CN114479111B

CN114479111B - Novel carrier for immobilizing horseradish peroxidase

Info

Publication number: CN114479111B
Application number: CN202210211725.XA
Authority: CN
Inventors: 汪丹; 黄睿; 王璐莹
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2022-03-04
Filing date: 2022-03-04
Publication date: 2023-03-07
Anticipated expiration: 2042-03-04
Also published as: CN114479111A

Abstract

The invention discloses a novel carrier for immobilizing horseradish peroxidase, which is prepared by the following steps: preparing a cobalt acetate solution and a 2-methylimidazole solution; adding the 2-methylimidazole solution into a cobalt acetate solution, stirring and mixing at normal temperature, and repeatedly washing and precipitating with water and methanol to obtain a primary ZIF-67 solid; dispersing the original ZIF-67 solid into absolute ethyl alcohol, adding nickel ions for etching, washing and drying the product to obtain the horseradish peroxidase immobilized carrier LDH @ ZIF-67. The carrier can efficiently load the horseradish peroxidase, has excellent pH stability and temperature stability, and can still keep higher enzyme activity after being repeatedly used for many times.

Description

Novel carrier for immobilizing horseradish peroxidase

Technical Field

The invention relates to a novel carrier for immobilizing horseradish peroxidase, belonging to the technical field of biology.

Background

The horseradish peroxidase is a glycoprotein complex enzyme containing a dark brown iron protoporphyrin prosthetic group, has biological activity, is widely distributed in the plant world, and has high content in horseradish. It contains abundant heme energy utilization H ₂ O ₂ Oxidizing a variety of organic and inorganic compounds. The horseradish peroxidase has the characteristics of high specific activity, stability, small molecular weight, easy preparation of pure enzyme and the like, and is widely applied.

The horseradish peroxidase can be used as a reagent for clinical diagnosis and immunoassay in medicine, and is also widely applied to degradation treatment of phenol-containing wastewater in industry. However, free enzyme catalysis has certain disadvantages, such as poor acid and alkali resistance, easy inactivation at high temperature, poor reusability and the like, and is easily affected by environment in the using process to lose self activity, and the free enzyme is not easy to separate after being used, so that the application of horseradish peroxidase in large-scale industrial production is greatly limited to a certain extent. Immobilizing enzymes on suitable carriers by enzyme immobilization techniques is an effective way to improve the usability of the enzymes.

Compared with free horseradish peroxidase, the immobilized horseradish peroxidase has the following characteristics: (1) Better catalytic activity, wider use temperature range, good acid and alkali resistance and reusability; (2) The immobilized horseradish peroxidase is easy to separate from a substrate and a product, and the product is easy to purify; (3) The immobilized horseradish peroxidase is more suitable for a multi-enzyme reaction system. At present, the research on the immobilized horseradish peroxidase not only has important value on the continuity and automation of industrial production, but also has great significance on the basic theoretical research in the biochemical fields of constructing a novel enzyme carrier, improving the enzymatic performance and the like.

The carrier property can obviously affect the enzyme loading capacity, the enzyme activity and the enzyme loss rate. At present, many carriers for immobilized enzymes have been reported, including inorganic materials such as silica gel, diatomaceous earth and alumina, and organic materials such as cellulose, chitosan and chitin. However, the existing carrier has the defects of single pore size, poor biocompatibility, large influence on enzyme activity and the like, and the ideal carrier for immobilizing the horseradish peroxidase still needs to be researched and found further.

Disclosure of Invention

The invention provides a novel carrier for immobilizing horseradish peroxidase. Firstly, synthesizing protogenic ZIF-67, dispersing the protogenic ZIF-67 into absolute ethyl alcohol, adding a nickel nitrate solution, and incubating in an ultrasonic bath at normal temperature to obtain a horseradish peroxidase immobilized carrier LDH @ ZIF-67, so that the horseradish peroxidase can be efficiently loaded, and the stability and the reusability of the horseradish peroxidase are improved.

The synthesis method of the novel carrier for immobilizing the horseradish peroxidase, which is provided by the invention, comprises the following steps of:

(1) Preparing a cobalt acetate solution and a 2-methylimidazole solution; and adding the 2-methylimidazole solution into the cobalt acetate solution, stirring and mixing at normal temperature, and repeatedly washing and precipitating with water and methanol to obtain the primary ZIF-67.

(2) Dispersing the original ZIF-67 solid into absolute ethyl alcohol, adding nickel ions for etching, washing and drying the product to obtain the horseradish peroxidase immobilized carrier LDH @ ZIF-67.

Preferably, the method comprises the following steps:

the cobalt acetate solution in the step (1) is prepared from cobalt acetate tetrahydrate and distilled water, and the concentration is 0.1-0.2M.

The 2-methylimidazole solution in the step (1) is prepared from dimethyl imidazole and distilled water, and the concentration is 0.7-0.8M.

In the step (1), the molar ratio of the cobalt acetate to the 2-methylimidazole is 1:45.

in the step (1), the cobalt acetate and the 2-methylimidazole are stirred to react for 6 hours.

The stirring condition in the step (1) is that the mixture is placed in a shaking table at the temperature of 30 ℃.

In the step (2), the washing solvent is water and methanol which are alternately used, and the washing times are 6-10 times.

And (3) dispersing the ZIF-67 solid in absolute ethyl alcohol in the step (2) to prepare a solution with the mass concentration of 1.5 g/L.

The nickel nitrate solution in the step (2) is prepared from nickel nitrate and absolute ethyl alcohol, and the concentration is 12g/L.

The mass ratio of the nickel nitrate to the native ZIF-67 solid in the step (2) is 1:12.

the incubation time in the step (2) is 1 hour.

The incubation condition in the step (2) is that the sample is placed in an ultrasonic bath at room temperature.

In the step (2), the washing solvent is water, and the washing times are 6-10.

In the step (2), the drying temperature is 80-100 ℃, and the drying time is 12-24 h.

Has the advantages that: the novel carrier for immobilizing the horseradish peroxidase can efficiently load the horseradish peroxidase, has excellent pH stability and temperature stability, and can still keep higher enzyme activity after being repeatedly used for many times.

Drawings

FIG. 1 is a scanning electron micrograph and a transmission electron micrograph (500 nm on a scale) of native ZIF-67 in a comparative example;

FIG. 2 is a scanning electron microscope and a transmission electron microscope (500 nm in scale) of the carrier obtained by nickel ion etching for 1 hour in example 1;

FIG. 3 is a scanning electron microscope and a transmission electron microscope (500 nm on a scale) of the carrier obtained by nickel ion etching for 2.5 hours in example 4;

FIG. 4 is an infrared spectrum of the immobilized enzyme in experiment one;

FIG. 5 shows the reusability of immobilized enzymes.

Detailed Description

The technical scheme of the invention is further explained by combining the attached drawings.

The novel carrier for immobilizing the horseradish peroxidase has a mesoporous structure and an LDH structure which are suitable for the molecular size of the horseradish peroxidase, the enzyme load can reach 48.63mg/g, and enzyme protein can be well protected, so that the immobilized enzyme has excellent pH, temperature stability and reusability.

The novel carrier for immobilizing the horseradish peroxidase is prepared by the following method:

(1) Native ZIF-67 solid synthesis: 0.3436mmol of cobalt acetate tetrahydrate and 15.46mmol of 2-methylimidazole are dissolved in 3ml and 20ml of deionized water, respectively. And then, quickly adding the 2-methylimidazole solution into a cobalt acetate tetrahydrate solution, stirring for 6 hours at normal temperature, centrifuging to obtain a ZIF-67 purple precipitate, washing the precipitate with water and methanol for three times, and drying in an oven at 60 ℃ overnight to obtain a primary ZIF-67 solid.

Load evaluation: the enzyme concentration of the supernatant was measured by coomassie brilliant blue method, the enzyme loading was calculated according to the following formula, three times per group of samples and averaged:

in the formula, q _E As solid loading, in milligrams per gram (mg/g);

c is the initial HRP solution concentration in milligrams per milliliter (mg/mL);

v is the initial HRP solution volume in milliliters (mL);

c' is the enzyme concentration in milligrams per milliliter (mg/mL) of the supernatant and wash after immobilization;

v' is the total volume of the supernatant and the washing solution after immobilization, and the unit is milliliter (mL);

m is the mass of the support in grams (g).

(2) Nickel ion etching

Comparative example

Native ZIF-67 solid: weighing 5mg of the original ZIF-67 solid, adding 1ml of enzyme solution with the concentration of 0.5mg/ml, and placing in a shaking table for incubation overnight at normal temperature; centrifuging and washing the supernatant to detect no enzyme activity; the obtained immobilized enzyme is dried in vacuum for 12h at 30 ℃.

Examples 1 to 4

Carriers for different etching times: weighing 30mg of native ZIF-67, dispersing into 20ml of absolute ethanol, adding 5ml of ethanol solution containing 60mg of nickel nitrate, uniformly stirring, placing in an ultrasonic bath for reacting for different times (1 h, 1.5h, 2h and 2.5 h), centrifuging, collecting precipitate, washing with ethanol for multiple times, and drying to obtain a series of immobilized enzyme carriers; weighing 5mg of carrier, adding 1ml of enzyme solution with the concentration of 0.5mg/ml, and placing in a shaking table for incubation at normal temperature overnight; centrifuging and washing the supernatant to detect no enzyme activity; the obtained immobilized enzyme is dried in vacuum for 12h at 30 ℃. As can be seen from table 1, example 1 has a significantly higher loading than the comparative example and other examples; the electron microscope images in the combined pictures of FIGS. 1-3 show that the carrier obtained in example 1 has obvious LDH structure, and the integral shape is complete, and no obvious collapse is seen. The vector obtained in example 1 is optimal in combination with the above results.

TABLE 1 analysis of the load of carriers for different etching times

Experiment-stability detection

The temperature and pH stability of the novel carrier for immobilizing horseradish peroxidase prepared in example 1 were compared. The experimental method is as follows:

(1) Taking 0.05ml of horseradish peroxidase solution or immobilized enzyme dispersion, adding 2.8ml of phosphate-phosphate buffer (pH =3.0, 7.4, 9) and 0.05ml of hydrogen peroxide (8 mmol/L), taking 0.1ml of guaiacol (20 mmol/L) as a substrate, measuring absorbance at the initial reaction time and after 2min at the position of 436nm by using an ultraviolet-visible spectrophotometer at 30 ℃, calculating an absorbance change value delta A, and calculating relative enzyme activity based on the highest enzyme activity.

(2) Taking 0.05ml of horseradish peroxidase solution or immobilized enzyme dispersion, adding 2.8ml of phosphate-phosphate buffer solution (pH = 7.4) and 0.05ml of hydrogen peroxide (8 mmol/L), taking 0.1ml of guaiacol (20 mmol/L) as a substrate, respectively measuring absorbance at the initial reaction and after 2min at the position of 436nm by using an ultraviolet-visible spectrophotometer at the temperature of 30 ℃, 45 ℃ and 60 ℃, representing the enzyme activity by using an absorbance change value delta A, and calculating the relative enzyme activity based on the highest enzyme activity.

Table 2 pH stability of the immobilized enzymes

TABLE 3 temperature stability of the immobilized enzymes

As can be seen from Table 2, the immobilized enzyme shows more excellent relative activity than the free enzyme under both acidic and alkaline conditions; from Table 3, the immobilized enzyme showed superior relative activity at a higher or lower temperature than the optimum temperature of the free enzyme. In conclusion, the novel carrier for immobilizing the horseradish peroxidase has better pH stability and temperature stability.

Experimental two-cycle reusability test

The reusability of the novel carrier for immobilizing horseradish peroxidase prepared in example 1 was compared. The experimental method is as follows:

7.78ml of PBS (pH = 7.4), 0.13ml of guaiacol (20 mmol/L), 0.09ml of hydrogen peroxide (8 mmol/L) and 2ml of the immobilized enzyme dispersion were mixed, and after completion of the reaction, the mixture was centrifuged, and the absorbance change of the supernatant was measured, and the value of this measurement was regarded as the maximum absorbance change and was regarded as the 1 st use. Washing the centrifuged precipitate with PBS buffer solution once to obtain recovered HRP @ LDH @ ZIF-67, testing the absorbance change value of the supernatant again according to the above conditions, and calculating the relative enzyme activity. The procedure was repeated for four times, and the relative enzyme activities were calculated and recorded as 2 nd to 5 th use.

As shown in FIG. 5, the activity of the immobilized enzyme was slightly decreased with the increase in the number of times of use, but was maintained at a high level. After the use for 4 times, the activity of the immobilized enzyme is not obviously reduced and can still be maintained at 85 percent of the initial activity; however, after the use times are increased again, the activity of the immobilized enzyme is reduced to about 65% of the initial activity due to the reasons that the product blocks the carrier pore channels or the horseradish peroxidase is eluted, and the like.

Claims

1. A carrier for immobilizing horseradish peroxidase, which is characterized by being prepared by the following method:

(1) Preparing cobalt acetate solution and 2-methylimidazole solution, mixing, centrifuging, recovering precipitate, washing,

drying to obtain primary ZIF-67 solid; the cobalt acetate solution is prepared from cobalt acetate tetrahydrate and distilled water, and the concentration is 0.1-0.2M; the 2-methylimidazole solution is prepared from dimethyl imidazole and distilled water, and the concentration is 0.7-0.8M; when mixing, the molar ratio of the cobalt acetate to the 2-methylimidazole is 1:45, a first step of;

(2) Preparing a nickel nitrate solution;

(3) Dispersing the primary ZIF-67 solid obtained in the step (1) into absolute ethyl alcohol to prepare a solution with the mass concentration of 1.5 g/L; mixing the obtained solution with the nickel nitrate solution prepared in the step (2), uniformly stirring, and incubating in an ultrasonic bath at normal temperature; centrifuging to obtain precipitate, washing and drying.

2. The carrier for immobilizing horseradish peroxidase according to claim 1, wherein the nickel nitrate solution in the step (2) is prepared from nickel nitrate and absolute ethyl alcohol, and the concentration is 12g/L; in the step (3), during mixing, the mass ratio of the nickel nitrate to the primary ZIF-67 solid is 1:12.

3. the carrier for immobilizing horseradish peroxidase according to claim 1, wherein the incubation time in step (3) is 1 hour.