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

Abstract

The invention discloses a urate oxidase mutant and application thereof in improving the thermal stability of urate oxidase, and belongs to the technical field of biology. The urate oxidase mutant disclosed by the invention is T68L / T75S / E222D / K299E, T68L / T75S / K299E or T75S / K299E, and the urate oxidase mutant disclosed by the invention is T68L / T75S / K299E. 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. And finally, a plurality of key sites and mutants influencing the stability are excavated, and the thermal stability is improved.

Description

[0001] This application is a divisional application of the invention patent application with the application number 202510315066.8, the application date of March 18, 2025, and the invention name of "Urate oxidase mutant and its application in improving the thermal stability and trypsin resistance of urate oxidase". TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, more particularly to a urate oxidase mutant and its application in improving the thermal stability of urate oxidase. BACKGROUND

[0003] Urate oxidase is one of the key enzymes in purine metabolism in living organisms, which can convert uric acid into allantoin, and has become a protein drug with excellent therapeutic effect. However, the 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.

[0004] The improvement of thermal stability can be achieved by combining directed evolution and high-throughput screening, and screening the random mutation library with microplate as the carrier. The B-factor strategy can also be used to improve stability by calculating the high flexibility area and rigidizing design. In recent years, many computational design platforms have been designed, such as Fireprot, PROSS, Grape, etc., which integrate consensus sequence analysis, unfolding free energy calculation and other modules to realize the rapid search of mutation sites, and have been experimentally and verified in the stability improvement of many enzymes.

[0005] The improvement of resistance to enzymatic hydrolysis is also a difficult problem to be solved in the design of protein drugs. At present, the improvement of resistance to enzymatic hydrolysis of protein molecules mainly includes direct mutation and indirect improvement. Direct mutation can find the cutting site directly contacted with protease through molecular docking and solvent accessible surface area calculation, and delay the protease hydrolysis process through mutation; indirect improvement can form a mask to the cutting site by mutating the residues near the cutting site, so as to reduce the probability of contact between the cutting site and protease, or block the contact between protease and its cutting site by artificial design of N-glycosylation modification, so as to resist protease hydrolysis.

[0006] Therefore, it is an urgent problem for those skilled in the art to provide a urate oxidase mutant and its application in improving the thermal stability of urate oxidase. SUMMARY

[0007] Therefore, the present application provides a urate oxidase mutant and its application in improving the thermal stability of urate oxidase.

[0008] The present application adopts the way of directed evolution and rational design to excavate key point mutations affecting the thermal stability of uric acid oxidase, and further improves the thermal stability of uric acid oxidase through combined mutation and iterative evolution. Meanwhile, the process also improves the activity and trypsin hydrolysis resistance of uric acid oxidase. Further, the strategy of solvent accessible surface area calculation is used to identify the cutting site which is probably exposed in the water environment, and the site affecting the trypsin hydrolysis resistance is excavated, so that the trypsin hydrolysis resistance is greatly improved through combined mutation.

[0009] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0010] The uric acid oxidase mutant is 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.

[0011] The amino acid sequence of the uric acid oxidase (UOX) is shown in SEQ ID NO. 1.

[0012] MSAVKAARYGKDNVRVYKVHKDEKTGVQTVYEMTVCVLLEGEIETSYTKADNSVIVATDSIKNTIYITAKQNPVTPPELFGSILGTHFIEKYNHIHAAHVNIVCHRWTRMDIDGKPHPHSFIRDSEEKRNVQVDVVEGKGIDIKSSLSGLTVLKSTNSQFWGFLRDEYTTLKETWDRILSTDVDATWQWKNFSGLQEVRSHVPKFDATWATAREVTLKTFAEDNSASVQATMYKMAEQILARQQLIETVEYSLPNKHYFEIDLSWHKGLQNTGKNAEVFAPQSDPNGLIKCTVGRSSLKSKL; SEQ ID NO. 1.

[0013] Further, the uric acid oxidase mutant is Y47F / T68L / T75W / D111G / K204F / F259I / K299E (M7), T68L / T75W / K204F / K299E (M4*) or T68L / T75W / K299E (M3*).

[0014] Further, the related biological material of the uricase mutant is at least one of the following (1)-(4):

[0015] (1) a nucleic acid molecule encoding the uricase mutant;

[0016] (2) an expression cassette containing the nucleic acid molecule of (1);

[0017] (3) a recombinant vector containing the nucleic acid molecule of (1) or an expression cassette containing (2);

[0018] (4) a recombinant microorganism containing the nucleic acid molecule of (1), an expression cassette containing (2), or a recombinant microorganism containing the recombinant vector of (3).

[0019] Further, the uricase mutant or the related biological material is used for improving the thermal stability of uricase.

[0020] Further, the uricase mutant is used for improving the thermal stability and trypsin resistance of uricase.

[0021] According to the technical solution, compared with the prior art, the uricase mutant and its application in improving the thermal stability of uricase are provided, the key sites affecting the thermal stability are mined by means of directed evolution combined with high-throughput screening, multiple virtual screening, etc., and the stability is improved by mutation; the key sites affecting the anti-enzymatic performance are mined by means of molecular dynamics simulation and solvent accessible surface area calculation, and the anti-enzymatic effect is improved by mutation. Finally, multiple key sites and mutants affecting the stability and anti-enzymatic performance are mined, and the thermal stability and anti-enzymatic performance are improved. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creative labor.

[0023] Figure 1 The figure is the anti-enzymatic half-life of WT, M3*, M4*, and M7 under trypsin treatment.

[0024] Figure 2 The figure is the electropherogram of the enzymatic products of WT, M4*, and M7 over time.

[0025] Figure 3The attached drawings are the changes of residual enzyme activity of M3*, M4* and M7 of the present application at 50℃. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0027] QuickMutation TM The gene random mutation kit is purchased from Shanghai Biyun Tian Biotechnology Co., Ltd. The High-Fidelity DNA Polymerase kit is purchased from New England Biolabs, and the Mut Express II Fast Mutagenesis Kit V2 kit is purchased from Jingnuozan Biotechnology Co., Ltd.

[0028] Example 1

[0029] 1) The key sites and mutants affecting thermal stability are excavated by the way of directed evolution combined with high-throughput screening, and the steps are as follows:

[0030] The uric acid oxidase (UOX) in the present application is from Aspergillus flavus, PDB DOI: https: / / doi.org / 10.2210 / pdb4D12 / pdb. The expression vector is pET-28a(+), and the enzyme cutting site is selected as BamH I and Hind III. The codon-optimized uric acid oxidase gene (the sequence is shown as SEQ ID NO. 2, synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.) is connected with the pET-28a(+) vector to obtain the expression plasmid pET-28a(+)-UOX.

[0031] The sequence of the codon-optimized uric acid oxidase gene is as follows:

[0032] ATGAGCGCGGTGAAAGCGGCGCGCTATGGCAAAGATAACGTGCGCGTGTATAAAGTGCATAAAGATGAAAAAACCGGCGTGCAGACCGTGTATGAAATGACCGTGTGCGTGCTGCTGGAAGGCGAAATTGAAACGAGCTATACCAAAGCGGATAACAGCGTGATTGTGGCGACCGATAGCATTAAAAACACCATTTATATTACCGCGAAACAGAACCCGGTGACCCCGCCGGAACTGTTTGGCAGCATTCTGGGCACCCATTTTATTGAAAAATATAACCATATTCATGCGGCGCATGTGAACATTGTGTGCCATCGCTGGACCCGCATGGATATTGATGGCAAACCGCATCCGCATAGCTTTATTCGCGATAGCGAAGAAAAACGCAACGTGCAAGTGGATGTGGTGGAAGGCAAAGGCATTGATATTAAAAGCAGCCTGAGCGGCCTGACCGTGCTGAAAAGCACCAACAGTCAGTTTTGGGGCTTTCTGCGCGATGAATATACCACCCTGAAAGAAACCTGGGATCGCATTCTGAGCACCGATGTGGATGCGACCTGGCAGTGGAAAAACTTTAGCGGCCTGCAAGAAGTGCGCAGCCATGTGCCGAAATTTGATGCCACCTGGGCGACGGCGCGCGAAGTGACCCTGAAAACCTTTGCGGAAGATAACAGCGCGAGCGTGCAAGCGACCATGTATAAAATGGCGGAACAGATTCTGGCGCGTCAGCAGCTGATTGAAACCGTGGAATATAGCCTGCCGAACAAACATTATTTTGAAATTGATCTGAGCTGGCATAAAGGCCTGCAGAACACCGGCAAAAACGCGGAAGTGTTTGCGCCGCAGAGCGATCCGAACGGCCTGATTAAATGCACCGTGGGCCGCAGCAGCCTGAAAAGCAAACTGTAA; SEQ ID NO. 2.

[0033] Based on the expression plasmid pET-28a(+)-UOX, the construction work of the random mutation library is as follows:

[0034] (1) Random mutation of the target gene:

[0035] The random mutation of the target gene was performed by using the QuickMutation Kit of Biyun Tian. TM Gene Random Mutation Kit.

[0036] Random mutation 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 11 μL, primer 2 21 μL, RandomMut polymerase 1 μL.

[0037] The primer sequences are as follows:

[0038] Primer 1: TAATACGACTCACTATAGGG; SEQ ID NO. 3;

[0039] Primer 2: GCTAGTTATTGCTCAGCGG; SEQ ID NO. 4.

[0040] Random mutation PCR reaction parameters: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 30 cycles; 72°C thorough extension for 10 min; 4°C temporary storage.

[0041] (2) Template removal: 1 μL of Dpn I was added to the amplification product, and the reaction was performed at 37°C for 2 h to remove the methylated template plasmid.

[0042] (3) DNA purification: Omega Cycle-Pure Kit was used for purification. 200 μL of CP Buffer was added to the product after template removal, and shaken to mix; centrifuged in a chromatography column, 1000 rpm for 1 min, and the filtrate was discarded; 700 μL of DNA Wash Buffer was added, centrifuged at 10000 rpm for 1 min, and the filtrate was discarded; centrifuged at 12000 rpm for 3 min, the residual liquid was collected and discarded; placed in a 55°C metal bath and dried for 2 min; 50 μL of Elution Buffer was added, and centrifuged at 12000 rpm for 1 min to collect the filtrate, which was the purified DNA.

[0043] (4) Large primer PCR:

[0044] The large primer PCR reaction system used High-Fidelity DNA Polymerase Kit.

[0045] Large primer PCR reaction system: ddH2O 32 μL, purified product 500 ng 5 μL, template (plasmid pET-28a (+)-UOX) 50 ng 1 μL, dNTP 1 μL, Q5 buffer 10 μL, Q5 High-Fidelity DNA Polymerase 1 μL.

[0046] Large primer PCR reaction parameters: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 3 min, 30 cycles; 72°C thorough extension for 2 min; 4°C temporary storage.

[0047] (5) Template removal: same as step (2).

[0048] (6) Recombination product obtained by circularization: circularization system: ddH2O 11 μL, 5x CE II Buffer 4 μL, large primer PCR product 1 μL, Exnase II 2 μL. 5x CE II Buffer and Exnase II are from Mut Express II FastMutagenesis Kit V2 kit.

[0049] The PCR instrument parameter is set as: 37°C, 30 min.

[0050] (7) Transformation of recombination product

[0051] Thaw E. coli BL21 (DE3) competent cells on ice, take 10 μL of recombination product and add it to 200 μL of thawed E. coli BL21 (DE3) competent cells, mix gently by tapping the tube wall, and stand on ice for 30 min; after 90 s of 42°C water bath heat shock, immediately put into ice for 5 min; add 500 μL of LB liquid medium, cultivate at 37°C for 1.5 h; centrifuge at 10000 rpm for 1 min, discard 450 μL of supernatant, and leave the remaining suspension. Add the resuspended bacterial solution to an LB solid plate with 50 mg / L kana resistance, and gently spread it with a sterile spreader, and cultivate at 37°C overnight. The random mutation library is obtained.

[0052] The UOX random mutation library with a mutation rate of 1.6‰ is obtained through the above steps, and a high-throughput screening experiment is carried out.

[0053] The specific experimental steps of high-throughput screening are as follows:

[0054] (1) Use Tecan Freedom EVO 100 pipetting workstation to dispense LB medium (containing 50 mg / L kana) into a 96-deep well plate, 600 μL of medium per well. As the subsequent primary and secondary culture medium.

[0055] (2) The work of monoclonal selection was completed by Kbiosystems K6-3, and the primary seed liquid was obtained by inoculating into the primary culture medium and culturing at 37°C and 1000 rpm for 8h.

[0056] (3) The primary seed liquid was inoculated into the secondary culture medium according to the inoculation amount of 2%, and cultured at 37°C and 1000 rpm; the remaining primary seed liquid was added into 50% glycerol according to the proportion of 1:1 and stored at-20°C.

[0057] (4) When the OD600 reached 0.6, IPTG was added by using a pipetting workstation, and the final concentration was 0.2mM. The culture condition was 30°C, 800 rpm, and the shaking culture was carried out for 12h.

[0058] (5) The bacteria were collected by using a refrigerated centrifuge, and the parameters were 3000 rpm, 4°C, and 5min.

[0059] (6) 200μL 50mM Tris-HCl (pH 8.5) was distributed into each well by using a pipetting workstation, and shaken to mix.

[0060] (7) The resuspended bacteria were placed in a-80°C refrigerator, and after freezing, they were taken out and thawed at room temperature. This step was repeated three times to complete the repeated freeze-thaw cell disruption operation.

[0061] (8) The crude enzyme and cell debris were separated by using a refrigerated centrifuge, and the parameters were 3000 rpm, 4°C, and 20min.

[0062] (9) The supernatant was collected, and the supernatant was the mutant enzyme liquid to be verified.

[0063] (10) The mutant enzyme liquid was heat treated at 40°C for 20min.

[0064] (11) 50μL of the treated mutant enzyme liquid was added to 150μL of uric acid solution (50mg / L), and placed in an enzyme marker, and the Abs293nm-time curve was detected by kinetic mode, and the residual enzyme activity after heat treatment was calculated. The higher the residual enzyme activity, the higher the potential stability of the mutant corresponding to the bacteria liquid, and the subsequent sequencing and shake flask expression verification.

[0065] 2) Expression, purification and enzymatic property determination of uric acid oxidase

[0066] (1) Wild type uricase and mutants were expressed in shake flasks. Inoculate 4 mL LB medium (containing 50 mg / L kana) at 1‰ inoculation amount, 37°C, 220 rpm for 8 h. Then inoculate 100 mL LB medium (containing 50 mg / L kana) at 1% inoculation amount, 37°C, 220 rpm for 8 h. When OD600 is 0.6-0.8, add IPTG as an inducer, final concentration is 0.2 mM. The culture conditions are 30°C, 170 rpm, and the culture time is 12 h.

[0067] (2) Uricase purification: centrifugal collection of bacterial precipitate, centrifuge parameters are 8000 rpm, 4°C, 5 min. Add 10 mL Lysis Buffer (10 mM imidazole, pH 8) to resuspend the precipitate. Use a high-pressure homogenizer to break the cell, parameters are 1000 pa, 4°C. After cell breaking, collect the supernatant again, centrifuge parameters are 8000 rpm, 4°C, 20 min. At this time, the supernatant is crude enzyme solution.

[0068] Use affinity chromatography to purify, add two column volumes of Lysis buffer to column equilibration. After pouring the crude enzyme solution, add Lysis Buffer and Washbuffer (30 mM imidazole, pH 8) to elute impurities, and add ElutionBuffer (300 mM imidazole, pH 8) to collect the target protein. Complete the affinity chromatography process.

[0069] Pour the collected protein into a 10KDa ultrafiltration centrifuge tube, centrifuge parameters are 3000g, 4°C, 15 min. After centrifugation, discard the filtrate and add 50 mM Tris-HCl (pH 8.5) buffer. This step is repeated three times, and the collected protein is the purified uricase.

[0070] (3) Enzyme activity detection: the substrate is uric acid solution, the concentration of uric acid solution is 50 mg / L, and 50 mM Tris-HCl (pH 8.5) is used as the solvent. Use 5M hydrochloric acid to stop the reaction. Set the metal bath to 30°C, 600 rpm, the reaction system is 1 mL, add 900 μL uric acid solution, add 50 μL of appropriately diluted purified uric acid oxidase to start the reaction, after 4 min of reaction, add 50 μL of hydrochloric acid to terminate the reaction. The control group is 50 μL of purified uric acid oxidase replaced by 50 μL of Tris-HCl buffer. Detect by enzyme label instrument, use the decrease of Abs293nm to represent enzyme activity.

[0071] The protein concentration of the purified uricase was detected by Coomassie brilliant blue method, with 0.2 mg / mL BSA protein as a standard, gradient dilution as a standard curve. Add 1X Coomassie brilliant blue, and detect the Abs595nm of the standard and uricase by an enzyme label instrument to obtain the protein concentration of uricase.

[0072] (4) Thermal stability detection: Put the pure uricase and the mutant into a metal bath at high temperature (the present application detects the stability at 40℃ and 50℃). Take samples over time, and determine the enzyme activity according to the enzyme activity detection steps, so as to obtain the curve of the residual enzyme activity changing with time. The time corresponding to the residual enzyme activity of 50% is the thermal stability half-life t 1 / 2 .

[0073] (5) After detection, four mutants with improved stability were obtained by directed evolution combined with high-throughput screening, as shown in Table 1.

[0074] Table 1 Mutants with improved thermal stability screened by directed evolution

[0075]

[0076] Note: T75S / K299E: The 75th amino acid of UOX is mutated from T to S, and the 299th amino acid is mutated from K to E.

[0077] 3) Key sites and mutants affecting thermal stability were mined by calculating the free energy of unfolded state, and the specific steps were as follows:

[0078] (1) UOX model construction by Alphafold2

[0079] Using Alphafold2

[0080] (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / Alpha Fold2.ipynb) to construct models for wild type and each mutant, MSA (Multiple Sequence Alignment) multiple sequence alignment uses MMseqs2_uniref_env. The template for structure prediction comes from the pdb100 database. Five structure models were generated, and were sorted according to the predicted local difference test (pLDDT: predicted Local Distance Difference Test) score. The model with the highest score over 90 was considered for mutant structure analysis.

[0081] (2) The unfolding free energy (ΔΔG) of the mutants was calculated and sorted by virtual screening using FoldX. The greater the absolute value of ΔΔG, the more likely the mutation is to enhance stability. FoldX is a plug-in of Yasara (Version 21.12.19), which optimizes the protein structure using the "repair object" and calculates using the "mutant object" module.

[0082] (3) According to the results of virtual screening, a total of 6 recommended mutants were obtained. They are T68L, T75W, D184W, K204F, A97K, and D134F.

[0083] According to the mutation design, the point mutation primer is shown in Table 2.

[0084] Table 2 Point mutation primer design

[0085]

[0086]

[0087] Point mutations were performed using Mut Express II FastMutagenesis Kit V2 kit from Vazyme, and the specific process is as follows:

[0088] Step 1: Amplification of the target gene;

[0089] 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 from Mut Express II Fast Mutagenesis Kit V2 kit.

[0090] Point mutation PCR reaction parameters: 95°C pre-denaturation for 30s; 95°C denaturation for 15s, 60°C annealing for 15s, 72°C extension for 6min, 30 cycles; 72°C thorough extension for 5min.

[0091] The second step is to remove the template: consistent with the random mutation template removal step.

[0092] The third step is linear DNA in vitro circularization: consistent with the random mutation circularization step.

[0093] The fourth step is to transform the recombination product: consistent with the random mutation recombination product transformation step.

[0094] The expression, purification, thermal stability test of the mutants were performed according to step 2). The mutants with improved thermal stability were obtained in Table 3.

[0095] Table 3 Mutants with improved thermal stability obtained by virtual screening

[0096]

[0097] 4) Combination mutation

[0098] The advantage mutants obtained in Table 1 and Table 3 were combined to obtain mutants with more improved thermal stability by superimposing advantage mutations. The synthesis of the combined mutants was completed by Anshengda Company. The transformation of the plasmid, and the expression, purification, thermal stability test of the combined mutants were the same as above, and finally the advantage mutants M3 (T68L / T75S / K299E), M4 (T68L / T75S / E222D / K299E), M3* (T68L / T75W / K299E) were obtained, and their stability was further improved. The results are shown in Table 4.

[0099] Table 4 Thermal stability half-life of wild type UOX and combined mutants M2, M3, M4, M3* at 40°C

[0100]

[0101] 5) Key sites affecting anti-enzymatic properties were mined by calculating solvent accessible surface area and improving anti-enzymatic effect by mutation, the specific steps are as follows:

[0102] (1) M3* was used as a template to construct a tetramer model by Alphafold2, and a stable conformation in a solvent environment was obtained by molecular dynamics simulation. This conformation was introduced into Discovery studio, and the relative solvent accessible surface area of trypsin cleavage sites: lysine (abbreviated as K or LYS), arginine (ARG) residues was calculated by Solvent accessible function. The larger the relative solvent accessible surface area, the greater the probability of the cleavage site exposed in the water environment, that is, the greater the possibility of being attacked by trypsin. The results are shown in Table 5.

[0103] Table 5 Solvent accessible surface area calculation of potential attack sites in M3*

[0104]

[0105]

[0106] (2) According to Table 5, it is found that K204 is the fourth in the solvent surface area ranking, which is a potential site for exposure in a solvent environment, and according to Table 3, K204F can enhance the thermal stability of the enzyme, so the mutation K204F is continued to be superimposed on the basis of M3* to obtain M4* (T68L / T75W / K204F / K299E), which is expected to enhance the stability and also enhance the anti-enzymatic performance of the enzyme. The mutant is synthesized by Anboda Company, and the expression and purification steps are the same as above.

[0107] (3) Anti-enzymatic detection: the wild-type uricase and its mutants are uniformly diluted to 0.5 mg / mL by using a phosphate solution with pH 6.8 (6.8 g of potassium dihydrogen phosphate is dissolved in 500 mL of water, and the pH value is adjusted to 6.8 by using a 0.1 mol / L sodium hydroxide solution). Trypsin (purchased from sigma: 1,000-2,000 BAEE units / mg) and aprotinin (purchased from aladdin: 3-8 TIU / mg) are configured to 2.5 mg / mL and 5 mg / mL respectively. A plurality of 100 μL of the wild-type uricase and its mutants are placed in a 30°C metal bath, and 10 μL of trypsin is added to each tube to start the enzymatic reaction. The samples are taken out over time, and 10 μL of aprotinin is added to stop the reaction. The enzyme activity is determined by using the enzyme activity detection step. In this way, the residual enzyme activity-time curve can be obtained. The time corresponding to the residual enzyme activity of 50% is the anti-enzymatic half-life. The anti-enzymatic half-life of the wild type is 28.84 min, the anti-enzymatic half-life of M3* is 75.5 min, and the anti-enzymatic half-life of M4* is 142.8 min( Figure 1 )。

[0108] 6) The key sites and mutants affecting the anti-enzymatic stability are mined by the way of directed evolution combined with high-throughput screening.

[0109] (1) The mutant library is constructed and screened based on M4*. The specific steps are shown in 1). The step in step (10) in step 1) is changed to adding trypsin with a final concentration of 0.25 mg / ml to the supernatant, and incubating at 30°C for 1 h. Then the subsequent processing is carried out. The rest of the steps remain unchanged.

[0110] (2) By the way of directed evolution combined with high-throughput screening, the present application finds the M7 mutant (Y47F / T68L / T75W / D111G / K204F / F259I / K299E) based on M4*. According to the detection, the anti-enzymatic half-life of M7 reaches 246.6 min( Figure 1 )。

[0111] (3) SDS-PAGE electrophoresis was performed simultaneously on the enzymatic digestion products of wild-type uricase, M3*, M4*, and M7 at different time points. The enzymatic digestion products were mixed with loading buffer at a ratio of 1:3 and boiled in water for 10 min. Then, 5 μL of marker and 10 μL of sample were loaded for detection. The mixture was first run at 80V for 20 min, then at 120V until the bottom was reached. FastBlue staining was performed for 20 min. Destaining was then performed, and the results were observed. See [link to results]. Figure 2 .according to Figure 2 It was found that the improved resistance to enzymatic degradation of M4* and M7 can indeed delay the degradation process of the key band near 35 kDa, which explains the improved resistance to enzymatic degradation half-life.

[0112] (4) At the same time, the activity and thermal stability of M4* and M7 were also tested 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 thermostable half-life t) 1 / 2 (and Table 6.)

[0113] Table 6 Summary of Mutants

[0114]

[0115] 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 uricase mutant, characterized in that, The corresponding models are T68L / T75S / E222D / K299E, T68L / T75S / K299E, or T75S / K299E. The amino acid sequence of the uricase is shown in SEQ ID NO.

1.

2. The biomaterials related to the uricase mutant of claim 1, characterized in that, It is at least one of the following (1)-(4): (1) Nucleic acid molecules encoding uricase mutants; (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).

3. The application of the uricase mutant of claim 1 or the related biomaterial of claim 2 in improving the thermal stability of uricase.

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