Novel urate oxidase capable of being used for detecting and degrading uric acid in vitro

Abstract

The invention discloses urate oxidase and application thereof. The amino acid sequence of the urate oxidase is shown as SEQ ID NO.01. The invention can effectively degrade uric acid in vitro, and has high activity and good stability.

Description

Technical Field

[0001] This invention belongs to the field of recombinant protein technology, specifically designing a urate oxidase for in vitro detection and degradation of uric acid. Background Technology

[0002] Uric acid oxidase is a crucial enzyme in purine metabolism, breaking down uric acid into allantoin, a more water-soluble form, thus facilitating its excretion. It is commonly used for uric acid detection and in the treatment of gout and hyperuricemia. Uric acid oxidase is found in most organisms, including animals, plants, yeast, bacteria, and fungi. Humans and some higher animals lack active uric acid oxidase, leading to high uric acid levels and increasing the risk of gout and other diseases. While uric acid oxidase has been widely used for detecting and degrading uric acid in recent years, it suffers from low activity and instability, necessitating the discovery and design of novel, highly active, and stable uric acid oxidases. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a highly active and stable urate oxidase.

[0004] Another object of the present invention is to provide the application of the above-mentioned urate oxidase.

[0005] The technical solution of the present invention is as follows:

[0006] A uricase oxidase, the amino acid sequence of which is shown in SEQ ID NO.01.

[0007] In a preferred embodiment of the present invention, its nucleotide sequence is shown in SEQ ID NO.02.

[0008] A recombinant expression vector loaded with and expressing uricase as shown in SEQ ID NO. 01.

[0009] In a preferred embodiment of the present invention, the nucleotide sequence of the urate oxidase is shown in SEQ ID NO.02.

[0010] In a preferred embodiment of the invention, it is based on the pET-22b(+) carrier.

[0011] A recombinant Escherichia coli loaded with the above-mentioned recombinant expression vector.

[0012] The above-mentioned uric acid oxidase is used to degrade uric acid in vitro.

[0013] The above-mentioned recombinant expression vectors are used in the in vitro degradation of uric acid.

[0014] The above-mentioned recombinant Escherichia coli was used to degrade uric acid in vitro.

[0015] The beneficial effects of this invention are: this invention can effectively degrade uric acid in vitro, with high activity and good stability. Attached Figure Description

[0016] Figure 1 This is a standard curve of uric acid in Example 3 of the present invention.

[0017] Figure 2 This is a diagram showing the optimal temperature for the product expressed in Example 4 of the present invention.

[0018] Figure 3 This is a diagram showing the optimal pH for the expression product in Example 5 of the present invention.

[0019] Figure 4 This is a graph comparing the specific activities of the expression product at different enzyme concentrations in Example 3 of the present invention.

[0020] Figure 5 This is a stability graph of the expressed product at 4°C and pH 9 in Example 6 of the present invention.

[0021] Figure 6 This is a stability graph of the expressed product in Example 6 of the present invention at room temperature (25℃-30℃) and pH 9.

[0022] Figure 7 This is a stability graph of the product expressed in Example 7 of the present invention at 36°C and pH 8.5. Detailed Implementation

[0023] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0024] Example 1: Construction of a recombinant vector for the uricase gene

[0025] Artificially synthesized uricase gene sequence:

[0026] Atgcatgaaattaaagaactgaacgtgaccgtgcagctgcgcggcgattttgatagcgcgcatagcgaaggcgataacaccaaagtggtgccgaccgataccgtgaaaaacaccgtgtatgcgaccgcgaaagatggcgtgggcagcccggaagaatttggcctgaaactgggcacccattttgtgggcgaatttgatcgcattaccggcggctatgtggaagtgacccagcatacctgggaacgcattcagaccgatggccagccgc atacccatagctttgtgcgcggcggcaccaaacgcaccaccaccgtgaccgtggatggcgatgattttaccgtgaccagcggcctgcaggatctgaaagtgctgaaaagcaccgaaagcggctttgtgggctatccgaccgatgaatataccaccctgccggaaaccgaagatcgcattctggcgaccagcgtgaccgcgaaatggacctataacaccgaagatctggattatgatgaagtgtgggcggatgtgcgccgcctgattctgaccacctttagcaccaactatagcccgagcctgcagcgcaccctgtatgaaatggcggaagcggtgctggaaacctgcccgaccattaccaccatttggtttagcatgccgaacaaacatcattttgtggtggatctgagccgcctgggcctggaaaacccgaacgaagtgttttatgcggcgccgaaaccgtatggcctgattgaagcgagcgtgacccgcgaaggcuag(SEQ ID NO.02)

[0027] The protein sequence of this gene is:

[0028] MHEIKELNVTVQLRGDFDSAHSEGDNTKVVPTDTVKNTVYATAKDGVGSP-EEFGLKLGTHFVGEFDRITGGYVEVTQHTWERIQTDGQPHTHSFVRGGTKRTTTVTVDGDDFTVTSGLQDLKVLKSTES GFVGYPTDEYTTLPETEDDRILATSVTAKWTYNTEDLDYDEVWADVRRLILTFSTNYSPSLQRTLYEMAEAVLETCPTITTIWFSMPNKHHFVVDLSRLGLENPNEVFYAAPKPYGLIEASVTREG(SEQ ID NO.01)

[0029] The artificially synthesized urate oxidase gene sequence was constructed into the pET-22b(+) vector via NcoI / Xhol restriction sites. The recombinant plasmid was then transformed into Escherichia coli BL21(DE3) competent cells. Since pET-22b(+) carries an ampicillin resistance gene, it was necessary to screen the cells using a medium resistant to ampicillin (final concentration: 100 μg / mL). After sequencing verification, the recombinant strain containing the urate oxidase gene was finally obtained.

[0030] Example 2: Recombinant expression and purification of the uricase gene

[0031] Expanded culture: 200 μL of recombinant Escherichia coli from Example 1 was taken from the bacterial culture bank and inoculated into 20 mL of LB medium containing 0.1 g / L ampicillin sodium. After overnight culture at 37°C and 200 rpm, it was inoculated into 200 mL of TB medium containing 0.1 g / L ampicillin sodium at a ratio of 1% for expanded culture at 37°C and 200 rpm for 4-5 h.

[0032] Induction of expression: IPTG (isopropyl-β-D-thiogalactose) was added to a final concentration of 0.5 mmol / L, and the mixture was cultured in a shaker at 180 rpm for 20 h at 18 °C.

[0033] Collect bacterial cells: Collect bacterial cells by centrifugation at 4000 rpm for 20 min. Resuspend in 20 mL of 50 mM Tris-HCl buffer at pH 8.5.

[0034] Cell disruption and centrifugation: After mixing the precipitate by pipetting, sonicate it. The power was 300W and the time was 20min. Then, the disrupted bacterial culture was centrifuged at 10000rpm for 30min at 4℃, and the supernatant was collected.

[0035] Protein purification: Take 5 mL of Ni-NTA-agarose gel pre-packed column, add 5 mL of Binding Buffer, and equilibrate 5 times; add 5 mL of supernatant from the top of the column each time until all supernatant has passed through the column; wash 4 times with 5 mL of Binding Buffer, and then wash 4 times with 5 mL of Washing Buffer; elute with 10-15 mL of Elution Buffer and collect in a clean centrifuge tube; finally, concentrate the collected purified protein using an ultrafiltration centrifuge tube (50 mL, 10 KD).

[0036] Example 3: Uric acid oxidase activity assay

[0037] Uric acid oxidase activity is defined as the amount of enzyme required to catalyze the conversion of 1 μmol of uric acid into allantoin per minute at 30°C and pH 8.5.

[0038] To construct a standard curve for uric acid: Prepare a stock solution of 4 mM uric acid. Sequentially prepare uric acid solutions of 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, and 0.20 mM. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 293 Measure the absorbance values ​​at various concentrations, plot a standard curve, and perform three routine experiments for each group. The results are as follows: Figure 1 As shown.

[0039] Uric acid oxidase activity assay: 0.1 mL of enzyme at a specific concentration and 0.8 mL of uric acid were added to 1.0 mL of Tris-HCl buffer (pH 8.5), and the mixture was incubated at 30 °C for 3 min. The OD value was then measured. 293 The absorbance at nm was used as a blank control, with buffer added instead of uric acid, and three replicates were performed for each group. Enzyme activity was calculated based on the uric acid standard curve.

[0040] The formula for calculating enzyme activity is as follows:

[0041]

[0042] Where U: enzyme activity; 1000: coefficient for converting mmol to μmol; W: amount of uric acid consumed in the enzymatic reaction, mg; Df: dilution factor of the enzyme solution; Mr: molecular weight of uric acid; t: reaction time, 3 min; V: amount of enzyme, 0.1 mL.

[0043] Using the uric acid oxidase activity assay method, enzyme solutions with concentrations of 0.05, 0.1, 0.5, 1, 2, and 3 mg / mL were prepared, and their activities were measured. The results are as follows: Figure 4 As shown, the enzyme with the highest specific activity was at a concentration of 0.05 mg / mL, with an enzyme activity of approximately 66.13 U / mg.

[0044] Example 4: Determination of the optimal temperature for uricase oxidase

[0045] Using the uric acid oxidase activity assay as the standard, the activity was measured at 20, 30, 40, 50, 60, and 70°C. The highest enzyme activity was defined as 100%, and the relative enzyme activities of the remaining groups were calculated. The temperature corresponding to the highest enzyme activity was the optimum temperature. Figure 2 As shown, the optimal temperature is 30℃.

[0046] Example 5: Determination of the optimal pH for uricase oxidase

[0047] Using the uric acid oxidase activity assay as the standard, the activity was measured at pH 7.5, 8, 8.5, 9, 10, and 11. The highest enzyme activity was defined as 100%, and the relative enzyme activities of the other groups were calculated. The pH corresponding to the highest enzyme activity was the optimal pH. Figure 3 As shown, the optimal pH is 9.

[0048] Example 6: Determination of the stability of uricase

[0049] Uric acid oxidase was incubated at 4°C. A portion of the incubated enzyme was taken out every 24 hours for activity determination. Initial enzyme activity was defined as 100%, and the remaining enzyme activity after different incubation times was calculated. Results are as follows: Figure 5 As shown, at 4°C, it retains approximately 100% activity after 4 days.

[0050] Uric acid oxidase was incubated at room temperature (25℃-30℃). A portion of the incubated enzyme was taken out at intervals for activity measurement. The initial enzyme activity was defined as 100%, and the remaining enzyme activity after different incubation times was calculated. The results are as follows: Figure 6 As shown, at room temperature, it still retains about 80% of its activity after one month.

[0051] Example 7: Determination of pH stability of uricase

[0052] Uric acid oxidase was incubated in a 36°C water bath at pH 8.5. A portion of the incubated enzyme was taken out at intervals for activity determination. Initial enzyme activity was defined as 100%, and the remaining enzyme activity after incubation at different pH values ​​and for different times was calculated. Results are as follows: Figure 7 As shown, under pH 8.5 conditions, more than 90% of the activity was still present after 10 days of incubation.

[0053] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A uricase oxidase, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

01.

2. The uricase according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

02.

3. A recombinant expression vector, characterized in that: It is loaded with and expresses uricase as shown in SEQ ID NO.

01.

4. The recombinant expression vector as described in claim 3, characterized in that: The nucleotide sequence of the urate oxidase is shown in SEQ ID NO.

02.

5. A recombinant expression vector as described in claim 3 or 4, characterized in that: It is based on the pET-22b(+) vector.

6. A recombinant Escherichia coli, characterized in that: Its loading includes the recombinant expression vector as described in any one of claims 3 to 5.

7. The use of the uricase according to claim 1 or 2 in the in vitro degradation of uric acid.

8. The use of the recombinant expression vector according to any one of claims 3 to 5 in the in vitro degradation of uric acid.

9. The application of the recombinant Escherichia coli according to claim 6 in the in vitro degradation of uric acid.

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