Heat-resistant uric acid oxidase TCI-UOX2 and application thereof

Abstract

The application discloses a heat-resistant uric acid oxidase TCI-UOX2 and application, and relates to the technical field of biology.The amino acid sequence of the heat-resistant uric acid oxidase TCI-UOX2 is shown as SEQ ID NO:1.The recombinant uric acid oxidase TCI-UOX2 shows the highest activity at 35 DEG C and pH 8, and still maintains about 100 percent relative activity at 5 DEG C to 40 DEG C.The enzyme still maintains about 100 percent residual activity after being incubated at the human physiological temperature of 37 DEG C for 12 hours.Under the anticoagulation environment of a high-uric-acid volunteer, the uric acid concentration can be reduced to the normal level of 360 micromoles per liter within 6 hours of reaction with the low-concentration 1 microgram per milliliter TCI-UOX2 enzyme solution.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a heat-resistant urate oxidase TC1-UOX2 and its applications. Background Technology

[0002] Uric acid (UA), the final product of purine metabolism, accumulates in serum and joints. Because humans lack the expression of uricase, this substance cannot be further metabolized. Uricase, an important member of the oxidoreductase family, plays a crucial role in the degradation of purines. This enzyme catalyzes the oxidative degradation of uric acid, producing allantoin as the main product, while generating H₂O₂ and CO₂ as byproducts. This catalytic process effectively inhibits the excessive accumulation of uric acid in the body, thus avoiding its potentially harmful effects. Uric acid oxidase is an endogenous enzyme present in most mammals, but not in humans. During primate evolution, the inactivation of the uricase gene in apes was caused by independent nonsense or frameshift mutations, undergoing a two-step degradation process involving the promoter and coding region. Two nonsense mutations were found in the human uricase gene, confirming at the molecular level that the human uricase gene is nonfunctional. When uricase is deficient, elevated uric acid levels can lead to hyperuricemia, which can damage kidney health and cause uric acid to bind with sodium, forming sodium urate crystals. These crystals accumulate in joints and serum, potentially triggering gout, an inflammatory disease. Gout is a chronic inflammatory arthritis caused by the deposition of monosodium urate (MSU) due to elevated serum uric acid (SU). When SU ​​levels consistently exceed the solubility limit of 6.8 mg / dL, MSU crystals precipitate from the solution and accumulate in joints and extra-articular spaces. When first- and second-line therapies for lowering SU fail at the medically acceptable maximum dose and gout symptoms continue to worsen, refractory or uncontrolled gout develops.

[0003] In recent years, the incidence of gout has been on the rise. According to the latest research, the global prevalence of gout ranges from 1% to 4%, while the annual incidence rate is between 0.1% and 0.3%. Frequent gout attacks can trigger a series of serious health problems, including but not limited to hypertension, hyperlipidemia, atherosclerosis, chronic kidney disease, osteoporosis, atrial fibrillation, and venous thromboembolism.

[0004] As research progresses, the application potential of Uox in uric acid detection and the treatment of gout and hyperuricemia is gradually being discovered. The method of detecting uric acid using Uox has the advantages of simplicity, speed, and high specificity, making it a commonly used clinical testing method. Furthermore, Uox can be directly injected during disease treatment to rapidly lower the uric acid level in the blood, and is used for long-term treatment of gout.

[0005] Regarding the potential role of uricase in the treatment of severe tophaceous gout, only a few case reports have been documented. Phillips and his team successfully treated three heart transplant patients with non-recombinant uricase, who had previously suffered from uncontrolled gout. After treatment, all patients experienced a reduction in tophi size and significant improvement in finger mobility. Furthermore, Phillips and his team treated a patient with severe tophaceous gout and end-stage renal disease with raburicase. This patient had previously been unresponsive to high-dose allopurinol treatment, but after raburicase treatment, their tophi significantly subsided. Notably, all patients treated with uricase tolerated the treatment well without any adverse reactions. These successful case reports provide strong support for the application of uricase in the treatment of gout.

[0006] A wide variety of organisms can produce uricase, including bacteria, fungi, plants, and animals other than humans. Microorganisms are prioritized for enzyme production primarily because of their significant simplicity in enzyme production, purification, and process optimization. Therefore, *E. coli* strains used for drug production have gained widespread acceptance both domestically and internationally due to their clear genetic background, short fermentation cycle, simple cultivation, mature gene modification techniques, and readily available gene editing tools, making them ideal strains for constructing recombinant uricase-producing strains.

[0007] This invention utilizes metagenomics to identify SOD functional genes in soil from the Tengchong Geothermal Experience Zone in China. Due to the constant temperature at the sampling site year-round, this environmental enzyme exhibited significant thermal stability under mesophilic conditions. Experiments demonstrated that the recombinant uricase showed activity over a wide range of temperatures, pH values, and in the presence of various denaturants and chemicals. Summary of the Invention

[0008] In view of this, the present invention provides a heat-resistant urate oxidase TC1-UOX2 and its application.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A thermostable uricase TC1-UOX2, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0011] A DNA molecule encoding the thermostable urate oxidase TC1-UOX2 of claim 1, wherein the nucleotide sequence of the DNA molecule is shown in SEQ ID NO: 2.

[0012] A recombinant vector comprising the DNA molecule of claim 2 and a regulatory sequence for expression operably linked to the DNA molecule.

[0013] The host cell comprises the DNA molecule of claim 2 or the recombinant vector of claim 3.

[0014] Application of a heat-resistant urate oxidase TC1-UOX2 in the degradation of uric acid.

[0015] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] This invention identified a urate oxidase gene from metagenomic data of soil from the Rehai geothermal area of ​​Tengchong City, Yunnan Province. Through gene synthesis, heterologous expression, and enzyme activity assays, a novel thermostable urate oxidase gene (named TC1-UOX2) was screened out. This gene showed the highest similarity (71.17%) to the urate oxidase from *Pyrinomonadaceae bacterium*, with a molecular weight of 31.7 kDa and a pI value of 5.89. The gene was heterologously expressed in *E. coli*, purified by Ni column affinity chromatography, and its enzymatic properties were determined. The results showed that the recombinant urate oxidase TC1-UOX2 exhibited good activity at 35℃ and pH... The enzyme exhibits peak activity at 8°C and maintains approximately 100% relative activity within the 5-40°C range. After incubation at the human physiological temperature of 37°C for 12 hours, it retains approximately 100% residual activity. In anticoagulant conditions in volunteers with high uric acid, reaction with a low concentration of 1 μg / mL TC1-UOX2 enzyme solution can reduce uric acid concentration to the normal level of 360 μmol / L within 6 hours. Furthermore, this enzyme is resistant to different concentrations of ions, denaturants, reducing agents, and inhibitors. Compared to most reported uricases, recombinant uricase TC1-UOX2 exhibits higher thermostability. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 The attached figure shows the phylogenetic tree obtained by the present invention based on amino acid sequences through maximum likelihood analysis;

[0019] Figure 2 The attached figure is an electrophoresis diagram of the TC1-UOX2 gene amplification of the present invention. Lanes 1-6 are for identification of TC1-UOX2 gene amplification.

[0020] Figure 3 The attached figure shows the PCR electrophoresis diagram of the TC1-UOX2 colonies of this invention. Lanes 1-4 are for identification of positive PCR clones of the colonies.

[0021] Figure 4 The attached figure shows the polyacrylamide gel electrophoresis of the recombinant protein TC1-UOX2 of this invention; M is a protein molecular weight marker labeled with mass; lane 1 shows the total protein of E. coli DH5α / pSHY211-TC1-UOX2; lane 2 shows the purified TC1-UOX2;

[0022] Figure 5 The attached figure illustrates the effects of temperature and pH on the activity of recombinant uricase TC1-UOX2. Figure 5 A represents the effect of temperature on the activity of TC1-UOX2; Figure 5 B represents the effect of pH on TC1-UOX2 activity; Figure 5 C represents the effect of temperature on stability; Figure 5 D represents the effect of pH on stability;

[0023] Figure 6 The attached figure shows the uric acid degradation curve of TC1-UOX2 under in vitro physiological conditions according to the present invention;

[0024] Figure 7 The attached figure shows the uric acid degradation curve of TC1-UOX2 under in vitro blood conditions according to the present invention. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1

[0027] Materials and Methods

[0028] Sample collection and metagenomic sequencing

[0029] Soil samples were collected from the Rehai Experience Area in Tengchong City, Baoshan City, Yunnan Province, at coordinates: latitude 24.95059613°N, longitude 98.43853158°E. Samples were rapidly frozen on dry ice and then enriched for laboratory DNA isolation and metagenomic sequencing. DNA isolation was performed using the MOBIO dnasy PowerSoil Kit (USA) according to the instruction manual. A HiSeq 2500GENWI instrument was used for metagenomic sequencing. De novo assembly was performed using the elvet assembly program version. The IMG website (https: / / img.jgi.doe.gov / cgi-bin / mer / main.cgi) was used for sequence lookup and analysis. The potential functions of individual genes and open reading frames (ORFs) were analyzed using the COG, KEGG, and Pfam databases.

[0030] Uric acid oxidase sequence prediction and sequence analysis (analysis of uricase genes predicted from hot springs)

[0031] Based on functional prediction, the sequence of a uricase gene (named TC1-UOX2) was isolated from a metagenomic database. The nucleotide sequence of the uricase (TC1-UOX2) gene was submitted to GenBank with accession number XXXXX. Sequence homology analysis was performed using the BLAST program provided by NCBI (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The signal peptide was predicted using SignalP (http: / / www.cbs.dtu.dk / services / SignalP / ). The primary structure of the amino acid sequence was derived and analyzed using the EXPASY tool (http: / / web.expasy.org / protparam / ). Phylogenetic analysis of the uricase gene (TC1-UOX2) was performed using MEGA 11 software, and a phylogenetic tree was constructed using the maximum likelihood (ML) method with a Poisson regression model. The sequence of the recombinase TC1-UOX2 was compared with protein structure data in a protein database (http: / / www.rcsb.org / ).

[0032] The amino acid sequence (281 residues) of the TC1-UOX2 enzyme is shown in SEQ ID NO: 1;

[0033] MPARIVQDNYGKSRVRLIKVDRSGPRHELQNLTVNIALEGDFTAIHTTGDNSLCLPTDTMKNTVYALAGETVHIEDADSFGRRLARYFLDNNAHVTAARIELIEHAWKRMKFDGTEHDHSFMCGSREKRMSWVNDTGNGVTIESGVEELIVLKTTRSGFVGFIKDKYTTLPETTDRIFATSIKARWRYGDPDAATAEAFRSIRETIIRVFAGHDSLSVQHTLYAMGNQVLEDFPQVEEIAFSLPNIHCLPIDMTKLGQENDNRIFVPTDEPHGLIEARLAR-;

[0034] The nucleotide sequence (846 bp) of the TC1-UOX2 gene, as shown in SEQ ID NO: 2;

[0035] ATGCCGGCCAGGATAGTTCAGGACAATTACGGCAAATCCCGTGTCCGGCTTATCAAGGTCGATCGCTCCGGCCCACGCCACGAACTCCAGAACCTTACGGTCAACATCGCTCTCGAAGGCGACTTCACTGCCATTCACACCACGGGCGATAACAGTCTGTGCCTGCCGACCGACACGATGAAGAACACGGTCTATGCCCTCGCGGGCGAGACCGTGCATATCGAGGACGCTGACAGTTTTGGGCGTCGACTGGCCCGCTATTTTCTCGACAACAACGCGCACGTCACCGCAGCGCGCATCGAGCTGATCGAGCACGCGTGGAAGCGGATGAAATTTGACGGCACCGAACACGATCATTCCTTTATGTGCGGGAGCCGCGAGAAACGAATGTCCTGGGTCAACGACACTGGCAACGGCGTGACGATCGAATCCGGTGTTGAAGAACTGATAGTCCTCAAGACGACCAGGTCGGGCTTTGTCGGATTCATTAAGGACAAGTACACTACGCTGCCCGAGACGACCGACCGCATCTTTGCGACATCGATCAAGGCCCGGTGGCGATACGGCGATCCTGATGCGGCCACAGCCGAAGCGTTTCGAAGCATTCGCGAAACGATCATCCGCGTATTCGCGGGCCACGACAGTCTGTCGGTCCAGCATACGCTCTACGCGATGGGCAACCAGGTGCTGGAGGATTTTCCGCAGGTCGAAGAGATCGCGTTCTCGCTGCCAAACATACACTGCCTGCCGATCGATATGACAAAGCTCGGCCAGGAAAACGACAACCGCATCTTTGTCCCGACCGACGAACCGCATGGCCTGATCGAGGCCCGGCTGGCCCGCTAA;

[0036] DNA samples obtained from soil (28.4℃, pH 3.0) in a geothermal experience area were sequenced. A search for uricase in the total sequence revealed a novel candidate uricase gene sequence, named TC1-UOX2. Nucleotide sequence analysis of the TC1-UOX2 gene showed an ORF length of 846 bp, encoding a uricase protein of 281 amino acid residues; no signal peptide sequence was found. The theoretical calculated molecular size of the recombinant protein TC1-UOX2 is 31.7 kDa, and the theoretical pI is 5.89. The amino acid sequence similarity of TC1-UOX2 to uricases from different genera was 86.01%, 86.01%, and 85.49%, respectively, originating from (NCBI accession NO.: MEO6051131.1 Pyrinomonadaceae bacterium); from (NCBI accession NO.: MBK8465393.1 Chloracidobacterium sp.); and from (NCBI accession NO.: HEV7646020.1 Pyrinomonadaceae bacterium). Phylogenetic analysis of the protein sequences from these genera showed that... Figure 1 TC1-UOX2 aggregates with factor-independenturate hydroxylase from Pyrinomonadaceae bacterium.

[0037] Figure 1 This is a phylogenetic tree obtained through maximum likelihood analysis based on amino acid sequences, showing the relative position of TC1-UOX2 with related uricases in the phylogenetic tree. Bootstrap values ​​are given at each node, expressed as a percentage of 1000 repetitions.

[0038] Amplification, molecular cloning, and heterologous expression of SOD enzyme genes

[0039] Amplification of the target gene for uricase oxidase

[0040] DNA extraction was performed using the metagenomic extraction kit (MOBIO dnasy PowerSoil Kit, USA) according to the instruction manual. The extracted DNA was then examined using agarose gel electrophoresis to verify the DNA extraction results (see [link to manual]). Figure 2 Based on the conserved sequence of the uricase gene known in the GenBank database, upstream and downstream primers were designed to amplify the uricase gene.

[0041] The full-length uricase gene was amplified by PCR using the following primers:

[0042] TC1-UOX2-F:

[0043] CATCATCATCATCATCATGAAATGCCGCCAGGATAGTTCAGG, as shown in SEQ ID NO: 3;

[0044] TC1-UOX2-R:

[0045] GTGCTCGAGTGCGGCCGCAAGGCGGGCCAGCCGGGCCTC, as shown in SEQ ID NO: 4.

[0046] PCR was performed using TransStarFastPfu Fly DNA polymerase (TransGen Biotech, China). The underlined sequence, homologous to the recombination fragment of the pSHY211 vector, was digested with BamHI and HindIII. The PCR program consisted of the following: denaturation at 95°C for 3 minutes, followed by 30 cycles of 98°C for 20 seconds, 65°C for 30 seconds, and 72°C for 45 seconds, and a final incubation at 72°C for 5 minutes for final extension. The PCR product was inserted into pSHY211 using the pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, China) to generate the expression plasmid pSHY211-TC1-UOX2. The generated vector was transformed into E. coli DH5α, and positive clones of pSHY211-TC1-UOX2 were screened and sequenced.

[0047] Cloning and molecular analysis of the TC1-UOX2 gene

[0048] Plasmid DNA was transformed into the host *E. coli* DH5α to construct recombinant engineered bacteria. After overnight incubation, colonies grew on LB agar plates. Positive clones were identified using standard colony PCR. The PCR system consisted of a Gold Mix (Green) and universal primers T7+ and T7-. Single clones from the LB agar plate were vortexed into 20 μL of sterile water. After thorough mixing, this mixture served as the template for colony PCR amplification. 0.5 μL of this mixture was added to the 20 μL PCR system for positive clone identification. Figure 3 It can be seen that the DNA fragment size of the urate oxidase functional gene TC1-UOX2 is close to 900bp, which is consistent with the expected theoretical value.

[0049] Escherichia coli colonies carrying pSHY211-TC1-UOX2 were inoculated into 100 mL LB broth containing 50 μg / mL kanamycin. The bacteria were cultured at 37°C and 180 rpm for 8 h in a shaker, followed by incubation at 25°C and 180 rpm for 12 h with shaking. Cells were collected by centrifugation at 4000 xg, and the lysate was sonicated. The supernatant was collected by centrifugation at 12000 xg for 20 min. Cell-free extracts were purified using a ni-chelate affinity column (Histrap, TransGen Biotech, China) according to previously reported methods. The purified protein was analyzed by 12% SDS-PAGE. Protein concentration was determined using a Bradford protein assay kit (Order No. C503031, Sangon Biotech, China), with bovine serum albumin as the standard.

[0050] Heterologous expression and purification of recombinant TC1-UOX2

[0051] 12% SDS-PAGE analysis showed that expression of his-labeled TC1-UOX2 in E. coli cells produced a protein band of approximately 35 kDa (see [link to analysis]). Figure 4 The experimental molecular weight of the expressed protein matches the predicted molecular weight of the monomer.

[0052] Uricase assay

[0053] The specific absorbance of uric acid is 290 nm. Uricase can oxidize uric acid to allantoin, thereby detecting the residual uric acid content and determining the enzyme activity based on the amount of uric acid reduced per unit time. Uricase activity is determined according to the kinetic assay of uricase activity, referring to the National Institutes for Food and Drug Control (Beijing, China).

[0054]

[0055] ΔA is the decrease in light density at 290 nm / min; VT (ml) is the total volume of the reaction solution; df is the dilution factor; 12.6 is the micromolar extinction coefficient at 290 nm wavelength; VE (ml) is the volume of enzyme solution added; and t is the reaction time (min).

[0056] Effects of pH and temperature on TC1-UOX2 activity

[0057] The biochemical characterization of TC1-UOX2 was determined using the redox allantoin method described above. The activity of purified TC1-UOX2 was measured at different pH values ​​(pH 3.0–8.0 prepared with citrate-disodium hydrogen phosphate buffer and pH 8.0–10.0 prepared with glycine-sodium hydroxide buffer) and temperatures (25–90 °C) to determine the optimal pH and temperature. To assess thermal and pH stability, the residual uricase activity of TC1-UOX2 was measured after continuous exposure to different temperatures (30, 35, 37, and 40 °C) for 5 days and at pH 3.0–11.0 for different durations (12 and 24 h).

[0058] Results of determination of the effects of temperature and pH on recombinant uricase oxidase TC1-UOX2

[0059] The activity of recombinant uricase TC1-UOX2 remains around 100% at temperatures ranging from 15 to 40°C, with optimal activity at 35°C. Figure 5 A) As the experimental temperature further increased to 80℃, TC1-UOX2 activity still retained more than 40% of its original value. TC1-UOX2 activity was measured within the pH range of 3.0-10.0, and the highest activity was observed at pH 8. Figure 5 B), close to the pH (7.4) under human physiological conditions.

[0060] Stability analysis results of recombinant uricase TC1-UOX2

[0061] The thermostability of the enzyme was tested at the optimum temperatures of 30℃, 35℃, 37℃, and 40℃. Experiments showed that after heating at the optimum temperature of 30℃ for 16 hours, the recombinant uricase TC1-UOX2 still retained nearly 100% of its activity. Figure 5 C), it exhibited excellent thermal stability when heated at the human physiological temperature of 37°C for up to 12 hours. pH stability analysis showed that ( Figure 5 As shown in D), after incubation at 4°C for 12 h and 24 h, it still retained at least 80% of its residual activity at pH 6.0-11.0.

[0062] Effects of metal ions and chemical reagents on the activity of TC1-UOX2

[0063] The 10 mM metal ion: Fe was evaluated in 50 mM potassium phosphate buffer (pH 7.8). 2+ Fe 3+ Mg 2+ Al 3+ K + Ca 2+ Mn 2+ Co2+ Ni 2+ Cu 2+ Zn 2+ Ag + Cd 2+ Ba 3+ Pb 2+ Effects of ions; 1% chemical reagents: DTT, SDS, PMSF, Urea; 10% chemical reagents: Tween-80, Triton X-100, DMSO, Ethanol, Methanol, IPA on the activity of TC1-UOX2.

[0064] Results of determination of the effects of metal ions and chemical reagents on the activity of TC1-UOX2

[0065] Table 1. Effects of metal ions, inhibitors, and organic solvents on the activity of recombinant uricase TC1-UOX2

[0066]

[0067]

[0068] This invention investigated the effects of different metal ions and chemical reagents on the activity of the recombinant enzyme TC1-UOX2 (Table 1). High concentrations of 10 mM Mg... 2+ Al 3+ K + Ca 2+ Mn 2+ Ba 2+ Pb 2+ It had almost no effect on TC1-UOX2, and the relative activity of the enzyme remained above 95%; 10 mM metal ion Fe 2+ Fe 3+ Cu 2+ Ag + Cd 2+ It has an inhibitory effect on enzyme activity, reducing the relative activity of the enzyme to below 20%. Under the influence of chemical reagents and organic solvents, 1% dithiothreitol, urea, ethylenediaminetetraacetic acid and 10% dimethyl sulfoxide and methanol have almost no effect on TC1-UOX2, and the relative activity of the enzyme remains above 90%; 1% SDS and 10% polysorbate 80, Triton X-100 and β-mercaptoethanol strongly inhibit the activity of recombinant enzyme TC1-UOX2.

[0069] Enzyme kinetic parameters

[0070] At the optimal reaction temperature and pH, using 0.2 mmol / L sodium borate as the reaction solution and different concentrations of uric acid (0.003–0.04 mol / L) as substrates, the reduction of substrate after 10 min of reaction was determined according to the standard curve. The Km and Vmax of the uric acid oxidase were calculated by plotting using the Lineweaver Burk method.

[0071] Enzyme kinetic parameter results

[0072] Kinetic parameters were calculated using uric acid at different concentrations (0.003–0.04 mol / L) as substrates at the optimal reaction temperature and pH. The enzyme activity of TC1-UOX2 was 84.27 U / mg, with an optimal Km value of 0.02 μM and a Vmax value of 96.15 μmol / min / mg.

[0073] Uric acid degradation capacity of enzymes under in vitro physiological conditions

[0074] In vitro, a high uric acid environment (pH 7.4) was set up using 0.9% NaCl buffer containing 600 μmol / L uric acid as the substrate. The enzymes were reacted with different concentrations (the concentration range of the enzymes used in the preliminary experiment) at 37°C. The reaction solution was removed after 20 min intervals to terminate the experiment. The remaining uric acid content was determined according to the uric acid standard curve, and the uric acid degradation curve was plotted.

[0075] Results of the enzyme's ability to degrade uric acid under in vitro physiological conditions

[0076] This experiment used a uric acid concentration below 360 μmol / L as the normal standard and set up a high uric acid environment of 0.9% NaCl with 600 μmol / L uric acid as the substrate. Different concentrations of TrUOX were added to simulate in vitro conditions (37℃, pH 7.4) for the reaction. Figure 6 After 60 minutes of reaction with a low concentration of 1 μg / mL TC1-UOX2, the uric acid concentration can reach the normal level of 360 μmol / L. A high concentration of 8 μg / mL TC1-UOX2 can reduce the uric acid concentration to the normal level within 20 minutes, and can degrade the uric acid concentration of 600 μmol / L to 0 μmol / L within 40 minutes.

[0077] Uric acid degradation capacity of enzymes under in vitro blood conditions

[0078] Based on the saline experiment, a minimum amount of enzyme was added to the anticoagulated blood samples of two volunteers with high uric acid, with 1 mL taken from each sample for the experiment. Three biological replicates were performed for each group. The uric acid content in the blood was continuously monitored every 1 hour using a uric acid analyzer (brand: Kefuyi uric acid analyzer; model: UA03-C).

[0079] Results of the enzyme's ability to degrade uric acid under in vitro blood conditions

[0080] Anticoagulated blood samples were selected from two volunteers with high uric acid, numbered 731 and 754, with uric acid concentrations of 618 μmol / L and 759 μmol / L, respectively. After reacting with 1 μg / mL TC1-UOX2 enzyme solution for 4 h and 6 h, respectively, the uric acid concentration reached the normal level of 360 μmol / L (see...). Figure 7 (420 μmol / L is the normal uric acid level for men, and 360 μmol / L is the normal uric acid level for women).

[0081] Statistical analysis

[0082] Unless otherwise stated, all trials were performed in triplicate, and the mean was used in all analyses. Results were statistically analyzed using SPSS 20.0 software and are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by the Tukey test to compare multiple treatment groups. In all comparisons, a p-value <0.05 was considered statistically significant.

[0083] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A heat-resistant uricase TC1-UOX2, characterized in that, The amino acid sequence of the heat-resistant uricase TC1-UOX2 is shown in SEQ ID NO:

1.

2. A DNA molecule encoding the thermostable uricase TC1-UOX2 of claim 1, characterized in that, The nucleotide sequence of the DNA molecule is shown in SEQ ID NO:

2.

3. A recombinant vector, characterized in that, The recombinant vector comprises the DNA molecule of claim 2 and a regulatory sequence for expression operably linked to the DNA molecule.

4. A host cell, characterized in that, The host cell comprises the DNA molecule of claim 2 or the recombinant vector of claim 3.

5. The use of the heat-resistant uricase TC1-UOX2 of claim 1 in the preparation of a medicament for treating gout.

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