Polypeptide having β-mannosidase activity

Abstract

The present invention addresses the problem of providing an enzyme that can be used for efficient mannan degradation. Provided is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 and having β-mannosidase activity.

Description

A polypeptide having β-mannosidase activity

[0001] The present invention relates to a polypeptide having β-mannosidase activity.

[0002] Mannan is a polysaccharide containing mannose as a constituent sugar, and is a major component of hemicellulose in plant cell walls. Woody biomass such as softwood contains a large amount of mannan, and decomposition of mannan is required when producing biofuel from woody biomass. Non-Patent Document 1 provides a review of the structure of mannan and enzymes that decompose mannan.

[0003] Furthermore, in the field of biofuel development, attempts have been made to use mannan as a sustainable resource, and for example, production of bioethanol from mannan has been reported (Non-Patent Document 2). In this document, *Saccharomyces cerevisiae*, a yeast that displays β-mannanase and β-mannosidase derived from *Aspergillus aculeatus* on its cell surface, is used.

[0004] Moreira et al., Appl Microbiol Biotechnol (2008) 79:165-178; Ishii et al., Biotechnol Biofuels (2016) 9:188

[0005] An object of the present invention is to provide an enzyme that can be used for efficient mannan decomposition.

[0006] The present invention, in order to solve the above problems, includes the following inventions: [1] A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, or a polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and having β-mannosidase activity. [2] The polypeptide according to [1], having an optimal pH of 4.5 to 6. [3] The polypeptide according to [1], exhibiting pH stability in the range of pH 3 to 10. [4] The polypeptide according to [1], having an optimal temperature of 20 to 40°C. [5] The polypeptide according to [1], exhibiting temperature stability in the range of 4 to 50°C. [6] A nucleic acid encoding the polypeptide according to [1]. [7] A vector containing the nucleic acid according to [6]. [8] A genetically modified cell containing the nucleic acid according to [6] or the vector according to [7]. [9] A method for producing a polypeptide having β-mannosidase activity, comprising the step of culturing the genetically modified cell according to [8].

[10] The method for producing a polypeptide having β-mannosidase activity, further comprising the step of recovering the polypeptide having β-mannosidase activity.

[11] An enzyme preparation having β-mannosidase activity, comprising the polypeptide described in [1].

[0007] The present invention provides an enzyme that can be used for efficient decomposition of mannan.

[0008] This is a schematic diagram of the plasmid created during the cloning of the Bifidobacterium denthium-derived β-mannosidase gene. This figure shows the nucleotide sequence (SEQ ID NO: 1) of the Bifidobacterium denthium-derived β-mannosidase gene. This figure shows the predicted amino acid sequence (SEQ ID NO: 2) of the Bifidobacterium denthium-derived β-mannosidase. This figure shows the nucleotide sequence (SEQ ID NO: 3) of a gene with codons optimized for E. coli based on the Bifidobacterium denthium-derived β-mannosidase gene. This is a schematic diagram of the plasmid created for E. coli expression of the Bifidobacterium denthium-derived β-mannosidase gene (pET23b-GH2-Man EcOPTThis is an electrophoresis graph showing the SDS-PAGE analysis results of proteins in the fraction before loading onto the Ni-NTA column (apply), the washing solution fraction after column passage (wash), and the elution fraction (elution) for the lysates of the Bifidobacterium denthium (BL21) and control strain (pET23b / BL21), as well as the β-mannosidase gene expression strain. This is a graph showing the measurement results of the optimal pH of Bifidobacterium denthium-derived β-mannosidase in Example 3 as the relative enzyme activity (%) at various pH levels. This is a graph showing the measurement results of the optimal pH and stable pH of Bifidobacterium denthium-derived β-mannosidase in Example 5 as the relative enzyme activity (%) at various pH levels. This is a graph showing the measurement results of the optimal temperature and stable temperature of Bifidobacterium denthium-derived β-mannosidase in Example 6 as the relative enzyme activity (%) at various temperatures.

[0009] The inventors screened numerous microorganisms for efficient mannan degradation and discovered that Bifidobacterium dentium possesses glucomannan metabolism capabilities. They also identified the polypeptide possessing β-mannosidase activity and the nucleic acid (gene) encoding it.

[0010] The present invention will be described in detail below.

[0011] [Explanation of Terms] In this specification, "mannan" refers to polysaccharides that contain mannose as a constituent sugar. In this specification, the term "mannan" comprehensively refers to polysaccharides that contain mannose as a constituent sugar, such as straight-chain mannans ("1,4-β-D-mannan") in which only mannose is linked by β-1,4 bonds, "glucomannan" in which mannose and glucose are linked by β-1,4 bonds, and "galactomannan" in which a galactose side chain is attached to a main chain made of mannose.

[0012] In this specification, "polysaccharides" refers to high-molecular-weight compounds composed of numerous monosaccharides, while "oligosaccharides" refers to compounds composed of a small number of monosaccharides (for example, 2 to 10, preferably 2 to 6, more preferably 2 to 4, but not limited to these), and the two are distinguished. In this specification, oligosaccharides containing mannose as a constituent sugar are sometimes referred to as "mann-oligosaccharides." The term "mann-oligosaccharide" comprehensively refers to oligosaccharides composed of only mannose linked by β-1,4 bonds, and oligosaccharides that contain mannose in addition to other sugars (for example, glucose) as constituent sugars.

[0013] In this specification, "β-mannosidase activity" means properties that satisfy at least the following (1) and (2): (1) Substrate specificity: It acts on 4-nitrophenyl (pNP)-β-D-mannose and β-1,4-mannobiose, releasing mannose from these substrates. On the other hand, it does not act on glucomannan or 1,4-β-D-mannan, and no mannose is released. (2) Activity: It has the ability to hydrolyze the β-glycosidic bond of pNP-β-D-mannose and the β-1,4 bond of β-1,4-mannobiose.

[0014] In this specification, "4-nitrophenyl (pNP)-β-D-mannose" is a compound in which a 4-nitrophenyl group is β-glycosidically bonded to the anomeric carbon (1st carbon) of D-mannose, and is also called 4-nitrophenyl β-D-mannopyranoside. "β-1,4-mannobiose" is a disaccharide in which two D-mannose molecules are linked by a β-1,4 bond. "Glucomannan" and "1,4-β-D-mannan" are as described above.

[0015] In this specification, "polypeptide having β-mannosidase activity" means that the polypeptide has properties that satisfy (1) and (2) above, but it is not intended that the substrate specificity and activity of the polypeptide are limited to these. β-mannosidase (EC 3.2.1.25), also known as β-D-mannoside mannohydrolase, belongs to the Glycoside Hydrolase Family 2 (GH2) and has the function of cleaving the β-glycosidic bond or β-1,4 bond from the non-reducing end of β-D-mannoside by hydrolysis, thereby cleaving β-D-mannose. "β-D-mannoside" refers to a form in which mannose is bonded to another molecule by a β-glycosidic bond at the 1st carbon (C1), and "other molecules" include sugar molecules such as mannose and non-sugar molecules (e.g., 4-nitrophenyl). Therefore, with respect to substrate specificity, the substrates of the polypeptide of the present invention are not necessarily limited to pNP-β-D-mannose and β-1,4-mannobiose. pNP-β-D-mannose is a type of β-D-mannoside and is a compound used as a substrate for measuring the enzymatic activity of β-mannosidase. Therefore, compounds similarly used as substrates for measuring the enzymatic activity of β-mannosidase can also be substrates. Manno-oligosaccharides can also be substrates. Not only oligosaccharides in which only mannose is linked by β-1,4 bonds (e.g., mannotriose (trisaccharide) and mannotetraose (tetrasaccharide)), but also oligosaccharides that contain β-1,4 bonds between mannose molecules and further contain other sugars (e.g., glucose) as constituent sugars can be substrates. Furthermore, regarding activity, the polypeptide of the present invention is not limited to hydrolyzing the β-glycosidic bond of pNP-β-D-mannose and the β-1,4 bond of β-1,4-mannobiose, but can also hydrolyze the β-1,4 bond within a manno-oligosaccharide substrate, for example. The polypeptide of the present invention can decompose manno-oligosaccharides from the non-reducing end in the exo form.

[0016] In this specification, "polypeptide" refers to a molecule composed of multiple amino acids linked by peptide bonds. Amino acids may be natural or unnatural. In this specification, the terms "polypeptide," "peptide," and "protein" are used interchangeably. Polypeptides may be modified as needed (e.g., glycosylation, phosphorylation, acylation, farnesylation, prenylation, sulfonation, etc.) to add functional groups, provided they exhibit β-mannosidase activity. In this specification, amino acids may be represented by commonly known three-letter or one-letter abbreviations. Furthermore, amino acid sequences are shown from left to right, from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus).

[0017] In this specification, the terms “nucleic acid” and “polynucleotide” are used interchangeably unless otherwise specified, and refer to polymers of nucleotides of any length. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Nucleotides may be natural or non-natural. “Nucleic acid” includes DNA (deoxyribonucleic acid), RNA (ribonucleic acid), heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids may be single-stranded or double-stranded and may be chemically modified. “DNA” may be either a sense strand or an antisense strand (e.g., usable as a probe), and may be genomic DNA, cDNA, or synthetic DNA. “RNA” is, for example, mRNA (messenger RNA). In this specification, the base sequence of a nucleic acid is shown from left to right, 5' to 3', using a generally known nucleic acid code, unless otherwise specified.

[0018] Furthermore, the terms “nucleic acid,” “polynucleotide,” and “gene” are used interchangeably in this specification. For example, in this specification, a nucleic acid that codes for a protein is also referred to as the gene of that protein.

[0019] In this specification, the percentage of "sequence identity" of a comparison amino acid sequence to a reference amino acid sequence (e.g., the amino acid sequence shown in SEQ ID NO: 2) is defined as the percentage of amino acid residues in the comparison sequence that are identical to amino acid residues in the reference sequence, after the sequences have been aligned to maximize the identity between the two sequences, and gaps have been introduced in one or both sequences as necessary. Sequence identity can be determined using homology search programs well known to those skilled in the art, such as BLAST (Basic Local Alignment Search Tool). BLAST is available from websites such as NCBI (National Center for Biotechnology Information). Sequence comparison and determination of sequence identity can be performed, for example, using the standard settings of NCBI's BLAST. Sequence identity of nucleotide sequences can also be determined in a similar manner.

[0020] In this specification, the "pH activity range," "optimal pH," "temperature activity range," and "optimal temperature" of a polypeptide represent the conditions under which the polypeptide (enzyme) reacts with a substrate, and can be evaluated using standard activity evaluation conditions. "Stable pH (pH stability)" and "stable temperature (temperature stability)" represent the storage or incubation conditions before the enzymatic reaction, and can be evaluated using standard stability evaluation conditions.

[0021] "Standard activity evaluation conditions" refers to a framework for evaluating activity by comparison, where either pH or temperature is set as a variable, and other factors (substrate, buffer, ionic strength, enzyme concentration, reaction time, detection method, etc.) are kept substantially identical. "Standard stability evaluation conditions" refers to a framework for measuring and comparing residual activity under standard activity evaluation conditions, where, after pretreatment (storage or incubation) with either pH or temperature set as a variable, the pH and temperature during the reaction are fixed to predetermined values. For comparative evaluation of activity or residual activity, relative activity, calculated with the maximum activity within the same measurement series set as 100%, can be used. In this specification, "relative activity" refers to the activity percentage calculated with the maximum activity within the same measurement series set as 100%, unless otherwise specified. Specific measurement conditions for each evaluation condition are shown below, but these are examples only and not limiting. Those skilled in the art can perform similar evaluations based on the descriptions herein using substantially equivalent conditions.

[0022] In this specification, "pH activity range" refers to the pH range in which activity can be detected under standard activity evaluation conditions with varying pH, and "optimal pH" refers to the pH or pH range in which the relative activity under the same conditions is a predetermined threshold (20% or more, preferably 60% or more, more preferably 90% or more, most preferably 100%). In this specification, "stable pH (pH stability)" refers to the pH or pH range in which the relative activity measured after storage or incubation according to standard stability evaluation conditions with varying pH is a predetermined threshold (preferably 60% or more, more preferably 90% or more).

[0023] Similarly, in this specification, "temperature activity range" means the temperature range in which activity can be detected under standard activity evaluation conditions with varying temperatures, "optimal temperature" means the temperature or temperature range in which the relative activity under the same conditions reaches a predetermined threshold (20% or more, preferably 60% or more, more preferably 90% or more, most preferably 100%), and "stable temperature (temperature stability)" means the temperature or temperature range in which the relative activity measured after storage or incubation is performed according to standard stability evaluation conditions with varying temperatures reaches a predetermined threshold (preferably 60% or more, more preferably 80% or more, and even more preferably 90% or more).

[0024] In this specification, a numerical range expressed using “~” means a range that includes the numbers at both ends as the upper and lower limits, unless otherwise specified. Where multiple candidate upper limits and multiple candidate lower limits are given for a parameter, the numerical range for that parameter may be constructed by combining any one candidate upper limit and any one candidate lower limit. Disclosing a numerical range in this specification also discloses any numbers or subranges that fall within that range, unless otherwise stated.

[0025] Furthermore, unless otherwise specified, the numerical values ​​described herein, whether preceded by the term "approximately," are to be interpreted as including tolerances, measurement errors, rounding errors, instrument accuracy, and variations due to measurement conditions (temperature, buffer composition, ionic strength, reagent lot differences, operator differences, etc.). These variations may be within ±1%, ±2%, ±5%, ±10%, or ±20% of the stated values. Even if the variation falls within these tolerances, the parameter is understood to be within the technical scope of the present invention as long as it satisfies the desired characteristics or produces the desired effects.

[0026] Furthermore, the optimal pH, optimal temperature, and activity level of the enzyme may vary slightly within a normal range depending on the storage period and conditions (temperature, buffer, ionic strength, etc.) after purification. These values ​​described herein should be understood to include such reasonable variations.

[0027] The terms used herein are explained above and below, but unless otherwise specified, the technical and scientific terms herein have the same meaning as those generally understood by those skilled in the art.

[0028] [Polypeptides] The present invention provides the following polypeptides (a) and (b): (a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2; (b) a polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and having β-mannosidase activity.

[0029] The polypeptide in (a) above is a polypeptide consisting of the amino acid sequence shown in Sequence ID No. 2. Sequence ID No. 2 (Figure 3) shows the amino acid sequence of a polypeptide having β-mannosidase activity derived from Bifidobacterium dentium. The polypeptide consisting of the amino acid sequence shown in Sequence ID No. 2 is a polypeptide having β-mannosidase activity. In this specification, "polypeptide having β-mannosidase activity" is also simply referred to as "β-mannosidase" or "enzyme". Furthermore, for convenience, the polypeptide in (a) above is also referred to as a polypeptide "derived from Bifidobacterium dentium", but in this specification, "derived from Bifidobacterium dentium" includes not only polypeptides, nucleic acids, etc. directly isolated from the bacterium, but also polypeptides, nucleic acids, etc. artificially produced by analyzing these.

[0030] The polypeptide in (b) above is defined by its sequence identity with the amino acid sequence of the polypeptide in (a) above. The polypeptide in (b) above is a polypeptide consisting of an amino acid sequence that has 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of the polypeptide in (a) above, insofar as it has β-mannosidase activity.

[0031] The polypeptide of the present invention may consist of an amino acid sequence modified by the substitution, addition, deletion, or insertion of one or more amino acids relative to the amino acid sequence of SEQ ID NO: 2, insofar as it possesses β-mannosidase activity. "Several" refers to any number as long as it is within the range of β-mannosidase activity, but preferably a number that satisfies the sequence identity described above. "Several" can refer to any number of bases in the range of 2 to 90, depending on the length of the reference sequence. "Substituted, deleted, added, or inserted base sequence" may refer to any one of substitution, deletion, addition, or insertion occurring alone, or any two or more of these occurring. Such mutant polypeptides are not limited to polypeptides with mutations artificially introduced by known mutation techniques (e.g., site-directed mutagenesis), but may also be obtained by isolating and purifying naturally occurring proteins. To maintain β-mannosidase activity, it is preferable that the substituted amino acids are conservative substitutions. A "conservative substitution" of amino acids refers to the substitution of amino acids with similar chemical properties when the amino acid sequence of a protein changes. Conservative substitutions include, but are not limited to, substitutions between the following combinations: leucine (Leu), isoleucine (Ile), and valine (Val) (all hydrophobic amino acids); aspartic acid (Asp) and glutamic acid (Glu) (both acidic amino acids); lysine (Lys) and arginine (Arg) (both basic amino acids); alanine (Ala) and glycine (Gly) (both hydrophobic amino acids); serine (Ser) and threonine (Thr) (both polar and containing a hydroxyl group); and phenylalanine (Phe) and tyrosine (Tyr) (both aromatic amino acids).

[0032] The presence of β-mannosidase activity in the polypeptide can be confirmed, for example, by following the method described in Example 4 or 7 below.

[0033] The polypeptide of the present invention has a molecular weight of 100 ± 10 kDa, as determined by SDS-PAGE. Molecular weight measurement by SDS-PAGE can be performed, for example, according to the method described in Example 2 below.

[0034] The polypeptide of the present invention can exhibit β-mannosidase activity between pH 3.5 and 7.5 (pH activity range), with an optimal pH of 4.5 to 6.5, preferably 5 to 6, and more preferably 5.5. This can be confirmed by measuring activity under standard activity evaluation conditions with varying pH. For example, under the conditions described in Example 3 or 5 below (enzyme 22 mg / L or 0.1 mg / mL, pNP-β-D-mannose 0.5 mM as substrate, 100 mM acetate buffer, reacted at 40°C), a pH series can be set and the activity at each pH can be measured (this can be done according to the measurement method described in Example 3), and evaluated by comparison. As an example of a buffer, citrate buffer can be used at pH 2.5 to 4.5, acetate buffer at pH 4.0 to 6.5, and phosphate buffer at pH 6.0 to 8.0, but is not limited to these. In this evaluation framework, pH 4.5–6.5, pH 5–6, and pH 5.5 may have relative activity of 20% or more, 60% or more, 90% or more, or 100%, respectively.

[0035] The polypeptide of the present invention may exhibit pH stability in the pH range of 3 to 10. The polypeptide of the present invention can be characterized as having a stable pH of pH 3 to 10 (preferably pH 3 to 8). The stable pH (pH stability) of the polypeptide of the present invention can be confirmed under standard stability evaluation conditions with varying pH. For example, it can be evaluated by measuring and comparing the activity under the conditions described in Example 5 below (after standing in 100 mM buffer at various pH values ​​at 4°C for 24 hours, reacting with 0.5 mM pNP-β-D-mannose as a substrate at pH 5.5 and 40°C). Within this evaluation framework, the polypeptide of the present invention may exhibit relative activity of 60% or more or 90% or more at pH 3 to 10 and pH 3 to 8.

[0036] The polypeptide of the present invention can exhibit β-mannosidase activity between 20 and 45°C (temperature activity range), with an optimal temperature of 20 to 40°C, preferably 20 to 30°C, and more preferably 25°C. This can be confirmed by measuring activity under standard activity evaluation conditions with varying temperatures. For example, under the conditions described in Example 6 below (reacting 0.004 to 0.02 mg / mL of enzyme with 0.5 mM pNP-β-D-mannose in 100 mM acetate buffer (pH 5.5)), a temperature series can be set up, and the activity at each temperature can be measured and compared. In this evaluation framework, the relative activity at 20 to 40°C, 20 to 30°C, and 25°C may be 20% or more, 60% or more, 90% or more, or 100%, respectively.

[0037] The polypeptide of the present invention may exhibit temperature stability in the range of 4 to 50°C. The polypeptide of the present invention can be characterized as having a stable temperature of 4 to 50°C (preferably 20 to 50°C, more preferably 20 to 40°C, and even more preferably 20 to 35°C). The stable temperature (temperature stability) of the polypeptide of the present invention can be confirmed under standard stability evaluation conditions with varying temperatures. For example, it can be evaluated by measuring and comparing the activity under the conditions described in Example 6 below (after standing for 30 minutes at the set temperature in 100 mM acetate buffer (pH 5.5), 0.002 mg / mL of enzyme is reacted with 0.5 mM pNP-β-D-mannose in 100 mM acetate buffer (pH 5.5) at 40°C). In this evaluation framework, the relative activity may be 60% or more, 80% or more, or 90% or more at 4 to 50°C, 20 to 50°C, 20 to 40°C, and 20 to 35°C.

[0038] Furthermore, the optimal pH, stable pH (pH stability), optimal temperature, and stable temperature (temperature stability) mentioned above are representative indicators of the characteristics of the polypeptide (enzyme) of the present invention and should not be interpreted restrictively.

[0039] [Nucleic Acids] The present invention further provides nucleic acids encoding the polypeptide of the present invention. The nucleic acids of the present invention include nucleic acids encoding the polypeptide of (a) above and nucleic acids encoding the polypeptide of (b) above.

[0040] SEQ ID NO: 1 (FIG. 2) shows DNA encoding β-mannosidase derived from Bifidobacterium dentium, which corresponds to a nucleic acid (gene) encoding the polypeptide of (a) above, that is, the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2. Since the genetic code is degenerate, and multiple codons can be used to encode a specific amino acid, the nucleic acid encoding the polypeptide of (a) above is not limited to SEQ ID NO: 1.

[0041] When producing a polypeptide by genetic engineering, codons can also be optimized for the host organism. For example, SEQ ID NO: 3 (FIG. 4) shows a sequence obtained by optimizing codons for Escherichia coli based on the nucleotide sequence set forth in SEQ ID NO: 1 (the amino acid sequence encoded by SEQ ID NO: 3 (SEQ ID NO: 4) is identical to the amino acid sequence set forth in SEQ ID NO: 2). A nucleic acid consisting of a nucleotide sequence with such optimized codons is also within the scope of the present invention.

[0042] The nucleic acid of the present invention may be a nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a nucleotide sequence complementary to any nucleotide sequence of a nucleic acid encoding the polypeptide of (a) above (e.g., SEQ ID NO: 1 or SEQ ID NO: 3) and a nucleic acid encoding the polypeptide of (b) above, and encodes a polypeptide having β-mannosidase activity. The term "stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, such conditions include conditions where two DNAs with high sequence identity, for example, two DNAs having 90% or higher sequence identity, hybridize, but two DNAs with lower sequence identity do not hybridize. Examples of stringent conditions include washing with a 0.1 × SSC, 0.1% SDS solution at 60°C to 70°C. Hybridization can be performed by methods well known to those skilled in the art, such as the method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory (2012).

[0043] The nucleic acid of the present invention can be prepared by any method known to those skilled in the art using a primer or probe prepared based on the nucleotide sequence disclosed herein. For example, the nucleic acid of the present invention can be obtained as a cDNA of a gene by PCR or other DNA amplification techniques using designed primers. Furthermore, the nucleic acid of the present invention can be synthesized by methods commonly used by those skilled in the art based on the sequence information disclosed herein. Alternatively, a probe can be prepared based on the nucleotide sequence disclosed herein and obtained by performing hybridization under stringent conditions as described above. As described above, the nucleic acid may be synthesized with codons optimized for the host organism, and mutant DNA can also be obtained.

[0044] The nucleic acid of the present invention can also be used to prepare an expression cassette by operably linking an expression regulatory element thereto. Examples of expression regulatory factors (or expression regulatory sequences) include a promoter (a sequence for initiating transcription of a gene), an enhancer (a sequence for enhancing promoter activity), a terminator (a sequence for terminating transcription), and the like. The expression cassette may comprise a nucleic acid encoding a secretion signal peptide for promoting extracellular secretion of the expressed polypeptide. Elements such as expression regulatory factors or secretion signals used in the expression cassette can be appropriately selected. As used herein, "operably linked" means that the elements contained in the expression cassette are functionally linked so as to enable transcription from the nucleic acid encoding the polypeptide of the present invention. Operable linkage can be performed using genetic recombination techniques known in the art.

[0045] [Vector] The present invention provides a vector comprising the nucleic acid of the present invention, that is, a vector comprising a nucleic acid encoding the polypeptide of the present invention.

[0046] In this specification, “vector” means a carrier for introducing a target gene into a host cell, and a vector may be designed to efficiently deliver the target gene into a host cell and express it in that host cell. A vector typically includes an origin (ori: a sequence that serves as the starting point for the vector to self-replicate in the host cell), a promoter, and a terminator. A vector may further include an enhancer and / or a polyA signal (a signal sequence for adding a polyA tail to the 3' end of mRNA, which improves mRNA stability and translation efficiency).

[0047] The vector may further contain selection markers. Selection markers are genes used to confirm the insertion of the nucleic acid of interest. Examples of selection markers include, but are not limited to, markers that confer selectable phenotypes, such as antibiotic or drug resistance (e.g., genes with resistance to hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol, or bialafos), and nutritional requirements (e.g., histidine (HIS3), tryptophan (TRP1), lysine (LYS2), methionine (MET17), adenine (ADE2), uracil (URA3), etc.). Genetically modified cells can be selected because, in an environment treated with a selection factor (e.g., the corresponding antibiotic or drug, or nutrient), only cells expressing the selection marker will survive or exhibit a different phenotype. The vector may also further include, for example, a reporter gene (a gene used to visually confirm the success of gene transfer, such as the GFP (green fluorescent protein) gene) or a tag sequence (a short amino acid sequence used to purify or detect the expressed protein, such as a His tag, FLAG tag, or HA tag).

[0048] Vectors can contain multiple cloning sites (MCS: regions containing multiple restriction enzyme cleavage sites). This allows for the insertion of the target gene or expression cassette into the vector. Vectors can also include nucleic acid sequences that encode secretion signals to enable extracellular secretion.

[0049] The vector can be appropriately selected depending on the host cell into which it will be introduced. Examples of vectors include plasmids, cosmids, viruses, and bacteriophages in their native or recombinant state. The vector can be suitable for introduction into bacterial cells, fungal cells, plant cells, or animal cells, for example. Commercially available vectors can be used as appropriate. For example, plasmid pET23b (Novagen) can be used for expression in E. coli. The elements (sequences) within the vector, as described above, may be those present in the commercially available vector, or they may be inserted into the commercially available vector from an external source.

[0050] [Genetically Modified Cells] The present invention provides genetically modified cells comprising the nucleic acid or vector of the present invention. Genetically modified cells of the present invention can be produced by introducing the nucleic acid or vector of the present invention into host cells.

[0051] In this specification, “host cell” means a cell that accepts and functions with foreign nucleic acids (i.e., nucleic acids introduced from outside) (e.g., the nucleic acids of the present invention), whether before or after gene transfer. In this specification, “genetically modified cell” means a cell that takes in nucleic acids (genes) introduced from outside and expresses those genes. Genetically modified cells include transformed cells, transduced cells, and gene-transfected cells, and these terms are used interchangeably in this specification. In this specification, “genetic modification” is also simply referred to as “recombination.” A “genetically modified cell” obtained by “introducing nucleic acids or vectors into a host cell” only needs to have the introduced nucleic acids present in the cell, and may be incorporated into the genome of the cell.

[0052] Genetically modified cells can be created by any method that involves introducing nucleic acids or vectors into host cells. This "introduction" can be, for example, by transformation, transduction, or transfection. The host cell is not particularly limited and can be, for example, a bacterial cell, a fungal cell, a plant cell, or an animal cell. Depending on the host cell, any known technique in the art may be appropriately selected. Techniques used for introducing nucleic acids or vectors into host cells include, but are not limited to, electroporation, microinjection, cationic lipid-mediated transformation or gene transfer (lipofection), chemically mediated gene transfer (e.g., using CaCl and / or CaP), lithium acetate-mediated transformation, gene gun (bioristic method), PEG-mediated transformation, protoplast fusion, liposome-mediated transformation, methods using Agrobacterium tumefaciens, and transformation or transduction by adenoviruses or other viruses or phages.

[0053] Host cells into which the target nucleic acid has been successfully introduced can be obtained by selection using the selection markers or reporter genes used during the creation of the genetically modified cells. Furthermore, the success of introducing the target nucleic acid into host cells (in other words, the presence of the target nucleic acid in the genetically modified cells) can be confirmed by methods such as PCR, Southern blotting, or Northern blotting. The expression of the polypeptide of the present invention in genetically modified cells can be confirmed by, for example, Western blotting. Such expression can also be confirmed by measuring the enzymatic activity exhibited by the polypeptide of the present invention according to the method described in Examples 4 or 7 below.

[0054] [Method for Producing Polypeptides] The present invention provides a method for producing polypeptides having β-mannosidase activity, and this method includes a step of culturing the genetically modified cells of the present invention. Through this culturing step, polypeptides having β-mannosidase activity are produced from the genetically modified cells of the present invention.

[0055] As genetically modified cells, those prepared as described above can be used. In the production of polypeptides having β-mannosidase activity, the host cells expressing the polypeptide are preferably bacterial or fungal cells, and more preferably bacteria (e.g., Escherichia (e.g., Escherichia coli), Bacillus (e.g., Bacillus subtilis), Pseudomonas (e.g., Pseudomonas putida), Brevibacterium, Streptococcus, Lactobacillus, Rhodococcus (e.g., Rhodococcus erythropolis and Rhodococcus opacus) (e.g., opacus), Streptomyces spp., Corynebacterium, etc.; or yeast (e.g., Saccharomyces (e.g., Saccharomyces cerevisiae), Kluyveromyces (e.g., Kluyveromyces marxianus), Schizosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Rhodotorula, Pichia, Candida, etc.).

[0056] In this specification, "cultivation" means growing genetically modified cells in a culture medium under appropriately controlled environmental conditions. The culture process is carried out using culture media and culture conditions known in the art, and can be appropriately adjusted depending on the type of cells used.

[0057] In this specification, "culture medium" means a liquid or solid substance containing nutrients necessary for culturing cells. The culture medium is preferably liquid. Examples of nutrients contained in the culture medium include carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, and vitamins.

[0058] Examples of carbon sources include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration may also be used. Any other carbon source in appropriate amounts can be used. These carbon sources may be used individually or in combination of two or more, but are not limited to these uses.

[0059] Examples of nitrogen sources include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn maceration, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, but are not limited to these.

[0060] Examples of phosphorus sources include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or sodium-containing salts equivalent to these, and either one or a combination of two or more of these may be used.

[0061] Examples of inorganic compounds include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. These can be used individually or in combination of two or more, but are not limited to these.

[0062] Examples of vitamins include B vitamins (e.g., B1, B2, B3, B5, B6, B7, B9, and B12) and vitamin C (ascorbic acid). These may be used individually or in combination of two or more, but are not limited to these.

[0063] Culture media commonly used for culturing host cells can be used. For example, LB medium (Lysogeny Broth) and Terrific Broth (TB medium) are known for E. coli, and YPD medium (Yeast Extract Peptone Dextrose) and SD medium (Synthetic Defined) are known for yeast. Commercially available media can also be used.

[0064] The culture medium may contain substances for selecting genetically modified cells. The culture medium may also contain substances for inducing the expression of the polypeptide of the present invention in genetically modified cells.

[0065] The pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in a suitable manner during cultivation. Furthermore, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble formation. Such substances can be added in batch or continuous order. To maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas may be injected into the medium, and to maintain an anaerobic and microaerobic state, no gas injection is necessary, but nitrogen, hydrogen, or carbon dioxide gas may be injected.

[0066] Culture conditions (temperature, pH, oxygen and carbon dioxide concentrations, osmotic pressure, humidity, etc.) can be appropriately set depending on the host cells. The culture temperature is, for example, 20°C to 55°C. The culture period is continued until the desired amount of polypeptide is produced, for example, 15 hours to 196 hours. Examples of culture methods include shaking culture, monolayer static culture, rotation culture, microcarrier culture, and tank culture. Furthermore, the culture method can be, for example, batch, continuous, or fed-batch culture.

[0067] Although the culture step of the manufacturing method of the present invention has been described above, it is not limited to the above as long as the genetically modified cells can grow and produce the polypeptide of the present invention.

[0068] The production method of the present invention may further include a step of recovering the polypeptide having β-mannosidase activity produced in the culture step described above. In one embodiment of the production method of the present invention, the polypeptide having β-mannosidase activity can be recovered from the culture medium or genetically modified cells after the culture step using methods known in the art. If necessary, the genetically modified cells may be lysed by methods commonly used by those skilled in the art. Also, if necessary, the recovered polypeptide may be purified. For the recovery and purification of the polypeptide, one or any combination of the following techniques may be used, but are not limited to: centrifugation, filtration, crystallization, treatment with protein precipitants (salting out), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and various other chromatography methods and HPLC. The polypeptide of the present invention can be obtained as a purified enzyme, for example, by purifying the supernatant of the culture medium or the lysate of genetically modified cells using affinity chromatography.

[0069] In another embodiment of the manufacturing method of the present invention, after the culture step described above, the recombinant cells themselves may be used as a source of polypeptides having β-mannosidase activity without separating the polypeptides having β-mannosidase activity from the culture medium or recombinant cells.

[0070] [Applications] Due to its activity, the polypeptide of the present invention can be used in a variety of applications. For example, the polypeptide of the present invention can be used in the process of decomposing mannan. For example, by using the polypeptide of the present invention together with an enzyme that decomposes mannan into manno-oligosaccharides (e.g., β-mannanase) or another means (e.g., acid hydrolysis, hot water hydrolysis, or alkaline treatment), mannan can be efficiently decomposed. Furthermore, the polypeptide of the present invention can be used in the process of producing mannose from mannan.

[0071] The polypeptide of the present invention can be used in the production of biofuels (e.g., bioethanol). For example, in the production of ethanol using woody biomass such as coniferous forests as a raw material, the polypeptide of the present invention can be used in the process of mannan decomposition. The polypeptide of the present invention can be used as a substitute or addition to β-mannosidase, which has been conventionally used in the production of bioethanol from mannan.

[0072] Furthermore, the polypeptides of the present invention can be used in processes involving the decomposition of mannan, for example, in the food industry, the feed industry, and the paper industry. For example, they can be used in the production of instant coffee, the production of oil from leguminous seeds (e.g., coconut), the improvement of beer quality, the production of pulp from wood such as coniferous trees, and pulp bleaching.

[0073] The polypeptide of the present invention may be used in combination with other enzymes as needed (e.g., mannanases (e.g., β-mannanase), glycosyl hydrolases (glucosidase, galactosidase, amylase, cellulase, xylanase, etc.)).

[0074] The present invention provides an enzyme preparation having β-mannosidase activity, comprising the polypeptide of the present invention as an active ingredient. The enzyme preparation of the present invention may be in any form that utilizes the enzymatic activity (β-mannosidase activity) of the polypeptide of the present invention. The enzyme preparation of the present invention may consist solely of the polypeptide of the present invention, or it may contain the polypeptide of the present invention as an enzymatically active component, and further contain other components. Other components include, but are not limited to, excipients, stabilizers, and salts. The dosage form is not particularly limited and may be liquid or solid (e.g., powder), or it may be in a form supported on a carrier. The enzyme preparation of the present invention can be manufactured using the polypeptide of the present invention by methods commonly used in the art.

[0075] The enzyme preparation of the present invention can be used in the above-described applications with respect to the polypeptide of the present invention in a form that provides the enzyme activity (β-mannosidase activity) of said polypeptide.

[0076] The present invention encompasses the following inventions: the polypeptide of the present invention for use as an enzyme having β-mannosidase activity; the use of the polypeptide of the present invention as an enzyme having β-mannosidase activity; a method for producing an enzyme preparation having β-mannosidase activity using the polypeptide of the present invention; the use of the polypeptide of the present invention in the production of an enzyme preparation having β-mannosidase activity; and compositions, formulations, and methods for producing the same comprising the polypeptide of the present invention.

[0077] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the compounds and reagents used in the examples of the present invention can be readily obtained from the market and used. Furthermore, technical matters not specifically mentioned in the examples can be easily understood and implemented by those skilled in the art.

[0078] [Example 1: Cloning of the β-mannosidase gene from Bifidobacterium denthium JCM1195 strain] (1-1. Extraction of the genome of Bifidobacterium denthium JCM1195 strain) Bifidobacterium denthium JCM1195 strain was purchased from the Microbial Materials Development Laboratory (JCM) of the RIKEN BioResource Research Center (BRC). The JCM1195 strain was inoculated into GAM medium (Shimadzu Diagnostics Corporation) and cultured overnight at 37°C. After culturing, 3 ml of the culture solution was centrifuged (17,000 g, 2 mins), and the supernatant was removed. The precipitate was washed twice with 1 ml of sucrose-EDTA (ethylenediaminetetraacetic acid) buffer (450 mM sucrose, 1 mM EDTA in 5 mM Tris-HCl [pH 8.0]) (17,000 g, 2 min), then resuspended in 450 μl of sucrose-EDTA buffer containing 5 μg / ml lysozyme (Sigma Aldrich Corp., St. Louis, MO, USA) and 40 U / ml mutanoricin (Sigma Aldrich Corp.), and allowed to stand at 37°C for 1 hour. Subsequently, genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Charbonnieres, France).

[0079] (1-2. Cloning and sequencing analysis of the β-mannosidase gene) Using the genomic DNA extracted in 1-1 as a template, PCR was performed using DNA polymerase PrimeSTAR Max (Takara Bio Inc.) and primers 5'-AAGGAGATATACATATGACTCACACTCCTAGC-3' (forward primer (pET23b-IF-RS00625-F): SEQ ID NO: 5 and Figure 1 (in Figure 1, lowercase letters represent sequences corresponding to the target coding sequence, and bold letters represent sequences of restriction enzyme sites)) and 5'-GCTCGAATTCGGATCCCACTCGCGCTTCAGA-3' (reverse primer (pET23b-IF-RS00625-R): SEQ ID NO: 6 and Figure 1 (in Figure 1, lowercase and bold letters are as described above)) to amplify the DNA fragments. This obtained the β-mannosidase gene from Bifidobacterium denthium strain JCM1195. Next, the obtained DNA fragment (β-mannosidase gene) was introduced into plasmid pET23b (Novagen Inc., Madison, WI, USA) that had been cleaved with NdeI-BamH1 using the In-Fusion HD PCR Cloning Kit (Clontech Laboratories, Inc., Palo Alto, CA, USA) (Figure 1).

[0080] The obtained gene sequences were analyzed using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Mix (Thermo Fisher Scientific, USA) with a SeqStudio genetic analyzer (Thermo Fisher Scientific). The resulting nucleotide sequence (SEQ ID NO: 1) is shown in Figure 2. Furthermore, the predicted amino acid sequence of the polypeptide encoded by this gene (SEQ ID NO: 2) is shown in Figure 3.

[0081] [Example 2: Expression of β-mannosidase from Bifidobacterium denthium] (2-1. Codon optimization of the β-mannosidase gene) Based on the β-mannosidase gene from Bifidobacterium denthium strain JCM1195 obtained in 1-2, GenScript Japan Co., Ltd. synthesized a gene with codons optimized for E. coli. The base sequence of the codon-optimized gene (SEQ ID NO: 3) is shown in Figure 4. The predicted amino acid sequence of the polypeptide encoded by this gene (SEQ ID NO: 4) is the same as the amino acid sequence (SEQ ID NO: 2) shown in Figure 3.

[0082] (2-2. Preparation of β-mannosidase gene expression strain) The DNA fragment of the gene synthesized in 2-1 was introduced into plasmid pET23b (Novagen) that had been cut with NdeI-Xho1 using the In-Fusion HD PCR Cloning Kit (Clontech Laboratories, Inc.) (Figure 5). The resulting plasmid (pET23b-GH2-Man) was then introduced. EcOPT ) was introduced into E. coli BL21 (DE3) and a genetically modified cell line containing the β-mannosidase gene ("β-mannosidase gene expression strain": pET23b-GH2-Man) was created. EcOPT We prepared pET23b (BL21). We also prepared a control strain (pET23b / BL21) by introducing pET23b into E. coli.

[0083] (2-3. Expression and Purification of β-Mannosidase) β-mannosidase gene-expressing strains were cultured in 50 ml of LB medium (lysogeny broth; Difco Laboratories, Detroit, MI, USA) containing 100 μg / ml ampicillin. Expression was induced by adding 0.1 mM isopropyl-β-D-thiogalaclopiranoside, and the culture was incubated with shaking at 20°C for 20 hours. The cells were then centrifuged (8,000 × g, 4°C for 30 minutes), washed twice with 20 mM sodium phosphate buffer (pH 7.5) containing 150 mM NaCl, suspended in 15 ml of the same buffer, and lysed using sonication (Qsonica LLC, Newton, CT, USA). The supernatant obtained by centrifugation (17,000 × g, 4°C for 15 minutes) was subjected to a Ni-NTA column (HisTrap HP; Global Life Science Technologies Japan Co., Ltd. (Cytiva)). This column was washed with 20 mM sodium phosphate buffer (pH 7.5) containing 0.5 M NaCl and 20 mM imidazole, and then eluted with 20 mM sodium phosphate buffer (pH 7.5) containing 0.5 M NaCl and 500 mM imidazole. The resulting eluted fraction was dialyzed to 20 mM sodium phosphate buffer (pH 7.5) to obtain purified enzyme.

[0084] β-mannosidase gene expression strain (pET23b-GH2-Man EcOPT Lysates of the β-mannosidase gene expression strain (pET23b / BL21) and control strain (pET23b / BL21), as well as the β-mannosidase gene expression strain, were analyzed by SDS-PAGE for the fraction before loading onto the Ni-NTA column (apply), the wash fraction after column passage (wash), and the elution fraction. SDS-PAGE was performed using the Blue Protein Standard (P7718S: NEB) according to the protocol described in the product instructions. The results are shown in Figure 6 (in Figure 6, "marker" indicates the lane of the molecular weight marker). β-mannosidase gene expression strain lysate (pET23b-GH2-Man EcOPTIn the lane for pET23b / BL21, a band was detected above and very close to the fourth band from the top of the molecular weight marker (95kDa), but this band was not detected in the lane for the control strain lysate (pET23b / BL21). The band at this position was detected in the β-mannosidase gene expression strain lysate (pET23b-GH2-Man). EcOPT The protein was detected in both the pre-load fraction (apply) and elution fraction lanes (BL21), and the results from the elution fraction lane indicated that a protein with a molecular weight of approximately 100 kDa was purified. Furthermore, the predicted amino acid sequence of the polypeptide encoded by this β-mannosidase gene predicted the molecular weight of this polypeptide to be 100071.24 Da.

[0085] Furthermore, the amount of enzyme in the elution fraction was measured using the bicinconic acid method (using the BCA Protein Assay Kit (Pierce)). The enzyme yield was 0.44 mg (0.22 mg / ml, 2.0 ml).

[0086] [Example 3: Optimal pH Measurement of β-Mannosidase Derived from Bifidobacterium Dentium] The optimal pH was investigated for the purified enzyme obtained in steps 2-3. 22 mg / L of the enzyme was reacted with 0.5 mM artificial substrate 4-nitrophenyl (pNP)-β-D-mannose (Sigma) in 100 mM buffers at 40°C. The p-nitrophenolate concentration in the reaction solution was measured, and one unit was defined as the activity that reacts with 1 μmol of substrate per minute to release the reaction product (i.e., p-nitrophenolate). The buffers used were sodium citrate buffers at pH 2.5, 3.0, 3.5, 4.0, and 4.5; sodium acetate buffers at pH 4.0, 4.5, 5.0, 5.5, 6.0, and 6.5; and sodium phosphate buffers at pH 6.0, 6.5, 7.0, 7.5, and 8.0.

[0087] The optimal pH was as shown in Figure 7. Figure 7 is a graph showing the relative activity of the enzyme at various pH levels. The enzyme activity at each pH level is expressed relatively, with the average activity at the pH where the highest activity was obtained set to 100%. The vertical axis shows the relative value (%) of the enzyme activity at each pH level, and the horizontal axis shows the pH test group. In the figure, white triangles represent the measurement results in sodium citrate buffer at pH 2.5, 3.0, 3.5, 4.0, and 4.5, black circles represent the measurement results in sodium acetate buffer at pH 4.0, 4.5, 5.0, 5.5, 6.0, and 6.5, and × represents the measurement results in sodium phosphate buffer at pH 6.0, 6.5, 7.0, 7.5, and 8.0. The highest activity was measured at pH 5.5. Therefore, in the following example, a pH of 5.5 condition was used during the enzyme reaction.

[0088] [Example 4: Measurement of activity of Bifidobacterium denthium-derived β-mannosidase against various substrates] The enzyme activity of the purified enzyme obtained in 2-3 was measured against each substrate. Four artificial substrates were used: 4-nitrophenyl (pNP)-β-D-mannose (Sigma), pNP-α-D-mannose (Sigma), pNP-β-D-glucose (Fujifilm Wako Pure Chemical Industries, Ltd.), and pNP-β-D-xylose (Nacalai Tesque Co., Ltd.), as well as three natural substrates: glucomannan (konjac, Megazyme, P-GLCML), 1,4-β-D-mannan (Megazyme, P-MANCB), and β-1,4-mannobiose (Megazyme, O-MBI). 2.2 mg / L of the enzyme was reacted with 0.5 mM of the artificial substrate or 5 mM of the natural substrate in 100 mM sodium acetate buffer (pH 5.5) at 40°C. The activity toward artificial substrates was measured by the concentration of p-nitrophenolate in the reaction solution. The activity toward natural substrates was measured by the concentration of free mannose in the reaction solution using the 3,5-dinitrosalicylic acid method. One unit was defined as the activity that reacts with 1 μmol of substrate per minute to release the reaction product (i.e., p-nitrophenolate or mannose). The results of the activity measurements toward each substrate are shown in Table 1 below.

[0089]

[0090] As shown in Table 1, enzyme activity was detected in the purified enzyme against pNP-β-D-mannose and β-1,4-mannobiose. The activity value against β-1,4-mannobiose was 0.21 units / mg. In the original application (Japanese Patent Application No. 2025-29055), Table 1 incorrectly stated the same activity value as 2.14 units / mg, so in this application, Table 1 has been replaced to correct this error. On the other hand, no enzyme activity was detected in the purified enzyme when D-glucose or D-xylose was β-glycosidically bonded to pNP, or when D-mannose was α-glycosidically bonded. Furthermore, no enzyme activity was detected against glucomannan or 1,4-β-D-mannan. From this, the following was determined about the purified enzyme: (1) Substrate specificity: This enzyme acts on pNP-β-D-mannose and β-1,4-mannobiose, releasing mannose from these substrates. On the other hand, it does not act on glucomannan or 1,4-β-D-mannan, and no mannose release is observed. (2) Activity: This enzyme has the ability to hydrolyze the β-glycosidic bond of pNP-β-D-mannose and the β-1,4 bond of β-1,4-mannobiose.

[0091] [Example 5: Measurement of optimal and stable pH of β-mannosidase derived from Bifidobacterium denthium] The optimal and stable pH of the purified enzyme obtained in steps 2-3 were measured, and the differences from known β-mannosidases were examined. β-mannosidase derived from Helix pomatia has been systematically reported by McCleary regarding its purification method, substrate specificity, specific activity, Km, and other properties, and has been shown to possess β-mannosidase activity (Carbohydr. Res., 111, 297-310, 1983).

[0092] (Method for measuring optimal pH) The enzyme reaction and enzyme activity were measured in accordance with the method described in Example 3, except that 0.1 mg / mL of enzyme was used in the reaction.

[0093] (Method for measuring stable pH) 1 mg / mL of enzyme was allowed to stand for 24 hours at 4°C in 100 mM of each buffer (i.e., sodium citrate buffers at pH 2.5, 3.0, 3.5, 4.0, and 4.5; sodium acetate buffers at pH 4.0, 4.5, 5.0, 5.5, 6.0, and 6.5; and sodium phosphate buffers at pH 6.0, 6.5, 7.0, 7.5, and 8.0). Then, 0.2 mg / L of enzyme was reacted with 0.5 mM pNP-β-D-β-mannose in 100 mM sodium acetate buffer (pH 5.5) at 40°C. The enzyme activity was measured according to the method described in Example 3.

[0094] (Results) Figure 8 shows the measurement results of the optimal pH and stable pH of the purified enzyme in this example. The enzyme activity at each pH is expressed relatively, with the average activity at pH 5.5, where maximum activity was observed, set to 100%. The vertical axis shows the relative value (%) of enzyme activity at each pH, ​​and the horizontal axis shows the pH test group. In the figure, the measurement results of the optimal pH are shown with white symbols, and the variation with the same type of buffer is connected with a dashed line. On the other hand, the measurement results of the stable pH are shown with black symbols, and the variation in the results with the same type of buffer is connected with a solid line. The types of symbols are as follows: diamonds represent measurement results with sodium citrate buffer at pH 2.5 to 4.5, triangles represent measurement results with sodium acetate buffer at pH 4.0 to 6.5, and circles represent measurement results with sodium phosphate buffer at pH 6.0 to 8.0.

[0095] Regarding the purified enzyme, in the optimal pH measurement of this example, as in Example 3, the highest enzyme activity was measured at an enzyme reaction pH of 5.5. Furthermore, from the stable pH measurement results, it was found that when the pH before reaction standing was 2.5, the enzyme exhibited relative activity of about 30% of the maximum activity, but in the pH range of 3.0 to 8.0, it showed a high relative activity of more than 90% of the maximum activity, confirming good pH stability in this range. In contrast, regarding the optimal pH, the β-mannosidase derived from Helix pomatia showed the highest activity at pH 4, and the activity decreased above pH 4.5. Regarding the stable pH, it showed almost maximum activity between pH 4.5 and 6, but the activity decreased sharply on the neutral to slightly alkaline side (above pH 7) (Carbohydr. Res., 111, 297-310, 1983). As described above, from both the optimal pH and stable pH perspectives, it became clear that this enzyme has different characteristics from known β-mannosidases derived from Helix pomatia. In particular, this enzyme was found to have significantly higher pH stability compared to known β-mannosidases derived from Helix pomatia.

[0096] [Example 6: Measurement of optimal and stable temperatures of β-mannosidase derived from Bifidobacterium denthium] The optimal and stable temperatures of the purified enzyme obtained in 2-3 were measured to examine the differences from known β-mannosidases (β-mannosidase derived from Helix pomatia).

[0097] (Method for determining optimal temperature) Using enzymes at concentrations of 0.004 to 0.02 mg / mL, the mixture was reacted with 0.5 mM pNP-β-D-mannose in 100 mM sodium acetate buffer (pH 5.5) at temperatures of 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, or 55°C. Enzyme activity was measured according to the method described in Example 3.

[0098] (Method for measuring stable temperature) 1 mg / mL of enzyme was allowed to stand for 30 minutes in 100 mM sodium acetate buffer (pH 5.5) at temperatures of 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, or 55°C, and then transferred to ice. 0.002 mg / L of enzyme was reacted with 0.5 mM pNP-β-D-mannose in 100 mM sodium acetate buffer (pH 5.5) at 40°C. Enzyme activity was measured according to the method described in Example 3.

[0099] (Results) Figure 9 shows the measurement results of the optimal temperature and stable temperature of the purified enzyme in this example. The enzyme activity at each temperature is expressed relatively, with the average activity at the temperature showing maximum activity set to 100%. The vertical axis shows the relative value (%) of enzyme activity at each temperature, and the horizontal axis shows the temperature test group. In the figure, the measurement results for the optimal temperature are shown with white circles, and the variation in the results is connected with dashed lines. On the other hand, the measurement results for the stable temperature are shown with black circles, and the variation in the results is connected with solid lines.

[0100] Measurements of the optimal temperature revealed that the enzyme exhibited the highest activity at a reaction temperature of 25°C. Furthermore, measurements of the stable temperature showed that the enzyme showed the highest activity when the pre-reaction standing temperature was 30°C, and exhibited high relative enzyme activity in the range of 20–40°C, particularly 20–35°C, confirming good temperature stability within this range. In contrast, β-mannosidase derived from Helix pomatia showed the highest activity at an optimal temperature of 55°C, and maintained maximum activity in the stable temperature range of 25–45°C (Carbohydr. Res., 111, 297-310, 1983). As described above, it became clear that this enzyme has different characteristics from known β-mannosidases derived from Helix pomatia, both in terms of optimal temperature and stable temperature.

[0101] [Example 7: Activity Measurement of Bifidobacterium denthium-derived β-mannosidase against Various Substrates] Based on the results of Examples 5 and 6, the reaction conditions with the substrates were set to pH 5.5 and 25°C for the purified enzyme obtained in 2-3, and the enzyme activity against each substrate was measured. Therefore, the measurement of enzyme activity against various substrates was carried out in accordance with the method described in Example 4, except that the temperature condition during reaction with the substrates was set to 25°C. The results of the activity measurement for each substrate are shown in Table 2 below.

[0102]

[0103] As shown in Table 2, enzyme activity was detected against pNP-β-D-mannose and β-1,4-mannobiose in the purified enzyme, while no enzyme activity was detected against other substrates. In this example, the same substrate specificity as in Example 4 was confirmed. Furthermore, the enzyme activity against pNP-β-D-mannose and β-1,4-mannobiose at a reaction temperature of 25°C was increased compared to the enzyme activity at a reaction temperature of 40°C (Example 4).

[0104] It should be noted that the present invention is not limited to the embodiments and examples described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included within the technical scope of the present invention. Furthermore, all academic and patent documents mentioned herein are incorporated herein by reference.

Claims

1. A polypeptide comprising the amino acid sequence shown in Sequence ID No. 2, or a polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 2, and possessing β-mannosidase activity.

2. The polypeptide according to claim 1, wherein the optimal pH is 4.5 to 6.

3. The polypeptide according to claim 1, which exhibits pH stability in the range of pH 3 to 10.

4. The polypeptide according to claim 1, wherein the optimal temperature is 20 to 40°C.

5. The polypeptide according to claim 1, which exhibits temperature stability in the range of 4 to 50°C.

6. A nucleic acid encoding the polypeptide described in claim 1.

7. A vector comprising the nucleic acid described in claim 6.

8. Genetically modified cells comprising the nucleic acid described in claim 6 or the vector described in claim 7.

9. A method for producing a polypeptide having β-mannosidase activity, comprising the step of culturing genetically modified cells as described in claim 8.

10. The method for producing a polypeptide having β-mannosidase activity, further comprising the step of recovering the polypeptide according to claim 9.

11. An enzyme preparation having β-mannosidase activity, comprising the polypeptide described in claim 1.

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