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A kind of alkaline xanthine dehydrogenase and its application in detection kit

A technology of xanthine dehydrogenase and xanthine, applied in the application field of Acinetobacter baumannii alkaline xanthine dehydrogenase, xanthine/hypoxanthine content, can solve the problem of high price, limited output, low activity, etc. problems, to achieve the effects of sensitive degradation and detection, high substrate affinity, and high catalytic efficiency

Active Publication Date: 2020-02-21
WUXI TMAXTREE BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, eukaryotic XDH derived from cattle and chickens can be obtained commercially, such as the bovine XDH provided by MyBioSource in the United States, but it is expensive, with limited yield and low activity, and no commercial microbial source XDH has been seen.

Method used

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  • A kind of alkaline xanthine dehydrogenase and its application in detection kit
  • A kind of alkaline xanthine dehydrogenase and its application in detection kit
  • A kind of alkaline xanthine dehydrogenase and its application in detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1, the acquisition of xanthine dehydrogenase and coding gene

[0103] 1. Acquisition of xanthine dehydrogenase coding gene and construction of recombinant plasmid pTRAN

[0104] 1. Extraction of Acinetobacter baumannii genomic DNA

[0105] Use an inoculation loop to pick a ring of Acinetobacter baumannii (purchased from the China Industrial Microbiology Culture Collection Center (CICC), the strain number is CICC 10254) preserved on the agar slant, and put it on a solid medium (formulation: peptone 5.0 g, beef extract 3.0g, NaCl 5.0g, agar 15.0g, distilled water 1.0L, pH7.0) streaked on the plate, and then inverted in a 37°C incubator for static culture for 16h; pick a single colony and inoculate it in 5ml liquid culture base (formula: 5.0 g of peptone, 3.0 g of beef extract, 5.0 g of NaCl, 1.0 L of distilled water, pH 7.0), cultivated overnight at 37 ° C and 220 rpm in a shaker; the bacterial genomic DNA extraction kit (OMEGA Corporation) , product code D33...

Embodiment 2

[0140] Embodiment 2, AbXDH as the characterization of xanthine dehydrogenase

[0141] 1. Assay method for xanthine dehydrogenase enzyme activity

[0142] Based on the following reactions catalyzed by xanthine dehydrogenase (reaction formula 1 and reaction formula 2), there is a strict stoichiometric relationship between the generation of NADH and the catalytic process of the enzyme, so the enzyme activity can be accurately characterized by the generation rate of the product NADH. Because NADH has specific absorption at 340nm, the present invention measures the enzyme activity by spectrophotometry.

[0143] The reaction system for the determination of 2mL xanthine dehydrogenase enzyme activity is shown in Table 1.

[0144] The 2mL reaction system composition of table 1 xanthine dehydrogenase activity assay

[0145]

[0146] 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 ...

Embodiment 3

[0171] Example 3, Application of Xanthine Dehydrogenase in Degrading Xanthine and Hypoxanthine and Processing Materials Containing Such Substrates

[0172] With reference to the construction method of the xanthine dehydrogenase reaction system in Table 1, construct the following a) to c) three groups of reaction systems:

[0173] a) 50mM Tris-HCl buffer solution (pH 7.5), containing EDTA at a final concentration of 1mM, 0.1mM potassium oxonate, 0.1mM nicotinamide adenine dinucleotide (NAD+), and 0.1mM xanthine And the xanthine dehydrogenase prepared by 0.216mg / L embodiment 1;

[0174] b) 50mM Tris-HCl buffer solution (pH 8.5), respectively containing EDTA with a final concentration of 1mM, 0.1mM potassium oxonate, 0.1mM nicotinamide adenine dinucleotide (NAD+), and 0.1mM xanthine , and the xanthine dehydrogenase prepared by 0.216mg / L embodiment 1;

[0175] c) 50mM Tris-HCl buffer solution (pH 8.5), containing EDTA at a final concentration of 1mM, 0.1mM potassium oxonate, 0.1...

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Abstract

The invention discloses alkaline xanthine dehydrogenase and application thereof in detection kits. A protein disclosed by the invention comprises a xanthine dehydrogenase large subunit and xanthine dehydrogenase small subunit. The alkaline xanthine dehydrogenase provided by the invention is reported for the first time, is acinetobacter baumannii xanthine dehydrogenase confirmed by experiments and is applicable to samples at alkali determination conditions. Compared with existing reports, the alkaline xanthine dehydrogenase has relatively high substrate affinity and the highest catalytic efficiency, is capable of sensitively degrading and detecting hypoxanthine / xanthine, PNP enzyme and 5'-nucleotidase and can be combined with PNP enzyme for detecting inorganic phosphate and adenosine deaminase, so that the catalytic efficiency is improved, the cost is lowered, and the industrial application process is benefited. The invention further provides a method for detecting the content of xanthine / hypoxanthine in samples including serum and urine by virtue the xanthine dehydrogenase. The invention further provides a detection kit for detecting the content of xanthine / hypoxanthine in the samples. The detection method and the detection kit are not influenced by dissolved oxygen in the samples; compared with the prior art, the alkaline xanthine dehydrogenase has the advantages that the detection range is relatively wide, the sensitivity is relatively high, and the detection method and the detection kit are suitable for popularization and application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a xanthine dehydrogenase and its application in the detection of hypoxanthine and xanthine-containing samples, in particular to an Acinetobacter baumannii alkaline xanthine dehydrogenase and its It is used in the degradation of hypoxanthine and xanthine and in the detection of xanthine / hypoxanthine content in samples such as blood and urine. 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, EC1.17.3 .2) and xanthine dehydrogenase (Xanthine dehydrogenase, referred to as XDH, EC 1.17.1.4) 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 v...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N1/15C12N1/19C12N1/21C12Q1/26
CPCC12N9/0093C12Q1/26C12Y117/01004C12Y117/03002
Inventor 邢新会王成华张翀苏楠
Owner WUXI TMAXTREE BIOTECHNOLOGY CO LTD
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