Urate oxidase and preparation and use thereof

Abstract

The application provides a uricase and preparation and application thereof. The uricase is based on an amino acid sequence of Candida utilis uricase, a molecular model is constructed by using a bioinformatics method, stability structure modification is carried out by using a mutation technology, an interchain disulfide bond is added between different subunits, the stability of the uricase is improved, and the uricase has good drug property, and has great application prospect in treatment of hyperuricemia or gout.

Description

Technical Field

[0001] This application relates primarily to the field of biotechnology, and in particular to a urate oxidase and its preparation and application. Background Technology

[0002] Uric acid, chemically known as trioxypurine, is a heterocyclic compound containing carbon, nitrogen, oxygen, and hydrogen. It is the final metabolic product of purine compounds in humans. Uric acid has no physiological function in the human body. Under normal circumstances, the total amount of uric acid in the human body is 0.9–1.6 g, with 750 mg produced daily. One-third of this uric acid comes from food, and two-thirds is synthesized within the body. Two-thirds of uric acid is excreted by the kidneys, and the remaining one-third is excreted through the intestines. Its production and excretion are in balance. If purine metabolism is disordered, resulting in excessive uric acid production or impaired uric acid excretion, abnormal purine metabolism will occur, leading to elevated blood uric acid levels and causing hyperuricemia (HUA). HUA is defined as a fasting blood uric acid level higher than 416 μmol / L for men and higher than 357 μmol / L for women on two separate days, under normal purine dietary conditions. When blood uric acid levels reach 475 μmol / L, it becomes clinically significant for diagnosis. In particular, those whose blood uric acid levels remain above 540 μmol / L have a 70% to 90% chance of developing gout.

[0003] Because the prevalence of hyperuricemia (HUA) is influenced by various factors such as environment, dietary habits, genetics, and statistical methods, reported rates vary across countries. The 2007-2008 National Health and Nutrition Examination Survey (NHANES) in the United States found a prevalence of 21.2% in men and 21.6% in women in the general population, 3.2% higher than the results of the 1988-1994 NHANES-III survey. The 2015-2016 NHANES statistics showed a prevalence of 20.2% in men and 20.0% in women in the United States. The organization conducted an overall statistical analysis of data from 2007-2016, showing that the prevalence remained stable at a high level without any improvement. A study by Engel et al. in Germany from 2004-2013, involving individuals over 50 years of age, found a HUA prevalence of 17%. A study by Ting et al. in Australia from 2008-2010 found a HUA prevalence of 16.6% in the general population. The 2016 Korean National Health and Nutrition Examination Survey (KNHANES) surveyed adults aged 19 and older, finding a prevalence of hyperuricemia (HUA) of 17.0% in men and 5.9% in women. Uaratanawong et al., in a study of individuals aged 35 and older who had lived in Bangkok for more than 10 years between 2009 and 2010, found a HUA prevalence of 24.4%. In my country, the overall prevalence of hyperuricemia from 2000 to 2014 was 13.3%. In recent years, with improved living standards and increased consumption of high-purine and high-protein diets, uric acid levels have been rising, and the prevalence of hyperuricemia is increasing year by year and showing a trend towards affecting younger people, making it another common metabolic disease after diabetes.

[0004] Currently, clinical treatment for gout primarily focuses on correcting hyperuricemia and controlling acute gout attacks. This is mainly achieved through the use of drugs that inhibit uric acid production, drugs that promote uric acid excretion, and uricase-inhibiting agents. Allopurinol is a leading example of a uric acid synthesis inhibitor. It works by inhibiting the activity of xanthine oxidase, thereby blocking uric acid formation. However, xanthine has lower solubility in urine than uric acid, increasing the renal excretory burden and failing to address the root cause of the problem. Commonly used uricase-inhibiting drugs include sulfadiazine, benzylsulfamethoxazole, and benzbromarone. However, long-term use of these drugs can lead to side effects such as kidney dysfunction, and the potential damage from cross-treatment is immeasurable. Due to the limitations in efficacy and safety of conventional gout medications, it has been reported that approximately 3% of the 3 million adult gout patients in the United States do not benefit from conventional treatment due to contraindications or poor efficacy. The search for newer treatments continues, and uricase-inhibiting agents have received increasing attention in recent years.

[0005] Many mammals possess the uricase gene, and the expressed uricase catalyzes the oxidation of uric acid into inactive purine metabolites allantoin, CO2, and H2O2. Allantoin is then excreted through the kidneys. Allantoin is known to be dozens of times more soluble than uric acid, making it easier to excrete through the kidneys. However, due to mutations in the uricase gene during human evolution, a stop codon was prematurely introduced into the coding sequence of the human uricase gene. Therefore, humans cannot synthesize active uricase themselves, and thus cannot further oxidize and decompose uric acid, produced by purine metabolism, into highly water-soluble allantoin, which is then excreted in urine. Therefore, supplementing with uricase to break down uric acid into allantoin and excrete it, thereby lowering uric acid levels, has become another strategy for treating hyperuricemia.

[0006] During the course of species evolution, all species except humans and the four great apes (chimpanzees, gorillas, orangutans, and gibbons) have retained the activity of uricase. Although many animals and microorganisms express uricase in their bodies, their activities vary, and the closer a species is to the top of the evolutionary tree, the lower its enzyme activity. For example, mammalian uricases are generally expressed in the cellular compartments of peroxisomes at high pH levels. These enzymes have low solubility and are unstable under physiological pH conditions. Their solubility gradually increases above pH 9, reaching a peak at around pH 10. Therefore, the drug development of these enzymes presents a significant challenge. Bomalaski et al. conducted specific activity studies on uricase from *Candida utilis*, *Arthrobacter globiformis*, *Aspergillus flavus*, and pigs under different pH conditions. They found that under human physiological conditions (pH = 7.4), *Candida utilis* uricase exhibited the highest specific activity (8.2 IU / mg), approximately 20.5 times that of pig uricase. Although *Candida utilis* uricase showed high activity under human physiological conditions, it was unstable; even at 4°C, aggregates could be detected after standing for half an hour. Summary of the Invention

[0007] To address the aforementioned technical problems, this application provides a novel structurally stable uricase. Through mutagenesis, interchain disulfide bonds are formed between different subunits, thereby effectively enhancing the stability of the uricase.

[0008] To achieve the above objectives, this application provides the following technical solution:

[0009] The first aspect of this application provides a mutated uricase subunit by introducing a cysteine ​​(Cys) mutation at one or more of the following sites in the wild-type uricase subunit as shown in SEQ ID NO:4: the 6th Ser position is mutated to Cys (S6C); the 7th Ser position is mutated to Cys (S7C); and the 8th Ser position is mutated to Cys (S21C).

[0010] A second aspect of this application provides a mutant uricase comprising the mutant uricase subunit.

[0011] A third aspect of this application provides a biomaterial selected from any of the following:

[0012] A1: An isolated nucleic acid molecule encoding the mutated uricase subunit as described in claim 1;

[0013] A2: An expression vector containing the nucleic acid molecules described in A1;

[0014] A3: Host cells containing the vector described in A2 or host cells whose genome integrates the nucleic acid molecules described in A1.

[0015] A fourth aspect of this application provides a conjugate comprising the mutant uricase subunit or the mutant uricase.

[0016] A fifth aspect of this application provides a pharmaceutical composition comprising the aforementioned mutated uricase and pharmaceutically acceptable excipients.

[0017] The sixth aspect of this application provides the use of the mutated uricase and the pharmaceutical composition in the preparation of a medicament for: 1) breaking down or lowering uric acid levels; 2) preventing or treating hyperuricemia or gout; and 3) treating tumor lysis syndrome.

[0018] The seventh aspect of this application provides a method for preparing the mutant uricase, comprising culturing the host cells to express the uricase, and isolating the uricase from the culture.

[0019] The eighth aspect of this application provides a method for obtaining the mutated uricase subunit, comprising molecular modeling of the uricase.

[0020] The mutated uricase in this application possesses good drug-like properties and shows great promise for the treatment of hyperuricemia or gout. Its beneficial technical effects include:

[0021] 1) Significantly improved stability: The mutated uricase in this application showed no aggregate formation after 24 hours of storage at 2–8°C, indicating high stability. In contrast, the wild-type uricase showed aggregate formation after 5 hours, and after 6 hours, the aggregate peak area accounted for more than 5% of the main peak.

[0022] 2) Specific activity retention: Specific activity greater than 1.8 U / mg.

[0023] The inventors discovered that introducing a Cys mutation at position 6 of uricase unexpectedly and significantly enhanced its stability, with a much greater reduction in aggregate formation than expected. Specifically, after 24 hours of incubation at 2–8°C, no aggregates were formed; in contrast, the wild-type showed an aggregate peak area exceeding 5% of the main peak after 6 hours and exhibited turbidity after 24 hours. Mutations at positions 7 and 8 of Cys also yielded similar effects. Furthermore, the modified uricase, due to its significant reduction in aggregates and improved stability, can extend the time window for subsequent processing steps, such as PEG coupling and purification, supporting further process optimization. Attached Figure Description

[0024] Figure 1 Crystal structure diagram of Candida utilis uricase.

[0025] Figure 2a Ramachandran Plots analysis of the molecular model of Candida utilis uricase showed that 95.48% of the amino acid residues fell into the Favored region.

[0026] Figure 2b The ProSA software analyzes the prediction model, and the score is represented by black dots, within the score range of proteins of similar size.

[0027] Figure 3a The molecular structure of Candida utilis uricase is shown, which is a compact barrel-shaped homotetramer structure.

[0028] Figure 3b The Cys at position 293 of the uricase in Candida utilis forms inter-chain disulfide bonds between different subunits with the mutant Cys at positions 6, 7, and 8.

[0029] Figure 4 Enzyme digestion maps of pUC18-CU plasmid and pET-28a vector.

[0030] Figure 5 PCR identification pattern of pET-28a-CU engineered bacteria: strains 1 to 8 are all positive.

[0031] Figure 6SDS-PAGE map of pET-28a-CU bacterial cell-induced expression; No. 10 is the Candida utilis uricase standard; strains 1-9 all expressed mutant uricase.

[0032] Figure 7 Stability study of uricase from Candida utilis at 2–8 °C, where 1–5 represent 0 h, 5 h, 6 h, 7 h, and 24 h of storage, respectively (x-axis: minutes, y-axis: AU).

[0033] Figure 8 Stability study of CUΔ6 urate oxidase at 2-8℃, 1-4 represent 0h, 5h, 7h and 24h respectively (x-axis minutes, y-axis AU). Detailed Implementation

[0034] The following embodiments and experimental examples are further illustrative of this application and should not be construed as limiting the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this application. The preferred embodiments and materials described herein are for illustrative purposes only.

[0035] The first aspect of this application provides a mutated uricase subunit by introducing a cysteine ​​(Cys) mutation at one or more of the following sites in the wild-type uricase subunit as shown in SEQ ID NO:4: the 6th Ser position is mutated to Cys (S6C); the 7th Ser position is mutated to Cys (S7C); and the 8th Ser position is mutated to Cys (S21C).

[0036] The mutation is used to form intermolecular (interchain) disulfide bonds. Preferably, the intermolecular disulfide bond refers to the -SS- bond formed between adjacent subunits.

[0037] In an optional embodiment, the uricase is derived from Candida utilis uricase.

[0038] In an optional embodiment, the subunit comprises an amino acid sequence as shown in SEQ ID NO: 1, 2, 3.

[0039] A second aspect of this application provides a mutant uricase comprising the mutant uricase subunit.

[0040] The uric acid oxidase mentioned refers to a class of oxidases that can catalyze the oxidation of uric acid to allantoin. Its natural form is a homotetramer, with each subunit having a molecular weight of approximately 30-35 kDa and a typical β-barrel fold structure.

[0041] In an optional embodiment, the uricase is a homologous or heterologous polymer, comprising at least one mutated uricase subunit. The C(Cys) at position 6, 7, or 8 of one mutated subunit forms an intermolecular (interchain) disulfide bond with the C(Cys) at position 293 of another subunit. Preferably, the other subunit is an adjacent subunit.

[0042] In an optional embodiment, the uricase comprises one, two, three, or four of the aforementioned mutant uricase subunits.

[0043] In an optional embodiment, the uricase is a homotetramer. Preferably, it has a compact barrel-shaped homotetramer structure.

[0044] In an optional implementation, each subunit contains an amino acid sequence SEQ ID NO:1, 2, or 3.

[0045] In an optional embodiment, the urate oxidase is a CUΔ6, CUΔ7, or CUΔ8 mutant.

[0046] In an optional implementation, the adjacent subunits are connected by disulfide bonds.

[0047] In an optional embodiment, the structure of the urate oxidase is as follows: Figure 3b As shown, the C6 / C7 / C8 of the first subunit forms disulfide bonds with the C293 of the adjacent second subunit; the C6 / C7 / C8 of the second subunit forms disulfide bonds with the C293 of the adjacent third subunit; the C6 / C7 / C8 of the third subunit forms disulfide bonds with the C293 of the adjacent fourth subunit; and the C6 / C7 / C8 of the fourth subunit forms disulfide bonds with the C293 of the first subunit. (The first, second, third, and fourth subunits are for descriptive purposes only.)

[0048] In an optional embodiment, the uricase has the following properties:

[0049] 1) Specific activity greater than 1.8 U / mg (detected by ultraviolet spectrophotometry);

[0050] 2) No urate oxidase polymers were formed after being placed at 2–8°C for 24 hours (detected by SEC-HPLC).

[0051] Preferably, the specific activity is greater than 2.5, 3, 4, 5, or 6 U / mg.

[0052] Uric acid oxidase polymers are high-molecular-weight abnormal aggregates, such as octamers or fibrous structures. These abnormal aggregates lead to reduced enzyme activity and may also trigger immunogenic reactions.

[0053] 1U is defined as the amount of enzyme required to hydrolyze 1 μmol of uric acid in 1 minute, and is detected by ultraviolet spectrophotometry at pH 7.2 and temperature 25±0.5℃.

[0054] A third aspect of this application provides a biomaterial selected from any of the following:

[0055] A1: An isolated nucleic acid molecule encoding the mutated uricase subunit as described in claim 1;

[0056] A2: An expression vector containing the nucleic acid molecules described in A1;

[0057] A3: Host cells containing the vector described in A2 or host cells whose genome integrates the nucleic acid molecules described in A1.

[0058] The nucleic acid molecules used in this application may be selected from the following group: DNA molecules, RNA molecules, or hybrid molecules thereof. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The sequences of the uricase and its subunit DNA molecules used in this application can be obtained using conventional techniques, such as PCR amplification. Alternatively, the relevant sequences can be synthesized artificially.

[0059] This application also relates to expression vectors comprising the aforementioned nucleic acid molecules and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins. The expression vectors can be viruses or plasmids, such as pUC18 vectors or pET-28a vectors.

[0060] This application also relates to host cells containing the aforementioned vector or genome-integrated nucleic acid molecules. Any host cell capable of effectively replicating and expressing the carried nucleic acid molecule is acceptable. The host cells include prokaryotic expression cells and eukaryotic expression cells. The prokaryotic cells may be selected from *Escherichia coli*, *Bacillus subtilis*, or *Corynebacterium glutamicum*, such as BL21(DE3). The eukaryotic cells may be selected from *Pichia pastoris*, *Aspergillus niger*, or *Streptomyces*.

[0061] A fourth aspect of this application provides a conjugate comprising the mutant uricase subunit or the mutant uricase.

[0062] In an optional implementation, the coupling compound is a polyethylene glycol-modified coupling compound.

[0063] A fifth aspect of this application provides a pharmaceutical composition comprising the aforementioned mutated uricase and pharmaceutically acceptable excipients.

[0064] The sixth aspect of this application provides the use of the mutated uricase and the pharmaceutical composition in the preparation of a medicament for: 1) breaking down or lowering uric acid levels; 2) preventing or treating hyperuricemia or gout; and 3) treating tumor lysis syndrome.

[0065] In an optional implementation, the gout is chronic gout or refractory gout.

[0066] In an optional implementation, the hyperuricemia is CKD complicated with hyperuricemia.

[0067] Tumor lysis syndrome (TLS) typically occurs during cancer treatment, especially in chemotherapy-sensitive, rapidly growing tumors such as leukemia and lymphoma. When a large number of tumor cells are rapidly killed, intracellular substances (such as uric acid) are suddenly released into the bloodstream, causing metabolic disturbances and potentially leading to serious complications such as acute kidney injury. Rasburicase's treatment for tumor lysis syndrome is currently in phase III clinical trials.

[0068] The seventh aspect of this application provides a method for preparing the mutant uricase, comprising culturing the host cells to express the uricase, and isolating the uricase from the culture.

[0069] The eighth aspect of this application provides a method for obtaining the mutated uricase subunit, comprising molecular modeling of the uricase.

[0070] In an optional implementation, the molecular modeling includes the following steps:

[0071] 1) The Swiss-model online software was used to perform homology modeling of Candida utilis uricase, with the modeling reference sequence being Aspergillus flavus uricase;

[0072] 2) Evaluate the model from step 1) using the GMQE model and the QMEAN Z-Scores model. The evaluation metrics are the GMQE value, the QMEAN Z-Scores score, and the Seq Identity value.

[0073] 3) Perform Swiss-Pdb Viewer analysis on the molecular model to assess its molecular conformational energy;

[0074] 4) Perform Ramachandran Plots analysis on the molecular model to evaluate its stereochemical characteristics;

[0075] 5) Perform ProSA analysis on the molecular model.

[0076] In an optional implementation, the method for obtaining the mutated uricase subunit further includes model-based analysis of the mutation pattern; and determining the mutation pattern through performance screening, such as specific activity and stability performance.

[0077] The present application will be further described below with reference to specific embodiments.

[0078] Experimental materials: Tryptone was purchased from Shanghai Guangmao Biochemical Products Co., Ltd.; Ammonium sulfate was purchased from Shenyang Chemical Reagent Factory; K2HPO4 was purchased from Shenyang Chemical Reagent Factory; NaH2PO4·H2O was purchased from Hunan Jiudian Pharmaceutical Co., Ltd.; NaCl was purchased from Tianjin Haiguang Pharmaceutical Co., Ltd.; Glycerol was purchased from Shenyang Chemical Reagent Factory; Isopropanol was purchased from Shenyang Chemical Reagent Factory; BamHI was purchased from NEB; HindIII was purchased from NEB; T4 ligase was purchased from Promega; Plasmid Mini Kit was purchased from QIAGEN; QIAquick Gel Extraction Kit was purchased from QIAGEN; SYBRsafe DNA gel stain was purchased from Invitrogen.

[0079] Unless otherwise specified in the embodiments, any techniques or conditions not described in the literature in this field shall be carried out in accordance with the techniques or conditions described in the literature. Reagents, drugs and instruments whose sources are not listed can be obtained through conventional commercial means and will not be described in detail here.

[0080] Example 1

[0081] 1. Construction of a molecular model of uricase

[0082] The amino acid sequence of Candida utilis uricase, obtained from GenBank, is shown in SEQ ID NO.4. Homology modeling of Candida utilis uricase was performed using the Swiss-model online software, with the reference sequence being Aspergillus flavus uricase (PDB ID: 6rgt). The GMQE (Global Model Quality Estimation) and QMEAN Z-Scores (Qualitative Model Energy Analysis) models were used. Evaluation metrics included GMQE value, QMEAN Z-Score, and Seq Identity value. GMQE values ​​ranged from 0 to 2, with higher values ​​indicating better modeling quality. QMEAN Z-Scores typically ranged from -4 to 0, with scores closer to 0 indicating a higher match between the model and the actual structure. Sequence homology (Seq Identity value) above 35% indicated that the aligned sequences belonged to the same family, i.e., homologous proteins. The results showed that the GMQE score of uricase from Aspergillus flavus was 0.82, the QMEANZ-Score was -1.40, and the Seq Identity was 48.82%, indicating successful modeling. The simulated crystal structure diagram is detailed below. Figure 1 The constructed molecular model was analyzed using Swiss-Pdb Viewer to assess whether its molecular conformation energy was minimum. The calculated total energy was -59930.469 kJ / mol, confirming that the model possesses a low molecular potential energy. Ramachandran Plots analysis of the molecular model showed that the amino acid residues in the Favored region accounted for more than 90% of the total protein, indicating that the model's conformation conforms to stereochemical rules. The results showed that 95.48% of the amino acid residues fell into the Favored region, and 0.79% fell into the Outlier region. The predicted model was analyzed using ProSA online software; the model structure score is represented by black dots. Within the score range of proteins of similar size, the results are detailed in Figure 2, further confirming the successful modeling.

[0083] MSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGGFDTSYTEADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRITDLYYKRSGDYKLSSAIKDLT VLKSTGSMFYGYNKCDFTTLQPTTDRILSTDVDATWVWDNKKIGTVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKL(SEQ ID NO.4)

[0084] 2. Structural modification for the stability of uricase

[0085] The uricase molecule *Candida utilis* has a compact barrel-shaped structure and poor thermal stability; noticeable aggregation can be detected after 5 hours at 2–8°C. This uricase molecule contains four Cys groups, located at positions 39, 168, 250, and 293 of the amino sequence. Based on the 3D molecular model and spatial accessibility analysis, it was found that the Cys group at position 293 is closest to the Ser groups at positions 6, 7, and 8 of another subunit. Therefore, positions 6, 7, and 8 were mutated to Cys, and the mutated sequences were named CUΔ6, CUΔ7, and CUΔ8, respectively. This promotes the formation of interchain disulfide bonds between different subunits, increasing the molecular stability. The molecular structure is shown in Figure 3. The modified amino acid sequences are shown in SEQ ID NOs 1, 2, and 3.

[0086] MSTTLCSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGGFDTSYTEADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRITDLYYKRSGDYKLSSAIKDLT VLKSTGSMFYGYNKCDFTTLQPTTDRILSTDVDATWVWDNKKIGTVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKL(SEQ ID NO.1)

[0087] MSTTLSCSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGGFDTSYTEADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRITDLYYKRSGDYKLSSAIKDLT VLKSTGSMFYGYNKCDFTTLQPTTDRILSTDVDATWVWDNKKIGTVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKL(SEQ ID NO.2)

[0088] MSTTLSSCTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGGFDTSYTEADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRITDLYYKRSGDYKLSSAIKDLT VLKSTGSMFYGYNKCDFTTLQPTTDRILSTDVDATWVWDNKKIGTVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKL(SEQ ID NO.3)

[0089] 3. Construction of engineered uricase bacteria

[0090] The three sequences were replaced with EMBOSS online software (https: / / www.ebi.ac.uk / Tools / st / emboss_backt ranseq / ) to replace the DNA sequences of E. coli with preferred codons. The whole genome sequence was synthesized by Suzhou Genewiz Company. The synthesized sequences were ligated into the pUC18 vector to obtain three engineered bacteria containing pUC18-CUΔ6, pUC18-CUΔ7 and pUC18-CUΔ8 plasmids, respectively. They were then stored in an ultra-low temperature freezer at -70℃.

[0091] 4. Expression of urate oxidase

[0092] The pUC18-CUΔ6, pUC18-CUΔ7, pUC18-CUΔ8, and pET-28a vector strains were removed from the -70℃ cryogenic freezer and inoculated onto LB agar plates, then cultured at 37℃ for 18 h. Single colonies were inoculated into 10 ml of LB liquid medium with antibiotics added, and cultured at 37℃ with shaking at 250 rpm for 18 h. The bacterial culture was then transferred to 10 ml centrifuge tubes and centrifuged at 10000×g for 1 min to collect the bacterial pellet. Using the Plasmid Mini Kit, 500 μl of P1 solution was added to resuspend the bacterial pellet, followed by 500 μl of P2 solution, which was inverted 4–6 times to mix thoroughly. Then, 700 μl of P3 solution was added and gently mixed. The pellet was centrifuged at 15000×g at 4℃ for 30 min, and the supernatant was added to an adsorption column and centrifuged at 12000×g for 1 min. Finally, 700 μl of QC solution was added, and the pellet was centrifuged at 12000×g for 1 min. Add 50 μl of QF solution, centrifuge at 12000×g for 1 min to elute and recover the plasmid.

[0093] The *Escherichia coli* BL21(DE3) strain stored in our laboratory was removed from the -70℃ ultra-low temperature freezer and inoculated onto LB agar plates, then cultured at 37℃ for 18 h. Single colonies were then inoculated into LB liquid medium and cultured at 37℃ with shaking at 250 rpm for 18 h to obtain the primary culture. The primary culture was then inoculated at a 1:50 ratio into 50 ml of LB liquid medium and cultured at 37℃ with shaking at 250 rpm until OD reached. 600 The value should be between 0.5 and 0.7. In a clean bench, transfer the bacterial suspension to a 50ml centrifuge tube and incubate on ice for 30 minutes. Centrifuge the chilled suspension at 4000×g, 4℃ for 15 minutes. Resuspend the precipitate in 50ml of pre-chilled 10% sterile glycerol and centrifuge at 4000×g, 4℃ for 15 minutes. Resuspend the precipitate again in 25ml of pre-chilled 10% sterile glycerol and centrifuge at 4000×g, 4℃ for 15 minutes. Aliquot 0.5ml of the pre-chilled 10% sterile glycerol resuspending solution and store at -70℃ for later use.

[0094] Take 17 μl each of pUC18-CU plasmid and pET-28a vector, add 2 μl of NE Buffer 2.1 restriction buffer, and add 1 μl each of BamHI and HindIII restriction endonucleases. Digest at 37℃ for 2 h. Perform agarose gel electrophoresis on the digested products. Weigh 0.37 g of low-melting-point agarose and place it in a shake flask. Add 30 ml of 1×TAE electrophoresis buffer and heat in a microwave oven until the agarose is completely melted. Add 3 μL of SYBR safe DNA gelstain, shake well, pour into a gel well, and insert a suitable comb. Use 1×TAE electrophoresis buffer and perform electrophoresis at a constant voltage of 100V. After electrophoresis, place the gel on a transilluminator plate and observe the band positions. See the enzyme digestion pattern for details. Figure 4 .

[0095] Using the QIAquick Gel Extraction Kit, gel strips containing uricase and pET-28a vector DNA bands were excised from the gel. The strips were weighed, and three volumes of QG buffer were added according to the weight. The gels were then placed in a 50°C water bath until completely melted, and one volume of isopropanol was added and mixed thoroughly. The mixture was then added to a purification column and centrifuged at 12000×g for 1 min. 500 μL of QG buffer was added to the purification column and centrifuged at 12000×g for 1 min. 750 μL of PE buffer was added to the purification column and centrifuged at 12000×g for 1 min. 50 μL of EB buffer was added and centrifuged at 12000×g for 1 min to elute and recover the target DNA fragment. Using T4 ligase, the uricase fragment and pET-28a vector were mixed at a 3:1 ratio and incubated overnight at 4°C to construct the pET-28a-CU expression plasmid. The expression plasmid was electroporated into *E. coli* BL21(DE3) to construct the engineered strain. PCR identification confirmed that all electroporation results were positive; details of the identification results can be found in [link to relevant documentation]. Figure 5 .

[0096] The engineered uric acid oxidase bacteria were removed from a -70°C cryogenic freezer and inoculated onto LB agar plates, then cultured at 37°C for 18 hours. Single colonies were inoculated into 10 ml of LB liquid medium with the addition of Kana antibiotic, and cultured at 37°C with shaking at 250 rpm for 18 hours to obtain the primary culture. The primary culture was then inoculated at a 1:50 ratio into 50 ml of LB liquid medium and cultured at 37°C with shaking at 250 rpm until OD reached [value missing]. 600 The values ​​were between 0.6 and 0.7. IPTG solution was added to a final concentration of 0.5 mmol / L, and the cells were incubated at 37°C with shaking at 250 rpm for 3 h to induce uricase expression. SDS-PAGE analysis of the expressing bacterial cells confirmed that all strains expressed uricase; detailed identification results can be found in [link to relevant documentation]. Figure 6 .

[0097] Example 2

[0098] 1. Protein content analysis of uricase:

[0099] The wavelength of the UV spectrophotometer was set to 595 nm. The standard was diluted with ultrapure water, and the uricase sample was also diluted. After reacting for 10 min, all samples were transferred to disposable cuvettes, and data were read on the UV spectrophotometer at 595 nm. The absorbance was zeroed using a blank solution, yielding the least squares equation y = kx (where k is the slope, x is the concentration, and y is the absorbance value at A595). The protein content of uricase was calculated using the formula C(mg / ml) = y ÷ k × A ÷ 1000.

[0100] 2. Detection of biological activity of urate oxidase:

[0101] Set the wavelength of the UV spectrophotometer to 293 nm and connect the flow cell to a water bath. Take 100 μl of 1200 μmol / L uric acid and add 800 μl of 100 mmol / L phosphate buffer (pH 7.2). Incubate at 25 °C for 10 min, then add 100 μl of uricase. Mix well and immediately pour the entire solution into a cuvette. Place the cuvette in the UV spectrophotometer and read the absorbance at 293 nm. At 25 °C, read the absorbance every 20 seconds for 10 min. Calculate the slope and correlation coefficient of the kinetic curve for the enzymatic reaction. Calculate the specific activity using the following formula.

[0102] (ΔA / t) / (ε·1)=ΔC / t=(Δμmol / minμts)=IU's

[0103] Uric acid oxidase activity (U / ml) = U's × dilution factor = (ΔA / t) × dilution factor ÷ (ε·1)

[0104] Specific activity (U / mg) = (ΔA / t) × dilution factor ÷ (ε·1) ÷ protein content, ε = 12.6 mmol / L -1 ·cm -1

[0105] The test results are detailed in Table 1 below:

[0106] Table 1

[0107]

[0108] 3. Stability study of uricase

[0109] The solutions of Candida utilis and CUΔ6 uricase were prepared in 20 mmol / L phosphate buffer (pH 7.5) and incubated at 2–8 °C for 5 h, 6 h, 7 h, and 24 h before liquid chromatography purity determination. A TSK-gel G3000SW (7.5 × 600 mm, 5 μm) column was used with a mobile phase of 13.37 mmol / L phosphate buffer, a flow rate of 1 mL / min, a column temperature of room temperature, and a UV detector at 280 nm. The injection volume was 40 μL, and the detection time was 30 min. The results for the detection of Candida utilis uricase are shown below. Figure 7Automatic integration of the liquid chromatography chromatograms using Empower software revealed that polymers of Candida utilis uricase could be detected after 5 hours of incubation, and after 6 hours, the proportion of the polymer peak area exceeded 5% of the main peak. Slight turbidity was observed in the sample after 24 hours. The detection results for CUΔ6 uricase are shown below. Figure 8 The automatic integration of the liquid phase spectrum using Empower software showed that no aggregates were formed even after the sample was left to stand for 24 hours, proving that CUΔ6 has significantly improved stability than Candidautilis uricase under conditions of 2–8 °C.

[0110] It should be understood that, within the scope of this application, the above-described technical features of this application and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

Claims

1. A mutated uricase subunit, characterized in that, Cysteine ​​(Cys) mutations are introduced at one or more of the following sites in the wild-type uricase subunit as shown in SEQ ID NO:4: Ser at position 6 is mutated to Cys (S6C); Ser at position 7 is mutated to Cys (S7C); Ser at position 8 is mutated to Cys (S21C).

2. The mutated uricase subunit as described in claim 1, characterized in that, The subunit comprises an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

3. A mutant uricase, characterized in that, It contains the uricase subunit as described in claim 1.

4. The mutated uricase as described in claim 3, characterized in that, The urate oxidase is a homotetramer.

5. The mutated uricase as described in claim 4, characterized in that, It has the following properties: 1) Specific activity greater than 1.8 U / mg (detected by ultraviolet spectrophotometry); 2) No urate oxidase polymers were formed after being placed at 2–8°C for 24 hours (detected by SEC-HPLC).

6. A biomaterial, characterized in that, Choose from any of the following: A1: An isolated nucleic acid molecule encoding the mutated uricase subunit as described in claim 1; A2: An expression vector containing the nucleic acid molecules described in A1; A3: A host cell containing the vector described in A2 or a host cell whose genome integrates the nucleic acid molecule described in A1.

7. A coupling, characterized in that, Contains the mutated uricase subunit as described in claim 1 or the mutated uricase as described in claim 3; preferably, the conjugate is a polyethylene glycol conjugate.

8. A pharmaceutical composition, characterized in that, It contains the mutated uricase as described in claim 3, or the conjugate as described in claim 7, and pharmaceutically acceptable excipients.

9. Use of the mutated uricase of claim 3 and the pharmaceutical composition of claim 8 in the preparation of a medicament, wherein the medicament is used for: 1) decomposing or lowering uric acid levels; 2) preventing or treating hyperuricemia or gout; 3) Treatment of tumor lysis syndrome.

10. A method for preparing the mutant uricase of claim 3, characterized in that, This includes culturing the host cells of claim 6 to express the uricase, and isolating the uricase from the culture.

11. A method for obtaining the mutated uricase subunit of claim 1, characterized in that, This includes molecular modeling of uricase; preferably, the molecular modeling includes the following steps: 1) Homology modeling of Candida utilis uricase was performed using Swiss-model, with the modeling reference sequence being Aspergillus flavus uricase; 2) Evaluate the model from step 1) using the GMQE model and the QMEAN Z-Scores model. The evaluation metrics are the GMQE value, the QMEAN Z-Scores score, and the Seq Identity value. 3) Perform Swiss-Pdb Viewer analysis on the molecular model to assess its molecular conformational energy; 4) Perform Ramachandran Plots analysis on the molecular model to evaluate its stereochemical characteristics; 5) Perform ProSA analysis on the molecular model.

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