Uraurate oxidase mutant and application thereof in improving thermal stability of urate oxidase and trypsin resistance

Abstract

The invention discloses a urate oxidase mutant and application thereof in improving the heat stability of urate oxidase and the trypsin resistance, and belongs to the technical field of biology. The urate oxidase mutant disclosed by the invention is Y47F / T68L / T75W / D111G / K204F / F259I / K299E, T68L / T75W / K204F / K299E or T68L / T75W / K299E. The invention further discloses a preparation method of the urate oxidase mutant. According to the method, the key sites influencing the thermal stability are mined in the modes of directed evolution, high-throughput screening, multiple virtual screening and the like, and the stability is improved through mutation; the key sites influencing the enzymolysis resistance are excavated through molecular dynamics simulation and solvent accessible surface area calculation, and the enzymolysis resistance effect is improved through mutation. Finally, a plurality of key sites and mutants influencing the stability and the enzymolysis resistance are excavated, and the thermal stability and the enzymolysis resistance are improved.

Description

Technical Field

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

[0002] Urate oxidase is one of the key enzymes in purine metabolism in organisms. It can convert uric acid into allantoin and has become a protein drug with excellent therapeutic effects. However, natural urate oxidase has problems such as poor thermal stability and poor resistance to protease hydrolysis. Therefore, it is necessary to improve the thermal stability and resistance to enzymatic hydrolysis of urate oxidase through molecular modification.

[0003] The modification of thermal stability can be achieved by combining directed evolution and high-throughput screening, using microplates as carriers to screen random mutation libraries; or the B-factor strategy can be used to improve stability by computationally searching for highly flexible regions and performing rigid design. In recent years, many computational design platforms have been designed, such as Fireprot, PROSS, and Grape, which integrate modules such as consensus sequence analysis and unfolding free energy calculation to achieve rapid search for mutation sites, and have been experimentally and verified in the stability modification of multiple enzymes.

[0004] Anti-enzymatic modification is also a difficult problem that needs to be solved in the process of protein drug design. At present, the anti-enzymatic modification of protein molecules themselves is mainly divided into two directions: direct mutation and indirect improvement. Direct mutation can find the cleavage site that is in direct contact with the protease through molecular docking and solvent accessible surface area calculation, and delay the protease hydrolysis process through mutation; indirect improvement can form a spatial masking of the cleavage site by mutating the residues near the cleavage site, thereby reducing the probability of contact between the cleavage site and the protease, and can also block the contact between the protease and its cleavage site through artificial design of N-glycosylation modification, so as to achieve the purpose of resisting protease hydrolysis.

[0005] Therefore, providing uricase oxidase mutants and use thereof in improving the thermal stability and trypsin resistance of uricase oxidase 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 oxidase mutant and its use in improving the thermal stability and trypsin resistance of uricase oxidase.

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

[0008] In order to achieve the above object, the present invention adopts the following technical solution: 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; The amino acid sequence of the urate oxidase (UOX) is shown in SEQ ID NO.1.

[0009] MSAVKAARYGKDNVRVYKVHKDEKTGVQTVYEMTVCVLLEGEIETSYTKADNSVIVATDSIKNTIYITAKQNPVTPPELFGSILGTHFIEKYNHIHAAHVNIVCHRWTRMDIDGKPHPHSFIRDSEEKRNVQVDVVEGKGIDIKSSLSGLTVL SEQ ID NO.1.

[0010] Furthermore, the uricase mutant is Y47F / T68L / T75W / D111G / K204F / F259I / K299E (M7), T68L / T75W / K204F / K299E (M4*) or T68L / T75W / K299E (M3*).

[0011] Furthermore, the biological material related to the urate oxidase mutant is at least one of the following (1)-(4): (1) a nucleic acid molecule encoding a uricase 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).

[0012] Furthermore, the uricase oxidase mutant or the related biomaterial is used to improve the thermal stability of uricase oxidase.

[0013] Furthermore, the uricase mutant is used to improve the thermal stability and trypsin resistance of uricase.

[0014] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses uricase mutants and their application in improving the thermal stability and trypsin resistance of uricase, and explores the key sites affecting thermal stability by means of directed evolution combined with high-throughput screening, multiple virtual screening, etc., and improves stability by mutation; explores the key sites affecting anti-enzymatic performance by means of molecular dynamics simulation and solvent accessible surface area calculation, and improves the anti-enzymatic effect by mutation. Finally, multiple key sites and mutants affecting stability and anti-enzymatic performance were explored, and the improvement of thermal stability and anti-enzymatic performance was obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0016] Figure 1 The attached figure shows the half-life of the anti-enzymatic hydrolysis of WT, M3*, M4* and M7 of the present invention after treatment with trypsin.

[0017] Figure 2 The attached figure is the electrophoresis diagram of the enzymatic hydrolysis products of WT, M4* and M7 of the present invention changing with time.

[0018] Figure 3 The attached figure shows the changes in thermal stability residual enzyme activity of M3*, M4* and M7 of the present invention at 50°C. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0020] The QuickMutation™ random mutation kit was purchased from Shanghai Bio-Tech 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.

[0021] Example 1 1) The key sites and mutants affecting thermal stability were discovered by combining directed evolution with high-throughput screening. The steps are as follows: The urate oxidase (UOX) in the present invention comes from Aspergillus flavus, PDB DOI: https: / / doi.org / 10.2210 / pdb4D12 / pdb. The expression vector is pET-28a (+), the restriction sites are BamHⅠ and HindⅢ, and the codon-optimized urate oxidase gene (sequence as shown in SEQ ID NO.2, synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.) is connected to the pET-28a (+) vector to obtain the expression plasmid pET-28a (+)-UOX.

[0022] Codon-optimized uricase gene sequence: ATGAGCGCGGTGAAAGCGGCGCGCTATGGCAAAGATAACGTGCGCGTGTATAAAGTGCATAAAGATGAAAAAACCGGCGTGCAGACCGTGTATGAAATGACCGTGTGCGTGCTGCTGGAAGGCGAAATTGAAACGAGCTATACCAAAGCGGATAACAGCGTGATTGTGGCGACCGATAGCATTAAAAACACCATTTATATTACCGCGAAACAGAACCCGGTGACCCCGCCGGAACTGTTTGGCAGCATTCTGGGCACCCATTTTATTGAAAAATATAACCATATTCATGCGGCGCATGTGAACATTGTGTGCCATCGCTGGACCCGCATGGATATTGATGGCAAACCGCATCCGCATAGCTTTATTCGCGATAGCGAAGAAAAACGCAACGTGCAAGTGGATGTGGTGGAAGGCAAAGGCATTGATATTAAAAGCAGCCTGAGCGGCCTGACCGTGCTGAAAAGCACCAACAGTCAGTTTTGGGGCTTTCTGCGCGATGAATATACCACCCTGAAAGAAACCTGGGATCGCATTCTGAGCACCGATGTGGATGCGACCTGGCAGTGGAAAAACTTTAGCGGCCTGCAAGAAGTGCGCAGCCATGTGCCGAAATTTGATGCCACCTGGGCGACGGCGCGCGAAGTGACCCTGAAAACCTTTGCGGAAGATAACAGCGCGAGCGTGCAAGCGACCATGTATAAAATGGCGGAACAGATTCTGGCGCGTCAGCAGCTGATTGAAACCGTGGAATATAGCCTGCCGAACAAACATTATTTTGAAATTGATCTGAGCTGGCATAAAGGCCTGCAGAACACCGGCAAAAACGCGGAAGTGTTTGCGCCGCAGAGCGATCCGAACGGCCTGATTAAATGCACCGTGGGCCGCAGCAGCCTGAAAAGCAAACTGTAA; SEQ ID NO.2。

[0023] Based on the expression plasmid pET-28a(+)-UOX, the construction of the random mutation library was carried out as follows: (1) Random mutation of target gene:

[0024] The random mutation of the target gene was performed using the Bio-Time QuickMutation™ Gene Random Mutation Kit.

[0025] 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.

[0026] The primer sequences are as follows: Primer 1: TAATACGACTCACTATAGGG; SEQ ID NO. 3; Primer 2: GCTAGTTATTGCTCAGCGG; SEQ ID NO.4.

[0027] Random mutation PCR reaction parameters: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles; complete extension at 72°C for 10 min; temporary storage at 4°C.

[0028] (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.

[0029] (3) DNA purification: Use Omega's Cycle-Pure Kit for purification. Add 200µL CP Buffer to the product after template elimination, shake and mix; pour into the chromatography column and centrifuge at 1000rpm for 1min, discard the filtrate; add 700µL DNA Wash Buffer, centrifuge at 10000rpm for 1min, discard the filtrate; centrifuge at 12000rpm for 3min, collect the remaining liquid and pour it out; dry in a 55℃ metal bath for 2min; add 50µL Elution Buffer, centrifuge at 12000rpm for 1min, collect the filtrate, which is the purified DNA.

[0030] (4) Megaprimer PCR: The megaprimer PCR reaction system used the Q5 ® High-Fidelity DNA Polymerase Kit.

[0031] Megaprimer PCR reaction system: ddH2O 32 µL, purified product 500ng 5 µL, template (plasmid pET-28a(+)-UOX) 50ng 1 µL, dNTP 1 µL, Q5 buffer 10 µL, Q5 High-Fidelity DNA Polymerase 1 µL.

[0032] The reaction parameters of the large primer PCR were as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 3 min, 30 cycles; complete extension at 72°C for 2 min; temporary storage at 4°C.

[0033] (5) Template elimination: Same as step (2).

[0034] (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 Mut Express IIFast Mutagenesis Kit V2.

[0035] The PCR instrument parameters were set to: 37°C, 30 min.

[0036] (7) Transformation of recombinant products Thaw E.coli BL21(DE3) competent cells on ice, take 10 µL of the recombinant product and add it to 200 µL of thawed E.coli BL21(DE3) competent cells, flick the tube wall to mix, and let it stand on ice for 30 minutes; heat shock in a 42℃ water bath for 90 seconds, and immediately put it in ice for 5 minutes; add 500 µL LB liquid culture medium, and culture at 37℃ for 1.5 hours; centrifuge at 10000rpm for 1 minute, discard 450 µL supernatant, and resuspend the rest. Add the resuspended bacterial solution to the LB solid plate with 50 mg / L kana resistance, spread it gently with a sterile coating stick, and culture it at 37℃ overnight. The random mutation library can be obtained.

[0037] 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.

[0038] The specific experimental steps of high-throughput screening are as follows: (1) Use the 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.

[0039] (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 solution.

[0040] (3) Inoculate the primary seed solution into the secondary culture medium at a rate of 2%, and culture at 37°C and 1000 rpm with high-speed shaking; add 50% glycerol to the remaining primary seed solution at a ratio of 1:1 and store at -20°C.

[0041] (4) When OD600 reaches 0.6, add IPTG using a pipetting workstation to a final concentration of 0.2 mM. The culture conditions are 30°C, 800 rpm, and shaking culture for 12 h.

[0042] (5) Use a refrigerated centrifuge to collect bacteria at 3000 rpm, 4°C, and 5 min.

[0043] (6) Use a pipetting workstation to dispense 200 µL of 50 mM Tris-HCl (pH 8.5) into each well and vortex to mix.

[0044] (7) Place the resuspended bacterial solution in a -80℃ refrigerator. After freezing, take it out and thaw it at room temperature. Repeat this step three times to complete the repeated freeze-thaw cell disruption operation.

[0045] (8) Use a refrigerated centrifuge to separate the crude enzyme and cell debris at 3000 rpm, 4°C, and 20 min.

[0046] (9) Collect the supernatant, which is the mutant enzyme solution to be verified.

[0047] (10) Heat treat the mutant enzyme solution at 40°C for 20 min.

[0048] (11) Take 50 µL of the treated mutant enzyme solution and add it to 150 µL of uric acid solution (50 mg / L), put it into the microplate reader, and detect the Abs293nm change curve over time in the kinetic mode to calculate the residual enzyme activity after heat treatment. The higher the residual enzyme activity, the more likely it is that the bacterial solution corresponding to this site is a mutant with improved stability, and subsequent sequencing and shake flask expression verification will be performed.

[0049] 2) Expression, purification and enzymatic properties of urate oxidase (1) The wild-type urate oxidase and mutants were expressed in shake flasks. 1‰ was inoculated into 4 mL LB medium (containing 50 mg / L kana) and cultured at 37℃ 220 rpm for 8 h. Then, 1% was inoculated into 100 mL LB medium (containing 50 mg / L kana) and cultured at 37℃ 220 rpm. When OD600 reached 0.6-0.8, IPTG was added as an inducer with a final concentration of 0.2 mM. The culture conditions were 30℃ 170 rpm for 12 h.

[0050] (2) Purification of urate oxidase: Collect 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 for resuspending. Use a high-pressure homogenizer to disrupt the cells at 1000 pa and 4°C. After cell disruption, centrifuge again to collect the supernatant at 8000 rpm, 4°C, and 20 min. The supernatant is now a crude enzyme solution.

[0051] Purification was performed by affinity chromatography. Two column volumes of Lysis buffer were added to the nickel agarose gel FF affinity chromatography column for column equilibration. After the crude enzyme solution was poured in, Lysis Buffer and Wash buffer (30 mM imidazole, pH 8) were added to elute the impurities, and Elution Buffer (300 mM imidazole, pH 8) was added to collect the target protein. The affinity chromatography process was completed.

[0052] The collected protein was poured into a 10KDa ultrafiltration centrifuge tube, and the centrifugation parameters were 3000g, 4°C, and 15min. After the centrifugation was completed, the filtrate was discarded and 50 mM Tris-HCl (pH 8.5) buffer was added. This step was performed three times, and the collected protein was the purified urate oxidase.

[0053] (3) Enzyme activity detection: The substrate is uric acid solution with a concentration of 50 mg / L, and 50 mM Tris-HCl (pH 8.5) is used as the solvent. The reaction is terminated with 5M hydrochloric acid. The metal bath is set to 30℃ 600rpm, the reaction system is 1mL, 900µL uric acid solution is added, and 50µL appropriately diluted purified urate oxidase is added to start the reaction. After 4 minutes of reaction, 50µL hydrochloric acid is added to terminate the reaction. The control group is 50µL purified urate oxidase replaced with 50µL Tris-HCl buffer. The detection is performed using an enzyme reader, and the reduction in Abs293nm is used to indicate the enzyme activity.

[0054] The protein concentration of the purified uricase was detected by the Coomassie brilliant blue method, with 0.2 mg / mL BSA protein as the standard and gradient dilution as the standard curve. 1X Coomassie brilliant blue was added, and the Abs595nm of the standard and uricase was detected by an ELISA reader to obtain the protein concentration of uricase.

[0055] (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 detection steps, so that a curve of the residual enzyme activity changing with time can be obtained. The time corresponding to the residual enzyme activity being 50% is the thermal stability half-life t 1 / 2 .

[0056] (5) After testing, four mutants with improved stability were obtained through directed evolution combined with high-throughput screening, as shown in Table 1.

[0057] Table 1 Directed evolution screening of mutants with improved thermal stability

[0058] 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.

[0059] 3) Mining the key sites and mutants that affect thermal stability through unfolding free energy calculation. The specific steps are as follows: (1) Using Alphafold2 to build the UOX model Alphafold2 (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynb) was used to build models for the wild type and each mutant, and MSA (Multiple Sequence Alignment) multiple sequence alignment used MMseqs2_uniref_env. The template used for structure prediction came from the pdb100 database. A total of five structural models were generated and ranked according to the predicted local difference test (pLDDT: predicted Local Distance Difference Test) score. The model with the highest score of more than 90 points was considered for mutant structure analysis.

[0060] (2) FoldX was used for virtual screening to calculate and sort the unfolding free energy (ΔΔG) of the mutants. The larger the negative ΔΔG and the larger the absolute value, the more likely the mutation is to enhance stability. FoldX is a plug-in for Yasara (Version 21.12.19). The "repair object" was used to optimize the protein structure, and the "mutant object" module was used for calculation.

[0061] (3) According to the virtual screening results, there are 6 recommended mutants, namely T68L, T75W, D184W, K204F, A97K, and D134F.

[0062] Primers were designed according to the mutations. The primers for point mutations are shown in Table 2.

[0063] Table 2 Primer design for point mutation

[0064] The point mutation was performed using Vazyme's Mut Express II Fast Mutagenesis Kit V2. The specific process is as follows:

[0065] Step 1: Amplification of target gene; 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 comes from Mut Express II Fast Mutagenesis Kit V2.

[0066] 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.

[0067] The second step is template elimination: the same as the random mutation template elimination step.

[0068] The third step is the in vitro circularization of the linear DNA: consistent with the random mutation circularization step.

[0069] The fourth step is the transformation of the recombination product: the same as the transformation step of the random mutation recombination product.

[0070] According to step 2), the mutants were expressed, purified, and tested for thermal stability. Mutants with improved thermal stability are shown in Table 3.

[0071] Table 3 Mutants with improved thermal stability through virtual screening

[0072] 4) Combined mutation The superior mutants obtained in Table 1 and Table 3 were combined, and mutants with more improved thermal stability were obtained by superimposing superior mutations. The synthesis of the combined mutants was completed by Anshengda. The transformation of plasmids, as well as the expression, purification, and thermal stability test of the combined mutants were the same as above, and finally the superior mutants M3 (T68L / T75S / K299E), M4 (T68L / T75S / E222D / K299E), and M3* (T68L / T75W / K299E) were obtained, and their stability was further improved. The results are shown in Table 4.

[0073] Table 4 Thermal stability half-life of wild-type UOX and combined mutants M2, M3, M4, M3* at 40°C

[0074] 5) Through the calculation of solvent accessible surface area, the key sites that affect the anti-enzymatic performance are discovered and the anti-enzymatic effect is improved through mutation. The specific steps are as follows: (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 area of ​​the trypsin cleavage site: 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 water environment, that is, the greater the possibility of being attacked by trypsin. The results are shown in Table 5.

[0075] Table 5 Calculation of solvent accessible surface area of ​​potential attack sites in M3*

[0076] (2) According to Table 5, K204 ranks fourth in terms of solvent surface area, which is 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 is superimposed on the basis of M3* to obtain M4* (T68L / T75W / K204F / K299E), hoping that it can enhance the enzyme's resistance to enzymolysis while enhancing stability. The construction of the mutant was synthesized by Anshengda Company, and the expression and purification steps were the same as above.

[0077] (3) Anti-enzymatic 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 using a 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 samples 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 reaction. Samples were taken out over time, and 10 µL of aprotinin was added to terminate the reaction. The enzyme activity was determined using the enzyme activity detection step. In this way, a curve of residual enzyme activity changing over time was obtained. The time corresponding to 50% of the residual enzyme activity was the anti-enzymatic half-life. The final half-life of the wild type against enzymatic hydrolysis was 28.84 min, the half-life of M3* against enzymatic hydrolysis was 75.5 min, and the half-life of M4* against enzymatic hydrolysis was 142.8 min ( Figure 1 ).

[0078] 6) Through directed evolution combined with high-throughput screening, key sites and mutants that affect anti-enzymatic stability are discovered.

[0079] (1) Use M4* as a template to construct a mutant library and screen it. The specific steps are as shown in 1). Change the step in step (10) of step 1) to add trypsin with a final concentration of 0.25 mg / ml to the supernatant and incubate at 30°C for 1 hour. Then perform the subsequent treatment. The remaining steps remain unchanged.

[0080] (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 246.6 min ( Figure 1 ).

[0081] (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 in 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. First, run at 80V for 20 minutes, then change to 120V until it reaches the bottom. Use FastBlue dye for staining for 20 minutes. Then decolorize and observe. For specific results, see Figure 2 .according to Figure 2It was found that the anti-enzymatic performance of M4* and M7 with improved anti-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.

[0082] (4) At the same time, the present invention also tested the activity and thermal stability of M4* and M7 according to step 2). The results are shown in Figure 3 (The time corresponding to the residual enzyme activity being 50% is the thermal stability half-life t 1 / 2 ) and Table 6.

[0083] Table 6 Summary of mutants

[0084] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 will not be limited to the embodiments shown herein, but rather 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, T68L / T75S / E222D / K299E, T68L / T75S / K299E, T75S / K299E, T75W, K299E, K91R, T68L, D184W, K204F, D134F, A97K, or E222D; The amino acid sequence of the uricase is shown in SEQ ID NO.

1.

2. The urate oxidase 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 biological material related to the uricase mutant according to claim 1, characterized in that: At least one of the following (1) to (4): (1) a nucleic acid molecule encoding a uricase 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 in improving the thermal stability of uricase.

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

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