CN112980808A

CN112980808A - Uricase, preparation method and application thereof

Info

Publication number: CN112980808A
Application number: CN201911273027.7A
Authority: CN
Inventors: 郭庆功; 林春娇; 张裕平
Original assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Current assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority date: 2019-12-12
Filing date: 2019-12-12
Publication date: 2021-06-18

Abstract

The invention relates to a mutated uricase, wherein at least one Y and/or at least one M in a binding site and a catalytic active site of the uricase are mutated, wherein the Y is mutated to any one of H, F, S and G, and the M is mutated to any one of V, L and I. The mutated uricase of the present invention is resistant to interference from the administration of acetaminophen. In addition, the invention also relates to a related preparation method, a detection method and application.

Description

Uricase, preparation method and application thereof

Technical Field

The invention relates to the field of uric acid detection, in particular to a novel mutated uricase.

Background

In organisms, Uric Acid (UA) is a metabolite of purine, and its solubility in serum is extremely low. In the absence of active uricase, the body is unable to metabolize uric acid to form other more soluble products (e.g., allantoin, urea, etc.). Generally, uric acid produced by the human body and uric acid cleared by the kidney can maintain balance. However, when the production of uric acid in the human body is excessive or the kidney has a reduced ability to remove uric acid, uric acid accumulates in the serum.

Currently, the most commonly used method for detecting serum uric acid levels is the uricase-peroxidase method based on the Trinder reaction. The basic principle of this method is to utilize uricase from other organisms (such as fungi, bacteria, etc.) to catalyze the production of allantoin, carbon dioxide and hydrogen peroxide from uric acid, water and oxygen. The generated hydrogen peroxide can generate quinone pigment and water together with 4-amino-azacyclo and chromogen under the catalysis of peroxidase. The quinone-based pigment produced therein has a distinct absorption peak in visible light of a specific wavelength. The amount of the quinone-based pigment produced can be determined by detecting the absorbance, and the contents of the hydrogen peroxide produced and urea in the sample can be estimated.

Paracetamol (also known as Paracetamol) is a common analgesic and antipyretic agent. It is reported that individuals who take acetaminophen have a problem of low serum uric acid test values.

In other words, for an individual taking acetaminophen, the uric acid content in the serum of the individual is evaluated by using a uric acid test item based on a uricase-peroxidase method, so that real data of the serum uric acid level of the individual cannot be obtained, and the judgment of a follow-up doctor is easily interfered. For example, for individuals exhibiting symptoms of redness, inflammation, etc., acetaminophen may be administered to relieve pain caused by redness or inflammation. However, taking acetaminophen may result in underestimation of individual uric acid levels, which may affect the physician's follow-up.

Based on this, in the field of uric acid detection, there is a strong need to eliminate interference caused by taking acetaminophen.

Disclosure of Invention

As described above, when uric acid is detected, there is a problem that the uric acid detection result is lower than the actual value due to the administration of acetaminophen. This problem may be due to: acetaminophen is oxidized in the liver in vivo to produce the intermediate NAPQI (N-acetyl-p-benzoquinone) which causes negative interference in the detection of serum uric acid levels by uricase-peroxidase method. However, the principle of this negative interference is not yet clear at present.

That is, even though the intermediate product NAPQI is considered to be a factor causing inaccurate uric acid detection results (in the case of taking acetaminophen), it is still unclear how the intermediate product NAPQI interferes with the uric acid detection process, so that it is difficult to eliminate the defect of inaccurate uric acid detection results caused by taking acetaminophen.

Through research, the inventor finds that: the second reaction in the uric acid detection method, namely the reaction of hydrogen peroxide and 4-amino-azacyclo-chromogen to generate the quinone pigment under the catalysis of peroxidase, has no obvious interference of NAPHQI. In fact, NAPHI interference is concentrated in the first reaction step of the uric acid detection method, i.e., the catalytic production of allantoin, carbon dioxide and hydrogen peroxide from uric acid, water and oxygen by uricase. Further, through a large number of experimental analyses, the interference of NAPHI on uric acid detection comes from its influence on the uricase enzyme activity in the first reaction.

Accordingly, in a first aspect, the present invention provides a mutated uricase, characterized in that at least one Y and/or at least one M of the binding site and the catalytically active site of the uricase is mutated, wherein the Y is mutated to any one of H, F, S and G and the M is mutated to any one of V, L and I.

The mutated uricase of the invention can replace the conventional uricase and be used in a uric acid detection reagent based on the Trinder reaction. Under the condition, the interference caused by NAPHI in the sample can be successfully resisted, so that the accuracy of the uric acid level is improved. Meanwhile, the reagent widens the application range of the detection reagent, and avoids the contraindication of taking acetaminophen by a subject.

It is understood that, according to sequence homology analysis of uricase (e.g., FIG. 1), the common natural homologous residue of tyrosine (Y) at the catalytic active site of uricase, such as the first Y in Motif A, is H; and phenylalanine (F) is structurally similar to tyrosine (Y). On the other hand, glycine (G) and serine (S) are both amino acid residues which have simple structures and can reduce steric hindrance of proteins. On the other hand, the common natural homologous residue of methionine (M) in the catalytic active site of uricase, such as M in Motif B, is I or V, and L is also an amino acid residue close in nature thereto.

In some embodiments, the Y is in Motif a of the uricase.

In a specific embodiment, said Y is first in Motif a of said uricase.

In some embodiments, the Y is mutated to H.

In other embodiments, the Y is mutated to G.

In some embodiments, the M is located in Motif B of the uricase.

In a specific embodiment, said M is at position 10 of Motif B of said uricase.

In some embodiments, the M is mutated to L.

In other embodiments, the M is mutated to I.

In some embodiments, the mutation to Y occurs at the catalytically active site of uricase.

In some embodiments, the mutation to M occurs at the catalytically active site of uricase.

In addition, the mutated uricase of the present invention may further comprise at least one additional mutation located at a non-binding site and at a non-catalytically active site.

In some embodiments, the additional mutation is a mutation of an amino acid residue selected from the group consisting of C, M, Y and W to an amino acid residue other than C, M, Y and W.

In still other embodiments, the additional mutation is a deletion of an amino acid residue selected from the group consisting of C, M, Y and W.

In a preferred embodiment, the additional mutation occurs in the vicinity of the binding site and/or the catalytically active site.

In some embodiments, the additional mutation occurs within 5 residues upstream and/or 5 residues downstream of each of the binding site and/or catalytically active site.

The present invention further improves the ability to eliminate NAPQI negative interference by introducing additional mutations at non-binding and non-catalytically active sites, particularly near them (e.g., within 5 residues upstream and/or 5 residues downstream).

In some embodiments, the uricase of the invention is derived from Aspergillus flavus, Bacillus sp.TB-90, or Candida utilis.

In an exemplary embodiment, the mutated uricase of the invention is derived from Aspergillus flavus and has the Y9H mutation; preferably, at least one additional mutation selected from the group consisting of Y17F, Y47F, Y66H, M232L and Y233F is further included.

In an exemplary embodiment, the mutated uricase of the present invention is derived from Bacillus sp.tb-90 and has the Y11H and M76I mutations; preferably, at least one additional mutation selected from the group consisting of M9Q, Y10R and Y254F is further included.

In an exemplary embodiment, the mutated uricase of the present invention is derived from Candida utilis and has the Y10H mutation; preferably, at least one additional mutation selected from the group consisting of Y49F, Y112V, and M238L is further included.

In a second aspect, the invention provides a nucleic acid encoding a mutated uricase of the invention.

In a third aspect, the invention provides a kit for detecting uric acid comprising the mutated uricase of the invention.

In a specific embodiment, the kit of the invention further comprises a chromogen, a peroxidase, and 4-AAP.

In a specific embodiment, the kit of the present invention may be in the form of:

a first reagent comprising a chromogen; and

a second reagent comprising 4-AAP and a mutated uricase of the invention,

wherein the kit further comprises a peroxidase enzyme present in the first reagent and/or the second reagent.

In one variant, the kit of the invention may be in the form of:

a first reagent comprising 4-AAP; and

a second reagent comprising a chromogen and a mutated uricase of the invention,

In a fourth aspect, the present invention provides a method of making a mutated uricase comprising the steps of:

1) obtaining a coding sequence of uricase;

2) mutating the coding sequence in step 1) such that Y in the binding site and the catalytically active site of the uricase is mutated to any one of H, F, S and G, and/or such that M in the binding site and the catalytically active site of the uricase is mutated to any one of V, L and I;

3) introducing the mutated coding sequence of step 2) into an expression vector and expressing.

In some embodiments, the Y is in Motif a of the uricase.

In a specific embodiment, said Y is first in Motif a of said uricase.

In some embodiments, the Y is mutated to H.

In other embodiments, the Y is mutated to G.

In some embodiments, the M is located in Motif B of the uricase.

In a specific embodiment, said M is at position 10 of Motif B of said uricase.

In some embodiments, the M is mutated to L.

In other embodiments, the M is mutated to I.

Additionally, said step 2) further comprises causing said uricase to comprise one or more additional mutations at a non-binding site and at a non-catalytically active site.

In some embodiments, the additional mutation mutates an amino acid residue selected from the group consisting of C, M, Y and W to an amino acid residue other than C, M, Y and W.

In still other embodiments, the additional mutation deletes an amino acid residue selected from the group consisting of C, M, Y and W.

In preferred embodiments, the additional mutation occurs within 5 residues upstream and/or 5 residues downstream of each of the binding site and the catalytically active site.

In particular embodiments, the uricase is derived from Aspergillus flavus, Bacillus sp.TB-90, or Candida utilis.

In an exemplary embodiment, the uricase is derived from Aspergillus flavus, step 2) is performed such that Y at position 9 of the uricase is mutated to H; preferably, the additional mutation is selected from one or more of the group consisting of Y17F, Y47F, Y66H, M232L and Y233F.

In an exemplary embodiment, the uricase is derived from Bacillus sp.tb-90, step 2) is performed such that the uricase has a Y mutation at position 11 to H and a M mutation at position 76 to I; preferably, the additional mutation is selected from one or more of the group consisting of M9Q, Y10R and Y254F.

In an exemplary embodiment, the uricase is derived from Candida utilis, and step 2) is performed such that the uricase has a Y mutation at position 10 to H; preferably, the additional mutation is selected from one or more of the group consisting of Y49F, Y112V and M238L.

In a fifth aspect, the present invention provides a method for detecting uric acid, comprising the steps of:

1) uniformly mixing and reacting a first reagent with a sample;

2) measuring a first absorbance;

3) adding a second reagent, uniformly mixing and reacting;

4) measuring the second absorbance; and

5) the concentration of the uric acid is calculated,

wherein the first reagent comprises a chromogen and the second reagent comprises 4-AAP and a mutated uricase of the invention, and the first reagent and/or the second reagent further comprise a peroxidase.

In one variant of the method of the invention, the method for detecting uric acid comprises the following steps:

1) uniformly mixing and reacting a first reagent with a sample;

2) measuring a first absorbance;

3) adding a second reagent, uniformly mixing and reacting;

4) measuring the second absorbance; and

5) the concentration of the uric acid is calculated,

wherein the first reagent comprises 4-AAP and the second reagent comprises a chromogen and a mutated uricase of the invention, and the first reagent and/or the second reagent further comprise a peroxidase.

In a sixth aspect, the mutated uricase of the invention, and the use of the kit of the invention for detecting uric acid are provided.

In a seventh aspect, the present invention provides the use of native uricase in the detection of uric acid, wherein neither of amino acid residues C, M, Y and W are present in the native uricase binding site nor the catalytically active site.

In a specific embodiment, the uric acid assay is performed based on a Trinder reaction.

In specific embodiments, the native uricase is derived from Oryza sativa, Brachypodium distachyon, or Elaeis guineensis.

In an exemplary embodiment, the native uricase is derived from Elaeis guineensis.

By specific selection of the natural uricase, the interference caused by taking acetaminophen is avoided in the process of detecting based on uric acid, and the detection accuracy is improved. Meanwhile, the invention widens the application range of the detection reagent and avoids the contraindication of taking acetaminophen by a subject.

Drawings

FIG. 1 shows the results of an amino acid sequence identity alignment of uricases from different sources.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

As used herein, "uricase" refers to the enzyme EC 1.7.3.3, which is capable of catalyzing uric acid, water, and oxygen to allantoin, carbon dioxide, and hydrogen peroxide. The uricase of the invention may be of natural or artificial origin, wherein artificial origin includes synthetic uricase or uricase that has been artificially mutated.

The uricase of the invention may be derived from a mammal, a plant, a fungus, or a bacterium. Preferably wherein the uricase is selected from the group consisting of: globiformis; favigen; racemifer; s.usitatus; rosae; chlorophyti; a. flavus; utilis; c.jadinii; norvegicus; m.musculus; taurus; lupus; cunicululus; dromedaries; l. japonicumas; linear; conn; boggoliensis; paenibacillus sp, such as Paenibacillus sp.jdr-2; bacillus sp, such as Bacillus sp.tb-90; ketdonobacter sp.

As used herein, "mutated uricase" refers to a mutant obtained by mutating the uricase of the present invention in accordance with the mutation pattern defined by the present invention.

As used herein, "binding site" refers to a uric acid binding site and/or a copper ion binding site on uricase. During catalysis, uricase binds to a substrate through its uric acid binding site; and the copper ions are combined with the copper ion binding sites on the uricase to play a role in transferring electrons.

As used herein, "catalytic site," also known as a conserved region, refers to Motif a and Motif B on uricase. Deletion of these conserved sites in uricase will result in loss of catalytic function of uricase.

For uricases of different origins, the person skilled in the art is able to derive their sequence information and the coding sequence from, for example, NCBI (http:// www.ncbi.nlm.nih.gov /). Also, the skilled person is able to determine the position of the binding site and the catalytically active site and their sequence. For example, the Uricase active sites are reported in the documents "Structure-based Characterization of Caninehuman clinical publications and iterative assays", "Molecular Cloning and Expression of uric Gene from organism microorganisms in Escherichia coli and Characterization of the Gene Product", and the like. Alternatively, the corresponding sites of uricase from different sources can be easily located in conjunction with the BLASTP computer program (also available via http:// www.ncbi.nlm.nih.gov /). For another example, ClustalX can be used to align homologous uricases to obtain binding sites and catalytically active sites of uricase, and fig. 1 shows the site information of some uricases aligned by ClustalX.

As shown in FIG. 1, for Aspergillus flavus uricase (accession number X61765.1 in Genbank), Motif A is located at positions 9-14 of the amino acid sequence, and the sequence is YGKDNV; the Motif B is positioned at 52 th to 63 th positions of an amino acid sequence of the Motif B, and the sequence is NSVIVATDSIKN; the copper ion binding site is positioned at 117 th to 121 th positions of the amino acid sequence of the copper ion binding site, and the sequence is HPHSF; the uric acid binding site is positioned at the 228 th to 229 th positions of the amino acid sequence of the uric acid binding site, and the sequence is VQ. For example, for uricase of Candida Utilis (accession number D32043.1 in Genbank), Motif A is located at positions 10-15 of the amino acid sequence, and the sequence is YGKDNV; the Motif B is positioned at the 54 th to 65 th positions of the amino acid sequence of the Motif B, and the sequence is NSSIVPTDTVKN; the copper ion binding site is located at 119 th to 123 th positions of the amino acid sequence of the copper ion binding site, and the sequence is HDHSF; the uric acid binding site is positioned at 234 th to 235 th positions of the amino acid sequence, and the sequence is VQ. From the exemplary alignment results of FIG. 1, it can be understood that one skilled in the art can determine the binding sites and catalytically active sites of uricases of different origins.

In another aspect, the binding sites and catalytically active sites of the invention typically have the following general sequences: motif a, y (h) -G-K-X-V; motif B, N-S-X-V (I) -a (p) -T-D-S (T) -I (M, V) -K-N; a copper ion binding site, H-X-H-X-F; uric acid binding site, V-Q.

Herein, the mutation of an amino acid residue is represented in the form of "pre-mutation residue type + mutation position + post-mutation residue type", for example, Y9H indicates that Y corresponding to position 9 of uricase is mutated to H; M232L indicates that M at position 232 of uricase is mutated to L; or in the form of "mutation position + type of residue before mutation → mutation position + type of residue after mutation", for example, 9Y → 9H indicates that Y corresponding to position 9 of uricase is mutated to H; 232M → 232L indicates that M at position 232 of uricase is mutated to L

As used herein, "non-binding and non-catalytically active sites" and "located at non-binding sites and at non-catalytically active sites" refer to the remaining regions of uricase other than the "binding sites" and the "catalytically active sites". As previously described, one skilled in the art can determine such regions by BLASTP or ClustalX. It is understood that such regions are considered non-core regions, and that mutation of such regions does not generally result in a major change in the catalytic activity of uricase.

In a preferred embodiment, the mutated uricase of the invention comprises at least one additional mutation within 5 residues upstream and/or 5 residues downstream of each of its binding site and catalytically active site. For example, in the case of uricase derived from Aspergillus flavus, Motif A is the 9 th to 14 th positions of uricase, and within 5 residues downstream thereof means the 15 th to 19 th positions of uricase, wherein the 17 th position is Y. In this case, Y at position 17 may be mutated to an amino acid residue other than C, M, Y and W (e.g., H), or Y at position 17 may be deleted. Similarly, additional mutations may also occur at positions 47, 66, 232 and/or 233 of uricase derived from Aspergillus flavus.

Herein, "amino acid residues other than C, M, Y and W" refer to amino acid residues selected from: G. a, V, L, I, F, D, N, E, K, Q, S, T, P, H and R.

The mutated uricase of the invention can be used for preparing a kit for detecting uric acid. In addition to uricase, other reactants for performing the uricase-peroxidase method may be included in the kit of the present invention, such as chromogens (e.g., TBHBA, TOOS), peroxidase, 4-AAP, and the like.

In addition, the kits of the invention may optionally include other common ingredients, including, but not limited to, buffers, such as phosphate buffer; potassium ferrocyanide; ascorbic acid oxidase, a stabilizer; and preservatives and the like.

The present invention is not particularly limited with respect to the concentrations of the respective components in the kit, and they may be present in the usual concentrations. For example, the concentration of uricase may be 500 to 10 KU/L; the concentration of the chromogen can be 0.2 to 1.4 mmol/L; the concentration of the peroxidase can be 2K to 20 KU/L; the concentration of 4-AAP can be 0.2 to 1.4 mmol/L; the buffer concentration of the buffer may be, for example, 10 to 300 mM; the pH of the buffer is 6.0 to 8.0; the concentration of potassium ferrocyanide can be 0.1 to 5 mmol/L; the concentration of ascorbic acid oxidase can be 1K to 10K U/L.

In the kit, the above components may be present in the form of a combination of two reagents.

For example, a chromogen and a buffer are included in the first reagent, and a buffer, uricase and 4-AAP are included in the second reagent, wherein peroxidase is also included in the first reagent and/or the second reagent. As another example, in the first reagent includes 4-AAP and buffer; and the second reagent comprises buffer solution, uricase and chromogen, wherein the first reagent and/or the second reagent also comprises peroxidase.

Further, the kit containing the mutated uricase of the invention can be used in uric acid detection, and therefore can resist the interference caused by taking acetaminophen, thereby truly and accurately reflecting the uric acid level of a subject.

The present invention is not particularly limited with respect to the detection steps and parameters, and those skilled in the art can select appropriate detection steps and parameters according to the specific constitution in the kit. An exemplary detection method may include the steps of:

1) uniformly mixing a first reagent containing the chromogen and a sample, and incubating for 2-10 min at about 37 ℃;

2) measuring a first absorbance at a wavelength of about 546nm (secondary wavelength 700 nm);

3) adding a second reagent containing peroxidase, 4-AAP and uricase, mixing uniformly and incubating for 2-10 min at about 37 ℃;

4) measuring the second absorbance at a wavelength of about 546nm (subwavelength 700 nm); and

5) the uric acid concentration was calculated.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

EXAMPLE 1 preparation of Aspergillus flavus uricase mutant M1

1) Synthesizing an exon part of the coding gene of Genbank X61765.1, replacing the 9 th amino acid coding sequence TAC in the coding gene by CAC, and respectively adding recognition sites TCTAGA and AAGCCT of exonuclease XbaI and HindIII at the upstream and downstream of the periphery of the X61765.1 coding gene;

2) digesting the nucleic acid sequence synthesized in step 1) with endonucleases XbaI and HindIII; the cleavage products were detected by agarose gel electrophoresis, and DNA having a fragment size of about 900bp was recovered using a gel recovery kit. The recovered product is marked as recovered product 1;

3) digesting plasmid pET-26b with endonuclease XbaI and HindIII; the cleavage products were detected by agarose gel electrophoresis, and DNA having a fragment size of about 5300bp was recovered using a gel recovery kit.

The recovered product is recorded as recovered product 2;

4) configuring the reaction system according to the reaction requirements of the T4 ligase, wherein the molar ratio of the recovered product 1 to the recovered product 2 is about 5: 1;

5) and transforming the connecting product into the Escherichia coli BL-21 strain in a chemical transformation mode to obtain the mutant uricase expressing strain.

The strain obtained was used to produce Aspergillus flavus uricase 9Y → 9H, and was designated uricase M1.

EXAMPLE 2 preparation of Aspergillus flavus uricase mutant M2

1) Synthesizing an exon part of the coding gene of X61765.1, replacing the amino acid coding sequence TAC at position 9 in the exon part with GGC, and adding recognition sites TCTAGA and AAGCCT of exonuclease XbaI and HindIII respectively at the upstream and downstream of the periphery of the coding gene of X61765.1;

steps 2) to 5) are the same as in example 1.

The strain obtained was used to produce 9Y → 9G Aspergillus flavus uricase, designated uricase M2.

EXAMPLE 3 preparation of Aspergillus flavus uricase mutant M3

1) Synthesizing an exon part of the coding gene of X61765.1, replacing the amino acid coding sequence TAC at position 9 in the exon part with TTC, and adding recognition sites TCTAGA and AAGCCT of exonuclease XbaI and HindIII respectively at the upstream and downstream of the periphery of the coding gene of X61765.1;

steps 2) to 5) are the same as in example 1.

The strain obtained was used to produce Aspergillus flavus uricase 9Y → 9F, designated uricase M3.

EXAMPLE 4 preparation of Aspergillus flavus uricase mutant M4

1) Synthesizing an exon part of the coding gene of X61765.1, replacing the amino acid coding sequence TAC at position 9 in the exon part with TCC, and adding recognition sites TCTAGA and AAGCCT of exonuclease XbaI and HindIII respectively at the upstream and downstream of the periphery of the coding gene of X61765.1;

steps 2) to 5) are the same as in example 1.

The strain obtained was used to produce 9Y → 9S Aspergillus flavus uricase, designated uricase M4.

EXAMPLE 5 preparation of Aspergillus flavus uricase mutant M5

1) Synthesizing an exon part of the coding gene of X61765.1, replacing a 9 th amino acid coding sequence TAC with CAC, a 17 th amino acid coding sequence TAC with TTC, a 47 th amino acid coding sequence TAC with TTC, a 66 th amino acid coding sequence TAC with CAC, a 232 nd amino acid coding sequence ATG with CTG, a 233 th amino acid coding sequence TAC with TTC, and adding recognition sites TCTAGA and AAGCCT of exonuclease XbaI and HindIII respectively at the upstream and downstream of the periphery of an X61765.1 coding gene;

steps 2) to 5) are the same as in example 1.

Aspergillus flavus uricase, 9Y → 9H, 17Y → 17F, 47Y → 47F, 66Y → 66H, 232M → 223L and 233Y → 233F, was produced by the obtained strain and was designated uricase M5.

Example 6 method for determining interference of NAPHQI on uricase enzyme Activity

When the detection of NAPHI interferes with the detection of uric acid, the maximum concentration of NAPHI in the sample is 100mg/L, and the sample amount is 5. mu.L. The dosage of the first reagent of the uric acid detection kit is generally 240 mu L, and the dosage of the second reagent is 60 mu L, wherein the uricase concentration is 7.5 kU/L. Through conversion, the uricase concentration in the final reaction system added with the uric acid detection kit is about 1.5kU/L, and the NAPHI concentration is up to 1.64 mg/L. Therefore, the influence of NAPHI on the activity of uricase is detected by the concentration through the specific steps of:

sample preparation:

uricase was dissolved in 70mmol/L of phosphate buffer solution of pH 7.8 to prepare a 1g/L solution. The enzyme activity of the strain is measured by a Micheli biochemical analyzer BS-800. If the enzyme activity of the uricase is lower than 1KU/L, the concentration of the uricase is increased until the enzyme activity of the uricase is between 1.2 and 2.0 KU/L; and if the activity of the uricase is higher than 10KU/L, diluting the uricase solution by using a phosphate buffer solution until the activity of the uricase is between 1.2 and 2.0 KU/L. Sample 1, sample 2, and sample 3 were obtained as described above.

Preparing a measuring reaction system:

the linear range of the activity of the uricase measured by the reaction system is 1-10 KU/L.

And (3) carrying out a determination reaction on a Mirey biochemical analyzer BS-800, determining the absorbance in the 20 th to 23 th reaction periods, and determining the activity of the uricase through the slope of a reaction curve.

Example 7 measurement of interference of NAPHQI on uricase Activity (Aspergillus flavus uricase)

The results of the assays carried out according to the method of example 6 on the unmutated Aspergillus flavus uricase (denoted wild type), and the Aspergillus flavus uricase mutants prepared in examples 1, 2 and 5 are shown in Table 1 below.

TABLE 1

Wherein "+++" represents that the absolute interference degree on the enzyme activity is more than 25 percent, "++" represents that the absolute interference degree is more than 15 percent and less than or equal to 25 percent, "+" represents that the absolute interference degree is more than 5 percent and less than or equal to 15 percent, and "-" represents that the absolute interference degree is less than or equal to 5 percent.

Example 8 method for determining interference of NAPHQI to uric acid detection

Preparing a uric acid detection kit:

a first reagent:

a second reagent:

assays Using formulated kits：

1) Setting the main wavelength of a Micheli biochemical analyzer BS-800 to be 546nm and the auxiliary wavelength to be 700 nm;

2) mix 240 μ l of the first reagent with 5 μ l of blank/calibration/sample and incubate for 5min at 37 ℃;

2) measuring the absorbance A1;

3) adding 60 μ l of the second agent, mixing and incubating at 37 ℃ for 5 min;

4) measuring the absorbance A2; and

5) the uric acid concentration was calculated.

Wherein, Delta A ═ [ (A2-A1)_{Calibrator tubes or sample tubes}]–[(A2－A1)_{Blank tube}](ii) a Uric acid concentration (Δ a)_Sample(s)/ΔA_{Calibration article}) Calibrator uric acid concentration.

Example 9 measurement of interference of NAPHQI on uric acid detection (Aspergillus flavus uricase)

Serum samples from a plurality of healthy subjects were collected and pooled, and NAPHQI was added to the serum to give final concentrations of 0mg/L, 20mg/L, 40mg/L, 60mg/L, 80mg/L and 100mg/L, respectively.

The results of the tests according to the method in example 8 were shown in tables 2 and 3 below, using non-mutated Aspergillus flavus uricase (denoted wild type), and Aspergillus flavus uricase mutants M1, M2 and M5 prepared in examples 1, 2 and 5, respectively, as uricase in a second reagent.

TABLE 2

Wherein "+++" represents that the absolute interference degree of UA detection is more than 40%, "++" represents that the absolute interference degree is more than 20% and less than or equal to 40%, "+" represents that the absolute interference degree is more than 10% and less than or equal to 20%, and "-" represents that the absolute interference degree is less than or equal to 10%.

TABLE 3

EXAMPLE 10 preparation of Bacillus sp.TB-90 uricase mutant

Following the procedure of example 1, using Genbank D49974.1 as the wild-type sequence, a Bacillus sp.TB-90 uricase, designated M11, was prepared 11Y → 11H and 76M → 76I.

According to the method in example 1, using Genbank D49974.1 as wild type sequence, 9M → 9Q, 10Y → 10R, 11Y → 11H, 76M → 76I and 254Y → 254F uricase of Bacillus sp.TB-90 was prepared, and is designated M12.

Example 11 measurement of interference of NAPHQI on uricase Activity (Bacillus sp.TB-90 uricase)

The unmutated Bacillus sp.TB-90 uricase (designated as wild type) was assayed according to the method of example 6, as well as the Bacillus sp.TB-90 uricase mutant prepared in example 10, and the results are shown in Table 4 below.

TABLE 4

Example 12 measurement of interference of NAPHQI on uric acid detection (Bacillus sp.TB-90 uricase)

Serum samples from a number of healthy subjects were collected and pooled, and NAPHQI was added to the serum to give final concentrations of 0mg/L and 100mg/L, respectively.

The non-mutated Bacillus sp.TB-90 uricase (denoted as wild type) and the Bacillus sp.TB-90 uricase mutants M11 and M12 prepared in example 10 were tested as uricase in a second reagent, respectively, according to the method in example 8, and the results are shown in Table 5 below.

TABLE 5

Example 13 preparation of Candida Utilis uricase mutant

Using Genbank D32043.1 as the wild-type sequence, Candida Utilis uricase 10Y → 10H was prepared according to the method of example 1 and is designated M21.

Using Genbank D32043.1 as the wild-type sequence, Candida Utilis uricase 10Y → 10H, 49Y → 49F, 112Y → 112V and 238M → 238L was prepared according to the method of example 1 and was designated M22.

Example 14 measurement of interference of NAPHQI on uricase Activity (Candida Utilis uricase)

The results of the measurements of the unmutated Candida Utilis uricase (designated as wild type) and the Candida Utilis uricase mutants prepared in example 13 according to the method of example 6 are shown in Table 6 below.

TABLE 6

Example 15 measurement of interference of NAPHQI on uric acid detection (Candida Utilis uricase)

As uricase in the second reagent, non-mutated Candida Utilis uricase (designated as wild type) and Candida Utilis uricase mutants M21 and M22 prepared in example 13 were tested according to the method of example 8, and the results are shown in Table 7 below.

TABLE 7

EXAMPLE 16 analysis and assay of other NAPHI-resistant uricases

Based on the above experimental data, we speculate that the absence of C, M, Y and W residues in the four active sites is critical for uricase to resist NAPHQI interference. Accordingly, we believe that wild-type uricase meeting the above conditions is particularly advantageous for anti-NAPHQI interference. From uricase sequence information that has been collected (see, for example, FIG. 1), it is presumed that uricases from Oryza sativa (Genbank. XM-015766705.2), Brachypodium distachyon (Genbank. XM-003564633.4) and Elaeis guineensis (Genbank. XM-010909500.3), respectively, also have an ability to resist interference by NAQPI.

Here, Elaeis guineensis uricase was used as an example to test the ability to combat NAPQI interference.

First, Elaeis guineensis uricase (genbank. xm — 010909500.3) was assayed according to the method of example 6, and the results are shown in table 8 below.

TABLE 8

Next, according to the method in example 8, Elaeis guineensis uricase (Genbank. XM-010909500.3) was used as uricase in the second reagent to perform the test, and the results are shown in Table 9 below.

TABLE 9

From the results in tables 8 and 9, it is clear that, consistent with our speculation, Elaeis guineensis uricase and other uricases that do not contain C, M, Y and W residues in the four active sites have the ability to resist NAPQI interference.

Claims

1. A mutated uricase, wherein at least one of Y and/or at least one M of a binding site and a catalytically active site of the uricase is mutated, wherein Y is mutated to any one of H, F, S and G, and M is mutated to any one of V, L and I.

2. The mutated uricase of claim 1, wherein the Y is located in Motif A of the uricase, such as position 1 of Motif A, and the M is located in Motif B of the uricase, such as position 10 of Motif B.

3. The mutated uricase of claim 1 or 2, wherein the Y is mutated to H or G.

4. The mutated uricase of claim 1 or 2, wherein the mutated uricase further comprises at least one additional mutation located at a non-binding site and at a non-catalytically active site.

5. The mutated uricase of claim 4, wherein the additional mutation is a mutation of an amino acid residue selected from C, M, Y and W to an amino acid residue other than C, M, Y and W, and/or a deletion of an amino acid residue selected from C, M, Y and W.

6. The mutated uricase of claim 4 or 5, wherein the additional mutation occurs in the vicinity of the binding site and/or the catalytically active site, e.g., within 5 residues upstream and/or 5 residues downstream of each of the binding site and/or the catalytically active site.

7. A mutated uricase according to any one of claims 1, 2 and 5, wherein the uricase is derived from Aspergillus flavus, Bacillus sp.TB-90 or Candida utilis.

8. The mutated uricase of claim 7, wherein the uricase is derived from Aspergillus flavus and has the Y9H mutation; optionally, the additional mutation is selected from the group consisting of Y17F, Y47F, Y66H, M232L and Y233F.

9. The mutated uricase of claim 7, wherein the uricase is derived from Bacillus sp.tb-90 and has Y11H and M76I mutations; optionally, the additional mutation is selected from the group consisting of M9Q, Y10R, and Y254F.

10. The mutated uricase of claim 7, wherein the uricase is derived from Candida utilis and has a Y10H mutation; optionally, the additional mutation is selected from the group consisting of Y49F, Y112V, and M238L.

11. A nucleic acid encoding the mutated uricase of any one of claims 1-10.

12. A uric acid detection kit comprising the mutated uricase of any one of claims 1-10.

13. The kit of claim 12, wherein the kit further comprises a chromogen, a peroxidase, and 4-AAP.

14. The kit of claim 13, wherein,

the kit comprises:

a first reagent comprising said chromogen; and

a second reagent, said 4-AAP and said mutated uricase,

wherein the kit further comprises a peroxidase enzyme present in the first reagent and/or the second reagent,

alternatively, the first and second electrodes may be,

the kit comprises:

a first reagent comprising said 4-AAP; and

a second reagent, said chromogen and said mutated uricase,

15. A method of making a mutated uricase comprising the steps of:

1) obtaining a coding sequence of uricase;

16. A method for detecting uric acid, comprising the steps of:

1) uniformly mixing and reacting a first reagent with a sample;

2) measuring a first absorbance;

3) adding a second reagent, uniformly mixing and reacting;

4) measuring the second absorbance; and

5) calculating uric acid concentration according to the first absorbance and the second absorbance,

wherein the first and second agents are as defined in claim 14.

17. Use of the urease according to any one of claims 1-10 or the kit according to any one of claims 12-14 for uric acid detection.

18. Use of uricase in uric acid detection, wherein neither of amino acid residues C, M, Y and W are present in the uricase binding site and the catalytically active site.

19. The use of claim 17 or 18, wherein said uric acid detection is performed based on a Trinder reaction.

20. The use of claim 18, wherein said uricase is derived from Oryza sativa, Brachypodium distachyon, or Elaeis guineensis.