Substance detection method using aminoacylase

Abstract

The present invention measures the glycine produced as a byproduct alongside the main product of a chemical reaction that uses hippuric acid and / or methyl hippuric acid as a starting material and is catalyzed by a hydrolytic enzyme and thereby detects the presence of at least the starting material. The hydrolytic enzyme is an aminoacylase that has the amino acid sequence of SEQ ID NO:1 or is a protein that has the amino acid sequence of SEQ ID NO:1 with one or more amino acid deletions, substitutions, or additions and can hydrolyze hippuric acid and methyl hippuric acid.

Description

Technical Field

[0001] The present invention relates to a method for detecting substances using aminoacylase, and particularly to a method for detecting hippuric acid or methyl hippuric acid by detecting glycine that is by-produced together with the main product through a chemical reaction in which hippuric acid or methyl hippuric acid as a starting material is catalyzed by aminoacylase.

Background Art

[0002] Since hippuric acid is known as a metabolite of toluene and methyl hippuric acid is known as a metabolite of xylene, hippuric acid or methyl hippuric acid is used as an exposure index for toluene or xylene. Therefore, detecting hippuric acid or methyl hippuric acid is useful in clinical examinations of workers handling these organic solvents.

[0003] Here, the present inventors have proposed a method for detecting substances using glycine oxidase disclosed in Patent Document 1. In this method, glycine that is by-produced together with the main product generated by the chemical reaction of the starting material is oxidized by a direct reaction that does not constitute an enzyme cycling reaction by the catalytic action of glycine oxidase, and the generated hydrogen peroxide is measured.

[0004] In the substance detection method disclosed in Patent Document 1, as a typical starting material, a chemical reaction of hippuric acid or methyl hippuric acid is cited. In this chemical reaction, hippuric acid or methyl hippuric acid is hydrolyzed by treatment with aminoacylase or an enzyme analogous thereto, and benzoic acid or methyl benzoic acid is produced as the main product and glycine is purified as the by-product.

[0005] As described above, hydrogen peroxide is generated by enzymatic treatment of the by-product glycine with glycine oxidase. Therefore, the starting material (hippuric acid or methylhippuric acid) can be detected by measuring this hydrogen peroxide. Accordingly, although the substance detection method disclosed in Patent Document 1 is not limited to the detection of hippuric acid or methylhippuric acid, it can be suitably used for the detection of hippuric acid or methylhippuric acid. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2019-187285 [Overview of the project] [Problems that the invention aims to solve]

[0007] When the present inventors further investigated the application of the substance detection method described in Patent Document 1 to the detection of hippuric acid or methylhippuric acid, it became clear that there are challenges in more accurately measuring the total amount of hippuric acid and methylhippuric acid in a sample.

[0008] As mentioned above, known enzymes for hydrolyzing hippuric acid or methylhippuric acid include aminoacylase (EC 3.5.1.14), hippuric acid hydrolase (EC 3.5.1.32), or N-acyl-D-amino acid deacylase (EC 3.5.1.81). Our own investigations have revealed that these known enzymes cannot effectively hydrolyze the ortho isomer of methylhippuric acid, one of its three isomers.

[0009] Methylhippuric acid exists in three isomers: ortho, meta, and para. While known aminoacylases can effectively hydrolyze hippuric acid, the meta-isomer 3-methylhippuric acid, and the para-isomer 4-methylhippuric acid, they cannot effectively hydrolyze the ortho-isomer 2-methylhippuric acid. If 2-methylhippuric acid cannot be effectively hydrolyzed, glycine derived from 2-methylhippuric acid will not be adequately produced as a by-product, making it difficult to appropriately quantify the total amount of hippuric acid and methylhippuric acid in a sample.

[0010] The present invention was made to solve these problems and aims to provide a substance detection method using aminoacylase that can quantify the total amount of at least hippuric acid and methylhippuric acid. [Means for solving the problem]

[0011] The substance detection method relating to this disclosure, in order to solve the above-mentioned problems, is a substance detection method that detects the presence of at least the starting material by measuring glycine produced as a by-product along with the main product generated by a chemical reaction catalyzed by a hydrolase of the starting material, wherein the starting material is hippuric acid and / or methylhippuric acid, and the hydrolase is an aminoacylase having the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, and is a protein having the activity to hydrolyze the hippuric acid and the methylhippuric acid.

[0012] According to the above configuration, by using an aminoacylase having the amino acid sequence shown in Sequence ID No. 1 or its homologous sequence as the enzyme for hydrolyzing hippuric acid and methylhippuric acid, it becomes possible to hydrolyze not only hippuric acid but also 2-methylhippuric acid, which could not be properly hydrolyzed conventionally. As a result, all three isomers of hippuric acid and methylhippuric acid can be properly hydrolyzed. This makes it possible not only to quantify the total amount of hippuric acid and methylhippuric acid, but also to quantify the amount of methylhippuric acid by subtracting the amount of hippuric acid from the total amount.

[0013] In the substance detection method of the above configuration, the measurement of glycine may be performed by measuring the hydrogen peroxide produced.

[0014] Furthermore, in the substance detection method with the above configuration, the enzymatic reaction that generates hydrogen peroxide may be an enzymatic reaction using glycine oxidase.

[0015] Furthermore, in the substance detection method of the above configuration, the measurement of hydrogen peroxide may be performed by reacting the hydrogen peroxide with aminoantipyrine and Trinder's reagent using the catalytic action of peroxidase, and measuring the degree of color development of the resulting quinone-based dye.

[0016] Furthermore, in the substance detection method having the above configuration, the main product may be benzoic acid or methylbenzoic acid.

[0017] Furthermore, in the substance detection method of the above configuration, the total amount of hippuric acid and methylhippuric acid contained in the sample to be detected may be quantified by measuring the hydrogen peroxide.

[0018] Furthermore, in the substance detection method of the above configuration, the amount of methylhippuric acid may be obtained by subtracting the amount of hippuric acid from the total amount of quantified hippuric acid and methylhippuric acid.

[0019] In addition, the present disclosure also includes a substance detection kit that is used in the substance detection method of the above configuration and contains, as an enzyme for hydrolyzing the starting material, an aminoacylase having the amino acid sequence shown in SEQ ID NO: 1, or a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1 and having an activity of hydrolyzing the hippuric acid and the methyl hippuric acid.

[0020] The above objects, other objects, features, and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments with reference to the accompanying drawings.

Effects of the Invention

[0021] In the present invention, with the above configuration, an effect is achieved in that a substance detection method using aminoacylase capable of quantifying at least the total amount of hippuric acid and methyl hippuric acid can be provided.

Brief Description of the Drawings

[0022] [Figure 1] It is a schematic diagram for explaining an outline of a substance detection method when the starting materials are hippuric acid and methyl hippuric acid, which is a representative example of the present disclosure. [Figure 2] It is a schematic diagram for explaining a representative example of a specific measurement method of glycine in the substance detection method described in FIG. 1. [Figure 3] It is a graph showing a comparison between the measured value of hippuric acid or methyl hippuric acid in a sample and the known concentration of hippuric acid or methyl hippuric acid in the sample by the substance detection method according to the present disclosure, which is an embodiment of the present disclosure. [Figure 4] It is a graph showing a comparison between the measured value of hippuric acid or methyl hippuric acid in a sample and the known concentration of hippuric acid or methyl hippuric acid in the sample when using a conventional aminoacylase, which is a comparative example of the present disclosure. [Figure 5]A graph showing the comparison between the measured values of hippuric acid or methyl hippuric acid in a sample and the known concentration of hippuric acid or methyl hippuric acid in the sample when using a conventional hippuric acid hydrolase, which is a comparative example of the present disclosure. [Embodiments for Carrying Out the Invention]

[0023] The substance detection method according to the present disclosure is a method for detecting the presence of a starting material or the production of a main product by measuring glycine by-produced together with the main product generated by a chemical reaction catalyzed by a hydrolase from the starting material. The starting materials include hippuric acid, methyl hippuric acid, or both hippuric acid and methyl hippuric acid. As the hydrolase (aminoacylase) that catalyzes these hippuric acid or methyl hippuric acid, an amidohydrolase derived from Staphylococcus aureus COL or a mutant enzyme thereof is used. This amidohydrolase has an activity of hydrolyzing hippuric acid and methyl hippuric acid, and particularly has an activity of hydrolyzing all three isomers of methyl hippuric acid.

[0024] [Basic Configuration of Substance Detection Method] First, the basic configuration of the substance detection method according to the present disclosure will be described. The starting material to be detected in the substance detection method according to the present disclosure is hippuric acid and / or methyl hippuric acid (that is, at least one of hippuric acid and methyl hippuric acid) as described above. In the substance detection method according to the present disclosure, a catalytic reaction (enzymatic reaction) of hydrolyzing hippuric acid or methyl hippuric acid with aminoacylase is utilized. Hippuric acid is known as a metabolite of toluene, and methyl hippuric acid is known as a metabolite of xylene.

[0025] Hippuric acid is known by the IUPAC name benzoylaminoacetic acid, and is also known by other names such as N-benzoylglycine. Methylhippuric acid has a structure in which the ortho, meta, or para position of the benzene ring structure of hippuric acid is modified with a methyl group. In IUPAC, taking the ortho position as an example, it is known as (2-methylbenzoylamino)acetic acid, and is also known by other names such as N-(o-Toluoyl)glycine.

[0026] Aminoacylase (EC 3.5.1.14) catalyzes the reaction that breaks down hippuric acid or methylhippuric acid (or both hippuric acid and methylhippuric acid) into benzoic acid or methylbenzoic acid and glycine. Other enzymes that catalyze this reaction (related enzymes to aminoacylase) include hippuric acid hydrolase (EC 3.5.1.32) and N-acyl-D-amino acid deacylase (EC 3.5.1.81). In this disclosure, as described later, amide hydrolase or a mutant enzyme derived from Staphylococcus aureus COL is used as the aminoacylase.

[0027] As described above, hippuric acid is a metabolite of toluene, and methylhippuric acid is a metabolite of xylene. When toluene or xylene is inhaled by workers handling these organic solvents, most of it is metabolized in the body to benzoic acid or methylbenzoic acid, which then undergoes glycine conjugation to become hippuric acid or methylhippuric acid, and is excreted in the urine. Therefore, hippuric acid or methylhippuric acid (hereinafter abbreviated as "hippuric acid, etc." as appropriate) is used as an indicator of toluene or xylene exposure. Accordingly, in this disclosure, biological samples such as urine that may contain hippuric acid, etc. are used as samples of chemical reactions that produce glycine as a by-product.

[0028] The sample used for the chemical reaction is not limited to urine, but may also be blood, saliva, bone marrow fluid, interstitial fluid, etc. In the case of blood, it may be whole blood or a blood sample containing only a portion of the blood components. The same applies to samples derived from bodily fluids such as bone marrow fluid and interstitial fluid. Furthermore, the source of the sample may be a worker handling organic solvents, i.e., a human. Therefore, the substance detection method according to this disclosure can be suitably used in health examinations to evaluate exposure to toluene or xylene in humans handling organic solvents. Note that the source of the sample is not limited to humans, but may also be other animals (mammals such as dogs, cats, pigs, cows, rats, and mice, or vertebrates other than mammals, etc.).

[0029] In the substance detection method according to this disclosure, as shown in Figure 1, first, a certain amount of a sample containing (potentially containing) hippuric acid, etc., is taken, and aminoacylase is added and mixed to hydrolyze the hippuric acid, etc. This produces benzoic acid or methylbenzoic acid (benzoic acid, etc.) and glycine. By measuring this glycine, the starting material, hippuric acid, etc., is detected. The method for measuring glycine in this disclosure is not particularly limited, and known methods can be suitably used. A typical method for measuring glycine is, for example, the method of measuring hydrogen peroxide produced by the catalytic action of glycine oxidase, as will be described later.

[0030] The enzyme used to hydrolyze the starting material in the substance detection method according to this disclosure may be any enzyme capable of hydrolyzing hippuric acid, etc., into benzoic acid, etc., and glycine, i.e., an aminoacylase. In particular, this disclosure mentions an aminoacylase having the amino acid sequence shown in SEQ ID NO: 1, or a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, and which has the activity to hydrolyze the aforementioned hippuric acid and the aforementioned methylhippuric acid. Typical examples of such aminoacylases include amide hydrolase or mutant enzymes derived from Staphylococcus aureus (S. zureus) COL.

[0031] [Composition of amide hydrolase] Next, we will specifically describe the amide hydrolase that is suitably used as a hydrolytic enzyme (aminoacylase) in the substance detection method according to this disclosure. Specifically, the amide hydrolase derived from S. zureus COL mentioned above can be, for example, the amide hydrolase described in Reference 1: Tavarekere S. Girish, Vivek B, Melwin Colaco, Sandra Misquith, B. Gopal, "Structure of an amidohydrolase, SACOL0085, from methicillin-resistant Staphylococcus aureus COL" Acta Crystallographica Section F, 69, pp.103-108, (2013). The contents described in Reference 1 are incorporated herein by reference and become part of this specification.

[0032] The amide hydrolase used in this disclosure is a protein having the amino acids shown as "SACOL0085" in Figure 2 of Reference 1, or the amino acid sequence shown in Sequence ID No. 1. Alternatively, the amide hydrolase used in this disclosure may be a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 1, and which has the activity to hydrolyze hippuric acid and methylhippuric acid. Here, one or more may be, for example, 1 to 20, 1 to 10, 1 to 8, 1 to 5, or 1 to 3.

[0033] Alternatively, the amide hydrolase used in this disclosure may be a protein having an amino acid sequence having 90% or more sequence homology with the amino acid sequence shown in SEQ ID NO: 1, and having the activity to hydrolyze hippuric acid and methylhippuric acid (hydrolytic activity of hippuric acid, etc.). Here, the amino acid sequence shown in SEQ ID NO: 1 may have 95% or more sequence homology, 97% or more sequence homology, 98% or more sequence homology, or 99% or more sequence homology.

[0034] Here, when the amide hydrolase used in this disclosure has an amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO: 1 and possesses hydrolytic activity for hippuric acid, etc., this hydrolytic activity for hippuric acid, etc. means the activity to hydrolyze all three isomers of hippuric acid and methylhippuric acid. As will be experimentally illustrated in the examples described later, the amide hydrolase used in this disclosure possesses hydrolytic activity not only for hippuric acid but also for 2-methylhippuric acid, 3-methylhippuric acid, and 4-methylhippuric acid. This makes it possible to easily quantify the total amount of hippuric acid and methylhippuric acid.

[0035] Furthermore, the amide hydrolase used in this disclosure may be a protein having the amino acid sequence shown in Sequence ID No. 1 or a homologous sequence (as described above, an amino acid sequence in which one or more amino acids are deleted, substituted, or added, or an amino acid sequence having 90% or more sequence homology), in which some amino acids have been altered to change the physical or chemical properties of the protein while still having the activity to hydrolyze hippuric acid and methylhippuric acid.

[0036] The aforementioned changes in the physical or chemical properties of proteins include, but are not limited to, improved thermal stability, altered substrate specificity, and changes in optimal pH. Furthermore, the aforementioned changes in amino acids include, but are not limited to, changes from L-amino acids to D-amino acids, changes to amino acids other than the 20-22 amino acids that make up proteins, and various post-translational modifications to amino acids.

[0037] The specific method for producing the amide hydrolase used in this disclosure is not particularly limited, and known methods can be suitably used. Typically, one method is to introduce DNA having the amino acid sequence shown in Sequence ID No. 1 or a homologous sequence into a host cell and overexpress it in the host cell.

[0038] Examples of DNA encoding amide hydrolase used in this disclosure include DNA having the nucleotide sequence shown in Sequence ID No. 2 of the sequence listing. This nucleotide sequence shown in Sequence ID No. 2 can be obtained, for example, by searching for "SACOL0085" using DBGET search on KEGG (Kyoto Encyclopedia of Genes and Genomes, URL: https: / / www.genome.jp / kegg / ) (https: / / www.genome.jp / dbget-bin / www_bget?sac:SACOL0085). The amino acid sequence shown in Sequence ID No. 1 can also be obtained using DBGET search.

[0039] The DNA encoding the amide hydrolase used in this disclosure may be any DNA encoding the amino acid sequence shown in SEQ ID NO: 1, and is not limited to DNA having the base sequence shown in SEQ ID NO: 2. For example, it may be DNA having a base sequence homologous to the base sequence shown in SEQ ID NO: 2, or it may be DNA having another base sequence encoding the amino acid sequence shown in SEQ ID NO: 1.

[0040] For example, a nucleotide sequence corresponding to the amino acid sequence shown in Sequence ID No. 1 may be obtained using known software or web services that convert amino acid sequences to nucleotide sequences, and DNA having this nucleotide sequence may be chemically synthesized using a known DNA synthesizer. At the time of filing this application, several commercial services are known that undertake the steps from obtaining a nucleotide sequence from a known amino acid sequence to synthesizing DNA, and in the examples described later, such commercial services are used to obtain the DNA encoding the amide hydrolase used in this disclosure.

[0041] When producing the amide hydrolase used in this disclosure, for example, a known self-replicating vector can be used. That is, a reproducible recombinant DNA can be constructed using the DNA encoding the amide hydrolase used in this disclosure and a self-replicating vector, and this recombinant DNA can be introduced into host cells.

[0042] Typical examples of autonomously replicating vectors used in this disclosure include known plasmid vectors. Specific plasmid vectors include pBR-type plasmids such as pBR322; pUC-type plasmids such as pUC18, pUC19, pUC118, pUC119; and pBlueScript II, pBluescript II SK(+ / -), pBluescript II KS(+ / -), pBluescript II XR, and pBluescript II Examples include pBS plasmids such as RI; pET plasmids such as pET-3a~3d, pET-11a~d, pET-14b, pET-15b, pET-21a~21d; pGEX plasmids such as pGEX-1, pGEX-2T, pGEX-3X; pTZ plasmids such as pTZ4, pTZ5, pTZ12, pTZ-18R, pTZ-19R; pSU plasmids such as pSU0, pSU7, pSU22, pSU23; Bacillus plasmids such as pUB110, pC194, pHY plasmids, pNU plasmids, pNY326, pNC plasmids; and shuttle vector plasmids such as pHV14, TRp7, YEp plasmids, and pBS7.

[0043] These plasmids can be appropriately selected depending on various conditions such as the type of host cell and the type of expression system. Furthermore, phage vectors or the like may be used as autonomously replicating vectors.

[0044] The method for inserting the DNA encoding the amide hydrolase used in this disclosure into an autonomously replicating vector is not particularly limited, and known methods can be suitably used. Generally, for example, the DNA (or gene) encoding the amide hydrolase and the vector are digested (cut) with a known type II restriction enzyme, and these DNA fragments and vector fragments are annealed as necessary, and then ligated using a DNA ligase or the like, but this method is not particularly limited.

[0045] The replicable recombinant DNA of the above configuration may also include DNA other than the DNA encoding the amide hydrolase used in this disclosure and the autonomously replicating vector. For example, it may include DNA encoding a regulatory sequence not included in the autonomously replicating vector, or DNA (or genes, etc.) encoding other proteins or peptides. In this case, the amide hydrolase used in this disclosure may be incorporated into the replicable recombinant DNA so as to constitute a chimeric protein together with other proteins and peptides.

[0046] The host cells used in producing the amide hydrolase used in this disclosure, that is, the host cells into which recombinant DNA having the DNA encoding the amide hydrolase used in this disclosure is introduced, are not particularly limited. Generally, microorganisms such as Escherichia coli, Bacillus subtilis, Actinomycetes, and yeast can be used, but are not limited thereto, and may also be plant cells or animal cells. In the examples described later, Escherichia coli is used as the host cell.

[0047] The method for introducing recombinant DNA into host cells, i.e., the transformation method, is not particularly limited, and known methods can be used depending on the type of host cell or the type of autonomously replicating vector. Typical transformation methods include, for example, electroporation or competent cell formation using calcium chloride for bacteria such as E. coli. If the host cell is yeast, methods such as partial removal of the cell wall of the yeast cell to form spheroplasts or lithium acetate can be used. Furthermore, if the host cell is a fungus, plant cell, or animal cell, particle gun or transfection methods can also be used.

[0048] A typical example of a method for producing the amide hydrolase used in this disclosure is, as described above, to produce transformants by introducing DNA encoding the amide hydrolase into host cells by various methods, and then culturing these transformants. Here, when producing the amide hydrolase used in this disclosure, the host cell culture scale is not particularly limited. For example, when using a liquid medium (culture medium), it may be a small-scale culture using test tubes or flasks, a large-scale culture using jar fermenters, or, at an industrial level, a large-scale culture using tanks.

[0049] The method for collecting the amide hydrolase used in this disclosure from cultured cells is not particularly limited, and known methods can be used. If the expressed amide hydrolase accumulates in the cells, the cultured cells can be collected, the cells can be disrupted by known methods to obtain a crude enzyme solution, and the amide hydrolase can be collected by purifying or concentrating this crude enzyme solution by known methods. If purification or concentration is not necessary, the crude enzyme solution can be used as the amide hydrolase according to this disclosure. Furthermore, if the expressed amide hydrolase is significantly secreted extracellularly, the amide hydrolase can be collected from the entire culture, including the cultured cells and culture medium.

[0050] The method for purifying the amide hydrolase used in this disclosure is not particularly limited, and known purification methods can be suitably used. Particularly suitable protein purification methods include, for example, Reference 2: Nishiya, Y., Yamamoto, M., Takemoto, J., Kano, S., and Nakano, S., "Monomeric sarcosine oxidase exhibiting high substrate affinity and thermostability" Int. J. Anal. Bio-Sci., 4, 55-62 (2016); Reference 3: Hiruta, M. and Nishiya, Y., "Creation of an L-mandelate oxidase via structure-guided design of engineered lactate oxidase" Int. J. Anal. Bio-Sci., 6, 25-29 (2018); Reference 4: Shimozawa, Y., Yoshida, S., Ikeda, K., Kato, Y., Toyama, F., and Nishiya, Y., "Easy preparation of a stable membrane-bound lactate dehydrogenase for application on lactate biosensor" Methods such as those described in Int. J. Anal. Bio-Sci., 8, 65-70 (2020) can be cited. The contents described in these references 2-4 are incorporated herein by reference and thus constitute part of this specification.

[0051] Furthermore, the specific production method of the amide hydrolase used in this disclosure can utilize various methods described in the publicly available literature, as well as commercial contract enzyme manufacturing services known at the time of filing this application. As mentioned above, the DNA encoding the amide hydrolase used in this disclosure can be manufactured using commercial services, but there are also commercial services known that undertake the steps up to constructing recombinant DNA containing the amide hydrolase encoding DNA or constructing an overexpression system, or the steps up to fermenting the protein using a microorganism that serves as a host cell. Therefore, this disclosure can also be implemented by utilizing such commercial services.

[0052] [Typical examples of glycine measurement methods] Next, we will describe a typical method for measuring glycine used in the substance detection method relating to this disclosure.

[0053] Glycine is the simplest amino acid, possessing no stereoisomerism between its D and L forms. It is a component of proteins, a raw material for the biosynthesis of various biomolecules, and is also known to be produced as a by-product in various chemical reactions.

[0054] In some chemical reactions, it can be difficult to directly measure the main product. Therefore, when glycine is produced as a byproduct, detecting the glycine makes it possible to indirectly determine whether the starting material is present in any given sample, or whether the starting material has been chemically reacted to produce the main product.

[0055] As previously stated, the present inventors have proposed a method for detecting substances using glycine oxidase disclosed in Patent Document 1. In this disclosure, the method for measuring glycine used in the substance detection method described in Patent Document 1 can be suitably used for detecting hippuric acid and the like. The contents described in Patent Document 1 (Japanese Patent Application Publication No. 2019-187285) are referred to herein and thus constitute part of this specification.

[0056] When the glycine measurement method disclosed in Patent Document 1 is applied to the substance detection method according to this disclosure, for example, as shown in Figure 1 or the upper part of Figure 2, a sample that may contain hippuric acid, etc., is first treated with the aforementioned aminoacylase (an amide hydrolase having the amino acid sequence shown in SEQ ID NO: 1 or a homologous sequence thereof). For convenience, the sample before treatment with aminoacylase is referred to as the "primary sample." If hippuric acid, etc., is present in the primary sample, benzoic acid, etc., and glycine are produced. In this disclosure, the hippuric acid, etc., which is the starting material, is detected (or measured or quantified) by measuring the produced glycine.

[0057] In this embodiment, as a specific method for measuring glycine, a method is used in which hydrogen peroxide produced by the catalytic action of glycine oxidase is measured, as shown in the middle and lower panels of Figure 2. For convenience, the sample obtained after treating the primary sample with aminoacylase is referred to as the "secondary sample."

[0058] The secondary sample, which has been treated with aminoacylase, i.e., the sample that (potentially) contains glycine, is then treated with glycine oxidase. For convenience, the sample obtained by treating the secondary sample with glycine oxidase is referred to as the "tertiary sample." If glycine is present in the secondary sample, it will be oxidized, and hydrogen peroxide will be directly generated along with glyoxylic acid and ammonia, as shown in the middle of Figure 2. In other words, the tertiary sample contains the directly generated hydrogen peroxide.

[0059] Next, 4-aminoantipyrine (4-AA), Trinder's reagent, and peroxidase are added to the tertiary sample treated with glycine oxidase and mixed, as shown in the lower part of Figure 2. If hydrogen peroxide is present in the tertiary sample (i.e., if glycine is present in the secondary sample before treatment with glycine oxidase), the hydrogen peroxide reacts with 4-AA and Trinder's reagent through the catalytic action of peroxidase to produce a quinone-based dye. For convenience, the sample obtained after treating the tertiary sample with peroxidase is referred to as the "quaternary sample."

[0060] Quinone dyes, for example, can be measured by their absorbance at 500-550 nm, so by measuring the absorbance of the quaternary sample, the amount of hydrogen peroxide produced in the tertiary sample can be quantified. If the hydrogen peroxide in the tertiary sample can be quantified, the glycine contained in the secondary sample, i.e., the sample before treatment with glycine oxidase, can be measured (quantified or detected). If the glycine in the secondary sample can be measured, then hippuric acid, etc., contained in the primary sample, i.e., the original sample before treatment with aminoacylase, can be measured (quantified or detected).

[0061] The glycine oxidase (EC 1.4.3.19) used in the glycine measurement method shown in Figure 2 catalyzes the deamination of glycine through oxidation. As shown in (1) below, oxidation of glycine releases ammonia from glycine, producing glyoxylic acid and hydrogen peroxide. Therefore, the oxidation of glycine can be determined by detecting hydrogen peroxide. Furthermore, as is clear from equation (1) below, 1 mole of hydrogen peroxide is produced when 1 mole of glycine is oxidized, so the amount of oxidized glycine (amount of glyoxylic acid produced) can be quantified by quantifying the amount of hydrogen peroxide produced. Glycine + H2O + O2 → Glyoxylic acid + NH3 + H2O2 ... (1)

[0062] The method for detecting hydrogen peroxide generated in the direct reaction of formula (1) above (method for measuring hydrogen peroxide) is not particularly limited, but generally, as shown in Figure 2, a method in which hydrogen peroxide, aminoantipyrine, and Trinder's reagent are reacted by the catalytic action of peroxidase, and the degree of color development of the resulting quinone-based dye is measured, known as the Trinder reaction, can be suitably used.

[0063] In the Trinder reaction, hydrogen peroxide in the sample is used as the oxidizing agent, and Trinder's reagent is used as the hydrogen donor to react with 4-aminoantipyrine (4-AA) via a catalytic reaction of an oxidase. This reaction generates a quinone-based dye in the sample through oxidative condensation, causing the sample to change color. By measuring the degree of color change using an absorbance spectrometer or similar device, the amount of hydrogen peroxide in the sample, and consequently the amount of glycine present before the enzymatic reaction, can be quantified.

[0064] The specific type of Trinder reagent is not particularly limited, and known compounds can be suitably used. Specifically, for example, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (TOOS), N-ethyl-N-sulfopropyl-3-methoxyaniline (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-(2-hydroxy-3-sulfopropyl)-3,5- Examples include aniline derivatives such as dimethoxyaniline (HDAOS) and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS); phenol derivatives such as phenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 3,5-dichlorophenol, 2,4-dibromophenol, 2,4,6-trichlorophenol, 2,4,6-tribromophenol, 3,5-dichloro-2-hydroxybenzenesulfonic acid, or 3-hydroxy-2,4,6-triiodobenzoyl acid; toluidine derivatives; and the like. In this disclosure, TOOS is particularly preferred.

[0065] Furthermore, the specific type of peroxidase used to produce quinone dyes from hydrogen peroxide is not particularly limited, and known types can be suitably used. Also, the structure of the produced quinone dye differs depending on the type of compound used as the trigger reagent, so it is not particularly limited. Known methods can also be used for measuring the quinone dye, so it is not limited to absorbance measurement under the conditions described above.

[0066] The glycine oxidase used in the glycine measurement method illustrated in this embodiment is not particularly limited, and known glycine oxidases can be suitably used. Specifically, examples include those derived from Bacillus cereus, Bacillus subtilis, and Geobacillus kaustophilus.

[0067] Alternatively, in the glycine measurement method described above, other enzymatic reactions can be used instead of the enzymatic reaction by glycine oxidase shown in the middle of Figure 2. Such enzymatic reactions are not particularly limited as long as they produce hydrogen peroxide. Specifically, other enzymatic reactions can be used, for example, the enzymatic reaction by S-adenosyl-L-methionine, glycine-N-methyltransferase, and sarcosine oxidase described in Reference 5: Japanese Patent No. 5978658. The contents described in Reference 5 are incorporated herein by reference.

[0068] In general, conventional methods amplify glycine through the glycine-sarcosine cycling reaction; therefore, it is necessary to quantify glycine using the rate method.

[0069] Methods for detecting (measuring) substances using enzymatic reactions can be classified into two types based on differences in analytical techniques: rate methods and endpoint methods.

[0070] The rate method is a method for quantitatively detecting a substance by measuring the rate of a chemical reaction that occurs when a substrate is catalyzed by an enzyme. In this method, for example, the rate during an enzyme-catalyzed reaction is measured as a change in absorbance or turbidity.

[0071] The endpoint method is a quantitative detection method for substances by chemically reacting a substrate using enzyme catalysis and measuring the total change before and after the reaction has reached its endpoint. In this method, for example, the reaction is allowed to proceed until the decrease in the starting material (substrate) or the increase in the product from the starting material substantially stops, and the amount of product produced is measured by absorbance or turbidity. The endpoint method has the advantage of being easier to apply to automated measurements using machines compared to the rate method because the production of products by the chemical reaction asymptotically approaches saturation.

[0072] In this disclosure, as shown in the upper part of Figures 1 and 2, equimolar glycine is produced as a by-product from hippuric acid, etc., as shown in the middle part of Figure 2, equimolar hydrogen peroxide is generated from glycine, and as shown in the lower part of Figure 2, hydrogen peroxide is used as an oxidizing agent to produce one molecule of quinone dye for every two molecules of hydrogen peroxide. As a result, a semi-equimolar amount of quinone dye is produced from hippuric acid, etc., making it possible to suitably use the endpoint method rather than the rate method. As described above, since the endpoint method is easy to apply to mechanical measurement, it is possible to automatically measure the detection (quantification) of hippuric acid, etc. by using the substance detection method according to this disclosure.

[0073] [Use of the substance detection method related to this disclosure] As described above, the substance detection method relating to this disclosure is a method for detecting a starting material by measuring the glycine produced as a by-product along with the main product generated by a chemical reaction catalyzed by a hydrolase, which is a hydrolase, of hippuric acid and / or methylhippuric acid, which is a starting material, with an amide hydrolase (aminoacylase) having the amino acid sequence shown in SEQ ID NO: 1 or a homologous sequence, using a known method.

[0074] According to the substance detection method of the above configuration, by using an amide hydrolase having the amino acid sequence shown in Sequence ID No. 1 or its homologous sequence as the enzyme for hydrolyzing hippuric acid or methylhippuric acid, all three isomers of methylhippuric acid, along with hippuric acid, can be effectively hydrolyzed. Therefore, if the sample to be detected is enzymatically treated with the amide hydrolase used in this disclosure, the sample will contain an amount of glycine corresponding to the hippuric acid and methylhippuric acid contained in the sample. This makes it possible to quantify the total amount of hippuric acid and methylhippuric acid contained in the sample by measuring hydrogen peroxide.

[0075] Furthermore, if the amount of hippuric acid contained in the sample can be obtained, the amount of methylhippuric acid can be obtained by subtracting the amount of hippuric acid from the total amount of quantified hippuric acid and methylhippuric acid. The method for quantifying the amount of hippuric acid to be subtracted is not particularly limited, and known methods can be suitably used. Typical examples include the method for measuring the concentrations of hippuric acid and methylhippuric acid in a biological sample, as described in Reference 6: Japanese Patent Publication No. 5751109. The contents described in Reference 6 are incorporated herein by reference and become part of this specification.

[0076] As shown in the examples described later, the amide hydrolase used in this disclosure shows a good correspondence between the known concentration in the sample and the measured value for hippuric acid, as well as 2-methylhippuric acid, 3-methylhippuric acid, and 4-methylhippuric acid. In contrast, when using the known aminoacylase PH1043 and the known hippuric acid hydrolase, the known concentration and the measured value show a good correspondence for hippuric acid, 3-methylhippuric acid, and 4-methylhippuric acid, but the known concentration and the measured value for 2-methylhippuric acid are far apart.

[0077] Reference 1, which reports the amide hydrolase used in this disclosure, does not mention the activity of this amide hydrolase in hydrolyzing hippuric acid and methylhippuric acid. The amide hydrolase used in this disclosure has amino acid sequence homology to the known aminoacylase PH1043, but the reason why the amide hydrolase used in this disclosure, unlike aminoacylase PH1043, also has enzymatic activity against 2-methylhippuric acid is not currently clear.

[0078] The substance detection method described herein is not particularly limited in its method for measuring glycine obtained by hydrolysis of hippuric acid or methylhippuric acid. However, typically, as described above, a method can be used to measure hydrogen peroxide produced by the catalytic action of glycine oxidase (or by the catalytic action of other enzymes). As described above, this measurement method allows the use of an endpoint method suitable for mechanical measurement, and therefore, by using the substance detection method described herein, it becomes possible to automatically measure (quantify) hippuric acid and the like.

[0079] This disclosure includes not only the substance detection method described above, but also various application fields of said substance detection method. For example, this disclosure includes kits (substance detection kits) used in the substance detection method described above.

[0080] The substance detection kit according to this disclosure only needs to contain, at a minimum, an amide hydrolase having the amino acid sequence shown in Sequence ID No. 1 or a homologous sequence, as an enzyme that hydrolyzes the starting materials, hippuric acid and methylhippuric acid. This amide hydrolase may be prepared as an enzyme solution by known methods when it is made into a kit. The specific composition of the enzyme solution is not particularly limited, and it only needs to contain the amide hydrolase in a known solvent at a concentration that is expected to be used under certain conditions.

[0081] Furthermore, the substance detection kit according to this disclosure may include other reagents or test equipment in addition to the amide hydrolase described above. As described above, the substance detection method according to this disclosure preferably uses a method in which hydrogen peroxide is generated from glycine produced as a by-product by the catalytic action of amide hydrolase through the catalytic action of glycine oxidase (or other enzymes) and this hydrogen peroxide is detected. For this reason, the substance detection kit according to this disclosure preferably also includes glycine oxidase (or other enzymes), and preferably also includes a reagent for detecting hydrogen peroxide.

[0082] Glycine oxidase (or other enzymes) can be prepared as an enzyme solution by known methods, similar to the amide hydrolase described above. Reagents for detecting hydrogen peroxide include the aforementioned 4-aminoantipyrine, Trinder's reagent, and peroxidase. 4-aminoantipyrine and Trinder's reagent may be prepared by dissolving them using known methods, and peroxidase may also be prepared as an enzyme solution using known methods.

[0083] As described above, the substance detection method relating to this disclosure is applicable to automated measurement systems. Therefore, the substance detection kit relating to this disclosure may include various test tools that can be used in automated measurement systems, and the amide hydrolase or other reagents may be prepared in known forms that can be used in automated measurement systems.

[0084] Furthermore, the substance detection kit according to this disclosure may also include other reagents such as sample diluents, enzyme reaction diluents, buffers, etc. These reagents are usually provided in suitable sealed containers. Alternatively, the substance detection kit according to this disclosure may include, but is not limited to, known disposable instruments used for sampling from samples, or microtubes for mixing the reagents. Furthermore, it may also include instructions describing protocols and other information necessary for using the substance detection kit according to this disclosure. Such instructions may be in printed form or recorded as data on a known recording medium.

[0085] Specific examples of substance detection kits relating to this disclosure include, for example, a kit for obtaining a sample from a worker who may handle toluene or xylene and detecting hippuric acid or methylhippuric acid, which are metabolites of toluene or xylene, contained in the sample, or for quantifying the total amount of hippuric acid and methylhippuric acid. Alternatively, the substance detection kit relating to this disclosure may be a research kit or investigation kit for detecting or quantifying hippuric acid or methylhippuric acid that are not metabolites.

[0086] Furthermore, the amide hydrolase having the amino acid sequence shown in Sequence ID No. 1 or its homologous sequence, used in the substance detection method or substance detection kit according to this disclosure, can also use hippuric acid and methylhippuric acid as detection reagents. As described above, the amide hydrolase used in this disclosure hydrolyzes hippuric acid, 3-methylhippuric acid, and 4-methylhippuric acid to produce benzoic acid or methylbenzoic acid, as well as glycine as a by-product, similar to conventional enzymes. In addition, it can also hydrolyze 2-methylhippuric acid, which conventional enzymes could not hydrolyze well.

[0087] Therefore, the aminoacylase (amide hydrolase) used in this disclosure can detect the total amount of hippuric acid and methylhippuric acid in a sample by quantifying glycine using the substance detection method described herein. However, it is also possible to detect the total amount of hippuric acid and methylhippuric acid in a sample by quantifying benzoic acid or methylbenzoic acid, for example. Accordingly, the hippuric acid and methylhippuric acid used in this disclosure can be used as detection reagents. [Examples]

[0088] The present invention will be described more specifically based on examples and comparative examples, but the present invention is not limited thereto. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

[0089] (Examples) First reagent R1 was prepared, containing 30 units / mL of amide hydrolase 4ewt, 7.5 units / mL of peroxidase, 4.5 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (TOOS), and 50 mM Tris buffer (pH 8.5). Second reagent R2 was prepared, containing 5 units / mL of glycine oxidase, 1.2 mM 4-aminoantipyrine, and 100 mM Tris buffer (pH 8.5).

[0090] Furthermore, as measurement samples, i.e., evaluation samples, as shown in Table 1, we prepared the following: Sample 1 containing hippuric acid, Sample 2 containing 2-methylhippuric acid, Sample 3 containing 3-methylhippuric acid, Sample 4 containing 4-methylhippuric acid, and Sample 0 as a negative control that does not contain hippuric acid or methylhippuric acid. As shown in Table 1, Samples 1 to 4 each consist of three types of samples containing 5 mM, 10 mM, or 20 mM hippuric acid or methylhippuric acid in physiological saline.

[0091] [Table 1]

[0092] To 2 μL of the sample dilution obtained by diluting these evaluation samples fivefold using an automated analyzer, 60 μL of the first reagent R1 was added and mixed, and the mixture was allowed to react thoroughly at 37°C. Then, 20 μL of the second reagent R2 was added and mixed, and the mixture was allowed to react thoroughly at 37°C. The absorbance of the reacted sample solution was measured using a JEOL Ltd. JCA-BM6050 automated analyzer at a primary wavelength of 545 nm and a secondary wavelength of 805 nm. From the obtained absorbance, the concentration of hippuric acid or methylhippuric acid in each evaluation sample was calculated and plotted on a graph. The results are shown in Figure 3.

[0093] In Figure 3, the horizontal axis represents the known concentration (in mM) of hippuric acid or methylhippuric acid in the evaluation sample, and the vertical axis represents the measured value (in mM) obtained by the automated analyzer. In Figure 3, the diamond symbol represents Sample Group 1, i.e., the evaluation sample containing hippuric acid; the square symbol represents Sample Group 2, i.e., the evaluation sample containing 2-methylhippuric acid; the triangle symbol represents Sample Group 3, i.e., the evaluation sample containing 3-methylhippuric acid; and the X symbol represents Sample Group 4, i.e., the evaluation sample containing 4-methylhippuric acid. The evaluation sample with a known concentration of 0 mM is Sample 0, but as mentioned above, it was used as a negative control in the measurements of Sample Groups 1 to 4, so the symbol corresponding to each sample group is used.

[0094] The amide hydrolase 4ewt used in this example was a gene chemically synthesized by converting the base sequence from the amino acid sequence shown in Sequence ID No. 1. Specifically, the 4ewt gene was produced by GenScript Japan Co., Ltd. (URL: https: / / www.genscript.jp / gene_synthesis.html), a commercial artificial gene synthesis service. As described above, recombinant DNA incorporating the 4ewt gene into a known vector was introduced into Escherichia coli (E. coli), the 4ewt protein was overexpressed, and the amide hydrolase 4ewt used in this example was produced by purifying it using the known method described in the aforementioned references 2-4.

[0095] (Comparative Example 1) Except for using the known aminoacylase PH1043 instead of the amide hydrolase used in the examples, the absorbance of each sample was measured in the same manner as in the examples, and the concentrations of hippuric acid or methylhippuric acid in each evaluation sample were calculated and plotted on a graph. The results are shown in Figure 4. The horizontal and vertical axes and symbols in Figure 4 are the same as in Figure 3.

[0096] The aminoacylase PH1043 used in this Comparative Example 1 was produced by Nipro Corporation, and for example, Reference 7: Satoru Imai, Kentaro Nagoshi, Shota Araki, Toshiaki Baba, Yoshiaki Nishiya, "Functional Modification of Hippuric Acid Hydrolase Derived from Pyrococcus horikoshii", The Society for Biotechnology, Japan (2018), Reference 8: Satoru Imai, Kentaro Nagoshi, Shota Araki, Toshiaki Baba, Yoshiaki Nishiya, "Enzymatic Measurement Method for Hippuric Acid and Development of Enzymes for Measurement", The Society for Biological Sample Analysis (2019), Reference 9: Satoru Imai, Kentaro Nagoshi, Shota Araki, Toshiaki Baba, Yoshiaki Nishiya, "Functional Improvement of Hippuric Acid Hydrolase", The Japan Society for Bioscience, Biotechnology, and Agrochemistry (2019), Reference 10: Satoru Imai, Kentaro Nagoshi, Shota Araki, Toshiaki Baba, Yoshiaki Nishiya, "Enzymatic Measurement Method for Hippuric Acid Hydrolase for Practical Application" Since this enzyme is a known enzyme described in "Improvement of the Function of the Basic Organism," The Molecular Biology Society of Japan (2019), Reference 11: Satoru Imai, Kentaro Nagoshi, Shota Araki, Toshiaki Baba, Yoshiaki Nishiya, "Development of an Enzymatic Measurement Method for Evaluating Total Exposure to Toluene and Xylene (1)," The Society for Biological Sample Analysis (2020), Reference 12: Takayuki Suginaka, Gento Torii, Satoru Imai, Toshiaki Baba, Yoshiaki Nishiya, "Development of an Enzymatic Measurement Method for Evaluating Total Exposure to Toluene and Xylene (2)," The Society for Biological Sample Analysis (2020), etc., a detailed explanation will be omitted. The contents described in these References 7-12 are incorporated into this specification by reference.

[0097] (Comparative Example 2) Except for using a known hippuric acid hydrolase (EC3.5.1.32, manufactured by Nipro Corporation) instead of the amide hydrolase used in the examples, the absorbance of each sample was measured in the same manner as in the examples, and the concentrations of hippuric acid or methylhippuric acid in each evaluation sample were calculated and plotted on a graph. The results are shown in Figure 5. The horizontal and vertical axes and symbols in Figure 5 are the same as in Figure 3 or Figure 4.

[0098] (Comparison of Examples and Comparative Examples) As is clear from the results of the examples, when using an amide hydrolase having the amino acid sequence shown in SEQ ID NO: 1, the measured values ​​for each sample in all of the sample groups 1 to 4 correspond well to the concentration of each sample.

[0099] In contrast, in both Comparative Example 1, which used aminoacylase PH1043, and Comparative Example 2, which used a known hippuric acid hydrolase, the measured values ​​and concentrations of each sample corresponded well for sample groups 1, 3, and 4, i.e., hippuric acid, 3-methylhippuric acid, and 4-methylhippuric acid. However, for sample group 2, i.e., 2-methylhippuric acid, the measured value was not significantly different from that of the negative control (sample 0).

[0100] This shows that the amide hydrolase used in this disclosure exhibits good reactivity with hippuric acid and all three types of methylhippuric acid, whereas conventional aminoacylase or hippuric acid hydrolase does not provide sufficient reactivity with 2-methylhippuric acid. Therefore, it can be seen that the substance detection method according to this disclosure can accurately quantify the total amount of hippuric acid and methylhippuric acid. Thus, using the substance detection method according to this disclosure, it is possible to quantify the amount of methylhippuric acid by subtracting the amount of hippuric acid from the total amount of hippuric acid and methylhippuric acid.

[0101] It should be noted that the present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments and multiple variations are also included in the technical scope of the present invention.

[0102] Furthermore, many improvements and other embodiments of the present invention will be apparent to those skilled in the art from the above description. Therefore, the above description should be interpreted as illustrative only and is provided for the purpose of teaching those skilled in the art the best mode of carrying out the invention. The details of its structure and / or function can be substantially modified without departing from the spirit of the invention. [Industrial applicability]

[0103] The present invention can be suitably used in fields where the presence of hippuric acid or methylhippuric acid, which are starting materials, can be detected by detecting glycine produced as a by-product of an enzymatic reaction by aminoacylase, or in fields where the total amount of hippuric acid and methylhippuric acid can be quantified.

Claims

1. A method for detecting the presence of at least the starting material by measuring glycine produced as a by-product along with the main product generated by a chemical reaction catalyzed by a hydrolytic enzyme using the starting material, The starting material is hippuric acid and / or methylhippuric acid, The hydrolase is characterized in that it is an aminoacylase having the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, and is a protein having the activity to hydrolyze hippuric acid and methylhippuric acid. Methods for detecting substances.

2. The measurement of glycine is performed by measuring the hydrogen peroxide produced. The method for detecting a substance according to claim 1.

3. The enzymatic reaction that produces hydrogen peroxide is an enzymatic reaction involving glycine oxidase. The method for detecting a substance according to claim 2.

4. The measurement of hydrogen peroxide is performed by reacting the hydrogen peroxide with aminoantipyrine and Trinder's reagent using the catalytic action of peroxidase, and measuring the degree of color development of the resulting quinone-based dye. The method for detecting a substance according to claim 3.

5. The main product is benzoic acid or methylbenzoic acid. A method for detecting a substance according to any one of claims 2 to 4.

6. The total amount of hippuric acid and methylhippuric acid contained in the sample to be detected is quantified by the measurement of hydrogen peroxide. A method for detecting a substance according to any one of claims 2 to 5.

7. The amount of methylhippuric acid is obtained by subtracting the amount of hippuric acid from the total amount of quantified hippuric acid and methylhippuric acid. The method for detecting a substance according to claim 6.

8. Used in the substance detection method according to any one of claims 1 to 7, The present invention is characterized by containing an aminoacylase having the amino acid sequence shown in Sequence ID No. 1, or a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 1, and having the activity to hydrolyze hippuric acid and methylhippuric acid, as an enzyme for hydrolyzing the starting material. Substance detection kit.

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