Heat-resisting urate oxidase gene of microbacterium category bacteria and application of same

A uric acid oxidase and heat-resistant technology, which is applied in genetic engineering, plant gene improvement, enzymes, etc., can solve the problems of unfavorable storage and use of enzymatic uric acid detection kits, short half-life, poor thermal stability, etc., and achieve excellent thermal stability. The effect of stability and catalytic properties

Inactive Publication Date: 2011-10-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Although the application of the existing urate oxidase in the above-mentioned fields has been reported, in actual production and application, due to the generally poor thermal stability of the existing urate oxidase, it can be used in the treatment of acute hyperuricemia and gout. If the half-life of the enzyme is too short in chronic hyperuricemia, it will also have adverse results on the storage and use of the enzymatic uric acid detection kit, thus affecting the effect of the enzyme application

Method used

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  • Heat-resisting urate oxidase gene of microbacterium category bacteria and application of same
  • Heat-resisting urate oxidase gene of microbacterium category bacteria and application of same
  • Heat-resisting urate oxidase gene of microbacterium category bacteria and application of same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the separation of producing thermostable urate oxidase bacterial strain:

[0034] Take 2.0 grams of soil (taken from the surrounding areas of Hangzhou) and inoculate it into 10 mL of enriched medium with uric acid as the only carbon source (uric acid, 5.0 g; yeast extract, 0.5 g; NaCl, 0.1 g; MgSO 4 .7H 2 O, 0.5g; K 2 HPO 4 , 2.0g; KH 2 PO 4 , 0.5g; H 2 O, the volume was adjusted to 1000 mL; pH 7.5, high-temperature and high-pressure sterilization was carried out according to the conventional method), and cultured on a shaking table at 30°C.

[0035] Three days later, 1 mL of the culture solution was inoculated on 10 ml of the same sterilized medium as above, and the culture was continued (shaking culture at 30° C. for 3 days).

[0036] Repeat this three times (i.e. repeat the above-mentioned continuous culture step 2 times) and then use an inoculation loop to dip a ring of enrichment culture, and in the solid separation medium (uric acid, 3.0g; yeas...

Embodiment 2

[0041] Embodiment 2, the purification of thermostable urate oxidase

[0042] With the enzyme fermentation condition (substratum consists of: uric acid, 3g; Corn steep liquor, 10g; K 2 HPO 4 · 3H 2 O, 2g; KH 2 PO 4 , 0.5g; MgSO 4 · 7H 2 O, 0.5 g; NaCl, 1 g; Water, 1000 mL; pH 7.5. Optimal culture condition is: culture temperature 30 ℃,) culture 10L fermented liquid, be specifically: get 10ml bacterial liquid and add in the culture medium of 100ml, ferment and cultivate 1 day at 30 ℃; Obtain 10L fermented liquid after merging.

[0043] The bacteria were collected by centrifugation at 5000rpm for 30min, and the cells were washed twice with 50mmol / L, pH7.5 phosphate buffer, and then suspended in 2 times the volume of buffer. After crushing the cells with a high-pressure cell disruptor, centrifuge at 5000 rpm for 50 min to remove cell debris to obtain a crude enzyme solution.

[0044] Add solid ammonium sulfate to the crude enzyme solution to make the saturation of solid ...

Embodiment 3

[0047] Embodiment 3: thermostability and optimal reaction temperature of enzyme:

[0048] The remaining enzyme activity was determined after treatment at different temperatures for 30 min in sodium borate buffer at pH 8.5. The result is as figure 2 As shown, the enzyme can retain 64% of the enzyme activity after being treated at 70°C for 30 minutes. Uric acid oxidase activity was measured at different temperatures, the results are shown in image 3 , the optimum temperature for enzyme action is 30°C.

[0049] The pH stability of the enzyme and the optimum pH of the enzyme reaction:

[0050] Different buffers at different pH values ​​(pH 3.0-6.0: 0.1M citric acid buffer; pH 6.0-8.5: 0.1M phosphate buffer; pH 8.5-9.5: 0.05M boric acid buffer; pH 9.5-11.0: 0.05M Borax buffer) was added to the enzyme solution, incubated at room temperature for 18 hours, and the remaining enzyme activity was determined. The result is as Figure 4 It was shown that the enzyme was stable at pH ...

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Abstract

The invention discloses the heat-resisting urate oxidase gene of microbacterium bacteria, comprising the nucleotide sequence presented in SEQ ID No.1. The invention also discloses a protein encoded by the gene, comprising the amino acid sequence presented in SEQ ID No.2. The invention also discloses the application of the heat-resisting urate oxidase gene of the microbacterium category bacteria: the gene is cloned in a vector so as to carry out induction expression, thus obtaining the heat-resisting urate oxidase with high activity.

Description

technical field [0001] The invention belongs to the field of microbial enzyme engineering. Specifically, the present invention isolates a heat-resistant uric acid oxidase (UOX, EC 1.7.3.3) gene from a strain of Microbacterium sp. genome. After the gene is cloned, it can be highly expressed in engineering bacteria to produce heat-resistant urate oxidase with excellent properties. Background technique [0002] Uric acid (2,4,6-trihydroxypurine, referred to as UA) is the direct product of hypoxanthine and xanthine metabolism. The level of uric acid in human blood is associated with gout, hypertension, diabetes, kidney disease and many clinical syndromes. Serum uric acid levels may reflect and integrate different risk factors and their possible interactions. Therefore, the analysis of uric acid indicators is very important in clinical practice. [0003] Uric acid oxidase is an enzyme in the purine degradation metabolic pathway in organisms. It uses oxygen as an acceptor and ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/70C12N9/06
Inventor 马晓航张鹏程周学来卢向锋李倩延孙玲艳
Owner ZHEJIANG UNIV
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