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Preparation method of lactate oxidase bioelectrode

A lactate oxidase and bioelectrode technology, applied in the field of bioengineering, can solve the problems of easy inactivation, difficult purification, and cannot be reused, and achieves the effects of high immobilization efficiency and good biocompatibility

Active Publication Date: 2020-08-11
NANJING UNIV OF TECH
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Problems solved by technology

[0003] As a room temperature enzyme, lactate oxidase has poor stability, is easily inactivated, and cannot be reused. It is mixed into the product after the reaction, making it difficult to purify, making it difficult to be widely used in detection. Therefore, an optimized immobilization method is required. ways to increase its usability
[0004] At present, the commonly used immobilization methods of lactate oxidase include cross-linking method, covalent binding method, microcapsule method, grid method, physical adsorption method, crystallization method, ion binding method, etc., but the application of bioelectrodes has reduced to a certain extent. Enzyme reaction activity and hinder enzyme electron transfer, so it is necessary to develop an optimized immobilization method of lactate oxidase

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  • Preparation method of lactate oxidase bioelectrode
  • Preparation method of lactate oxidase bioelectrode
  • Preparation method of lactate oxidase bioelectrode

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preparation example Construction

[0036] A first aspect of the present invention provides a method for preparing a lactate oxidase bioelectrode, comprising the steps of:

[0037] (1) Electrochemical oxidation treatment of Pb electrode;

[0038] (2) Use immobilized carrier to immobilize the enzyme.

[0039] In one embodiment, the electrochemical oxidation treatment of the Pb electrode comprises the steps of:

[0040] A1: The Pb electrode is placed in a mixed solution of nitric acid and potassium dichromate for electrochemical oxidation treatment;

[0041] A2: Use cyclic voltammetry to scan a circle, and perform electrochemical oxidation treatment.

[0042] In one embodiment, the mass concentration of nitric acid is 8-12%; the mass concentration of potassium dichromate is 1.5-3.5%; preferably, the mass concentration of nitric acid is 9-11%; the mass concentration of potassium dichromate 2.1-2.8%; more preferably, the mass concentration of nitric acid is 10%; the mass concentration of potassium dichromate is 2...

Embodiment 1

[0064] Embodiment 1 of the present invention provides a kind of lactate oxidase, and its preparation method is as follows:

[0065] B1: Extract the entire gene of Yarrowia, and design a pair of primers for PCR amplification of the target gene LOX to obtain the target gene sequence;

[0066] B2: Using the method of homologous recombination, using a one-step cloning kit, 37 ° C water bath for 30 minutes to connect the target gene obtained in step (1) with the starting vector pET-28a(+) to construct an expression vector;

[0067] B3: Use the host strain E.coli BL21(DE3) to construct the genetically engineered strain E.coliBL21(DE3-pET-LOX) containing the LOX gene, insert the constructed LOX strain into an LB shaker flask, and culture it at 37°C for 3.5h. Then 25 μL of 50 mM lactose solution was added, cultured at 20° C. for 24 hours, and the lactate oxidase gene was expressed. Ultrasonic crushing is carried out again, the supernatant obtained by crushing is a crushed supernatant...

Embodiment 2

[0070] Embodiment 2 of the present invention provides bioelectrode material, and its preparation method is as follows:

[0071] (1) Electrochemical oxidation treatment of Pb electrode;

[0072] A1: The Pb electrode is placed in the mixed solution of nitric acid and potassium dichromate for electrochemical oxidation treatment; the mass concentration of nitric acid is 10%; the mass concentration of potassium dichromate is 2.6%; the mass concentration of nitric acid and potassium dichromate The ratio is 0.82;

[0073] A2: Use cyclic voltammetry to scan a circle for electrochemical oxidation treatment;

[0074] (2) Use the immobilized carrier to immobilize the enzyme, and the immobilization method is:

[0075] Mix 1.0 U of enzyme solution with the immobilized carrier, drop it onto the surface of the electrode obtained in step (1), and place it in the dark at 4°C until dry; the volume ratio of the enzyme solution to the immobilized carrier is 1:1;

[0076] The enzyme liquid is t...

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Abstract

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 the preparation method of the lactate oxidase bioelectrode. The preparation method comprises the following steps: carrying out electrochemical oxidation treatment on a Pb electrode; and immobilizing an enzyme byusing an immobilized carrier. The invention provides the preparation method of the lactate oxidase bioelectrode. The method for preparing the bioelectrode through the electrochemical oxidation treatment on the Pb electrode and immobilization on a specific enzyme solution solves problems of low lactate oxidase immobilization rate and poor biocompatibility of an immobilization material. The methodrealizes high efficiency and repeatability of the lactate oxidase bioelectrode, and is an enzyme immobilization technology with high immobilization efficiency and good biocompatibility.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically, the invention provides a preparation method of a lactate oxidase bioelectrode. Background technique [0002] Lactate oxidase is a flavoprotein that uses oxygen as a substrate and FMN and FAD as cofactors to convert lactate directly into pyruvate. FMN and FAD are firmly combined with the enzyme protein, and no external cofactors are needed. The production of pyruvate by lactate oxidase is one of the important means of industrially producing pyruvate. In addition, when there is a large amount of lactic acid in the human body and cannot be metabolized, symptoms of lactic acidosis will appear, so lactate oxidase is of great significance for the detection of lactic acid in the human body. [0003] As a room temperature enzyme, lactate oxidase has poor stability, is easily inactivated, and cannot be reused. It is mixed into the product after the reaction, making it diffi...

Claims

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Application Information

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IPC IPC(8): C12Q1/00C12N11/10C12N11/02C12N11/096C12N15/53C12N9/04C12N15/70G01N27/327
CPCC12Q1/001C12N11/10C12N11/02C12N11/096C12N9/0006C12N15/70G01N27/3271
Inventor 姜岷马婕妤周杰董维亮储振宇金万勤
Owner NANJING UNIV OF TECH
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