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Xanthine dehydrogenase intercepting body and application thereof

A technology of xanthine dehydrogenase and recombinant vector, which is applied in the fields of application, oxidoreductase, biochemical equipment and methods, etc., can solve the problems of high price, unseen commercial microbial source XDH, limited yield, etc., and achieve simplification Production process, wide pH range and tolerance, and the effect of reducing production costs

Active Publication Date: 2016-10-05
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, XDH from cattle and chicken sources can be obtained commercially so far, such as the cattle XDH provided by MyBioSource in the United States, but the price is expensive and the output is very limited, and commercialized microbial source XDH has not yet been seen.

Method used

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  • Xanthine dehydrogenase intercepting body and application thereof
  • Xanthine dehydrogenase intercepting body and application thereof
  • Xanthine dehydrogenase intercepting body and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, the acquisition of xanthine dehydrogenase truncated body and coding gene

[0056] 1. Acquisition of Xanthine Dehydrogenase Truncated Body and Its Encoding Gene

[0057] 1. Construction of theoretical calculation model of wild-type xanthine dehydrogenase

[0058] Using the crystal structure of xanthine dehydrogenase from Rhodobacter capsulata (PDB ID: 2W3S) analyzed in the protein crystal structure database as a template, the homology models of the large and small subunits of wild-type xanthine dehydrogenase were constructed respectively; The operation is completed with the help of the Swiss-Model server (http: / / www.swissmodel.expasy.org / interactive), or SPDBV software (http: / / spdbv.vital-it.ch) can be used to build a homology model on the local computer .

[0059] With the help of SPDBV software, import the 2W3S PDB file and the theoretical model PDB file of the large and small subunits of the wild type; with the help of the structural comparison functio...

Embodiment 2

[0093] Embodiment 2, xanthine dehydrogenase truncated body is used as the characterization of xanthine dehydrogenase

[0094] 1. Assay method for xanthine dehydrogenase enzyme activity

[0095] The reaction system for the determination of 2mL enzyme activity is shown in Table 1.

[0096] Table 1 Composition of 2mL reaction system for xanthine dehydrogenase activity assay

[0097]

[0098] Mix the remaining reagents in the 2mL reaction system in Table 1 except the xanthine dehydrogenase aqueous solution and place in a 40°C water bath for 5 minutes, then add 100 μL xanthine dehydrogenase aqueous solution to start the reaction. Record the OD within 3-5min of the reaction while reacting at 40°C 295 Absorbance value change, calculate the rate of change of absorbance with time in the initial linear part of the reaction curve (ΔOD 295 / min), the rate of change of absorbance with time (ΔOD 295 / min) reflects the rate at which xanthine dehydrogenase aqueous solution generates ur...

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PUM

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Abstract

The invention discloses a xanthine dehydrogenase intercepting body and an application thereof. The protein comprises xanthine dehydrogenase intercepting body small subunit, xanthine dehydrogenase intercepting body intermediate subunit, and xanthine dehydrogenase intercepting body large subunit. The affinity of xanthine dehydrogenase intercepting body is increased by 19% by comparing a wild substrate (xanthine), the conversion number is increased by 115%, the catalysis efficiency is increased by 166%, and the temperature toleration is increased by 11 DEG C. The xanthine dehydrogenase intercepting body is in favor of developing the novel application field by combining with other enzymes, and is suitable for sample detection for detecting xanthine and hypoxanthine, the xanthine dehydrogenase intercepting body can be used for detecting inorganic phosphoric acid by combining with PNP enzyme, can be used for detecting adenosine deaminase by combining with PNP, XOD and POD enzymes and detecting 5'-nucleotidase, and is suitable for industrial application. The xanthine dehydrogenase intercepting body is in favor of increasing enzyme catalysis efficiency and reducing cost, and is in favor of industrial application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a xanthine dehydrogenase truncated body and application thereof. Background technique [0002] Xanthine oxidoreductase (XOR for short) is a flavoprotein oxidoreductase containing iron-sulfur clusters and molypterin prosthetic groups. It has xanthine oxidase (XOD for short, EC 1.17. 3.2) and xanthine dehydrogenase (Xanthine dehydrogenase, referred to as XDH, EC1.17.1.4) in two different forms. XOR can not only use naturally occurring substances such as oxygen, NAD, and nitrate as electron acceptors, but also use artificial dyes such as PMS and methylene blue as electron acceptors to catalyze the oxidation of purines, pteridines, and aldehydes. The sp2 hybridized carbon atom of a heterocyclic molecule (Li Lishu, Chen Xianhua, Shao Yebo, Liu Xuan, Xu Ping, "Structure, Function and Action of Xanthine Oxidoreductase". Therefore, XOR has important commercial application value and is used ...

Claims

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Application Information

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