β-(1,3)(1,4)-glucan binding polypeptides, detection methods of β-(1,3)(1,4)-glucan, β-(1,3)(1,4)-glucan detection kits, and β-1,3-glucan binding polypeptides, β-1,3-glucan detection methods, β-1,3-glucan detection kits, recombinant microorganisms or cells

Abstract

To provide β-(1,3)(1,4)-glucan binding polypeptides that bind to MLG but not to 13BG.SOLUTION: The disclosed β-(1,3)(1,4)-glucan binding polypeptide comprises an amino acid sequence having 80% or more sequence identity to an amino acid sequence W1 with mutation, and having a specific binding activity to β-(1,3)(1,4)-glucan, where the amino acid sequence W1 is the sequence of a polypeptide constituting a catalytic domain (Cat D) of wild type β-(1,3)(1,4)-glucanase that belongs to GH16 type enzyme, and comprises two motif sequences SEQ ID NO:44: EXDXE (X may be any amino acid) or SEQ ID NO:45: EXDXXE (X may be any amino acid), where in the disclosed β-(1,3)(1,4)-glucan binding polypeptide, at least one of two glutamic acid (E) present in each motif is substituted with a different amino acid.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a β-(1,3)(1,4)-glucan-binding polypeptide, a method for detecting β-(1,3)(1,4)-glucan, a β-(1,3)(1,4)-glucan detection kit, and a recombinant microorganism or cell. Further, the present invention relates to a β-1,3-glucan-binding polypeptide, a method for detecting β-1,3-glucan, a β-1,3-glucan detection kit, a recombinant microorganism or cell.

Background Art

[0002] β-1,3-glucan (hereinafter sometimes referred to as "13BG") is known as one of β-glucans, and has a repeating structure in which glucose is linked in a large number by β-1,3-linkages, and is a major polysaccharide constituting the cell walls of various fungi. β-glucans include those having a branched structure in which glucose is further β-1,6-linked or β-1,4-linked with 13BG as the main chain. In addition, it may take a complex form with other polysaccharides, proteins, etc. Since it is not synthesized in the human body, when deep-seated fungal infections are suspected, it is a common method to measure the 13BG concentration in the blood. In particular, since 13BG is an extracellular polysaccharide of fungi such as Candida and Aspergillus, the β-D-glucan test is frequently used for blood tests of patients suspected of having fungal infections and for monitoring in vivo 13BG for risk assessment after organ transplantation.

[0003] As kits for diagnosing deep-seated pathogenic fungal infections, several highly sensitive detection reagents for blood 13BG are used. For example, β-D-glucan tests using hemocyte extracts of horseshoe crabs (Amoebocyte lysate), ELISA kits using antibodies, etc. have been developed (for example, Patent Document 1). Among them, the β-D-glucan test using horseshoe crabs as a raw material is a serodiagnostic agent used in many countries and is frequently used in clinical practice.

[0004] However, the β-D-glucan test also mainly reacts with MLG derived from plants and the like, and there are not a few examples showing false positives. This problem is caused by the function of the serine protease precursor (Factor G) contained in Amoebocyte lysate. Factor G is a protein that has the function of directly binding to 1,3-β-D-glucan (13BG), and is activated by binding to 13BG and functions as a protease. In the presence of 13BG, activated Factor G further activates the pro-clotting enzyme and converts it into a clotting enzyme (Non-Patent Document 1). By using a synthetic peptide substrate or the like that can be cleaved by this clotting enzyme, it becomes possible to measure the 13BG concentration in the sample.

[0005] Regarding the activation of Factor G by 13BG, it is known that Factor G is strongly activated by various 13BGs, while it is not activated by Carboxymetyl Cellulose (a β-1,4-glucan derivative) (Non-Patent Document 2). Furthermore, Factor G is also known to be activated by β-(1,3)(1,4)-glucans such as Lichenan and Barley β-glucan (hereinafter sometimes referred to as "MLG") (Non-Patent Document 2). The activation of Factor G by MLG is considered to be one of the causes of false positive reactions in the clinical field. Also, in the same document, it is shown that the reactivity with some 13BGs dissolved in water (such as Laminaran oligosaccharides, Partially degraded curdlans, Schizophyllan, etc.) is lower than that of other 13BGs, and these are also related to false negative reactions in the clinical field.

[0006] In addition, the β-D-glucan test may show positive results, for example, due to 1,3-β-D-glucan derived from food that has migrated to the bronchi and lungs during aspiration, or MLG derived from grains (Non-Patent Document 3). Normally, it is considered that high-molecular polysaccharides such as 1,3-β-D-glucan hardly migrate from the digestive tract into the blood, but it has been shown that they can efficiently migrate from the bronchial and lung mucosa into the blood (Patent Document 2). Therefore, since Factor G can be activated not only by 1,3-β-D-glucan derived from pathogenic fungi that grow in the body but also by MLG derived from plants, etc., a certain number of false positive reactions can occur in the mycosis test, and it can be said that this is a problem that is difficult to avoid.

[0007] The β-D-glucan test used in clinical diagnosis uses horseshoe crabs as raw materials. Since horseshoe crabs and their eggs occupy an important position in the ecosystem and are a nutrient source for many organisms, there are concerns about the decline and depletion of related marine resources (Non-Patent Document 4). To avoid the impact on marine resources, a method for preparing the protein required for the β-D-glucan test as a recombinant protein has also been developed (Patent Document 3). However, since the final reactivity depends on the polysaccharide recognition function of Factor G, it is easily predicted that the β-D-glucan test prepared using recombinant protein can also induce the activation of Factor G by polysaccharides such as MLG other than 1,3-β-D-glucan. Furthermore, as described above, the fact that different reactivities are shown for 1,3-β-D-glucan with different branched structures and molecular weights is also a problem in the β-D-glucan test using horseshoe crabs as raw materials. Therefore, it can be said that a method for measuring the blood 1,3-β-D-glucan concentration that does not depend on the polysaccharide recognition function of Factor G is useful.

[0008] When detecting and quantifying 13BG, multiple methods using 13BG-binding proteins other than Factor G have been developed. For example, methods using anti-13BG antibodies (Patent Document 1, Patent Document 4, Non-Patent Document 5), methods using recombinant proteins of the extracellular domain of Dectin-1, which is a mammalian 13BG receptor (Non-Patent Document 6), methods using recombinant proteins of the 13BG-binding domain of β-glucan recognition protein (BGRP) possessed by insects such as silkworms (Patent Document 5), etc. have been developed.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0011] However, since the 13BG to be measured has extremely diverse microstructures, it has been difficult to efficiently detect and quantify 13BG by conventional 13BG detection and quantification methods such as Patent Documents 1-5 and Non-Patent Documents 1-6 without depending on the origin of 13BG.

[0012] Furthermore, in conventional 13BG detection and quantification methods, there is no means to distinguish between fungal-derived 13BG and plant-derived MLG. In clinical tests, it has not been easy to determine whether it is a reaction dependent on the presence of pathogenic fungi in the body or a reaction dependent on 13BG or MLG that has migrated into the blood through other pathways.

[0013] The present invention has been made in view of the above circumstances, and provides a β-1,3-glucan-binding polypeptide that has no activity to bind to MLG and has the property of binding to 13BG with diverse microstructures. Another object is to provide a method for detecting β-1,3-glucan using the same, a β-1,3-glucan detection kit, a recombinant microorganism or cell.

[0014] Furthermore, the present invention provides a β-(1,3)(1,4)-glucan-binding polypeptide that does not bind to 13BG and has the property of binding to MLG, a method for detecting β-(1,3)(1,4)-glucan using the same, a β-(1,3)(1,4)-glucan detection kit, and a recombinant microorganism or cell.

Means for Solving the Problems

[0015] In order to solve the above problems, the following β-(1,3)(1,4)-glucan-binding polypeptide, method for detecting β-(1,3)(1,4)-glucan, β-(1,3)(1,4)-glucan detection kit, and β-1,3-glucan-binding polypeptide, method for detecting β-1,3-glucan, β-1,3-glucan detection kit, recombinant microorganism or cell are provided. [1] The following two motif sequences contained in the amino acid sequence W1 of the polypeptide constituting the catalytic domain (CatD) of wild-type β-(1,3)(1,4)-glucanase belonging to the GH16 type enzyme: SEQ ID NO: 44: EXDXE (X represents any amino acid), or SEQ ID NO: 45: EXDXXE (X represents any amino acid) wherein at least one of the two glutamic acids (E) in each of the motif sequences is substituted with another amino acid, and the amino acid sequence contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence W1, and having specific binding activity with β-(1,3)(1,4)-glucan (MLG), β-(1,3)(1,4)-glucan-binding polypeptide. [2] The other amino acid is any one of glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), serine (S), threonine (T), aspartic acid (D), asparagine (N), glutamine (Q), The β-(1,3)(1,4)-glucan-binding polypeptide of [1] above. [3] The amino acid sequence W1 is the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 6. The β-(1,3)(1,4)-glucan-binding polypeptide of [1] or [2] above. [4] A method for detecting β-(1,3)(1,4)-glucan, comprising the step of contacting a test sample with a reagent containing any one of the β-(1,3)(1,4)-glucan-binding polypeptides of [1] to [3] above. A method for detecting β-(1,3)(1,4)-glucan. [5] A reagent containing any one of the β-(1,3)(1,4)-glucan-binding polypeptides of [1] to [3] above. A β-(1,3)(1,4)-glucan detection kit. [6] A recombinant microorganism or cell containing a nucleotide sequence encoding any one of the β-(1,3)(1,4)-glucan-binding polypeptides of [1] to [3] above. [7] The following motif sequence contained in the amino acid sequence W2 of the polypeptide constituting the catalytic domain (CatD) of wild-type β-1,3-glucanase belonging to the GH64 type enzyme: SEQ ID NO: 48: XEXTX (X represents any amino acid) wherein the glutamic acid (E) in the above is substituted with another amino acid, and the amino acid sequence has a sequence identity of 80% or more with the amino acid sequence W2, having specific binding activity with β-1,3-glucan (13BG), A β-1,3-glucan-binding polypeptide. [8] The following motif sequence contained in the amino acid sequence W3 of the polypeptide constituting the catalytic domain (CatD) of wild-type β-1,3-glucanase belonging to the GH81 type enzyme: SEQ ID NO: 49: ESXSE (X represents any amino acid) wherein at least the glutamic acid (E) located on the N-terminal side of the two glutamic acids (E) in the above is substituted with another amino acid, and the amino acid sequence has a sequence identity of 80% or more with the amino acid sequence W3, having specific binding activity with β-1,3-glucan (13BG), A β-1,3-glucan-binding polypeptide. [9] wherein the other amino acid is glutamine (Q), the β-1,3-glucan-binding polypeptide of [7] or [8].

[10] wherein the amino acid sequence W2 is the amino acid sequence represented by SEQ ID NO: 17, the β-1,3-glucan-binding polypeptide of [7].

[11] wherein the amino acid sequence W3 is the amino acid sequence represented by SEQ ID NO: 32, the β-1,3-glucan-binding polypeptide of [8].

[12] comprising a polypeptide constituting a carbohydrate-binding module (CBM) of a wild-type β-1,3-glucanase belonging to the GH81 type enzyme, the β-1,3-glucan-binding polypeptide of [8].

[13] comprising a step of contacting a test sample with a reagent comprising any one of the β-1,3-glucan-binding polypeptides of [7] to

[12] , A method for detecting β-1,3-glucan.

[14] having a reagent comprising any one of the β-1,3-glucan-binding polypeptides of [7] to

[12] , A β-1,3-glucan detection kit.

[15] a recombinant microorganism or cell comprising a nucleotide sequence encoding any one of the β-1,3-glucan-binding polypeptides of [7] to

[12] . [Effect of the Invention]

[0016] The β-(1,3)(1,4)-glucan-binding polypeptide of the present invention does not bind to 13BG and has the property of binding to MLG. The method for detecting β-(1,3)(1,4)-glucan and the β-(1,3)(1,4)-glucan detection kit of the present invention can specifically detect MLG. The β-1,3-glucan-binding polypeptide of the present invention does not have the activity of binding to MLG and has the property of binding to 13BG with various fine structures. The method for detecting β-1,3-glucan and the β-1,3-glucan detection kit of the present invention can detect 13BG with various fine structures. According to the recombinant microorganism or cell of the present invention, the β-(1,3)(1,4)-glucan-binding polypeptide or β-1,3-glucan-binding polypeptide of the present invention can be obtained simply and efficiently.

Brief Description of Drawings

[0017]

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Figure 17

Modes for Carrying Out the Invention

[0018] As enzymes that specifically decompose BG, β-glucanases belonging to EC 3.2.1.6, EC 3.2.1.39, EC 3.2.1.73, etc. are known. Among these, the enzyme belonging to EC 3.2.1.6 is an endo 1,3(4)-β-glucanase, and for example, a part of the enzymes belonging to GH16 by glycoside hydrolase (GH) classification is known and is used as an enzyme that decomposes 13BG and MLG. In addition, the enzyme belonging to EC 3.2.1.73 is an endo-β-(1,3)(1,4)-glucanase, and for example, a part of the enzymes belonging to GH16 is also distributed as a recombinant protein expected to have MLG-degrading activity and is widely used. Furthermore, the enzyme belonging to EC 3.2.1.39 is an endo-β-1,3-glucanase classified into the GH16, GH17, GH64, GH81, GH128 families, etc., and is widely used expecting its activity to decompose 13BG. These glucanases have a catalytic domain (CatD) with BG-degrading activity, and furthermore, may have a carbohydrate binding module (CBM) with binding activity to BG. Therefore, it is considered that the development of a BG-binding polypeptide using only the CBM region is relatively easy.

[0019] In contrast, the present inventors focused on CatD, which is originally used expecting BG-degrading activity, and attempted to modify its function. And it was found that some mutants of endo-β-(1,3)(1,4)-glucanase belonging to GH16 do not have binding activity to 13BG but have strong binding activity to MLG. And it was shown that this β-(1,3)(1,4)-glucanase mutant can be used as a novel MLG-specific binding probe that can be expressed in Escherichia coli, and a method for specifically detecting MLG was developed using this.

[0020] Furthermore, the present inventors found that some mutants of endo-β-1,3-glucanases belonging to GH64 or GH81 do not have binding activity to MLG but have binding activity to 13BG derived from various species. In particular, some mutants of endo-β-1,3-glucanases belonging to GH81, and mutants containing both a mutant CatD and a CBM, were found to have strong binding activity to 13BG derived from various species. Then, it was shown that this β-1,3-glucanase mutant can be used as a novel 13BG-specific binding probe that can be expressed in Escherichia coli, and a 13BG-specific detection method was developed using this probe.

[0021] Hereinafter, one embodiment of the β-(1,3)(1,4)-glucan-binding polypeptide, the method for detecting β-(1,3)(1,4)-glucan, the β-(1,3)(1,4)-glucan detection kit, and the β-1,3-glucan-binding polypeptide, the method for detecting β-1,3-glucan, the β-1,3-glucan detection kit, the recombinant microorganism or cell of the present invention will be described.

[0022] <β-(1,3)(1,4)-Glucan-Binding Polypeptide> The β-(1,3)(1,4)-glucan-binding polypeptide of the present invention does not exhibit degrading activity against β-(1,3)(1,4)-glucan and has specific binding activity to β-(1,3)(1,4)-glucan.

[0023] The β-(1,3)(1,4)-glucan-binding polypeptide of the present invention includes mutants in which a predetermined position of the amino acid sequence of the polypeptide constituting the catalytic domain (CatD) of wild-type β-(1,3)(1,4)-glucanase belonging to the GH16 type enzyme (hereinafter sometimes referred to as "amino acid sequence W1" or "wild-type amino acid sequence W1") is substituted with another amino acid.

[0024] In one aspect, the polypeptide having the catalytic region CatD of endo-β-(1,3)(1,4)-glucanase is selected from the group consisting of β-(1,3)(1,4)-glucanases having a catalytic region (CatD) characterized by the GH16 type enzyme family. Examples of such β-(1,3)(1,4)-glucanases include the amino acid sequence (GenBank Accession number; CAA86922.1) of β-(1,3)(1,4)-glucanase (EC 3.2.1.73) derived from Bacillus subtilis.

[0025] Examples of the wild-type sequence of the catalytic region CatD of β-(1,3)(1,4)-glucanase include the amino acid sequences (SEQ ID NO: 2, 6) of β-(1,3)(1,4)-glucanase CatD of Bacillus bacteria.

[0026] As exemplified in SEQ ID NO: 1, 2, 5, 6, in the amino acid sequence W1 of wild-type β-(1,3)(1,4)-glucanases from different origins, EXDXE (SEQ ID NO: 44), or EXDXXE (SEQ ID NO: 45) Any of the motif sequences represented by is commonly included at the corresponding position of each wild-type amino acid sequence W1. Here, "X" in the motif sequences represented by SEQ ID NO: 44, 45 represents any amino acid.

[0027] And in the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention, at least one (one or two) of the two glutamic acids (E) in the motif sequence (EXDXE (SEQ ID NO: 44), or EXDXXE (SEQ ID NO: 45)) contained in the amino acid sequence W1 is substituted with another amino acid.

[0028] The amino acid substituted for glutamic acid (E) in the motif sequences represented by SEQ ID NOs: 44 and 45 is not particularly limited, but is preferably glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), serine (S), threonine (T), aspartic acid (D), asparagine (N), glutamine (Q), and more preferably alanine (A), methionine (M), serine (S), threonine (T), aspartic acid (D), asparagine (N), glutamine (Q).

[0029] Furthermore, taking the amino acid sequences of wild-type β-(1,3)(1,4)-glucanases (Bacteroides ovatus, Bacillus subtilis) represented by SEQ ID NOs: 1 and 5 as examples, the amino acid substitution positions in one form of the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention will be described. SEQ ID NOs: 1 and 5 are the full-length amino acid sequences of wild-type β-(1,3)(1,4)-glucanases.

[0030] In one form of the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention, the amino acid substitution position with respect to the wild-type amino acid sequence W1 of CatD of the wild-type β-(1,3)(1,4)-glucanase belonging to the GH16 type enzyme is at least one of positions 143 and 148 of the glutamic acid (E) in the full-length amino acid sequence represented by SEQ ID NO: 1. That is, the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention can also be described as having at least one of the amino acids (glutamic acid (E)) at positions corresponding to positions 143 and 148 based on the amino acid sequence shown in SEQ ID NO: 1 substituted in the amino acid sequence of CatD.

[0031] Similarly, the amino acid substitution positions of the wild-type β-(1,3)(1,4)-glucanase belonging to the GH16 type enzyme for the wild-type amino acid sequence W1 of CatD are at least one of the glutamic acids (E) at positions 133 and 137 of the full-length amino acid sequence represented by SEQ ID NO: 5. That is, the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention can also be described as having at least one of the amino acids (glutamic acid (E)) at positions corresponding to positions 133 and 137 when the amino acid sequence shown in SEQ ID NO: 5 is used as a reference in the amino acid sequence of CatD being substituted.

[0032] 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 BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) sequence analysis software or the like.

[0033] Furthermore, the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention includes an amino acid sequence having 80% or more sequence identity with the wild-type amino acid sequence W1 (hereinafter sometimes referred to as "amino acid sequence X1"). That is, the amino acid sequence of the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention may have substitutions, deletions, insertions, additions, or combinations thereof of one or more amino acid residues, other than the substitution positions (glutamic acid (E)) in the motif sequences of SEQ ID NOs: 44 and 45. When the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention includes substitutions, deletions, or insertions of amino acid residues, other than the substitution positions (glutamic acid (E)) in the motif sequences of SEQ ID NOs: 44 and 45, the amino acid sequence X1 may have 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more sequence identity with the wild-type amino acid sequence W1 (for example, SEQ ID NOs: 2 and 6). Also, as described above, the amino acid sequence of the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention only needs to include an amino acid sequence X1 in which the glutamic acid (E) in the motif sequences of SEQ ID NOs: 44 and 45 contained in the wild-type amino acid sequence W1 is substituted with another amino acid and has 80% or more sequence identity with the amino acid sequence W1. As long as the function of the β-(1,3)(1,4)-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 X1 are also included. Specifically, for example, the β-(1,3)(1,4)-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 X1.

[0034] More specifically, with respect to the amino acid sequence of the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention, when amino acid residues are substituted, deleted, inserted, or added at positions other than the two glutamic acids (E) of the motif sequences of SEQ ID NOs: 44 and 45 with respect to the wild-type amino acid sequence W1, the number of such amino acid residues is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. Further, in one form of the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention, when, for example, an amino acid sequence related to a heterologous protein is added to the end of the amino acid sequence X1, the added amino acid residues may be, for example, 5 to 100, 5 to 1000, or more.

[0035] In one form of the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention, it is preferable to include, for example, the amino acid sequences represented by SEQ ID NOs: 3, 4, 7, and 8.

[0036] The polypeptides represented by SEQ ID NOs: 3, 4, 7, and 8 are examples in which the glutamic acid (E) in the motif sequences of SEQ ID NOs: 44 and 45 is substituted with glutamine (Q) and has no other mutations (substitutions, deletions, insertions, additions of amino acid residues).

[0037] The method for producing the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention is not particularly limited. The β-(1,3)(1,4)-glucan-binding polypeptide of the present invention can be produced by conventionally known methods such as, for example, a method using cell engineering techniques or a method of synthesizing using a peptide synthesizer.

[0038] The recombinant microorganism or cell of the present invention contains a nucleotide sequence encoding the β-(1,3)(1,4)-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 β-(1,3)(1,4)-glucan-binding polypeptide of the present invention can be efficiently obtained.

[0039] The β-(1,3)(1,4)-glucan-binding polypeptide of the present invention does not have the activity of degrading MLG. And the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention does not have the binding activity to 13BG and has excellent specific binding activity to MLG. Therefore, the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention can detect MLG simply and accurately. Further, the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention can detect MLG regardless of its properties such as purification purity and solubility / insolubility.

[0040] <Method for Detecting β-(1,3)(1,4)-Glucan and Kit for Detecting β-(1,3)(1,4)-Glucan> The method for detecting β-(1,3)(1,4)-glucan of the present invention includes the step of bringing a test sample into contact with a reagent containing the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention. The method for detecting β-(1,3)(1,4)-glucan of the present invention includes various forms of detection methods that utilize the specific binding activity of the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention.

[0041] The conditions such as temperature and pH in the above step of the method for detecting β-(1,3)(1,4)-glucan of the present invention are not particularly limited. For example, in one embodiment of the method for detecting β-(1,3)(1,4)-glucan of the present invention, from the perspective of the binding activity of the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention, for example, the temperature may be 90°C or lower, preferably 40°C or lower. From the same perspective, for example, the pH may be in the range of 4.8 to 8.0.

[0042] In the present invention, the term "detection" is intended to include the concept of "quantification". Specifically, the method for detecting β-(1,3)(1,4)-glucan may be a method for measuring the amount of β-(1,3)(1,4)-glucan contained in a test sample.

[0043] One form of the method for detecting β-(1,3)(1,4)-glucan of the present invention includes the following steps: (i) contacting a test sample containing β-(1,3)(1,4)-glucan (MLG) with a reagent to obtain a product formed by the test sample and the reagent, and (ii) detecting the product formed in step (i). It includes.

[0044] The reagent contains the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention. Further, the reagent preferably contains a reporter molecule.

[0045] In step (i), the test sample and the reagent are contacted. The test sample includes an unpurified test sample, a purified test sample, or a crudely purified test sample. The test sample containing MLG may be soluble or insoluble in water.

[0046] The origin of MLG contained in the test sample is not particularly limited, and may be, for example, cell walls, extracts, β-(1,3)(1,4)-glucan secreted outside the cells of fungi, basidiomycetes such as mushrooms, bacteria, yeast, lichens, etc. It may be artificially synthesized MLG. Furthermore, it may be MLG present in environments such as indoors, soil, rivers, seawater, the atmosphere, outer space, etc.

[0047] 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 included. Examples of luminescence include fluorescence and phosphorescence, and typically fluorescence emission is used for detection. When the reporter molecule exhibits luminescence, examples of such a molecule include fluorescent dyes, luminescent proteins (such as luciferase and fluorescent proteins), and proteins that catalyze the luminescence reaction of the molecule. When the reporter molecule exhibits radiation, examples of such a molecule include radioisotopes. When the reporter molecule exhibits color development, examples of such a molecule 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.

[0048] In one form of the method for detecting β-(1,3)(1,4)-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 bind to each other to form a pair.

[0049] Specifically, the first and second split reporter molecules do not exhibit the function as a reporter molecule when they exist separately, but when these split reporter molecules are close to or bind to each other, they come to exhibit the function as 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 bind to each other.

[0050] The split reporter molecule may be configured such that when the first and second split reporter molecules are close to or bind to each other, structural complementarity is promoted and an active reporter molecule is formed.

[0051] Also, the reporter molecule is preferably a reporter protein.

[0052] 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 bind 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 bind to each other to cause BRET or FRET. 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.

[0053] 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.

[0054] 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 modified forms of these enzymes. Examples of fluorescent proteins include, for example, green fluorescent protein (GFP) and modified forms of GFP.

[0055] Also, in one form of the method for detecting β-(1,3)(1,4)-glucan of the present invention, the reagent is (1) a first fusion protein comprising a first polypeptide selected from the β-(1,3)(1,4)-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 β-(1,3)(1,4)-glucan-binding polypeptides of the present invention and a second split reporter molecule, wherein the first split reporter molecule and the second split reporter molecule can form an active reporter molecule as a pair, In step (i), β-(1,3)(1,4)-glucan (MLG) binds to the first fusion protein and the second fusion protein, thereby forming an active reporter molecule with the first split reporter molecule and the second split reporter molecule. In step (ii), the active reporter molecule formed in step (i) is detected.

[0056] In this case, when fusing the β-(1,3)(1,4)-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 can be those similar to the amino acid sequences of general 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: 46), and an EAAAK linker consisting of a repeating sequence of EAAAK (SEQ ID NO: 47) 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 β-(1,3)(1,4)-glucan-binding polypeptide. Also, a fusion protein fused to both the N-terminal side and the C-terminal side may be used.

[0057] In this specification, regarding the split reporter molecule and the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention, "fused" includes not only the mode in which the split reporter molecule is directly linked to the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention, but also the mode in which the split reporter molecule is arthroscopically linked to the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention via another molecule.

[0058] Furthermore, a fusion protein can also be used by binding a split reporter molecule to an antibody (e.g., His-tag antibody) that detects the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention. In this case, examples of the split reporter molecule include luciferase.

[0059] 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 C-terminal side of a 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 C-terminal side of a second polypeptide.

[0060] 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 a 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 a 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 a 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 a second polypeptide.

[0061] In yet another aspect, · The first fusion protein is a fusion protein in which a first split reporter molecule is fused to both the N-terminal side and C-terminal side of a first polypeptide, and / or · The second fusion protein is a fusion protein in which a second split reporter molecule is fused to both the N-terminal side and C-terminal side of a second polypeptide.

[0062] In step (i), β-(1,3)(1,4)-glucan binds to the first fusion protein and the second fusion protein, thereby forming an active reporter molecule with 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.

[0063] 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, when the activity of the formed active reporter protein is high compared to the absence of β-(1,3)(1,4)-glucan, it indicates that β-(1,3)(1,4)-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, when the fluorescence emitted from the fluorescent substance is high compared to the absence of β-(1,3)(1,4)-glucan, it indicates that β-(1,3)(1,4)-glucan is present in the test sample.

[0064] As described later in the examples, the difference in activity (background) between the inactivated reporter protein observed in the absence of β-(1,3)(1,4)-glucan having the target structure and the activity of the reporter protein reconstituted in the presence of a sufficient amount of β-(1,3)(1,4)-glucan shows high sensitivity of about 30 times or more.

[0065] 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 methods for quantitatively analyzing an image detected by a luminometer, a spectrophotometer, a fluorometer, a flow cytometer, a multiplex, chromatography, a CCD camera, etc. with a computer, and methods for quantitatively analyzing by identifying physical contact with a sensor chip.

[0066] In steps (i) and (ii), the reagent containing the first fusion protein and the second fusion protein can be directly supplied into a test tube containing a test specimen, or it can be carried out using cells expressing these fusion proteins or the like. 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.

[0067] 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, etc. Examples of the cells used here include animal cells, insect cells, and plant cells, and established cell lines are easy to use and preferred.

[0068] In addition, in one form of the method for detecting β-(1,3)(1,4)-glucan of the present invention, for example, it can be carried out according to known methods such as direct adsorption method, sandwich method, competitive method, etc. in ELISA using the β-(1,3)(1,4)-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. Further, 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 or luminescence generated by the enzyme reaction can be measured using an absorptiometer, luminometer, etc. Furthermore, for example, when ALP is used as the labeling substance, a luminescent substrate such as 3-(4-methoxypyro(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.

[0069] The β-(1,3)(1,4)-glucan detection kit of the present invention has a reagent containing the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention.

[0070] The reagent can contain a reporter molecule, a labeling substance, etc.

[0071] Regarding the details of the reagents, fusion proteins, and reporter molecules in the detection kit, they are the same as those described in the detection method.

[0072] In addition, the detection kit can include various materials, devices, etc. for the detection of β-(1,3)(1,4)-glucan.

[0073] According to the method for detecting β-(1,3)(1,4)-glucan or the detection kit for β-(1,3)(1,4)-glucan of the present invention, it is possible to detect MLG contained in a test sample with high throughput without requiring complicated washing operations or the like.

[0074] Also, according to the method for detecting β-(1,3)(1,4)-glucan or the detection kit for β-(1,3)(1,4)-glucan of the present invention, it is possible to estimate the MLG content as a functional molecule in food. In addition, an increase in the blood concentration of MLG due to aspiration can be observed.

[0075] Furthermore, as described above, by applying the β-(1,3)(1,4)-glucan-binding polypeptide of the present invention to an analytical method such as ELISA, a tool for easily detecting MLG can be provided. Also, by using the method for detecting β-(1,3)(1,4)-glucan or the detection kit for β-(1,3)(1,4)-glucan of the present invention, by quantifying the concentration of MLG contained in the blood, secretions, organs, washing liquids obtained by washing organs or tissues (for example, bronchoalveolar lavage fluid, oral washing liquid, and peritoneal washing liquid) 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 the possibility of aspiration.

[0076] The β-(1,3)(1,4)-glucan-binding polypeptide, the method for detecting β-(1,3)(1,4)-glucan, the β-(1,3)(1,4)-glucan detection kit, recombinant microorganism or cell of the present invention are not limited to the above embodiments.

[0077] <β-1,3-glucan-binding polypeptide> The β-1,3-glucan-binding polypeptide of the present invention does not exhibit β-1,3-glucan-degrading activity and has specific binding activity with β-1,3-glucan.

[0078] The polypeptide having the catalytic region CatD of β-1,3-glucanase is selected from the group consisting of β-1,3-glucanases having a catalytic domain (CatD) characterized by the GH64 type or GH81 enzyme family.

[0079] (First Embodiment) In one form of the β-1,3-glucan-binding polypeptide of the present invention, it includes a mutant in which a predetermined position of the amino acid sequence of the polypeptide constituting the catalytic domain (CatD) of the wild-type β-1,3-glucanase belonging to the GH64 type enzyme (hereinafter sometimes referred to as "amino acid sequence W2" or "wild-type amino acid sequence W2") is substituted with another amino acid.

[0080] As the full-length wild-type amino acid sequence of the GH64 type β-1,3-glucanase, for example, the amino acid sequence (SEQ ID NO: 16) of β-1,3-glucanase of bacteria belonging to the genus Paenibacillus can be exemplified.

[0081] SEQ ID NOs: 17, 20, 23, 26, 29 are examples of the wild-type amino acid sequence W2 of the polypeptide constituting the catalytic domain (CatD) of the wild-type β-1,3-glucanase belonging to the GH64 type enzyme.

[0082] As exemplified in SEQ ID NOs: 17, 20, 23, 26, 29, in the wild-type amino acid sequence W2 of the catalytic domain (CatD) of wild-type β-1,3-glucanases from different origins, the motif sequence represented by XEXTX (SEQ ID NO: 48) is commonly included at the corresponding position of the wild-type amino acid sequence W2. Here, "X" in the motif sequence represented by SEQ ID NO: 48 represents any amino acid.

[0083] Moreover, in the β-1,3-glucan-binding polypeptide of the present invention, the glutamic acid (E) in the motif sequence: XEXTX (SEQ ID NO: 48) contained in the wild-type amino acid sequence W2 is substituted with another amino acid.

[0084] The amino acid substituted for glutamic acid (E) in the motif sequence represented by SEQ ID NO: 48 is not particularly limited, but for example, glutamine (Q) is preferable.

[0085] Furthermore, taking the amino acid sequence of the wild-type β-1,3-glucanase represented by SEQ ID NO: 16 as an example, the amino acid substitution positions in one form of the β-1,3-glucan-binding polypeptide of the present invention will be described. SEQ ID NO: 16 is the full-length amino acid sequence of the wild-type β-1,3-glucanase.

[0086] In one form of the β-1,3-glucan-binding polypeptide of the present invention, the amino acid substitution position with respect to the wild-type amino acid sequence W2 of CatD of the wild-type β-1,3-glucanase belonging to the GH64 type enzyme is the 264th position of the full-length amino acid sequence represented by SEQ ID NO: 16. That is, it can also be said that in the amino acid sequence of CatD of the β-1,3-glucan-binding polypeptide of the present invention, the amino acid (glutamic acid (E)) at the position corresponding to the 264th position based on the amino acid sequence shown in SEQ ID NO: 16 is substituted.

[0087] Furthermore, the β-1,3-glucan-binding polypeptide of this embodiment includes an amino acid sequence (hereinafter sometimes referred to as "amino acid sequence X2") having a sequence identity of 80% or more with the wild-type amino acid sequence W2. That is, the amino acid sequence of the β-1,3-glucan-binding polypeptide of the present invention may have substitutions, deletions, insertions, additions, or combinations thereof of one or more amino acid residues, in addition to the substitution position (glutamic acid (E)) in the motif sequence of SEQ ID NO: 48. In the β-1,3-glucan-binding polypeptide of this embodiment, in addition to the substitution position (glutamic acid (E)) in the motif sequence of SEQ ID NO: 48, when including substitutions, deletions, or insertions of amino acid residues, the amino acid sequence X2 may have a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more with the wild-type amino acid sequence W2 (for example, SEQ ID NO: 17). Also, as described above, the amino acid sequence of the β-1,3-glucan-binding polypeptide of this embodiment only needs to include an amino acid sequence X2 in which the glutamic acid (E) of the motif sequence of SEQ ID NO: 48 contained in the wild-type amino acid sequence W2 is substituted with another amino acid and which has a sequence identity of 80% or more with the wild-type amino acid sequence W2. As long as the function of the β-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 X2 are also included. Specifically, for example, the β-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 X2.

[0088] More specifically, when amino acid residues are substituted, deleted, inserted, or added to the amino acid sequence of the β-1,3-glucan-binding polypeptide of this embodiment at positions other than glutamic acid (E) in the motif sequence of SEQ ID NO: 48 with respect to the wild-type amino acid sequence W2, the number of such amino acid residues is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. Further, in the β-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 number of added amino acid residues may be, for example, 5 to 100, 5 to 1000, or more.

[0089] In one form of the β-1,3-glucan-binding polypeptide of the present invention, for example, it preferably contains the amino acid sequence represented by SEQ ID NO: 18. In this case, it shows excellent detection sensitivity particularly for SPG.

[0090] The polypeptide represented by SEQ ID NO: 18 is an example in which glutamic acid (E) in the motif sequence represented by SEQ ID NO: 48 is substituted with glutamine (Q) in the wild-type amino acid sequence represented by SEQ ID NO: 17, and has no other mutations (substitution, deletion, insertion, addition of amino acid residues).

[0091] The method for producing the β-1,3-glucan-binding polypeptide of the present invention is not particularly limited. The β-1,3-glucan-binding polypeptide of the present invention can be produced by conventionally known methods such as a method using cell engineering techniques or a method of synthesizing using a peptide synthesizer.

[0092] The recombinant microorganism or cell of the present invention contains a nucleotide sequence encoding the β-1,3-glucan-binding polypeptide of the present invention. Here, the cell refers to a cell line widely and commonly 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 β-1,3-glucan-binding polypeptide of the present invention can be efficiently obtained.

[0093] The β-1,3-glucan-binding polypeptide of this embodiment does not have degrading activity against 13BG, and the β-1,3-glucan-binding polypeptide of the present invention does not have binding activity against MLG but has binding activity against various 13BGs. Therefore, the β-1,3-glucan-binding polypeptide of the present invention can simply and accurately detect β-1,3-glucan. Further, the β-1,3-glucan-binding polypeptide of the present invention can detect 13BG regardless of its properties such as purification purity and solubility / insolubility.

[0094] <Second Embodiment>

[0095] In one form of the β-1,3-glucan-binding polypeptide of the present invention, it includes a mutant in which a predetermined position of the amino acid sequence of the polypeptide constituting the catalytic domain (CatD) of the wild-type β-1,3-glucanase belonging to the GH81 type enzyme (hereinafter, may be referred to as "amino acid sequence W3" or "wild-type amino acid sequence W3") is substituted with another amino acid.

[0096] Examples of the wild-type amino acid sequence of the GH81 type β-1,3-glucanase include, for example, the wild-type amino acid sequence (SEQ ID NO: 31) of the β-1,3-glucanase of bacteria belonging to the genus Alkalihalobacillus. Examples of the wild-type sequence of the catalytic region CatD of the GH81 type β-1,3-glucanase include, for example, the wild-type amino acid sequence W3 (SEQ ID NO: 32) of the polypeptide constituting the β-1,3-glucanase CatD of bacteria belonging to the genus Alkalihalobacillus.

[0097] As exemplified in SEQ ID NO: 32, in the wild-type amino acid sequence W3 of the wild-type β-1,3-glucanase, ESXSE (SEQ ID NO: 49) the motif sequence represented by is commonly included at the corresponding position of the wild-type amino acid sequence W3. Here, "X" in the motif sequence represented by SEQ ID NO: 49 represents any amino acid.

[0098] Furthermore, in the β-1,3-glucan-binding polypeptide of the present invention, at least the glutamic acid (E) on the N-terminal side among the two glutamic acids in the motif sequence: ESXSE (SEQ ID NO: 49) contained in the wild-type amino acid sequence W3 is substituted with another amino acid. In addition, among the two glutamic acids (E) in ESXSE (SEQ ID NO: 49), the glutamic acid (E) on the C-terminal side together with the glutamic acid (E) on the N-terminal side may also be substituted with another amino acid.

[0099] The amino acid substituted for the glutamic acid (E) in the motif sequence represented by SEQ ID NO: 49 is not particularly limited, but for example, glutamine (Q) is preferable.

[0100] Furthermore, taking the wild-type β-1,3-glucanase amino acid sequence represented by SEQ ID NO: 31 as an example, the amino acid substitution position in one form of the β-1,3-glucan-binding polypeptide of the present invention will be described. SEQ ID NO: 31 is the amino acid sequence of the full length (including CBM) of a wild-type β-1,3-glucanase (Alkalihalobacillus halodurans) belonging to the GH81 type enzyme.

[0101] In one form of the β-1,3-glucan-binding polypeptide of the present invention, the amino acid substitution position with respect to the amino acid sequence W3 of the CatD of the wild-type β-1,3-glucanase belonging to the GH81 type enzyme is position 542 of the full-length amino acid sequence represented by SEQ ID NO: 31. That is, it can also be said that in the amino acid sequence of CatD of the β-1,3-glucan-binding polypeptide of the present invention, the amino acid (glutamic acid (E)) at the position corresponding to position 542 based on the amino acid sequence shown in SEQ ID NO: 31 is substituted.

[0102] Furthermore, the β-1,3-glucan-binding polypeptide of this embodiment includes an amino acid sequence having 80% or more sequence identity with the wild-type amino acid sequence W3 (hereinafter sometimes referred to as "amino acid sequence X3"). That is, the amino acid sequence of the β-1,3-glucan-binding polypeptide of this embodiment may have substitutions, deletions, insertions, additions, or combinations thereof of one or more amino acid residues, other than the substitution position (glutamic acid (E)) in the motif sequence represented by SEQ ID NO: 49. When the β-1,3-glucan-binding polypeptide of this embodiment includes substitutions, deletions, or insertions of amino acid residues, other than the substitution position (glutamic acid (E)) in the motif sequence represented by SEQ ID NO: 49, the amino acid sequence X3 may have 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more sequence identity with the wild-type amino acid sequence W3 (for example, SEQ ID NO: 32). Also, as described above, the amino acid sequence of the β-1,3-glucan-binding polypeptide of this embodiment only needs to include an amino acid sequence X3 in which the glutamic acid (E) of the motif sequence of SEQ ID NO: 49 included in the wild-type amino acid sequence W3 is substituted with another amino acid and which has 80% or more sequence identity with the amino acid sequence W. As long as the function of the β-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 X3 are also included. Specifically, for example, the β-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 X3.

[0103] More specifically, when amino acid residues are substituted, deleted, inserted, or added to the amino acid sequence of the β-1,3-glucan-binding polypeptide of the present invention at positions other than glutamic acid (E) in the motif sequence represented by SEQ ID NO: 49 with respect to the wild-type amino acid sequence W3, the number of such amino acid residues is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. Further, in the β-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 X3, the number of added amino acid residues may be, for example, 5 to 100, 5 to 1000, or more.

[0104] Also, the β-1,3-glucan-binding polypeptide of this embodiment preferably contains a carbohydrate-binding module (CBM) of a wild-type β-1,3-glucanase belonging to the GH81 type enzyme. That is, the β-1,3-glucan-binding polypeptide of this embodiment preferably contains the mutant CatD of the above-described β-1,3-glucanase and the amino acid sequence of the carbohydrate-binding module (CBM). In the β-1,3-glucan-binding polypeptide of this embodiment, the amino acid sequence of the carbohydrate-binding module (CBM) is, for example, in the range of positions 791 to 924, or positions 925 to 1020, of the amino acid sequence represented by SEQ ID NO: 31.

[0105] In one form of the β-1,3-glucan-binding polypeptide of this embodiment, for example, it preferably contains the amino acid sequence represented by SEQ ID NO: 33 or SEQ ID NO: 34.

[0106] The polypeptides represented by SEQ ID NOs: 33 and 34 are examples in which glutamic acid (E) in the motif sequence of SEQ ID NO: 49 is substituted with glutamine (Q) and has no other mutations (substitution, deletion, insertion, addition of amino acid residues).

[0107] The method for producing the β-1,3-glucan-binding polypeptide of the present invention is not particularly limited. The β-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.

[0108] The recombinant microorganism or cell of the present invention contains a nucleotide sequence encoding the β-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 β-1,3-glucan-binding polypeptide of the present invention can be efficiently obtained.

[0109] The β-1,3-glucan-binding polypeptide of this embodiment does not have degrading activity against 13BG, and the β-1,3-glucan-binding polypeptide of this embodiment does not have binding activity against MLG and has binding activity against various 13BG. Therefore, the β-1,3-glucan-binding polypeptide of this embodiment can simply and accurately detect β-1,3-glucan. In addition, the β-1,3-glucan-binding polypeptide of this embodiment can detect 13BG regardless of properties such as purification purity and solubility / insolubility. Further, when the β-1,3-glucan-binding polypeptide of this embodiment contains the mutant CatD of the above-described β-1,3-glucanase and the amino acid sequence of the carbohydrate-binding module (CBM), it can detect a wider variety of 13BG more efficiently compared to the form without the carbohydrate-binding module (CBM).

[0110] Regarding the method for detecting β-1,3-glucan, the β-1,3-glucan detection kit, the recombinant microorganism or cell of the present invention, they can include forms similar to the embodiments of the method for detecting β-(1,3)(1,4)-glucan and the β-(1,3)(1,4)-glucan detection kit of the present invention described above. For example, in one aspect of the method for detecting β-1,3-glucan of the present invention, from the perspective of the binding activity of the β-1,3-glucan-binding polypeptide of the present invention, for example, the temperature may be 50°C or lower. From the same perspective, for example, the pH may be in the range of 4.8 to 8.0. Other detailed descriptions regarding the method for detecting β-1,3-glucan, the β-1,3-glucan detection kit, the recombinant microorganism or cell of the present invention are omitted.

[0111] Note that as will be described later in the examples, in one form of the method for detecting β-1,3-glucan and the β-1,3-glucan detection kit of the present invention, the difference between the activity (background) of the inactivated reporter protein observed in the absence of β-1,3-glucan having the target structure and the activity of the reporter protein reconstituted in the presence of a sufficient amount of β-1,3-glucan shows high sensitivity of about 400 times or more.

[0112] The method for detecting β-1,3-glucan includes a step of contacting a test sample with a reagent containing the β-1,3-glucan-binding polypeptide of the present invention.

[0113] The β-1,3-glucan detection kit of the present invention has a reagent containing the β-1,3-glucan-binding polypeptide of the present invention.

[0114] The recombinant microorganism or cell of the present invention contains a nucleotide sequence encoding the β-1,3-glucan-binding polypeptide of the present invention.

[0115] The β-(1,3)(1,4)-glucan-binding polypeptide, method for detecting β-(1,3)(1,4)-glucan, β-(1,3)(1,4)-glucan detection kit, β-1,3-glucan-binding polypeptide, method for detecting β-1,3-glucan, β-1,3-glucan detection kit, recombinant microorganism or cell of the present invention are not limited to the above embodiments.

Example

[0116] Hereinafter, the present invention will be described more specifically together with examples, but the present invention is not limited to these examples at all.

[0117] <1> Materials and methods (1) Materials Barley BG (β-(1,3)(1,4)-glucan: MLG), Laminarin (β-1,3-D-glucan), mannan derived from Saccharomyces cerevisiae (α-1,6- / α-1,2-mannan, α-1,3-mannan), bovine serum albumin (BSA), and imidazole were purchased from Sigma-Aldrich. Yeast BG and xylan derived from Birchwood (β-1,4-xylan) were obtained from Megazyme, and SPG (Schizophyllan) was obtained from Kaken Pharmaceutical Co., Ltd. Dextran T10 (α-1,4- / α-1,6-glucan) was from Pharmacia. D-PBS(-) (Dulbecco's phosphate-buffered solution), Tween 20, Starch (α-1,6- / α-1,4-glucan), Pachyman (β-1,3-D-glucan), ethylene glycol chitin (EG-Chitin, water-soluble poly-β-1,4-N-acetylglucosamine), hydroxyethyl cellulose (HE-Cellulose, water-soluble β-1,4-glucan), and heparin sodium were obtained from FUJIFILM Wako Pure Chemical Corporation. 0.5 mol / L-EDTA Solution (pH 8.0) was purchased from Nacalai Tesque. Pullulan (β-1,6-D-glucan) was purchased from InvivoGen. Disaccharide (Laminaribiose) in which two molecules of glucose are linked by β-1,3 bond, trisaccharide (Laminaritriose) with three molecules linked, tetrasaccharide (Laminaritetraose) with four molecules linked, pentasaccharide (Laminaripentaose) with five molecules linked, hexasaccharide (Laminarihexaose) with six molecules linked, and heptasaccharide (Laminariheptaose) with seven molecules linked were obtained from Seikagaku Corporation. 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. Commercial products were used for staple bread (edible bread), white rice (packaged cooked rice), and dried short pasta (fusilli).

[0118] (2) Plasmid construction The pCold I DNA vector (Takara Bio, GenBank: AB186388.1) was used with the nucleotide sequences of 1 - 300 and 361 - 4407, into which a DNA sequence encoding a GS linker peptide (Gly - Gly - Ser - Gly - Gly - Gly - Ser - Gly - Gly - Ser - Gly sequence) was inserted between 300 and 361. 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. On the C - terminal side of the GS linker peptide, DNA sequences encoding the following respective amino acid sequences were inserted to construct plasmids for preparing HiBiT - tagged fusion proteins. (1) BoMLG - WT (SEQ ID NO: 2), an endo - 1,3(4) - β - glucanase (EC 3.2.1.6) belonging to GH16, and its variants, namely GH16 - BoMLG - E143Q (SEQ ID NO: 3) and GH16 - BoMLG - E148Q (SEQ ID NO: 4) having a mutation (Gln) at the position corresponding to the 143rd or 148th position in the amino acid sequence of SEQ ID NO: 1 (2) GH16 - LicS - WT (SEQ ID NO: 6), an endo - β-(1,3)(1,4) - glucanase (EC 3.2.1.73) belonging to GH16, and its variants, namely GH16 - LicS - E133Q (SEQ ID NO: 7) and GH16 - LicS - E137Q (SEQ ID NO: 8) having a mutation (Gln) at the position corresponding to the 133rd or 137th position in the amino acid sequence of SEQ ID NO: 5 (3) GH17 - CJP38 - WT (SEQ ID NO: 10), an endo - β - 1,3 - glucanase (EC 3.2.1.39) belonging to GH17, and its variants, namely GH17 - CJP38 - E124Q (SEQ ID NO: 11) and GH17 - CJP38 - E267Q (SEQ ID NO: 12) having a mutation (Gln) at the position corresponding to the 124th or 267th position in the amino acid sequence of SEQ ID NO: 9 (4) The catalytic domain of endo-β-1,3-glucanase (EC 3.2.1.39) belonging to GH16, GH16-ZgLamC-CatD-WT (SEQ ID NO: 14), and variants thereof, namely GH16-ZgLamC-CatD-E142Q (SEQ ID NO: 15) having a mutation (Gln) at the position corresponding to the 142nd position in the amino acid sequence of SEQ ID NO: 13, (5) Endo-β-1,3-glucanase (EC 3.2.1.39) belonging to GH64, GH64-Pb-WT (SEQ ID NO: 17), and variants thereof, namely GH64-Pb-E264Q (SEQ ID NO: 18) having a mutation (Gln) at the position corresponding to the 264th position in the amino acid sequence of SEQ ID NO: 16, (6) Endo-β-1,3-glucanase (EC 3.2.1.39) belonging to GH64, GH64-Sm-WT (SEQ ID NO: 20), and variants thereof, namely GH64-Sm-E154Q (SEQ ID NO: 21) having a mutation (Gln) at the position corresponding to the 154th position in the amino acid sequence of SEQ ID NO: 19, (7) Endo-β-1,3-glucanase (EC 3.2.1.39) belonging to GH64, GH64-Kf-WT (SEQ ID NO: 23), and variants thereof, namely GH64-Kf-E149Q (SEQ ID NO: 24) having a mutation (Gln) at the position corresponding to the 149th position in the amino acid sequence of SEQ ID NO: 22, (8) The catalytic domain of endo-β-1,3-glucanase (EC 3.2.1.39) belonging to GH64, GH64-Art-CatD-WT (SEQ ID NO: 26), and variants thereof, namely GH64-Art-CatD-E153Q (SEQ ID NO: 27) having a mutation (Gln) at the position corresponding to the 153rd position in the amino acid sequence of SEQ ID NO: 25, (9) Endo-β-1,3-glucanase (EC 3.2.1.39) belonging to GH64, GH64-Le-WT (SEQ ID NO: 29), and variants thereof, namely GH64-Le-E145Q (SEQ ID NO: 30) having a mutation (Gln) at the position corresponding to the 145th position in the amino acid sequence of SEQ ID NO: 28, (10) GH81-BH0236-WT (SEQ ID NO: 32), an endo-β-1,3-glucanase (EC 3.2.1.39) belonging to GH81, and variants thereof, namely GH81-BH0236-E542Q (SEQ ID NO: 33) having a mutation (Gln) at the position corresponding to the 542nd position in the amino acid sequence of SEQ ID NO: 31, and GH81-BH0236-CatD-E542Q (SEQ ID NO: 34), a variant of its catalytic domain, (11) GH128-I-Am-WT (SEQ ID NO: 36), an endo-β-1,3-glucanase (EC 3.2.1.39) belonging to GH128, and variants thereof, namely GH128-I-Am-E199Q (SEQ ID NO: 37) having a mutation (Gln) at the position corresponding to the 199th position in the amino acid sequence of SEQ ID NO: 35, (12) GH128-II-Pv-WT (SEQ ID NO: 39), an endo-β-1,3-glucanase (EC 3.2.1.39) belonging to GH128, and variants thereof, namely GH128-II-Pv-E214Q (SEQ ID NO: 40) having a mutation (Gln) at the position corresponding to the 214th position in the amino acid sequence of SEQ ID NO: 38, (13) GH128-III-Bg-WT (SEQ ID NO: 42), an endo-β-1,3-glucanase (EC 3.2.1.39) belonging to GH128, and variants thereof, namely GH128-III-Bg-E208Q (SEQ ID NO: 43) having a mutation (Gln) at the position corresponding to the 208th position in the amino acid sequence of SEQ ID NO: 41, (14) An artificial protein (sBGRP) prepared based on the N-terminal region of an insect-derived β-glucan recognition protein (BGRP) developed in the past (Patent Document 5)

[0119] Similarly, a pCold I DNA vector was prepared by inserting a DNA fragment encoding an SmBiT tag, which is one of the C-terminal subunits (11 amino acid residues) of NanoLuc, into the N-terminal side of the GS linker peptide. On the C-terminal side of the GS linker peptide GH16-LicS-E137Q (SEQ ID NO: 8), GH64-Pb-E264Q (SEQ ID NO: 18), GH81 - BH0236 - E542Q (SEQ ID NO: 33), or GH81 - BH0236 - CatD - E542Q (SEQ ID NO: 34), A DNA sequence encoding each of the amino acid sequences was inserted.

[0120] 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), a strep - tag II (ST - II) consisting of Ser - Ala residues (Reference 1) into the N - terminal side of the GS linker peptide, and GH16 - LicS - E137Q (SEQ ID NO: 8) A DNA sequence encoding the amino acid sequence was inserted.

[0121] 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 GH16 - LicS - E137Q (SEQ ID NO: 8), GH64 - Pb - E264Q (SEQ ID NO: 18), GH81 - BH0236 - E542Q (SEQ ID NO: 33), or GH81 - BH0236 - CatD - E542Q (SEQ ID NO: 34), A DNA sequence encoding each of the amino acid sequences was inserted.

[0122] The DNA sequences for the expression of β - glucanase - related polypeptide chains were 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.

[0123] (3) Preparation of Protein The expression vector was transformed into Escherichia coli SHuffle (New England Biolabs) for protein expression and cultured at 37°C in LB medium supplemented with ampicillin. After preculture, it was cooled to 15°C, IPTG (0.1 mM) was added, and the culture was continued for 4 to 72 hours to induce the expression of the recombinant protein. After washing, the cell suspension supplemented with protease inhibitors 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 of each solution was calculated using a commercially available measurement kit (Bradford method, Bio-Rad).

[0124] (4) 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 Barley BG or Laminarin (0 - 4 μg / mL) for blocking. After washing, for the Yeast BG immobilized plate, GH16-LicS-WT-HiBiT, GH16-LicS-E133Q-HiBiT, GH16-LicS-E137Q-HiBiT, GH16-BoMLG-WT-HiBiT, GH16-BoMLG-E143Q-HiBiT, GH16-BoMLG-E148Q-HiBiT diluted to 1 μg / mL in BPBST was added, while for the Laminarin immobilized plate, GH17-CJP38-WT-HiBiT, GH17-CJP38-E124Q-HiBiT, or GH17-CJP38-E267Q-HiBiT diluted to 1 μg / mL in BPBST was added respectively, and incubated at room temperature. After washing, to evaluate the binding ability of each protein to MLG or 13BG, 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).In addition, BPBST was added to 96-well white ELISA plates coated with various glucans (Yeast BG, SPG, Laminarin or Paramylon) diluted to 4 μg / mL or D-PBS(-) for blocking. After washing, various HiBiT-tag fusion probes (sBGRP, GH16-PcLam16A-E140Q, GH16-ZgLamC-CatD-E142Q, GH17-JPC38-E124Q, GH64-Pb-E264Q, GH64-Sm-E154Q, GH64-Kf-E149Q, GH64-Art-CatD-E153Q, GH64-Le-E145Q, GH81-BH0236-E542Q, GH128-I-Am-E199Q, GH128-II-Pv-E214Q, GH128-III-Bg-E208Q) diluted to 2 μg / mL in BPBST were added to each plate and incubated at room temperature. After washing, to evaluate the binding ability of each protein to various 13BGs, the signal derived from the HiBiT tag fused to each protein was measured in the same procedure as above, and the ratio of the luminescence signal shown by Blank [D-PBS(-)] to the signal dependent on the immobilized polysaccharide was calculated and shown in a graph. Furthermore, BPBST was added to 96-well white ELISA plates coated with Yeast BG, SPG, Laminarin or Paramylon (0 - 4 μg / mL) for blocking. After washing, GH81-BH0236-E542Q-HiBiT or GH81-BH0236-CatD-E542Q-HiBiT diluted to 2 μg / mL in BPBST was added and incubated at room temperature. After washing, to evaluate the binding ability of each protein to various 13BGs, the signal derived from the HiBiT tag fused to each protein was measured in the same procedure as above.

[0125] (5) Sandwich ELISA-like test A 96-well white plate for ELISA was coated with GH16-LicS-E137Q-ST-II (2 μg / ml) and blocked with D-PBS(-) (BPBST) containing BSA and Tween20. After washing, the test sample (Barley BG, 0 - 22.2 ng / mL) was added to each well and incubated at room temperature. The plate was washed, and GH16-LicS-E137Q-HiBiT (2 μg / ml) was added and incubated at room temperature. 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).

[0126] (6) Luciferase fragment complementation assay (SLCA) A mixed solution (5 μL) of NanoLuc fragment fusion glucan-binding probes (SmBiT fusion and LgBiT fusion, 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 fusion probe mixed solution, combinations of GH16-LicS-E137Q-LgBiT and GH16-LicS-E137Q-SmBiT, GH81-BH0236-E542Q-LgBiT and GH81-BH0236-E542Q-SmBiT, GH81-BH0236-CatD-E542Q-LgBiT and GH81-BH0236-CatD-E542Q-SmBiT, sBGRP-LgBiT and sBGRP-SmBiT, GH64-Pb-E264Q-LgBiT and GH64-Pb-E264Q-SmBiT were used. After shaking, 15 μL of the furimazine substrate solution (Promega) was added to the well, and the luminescence signal from the reconstituted NanoLuc was measured using a GloMax luminometer (Promega).

[0127] (7) Evaluation of the reactivity between GH81-BH0236 mutants and oligosaccharides First, to clarify the degree of polymerization of oligosaccharides recognizable by the mutant of the catalytic domain of GH81-BH0236, a 96-well white ELISA plate coated with Yeast BG (200 ng / mL) was blocked with D-PBS(-) containing BSA and Tween20 (BPBST), washed, and then GH81-BH0236-CatD-E542Q-HiBiT (final concentration 100 ng / mL at the time of reaction) was added in the presence or absence of Laminari-oligosaccharide (from dimer to heptamer, final concentration 250 μM at the time of reaction) and incubated at room temperature. After washing, to evaluate the binding of each protein to Yeast BG 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). Subsequently, to clarify the degree of polymerization of oligosaccharides recognizable by the full-length mutant of GH81-BH0236, a 96-well white ELISA plate coated with SPG (1 μg / mL) was blocked with BPBST, washed, and then GH81-BH0236-E542Q-HiBiT (final concentration 100 ng / mL at the time of reaction) was added in the presence or absence of Laminari-oligosaccharide (from dimer to heptamer, final concentration 250 μM at the time of reaction) and incubated at room temperature. After washing, to evaluate the binding of each protein to SPG on the plate, the signal derived from the HiBiT tag fused to each protein was measured in the same procedure.

[0128] (8) Preparation of cereal heating suspension Commercially available edible staple bread (sandwich bread), white rice (packaged cooked rice), and a portion of them were placed in a 2 mL tube. For dried short pasta (fusilli), a portion was taken after boiling and placed in a 2 mL tube. D-PBS(-) was added to make a concentration of 100 mg / mL, metal beads were added, and the mixture was processed with a bead crusher (FastPrep-24; MP Biomedicals). The samples were autoclaved (121 °C, 20 minutes), and then processed again with the bead crusher. Each sample was diluted 10-fold with D-PBS(-) containing BSA and Tween 20 (BPBST) (final wet weight 10 mg / mL), and the suspension was used for evaluating the reactivity with each luciferase fragment complementation assay.

[0129] (9) Preparation of Candida culture supernatant sample Yeast-like cells (1 × 10^5 cells) of Candida albicans NBRC 1385 (NITE Biological Resource Center) were added to 1 mL of RPMI1640 medium (Thermo Fisher Scientific) and cultured at 37 °C for 24 hours. After centrifugation, the supernatant was collected, and the fraction filtered through a 0.22 μm filter was further heated at 90 °C for 10 minutes to obtain a Candida culture supernatant sample. A portion of the culture supernatant was further treated with commercially available endo-β-1,3-glucanase (zymolyase 100T) (final concentration 2 μg / mL) at 37 °C for 1 hour, and then heated at 90 °C for 10 minutes to be used as an enzyme-treated solution.

[0130] <2> Results (1) Binding activity of various β-glucanase mutants to Barley BG (Figure 1) Endo-1,3(4)-β-glucanase (EC 3.2.1.6) belonging to the GH16 family, GH16-BoMLG (enzyme derived from Bacteroides ovatus, amino acid range 21-271) (SEQ ID NOs: 2-4), which is known as an enzyme that degrades Barley BG, and the wild-type or mutant sequences (SEQ ID NOs: 6-8) of GH16-LicS (enzyme derived from Bacillus subtilis, amino acid range 29-242), an endo-β-(1,3)(1,4)-glucanase (EC 3.2.1.73) belonging to the GH16 family, were expressed in E. coli as HiBiT fusion proteins. The binding activity of these proteins to Barley BG immobilized on a plate was evaluated by a direct ELISA-like test. As a result, neither GH16-BoMLG-WT-HiBiT nor GH16-LicS-WT-HiBiT, which were fused with the wild-type enzyme, bound to Barley BG, whereas GH16-BoMLG-E143Q-HiBiT, GH16-BoMLG-E148Q-HiBiT (Figure 1A), GH16-LicS-E133Q-HiBiT, and GH16-LicS-E137Q-HiBiT showed binding activity to Barley BG (Figure 1B). Among these, GH16-BoMLG-E143Q, GH16-BoMLG-E148Q, and GH16-LicS-E133Q remained weak in binding, but extremely strong binding activity was observed in GH16-LicS-E137Q.

[0131] (2) Concentration-dependent reactivity of MLG by SLCA or sandwich ELISA-like test using LicS mutants (Figure 2) To achieve highly sensitive detection of MLG, GH16-LicS-E137Q, which exhibits strong binding activity to MLG, was fused with the STII tag and expressed. This was immobilized on a plate to construct a sandwich ELISA-like test. As a result of verifying the reaction with MLG, an increase in the luminescence level of luciferase was observed in a concentration-dependent manner in the range of 30.4 pg / mL - 22.2 ng / mL (left in Figure 2). On the other hand, when GH16-LicS-E137Q was fused with LgBiT and SmBiT, which are luciferase fragments, and SLCA was constructed, an increase in the luminescence level of luciferase was observed in a concentration-dependent manner in the range of 823 pg / mL - 22.2 ng / mL (right in Figure 2). From these results, it was shown that when detecting and quantifying soluble MLG using GH16-LicS-E137Q, ELISA can detect MLG with higher sensitivity than SLCA.

[0132] (3) Binding activity of β-1,3-glucanase mutants to laminarin (Figure 3) Wild-type or mutant sequences (SEQ ID NOs: 10 - 12) of endo-β-1,3-glucanase (EC 3.2.1.39), which belongs to the GH17 family and is known as an enzyme that degrades 1,3-β-glucan (13BG), and is derived from Cryptomeria japonica (amino acid range 29 - 348), and HiBiT fusion proteins were expressed in E. coli. The binding activity of the above proteins to laminarin immobilized on a plate was evaluated by a direct ELISA-like test. As a result, GH17-CJP38-WT-HiBiT and GH17-CJP38-E124Q-HiBiT did not bind to laminarin immobilized on the plate, while GH17-CJP38-E267Q-HiBiT showed binding activity to laminarin (Figure 3), indicating that some proteins among the modified endo-β-1,3-glucanases may be available as 13BG-binding molecules.

[0133] (4) Comparison of binding activities of various β-1,3-glucanase mutants to various 13BGs (Figure 4) Although the above-mentioned GH17-CJP38-E267Q-HiBiT showed binding activity to laminarin, its binding activity was extremely weak. Therefore, attention was paid to representative endo-β-1,3-glucanases (EC 3.2.1.39) belonging to families other than GH17, such as the GH16, GH64, GH81, and GH128 families, and HiBiT fusion proteins with mutant sequences that suppressed glucolytic activity were expressed in Escherichia coli. For comparison, an artificial protein (sBGRP) (Patent Document 5) prepared based on the N-terminal region of an insect-derived β-glucan recognition protein (BGRP) developed in the past was also prepared as a HiBiT fusion protein in the same manner.Various 13BG immobilized on an ELISA plate was evaluated for reactivity with each protein (2 μg / mL) fused with the HiBiT tag, specifically, sBGRP, the mutant GH16-ZgLamC-CatD-E142Q (SEQ ID NO: 15) of GH16-ZgLamC-CatD (enzyme derived from Zobellia galactanivorans, amino acid range 31-257), GH17-JPC38-E124Q, the mutant GH64-Pb-E264Q (SEQ ID NO: 18) of GH64-Pb (enzyme derived from Paenibacillus barengoltzii, amino acid range 29-449), the mutant GH64-Sm-E154Q (SEQ ID NO: 21) of GH64-Sm (enzyme derived from Streptomyces matensis, amino acid range 19-401), the mutant GH64-Kf-E149Q (SEQ ID NO: 24) of GH64-Kf (enzyme derived from Kribbella flavida, amino acid range 32-396), the mutant GH64-Art-CatD-E153Q (SEQ ID NO: 27) of GH64-Art-CatD (enzyme derived from Arthrobacter sp., amino acid range 37-421), the mutant GH64-Le-E145Q (SEQ ID NO: 30) of GH64-Le (enzyme derived from Lysobacter enzymogenes, amino acid range 27-395), the mutant GH81-BH0236-E542Q (SEQ ID NO: 33) of GH81-BH0236 (enzyme derived from Alkalihalobacillus halodurans, amino acid range 26-1020), the mutant GH128-I-Am-E199Q (SEQ ID NO: 37) of GH128-I-Am (enzyme derived from Amycolatopsis mediterranei, amino acid range 28-269), the mutant GH128-II-Pv-E214Q (SEQ ID NO: 40) of GH128-II-Pv (enzyme derived from A, amino acid range 28-290), and the mutant GH128-III-Bg-E208Q (SEQ ID NO: 43) of GH128-III-Bg (enzyme derived from Blastomyces gilchristii, amino acid range 23-262).As a result, binding activity to Yeast BG was observed in sBGRP, each mutant of the GH64 enzyme, and GH81-BH0236-E542Q (Figure 4A), and binding activity to SPG was confirmed in sBGRP and GH64-Pb-E264Q, GH81-BH0236-E542Q (Figure 4B). In addition, binding activity to Laminarin was significantly observed in GH81-BH0236-E542Q (Figure 4C), and binding activity to Paramylon was similar to the reactivity found in Yeast BG and was confirmed in sBGRP, each mutant of the GH64 enzyme, and GH81-BH0236-E542Q (Figure 4D). As a result of these comparisons, it was confirmed that some mutants exhibited relatively strong binding activity to 13BG, and in particular, strong binding activity was shown in GH81-BH0236-E542Q.

[0134] (5) Comparison of the binding activities of the GH81-BH0236 mutant and the GH81-BH0236-CatD mutant with various BGs (Figure 5) GH81-BH0236-E542Q used for the comparative evaluation above is the full-length molecule (FL) of an endo-β-1,3-glucanase belonging to the GH81 family, and CBM6 and CBM56 are present on the C-terminal side. In the above examination, since strong binding activity was shown against 13BGs such as SPG and Laminarin with different three-dimensional structures, it was considered that the CBM region is also important in binding to any BG. Therefore, mutants in the CatD region of GH81-BH0236 (not including the CBM region) were prepared as HiBiT-tag fusion proteins, and their binding to various BGs was evaluated. As a result, no significant difference in binding activity was observed for Yeast BG, Laminarin, and Paramylon (Figures 5A, C, and D). On the other hand, for SPG with a triple helix structure, a significant decrease in binding activity was observed in GH81-BH0236-CatD-E542Q without the CBM region (Figure 5B). From this result, it was shown that it is effective to use mutants of the full-length enzyme of GH81-BH0236 in order to efficiently detect a wide variety of 13BGs.

[0135] (6) Comparison of the binding activities of the GH81-BH0236 variant and the GH81-BH0236-CatD variant with 13BG oligosaccharides (Figure 6) Since the binding activities of GH81-BH0236-E542Q and GH81-BH0236-CatD-E542Q to SPG were significantly different, it was predicted that the sizes of 13BG that could be recognized by each were different. Therefore, the binding activities of various 13BG oligosaccharides with the GH81-BH0236 variant and the GH81-BH0236-CatD variant were evaluated by a competitive ELISA method. GH81-BH0236-CatD-E542Q-HiBiT was added together with Laminari-oligo (degree of polymerization 2-7) to an ELISA plate coated with Yeast BG, and the luminescence signal derived from the HiBiT tag of GH81-BH0236-CatD-E542Q bound to the Yeast BG fixed on the plate was measured and evaluated. As a result, in the wells to which DP3-DP7 of 13BG were added, the luminescence signal decreased significantly (Figure 6A). On the other hand, when GH81-BH0236-E542Q-HiBiT was added together with Laminari-oligo (degree of polymerization 2-7) to an ELISA plate coated with SPG, in the wells to which 13BG of DP2-DP7 (degree of polymerization 2-7) were added, the luminescence signal decreased significantly (Figure 6B). From these results, it was shown that full-length GH81-BH0236-E542Q could recognize 13BG with a degree of polymerization of 2 or more, while GH81-BH0236-CatD-E542Q could bind to 13BG with a degree of polymerization of 3 or more.

[0136] (7) Results of comparative reactivity of SLCA with various polysaccharides using 13BG-binding probes (Figure 7) GH81-BH0236-E542Q and GH16-LicS-E137Q were each fused with a luciferase fragment (SmBiT tag and LgBiT) and expressed to construct SLCA. The structural specificity of each SLCA for polysaccharides was evaluated. As a result, SLCA using GH81-BH0236-E542Q showed no binding activity for polysaccharides other than 13BG (Yeast BG), indicating that it is a highly specific polysaccharide detection method for 13BG (Figure 7A). In addition, for SLCA using GH81-BH0236-E542Q against Yeast BG (25 μg / mL), its luminescence signal showed a value about 400 - 500 times higher compared to Blank. Subsequently, SLCA using GH16-LicS-E137Q showed no binding activity for polysaccharides other than MLG (Barley BG), indicating that it is a highly specific polysaccharide detection method for MLG (Figure 7B). In addition, for SLCA using GH16-LicS-E137Q against Barley BG (25 μg / mL), its luminescence signal showed a value about 30 - 40 times higher compared to Blank.

[0137] (8) Comparative concentration-dependent reactivity of various BGs by SLCA using 13BG-specific binding probes (Figure 8) β-glucanase mutants (GH64-Pb-E264Q, GH81-BH0236-E542Q, GH81-BH0236-CatD-E542Q) and sBGRP (Non-Patent Document 7) that showed strong binding activity to 13BG were each fused with luciferase fragments (SmBiT tag and LgBiT) for expression to construct SLCA. The detection sensitivities of various 13BGs in each SLCA were compared and evaluated. As a result, it was shown that the SLCA using GH81-BH0236-E542Q had the highest detection sensitivity for Yeast BG, Laminarin, and Paramylon (Figs. 8A, C, D). On the other hand, for SPG with a triple helix structure, it was shown that the SLCA using GH64-Pb-E264Q had the highest detection sensitivity, and next, the detection sensitivity of the SLCA using GH81-BH0236-E542Q was high (Fig. 8B). On the other hand, none of the SLCAs showed a reaction to Barley BG (MLG), and it was proven that these SLCAs are tools capable of specifically detecting and quantifying 13BG (Fig. 8E).

[0138] (9) Results of reactivity evaluation of SLCA using heated suspensions of various grains and 13BG binding probe (Fig. 9) Since we were able to construct an SLCA that can specifically detect and quantify 13BG and MLG respectively, we examined whether a method for specifically detecting MLG can be used as a means to solve the problem of false positive reactions in conventional 13BG detection. That is, in the elderly, polysaccharides are taken up into the blood at high concentrations via the airway mucosa and lungs due to aspiration, which affects the 13BG detection method, causing some false positive reactions. Although it is difficult to avoid these false positive reactions, combining them with the MLG-specific detection method makes it easier to estimate the cause. Using the 13BG·MLG-specific SLCA constructed this time, the reactivity with heated suspensions of typical grains frequently consumed in normal diets was evaluated. As a result, the SLCA using GH81-BH0236-E542Q showed strong reactivity with heated suspensions of staple bread (commercial bread), white rice (packaged cooked rice), and short pasta, indicating that 13BG was contained in all of these food ingredients (Figure 9A). On the other hand, the SLCA using GH16-LicS-E137Q also similarly showed strong reactivity with heated suspensions of staple bread (commercial bread), white rice (packaged cooked rice), and short pasta, indicating that MLG was contained in all of these food ingredients (Figure 9B). From these results, it was shown that it is possible to predict the transfer of food-derived polysaccharides into the blood by combining the 13BG-specific detection method and the MLG-specific detection method.

[0139] (10) Results of reactivity evaluation of SLCA using Candida culture supernatant and 13BG-binding probe (Figure 10) Using SLCA that can specifically detect and quantify 13BG and MLG respectively, an attempt was made to detect 13BG in the culture supernatant of Candida albicans, which is a clinically problematic pathogenic fungus. As a result, the SLCA using GH81 - BH0236 - E542Q showed strong reactivity with the culture supernatant of Candida, while no increase in the luminescence signal was observed with the SLCA using GH16 - LicS - E137Q (Figure 10A). Furthermore, the increase in the luminescence signal observed in the reaction between the SLCA using GH81 - BH0236 - E542Q and the Candida culture supernatant almost completely disappeared by adding commercially available end - β - 1,3 - glucanase (zymolyase 100T), which proved that the SLCA using GH81 - BH0236 - E542Q specifically detected 13BG in the fungal culture supernatant (Figure 10B).

[0140] (11) Results of reactivity evaluation of SLCA using LicS mutants and GH81 - BH0236 mutants in the presence of EDAT or heparin (Figure 11) The polysaccharide detection method using GH16-LicS-E137Q or GH81-BH0236-E542Q was verified for the possibility of being affected by anticoagulants (such as EDTA and Heparin) frequently used during blood collection at the clinical site. D-PBS(-) (Control), Heparin (100 μg / mL, final concentration), or EDTA (10 mM, final concentration) was added to Barley BG (0 - 1000 ng / mL), and the reactivity with SLCA using GH16-LicS-E137Q was evaluated. As a result, it was proved that SLCA using GH16-LicS-E137Q was not affected by EDTA or Heparin in MLG detection (Figure 11A). Also, when D-PBS(-) (Control), Heparin (100 μg / mL, final concentration), or EDTA (10 mM, final concentration) was added to Yeast BG (0 - 1000 ng / mL) and the reactivity with SLCA using GH81-BH0236-E542Q was evaluated, it was proved that SLCA using GH81-BH0236-E542Q was not affected by EDTA or Heparin in 13BG detection (Figure 11B). From these results, it was shown that the 13BG-binding probe and MLG-binding probe obtained in the present invention functioned normally even in the presence of anticoagulants used during blood collection, and that variants of each enzyme could be used as candidates for MLG- and 13BG-binding proteins.

[0141] (12) Thermal stability (Figure 12) and pH stability (Figure 13) of GH16-LicS-E137Q For thermal stability evaluation, GH16-LicS-E137Q-HiBiT diluted with D-PBS(-) was dispensed into microtubes and treated at each temperature from 20 to 90 °C for 10 minutes. After cooling, an equal volume of D-PBS(-) (BPBST) containing BSA and Tween20 was added to make it 0.5 μg / mL. Also, for pH stability evaluation, GH16-LicS-E137Q-HiBiT was dispensed into microtubes and diluted (0.5 μg / mL) with McIlvaine buffer (pH 2.4 - 8.0) containing BSA. BPBST was added to a 96-well white ELISA plate coated with Barley BG diluted to 0.5 μg / mL for blocking. After washing, GH16-LicS-E137Q-HiBiT from each microtube was added to the plate and incubated at room temperature. After washing, to evaluate the binding of each protein to Barley BG 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).

[0142] As a result of evaluating the thermal stability of GH16-LicS-E137Q, although GH16-LicS-E137Q showed a slight decrease in reactivity when heat-treated at 50 - 80 °C with respect to binding to Barley BG, it retained its binding activity (Figure 12). This binding was also retained when treated at 90 °C.

[0143] Also, as a result of evaluating the pH stability of GH16-LicS-E137Q, it was revealed that GH16-LicS-E137Q had binding activity in the pH range of 4.8 - 8.0 with respect to binding to Barley BG, and it was shown to be particularly suitable for the detection of β-(1,3)(1,4)-glucan under these conditions (Figure 13).

[0144] (13) Examination of substituted amino acids in GH16-LicS-E137 (Methods and Results) Based on GH16-LicS-E137Q (SEQ ID NO: 8) that showed strong binding activity to Barley BG, the amino acid substitution of E137 was replaced with all amino acids, and its binding activity was evaluated again. The 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 further cultured to induce the expression of GH16-LicS-E137X (X includes all amino acids). All proteins were extracted, purified, and dialyzed. For confirmation of their expression, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a commercially available kit. When the proteins separated by SDS-PAGE were stained with CBB dye (Quick Blue Staining Solution; BioDynamics Laboratory), among all the samples analyzed, the mutant substituted with proline (GH16-LicS-E137P) was difficult to express, and the signal obtained depending on the amount of HiBiT was also extremely small. Single bands of proteins showing the expected molecular sizes were confirmed for the other mutants.

[0145] Next, a 96-well white ELISA plate coated with Barley BG (0.5 μg / mL) or D-PBS(-) was blocked with D-PBS(-) containing BSA and Tween 20 (BPBST), washed, and GH16-LicS-E137X-amino acid mutant-HiBiT (200 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).

[0146] Figure 14 shows the values obtained by dividing the luminescence levels derived from each of the obtained mutants by the background level of D-PBS(-). As a result, some mutants (alanine (A), methionine (M), serine (S), threonine (T), aspartic acid (D), asparagine (N)) actually showed stronger activity than GH16-LicS-E137Q. In particular, the GH16-LicS-E137X mutants substituted with serine (S) and asparagine (N) showed strong binding activity (Figure 14).

[0147] (14) Thermal stability (Figure 15) and pH stability (Figure 16) of GH81-BH0236-E542Q For thermal stability evaluation, GH81-BH0236-E542Q-HiBiT diluted with D-PBS(-) was dispensed into microtubes and treated at each temperature from 20 to 90 °C for 5 minutes. After cooling, an equal volume of D-PBS(-) containing BSA and Tween 20 (BPBST) was added to make it 0.5 μg / mL. For pH stability evaluation, GH81-BH0236-E542Q-HiBiT was dispensed into microtubes and diluted (0.5 μg / mL) with McIlvaine buffer (pH 2.4 - 8.0) containing BSA. BPBST was added to a 96-well white ELISA plate coated with Yeast BG diluted to 4 μg / mL for blocking. After washing, GH81-BH0236-E542Q-HiBiT from each microtube was added to the plate and incubated at room temperature. After washing, to evaluate the binding of each protein to Yeast BG 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).

[0148] As a result of evaluating the thermal stability of GH81 - BH0236 - E542Q, it was found that GH81 - BH0236 - E542Q showed a decrease in reactivity when heated at 50°C for binding to Yeast BG, and the binding activity almost completely disappeared after treatment at 60°C or higher (Figure 15). Therefore, it was shown that it can be stably used for the detection of β - 1,3 - glucan (13BG) in an environment of around 40°C or lower.

[0149] Also, as a result of evaluating the pH stability of GH81 - BH0236 - E542Q, it became clear that GH81 - BH0236 - E542Q has binding activity in the range of pH 4.8 - 8.0 for binding to Yeast BG, and it was shown that it is particularly suitable for the detection of β - 1,3 - glucan (13BG) under these conditions (Figure 16).

[0150] (15) Examination of substituted amino acids in GH81 - BH0236 - E542 (Methods and Results) Based on GH81 - BH0236 - E542Q (SEQ ID NO: XX) which showed strong binding activity to Yeast BG, the amino acid substitution of E542 was replaced with all amino acids, 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 and IPTG (0.1 mM) was added, and further cultured to induce the expression of GH81 - BH0236 - E542X (X includes all amino acids). All proteins were extracted, purified, and dialyzed, and for confirmation of their expression, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS - PAGE) was performed using a commercially available kit. When the proteins separated by SDS - PAGE were stained with CBB dye (Quick Blue Staining Solution; BioDynamics Laboratory), a single band of protein showing the theoretical molecular size was confirmed for all the mutant samples analyzed.

[0151] Next, a 96-well white ELISA plate coated with Yeast BG (0.5 μg / mL) or D-PBS(-) was blocked with D-PBS(-) containing BSA and Tween20 (BPBST). After washing, GH81-BH0236-E542X-amino acid mutant-HiBiT (200 ng / ml) was added and incubated at room temperature. After washing, to evaluate the binding of each protein to β-1,3-glucan (13BG) 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). The graph shows the luminescence levels obtained from each mutant, divided by the background level of D-PBS(-).

[0152] As a result, although some mutants (tyrosine (Y), phenylalanine (F), histidine (H), lysine (K), arginine (R), asparagine (N)) showed slightly weak binding activity, all amino acid mutants except the wild type (E) were shown to bind to β-1,3-glucan (13BG) on the plate. Among the GH81-BH0236-E542X mutants, GH81-BH0236-E542Q (SEQ ID NO: 33) substituted with glutamine (Q) showed the strongest binding activity (Figure 17).

[0153] (References) 1. Schmidt TGM., et. al., Nature Protocols. 2007;2(6):1528-35.

[0154] In the following SEQ LISTING, "FL" indicates a full-length amino acid sequence, and "WT" indicates a wild-type amino acid sequence. Also, "E**Q" indicates that the glutamic acid (E) at position ** in the full length has been substituted with glutamine (Q). Accession number 1: GH16-BoMLG-FL (Bacteroides ovatus) GenBank: ALJ47757.1 MKKVALFLFLSVCFAQQSCS SDSVGTEPEENPQDILFKDDFNFFDEKVWTKETHEPGWTNQELQAYDAAHVSVGKDGDKSVLILTAERKGNKIYSGRINSKGKKSFKYRKIEASIKLPKTNGGLWPAFWMMGDNDKQWPACG EIDIME MGEQSGMAAGDSEKQVNTAIHYGPSAAAHEQQYYKANVANSLQDGNYHTYSLDWDENNLTISIDNVKFHTFDISSNTYFHDNFYILFNLAVGGAFTGITDINKLTGLKDGQKVNMYIDWVKIL Accession number 2: From GH16-BoMLG-WT (Bacteroides ovatus) GenBank: ALJ47757.1 SDSVGTEPEENPQDILFKDDFNFFDEKVWTKETHEPGWTNQELQAYDAAHVSVGKDGDKSVLILTAERKGNKIYSGRINSKGKKSFKYRKIEASIKLPKTNGGLWPAFWMMGDNDKQWPACG EIDIME MGEQSGMAAGDSEKQVNTAIHYGPSAAAHEQQYYKANVANSLQDGNYHTYSLDWDENNLTISIDNVKFHTFDISSNTYFHDNFYILFNLAVGGAFTGITDINKLTGLKDGQKVNMYIDWVKIL Accession number 3: GH16-BoMLG-E143Q (Bacteroides ovatus) From GenBank: ALJ47757.1 SDSVGTEPEENPQDILFKDDFNFFDEKVWTKETHEPGWTNQELQAYDAAHVSVGKDGDKSVLILTAERKGNKIYSGRINSKGKKSFKYRKIEASIKLPKTNGGLWPAFWMMGDNDKQWPACG QIDIMEMGEQSGMAAGDSEKQVNTAIHYGPSAAAHEQQYYKANVANSLQDGNYHTYSLDWDENNLTISIDNVKFHTFDISSNTYFHDNFYILFNLAVGGAFTGITDINKLTGLKDGQKVNMYIDWVKIL Accession No. 4: GH16-BoMLG-E148Q (Bacteroides ovatus) From GenBank: ALJ47757.1 SDSVGTEPEENPQDILFKDDFNFFDEKVWTKETHEPGWTNQELQAYDAAHVSVGKDGDKSVLILTAERKGNKIYSGRINSKGKKSFKYRKIEASIKLPKTNGGLWPAFWMMGDNDKQWPACG EIDIMQ MGEQSGMAAGDSEKQVNTAIHYGPSAAAHEQQYYKANVANSLQDGNYHTYSLDWDENNLTISIDNVKFHTFDISSNTYFHDNFYILFNLAVGGAFTGITDINKLTGLKDGQKVNMYIDWVKIL Accession No. 5: GH16-LicS-FL (Bacillus subtilis) GenBank: CAA86922.1 MPYLKRVLLLLVTGLFMSLFAVTATASA QTGGSFFDPFNGYNSGFWQKADGYSNGNMFNCTWRANNVSMTSLGEMRLALTSPAYNKFDCGENRSVQTYGYGLYEVRMKPAKNTGIVSSFFTYTGPTDGTPW DEIDIE FLGKDTTKVQFNYYTNGAGNHEKIVDLGFDAANAYHTYAFDWQPNSIKWYVDGQLKHTATNQIPTTPGKIMMNLWNGTGVDEWLGSYNGVNPLYAHYDWVRYTKK Accession No. 6: GH16-LicS-WT (Bacillus subtilis) From GenBank: CAA86922.1 QTGGSFFDPFNGYNSGFWQKADGYSNGNMFNCTWRANNVSMTSLGEMRLALTSPAYNKFDCGENRSVQTYGYGLYEVRMKPAKNTGIVSSFFTYTGPTDGTPW DEIDIE FLGKDTTKVQFNYYTNGAGNHEKIVDLGFDAANAYHTYAFDWQPNSIKWYVDGQLKHTATNQIPTTPGKIMMNLWNGTGVDEWLGSYNGVNPLYAHYDWVRYTKK SEQ ID NO: 7: from GH16-LicS-E133Q (Bacillus subtilis), GenBank: CAA86922.1 QTGGSFFDPFNGYNSGFWQKADGYSNGNMFNCTWRANNVSMTSLGEMRLALTSPAYNKFDCGENRSVQTYGYGLYEVRMKPAKNTGIVSSFFTYTGPTDGTPW DQIDIE FLGKDTTKVQFNYYTNGAGNHEKIVDLGFDAANAYHTYAFDWQPNSIKWYVDGQLKHTATNQIPTTPGKIMMNLWNGTGVDEWLGSYNGVNPLYAHYDWVRYTKK SEQ ID NO: 8: from GH16-LicS-E137Q (Bacillus subtilis), GenBank: CAA86922.1 QTGGSFFDPFNGYNSGFWQKADGYSNGNMFNCTWRANNVSMTSLGEMRLALTSPAYNKFDCGENRSVQTYGYGLYEVRMKPAKNTGIVSSFFTYTGPTDGTPW DEIDIQ FLGKDTTKVQFNYYTNGAGNHEKIVDLGFDAANAYHTYAFDWQPNSIKWYVDGQLKHTATNQIPTTPGKIMMNLWNGTGVDEWLGSYNGVNPLYAHYDWVRYTKK SEQ ID NO: 9: GH17-CJP38-FL (Cryptomeria japonica) GenBank: BAD93486.1 MELLKQHRYMFLLISCIVILLNSMHADCEQIGVNYGMDGNNLPSAGDVVSLMKKNNIGKMRIFGPNADVLRAFANSRIEVIVGVENKGLEAVASSQDSANGWVNDNIKPFYPSTNIKYIA VGNE VLEMPDNAQYVSFLVPAIKNIQTALENANLQNNIKVSTAHAMTVIGTSSPPSKGTFKDAVKDSMSSILQFLQDHGSPFMANVYPYFSYDGDRSIKLDYALFNPTPPVVDEGLSYTNLFDAMVDAVLSAMESLGHPNIPIVIT ESGWPS AGKDVATIENAQTYNNNLIKHVLSNAGTPKRPGSSIETYIFALFNENLKGPAEVEKHFGLFNPDEQPVYPVKFSLN Accession No. 10: GH17 - CJP38 - WT (Cryptomeria japonica) From GenBank: BAD93486.1 EQIGVNYGMDGNNLPSAGDVVSLMKKNNIGKMRIFGPNADVLRAFANSRIEVIVGVENKGLEAVASSQDSANGWVNDNIKPFYPSTNIKYIA VGNE VLEMPDNAQYVSFLVPAIKNIQTALENANLQNNIKVSTAHAMTVIGTSSPPSKGTFKDAVKDSMSSILQFLQDHGSPFMANVYPYFSYDGDRSIKLDYALFNPTPPVVDEGLSYTNLFDAMVDAVLSAMESLGHPNIPIVIT ESGWPS AGKDVATIENAQTYNNNLIKHVLSNAGTPKRPGSSIETYIFALFNENLKGPAEVEKHFGLFNPDEQPVYPVKFSLN Accession No. 11: GH17 - CJP38 - E124Q (Cryptomeria japonica) From GenBank: BAD93486.1 EQIGVNYGMDGNNLPSAGDVVSLMKKNNIGKMRIFGPNADVLRAFANSRIEVIVGVENKGLEAVASSQDSANGWVNDNIKPFYPSTNIKYIAVGNQ VLEMPDNAQYVSFLVPAIKNIQTALENANLQNNIKVSTAHAMTVIGTSSPPSKGTFKDAVKDSMSSILQFLQDHGSPFMANVYPYFSYDGDRSIKLDYALFNPTPPVVDEGLSYTNLFDAMVDAVLSAMESLGHPNIPIVIT ESGWPS AGKDVATIENAQTYNNNLIKHVLSNAGTPKRPGSSIETYIFALFNENLKGPAEVEKHFGLFNPDEQPVYPVKFSLN SEQ ID NO: 12: GH17-CJP38-E267Q (Cryptomeria japonica) From GenBank: BAD93486.1 EQIGVNYGMDGNNLPSAGDVVSLMKKNNIGKMRIFGPNADVLRAFANSRIEVIVGVENKGLEAVASSQDSANGWVNDNIKPFYPSTNIKYIA VGNE VLEMPDNAQYVSFLVPAIKNIQTALENANLQNNIKVSTAHAMTVIGTSSPPSKGTFKDAVKDSMSSILQFLQDHGSPFMANVYPYFSYDGDRSIKLDYALFNPTPPVVDEGLSYTNLFDAMVDAVLSAMESLGHPNIPIVIT QSGWPS AGKDVATIENAQTYNNNLIKHVLSNAGTPKRPGSSIETYIFALFNENLKGPAEVEKHFGLFNPDEQPVYPVKFSLN SEQ ID NO: 13: GH16-ZgLamC-FL (Zobellia galactanivorans) GenBank: CAZ95067.1 MKKIITYLLMVLCFGMTPILSAQDYNLVWQ DEFDDGIGPDWVFETGMGYNGWGNNELQYYRRENAAVENGNLVITAKHENFGGAQYTSARMKTQGRKSFKYGKIEARIALPSGQGLWPAFWMLGNNITSVSWPACG EIDIMERINNALQTHGTIHWSDQNGDHASYGDDVGVSDPGQYHIYSVEWDANSIKWFVDGQQFNEVDISNGVNGTGEFQNEFFILLNMAVGGDWPGFDVDQSKLPAQMLVDYVRVYQKGDD NDSANTLKIEAESYLYSNDVQKEPCSEGGENVGYINNGSWMSYPGINFPSSGNYLIEYRVASAVDGGRFSSDLEAGETVLGELSVPNTGGWQNWTTVSQTVNVSAGTYQFGLYSISGGWNINWIRITKQGTASAATALNSSLIASDNGISQEIRVYPNPFTEYVSVNFDGEAANLTLQDMLGTVIFSKSGVSADESVDLSGLKSGVYFLTIEQDGKSTVRQLIKE Accession No. 14: GH16-ZgLamC-WT (Zobellia galactanivorans) From GenBank: CAZ95067.1 DEFDDGIGPDWVFETGMGYNGWGNNELQYYRRENAAVENGNLVITAKHENFGGAQYTSARMKTQGRKSFKYGKIEARIALPSGQGLWPAFWMLGNNITSVSWPACG EIDIME RINNALQTHGTIHWSDQNGDHASYGDDVGVSDPGQYHIYSVEWDANSIKWFVDGQQFNEVDISNGVNGTGEFQNEFFILLNMAVGGDWPGFDVDQSKLPAQMLVDYVRVYQKGDD Accession No. 15: GH16-ZgLamC-CatD-E142Q (Zobellia galactanivorans) From GenBank: CAZ95067.1 DEFDDGIGPDWVFETGMGYNGWGNNELQYYRRENAAVENGNLVITAKHENFGGAQYTSARMKTQGRKSFKYGKIEARIALPSGQGLWPAFWMLGNNITSVSWPACG EIDIMQ RINNALQTHGTIHWSDQNGDHASYGDDVGVSDPGQYHIYSVEWDANSIKWFVDGQQFNEVDISNGVNGTGEFQNEFFILLNMAVGGDWPGFDVDQSKLPAQMLVDYVRVYQKGDD Accession No. 16: GH64-Pb-FL (Paenibacillus barengoltzii) GenBank: WP_016313499.1 MTTKWVQRFSLFFIVFLLSISVNISASAADFTQGADVSGNNVTLWFKSSVNTTWVDVHYKVNSGVQQNVRMSFNAGAARFEHTILSAAQAEIEYFFTYNNGVPAYDTTTFTYRSGQPDPEPSTNSIYSIPASSIPQPSEGGVSLKVMNGTGGAYTDDQIYWGVIGINPVNGKWSYLDLAGRLLPISSDLNNAPGHLTKDGINYANIYHKISDANWVNLPKIESGRLFLSVGSPLYMKTFDDGFAGPDLNNPTNPNLNVIFDF VEFTV DKDGYHGNTTRVDQFGFPIQHRLVNLAGNYDRTVGELESETRSGLFAKYVNEVPHEFKSLGTLQAPYRILSPMKGPFQEGGAYENYFAGYSSISTQDILLGVGEASNPEVCAALNRHVYTEPDNWNRVDQYYQAAPANYYAKFWHDHSIDGLAYGFCYDDVNGQAAYLEVGDPKGLIVRVGW Sequence number 17: GH64-Pb-WT (Paenibacillus barengoltzii) From GenBank: WP_016313499.1 ADFTQGADVSGNNVTLWFKSSVNTTWVDVHYKVNSGVQQNVRMSFNAGAARFEHTILSAAQAEIEYFFTYNNGVPAYDTTTFTYRSGQPDPEPSTNSIYSIPASSIPQPSEGGVSLKVMNGTGGAYTDDQIYWGVIGINPVNGKWSYLDLAGRLLPISSDLNNAPGHLTKDGINYANIYHKISDANWVNLPKIESGRLFLSVGSPLYMKTFDDGFAGPDLNNPTNPNLNVIFDF VEFTV DKDGYHGNTTRVDQFGFPIQHRLVNLAGNYDRTVGELESETRSGLFAKYVNEVPHEFKSLGTLQAPYRILSPMKGPFQEGGAYENYFAGYSSISTQDILLGVGEASNPEVCAALNRHVYTEPDNWNRVDQYYQAAPANYYAKFWHDHSIDGLAYGFCYDDVNGQAAYLEVGDPKGLIVRVGW Accession No. 18: GH64-Pb-E264Q (Paenibacillus barengoltzii) From GenBank: WP_016313499.1 ADFTQGADVSGNNVTLWFKSSVNTTWVDVHYKVNSGVQQNVRMSFNAGAARFEHTILSAAQAEIEYFFTYNNGVPAYDTTTFTYRSGQPDPEPSTNSIYSIPASSIPQPSEGGVSLKVMNGTGGAYTDDQIYWGVIGINPVNGKWSYLDLAGRLLPISSDLNNAPGHLTKDGINYANIYHKISDANWVNLPKIESGRLFLSVGSPLYMKTFDDGFAGPDLNNPTNPNLNVIFDF VQFTV DKDGYHGNTTRVDQFGFPIQHRLVNLAGNYDRTVGELESETRSGLFAKYVNEVPHEFKSLGTLQAPYRILSPMKGPFQEGGAYENYFAGYSSISTQDILLGVGEASNPEVCAALNRHVYTEPDNWNRVDQYYQAAPANYYAKFWHDHSIDGLAYGFCYDDVNGQAAYLEVGDPKGLIVRVGW Accession No. 19: GH64-Sm-FL (Streptomyces matensis) GenBank: BAA34349.1 MLRTLRRRVTAVALGLAT ALGGGWLAAGVPSPAHAAVPATIPLTITNNSGRAEQIHIYNLGTELSSGRQGWADASGAFHPWPAGGNPPTPAPDASIPGPAPGRSTTIQIPKFSGRIYFSYGRKMEFRLTTGGLVQPAVQNPTDPNRDILFNW SEYTLNDSGLWINSTQVDMFSAPYTVGVRRGDGTTLSTGKLRPGGYNGVFNALRGQSGGWANLIQTRSDGTVLRALSPLYGVETGALPASVMDDYINRVWNKYTGTDLIVTPFADRPDVRYTGRVSGGVLRFTDGSGAVVTTFQKPDASSVFGCHRLLDAPNDQVRGPISRTLCAGFNRTTLLANPHQPDRSAAGFYQEPVTNHYARIIHAHMADGKAYGFAFDDVGHHESLVHDGDPRGASLTLDPFD SEQ ID NO:20: from GH64-Sm-WT (Streptomyces matensis) GenBank: BAA34349.1 ALGGGWLAAGVPSPAHAAVPATIPLTITNNSGRAEQIHIYNLGTELSSGRQGWADASGAFHPWPAGGNPPTPAPDASIPGPAPGRSTTIQIPKFSGRIYFSYGRKMEFRLTTGGLVQPAVQNPTDPNRDILFNW SEYTL NDSGLWINSTQVDMFSAPYTVGVRRGDGTTLSTGKLRPGGYNGVFNALRGQSGGWANLIQTRSDGTVLRALSPLYGVETGALPASVMDDYINRVWNKYTGTDLIVTPFADRPDVRYTGRVSGGVLRFTDGSGAVVTTFQKPDASSVFGCHRLLDAPNDQVRGPISRTLCAGFNRTTLLANPHQPDRSAAGFYQEPVTNHYARIIHAHMADGKAYGFAFDDVGHHESLVHDGDPRGASLTLDPFD SEQ ID NO:21: GH64-Sm-E154Q (Streptomyces matensis) from GenBank: BAA34349.1 ALGGGWLAAGVPSPAHAAVPATIPLTITNNSGRAEQIHIYNLGTELSSGRQGWADASGAFHPWPAGGNPPTPAPDASIPGPAPGRSTTIQIPKFSGRIYFSYGRKMEFRLTTGGLVQPAVQNPTDPNRDILFNW SQYTLNDSGLWINSTQVDMFSAPYTVGVRRGDGTTLSTGKLRPGGYNGVFNALRGQSGGWANLIQTRSDGTVLRALSPLYGVETGALPASVMDDYINRVWNKYTGTDLIVTPFADRPDVRYTGRVSGGVLRFTDGSGAVVTTFQKPDASSVFGCHRLLDAPNDQVRGPISRTLCAGFNRTTLLANPHQPDRSAAGFYQEPVTNHYARIIHAHMADGKAYGFAFDDVGHHESLVHDGDPRGASLTLDPFD Accession number 22: GH64-Kf-FL (Kribbella flavida) GenBank: ADB34580.1 MRIKPKLLAVLAAAATVAAGLTATVSAPAEA VPATIPLKITNNSGRGEPVYIYNLGTNLATGQQGWADANGTFHPWPAGGNPPTPAPDASIAGPANGQSITLRMPKFSGRVYFSYGQKLVFKLTTGGLVQPAVQNPSDPNRNILFNW TEYTL NDSGLWINSTQV D MFSAPYAVGVQRADGTTKVTGHLKPGGYNGFFTALRGQPGGWANLIQTAPNGTVLRALAPSYGVEIGALPATVMDDYVNRVWSKYSTQTLTVTPFTDQPNIKYFGRVSGNVMNFTNSSGQVVTSFQKPNSASIFGCAGLLDAPNDLVRGPISRTLCAGFNRSTLLTNPNQPDSSNVDFYKDVVTNHYSRKIHAQMADGKAYGFAFDDVGAHESLVHDGNPQQAYITLDPFN Accession number 23: GH64-Kf-WT (Kribbella flavida) From GenBank: ADB34580.1 VPATIPLKITNNSGRGEPVYIYNLGTNLATGQQGWADANGTFHPWPAGGNPPTPAPDASIAGPANGQSITLRMPKFSGRVYFSYGQKLVFKLTTGGLVQPAVQNPSDPNRNILFNW TEYTL NDSGLWINSTQVD MFSAPYAVGVQRADGTTKVTGHLKPGGYNGFFTALRGQPGGWANLIQTAPNGTVLRALAPSYGVEIGALPATVMDDYVNRVWSKYSTQTLTVTPFTDQPNIKYFGRVSGNVMNFTNSSGQVVTSFQKPNSASIFGCAGLLDAPNDLVRGPISRTLCAGFNRSTLLTNPNQPDSSNVDFYKDVVTNHYSRKIHAQMADGKAYGFAFDDVGAHESLVHDGNPQQAYITLDPFN Sequence number 24: From GH64-Kf-E149Q (Kribbella flavida), GenBank: ADB34580.1 VPATIPLKITNNSGRGEPVYIYNLGTNLATGQQGWADANGTFHPWPAGGNPPTPAPDASIAGPANGQSITLRMPKFSGRVYFSYGQKLVFKLTTGGLVQPAVQNPSDPNRNILFNW TQYTL NDSGLWINSTQV D MFSAPYAVGVQRADGTTKVTGHLKPGGYNGFFTALRGQPGGWANLIQTAPNGTVLRALAPSYGVEIGALPATVMDDYVNRVWSKYSTQTLTVTPFTDQPNIKYFGRVSGNVMNFTNSSGQVVTSFQKPNSASIFGCAGLLDAPNDLVRGPISRTLCAGFNRSTLLTNPNQPDSSNVDFYKDVVTNHYSRKIHAQMADGKAYGFAFDDVGAHESLVHDGNPQQAYITLDPFN Sequence number 25: GH64-Art-FL (Arthrobacter sp.) GenBank: BAA04892.1 MPHDRKNSSRRAWAALCAAVLAVSGALVGVAAPASA VPATIPLTITNDSGRGPIYLYVLGERDGVAGWADAGGTFHPWPGGVGPVPVPAPDASIAGPGPGQSVTIRLPKLSGRVYYSYGQKMTFQIVLDGRLVQPAVQNDSDPNRNILFNW TEYTL NDGGLWINSTQV DHWSAPYQVGVQRADGQVLSTGMLKPNGYEAFYTALESAGWGGLVQRAPDGSRLRALNPSHGIDVGKISSASIDSYVTEVWNSYRTRDMCVTPFSHEPGTQFRGRVDGDWFRFRNGSGQEVAAFKKPDASSVYGCHKDLQAPNDHVVGPIARTLCAALVRTTALTNPNQPDANSAGFYQDARTNVYAKLAHQQMANGKAYAFAFDDVGAHESLVHDGNPQAAYIKLDPFTGTATPIANGGSTEQPGTPGGLPA GTGALRIGSTLCLDVPWADPTDTNQVQLATCSGNAAQQWTRGTDGTVRALGKCLDVARSGTADGTAVWIYTCNGTGAQKWTYDSATKALRNPQSGKCLDAQGGAPLRDGQKVQLWTCNQTEAQRWTL Accession number 26: GH64-Art-WT (Arthrobacter sp.) From GenBank: BAA04892.1 VPATIPLTITNDSGRGPIYLYVLGERDGVAGWADAGGTFHPWPGGVGPVPVPAPDASIAGPGPGQSVTIRLPKLSGRVYYSYGQKMTFQIVLDGRLVQPAVQNDSDPNRNILFNW TEYTL NDGGLWINSTQV D HWSAPYQVGVQRADGQVLSTGMLKPNGYEAFYTALESAGWGGLVQRAPDGSRLRALNPSHGIDVGKISSASIDSYVTEVWNSYRTRDMCVTPFSHEPGTQFRGRVDGDWFRFRNGSGQEVAAFKKPDASSVYGCHKDLQAPNDHVVGPIARTLCAALVRTTALTNPNQPDANSAGFYQDARTNVYAKLAHQQMANGKAYAFAFDDVGAHESLVHDGNPQAAYIKLDPFTGTATPIANGGSTEQPGTPGGLPA Accession number 27: GH64-Art-E153Q (Arthrobacter sp.) From GenBank: BAA04892.1 VPATIPLTITNDSGRGPIYLYVLGERDGVAGWADAGGTFHPWPGGVGPVPVPAPDASIAGPGPGQSVTIRLPKLSGRVYYSYGQKMTFQIVLDGRLVQPAVQNDSDPNRNILFNWTQYTL NDGGLWINSTQV D HWSAPYQVGVQRADGQVLSTGMLKPNGYEAFYTALESAGWGGLVQRAPDGSRLRALNPSHGIDVGKISSASIDSYVTEVWNSYRTRDMCVTPFSHEPGTQFRGRVDGDWFRFRNGSGQEVAAFKKPDASSVYGCHKDLQAPNDHVVGPIARTLCAALVRTTALTNPNQPDANSAGFYQDARTNVYAKLAHQQMANGKAYAFAFDDVGAHESLVHDGNPQAAYIKLDPFTGTATPIANGGSTEQPGTPGGLPA Sequence number 28: GH64-Le-FL (Lysobacter enzymogenes) GenBank: AAN77504.1 MVTRRTFLGASAAALAAPLLPRGALA ATPARFNLALLNASGQNTAYAYVTGFDNGRPLFVRADGSAYYPPSPSAPVTPLGADCAIPLGANGSTVRVSVPRMYGARIYLVTGSRLDFYVNPGPAVVHPSFLNTSDTNFNKNWTF AEFTF NEYELFSNISYVDFVAAPLGLSLRSLSGRVETIPGLPAASLDPICYSLQQQASQEGSAWNSLIQRGPDGRNLRAMSAHYQAARFQNYLAGYIDACWNKYRGTTLTVDTQSGFGVLTARVGGDNLLRFNNGEAFAKPSTADVLSCDSGPFSLGGASEVRKAIIPRLAAALNRTTLLDNPNQPNGEVASRFYRNAQTNHYARLVHERLPDNRGYAFPYDDVTASGGPDFSGAARSGDPDTLTVTLRALR Sequence number 29: GH64-Le-WT (Lysobacter enzymogenes) From GenBank: AAN77504.1 ATPARFNLALLNASGQNTAYAYVTGFDNGRPLFVRADGSAYYPPSPSAPVTPLGADCAIPLGANGSTVRVSVPRMYGARIYLVTGSRLDFYVNPGPAVVHPSFLNTSDTNFNKNWTF AEFTF NEYELFSNISYVDFVAAPLGLSLRSLSGRVETIPGLPAASLDPICYSLQQQASQEGSAWNSLIQRGPDGRNLRAMSAHYQAARFQNYLAGYIDACWNKYRGTTLTVDTQSGFGVLTARVGGDNLLRFNNGEAFAKPSTADVLSCDSGPFSLGGASEVRKAIIPRLAAALNRTTLLDNPNQPNGEVASRFYRNAQTNHYARLVHERLPDNRGYAFPYDDVTASGGPDFSGAARSGDPDTLTVTLRALR Sequence number 30: GH64-Le-E145Q (Lysobacter enzymogenes) From GenBank: AAN77504.1 ATPARFNLALLNASGQNTAYAYVTGFDNGRPLFVRADGSAYYPPSPSAPVTPLGADCAIPLGANGSTVRVSVPRMYGARIYLVTGSRLDFYVNPGPAVVHPSFLNTSDTNFNKNWTF AQFTF NEYELFSNISYVDFVAAPLGLSLRSLSGRVETIPGLPAASLDPICYSLQQQASQEGSAWNSLIQRGPDGRNLRAMSAHYQAARFQNYLAGYIDACWNKYRGTTLTVDTQSGFGVLTARVGGDNLLRFNNGEAFAKPSTADVLSCDSGPFSLGGASEVRKAIIPRLAAALNRTTLLDNPNQPNGEVASRFYRNAQTNHYARLVHERLPDNRGYAFPYDDVTASGGPDFSGAARSGDPDTLTVTLRALR Sequence number 31: GH81-BH0236-FL (Alkalihalobacillus halodurans) GenBank: BAB03955.1 MKGKNVQLLFALVVIILLFPTGASA SPHAVSVGKGSYATEFPEIDFGGINDPGFRDQQGEPPATIYRSDRVTGPMQTNSWWGSLAVDRFSMNQYPHPFSVRHRAEGLHVFYDAPHNMVVHENREAGTWHIHGAIGTDFTIKHSGTANFEQAVVDDYNDWYVRGLLENGAHQMAITYGVGSPYIFVEYEDGSAVLDFDIAPDVWEMNGHVIGFSTHDHKHYAAFAPPGQNWSGIGSKTLTNNADYIAIAKLPEKDGNMLAKFEQYAYSVVRDAVADWTYDEATGTVTTTFEVTTEAKVQGAPDGTIFALYPHQYRHLASSSENQLLQNYQYEIIRGTMIGLEGKRFTTELTYPGVLPSLPDLGDYDRERLIGYLHDATSDYPTGSDTYELGKYIGKLATLAPIADQMGEYELAEQFRGELKDILEDWLQATNASGQLKGKNLFYYNENWGTILGYHAAHSSATRIN D HHFHYGYFVKAAAEIARADQEWAKSENWGGMIDLLIRDFMADRDDDLFPYLRMFDPYSGNSWADGLATFDAGNNQ ESSSE AMHAWTNVILWAEATGNKALRDRAIYLYTTEMSAINEYFFDVHQEIFPEEYGPEIVTINWGGKMDHATWWNSGKVEKYAINWLPFHGGSLYLGHHPDYVDRAYEELRRDIGSTDWNLWSNLVWMYRAFTNPDDALQQMEASIDDYGLFDPGNEKIIERGSTKAQTYHWIHNLAELGRVDPTVTANHPIYAVFNKNGNRTYIVYNFSDSPITVQFSDGHSIQVEPHSFNIGNGDGPTNPDPSEP D LKNPYERIQAEAYDAMSGIQTEGTDDDGGGDNIGWINDGDWVKYERVHFERDASSIEVRVASDTPGGRIEIRTGSPTGTLLGDVQVPNTGGWQQWQTVTGNVQIQPGTYDVYLVFKGSPEYDLMNVNWFVFRAN GQGNGDSHTHPDYTAGIRGITGNEVTIFFAPTTEARYVDVHLKVNNGQQLNYRMTERNGEWERVVENLSSGDVLEYSFTYEKLGPQYTTEWFTYSR Accession No. 32: GH81 - BH0236 - CatD - WT (Alkalihalobacillus halodurans) From GenBank: BAB03955.1 SPHAVSVGKGSYATEFPEIDFGGINDPGFRDQQGEPPATIYRSDRVTGPMQTNSWWGSLAVDRFSMNQYPHPFSVRHRAEGLHVFYDAPHNMVVHENREAGTWHIHGAIGTDFTIKHSGTANFEQAVVDDYNDWYVRGLLENGAHQMAITYGVGSPYIFVEYEDGSAVLDFDIAPDVWEMNGHVIGFSTHDHKHYAAFAPPGQNWSGIGSKTLTNNADYIAIAKLPEKDGNMLAKFEQYAYSVVRDAVADWTYDEATGTVTTTFEVTTEAKVQGAPDGTIFALYPHQYRHLASSSENQLLQNYQYEIIRGTMIGLEGKRFTTELTYPGVLPSLPDLGDYDRERLIGYLHDATSDYPTGSDTYELGKYIGKLATLAPIADQMGEYELAEQFRGELKDILEDWLQATNASGQLKGKNLFYYNENWGTILGYHAAHSSATRIN D HHFHYGYFVKAAAEIARADQEWAKSENWGGMIDLLIRDFMADRDDDLFPYLRMFDPYSGNSWADGLATFDAGNNQ ESSSEAMHAWTNVILWAEATGNKALRDRAIYLYTTEMSAINEYFFDVHQEIFPEEYGPEIVTINWGGKMDHATWWNSGKVEKYAINWLPFHGGSLYLGHHPDYVDRAYEELRRDIGSTDWNLWSNLVWMYRAFTNPDDALQQMEASIDDYGLFDPGNEKIIERGSTKAQTYHWIHNLAELGRVDPTVTANHPIYAVFNKNGNRTYIVYNFSDSPITVQFSDGHSIQVEPHSFNIGNGDGPTNPDPSEP D LKNPYERIQAEAYDAMSGIQTEGTDDDGGGDNIGWINDGDWVKYERVHFERDASSIEVRVASDTPGGRIEIRTGSPTGTLLGDVQVPNTGGWQQWQTVTGNVQIQPGTYDVYLVFKGSPEYDLMNVNWFVFRAN G QGNGDSHTHPDYTAGIRGITGNEVTIFFAPTTEARYVDVHLKVNNGQQLNYRMTERNGEWERVVENLSSGDVLEYSFTYEKLGPQYTTEWFTYSR Sequence number 33: GH81 - BH0236 - E542Q (Alkalihalobacillus halodurans) From GenBank: BAB03955.1 SPHAVSVGKGSYATEFPEIDFGGINDPGFRDQQGEPPATIYRSDRVTGPMQTNSWWGSLAVDRFSMNQYPHPFSVRHRAEGLHVFYDAPHNMVVHENREAGTWHIHGAIGTDFTIKHSGTANFEQAVVDDYNDWYVRGLLENGAHQMAITYGVGSPYIFVEYEDGSAVLDFDIAPDVWEMNGHVIGFSTHDHKHYAAFAPPGQNWSGIGSKTLTNNADYIAIAKLPEKDGNMLAKFEQYAYSVVRDAVADWTYDEATGTVTTTFEVTTEAKVQGAPDGTIFALYPHQYRHLASSSENQLLQNYQYEIIRGTMIGLEGKRFTTELTYPGVLPSLPDLGDYDRERLIGYLHDATSDYPTGSDTYELGKYIGKLATLAPIADQMGEYELAEQFRGELKDILEDWLQATNASGQLKGKNLFYYNENWGTILGYHAAHSSATRIN D HHFHYGYFVKAAAEIARADQEWAKSENWGGMIDLLIRDFMADRDDDLFPYLRMFDPYSGNSWADGLATFDAGNNQ QSSSE AMHAWTNVILWAEATGNKALRDRAIYLYTTEMSAINEYFFDVHQEIFPEEYGPEIVTINWGGKMDHATWWNSGKVEKYAINWLPFHGGSLYLGHHPDYVDRAYEELRRDIGSTDWNLWSNLVWMYRAFTNPDDALQQMEASIDDYGLFDPGNEKIIERGSTKAQTYHWIHNLAELGRVDPTVTANHPIYAVFNKNGNRTYIVYNFSDSPITVQFSDGHSIQVEPHSFNIGNGDGPTNPDPSEP D LKNPYERIQAEAYDAMSGIQTEGTDDDGGGDNIGWINDGDWVKYERVHFERDASSIEVRVASDTPGGRIEIRTGSPTGTLLGDVQVPNTGGWQQWQTVTGNVQIQPGTYDVYLVFKGSPEYDLMNVNWFVFRAN GQGNGDSHTHPDYTAGIRGITGNEVTIFFAPTTEARYVDVHLKVNNGQQLNYRMTERNGEWERVVENLSSGDVLEYSFTYEKLGPQYTTEWFTYSR Accession No. 34: GH81 - BH0236 - CatD - E542Q (Alkalihalobacillus halodurans) From GenBank: BAB03955.1 SPHAVSVGKGSYATEFPEIDFGGINDPGFRDQQGEPPATIYRSDRVTGPMQTNSWWGSLAVDRFSMNQYPHPFSVRHRAEGLHVFYDAPHNMVVHENREAGTWHIHGAIGTDFTIKHSGTANFEQAVVDDYNDWYVRGLLENGAHQMAITYGVGSPYIFVEYEDGSAVLDFDIAPDVWEMNGHVIGFSTHDHKHYAAFAPPGQNWSGIGSKTLTNNADYIAIAKLPEKDGNMLAKFEQYAYSVVRDAVADWTYDEATGTVTTTFEVTTEAKVQGAPDGTIFALYPHQYRHLASSSENQLLQNYQYEIIRGTMIGLEGKRFTTELTYPGVLPSLPDLGDYDRERLIGYLHDATSDYPTGSDTYELGKYIGKLATLAPIADQMGEYELAEQFRGELKDILEDWLQATNASGQLKGKNLFYYNENWGTILGYHAAHSSATRIN D HHFHYGYFVKAAAEIARADQEWAKSENWGGMIDLLIRDFMADRDDDLFPYLRMFDPYSGNSWADGLATFDAGNNQ QSSSEAMHAWTNVILWAEATGNKALRDRAIYLYTTEMSAINEYFFDVHQEIFPEEYGPEIVTINWGGKMDHATWWNSGKVEKYAINWLPFHGGSLYLGHHPDYVDRAYEELRRDIGSTDWNLWSNLVWMYRAFTNPDDALQQMEASIDDYGLFDPGNEKIIERGSTKAQTYHWIHNLAELGRVDPTVTANHPIYAVFNKNGNRTYIVYNFSDSPITVQFSDGHSIQVEPHSFNIGNGDGPTNPDPSEP D Sequence number 35: GH128-I-Am-FL (Amycolatopsis mediterranei) GenBank: AEK42318.1 MNQFRKLLVLVTVAIAAIGGLAAPATA AGTKKGVSAAAFSGVTAALGDVGARWFYTWAADPQGITAPAGTEFVPMIWGRDSVTADQLQRAKAAGSTLLAFN E PDLAGQANMSVETALDLWPQLQATGMRLGAPAVAYGGDTPGGWLDRFMSGAAARGYRVDFIPLHWYGGDFSAAATGQLQSYLQAVYNRYHRPIWLT E YALTDFSGSTPRYPSAAEQADFVSRSTAMLNGLSFVERYAWFSLSTSTTPTGLYTGTTPNSSGVAYRAAG Sequence number 36: GH128-I-Am-WT (Amycolatopsis mediterranei) From GenBank: AEK42318.1 AGTKKGVSAAAFSGVTAALGDVGARWFYTWAADPQGITAPAGTEFVPMIWGRDSVTADQLQRAKAAGSTLLAFN E PDLAGQANMSVETALDLWPQLQATGMRLGAPAVAYGGDTPGGWLDRFMSGAAARGYRVDFIPLHWYGGDFSAAATGQLQSYLQAVYNRYHRPIWLT EYALTDFSGSTPRYPSAAEQADFVSRSTAMLNGLSFVERYAWFSLSTSTTPTGLYTGTTPNSSGVAYRAAG Accession number 37: GH128-I-Am-E199Q (Amycolatopsis mediterranei) From GenBank: AEK42318.1 AGTKKGVSAAAFSGVTAALGDVGARWFYTWAADPQGITAPAGTEFVPMIWGRDSVTADQLQRAKAAGSTLLAFN E PDLAGQANMSVETALDLWPQLQATGMRLGAPAVAYGGDTPGGWLDRFMSGAAARGYRVDFIPLHWYGGDFSAAATGQLQSYLQAVYNRYHRPIWLT Q YALTDFSGSTPRYPSAAEQADFVSRSTAMLNGLSFVERYAWFSLSTSTTPTGLYTGTTPNSSGVAYRAAG Accession number 38: GH128-II-Pv-FL (Pseudomonas viridiflava) GenBank: SMS09959.1 MILRKALQTRHALIAFALLLSVLSESA WTATKSVKRGVAYDVASPADLSALSTGMSWWYNWSPKPHDRLAAYDYAGQYNVDFVPMVWNANLDDGQLKLYLLAHPGIRYLLVIN E PNLVDQANMTPQAAAQLWPRLEQISAQTGVKLVGPAMNWGTMTGYGDPVAWLDAFYAAYASAHQGRDPQIDYLAFHWYDYGLSSMLDRLSRYGKPFWVT E FANWHTLDDGLQIDSLEKQKQQMAEMVTMLERRSDVFRYAWFTGRMTPDPHFSSLLDAEGRLTELGQYYLSLPYSE Accession number 39: GH128-II-Pv-WT (Pseudomonas viridiflava) From GenBank: SMS09959.1 WTATKSVKRGVAYDVASPADLSALSTGMSWWYNWSPKPHDRLAAYDYAGQYNVDFVPMVWNANLDDGQLKLYLLAHPGIRYLLVIN E PNLVDQANMTPQAAAQLWPRLEQISAQTGVKLVGPAMNWGTMTGYGDPVAWLDAFYAAYASAHQGRDPQIDYLAFHWYDYGLSSMLDRLSRYGKPFWVT E FANWHTLDDGLQIDSLEKQKQQMAEMVTMLERRSDVFRYAWFTGRMTPDPHFSSLLDAEGRLTELGQYYLSLPYSE Sequence number 40: GH128-II-Pv-E214Q (Pseudomonas viridiflava) From GenBank: SMS09959.1 WTATKSVKRGVAYDVASPADLSALSTGMSWWYNWSPKPHDRLAAYDYAGQYNVDFVPMVWNANLDDGQLKLYLLAHPGIRYLLVIN E PNLVDQANMTPQAAAQLWPRLEQISAQTGVKLVGPAMNWGTMTGYGDPVAWLDAFYAAYASAHQGRDPQIDYLAFHWYDYGLSSMLDRLSRYGKPFWVT Q FANWHTLDDGLQIDSLEKQKQQMAEMVTMLERRSDVFRYAWFTGRMTPDPHFSSLLDAEGRLTELGQYYLSLPYSE Sequence number 41: GH128-III-Bg-FL (Blastomyces gilchristii) GenBank: OAT07255.1 MVSFKSLLVTSALACAVSCAPA ADYYTIAANPGSGKRGLAYNNINLLTAFEGGPFSWSYNWEPRPGGYTAGIEYVPMLWGPRGYGSWNADAEAGIAAGSKNLLAFN EPDIASQANMSPEAAAAAYQKYMNPYAARARLGSPAVSNGAPPKGLGWMQGFLDVCAGNCKIDFLAVHWHGPSGNVDDFKRYVSEAIALGQKYGIGTVWVT E FEGQGDEEAQVNFLKEVLPWLDSNAGVERYASFFVDNLVKGGALTSVGKAYKTI Sequence number 42: GH128-III-Bg-WT (Blastomyces gilchristii) From GenBank: OAT07255.1 ADYYTIAANPGSGKRGLAYNNINLLTAFEGGPFSWSYNWEPRPGGYTAGIEYVPMLWGPRGYGSWNADAEAGIAAGSKNLLAFN E PDIASQANMSPEAAAAAYQKYMNPYAARARLGSPAVSNGAPPKGLGWMQGFLDVCAGNCKIDFLAVHWHGPSGNVDDFKRYVSEAIALGQKYGIGTVWVT E FEGQGDEEAQVNFLKEVLPWLDSNAGVERYASFFVDNLVKGGALTSVGKAYKTI Sequence number 43: GH128-III-Bg-E208Q (Blastomyces gilchristii) From GenBank: OAT07255.1 ADYYTIAANPGSGKRGLAYNNINLLTAFEGGPFSWSYNWEPRPGGYTAGIEYVPMLWGPRGYGSWNADAEAGIAAGSKNLLAFN E PDIASQANMSPEAAAAAYQKYMNPYAARARLGSPAVSNGAPPKGLGWMQGFLDVCAGNCKIDFLAVHWHGPSGNVDDFKRYVSEAIALGQKYGIGTVWVT Q FEGQGDEEAQVNFLKEVLPWLDSNAGVERYASFFVDNLVKGGALTSVGKAYKTI Sequence number 44: EXDXE Sequence number 45: EXDXXE Sequence number 46: DDAKK Sequence number 47: EAAAK Sequence number 48: XEXTX Sequence number 49: ESXSE

Claims

1. The following two motif sequences contained in the amino acid sequence W1 of the polypeptide constituting the catalytic domain (CatD) of wild-type β-(1,3)(1,4)-glucanase belonging to GH16 type enzyme: SEQ ID NO: 44: EXDXE (X represents any amino acid), or SEQ ID NO: 45: EXDXXE (X represents any amino acid) wherein at least one of the two glutamic acids (E) in each of the said motif sequences is substituted with another amino acid, and the amino acid sequence contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence W1, having specific binding activity with β-(1,3)(1,4)-glucan (MLG), β-(1,3)(1,4)-glucan-binding polypeptide.

2. The said another amino acid is any one of glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), serine (S), threonine (T), aspartic acid (D), asparagine (N), glutamine (Q), The β-(1,3)(1,4)-glucan-binding polypeptide according to Claim 1.

3. The amino acid sequence W1 is the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 6, The β-(1,3)(1,4)-glucan-binding polypeptide according to Claim 1.

4. A method for detecting β-(1,3)(1,4)-glucan, comprising the step of contacting a test sample with a reagent containing the β-(1,3)(1,4)-glucan-binding polypeptide according to any one of Claims 1 to 3. Method for detecting β-(1,3)(1,4)-glucan.

5. A reagent kit for detecting β-(1,3)(1,4)-glucan, having a reagent containing the β-(1,3)(1,4)-glucan-binding polypeptide according to any one of Claims 1 to 3. Reagent kit for detecting β-(1,3)(1,4)-glucan.

6. A recombinant microorganism or cell containing a nucleotide sequence encoding the β-(1,3)(1,4)-glucan-binding polypeptide according to any one of Claims 1 to 3.

7. The following motif sequence contained in the amino acid sequence W2 of the polypeptide constituting the catalytic domain (CatD) of wild-type β-1,3-glucanase belonging to GH64 type enzyme: SEQ ID NO: 48: XEXTX (X represents any amino acid) wherein the glutamic acid (E) is substituted with another amino acid, and the amino acid sequence contains an amino acid sequence having 80% or more sequence identity with the amino acid sequence W2, having specific binding activity with β-1,3-glucan (13BG), β-1,3-glucan-binding polypeptide.

8. The following motif sequence contained in the amino acid sequence W3 of the polypeptide constituting the catalytic domain (CatD) of wild-type β-1,3-glucanase belonging to GH81 type enzyme: SEQ ID NO: 49: ESXSE (X represents any amino acid) wherein at least the glutamic acid (E) located on the N-terminal side of the two glutamic acids (E) is substituted with another amino acid, and the amino acid sequence has a sequence identity of 80% or more with the amino acid sequence W3, and having specific binding activity with β-1,3-glucan (13BG), β-1,3-glucan-binding polypeptide.

9. wherein the other amino acid is glutamine (Q), The β-1,3-glucan-binding polypeptide according to Claim 7 or 8.

10. wherein the amino acid sequence W2 is the amino acid sequence represented by SEQ ID NO: 17, The β-1,3-glucan-binding polypeptide according to Claim 7.

11. wherein the amino acid sequence W3 is the amino acid sequence represented by SEQ ID NO: 32, The β-1,3-glucan-binding polypeptide according to Claim 8.

12. comprising a polypeptide constituting the carbohydrate-binding module (CBM) of wild-type β-1,3-glucanase belonging to GH81 type enzyme, The β-1,3-glucan-binding polypeptide according to Claim 8.

13. A method for detecting β-1,3-glucan, comprising the step of contacting a test sample with a reagent containing the β-1,3-glucan-binding polypeptide according to Claim 7 or 8. Method for detecting β-1,3-glucan.

14. A β-1,3-glucan detection kit having a reagent containing the β-1,3-glucan-binding polypeptide according to Claim 7 or 8. β-1,3-glucan detection kit.

15. A recombinant microorganism or cell comprising a nucleotide sequence encoding the β-1,3-glucan-binding polypeptide according to Claim 7 or 8.

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