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Process for preparing aspergillus flavus urate oxidase

A technology for uric acid oxidase and Aspergillus flavus, which is applied in the field of preparation of Aspergillus flavus uric acid oxidase, can solve the problems of inability to large-scale production, low expression amount, long culture period and the like, and achieves simple expression scheme, simple purification process, and high purification efficiency. Yield-enhancing effect

Active Publication Date: 2007-08-29
CHANGCHUN SHENGJINNUO BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The main problem of the current Aspergillus flavus uric acid oxidase E. coli expression system is that the expression level is not high and cannot be produced on a large scale
The expression level of the product expressed by yeast is up to 13%, the culture period is long and the cost is high

Method used

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  • Process for preparing aspergillus flavus urate oxidase
  • Process for preparing aspergillus flavus urate oxidase
  • Process for preparing aspergillus flavus urate oxidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: 1. Obtaining of Aspergillus flavus urate oxidase gene

[0023] Table 1: Aspergillus flavus urate oxidase cDNA sequence

[0024] 001

061

121

181

241

301

361

421

481

541

601

661

721

781

841

901

gag catatg t ccgcggtTaa agcagcccgc tacggcaaGg acaacgttcg cgtgtacaag

gttcacaaag acgagaagac cggtgtccag acggtgtacg agatgaccgt ctgtgtgctt

ctggagggtg agattgagac cAGttacacc aaggccgaca acagcgtcat tgtcgcaacc

gacAGcatta agaacaccat ttacatcacc gccaagcaga accccgttac tcctcccgag

ctgttcggct ccatcctggg cacacacttc attgagaagt acaaccacat ccatgccgct

cacgtcaaca ttgtctgcca ccgctggacc cggatggaca ttgacggcaa gccacacccG

cacAGcttca tccgcgacag cgaggagaag cggaatgtgc aggtggacgt ggtcgagggc

aagggcatcg atatcaagtc gtctctgtcc ggcctgaccg tgctgaagag caccaactcg

cagttctggg gcttcctgcg tgacgagtac accacactta aggagacctg ggaccgtatc

ctgagcaccg acgtcgatgc cacttggcag tggaagaatt tcagtggact ccaggaggtc

cgctcgcacg tgccta...

Embodiment 2

[0036] Embodiment 2: Aspergillus flavus urate oxidase activity analysis

[0037] The method (Legoux R.et al., J.Biol.Chem., 1992,267, (12), 8565-8570) analysis enzyme activity of the Aspergillus flavus uric acid oxidase after the purification, 3ml buffer system (TEA buffer, 7.5g triethanolamine; 0.38g EDTA) containing 0.18μmol uric acid, pH 8.9, 30°C incubation, detection wavelength 292nm, the amount of enzyme required to oxidize 1μmol uric acid to allantoic acid per minute is an enzyme activity unit. Add 1 mg / mL enzyme 1 μL, 2 μL, 5 μL, 10 μL, measure the absorbance value after the reaction, the results are shown in Figure 6.

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Abstract

The invention relates to the biotechnology field, in particular to a method for preparation of aspergillus flavus uricoxidase, which contains the following steps: (1), obtaining the gene of aspergillus flavus uricoxidase; (2), constructing the expression carrier and converting colibacillus work bacterium; (3), sifting, planting and induced expressing the positive clone; (4), separating and purifying the fused protein; (5), enzyme-cutting and purifying the fused protein; and the related expression carrier plasmid being to pET series carrier plasmid. It is fit to mass preparation, the expression scheme simple while expression yield high, the purifying craft simple while yield ratio high, the expression product soluble and purified directly by chromatography, and the purifying yield increased greatly.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing uric acid oxidase from Aspergillus flavus. Background technique [0002] Uric acid oxidase (uric acid oxidoreductase, EC 1.7.3.3, Urate Oxidase, Uricase) is a protease that can oxidize uric acid to allantoin. The product xanthine produced by the decomposition of purine nucleotides in the body produces uric acid under the action of xanthine oxidase, and uric acid is further decomposed into CO under the action of urate oxidase, allantoinase, allantoinase, and urease 2 and NH 3 . Different species have different degrees of uric acid decomposition. Most of the hypoxanthine and guanine produced in mammals are converted into inosine monophosphate and guanosine monophosphate by phosphoribosyltransferase, but 10% still have to be decomposed, excluding in vitro. In the human body, uric acid is the final product of purine degradation and is excreted with urine. [0...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N9/04C12N15/53C12N15/63C12N15/70C12P21/02
Inventor 刘国安方永强张玉良
Owner CHANGCHUN SHENGJINNUO BIOLOGICAL PHARMA
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