阅读说明：本技术 一种乳酸氧化酶生物电极的制备方法 (Preparation method of lactate oxidase bioelectrode ) 是由 姜岷 马婕妤 周杰 董维亮 储振宇 金万勤 于 2020-06-09 设计创作，主要内容包括：本发明属于生物工程技术领域,更具体地,本发明提供一种乳酸氧化酶生物电极的制备方法。本发明的第一方面提供一种乳酸氧化酶生物电极的制备方法,包括步骤：Pb电极的电化学氧化处理；利用固定化载体进对酶进行固定。本发明提供了一种乳酸氧化酶生物电极的制备方法,通过对Pb电极的电化学氧化处理以及对特定酶液的固定制备生物电极的方法解决了乳酸氧化酶固定化率低以及固定化材料生物相容性差的问题；实现了乳酸氧化酶生物电极的高效性和可重复性,是一种固定化效率高、生物相容性好的酶固定化技术。(The invention belongs to the technical field of bioengineering, and particularly provides a preparation method of a lactate oxidase bioelectrode. The first aspect of the invention provides a preparation method of a lactate oxidase bioelectrode, which comprises the following steps: electrochemical oxidation treatment of the Pb electrode; the enzyme is immobilized by using an immobilization carrier. The invention provides a preparation method of a lactate oxidase bioelectrode, which solves the problems of low immobilization rate of lactate oxidase and poor biocompatibility of immobilized materials by electrochemical oxidation treatment of a Pb electrode and a method for preparing the bioelectrode by immobilizing specific enzyme solution; realizes the high efficiency and repeatability of the lactate oxidase bioelectrode, and is an enzyme immobilization technology with high immobilization efficiency and good biocompatibility.)

1. A preparation method of a lactate oxidase bioelectrode is characterized by comprising the following steps:

(1) electrochemical oxidation treatment of the Pb electrode;

(2) the enzyme is immobilized by using an immobilization carrier.

2. The method for preparing lactate oxidase bioelectrode according to claim 1, wherein the electrochemical oxidation treatment of the Pb electrode comprises the steps of:

a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment;

a2: and scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment.

3. The method for preparing the lactate oxidase bioelectrode according to claim 2, wherein the mass concentration of the nitric acid is 8-12%; the mass concentration of the potassium dichromate is 1.5-3.5%.

4. The method for preparing lactate oxidase bioelectrode according to any of claims 1-3, wherein the immobilized carrier is selected from any one or more of glutaraldehyde, chitosan, polylysine, and perfluorosulfonic acid; preferably chitosan.

5. The method for preparing lactate oxidase bioelectrode according to claim 4, wherein the chitosan is stored at 3-6 ℃ at a volume concentration of 1.00-1.50%.

6. The method for preparing lactate oxidase bioelectrode according to claim 4, wherein the immobilization of the enzyme by the immobilization carrier is performed by:

c1: dropwise adding enzyme solution to the surface of the electrode obtained in the step (1), and placing the electrode in a dark place at 3-6 ℃ until the electrode is dried;

c2: and D, dropwise adding the immobilized carrier, covering and titrating the immobilized carrier on the surface of the electrode obtained in the step C1, and placing the immobilized carrier in the dark at the temperature of 3-6 ℃ until the immobilized carrier is dried.

7. The method for preparing lactate oxidase bioelectrode according to claim 4, wherein the volume ratio of the enzyme solution to the immobilized carrier is 1: (0.8 to 1.2).

8. The method for producing lactate oxidase bioelectrode according to any of claims 1 to 3, wherein the method for producing the enzyme solution comprises:

b1: carrying out whole-gene extraction on yarrowia, and designing a pair of primers to carry out PCR amplification on a target gene LOX to obtain a target gene sequence;

b2: connecting the target gene obtained in the step (1) and a starting vector pET-28a (+) by using a homologous recombination method and a one-step cloning kit in water bath at 37 ℃ for 30min to construct an expression vector;

b3: constructing a gene engineering bacterium E.coliBL21 containing LOX genes by using a host bacterium E.coliBL21, inoculating the constructed LOX strain into an LB shake flask, culturing at 34-39 ℃ for 2.5-4.5 h, adding 50mM lactose solution, culturing at 18-22 ℃ for 22-26 h, and expressing a lactate oxidase gene;

b4: and D, purifying the substance obtained in the step B3 by using a nickel ion column, and concentrating to obtain a LOX protease liquid.

9. A bioelectrode obtained by the method for producing a lactate oxidase bioelectrode according to any one of claims 1 to 8.

10. A lactate oxidase biosensor comprising the bioelectrode of claim 9.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly provides a preparation method of a lactate oxidase bioelectrode.

Background

Lactate oxidase is a flavoprotein, uses oxygen as a substrate, uses FMN and FAD as cofactors, and can directly convert lactic acid into pyruvic acid. FMN and FAD are firmly combined with enzyme protein, and no cofactor is required to be added from an external source. The production of pyruvic acid by using lactate oxidase is one of the important means for the industrial production of pyruvic acid. In addition, lactic acid oxidase is of great significance for the detection of lactic acid in humans because lactic acid acidosis symptoms occur when large amounts of lactic acid are present in humans and cannot be metabolized.

Lactate oxidase, as a normal temperature enzyme, has poor stability, is easy to inactivate and can not be reused, and the product is mixed after the reaction, so that the purification is difficult, and the wider application in detection is difficult, therefore, an optimized immobilization mode is needed to improve the availability of the lactate oxidase.

At present, the commonly used immobilization methods of the lactate oxidase include a cross-linking method, a covalent bonding method, a microencapsulation method, a grid method, a physical adsorption method, a crystallization method, an ion bonding method and the like, but the enzyme reaction activity is reduced to a certain extent and the enzyme electron transfer is blocked in the application of a bioelectrode, so that the development of an optimized immobilization method of the lactate oxidase is necessary.

Disclosure of Invention

In order to solve the above technical problems, a first aspect of the present invention provides a method for preparing a lactate oxidase bioelectrode, comprising the steps of:

(1) electrochemical oxidation treatment of the Pb electrode;

(2) the enzyme is immobilized by using an immobilization carrier.

As a preferable aspect of the present invention, the electrochemical oxidation treatment of the Pb electrode includes the steps of:

a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment;

a2: and scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment.

As a preferred technical scheme, the mass concentration of the nitric acid is 8-12%; the mass concentration of the potassium dichromate is 1.5-3.5%.

As a preferred technical scheme of the invention, the immobilized carrier is selected from any one or combination of more of glutaraldehyde, chitosan, polylysine and perfluorosulfonic acid (Nafion); preferably chitosan.

As a preferred technical scheme, the volume concentration of the chitosan is 0.25-1.50%, and the chitosan is stored at the temperature of 3-6 ℃.

As a preferred technical scheme of the invention, the immobilization mode of immobilizing the enzyme by utilizing the immobilization carrier comprises the following steps:

c1: dropwise adding enzyme solution to the surface of the electrode obtained in the step (1), and placing the electrode in a dark place at 3-6 ℃ until the electrode is dried;

c2: and D, dropwise adding the immobilized carrier, covering and titrating the immobilized carrier on the surface of the electrode obtained in the step C1, and placing the immobilized carrier in the dark at the temperature of 3-6 ℃ until the immobilized carrier is dried.

As a preferred technical scheme of the invention, the volume ratio of the enzyme solution to the immobilized carrier is 1: (0.8 to 1.2).

As a preferable technical scheme of the invention, the preparation method of the enzyme solution comprises the following steps:

b1: carrying out whole-gene extraction on yarrowia, and designing a pair of primers to carry out PCR amplification on a target gene LOX to obtain a target gene sequence;

b2: connecting the target gene obtained in the step (1) and a starting vector pET-28a (+) by using a homologous recombination method and a one-step cloning kit in water bath at 37 ℃ for 30min to construct an expression vector;

b3: constructing a gene engineering bacterium E.coli BL21 containing LOX gene by using a host bacterium E.coli BL21, inoculating the constructed LOX strain into an LB shake flask, culturing at 34-39 ℃ for 2.5-4.5 h, adding 50mM lactose solution, culturing at 18-22 ℃ for 22-26 h, and expressing the lactate oxidase gene;

b4: and D, purifying the substance obtained in the step B3 by using a nickel ion column, and concentrating to obtain a LOX protease liquid.

The second aspect of the present invention provides a bioelectrode prepared according to the method for preparing a lactate oxidase bioelectrode.

In a third aspect, the present invention provides a lactate oxidase biosensor comprising the bioelectrode.

Has the advantages that: the invention provides a preparation method of a lactate oxidase bioelectrode, which solves the problems of low immobilization rate of lactate oxidase and poor biocompatibility of immobilized materials by electrochemical oxidation treatment of a Pb electrode and a method for preparing the bioelectrode by immobilizing specific enzyme solution; realizes the high efficiency and repeatability of the lactate oxidase bioelectrode, and is an enzyme immobilization technology with high immobilization efficiency and good biocompatibility.

Drawings

FIG. 1 is an SDS-PAGE pattern of lactate oxidase;

FIG. 2 is a graph showing the influence of different immobilization carriers on the enzyme activity of immobilized lactate oxidase;

FIG. 3 is a graph showing the comparison of immobilization rates of different immobilization carriers;

FIG. 4 is a graph showing comparative immobilization curves for different curing modes;

FIG. 5: comparing the tolerance curves of the enzyme at different temperatures and pH values;

FIG. 6 is a graph showing the reproducibility of lactate oxidase electrodes.

Detailed Description

The technical features of the technical solutions provided by the present invention are further clearly and completely described below with reference to the specific embodiments, and the scope of protection is not limited thereto.

The words "preferred", "more preferred", and the like, in the present invention refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range from "1 to 10" should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10. Exemplary subranges of the range 1 to 10 include, but are not limited to, 1 to 6.1, 3.5 to 7.8, 5.5 to 10, and the like.

The invention mainly provides a preparation method of the lactate oxidase bioelectrode, which is used for solving the problems of low lactate oxidase bioelectrode fixing efficiency and poor sensitivity caused by poor stability, easy inactivation and incapability of being reused of lactate oxidase and improving the high efficiency and reusability of the lactate oxidase bioelectrode.

The first aspect of the invention provides a preparation method of a lactate oxidase bioelectrode, which comprises the following steps:

(1) electrochemical oxidation treatment of the Pb electrode;

(2) the enzyme is immobilized by using an immobilization carrier.

In one embodiment, the electrochemical oxidation treatment of the Pb electrode includes the steps of:

a1: placing a Pb electrode in a mixed solution of nitric acid and potassium dichromate, and performing electrochemical oxidation treatment;

a2: and scanning for one circle by cyclic voltammetry to perform electrochemical oxidation treatment.

In one embodiment, the mass concentration of the nitric acid is 8-12%; the mass concentration of the potassium dichromate is 1.5-3.5%; preferably, the mass concentration of the nitric acid is 9-11%; the mass concentration of the potassium dichromate is 2.1-2.8%; more preferably, the mass concentration of nitric acid is 10%; the mass concentration of potassium dichromate is 2.6%.

In one embodiment, the mass ratio of the nitric acid to the potassium dichromate is 0.6 to 1; preferably 0.7 to 0.9; more preferably 0.82.

In one embodiment, the immobilization support is selected from the group consisting of glutaraldehyde, chitosan, polylysine, a combination of any one or more of perfluorosulfonic acids (Nafion); preferably chitosan; further preferably, the volume concentration of the chitosan is 0.25-1.5%; further preferably, the volume concentration of the chitosan is 0.5-1.25%; further preferably, the volume concentration of the chitosan is 0.75-1.25%; further preferably, the volume concentration of the chitosan is 1.0-1.25%; more preferably, the chitosan is stored at 3-6 ℃.

In one embodiment, the immobilization of the enzyme by the immobilization carrier is performed by:

c1: dropwise adding the enzyme solution to the surface of the electrode obtained in the step (1), and placing the electrode in a dark place at the temperature of 3-6 ℃ until the electrode is dried;

c2: and D, covering and dropwise adding the immobilized carrier on the surface of the electrode obtained in the step C1, and placing the electrode at 3-6 ℃ in a dark place until the electrode is dried.

In another embodiment, the immobilization of the enzyme by the immobilization carrier is performed by: and (3) mixing the enzyme solution with an immobilized carrier, dropwise adding the mixture to the surface of the electrode obtained in the step (1), and placing the mixture at 4 ℃ in the dark until the mixture is dried.

In one embodiment, the volume ratio of the enzyme solution to the immobilization carrier is 1: (0.8 to 1.2); preferably, the volume ratio of the enzyme solution to the immobilization carrier is 1: (0.9 to 1.1); more preferably, the volume ratio of the enzyme solution to the immobilization carrier is 1: 1.

in one embodiment, the amount of the enzyme solution in the step C1 is 0.8-1.2U; preferably 0.9-1.1U; more preferably 1.0U.

In one embodiment, the method of preparing an enzyme solution comprises:

b1: carrying out whole-gene extraction on yarrowia, and designing a pair of primers to carry out PCR amplification on a target gene LOX to obtain a target gene sequence;

b2: connecting the target gene obtained in the step (1) and a starting vector pET-28a (+) by using a homologous recombination method and a one-step cloning kit in water bath at 37 ℃ for 30min to construct an expression vector;

b3: constructing a gene engineering bacterium E.coliBL21(DE3-pET-LOX) containing LOX genes by using a host bacterium E.colibl21 (DE3), inoculating the constructed LOX strain into an LB shake flask, culturing for 2.5-4.5 h at 34-39 ℃, adding 50mM lactose solution, culturing for 22-26 h at 18-22 ℃, and expressing a lactate oxidase gene;

b4: and D, purifying the substance obtained in the step B3 by using a nickel ion column, and concentrating to obtain a LOX protease liquid.

Preferably, the preparation method of the enzyme solution comprises:

b1: carrying out whole-gene extraction on yarrowia, and designing a pair of primers to carry out PCR amplification on a target gene LOX to obtain a target gene sequence;

b2: connecting the target gene obtained in the step (1) and a starting vector pET-28a (+) by using a homologous recombination method and a one-step cloning kit in water bath at 37 ℃ for 30min to construct an expression vector;

b3: constructing a gene engineering bacterium E.coliBL21 (recorded as DE3-pET-LOX) containing LOX gene by using a host bacterium E.colibl21 (recorded as DE3), inoculating the constructed LOX strain into an LB shake flask, culturing for 3.5h at 37 ℃, adding 25 mu L of 50mM lactose solution, and culturing for 24h at 20 ℃ to express a lactate oxidase gene;

b4: and D, adsorbing the substance obtained in the step B3 by using a nickel ion column, and concentrating by using an ultrafiltration tube to obtain the LOX protease liquid.

The second aspect of the present invention provides a bioelectrode prepared according to the method for preparing a lactate oxidase bioelectrode.

In a third aspect, the present invention provides a lactate oxidase biosensor comprising the bioelectrode.