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Mutant xanthine dehydrogenase and applications thereof

A technology of xanthine oxidase and xanthine, which is applied in the application field of variant xanthine dehydrogenase, degrading samples containing hypoxanthine and xanthine, and can solve problems such as XDH transformation of Rhodobacter capsularis

Active Publication Date: 2018-10-09
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with milk XDH, which is partially enzymatically hydrolyzed by proteases such as trypsin or oxidized to form a disulfide bond, it can be converted into XOD spontaneously, but neither treatment method can convert Rhodobacter capsularis XDH into XOD, that is, Rhodobacter capsularis XDH is a Pure XDH

Method used

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  • Mutant xanthine dehydrogenase and applications thereof
  • Mutant xanthine dehydrogenase and applications thereof
  • Mutant xanthine dehydrogenase and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1, the preparation of variant xanthine dehydrogenase

[0079] 1. Construction of mutant xanthine dehydrogenase recombinant plasmid

[0080] 1. Selection of mutation sites and design of mutation primers

[0081] The fundamental difference between xanthine dehydrogenase and xanthine oxidase lies in the difference in the structure of the electron acceptor region, resulting in different redox environments and thus different selectivities for NAD and oxygen. The present invention selects D358, F270, and S362 sites that contribute greatly to the oxidation-reduction potential of the electron acceptor region for site-directed mutation, and constructs a mutant xanthine dehydrogenase that utilizes oxygen as an electron acceptor to enhance activity.

[0082] The mutation method mediated by PCR primers was used to construct mutants using the recombinant plasmid pTRAN as a template. The sequences of the designed primers were as follows:

[0083] Primer pairs for constru...

Embodiment 2

[0117] Embodiment 2, the characterization of variant xanthine dehydrogenase as xanthine oxidase and xanthine dehydrogenase

[0118] 1. Determination of xanthine oxidase activity

[0119] The xanthine oxidase activity of variant xanthine dehydrogenases can be precisely characterized by two methods.

[0120] The first method is based on xanthine oxidase catalyzing the following reactions (reaction formulas 1 and 2). Uric acid has specific absorption at 295 nm, so the enzyme activity can be characterized by measuring the generation of product uric acid by spectrophotometry.

[0121]

[0122]

[0123] In the present invention, the reaction system for measuring xanthine oxidase enzyme activity is shown in Table 1.

[0124] The 2mL reaction system composition of table 1 xanthine oxidase activity assay

[0125] Numbering

sample name

Mother solution concentration (mM)

Amount added (μL)

Final concentration (mM)

1

Tris-HCl aqueous solution at p...

Embodiment 3

[0181] Example 3. Application of mutant xanthine dehydrogenase as xanthine oxidase in degrading xanthine and hypoxanthine and processing materials containing such substrates

[0182] 1. Method 1

[0183] With reference to the construction method of the xanthine oxidase reaction system in Table 2, construct the following a) to j) 8 groups of reaction systems:

[0184] a) 50mM Tris-HCl buffer solution (pH 8.5), respectively containing EDTA with a final concentration of 1mM, 0.1mM potassium oxonate, 0.1mM xanthine, 0.0033% 4-AAP, 0.2% phenol, 2.68U The variant xanthine dehydrogenase F270L prepared by / ml horseradish peroxidase (HRP) and 1.56mg / L embodiment 1;

[0185] b) 50mM Tris-HCl buffer solution (pH 9.5), respectively containing final concentration of 1mM EDTA, 0.1mM potassium oxonate, 0.1mM xanthine, 0.0033% 4-AAP, 0.2% phenol, 2.68U The variant xanthine dehydrogenase D358Insert prepared by / ml horseradish peroxidase (HRP) and 1.23mg / L embodiment 1;

[0186] c) 50mM Tris-...

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Abstract

The invention discloses a mutant xanthine dehydrogenase and applications thereof. The mutant xanthine dehydrogenase is obtained through any of the following mutations of a wild mutant xanthine dehydrogenase: 1) a polypeptide fragment with 15 amino acids with the length being made up of QDISAVCYKLSKRFE is inserted between D358 and E359 sites of an alpha subunit of the wild mutant xanthine dehydrogenase; 2) an F270L mutation is carried out on the alpha subunit of the wild mutant xanthine dehydrogenase; and 3) an S362A mutation is carried out on the alpha subunit of the wild mutant xanthine dehydrogenase. Xanthine oxidase activity of the mutant xanthine dehydrogenase is significantly enhanced. Compared with the wild mutant xanthine dehydrogenase, the activity of catalytic oxidation of a xanthine substrate is increased by 2125 times, 63 times and 122 times by using oxygen in air as an electron acceptor, and so the production cost can be reduced, and the mutant xanthine dehydrogenase is more suitable for industrial production applications.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a variant xanthine dehydrogenase and its application, in particular to a variant xanthine dehydrogenase with enhanced xanthine oxidase activity through point mutation and its ability to degrade hypoxanthine-containing and application of xanthine samples. Background technique [0002] Xanthine oxidase (XOD for short, EC 1.17.3.2) is a flavoprotein oxidoreductase containing iron-sulfur clusters and molypterin prosthetic groups. It uses oxygen in the air as an electron acceptor to catalyze oxidation Including sp2 hybridized carbon atoms of various heterocyclic molecules such as purine, pteridine and aldehydes, accompanied by the generation of active oxygen species such as hydrogen peroxide and superoxide radicals (Li Lishu, Chen Xianhua, Shao Yebo, Liu Xuan, Xu Ping, " Structure, function and role of xanthine oxidoreductases"). XOD has important commercial application value and is use...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53A62D3/02A62D101/28
CPCA62D3/02A62D2101/28C12N9/0093C12Y117/03002
Inventor 邢新会王成华张翀
Owner TSINGHUA UNIV
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