Mammalian orthogonal enzymes and use thereof

Abstract

Provided is a method for analyzing a target using a labeled target-binding substance, wherein at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, or both β-glucosidase and at least one enzyme selected from the group consisting of the aforementioned enzymes, are used as a labeling material.

Description

Mammalian orthogonal enzymes and their uses

[0001] The present invention relates to a group of mammalian enzymes and their uses. The enzymes disclosed in the present application are useful for analyzing target substances in mammalian cells, tissues, or individuals.

[0002] In the enzyme-sensitized method, a substance that specifically binds to a target substance is labeled with an enzyme, and the presence or absence and amount of the target substance are analyzed by detecting and measuring the color development of the substrate degradation product produced by the enzyme-substrate reaction. Well-known and widely used enzymes include horseradish peroxidase, alkaline phosphatase from bovine small intestine, and β-galactosidase from E. coli.

[0003] Several other enzymes are known. For example, Patent Document 1 discloses an analytical method using an antibody labeled with an enzyme, and lists alkaline phosphatase, acid phosphatase, alpha- and beta-galactosidase, alpha- and beta-glucosidase, esterase, and beta-lactamase as examples of the labeled enzyme. Patent Documents 2 to 7 disclose analytical methods using target-binding substances labeled with an enzyme, and lists glucosidase as an example of the labeled enzyme in Patent Documents 2, 3, and 5, glucosidase and glucuronidase in Patent Document 4, and β-glucuronidase in Patent Documents 6 and 7.

[0004] Japanese Patent No. 4648902 Japanese Patent No. 5265816 Japanese Patent No. 6765232 Japanese Patent No. 4434015 Japanese Patent Publication No. 2017-176039 Japanese Patent No. 6099978 Japanese Patent Publication No. 2008-64475

[0005] However, since the above-mentioned general-purpose enzymes have similar endogenous activity in mammals, when they are used to analyze cells and tissues derived from mammals, the endogenous activity must be inactivated, and they cannot be used in living organisms or cells. Furthermore, since fluorescent substances such as NADH and FMN are naturally present in mammalian cells, when analyzing using the above-mentioned enzymes, it is necessary to generate a certain amount of fluorescence or more, and the analytical targets are limited to substances that are relatively abundant in a single cell.

[0006] On the other hand, it would be convenient if the enzyme-sensitized method could be easily adapted to multiple colors for simultaneous detection of multiple targets. However, the existing multicolor method (PerkinElmer OPAL method) is cumbersome because it requires repeated staining for each target antigen, and each repetition causes color bleeding and a decrease in staining efficiency due to antigen denaturation.

[0007] The present invention relates to an enzyme-enhanced method for the analysis of mammals, which also allows for multicolor analysis. The inventors have screened several enzymes that can be used in such analyses, based on the following criteria: (1) enzyme activity on relatively bulky substrates (substrates that can produce fluorescent degradation products), (2) no endogenous activity in mammals, (3) no cross-reactivity with each other, and (4) the ability to react under the same biocompatible conditions (neutral pH, 4-37°C).

[0008] As a result, the present inventors have identified α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, α-glucuronidase, and β-glucosidase as enzymes for an enzyme-sensitized method that is also capable of multicolor analysis for the analysis of mammalian animals, and have completed the present invention.

[0009] The present invention provides the following: [1] A method for analyzing a target using a labeled target-binding substance, wherein the labeling substance is at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, or at least one enzyme selected from the group consisting of these and β-glucosidase. [2] The analytical method according to 1, which is carried out on mammalian cells, tissues, or individuals. [3] The analytical method according to 1 or 2, wherein the target-binding substance is an antibody. [4] The analytical method according to 3, wherein the presence or amount of a target is detected by fluorescence derived from a substrate degradation product produced by an enzymatic reaction, and a multicolor analysis is performed using two or more target-binding substances labeled with two or more enzymes selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase; or a combination of an enzymatic preparation containing at least one enzyme selected from the group consisting of these and an enzymatic preparation containing β-glucosidase. [6] A conjugate of a target-binding substance with at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase; or a combination of a conjugate of a target-binding substance with at least one enzyme selected from the group consisting of these enzymes, and a conjugate of β-glucosidase and a target-binding substance. [7] The conjugate or combination according to 6, wherein the target-binding substance is an antibody. [8] A kit for detecting a target substance, comprising:[9] The kit according to 8, which is for ELISA, comprising at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, or a combination of at least one enzyme selected from the group consisting of them and β-glucosidase.

[10] The analytical method according to any one of items 1 to 4, the enzyme preparation or combination of enzyme preparations according to item 5, the conjugate or combination of conjugates according to item 6 or 7, or the kit according to item 8 or 9, wherein the α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, or β-glucosidase is selected from the following: (1) α-arabinofuranosidase derived from Clostridium thermocellum, (2) α-rhamnosidase derived from Sphingomonas paucimobilis or Streptomyces avermitilis, (3) β-xylosidase derived from the genus Geobacillus, (4) α-sulfoquinovosidase derived from Escherichia coli, (5) α-galacturonidase derived from Bacillus subtilis, or (6) Eisenbergiella (1) β-galacturonidase derived from Azotobacter tayi or Faecalibacterium sp., (2) β-galacturonidase derived from Azotobacter tayi or Faecalibacterium sp., (3) β-galacturonidase derived from Azotobacter tayi or Faecalibacterium sp., (4) β-glucuronidase derived from Azotobacter tayi or Faecalibacterium sp., (5) β-galacturonidase derived from Azotobacter tayi or Faecalibacterium sp., (6) β-glucuronidase derived from Azotobacter tayi or Faecalibacterium sp., (7) α-glucuronidase derived from Thermotoga Maritima, (8) β-glucosidase derived from Terrabacter Ginsenosidimutans.

[11] The method according to any one of (1) to (4), the enzyme preparation or combination of enzyme preparations according to (5), the conjugate or combination of conjugates according to (6) or (7), or the kit according to (8) or (9), wherein the α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, or β-glucosidase is selected from the following: (1) α-arabinofuranosidase: (1a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or a multimer thereof;(1b) a protein or multimer thereof consisting of an amino acid sequence set forth in SEQ ID NO: 1 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has α-arabinofuranosidase activity; (1c) a protein or multimer thereof consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1, and which has α-arabinofuranosidase activity, (2) α-rhamnosidase: (2a) a protein or multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 9; (2b) a protein or multimer thereof consisting of an amino acid sequence set forth in SEQ ID NO: 2 or 9 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has α-rhamnosidase activity; (2c) a protein or multimer thereof consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 2 or 9, and which has α-rhamnosidase activity, (3) β-xylosidase: (3a) a protein or multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 3; (3b) a protein or multimer thereof consisting of an amino acid sequence set forth in SEQ ID NO: 3, in which one or more amino acids have been substituted, deleted, inserted, and / or added, said protein having β-xylosidase activity; (3c) a protein or multimer thereof consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 3, and having β-xylosidase activity, (4) α-sulfoquinovosidase: (4a) a protein or multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 4; (4b) a protein or multimer thereof consisting of an amino acid sequence set forth in SEQ ID NO: 4, in which one or more amino acids have been substituted, deleted, inserted, and / or added, said protein having α-sulfoquinovosidase activity; (4c) a protein or multimer thereof consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 4, and having α-sulfoquinovosidase activity, (5) α-galacturonidase: (5a) a protein or multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 5;(5b) a protein or multimer thereof consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 5, and having α-galacturonidase activity; (5c) a protein or multimer thereof consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 5, and having α-galacturonidase activity, (6) β-galacturonidase: (6a) a protein or multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 6 or 12; (6b) a protein or multimer thereof consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 6 or 12, and having β-galacturonidase activity; (6c) a protein or multimer thereof consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 6 or 12, and having β-galacturonidase activity, (7) α-glucuronidase: (7a) A protein or multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 7; (7b) A protein or multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 7 in which one or more amino acids have been substituted, deleted, inserted, and / or added, said protein having α-glucuronidase activity; (7c) A protein or multimer thereof consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 7, said protein having α-glucuronidase activity, (8) β-glucosidase: (8a) A protein or multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 8; (8b) A protein or multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 8 in which one or more amino acids have been substituted, deleted, inserted, and / or added, said protein having β-glucosidase activity; (8c) A protein or multimer thereof consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 8, said protein having β-glucosidase activity.

[0010] The enzymes disclosed herein have no activity in mammalian cells and are mammalian orthogonal (they selectively react only with specific substrates without reacting with reactive biomolecules present in excess in contaminated biological systems). Therefore, they are useful as sensitizing enzymes for use in bioanalysis. Furthermore, the enzymes disclosed herein can be selected in combination so that they do not cross-react with each other. Therefore, they are useful for multicolor analysis.

[0011] Endogenous enzyme activity in cells: 1.0 x 10 5 Enzyme reactivity after incubation of cells in medium containing 0.3 mM of each substrate for 18 hours. (a) JY25, (b) HeLa, (c) HepG2. Endogenous enzyme activity in JY25 cells. SDS-PAGE after HiTrap Q HP column chromatography. Lineweaver-Burk plot. Substrate specificity. ELISA results. (a) Primary antibody concentration dependence, (b) enzyme concentration dependence. Cell ELISA results. (a) JY25, (b) HeLa, (c) HepG2. Simultaneous detection of EpCAM and PD-L1 in HepG2 cells. Activity of Sphingomonas paucimobilis-derived rhamnosidase (SpRam). Activity of Streptomyces avermitilis-derived rhamnosidase (SaRam). Enzyme activity of SpRam. Enzyme activity of SaRam. (a) pNPR, (b) MURS. Cross-reactivity of pRam. Cross-reactivity of SaRam. Non-specific adsorption of the enzyme to cells. 1 HNMR spectrum SDS-PAGE results Lineweaver-Burk plot SDS-PAGE results of enzyme-modified antibody Cell-ELISA results using α-SQ-modified antibody SDS-PAGE results (a) EtGalUAse, (b) FcGalUAse Lineweaver-Burk plot (a) EtGalUAse, (b) FcGalUAse Cross-reactivity assessment (a) EtGalUAse, (b) FcGalUAse Non-specific adsorption of galacturonidase to cells (a) HeLa, (b) JY25 SDS-PAGE results 3-color cell ELISA results Sequences listed in the sequence table

[0012] The present invention relates to a method for analyzing a target using a labeled target-binding substance, in which the labeling substance is at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, α-glucuronidase, and β-glucosidase.

[0013] In a preferred embodiment, at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, or at least one enzyme selected from the group consisting of these enzymes and β-glucosidase, is used as the labeling substance. Note that α- and β- may be represented as a- and b- for convenience.

[0014] With respect to the enzymes used in the present invention, the term "multimer" refers to a molecule or supramolecule formed by two or more identical molecules or subunits held together by physical or chemical forces. The formation of a multimer is called multimerization. The multimer may be a dimer to an octamer. The enzymes used in the present invention may also contain small molecular components (sometimes called cofactors) other than proteins. Examples of such components include non-protein organic compounds, metal ions, and metal-containing organic compounds.

[0015] The analytical method of the present invention is carried out on cells, tissues, or individuals of mammals, including humans, rhesus monkeys, rats, mice, hamsters, rabbits, goats, sheep, dogs, cats, horses, cows, and pigs.

[0016] The labeled binding substance used in the analytical method of the present invention is not particularly limited as long as it can bind to the target analyte. Examples of labeled binding substances include antibodies, minibodies, such as single-chain antibodies (single-chain variable fragments, scFv), diabodies, peptides, ligand proteins, small ligand molecules, affibodies, nanobodies, monobodies, and aptamers. In a preferred embodiment, the target-binding substance is an antibody.

[0017] The analytical method of the present invention detects the presence or amount of a target based on fluorescence derived from substrate degradation products produced by an enzymatic reaction, and multicolor analysis can be performed by using two or more target-binding substances labeled with two or more enzymes selected from the group.

[0018] [Arabinofuranosidase] Arabinofuranosidase (EC 3.2.1.55) is an enzyme that acts on α-L-arabinofuranosides, α-L-arabinans containing (1,3)- or (1,5)-linkages, arabinoxylans, and arabinogalactans.

[0019] In a preferred embodiment, α-arabinofuranosidase from Clostridium thermocellum is used.

[0020] In another preferred embodiment, any of the following enzymes is used: (1a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or a multimer thereof, such as a hexamer; (1b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which one or more amino acids have been substituted, deleted, inserted, and / or added, or a multimer thereof, such as a hexamer, which has α-arabinofuranosidase activity; (1c) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 or a multimer thereof, such as a hexamer, which has α-arabinofuranosidase activity.

[0021] [α-Rhamnosidase] α-Rhamnosidase (EC 3.2.1.40) is found in plants, yeast, fungi, bacteria, etc. and utilizes a reverse hydrolysis mechanism to release β-L-rhamnose. The substrates are many natural glycosides with terminal α-L-rhamnose, such as naringin, rutin, quercitrin, hesperidin, diostin, and terpenyl glycosides.

[0022] In a preferred embodiment, α-rhamnosidase derived from Sphingomonas paucimobilis or Streptomyces avermitilis is used.

[0023] In another preferred embodiment, any of the following enzymes is used: (2a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 9 or a multimer thereof; (2b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 9 in which one or more amino acids have been substituted, deleted, inserted, and / or added, or a multimer thereof, which has α-rhamnosidase activity; or (2c) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 2 or 9 or a multimer thereof, which has α-rhamnosidase activity.

[0024] β-Xylosidase β-Xylosidase (EC 3.2.1.47) is an enzyme found in viruses and cellular organisms that hydrolyzes alpha-D-galactosyl-(1->4)-beta-D-galactosyl-(1->4)-beta-D-glucosyl-(1<->1)-ceramide to produce D-galactose and beta-D-galactosyl-(1->4)-beta-D-glucosyl-(1<->1)-ceramide.

[0025] In a preferred embodiment, β-xylosidase derived from the genus Geobacillus is used, and in a particularly preferred embodiment, β-xylosidase derived from Geobacillus sp. is used.

[0026] In another preferred embodiment, any of the following enzymes is used: (3a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 or a multimer thereof, for example a dimer; (3b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 in which one or more amino acids have been substituted, deleted, inserted, and / or added, or a multimer thereof, for example a dimer, which has β-xylosidase activity; (3c) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 3 or a multimer thereof, for example a dimer, which has β-xylosidase activity.

[0027] [α-Sulfoquinovosidase] α-Sulfoquinovosidase (EC 3.2.1.199) is present in Escherichia coli and Pseudomonas putida, and hydrolyzes the terminal non-reducing α-sulfoquinovoside residue contained in α-sulfoquinovyl diacylglyceride and α-sulfoquinovyl glycerol.

[0028] In a preferred embodiment, α-sulfoquinovosidase derived from Escherichia coli is used.

[0029] In another preferred embodiment, any of the following enzymes is used: (4a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 or a multimer thereof, for example, a dimer; (4b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which one or more amino acids have been substituted, deleted, inserted, and / or added, or a multimer thereof, for example, a dimer, which has α-sulfoquinovosidase activity; (4c) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 4 or a multimer thereof, for example, a dimer, which has α-sulfoquinovosidase activity.

[0030] [α-Galacturonidase] α-Galacturonidase (EC 3.2.1.22) catalyzes the hydrolysis of terminal non-reducing α-D-galactose residues in α-D-galactosides, including galacto-oligosaccharides, galactomannans, and galactolipids.

[0031] In a preferred embodiment, α-galacturonidase derived from Bacillus subtilis is used.

[0032] In another preferred embodiment, any of the following enzymes is used: (5a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 5 or a multimer thereof, such as a tetramer; (5b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which one or more amino acids have been substituted, deleted, inserted, and / or added, or a multimer thereof, such as a tetramer, which has α-galacturonidase activity; (5c) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 5 or a multimer thereof, such as a tetramer, which has α-galacturonidase activity.

[0033] β-Galacturonidase β-Galacturonidase (3.2.1.31) catalyzes the hydrolysis of β-D-glucuronoside to produce D-glucuronic acid and an alcohol.

[0034] In a preferred embodiment, β-galacturonidase derived from Eisenbergiella tayi or Faecalibacterium sp. is used.

[0035] In another preferred embodiment, any of the following enzymes is used: (6a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 or 12 or a multimer thereof, such as a tetramer; (6b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 or 12 in which one or more amino acids have been substituted, deleted, inserted, and / or added or a multimer thereof, such as a tetramer, which has β-galacturonidase activity; (6c) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 6 or 12 or a multimer thereof, such as a tetramer, which has β-galacturonidase activity;

[0036] [α-Glucuronidase] α-Glucuronidase (EC 3.2.1.139) catalyzes the hydrolysis of α-D-glucuronoside to produce D-glucuronic acid and alcohol.

[0037] In a preferred embodiment, α-glucuronidase from Thermotoga Maritima is used.

[0038] In another preferred embodiment, any of the following enzymes is used: (7a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 7 or a multimer thereof; (7b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 7 with one or more amino acid substitutions, deletions, insertions, and / or additions or a multimer thereof, which has α-glucuronidase activity; or (7c) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 7 or a multimer thereof, which has α-glucuronidase activity.

[0039] [β-Glucosidase] β-Glucosidase (EC 3.2.1.191) catalyzes the hydrolysis of protopanaxadiol-type ginsenosides having one glucosyl residue at the 3-position to produce protopanaxadiol-type ginsenosides without glycosidic modification at the 3-position and D-glucopyranose.

[0040] In a preferred embodiment, β-glucosidase derived from Terrabacter Ginsenosidimutans is used.

[0041] In another preferred embodiment, any of the following enzymes is used: (8a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8 or a multimer thereof, such as a trimer; (8b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8 in which one or more amino acids have been substituted, deleted, inserted, and / or added, or a multimer thereof, such as a trimer, which has β-glucosidase activity; (8c) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 8 or a multimer thereof, such as a trimer, which has β-glucosidase activity.

[0042] Enzymes used in particularly preferred embodiments are shown in the table below.

[0043] [Method for selecting a mammalian orthogonal enzyme] The present invention also provides a method for selecting a mammalian orthogonal enzyme that is useful for analyzing a target substance in a mammalian cell, tissue, or individual.

[0044] One of the criteria for selecting a mammalian orthogonal enzyme is that it is a glycosidase (EC 3.2.-), preferably a glycoside bond hydrolase or glycosidase (EC 3.2.1.-).

[0045] Another criterion for selection is that the enzyme has no endogenous activity in mammals, preferably humans. This is because there is no need to inactivate endogenous activity when using it to analyze cells and tissues derived from mammals. Such enzymes can be selected using a database to identify enzymes whose expression in humans has not been confirmed. Furthermore, enzymes can be selected by focusing on the substrate and excluding enzymes belonging to enzyme classes whose substrates are likely to be endogenous in humans.

[0046] Another criterion for selection is the possibility of exoenzyme activity. Even enzymes belonging to enzyme classes for which only endoenzyme activity has been reported may have exoenzyme activity. Even enzymes without endoenzyme activity may be applicable to mammalian assays depending on the substrate structure.

[0047] Other criteria for selection are sufficient activity under biocompatible conditions, i.e., at 25-45°C and in a weakly acidic to neutral pH range, for example, pH 6.5-7.5, more particularly pH 6.8-7.2. An enzyme may have an optimum temperature and optimum pH outside these ranges, as long as it has sufficient activity under biocompatible conditions. An enzyme with sufficient activity can be selected, for example, from an enzyme with an optimum temperature of 27-70°C and an optimum pH of 6.0-7.5, whose kcat / Km value is equivalent to that of enzymes used in conventional bioanalysis. A kcat / Km value equivalent to that of enzymes used in conventional bioanalysis means that the kcat / Km value is 1.0 x 10 3 ~9.0×10 6 s -1 M -1 Preferably, it is 2.5 x 10 3 ~6.0×10 6 s -1 M -1 This means that...

[0048] When selecting multiple enzymes for multicolor analysis, it is advisable to select enzymes from a group of candidate enzymes that have substrate structures that are dissimilar and unlikely to cross-react.

[0049] At least one of the kcat value and the Km value may be used as a criterion for selection. The kcat value criterion may be, for example, several tens of s-1 or more, and the Km value criterion may be, for example, several hundreds of μM or less.

[0050] [Enzyme Preparation] The present invention also provides an enzyme preparation containing at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, or a combination of an enzyme preparation containing at least one enzyme selected from the group consisting of these enzymes and an enzyme preparation containing β-glucosidase. Such an enzyme preparation or combination of enzyme preparations is suitable for use in enzyme sensitization methods.

[0051] The agent refers to a composition. The agent can be in various forms as long as it does not affect the stability of the enzyme or the enzymatic reaction. Examples include liquids, powders, and granules.

[0052] The enzyme preparation may contain other ingredients in addition to the enzyme as the active ingredient, as long as they do not affect the stability of the enzyme or the enzymatic reaction. The content of the active ingredient in the enzyme preparation is not particularly limited, but the enzyme preparation can be prepared so that the enzymatic activity per gram of the enzyme preparation is 1 U to 1000 U, preferably 10 U to 300 U.

[0053] Examples of other ingredients contained in enzyme preparations include excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, and physiological saline. Examples of excipients include lactose, sorbitol, D-mannitol, maltodextrin, and sucrose. Examples of buffers include phosphates, citrates, and acetates. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol.

[0054] [Method for Producing Enzymes] The enzymes used in the present invention can be prepared by those skilled in the art using conventional techniques. There are no particular limitations on the method as long as the desired enzyme can be produced, and the enzyme can be produced by a culture method using a microorganism that biosynthesizes the enzyme, or by genetic engineering techniques. Commercially available enzymes may also be used in the present invention.

[0055] When a culture method is used, the culture conditions and culture method are not particularly limited as long as the target enzyme is produced. Genetic engineering techniques can be used, for example, to produce the target enzyme by designing and cloning a polynucleotide encoding the target enzyme (e.g., a polynucleotide encoding any one of the amino acid sequences of SEQ ID NOS: 1 to 9), transforming a suitable host cell (e.g., Escherichia coli), and recovering the target enzyme protein produced by the transformant.

[0056] [Conjugate] The present invention also provides a conjugate of a target-binding substance with at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase; or a combination of a conjugate of a target-binding substance with at least one enzyme selected from the group consisting of these enzymes, and a conjugate of β-glucosidase with a target-binding substance.

[0057] Examples of labeled binding substances include antibodies, minibodies, such as single-chain antibodies (single-chain variable fragments, scFv), diabodies, peptides, ligand proteins, small ligand molecules, affibodies, nanobodies, monobodies, and aptamers. The target-binding substance is typically an antibody.

[0058] Antibodies may be polyclonal or monoclonal. Examples of polyclonal antibodies include those derived from rabbits, guinea pigs, goats, sheep, rats, mice, and chickens, while examples of monoclonal antibodies include those produced by hybridomas obtained from animal species such as rats, mice, chickens, rabbits, and humans.

[0059] Enzyme-labeled antibodies may be used to directly bind to the substance (antigen) to be analyzed, or may be used as a secondary antibody in analytical methods such as ELISA, which will be described later.

[0060] [Use of Enzymes, etc.] The enzymes discovered by the present invention, i.e., at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, can be used in an analytical method that uses an antibody modified with the enzyme in combination with a fluorescent dye having a fluorescent group that serves as a substrate for the enzyme, i.e., a group that is cleaved by an enzymatic reaction and emits fluorescence.

[0061] The enzyme discovered by the present invention, i.e., at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, can be used in a cell staining method using a cell-retaining fluorescent compound (CLAMP method, see WO / 2020 / 250998), which enables specific and highly sensitive detection of only cells expressing a target cell surface protein. Examples of the cell-retaining fluorescent compound include the following compounds (1) to (10).

[0062]

[0063] The present invention also provides a kit for detecting a target substance, comprising: at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase; or a combination of at least one enzyme selected from the group consisting of them with β-glucosidase. The kit is preferably a kit for ELISA (Enzyme Linked Immunosolvent Assay). Such a kit may comprise an enzyme-antibody conjugate, a well microplate, a dilution buffer, a washing buffer, a chromogenic substrate, a stop solution, and instructions.

[0064] The enzyme discovered by the present invention, i.e., at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, may be used in combination with enzymes used in conventional biochemical analyses, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO).

[0065] [Multicolor analysis] By using two or more enzymes selected from the group consisting of the enzymes discovered by the present invention, namely, α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, multiple targets can be simultaneously detected in multiple colors. By using two or more enzymes selected from this group of enzymes, multicolor analysis can be performed even for targets expressed in trace amounts in cells.

[0066] The two or more enzymes used in the multicolor analysis can be arbitrarily selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase. The number of enzymes may be two, three, or four or more, and can be selected depending on the number of targets to be analyzed. Furthermore, the combination of two or more enzymes used in the multicolor analysis is not particularly limited as long as they are selected from this enzyme group, but is preferably selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, and β-galacturonidase. Particularly preferably, the two or more enzymes used in the multicolor analysis are selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, and β-galacturonidase.

[0067] The combination of enzymes when there are two types of targets is not particularly limited, but for example, a combination of β-galacturonidase and α-arabinofuranosidase can be selected.The combination of enzymes when there are three types of targets is not particularly limited, but for example, a combination of α-arabinofuranosidase, α-rhamnosidase, and β-galacturonidase can be selected.

[0068] [Characteristics of the enzyme of the present invention] (Enzyme activity) The enzyme used in the present invention has sufficient activity to be used in the enzyme sensitization method. Sufficient activity means an enzyme activity equivalent to that of a conventional sensitized enzyme. Alternatively, for example, under optimal conditions, the enzyme has an activity of 10 3 s -1 M -1 More than 10, preferably 4 s -1 M -1 More than 10, particularly preferably 5 s -1 M -1 This means that the enzyme has a kcat / Km value of at least 1.

[0069] Those skilled in the art can measure enzyme activity using known methods, and the method is not particularly limited as long as it allows the measurement of the target enzyme activity. For example, a method for measuring enzyme activity can be used in which a quenching substrate that is cleaved by the enzyme to develop color or emit fluorescence is reacted with the enzyme, and the absorbance or fluorescence from the substrate degradation product is measured.

[0070] (Endogenous Enzyme Activity) The enzymes discovered by the present invention, i.e., α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, are not endogenously present in mammals, preferably humans, or even if present, their activity is barely detectable. Therefore, when these enzymes are used as labeling substances to analyze cells and tissues derived from mammals, there is no need to inactivate endogenous enzyme activity.

[0071] The endogenous enzyme activity is not particularly limited, but can be determined, for example, by adding a synthetic substrate in which pNP is bound to a substrate specific for α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, α-glucuronidase, or β-glucosidase to mammalian, preferably human-derived cells, culturing the cells for 12 to 24 hours, preferably 18 hours, and then measuring the absorbance of the culture supernatant. Examples of substrates specific to α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, α-glucuronidase, or β-glucosidase include α-L-arabinofuranoside, α-L-rhamnopyranoside, β-D-xylopyranoside, α-D-sulfoquinovoside, α-D-galactopyranoside, β-D-galactopyranoside, α-D-glucuronide, and β-D-glucoside.

[0072] (Cross-reactivity) The enzymes discovered by the present invention, i.e., α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, exhibit high activity toward substrates specific to each enzyme, but exhibit no or negligible reactivity toward substrates specific to other enzymes or β-glucosidase. Thus, two or more enzymes selected from this group can be used as labeling substances when multiple targets are simultaneously analyzed.

[0073] Cross-reactivity can be investigated by measuring the reactivity of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, α-glucuronidase, or β-glucosidase with a substrate specific to each enzyme and with a substrate specific to the other enzyme, and comparing the activities. Examples of substrates specific to α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, α-glucuronidase, or β-glucosidase include α-L-arabinofuranoside, α-L-rhamnopyranoside, β-D-xylopyranoside, α-D-sulfoquinovoside, α-D-galactopyranoside, β-D-galactopyranoside, α-D-glucuronide, and β-D-glucoside.

[0074] (Nonspecific Adsorption to Cells) The enzymes discovered by the present invention, namely, α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, hardly exhibit nonspecific adsorption to cell surfaces. Therefore, by using these enzymes as labeling substances in enzyme-sensitized methods, it becomes difficult for fluorescent signals to be generated from sources other than the target, thereby improving the accuracy of target detection.

[0075] Non-specific adsorption to cells can be examined by adding an enzyme to the cells, leaving them to stand for 30 minutes to 2 hours, preferably 1 hour, washing them, and then measuring the enzyme activity of the cells.

[0076] [Regarding identity of amino acid sequences, etc.] In the present invention, when an "amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added" is mentioned, the number of amino acids to be substituted, etc. is not particularly limited in any protein, unless otherwise specified, as long as the protein consisting of that amino acid sequence has the desired function, but may be about 1 to 9 or 1 to 4 amino acids, or may include an even greater number of substitutions, etc., as long as the substitutions are with amino acids with similar properties. Means for preparing polynucleotides or proteins with such amino acid sequences are well known to those skilled in the art.

[0077] In the present invention, the term "identity" in relation to amino acid sequences or nucleotide sequences (sometimes referred to as nucleotide sequences) refers to the percentage of identical amino acids or nucleotides shared between two sequences when the two sequences are aligned in an optimal manner, unless otherwise specified. Identity can be calculated as follows: (number of identical positions / total number of positions) × 100, and can be calculated using commercially available algorithms. Such algorithms are incorporated into the NBLAST and XBLAST programs described in Altschul et al., J. Mol. Biol. 215 (1990) 403-410. More specifically, searches and analyses of nucleotide or amino acid sequence identity can be performed using algorithms or programs well known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, ClustalW). When using a program, parameters can be appropriately set by those skilled in the art, or the default parameters of each program can be used. Specific techniques for these analysis methods are also well known to those skilled in the art. The identity may be calculated using genetic information processing software GENETIX (registered trademark) (Genetics Corporation).

[0078] As used herein, when referring to amino acids or nucleotides, identity refers to sequence identity of at least 75% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97.5% or more, even more preferably 99% or more, and even more preferably 99.8% or more, unless otherwise specified.

[0079] 1. Enzyme Selection By focusing on sugar hydrolases, the 7,000 enzyme classes were narrowed down to 200. Subsequently, a complete search was performed using the database at ExPASy (Swiss Institute of Bioinformatics), and 155 enzymes not found to be expressed in humans were selected (Table 1). Next, the Human Protein Atlas (HPA) database (SciLifeLab, Sweden) was used to exclude enzyme classes likely to be present in humans based on their substrates, narrowing the list down to 86 enzymes.

[0080] From here on, we used the BRENDA (BRaunschweig ENzyme DAtabase) database. First, we focused on the section that lists substrates and their activity, and selected 25 classes containing enzymes with reported exo activity. However, for classes where only endo activity has been reported, there is a possibility that they may also have exo activity, and depending on the substrate structure, they may be applicable to this method. While we cannot definitively conclude that there is no future applicability, more detailed investigation of these classes is required.

[0081] Among these, the enzyme's k at around 37 °C and pH 7.0 cat The Km values ​​were compared to those of conventional enzymes for bioanalysis, and the Km values ​​were 10 4 s -1 M -1 From these, enzyme classifications that are not very similar in substrate structure and are unlikely to cross-react were selected, narrowing it down to eight types.

[0082] From the final remaining enzyme classifications, we comprehensively evaluated various literature that appears to use the same enzyme to select the enzyme with the highest applicability. cat The value is several tens of seconds -1 The Km value was set to several hundred μM or less.

[0083] Co-crystal structure analysis with the substrate was used to first confirm the size of the active center and determine the applicability of this method to the substrate. We also considered that the molecular weight should not be too large, that the isoelectric point should be acidic, and that nonspecific adsorption to cells should be minimized. Finally, we considered literature regarding substrate specificity and ensuring minimal overlap in glycoside hydrolase (GH) classification, and selected combinations that did not cross-react. The following eight enzymes were concluded to be optimal for each of the eight enzyme classifications (Table 2).

[0084]

[0085]

[0086] 2. Evaluation of endogenous enzyme activity 2.1 Evaluation of endogenous enzyme activity (1) [Experiment] This evaluation used HepG2 cells (human hepatoma cells) and HeLa cells (human cervical cancer cells) as human-derived adherent cells, and JY25 cells (human Epstein-Barr virus-immortalized B cells) as human-derived suspension cells. The substrates used were NPA, 4-nitrophenylphosphate, 4-nitrophenyl β-D-galactopyranoside, 4-nitrophenyl α-L-rhamnopyranoside, 4-nitrophenyl β-D-xylopyranoside, 4-nitrophenyl α-D-sulfoquinovoside, 4-nitrophenyl α-D-galacturonide, 4-nitrophenyl β-D-galacturonide, 4-nitrophenyl α-D-glucuronide, and 4-nitrophenyl β-D-glucopyranoside.

[0087] Adherent cells were washed with serum-containing medium and then cultured at 1.0 × 10 5The cells were suspended in serum-free medium at a density of 1.0 × 10 cells / 200 μL. 200 μL of the cell suspension was dispensed into a 96-well plate and cultured overnight at 37°C under 5% CO2 conditions to allow adhesion. After that, the supernatant was removed and the cells were washed with serum-free medium, followed by the addition of 90 μL of 10 mM HEPES / 300 mM mannitol (pH 7.4) solution. 1.0 × 10 cells / 200 μL of serum-free medium were added. 7 RIPA buffer was added to the cells at a concentration of 90 μL, and the mixture was stirred for 15 minutes using a vortex mixer and then centrifuged. 5 The cells were suspended in 100 mM citric acid (pH 4.5) solution and 10 mM HEPES / 300 mM mannitol (pH 7.4) solution at a concentration of 90 μL per well, and 90 μL of the suspension was dispensed into a 96-well plate.

[0088] The suspension cells were washed with serum-free medium and then cultured at 4.0 × 10 5 The cells were suspended in 10 mM HEPES / 300 mM mannitol (pH 7.4) solution at a concentration of 1.0 × 10 cells / 90 μL. 90 μL of the suspension was added to a 96-well plate. 7 RIPA buffer was added to the cells at a concentration of 90 μL, and the mixture was stirred for 15 minutes using a vortex mixer and then centrifuged. 5 The cells were suspended in 100 mM citric acid (pH 4.5) solution and 10 mM HEPES / 300 mM mannitol (pH 7.4) solution at a concentration of 90 μL per well, and 90 μL of the suspension was dispensed into a 96-well plate.

[0089] 10 μL of 3 mM substrate dissolved in 10 mM HEPES (pH 7.4) was added to each sample to a final concentration of 0.3 mM, and the cells were incubated for 18 hours. After centrifugation, the supernatants were transferred to 96-well plates, and the pH was adjusted by adding 0.1 mM NaOH solution. The absorbance change at 410 nm for each solution was measured using an Infinite 200 PRO (Tecan) (see figure below). The reaction rate was calculated assuming 100% absorbance for a sample containing NPs under the same conditions. The protein concentration at the time of lysing was also measured using the Pierce BCA Protein Assay to confirm successful lysing. Results are expressed as the mean ± standard deviation of triplicate experiments.

[0090]

[0091] [Results] Figure 1-1 shows the endogenous activity evaluation results for HepG2, HeLa, and JY25 cells and their lysates. Phosphatase (Phos), which has been confirmed to be expressed in HepG2, HeLa, and JY25 cells, was confirmed to be active. The stronger reaction in the lysate (pH 4.5) is due to the presence of acid phosphatase. Furthermore, the activity of β-galactosidase (β-Gal), which has been confirmed to be expressed in lysosomes in both cell types, was predominantly observed in the lysate (pH 4.5). On the other hand, the enzymes of interest in this study, α-Abf, α-Ram, β-Xyl, α-SQ, α-GalUA, β-GalUA, and α-GluUA, were barely detected, suggesting that they are highly likely not endogenous. Furthermore, β-Glu showed weak activity, especially under live cell conditions. This was thought to be due to the very small amount of β-Glu present on the cell membrane surface.

[0092] 2.2 Evaluation of endogenous enzyme activity (2) [Experiment] The enzyme activities of α-arabinofuranosidase (α-Abf), α-galacturonidase (α-GalUA), α-glucuronidase (α-GluUA), α-sulfoquinovosidase (α-SQ), α-rhamnosidase (α-Ram), β-galacturonidase (β-GalUA), β-glucosidase (β-Glu), and β-xylosidase (β-Xyl) in JY25 cells were evaluated using the enzymes reported as endogenous enzymes in the HPA. The enzyme activities of ALP, β-Gal, α-D-galactosidase (α-gal), α-L-fucosidase (α-fco), α-D-galactosidase (α-galUA), α-D-mannosidase (α-man), β-D-gulcuronidase (β-gulUA), β-D-fucosidase (β-fco), and β-D-mannosidase (β-man) were compared. The experimental procedures were performed in the same manner as in "2.1 Evaluation of endogenous enzyme activity (1)" above, except that specific substrates for each enzyme were used.

[0093] [Results] The results are shown in Figure 1-2. For the enzymes we focused on in this study, α-Abf, α-Ram, β-Xyl, α-SQ, α-GalUA, β-GalUA, and α-GluUA, almost no activity was observed, suggesting that they are highly likely not endogenous.

[0094] 3. Arabinofuranosidases 3.1 Selection of Arabinofuranosidases Of these eight enzymes, CtAbf is expected to have the most information available and the greatest potential for application. First, α-Abf hydrolyzes α-L-arabinofuranose, a naturally occurring monosaccharide with a high abundance among sugars. Therefore, numerous CAZy-derived enzymes have been reported, totaling 326. Furthermore, from among those reported in BRENDA, three enzymes were selected that retained sufficiently high exo-activity under mild conditions. Among these, CtAbf has been cocrystallized with α-1,5-arabinoxylose and other molecules, and the structure of its substrate molecules, which have been confirmed to be active, sufficiently suggests a large active center. Furthermore, the molecular weight, isoelectric point, hydrophilicity, and presence or absence of a signal peptide were all consistent with those of enzymes previously used in analyses, indicating no problems. In addition, among the eight enzymes selected, none of them reacted with β-D-xylopyranoside, which has the most similar substrate structure, and the enzymes in the same GH classification also suggest that they retain high substrate specificity, and are therefore considered to be applicable.

[0095] 3.2 Preparation of arabinofuranosidase [Experiment] The target protein is α-L-arabinofuranosidase (CtAbf) derived from the thermophilic anaerobic bacterium Clostridium thermocellum. Clostridium thermocellum is abundant in soil and has been attracting attention for its hydrolytic activity, including that of cellulose, and research has been progressing accordingly (Nakane K. Barry P., J. Histochem. Cytochem., 14, 12, 929-931 (1966)). In this study, we attempted to express CtAbf with (His)6 introduced as a purification tag at its C-terminus. The amino acid sequence is shown in the sequence listing as SEQ ID NO:1.

[0096] The CtAbf expression plasmid was constructed by our collaborators as follows: Genomic DNA was obtained from the National Institute for Product Evaluation and Research (NIER) and amplified from Clostridium thermocellum genomic DNA using Phusion DNA polymerase with the primer combination 5-CATATGAAAAAAGCCAGAATGACCGTTGACA-3 (SEQ ID NO: 10) and 5-CTCGAGTTTACCTATCCGAATTACATTCCAAGAGGCT-3 (SEQ ID NO: 11). The amplified gene fragment was cloned into pBluescript II, digested with NdeI and XhoI, and inserted into the cloning site of pET22 to construct the CtAbf expression plasmid.

[0097] E. coli BL21 (DE3) was transformed with the CtAbf expression plasmid on agar plates containing 100 mg / L ampicillin. After transformation, the transformed cells were inoculated into 40 mL of Luria Bertani medium containing 100 mg / L ampicillin and grown overnight with shaking at 180 rpm at 37°C. The culture was then transferred to 1 L of Terrific Broth containing 100 mg / L ampicillin and grown with shaking at 180 rpm at 37°C until the OD600 reached 0.8. The culture was cooled to 27°C, supplemented with IPTG to a final concentration of 0.5 mM, and incubated with shaking for 48 hours.

[0098] E. coli cells were harvested by centrifugation (4500 g), washed with 100 mL of Dulbecco's Phosphate-Buffered Saline (DPBS), resuspended in 100 mL of DPBS, and sonicated. The resulting spheroplasts were harvested by centrifugation (8000 g) and subjected to His tag affinity chromatography at 4 °C using a BioLogic DuoFlow medium-pressure chromatography system (Bio-Rad) equipped with a HisTrap FF column (Cytiva). The His tag affinity chromatography was performed using a 20 mM Tris-HCl / 0.5 M NaCl / 0.5 M imidazole (pH 7.4) solution. The spheroplasts were then desalted using a PD-10 column (Cytiva) and further purified by anion exchange chromatography using a BioLogic DuoFlow medium-pressure chromatography system equipped with a HiTrap Q HP column (Cytiva). Here, a 20 mM Tris-HCl / 0.5 M NaCl (pH 8.0) solution was used. The fractions were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (see figure below). After chromatography, the purification was completed by ultrafiltration using DPBS, and the concentration was determined based on the absorbance at 280 nm.

[0099]

[0100] [Results] The purified fraction was analyzed by SDS-PAGE and confirmed to have a molecular weight equivalent to that of CtAbf (Figure 2). The yield was approximately 10 mg.

[0101] 3.3 Arabinofuranosidase activity [Experiment] The activity of the expressed and purified CtAbf was evaluated using NPA and MUA as substrates (see figure below). 40 μL of CtAbf dissolved in DPBS at a final concentration of 8.5 nM was mixed with 40 μL of 0 to 4 mM substrate in a 96-well plate. The mixed solution was incubated at 37°C for a maximum of 60 minutes, and the change in absorbance at 410 nm or 360 nm for each solution was measured over time using an Infinite 200 PRO. The reaction amount of the substrate per specified time was determined using a calibration curve prepared in advance using NP and MU, and Vmax, Km, and k were calculated by creating a Michaelis-Menten plot and a Lineweaver-Burk plot. cat and k cat In addition, similar measurements were performed at 4°C and 27°C using NPA as the substrate to evaluate the temperature dependence.

[0102] In addition, 40 μL of CtAbf dissolved in DPBS at final concentrations of 0 and 8.5 nM and 40 μL of 1 mM substrate were mixed in a 96-well plate and the pH was adjusted from 7.4 to 12.5 using NaOH solution. The mixed solutions were incubated at 37°C for up to 60 minutes, and the change in absorbance at 410 nm for each solution was measured over time using an Infinite 200 PRO. The amount of substrate reacted per specified time was calculated using a calibration curve prepared in advance for each pH using NP, and pH dependence and optimal reaction termination conditions were evaluated. Results are expressed as the mean ± standard deviation of triplicate experiments.

[0103]

[0104] [Results] The Lineweaver-Burk plot results obtained from the experiment at 37°C are shown in Figure 3. The kinetic constants calculated from these experimental results and literature values ​​are summarized in the table below. Km, k cat The results were almost identical to those in the literature. In addition, the enzyme showed high activity against MUA, suggesting that, as expected, the active center is relatively large and that it is possible to take rigid and bulky molecules as substrates. cat / Km was found to have high enzymatic activity comparable to that of conventional sensitized enzymes.

[0105]

[0106]

[0107] 3.4 Cross-reactivity of arabinofuranosidases [Experiment] The substrate specificity of CtAbf was evaluated using NPA, 4-Nitrophenyl β-D-galactopyranoside, 4-Nitrophenyl α-L-rhamnopyranoside, 4-Nitrophenyl β-D-xylopyranoside, 4-Nitrophenyl α-D-sulfoquinovoside, 4-Nitrophenyl α-D-galacturonide, 4-Nitrophenyl β-D-galacturonide, 4-Nitrophenyl α-D-glucuronide, and 4-Nitrophenyl β-D-glucopyranoside as substrates (see figure below).

[0108] 40 μL of CtAbf (final concentration 8.5 nM) dissolved in DPBS and 40 μL of 1 mM substrate were mixed in a 96-well plate. The mixture was incubated at 37°C for 18 hours, and the absorbance of each solution at 410 nm was measured using an Infinite 200 PRO. The amount of substrate reacted per specified time was calculated assuming 100% absorbance for a sample containing NP under the same conditions. Results are expressed as the mean ± standard deviation of triplicate experiments.

[0109]

[0110] [Results] The experimental results for substrate specificity for CtAbf are shown in Figure 4. NPA, used as a control, was confirmed to react at approximately 90%. On the other hand, the following 4-Nitrophenyl α-L-rhamnopyranoside, 4-Nitrophenyl β-D-xylopyranoside, 4-Nitrophenyl α-D-sulfoquinovoside, 4-Nitrophenyl α-D-galacturonide, 4-Nitrophenyl β-D-galacturonide, 4-Nitrophenyl α-D-glucuronide, 4-Nitrophenyl β-D-glucopyranoside, and 4-Nitrophenyl β-D-galactopyranoside were not degraded at all. This suggests that α-Ram, β-Xyl, α-SQ, α-GalUA, β-GalUA, α-GluUA, β-Glu, and β-Gal can be simultaneously utilized with CtAbf. In particular, for β-Xyl, β-GalUA, β-Glu, and β-Gal, the three-dimensional structure of the hydroxyl groups up to the third position is identical, but they are highly orthogonal, and among the reported α-Abfs, CtAbf is thought to have particularly high substrate specificity.

[0111] 3.5 Cell ELISA for Arabinofuranosidase [Experiment] (Reagent) GlycoYELLOW TMβ-Gal (GYG) was purchased from Goryo Chemical Co., Ltd. Avidin, Hanks' Balance Salt Solution (-) without Ca, Mg, and Phenol Red (HBSS) were purchased from Nacalai Tesque, Inc. Human anti-human HER2 antibody (Anti-human HER2 antibody) was purchased from Chugai Pharmaceutical Co., Ltd. Goat anti-mouse IgG antibody-biotin (Anti-mouse IgG antibody-biotin), Fluorescein (FLU), 4-Hydroxyazobenzene 2-carboxylic acid (HABA), and 4-Methylumbelliferone (MU) were purchased from Tokyo Chemical Industry Co., Ltd. Biotin Labeling Kit-NH 2 were purchased from Dojin Chemical Research Institute Co., Ltd. EDTA, Escherichia coli-derived β-Gal (EcGal), Fluorescein di β-D-galactopyranoside (FDG), Normal Mouse IgG, Streptavidin (SA), Sodium hydrogen carbonate, and Tween 20 were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. Rat anti-mouse / human CD44 antibody-biotin (Anti CD44-biotin), mouse anti-human EpCAM antibody-biotin (Anti EpCAM-biotin), and mouse anti-human PD-L1 antibody-biotin (Anti PD-L1-biotin) were purchased from BioLegend. Human anti-human CD20 antibody was purchased from Novartis. Human anti-human EGFR antibody was purchased from Merck. Bovine serum albumin, 4-methylumbelliferyl β-D-galactopyranoside (MUG), and sodium carbonate were purchased from Sigma-Aldrich.

[0112] (Biotinylation) Anti-human CD20 antibody, anti-human EGFR antibody, anti-human HER2 antibody, EcGal, and CtAbf were biotinylated using the Biotin Labeling Kit-NH2 to prepare anti-CD20-biotin, anti-EGFR-biotin, anti-HER2-biotin, EcGal-biotin, and CtAbf-biotin for Cell ELISA. The biotinylation rate was calculated using the Pierce BCA Protein Assay and HABA Assay.

[0113] (ELISA) The enzyme used was CtAbf. NPA or MUA was used as the substrate. A UV-Vis-IR spectrophotometer V-670 (JASCO) was used to measure the absorption and fluorescence spectra of these substrate molecules.

[0114] An enzyme-antibody conjugate was prepared by premixing anti-mouse IgG antibody-biotin, 5 μg / mL SA, and 1 μg / mL CtAbf-biotin to final concentrations of 1 μg / mL. 100 μL of normal mouse IgG dissolved in 0.05 M carbonate / bicarbonate buffer (pH 9.6) was added to a 96-well plate and incubated overnight at 4°C. The plate was then washed twice with PBS-T (DPBS containing 0.05% Tween 20). 200 μL of blocking buffer (PBS-T containing 1% BSA, 1 mM EDTA) was added and incubated for 2 hours at 37°C to block nonspecific binding sites. The plate was then washed three times with PBS-T. 100 μL of previously prepared enzyme-antibody conjugate was then added to the plate and incubated for 1 hour at 4°C. The plate was then washed five times with PBS-T. 100 μL of 0.1 mM NPA or MUA dissolved in DPBS was added, incubated at 27°C, and the changes in absorbance or fluorescence intensity of each solution were measured over time using an Infinite 200 PRO (Nakane et al., 1966). Results are expressed as the mean ± standard deviation from triplicate experiments.

[0115]

[0116] (Cell ELISA) The enzymes used were CtAbf and EcGal. GYG or FDG was used as the substrate for EcGal. A UV-Vis-IR spectrophotometer V-670 was used to measure the absorption and fluorescence spectra of these substrate molecules.

[0117] The final concentrations of antibody-biotin, SA, and enzyme-biotin were 10 μg / mL, 50 μg / mL, and 10 μg / mL, respectively, to prepare an enzyme-antibody complex. After washing the cells twice, 1.0 × 10 6The cells were suspended at a concentration of 100 μL per 100 μL. After centrifugation, the supernatant was removed, and 100 μL of a previously prepared enzyme-antibody conjugate was added. The mixture was then left to stand at 4°C for 1 hour. After washing three times, 300 μL of substrate (final concentration: 0.1 mM) was added and the mixture was suspended. 100 μL of each solution was plated on a 96-well plate and incubated at 27°C. The changes in absorbance or fluorescence intensity of each solution were measured over time using an Infinite 200 PRO. Results are expressed as the mean ± standard deviation of triplicate experiments.

[0118]

[0119] (Multicolor cell ELISA) Antibody-biotin, SA, and enzyme-biotin were mixed in advance to a final concentration of 10 μg / mL, and 50 μg / mL, respectively, to prepare an enzyme-antibody complex. Biotin solution was added to the prepared enzyme-antibody complex solution to a final concentration of 0.1 mM to block any remaining SA binding sites. After washing the cells twice, 1.0 × 10 6 The cells were suspended at a concentration of 100 μL per 100 μL. After centrifugation, the supernatant was removed, and 100 μL of a previously prepared enzyme-antibody conjugate was added simultaneously. The mixture was then left to stand at 4°C for 1 hour. After washing three times, 300 μL of substrate (final concentration 0.1 mM) was added simultaneously and suspended. 100 μL of this mixture was plated in 96-well plates and incubated at 27°C. The changes in absorbance or fluorescence intensity of each solution were measured over time using an Infinite 200 PRO (see figure below). Results are expressed as the mean ± standard deviation of triplicate experiments.

[0120]

[0121] [Results and Discussion] (Biotinylation) The biotinylation rates calculated using the Pierce BCA Protein Assay and HABA Assay were 14.3 biotin / unit for Anti-CD20-biotin, 0.7 biotin / unit for Anti-EGFR-biotin, 5.3 biotin / unit for Anti-HER2-biotin, 12.4 biotin / unit for EcGal-biotin, and 3.6 biotin / unit for CtAbf-biotin. Since it was confirmed that these could be biotinylated, we decided to use these for the following experiments.

[0122] (ELISA) Quantitative evaluation was performed by ELISA using MUA (Figure 5a). When the seeding concentration of the target was changed, a linear increase in signal was observed in a concentration-dependent manner. Even 100 ng / mL of the target was detected with a sufficient S / N ratio.

[0123] The detection results for different enzyme-antibody conjugate concentrations are also shown (Figure 5b). The higher the concentration, the greater the signal. At the same time, nonspecific adsorption to the plate was almost nonexistent, demonstrating the high applicability of CtAbf.

[0124] (Cell ELISA) EpCAM, an adhesion molecule receptor of the human hepatocyte cell line HepG2, and CD20, a B cell marker of the EB virus-infected B cell line JY25, were detected (Figures 6a and 6b). Detection was possible while maintaining a high signal-to-noise ratio. PD-L1, a programmed cell death protein ligand of HepG2, was expressed in lower amounts than the above two proteins, resulting in a poorer signal-to-noise ratio. However, detection was possible with significant differences between the presence and absence of IFNγ stimulation (Figure 6c).

[0125] (Multicolor cell ELISA) Multicolor cell ELISA was performed targeting EpCAM and PD-L1, which is expressed at very low levels. Detection was attempted using EcGal and FDG for EpCAM, and CtAbf and MUA for PD-L1. The results are shown in Figure 7. For both targets, no signal was detected without antibody addition. However, signal was detected with antibody addition, demonstrating successful simultaneous detection of EpCAM and PD-L1. The signal-to-noise ratios (S / N) before IFN-γ stimulation were 8.6 and 7.4, respectively, and 15.9 and 22.7 after stimulation, respectively. This demonstrates that simultaneous protein detection is possible regardless of expression level.

[0126] 4. Rhamnosidase 4.1 Selection of rhamnosidase α-L-Rham was selected using enzyme databases, cazy and BRENDA. Commercially available α-L-Rham was first selected. Subsequently, those with reported crystal structures and high enzymatic activity under mild reaction conditions (neutral pH and 37°C) were selected.

[0127] The results of the database and literature survey are shown in the table below. From the database, it was found that 40 types of rhamnosidases have been investigated among all rhamnosidases. Of these, six are available for purchase. We selected the available types because we thought that this enzyme should be available to anyone for medical applications. After that, we investigated the enzymes that have high enzymatic activity under mild reaction conditions (kcat / KM of 1.0 × 10 at neutral pH and 37°C). 4 (s -1 M -1 We selected two rhamnosidases, Sphingomonas paucimobilis (SpRam) and Streptomyces avermitilis (SaRam), which have the same or higher molecular weight (Mg) and whose crystal structures have been reported.

[0128] Of these two, we selected rhamnosidase (SpRam) from Sphingomonas paucimobilis, which has high enzymatic activity under milder reaction conditions.

[0129]

[0130]

[0131] 4.2.1 Preparation of rhamnosidase (Sphingomonas paucimobilis) (SpRam) [Experiment] A (His)8 purification tag and a Nus-tag solubility tag were introduced to the N-terminus of rhamnosidase. Purification was performed using a His-tag column with a His TrapFF crude column. The target product was then confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

[0132] [Results] As shown in Figure 8-1, the expression of the target product of 167 kDa was confirmed.

[0133] 4.2.2 Construction of Streptomyces avermitilis-derived rhamnosidase (SaRam) SaRam contained a His tag at the C-terminus. Purification was performed using a His-Trap FF crude column, a His-Trap Q HP column, and a size-exclusion column on a HiLoad Superdex 75 column. The target product was then confirmed by 8 M SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 8-2).

[0134] 4.3.1 SpRam activity [Experiment] pNPR (final concentrations: 0.125, 0.25, 0.5, 1, 2, 3 mM) was added to α-L-Rham (final concentration: 1.8 nM) dissolved in Dulbecco's Phosphate-Buffered Saline (DPBS). After 600 seconds, the optical density at 410 nm of each solution was measured using a plate reader. The substrate reaction amount was calculated from a previously prepared p-nitrophenol calibration curve, and enzyme activity was evaluated by creating a Lineweaver-Burk plot.

[0135]

[0136] [Results] The Lineweaver-Burk plot results obtained from the experiment are shown in Figure 9-1. The table below summarizes the kinetic constants calculated from the experimental results and the kinetic constants of conventional sensitized enzymes. The KM, kcat, and kcat / KM of SpRam were 0.67 mM and 371 s, respectively. -1 , 5.5 × 10 5 s -1 M -1 The calculated kcat / KM ratio showed that SpRam had high enzymatic activity comparable to that of conventional sensitized enzymes. Furthermore, when comparing the kcat and KM values ​​of the conventional enzymes, SpRam had a higher kcat value than the three enzymes, demonstrating a high efficiency in converting substrates to products. The KM value was comparable to that of HRP, but higher than that of AP and β-gal.

[0137]

[0138] 4.3.2 SaRam Activity [Experimental] The activity of α-rham was measured for pNP-α-L-rhamnopyranoside or 4-methylumbelliferone-α-L-rhamnopyranoside substrates. The substrates were reacted with α-rham in DPBS (pH 7.4) at 37°C, and the reaction rate was calculated by measuring the absorbance of pNP (405 nm) or the fluorescence of MU (ex. 360 nm, em. 450 nm). Measurements were performed within a time period where the reaction rate was considered constant at sufficiently low enzyme concentrations. Rate constants were calculated from Lineweaver-Burk plots.

[0139]

[0140]

[0141] [Results] The Lineweaver-Burk plot results obtained from the experiment are shown in Figure 9-2. The kinetic constants calculated from the experimental results are summarized in the table below.

[0142]

[0143] 4.4.1 Cross-reactivity of SpRam [Experiment] pNPR, potassium p-nitrophenyl α-D-sulfoquinovoside, p-nitrophenyl α-D-galacturonide, p-nitrophenyl α-D-glucuronide, p-nitrophenyl β-D-galactopyranoside, p-nitrophenyl α-L-arabinofuranoside, p-nitrophenyl β-D-xylopyranoside, and p-nitrophenyl β-D-glucopyranoside (final concentration: 1 mM) were added to α-L-Ram dissolved in Dulbecco's Phosphate-Buffered Saline (DPBS) (final concentration: 14.5 nM). After 18 hours, the optical density of each solution was measured at 410 nm using a plate reader. The reaction rate of each substrate was calculated based on the optical density of p-nitrophenol.

[0144]

[0145] [Results] Figure 10-1 shows the reaction rates of SpRam with eight different substrates. The reaction rate was high with the rhamnosidase substrate. On the other hand, almost no reaction was observed with the substrates of α-D-sulfoquinovosidase, α-D-galacturonidase, α-D-glucuronidase, β-D-galactosidase, α-L-arabinofuranosidase, β-D-xylosidase, and β-D-glucosidase. This indicates that SpRam does not cross-react with the substrates of the seven enzymes that may be used simultaneously.

[0146] 4.4.2 Cross-reactivity of SaRam [Experiment] α-rham (20 nM or less) was reacted with pNP substrates (0.1 mM) of α-araf, α-galUA, α-gluUA, α-sq, α-rham, β-galUA, and β-glu at 37°C in DPBS at pH 7.4 for 18 hours. Afterwards, the absorbance at 405 nm was measured, and the relative reaction rates were calculated by setting the intrinsic reaction rates of the other pNP substrates at 1.

[0147] [Results] The relative reaction rates for each substrate were calculated, with the reaction rate for the original substrate being set at 1. As a result, α-rham did not cross-react with the substrates for the other enzymes (FIG. 10-2).

[0148] 4.5 Nonspecific adsorption of rhamnosidase to cells [Experiment] 1.0 x 10 6 100 μL of α-rham (0.1-10 μg / mL) was added to each JY25 cell, incubated at room temperature for 1 hour, and then washed three times with DPBS. MU substrate was added to the cells at a final concentration of 0.1 mM at 37°C for 1 hour. Fluorescence intensity at 450 nm was then measured.

[0149] [Results] Conventional bioanalytical sensitizing enzymes such as HRP, ALP, and β-gal adsorb nonspecifically to cell surfaces to some extent, which hinders the detection of proteins expressed at low levels on cells. Therefore, we evaluated the nonspecific adsorption of a new enzyme to the cell surface. 1 × 10 JY25 cells, a human B cell line, were used. 6 Each enzyme was added to the plate at a concentration of 0.1-10 μg / mL and allowed to stand at 4°C for 1 hour. After washing three times, 0.1 mM MU-type substrate was added and the reaction was allowed to proceed at 37°C for 1 hour. The results showed that α-rham was less adsorbed than β-gal (Figure 11).

[0150] 4.6 Rhamnosidase Substrate Synthesis [Experiment] (Synthesis of 1,2,3,4-tetra-O-acetyl-L-rhamnopyranoside) 5.00 g of L(+)-rhamnose monohydrate was dissolved in 26.0 mL of acetic anhydride and cooled to 0°C on ice. Then, HSO was added under ice cooling. 4 Six drops of 1,2,3,4-O-acetyl-α-L-rhamnopyranoside were added and stirred for 1 hour. After stirring for an additional 3 hours at room temperature, saturated aqueous NaHCO3 was added until the reaction mixture became neutral to quench the reaction. The reaction mixture was extracted three times with CHCl3. After drying over Na2SO4 and removing the solvent, the mixture was concentrated to give 8.70 g of 1,2,3,4-O-acetyl-α-L-rhamnopyranoside as a clear syrup. No further purification was performed, and the yield was 96%. The product was 1 The reaction was confirmed by H NMR (400 MHz, CDCl3).

[0151] (Synthesis of resorufin 2,3,4-tetra-O-acetyl-L-rhamnopyranoside) 1g (1eq.) of 1,2,3,4-tetra-O-acetyl-L-rhamnopyranoside and 6.4x10 resorufin 2 mg (2 eq.) of the mixture was dissolved in a mixture of 10 mL of CHCl (super-anhydrous) and 30 mL of CHCN (super-anhydrous) under a N atmosphere, and BF·EtO 3.1×10 2 The reaction was initiated by adding 2.5 eq. of NaHCO3. After 20 hours, the reaction was stopped by adding NaHCO3, and the mixture was extracted three times with CHCl. ​​The mixture was dried over NaSO4, the solvent was removed, and the product was purified by gel filtration chromatography using CHCl as an eluent to obtain 2.8 mg of an orange syrup. The yield was approximately 3%. 1 The reaction was confirmed by H NMR (400 MHz, CDCl3).

[0152] (Synthesis of Resorufin α-L-rhamnopyranoside) Resorufin 2,3,4-tetra-O-acetyl-L-rhamnopyranoside was reacted with 50 mM K2CO3 / MeOH solution at room temperature for 35 minutes, and then neutralized with AcOH. The crude product was purified by column chromatography using CHCl3 and MeOH as eluents to obtain approximately 2 mg of dark orange crystals. The yield was over 90%. 1 The product was confirmed by H NMR (400 MHz, DMSO-d6, 90°C).

[0153] [Results] Resorufin α-L-rhamnoside was synthesized according to the scheme shown below, and the compounds shown in Figures 12a and 12b were obtained. 1 H NMR spectra were obtained and the desired product was obtained in an overall yield of approximately 3%.

[0154]

[0155] 5. Sulfoquinovosidase 5.1 Selection of Sulfoquinovosidase We focused on sugars found in plant cell walls and chloroplasts, organelles not present in mammalian cells. Specifically, we selected α-sulfoquinovosidase (α-SQ, EC 3.2.1.199), which degrades the highly hydrophilic α-sulfoquinovose. High substrate hydrophilicity is important for ensuring water solubility even when modified with hydrophobic fluorescent dyes. α-Sulfoquinovose exists as a sulfoglycolipid in the thylakoid membrane of chloroplasts. While α-sulfoquinovose is abundant in the environment, its absence in mammalian cells suggests that its enzymatic activity is not endogenous to mammalian cells. α-SQ is also present in plants and prokaryotes and has been reported to have high substrate specificity and enzymatic activity. The sequence of α-SQ is shown in the Sequence Listing as SEQ ID NO:4.

[0156] 5.2 Construction of sulfoquinovosidase α-SQ contained (His)8 as a purification tag at the C-terminus. Purification was performed using a His-Trap FF crude column, a His-Trap Q HP column, and a size-exclusion column on a HiLoad Superdex 75 column. The target product was then confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 13).

[0157] 5.3 Reactivity of sulfoquinovosidase [Experiment] PNPSQ (final concentrations: 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8 mM) was added to a-SQ (final concentration: 13.6 nM) dissolved in DPBS. After 10 min, Na2CO3 solution (final concentration: 0.5 mM) was added to stop the reaction, and the optical density at 405 nm of each solution was measured using an AD200 microplate reader (Beckman Coulter Inc.). The substrate reaction amount was calculated from a previously prepared p-nitrophenol calibration curve, and enzyme activity was evaluated by creating a Lineweaver-Burk plot.

[0158] [Results] The Lineweaver-Burk plot results obtained from the experiment are shown in Figure 14.M , k cat are 0.32 mM and 16 s, respectively. -1 The calculated value is almost the same as that in the literature (see table below). cat / K M is 5.0 × 10 4 s -1 M -1 It was found that the enzyme had high enzymatic activity comparable to that of conventional sensitized enzymes.

[0159] Here, from the Michaelis-Menten plot, we can see that when the substrate concentration is 4 mM or higher, the enzyme reaction rate asymptotically approaches the maximum reaction rate Vmax and becomes constant. Conversely, when an enzyme reaction is carried out with a substrate concentration of 4 mM or less, there is no linearity between the product concentration and reaction time. However, it is thought that linear approximation is possible in the short reaction time range. Therefore, when linearity between the number of enzymes and the product concentration is required, as in ELISA, it is necessary to create a calibration curve in advance and determine a reaction time that ensures quantitation. To avoid this kind of manipulation, it is necessary to use a K M strategy using enzymes small enough to 37 It is thought that a strategy such as defining quantitation by the number of wells in which an enzyme reaction occurred would be effective.

[0160]

[0161] 5.4 Cell ELISA for Sulfoquinovosidase [Experiment] (Preparation of Modified Antibody) To remove the preservative, the anti-IgG antibody solution was placed in a microdialyzer and dialyzed against 500 mL of Buffer A (100 mM sodium phosphate, 150 mM NaCl, pH 7.2) at 4°C overnight. The anti-IgG antibody solution (5 mg / mL, 110 μL, Buffer A) was then prepared by ultrafiltration using an Amicon Ultra-0.5 device (NMWL:50K). A stock solution (10 mg / mL) of SM(PEG)4 (PEGylated SMCC crosslinker) dissolved in dehydrated dimethyl sulfoxide was diluted 10-fold with Buffer A, and immediately added 33.9 μL to the anti-IgG antibody solution and allowed to stand at room temperature for 1 hour (anti-IgG:SM(PEG)4 = 1:18). Then, 2.36 mL of buffer B (100 mM sodium phosphate, 150 mM NaCl, pH 6.0) was added to bring the total volume to 2.5 mL, and the mixture was purified on a PD-10 column previously purged with buffer B. The mixture was then ultrafiltered with an Amicon Ultra-0.5 device (NMWL: 50K) to a concentration of 5 mg / mL while replacing with buffer A.

[0162] A-SQ solution (10 mg / mL, 100 μL) was prepared by ultrafiltration using an Amicon Ultra-0.5 device (NMWL: 50K) into Buffer C (100 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, pH 7.8). 4.66 μL of a 35 mM solution of 2-iminothiolane dissolved in Buffer C was added to the a-SQ solution and allowed to stand at room temperature for 1 hour (a-SQ:2-iminothiolane = 1:35). Then, 2.40 mL of Buffer C was added to bring the total volume to 2.5 mL, and the a-SQ was purified on a PD-10 column purging with Buffer C. A 0.5 mL aliquot was taken for SDS-PAGE analysis. Thereafter, the remaining 3.0 ml was replaced with buffer A and concentrated to about 5 mg / mL using an Amicon Ultra-0.5 device (NMWL: 50K).

[0163] The antibody solution and enzyme solution prepared above were mixed and left to stand for one hour (a-SQ:Ab = 2:1). The reaction was then stopped by adding N-ethylmaleimide to a final concentration of 10 μM. Buffer A was added to bring the total volume to 2.5 mL, and the mixture was purified using a PD-10 column purged with buffer A. At this point, the total volume was 3.5 mL, of which 0.5 mL was removed for use in SDS-PAGE. The remaining 3.0 mL was then concentrated to 1 mg / mL using an Amicon Ultra-0.5 device (NMWL: 50K).

[0164]

[0165] SDS-PAGE was used to confirm the enzyme-modified antibody. For molecular weight measurement by SDS-PAGE, first, a solution was taken from the ultrafiltrated sample so that the amount of protein was 10 μg, and then subjected to NuPAGE. TM LDS Sample Buffer (4X) 5 μl (Invitrogen) and NuPAGE TMSample Reducing Agent (10X) 2 μl (Invitrogen) was added to prepare a sample (reducing condition) and boiled for 5 minutes. TM Samples without adding 2 μl of Sample Reducing Agent (10X) (Invitrogen) were prepared (non-reducing conditions) and boiled for 5 minutes. After boiling, 10 μl of each sample was added to wells on an acrylamide gel and electrophoresed. The gel was then stained with Coomassie Blue (CBB staining) to confirm bands. Instant Blue (Expedeon) was used for CBB staining.

[0166] (ELISA) To remove the preservative, the primary antibody solution was added to a microdialyzer and dialyzed against 500 mL of DPBS (4°C overnight). The primary antibody was replaced with coating buffer (carbonate / bicarbonate buffer, pH 9.6) at a concentration of 3 μg / mL. 100 μL of this solution was added to a 96-well ELISA plate and incubated overnight at 4°C. The plate was washed twice with washing buffer (DPBS, 0.05% Tween 20), and 200 μL of blocking buffer (DPBS, 0.05% Tween 20, 5% BSA, 1 mM EDTA) was added and incubated at room temperature for 5 hours. Immediately after removing the blocking buffer, 100 μL of the a-SQ-modified antibody and a-SQ prepared in 2-2-5. at concentrations ranging from 0.03 to 30 μg / mL were added to each well and incubated at room temperature for 2 hours. After washing four times with washing buffer, 100 μL of PNPaSQ (5 mM, DPBS) was added to each well, and the optical density of each solution was measured after 30 minutes using an infinite 200PRO M Plex plate reader (Tecan Group Ltd.).

[0167]

[0168] (Cell ELISA) A549 cells (human alveolar basal epithelial adenocarcinoma cells) (CD44+) were used as the target for CD44 detection, and HepG2 cells (human hepatoma-derived cells) (CD44-) served as the negative control. Each cell was washed with DPBS (2 x 2 mL), and Trypsin EDTA (1 mL) was added. The cells were then cultured at 37°C and 5% CO2 for 5-10 minutes to detach the cells. After washing twice with serum-free DMEM medium (5% FBS), 1.0 x 10 cells were cultured. 5 The cells were suspended in serum-containing medium (5% FBS) at a concentration of 100 cells / 100 μL. 100 μL of the cell suspension was dispensed into a 96-well plate and cultured overnight at 37°C under 5% CO2 conditions to allow adhesion.

[0169] After confirming cell adhesion under a microscope, the supernatant was removed. Immediately, DMEM solution (2% FBS, 1-10 μg / ml) for primary antibody staining was added and the plate was incubated at 4°C for 30 minutes. After washing four times with DMEM solution (1% FBS), DMEM solution (2% FBS, 1-10 μg / ml) for a-SQ-modified antibody staining was immediately added and the plate was incubated at 4°C for 30 minutes. After washing five times with DMEM solution (1% FBS), the substrate PNPSQ (1 mM, DPBS) was added and the plate was incubated at 37°C for 5 hours for the enzyme reaction. The optical density of each solution was measured using an infinite 200PRO M Plex plate reader (Tecan Group Ltd.).

[0170]

[0171] [Results] (ELISA) The a-SQ-modified antibody exhibited higher enzyme activity than a-SQ alone (Figure 15), suggesting that a-SQ was able to modify the antibody while maintaining its antigen-recognition ability.

[0172] (Cell ELISA) The results of detecting the membrane protein CD44 by Cell-ELISA are shown (Figure 16). No signal was detected in HepG2 (CD44-). On the other hand, a large signal was detected in A-549 (CD44+) under certain conditions using both the primary antibody and the enzyme-conjugated secondary antibody, demonstrating successful detection of CD44.

[0173] 6. Galacturonidase 6.1 Selection of galacturonidase The results of a literature survey are shown in the table below (Table 11). The literature survey revealed that there are currently 10 types of β-GalUAse. These enzymes are derived from bacteria of the Lachnospiraceae family. Among them, one enzyme has high activity under mild conditions (neutral pH, room temperature to 37°C, kcat / KM, an index of activity, is 1.0 x 10 4 (s -1 ・M -1 ) or more) and whose crystal structures have been reported, two types of β-GalUAse were selected: one derived from Eisenbergiella tayi, a bacterium present in human blood (EtGalUAse), and one derived from Faecalibacterium sp., a bacterium present in the human large intestine, which has high activity under mild conditions but whose crystal structure has not been reported (Table 12).

[0174]

[0175]

[0176] 6.2 Preparation of Galacturonidases These enzymes were β-D-galacturonidase (EtGalUAse) derived from the human blood bacterium Eisenbergiella tayi and β-D-galacturonidase (FcGalUAse) derived from the large intestine bacterium Faecalibacterium sp. Expression of EtGalUAse was attempted with a (His)6 purification tag at the N-terminus and the SSGVDLGTENLYFQSNA sequence inserted to allow cleavage with TEV protease after purification. Expression of FcGalUAse was attempted with a (His)6 purification tag inserted at the N-terminus. The amino acid sequences are shown in SEQ ID NO: 13 or 14 in the Sequence Listing. The SSGVDLGTENLYFQSNA sequence inserted into the EtGalUAse sequence corresponds to amino acids 8 to 24 of SEQ ID NO: 13.

[0177] EtGalUAse and FcGalUAse were subjected to His-tag affinity chromatography and then analyzed by SDS-PAGE (Fig. 17a, b).

[0178] 6.3 Galacturonidase reactivity The Lineweaver-Burk plots of EtGalUAse and FcGalUAse obtained from the experiment are shown in Figure 18a and b. The KM, kcat, and kcat / KM of EtGalUAse are 2.6 × 10 2 μM, 10 s -1 , 4.0×10 4 s -1 M -1 The KM, kcat, and kcat / KM of FcGalUAse were calculated to be 3.6 × 10 2 μM, 23 s -1 , 6.5×10 4 s -1 M -1 This was calculated as kcat / KM = 1.0 × 10 4 This was a sufficient activity value for use in highly sensitive protein detection.

[0179] 6.4 Cross-reactivity of galacturonidase [Experiment] EtGalUAse (final concentration: 1.3 nM) and FcGalUAse (final concentration: 1.6 nM) dissolved in Dulbecco's Phosphate-Buffered Saline (DPBS) were mixed with pNP-type substrates (pNP-β-GalUA, p-Nitrophenyl α-D-galactopyranoside, p-Nitrophenyl β-D-galactopyranoside, p-Nitrophenyl α-D-galacturonide, p-Nitrophenyl α-L-fucopyranoside, p-Nitrophenyl β-D-fucopyranoside, p-Nitrophenyl α-L-arabinofuranoside, p-Nitrophenyl β-L-arabinofuranoside, p-Nitrophenyl α-D-glucoside, p-Nitrophenyl β-D-glucoside, p-Nitrophenyl α-D-glucuronide, p-Nitrophenyl β-D-glucuronide, p-Nitrophenyl α-D-mannopyranoside, p-Nitrophenyl β-D-mannnopyranoside, p-Nitrophenyl α-L-rhamnopyranoside, p-Nitrophenyl β-D-xylopyranoside, p-Nitrophenyl sulfoquinovoside, or p-Nitrophenylphospate (final concentration: 1 mM) were added, and after 18 hours, the optical density of each solution at 410 nM was measured using a plate reader. The reaction volume of the substrate was calculated using a previously prepared p-Nitrophenol standard curve and compared with that of pNP-β-GalUA.

[0180] [Results] The reactivity of EtGalUAse and FcGalUAse with 18 different substrates is shown in Figure 19a and b. Both EtGalUAse and FcGalUAse showed high reactivity with β-GalUA, but little reactivity with the other 17 substrates. Therefore, EtGalUAse and FcGalUAse hardly cross-react with substrates that may be used simultaneously, demonstrating their high substrate specificity.

[0181] 6.5 Nonspecific Adsorption of Galacturonidase [Experiment] To evaluate the nonspecific adsorption of EtGalUAse and FcGalUAse to cell surfaces, HeLa cells (human cervical cancer cells) were used as human adherent cells, and JY25 cells (human Epstein-Barr virus-immortalized B cells) were used as human suspension cells. β-D-galactosidase (EcGal) from E. coli was used as a negative control. pNP-β-D-garactopyranoside and pNP-β-GalUA were used as substrates for each enzyme.

[0182] HeLa cells and JY25 cells were washed three times with serum-free DMEM or RPMI1640 at 150 g for 3 minutes at 4°C. After washing, 1.0 × 10 6 The cells were suspended in serum-free medium at a concentration of 100 μL of cells / 100 μL. 100 μL of the cell suspension was added to an Eppendorf tube and centrifuged at 1000 g for 4 minutes at 4°C. The supernatant was removed. The precipitated cells were suspended in 100 μL of enzyme (0, 2.5, 5.0, 7.5, 10 μg / mL) and incubated for 60 minutes at 4°C. After incubation, the cells were centrifuged at 1000 g for 4 minutes at 4°C and washed three times with HBSS. After washing, the supernatant was removed and 400 μL of 1 mM substrate was added and suspended. After suspension, 100 μL of the solution was transferred to a 96-well plate and the optical density at 410 nm was measured.

[0183] [Results] The results of the evaluation of nonspecific adsorption of EcGal, EtGalUAse, and FcGalUAse to human cells are shown in Figure 20a and b. EtGalUAse and FcGalUAse, like the negative control EcGal, showed almost no nonspecific adsorption to HeLa cells and JY25 cells. This suggests that EtGalUAse and FcGalUAse are unlikely to adsorb nonspecifically to human cells.

[0184] 7. Xylosidase 7.1 Selection of Xylosidase β-xylosidase was selected using the following procedure. 900 types of β-xylosidases were registered in the database. First, commercially available β-xylosidases derived from Geobacillus thermodenitrificans and Bacillus pumilus were evaluated. The former showed significantly lower activity, and the latter showed cross-reactivity with α-araf. Therefore, a β-xylosidase derived from Geobacillus sp. was selected as it had high activity and did not cross-react with α-araf. After genetic modification, it was expressed in E. coli and purified (Figure 21).

[0185] 7.2 Construction of Xylosidase A β-xyl expression plasmid was constructed by inserting the gene into the pET22 cloning site. The β-xyl expression plasmid was transformed into E. coli BL21 and placed on agar medium containing 100 mg / L ampicillin. After transformation, the transformed cells were inoculated into 40 mL of Luria Bertani medium containing 100 mg / L ampicillin and grown overnight with shaking at 180 rpm at 37°C. The culture was then transferred to 1 L of Terrific Broth medium containing 100 mg / L ampicillin. The culture was grown with shaking at 180 rpm at 37°C until the OD600 reached 0.8. The culture was cooled to 27°C, supplemented with IPTG to a final concentration of 0.5 mM, and incubated with shaking for 48 hours. After harvesting, the cells were purified using a HisTrap FF column (Cytiva) at 4°C. Here, a 20 mM Tris-HCl / 0.5 M NaCl / 0.5 M imidazole (pH 7.4) solution was used. The fractions were then analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 21). After chromatography, the purification was completed by ultrafiltration using DPBS, and the concentration was determined based on the absorbance at 280 nm.

[0186] 7.3 Xylosidase Reactivity [Experiment] The activity of β-xylase toward pNP β-D-xylopyranoside or 4-methylumbelliferone β-D-xylopyranoside was measured. The substrate was reacted with β-xylase in DPBS (pH 7.4) at 37°C, and the reaction rate was calculated by measuring the absorbance of pNP (405 nm) or the fluorescence of MU (ex. 360 nm, em. 450 nm). Measurements were performed within a time period where the reaction rate was considered constant at sufficiently low enzyme concentrations. The rate constant was calculated from a Lineweaver-Burk plot.

[0187] [Results] The activity of β-xyl toward the fluorescent substrate 4-methylumbelliferone (MU) was measured in DPBS buffer (pH 7.4) at 37°C (Table 13). As a result, the Km / kcat value was comparable to that of conventional sensitized enzymes.

[0188]

[0189] 7.4 Cross-reactivity of xylosidases [Experiment] β-xyl (20 nM or less) was reacted with pNP substrates (0.1 mM) of α-araf, α-galUA, α-gluUA, α-sq, α-rham, β-galUA, and β-glu at 37°C in DPBS at pH 7.4 for 18 hours. The absorbance at 405 nm was then measured, and the relative reaction rates were calculated by setting the intrinsic reaction rates of the other pNP substrates at 1.

[0190] [Results] The relative reaction rates for each substrate were calculated, with the reaction for the original substrate being set at 1. As a result, β-xyl did not cross-react with the substrates for the other enzymes (Figure 10-2).

[0191] 7.5 Nonspecific adsorption of xylosidase to cells [Experiment] 1.0 x 10 6 100 μL of β-xyl was added to each JY25 cell at a concentration of 0.1–10 μg / mL, and the cells were incubated at room temperature for 1 hour. After incubation, the cells were washed three times with DPBS. MU substrate was added to the cells at a final concentration of 0.1 mM at 37°C for 1 hour. Fluorescence intensity at 450 nm was then measured.

[0192] [Results] Conventional bioanalytical sensitizing enzymes such as HRP, ALP, and β-gal adsorb nonspecifically to cell surfaces to some extent, which hinders the detection of proteins expressed at low levels on cells. Therefore, we evaluated the nonspecific adsorption of a new enzyme to the cell surface. 1 × 10 JY25 cells, a human B cell line, were used. 6 Each enzyme was added to the column at a concentration of 0.1-10 μg / mL and allowed to stand at 4°C for 1 hour. After three washes, 0.1 mM MU-type substrate was added and the reaction was allowed to proceed at 37°C for 1 hour. The results showed that β-xyl was less adsorbed than β-gal (Figure 11).

[0193] 8. Three-Color Cell ELISA [Experiment] SK-BR-3 cells (human breast cancer cells) were used to detect EGFR, EpCAM, and HER2. An enzyme-antibody conjugate was prepared by premixing antibody-biotin (5 μg / mL), SA (25 μg / mL), and enzyme-biotin (5 μg / mL) to a final concentration of 5 μg / mL. Biotin solution was added to the enzyme-antibody conjugate solution to a final concentration of 0.1 mM to block any remaining SA binding sites. After washing twice, the cells were resuspended at 1.0 × 106 cells / 100 μL. After centrifugation, the supernatant was removed, and 100 μL of pre-prepared enzyme-antibody conjugate was added simultaneously. The cells were then incubated at 4°C for 1 hour. After washing three times, 300 μL of substrate (0.1 mM final concentration) was added simultaneously and resuspended. 100 μL of each solution was plated onto a 96-well plate and incubated at 27°C. The changes in absorbance or fluorescence intensity of each solution were measured over time using an Infinite 200 PRO. The results are expressed as the mean ± standard deviation of triplicate experiments.

[0194]

[0195] [Results] Multicolor cell ELISA was performed targeting HER2, EpCAM, and the very low-expression EGFR. Detection was attempted using EcGal and FDG for EpCAM, α-rham and resorufin α-L-rhamnopyranoside (RER) for HER2, and CtAbf and MUA for EGFR. The results are shown in Figure 22. No signal was detected without antibody addition for any of the targets. However, a signal was detected with antibody addition, demonstrating successful simultaneous detection of EpCAM, HER2, and EGFR.

[0196] SEQ ID NO:1 α-arabinofuranosidase SEQ ID NO:2 α-rhamnosidase, Sphingomonas paucimobilis SEQ ID NO:3 β-xylosidase SEQ ID NO:4 α-sulfoquinovosidase SEQ ID NO:5 α-galacturonidase SEQ ID NO:6 β-galacturonidase, Eisenbergiella tayi SEQ ID NO:7 α-glucuronidase SEQ ID NO:8 β-glucosidase SEQ ID NO:9 α-rhamnosidase, Streptomyces avermitilis SEQ ID NO:10 PCR forward primer SEQ ID NO:11 PCR reverse primer SEQ ID NO:12 β-galacturonidase, Faecalibacterium sp. SEQ ID NO:13 Tagged EtGalUAse SEQ ID NO:14 Tagged FcGalUAse

Claims

1. A method for analyzing a target using a labeled target-binding substance, comprising: At least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, or At least one enzyme selected from the group consisting of these and β-glucosidase A method of analyzing a target using the method.

2. The analytical method according to claim 1 , which is carried out on a cell, tissue, or individual of a mammalian animal.

3. The method of claim 1 , wherein the target binding substance is an antibody.

4. The analytical method according to claim 3, wherein the presence or amount of a target is detected by fluorescence derived from a substrate degradation product produced by an enzymatic reaction, and a multicolor analysis is performed using two or more target-binding substances each labeled with two or more enzymes selected from the group.

5. For use in enzyme-sensitized methods, An enzyme preparation comprising at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase; or A combination of an enzyme preparation containing at least one enzyme selected from the group consisting of these and an enzyme preparation containing β-glucosidase.

6. A conjugate of at least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase with a target-binding substance; or A combination of a conjugate of at least one enzyme selected from the group consisting of these and a target-binding substance, and a conjugate of β-glucosidase and a target-binding substance.

7. The conjugate or combination according to claim 6, wherein the target binding substance is an antibody.

8. A kit for detecting a target substance, comprising: At least one enzyme selected from the group consisting of α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and α-glucuronidase, or A combination of at least one enzyme selected from the group consisting of these and β-glucosidase.

9. The kit according to claim 8 for ELISA.

10. The method of analysis according to any one of claims 1 to 4, the enzyme preparation or combination of enzyme preparations according to claim 5, the conjugate or combination of conjugates according to claim 6 or 7, or the kit according to claim 8 or 9, wherein α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, and β-glucosidase are selected from the following: (1) α-arabinofuranosidase derived from Clostridium thermocellum, (2) α-rhamnosidase derived from Sphingomonas paucimobilis or Streptomyces avermitilis, (3) β-xylosidase derived from Geobacillus genus, (4) α-sulfoquinovosidase derived from Escherichia coli, (5) α-galacturonidase derived from Bacillus subtilis, (6) β-galacturonidase derived from Eisenbergiella tayi or Faecalibacterium sp., (7) α-glucuronidase from Thermotoga Maritima, (8) β-glucosidase derived from Terrabacter Ginsenosidimutans.

11. The method according to any one of claims 1 to 4, the enzyme preparation or combination of enzyme preparations according to claim 5, the conjugate or combination of conjugates according to claim 6 or 7, or the kit according to claim 8 or 9, wherein α-arabinofuranosidase, α-rhamnosidase, β-xylosidase, α-sulfoquinovosidase, α-galacturonidase, β-galacturonidase, β-glucosidase are selected from the following: (1) α-arabinofuranosidase: (1a) a protein consisting of the amino acid sequence set forth in SEQ ID NO:1 or a multimer thereof; (1b) A protein or a multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO:1 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has α-arabinofuranosidase activity; (1c) a protein or a multimer thereof having an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:1 and having α-arabinofuranosidase activity; (2) α-Rhamnosidase: (2a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 9, or a multimer thereof; (2b) a protein or a multimer thereof consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2 or 9, and having α-rhamnosidase activity; (2c) a protein or a multimer thereof having an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 2 or 9 and having α-rhamnosidase activity; (3) β-xylosidase: (3a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 or a multimer thereof; (3b) a protein or a multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 3 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has β-xylosidase activity; (3c) a protein or a multimer thereof having an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 3 and having β-xylosidase activity; (4) α-Sulfoquinovosidase: (4a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 or a multimer thereof; (4b) A protein or a multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has α-sulfoquinovosidase activity; (4c) A protein or a multimer thereof having an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 4 and having α-sulfoquinovosidase activity. (5) α-galacturonidase: (5a) a protein consisting of the amino acid sequence set forth in SEQ ID NO:5 or a multimer thereof; (5b) A protein or a multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO:5 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has α-galacturonidase activity; (5c) A protein or a multimer thereof having an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:5 and having α-galacturonidase activity. (6) β-galacturonidase: (6a) a protein or a multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 6 or 12; (6b) A protein or a multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 6 or 12 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has β-galacturonidase activity; (6c) A protein or a multimer thereof having an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 6 or 12, and having β-galacturonidase activity. (7) α-glucuronidase: (7a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 7 or a multimer thereof; (7b) A protein or a multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 7 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has α-glucuronidase activity; (7c) A protein or a multimer thereof having an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO: 7 and having α-glucuronidase activity. (8) β-glucosidase: (8a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8 or a multimer thereof; (8b) A protein or a multimer thereof consisting of the amino acid sequence set forth in SEQ ID NO: 8 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has β-glucosidase activity; (8c) A protein or a multimer thereof having an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO:8 and having β-glucosidase activity.