Catechol-O-methyltransferase mutant as well as preparation method and application thereof

Abstract

The invention provides a catechol-O-methyltransferase mutant as well as a preparation method and application thereof, and relates to the technical field of biology. The catechol-O-methyltransferase mutant contains 79P mutation relative to wild type catechol-O-methyltransferase, the catechol-O-methyltransferase mutant can be expressed in vitro through recombinant cells, and the problems that in the prior art, a catechol-O-methyltransferase preparation process is poor in stability and high in cost are solved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a catechol-O-methyltransferase mutant, its preparation method, and its application. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Catecholamines are a collective term for adrenaline, noradrenaline, and dopamine, primarily produced and released by the sympathetic nervous system and adrenal medulla. The adrenal medulla mainly produces adrenaline and a small amount of noradrenaline, while the sympathetic nervous system primarily secretes noradrenaline. Both are metabolized through two different pathways by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) to ultimately produce 3-methoxy-4-hydroxymandelic acid (VMA, also known as vanillic acid), which combines with glucuronic acid or sulfate to form esters and is excreted in the urine. In pathological conditions, especially in chromaffin tumors, large amounts of adrenaline and noradrenaline are secreted, leading to elevated blood pressure.

[0004] COMT is one of the main mammalian enzymes involved in the metabolism and degradation of catecholamine neurotransmitters (such as adrenaline, noradrenaline, isoproterenol, and dopamine). Its mechanism of action involves transferring a methyl group from the methyl donor S-adenosylmethionine (SAM) to the 3- and 4-hydroxyl groups of the catecholamine, thereby inactivating the neurotransmitter. Besides its role in the metabolism of endogenous substances, COMT is a key target in the development of catecholamine drugs for hypertension, asthma, and Parkinson's disease. COMT exists widely in various mammalian tissues in two forms: membrane-bound and secreted. These two forms differ in molecular sequence and function, and their tissue distribution also varies.

[0005] COMT plays an important role in the field of medical testing, especially in the research on the development of a test kit for three catecholamines (dopamine, adrenaline, and noradrenaline). By converting catecholamines into methylated substances that are more conducive to analysis and detection, the kit can monitor physiological components related to diseases such as secondary hypertension and pheochromocytoma.

[0006] Currently, diagnostic kits primarily utilize COMT extracted using a natural extraction process. This process mainly involves the separation, purification, and freeze-drying of COMT from fresh pig liver. The steps include obtaining the pig liver, disrupting the liver tissue, lysing and extracting cells, clarifying after acidification, precipitating with ammonium sulfate, ultrafiltration concentration and liquid exchange, chromatographic separation, and freeze-drying of the formulation. The main drawbacks of this process are: 1) Batch and source control of pig liver: Due to the inability to effectively control the source of the pig liver, there are significant differences in COMT content between different batches, leading to reduced stability in subsequent preparation processes; 2) Separation and purification process control: The process requires acid precipitation and ammonium sulfate precipitation, adding two preliminary purification steps, resulting in a longer process route and increasing the risk of instability during enzyme preparation; 3) Finished product formulation process control: As a directly used component in the kit, the formulation is based on a fundamental formula, requiring improved control over the appearance of the finished product during freeze-drying; 4) Overall production cost control: Due to the long process route, the costs of raw materials, consumables, and time are increased, resulting in a low yield and an estimated production cost exceeding 200 yuan / mg. Therefore, it is necessary to improve the stability of the COMT preparation process and reduce production costs.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The purpose of this invention is to provide a catechol-O-methyltransferase mutant and its preparation method, so as to alleviate the problems of poor stability and high cost of catechol-O-methyltransferase in the preparation process in the prior art.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect, a catechol-O-methyltransferase mutant is provided, wherein the catechol-O-methyltransferase mutant, relative to the wild-type catechol-O-methyltransferase, contains the following mutation: 79P; the position of the amino acid residue is determined with reference to the amino acid sequence shown in SEQ ID NO.1.

[0010] Secondly, a biomaterial is provided, said biomaterial being selected from any one of (i) to (iii): (i) A polynucleotide, said polynucleotide encoding the catechol-O-methyltransferase mutant described in the first aspect; (ii) A vector carrying the polynucleotide described in (i); (iii) Recombinant cells, said recombinant cells carrying the polynucleotides described in (i), or containing the vector described in (ii), or expressing the catechol-O-methyltransferase mutant described in the first aspect.

[0011] Thirdly, a method for preparing the catechol-O-methyltransferase mutant described in the first aspect is provided, the method comprising culturing the recombinant cells described in the second aspect and then isolating the catechol-O-methyltransferase mutant.

[0012] Fourthly, the application of the catechol-O-methyltransferase mutant described in the first aspect, or the biomaterial described in the second aspect, or the preparation method described in the third aspect, in any one of (I) to (III) is provided: (I) Detection of catecholamines; (II) Preparation of products for the detection of catecholamines; (III) Prepare products for detecting diseases related to abnormal catecholamines or abnormal catecholamine metabolites; Fifthly, a kit for the detection of catecholamines is provided, the kit comprising the catechol-O-methyltransferase mutant described in the first aspect.

[0013] In a sixth aspect, a method for detecting catecholamines is provided, the method comprising methylating catecholamines in a sample using the catechol-O-methyltransferase mutant described in the first aspect.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes mutational design to screen and obtain a catechol-O-methyltransferase mutant with improved enzyme activity, purity, and stability compared to the wild type. By expressing this catechol-O-methyltransferase mutant in vitro, the pretreatment steps in natural processes are reduced, simplifying process control. Compared to natural extraction processes, the risk of instability in raw material (pig liver) preparation is avoided. Compared to samples obtained through natural processes, the catechol-O-methyltransferase mutant obtained using the method of this invention exhibits significantly improved purity and enzyme activity. The preparation process is simple and highly controllable. In the optimized production process, due to the simplified process route, reduced raw material, consumable, and time costs, and higher yield, production costs are significantly reduced. It is estimated that the cost of preparation from natural processes (200 RMB / mg) can be reduced to 3.5 RMB / mg, resulting in an overall production cost reduction of ≥50 times, while significantly improving performance satisfaction. Compared with naturally processed enzyme products, the catechol-O-methyltransferase mutant and its preparation method provided by this invention avoid the risk of batch quality instability caused by inconsistent pig liver sources, improve the degree of control over material storage conditions, and eliminate the problem of preserving fresh pig liver. This invention significantly reduces detection background, improves enzyme stability, and reduces the cost of reagent kit raw materials, thereby improving reagent kit performance and expanding the application scope of the reagent kit. Attached Figure Description

[0015] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 The SDS-PAGE results are for COMT wild-type, mutant 3, His mutant 3, and Flag mutant 3 in Example 2. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0019] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual such relationship, order, or importance between these entities or behaviors, such as first, second... ninth, etc.

[0020] In this text, the articles “a / an,” “a / an,” and “the” include plural references unless the context clearly indicates otherwise.

[0021] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0022] In this document, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0023] In this article, "each...independently selected" and "...independently selected" are interchangeable and should all be interpreted broadly. They refer to the range or options that each member of a set of variables or components can choose independently, that is, the choice of each variable or component is independent and is not affected by the choice of other variables or components.

[0024] In this article, peptides, polypeptides, and proteins are not strictly distinguished and can be used interchangeably in some cases. Generally, peptides refer to polymers composed of amino acids linked by peptide bonds. They can be natural polymers, synthetic polymers, or combinations thereof. Polypeptides can also contain non-amino acid components.

[0025] In this article, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common naturally occurring amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0026] In this document, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The alignment of amino acid sequence identity percentages can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA. Those skilled in the art can determine appropriate parameters for the aligned sequences, including any algorithms required to achieve maximum alignment of the full length of the compared sequences. Unless otherwise specified, differences between any two sequences include, but are not limited to, insertions, deletions, or mutations.

[0027] In this document, "conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue that is physicochemically similar, such that the substitution does not alter or minimally alters the properties and function of the entire polypeptide or protein. Conservative amino acid substitution is well known to those skilled in the art. Families of amino acid residues with similar side chains are known in the art, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Specific forms of conserved amino acid substitution include those that replace amino acids not in the normal 20 amino acids encoded by the genetic code.

[0028] Unless otherwise specified, the amino acid positions of catechol-O-methyltransferases and their mutants herein refer to the amino acid sequence shown in SEQ ID NO.1, with the positions arranged from the N-terminus to the C-terminus, and the first amino acid residue at the N-terminus of the amino acid sequence shown in SEQ ID NO.1 designated as position 1. However, it should be noted, and those skilled in the art will understand, that different sequences may have different numbering systems, for example, if additional amino acid residues are added or removed compared to SEQ ID NO.1. Therefore, when referring to a specific amino acid residue by its number, this description is not limited to the amino acid precisely located at that numbered position when counting from the amino acid sequence of SEQ ID NO.1, but also refers to equivalent / corresponding amino acid residues in any and all sequences, the equivalent / corresponding positions of which can be obtained by comparison in accordance with the above definition of "identity," even if the residue is not at the same precise numbered position. For example, if the sequence is shorter or longer than SEQ ID NO.1, or has insertions or deletions compared to SEQ ID NO.1. For example, if sequence X is missing two amino acid residues at the N-terminus compared to the sequence in SEQ ID NO. 1, then position 1 of sequence X is equivalent to position 3 of the sequence in SEQ ID NO. 1. Unless otherwise specified, the third amino acid residue mentioned refers to position 1 when referring to the amino acid position in sequence X. It should be noted that when all or part of catechol-O-methyltransferase and its mutants are fused with other polypeptides, the positions of the amino acid residues belonging to the catechol-O-methyltransferase and its mutant portions in the fusion protein are determined with reference to SEQ ID NO. 1. For example, if the fusion protein contains catechol-O-methyltransferase or its mutants starting from position 101, then position 3 of the catechol-O-methyltransferase or its mutant portion (i.e., position 103 of the fusion protein) is equivalent to position 3 of the sequence in SEQ ID NO. 1.

[0029] In this document, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, it may optionally encode the sense or antisense strand. Nucleic acid molecules can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0030] In this document, the term "vector" refers to a delivery system that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) into and express that genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. Examples of vectors include: plasmids, episome plasmids, microcircular DNA, phage particles, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Vectors may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication origin site. Vectors can be expression vectors or cloning vectors. In some embodiments, the vectors (e.g., expression vectors) provided in this disclosure contain a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof as described in this disclosure, and at least one promoter operatively linked to said nucleic acid sequence (e.g., SV40, CMV, EF). 1α), and at least one selection marker.

[0031] In this document, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Progeny may not be identical to primary cells due to natural, accidental, or intentional mutations, and may differ from primary cells morphologically and / or in genomic DNA. “Recombinant cells” include primary modified cells and cultures derived therefrom, where modification includes gene insertion and / or deletion, with the inserted gene either integrated or not integrated into the genome. Any of the aforementioned cells can be prokaryotic or eukaryotic. Prokaryotic cells include, but are not limited to, *Escherichia coli*, *Bacillus*, or *Staphylococcus*. Eukaryotic cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells.

[0032] This invention utilizes the publicly available porcine-derived soluble S-COMT gene for mutation at different sites, including single-point mutations, double-point mutations, and multi-point mutations, to screen and optimize the gene. A tag-free insect expression vector is constructed and expressed, and dominant mutations are screened using enzyme activity indicators from the culture. After determining the mutation sequence, the sequence is constructed into three expression vectors: tag-free, His, and Flag, and expressed. Dominant tags are screened using the activity indicators of the purified target protease. It was found that compared to the wild-type catechol-O-methyltransferase mutant, the mutant enzyme with a mutation of P at position 79 exhibits higher enzyme activity; furthermore, combining it with a suitable tag can further enhance enzyme activity. Based on these findings, the following technical solution is proposed: In a first aspect, a catechol-O-methyltransferase mutant (hereinafter also referred to as the COMT mutant) is provided, wherein the COMT mutant, relative to the wild-type COMT, contains the following mutation: 79P (the 79th position is mutated to P); the position of the amino acid residue is determined with reference to the amino acid sequence shown in SEQ ID NO.1.

[0033] In an optional implementation, the wild-type COMT includes COMTs derived from mammals, including but not limited to humans, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, monkeys, pigs, goats, cattle, or horses.

[0034] In an optional implementation, the wild-type COMT includes COMT derived from pigs.

[0035] In an optional embodiment, the amino acid sequence of the wild-type COMT contains the sequence shown in SEQ ID NO.1. The wild-type shown in SEQ ID NO.1 is derived from porcine COMT and can be expressed in a soluble manner. Mutating based on it helps to obtain a porcine COMT mutant that can be expressed in a soluble manner.

[0036] In an alternative implementation, the mutation relative to wild-type COMT is 79P.

[0037] In an optional embodiment, the COMT mutant contains the amino acid sequence shown in SEQ ID NO.2.

[0038] In an optional embodiment, the amino acid sequence of the COMT mutant is shown in SEQ ID NO.2.

[0039] In an optional embodiment, the COMT mutant amino acid sequence contains a sequence having at least 90% (e.g., but not limited to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO. 2, and the amino acid sequence has N at position 19, Q at position 56, A at position 116, W at position 132T, W at position 141, C at position 171, G at position 183, C at position 186, and T at position 190. Further optionally, the sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO. 2 differs from SEQ ID NO. 2 in that it contains conserved amino acid substitutions.

[0040] In an optional embodiment, the COMT mutant is further linked to a marker protein.

[0041] In an optional embodiment, the marker protein includes a tag protein, and exemplary tag proteins include, but are not limited to, one or more of the following: His tag, GST tag, FLAG tag, SUMO tag, MBP tag and Strep tag, preferably His tag.

[0042] In an optional embodiment, the COMT mutant amino acid sequence is shown in SEQ ID NO.2 and is tagged with His. Through comparison of molecular weight, purity, activity, and kit performance, the COMT mutant with this amino acid sequence showed the highest performance in binding to His-tagged recombinant proteins and achieved the best results in kit applications.

[0043] Secondly, a biomaterial is provided, said biomaterial being selected from any one of (i) to (iii): (i) A polynucleotide, said polynucleotide encoding the COMT mutant described in the first aspect.

[0044] (ii) A vector carrying the polynucleotide of (i); in an optional embodiment, the vector comprises a baculovirus.

[0045] (iii) Recombinant cells, wherein the recombinant cells carry the polynucleotides described in (i), or contain the vector described in (ii), or express the COMT mutant described in the first aspect; in an optional embodiment, the recombinant cells are insect cells, preferably Sf9 cells.

[0046] Thirdly, a method for preparing the COMT mutant described in the first aspect is provided, the method comprising culturing the recombinant cells described in the second aspect and then isolating the COMT mutant.

[0047] In an optional embodiment, the preparation method includes expressing the COMT mutant using a baculovirus-insect cell expression system (BEVS). The baculovirus-insect cell expression system meets the performance requirements of the kit, has low manufacturing costs, and good scalability.

[0048] In an optional embodiment, the preparation method includes constructing a baculovirus vector and expanding the baculovirus vector to obtain at least a third-generation baculovirus vector, then using the at least third-generation baculovirus vector to infect insect cell diseases and expand the culture to obtain the COMT mutant. The multi-stage seed preparation and culture process is simple to operate and provides better stability in seed state control. Using a more stable seed library as the starting point for production increases batch-to-batch stability at the initial stage of the process. Simultaneously, the insect host cell expansion culture process is simple to operate and provides better stability in cell state control.

[0049] In an optional embodiment, the preparation method further includes isolating and purifying the COMT mutant expressed in insect cells.

[0050] In an optional embodiment, the preparation method further includes freeze-drying the COMT mutant. The specific process selection for freeze-drying, as well as the specific operating steps, process parameters, and equipment involved, can be based on methods well-known in the art, such as those described in various general and more specific textbooks, references, process manuals, product instructions, standard documents, and equipment manuals; this invention does not limit these methods. Exemplary freeze-drying steps include sample aliquoting before freeze-drying, partial stoppering, freeze-drying, unloading, full stoppering, and capping. Conventional lyophilization process steps can be selected for freeze-drying, which is simple and controllable. Exemplary freeze-drying steps are shown in Table 4 of Example 4.

[0051] In an optional embodiment, the freeze-drying protectant includes trehalose, and the excipient includes mannitol.

[0052] In an optional embodiment, the working concentration of trehalose is 0.5~2%w / v, for example, but not limited to 0.5%w / v, 1%w / v, 1.5%w / v or 2%w / v, preferably 1%wt.

[0053] In optional embodiments, the working concentration of mannitol is 1 to 10% w / v, for example, but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% w / v, preferably 5% w / v.

[0054] Compared to natural processes, introducing the two-component sugars trehalose and mannitol into the finished product system avoids risks such as protein inactivation and appearance collapse during freeze-drying. Combined with an optimized vacuum freeze-drying profile, the storage stability and appearance control of this COMT mutant can be improved. Compared to the non-freeze-dried form, the freeze-dried form offers advantages in terms of shelf life and usage concentration.

[0055] In an optional embodiment, the preparation method includes isolating and purifying the COMT mutant expressed in insect cells, and freeze-drying the COMT mutant, wherein trehalose is used as a freeze-drying protectant and mannitol is used as an excipient; the isolation and purification of the COMT mutant expressed in insect cells includes the following steps: (A) Resuspend the cell pellet in the first buffer solution, then sonicate, collect the supernatant and filter, and collect the filtrate; (B) Chromatographic purification step (A) The filtrate collected in step (A) is used to equilibrate the chromatography column with the second buffer and to elute the sample with the third buffer; (C) Use an ultrafiltration hollow fiber membrane to perform ultrafiltration replacement of the eluent from step (B), with the replacement medium being the fourth buffer solution; The first, second, third, and fourth buffer solutions each contain trehalose and / or mannitol independently. The remaining components of the first, second, third, and fourth buffer solutions can be selected according to their intended use and common knowledge in the art, for example, according to methods well known in the art and described in various general and more specific textbooks, references, process manuals, product specifications, standard documents, equipment manuals, etc., and the present invention does not limit them in this regard.

[0056] The preferred method described above employs a highly controllable and stable separation and purification process. The cell resuspension and disruption processes are simple and highly operable, while the chromatography process offers high resolution and effectively removes numerous impurities. This process reduces the pretreatment steps required in natural processes, simplifying process control. Furthermore, the necessary stabilizing components (trehalose and mannitol) are directly introduced into the finished buffer system throughout the entire process, increasing sample stability during purification. Compared to natural processes, this avoids the risks of sample degradation and loss of activity during separation and purification.

[0057] In an optional embodiment, the first buffer solution contains mannitol and trehalose, preferably 5% wt mannitol and 1% trehalose; In an optional embodiment, the second buffer solution contains trehalose, preferably 1% trehalose.

[0058] In an optional embodiment, the third buffer solution contains trehalose, preferably 1% trehalose.

[0059] In an optional embodiment, the fourth buffer solution contains mannitol and trehalose, preferably 5% wt mannitol and 1% trehalose.

[0060] In an optional embodiment, the COMT mutant is not tagged, and step (B) uses DEAE anion exchange chromatography. An exemplary method for separating and purifying untagged COMT mutants is as follows: after lysing and resuspending fermentation culture cells, high-pressure disruption is performed, followed by centrifugation, filtration for clarification, anion exchange chromatography, and ultrafiltration concentration. In the separation and purification stage, frozen cells are thawed after being removed from -80°C, and the sample is diluted with cell disruption buffer. Pressure and disruption time parameters are set according to standard cell high-pressure disruption conditions. High-speed centrifugation and membrane filtration are used to remove cell debris. In DEAE anion exchange chromatography, linear separation mode is used to remove aggregates and other impurities. The target component sample is directly concentrated by hollow fiber ultrafiltration. The protein concentration and conductivity are adjusted before freeze-drying to complete the separation and purification process.

[0061] In an optional embodiment, the COMT mutant is tagged with a His tag, and step (B) involves chromatography using Ni metal ion affinity packing material. An exemplary method for separating and purifying the His-tagged COMT mutant is as follows: referring to the above method for separating and purifying the untagged COMT mutant, the DEAE anion exchange chromatography process is replaced with Ni chromatography, while other process routes remain consistent. Compared to untagged COMT mutants, tagged molecules can be purified using Ni chromatography, eliminating the need for complex separation and purification processes such as anion exchange chromatography. This results in higher process efficiency and product purity, and the COMT mutant structure is not affected by the His tag incorporation.

[0062] In an optional embodiment, the COMT mutant is tagged with a Flag label, and step (B) uses Flag affinity chromatography. An exemplary method for separating and purifying Flag-tagged COMT mutants is as follows: referring to the above method for separating and purifying untagged COMT mutants, the DEAE anion exchange chromatography process is changed to a Flag chromatography process, while other process routes remain the same.

[0063] In an optional implementation, step (C) uses a 5KD ultrafiltration hollow fiber membrane for fluid exchange.

[0064] Fourthly, the application of the COMT mutant described in the first aspect, or the biomaterial described in the second aspect, or the preparation method described in the third aspect, in any one of (I) to (III) is provided: (I) Detection of catecholamines; (II) Preparation of products for the detection of catecholamines; (III) Prepare products for detecting diseases associated with abnormal catecholamines or abnormal catecholamine metabolites.

[0065] In an optional embodiment, the catecholamine includes one or more of adrenaline, noradrenaline, isoproterenol, and dopamine.

[0066] In an optional embodiment, the condition associated with catecholamine abnormalities or abnormalities in catecholamine metabolites includes secondary hypertension or pheochromocytoma.

[0067] In an optional implementation, (I) is for non-diagnostic and non-therapeutic purposes.

[0068] Fifthly, a kit for the detection of catecholamines is provided, the kit comprising the COMT mutant described in the first aspect.

[0069] In an optional embodiment, the catechol-O-methyltransferase mutant in the kit is a lyophilized formulation.

[0070] In an optional embodiment, the catechol-O-methyltransferase mutant in the kit is a lyophilized formulation obtained by the freeze-drying method in the preparation method described in the third aspect.

[0071] In an optional embodiment, the kit may also include reagents and / or consumables known to those skilled in the art for the detection of catecholamines. Those skilled in the art may formulate reagents or other reagents in the kit according to the corresponding detection method, and the present invention does not limit this.

[0072] In a sixth aspect, a method for detecting catecholamines is provided, the method comprising methylating catecholamines in a sample using the COMT mutant described in the first aspect.

[0073] In optional embodiments, the detection method further includes detecting methylated catecholamines, for example, but not limited to, using methods such as chromatography, product analysis, or chromatography-tandem mass spectrometry to detect methylated catecholamines.

[0074] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0075] Example 1 1. Screening for different mutant COMT genes: The COMT sequence from wild-type pigs was selected as the target for site-directed amino acid mutagenesis. Mutations were performed on key amino acid sites, and dominant gene sequences were screened using enzyme activity analysis. The wild-type COMT amino acid sequence is shown below: DTKEQRILNHVLQHAEPGNAQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEHQPSVLLELGAYCGYSAVRMARLLSPGARLITIEINPDCAAITQRMVDFAGV KDKVTLVVGASQDIIPQLKKKYDVDTLDMVFLDHWKDRYLPDTLLLEECGLLRKGTVLLADNVICPGAPDFLAHVRGSSCFECTHYQSFLEYREVVDGLEKAIYKG The original COMT amino acid sequence was mutated using site-directed mutagenesis. Ten mutant COMT amino acid sequences are shown below. Underlined amino acid residues indicate mutation sites, and a summary of mutation sites is shown in Table 1. All gene sequences were obtained through whole-genome synthesis and the insect baculovirus vector pFastBac1 was constructed using standard gene manipulation methods.

[0076] Table 1

[0077] Sequence 1: A116D DTKEQRILNHVLQHAEPGNAQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEHQPSVLLELGAYCGYSAVRMARLLSPGARLITIEINPDCAAITQRMVDFAGVKDKVTLVVG D SQDIIPQLKKKYDVDTLDMVFLDHWKDRYLPDTLLLEECGLLRKGTVLLADNVICPGAPDFLAHVRGSSCFECTHYQSFLEYREVVDGLEKAIYKG Sequence 2: N19D DTKEQRILNHVLQHAEPG D AQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEHQPSVLLELGAYCGYSAVRMARLLSPGARLITIEINPDCAAITQRMVDFAGVKDKVTLVVGASQDIIPQLKKKYDVDTLDMVFLDHWKDRYLPDTLLLEECGLLRKGTVLLADNVICPGAPDFLAHVRGSSCFECTHYQSFLEYREVVDGLEKAIYKG Sequence 3: S79P DTKEQRILNHVLQHAEPGNAQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEHQPSVLLELGAYCGYSAVRMARLL P PGARLITIEINPDCAAITQRMVDFAGVKDKVTLVVGASQDIIPQLKKKYDVDTLDMVFLDHWKDRYLPDTLLLEECGLLRKGTVLLADNVICPGAPDFLAHVRGSSCFECTHYQSFLEYREVVDGLEKAIYKG Sequence 4: N19D S79P A116D DTKEQRILNHVLQHAEPG D AQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEHQPSVLLELGAYCGYSAVRMARLL P PGARLITIEINPDCAAITQRMVDFAGVKDKVTLVVGD SQDIIPQLKKKYDVDTLDMVFLDHWKDRYLPDTLLLEECGLLRKGTVLLADNVICPGAPDFLAHVRGSSCFECTHYQSFLEYREVVDGLEKAIYKG Sequence 5: N19D Q56R S79P A116D DTKEQRILNHVLQHAEPG D AQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEH R PSVLLELGAYCGYSAVRMARLL P PGARLITIEINPDCAAITQRMVDFAGVKDKVTLVVG D SQDIIPQLKKKYDVDTLDMVFLDHWKDRYLPDTLLLEECGLLRKGTVLLADNVICPGAPDFLAHVRGSSCFECTHYQSFLEYREVVDGLEKAIYKG Sequence 6: N19D Q56R S79P A116D T132K C171R DTKEQRILNHVLQHAEPG D AQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEH R PSVLLELGAYCGYSAVRMARLLPPGARLITIEINPDCAAITQRMVDFAGVKDKVTLVVG D SQDIIPQLKKKYDVD K LDMVFLDHWKDRYLPDTLLLEECGLLRKGTVLLADNVI R PGAPDFLAHVRGSSCFECTHYQSFLEYREVVDGLEKAIYKG Sequence 7: N19D Q56R S79P A116D T132K C171R T190E DTKEQRILNHVLQHAEPG D AQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEH R PSVLLELGAYCGYSAVRMARLL P PGARLITIEINPDCAAITQRMVDFAGVKDKVTLVVG D SQDIIPQLKKKYDVDK LDMVFLDHWKDRYLPDTLLLEECGLLRKGTVLLADNVI R PGAPDFLAHVRGSSCFEC E HYQSFLEYREVVDGLEKAIYKG Sequence 8: N19D Q56R S79P A116D T132K C171R G183R T190E DTKEQRILNHVLQHAEPG D AQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEH R PSVLLELGAYCGYSAVRMARLL P PGARLITIEINPDCAAITQRMVDFAGVKDKVTLVVG D SQDIIPQLKKKYDVD K LDMVFLDHWKDRYLPDTLLLEECGLLRKGTVLLADNVI R PGAPDFLAHVR R SSCFEC E HYQSFLEYREVVDGLEKAIYKG Sequence 9: N19D Q56R S79P A116D T132K W141P C171R T190E DTKEQRILNHVLQHAEPG D AQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEH R PSVLLELGAYCGYSAVRMARLL P PGARLITIEINPDCAAITQRMVDFAGVKDKVTLVVG D SQDIIPQLKKKYDVD K LDMVFLDH P KDRYLPDTLLLEECGLLRKGTVLLADNVI R PGAPDFLAHVRGSSCFEC E HYQSFLEYREVVDGLEKAIYKG Sequence 10: N19D Q56R S79P A116D T132K C171R G183R C186R DTKEQRILNHVLQHAEPG DAQSVLEAIDTYCEQKEWAMNVGDKKGKIVDAVIQEH R PSVLLELGAYCGYSAVRMARLL P PGARLITIEINPDCAAITQRMVDFAGVKDKVTLVVG D SQDIIPQLKKKYDVD K LDMVFLDHWKDRYLPDTLLLEECGLLRKGTVLLADNVI R PGAPDFLAHVR R SS R FECTHYQSFLEYREVVDGLEKAIYKG 2. Cultivate expression: The above 11 vectors (wild-type and mutant) were transfected into insect Sf9 cells according to the following ratio: plasmid: liposome: Sf9 cells (1.2 × 10⁻⁶ cells / cells). 6 Transfection was performed using a ratio of 4 μg: 8 μl: 2 ml. Cells were cultured at 27°C and 100 rpm for 120 h, then centrifuged at 3500 rpm for 5 min. The supernatant was collected to prepare the P1 generation virus. The P1 generation virus volume was calculated as follows: SB Expi Sf9 cells (0.8 × 10⁻⁶ cells / mL). 6 Inoculate with the virus at a ratio of 1:20 (cells / mL) and culture volume of SB ExpiSf9 cells (0.8 × 10⁻⁶ cells / mL). Incubate at 27°C, 100 rpm for 72 h, centrifuge at 3500 rpm for 5 min, collect the supernatant, and prepare the P2 generation virus stock. The P2 generation virus stock volume is: SB ExpiSf9 cells (0.8 × 10⁻⁶ cells / mL) = 1:20. 6 Infect cells with the virus at a ratio of 1:30 (cells / mL) at 27°C and 100 rpm for 72 h. Centrifuge at 3500 rpm for 5 min, collect the supernatant, and prepare the P3 generation virus strain. After the P3 generation virus strain is expanded, collect the centrifuged cells and store them at -80°C for up to one year.

[0078] During the expansion culture phase, 40 ml of SB ExpiSf9 cell culture was taken, and 0.8 × 10⁻⁶ cells were added. 6 Cells / ml were passaged and cultured at 27℃ and 100 rpm for 72 h, reaching a cell density of 4.5 × 10⁻⁶ cells / ml. 6 When cell count / ml is ≥90%, the dosage is calculated based on the P3 generation virus volume: SB ExpiSf9 cells (4.5 × 10⁻⁶ cells / ml). 6 Infect the cells / mL culture volume at a ratio of 1:15, incubate at 27℃ and 100 rpm for 72 h, centrifuge at 8000 rpm for 20 min, and collect the precipitate.

[0079] 3. Cell disruption: The cell pellets harvested from each group by centrifugation were resuspended in 2 ml of 0.02 M Tris + 5% mannitol + 1% trehalose + 1 mM EDTA + 1 mM MPMSF pH 8.0 buffer, sonicated, and the supernatant was collected and filtered through a 0.45 μm filter membrane.

[0080] 4. Enzyme activity assay: Protein concentration and activity were measured in wild-type and mutant samples. Dominant molecules and mutant strains were screened and confirmed based on enzyme activity. Sample numbers were 0-10, corresponding to wild-type, mutant 1, mutant 2, mutant 3, mutant 4, mutant 5, mutant 6, mutant 7, mutant 8, mutant 9, and mutant 10.

[0081] Table 2

[0082] Based on the above results, compared with wild-type COMT, the sequence 3 (mutant 3) molecules among the 10 mutant groups showed higher total protein expression and enzyme activity. Therefore, the single mutation site molecule with sequence 3 was selected as the dominant amino acid sequence.

[0083] Example 2 Screening for dominant mutant COMT genes incorporated into tags: 1. Construction of tagless vectors for superior COMT mutant molecules: The COMT mutant and wild type of Example 1 were selected, and the gene encoding it was constructed into an insect baculovirus vector without the His tag (which has already been constructed in Example 1). The COMT mutant of Example 1 was constructed into an insect baculovirus vector with the His tag and an insect baculovirus vector with the Flag tag. The tag was located at the C-terminus of the COMT mutant or the wild type, and a total of 4 insect expression vectors were constructed.

[0084] 2. Cultivate expression: The above four vectors (wild-type untagged, mutant 3 untagged, and two tagged vectors of mutant 3) were transfected into insect Sf9 cells according to the following ratio: plasmid: liposome: Sf9 cells (1.2 × 10⁻⁶ cells). 6 Transfection was performed using a ratio of 4 μg: 8 μl: 2 ml. Cells were cultured at 27°C and 100 rpm for 120 h, then centrifuged at 3500 rpm for 5 min. The supernatant was collected to prepare the P1 generation virus. The P1 generation virus volume was calculated as follows: SB Expi Sf9 cells (0.8 × 10⁻⁶ cells / mL). 6Inoculate with the virus at a ratio of 1:20 (cells / mL) and culture volume of SBExpiSf9 cells (0.8 × 10⁻⁶ cells / mL). Incubate at 27°C, 100 rpm for 72 h, centrifuge at 3500 rpm for 5 min, collect the supernatant, and prepare the P2 generation virus stock. The P2 generation virus stock volume is calculated as follows: SBExpiSf9 cells (0.8 × 10⁻⁶ cells / mL) = 1:20. 6 Inoculate with the virus at a ratio of 1:30 (cells / mL) and culture volume. Incubate at 27°C and 100 rpm for 72 h. Centrifuge at 3500 rpm for 5 min, collect the supernatant, and prepare the P3 generation virus strain. After the P3 generation virus strain is expanded, collect the centrifuged cells and store at -80°C.

[0085] During the expansion culture phase, 400 ml of SB ExpiSf9 cell culture was taken, and 0.8 × 10⁻⁶ cells were added. 6 Cells / ml were passaged and cultured at 27℃ and 100 rpm for 72 h, reaching a cell density of 4.5 × 10⁻⁶ cells / ml. 6 When cell count / ml is ≥90%, the dosage is calculated based on the P3 generation virus volume: SB ExpiSf9 cells (4.5 × 10⁻⁶ cells / ml). 6 Infect the cells / mL culture volume at a ratio of 1:15, incubate at 27℃ and 100 rpm for 72 h, centrifuge at 8000 rpm for 20 min, and collect the precipitate.

[0086] 3. Cell disruption: The cell pellets harvested from centrifugation in each group were resuspended in the first buffer (20 ml 0.02 M Tris + 5% mannitol + 1% trehalose + 1 mM EDTA + 1 mM PMSF pH 8.0 buffer), sonicated, and the supernatant was collected and filtered through a 0.45 μm filter membrane.

[0087] 4. Tag-free molecule separation and purification: Using 20 mL of DEAE anion exchange chromatography packing material, equilibrate the column to 3 column volumes with the second buffer (0.02 M Tris + 1% trehalose + 1 mM EDTA + 1 mM PMSF pH 8.0) at a flow rate of 4 mL / min. Inject 20 mL of sample into the column at a flow rate of 4 mL / min. Equilibrate the column to 3 column volumes with the second buffer at a flow rate of 4 mL / min. Elute the sample with 10% third buffer (0.02 M Tris + 1% trehalose + 1 mM EDTA + 1 mM PMSF + 1 M NaCl pH 8.0) for 5 CV at a flow rate of 4 mL / min. Collect 100 mL of the chromatographic sample.

[0088] A 5KD ultrafiltration hollow fiber membrane was used to ultrafilter the collected samples. The buffer solution was changed after the buffer change to a fourth buffer (0.02 M Tris + 5% mannitol + 1% trehalose + 60 mM NaCl, pH 8.0). The sample concentration was adjusted to 5.0 mg / ml, and enzyme activity was measured and SDS-PAGE (reduction) purity analysis was performed. The results are as follows: Figure 1 As shown in Table 3.

[0089] 5. His-tagged molecule isolation and purification: Using 20 mL of Ni metal ion affinity packing material, equilibrate the column to 3 column volumes with the second buffer (0.02 M Tris + 1% trehalose + 1 mM EDTA + 1 mM PMSF pH 8.0) at a flow rate of 4 mL / min. Inject 20 mL of sample into the column at a flow rate of 4 mL / min. Equilibrate the column to 3 column volumes with the second buffer at a flow rate of 4 mL / min. Elute the sample with 5 CV of 25% third buffer (0.02 M Tris + 1% trehalose + 1 mM EDTA + 1 mM PMSF + 1 M imidazole pH 8.0) at a flow rate of 4 mL / min. Collect 100 mL of the chromatographic sample.

[0090] A 5KD ultrafiltration hollow fiber membrane was used to ultrafilter the collected samples. The buffer solution was changed after the buffer change to buffer IV (0.02 M Tris + 5% mannitol + 1% trehalose + 60 mM NaCl, pH 8.0). The sample concentration was adjusted to 5.0 mg / ml, and enzyme activity was measured and SDS-PAGE (reduction) purity analysis was performed. The results are as follows: Figure 1 As shown in Table 3.

[0091] 6. Flag-tagged molecule isolation and purification: Using 20 mL Flag affinity packing material, equilibrate the column to 3 column volumes with the second buffer (0.02 M Tris + 1% trehalose + 1 mM EDTA + 1 mM PMSF pH 8.0) at a flow rate of 4 mL / min. Inject 20 mL of sample into the column at a flow rate of 4 mL / min. Equilibrate the column to 3 column volumes with the second buffer at a flow rate of 4 mL / min. Elute the sample with 5 CV of 60% third buffer (0.02 M Tris + 1% trehalose + 1 mM EDTA + 1 mM PMSF + 0.5 M competitive peptide pH 8.0) at a flow rate of 4 mL / min. Collect 100 mL of the chromatographic sample.

[0092] A 5KD ultrafiltration hollow fiber membrane was used to ultrafilter the collected samples. The buffer solution was changed after the buffer change to a fourth buffer (0.02 M Tris + 5% mannitol + 1% trehalose + 60 mM NaCl, pH 8.0). The sample concentration was adjusted to 5.0 mg / ml, and enzyme activity was measured and SDS-PAGE (reduction) purity analysis was performed. The results are as follows: Figure 1 As shown in Table 3.

[0093] Table 3

[0094] Based on the above results, compared with untagged molecules, His-tagged molecules have higher purity and higher enzyme activity. Therefore, the His tag was chosen as the integration tag.

[0095] Example 3 Performance evaluation of the superior mutant M3-COMT gene + His-integrated tag sample kit 1. Cultivate expression: Take 800ml of SB ExpiSf9 cell culture, 0.8×10 6 Cells / ml were passaged and cultured at 27℃ and 100 rpm for 72 h, reaching a cell density of 4.5 × 10⁻⁶ cells / ml. 6 When the cell viability is ≥90%, take 53 ml of His-M3 mutant strain P3 and divide it according to the following ratio: P3 strain volume: SB ExpiSf9 cells (4.5 × 10⁻⁶ cells / ml). 6 Infect the cells / mL culture volume at a ratio of 1:15, incubate at 27℃ and 100rpm for 72h, centrifuge at 8000rpm for 20min, and collect the precipitate.

[0096] 2. Cell disruption: The centrifuged cell pellet was resuspended in 40 ml of 0.02 M Tris + 5% mannitol + 1% trehalose + 1 mM EDTA + 1 mM PMSF pH 8.0 buffer, sonicated, and the supernatant was collected and filtered through a 0.45 μm filter membrane.

[0097] 3. His-tagged molecule isolation and purification: Using 40 mL of Ni metal ion affinity packing material, equilibrate the column to 3 column volumes with buffer A (0.02 M Tris + 1% trehalose + 1 mM EDTA + 1 mM PMSF pH 8.0) at a flow rate of 4 mL / min. Inject 40 mL of sample into the column at a flow rate of 4 mL / min. Equilibrate the column to 3 column volumes with buffer A at a flow rate of 4 mL / min. Elute the sample with 25% buffer B (0.02 M Tris + 1% trehalose + 1 mM EDTA + 1 mM PMSF + 1 M imidazole pH 8.0) for 5 CV at a flow rate of 4 mL / min. Collect 100 mL of the chromatographic sample.

[0098] A 5KD ultrafiltration hollow fiber membrane was used to perform ultrafiltration and medium replacement on the chromatographically collected samples. After medium replacement, the system was 0.02 M Tris + 5% mannitol + 1% trehalose + 60 mM NaCl, pH 8.0. The sample concentration was adjusted to 5.0 mg / ml.

[0099] 4. Freeze-drying of formulations: After adding 1 ml of ultrafiltration solution to a 5 ml brown vial, the sample was freeze-dried. The freeze-drying parameters are shown in Table 4.

[0100] Table 4

[0101] 5. Reagent kit performance testing: One vial each of wild-type, mutant 3, and His-mutant 3 freeze-dried products were taken and tested. The detection performance for dopamine, adrenaline, and noradrenaline was tested. COMT catalyzes the enzymatic reaction of catecholamine substrates in the sample, resulting in methylation, followed by enzyme immunoassay. The enzyme immunoassay uses a two-step sandwich method to determine the methylated analyte. Goat anti-rabbit IgG is coated on a solid-phase carrier, and the sample and specific antibody are added. After reaction and washing, the enzyme-labeled component is added again, and the reaction is performed. The luminescence value is detected. The higher the luminescence value, the higher the concentration of the analyte; P0 is the background detection value.

[0102] Table 5. Dopamine Detection Results

[0103] Table 6 Adrenaline Detection Results

[0104] Table 7 Norepinephrine Detection Results

[0105] Based on the above results, compared with wild-type and untagged mutant 3, His-mutant 3 meets the substrate transformation requirements for the kit application. Therefore, the His-mutant 3 recombinant molecule preparation process can meet the substrate transformation requirements of the kit, and the production process is simple with significantly higher process control stability than the natural molecule process route.

[0106] In the above embodiments, SDS-PAGE reduction electrophoresis was performed according to the "Standard Operating Procedure for SDS-PAGE Electrophoresis". The purity determination method for SDS-PAGE reduction electrophoresis was as follows: After dissolving the freeze-dried sample, the concentration was adjusted to 1 mg / ml with purified water. A volume of 20 μL was taken and subjected to 15% SDS-PAGE reduction gel detection. After Coomassie brilliant blue staining, purity analysis was performed using a gel imaging system.

[0107] In the above embodiments, the enzyme activity assay was performed according to the method described in "Study on the determination of catechol-O-methyltransferase activity in rat liver tissue by RP-HPCL method" (2009.1:59-61).

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A catechol-O-methyltransferase mutant, characterized in that, Compared to wild-type catechol-O-methyltransferase, it contains the following mutation: 79P; the position of the amino acid residues is determined with reference to the amino acid sequence shown in SEQ ID NO.

1.

2. The catechol-O-methyltransferase mutant according to claim 1, characterized in that, The wild-type catechol-O-methyltransferase includes catechol-O-methyltransferases derived from mammals; Optionally, the wild-type catechol-O-methyltransferase includes catechol-O-methyltransferase derived from pigs; Optionally, the amino acid sequence of the wild-type catechol-O-methyltransferase contains the sequence shown in SEQ ID NO.1; Alternatively, the mutation relative to wild-type catechol-O-methyltransferase is 79P; Optionally, the catechol-O-methyltransferase mutant contains the amino acid sequence shown in SEQ ID NO.2; or, contains an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO.2, wherein the amino acid residues at positions 19, 56, 116, 132, 141, 171, 183, 186 and 190 of the amino acid sequence are N, Q, A, T, W, C, G, C and T, respectively. Optionally, the amino acid sequence of the catechol-O-methyltransferase mutant is shown in SEQ ID NO.

2.

3. The catechol-O-methyltransferase mutant according to claim 1 or 2, characterized in that, It is also linked to a marker protein; Optionally, the marker protein includes a tag protein; Optionally, the tag protein includes one or more of the following: His tag, GST tag, Flag tag, SUMO tag, MBP tag, and Strep tag, preferably the His tag.

4. A biomaterial, characterized in that, Choose from any one of (i) to (iii): (i) A polynucleotide, said polynucleotide encoding the catechol-O-methyltransferase mutant according to any one of claims 1 to 3; (ii) A vector carrying the polynucleotide described in (i); (iii) Recombinant cells, said recombinant cells carrying the polynucleotide of (i), or containing the vector of (ii), or expressing the catechol-O-methyltransferase mutant of any one of claims 1 to 3.

5. The method for preparing the catechol-O-methyltransferase mutant according to any one of claims 1 to 3, characterized in that, The recombinant cells as described in claim 4 are cultured, and then the catechol-O-methyltransferase mutant is isolated.

6. The preparation method according to claim 5, characterized in that, The preparation method includes expressing the catechol-O-methyltransferase mutant using a baculovirus-insect cell expression system; Optionally, the preparation method includes constructing a baculovirus vector and expanding the baculovirus vector to obtain at least a third-generation baculovirus vector, and then using the at least third-generation baculovirus vector to infect insect cell diseases for further expansion culture to obtain the catechol-O-methyltransferase mutant.

7. The preparation method according to claim 5 or 6, characterized in that, The preparation method further includes isolating and purifying the catechol-O-methyltransferase mutant expressed in insect cells; and / or, the preparation method further includes freeze-drying the catechol-O-methyltransferase mutant. Optionally, the freeze-drying protectant includes trehalose, and the excipient includes mannitol; Optionally, the working concentration of the trehalose is 0.5-2% w / v, and / or the working concentration of the mannitol is 1-10% w / v; Optionally, the working concentration of the trehalose is 1% w / v, and / or the working concentration of the mannitol is 5% w / v; Optionally, the isolation and purification of the catechol-O-methyltransferase mutant expressed in insect cells includes the following steps: (A) Resuspend the cell pellet in the first buffer solution, then sonicate, collect the supernatant and filter, and collect the filtrate; (B) Chromatographic purification step (A) The filtrate collected in step (A) is used to equilibrate the chromatography column with the second buffer and to elute the sample with the third buffer; (C) Use an ultrafiltration hollow fiber membrane to perform ultrafiltration replacement of the eluent from step (B), with the replacement medium being the fourth buffer solution; The first, second, third, and fourth buffer solutions each contain trehalose and / or mannitol independently.

8. The application of the catechol-O-methyltransferase mutant according to any one of claims 1 to 3, or the biomaterial according to claim 4, or the preparation method according to any one of claims 5 to 7 in any one of (I) to (III): (I) Detection of catecholamines; (II) Preparation of products for the detection of catecholamines; (III) Prepare products for detecting diseases related to abnormal catecholamines or abnormal catecholamine metabolites; Optionally, the catecholamines include one or more of adrenaline, noradrenaline, isoproterenol, and dopamine; Optionally, the conditions associated with catecholamine abnormalities or abnormalities in catecholamine metabolites include secondary hypertension or pheochromocytoma.

9. A kit for the detection of catecholamines, characterized in that, It includes the catechol-O-methyltransferase mutant according to any one of claims 1 to 3; Optionally, the catechol-O-methyltransferase mutant in the kit is a lyophilized formulation; Optionally, the catechol-O-methyltransferase mutant in the kit is a lyophilized formulation obtained according to the preparation method of claim 7.

10. A method for detecting catecholamines, characterized in that, This includes using the catechol-O-methyltransferase mutant according to any one of claims 1 to 3 to methylate catecholamines in a sample.

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