阅读说明：本技术 一种有机-无机杂化纳米花及其制备方法 (Organic-inorganic hybrid nano flower and preparation method thereof ) 是由 武晓鹂 任珂 王孟武 贺泽文 彭少玲 宁继禅 刘远立 于 2019-09-10 设计创作，主要内容包括：本发明提供了一种有机-无机杂化纳米花及其制备方法,涉及酶固定化技术领域。本发明提供的有机-无机杂化纳米花是以稀土层状化合物为无机载体、以生物酶为有机组分经自组装复合而成的花状固定化酶；所述稀土层状化合物为Ln<Sub>2</Sub>(OH)<Sub>5</Sub>NO<Sub>3</Sub>·nH<Sub>2</Sub>O,其中,Ln为La、Ce、Pr、Nd、Sm、Eu、Gd、Tb、Dy、Ho、Er、Tm、Yb和Y中的一种或几种,n=1.1~2.5；所述生物酶为a-淀粉酶、辣根过氧化物酶和漆酶中的一种或几种。本发明首次以稀土层状化合物作为无机载体负载生物酶并形成花状固定化酶,与游离酶相比,所得固定化酶的稳定性和催化性能更好；且制备方法条件温和、过程简单、耗时较短。(The invention provides an organic-inorganic hybrid nano flower and a preparation method thereof, relating to the technical field of enzyme immobilization. The organic-inorganic hybrid nano flower provided by the invention is a flower-shaped immobilized enzyme which is formed by self-assembling and compounding rare earth lamellar compound serving as an inorganic carrier and biological enzyme serving as an organic component; the rare earth layered compound is Ln 2 (OH) 5 NO 3 ·nH 2 The immobilized enzyme is a flower-like immobilized enzyme which is formed by loading a biological enzyme by taking a rare earth layered compound as an inorganic carrier for the first time, and has better stability and catalytic performance compared with a free enzyme, and the preparation method has the advantages of mild conditions, simple process and shorter time consumption.)

1. An organic-inorganic hybrid nano flower is characterized in that the organic-inorganic hybrid nano flower is a flower-shaped immobilized enzyme which is formed by self-assembling and compounding rare earth layered compounds serving as inorganic carriers and biological enzymes serving as organic components;

the rare earth layered compound is Ln₂(OH)₅NO₃·nH₂O, wherein Ln is one or more of La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Y, and n =1.1 ~ 2.5.5;

the biological enzyme is one or more of a-amylase, horseradish peroxidase and laccase.

2. The method for preparing organic-inorganic hybrid nanoflowers according to claim 1, comprising the steps of:

(1) mixing the rare earth nitrate aqueous solution with biological enzyme to obtain a mixed solution; the rare earth ions in the rare earth nitrate are one or more of La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Y;

(2) sequentially adding ammonium nitrate and ammonia water into the mixed solution, and then aging to obtain an aging solution;

(3) and sequentially centrifuging, washing and drying the aging solution to obtain the organic-inorganic hybrid nanoflower.

3. The method according to claim 2, wherein the molar concentration of the rare earth ions in the aqueous rare earth nitrate solution is 0.005 ~ 1 mol/L.

4. The method according to claim 2, wherein the concentration of the biological enzyme in the rare earth nitrate aqueous solution is 0.001 ~ 3 mg/mL.

5. The method of claim 2, wherein the temperature of the mixing is 15 ~ 60 ℃.

6. The method of claim 2, wherein the molar ratio of ammonium nitrate to rare earth ions is 1 ~ 10: 1.

7. The method according to claim 2, wherein the pH of the mixture obtained after adding aqueous ammonia is 5 ~ 8.

8. The method of claim 2, wherein the aging time is 12 ~ 72 hours.

9. The method according to claim 2, wherein the washing is carried out by washing with water and absolute ethanol in this order.

10. The method of claim 2, wherein the drying temperature is 30 ~ 60 ℃.

Technical Field

The invention relates to the technical field of enzyme immobilization, in particular to an organic-inorganic hybrid nano flower and a preparation method thereof.

Background

Biological enzymes are a very important class of biocatalysts, with high specificity and high catalytic properties. However, industrial application of enzymes is still challenging due to limitations of the enzyme itself or related process parameters, such as poor stability, difficulty in recovery, and high production costs.

Enzyme immobilization is a technique that effectively overcomes these limitations. Compared with free enzyme, immobilized enzyme has the following advantages: can improve the stability of free enzyme, improve the reusability of enzyme and facilitate the separation of products. However, since most of the synthesis of immobilized enzymes is performed under severe conditions, such as high temperature and high pressure or the use of toxic organic solvents, etc., the catalytic performance of immobilized biological enzymes is reduced, which prevents the wide application of these biocatalytic systems. Therefore, how to improve the activity of the immobilized enzyme becomes a problem to be solved urgently.

Disclosure of Invention

In view of the above, the present invention aims to provide an organic-inorganic hybrid nanoflower and a preparation method thereof. According to the invention, a rare earth lamellar compound is used as an inorganic carrier to load biological enzyme and form a flower-shaped immobilized enzyme, and the obtained immobilized enzyme has good stability and higher catalytic performance than corresponding free enzyme; the preparation method provided by the invention has the advantages of mild conditions, simple process and short time consumption.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an organic-inorganic hybrid nano flower, which is a flower-shaped immobilized enzyme formed by self-assembling and compounding rare earth layered compound serving as an inorganic carrier and biological enzyme serving as an organic component;

the rare earth layered compound is Ln₂(OH)₅NO₃·nH₂O, wherein Ln is one or more of La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Y, and n =1.1 ~ 2.5.5;

the biological enzyme is one or more of a-amylase, horseradish peroxidase and laccase.

The invention provides a preparation method of the organic-inorganic hybrid nanometer flower, which comprises the following steps:

(1) mixing the rare earth nitrate aqueous solution with biological enzyme to obtain a mixed solution; the rare earth ions in the rare earth nitrate are one or more of La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Y;

(2) sequentially adding ammonium nitrate and ammonia water into the mixed solution, and then aging to obtain an aging solution;

(3) and sequentially centrifuging, washing and drying the aging solution to obtain the organic-inorganic hybrid nanoflower.

Preferably, the molar concentration of the rare earth ions in the rare earth nitrate aqueous solution is 0.005 ~ 1 mol/L.

Preferably, the concentration of the biological enzyme in the rare earth nitrate aqueous solution is 0.001 ~ 3 mg/mL.

Preferably, the temperature of the mixing is 15 ~ 60 ℃.

Preferably, the molar ratio of ammonium nitrate to rare earth ions is 1 ~ 10: 10.

Preferably, the pH of the mixture obtained after addition of aqueous ammonia is 5 ~ 8.

Preferably, the aging time is 12 ~ 72 h.

Preferably, the washing is carried out by sequentially using water and absolute ethyl alcohol.

Preferably, the temperature of the drying is 30 ~ 60 ℃.

The invention provides an organic-inorganic hybrid nano flower, which is a flower-shaped immobilized enzyme formed by self-assembling and compounding rare earth layered compound serving as an inorganic carrier and biological enzyme serving as an organic component; the rare earth layered compound is Ln₂(OH)₅NO₃·nH₂O, wherein Ln is one or more of La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Y, n =1.1 ~ 2.5.5, and the biological enzyme is one or more of a-amylase, horseradish peroxidase and laccaseThe inorganic carrier of the biological enzyme, the rare earth ions in the rare earth lamellar compound provide empty tracks for coordinating free enzyme, the special lamellar structure provides space for adsorbing the enzyme, and meanwhile, the synergistic effect between the rare earth ions and the biological enzyme can improve the catalytic performance of the immobilized enzyme; in addition, the flower-like morphology has higher specific surface area and surface energy than zero-dimensional nanoparticles and one-dimensional nanotubes or nanorods, and increases mass transfer between immobilized enzyme and reaction substrate, thereby further improving the catalytic performance of the enzyme. Therefore, the invention takes the rare earth lamellar compound as the inorganic carrier to load the biological enzyme and form the flower-shaped immobilized enzyme, compared with the free enzyme, the obtained immobilized enzyme has good stability, is more beneficial to storage and is convenient to be separated from the product in practical application, and the obtained immobilized enzyme has better performance

High catalytic performance.

The invention provides a preparation method of organic-inorganic hybrid nanoflower. The organic-inorganic hybrid nanoflower is prepared by a coprecipitation method at a lower temperature, the conditions are mild, the process is simple, the time consumption is short, and the temperature range of enzyme activity is kept.

Drawings

Fig. 1 is an SEM image of organic-inorganic hybrid nanoflower prepared in example 1, wherein left and right sides of fig. 1 are SEM images at different magnifications;

FIG. 2 is an FT-IR spectrum of the organic-inorganic hybrid nanoflower prepared in example 1;

FIG. 3 is a graph showing a control curve of stability of the organic-inorganic hybrid nanoflower prepared in example 1 against free enzyme, wherein in FIG. 3, the upper curve represents immobilized enzyme and the lower curve represents free enzyme;

FIG. 4 is a graph showing a comparison of catalytic performance between the organic-inorganic hybrid nanoflower prepared in example 1 and free enzyme, wherein in FIG. 4, the upper graph represents immobilized enzyme and the lower graph represents free enzyme;

FIG. 5 is a graph showing a control curve of the stability of the organic-inorganic hybrid nanoflower prepared in example 2 against free enzyme, wherein in FIG. 5, the upper curve represents immobilized enzyme and the lower curve represents free enzyme;

FIG. 6 is a comparative graph of catalytic performance of the organic-inorganic hybrid nanoflower prepared in example 2 and free enzyme, wherein in FIG. 6, the upper curve represents immobilized enzyme and the lower curve represents free enzyme;

FIG. 7 is a graph showing the control of the stability of the organic-inorganic hybrid nanoflower prepared in example 3 with respect to free enzyme, wherein the upper graph in FIG. 7 represents immobilized enzyme and the lower graph represents free enzyme;

FIG. 8 is a graph showing a comparison of catalytic performance between the organic-inorganic hybrid nanoflower prepared in example 3 and free enzyme, wherein in FIG. 8, the upper graph represents immobilized enzyme and the lower graph represents free enzyme;

fig. 9 is a stability control curve of the organic-inorganic hybrid nano flowers prepared in example 4 with free enzyme, in fig. 9, the upper curve represents immobilized enzyme and the lower curve represents free enzyme;

FIG. 10 is a graph showing a comparison of catalytic performance between the organic-inorganic hybrid nanoflower prepared in example 4 and free enzyme, wherein in FIG. 10, the upper graph represents immobilized enzyme and the lower graph represents free enzyme;

FIG. 11 is a graph showing the control of the stability of the organic-inorganic hybrid nanoflower prepared in example 5 against free enzyme, in FIG. 11, the upper graph represents immobilized enzyme and the lower graph represents free enzyme;

FIG. 12 is a graph showing a comparison of catalytic performance between the organic-inorganic hybrid nanoflower prepared in example 5 and free enzyme, wherein in FIG. 12, the upper graph represents immobilized enzyme and the lower graph represents free enzyme;

fig. 13 is a stability control curve of the organic-inorganic hybrid nanoflower prepared in example 6 with free enzyme, in fig. 13, the upper curve represents immobilized enzyme and the lower curve represents free enzyme;

fig. 14 is a graph comparing catalytic performance of the organic-inorganic hybrid nanoflower prepared in example 6 with that of free enzyme, and in fig. 14, the upper curve represents immobilized enzyme and the lower curve represents free enzyme.

Detailed Description

The invention provides an organic-inorganic hybrid nano flower, which is a flower-shaped immobilized enzyme formed by self-assembling and compounding rare earth layered compound serving as an inorganic carrier and biological enzyme serving as an organic component;

the rare earth layered compound is Ln₂(OH)₅NO₃·nH₂O, wherein Ln is one or more of La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Y, and n is =1.1 ~ 2.5.5;

the biological enzyme is one or more of a-amylase, horseradish peroxidase and laccase.

The invention takes the rare earth lamellar compound as the inorganic carrier of the organic biological enzyme for the first time, the rare earth ions in the rare earth lamellar compound provide empty tracks for coordinating free enzyme, the special lamellar structure provides space for adsorbing enzyme, and meanwhile, the synergistic action between the rare earth ions and the biological enzyme can improve the catalytic performance of the immobilized enzyme; in addition, the flower-like morphology has higher specific surface area and surface energy than zero-dimensional nanoparticles and one-dimensional nanotubes or nanorods, and increases mass transfer between immobilized enzyme and reaction substrate, thereby further improving the catalytic performance of the enzyme. The invention takes the rare earth lamellar compound as the inorganic carrier to load the biological enzyme and form the flower-shaped immobilized enzyme, compared with the free enzyme, the obtained immobilized enzyme has good stability and is more beneficial to

The immobilized enzyme is convenient to separate from the product in practical application due to preservation, and the obtained immobilized enzyme has higher catalytic performance.

The invention provides a preparation method of the organic-inorganic hybrid nanometer flower, which comprises the following steps:

(1) mixing the rare earth nitrate aqueous solution with biological enzyme to obtain a mixed solution; the rare earth ions in the rare earth nitrate are one or more of La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Y;

(2) sequentially adding ammonium nitrate and ammonia water into the mixed solution, and then aging to obtain an aging solution;

(3) and sequentially centrifuging, washing and drying the aging solution to obtain the organic-inorganic hybrid nanoflower.

The invention mixes the rare earth nitrate water solution with the biological enzyme to obtain the mixed solution. In the present invention, theThe aqueous solution of rare earth nitrate is preferably obtained by dissolving rare earth nitrate in water. In the present invention, the rare earth nitrate is preferably Ln (NO)₃)₃·6H₂O; ln (NO) as defined above₃)₃·6H₂Ln in O is one or more of La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Y in the invention, the water is preferably deionized water, the molar concentration of rare earth ions (Ln ions) in the rare earth nitrate aqueous solution is preferably 0.005 ~ 1mol/L, more preferably 0.1 ~ 0.5.5 mol/L.

In the invention, the concentration of the biological enzyme in the rare earth nitrate aqueous solution is preferably 0.001 ~ 3mg/mL, more preferably 0.5 ~ 2.5.5 mg/mL, in the invention, the temperature of mixing the rare earth nitrate aqueous solution and the biological enzyme is preferably 15 ~ 60 ℃, more preferably 20 ~ 40 ℃, and most preferably 30 ℃, in the invention, the organic-inorganic hybrid nanoflower is prepared under the condition of lower temperature, the condition is mild, and the temperature range of enzyme activity is kept.

The method comprises the steps of obtaining a mixed solution, adding ammonium nitrate and ammonia water into the mixed solution in sequence, and then aging to obtain an aged solution, wherein the molar ratio of the ammonium nitrate to rare earth ions is preferably 1 ~: 1, more preferably 5 ~: 1, preferably adding the ammonium nitrate under stirring, the stirring speed and time are not particularly required, and the ammonium nitrate can be uniformly mixed in the mixed solution

In the present invention, the aqueous ammonia is used on the one hand to adjust the pH of the solution and on the other hand to provide sufficient OH^-The aging time is preferably 12 ~ 72 h, more preferably 20 ~ 60h, the aging temperature is preferably 15 ~ 60 ℃, more preferably 20 ~ 40 ℃, the aging is favorable for forming the rare earth layered compound, and the self-assembly of the biological enzyme and the rare earth layered compound into the organic-inorganic hybrid nano flower is facilitated₂(OH)₅NO₃·nH₂And (3) uniformly and automatically assembling the O to generate the organic-inorganic hybrid nano flower.

The method has no special requirement on the centrifugation method, and can separate the solid from the liquid of the aging solution by adopting a method well known in the field.

The invention provides a preparation method of the organic-inorganic hybrid nanoflower, which has the advantages of mild conditions, simple process and short time consumption, and is in the temperature range of enzyme activity maintenance.

The following examples are provided to illustrate the organic-inorganic hybrid nanoflower and the preparation method thereof

These are described in detail, but they should not be construed as limiting the scope of the invention.