Genetically engineered urate oxidase mutant and application thereof

Abstract

The invention relates to a genetically engineered urate oxidase mutant and application thereof, and belongs to the field of biotechnology and bioengineering. According to the invention, a wild urate oxidase gene is used as a template, a protein engineering strategy is adopted to obtain a urate oxidase mutant library, and a high-throughput screening mode of developing qualitative primary selection and absorbance method quantitative screening is established to screen out a plurality of genetic engineering urate oxidase mutants capable of efficiently degrading uric acid. Compared with a wild type, the urate oxidase mutant with high enzyme activity has important industrial production and medical application values, and has a good prospect in developing novel uric acid reducing drugs or drug combinations.

Description

Technical Field

[0001] The present application relates to a genetically engineered urate oxidase mutant and its application, belonging to the technical fields of biotechnology and bioengineering. Background Art

[0002] Urate oxidase is a very important enzyme in the purine metabolism pathway of organisms. It can catalyze the oxidation and decomposition of uric acid to generate allantoin, which is highly soluble in water, and its water solubility is nearly a hundred times that of uric acid. Therefore, urate oxidase can rapidly and directly reduce the uric acid level in organisms and relieve uric acid accumulation. However, during the long process of evolution, the urate oxidase of hominids has gradually lost its activity and become a pseudogene, resulting in the absence of functionally active urate oxidase in the human body. When the uric acid concentration in the human body remains at a high level for a long time, it will cause hyperuricemia. Excessive blood uric acid levels will also cause urate crystals to deposit in joints, leading to gout.

[0003] Urate oxidase is a novel drug for the treatment of hyperuricemia and gout. It can not only rapidly reduce the blood uric acid level in humans, but also promote the dissolution of existing tophi, and has good application prospects for the treatment of hyperuricemia and gout. Currently, there are mainly two types of marketed urate oxidase drugs. One type is recombinant urate oxidase (AfUOX) derived from Aspergillus flavus, namely rasburicase and ELITEK, which were respectively launched abroad in 2001 and 2002 for the treatment of hyperuricemia caused by tumor lysis syndrome. Although these two drugs can effectively reduce the uric acid level in the blood and reduce the deposition of uric acid crystals in joints and soft tissues, they have problems such as low activity, large dosage, easy occurrence of infusion reactions and allergic reactions. The other type is mammalian-derived, PEGylated porcine-baboon chimeric urate oxidase (Pegloticase) launched in the United States in 2010. Although its allergic reaction is relatively small, its relative activity is even lower, the infusion reaction is severe, and at the same time, 40% of patients produce anti-PEG antibodies, resulting in a decrease in the uric acid-lowering effect. Existing urate oxidase drugs generally have disadvantages such as low activity, large clinical dosage, high price, and short in vivo half-life, which limit the application of urate oxidase drugs in the treatment of gout. To improve the activity of urate oxidase, Zhang Jinlong et al. (Chinese Journal of Biotechnology, 2010, 26(8): 1102-1107) once explored its activity by mutating the TGC (Cys) at positions 307-309 of the Aspergillus flavus urate oxidase gene to GCC (Ala). However, the enzyme activity of this single-point mutation only increased by about 60%, and its stability was unknown.

[0004] Therefore, it is necessary to develop more Aspergillus flavus urate oxidases with high enzyme activity and high stability, so as to reduce the dosage, slow down allergic reactions and infusion reactions, and improve the efficiency of Aspergillus flavus urate oxidase in the treatment of hyperuricemia and gout. Summary of the Invention

[0005] To solve the above problems, the present invention provides a genetically engineered urate oxidase mutant and its application. Using the Aspergillus flavus urate oxidase gene as a template, combinatorial protein engineering modifications including error-prone PCR are carried out to obtain an Aspergillus flavus urate oxidase mutant library. Multiple mutant recombinant strains with high efficiency in degrading uric acid are screened out, and mutant Aspergillus flavus urate oxidase with high enzyme activity compared to the wild type is obtained, which has important industrial production and economic value.

[0006] According to one aspect of the present application, a genetically engineered urate oxidase is provided, and the genetically engineered urate oxidase comprises substitution at one or more sites among the amino acids at positions 5, 22, 24, 49, 56, 73, 97, 101, 139, 148, 190, 198, 199, 218, 226, 237, 239, 242, 244, 274, 275, 287, 299, 300 and 302, wherein the amino acid positions are referenced to SEQ ID: No.1.

[0007] Optionally, the genetically engineered urate oxidase comprises substitution at two, three, four, five, or six of the above amino acid sites or a combination thereof, wherein the amino acid positions are referenced to SEQ ID: No.1.

[0008] Optionally, the genetically engineered urate oxidase is based on wild-type urate oxidase and comprises at least one substitution or substitution set, the substitution or substitution set comprising 56 / 275, 49 / 226, 24, 299, 73, 198, 300, 218, 97, 275, 139 / 190 / 274 / 287, 237, 5 / 239, 49, 199, 148, 274, 199 / 244 / 302, 22 / 237 / 274, 24 / 242 / 101, 24 / 101 / 239 / 242, 22 / 237 / 274 / 239, 5 / 239 / 49, 5 / 239 / 199, 5 / 239 / 274, 5 / 239 / 148, 5 / 239 / 148 / 49 / 199 / 274 and 5 / 239 / 274 / 242, wherein the amino acid positions are referenced to SEQ ID: No.1.

[0009] Optionally, the genetically engineered urate oxidase includes at least one substitution or set of substitutions based on wild-type urate oxidase, and the substitution or set of substitutions includes 5 / 239 / 274, 24 / 242 / 101, 5 / 239, 199 / 244 / 302, 22 / 237 / 274, 148, 199, 274, 5 / 239 / 148 / 49 / 199 / 274, and 5 / 239 / 274 / 242, where the amino acid positions are numbered with reference to SEQ ID: No. 1.

[0010] Optionally, the genetically engineered urate oxidase includes at least one substitution or set of substitutions based on wild-type urate oxidase, and the substitution or set of substitutions includes K5I / 239 / 274, K5I / 239, R199H / Q244E / L302Q, D22G / E237D / 274, S148I, R199S, K274N, K24E / 242 / N101I, K5I / 239 / S148I / K49N / R199S / 274, and K5I / 239 / 274 / 242, where the amino acid positions are numbered with reference to SEQ ID: No. 1. Taking K5I as an example, it represents replacing the lysine K at position 5 with isoleucine I. For those not specifying the substituted amino acid above, any amino acid can be used for substitution.

[0011] Optionally, the isoleucine I at position 239 is replaced with one of the amino acids A, N, C, Q, L, F, S, T, V.

[0012] Optionally, the arginine R at position 242 is replaced with one of A, N, D, C, Q, E, G, I, L, K, M, F, T, W, Y, V.

[0013] Optionally, the lysine K at position 274 is replaced with one of A, R, D, C, Q, E, G, I, H, L, M, F, P, S, T, W, Y, V, N.

[0014] According to another aspect of the present application, a high-throughput screening method for a high-enzymatic-activity urate oxidase mutant is provided, and the method includes the following steps:

[0015] 1) Establish a mutant library: Using urate oxidase as a template, a mutant library is obtained by adopting protein engineering strategies;

[0016] 2) Preliminary screening by color reaction: The transformant clones are reacted with uric acid and Amplex Red-HRP reagent in the dark to observe color changes. Those showing a pink color are positive transformants;

[0017] 3) High-throughput screening: In the reaction substrate of urate oxidase, a solution containing the urate oxidase mutant is added for catalytic reaction. After terminating the reaction, high-enzymatic-activity mutants are screened by measuring the absorbance.

[0018] Optionally, the primary screening of the color reaction in step 2) includes the following steps: coating the transformed bacterial solution on an IPTG plate and culturing overnight, covering the transferred bacteria with filter paper and then subjecting them to freezing treatment, and subsequently spreading them in a liquid mixed with uric acid and Amplex Red-HRP reagent, observing the color change under light protection, and those showing pink are positive transformants.

[0019] Optionally, the combinatorial protein engineering strategy includes error-prone PCR, combinatorial mutation, and saturation mutation. It can be understood by those skilled in the art that other conventional methods in the art can also be used as long as they can be used to obtain a mutant library.

[0020] According to another aspect of the present application, there is provided the use of the high-throughput screening method for any of the above-mentioned high-enzymatic-activity urate oxidase mutants in the preparation of a urate oxidase activity detection reagent or kit.

[0021] According to another aspect of the present application, there is provided the use of any of the above-mentioned genetically engineered urate oxidase in the production of a urate oxidase preparation or a composition for reducing uric acid activity.

[0022] Optionally, the urate oxidase preparation or the composition for reducing uric acid activity includes pure enzyme, modified enzyme, immobilized enzyme, crude enzyme, whole cell catalyst, uric acid-lowering drug, uric acid detection reagent or kit. It can be understood by those skilled in the art that in addition to the above-listed products, other products containing any of the above-mentioned genetically engineered urate oxidase should also be within the protection scope of the present application.

[0023] According to another aspect of the present application, there is provided a nucleic acid molecule encoding any of the above-mentioned genetically engineered urate oxidase, or a recombinant vector, recombinant cell, recombinant bacterium, reagent or kit containing the nucleic acid molecule. It can be understood by those skilled in the art that in addition to the above-listed products, other products containing any of the above-mentioned nucleic acid molecules should also be within the protection scope of the present application.

[0024] Optionally, the nucleic acid molecule includes a DNA molecule and / or an RNA molecule.

[0025] Optionally, the recombinant vector includes a viral vector and / or a non-viral vector.

[0026] Optionally, the viral vector includes an adeno-associated virus vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, and / or an oncolytic virus vector; the non-viral vector includes a cationic polymer, a liposome, and / or a plasmid vector.

[0027] According to another aspect of the present application, there is provided the use of the above nucleic acid molecule, recombinant vector, recombinant cell, recombinant bacterium, reagent or kit in the production of urate oxidase preparation or a composition with uric acid-lowering activity.

[0028] The beneficial effects of the present application include but are not limited to:

[0029] 1. The genetically engineered urate oxidase according to the present application has the advantages of high enzyme activity and high stability, can improve the efficiency of Aspergillus flavus urate oxidase in treating hyperuricemia and / or gout, and has important industrial production and pharmaceutical application values.

[0030] 2. For the genetically engineered urate oxidase according to the present application, the enzyme activity of Z5-R242W can reach 3.2 times that of the wild-type enzyme activity of AfUOX. In addition, 18A6 and Z5-R242W also have significant improvements compared to the wild-type enzyme activity of AfUOX, and have important application values for reducing blood uric acid levels, treating hyperuricemia and / or gout in the prior art.

[0031] 3. For the high-throughput screening method of the high-enzyme-activity urate oxidase mutant according to the present application, a mutant library is constructed by protein engineering transformation including error-prone PCR, and a preliminary screening method using Amplex Red-HRP as a chromogenic agent is established to preliminarily screen out mutants inactivated by random mutations. Then, a high-throughput screening method based on measuring OD293 with a microplate reader is used to screen mutant recombinant strains that can efficiently degrade uric acid. The above method provided by the present application can significantly improve the screening throughput and screening efficiency of the mutant library, and has good application values. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings described herein are used to provide a further understanding of the present application, and constitute a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation to the present application. In the drawings:

[0033] Figure 1 It is a graph showing the enzyme activity results of different combination mutant strains of AfUOX involved in Example 5 of the present application;

[0034] Figure 2 It is the pH stability result of the wild-type AfUOX and its mutants involved in Example 6 of the present application;

[0035] Figure 3 It is the temperature stability result of the wild-type AfUOX and its mutants involved in Example 6 of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] The present application will be described in detail below in conjunction with embodiments. However, the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present application are all purchased through commercial channels.

[0037] In the present invention, the AfUOX (Aspergillus flavus uricase) gene is used as a template, and a random mutation library is established through protein engineering modification including random error-prone PCR. The plate color reaction method is used for preliminary screening, and then high-throughput screening in a 96-well plate is carried out to screen out high-activity mutants.

[0038] Example 1 Establishment of a random mutation library of Aspergillus flavus uricase

[0039] Using the AfUOX gene as a template, BGI was commissioned to synthesize the coding gene DNA optimized for Escherichia coli codons, which was digested and ligated to the pET28b(+) vector to obtain the AfUOX-pET28b recombinant expression vector. Using this vector as a template, error-prone PCR was carried out with the error-prone PCR enzyme GeneMorphII enzyme. Then, the processed error-prone PCR product was transferred into BL21(DE3) to obtain a mutant library plate of AfUOX (with mutations)-pET28b-BL21(DE3) recombinant expression bacteria. Monoclonal colonies of the library were transferred, preserved, induced, and harvested using common methods in the field.

[0040] Example 2 Screening by plate color reaction

[0041] The transformed error-prone PCR mutant library usually has a protein inactivation rate of about 40%. In order to improve the screening efficiency and reduce the screening workload, the present invention has established a simple and rapid colorimetric primary screening method to exclude the transformant clones with protein inactivation, greatly improving the screening efficiency and screening throughput. The specific screening method is as follows: The transformed bacterial solution is spread on an LB-Kan-0.05 mM IPTG plate and cultured overnight at 37°C. After covering the transferred bacteria with filter paper, it is taken out after freezing at -80°C for 30 min, and then spread in a liquid mixed with 2 mM uric acid and Amplex Red-HRP reagent, and the color change is observed under dark reaction at 37°C. The positive transformants are those showing pink color.

[0042] Example 3 High-throughput screening method

[0043] For the positive transformants after primary screening, the detailed steps of the high-throughput screening method after culturing and inducing expression in a 96-deep well plate are as follows:

[0044] 1) Whole-cell lysozyme treatment

[0045] The present invention compared the activity differences with different addition amounts of lysis buffer after lysozyme treatment. The collected bacterial cells were added to 450 μL of 50 mM Tris-HCl, pH 8.5, and 50 μL of lysis solution (100 mg of lysozyme in 10 mL of lysis solution). After lysis at 37 °C and 120 rpm for 1 h, 50 μL of the supernatant was taken by centrifugation and diluted with 450 μL of 50 mM Tris-HCl, pH 8.5. During the reaction, 1, 2, 3, 4, and 5 μL of the diluted crude enzyme solution were added respectively. The results showed that the uric acid consumption was 157.6 μM after adding 5 μL of AfUOX and reacting for 10 min, which was about 25% of the total uric acid amount (600 μM), and this could be used as a screening condition.

[0046] 2) Whole-cell freeze-thaw treatment

[0047] In order to further simplify the screening method, the effects of different freezing times on the whole-cell enzyme activity reaction were detected. The collected bacterial cells were treated as follows: a) resuspended with 50 mM Tris-HCl, pH 8.5 and then frozen; b) the bacterial sludge was frozen once; c) the bacterial sludge was frozen twice; d) the bacterial sludge was frozen three times, with a total of four groups of treatments. Subsequently, 1 μL, 2 μL, and 5 μL of the bacterial suspension (resuspended in 1 mL) were added to the reaction system (the final concentrations of each component in the 200 μL reaction system were as follows: 50 mM Tris-HCl with pH 8.5, 600 μM of uric acid) for enzyme activity determination. The experimental results showed that the enzyme activity retention was the best in the case of a) freezing after resuspension. This method was selected for subsequent high-throughput activity screening in 96-well microplates.

[0048] Example 4 Enzyme activity detection of Aspergillus flavus uricase

[0049] 1) Determination of whole-cell catalytic enzyme activity

[0050] After determining the treatment method of the bacterial cells, the enzyme activity screening and determination in 96-well microplates were continued. The final concentrations of each component in the 200 μL reaction system were as follows: 50 mM Tris-HCl with pH 8.5, 600 μM of uric acid, the resuspended and freeze-thawed bacterial solution with an OD of 0.04. At 37 °C and 900 rpm, the catalytic reaction was carried out for 10 minutes. 10 μL of 3 M H2SO4 was added to the reaction system to terminate the reaction, and then the absorbance value at A293 of the reaction system was detected, and the uric acid consumption rate per unit time was calculated and compared with the wild-type AfUOX to screen out candidate strains with significantly improved uricase enzyme activity for pure enzyme level enzyme activity determination.

[0051] 2) Determination of pure enzyme activity and catalytic rate

[0052] The candidate strains were cultured in 10-ml test tubes, induced for expression, and purified to obtain pure enzymes for enzyme activity assays. The reaction conditions were the same as those for the aforementioned whole-cell catalysis. The amount of pure enzyme used was 0.1 - 0.5 μg. By detecting the absorbance value at A293 in the reaction system, the uric acid consumption rate per unit time was calculated and compared with the enzyme activity of the wild-type pure enzyme of AfUOX to further confirm and screen out candidate strains with significantly improved enzyme activity.

[0053] After screening approximately 5,000 library transformants, 20 positive mutants with significantly improved activity were obtained. The further results of pure enzyme activity assays are shown in Table 1.

[0054] Table 1 Efficiency of improved enzyme activity of mutants

[0055] Number Mutated amino acid Enzyme activity improvement efficiency (taking wild type as 100%) AfUOX Wild type 100.00％ 18A6 K24E R242C N101I 254.96％ 2-9E R199S 162.49％ 2-2C K49N 196.13％ 1E K5I I239V 334.09％ 2-5D R199H Q244E L302Q 229.31％ 2-5G V56I N275D 197.59％ 2-2H K49N A226V 149.49％ 2-8E D22G E237D K274E 287.93％ 2-6E E173D 76.29％ 10E K24E 194.65％ 2-4B K299N 162.39％ 2-4F V73P 194.77％ 4F A198V 149.92％ 8F S300T 150.96％ 12H K218N 119.98％ 2-6H A97V 119.78％ 2-11E N275S 141.01％ 2-12F K139E K190I K274T G287E 151.58％ 8E E237D 196.50％ 2-8F S148I 250.61％ 2-12G K274N 209.48％

[0056] Example 5 Further modification of Aspergillus flavus uricase mutants

[0057] 1) Combinatorial mutagenesis

[0058] Eight highly active mutants (1E, 2-2C, 2-9E, 2-8F, 2-12G, 2-5D, 2-8E, 18A6) screened in Example 4 above were selected for the following combinatorial mutagenesis to obtain combinatorial mutants Z1-Z7. The enzyme activity results are as Figure 1 and shown in Table 2.

[0059] Table 2 Efficiency of improved enzyme activity of mutants

[0060] Number Mutated amino acid Enzyme activity improvement efficiency (taking wild type as 100%) AfUOX Wild type 100.00％ Z1 K24E N101I I239V R242C 191.48％ Z2 D22G E237D K274E I239V 164.01％ Z3 K5I I239V K49N 178.34％ Z4 K5I I239V R199S 209.14％ Z5 K5I I239V K274N 280.69％ Z6 K5I I239V S148I 208.46％ Z7 K5I I239V S148I K49N R199S K274N 261.42％

[0061] The pure enzyme activities of the combinatorial mutants were assayed. The results are shown in Table 2. The enzyme activity of Z5 was the best, reaching 2.81 times that of the wild-type of AfUOX.

[0062] 2) Saturation mutagenesis

[0063] Taking the screened Z5 mutant strain as a template, and taking the construction of the I239X saturation mutagenesis mutant as an example, on the basis of the Z5 mutant strain containing three mutations of K5I, I239V, and K274N, the 239th amino acid was replaced with other amino acids except isoleucine (I) to obtain 18 recombinant uricase strains with 239-site saturation mutagenesis. The pure enzyme activities of these mutants were detected.

[0064] Using the method in step 2) of Example 4, the enzyme activities of the saturation mutagenesis strains were assayed. Among the Z5-I239X saturation mutagenesis mutants, 8 mutants had improved activities compared with the wild-type. The construction and enzyme activity detection of mutants with saturation mutagenesis of K274N and R242C were carried out in the same way. The results are shown in Tables 3, 4, and 5.

[0065] Table 3 Efficiency of improved enzyme activity of mutants

[0066]

[0067]

[0068] Table 4 Efficiency of improved enzyme activity of mutants

[0069]

[0070]

[0071] Table 5 Efficiency of improved enzyme activity of mutants

[0072]

[0073]

[0074] The results showed that the enzyme activities of the K274X saturation mutants of Z5 all exceeded those of the wild-type AfUOX. Among them, the activities of 5 mutants were equivalent to that of Z5, which were 2.83 - 2.99 times that of the wild-type. In the R242X saturation mutants of Z5, the enzyme activities of most mutants exceeded those of the wild-type AfUOX. Among them, the activities of 2 mutants were equivalent to that of Z5, and the activity of the Z5-R242W mutant exceeded that of the Z5 mutant, which was 1.1 times that of the Z5 mutant.

[0075] Example 6 Detection of the stability of Aspergillus flavus urate oxidase mutants

[0076] The pH stability and temperature stability of representative mutants were detected respectively.

[0077] 1) pH stability

[0078] The test operation included the following steps: incubate the pure enzyme at different pH values under the conditions of 4°C for 20 h. Then, perform enzyme activity detection under the conditions of 50 mM Tris-HCl with a pH of 8.5, 600 μM of the substrate uric acid, 0.1 μg of pure enzyme, 37°C, 900 rpm, and a catalytic reaction for 10 minutes. Taking the enzyme reaction for 10 min without incubation as 100% enzyme activity. The results showed that under these detection conditions, the Z5-R242W and 18A6 had the highest activity retention (80%) at each pH (7.0 - 10.5), which was significantly stronger than that of the wild-type (40%).

[0079] The pH stability results of the wild-type AfUOX and its mutants are as Figure 2 shown.

[0080] 2) Temperature stability

[0081] The test operation includes the following steps: incubate the Aspergillus flavus uricase mutant at different temperatures (0°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C) for 10 min, and then perform enzyme activity detection. The detection conditions are 50 mM Tris-HCl with a pH of 8.5, 600 μM of the substrate uric acid, 0.1 μg of pure enzyme, 37°C, 900 rpm, and a catalytic reaction for 10 minutes. The enzyme activity of the non-incubated sample is taken as 100%. The detection results show that the mutant Z5-R242W still retains 20% activity after incubation at the high temperature of 60°C, while the wild type completely loses its activity at this temperature.

[0082] The temperature stability results of the AfUOX wild type and its mutants are as Figure 3 shown.

[0083] As mentioned above, only the embodiments of this application are described. The protection scope of this application is not limited by these specific embodiments, but is determined by the claims of this application. For those skilled in the art, various changes and modifications can be made to this application. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of this application shall be included within the protection scope of this application.

Claims

1. A genetically engineered urate oxidase, characterized in that, The genetically engineered urate oxidase includes substitution at one or more sites among the amino acids at positions 5, 22, 24, 49, 56, 73, 97, 101, 139, 148, 190, 198, 199, 218, 226, 237, 239, 242, 244, 274, 275, 287, 299, 300, and 302, wherein the amino acid positions are referenced according to SEQ ID: No.

1.

2. The genetically engineered urate oxidase according to claim 1, wherein The genetically engineered urate oxidase includes substitution at two, three, four, five, or six sites among the amino acid sites described in claim 1, or a combination thereof, wherein the amino acid positions are referenced according to SEQ ID: No.

1.

3. The genetically engineered urate oxidase according to claim 1 or 2, characterized in that, The genetically engineered urate oxidase has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID: No.

1.

4. A high-throughput screening method for urate oxidase mutants with high enzyme activity, characterized in that, The method includes the following steps: 1) Establishing a mutant library: Using urate oxidase as a template, a mutant library is obtained by adopting protein engineering strategies. 2) Primary screening by color reaction: The transformant clones are reacted with uric acid and Amplex Red-HRP reagent in the dark to observe color changes. Those showing a pink color are positive transformants. 3) High-throughput screening: In the reaction substrate of urate oxidase, a solution containing urate oxidase mutants is added for catalytic reaction. After terminating the reaction, high-activity mutants are screened by measuring the absorbance.

5. The high-throughput screening method for the urate oxidase mutant with high enzyme activity according to claim 4, wherein The primary screening by color reaction in step 2) includes the following steps: The transformed bacterial solution is spread on an IPTG plate and cultured overnight. After covering the transferred bacteria with filter paper and then freezing treatment, it is then spread in a liquid mixed with uric acid and Amplex Red-HRP reagent, and the color changes are observed in the dark. Those showing a pink color are positive transformants.

6. Use of the high-throughput screening method for high-activity urate oxidase mutants as described in claim 4 or 5 in the preparation of a reagent or kit for detecting urate oxidase activity.

7. Use of the genetically engineered urate oxidase as described in any one of claims 1 to 3 in the production of a urate oxidase preparation or a composition with uric acid-lowering activity.

8. The application according to claim 7, wherein The preparation or the composition includes pure enzyme, modified enzyme, immobilized enzyme, crude enzyme, whole-cell catalyst, uric acid-lowering drug, uric acid detection reagent or kit.

9. A nucleic acid molecule encoding the genetically engineered urate oxidase as described in any one of claims 1 to 3, or a recombinant vector, recombinant cell, recombinant bacterium, reagent, or kit containing the nucleic acid molecule.

10. Use of the nucleic acid molecule, recombinant vector, recombinant cell, recombinant bacterium, reagent, or kit as described in claim 9 in the production of a urate oxidase preparation or a composition with uric acid-lowering activity.

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