Carbohydrate-degrading enzyme, acid-fast bacterium detection kit, and in vitro acid-fast bacterium detection method

Abstract

To provide saccharide-degrading enzymes capable of cleaving the β-linkage at the non-reducing terminal of D-arabinan chain and kits and methods for detecting acid-fast bacteria using the enzyme.SOLUTION: The saccharide-degrading enzyme comprises amino acid sequence (a) or (b) shown below, and has an exo-β-D-arabinofuranosidase activity: (a) an amino acid sequence of a specific amino acid sequence; or (b) the above specific amino acid sequence in which one or several amino acids substituted, deleted, inserted or added.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a glycolytic enzyme, a detection kit for acid-fast bacteria, and a method for detecting acid-fast bacteria. In vitro It relates to a detection method. [Background technology]

[0002] Infectious diseases caused by Mycobacterium bacteria include tuberculosis, nontuberculous mycobacterial infections, and MAC disease. It was thought that tuberculosis could be eradicated with the introduction of antituberculosis drugs such as streptomycin and rifampicillin and widespread use of BCG vaccination. However, infections with multidrug-resistant Mycobacterium tuberculosis are increasing in developing countries. Two billion people, or one-third of the world's population, are infected with tuberculosis, and two million people die from the disease each year. In Japan, more than 20,000 cases of infection are confirmed each year. Because so many people are infected with Mycobacterium bacteria, research into Mycobacterium bacteria is important.

[0003] The cell walls of mycobacteria, such as Mycobacterium tuberculosis, contain a glycolipid called lipoarabinomannan (LAM). LAM contains lipomannan (LM), a glycolipid in which phosphatidylinositol is modified with mannan chains, and LM is modified with D-arabinan chains. The D-arabinan chain has a basic structure in which the main chain is composed of α-1,5-linked D-arabinofuranose (D-Araf) and contains D-Arafs branched by α-1,3-linkages. The non-reducing end of the D-arabinan chain is modified with β-1,2-linked D-Arafs. The arabinogalactan chains (AG) that make up the cell walls of mycobacteria are also modified with D-arabinan chains similar to LAM.

[0004] The structure of LAM varies depending on the type of mycobacterium. Therefore, sensitive detection of mycobacteria or determination of the type of mycobacterium is possible based on the products produced by LAM degradation. Furthermore, since LAM has immunostimulatory properties, knowledge about the substances obtained by LAM degradation is expected to advance the development of immunostimulatory agents.

[0005] Patent Document 1 discloses endo-α-D-arabinase (endo-αDA) and exo-α-D-arabinofuranosidase (exo-αDA), which cleave α-1,5-linkages. Endo-αDA is an enzyme that acts endo-type on the D-arabinan chains of LAM and AG to release α-D-arabinooligosaccharides and degrade LAM into LM. Exo-αDA is an enzyme that acts exo-type on the α-D-arabinooligosaccharides released by endo-αDA to release D-arabinose (D-Ara). Because the non-reducing end of the D-arabinan chain is modified with β-1,2-linked D-Araf (β-D-Araf), an exo-type enzyme that cleaves the β-linkage at the non-reducing end is required to completely degrade LAM into monosaccharides. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2019-17358 Summary of the Invention [Problem to be solved by the invention]

[0007] To date, no glycolytic enzymes that act on exo-type glycosidases to cleave β-D-arabinofuranoside bonds have been reported.

[0008] The present invention has been made in view of the above circumstances, and provides a glycolytic enzyme capable of cleaving the β-bond at the non-reducing end of a D-arabinan chain, as well as a detection kit for mycobacteria and a method for detecting mycobacteria using the glycolytic enzyme. In vitro The object is to provide a detection method. [Means for solving the problem]

[0009] The carbohydrate-degrading enzyme according to the first aspect of the present invention is It consists of either the following amino acid sequence (a) or (b): It has exo-β-D-arabinofuranosidase activity. (a) the amino acid sequence shown in SEQ ID NO: 1 (b) an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 1

[0010] The acid-fast bacillus detection kit according to the second aspect of the present invention comprises: The present invention comprises the carbohydrate-degrading enzyme according to the first aspect of the present invention.

[0011] The acid-fast bacillus detection kit according to the second aspect of the present invention comprises: Further comprising at least one of exo-α-D-arabinofuranosidase and endo-α-D-arabinofuranosidase, This may also be the case.

[0012] Acid-fast bacteria according to the third aspect of the present invention In vitro The detection method is a treatment step of treating a sample with the carbohydrate-degrading enzyme according to the first aspect of the present invention; a detection step for detecting a product produced in the processing step; Includes. [Effects of the Invention]

[0013] The glycolytic enzyme of the present invention can cleave the β-bond at the non-reducing end of the D-arabinan chain. In vitro A detection method is provided. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows a β-D-Araf bond cleaved by a carbohydrate-degrading enzyme according to embodiment 1 of the present invention. [Figure 2] FIG. 1 is a diagram showing a schematic diagram of a LAM. [Figure 3] FIG. 1 shows bands in SDS (Sodium dodecyl sulfate)-polyacrylamide gel electrophoresis (SDS-PAGE) according to Example 1. [Figure 4] FIG. 1 shows thin layer chromatography (TLC) spots according to Test Example 1. [Figure 5] FIG. 1 shows the optimum pH of the carbohydrate-degrading enzyme in Test Example 2. [Figure 6] FIG. 1 is a graph showing the optimum temperature of a carbohydrate-degrading enzyme in Test Example 2. [Figure 7] FIG. 10 is a diagram showing the results of HPAEC-PAD (High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection) in Test Example 3. [Figure 8] FIG. 1 shows the results of HPAEC-PAD for LAM derived from Mycobacterium smegmatis (M. smegmatis) in Test Example 4. [Figure 9] FIG. 1 shows the results of silver pass staining of LAM derived from M. smegmatis in Test Example 4. [Figure 10] FIG. 1 shows the results of HPAEC-PAD for LAM derived from Mycobacterium tuberculosis (M. tuberculosis) in Test Example 4. [Figure 11] FIG. 1 shows the results of silver pass staining of LAM derived from M. tuberculosis in Test Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0015] Embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0016] (Embodiment 1) The glycolytic enzyme according to this embodiment consists of the amino acid sequence shown in SEQ ID NO: 1. The glycolytic activity of this glycolytic enzyme is exo-β-D-arabinofuranosidase activity, which cleaves the β-bond at position 1 of β-D-Araf, as indicated by the arrow in Figure 1.

[0017] The glycolytic enzyme according to the present embodiment acts on the exo-type. As shown in Figure 2, the non-reducing end of LAM has a β-1,2-linked D-Araf. Because the glycolytic enzyme acts on the exo-type, it cleaves the β-bond at the carbon atom 1 of β-D-Araf at the terminal of the glycan of LAM or the like, thereby liberating D-Ara.

[0018] The amino acid sequence of the glycolytic enzyme according to this embodiment may be SEQ ID NO: 1 with one or several amino acids substituted, deleted, inserted, or added, as long as the exo-β-D-arabinofuranosidase activity is not lost. The number of amino acids substituted or mutated varies depending on the position or type of the amino acid residue in the three-dimensional structure of the protein. The number of amino acids mutated by substitution or the like is preferably 1 to 30, more preferably 1 to 20, even more preferably 1 to 10, and particularly preferably 1 to 5.

[0019] In the case of substitution, preferably, an acidic amino acid is substituted with any other acidic amino acid as long as the exo-β-D-arabinofuranosidase activity is not lost. Similarly, it is preferred that a basic amino acid, an aromatic amino acid, an aliphatic amino acid, or an oxyamino acid be substituted with another basic amino acid, an aromatic amino acid, an aliphatic amino acid, or an oxyamino acid, respectively. Unless otherwise specified, amino acids are in the L-form.

[0020] The carbohydrate-degrading enzyme of this embodiment may be a protein consisting of an amino acid sequence that has 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, so long as exo-β-D-arabinofuranosidase activity is not lost. Generally, when the amino acid sequence identity between two proteins is 80% or more, the three-dimensional structures are very similar and function is maintained. Since the higher the sequence identity between the amino acid sequences, the higher the similarity in the three-dimensional structures. Therefore, the sequence identity is preferably 85% or more or 90% or more, and more preferably 95% or more, so long as exo-β-D-arabinofuranosidase activity is not lost.

[0021] The glycosidase according to the present embodiment can be synthesized by known methods. For example, the glycosidase may be synthesized by chemical polypeptide synthesis or by genetic engineering using Escherichia coli or the like. Examples of polypeptide synthesis methods include liquid-phase and solid-phase methods. The liquid-phase method involves carrying out a reaction in solution, isolating and purifying the product from the reaction mixture, and using the resulting product as an intermediate in the subsequent peptide elongation reaction. On the other hand, the solid-phase method involves binding amino acids to a solid support that is insoluble in the reaction solvent, and sequentially subjecting the bound amino acids to condensation reactions to elongate the peptide. The glycosidase can also be synthesized by condensing a partial peptide or amino acid constituting a protein with the remaining portion, and, if the product has a protecting group, removing the protecting group.

[0022] In a genetic engineering method, for example, a carbohydrate-degrading enzyme can be obtained by transforming a host cell with an expression vector that expresses a gene encoding the carbohydrate-degrading enzyme and culturing the transformant. The nucleotide sequence of the gene encoding the carbohydrate-degrading enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1 is shown, for example, in SEQ ID NO: 2. This gene is contained in Aureobacterium sp. M2.

[0023] An expression vector suitable for a host cell can be constructed using various regulatory elements (such as promoters, ribosome binding sites, terminators, enhancers, and various cis-elements that control expression levels) for expressing the gene encoding the above-mentioned glycosidase in the host cell. The constructed expression vector can be introduced into host cells such as Escherichia coli by a method such as electroporation. By culturing the host cells under specified conditions, the glycosidase is expressed in the host cells, and the glycosidase can be extracted.

[0024] Aureobacterium sp. M2 has a gene encoding the carbohydrate-degrading enzyme according to this embodiment. The gene encoding the carbohydrate-degrading enzyme according to this embodiment may be obtained from the genomic DNA of a bacterium having a homologue of the gene whose nucleotide sequence is set forth in SEQ ID NO: 2.

[0025] The obtained carbohydrate-degrading enzyme may be purified and analyzed by, for example, ion exchange chromatography, high performance liquid chromatography, reverse phase chromatography, affinity chromatography, Edman degradation method, or the like.

[0026] The conditions under which the carbohydrate-degrading enzyme according to this embodiment exhibits its enzymatic activity are not particularly limited, but for example, the temperature is 25 to 45° C., preferably 30 to 40° C., and more preferably 35 to 40° C. The pH is 5.5 to 7.5, preferably 5.8 to 7, and more preferably 5.8 to 6.5.

[0027] The glycolytic activity of a glycolytic enzyme can be confirmed, for example, by the following method. A glycolytic enzyme is reacted with paranitrophenyl-β-D-arabinofuranoside (pNP-β-D-Araf), in which a paranitrophenyl group is bound to R (see Figure 1). The resulting monosaccharides are detected by chromatography, such as TLC and HPAEC-PAD, and the enzyme activity can be calculated from the amount of monosaccharides. Alternatively, instead of pNP-β-D-Araf, glycolytic enzymes may be reacted with LAM from mycobacteria such as M. smegmatis to detect D-Ara.

[0028] The glycosidase according to the present embodiment can cleave the β-bond at the non-reducing end of a D-arabinan chain. This is a novel enzyme that has not been reported to date as being capable of cleaving the β-bond at the non-reducing end of a D-arabinan chain. This glycosidase is useful for analyzing the terminal structure of LAM, etc.

[0029] (Embodiment 2) The method for detecting acid-fast bacteria according to this embodiment includes a processing step and a detection step. In the processing step, a sample is treated with the carbohydrate-degrading enzyme according to embodiment 1. The sample is, for example, a specimen from a subject suspected of containing acid-fast bacteria, specifically, a body fluid such as blood, serum, urine, or saliva. The sample may also be a sample prepared from a body fluid by a known method. The acid-fast bacteria are not particularly limited as long as they are acid-fast bacteria such as Mycobacterium tuberculosis and Mycobacterium leprae, which have LAM. Examples of acid-fast bacteria include M. smegmatis, M. tuberculosis, Mycobacterium chelonae, and Mycobacterium bovis.

[0030] In the treatment step, a sample is exposed to the glycolytic enzyme according to embodiment 1 under conditions under which the glycolytic enzyme exhibits glycolytic activity. If the sample contains β-1,2-linked D-Araf at the terminal of the glycan that is cleaved by the glycolytic enzyme, the β-1,2-linked D-Araf in the sample is cleaved in the treatment step to produce D-Ara.

[0031] In the treatment step, at least one of exo-αDA and endo-αDA may be further used in addition to the glycosidase of embodiment 1. For example, in the case of the LAM shown in Figure 2, the α-1,5-bond can be cleaved by endo-αDA and exo-αDA, and therefore more D-Ara can be produced than when only the glycosidase of embodiment 1 is used.

[0032] In the detection step, the product produced in the treatment step, such as D-Ara, is detected. The product can be detected by the above-mentioned chromatography method or the like. In this case, the product can be identified by using a standard sample of the target product as a control. Depending on the detection method used, the amount of product produced may be evaluated in the detection step. It is preferable to subject a sample not treated with the carbohydrase in the treatment step to the same method and compare the results with those of the product produced by treatment with the carbohydrase. This makes it possible to confirm the presence or absence of the product contained in the sample before treatment in the treatment step.

[0033] The type of acid-fast bacterium or species of acid-fast bacterium contained in a sample can be detected based on the type of product detected in the detection step, as well as the amount of the product produced. In the case of M. smegmatis, which has an LAM with the structure shown in FIG. 2, D-Ara cleaved by the glycosidase of embodiment 1 is detected. On the other hand, for example, in LAM derived from M. tuberculosis, β-1,2-linked D-Araf is further linked to mannose (Man-capped), and therefore, β-1,2-linked D-Araf, which cannot be cleaved by the glycosidase of embodiment 1, is thought to be greater in LAM than in M. smegmatis. Therefore, based on the amount of D-Araf detected in the detection step, it can be determined whether the acid-fast bacterium contained in a sample is M. smegmatis or M. tuberculosis. Furthermore, by analyzing the products obtained by combining exo-αDA and endo-αDA, many types of acid-fast bacterium can be identified.

[0034] The method for detecting mycobacteria according to this embodiment uses a novel glycolytic enzyme with exo-β-D-arabinofuranosidase activity, making it useful for detecting LAMs and other molecules containing β-1,2-linked D-Araf at their termini. Furthermore, the use of other glycolytic enzymes, such as exo-αDA and endo-αDA, in combination can improve the efficiency of LAM detection and identification.

[0035] In another embodiment, there is provided a detection kit for acid-fast bacteria, comprising the carbohydrate-degrading enzyme according to embodiment 1. The detection kit may further comprise buffers, reagents, etc. used in the above-mentioned processing and detection steps. The detection kit may also further comprise at least one of exo-αDA and endo-αDA. [Example]

[0036] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0037] Example 1: Construction of recombinant enzymes The MA1061 gene, a glycoside hydrolase family GH116 homologue from Aureobacterium sp. M2, was amplified as follows. First, to prepare the vector, the genome was amplified by PCR using PrimeSTAR™ (Takara Bio). The PCR reaction solution (50 μl) consisted of 10 μl of 5x PrimeSTAR™ buffer, 4 μl of dNTP mixture, 1 μl of forward primer A (10 pmol / μl), 1 μl of reverse primer A (10 pmol / μl), 1 μl of template (pET23d vector (Novagen)), 0.5 μl of PrimeSTAR™ HS DNA polymerase, and 32.5 μl of double-distilled water (ddH2O). The nucleotide sequences of forward primer A and reverse primer A are shown in SEQ ID NOs: 3 and 4, respectively. The PCR reaction consisted of 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 4 minutes.

[0038] A primer mix for amplifying the MA1061 gene was prepared. The primer mix consisted of 1 μl of forward primer B (100 pmol / μl), 1 μl of reverse primer B (100 pmol / μl), and 18 μl of ddH2O. The nucleotide sequences of forward primer B and reverse primer B are shown in SEQ ID NOs: 5 and 6, respectively.

[0039] Next, the full-length MA1061 gene was amplified by PCR using PrimeSTAR™ (Takara Bio) with the Aureobacterium sp. M2 genome prepared using the FastPure™ DNA kit (Takara Bio). The PCR reaction mixture (50 μl) consisted of 25 μl of 2× Gflex PCR buffer, 2 μl of primer mix, 1 μl of template (52.6 ng / μl), 1 μl of Tks Gflex DNA polymerase, and 21 μl of ddH2O. The PCR reaction consisted of 30 cycles of 98°C for 10 seconds, 58°C for 15 seconds, 60°C for 15 seconds, 62°C for 15 seconds, and 72°C for 2.5 minutes.

[0040] The purified PCR product was used to prepare an infusion reaction solution using the In-Fusion HD cloning kit (Clontech). The infusion reaction solution consisted of 1.0 μl of 5x Infusion HD Enzyme Premix, 2 μl of vector, and 2 μl of PCR product, and ddH2O was added to make up a total volume of 5 μl. The infusion reaction solution was incubated at 50°C for 15 minutes and then cooled on ice.

[0041] Colony PCR was performed using the infusion reaction mixture. 2.5 μl of the infusion reaction mixture was added to 50 μl of competent DH5α cells, vortexed, and incubated on ice for 5 minutes. The mixture was then incubated at 42°C for 45 seconds, and 50 μl of the reaction mixture was evenly spread onto an L Bamp plate. The L Bamp plate was incubated at 37°C for 12 to 16 hours. Eight colonies were randomly selected and used as templates for PCR using Emerald PCR Master Mix (Takara Bio). The amplified MA1061 was electrophoresed on a 1% agarose gel to confirm the size of the DNA fragment.

[0042] Colonies with DNA of the desired size were cultured, and the entire volume was centrifuged at 12,000 × g for 10 minutes to collect the cells. Plasmids were extracted using the NucleoSpin™ Plasmid EasyPure Kit (Takara Bio Inc.), and the DNA concentration was measured. The plasmids were digested with restriction enzymes and electrophoresed on a 1% agarose gel to confirm the plasmid length. Escherichia coli BL21 (DE3) was transformed with the prepared plasmids using standard methods.

[0043] The enzyme was induced in the transformed E. coli using the Overnight Express Autoinduction System (Merck). The intracellular enzyme was extracted using BugBuster™ protein extraction reagent (Merck). The protein was then purified using a His-tag attached to the C-terminus of the protein. For purification, 600 μl of crude enzyme was added to 1.2 ml of equilibration buffer and loaded onto a column packed with Metal Affinity Resin (1 ml). A gradient eluate of imidazole solution, prepared by mixing appropriate amounts of equilibration buffer and elution buffer, was loaded onto the column, and the eluted fraction was collected. The equilibration buffer was 50 mM sodium phosphate and 300 mM sodium chloride (pH 7.0), and the elution buffer was 50 mM sodium phosphate, 300 mM sodium chloride, and 100 mM imidazole (pH 7.0). The resulting protein (MA1061 protein) was evaluated by standard SDS-PAGE.

[0044] (result) The molecular weight of the MA1061 protein, including the His-tag, predicted from the amino acid sequence (SEQ ID NO: 1) encoded by the MA1061 gene is 95.0 kDa. As shown in Figure 3, a single band was confirmed for the MA1061 protein in SDS-PAGE at approximately 95.0 kDa. The MA1061 protein solution (83.9 ng / μl) obtained by desalting and concentrating the protein using an ultrafiltration membrane was used in the following test examples.

[0045] (Test Example 1: Examination of Enzyme Activity) MA1061 protein was reacted with pNP-β-D-Araf as a substrate. The reaction mixture consisted of 5 μl of 20 mM pNP-β-D-Araf, 0.5 μl of 1 M acetate buffer (pH 6.0), 1 μl of MA1061 protein, and 3.5 μl of HO. Water was added instead of MA1061 protein as a control.

[0046] The products contained in the reaction solution were confirmed by TLC. The reaction solution and a standard were spotted onto a plate (TLC aluminum sheet, silicagel 60) and air-dried. An appropriate amount of developing solvent was poured onto the developing layer, and the plate was immersed in it to develop. When the developing solvent rose to the top, the plate was removed and air-dried in a fume hood. A color developer was sprayed onto the plate, which was then air-dried again and heated at 130°C for approximately 1 minute. The developing solvent was prepared by mixing n-propanol, ethanol, and water in a volume ratio of 7:1:2. The standard was a 1 mM D-Ara solution. Orcinol-sulfuric acid (a 10:1 volume mixture of 10% sulfuric acid containing 1% FeCl3 and 6% orcinol) was used as the color developer.

[0047] (result) As shown in Figure 4, a D-Ara spot was confirmed in the reaction mixture by TLC, demonstrating that the MA1061 protein possesses exo-β-D-arabinofuranosidase activity. Hereinafter, the MA1061 protein will be referred to as exo-βDA.

[0048] (Test Example 2: Enzyme reaction conditions for exo-βDA) The enzymatic reaction was carried out using pNP-β-D-Araf as a substrate as described below, and the optimal pH and temperature were determined by quantifying the paranitrophenol released by hydrolysis under alkaline conditions.

[0049] To determine the optimal pH, a mixture of 5 μL of 20 mM pNP-β-D-Araf solution and 10 μL of 0.1 M acetate buffer (pH 4-6) or 0.1 M sodium phosphate buffer (pH 6-8) was incubated at 37°C for 5 minutes, and 5 μL of the exo-βDA solution (83.9 ng / μL diluted 20-fold with 5 mM phosphate buffer) obtained in Test Example 1 was added. The reaction was continued at 37°C for 20 minutes, and 30 μL of 120 mM NaCO3 was added to terminate the reaction. The absorbance at 400 nm was measured.

[0050] To determine the optimal temperature, a mixture of 5 μL of 20 mM pNP-β-D-Araf solution and 10 μL of 0.1 M sodium phosphate buffer (pH 6) was incubated at 37°C for 5 minutes, to which 5 μL of the exo-βDA solution (83.9 ng / μL diluted 20-fold with 5 mM phosphate buffer) obtained in Test Example 1 was added. The reaction was continued at each temperature for 20 minutes, and the reaction was stopped by adding 30 μL of 120 mM NaCO3. The absorbance at 400 nm was measured.

[0051] (result) As shown in Figure 5, the optimum pH of exo-βDA was 6. As shown in Figure 6, the optimum temperature of exo-βDA was 35-40°C. In addition, enzyme kinetic analysis showed that the Km value was 3.2 mM and the k cat is 13.2 / sec, k cat / Km was 4.14 / mM·sec.

[0052] (Test Example 3: Reactivity with synthetic substrates) The reactivity was examined using a synthetic substrate, A22BβT, which is partially branched and has 22 D-arabinose residues. A22BβT has an acetonide tag at the reducing end, and the D-arabinose residues at the non-reducing end are linked by α-linkages, except for the D-arabinose linked by β-linkages. The enzymes used were exo-βDA, exo-αDA, and endo-αDA.

[0053] The reaction mixture consisted of 2 μl of A22BβT (2 mg / ml), 5 μl of 1 M sodium phosphate buffer (pH 6.0), 1 μl of enzyme (1 μl of each enzyme if multiple enzymes were used), and 91 μl of water. No enzyme was added to the control.

[0054] Approximately 100 μl of the undiluted enzyme reaction mixture was measured by HPAEC-PAD. An ICS-3000 (Dionex) was used for HPAEC-PAD. A pulsed amperometric detector was used, and a CarboPack PA-1 column was used. The flow rate was 1.00 mL / min, the temperature was 30°C, the acquisition time was 60 min, and the sample volume was 25 μl. Eluent A was 100 mM NaOH, and eluent B was 100 mM NaOH containing 500 mM CH₂COONa. The elution conditions were as follows: 100% eluent A from 0 to 5 min, 100% eluent A to 0% and 0% to 100% eluent B from 5 to 45 min, 100% eluent B from 45 to 50 min, and 100% eluent A from 50 to 60 min.

[0055] (result) As shown in Figure 7, the area of ​​the peak representing the monosaccharide D-Ara increased from 8.04 with exo-βDA alone to 10.41 with the addition of endo-αDA, and then to 44.49 with the addition of exo-αDA. This indicates that only the terminal β-bond was cleaved with exo-βDA alone, while the addition of endo-αDA partially cleaved the α-bond. Furthermore, the addition of exo-αDA cleaved all bonds, resulting in degradation to the monosaccharide D-Ara. The letters "A," "L," "T," "B," and "β" used to represent the peaks in Figure 7 represent D-Ara, linear, acetonide tag, branched, and β-bond, respectively. For example, "A22BβT" indicates that the protein contains 22 D-Ara and acetonide tags, and is branched and β-bonded. Furthermore, "A3LT" indicates that the protein contains three D-Ara and acetonide tags, and is linear (no branching).

[0056] (Test Example 4: Examination of decomposition of LAM) Each enzyme was reacted with LAM derived from M. smegmatis or LAM derived from M. tuberculosis (manufactured by Nakarai). The reaction mixture consisted of 5 μl of LAM (1 mg / ml), 1 μl of 1 M sodium phosphate buffer (pH 6.0), 1 μl of enzyme (1 μl of each enzyme if multiple enzymes were used), and 13 μl of water. No enzyme was added to the control.

[0057] The enzyme reaction mixture was measured by HPAEC-PAD in the same manner as in Test Example 3. Furthermore, the enzyme reaction mixture was examined by silver pass staining using a Silvest Stain Neo (protein and nucleic acid staining) kit (manufactured by Nacalai Tesque). The sample composition for silver pass staining consisted of 10 μl of enzyme reaction mixture and 2.5 μl of 5x SDS sample buffer.

[0058] (result) As shown in Figure 8, in the case of M. smegmatis-derived LAM, exo-βDA alone only slightly released D-Ara, but the combined use of exo-αDA and endo-αDA, which cleave α-linkages, increased the amount of released D-Ara by up to 14-fold. Silver-path staining, as shown in Figure 9, demonstrated that endo-αDA (Endo-α) cleaved the sugars from LAM and degraded it to lipomannan, but exo-type enzymes, i.e., exo-βDA (Exo-β) and exo-αDA (Exo-α), did not degrade LAM.

[0059] As shown in Figure 10, an increase in liberated D-Ara was confirmed in M. tuberculosis-derived LAM by combining exo-αDA and endo-αDA. Compared to M. smegmatis-derived LAM, M. tuberculosis-derived LAM showed a smaller increase in D-Ara, i.e., a lower reactivity with exo-βDA. This is thought to be due to the Man cap at the end of M. tuberculosis-derived LAM. As shown in Figure 11, silver-path staining of M. tuberculosis-derived LAM revealed that LAM was degraded to lipomannan by endo-αDA.

[0060] The above-described embodiments are intended to explain the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not by the embodiments. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention. [Industrial Applicability]

[0061] The present invention is useful as a research reagent and for detecting acid-fast bacteria.

Claims

1. It consists of either the following amino acid sequence (a) or (b): having exo-β-D-arabinofuranosidase activity, Carbohydrate-degrading enzyme. (a) the amino acid sequence shown in SEQ ID NO: 1 (b) an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 1;

2. The carbohydrate-degrading enzyme according to claim 1 is provided. Acid-fast bacillus detection kit.

3. Further comprising at least one of exo-α-D-arabinofuranosidase and endo-α-D-arabinofuranosidase, The acid-fast bacterium detection kit according to claim 2.

4. a treatment step of treating a sample with the carbohydrate-degrading enzyme according to claim 1; a detection step for detecting a product produced in the processing step; An in vitro method for detecting acid-fast bacilli, comprising:

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