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Alkaline urate oxidase and applications thereof to detection kit and reduction of uric acid in foods

A uric acid oxidase and detection kit technology, applied in the biological field, can solve the problems of narrow pH applicable range, narrow temperature applicable range, poor storage stability, etc., to reduce enzyme usage, improve work efficiency, and high substrate affinity. sexual effect

Active Publication Date: 2019-04-02
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, most urate oxidases can only maintain high activity in a weak alkaline environment, and there are problems such as narrow pH range, narrow temperature range, poor storage stability in alkaline environment, and rapid loss of enzyme activity.

Method used

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  • Alkaline urate oxidase and applications thereof to detection kit and reduction of uric acid in foods
  • Alkaline urate oxidase and applications thereof to detection kit and reduction of uric acid in foods
  • Alkaline urate oxidase and applications thereof to detection kit and reduction of uric acid in foods

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Experimental program
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Embodiment 1

[0074] Acquisition of urate oxidase and its coding gene

[0075] 1. Acquisition of urate oxidase-encoding gene and construction of recombinant plasmid pSEUOX

[0076] 1. Extraction of yeast genomic DNA

[0077] Add 5ml YED liquid medium, the formula is: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, add to Arxula adeninivorans freeze-dried powder, culture for 12h; draw a line on the solid medium plate, the formula is: Yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agar powder 15g / L, and then cultured upside down in a 24°C incubator for 24h; pick a single colony and inoculate it in 5ml liquid medium, and incubate at 24°C, 180r Cultivate overnight in a shaker / min; use the Yeast Genomic DNA Extraction Kit, OMEGA Company, product code D3370-01, to extract the total genomic DNA.

[0078] 2. Design and synthesize primers

[0079] According to the urate oxidase gene data in Arxula adeninivorans published in NCBI, the following primers were designed to amplify the urate oxi...

Embodiment 2

[0105] Characterization of urate oxidase

[0106] 1. Determination of urate oxidase enzyme activity

[0107] Based on the following reaction catalyzed by urate oxidase (reaction formula 1), there is a strict stoichiometric relationship between the uric acid participating in the reaction and the catalytic process of the enzyme, so the enzyme activity can be accurately characterized by the reaction rate of the reactant uric acid. Because uric acid has specific absorption at 295nm, the present invention measures the enzyme activity by spectrophotometry.

[0108]

[0109] The reaction system for the determination of 2 mL urate oxidase enzyme activity is shown in Table 1.

[0110] The 2mL reaction system composition of table 1 urate oxidase enzyme activity assay

[0111]

[0112] Mix the remaining reagents in the 2 mL reaction system in Table 1 except the urate oxidase aqueous solution, and then add 100 μL urate oxidase solution to start the reaction. Record the OD within ...

Embodiment 3

[0138] Application of uricase oxidase in degrading uric acid and processing materials containing such substrates.

[0139] With reference to the construction method of the urate oxidase reaction system in Table 1, the following reaction system is constructed:

[0140] 50mmol / L Tris-HCl buffer solution (pH 8.5), 1mmol / L EDTA, the urate oxidase prepared by 0.1mmol / L uric acid and embodiment 1;

[0141] Add and mix all the reagents in the reaction system except urate oxidase, place in a water bath at 40°C for 5 minutes, then add urate oxidase to start the reaction, record the change of absorbance at 295nm within 3 minutes while reacting, and draw the absorbance ( ΔOD 295 ) with time, and calculate the rate of change of absorbance with time in the initial linear part of the reaction curve (ΔOD 295 / min), and its degradation was detected by the change of reactant uric acid. Experimental results such as Figure 5 shown.

[0142] Figure 5 It was shown that urate oxidase could ...

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Abstract

The invention relates to an alkaline urate oxidase and applications thereof to a detection kit and the reduction of uric acid in foods. The nucleotide sequence is as shown in the sequence SEQ ID No.1,and the amino acid sequence is as shown in the sequence SEQ ID No.2. A yeast urate oxidase is recombined into the highly-activity alkaline urate oxidase which is capable of degrading, measuring and detecting samples in an alkaline environment, has a wider alkaline measurement range and higher catalytic efficiency than previously-reported enzymes, and is advantageous for improving production efficiency, reducing costs and facilitating industrial application better. According to the kit which utilizes the alkaline urate oxidase and detects uric acid content by a colorimetric method, the kit hasstrong specificity, high sensitivity and simple method. The alkaline urate oxidase is used to develop low-purine foods, and has high application value in low-purine foods.

Description

technical field [0001] The invention relates to the fields of biotechnology and food, in particular to an alkaline urate oxidase and its application in a detection kit and reducing uric acid in food. Background technique [0002] Urate oxidase, Urate oxidase, referred to as UOX; EC.1.7.3.3, is an enzyme in the purine metabolic pathway in organisms, which catalyzes the decomposition of uric acid, water and oxygen into allantoin and hydrogen peroxide. UOX has important commercial application value. UOX has strong specificity and high catalytic efficiency. It is used for clinical detection of serum uric acid, uric acid sensor detection based on immobilized urate oxidase, uric acid lowering treatment for gout patients, and thrombolysis for cerebral infarction. Treatment, etc. (Kai Lei, Yue Ke, Ma Xiaohang. Research and Application of Urate Oxidase. Science and Technology Bulletin, 2008(02):183-187). [0003] Uric acid is the final product of purine metabolism in the human body ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/06C12Q1/62C12Q1/28C12Q1/26A23L5/20
CPCA23L5/25C12N9/0048C12Q1/26C12Q1/28C12Q1/62C12Y107/03003C12Q2326/50C12Q2326/96
Inventor 王成华张冉高世珏孙毓刘小玲
Owner GUANGXI UNIV
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