A uricase mutant of Myceliophthora thermophila and its application

Abstract

The present invention discloses a Myceliophthora thermophila uricase mutant and its application. The Myceliophthora thermophila uricase mutant comprises one or more mutations in combination on the original wild-type Myceliophthora thermophila uricase (as shown in SEQ ID NO.1) or the wild-type Myceliophthora thermophila uricase with amino acids 297-304 removed (as shown in SEQ ID NO.3), and can be used to treat diseases associated with elevated uric acid, such as hyperuricemia, hyperuricosuria, and gout. The Myceliophthora thermophila uricase mutant provided by the present invention has higher catalytic activity and better thermal stability and stability against enzymatic digestion.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a Myceliophthora thermophila uricase mutant and application thereof. Background Art

[0002] Hyperuricemia is a metabolic syndrome caused by impaired purine metabolism. It is the second most common metabolic disease after diabetes in terms of number of patients. Hyperuricemia is also associated with many complications, such as obesity, hypertension, hyperlipidemia, impaired renal function, diabetes, and coronary artery disease. Traditional uric acid-lowering medications can increase the burden on the kidneys and further exacerbate the condition. During chemotherapy for hematologic malignancies such as leukemia and lymphoma, massive tumor cell death and lysis, coupled with extensive degradation of intracellular nucleic acids, lead to a sharp increase in plasma uric acid. Over a short period of time, large amounts of uric acid accumulate in the kidneys, causing acute renal failure and the development of tumor lysis syndrome (TLS), typified by hyperuricemia.

[0003] Uric acid is the end product of purine metabolism in the human body. Excessive uric acid levels can lead to diseases such as gout and hyperuricemia. Current clinical treatments for hyperuricemia and gout primarily include drugs that inhibit uric acid production, such as xanthine oxidase inhibitors like allopurinol and febuxostat; drugs that promote uric acid excretion, such as benzbromarone, which inhibit urate reabsorption by targeting the renal tubular epithelium; immunosuppressants that alleviate acute gout attacks; and uricase drugs that directly break down uric acid. Long-term use of drugs like benzbromarone and febuxostat can easily damage organs such as the liver and kidneys.

[0004] Uricase, also known as urate oxidase (UOX, EC 1.7.3.3), is widely found in microorganisms (such as Bacillus fastidious and Aspergillus flavus), plants (such as soybeans), and animals (such as pigs, dogs, and baboons). In the presence of oxygen, it oxidizes uric acid into 5-hydroxyisouric acid, which is then converted to allantoin. Allantoin is an easily eliminated, inert, water-soluble purine metabolite that is primarily excreted via the kidneys. Due to its high specificity and catalytic efficiency, clinical studies have demonstrated that uricase can rapidly and efficiently lower serum uric acid with virtually no toxic side effects, demonstrating promising therapeutic effects in patients with hyperuricemia and refractory gout. However, drawbacks such as poor stability, easy clearance after administration, and immunogenicity significantly limit its clinical application.

[0005] Therefore, it is of great significance to develop a stable and catalytically efficient uricase for the treatment of hyperuricemia-related diseases.

[0006] Explanation of terms

[0007] Mutations are represented by triplets: letter-number-letter, where the number indicates the position of the mutated amino acid, the letter before the number corresponds to the amino acid for which the mutation is designed, and the letter after the number indicates the amino acid used to replace the amino acid before the number. Summary of the Invention

[0008] The first objective of the present invention is to provide a recombinant mutant Myceliophthora thermophila uricase with high activity and good stability, which is expected to reduce the therapeutic dosage of uricase and reduce the immunogenicity caused by exogenous proteins.

[0009] The present invention adopts the following technical solutions:

[0010] A Myceliophthora thermophila uricase mutant comprising at least one mutation, or a combination of two or more mutations, at positions corresponding to the wild-type Myceliophthora thermophila uricase of SEQ ID NO: 1 or the wild-type Myceliophthora thermophila uricase with amino acids 297-304 truncated at the C-terminus (as shown in SEQ ID NO: 3); wherein the mutation is selected from:

[0011] (a) At position 129, arginine was replaced by alanine (R129A);

[0012] (b) at position 243, alanine was replaced by aspartic acid (A243D);

[0013] (c) at position 248, aspartic acid is replaced by glutamic acid (D248E);

[0014] (d) at position 212, serine was replaced by leucine (S212L);

[0015] (e) at position 55, valine was replaced by leucine and isoleucine (V55I);

[0016] (f) at position 160, alanine was replaced by serine (A160S);

[0017] (g) At position 208, lysine was replaced by alanine (K208A)

[0018] (h) at position 191, lysine was replaced by asparagine (K191N);

[0019] (i) At position 24, lysine was replaced by glutamine (K24Q);

[0020] (j) at position 275, lysine was replaced by asparagine (K275N);

[0021] (k) at position 202, arginine was replaced by histidine (R202H);

[0022] (l) at position 166, arginine was replaced by isoleucine (R166I);

[0023] (n) at position 68, lysine was replaced by isoleucine or alanine (K68I, K68A);

[0024] (o) at position 137, arginine was replaced by glutamic acid (R137E);

[0025] (p) at position 145, lysine was replaced by serine (K145S);

[0026] (q) at position 290, isoleucine was replaced by phenylalanine or valine (I290F, I290V);

[0027] (r) At position 179, isoleucine was replaced by valine (I179V).

[0028] Preferably, the Myceliophthora thermophila uricase mutant further comprises one or two or more of the following mutations in combination:

[0029] (s) at position 96, serine was replaced by aspartic acid (S96D);

[0030] (t) At position 227, proline was replaced by glutamine (P227Q).

[0031] Preferably, the Myceliophthora thermophila uricase mutant further comprises one or more of the following mutations in combination:

[0032] (u) at position 228, serine was replaced by glycine (S228G);

[0033] (v) At position 290, isoleucine was replaced by tyrosine (I290Y).

[0034] In certain embodiments, the Myceliophthora thermophila uricase mutant comprises at least one of the following mutations: S96D, K191N, I179V, A160S, S212L, V55I.

[0035] In certain other embodiments, the Myceliophthora thermophila uricase mutant comprises at least one of the following mutations: S96D, K191N, I179V, A160S, S212L, V55I, K145S, R202H.

[0036] Compared with the wild-type uricase, the activity of the Myceliophthora thermophila uricase mutant is increased by 103%.

[0037] The Myceliophthora thermophila uricase mutant may, for example, have 10-12 times, 5-9 times, or 2-5 times higher stability in the presence of trypsin than wild-type uricase.

[0038] The second object of the present invention is to provide use of the Myceliophthora thermophila uricase mutant in the preparation of a drug for treating hyperuricemia and / or hyperuricosuria.

[0039] Preferably, it is mainly used for oral intestinal uricase-lowering new drugs.

[0040] The third object of the present invention is to provide the use of the above-mentioned Myceliophthora thermophila uricase mutant in the preparation of a drug for treating gout.

[0041] The fourth object of the present invention is to provide a drug for treating hyperuricemia, hyperuricosuria or gout, wherein the active ingredient is the above-mentioned Myceliophthora thermophila uricase mutant.

[0042] Preferably, the medicament may contain an agent that increases pH.

[0043] Preferably, the Myceliophthora thermophila uricase mutant may be in a soluble form or a crystalline form.

[0044] In addition, the present invention also provides a nucleic acid sequence encoding the Myceliophthora thermophila uricase mutant, an expression vector comprising the nucleic acid sequence, and a host cell comprising the expression vector, such as an Escherichia coli cell.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The present invention provides a thermophilic Myceliophthora uricase mutant by mutating multiple key amino acid sites. Compared with the wild-type uricase, the mutant has significantly improved activity and stability. Through mutation at specific sites, the mutant's uric acid degradation ability is enhanced, and its enzyme activity is increased by 103%; in addition, in the presence of pancreatic enzymes, the mutant's stability is increased by 2-12 times, that is, it is more tolerant to the digestive tract environment and is suitable for oral preparations. Compared with traditional intravenous injection of uricase, it is more convenient to use and has higher patient compliance.

[0047] In summary, the Myceliophthora thermophila uricase mutant of the present invention has significant advantages in uric acid degradation activity, stability, and drug application potential. It can provide a more effective and convenient treatment plan for patients with gout, hyperuricemia, and hyperuricosuria, and has broad application prospects in the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1This is the SDS-PAGE diagram of the recombinant uricase purification process, which from left to right are: before induction; after induction; supernatant; precipitate; flow-through; elution; Marker.

[0049] Figure 2 Summary of the ability of single-point mutant Myceliophthora thermophila uricase to resist trypsin digestion.

[0050] Figure 3 Summary of the ability of the double mutant Myceliophthora thermophila uricase to resist trypsin digestion.

[0051] Figure 4 Summary of the ability of the triple mutant Myceliophthora thermophila uricase to resist trypsin digestion.

[0052] Figure 5 This is the SDS-PAGE gel image of the ability of the recombinant mutant Myceliophthora thermophila uricase to resist trypsin digestion.

[0053] Figure 6 This is the enzyme activity curve of the recombinant mutant Myceliophthora thermophila uricase. DETAILED DESCRIPTION

[0054] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, without departing from the concept of the present invention, several variations and improvements can be made, and these all fall within the scope of protection of the present invention.

[0055] The amino acid sequence of the wild-type Myceliophthora thermophila uricase (mtUOX) is shown in SEQ ID NO: 1: MPNLASARYGKDNVRVCKVQRDEKTGFHTVTEMTVSCLLEGQIETSYTAADNSVVVATDSIKNTIYIKAKEHPVNPPELYASLLGQHFLDKYPHISTAHVNLAVHRWTRLEVDGAPHPHSFYRDGQETRNVEVAVSRGPGGIRIKSSIVGLSVFKSTGSAFHGFVRDEYTTLPETWDRILSTDVDCSWTWKPFDTVEAVQQRVERFDKAWESARGIILRLFAVEESPSVQNTMYKMSEQILEAVPEVDRVDFALPNKHYFELDLSWHKGIKNTGKDAEVYVPQSGPNGLIKCEVARDETTLAHR

[0056] Its nucleotide sequence is shown as SEQ ID NO:2 below: ATGCCGAACTTGGCATCCGCTCGTTACGGCAAAGATAACGTTCGTGTTTGCAAAGTTCAGCGTGATGAAAAAACCGGTTTCCACACCGTTACCGAAATGACTGTTAGCTGTCTGCTGGAAGGTCAGATCGAAACTTCTTACACTGCAGCGGACAACTCCGTTGTTGTTGCGACCGATTCCATCAAGAATACCATCTACATCAAAGCGAAAGAACATCCGGTAAACCCACCGGAACTGTACGCTTCTCTGCTGGGTCAGCACTTCTTGGACAAATATCCACACATCTCTACCGCTCACGTTAACCTGGCTGTGCACCGTTGGACTCGTCTGGAAGTTGATGGTGCACCGCATCCGCACTCTTTCTACCGTGACGGCCAAGAAACTCGTAACGTTGAAGTTGCTGTTTCTCGTGGTCCAGGTGGTATCCGCATCAAATCCAGCATCGTGGGCCTGTCTGTGTTCAAATCTACCGGTTCTGCGTTCCACGGTTTCGTTCGTGACGAATACACCACTCTGCCGGAAACCTGGGACCGTATCTTGTCTACCGACGTTGACTGCTCTTGGACCTGGAAACCGTTCGATACCGTTGAAGCGGTTCAGCAGCGTGTTGAACGTTTCGACAAGGCGTGGGAATCCGCGCGTGGTATCATCTTGCGTCTGTTCGCGGTAGAAGAATCTCCGTCCGTTCAGAACACCATGTACAAGATGAGCGAACAGATCTTGGAAGCAGTACCGGAAGTTGACCGTGTTGATTTCGCTTTGCCGAACAAACACTACTTCGAACTGGACCTGTCTTGGCACAAAGGTATCAAGAACACCGGTAAAGACGCTGAAGTTTACGTACCGCAGTCTGGTCCGAACGGTCTGATCAAATGCGAAGTAGCACGTGACGAAACCACTCTGGCGCACCGT

[0057] The present invention also provides a recombinant Myceliophthora thermophila uricase F1B2, the amino acid sequence of which is shown in SEQ ID NO: 3: MPNLASARYGKDNVRVCKVQRDEKTGFHTVTEMTVSCLLEGQIETSYTAADNSVVVATDSIKNTIYIKAKEHPVNPPELYASLLGQHFLDKYPHISTAHVNLAVHRWTRLEVDGAPHPHSFYRDGQETRNVEVAVSRGPGGIRIKSSIVGLSVFKSTGSAFHGFVRDEYTTLPETWDRILSTDVDCSWTWKPFDTVEAVQQRVERFDKAWESARGIILRLFAVEESPSVQNTMYKMSEQILEAVPEVDRVDFALPNKHYFELDLSWHKGIKNTGKDAEVYVPQSGPNGLIKCEVARD

[0058] Its nucleotide sequence is as shown in SEQ ID NO:4: ATGCCGAACTTGGCATCCGCTCGTTACGGCAAAGATAACGTTCGTGTTTGCAAAGTTCAGCGTGATGAAAAAACCGGTTTCCACACCGTTACCGAAATGACTGTTAGCTGTCTGCTGGAAGGTCAGATCGAAACTTCTTACACTGCAGCGGACAACTCCGTTGTTGTTGCGACCGATTCCATCAAGAATACCATCTACATCAAAGCGAAAGAACATCCGGTAAACCCACCGGAACTGTACGCTTCTCTGCTGGGTCAGCACTTCTTGGACAAATATCCACACATCTCTACCGCTCACGTTAACCTGGCTGTGCACCGTTGGACTCGTCTGGAAGTTGATGGTGCACCGCATCCGCACTCTTTCTACCGTGACGGCCAAGAAACTCGTAACGTTGAAGTTGCTGTTTCTCGTGGTCCAGGTGGTATCCGCATCAAATCCAGCATCGTGGGCCTGTCTGTGTTCAAATCTACCGGTTCTGCGTTCCACGGTTTCGTTCGTGACGAATACACCACTCTGCCGGAAACCTGGGACCGTATCTTGTCTACCGACGTTGACTGCTCTTGGACCTGGAAACCGTTCGATACCGTTGAAGCGGTTCAGCAGCGTGTTGAACGTTTCGACAAGGCGTGGGAATCCGCGCGTGGTATCATCTTGCGTCTGTTCGCGGTAGAAGAATCTCCGTCCGTTCAGAACACCATGTACAAGATGAGCGAACAGATCTTGGAAGCAGTACCGGAAGTTGACCGTGTTGATTTCGCTTTGCCGAACAAACACTACTTCGAACTGGACCTGTCTTGGCACAAAGGTATCAAGAACACCGGTAAAGACGCTGAAGTTTACGTACCGCAGTCTGGTCCGAACGGTCTGATCAAATGCGAAGTAGCACGTGAC

[0059] The present invention also provides a recombinant Myceliophthora thermophila uricase F1B2-S96D, the amino acid sequence of which is shown in SEQ ID NO: 5: MPNLASARYGKDNVRVCKVQRDEKTGFHTVTEMTVSCLLEGQIETSYTAADNSVVVATDSIKNTIYIKAKEHPVNPPELYASLLGQHFLDKYPHIDTAHVNLAVHRWTRLEVDGAPHPHSFYRDGQETRNVEVAVSRGPGGIRIKSSIVGLSVFKSTGSAFHGFVRDEYTTLPETWDRILSTDVDCSWTWKPFDTVEAVQQRVERFDKAWESARGIILRLFAVEESPSVQNTMYKMSEQILEAVPEVDRVDFALPNKHYFELDLSWHKGIKNTGKDAEVYVPQSGPNGLIKCEVARD

[0060] Its nucleotide sequence is as shown in SEQ ID NO:6: ATGCCGAACTTGGCATCCGCTCGTTACGGCAAAGATAACGTTCGTGTTTGCAAAGTTCAGCGTGATGAAAAAACCGGTTTCCACACCGTTACCGAAATGACTGTTAGCTGTCTGCTGGAAGGTCAGATCGAAACTTCTTACACTGCAGCGGACAACTCCGTTGTTGTTGCGACCGATTCCATCAAGAATACCATCTACATCAAAGCGAAAGAACATCCGGTAAACCCACCGGAACTGTACGCTTCTCTGCTGGGTCAGCACTTCTTGGACAAATATCCACACATCGACACCGCTCACGTTAACCTGGCTGTGCACCGTTGGACTCGTCTGGAAGTTGATGGTGCACCGCATCCGCACTCTTTCTACCGTGACGGCCAAGAAACTCGTAACGTTGAAGTTGCTGTTTCTCGTGGTCCAGGTGGTATCCGCATCAAATCCAGCATCGTGGGCCTGTCTGTGTTCAAATCTACCGGTTCTGCGTTCCACGGTTTCGTTCGTGACGAATACACCACTCTGCCGGAAACCTGGGACCGTATCTTGTCTACCGACGTTGACTGCTCTTGGACCTGGAAACCGTTCGATACCGTTGAAGCGGTTCAGCAGCGTGTTGAACGTTTCGACAAGGCGTGGGAATCCGCGCGTGGTATCATCTTGCGTCTGTTCGCGGTAGAAGAATCTCCGTCCGTTCAGAACACCATGTACAAGATGAGCGAACAGATCTTGGAAGCAGTACCGGAAGTTGACCGTGTTGATTTCGCTTTGCCGAACAAACACTACTTCGAACTGGACCTGTCTTGGCACAAAGGTATCAAGAACACCGGTAAAGACGCTGAAGTTTACGTACCGCAGTCTGGTCCGAACGGTCTGATCAAATGCGAAGTAGCACGTGACGAAACCACTCTGGCGCACCGT

[0061] The present invention also provides a recombinant Myceliophthora thermophila uricase F1B2-A160S-V55I, the amino acid sequence of which is shown in SEQ ID NO: 7: MPNLASARYGKDNVRVCKVQRDEKTGFHTVTEMTVSCLLEGQIETSYTAADNSVIVATDSIKNTIYIKAKEHPVNPPELYASLLGQHFLDKYPHISTAHVNLAVHRWTRLEVDGAPHPHSFYRDGQETRNVEVAVSRGPGGIRIKSSIVGLSVFKSTGSFHGFVRDEYTTLPETWDRILSTDVDCSWTWKPFDTVEAVQQRVERFDKAWESARGIILRLFAVEESPSVQNTMYKMSEQILEAVPEVDRVDFALPNKHYFELDLSWHKGIKNTGKDAEVYVPQSGPNGLIKCEVARD

[0062] Its nucleotide sequence is shown as SEQ ID NO:8: ATGCCGAACTTGGCATCCGCTCGTTACGGCAAAGATAACGTTCGTGTTTGCAAAGTTCAGCGTGATGAAAAAACCGGTTTCCACACCGTTACCGAAATGACTGTTAGCTGTCTGCTGGAAGGTCAGATCGAAACTTCTTACACTGCAGCGGACAACTCCGTTATCGTTGCGACCGATTCCATCAAGAATACCATCTACATCAAAGCGAAAGAACATCCGGTAAACCCACCGGAACTGTACGCTTCTCTGCTGGGTCAGCACTTCTTGGACAAATATCCACACATCTCTACCGCTCACGTTAACCTGGCTGTGCACCGTTGGACTCGTCTGGAAGTTGATGGTGCACCGCATCCGCACTCTTTCTACCGTGACGGCCAAGAAACTCGTAACGTTGAAGTTGCTGTTTCTCGTGGTCCAGGTGGTATCCGCATCAAATCCAGCATCGTGGGCCTGTCTGTGTTCAAATCTACCGGTTCTTCTTTCCACGGTTTCGTTCGTGACGAATACACCACTCTGCCGGAAACCTGGGACCGTATCTTGTCTACCGACGTTGACTGCTCTTGGACCTGGAAACCGTTCGATACCGTTGAAGCGGTTCAGCAGCGTGTTGAACGTTTCGACAAGGCGTGGGAATCCGCGCGTGGTATCATCTTGCGTCTGTTCGCGGTAGAAGAATCTCCGTCCGTTCAGAACACCATGTACAAGATGAGCGAACAGATCTTGGAAGCAGTACCGGAAGTTGACCGTGTTGATTTCGCTTTGCCGAACAAACACTACTTCGAACTGGACCTGTCTTGGCACAAAGGTATCAAGAACACCGGTAAAGACGCTGAAGTTTACGTACCGCAGTCTGGTCCGAACGGTCTGATCAAATGCGAAGTAGCACGTGACGAAACCACTCTGGCGCACCGT

[0063] Example

[0064] Example 1: Construction of recombinant expression vectors for a series of mutants of Myceliophthora thermophila uricase

[0065] The proposed mutation site was selected, the target gene was amplified by PCR, and after verification by nucleic acid gel, it was transformed into Escherichia coli DH5α. The mutant coding sequence was determined by picking single colonies on conventional kanamycin plates to confirm that the thermophilic Myceliophthora uricase mutant was obtained.

[0066] Example 2: Expression and purification of Myceliophthora thermophila uricase mutants

[0067] The target protein was expressed using the Escherichia coli BL21 (DE3) prokaryotic expression system. The transformed E. coli cells were cultured in LB medium (containing 0.1 g / L kanamycin) for 4-6 h, induced with 200 μM isopropyl-β-D-thiogalactopyranoside (IPTG) at 17°C for 12 h, and centrifuged at 5000 rpm for 10 min to collect the cells for purification of the uricase mutant.

[0068] Purification was performed using affinity chromatography. The supernatant of the disrupted bacterial solution was mixed with a nickel medium. The target protein was specifically bound to the nickel medium through a 6×His tag. After washing with low-concentration imidazole and eluting with high-concentration imidazole, a high-purity target protein solution was obtained. The purity of the target protein was tested by SDS-PAGE. The results were as follows: Figure 1 The molecular weight of mtUOX and its mutants is approximately 34.2 kDa; mtUOX and its mutants are stable in Tris-HCl pH 8.0, 150 mM NaCl solution.

[0069] Example 3: Stability determination of a series of mutants of Myceliophthora thermophila uricase against trypsin digestion

[0070] Trypsin stability assay steps: 100 μM uricase solution and 1 mg / mL trypsin were mixed in equal volumes and incubated at 37°C for a certain period of time. 20 μL of samples were taken at 0 min, 10 min, 20 min, 30 min, 45 min, and 60 min of incubation, and 5× SDS was added. The samples were immediately denatured at 95°C for 10 min. The degradation of the protein after incubation with trypsin was verified by SDS-PAGE.

[0071] Twenty-one mutant Myceliophthora thermophila uricases were designed, each with a single amino acid substitution corresponding to the wild-type sequence (SEQ ID NO. 1 or SEQ ID NO. 3). In this example, the more active wild-type Myceliophthora thermophila uricase with C-terminal truncation at amino acids 297-304 (as shown in SEQ ID NO: 3) was used as the parent mutant, designated F1B2 and labeled R1. The specific activity, percentage increase in trypsin resistance, and fold increase of all subsequent mutants were based on R1.

[0072] Table 1 shows the amino acid substitutions of the recombinant mutant uricases from Myceliophthora thermophila, as well as the specific activity of each enzyme (μM of uric acid consumed in 1 min per μM enzyme), and the trypsin stability (the amount of protein remaining after 30 min of trypsin digestion). “-” indicates that the mutant has lower specific activity or resistance to trypsin digestion than the wild-type uricase.

[0073] Table 1

[0074] clone replace Remaining after 30 min of trypsin digestion (%) Increase multiples Specific activity Improvement percentage (%) WT 6.49 / 30.33 / R1 F1B2 10.28 / 100.08 / R1-M1 S96D 27.39 4.22 136.88 36.77 R1-M2 K191N 21.74 3.35 100.12 0.04 R1-M3 I179V 18.82 2.90 185.70 85.55 R1-M4 A160S 18.63 2.87 100.79 0.71 R1-M5 I290F 17.98 2.77 34.65 - R1-M6 S212L 15.97 2.46 101.83 1.75 R1-M7 A243D 12.92 1.99 106.34 6.25 R1-M8 R202H 12.53 1.93 148.83 48.71 R1-M9 I290V 11.42 1.76 103.35 3.27 R1-M10 K145S 11.23 1.73 111.28 11.19 R1-M11 P227Q 9.60 1.48 62.54 - R1-M12 K24Q - - 154.97 54.85 R1-M13 K275N - - 153.68 53.56 R1-M14 R129A - - 145.65 45.53 R1-M15 R166I - - 144.92 44.80 R1-M16 K68A - - 136.77 36.66 R1-M17 K68I - - 134.51 34.40 R1-M18 R137E - - 117.88 17.79 R1-M19 V55I - - 117.88 17.79 R1-M20 K208A - - 117.79 17.70 R1-M21 D248E - - 114.57 14.48

[0075] The ability of single-point mutant Myceliophthora thermophila uricase to resist trypsin digestion is summarized as follows: Figure 2 shown.

[0076] The above results indicate that S96D, K191N, I179V, A160S, S212L, and V55I are key mutation sites for improving enzyme activity or resistance to trypsin digestion. Therefore, the thermophila uricase containing these six mutations was used as the parent in the second round of design of the thermophila uricase, denoted as R2.

[0077] Table 2 depicts the amino acid substitutions of the mutant uricases, as well as the specific activity of each enzyme (μM of uric acid consumed in 1 min per μM enzyme), and trypsin stability (the amount of protein remaining after 30 min of trypsin digestion). “-” indicates that the specific activity or resistance to trypsin digestion is lower than that of the wild-type uricase.

[0078] Table 2

[0079] clone replace Remaining after 30 min of trypsin digestion (%) Increase multiples Specific activity Improvement percentage (%) R2-M1 A160S-V55I 78.72 12.13 101.98 1.90 R2-M2 A160S-S212L 60.16 9.27 157.12 - R2-M3 S212L-V55I 56.07 8.64 111.51 11.42 R2-M4 I179V-V55I 47.64 7.34 175.16 - R2-M5 S96D-A160S 46.60 7.18 107.06 6.97 R2-M6 S96D-V55I 44.78 6.90 105.46 5.38 R2-M7 A243D-V55I 41.54 6.40 109.06 8.97 R2-M8 A160S-A243D 39.39 6.07 100.41 0.33 R2-M9 S228G-I290Y 39.20 6.04 - - R2-M10 K191N-V55I 36.86 5.68 101.26 1.18 R3-M11 S212L-A243D 30.50 4.70 172.39 10.38 R2-M12 S212L-K191N 12.68 3.96 101.84 1.76 R2-M13 S96D-A243D 16.87 2.60 120.95 20.85 R2-M14 K191N-I179V 16.55 2.55 100.17 0.09 R2-M15 S96D-S212L 15.71 2.42 148.64 48.52 R2-M16 S96D-K191N 15.58 2.40 149.78 49.66 R2-M17 K191N-A160S 14.54 2.24 100.48 0.40 R2-M18 I179V-A160S 13.95 2.15 154.77 54.65 R2-M19 S96D-I179V 13.76 2.12 203.65 103.49 R2-M20 S212L-R129A 12.27 1.89 167.67 67.54 R2-M21 K191N-A243D 10.32 1.59 - - R2-M22 I179V-A243D - - 140.49 40.38 R2-M23 I179V-S212L - - 124.38 24.28

[0080] The resistance of the double mutant Myceliophthora thermophila uricase to trypsin digestion is summarized as follows: Figure 3 shown.

[0081] The above results indicate that A160S-V55I, A160S-S212L, S212L-V55I, I179V-V55I, S96D-A160S, S96D-V55I, A243D-V55I, and A160S-A243D are key mutation sites for improving enzyme activity or resistance to trypsin digestion. Therefore, the thermophila myceliac uricase containing these eight mutations was used as the parent in the third round of design of the thermophila myceliac uricase, denoted as R3.

[0082] Table 3 depicts the amino acid substitutions of the mutant uricases, as well as the specific activity of each enzyme (μM of uric acid consumed in 1 min per μM enzyme), and trypsin stability (the amount of protein remaining after 30 min of trypsin digestion). “-” indicates that the specific activity or resistance to trypsin digestion is lower than that of the wild-type uricase.

[0083] Table 3

[0084] clone replace Remaining after 30 min of trypsin digestion (%) Increase multiples Specific activity Improvement percentage (%) R3-M1 A160S-V55I-S96D 71.20 10.97 112.17 12.08 R3-M2 A160S-V55I-I179V 37.71 5.81 103.47 3.39 R3-M3 A160S-V55I-K191N 35.24 5.43 103.42 - R3-M4 A160S-V55I-S212L 31.15 4.80 109.96 - R3-M5 A160S-V55I-A243D 28.43 4.38 114.63 14.54 R3-M6 A160S-V55I -R202H 24.40 3.76 246.07 - R3-M7 S212L-V55I-R202H 18.11 2.79 247.37 - R3-M8 S212L-V55I K145S 17.91 2.76 104.03 3.95 R3-M9 S96D-V55I -R202H 17.46 2.69 123.25 23.15 R3-M10 S96D-A160S-K145S 16.10 2.48 246.64 - R3-M11 I179V-V55I-R202H 15.77 2.43 110.47 - R3-M12 A160S-S212L-R202H 13.76 2.12 230.01 - R3-M13 S96D-V55I-K145S 13.69 2.11 124.59 24.49 R3-M14 S96D-A160S-R202H 13.56 2.09 240.10 - R3-M15 A243D-V55IK-145S 11.81 1.82 119.84 19.74 R3-M16 A160S-A243D-K145S 11.16 1.72 184.94 - R3-M17 A243D-V55I-R202H 10.84 1.67 275.82 - R3-M18 I179V-V55I-K145S - - 166.34 66.21 R3-M19 A160S-A243D-R202H - - 111.86 11.77

[0085] The ability of the triple mutant Myceliophthora thermophila uricase to resist trypsin digestion is summarized as follows: Figure 4 shown.

[0086] Table 4 depicts the amino acid substitutions of all mutant uricases, as well as the specific activity of each enzyme (μM of uric acid consumed in 1 min per μM enzyme), and trypsin stability (the amount of protein remaining after 30 min of trypsin digestion). “-” indicates that the specific activity or resistance to trypsin digestion is lower than that of the wild-type uricase.

[0087] Table 4

[0088] Protein Remaining after 30 min of trypsin digestion (%) Increase multiples Specific activity Improvement percentage (%) R2-M1 78.72 12.13 101.98 1.9 R3-M1 71.20 10.97 112.17 12.08 R2-M2 60.16 9.27 157.12 - R2-M3 56.07 8.64 111.51 11.42 R2-M4 47.64 7.34 175.16 - R2-M5 46.60 7.18 107.06 6.97 R2-M6 44.78 6.90 105.46 5.38 R2-M7 41.54 6.40 109.06 8.97 R2-M8 39.39 6.07 100.41 0.33 R2-M9 39.20 6.04 - - R3-M2 37.71 5.81 103.47 3.39 R2-M10 36.86 5.68 101.26 1.18 R3-M3 35.24 5.43 103.42 - R3-M4 31.15 4.80 109.96 - R3-M11 30.50 4.70 172.39 10.38 R3-M5 28.43 4.38 114.63 14.54 R1-M1 27.39 4.22 136.88 36.77 R2-M12 12.68 3.96 101.84 1.76 R3-M6 24.40 3.76 246.07 - R1-M2 21.74 3.35 100.12 0.04 R1-M3 18.82 2.90 185.7 85.55 R1-M4 18.63 2.87 100.79 0.71 R3-M7 18.11 2.79 247.37 - R1-M5 17.98 2.77 34.65 - R3-M8 17.91 2.76 104.03 3.95 R3-M9 17.46 2.69 123.25 23.15 R2-M13 16.87 2.60 120.95 20.85 R2-M14 16.55 2.55 100.17 0.09 R3-M10 16.10 2.48 246.64 - R1-M6 15.97 2.46 101.83 1.75 R3-M11 15.77 2.43 110.47 - R2-M15 15.71 2.42 148.64 48.52 R2-M16 15.58 2.40 149.78 49.66 R2-M17 14.54 2.24 100.48 0.4 R2-M18 13.95 2.15 154.77 54.65 R2-M19 13.76 2.12 203.65 103.49 R3-M12 13.76 2.12 230.01 - R3-M13 13.69 2.11 124.59 24.49 R3-M14 13.56 2.09 240.1 - R1-M7 12.92 1.99 106.34 6.25 R1-M8 12.53 1.93 148.83 48.71 R2-M20 12.27 1.89 167.67 67.54 R3-M15 11.81 1.82 119.84 19.74 R1-M9 11.42 1.76 103.35 3.27 R1-M10 11.23 1.73 111.28 11.19 R3-M16 11.16 1.72 184.94 - R3-M17 10.84 1.67 275.82 - R2-M21 10.32 1.59 - - R1-M11 9.60 1.48 62.54 - R3-M18 - - 166.34 66.21 R1-M12 - - 154.97 54.85 R1-M13 - - 153.68 53.56 R1-M14 - - 145.65 45.53 R1-M15 - - 144.92 44.8 R2-M22 - - 140.49 40.38 R1-M16 - - 136.77 36.66 R1-M17 - - 134.51 34.4 R2-M23 - - 124.38 24.28 R1-M18 - - 117.88 17.79 R1-M19 - - 117.88 17.79 R1-M20 - - 117.79 17.7 R1-M21 - - 114.57 14.48 R3-M19 - - 111.86 11.77

[0089] The results of SDS-PAGE test in this example are as follows Figure 5 shown by Figure 5 It can be seen that the A160S-V55I, A160S-V55I-S96D and A160S-S212L mutants have strong resistance to trypsin digestion and slow digestion rate, while the wild-type Myceliophthora thermophila uricase is easily digested by trypsin.

[0090] Example 4: Activity determination of a series of mutants of Myceliophthora thermophila uricase

[0091] Activity determination definition: The amount of enzyme required to oxidize 1 μmol of substrate uric acid per minute is one activity unit.

[0092] Enzyme activity assay steps: Dissolve uric acid powder in sodium tetraborate solution (pH 8.6) to prepare a 300 μM uric acid solution. Dilute the appropriate uricase solution to 5 μM using SEC buffer (Tris-HCl pH 8.0, 150 mM NaCl).

[0093] Set the detection wavelength of the microplate reader to 293 nm and 37°C. Add 5 μL of uricase solution to each well of a 96-well plate without UV absorption. Mix 195 μL of uric acid solution preheated to 37°C with the uricase solution. Initiate the reaction and continuously measure the absorbance change at 293 nm for 20 minutes, measuring the value every 1 minute. Use a blank control with a solution containing the same uric acid concentration but no enzyme.

[0094] The catalytic reaction activity was compared by comparing the rate of decrease in uric acid concentration. The enzyme activity results of some mutants are as follows: Figure 6 The results show that the mutant of the present invention has good enzyme activity.

[0095] The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments. As long as the requirements of the present invention are met, they are within the scope of protection of the present invention. In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached hereto.

Claims

1. A Myceliophthora thermophila uricase mutant, characterized in that: The mutation site on the wild-type Myceliophthora thermophila uricase with amino acids 297-304 removed is A160S. The amino acid sequence of the wild-type Myceliophthora thermophila uricase with amino acids 297-304 removed is shown in SEQ ID NO.

3.

2. The Myceliophthora thermophila uricase mutant according to claim 1, characterized in that The mutant has the mutation site of A160S-V55I, A160S-S212L, A160S-S96D or A160S-A243D on the wild-type Myceliophthora thermophila uricase with amino acids 297-304 removed.

3. The Myceliophthora thermophila uricase mutant according to claim 2, characterized in that The mutant has the following mutation sites on the wild-type Myceliophthora thermophila uricase with amino acids 297-304 removed: A160S-V55I-S96D, A160S-V55I-I179V, A160S-V55I-K191N, A160S-V55I-S212L, A160S-V55I-A243D or A160S-V55I-R202H.

4. Use of the Myceliophthora thermophila uricase mutant according to any one of claims 1 to 3 in the preparation of a medicament for treating hyperuricemia and / or hyperuricosuria.

5. The use according to claim 4, characterized in that The drug is administered orally and acts on the intestine.

6. Use of the Myceliophthora thermophila uricase mutant according to any one of claims 1 to 3 in the preparation of a drug for treating gout.

7. A drug for treating hyperuricemia, hyperuricosuria or gout, characterized in that: The active ingredient is the Myceliophthora thermophila uricase mutant according to any one of claims 1 to 3.

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