Urate oxidase mutants and their application in improving the thermostability and trypsin resistance of urate oxidase

Abstract

The present invention discloses uricase mutants and their applications in improving the thermal stability and trypsin resistance of uricase, belonging to the field of biotechnology. The uricase mutants disclosed in the present invention are Y47F / T68L / T75W / D111G / K204F / F259I / K299E, T68L / T75W / K204F / K299E or T68L / T75W / K299E. The present invention mines key sites that affect thermal stability through directed evolution combined with high-throughput screening, multiple virtual screening, etc., and improves stability through mutation; mines key sites that affect anti-enzymatic performance through molecular dynamics simulation and solvent accessible surface area calculation, and improves anti-enzymatic effect through mutation. Finally, multiple key sites and mutants that affect stability and anti-enzymatic performance are excavated, and improvements in thermal stability and anti-enzymatic performance are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a uricase mutant and an application thereof in improving the thermal stability and trypsin resistance of uricase. Background Art

[0002] Urate oxidase is a key enzyme in purine metabolism, converting uric acid into allantoin, making it a promising protein-based drug. However, natural urate oxidase suffers from poor thermal stability and resistance to proteolysis. Therefore, molecular engineering is needed to enhance the thermal stability and resistance to proteolysis of urate oxidase.

[0003] Thermal stability can be modified through a combination of directed evolution and high-throughput screening, using microplates as a carrier for screening random mutation libraries. Alternatively, the B-factor strategy can be employed to computationally identify highly flexible regions and then rigidify them to enhance stability. In recent years, numerous computational design platforms have been developed, such as Fireprot, PROSS, and Grape. These platforms integrate modules such as consensus sequence analysis and unfolding free energy calculations to rapidly identify mutation sites, and have been experimentally validated in the stability modification of multiple enzymes.

[0004] Enzyme-resistance modification is also a difficult problem that needs to be solved in the design of protein drugs. Currently, the enzymatic resistance modification of protein molecules themselves is mainly divided into two directions: direct mutation and indirect enhancement. Direct mutation can be used through molecular docking and solvent-accessible surface area calculation to find the cleavage site that directly contacts the protease, and then mutate to delay the protease hydrolysis process. Indirect enhancement can be achieved by mutating residues near the cleavage site to spatially mask the cleavage site, thereby reducing the probability of contact between the cleavage site and the protease. It can also be achieved by artificially designing N-glycosylation modifications to block the contact between the protease and its cleavage site, thereby achieving the purpose of protease hydrolysis.

[0005] Therefore, providing uricase mutants and their use in improving the thermal stability and trypsin resistance of uricase is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a uricase mutant and its use in improving the thermal stability and trypsin resistance of uricase.

[0007] The present invention uses directed evolution and rational design to explore key point mutations that affect the thermal stability of uricase, and then significantly improves the thermal stability of uricase through combined mutations and iterative evolution. At the same time, this process also improves the activity and resistance to trypsin hydrolysis of uricase. Further, a strategy of solvent accessible surface area calculation is used to identify cleavage sites with a high probability of exposure to water environments, explore sites that affect resistance to trypsin hydrolysis, and significantly improve the resistance to enzymatic hydrolysis through combined mutations.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] Urate oxidase mutants, Y47F / T68L / T75W / D111G / K204F / F259I / K299E (M7), T68L / T75W / K204F / K299E (M4*), T68L / T75W / K299E (M3*), T68L / T75S / E222D / K299E (M4), T68L / T75S / K299E (M3), T75S / K299E (M2), T75W, K299E, K91R, T68L, D184W, K204F, D134F, A97K, or E222D;

[0010] The amino acid sequence of the urate oxidase (UOX) is shown in SEQ ID NO.1.

[0011] MSAVKAARYGKDNVRVYKVHKDEKTGVQTVYEMTVCVLLEGEIETSYTKADNSVIVATDSIKNTIYITAKQNPVTPPELFGSILGTHFIEKYNHIHAAHVNIVCHRWTRMDIDGKPHPHSFIRDSEEKRNVQVDVVEGKGIDIKSSLSGLTVL SEQ ID NO.1.

[0012] Furthermore, the uricase mutant is Y47F / T68L / T75W / D111G / K204F / F259I / K299E (M7), T68L / T75W / K204F / K299E (M4*) or T68L / T75W / K299E (M3*).

[0013] Furthermore, the biological material related to the urate oxidase mutant is at least one of the following (1)-(4):

[0014] (1) a nucleic acid molecule encoding a urate oxidase mutant;

[0015] (2) an expression cassette containing the nucleic acid molecule described in (1);

[0016] (3) a recombinant vector containing the nucleic acid molecule described in (1) or a recombinant vector containing the expression cassette described in (2);

[0017] (4) A recombinant microorganism containing the nucleic acid molecule described in (1), a recombinant microorganism containing the expression cassette described in (2), or a recombinant microorganism containing the recombinant vector described in (3).

[0018] Furthermore, the use of the uricase mutant or the related biomaterial in improving the thermal stability of uricase.

[0019] Furthermore, the uricase mutant is used to improve the thermal stability and trypsin resistance of uricase.

[0020] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses uricase mutants and their use in improving the thermostability and trypsin resistance of uricase. Through directed evolution combined with high-throughput screening and multiple virtual screening, key sites affecting thermostability are discovered, and stability is improved through mutation. Furthermore, key sites affecting resistance to enzymatic hydrolysis are discovered through molecular dynamics simulation and solvent-accessible surface area calculation, and resistance to enzymatic hydrolysis is improved through mutation. Ultimately, multiple key sites and mutants affecting stability and resistance to enzymatic hydrolysis are discovered, and improvements in thermostability and resistance to enzymatic hydrolysis are achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0022] Figure 1 The accompanying figure shows the half-life of WT, M3*, M4*, and M7 of the present invention after trypsin treatment.

[0023] Figure 2 The accompanying drawings are electrophoretic diagrams of the enzymatic hydrolysis products of WT, M4*, and M7 of the present invention that change over time.

[0024] Figure 3The accompanying figure shows the changes in thermal stability residual enzyme activity of M3*, M4*, and M7 of the present invention at 50°C. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] The QuickMutation™ random gene mutation kit was purchased from Shanghai Biotech Co., Ltd.; the Q5® High-Fidelity DNA Polymerase kit was purchased from New England Biolabs; and the Mut Express II Fast Mutagenesis Kit V2 was purchased from Beijing Novogene Biotech Co., Ltd.

[0027] Example 1

[0028] 1) Directed evolution combined with high-throughput screening was used to identify key sites and mutants that affect thermal stability. The steps are as follows:

[0029] The urate oxidase (UOX) in this invention is derived from Aspergillus flavus (PDB DOI: https: / / doi.org / 10.2210 / pdb4D12 / pdb). The expression plasmid pET-28a(+) was ligated with the pET-28a(+) vector, using the BamHI and HindIII restriction sites. The codon-optimized urate oxidase gene (sequence shown in SEQ ID NO. 2, synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.) was synthesized and ligated with the pET-28a(+) vector to generate the expression plasmid pET-28a(+)-UOX.

[0030] Codon-optimized urate oxidase gene sequence:

[0031] ATGAGCGCGGTGAAAGCGGCGCGCTATGGCAAAGATAACGTGCGCGTGTATAAAGTGCATAAAGATGAAAAAACCGGCGTGCAGACCGTGTATGAAATGACCGTGTGCGTGCTGCTGGAAGGCGAAATTGAAACGAGCTATACCAAAGCGGATAACAGCGTGATTGTGGCGACCGATAGCATTAAAAACACCATTTATATTACCGCGAAACAGAACCCGGTGACCCCGCCGGAACTGTTTGGCAGCATTCTGGGCACCCATTTTATTGAAAAATATAACCATATTCATGCGGCGCATGTGAACATTGTGTGCCATCGCTGGACCCGCATGGATATTGATGGCAAACCGCATCCGCATAGCTTTATTCGCGATAGCGAAGAAAAACGCAACGTGCAAGTGGATGTGGTGGAAGGCAAAGGCATTGATATTAAAAGCAGCCTGAGCGGCCTGACCGTGCTGAAAAGCACCAACAGTCAGTTTTGGGGCTTTCTGCGCGATGAATATACCACCCTGAAAGAAACCTGGGATCGCATTCTGAGCACCGATGTGGATGCGACCTGGCAGTGGAAAAACTTTAGCGGCCTGCAAGAAGTGCGCAGCCATGTGCCGAAATTTGATGCCACCTGGGCGACGGCGCGCGAAGTGACCCTGAAAACCTTTGCGGAAGATAACAGCGCGAGCGTGCAAGCGACCATGTATAAAATGGCGGAACAGATTCTGGCGCGTCAGCAGCTGATTGAAACCGTGGAATATAGCCTGCCGAACAAACATTATTTTGAAATTGATCTGAGCTGGCATAAAGGCCTGCAGAACACCGGCAAAAACGCGGAAGTGTTTGCGCCGCAGAGCGATCCGAACGGCCTGATTAAATGCACCGTGGGCCGCAGCAGCCTGAAAAGCAAACTGTAA; SEQ ID NO.2。

[0032] Based on the expression plasmid pET-28a(+)-UOX, the construction of the random mutation library was carried out as follows:

[0033] (1) Random mutation of target gene:

[0034] The random mutagenesis of the target gene was performed using the Beyotime QuickMutation™ random gene mutagenesis kit.

[0035] Random mutagenesis reaction system: ddH2O 27.75 µL, Mutant enhancer 5 µL, RandomMut buffer 5 µL, dNTP 1.25 µL, template (plasmid pET-28a(+)-UOX) 1000 ng 8 µL, primer 1 1 µL, primer 2 1 µL, RandomMut polymerase 1 µL.

[0036] The primer sequences are as follows:

[0037] Primer 1: TAATACGACTCACTATAGGG; SEQ ID NO. 3;

[0038] Primer 2: GCTAGTTATTGCTCAGCGG; SEQ ID NO. 4.

[0039] Random mutagenesis PCR reaction parameters were as follows: pre-denaturation at 94°C for 3 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min; complete extension at 72°C for 10 min; and storage at 4°C.

[0040] (2) Template removal: Add 1 µL of DpnⅠ to the amplified product and incubate at 37°C for 2 h to remove the methylated template plasmid.

[0041] (3) DNA purification: Purify using Omega's Cycle-Pure Kit. Add 200µL of CP Buffer to the template-eliminator product and vortex to mix thoroughly. Pour the product into a chromatography column and centrifuge at 1000rpm for 1 min. Discard the filtrate. Add 700µL of DNA Wash Buffer and centrifuge at 10000rpm for 1 min. Discard the filtrate. Centrifuge at 12000rpm for 3 min. Collect and discard the remaining liquid. Dry in a 55℃ metal bath for 2 min. Add 50µL of Elution Buffer and centrifuge at 12000rpm for 1 min. Collect the filtrate, which is the purified DNA.

[0042] (4) Megaprimer PCR:

[0043] The megaprimer PCR reaction system used the Q5 ® High-Fidelity DNA Polymerase Kit.

[0044] Megaprimer PCR reaction system: ddH2O 32 µL, purified product 500 ng 5 µL, template (pET-28a(+)-UOX) 50 ng 1 µL, dNTP 1 µL, Q5 buffer 10 µL, Q5 High-Fidelity DNA Polymerase 1 µL.

[0045] The reaction parameters of the large primer PCR were as follows: pre-denaturation at 98°C for 30 s; 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 3 min; complete extension at 72°C for 2 min; and temporary storage at 4°C.

[0046] (5) Eliminate template: Same as step (2).

[0047] (6) Circularization to obtain recombinant product: Circularization system: ddH2O 11 µL, 5x CE II Buffer 4 µL, megaprimer PCR product 1 µL, Exnase II 2 µL. 5x CE II Buffer and Exnase II are from the Mut Express II Fast Mutagenesis Kit V2.

[0048] The PCR instrument parameters were set to: 37°C, 30 min.

[0049] (7) Transformation of recombinant products

[0050] Thaw E. coli BL21(DE3) competent cells on ice. Add 10 µL of the recombinant product to 200 µL of thawed E. coli BL21(DE3) competent cells, gently flick the tube to mix, and incubate on ice for 30 minutes. Heat shock the tube in a 42°C water bath for 90 seconds, then immediately place the tube on ice for 5 minutes. Add 500 µL of LB liquid medium and incubate at 37°C for 1.5 hours. Centrifuge at 10,000 rpm for 1 minute, discard 450 µL of the supernatant, and resuspend the remaining culture. Plate the resuspended culture onto a solid LB plate containing 50 mg / L kana resistance. Gently spread the suspension evenly with a sterile spreader, and incubate at 37°C overnight. This will generate a random mutagenesis library.

[0051] Through the above steps, a UOX random mutation library with a mutation rate of 1.6‰ was obtained, and a high-throughput screening experiment was performed.

[0052] The specific experimental steps of high-throughput screening are as follows:

[0053] (1) Use a Tecan Freedom EVO 100 pipetting workstation to dispense LB culture medium (containing 50 mg / L kana) into a 96-deep-well plate, with 600 µL of culture medium dispensed into each well. This will serve as the subsequent primary and secondary culture medium.

[0054] (2) Use Kbiosystems K6-3 to complete the work of single clone selection, inoculate into the primary culture medium, and culture with high-speed shaking at 37°C and 1000 rpm for 8 hours to obtain the primary seed liquid.

[0055] (3) Inoculate the first-level seed liquid into the second-level culture medium at a 2% inoculation rate and culture at 37°C and 1000 rpm with high-speed shaking; add 50% glycerol to the remaining first-level seed liquid at a ratio of 1:1 and store at -20°C.

[0056] (4) When OD600 reaches 0.6, add IPTG using a pipetting workstation to a final concentration of 0.2 mM. Incubate at 30°C, 800 rpm, and shake for 12 h.

[0057] (5) Use a refrigerated centrifuge to collect the bacteria at the following parameters: 3000 rpm, 4°C, and 5 min.

[0058] (6) Use a pipetting workstation to dispense 200 µL of 50 mM Tris-HCl (pH 8.5) into each well and shake to mix.

[0059] (7) Place the resuspended bacterial solution in a -80℃ freezer. Once frozen solid, take it out and thaw it at room temperature. Repeat this step three times to complete the freeze-thaw cycle.

[0060] (8) Use a refrigerated centrifuge to separate the crude enzyme and cell debris at 3000 rpm, 4°C, and 20 min.

[0061] (9) Collect the supernatant, which is the mutant enzyme solution to be verified.

[0062] (10) Heat-treat the mutant enzyme solution at 40°C for 20 min.

[0063] (11) Take 50µL of the treated mutant enzyme solution and add it to 150µL of uric acid solution (50mg / L). Place it in a microplate reader and use the kinetic mode to detect the Abs293nm change curve over time to calculate the residual enzyme activity after heat treatment. The higher the residual enzyme activity, the more likely the bacterial solution corresponding to the site is a mutant with improved stability. Sequencing and shake flask expression verification will be performed later.

[0064] 2) Expression, purification, and enzymatic characterization of urate oxidase

[0065] (1) Wild-type urate oxidase and mutants were expressed in shake flasks. 1‰ of the inoculum was inoculated into 4 mL of LB medium (containing 50 mg / L kana) and cultured at 37°C, 220 rpm, for 8 h. Then, 1% of the inoculum was inoculated into 100 mL of LB medium (containing 50 mg / L kana) and cultured at 37°C, 220 rpm, and shaken. When the OD600 reached 0.6-0.8, IPTG was added as an inducer to a final concentration of 0.2 mM. The culture was cultured at 30°C, 170 rpm, and incubated for 12 h.

[0066] (2) Purification of urate oxidase: Collect the bacterial precipitate by centrifugation at 8000 rpm, 4°C, and 5 min. Add 10 mL of Lysis Buffer (10 mM imidazole, pH 8) to the precipitate and resuspend it. Use a high-pressure homogenizer to lyse the cells at 1000 Pa and 4°C. After lysis, centrifuge again at 8000 rpm, 4°C, and 20 min to collect the supernatant. The supernatant is now the crude enzyme solution.

[0067] Purify using affinity chromatography. Equilibrate a nickel-agarose gel FF affinity column with two column volumes of lysis buffer. Add the crude enzyme solution, then add lysis buffer and wash buffer (30 mM imidazole, pH 8) to elute contaminants. Then, add elution buffer (300 mM imidazole, pH 8) to collect the target protein. This completes the affinity chromatography process.

[0068] Pour the collected protein into a 10 kDa ultrafiltration centrifuge tube and centrifuge at 3000 g, 4°C, for 15 minutes. After centrifugation, discard the filtrate and add 50 mM Tris-HCl (pH 8.5) buffer. Repeat this step three times to collect the purified urate oxidase.

[0069] (3) Enzyme activity assay: The substrate was uric acid solution at a concentration of 50 mg / L, and 50 mM Tris-HCl (pH 8.5) was used as the solvent. The reaction was terminated with 5M hydrochloric acid. The metal bath was set to 30°C and 600 rpm. The reaction system was 1 mL. 900 µL of uric acid solution was added, followed by 50 µL of appropriately diluted purified urate oxidase to initiate the reaction. After 4 minutes of reaction, 50 µL of hydrochloric acid was added to terminate the reaction. The control group consisted of 50 µL of purified urate oxidase replaced with 50 µL of Tris-HCl buffer. The enzyme activity was measured using a microplate reader, and the decrease in Abs293 nm was used to indicate the enzyme activity.

[0070] The protein concentration of purified urate oxidase was determined using the Coomassie Brilliant Blue assay, using 0.2 mg / mL BSA protein as the standard and a gradient dilution as the calibration curve. 1X Coomassie Brilliant Blue was added, and the Abs 595 nm of the standard and urate oxidase were measured using a microplate reader to determine the protein concentration.

[0071] (4) Thermal stability test: Pure urate oxidase and mutants are placed in a high-temperature metal bath (the present invention tests the stability at 40°C and 50°C). Samples are taken over time, and the enzyme activity is measured according to the enzyme activity test steps. In this way, a curve of the residual enzyme activity changing with time can be obtained. The time corresponding to the residual enzyme activity reaching 50% is the thermal stability half-life t 1 / 2 .

[0072] (5) After testing, four mutants with improved stability were obtained through directed evolution combined with high-throughput screening, as shown in Table 1.

[0073] Table 1 Mutants with improved thermal stability screened by directed evolution

[0074]

[0075] Note: T75S / K299E: The 75th position of the UOX amino acid sequence mutated from T to S, and the 299th position mutated from K to E.

[0076] 3) Calculate the unfolded free energy to identify key sites and mutants that affect thermal stability. The specific steps are as follows:

[0077] (1) Using Alphafold2 to build the UOX model

[0078] Models of the wild type and each mutant were constructed using AlphaFold2 (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynb). Multiple sequence alignment (MSA) was performed using MMseqs2_uniref_env. Templates for structure prediction were obtained from the pdb100 database. Five structural models were generated and ranked according to their predicted local distance difference test (pLDDT) scores. Models with scores exceeding 90 were considered for mutant structural analysis.

[0079] (2) FoldX was used for virtual screening to calculate and rank the unfolding free energy (ΔΔG) of the mutants. A negative ΔΔG with a larger absolute value indicates that the mutation tends to enhance stability. FoldX is a plug-in for Yasara (Version 21.12.19). The protein structure was optimized using the "repair object" module and the "mutant object" module was used for calculations.

[0080] (3) According to the virtual screening results, there are 6 recommended mutants, namely T68L, T75W, D184W, K204F, A97K, and D134F.

[0081] Primers were designed according to the mutations. The primers for point mutations are shown in Table 2.

[0082] Table 2 Point mutation primer design

[0083]

[0084] Point mutagenesis was performed using Vazyme's Mut Express II Fast Mutagenesis Kit V2. The specific procedure is as follows:

[0085] Step 1: target gene amplification;

[0086] Point mutation PCR reaction system: ddH2O 18 µL, 2x Max Buffer 25 µL, dNTP Mix 1 µL, template (pET-28a(+)-UOX) 1 µL, Primer F 2 µL, Primer R 2 µL, DNA Polymerase 1 µL. 2x Max Buffer was used from the Mut Express II Fast Mutagenesis Kit V2.

[0087] Point mutation PCR reaction parameters: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 6 min, 30 cycles; complete extension at 72°C for 5 min.

[0088] The second step is template elimination: the same as the random mutation template elimination step.

[0089] The third step is the in vitro circularization of the linear DNA: consistent with the random mutation circularization step.

[0090] The fourth step is the transformation of the recombination product: the same as the transformation step of the random mutation recombination product.

[0091] Following step 2), mutants were expressed, purified, and thermally stabilized. Mutants with improved thermal stability are listed in Table 3.

[0092] Table 3 Mutants with improved thermal stability through virtual screening

[0093]

[0094] 4) Combination mutation

[0095] The dominant mutants obtained in Tables 1 and 3 were combined to obtain mutants with further enhanced thermostability by superimposing the dominant mutations. Synthesis of these combined mutants was performed by Anshengda. Plasmid transformation, expression, purification, and thermostability testing of these combined mutants were performed as described above. Ultimately, the dominant mutants M3 (T68L / T75S / K299E), M4 (T68L / T75S / E222D / K299E), and M3* (T68L / T75W / K299E) were obtained, demonstrating further enhanced stability. The results are shown in Table 4.

[0096] Table 4 Thermal stability half-life of wild-type UOX and combined mutants M2, M3, M4, and M3* at 40°C

[0097]

[0098] 5) Calculate the solvent accessible surface area to identify key sites that affect the resistance to enzymatic hydrolysis and improve the resistance to enzymatic hydrolysis through mutation. The specific steps are as follows:

[0099] (1) Using M3* as a template, a tetramer model was constructed using Alphafold2, and a stable conformation in a solvent environment was obtained through molecular dynamics simulation. This conformation was imported into Discovery Studio, and the SolventAccessible function was used to calculate the relative solvent-accessible surface areas of the trypsin cleavage sites: lysine (abbreviated as K or LYS) and arginine (ARG) residues. The larger the relative solvent-accessible surface area, the greater the probability that the cleavage site is exposed to the aqueous environment, that is, the greater the possibility of being attacked by trypsin. The results are shown in Table 5.

[0100] Table 5 Calculation of solvent accessible surface area of ​​potential attack sites in M3*

[0101]

[0102] (2) According to Table 5, K204 ranked fourth in terms of solvent surface area, making it a potential site with a high probability of being exposed to the solvent environment. According to Table 3, K204F can enhance the thermal stability of the enzyme. Therefore, the mutation K204F was superimposed on M3* to obtain M4* (T68L / T75W / K204F / K299E), hoping that it would enhance the stability while also enhancing the enzyme's resistance to enzymatic hydrolysis. The construction of the mutant was synthesized by Anshengda Company, and the expression and purification steps were the same as above.

[0103] (3) Anti-enzymatic hydrolysis test: Wild-type uricase and its mutants were uniformly diluted to 0.5 mg / mL using a phosphate solution of pH 6.8 (6.8 g of potassium dihydrogen phosphate was taken and dissolved in 500 mL of water, and the pH value was adjusted to 6.8 with 0.1 mol / L sodium hydroxide solution). 2.5 mg / mL of trypsin (purchased from Sigma: 1,000-2,000 BAEE units / mg) and 5 mg / mL (purchased from Aladdin: 3-8 TIU / mg) of aprotinin were prepared using the phosphate solution. Multiple 100 µL aliquots of wild-type uricase and its mutants were placed in a 30°C metal bath, and 10 µL of trypsin was added to each tube to start the enzymatic hydrolysis reaction. Samples were taken out over time, and 10 µL of aprotinin was added to stop the reaction. The enzyme activity was then measured using the enzyme activity detection step. In this way, a curve of the residual enzyme activity changing with time was obtained. The time corresponding to 50% of the residual enzyme activity was the anti-enzymatic hydrolysis half-life. The final anti-enzymatic half-life of the wild type was 28.84 min, the anti-enzymatic half-life of M3* was 75.5 min, and the anti-enzymatic half-life of M4* was 142.8 min ( Figure 1 ).

[0104] 6) Through directed evolution combined with high-throughput screening, key sites and mutants that affect anti-enzymatic stability are discovered.

[0105] (1) Construct a mutant library using M4* as a template and screen it. The specific steps are as shown in 1). In step 1), the procedure in step (10) is modified to add trypsin to the supernatant at a final concentration of 0.25 mg / ml and incubate at 30°C for 1 hour. Then proceed with the subsequent treatment. The remaining steps remain unchanged.

[0106] (2) Through directed evolution combined with high-throughput screening, the present invention discovered the M7 mutant (Y47F / T68L / T75W / D111G / K204F / F259I / K299E) based on M4*. According to the test, the half-life of M7 against enzymatic hydrolysis reached 246.6min ( Figure 1 ).

[0107] (3) At the same time, SDS-PAGE electrophoresis was performed on the enzymatic hydrolysis products of wild-type urate oxidase, M3*, M4*, and M7 at different times. The enzymatic hydrolysis products were mixed with the loading buffer at a ratio of 1:3 and boiled in boiling water for 10 minutes. Then, the sample was loaded for detection, with 5µL of marker and 10µL of sample loaded. First, run at 80V for 20 minutes, then change to 120V until it reaches the bottom. Use FastBlue dye for staining, and the staining time is 20 minutes. Then decolorize and observe. For specific results, see Figure 2 .according to Figure 2 It was found that the anti-enzymatic performance of M4* and M7 with improved enzymatic performance can indeed delay the degradation process of the key band near 35KDa, which also explains the reason for the improvement of the anti-enzymatic half-life.

[0108] (4) At the same time, the present invention also tested the activity and thermal stability of M4* and M7 according to step 2), and the results are shown in Figure 3 (The time corresponding to when the residual enzyme activity is 50% is the thermal stability half-life t 1 / 2 ) and Table 6.

[0109] Table 6 Summary of mutants

[0110]

[0111] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one 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 present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A urate oxidase mutant, characterized in that: Y47F / T68L / T75W / D111G / K204F / F259I / K299E, T68L / T75W / K204F / K299E, T68L / T75W / K299E, T75W, or T68L; The amino acid sequence of the uricase is shown in SEQ ID NO.

1.

2. The uricase mutant according to claim 1, characterized in that The uricase mutant is Y47F / T68L / T75W / D111G / K204F / F259I / K299E, T68L / T75W / K204F / K299E or T68L / T75W / K299E.

3. The biomaterial related to the uricase mutant according to claim 1, characterized in that: At least one of the following (1)-(4): (1) a nucleic acid molecule encoding a urate oxidase mutant; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1) or a recombinant vector containing the expression cassette described in (2); (4) A recombinant microorganism containing the nucleic acid molecule described in (1), a recombinant microorganism containing the expression cassette described in (2), or a recombinant microorganism containing the recombinant vector described in (3).

4. Use of the uricase mutant according to any one of claims 1 to 2 or the related biomaterial according to claim 3 for improving the thermal stability of uricase.

5. Use of the uricase mutant according to claim 2 for improving the thermal stability and trypsin resistance of uricase.

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