α-1,3-glucan binding polypeptide, α-1,3-glucan detection method, α-1,3-glucan detection kit, recombinant microorganism or cell

Abstract

To provide an α-1, 3-glucan binding polypeptide having no α-1,3-gulucan cleaving activity and excellent in specific binding activity to α-1,3-gulucan.SOLUTION: An α-1, 3-glucan-binding polypeptide consists of an amino acid sequence in which at least one of the three aspartic acids (D) in the following two motif sequences contained in the amino acid sequence W of the polypeptide constituting the catalytic domain (CatD) of wild-type α-1,3-glucanase belonging to the GH87 type enzyme, SEQ ID NO: 11: ADXXN (X represents any amino acid) and SEQ ID NO: 12: RXXGDD (X represents any amino acid), is replaced with another amino acid, and the sequence identity with the amino acid sequence W is 80% or more.SELECTED DRAWING: Figure 2A

Description

Technical Field

[0001] The present invention relates to an α-1,3-glucan-binding polypeptide that does not exhibit α-1,3-glucan-degrading activity and has specific binding activity to α-1,3-glucan, a method for detecting α-1,3-glucan using this α-1,3-glucan-binding polypeptide, an α-1,3-glucan detection kit, a recombinant microorganism or cell.

Background Art

[0002] α-1,3-glucan is a polysaccharide in which glucose is linked by α-1,3-bonds. Generally, it is known as a water-insoluble polymer and exists as a polysaccharide constituting the cell wall of fungi or as an exopolysaccharide of bacteria. In recent years, commercial utilization methods of α-1,3-glucan and its derivatives have also been studied.

[0003] In addition, bacteria of the genus Streptococcus present in the oral cavity of mammals synthesize α-1,3-glucan outside the cells using sucrose as a raw material. In the oral cavity, α-1,3-glucan is widely recognized as an insoluble polymer constituting an adhesive polysaccharide (mutan).

[0004] In addition, α-1,3-glucan is also known as a polysaccharide constituting the cell wall of fungi such as mushrooms, molds, and yeasts. In particular, in fungi that infect plants, α-1,3-glucan plays a role in inhibiting the activation of the host immune system and plays an important role during infection.

[0005] In addition, for example, it is known that α-1,3-glucan is present in the extracellular polysaccharides of Aspergillus fungi (Non-Patent Document 1). Since α-1,3-glucan is a polysaccharide not synthesized by humans, it may be available as a biomarker for deep mycosis. Furthermore, among pathogenic fungi that infect humans, for the causative fungi of histoplasmosis, paracoccidioidomycosis, coccidioidomycosis, blastomycosis, as well as fungi of the genus Cryptococcus, Trichosporon, and Schizophyllum (Suehirotake), it is said that α-1,3-glucan is present in the cell wall.

[0006] In addition, in order to provide a kit for detecting and quantifying α-1,3-glucan contained in an unpurified sample, it is required that the reagent contains a molecule that can specifically bind to α-1,3-glucan. So far, as molecules that bind to α-1,3-glucan, for example, antibodies (mouse IgM: MOPC104e) (for example, Non-Patent Document 2), lectins (banana-derived BanLec) (for example, Non-Patent Document 3), carbohydrate-binding modules (CBM6) (for example, Non-Patent Document 4), etc. have been reported. Some of these molecules can be used to detect the cell wall α-1,3-glucan of fungi.

[0007] In addition, attempts have also been made to detect α-1,3-glucan using the α-1,3-glucan-binding module of some α-1,3-glucanases (EC 3.2.1.59). α-1,3-glucanase (EC 3.2.1.59) can be divided into GH71-type enzymes and GH87-type enzymes according to the glycoside hydrolase (GH) classification. Enzymes belonging to either type have a catalytic domain (hereinafter sometimes referred to as "CatD") with α-1,3-glucan-degrading activity, and there are also many enzymes that have a carbohydrate-binding module (CBM) with binding activity to α-1,3-glucan.

[0008] In α-1,3-glucanase (EC 3.2.1.59), the region containing the carbohydrate binding module (CBM) is responsible for α-1,3-glucan binding activity, and the catalytic domain is recognized as having no α-1,3-glucan binding activity. A method using CBM for the detection of α-1,3-glucan has been developed. Specifically, for example, α-1,3-glucanase CBM (AglKA-DS1CB6DS2, corresponding to SEQ ID NO: 14) with a fluorescent molecule fused and expressed is known to be available for the detection of α-1,3-glucan present in the cell wall of Aspergillus cells (Non-Patent Document 5).

[0009] Technology

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0011] However, the conventional molecules as described above also have binding properties with polysaccharides having structures other than α-1,3-glucan. Therefore, in the presence of a large amount of contaminants, it is not suitable for accurately detecting only α-1,3-glucan. For example, it is known that the CBM of α-1,3-glucanase also binds to polysaccharides such as xylan, and there is a problem of low specific binding affinity to α-1,3-glucan. That is, in order to detect and quantify trace amounts of α-1,3-glucan contained in an unpurified measurement sample with high sensitivity regardless of its solubility, it is required to have excellent specific binding activity to α-1,3-glucan compared to conventional molecules.

[0012] An object of the present invention is to provide an α-1,3-glucan-binding polypeptide (α-1,3-glucanase mutant) that does not have α-1,3-glucan cleavage activity and has excellent specific binding activity to α-1,3-glucan, and a method for detecting α-1,3-glucan, an α-1,3-glucan detection kit, a recombinant microorganism or cell using this α-1,3-glucan-binding polypeptide.

Means for Solving the Problems

[0013] In order to solve the above problems, the following α-1,3-glucan-binding polypeptide, method for detecting α-1,3-glucan, α-1,3-glucan detection kit, recombinant microorganism or cell are provided. [1] It does not show α-1,3-glucan-degrading activity and has specific binding activity with α-1,3-glucan, The following two motif sequences contained in the amino acid sequence W of the polypeptide constituting the catalytic domain (CatD) of wild-type α-1,3-glucanase belonging to the GH87 type enzyme, SEQ ID NO: 11: ADXXN (X represents any amino acid), and, SEQ ID NO: 12: RXXGDD (X represents any amino acid) At least one of the three aspartic acids (D) is substituted with another amino acid, and it has 80% or more sequence identity with the amino acid sequence W, an amino acid sequence containing α-1,3-glucan-binding polypeptide. [2] The other amino acids are one to three of methionine (M), isoleucine (I), leucine (L), histidine (H), phenylalanine (F), valine (V), tryptophan (W), asparagine (N), alanine (A), glycine (G), glutamine (Q), serine (S), proline (P), threonine (T), lysine (K), The α-1,3-glucan-binding polypeptide of [1] above. [3] The amino acid sequence W is any one of the amino acid sequences represented by SEQ ID NOs: 1 to 10, The α-1,3-glucan-binding polypeptide of [1] above. [4] A method for detecting α-1,3-glucan, comprising a step of contacting a test sample with a reagent containing the α-1,3-glucan-binding polypeptide according to any one of [1] to [3]. Method for detecting α-1,3-glucan. [5] The α-1,3-glucan is a polymer composed of three or more glucoses, The method for detecting α-1,3-glucan of [4] above. [6] A reagent containing the α-1,3-glucan-binding polypeptide according to any one of [1] to [3], α-1,3-glucan detection kit. [7] A recombinant microorganism or cell containing a nucleotide sequence encoding the α-1,3-glucan-binding polypeptide according to any one of [1] to [3].

Advantages of the Invention

[0014] The α-1,3-glucan-binding polypeptide of the present invention has no activity to cleave α-1,3-glucan and has excellent specific binding activity to α-1,3-glucan. Further, according to the method for detecting α-1,3-glucan and the α-1,3-glucan detection kit of the present invention, α-1,3-glucan can be accurately detected even in the presence of contaminants, for example. Furthermore, according to the recombinant microorganism or cell of the present invention, the α-1,3-glucan-binding polypeptide of the present invention can be produced.

Brief Description of Drawings

[0015]

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[0016] The inventors of the present invention focused on the catalytic domain (CatD) of α-1,3-glucanase (EC 3.2.1.59), which has a role in decomposing α-1,3-glucan and does not have a binding force to α-1,3-glucan, rather than the CBM that has been shown to have the binding activity of α-1,3-glucan and has been used for the detection of α-1,3-glucan. When the amino acid sequence of this catalytic domain (CatD) was mutated to a sequence different from the wild type, although it is difficult in GH71-type enzymes, on the other hand, in GH87-type enzymes, it was newly found that an α-1,3-glucan-binding polypeptide (α-1,3-glucanase mutant) that loses the decomposition activity against α-1,3-glucan and has a strong α-1,3-glucan-binding activity can be obtained. Furthermore, the characteristics of these α-1,3-glucan-binding polypeptides were analyzed, and by applying this α-1,3-glucan-binding polypeptide, it was found that α-1,3-glucan can be accurately detected with a simple operation regardless of whether the test sample contains impurities or is a purified sample, and regardless of the properties such as soluble or insoluble. The present invention has been completed based on these findings.

[0017] (α-1,3-glucan-binding polypeptide) Hereinafter, one embodiment of the α-1,3-glucan-binding polypeptide of the present invention will be described.

[0018] The α-1,3-glucan-binding polypeptide of the present invention does not exhibit α-1,3-glucan-decomposing activity and has specific binding activity with α-1,3-glucan.

[0019] The α-1,3-glucan-binding polypeptide of the present invention includes a variant in which a predetermined position of the amino acid sequence of the polypeptide constituting the catalytic domain (CatD) of wild-type α-1,3-glucanase belonging to the GH87 type enzyme (hereinafter sometimes referred to as "amino acid sequence W") is substituted with another amino acid.

[0020] The wild-type α-1,3-glucanase (EC 3.2.1.59) belonging to the GH87 type enzyme is not particularly limited in its origin, and may be, for example, α-1,3-glucanase derived from bacteria such as the genus Niallia, the genus Paenibacillus, the genus Paenibacillus, and the genus Streptomyces. More specifically, for example, it may be derived from Niallia circulans, Paenibacillus curdlanolyticus, Paenibacillus glycanilyticus, Paenibacillus humicus, Paenibacillus sp., Streptomyces thermodiastaticus. The amino acid sequences of these α-1,3-glucanases are known, for example, as GenBank Accession numbers; BAE98302.1, ADT91063.1, BAP10900.2, BAP10901.1, BAI23187.1, BAG15879.1, BAH10514.1, BAG15878.1, BAF56208.1, BCK59654.1, etc.

[0021] SEQ ID NOs: 1 to 10 (FIGS. 11 to 14) are an example of the amino acid sequence W of the polypeptide constituting the catalytic domain (CatD) of wild-type α-1,3-glucanase belonging to the GH87 type enzyme.

[0022] As exemplified in SEQ ID NOs: 1 to 10, in the amino acid sequence W of wild-type α-1,3-glucanases with different origins, two motif sequences represented by ADXXN (SEQ ID NO: 11) and RXXGDD (SEQ ID NO: 12) are commonly included at the corresponding positions of each amino acid sequence W. Here, "X" in the motif sequences represented by SEQ ID NOs: 11 and 12 represents any amino acid.

[0023] And, in the α-1,3-glucan-binding polypeptide of the present invention, at least any one of the three aspartic acids (D) in the motif sequences (ADXXN (SEQ ID NO: 11), RXXGDD (SEQ ID NO: 12)) contained in the amino acid sequence W is substituted with another amino acid. In other words, the aspartic acid (D) contained in SEQ ID NO: 11 is D 1 and the two aspartic acids (D) contained in SEQ ID NO: 12 are D from the N-terminal side 2 and D 3 when taken as 1 and D 2 and D 3 one, two, or three of them are substituted with other amino acids. In the case of an embodiment in which a plurality of aspartic acids (D) are substituted with other amino acids, the other amino acids to be substituted may be the same or different.

[0024] The amino acids substituted for the aspartic acid (D) in the motif sequences represented by SEQ ID NOs: 11 and 12 are not particularly limited, and examples thereof include one, two, or three of methionine (M), isoleucine (I), leucine (L), histidine (H), phenylalanine (F), valine (V), tryptophan (W), asparagine (N), alanine (A), glycine (G), glutamine (Q), serine (S), proline (P), threonine (T), lysine (K), and the like. Among them, the amino acids substituted for the aspartic acid (D) are preferably methionine (M), isoleucine (I), leucine (L), serine (S), histidine (H), phenylalanine (F), asparagine (N), alanine (A), and threonine (T). When the amino acid to be substituted is one of these amino acids, the resulting polypeptide (α-1,3-glucanase mutant) does not have the cleavage activity of α-1,3-glucan and has excellent specific binding activity to α-1,3-glucan.

[0025] Furthermore, taking the amino acid sequence of wild-type α-1,3-glucanase (Niallia circulans) represented by SEQ ID NO: 13 as an example, the amino acid substitution positions in one form of the α-1,3-glucan-binding polypeptide of the present invention will be described. SEQ ID NO: 13 is the full-length amino acid sequence of wild-type α-1,3-glucanase, which includes the amino acid sequence of CatD represented by SEQ ID NO: 1 and the amino acid sequence of CBM represented by SEQ ID NO: 14.

[0026] In one form of the α-1,3-glucan-binding polypeptide of the present invention, the amino acid substitution positions for the amino acid sequence W of CatD of wild-type α-1,3-glucanase belonging to the GH87 type enzyme are at least any one of the aspartic acids (D) at positions 1067, 1090, and 1091 of the full-length amino acid sequence represented by SEQ ID NO: 13. That is, the α-1,3-glucan-binding polypeptide of the present invention can also be described as having at least any one of the amino acids (aspartic acid (D)) at positions corresponding to positions 1067, 1090, and 1091 of the amino acid sequence shown in SEQ ID NO: 13 substituted in the amino acid sequence of CatD. Here, in the present invention, the "amino acid at the corresponding position" refers to the amino acid residue at the position corresponding to a specific position in the reference sequence when aligned with the reference amino acid sequence (for example, the reference sequence of SEQ ID NO: 1) using sequence analysis software such as BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0027] Therefore, the α-1,3-glucan-binding polypeptide of the present invention can also be described as having the amino acids at positions corresponding to positions 1067, 1090, and 1091 of the amino acid sequence of SEQ ID NO: 13 substituted with different amino acids as appropriate in the amino acid sequences shown in SEQ ID NOs: 2 to 10, for example.

[0028] Furthermore, the α-1,3-glucan-binding polypeptide of the present invention includes an amino acid sequence (hereinafter sometimes referred to as "amino acid sequence X") having 80% or more sequence identity with amino acid sequence W. That is, the amino acid sequence of the α-1,3-glucan-binding polypeptide of the present invention may have substitution, deletion, insertion, addition, or a combination thereof of one or more amino acid residues, except at the substitution positions (aspartic acid (D)) in the motif sequences of SEQ ID NOs: 11 and 12. When the α-1,3-glucan-binding polypeptide of the present invention includes substitution, deletion, or insertion of amino acid residues, except at the substitution positions (aspartic acid (D)) in the motif sequences of SEQ ID NOs: 11 and 12, the amino acid sequence X may have 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more sequence identity with amino acid sequence W (for example, SEQ ID NOs: 1-10). Also, as described above, the amino acid sequence of the α-1,3-glucan-binding polypeptide of the present invention only needs to include an amino acid sequence X in which the aspartic acid (D) in the motif sequences of SEQ ID NOs: 11 and 12 contained in amino acid sequence W is substituted with another amino acid and which has 80% or more sequence identity with amino acid sequence W. As long as the function of the α-1,3-glucan-binding polypeptide of the present invention is not impaired, forms in which an amino acid sequence of an appropriate length is added to this amino acid sequence X are also included. Specifically, for example, the α-1,3-glucan-binding polypeptide of the present invention may have an amino acid sequence related to a heterologous protein such as a functional tag (for example, luciferase or alkaline phosphatase) added to the amino acid sequence X.

[0029] More specifically, in the amino acid sequence of the α-1,3-glucan-binding polypeptide of the present invention, when amino acid residues are substituted, deleted, inserted, and added at positions other than the three aspartic acids (D) of the motif sequences of SEQ ID NOs: 11 and 12 with respect to the amino acid sequence W, the number of the amino acid residues is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. Further, in the α-1,3-glucan-binding polypeptide of the present invention, for example, when an amino acid sequence related to a heterologous protein is added to the end of the amino acid sequence X, the added amino acid residues may be, for example, 5 to 100, 5 to 1000, or more.

[0030] In one form of the α-1,3-glucan-binding polypeptide of the present invention, for example, the amino acid sequence is represented by SEQ ID NOs: 15 to 21.

[0031] The polypeptide represented by SEQ ID NOs: 15 to 21 is an example in which aspartic acid (D) in the motif sequences of SEQ ID NOs: 11 and 12 is substituted with asparagine (N) or alanine (A) and has no other mutations (substitution, deletion, insertion, addition of amino acid residues).

[0032] The α-1,3-glucan-binding polypeptide of the present invention is a soluble molecule. Further, the α-1,3-glucan-binding polypeptide of the present invention specifically binds to a region containing three or more glucose polymers in α-1,3-glucan. The α-1,3-glucan-binding polypeptide of the present invention, for example, when measuring the affinity for α-1,3-glucan by the Bio-layer interferometry (BLI) method, binds to α-1,3-glucan with a dissociation constant (KD) of 1.0×10 -6 M or less, 1.0×10 -7 M or less, 1.0×10 -8 M or less, or 1.0×10 -9 M or less.

[0033] The method for producing the α-1,3-glucan-binding polypeptide of the present invention is not particularly limited. The α-1,3-glucan-binding polypeptide of the present invention can be produced by a conventionally known method such as a method using cell engineering techniques or a method of synthesis using a peptide synthesizer.

[0034] The recombinant microorganism or cell of the present invention contains a nucleotide sequence encoding the α-1,3-glucan-binding polypeptide of the present invention. Here, the cell refers to a cell line widely and generally used in cell engineering techniques, and examples include CHO cells, HEK293T cells, and insect cells in the case of mammalian cells. According to the recombinant microorganism or cell of the present invention, the α-1,3-glucan-binding polypeptide of the present invention can be efficiently obtained.

[0035] Since the α-1,3-glucan-binding polypeptide of the present invention does not have α-1,3-glucan cleavage activity and has excellent specific binding activity to α-1,3-glucan, α-1,3-glucan can be detected simply and accurately. In addition, the α-1,3-glucan-binding polypeptide of the present invention can detect α-1,3-glucan regardless of the properties such as the purification purity and solubility / insolubility of α-1,3-glucan.

[0036] (Method for detecting α-1,3-glucan · Kit for detecting α-1,3-glucan) The method for detecting α-1,3-glucan of the present invention includes a step of bringing a test sample into contact with a reagent containing the α-1,3-glucan-binding polypeptide of the present invention. The method for detecting α-1,3-glucan of the present invention includes various forms of detection methods that utilize the specific binding activity of the α-1,3-glucan-binding polypeptide of the present invention.

[0037] In the present invention, the term "detection" is intended to include the concept of "quantification". Specifically, the method for detecting α-1,3-glucan may be a method for measuring the amount of α-1,3-glucan contained in a test sample.

[0038] The α-1,3-glucan to be detected is preferably a polymer composed of three or more glucoses, more preferably a polymer composed of four or more glucoses, and even more preferably a polymer composed of five or more glucoses.

[0039] One form of the method for detecting α-1,3-glucan of the present invention comprises the following steps: (i) a step of contacting a test sample containing α-1,3-glucan with a reagent to obtain a product formed by the test sample and the reagent, and (ii) a step of detecting the product formed in the step (i) and includes.

[0040] The reagent contains the α-1,3-glucan-binding polypeptide of the present invention. Further, the reagent preferably contains a reporter molecule.

[0041] In step (i), the test sample and the reagent are brought into contact. The test sample includes an unpurified test sample, a purified test sample, or a crudely purified test sample. The test sample containing α-1,3-glucan may be soluble or insoluble in water.

[0042] The origin of the α-1,3-glucan contained in the test sample is not particularly limited, and may be, for example, the cell wall, extract, or α-1,3-glucan secreted outside the cell of fungi, basidiomycetes such as mushrooms, bacteria, yeast, lichens, etc. It may be an artificially synthesized α-1,3-glucan. Furthermore, it may be α-1,3-glucan present in environments such as indoors, soil, rivers, seawater, the atmosphere, and outer space.

[0043] Examples of fungi include, for example, in classical classification, Ascomycota, Zygomycota, Ascomycetes, Basidiomycetes, and Deuteromycetes. Specifically, examples of fungi include genera such as Aspergillus, Auricularia, Botrytis, Cochliobolus, Coprinopsis, Cryptococcus, Fomitiporia, Histoplasma, Laccaria, Neurospora, Penicillium, Puccinia, Punctularia, Schizophyllum, Schizosaccharomyces, Sordaria, Tremella, and Trichosporon.

[0044] Examples of bacteria include, for example, bacteria belonging to the genera Streptococcus, Lactobacillus, Leuconostoc, and the like.

[0045] A reporter molecule has a function of presenting an index for detection. Here, the function of the reporter molecule means directly or indirectly bringing about a detectable signal that is visually observed, electrically detected, or recorded by other means, and specifically, for example, changes in luminescence, radiation, color development, aggregation, magnetic and electrical properties are mentioned. Luminescence includes fluorescence and phosphorescence, and typically fluorescence emission is used for detection. When the reporter molecule exhibits luminescence, examples of such molecules include fluorescent dyes, luminescent proteins (such as luciferase and fluorescent proteins, etc.), and proteins that catalyze the luminescence reaction of the molecule. When the reporter molecule exhibits radiation, examples of such molecules include radioactive isotopes. When the reporter molecule exhibits color development, examples of such molecules include chromogenic dyes, or enzymes (such as peroxidase, alkaline phosphatase, galactosidase, etc.) that can amplify the signal by color development. In addition, colored high-molecular-weight colloidal substances, for example, particulate substances such as latex beads, magnetic and paramagnetic beads can also be used. Furthermore, these can also be used in combination with a method of amplifying the signal by interaction with a biosensor.

[0046] In one form of the method for detecting α-1,3-glucan of the present invention, it is preferable that the reporter molecule includes a first split reporter molecule and a second split reporter molecule. The first split reporter molecule and the second split reporter molecule can form an active reporter molecule by forming a pair. Here, "forming a pair" means that the first split reporter molecule and the second split reporter molecule are close to or bonded to each other to form a pair.

[0047] Specifically, the first and second split reporter molecules do not exhibit the function of a reporter molecule when they exist separately, but when these split reporter molecules are close to or bound to each other, they come to exhibit the function of a reporter molecule. The structure of the split reporter molecule is not specifically limited, as long as the first and second split reporter molecules can identify the formed active reporter molecule when they are close to or bound to each other.

[0048] The split reporter molecule may be configured such that when the first and second split reporter molecules are close to or bound to each other, structural complementarity is promoted and an active reporter molecule is formed.

[0049] Also, the reporter molecule is preferably a reporter protein.

[0050] The configuration when the split reporter molecule is a split reporter protein will be described below. For example, when the split reporter protein includes a first split reporter protein and a second split reporter protein, it may be configured such that bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when the first split reporter protein and the second split reporter protein are close to or bound to each other. In this case, the active reporter protein refers to the state in which the first and second split reporter proteins are close to or bound to each other and BRET or FRET occurs. When the reporter protein is one that causes BRET or FRET, it may be configured to bind a fluorescent substance to one or the other of the split reporter proteins.

[0051] Furthermore, the split reporter protein may be configured such that when the first and second split reporter proteins are in proximity or bound, a protein-spliced active reporter protein is formed. This can be achieved by fusing a protein-splicing domain to each of the first and second split reporter proteins. Examples of protein-splicing domains include, for example, combinations such as DnaEn and DnaEc, which are protein-splicing domains derived from the DnaE gene of Synechocystis sp.

[0052] Specific examples of reporter proteins include, for example, enzymes and fluorescent proteins. Examples of enzymes include, for example, luciferase, alkaline phosphatase, horseradish peroxidase, invertase, β-galactosidase and β-glucuronidase, as well as variants of these enzymes. Examples of fluorescent proteins include, for example, green fluorescent protein (GFP) and variants of GFP.

[0053] In one form of the method for detecting α-1,3-glucan of the present invention, the reagent is (1) a first fusion protein comprising a first polypeptide selected from the α-1,3-glucan-binding polypeptides of the present invention and a first split reporter molecule, and (2) a second fusion protein comprising a second polypeptide selected from the α-1,3-glucan-binding polypeptides of the present invention and a second split reporter molecule and includes the first split reporter molecule and the second split reporter molecule can form an active reporter molecule as a pair, in step (i), when α-1,3-glucan binds to the first fusion protein and the second fusion protein, an active reporter molecule is formed by the first split reporter molecule and the second split reporter molecule, In step (ii), the active reporter molecule formed in step (i) is detected.

[0054] In this case, when fusing the α-1,3-glucan-binding polypeptide and the split reporter protein in the first fusion protein and the second fusion protein, it is preferable to insert a linker peptide with an appropriately adjusted length to avoid steric hindrance. The linker peptide consists of a peptide of 1 residue to about 20 residues, and examples of its amino acid sequence include those similar to the amino acid sequences of common linkers used in the production of fusion proteins. Specifically, for example, a GS linker consisting of a repeating sequence containing Gly-Ser (GS), a DDAKK linker consisting of a repeating sequence of DDAKK (SEQ ID NO: 22), and an EAAAK linker consisting of a repeating sequence of EAAAK (SEQ ID NO: 23) can be mentioned. Also, in the first and second fusion proteins, the region where the split reporter protein is fused is not limited to either the N-terminal side or the C-terminal side, and it is preferable to determine the fusion region according to the characteristics of the α-1,3-glucan-binding polypeptide. Also, a fusion protein fused to both the N-terminal side and the C-terminal side may be used.

[0055] In this specification, regarding the split reporter molecule and the α-1,3-glucan-binding polypeptide of the present invention, "fused" includes not only the mode in which the split reporter molecule is directly linked to the α-1,3-glucan-binding polypeptide of the present invention, but also the mode in which the split reporter molecule is arthroscopically linked to the α-1,3-glucan-binding polypeptide of the present invention via another molecule.

[0056] Furthermore, a split reporter molecule can be bound to an antibody (for example, a His-tag antibody) that detects the α-1,3-glucan-binding polypeptide of the present invention and a fusion protein can be used. In this case, examples of the split reporter molecule include luciferase.

[0057] In one aspect, · The first fusion protein is a fusion protein in which a first split reporter molecule is fused to the N-terminal side or the C-terminal side of the first polypeptide, · The second fusion protein is a fusion protein in which a second split reporter molecule is fused to the N-terminal side or the C-terminal side of the second polypeptide.

[0058] In another aspect, · The first fusion protein is a fusion protein in which a first split reporter molecule is fused to the N-terminal side of the first polypeptide, and the second fusion protein is a fusion protein in which a second split reporter molecule is fused to the C-terminal side of the second polypeptide, or Or, · The first fusion protein is a fusion protein in which a first split reporter molecule is fused to the C-terminal side of the first polypeptide, and the second fusion protein is a fusion protein in which a second split reporter molecule is fused to the N-terminal side of the second polypeptide.

[0059] In yet another aspect, · The first fusion protein is a fusion protein in which a first split reporter molecule is fused to the N-terminal side and the C-terminal side of the first polypeptide, and / or · The second fusion protein is a fusion protein in which a second split reporter molecule is fused to the N-terminal side and the C-terminal side of the second polypeptide.

[0060] In step (i), α-1,3-glucan binds to the first fusion protein and the second fusion protein, thereby forming an active reporter molecule by the first split reporter molecule and the second split reporter molecule. In subsequent step (ii), the active reporter molecule formed in step (i) is detected.

[0061] In step (ii), the product formed in step (i) is detected. When the reporter molecule is a reporter protein, the activity of the formed active reporter protein is detected. For example, if the activity of the formed active reporter protein is high as compared to the case in the absence of α-1,3-glucan, it indicates that α-1,3-glucan is present in the test sample. When the reporter molecule is a fluorescent substance, the formed active reporter molecule is irradiated with excitation light, and the emitted fluorescence is detected. For example, if the fluorescence emitted from the fluorescent substance is high as compared to the case in the absence of α-1,3-glucan, it indicates that α-1,3-glucan is present in the test sample.

[0062] As will be described later in the examples, the difference in the activity between the inactivated reporter protein (background) observed in the absence of α-1,3-glucan having the target structure and the activity of the reporter protein reconstituted in the presence of a sufficient amount of α-1,3-glucan shows high sensitivity of about 100-fold or more.

[0063] For the detection in step (ii), the detection means is not particularly limited, and a known method or apparatus can be appropriately selected according to the type and structure of the reporter molecule. Examples of the detection method and detection apparatus include a method of quantitatively analyzing an image detected by a luminescence photometer, a spectroscopic altimeter, a fluorescence photometer, a flow cytometer, a multiplex, chromatography, a CCD camera, etc. with a computer, and a method of identifying and quantitatively analyzing physical contact with a sensor chip.

[0064] In steps (i) and (ii), it can be carried out by directly supplying a reagent containing the first fusion protein and the second fusion protein and a test sample into a test tube, or it can also be carried out using cells expressing these fusion proteins. In this case, the expression of the first fusion protein and the second fusion protein may be transient expression or constitutive expression. That is, stable expression cell lines of the first and second fusion proteins can be used.

[0065] Specifically, the first and second fusion proteins can be exemplified by those produced in, for example, Escherichia coli, yeast, plants, animal cells, and cell-free expression systems, and those produced in large quantities in Escherichia coli are particularly preferred. Also, a crude purification solution such as a cell lysate expressing the first and second fusion proteins may be used, but it is preferable to use a fusion protein that is fused with a general low-molecular-weight peptide tag and purified to high purity by column purification or the like. In this case, examples of the peptide tag include GST tag, Protein A tag, antibody Fc region, polyhistidine tag, V5 tag, Myc tag, SBP tag, Halo tag, Strep tag, and the like. Also, examples of the cells used here include animal cells, insect cells, and plant cells, and established cell lines are easy to use and preferred.

[0066] In addition, in one embodiment of the method for detecting α-1,3-glucan of the present invention, for example, it can be carried out according to known methods such as the direct adsorption method, sandwich method, and competitive method in ELISA using the α-1,3-glucan-binding polypeptide of the present invention. As the labeling substance and carrier, those conventionally known can be appropriately used. Examples of the labeling substance include enzymes (e.g., horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), firefly luciferase, etc.), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), etc.), fluorescent proteins (allophycocyanin (APC), phycoerythrin (R-PE), etc.), radioisotopes such as 125I, magnetic particles, latex particles (e.g., polystyrene, styrene-butadiene copolymer, etc.), metal colloid particles (e.g., gold, silver, copper, iron, platinum, palladium, etc., or mixtures thereof), avidin, biotin, etc. When an enzyme is used as the labeling substance, for example, a substrate such as a chromogenic substrate, fluorescent substrate, or luminescent substrate corresponding to the enzyme is added to the measurement system, and signals such as color development and luminescence generated by the enzyme reaction can be measured using an absorptiometer, luminometer, etc. Further, for example, when ALP is used as the labeling substance, a luminescent substrate such as 3-(4-methoxyspiro(1,2-dioxetane-3,2'-tricyclo[3.3.1.13,7]decane)-4-yl)phenyl phosphate disodium (e.g., trade name AMPPD (registered trademark)) can be used. When biotin or hapten is used as the labeling substance, streptavidin or hapten antibody conjugated with an enzyme, fluorescent substance, chemiluminescent substance, staining substance, or radioactive substance, etc. can be used.

[0067] The α-1,3-glucan detection kit of the present invention has a reagent containing the α-1,3-glucan-binding polypeptide of the present invention.

[0068] The reagent can contain a reporter molecule, a labeling substance, etc.

[0069] Regarding the details of the reagents, fusion proteins, and reporter molecules in the detection kit, they are the same as those described for the detection method.

[0070] In addition, the detection kit can include various materials, devices, etc. for the detection and quantification of α-1,3-glucan.

[0071] According to the method for detecting α-1,3-glucan or the detection kit for α-1,3-glucan of the present invention, α-1,3-glucan contained in a test sample can be detected with high throughput without requiring complicated washing operations, etc. By applying the method for detecting α-1,3-glucan of the present invention, it is expected that α-1,3-glucan derived from various biological species and purification methods and showing various forms such as soluble / insoluble solubility, gel-like and particulate forms can be screened. Also, the detection method of the present invention does not require a washing step, etc. of the test sample, and can simply and accurately detect α-1,3-glucan.

[0072] Also, according to the method for detecting α-1,3-glucan or the detection kit for α-1,3-glucan of the present invention, it is possible to monitor the presence of pathogenic fungi such as Aspergillus in the living environment. That is, by detecting α-1,3-glucan in the living environment, the risk of developing lung infections caused by fungi such as Aspergillus can be predicted.

[0073] Furthermore, as described above, by applying the α-1,3-glucan-binding polypeptide of the present invention to an analysis method such as ELISA, a tool for simply detecting α-1,3-glucan can be provided. Also, by the method for detecting α-1,3-glucan or the detection kit for α-1,3-glucan of the present invention, by quantifying the concentration of α-1,3-glucan contained in the blood, secretion, organ, washing solution obtained by washing an organ or organ (for example, alveolar lavage fluid, oral washing solution, and peritoneal washing solution) or urine of a subject, it is expected to be possible to predict the possibility of developing infectious diseases such as mycosis and bacteriosis, and to observe the in-vivo state before, during, and after the onset of the infectious disease.

[0074] The α-1,3-glucan-binding polypeptide, α-1,3-glucan detection method, α-1,3-glucan detection kit, recombinant microorganism or cell of the present invention are not limited to the above embodiments.

Examples

[0075] Hereinafter, the present invention will be described more specifically together with examples, but the present invention is not limited to these examples at all.

[0076] <1> Materials and methods (1) Materials Bovine serum albumin (BSA), imidazole, Laminarin (β-1,3-D-glucan), mannan from Saccharomyces cerevisiae (α-1,6- / α-1,2-mannan, α-1,3-mannan) were purchased from Sigma-Aldrich. Isolichenan (α-1,3- / α-1,4-glucan) was purchased from ELICITYL. Dextran T10 (α-1,4- / α-1,6-glucan) was from Pharmacia. D-PBS(-) (Dulbecco's phosphate buffered solution), Tween20, Starch (α-1,6- / α-1,4-glucan), curdlan (β-1,3-D-glucan), chitin (poly-β-1,4-N-acetylglucosamine), D-Glucose, Sucrose, Heparin Sodium were obtained from FUJIFILM Wako Pure Chemical Corporation. 0.5 mol / L-EDTA Solution (pH8.0) was purchased from Nacalai Tesque. Pustulan (β-1,6-D-glucan) was purchased from InvivoGen. The disaccharide (Nigerose) in which two molecules of glucose are linked by an α-1,3 bond, the trisaccharide (Nigerotriose) in which three molecules are linked, the tetrasaccharide (Nigerotetraose) in which four molecules are linked, the pentasaccharide (Nigeropentaose) in which five molecules are linked, and Birchwood-derived Xylan (β-1,4-xylan) were purchased from Megazyme. Chitosan Oligosaccharides (poly-β-1,4-glucosamine) were purchased from Tokyo Chemical Industry Co., Ltd. Endo-β-1,3-glucanase (zymolyase 100T) was purchased from Seikagaku Corporation.

[0077] (2) Amplification and purification of the GtfB gene fragment Streptococcus mutans Clarke 1924 NBRC 13955 (NITE Biological Resource Center) was added to 5 mL of brain heart infusion liquid medium and statically cultured overnight at 37°C. The grown cells were washed with D-PBS(-), and genomic DNA was purified using the Maxwell 16 Cell LEV DNA Purification Kit (Promega). Among the whole genome sequence (GenBank: AP019720.1), the DNA sequence encoding the region excluding the signal sequence of GtfB (Protein ID: BBK80291.1), which is Glucosyltransferase-I (EC 2.4.1.5) belonging to GH70, was amplified by PCR, and the DNA fragment was recovered using a commercially available purification kit. When the sequence was confirmed by DNA Sequence, a DNA sequence encoding an amino acid sequence that matches the region excluding the signal sequence (from position 39 to position 1476) of GtfB (NCBI Reference Sequence: WP_111694388.1) of Streptococcus mutans was recovered.

[0078] (3) Plasmid construction The pCold I DNA vector (Takara Bio, GenBank: AB186388.1) uses the nucleotide sequences of 1-300 and 361-4407, and a DNA sequence encoding a GS linker peptide (Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly (SEQ ID NO: 24: GGSGGGSGGSG)) was inserted between 300 and 361. A DNA sequence encoding the amino acid sequence of the catalytic domain AglKA-CatD-WT (SEQ ID NO: 1) of α-1,3-glucanase (EC 3.2.1.59) belonging to GH86 was inserted on the C-terminal side of the GS linker peptide to construct a plasmid for the preparation of AglKA-CatD-WT-His. Similarly, a DNA sequence encoding the amino acid sequence of the region excluding the signal peptide of Glucosyltransferase-I (EC 2.4.1.5) belonging to GH70 (from position 39 to position 1476) was inserted on the C-terminal side of the GS linker peptide to construct a plasmid for the preparation of GtfB-His. In addition, a DNA fragment encoding the HiBiT tag, which is one of the C-terminal subunits (11 amino acid residues) of NanoLuciferase (NanoLuc) derived from Oplophorus gracilirostris, was inserted on the N-terminal side of the GS linker peptide to prepare a pCold I DNA vector, and on the C-terminal side of the GS linker peptide, AglKA-CBM (up to 31-466 of the sequence shown in SEQ ID NO: 12), AglKA-CatD-WT (SEQ ID NO: 1) and its mutants, that is, mutations (Ara or Asn) at positions corresponding to positions 1067, 1090 or 1091 in the amino acid sequence of SEQ ID NO: 12 AglKA-CatD (SEQ ID NOs: 15-17), as well as AglST-CatD-D376A (SEQ ID NO: 20), AglFH1-CatD-D1068A (SEQ ID NO: 21), and the wild-type amino acid sequence AGN1-WT (SEQ ID NO: 25) of α-1,3-glucanase (EC 3.2.1.59) belonging to GH71 and its mutants AGN1-D259N (SEQ ID NO: 26), AGN1-E262Q (SEQ ID NO: 27) were inserted.Similarly, a pCold I DNA vector was prepared by inserting a DNA fragment encoding a SmBiT tag, which is one of the C-terminal subunits of NanoLuc (11 amino acid residues), into the N-terminal side of the GS linker peptide, and a DNA sequence encoding each amino acid sequence of AglKA-CBM (from 31 to 466 of the sequence shown in SEQ ID NO: 14) or AglKA-CatD-D1090A (SEQ ID NO: 16) was inserted into the C-terminal side of the GS linker peptide. Similarly, a pCold I DNA vector was prepared by inserting a DNA sequence encoding an 8-residue peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 28: WSHPQFEK), strep-tag II (ST-II) consisting of Ser-Ala residues (Reference 2)) into the N-terminal side of the GS linker peptide, and a DNA sequence encoding the amino acid sequence of AglKA-CatD-D1090A (SEQ ID NO: 16) was inserted into the C-terminal side of the GS linker peptide. Also, a pCold I DNA vector was prepared by inserting a DNA fragment encoding the N-terminal subunit of NanoLuc (LgBiT, 18 kDa) into the C-terminal side of the GS linker peptide, and a DNA sequence encoding the amino acid sequence of AglKA-CatD-D1090A (SEQ ID NO: 16) was inserted into the N-terminal side of the GS linker peptide. The DNA sequence for the expression of the α-1,3-glucanase-related polypeptide chain was synthesized with codon optimization for E. coli expression. All protein expression plasmid vectors were transformed into E. coli DH5α competent cells (BioDynamics Laboratory), grown, and the plasmid vectors were purified.

[0079] (4) Preparation of Protein The expression vector was transformed into Escherichia coli SHuffle (New England Biolabs) for protein expression, and the cells were cultured at 37 °C in LB medium supplemented with ampicillin. After preculture, the cells were cooled to 15 °C, IPTG (0.1 mM) was added, and the cells were cultured for 24 to 72 hours to induce the expression of the recombinant protein. After washing, the cell suspension supplemented with protease inhibitor was sonicated on ice. The soluble fraction collected by centrifugation was bound to a cobalt column (Takara Bio Inc.) for His-tag protein purification and washed with PBS containing imidazole (15 mM). The protein bound to the column was eluted with PBS containing imidazole (150 mM) and dialyzed to remove imidazole. The protein concentration was measured using a commercially available kit (Bradford method). For confirmation of the expression of the recombinant protein, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a commercially available kit. The protein separated by SDS-PAGE was stained with CBB dye (Quick Blue Staining Solution; BioDynamics Laboratory Inc.), and the band of the molecular size of the theoretical value was confirmed.

[0080] (5) ELISA-like test by direct solid-phase immobilization method of polysaccharide 50 μL of D-PBS(-) (BPBST) containing BSA and Tween 20 was added to a 96-well white ELISA plate coated with Isolichenan (4 μg / mL) or D-PBS(-) for blocking. After washing, AglKA-CBM-HiBiT, AglKA-CatD-WT-HiBiT, AglKA-CatD-D1067A-HiBiT, AglKA-CatD-D1090A-HiBiT, AglKA-CatD-D1091A-HiBiT diluted to 2 μg / mL in BPBST were added respectively and incubated at room temperature. After washing, to evaluate the binding ability of each protein to Isolichenan, the signal derived from the HiBiT tag fused to each protein was detected by adding 25 μL of the LgBiT and NanoLuc substrate mixture (Nano-Glo HiBiT Lytic Detection System, Promega), and the reconstituted luciferase activity was measured using a microplate reader (GloMax, Promega). Also, a 96-well white ELISA plate coated with Isolichenan or Dextran T10 (0 - 4 μg / mL) was blocked with BPBST, and after washing, AglKA-CBM-HiBiT or AglKA-CatD-D1090A-HiBiT (2 μg / mL) was added and incubated at room temperature. After washing, the signal derived from the HiBiT tag was measured in the same procedure as above.

[0081] (6) Preparation of heat-killed Aspergillus cells Conidia (1×10^5) of Aspergillus nidulans NBRC 8003 (NITE Biological Resource Center) were added to 5 mL of potato dextrose liquid medium and cultured with shaking at 37°C and 200 rpm / min for 5 days. The cells were autoclaved (121°C, 20 minutes), 25 mL of D-PBS(-) was added, and the washing operation using a PET mesh (EASYstrainer, Greiner Bio-One) was repeated twice. D-PBS(-) containing BSA and Tween20 (BPBST) was added at 1 mL per 200 mg of the wet weight of the cells, and the mixture with metal beads treated with a bead crusher (FastPrep-24; MP Biomedicals) was used as heat-killed Aspergillus cells. A part of the heat-killed cell solution was diluted to 20 mg / mL, AglKA-CatD-WT-His (final concentration 1 μg / mL) was added, and the mixture was treated with the enzyme at 37°C for 16 hours and then heated at 90°C for 5 minutes to be used as enzyme-treated cells. As a negative control for the enzyme-treated cells, a sample to which an enzyme inactivated by heating at 90°C for 5 minutes in advance was added was prepared in the same manner.

[0082] (7) Preparation of Aspergillus culture supernatant sample Conidia (1×10^5) of Aspergillus nidulans NBRC 8003 were added to 5 mL of RPMI1640 medium (Thermo Fisher Scientific) and cultured with shaking at 37°C and 200 rpm / min for 65 hours. The supernatant was collected, heated at 90°C for 10 minutes, and the fraction from which precipitates were removed with a 0.45 μm filter was used as the Aspergillus culture supernatant sample.

[0083] (8) Evaluation of binding activity to heat-killed Aspergillus cells 25 μL of heat-killed Aspergillus cells (10 mg / mL) suspended in BPBST was mixed with each HiBiT fusion protein (1 μg / mL) diluted in BPBST for 10 minutes, and unbound proteins were removed by washing three times. Then, 30 μL of the LgBiT and NanoLuc substrate mixture (Nano-Glo HiBiT Lytic Detection System, Promega) was added, and after transferring to a 96-well white plate, the signal derived from the HiBiT tag fused to each protein bound to the cells was measured using a microplate reader (GloMax, Promega). Also, to clarify the binding specificity to α-1,3-glucan in the binding of AglKA-CatD-D1090A-HiBiT to heat-killed cells, heat-killed cells treated with α-1,3-glucanase (AglKA-CatD-WT-His; 13AGase) or heat-treated α-1,3-glucanase (Boiled) were used and examined in the same manner as above.

[0084] (9) Staining and Fluorescence Microscopy of Heat-Killed Aspergillus Cells 10 μL of heat-killed Aspergillus cells (untreated sample, enzyme-treated sample, heat-inactivated enzyme-treated sample, all at 10 mg / mL) suspended in D-PBS(-) (BPBST) containing BSA and Tween 20 was taken, mixed with 0.5 μg of AglKA-CatD-D1090A-HiBiT, and incubated at room temperature for 30 minutes. Unbound proteins were washed, and then Alexa Fluor 647-labeled anti-His tag monoclonal antibody (MBL Life Science) was added and incubated at room temperature for 20 minutes. After washing, imaging was performed using an EVOS FL Cell Imaging System (Thermo Fisher Scientific).

[0085] (10) Preparation of α-1,3-Glucan GtfB-His (10 μg / mL) was added to a 20% sucrose solution [D-PBS(-)], and the mixture was shaken at 30 °C for 48 hours. The resulting insoluble fraction was washed with water, then with ethanol and acetone, and dried. The obtained dried product was weighed, suspended in D-PBS(-), and treated with metal beads using a bead crusher (FastPrep-24; MP Biomedicals) and used as α-1,3-glucan particles (13AG-particle) in each test. Also, dextran T10 added to a 20% sucrose solution [D-PBS(-)] was adjusted in the same manner. After shaking at 30 °C for 48 hours, the insoluble fraction was removed, heated at 90 °C for 10 minutes, ethanol was added to make it 70%, and after centrifugation, the obtained precipitate fraction was washed again with ethanol and acetone, dissolved in sterile water after drying, and freeze-dried. The obtained dried product (s13AG-Dex) was weighed, D-PBS(-) was added, and it was used in tests such as coating of ELISA plates.

[0086] (11) Thermal and pH stability of the AglKA-CatD mutant For thermal stability evaluation, AglKA-CatD-D1090A-HiBiT diluted with D-PBS(-) was dispensed into microtubes and treated at each temperature from 20 to 90 °C for 5 minutes. After cooling, it was diluted (0.5 μg / mL) with D-PBS(-) (BPBST) containing BSA and Tween20. Also, for pH stability evaluation, AglKA-CatD-D1090A-HiBiT was dispensed into microtubes and diluted (0.5 μg / mL) with McIlvaine buffer (pH 2.4 - 8.0) containing BSA. Subsequently, a 96-well white ELISA plate coated with s13AG-Dex (0 - 5 μg / mL) was blocked with BPBST, washed, and then AglKA-CatD-D1090A-HiBiT from each microtube was added to the plate and incubated at room temperature. After washing, to evaluate the binding of each protein to α-1,3-glucan on the plate, the signal derived from the HiBiT tag fused to each protein was detected by adding 25 μL of the LgBiT and NanoLuc substrate mixture (Nano-Glo HiBiT Lytic Detection System, Promega), and the reconstituted luciferase activity was measured using a microplate reader (GloMax, Promega).

[0087] (12) Evaluation of the reactivity between the AglKA-CatD mutant and oligosaccharides A 96-well white ELISA plate coated with s13AG-Dex (0 - 5 μg / mL) was blocked with D-PBS(-) (BPBST) containing BSA and Tween 20. After washing, AglKA-CatD-D1090A-HiBiT (100 ng / ml) was added in the presence or absence of glucose, Nigero-oligosaccharide (from dimer to pentamer, 250 μm) or s13AG-Dex (500 μg / ml) and incubated at room temperature. After washing, to evaluate the binding of each protein to α-1,3-glucan on the plate, the signal derived from the HiBiT tag fused to each protein was detected by adding 25 μL of the LgBiT and NanoLuc substrate mixture (Nano-Glo HiBiT Lytic Detection System, Promega), and the reconstituted luciferase activity was measured using a microplate reader (GloMax, Promega).

[0088] (13) Measurement of binding affinity by Bio-layer Interferometry (BLI) The binding affinity of AglKA-CatD-D1090A for α-1,3-glucan was calculated using a BLI biosensor (BLItz system; Pall ForteBio). A biotinylated s13AG-Dex solution was reacted with a streptavidin sensor chip for 120 seconds to bind. Serial two-fold diluted AglKA-CatD-D1090A-HiBiT (65 kDa) was reacted with the sensor chip to calculate the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD). The data were analyzed using BLItz Pro software (Pall ForteBio).

[0089] (14) Luciferase fragment complementation assay (SLCA) A mixed solution (5 μL) of NanoLuc fragment - fused α - 1,3 - glucan - binding probes (SmBiT - fused and LgBiT - fused, 200 nM each) was added to the test sample (10 μL) in a 96 - well white plate and incubated for 30 minutes under shaking conditions. As the NanoLuc fragment - fused probe mixed solution, combinations of AglKA - CatD - D1090A - LgBiT with AglKA - CatD - D1090A - SmBiT or AglKA - CBM - SmBiT were used. After shaking, 15 μL of furimazine substrate solution (Promega) was added to the well, and the luminescence signal from reconstituted NanoLuc was measured using a GloMax luminometer (Promega). Also, in the kinetic measurement using this assay method, with the complex of the test specimen and the NanoLuc fragment - fused α - 1,3 - glucan - binding probe formed, various glycoside hydrolases were added to the furimazine substrate solution at a concentration of 0.1 μg / mL, and the luminescence signal was measured every minute for 30 minutes. The luminescence signal level when the control group was set to 1 was determined to evaluate the effect of the added enzyme.

[0090] (15) Sandwich ELISA - like test A 96 - well white plate for ELISA was coated with AglKA - CatD - D1090A - ST - II (2 μg / ml) and blocked with D - PBS(-) (BPBST) containing BSA and Tween20. After washing, the test specimen was added to each well and incubated for 1 hour. The plate was washed, and AglKA - CatD - D1090A - HiBiT (1 μg / ml) was added and incubated for 1 hour. After sufficient washing, the signal derived from the HiBiT tag fused to each protein was detected by adding 25 μL of the LgBiT and NanoLuc substrate mixture (Nano - Glo HiBiT Lytic Detection System, Promega), and the reconstituted luciferase activity was measured using a microplate reader (GloMax, Promega).

[0091] <2> Results (1) Binding activity of α - 1,3 - glucanase - related molecules to Isolichenan (Figure 1)A plasmid vector containing a DNA sequence encoding region CBM (Reference: Non-Patent Document 4) excluding the catalytic domain of endo-α-1,3-glucanase AglKA, known as an α-1,3-glucan-binding protein, was prepared, expressed in Escherichia coli as a HiBiT fusion protein (AglKA-CBM-HiBiT), and after purification, its binding activity to commercially available Isolichenan (derived from Cetraria islandica, a type of lichen) was evaluated. Furthermore, the binding activity of the catalytic domain of endo-α-1,3-glucanase, which had not been used as an α-1,3-glucan-binding protein until now, to Isolichenan was also evaluated simultaneously by a direct ELISA-like test. First, as the catalytic domain used for the evaluation, AglKA, which is the full-length enzyme of AglKA-CBM derived from Niallia circulans, a known α-1,3-glucan-binding protein, was selected. Plasmids encoding the wild-type sequence of AglKA-CatD (SEQ ID NO: 1) and AglKA-CatD mutants in which aspartic acid on the ADXXN motif sequence (SEQ ID NO: 11) and RXXGDD motif sequence (SEQ ID NO: 12) involved in its α-1,3-glucan-degrading activity was substituted with alanine were prepared and expressed in Escherichia coli as HiBiT fusion proteins (AglKA-CatD-WT-HiBiT, AglKA-CatD-D1067A-HiBiT, AglKA-CatD-D1090A-HiBiT, AglKA-CatD-D1091A-HiBiT), and were used for evaluation after purification. As a result of the direct ELISA-like test, it was shown that AglKA-CBM-HiBiT strongly binds to Isolichenan immobilized on the plate (left in Fig. 1A). On the other hand, among the AglKA-CatD mutants, AglKA-CatD-D1090A-HiBiT and AglKA-CatD-D1091A-HiBiT showed a significant increase in signal compared to the non-immobilized well in the Isolichenan-immobilized well. However, when comparing the binding activities of AglKA-CBM and AglKA-CatD-D1090A to Isolichenan at each concentration, it was shown that the binding activity of AglKA-CatD-D1090A was extremely weak (right in Fig. 1A).Moreover, AglKA-CBM and AglKA-CatD-D1090A did not show binding activity to Dextran immobilized on the ELISA plate, demonstrating that the results obtained above were not due to non-specific binding (Figure 1B).

[0092] (2) Binding activity of α-1,3-glucanase-related molecules to heat-killed cells of Aspergillus (Figure 2) The binding activity shown above was evaluated for the activity against the complex polysaccharide derived from lichen, leaving the possibility that it may be different from the binding activity against the α-1,3-glucan derived from fungi. Therefore, when the binding activity of each α-1,3-glucanase-related molecule against heat-killed Aspergillus cells was evaluated, almost no binding activity of AglKA-CBM was observed, while all AglKA-CatD mutants showed strong binding activity (left in Fig. 2A). Since the binding activity against heat-killed Aspergillus cells was not observed with wild-type AglKA-CatD, it was shown that this binding activity is an activity exhibited only by the mutants. This AglKA is known as an endo-α-1,3-glucanase belonging to GH87, but enzymes capable of degrading α-1,3-glucan also exist in GH71. Therefore, the wild-type amino acid sequence of AGN1-WT (SEQ ID NO: 24), which is a GH71-type α-1,3-glucanase, and AGN1-D259N (SEQ ID NO: 25) and AGN1-E262Q (SEQ ID NO: 26), in which Asp259 or Glu262 involved in its α-1,3-glucan-degrading activity was replaced with asparagine or glutamine, respectively, were expressed in Escherichia coli as HiBiT fusion proteins and evaluated in the same manner. As a result, no binding activity against heat-killed Aspergillus cells was observed for AGN1-WT (SEQ ID NO: 24), AGN1-D259N (SEQ ID NO: 25), and AGN1-E262Q (SEQ ID NO: 26) (right in Fig. 2A). In addition, the binding of AglKA-CatD-D1090A to heat-killed Aspergillus cells was attenuated by pre-treating the cells with α-1,3-glucanase (AglKA-CatD-WT-His) (13AGase), and its reactivity was not affected by treatment with α-1,3-glucanase (13AGase Boiled) inactivated by heat treatment. Therefore, it was shown that the AglKA-CatD mutant specifically recognizes the α-1,3-glucan on the surface of heat-killed Aspergillus cells (Fig. 2B).Furthermore, it was confirmed under a microscope that AglKA-CatD-D1090A binds directly to the surface of heat-killed Aspergillus cells, that this binding disappears upon treatment with α-1,3-glucanase (13AGase), and that it does not disappear when treated with heat-inactivated α-1,3-glucanase (13AGase Boiled) (Figure 2C). Furthermore, as AglKA-CatD mutants in which aspartic acid conserved in two motif sequences (SEQ ID NOs: 11 and 12) related to α-1,3-glucan-degrading activity was substituted with an amino acid other than alanine, for example, AglKA-CatD-D1067N (SEQ ID NO: 18) and AglKA-CatD-D1090N (SEQ ID NO: 19) were expressed in E. coli as HiBiT fusion proteins, and their binding activity to α-1,3-glucan was evaluated. As a result, binding activity to α-1,3-glucan on the ELISA plate was also confirmed in mutants in which aspartic acid was substituted with asparagine (Figure 2D).

[0093] (3) pH, temperature stability, and size-dependent binding of AglKA-CatD-D1090A to α-1,3-glucan (Figure 3) In the above, with respect to Aspergillus-derived α-1,3-glucan, it was revealed that the AglKA-CatD variant, which had not been used hitherto, showed stronger binding activity than the known α-1,3-glucan-binding protein AglKA-CBM. Therefore, the characteristics of the AglKA-CatD variant as an α-1,3-glucan-binding protein were examined. Using a direct ELISA-like test for α-1,3-glucan prepared by enzymatic synthesis, it was clarified that AglKA-CatD-D1090A was not completely inactivated by treatment at 50°C and lost its function upon heat treatment at 60°C or higher (Figure 3A), and further that it showed binding activity in the pH range of 4.8 - 8.0 (Figure 3B). Next, to clarify the minimum α-1,3-glucan unit necessary for the binding of AglKA-CatD-D1090A, a competitive ELISA-like test using an α-1,3-glucan-immobilized ELISA plate was conducted. The binding of AglKA-CatD-D1090A to α-1,3-glucan on the plate was significantly inhibited by glucose units and α-1,3-glucan having a size of trimeric α-1,3-glucan (Nigero-oligo DP3) or more (Figure 3C).

[0094] (4) Binding affinity of AglKA-CatD-D1090A for α-1,3-glucan (Figure 4) By the BLI method, the binding force (KD value) of AglKA-CatD-D1090A-HiBiT for α-1,3-glucan immobilized on the sensor chip was calculated. From the reaction curves at each concentration of AglKA-CatD-D1090A, the affinity (KD) shown by calculation using BLI software was 2.43×10 -8 M (Figure 4). From the above, the appropriately modified CatD of GH87-type AglKA does not have α-1,3-glucan-degrading activity, but particularly retains strong binding activity for fungal-derived α-1,3-glucan, indicating that it can be a new candidate for an α-1,3-glucan-binding protein.

[0095] (5) Construction of a luciferase fragment complementation assay for the detection and quantification of α-1,3-glucan (Figure 5) Subsequently, to detect and quantify α-1,3-glucan in the crude sample with high sensitivity, the AglKA-CatD mutant was used in the luciferase fragment complementation assay (SLCA), which is a homogeneous measurement system. When SmBiT and LgBiT, which are fragments of NanoLuc, were fused to AglKA-CatD-D1090A, no luciferase luminescence was observed when each was mixed with α-1,3-glucan alone. However, strong luminescence was confirmed only when both the SmBiT fusion and the LgBiT fusion were mixed with α-1,3-glucan (Figure 5A). This luminescence did not occur in the absence of the ligand α-1,3-glucan, indicating that luciferase was reconstituted only in the presence of the α-1,3-glucan to be detected. Furthermore, by using this measurement method, concentration-dependent luminescence of α-1,3-glucan particles (13AG-particle) could be confirmed in a test of only about 30 minutes (Figure 5B). At this time, the difference in the luminescence level of luciferase (background) observed in the absence of α-1,3-glucan and the activity of luciferase reconstituted in the presence of a sufficient amount of α-1,3-glucan (10 μg / mL) was more than about 100-fold. This means that by applying the AglKA-CatD mutant as a new α-1,3-glucan-binding protein, the measurement of the amount of α-1,3-glucan, which conventionally required time and effort for quantification, can now be carried out by an extremely simple method. In particular, this measurement method enables the quantitative detection of α-1,3-glucan even at a low concentration of 1 μg / mL or less.

[0096] (6) Functional evaluation of the luciferase fragment complementation assay using the AglKA-CatD mutant (Figure 6) The structural specificity in ligand recognition of the luciferase fragment complementation assay using the AglKA-CatD variant was verified using commercially available reagents. The test was evaluated for the reactivity of each sample by dividing the signal from the test sample (10 μL, 25 μg / mL each) in the plate by the signal from Blank [D-PBS(-) 10 μL] (background value). As a result, AglKA-CatD-D1090A showed strong reactivity only to α-1,3-glucan particles (13AG-particle) and did not react with reagents such as Isolichenan, Dextran T10, Starch, Curdlan, Laminarin, Pustulan, Chitin, Chitosan Oligo, Xylan (Birchwood), Mannan (Figure 6A). Furthermore, when evaluating the reactivity of the luciferase fragment complementation assay using AglKA-CatD-D1090A to heat-killed Aspergillus cells, an increase in luciferase signal dependent on the addition concentration of dead cells (0 - 5000 μg / mL) was observed (Figure 6B). This reaction disappeared by pre-treating the cells with α-1,3-glucanase (AglKA-CatD-WT-His) (13AGase) and was also shown to be unaffected when treated with heat-inactivated α-1,3-glucanase (13AGase Boiled). From these results, it was proven that the luciferase fragment complementation assay using AglKA-CatD-D1090A can detect and quantify only α-1,3-glucan with strict ligand specificity. Subsequently, the possibility that the luciferase fragment complementation assay using AglKA-CatD-D1090A is affected by anticoagulants (such as EDTA and Heparin) frequently used during blood collection in clinical settings was also verified. D-PBS(-) (Control), Heparin (100 μg / mL, final concentration), or EDTA (10 mM, final concentration) was added to α-1,3-glucan particles (13AG-particle) (0 - 5000 ng / mL), and a luciferase fragment complementation assay was performed.As a result, the luciferase fragment complementation assay using AglKA-CatD-D1090A was shown to be inhibited by EDTA treatment (right in Fig. 6C), but not affected by Heparin treatment (left in Fig. 6C). Also, the reactivity to heat-killed Aspergillus cells was compared between the case of performing the luciferase fragment complementation assay using a fusion of AglKA-CBM, a conventional α-1,3-glucan binding protein, and SmBiT, and the case of using AglKA-CatD-D1090A-SmBiT. AglKA-CBM-SmBiT or AglKA-CatD-D1090A-SmBiT (both 200 nM) was mixed with a mixture of AglKA-CatD-D1090A-LgBiT (200 nM) and heat-killed Aspergillus cells (0 - 5000 μg / mL), and the luminescence level of the reconstituted luciferase was measured 30 minutes later. As a result, AglKA-CatD-D1090A-SmBiT showed stronger reactivity than AglKA-CBM-SmBiT (Fig. 6D). From this, it was shown that the method using the AglKA-CatD mutant exhibited excellent detection sensitivity in the detection of fungal α-1,3-glucan by the luciferase fragment complementation assay.

[0097] (7) Detection of extracellular α-1,3-glucan in Aspergillus (Fig. 7) Using the luciferase fragment complementation assay with AglKA-CatD-D1090A, we attempted to detect extracellular α-1,3-glucan of the filamentous fungus Aspergillus. Aspergillus nidulans was cultured in a medium (RPMI1640) without α-1,3-glucan, and its supernatant was collected and verified for reaction with the luciferase fragment complementation assay. As a result, an increase in luminescence was observed in the undiluted supernatant, its 3-fold diluted solution, and 9-fold diluted solution compared to the Control RPMI1640 medium (left in Fig. 7A), indicating the presence of α-1,3-glucan in the fungal culture supernatant. Furthermore, to achieve highly sensitive detection of fungal soluble α-1,3-glucan, a sandwich ELISA-like test using AglKA-CatD-D1090A was constructed and similarly verified for reaction with the culture supernatant. As a result, an increase in luminescence was observed in the undiluted supernatant, its 3-fold diluted solution, 9-fold diluted solution, 27-fold diluted solution, and 81-fold diluted solution compared to the Control RPMI1640 medium (right in Fig. 7A). From this comparison result, it became clear that when detecting α-1,3-glucan in the fungal culture supernatant, the sandwich ELISA-like test showed better detection sensitivity than the luciferase fragment complementation assay. Furthermore, to verify whether AglKA-CatD-D1090A correctly detected fungal soluble α-1,3-glucan in the culture supernatant, various carbohydrate hydrolases were added simultaneously with the NanoLuc substrate during the luciferase fragment complementation assay using the undiluted culture supernatant, and the change in the luminescence signal over time was evaluated. As a result, it was shown that the luminescence signal decreased over time only when α-1,3-glucanase (AglKA-CatD-WT-His) (13AGase) was added, while the luminescence signal was not affected when heat-inactivated α-1,3-glucanase (13AGase Boiled) or endo-β-1,3-glucanase (zymolyase 100T) (13BGase) was added (Fig. 7B), proving that AglKA-CatD-D1090A specifically detected fungal soluble α-1,3-glucan in the culture supernatant.

[0098] (8)Evaluation of the binding activity of GH87 type α-1,3-glucanase mutants other than AglKA to α-1,3-glucan (Figure 8) Among endo-α-1,3-glucanases (EC 3.2.1.59), mutants in the catalytic region of GH71 type enzymes have been shown to be not available as α-1,3-glucan specific binding proteins. On the other hand, multiple enzymes other than AglKA are known for GH87 type α-1,3-glucanases, and among them, the functionality of 10 kinds has been evaluated as α-1,3-glucanases. Therefore, mutants in the catalytic region of GH87 type α-1,3-glucanases other than AglKA, for example, mutant AglST-CatD-D376A (SEQ ID NO: 20) in the catalytic region of AglST derived from Streptomyces thermodiastaticus and mutant AglFH1-CatD-D1068A (SEQ ID NO: 21) in the catalytic region of AglFH1 derived from Paenibacillus glycanilyticus were expressed in Escherichia coli as HiBiT fusion proteins, and their binding activity to α-1,3-glucan was evaluated. As a result, binding activity to α-1,3-glucan on ELISA plates was also confirmed in GH87 type α-1,3-glucanase mutants other than AglKA (Figure 8), indicating that they can be used as α-1,3-glucan binding polypeptides.

[0099] As described above, it was confirmed that the polypeptides of SEQ ID NOs: 15 to 21 (α-1,3-glucan binding polypeptides) are excellent in specific binding activity to α-1,3-glucan and can accurately detect α-1,3-glucan. Further, according to the method for detecting α-1,3-glucan and the α-1,3-glucan detection kit using these α-1,3-glucan binding polypeptides, for example, α-1,3-glucan can be accurately detected even in the presence of contaminants. Therefore, it was confirmed that by substituting one, two, or three of the three aspartic acids (D) in the motif sequences (SEQ ID NO: 11: ADXXN, SEQ ID NO: 12: RXXGDD) contained in the amino acid sequence W of CatD of wild-type α-1,3-glucanase belonging to the GH87 type enzyme with one, two, or three other amino acids, α-1,3-glucan binding polypeptides having the above effects can be obtained.

[0100] (9) Consideration of substituted amino acids Methods and results: Based on AglKA-CatD-D1090A (SEQ ID NO: 16) which showed strong binding activity to α-1,3-glucan, the amino acid substitution of D1090 was replaced with all amino acids including alanine, and its binding activity was evaluated again. An expression vector (pCold-I vector) containing the DNA encoding each mutant was transformed into Escherichia coli SHuffle for protein expression, and the cells were cultured at 37 °C in LB medium supplemented with ampicillin. After preculture, the cells were cooled to 15 °C, IPTG (0.1 mM) was added, and the expression of AglKA-CatD-D1090X (X includes all amino acids) was induced by culturing for 24 to 72 hours. All proteins were extracted, purified, and dialyzed, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a commercially available kit for confirmation of their expression. When the proteins separated by SDS-PAGE were stained with CBB dye (Quick Blue staining solution; BioDynamics Laboratory), a single band of the protein showing the theoretical molecular size was confirmed in all the samples analyzed.

[0101] Next, 96-well white ELISA plates coated with α-1,3-glucan (500 ng / ml) or D-PBS(-) were blocked with D-PBS(-) containing BSA and Tween 20 (BPBST), washed, and then AglKA-CatD-D1090X-amino acid mutant-HiBiT (400 ng / ml) was added and incubated at room temperature. After washing, to evaluate the binding of each protein to α-1,3-glucan on the plate, the signal derived from the HiBiT tag fused to each protein was detected by adding 25 μL of the LgBiT and NanoLuc substrate mixture (Nano-Glo HiBiT Lytic Detection System, Promega), and the reconstituted luciferase activity was measured using a microplate reader (GloMax, Promega). As a result, some mutants (methionine (M), isoleucine (I), leucine (L), serine (S), histidine (H), phenylalanine (F), asparagine (N), threonine (T)) actually showed stronger activity than AglKA-CatD-D1090A, and in particular, the AglKA-CatD-D1090M mutant substituted with methionine (M) showed the strongest binding activity (Figure 9).

[0102] Furthermore, to calculate the dissociation constant of the binding of AglKA-CatD-D1090M to α-1,3-glucan, analysis using a BLI biosensor (BLItz system; Pall ForteBio) showed that the affinity (KD) indicated by calculation using BLI software from the reaction curve of AglKA-CatD-D1090M-HiBiT was 5.06×10 -9 M, and a clear improvement in binding force was observed compared to AglKA-CatD-D1090A-HiBiT.

[0103] Furthermore, by using AglKA-CatD-D1090M-HiBiT, which showed strong binding activity to α-1,3-glucan, as a detection probe, it was verified whether the detection sensitivity of α-1,3-glucan sandwich ELISA was improved. A 96-well white plate for ELISA was coated with AglKA-CatD-D1090A-ST-II (2 μg / ml) and blocked with D-PBS(-) (BPBST) containing BSA and Tween20. After washing, the supernatant (undiluted, 3-fold diluted, 9-fold diluted) of Aspergillus nidulans NBRC8003 cultured in RPMI1640 medium for 7 days or the control medium (Blank) was added to each well and incubated for 1 hour. The plate was washed, and AglKA-CatD-D1090A-HiBiT or AglKA-CatD-D1090M-HiBiT (1 μg / ml) was added and incubated for 1 hour. After sufficient washing, the signal derived from the HiBiT tag fused to each protein was detected by adding 25 μL of the LgBiT and NanoLuc substrate mixture (Nano-Glo HiBiT Lytic Detection System, Promega), and the reconstituted luciferase activity was measured using a microplate reader (GloMax, Promega).

[0104] As a result, when using AglKA-CatD-D1090M-HiBiT, it was shown that the detection power of α-1,3-glucan contained in the Aspergillus culture supernatant increased compared to the case of detection using AglKA-CatD-D1090A-HiBiT (Figure 10).

[0105] (References) 1. Schmidt TGM., et. al., Nature Protocols. 2007;2(6):1528-35.

[0106] (SEQ ID NOs: 1 to 28) SEQ ID NO: 1: AglKA-CatD-WT (Niallia circulans) From GenBank: BAE98302.1 NLVVYAQRGASMPYTRYDTDDAARGGGATLQSAPNFDQALTASEASGQRYIALPSNGSYAQWTIRPGEGGDGVTMRFTMPDSANGMGLNGSLDVYVNGVKAKTVPLTSYYSWQYFSSDHPADTPAGGRPLFRFDEVHWKMDTPLQPGDTIRIQKSGADSLEYGVDFLEIEAVPAAIARPANSVSVTDFGAVANDGQDDLAAFEAAVNAAVTSGKILYIPAGTFHLGNMWKIGSVANKINNITIMGAGIWHTNIQFTNPNQASGGISFRVTGQLDFSHIYMNSNLRSRYGEQAVYKGFMDNFGTNSKVHNVWVEHFECGFWVGDYAHTPAIIANGLVIENSRIRNNLA D GVNFAQGTSNSTVRNSSIRNNG DD GLAVWTSNVNGAPAGVNNTFSYNTIENNWRAAGIAFFGGSGHKATHNLIVDTVGGSAIRMNTVFPGYHFQNNTGIVFSDTTIINSGTSRDLYNGERGAIDLEASNDPIKNVTFTNIDIINTQRSAIQFGYGGGFENIVFNNININGAGKDGVLTSRFSSPHPGAAIYTYTGNGSATFNNLTTNDIAHPNLYFIQNGFNLTIQ Accession No. 2: AglST-CatD-WT (Streptomyces thermodiastaticus) From GenBank: BCK59654.1 APAAQAAPAAQAATAGADLPFTSVEAESATTTGTKIGPDYTQGTLASEASGRQAVRLDAGQRVEFTVPRAANALTVAYSVPDGQSGTLDVYVNGTKLDRSLTVTSKYSYVDTGWIPGAKTHHFYDNTRLLLGRDVQAGDTVTLQATNVQVTVDVADFEQVSAAAGQPAGSVSVTDKGADPTGQGDSTQAFRDAIAAAQGGVVWIPPGDYRITGPLSGVQNVTLQGAGSWYSVVHSSHFIDQTDSAGHVHLKDFAVIGEVTERVDSSPDNFVNGSLGPGSSVSGMWIQHVKVGLWLTGTNDDLVVENNRILDTTA D GLNLNGTAKNVTVRDNFLRNQG DD ALAMWSLYAPDTDCRFENNTITQPNLANGIAIYGGTDITVKGNLISDTNALGSGIAISNQKFAEPFHPLAGTITVDGNTLVRTGAINPNWNHPMGALRVDSYDSAIEARVDITDTTITDSPYSAFEFVSGGGQGHAVKNVTVDGAAVKNTGTVVVQAEAPGEATFRNVTATGTGAAGIYNCPFPSGSGTFTVTDGGGNSGWDTTWSDCSTWPQP Sequence number 3: AglFH1 - CatD - WT (Paenibacillus glycanilyticus) From GenBank: BAP10900.2 TVSLYSGRGANMPFTIMEAESTSNATNGTKLTPNFKPGDYAGEASGRSSVYLDATGEYVEFTLTSPANAFVLRNAVAENTTGTVSIYADGVSKGKFNVSSKFSYLYATPSTLGRLGYDNAPGAGLTAYWLYEDAQLMLDQVYPAGTKIKIQKDAGDVSWIYVDLLETENVAPPQANPDPTKYVAVSASKSIDQALTEFRQDNTKKGIYIPAGEWTINSKIFLYGRATEIVGAGPWYTKLVAPQSQSNTDVGFNISAAANGSTIRDLSAWGNYINRVDGPGKFIDGNGMQNVTVQNIWVEHFVCLYWGVNSSYNTFKNNRIKNTFA D GINMTNGSSYNVIDNNYARGTG DD SFALFSATDSGGSYNVGNKYTNLTATNVRRAAAFAVYGGSDNLFQNLYGADTLTYPGITISSYSFGYNTLGFGDQDTVIDGATLDRTGGDFWTSVGADDKINEYQNFGAIWIYGGDRAIKNILIKNVDINNPVYFGLMFQSMSPNNMVMQNIRVENVNINNPSRYGIKLVVRAEQGQGPAYGGASFTNVKVNNPGISAIYGEAQSPNFTVTRVSGNNW Accession No. 4: Mutanase (MuB)-CatD-WT (Paenibacillus sp.) From GenBank: BAF56208.1 SLNVYAARGAVMPYSRYDTEDATLGGGAVLKSAPTFDQALTASEASGQKYVALPSNGSSVQWTIRQGQGGAGVTMRFTMPDSSDGMGLNGSLDAYVNGAKVKTIPLTSYYNWQYFSGDMPADAPSGGRPLFRFDEVHFKLDQALQPGDTIRIQKTNGDNLEYGVDFLEIEPVPTEIARPANAVSVTDYGAVANDGQDDLAAFKAAVNAAVAGGKTLYIPAGTFHLSSMWEIGSASNMINNITITGAGIWHTNIQFTNPNAAGGGISLRISGKLDFSNLYMNSNLRSRYGQNAIYKGFMDNFGNNSVIRDVWVEHFECGFWVGDYAHTPAIYANGLTIENSRIRNNLA D GVNFAQGTSNSIVRNSNLRNNG DD ALAVWTSNTNGAPEGVNNTFSYNTIENNWRAGGIAFFGGSGHKADHNYIIDCVGGSGIRMNTVFPGYHFANNTGIVFLDNTIVNSGTSKDLYNGERGAIDLEASNDAIKNVTFTNIDIINSQRDAIQFGYGGGFQNIVFNQITIHGTGLDGITTSRFSGPHLGAAIFTYTGNGAAIFNNLTTSNIAYPNKYYIQNGFNLVIQ Accession No. 5: Mutanase (MuE)-CatD-WT (Paenibacillus sp.) From GenBank: BAH10514.1 NLVVYAARGASMPYKRYDTEDATRGGGATLQSAPTFDQALTASEASGQSYIALPSNGSYLQWTVRPGEGGAGVTMRFTLPDSANGMGLNGSLDVYVNGTKAKTIPLTSYYNWQYFSSDQPADAPGGGRPLFRFDEVHWKLDTPLQAGDTIRIQKSNADTLEYGVDFIEIEPVPTAIPRPANSVSVTDFGAVANDGNDDLAAFEAAVTAAVSTGKTLYIPEGTFHLSNMWKIGTVSNKINDITILGAGIWHTNIQFTNPNIAGGGISFRVQGKLDFSNVYMNSVYMNSNLRSRYHEGAIYKAFMDNFGKNSKVHNVWVEHFECGFWVADYAHTPAIYADGLVIENSRIRNNLA D GVNFAQGTSNSTVRNSSIRNNG DD GLAVWTSNVNAAPAGVNNTFSNNTIENNWRAAAIAFFGGSGHKATNNLIVDTVGGSAFRMNTVFPGYHFADNTGIQFSDSTIINSGTSKDLYNGERGAIDLEASGDSIKNVTFTNIDILNTQRSAIQFGYGGGFQNIVFNNIKIDGTGKDGILTSRFTTPHPGAAIYTYTGNGSATFHNLTTSNIAHPNVNFIQQGFNLVIQ Accession number 6: AglFH2-CatD-WT (Paenibacillus glycanilyticus) From GenBank: BAP10901.1 NLVVYSVRGASVPYTRYDTDDAIRGGGATLQSAPTFDQALTASEASGQKYIALPSSGSYLQWTVRQGEGGAGVTMRFTMPDSSDGMGLSGSLDVYVNNVKVKTVALSSYYNWQYFSGDMPADAPGGGRPLFRFDEVHWKLDTPLQPGDTVRIQKGNDSIEYGVDFIEVEQVPTAIAQPANSVSVTDYGAIANDGLDDLTAFKAAVNAAVAANKTLYIPAGTFHLGNMWEIGSVSNMINNLTITGAGYWYTNIQFTNPNAAGGGISLRITGKLDFSNVYLNSKLRSRYGQNAIYKGFMDNFGTNSKIHDVWVEHFECGFWVGDYAHTPSIYANGLTIENSRIRNNLA D GVNFSQGTSNSIVRNSNLRNNG DD ALAVWTSNTNGAPAGVNNTFSYNTIENNWRAAGIAFFGGSGHKADHNVIIDTVGGSGVRLNTVFPGYHFQNNTGITISDTTIINSGTSQDLYNGERGAIDLEASNDSIKNVTFTNIDILNAQRDGIQFGYGGGFENIVFNNVTIDGTGKDGVTTSRFSGPHLGAAIYTYTGNGSATFNNLVTRNIAYPNVNYVQSGFTLTFN Sequence number 7: Mutanase (Mut)-CatD-WT (Paenibacillus humicus) From GenBank: BAI23187.1 SLTVYSARGASMPYSRYDTEDAVLGGGAVLRTAPTFDQSLIASEASGQKYAALPSNGSSLQWTVRQGQGGAGVTMRFTMPDTSDGMGQNGSLDVYVNGTKAKTVSLTSYYSWQYFSGDMPADAPGGGRPLFRFDEVHFKLDTALKPGDTIRVQKGGDSLEYGVDFIEIEPIPAAVARPANSVSVTEYGAVANDGKDDLAAFKAAVTAAVAAGKSLYIPEGTFHLSSMWEIGSATSMIDNFTVTGAGIWYTNIQFTNPNASGGGISLRIKGKLDFSNIYMNSNLRSRYGQNAVYKGFMDNFGTNSIIHDVWVEHFECGMWVGDYAHTPAIYASGLVVENSRIRNNLA D GINFSQGTSNSTVRNSSIRNNG DD GLAVWTSNTNGAPAGVNNTFSYNTIENNWRAAAIAFFGGSGHKADHNYIIDCVGGSGIRMNTVFPGYHFQNNTGITFSDTTIINSGTSQDLYNGERGAIDLEASNDAIKNVTFTNIDIINAQRDGVQIGYGGGFENIVFNNITIDGTGRDGISTSRFSGPHLGAAIYTYTGNGSATFNNLVTRNIAYAGGNYIQSGFNLTIK Sequence number 8: Mutanase (MutP)-CatD-WT (Paenibacillus curdlanolyticus) From GenBank: ADT91063.1 NLVVYAQRGASMPYSRYDTEDATRGGGATLQTAPTFNQAQIASEASGQSYIALPSNGSSAQWTVRQGQGGAGVTMRFTMPDSTDGMGLNGSLDVYVNGVKVKTVSLTSYYSWQYFSGDMPGDAPSAGRPLFRFDEVHWKLDTPLQPGDTIKIQKGNGDSLEYGIDFLEIEPVPTAIAKPANSLSVTEYGAVANDGQDDLAAFKATVTAAVAAGKSVYIPAGTFNLSSMWEIGSANNMINNITITGAGYWHTNIQFTNPNAAGGGISLRISGQLDFSNVYMNSNLRSRYGQNAIYKGFMDNFGTNSKIHDVWVEHFECGMWVGDYAHTPAIYATGLVVENSRIRNNLA D GINYSQGTSNSIVRNSSIRNNG DD GLAVWTSNTNGAPAGVNNTFSYNTIENNWRAGGIAFFGGGGHKADHNLIVDTVGGSGIRMNTVFPGYHFQNNTGITFSDNTLINTGTSQDLYNGERGAIDLEASNDAIKNVTFTNIDIINTQRDAIQFGYGGGFENIVFNNININGTGLDGVTTSRFAGPHKGAAIYTYTGNGSATFNNLTTSNVAYPGLNFIQQGFNLVIQ Accession No. 9: Mutanase (MuC1)-CatD-WT (Paenibacillus sp.) From GenBank: BAG15879.1 NLVVYAARGASMPYSRYDTEDATLGGGATLKSAPTFDQALPASEASGQRYIALPSNGSNLEWTVRQGQGGAGVTMRFTMPDSADGMGLNGSLDVYVNGAKVKTVSLTSYYSWQYFSGDMPGDTPSAGRPLFRFDEVHWKMDTPLQPGDKIRIQKNNGDSLEYGVDFIEIESVPTAIARPANSVSVTDYGAVANDGQDDLAAFKATVNAAVASGKSIYIPAGTFNLSSMWEIGSASNMINNLTITGAGLWHTNIQFTNPNAAGGGISLRISGKLDFSNVYMNSNLRSRYGQNAIYKGFMDNFGTNSIIHDVWVEHFECGMWVGDYAHTPAIYATGLVVENSRIRNNLA D GINFSQGTSNSIVRNSSVRNNG DD GLAVWTSNTNGAPAGVNNTFSYNTIENNWRAAAIAFFGGSGHKADHNYIIDTVGGSGIRMNTVFPGYHFQNNTGVVFSDTTIINSGTSKDLYGGERGAIDLEASNDAIKNVTFNNIDIINTQRDAIQFGYGGGFENIVFNNININGTGLDEITTSRFSGPHKGAAIYTYTGNGAATFNNLTTSNIAYPNLNYIQSGFNLTIH Accession No. 10: Mutanase (MuA)-CatD-WT (Paenibacillus sp.) From GenBank: BAG15878.1 TQSLFIGRGANMPYDMYEAEDGVIGGGAVKLSANRTIGDPAGEASGRRAVTLNTTGSYVEFTTKASTNTLVTRFSIPDSASGDGTNATLNIYVNGVFSKAINLTSKYAWLYGSETSPGNSPSAGSPRHIYDEANIMFDSTIPAGSTIRLQKDSANTSQYAIDFISLEQVSPIANPDPAKYTVPAGFTHQDVQNALDKVRMDTTGNLVGVYLPPGNYQTSNKFQVYGKAVKVIGAGPWYTRFIAPTNQENTDVGFRASDTANGSTFANFAYFGNYTSRIDGPGKVFDFSNVANITIDNIWTEHQVCMYWGANTDNMVIKNSRIRNTFA D GINMTNGSTNNLVSNIEARATG DD SFALFSAIDSGGADMKDNVYENLTSILTWRAAGVAVYGGYANTFRNIYIADTLCYSGITISSLDFGYPMNGFGASPTTNFENISIVRAGGHFWGAQTFPAIWVFSASKVFQGIRVNDVDIIDPTYHGIMFQTNYVGSTPQFPVTDTIFNNVTITGAQKSGDAFDSKSGVGIWVNEAAEAGQGPARGSATFNNLKITNTVTNIKNNTSTFTINVNP Sequence number 11: ADXXN Sequence number 12: RXXGDD Sequence number 13: AglKA-WT (Niallia circulans) From GenBank: BAE98302.1 MRTKYVAWSLIAALLITTLFQSVGPGEPVE AAGGPNLTLGKTITASGQSQTYSPNNVKDSNQGTYWESTNNAFPQWIQADLGASTSIDQIVLKLPANWETRTQTLTVQGSSNGSTYSTIVGSANYVFNPAVAGNSVTINFDPTSTRYVRLQVTNNTGWPAAQLSEFEIYGASGPTPTPSATPQPSGTYQAEAAALSGGAKVNTDHSGYAGTGFVDGYWTQGAATTFTVQAQAGGNHNVTLKYANASGSDKTLSLYVNGTKLRQTNLPNLANWDTWGNKAETVNLNAGSNTIAYKYDSGDSGNVNLDQITVEAASSTPTPTPTSTPTPTPTPTPTPTPTPTPTPTPTPTPTATPTPPPGSNIAVGKPITASSSTFTFVAANANDNSTDTYWEGGGNPSTLTLDLGSNHDVTSIVLKLNPSTAWSTRTQTIQVLGHNQSTTTFSNLVSSQSYTFNPASGNTVTIPVTATVKRLQLNFTANSGASAGQVAEFQVFGTPSANPDLIITDMSWSPASPIETNAITLNATVKNNGTAPSGATTVNFYLNNTLAGSAPVNGLAAGASTTVSLNAGTRTAASYTVNAKVDENNSVIELNESNNSYTHATPLVVAAVSSSDLVGTVSWTPSNPVAGNAVALTVNLKNQGNIASAGGAHGVTVALKNPAGSTLQSFSGSYTGVLAAGASVNVTLPGTWTASPGTYTLTTTIAADGNELPIKQSNNVSNTNLVVYAQRGASMPYTRYDTDDAARGGGATLQSAPNFDQALTASEASGQRYIALPSNGSYAQWTIRPGEGGDGVTMRFTMPDSANGMGLNGSLDVYVNGVKAKTVPLTSYYSWQYFSSDHPADTPAGGRPLFRFDEVHWKMDTPLQPGDTIRIQKSGADSLEYGVDFLEIEAVPAAIARPANSVSVTDFGAVANDGQDDLAAFEAAVNAAVTSGKILYIPAGTFHLGNMWKIGSVANKINNITIMGAGIWHTNIQFTNPNQASGGISFRVTGQLDFSHIYMNSNLRSRYGEQAVYKGFMDNFGTNSKVHNVWVEHFECGFWVGDYAHTPAIIANGLVIENSRIRNNLA D GVNFAQGTSNSTVRNSSIRNNG DD GLAVWTSNVNGAPAGVNNTFSYNTIENNWRAAGIAFFGGSGHKATHNLIVDTVGGSAIRMNTVFPGYHFQNNTGIVFSDTTIINSGTSRDLYNGERGAIDLEASNDPIKNVTFTNIDIINTQRSAIQFGYGGGFENIVFNNININGAGKDGVLTSRFSSPHPGAAIYTYTGNGSATFNNLTTNDIAHPNLYFIQNGFNLTIQ Sequence number 14: AglKA-CBM (Niallia circulans) From GenBank: BAE98302.1 AAGGPNLTLGKTITASGQSQTYSPNNVKDSNQGTYWESTNNAFPQWIQADLGASTSIDQIVLKLPANWETRTQTLTVQGSSNGSTYSTIVGSANYVFNPAVAGNSVTINFDPTSTRYVRLQVTNNTGWPAAQLSEFEIYGASGPTPTPSATPQPSGTYQAEAAALSGGAKVNTDHSGYAGTGFVDGYWTQGAATTFTVQAQAGGNHNVTLKYANASGSDKTLSLYVNGTKLRQTNLPNLANWDTWGNKAETVNLNAGSNTIAYKYDSGDSGNVNLDQITVEAASSTPTPTPTSTPTPTPTPTPTPTPTPTPTPTPTPTPTATPTPPPGSNIAVGKPITASSSTFTFVAANANDNSTDTYWEGGGNPSTLTLDLGSNHDVTSIVLKLNPSTAWSTRTQTIQVLGHNQSTTTFSNLVSSQSYTFNPASGNTVTIPVTA Accession number 15: AglKA-CatD-D1067A (Niallia circulans) From GenBank: BAE98302.1 NLVVYAQRGASMPYTRYDTDDAARGGGATLQSAPNFDQALTASEASGQRYIALPSNGSYAQWTIRPGEGGDGVTMRFTMPDSANGMGLNGSLDVYVNGVKAKTVPLTSYYSWQYFSSDHPADTPAGGRPLFRFDEVHWKMDTPLQPGDTIRIQKSGADSLEYGVDFLEIEAVPAAIARPANSVSVTDFGAVANDGQDDLAAFEAAVNAAVTSGKILYIPAGTFHLGNMWKIGSVANKINNITIMGAGIWHTNIQFTNPNQASGGISFRVTGQLDFSHIYMNSNLRSRYGEQAVYKGFMDNFGTNSKVHNVWVEHFECGFWVGDYAHTPAIIANGLVIENSRIRNNLA AGVNFAQGTSNSTVRNSSIRNNGDDGLAVWTSNVNGAPAGVNNTFSYNTIENNWRAAGIAFFGGSGHKATHNLIVDTVGGSAIRMNTVFPGYHFQNNTGIVFSDTTIINSGTSRDLYNGERGAIDLEASNDPIKNVTFTNIDIINTQRSAIQFGYGGGFENIVFNNININGAGKDGVLTSRFSSPHPGAAIYTYTGNGSATFNNLTTNDIAHPNLYFIQNGFNLTIQ Accession No. 16: AglKA-CatD-D1090A (Niallia circulans) From GenBank: BAE98302.1 NLVVYAQRGASMPYTRYDTDDAARGGGATLQSAPNFDQALTASEASGQRYIALPSNGSYAQWTIRPGEGGDGVTMRFTMPDSANGMGLNGSLDVYVNGVKAKTVPLTSYYSWQYFSSDHPADTPAGGRPLFRFDEVHWKMDTPLQPGDTIRIQKSGADSLEYGVDFLEIEAVPAAIARPANSVSVTDFGAVANDGQDDLAAFEAAVNAAVTSGKILYIPAGTFHLGNMWKIGSVANKINNITIMGAGIWHTNIQFTNPNQASGGISFRVTGQLDFSHIYMNSNLRSRYGEQAVYKGFMDNFGTNSKVHNVWVEHFECGFWVGDYAHTPAIIANGLVIENSRIRNNLADGVNFAQGTSNSTVRNSSIRNNG A DGLAVWTSNVNGAPAGVNNTFSYNTIENNWRAAGIAFFGGSGHKATHNLIVDTVGGSAIRMNTVFPGYHFQNNTGIVFSDTTIINSGTSRDLYNGERGAIDLEASNDPIKNVTFTNIDIINTQRSAIQFGYGGGFENIVFNNININGAGKDGVLTSRFSSPHPGAAIYTYTGNGSATFNNLTTNDIAHPNLYFIQNGFNLTIQ Accession No. 17: AglKA-CatD-D1091A (Niallia circulans) From GenBank: BAE98302.1 From GenBank: BAE98302.1 NLVVYAQRGASMPYTRYDTDDAARGGGATLQSAPNFDQALTASEASGQRYIALPSNGSYAQWTIRPGEGGDGVTMRFTMPDSANGMGLNGSLDVYVNGVKAKTVPLTSYYSWQYFSSDHPADTPAGGRPLFRFDEVHWKMDTPLQPGDTIRIQKSGADSLEYGVDFLEIEAVPAAIARPANSVSVTDFGAVANDGQDDLAAFEAAVNAAVTSGKILYIPAGTFHLGNMWKIGSVANKINNITIMGAGIWHTNIQFTNPNQASGGISFRVTGQLDFSHIYMNSNLRSRYGEQAVYKGFMDNFGTNSKVHNVWVEHFECGFWVGDYAHTPAIIANGLVIENSRIRNNLADGVNFAQGTSNSTVRNSSIRNNGD A GLAVWTSNVNGAPAGVNNTFSYNTIENNWRAAGIAFFGGSGHKATHNLIVDTVGGSAIRMNTVFPGYHFQNNTGIVFSDTTIINSGTSRDLYNGERGAIDLEASNDPIKNVTFTNIDIINTQRSAIQFGYGGGFENIVFNNININGAGKDGVLTSRFSSPHPGAAIYTYTGNGSATFNNLTTNDIAHPNLYFIQNGFNLTIQ Accession No. 18: AglKA-CatD-D1067N (Niallia circulans) From GenBank: BAE98302.1 NLVVYAQRGASMPYTRYDTDDAARGGGATLQSAPNFDQALTASEASGQRYIALPSNGSYAQWTIRPGEGGDGVTMRFTMPDSANGMGLNGSLDVYVNGVKAKTVPLTSYYSWQYFSSDHPADTPAGGRPLFRFDEVHWKMDTPLQPGDTIRIQKSGADSLEYGVDFLEIEAVPAAIARPANSVSVTDFGAVANDGQDDLAAFEAAVNAAVTSGKILYIPAGTFHLGNMWKIGSVANKINNITIMGAGIWHTNIQFTNPNQASGGISFRVTGQLDFSHIYMNSNLRSRYGEQAVYKGFMDNFGTNSKVHNVWVEHFECGFWVGDYAHTPAIIANGLVIENSRIRNNLA N GVNFAQGTSNSTVRNSSIRNNGDDGLAVWTSNVNGAPAGVNNTFSYNTIENNWRAAGIAFFGGSGHKATHNLIVDTVGGSAIRMNTVFPGYHFQNNTGIVFSDTTIINSGTSRDLYNGERGAIDLEASNDPIKNVTFTNIDIINTQRSAIQFGYGGGFENIVFNNININGAGKDGVLTSRFSSPHPGAAIYTYTGNGSATFNNLTTNDIAHPNLYFIQNGFNLTIQ Accession No. 19: AglKA-CatD-D1090N (Niallia circulans) From GenBank: BAE98302.1 NLVVYAQRGASMPYTRYDTDDAARGGGATLQSAPNFDQALTASEASGQRYIALPSNGSYAQWTIRPGEGGDGVTMRFTMPDSANGMGLNGSLDVYVNGVKAKTVPLTSYYSWQYFSSDHPADTPAGGRPLFRFDEVHWKMDTPLQPGDTIRIQKSGADSLEYGVDFLEIEAVPAAIARPANSVSVTDFGAVANDGQDDLAAFEAAVNAAVTSGKILYIPAGTFHLGNMWKIGSVANKINNITIMGAGIWHTNIQFTNPNQASGGISFRVTGQLDFSHIYMNSNLRSRYGEQAVYKGFMDNFGTNSKVHNVWVEHFECGFWVGDYAHTPAIIANGLVIENSRIRNNLADGVNFAQGTSNSTVRNSSIRNNG N DGLAVWTSNVNGAPAGVNNTFSYNTIENNWRAAGIAFFGGSGHKATHNLIVDTVGGSAIRMNTVFPGYHFQNNTGIVFSDTTIINSGTSRDLYNGERGAIDLEASNDPIKNVTFTNIDIINTQRSAIQFGYGGGFENIVFNNININGAGKDGVLTSRFSSPHPGAAIYTYTGNGSATFNNLTTNDIAHPNLYFIQNGFNLTIQ Accession No. 20: AglST-CatD-D376A (Streptomyces thermodiastaticus) From GenBank: BCK59654.1 APAAQAAPAAQAATAGADLPFTSVEAESATTTGTKIGPDYTQGTLASEASGRQAVRLDAGQRVEFTVPRAANALTVAYSVPDGQSGTLDVYVNGTKLDRSLTVTSKYSYVDTGWIPGAKTHHFYDNTRLLLGRDVQAGDTVTLQATNVQVTVDVADFEQVSAAAGQPAGSVSVTDKGADPTGQGDSTQAFRDAIAAAQGGVVWIPPGDYRITGPLSGVQNVTLQGAGSWYSVVHSSHFIDQTDSAGHVHLKDFAVIGEVTERVDSSPDNFVNGSLGPGSSVSGMWIQHVKVGLWLTGTNDDLVVENNRILDTTADGLNLNGTAKNVTVRDNFLRNQG A DALAMWSLYAPDTDCRFENNTITQPNLANGIAIYGGTDITVKGNLISDTNALGSGIAISNQKFAEPFHPLAGTITVDGNTLVRTGAINPNWNHPMGALRVDSYDSAIEARVDITDTTITDSPYSAFEFVSGGGQGHAVKNVTVDGAAVKNTGTVVVQAEAPGEATFRNVTATGTGAAGIYNCPFPSGSGTFTVTDGGGNSGWDTTWSDCSTWPQP Sequence number 21: AglFH1-CatD-D1068A (Paenibacillus glycanilyticus) From GenBank: BAP10900.2 TVSLYSGRGANMPFTIMEAESTSNATNGTKLTPNFKPGDYAGEASGRSSVYLDATGEYVEFTLTSPANAFVLRNAVAENTTGTVSIYADGVSKGKFNVSSKFSYLYATPSTLGRLGYDNAPGAGLTAYWLYEDAQLMLDQVYPAGTKIKIQKDAGDVSWIYVDLLETENVAPPQANPDPTKYVAVSASKSIDQALTEFRQDNTKKGIYIPAGEWTINSKIFLYGRATEIVGAGPWYTKLVAPQSQSNTDVGFNISAAANGSTIRDLSAWGNYINRVDGPGKFIDGNGMQNVTVQNIWVEHFVCLYWGVNSSYNTFKNNRIKNTFADGINMTNGSSYNVIDNNYARGTG A DSFALFSATDSGGSYNVGNKYTNLTATNVRRAAAFAVYGGSDNLFQNLYGADTLTYPGITISSYSFGYNTLGFGDQDTVIDGATLDRTGGDFWTSVGADDKINEYQNFGAIWIYGGDRAIKNILIKNVDINNPVYFGLMFQSMSPNNMVMQNIRVENVNINNPSRYGIKLVVRAEQGQGPAYGGASFTNVKVNNPGISAIYGEAQSPNFTVTRVSGNNW Sequence number 22: DDAKK Sequence number 23: EAAAK Sequence number 24: GGSGGGSGGSG Sequence number 25: AGN1-WT (Schizosaccharomyces pombe) From GenBank: AAT84064.1 DKMVVAHFIVGNTYPYTVSNWEEDIQDAIAVGIDGFALNMGSDAWQVERIEDAYDAAASVSSDFKLFISFDMSIISADADFIEGVVRRFADKPNQLYYDGKVFVSTFAGETDTFGYSDVSTGWDSAVKEPLASAGYPIYFVPSWTSLGQGALEESVADGFLSWNAWPTTDADMNDNDDIGYQNLANSLGKLYVAPVSPWFYTHLSYKNWAYKSDWLIIDRWNEMLSVQPDMIEVLTWN D YG E SHYIGNIQGALPAGSEGYVDGFDHTAWRYLMSPYISAYKLGLSEPYINFESLFYWYRPTPKSATATADSLSYPSGGDYMEDEIFVLVYLLQSAEVTVTCGSTTQTFSGVPGVNQFTIPMETNASPSFTVARQGGTLASGTGPEIVDSLSIYNFNAYTGVLYF Sequence number 26: AGN1-D259A (Schizosaccharomyces pombe) From GenBank: AAT84064.1 DKMVVAHFIVGNTYPYTVSNWEEDIQDAIAVGIDGFALNMGSDAWQVERIEDAYDAAASVSSDFKLFISFDMSIISADADFIEGVVRRFADKPNQLYYDGKVFVSTFAGETDTFGYSDVSTGWDSAVKEPLASAGYPIYFVPSWTSLGQGALEESVADGFLSWNAWPTTDADMNDNDDIGYQNLANSLGKLYVAPVSPWFYTHLSYKNWAYKSDWLIIDRWNEMLSVQPDMIEVLTWN A YGESHYIGNIQGALPAGSEGYVDGFDHTAWRYLMSPYISAYKLGLSEPYINFESLFYWYRPTPKSATATADSLSYPSGGDYMEDEIFVLVYLLQSAEVTVTCGSTTQTFSGVPGVNQFTIPMETNASPSFTVARQGGTLASGTGPEIVDSLSIYNFNAYTGVLYF Accession No. 27: AGN1-E262Q (Schizosaccharomyces pombe) From GenBank: AAT84064.1 DKMVVAHFIVGNTYPYTVSNWEEDIQDAIAVGIDGFALNMGSDAWQVERIEDAYDAAASVSSDFKLFISFDMSIISADADFIEGVVRRFADKPNQLYYDGKVFVSTFAGETDTFGYSDVSTGWDSAVKEPLASAGYPIYFVPSWTSLGQGALEESVADGFLSWNAWPTTDADMNDNDDIGYQNLANSLGKLYVAPVSPWFYTHLSYKNWAYKSDWLIIDRWNEMLSVQPDMIEVLTWNDYG Q SHYIGNIQGALPAGSEGYVDGFDHTAWRYLMSPYISAYKLGLSEPYINFESLFYWYRPTPKSATATADSLSYPSGGDYMEDEIFVLVYLLQSAEVTVTCGSTTQTFSGVPGVNQFTIPMETNASPSFTVARQGGTLASGTGPEIVDSLSIYNFNAYTGVLYF Accession No. 28: WSHPQFEK

Claims

1. It does not exhibit α-1,3-glucan decomposition activity and has specific binding activity to α-1,3-glucan, The following two motif sequences contained in the amino acid sequence W of the polypeptide constituting the catalytic domain (CatD) of wild-type α-1,3-glucanase belonging to the GH87 type enzyme: SEQ ID NO: 11: ADXXN (wherein X represents any amino acid), and SEQ ID NO: 12: RXXGDD (X represents any amino acid) At least one of the three aspartic acids (D) in the formula (I) is substituted with another amino acid; and The sequence identity with the amino acid sequence W is 80% or more. Contains an amino acid sequence, α-1,3-glucan binding polypeptide.

2. The other amino acids are 1 to 3 of methionine (M), isoleucine (I), leucine (L), histidine (H), phenylalanine (F), valine (V), tryptophan (W), asparagine (N), alanine (A), glycine (G), glutamine (Q), serine (S), proline (P), threonine (T), and lysine (K). The α-1,3-glucan-binding polypeptide of claim 1.

3. The amino acid sequence W is any one of the amino acid sequences represented by SEQ ID NOs: 1 to 10. The α-1,3-glucan-binding polypeptide of claim 1.

4. The method comprises the step of contacting a test sample with a reagent comprising an α-1,3-glucan binding polypeptide according to any one of claims 1 to 3. Method for detecting α-1,3-glucan.

5. The method for detecting α-1,3-glucan according to claim 4, wherein the α-1,3-glucan is a polymer composed of three or more glucose units.

6. A reagent comprising the α-1,3-glucan binding polypeptide of any one of claims 1 to 3. α-1,3-glucan detection kit.

7. A recombinant microorganism or cell comprising a base sequence encoding the α-1,3-glucan-binding polypeptide of any one of claims 1 to 3.

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