanning electron micrograph of the prussian blue copper-based analogue-tyrosinase complex obtained in this example is shown in fig. 7, and is a regular cubic lamellar structure.
The activity of the prussian blue copper-based analogue-tyrosinase complex prepared in this example was measured. The specific operation steps are as follows: 50 mu L of Prussian blue copper-based analogue-tyrosinase complex, equivalent amount of free tyrosinase and equivalent amount of Prussian blue copper-based analogue solution are respectively added into 950 mu L L-dopa (10mM) phosphate buffer solution (50mM, pH 6.0) to be rapidly mixed, and an ultraviolet spectrophotometer is used for measuring and detecting the dynamic change of 475nm absorbance within 1 min. The enzyme activity is compared by the slope of the light absorption value and the time. Activity determination shows that the Prussian blue copper-based analogue has catalytic monophenol oxidation activity, the activity of a Prussian blue copper-based analogue-tyrosinase compound obtained by compounding with tyrosinase is higher than the sum of the activities of an equivalent Prussian blue copper-based analogue and an equivalent tyrosinase, and the activity is improved by 1 time.
The Prussian blue copper-based analogue-tyrosinase complex (CuFe @ TYR) prepared in the example and an equal amount of Tyrosinase (TYR) were dispersed in 5mL of phosphate buffer, treated at a high temperature of 80 ℃, and measured by an ultraviolet spectrophotometer to detect the dynamic change of the absorbance value at 475nm within 1 min. The enzyme activity is compared by the slope of the light absorption value and the time. The Prussian blue copper-based analogue-tyrosinase complex and free tyrosinase were treated at a high temperature of 80 ℃ and the activity was retained as shown in FIG. 8. By taking the activity of the free tyrosinase before treatment as 100 percent reference, it can be seen that the activity of the free tyrosinase is reduced to 20 percent after the free tyrosinase is treated at the high temperature of 80 ℃ for 2 hours, the Prussian blue copper-based analogue-tyrosinase compound can still retain 80 percent of activity, and the thermal stability of the tyrosinase is obviously improved after the tyrosinase is immobilized.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for preparing a Prussian blue structure-enzyme complex is characterized by comprising the following steps: the preparation method comprises the following preparation steps:
(1) respectively preparing metal ion solution and K4[Fe(CN)6]Solutions, surfactant solutions and enzyme solutions;
(2) mixing a metal ion solution, a surfactant solution and an enzyme solution, stirring and preheating;
(3) adding K dropwise into the mixed solution obtained in the step (2)4[Fe(CN)6]Reacting the solution to generate precipitate particles, and then cooling to 4-20 ℃ for continuous reaction;
(4) and (4) carrying out centrifugal separation, washing and drying on the precipitate particles obtained in the step (3) to obtain the Prussian blue structure-enzyme complex.
2. The method for preparing a prussian blue structure-enzyme complex according to claim 1, wherein: the metal ions in the step (1) comprise at least one of iron ions, copper ions, nickel ions, zinc ions, cobalt ions, calcium ions and magnesium ions; the concentration of the metal ion solution is 2-50 mM.
3. The method for preparing a prussian blue structure-enzyme complex according to claim 1, wherein: said K in step (1)4[Fe(CN)6]The concentration of the solution is 2-200 mM.
4. The method for preparing a prussian blue structure-enzyme complex according to claim 1, wherein: the surfactant in the step (1) comprises at least one of polyvinylpyrrolidone, sodium dodecyl benzene sulfonate and pluronic.
5. The method for preparing a prussian blue structure-enzyme complex according to claim 1, wherein: the enzyme in the step (1) comprises at least one of cytochrome C, horseradish peroxidase, lipase, carbonic anhydrase, amylase, sucrase, tyrosinase, superoxide dismutase, glucose oxidase, alcohol dehydrogenase, laccase, aldehyde ketone reductase and trypsin; the concentration of the enzyme solution is 0.05-10 mg/mL.
6. The method of claim 1A method for preparing a Prussian blue structure-enzyme complex is characterized by comprising the following steps: the preheating temperature in the step (2) is 30 DEG C～Preheating time is 1-30 min at 60 ℃.
7. The method for preparing a prussian blue structure-enzyme complex according to claim 1, wherein: metal ions in step (2) and K in step (3)4[Fe(CN)6]The amount ratio of the substances (A) is 1:4 to 4: 1.
8. The method for preparing a prussian blue structure-enzyme complex according to claim 1, wherein: and (4) cooling to 4-20 ℃ in the step (3) and continuously reacting for 0.5-24 h.
9. The method for preparing a prussian blue structure-enzyme complex according to claim 1, wherein: the drying mode in the step (4) is freeze drying, vacuum drying or a combination of the freeze drying and the vacuum drying.
10. A prussian blue structure-enzyme complex characterized by: prepared by the method of any one of claims 1 to 9.
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| CN110628755B (en) | 2022-12-16 |
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