Galactose epimerase

Abstract

The purpose of the present invention is to provide a novel galactose epimerase.　Provided is a galactose epimerase comprising a polypeptide represented by any of (1) to (3) below. (1) A polypeptide comprising the amino acid sequence indicated in SEQ ID NO: 1. (2) A polypeptide that has galactose epimerase activity and is obtained by substituting, adding, inserting, or deleting one or more amino acid residues in the amino acid sequence indicated in SEQ ID NO: 1. (3) A polypeptide that has galactose epimerase activity and has 90% or more sequence identity to the amino acid sequence indicated in SEQ ID NO: 1.

Description

Galactose epimerase

[0001] This technology relates to galactose epimerase. More specifically, this technology relates to a novel galactose epimerase, DNA encoding the galactose epimerase, an expression cassette or recombinant vector containing the DNA, a transformant, a method for producing galactose epimerase, a method for producing talose, a method for producing food and drink products, a method for reducing galactose in food and drink products, food and drink products, an enzyme agent for producing talose and / or reducing galactose, and the use of a specific polypeptide.

[0002] In order to produce various isomerized sugars, the development of enzymes having the activity of epimerizing sugars has been underway. For example, in Patent Document 1, a protein consisting of a specific amino acid sequence is disclosed, which has few by-products, low substrate specificity, can catalyze the epimerization of various sugars, and is excellent in heat resistance and suitable for use on an industrial scale.

[0003] Further, in Patent Document 2, a protein having the epimerization activity of glucose and mannose, which contains a specific specific insertion sequence and can efficiently produce mannose using a relatively inexpensive raw material, is disclosed.

[0004] Japanese Unexamined Patent Application Publication No. 2020 - 137485, Japanese Unexamined Patent Application Publication No. 2019 - 033702

[0005] Galactose epimerase, which is a kind of enzyme having the activity of epimerizing sugars, is an enzyme that catalyzes the epimerization of galactose and talose. Talose is known to be a rare sugar with functionality, and talose and its derivatives are known to have antibacterial and anti-inflammatory effects. Thus, galactose epimerase is an enzyme that can be expected to contribute to various applications.

[0006] Therefore, the main object of this technology is to provide a novel galactose epimerase.

[0007] This technology first provides a galactose epimerase comprising a polypeptide as shown in any of the following (1) to (3): (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (2) a polypeptide having galactose epimerase activity, wherein one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 are substituted, added, inserted, deleted, or inverted; and (3) a polypeptide having galactose epimerase activity, wherein the amino acid sequence shown in SEQ ID NO: 1 has a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1. The galactose epimerase relating to this technology may be derived from microorganisms of the class Anaerolineae.

[0008] This technology provides DNA comprising one of the following polynucleotides: (1) a polynucleotide encoding galactose epimerase according to this technology; (2) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2; and (3) a polynucleotide comprising a sequence equivalent to the base sequence shown in SEQ ID NO: 2 and encoding a polypeptide having galactose epimerase activity. This technology also provides an expression cassette or recombinant vector comprising the DNA according to this technology. This technology also provides a transformant obtained by transforming a host with the expression cassette or recombinant vector according to this technology.

[0009] This technology also provides a method for producing galactose epimerase, comprising: a culture step of culturing an Anaerolineae microorganism or a transformant related to this technology; and a recovery step of recovering galactose epimerase related to this technology from the culture obtained in the culture step.

[0010] This technology further provides a method for producing talose, comprising the step of reacting galactose with galactose epimerase according to this technology. This technology also provides a method for producing food and beverages, comprising the step of reacting galactose-containing food and beverages and / or their raw materials with galactose epimerase according to this technology. In addition, this technology provides a method for reducing galactose in food and beverages, comprising the step of reacting galactose-containing food and beverages and / or their raw materials with galactose epimerase according to this technology. This technology also provides food and beverages obtained using galactose epimerase according to this technology. This technology also provides an enzyme preparation for producing talose and / or reducing galactose, which includes galactose epimerase according to this technology.

[0011] In addition, this technology provides the use of any polypeptide shown in (1) to (3) above as a galactose epimerase. This technology also provides the use of any polypeptide shown in (1) to (3) above for the production of talose. This technology also provides the use of any polypeptide shown in (1) to (3) above for reducing galactose in food and beverages.

[0012] The following is an explanation of the technical terms used in this technology. <Amino Acid Notation> The 20 amino acid residues in an amino acid sequence are sometimes represented by a single-letter abbreviation. In other words, glycine (Gly) is sometimes represented by G, alanine (Ala) by A, valine (Val) by V, leucine (Leu) by L, isoleucine (Ile) by I, phenylalanine (Phe) by F, tyrosine (Tyr) by Y, tryptophan (Trp) by W, serine (Ser) by S, threonine (Thr) by T, cysteine ​​(Cys) by C, methionine (Met) by M, aspartic acid (Asp) by D, glutamic acid (Glu) by E, asparagine (Asn) by N, glutamine (Gln) by Q, lysine (Lys) by K, arginine (Arg) by R, histidine (His) by H, and proline (Pro) by P.

[0013] <Amino Acid Sequence> In this technology, the displayed amino acid sequence has the N-terminus on the left and the C-terminus on the right.

[0014] <Types of Amino Acids> In this technology, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "Acidic amino acids" include aspartic acid and glutamic acid. "Basic amino acids" include lysine, arginine, and histidine.

[0015] <Sequence Modification (Substitution, Addition, Insertion, Deletion, Inversion, etc.)> In this technology, modification of amino acid sequences and / or base sequences includes not only artificially performed modifications (substitution, addition, insertion, deletion, inversion, etc.) but also naturally occurring modifications (substitution, addition, insertion, deletion, inversion, etc.), i.e., cases where the sequences were originally different.

[0016] <Separation and Purification> In this technology, the term "separated" is used interchangeably with "purified." The term "separated" is used to distinguish it from its natural state, that is, the state in which it exists in nature. The artificial operation of "separation" results in a "separated state," which is a state different from the natural state. The separated product is clearly and definitively different from the natural product itself.

[0017] <Equivalent Sequences> In this technology, an "equivalent sequence" refers to an amino acid sequence or nucleotide sequence that differs in part from a reference amino acid sequence (SEQ ID NO: 1 in this technology) or nucleotide sequence (SEQ ID NO: 2 in this technology), but whose differences do not substantially affect the function of the protein (galactose epimerase activity in this technology). Therefore, in this technology, enzymes having a polypeptide chain consisting of an equivalent amino acid sequence, or enzymes encoded by polynucleotides consisting of an equivalent nucleotide sequence, exhibit galactose epimerase activity.

[0018] <Talose> Talos is a type of monosaccharide and the 2-position epimer of galactose. Talos is also a rare sugar that is found in small quantities in nature and is expected to have various physiological functions. In industry, applications of talose are expected in functional polymers, carbon materials, nanocarbon materials, etc.

[0019] This is a photograph showing the SDS-PAGE results of galactose epimerase 1 in the example. This is a graph showing the talose production rate of galactose epimerase 1 in the example. This is a photograph showing the TLC analysis results of galactose epimerase 1 in the example. This is a graph showing the optimal temperature of galactose epimerase 1 in the example. This is a graph showing the optimal pH of galactose epimerase 1 in the example.

[0020] The following describes preferred embodiments for implementing this technology. Note that the embodiments described below are merely examples of typical embodiments of this technology, and this should not be interpreted as narrowing the scope of this technology.

[0021] 1. Galactose Epimerase The galactose epimerase related to this technology will be described in detail below.

[0022] [Amino Acid Sequence] The galactose epimerase relating to this technology comprises a polypeptide as shown in any of the following (1) to (3): (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (2) a polypeptide having galactose epimerase activity, wherein one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 are substituted, added, inserted, deleted, or inverted; and (3) a polypeptide having galactose epimerase activity, wherein the amino acid sequence shown in SEQ ID NO: 1 has a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1.

[0023] In the polypeptide of (2) above, the modification of amino acids may include only one type of modification (e.g., only substitution) from among substitution, addition, insertion, deletion, and inversion, or it may include two or more types of modifications (e.g., substitution and insertion). In the polypeptide of (2) above, the number of amino acid differences at any difference site may be one or several, for example, 1 to 80, preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40, or 1 to 30, more preferably 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, even more preferably 1 to 3, and particularly preferably 1 or 2 or 1.

[0024] In the polypeptide of (3) above, the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 should be 90% or more, but preferably 93% or more, more preferably 95% or more, even more preferably 96% or more, and even more preferably 97% or more, 98% or more, or 99% or more.

[0025] Here, in the polypeptide of (3) above, the sequence identity for each amino acid sequence shown in SEQ ID NO: 1 is the sequence identity calculated by comparing it with the amino acid sequence shown in SEQ ID NO: 1. Furthermore, "sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) of BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0026] Furthermore, it is desirable not to introduce substitutions or deletions to the active site in the polypeptides of (2) or (3) above. The active site of SEQ ID NO: 1 is presumed to be the 281st (histidine residue) and 412th (histidine residue) of the amino acid sequence.

[0027] The polypeptide of this technology may be part of a larger protein (e.g., a fusion protein). Examples of sequences that can be added to a fusion protein include sequences useful for purification, such as multiple histidine residues, and sequences that ensure stability during recombinant production.

[0028] In the polypeptide of (2) or (3) above, when an amino acid substitution is introduced relative to SEQ ID NO: 1, a preferred embodiment of the introduced amino acid substitution is a conservative substitution. That is, examples of substitutions in the polypeptide of (2) or (3) above include substitution with another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution with another noncharged amino acid if the amino acid before substitution is an uncharged amino acid, substitution with another acidic amino acid if the amino acid before substitution is an acidic amino acid, and substitution with another basic amino acid if the amino acid before substitution is a basic amino acid.

[0029] It is preferable that the galactose epimerase containing the polypeptide of (2) or (3) has galactose epimerase activity equal to or greater than that of the galactose epimerase containing the polypeptide of (1). Specifically, the relative activity of the galactose epimerase containing the polypeptide of (2) or (3) is 0.7 times or more, 0.8 times or more, 0.9 times or more, 1.0 times or more, 1.1 times or more, preferably 1.15 times or more, more preferably 1.2 times or more, and even more preferably 1.25 times or more, than the relative activity of the galactose epimerase containing the polypeptide of (1).

[0030] In addition to being used as galactose epimerase, any of the polypeptides described above in (1) to (3) can also be suitably used for related purposes, such as for the production of talose and for reducing galactose in food and beverages, as will be described later.

[0031] [Origin] The origin of the galactose epimerase related to this technology is not particularly limited, as long as it contains the polypeptide described above. For example, galactose epimerase derived from microorganisms of the class Anaerolineae is used. Examples of microorganisms of the class Anaerolineae include Anaerolinea thermophila.

[0032] Here, "galactose epimerase derived from Anaerolineae microorganisms" refers to galactose epimerase produced by Anaerolineae microorganisms (whether wild-type or mutant), or galactose epimerase obtained by genetic engineering using the galactose epimerase gene. Therefore, recombinants produced by host microorganisms into which a galactose epimerase gene (or a modified version thereof) obtained from Anaerolineae microorganisms has been introduced also fall under the category of "galactose epimerase derived from Anaerolineae microorganisms."

[0033] The strains of microorganisms belonging to the class Anaerolineae are not particularly limited as long as they do not impair the action or effect of this technology, and strains that are generally available from biological genetic resource institutions, etc., can be used.

[0034] More specifically, the galactose epimerase containing the polypeptide consisting of the amino acid sequence shown in Sequence ID No. 1 is a galactose epimerase derived from microorganisms of the class Anaerolineae.

[0035] Furthermore, the galactose epimerase related to this technology may be an artificial protein artificially designed based on the amino acid sequences described in (1) to (3) above.

[0036] [Enzymatic Chemical Properties] (1) Action The galactose epimerase related to this technology is an enzyme that produces talose using galactose as a substrate. In addition, the galactose epimerase related to this technology may also be an enzyme that has other actions, as long as it has galactose epimerase activity.

[0037] The conversion rate from galactose to talose in the galactose epimerase according to this technology is not particularly limited, but is, for example, 5% or more, preferably 8% or more, and more preferably 10% or more. The upper limit of the conversion rate from galactose to talose in the galactose epimerase according to this technology is also not particularly limited and may be 100%. Specifically, the conversion rate from galactose to talose of the galactose epimerase containing a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is 14.7% at 37°C and 48 hours of reaction.

[0038] Furthermore, galactose epimerase containing the polypeptide with the amino acid sequence shown in Sequence ID No. 1 also has a very slight ability to produce mannose using glucose as a substrate, but the conversion rate from glucose to mannose is extremely low at 0.4% after 48 hours of reaction at 37°C.

[0039] (2) Molecular weight The molecular weight of the galactose epimerase relating to this technology is 45 to 55 kDa. As a specific example, the molecular weight of the galactose epimerase containing a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is 48.9 kDa.

[0040] The molecular weight is calculated from the amino acid sequence.

[0041] (3) Optimal temperature The optimal temperature for the galactose epimerase related to this technology is 30 to 40°C. Furthermore, the galactose epimerase related to this technology exhibits more than 80% of its activity at the optimal temperature at 30 to 40°C.

[0042] The optimal temperature is the value obtained when using 0.5 mol / L acetate buffer at pH 6.0.

[0043] (4) Optimal pH The galactose epimerase according to the present technology has an optimal pH in the range of pH 6.0 to 7.0. Further, the galactose epimerase according to the present technology has an activity of 80% or more of the activity at the optimal pH at pH 6.0 to 7.0.

[0044] The optimal pH is a value measured using 50 mM acetate buffer (pH 3.0 to 6.0) and 50 mM phosphate buffer (pH 7.0 to 10.0).

[0045] 2. DNA encoding galactose epimerase The DNA according to the present technology contains a polynucleotide shown in any of the following (1) to (3). (1) A polynucleotide encoding the galactose epimerase according to the present technology, (2) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2, and (3) A polynucleotide containing a sequence equivalent to the nucleotide sequence shown in SEQ ID NO: 2 and encoding a polypeptide having galactose epimerase activity

[0046] The "polynucleotide encoding the galactose epimerase according to the present technology" in (1) above refers to a polynucleotide from which the galactose epimerase according to the present technology can be obtained when expressed, and includes not only a polynucleotide having a nucleotide sequence corresponding to the amino acid sequence of the galactose epimerase according to the present technology, but also a polynucleotide obtained by adding a sequence that does not encode an amino acid sequence to such a polynucleotide. It also includes polynucleotides with degenerate codons.

[0047] As a specific example of the polynucleotide encoding the galactose epimerase according to the present technology, it is shown in SEQ ID NO: 2 (the genomic DNA sequence encoding the amino acid sequence of SEQ ID NO: 1).

[0048] The DNA according to the present technology includes a polynucleotide (hereinafter also referred to as an "equivalent sequence") having a nucleotide sequence that is different in part but has an equivalent function of the protein it encodes when compared with the nucleotide sequence of the polynucleotide encoding the galactose epimerase according to the present technology in (1) above.

[0049] Specific examples of equivalent sequences include polynucleotides that encode polypeptides having galactose epimerase activity, comprising a base sequence that includes one or more base substitutions, additions, insertions, deletions, or inversions based on the base sequence of a polynucleotide encoding galactose epimerase related to this technology (for example, Sequence ID No. 2).

[0050] Modifications to the bases may occur at multiple locations in the sequence. The term "multiple" here varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the DNA, but for example, it is 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases.

[0051] Furthermore, specific examples of equivalent sequences include sequences having, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably about 90% or more, even more preferably 95% or more, and particularly preferably 99% or more sequence identity with respect to the base sequence of the polynucleotide encoding the galactose epimerase related to this technology (for example, Sequence ID No. 2).

[0052] Here, "sequence identity" can be calculated using publicly available or commercially available software that has an algorithm for comparing a reference sequence with a query sequence. Specifically, BLAST, FASTA, or GENETYX (manufactured by Software Development Co., Ltd.) can be used, and these should be used with their default parameters set.

[0053] In this technology, DNA that hybridizes under stringent conditions with DNA having a complementary base sequence to the DNA shown in Sequence ID No. 2 is also included in the DNA related to this technology, as long as it encodes a polypeptide having galactose epimerase activity.

[0054] Here, "stringent conditions" refers to conditions in which the samples are incubated at 50°C to 65°C for 4 hours to overnight in a 6x SSC (1x SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5x Denhartz's (0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficol 400), and 100 μg / ml salmon sperm DNA.

[0055] Hybridization under stringent conditions is specifically performed by the following method: A nylon membrane immobilized with a DNA library or cDNA library is prepared, and the nylon membrane is blocked at 65°C in a pre-hybridization solution containing 6×SSC, 0.5% SDS, 5×Denharts, and 100 μg / mL salmon sperm DNA. Then, each probe labeled with 32P is added, and the membrane is incubated overnight at 65°C. After washing the nylon membrane in 6×SSC at room temperature for 10 minutes, in 2×SSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2×SSC containing 0.1% SDS at 45°C for 30 minutes, autoradiography can be performed to detect DNA that has specifically hybridized with the probe.

[0056] [Method for obtaining DNA related to this technology] The DNA related to this technology can be prepared in an isolated state by referring to the sequence information disclosed in this specification or the attached sequence listing, using standard genetic engineering methods, molecular biological methods, biochemical methods, chemical synthesis, PCR (e.g., overlap PCR), or a combination thereof.

[0057] Furthermore, the DNA related to this technology can also be isolated from microorganisms that produce polypeptides having galactose epimerase activity. For example, the target DNA can be isolated from the genome of the microorganism by PCR or hybridization using primers or probes designed considering gene degeneracy from known amino acid sequence information, or primers or probes designed based on known base sequence information.

[0058] The DNA related to this technology encompasses various types of DNA derived from codon degeneracy. Artificially creating multiple types of DNA encoding the same amino acid sequence can be easily done using known genetic engineering techniques. For example, in the production of genetically engineered proteins, if the codons used in the original gene encoding the target protein are infrequently used in the host, the protein expression level may be low. In such cases, high expression of the target protein can be achieved by optimizing the codon utilization frequency for the host without altering the encoded amino acid sequence.

[0059] As an indicator of codon utilization frequency, the sum of the host-optimal codon utilization frequencies for each codon can be adopted. An optimal codon is defined as the codon with the highest utilization frequency among codons corresponding to the same amino acid. Codon utilization frequency is not particularly limited as long as it is optimized for the host, but the following are examples of optimal codons in E. coli. F: Phenylalanine (ttt), L: Leucine (ctg), I: Isoleucine (att), M: Methionine (atg), V: Valine (gtg), Y: Tyrosine (tat), Stop codon (taa), H: Histidine (cat), Q: Glutamine (cag), N: Asparagine (aat), K: Lysine (aaa), D: Aspartic acid (gat), E: Glutamic acid (gaa), S: Serine (agc), P: Proline (ccg), T: Threonine (acc), A: Alanine (gcg), C: Cysteine ​​(tgc), W: Tryptophan (tgg), R: Arginine (cgc), G: Glycine (ggc).

[0060] Methods for introducing mutations into genes and artificially modifying amino acid sequences include known methods such as the Kunkel method and the Gapped duplex method, as well as mutation introduction kits utilizing site-directed mutagenesis, such as the QuickChange™ Site-Directed Mutagenesis Kit (Stratagene), GeneTailor™ Site-Directed Mutagenesis System (Invitrogen), and the TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio).

[0061] The base sequence of DNA can be confirmed by sequencing using conventional methods. For example, it can be done by dideoxynucleotide chain termination (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463). Alternatively, the sequence can be analyzed using a suitable DNA sequencer.

[0062] To confirm whether the obtained DNA encodes the target polypeptide, the determined base sequence can be compared with the base sequence described in Sequence ID No. 2. Alternatively, the amino acid sequence predicted from the determined base sequence can be compared with the amino acid sequence described in Sequence ID No. 1.

[0063] [Uses of DNA related to this technology] The DNA related to this technology can be used as a probe for identifying the gene encoding galactose epimerase related to this technology, the DNA encoding galactose epimerase related to this technology, or as a primer for amplifying or mutating the DNA encoding galactose epimerase related to this technology.

[0064] The DNA related to this technology can be used to prepare the galactose epimerase related to this technology. A genetic engineering preparation method using the DNA related to this technology makes it possible to obtain a more homogeneous galactose epimerase related to this technology. Furthermore, this method is suitable for preparing large quantities of the galactose epimerase related to this technology. If the DNA related to this technology is a polynucleotide DNA that does not contain a start codon, the galactose epimerase related to this technology can be obtained by adding a start codon or a signal peptide containing a start codon and then expressing it.

[0065] The DNA related to this technology can also be used as an experimental tool for elucidating the mechanism of action of the galactose epimerase related to this technology, or as a tool for designing or creating mutants (modified versions) of the galactose epimerase related to this technology.

[0066] 3. Expression Cassette and Recombinant Vector The expression cassette and recombinant vector related to this technology include the DNA related to this technology as described above.

[0067] The expression cassette and recombinant vector related to this technology may contain, as necessary, transcription elements such as enhancers, CCAAT boxes, TATA boxes, and SPI sites, in addition to promoters and terminators, as regulatory factors. These regulatory factors only need to be operablely ligated to the DNA of this technology. Operable ligation means that the various regulatory factors that regulate the DNA of this technology and the DNA of this technology are ligated in a manner that allows them to function in the host cell.

[0068] Regarding the recombinant vectors related to this technology, preferred expression vectors are those constructed for genetic recombination from phages, plasmids, or viruses that can autonomously proliferate within a host. Such expression vectors are well known, and commercially available examples include pQE vectors (Qiagen Co., Ltd.), pDR540, pRIT2T (GE Healthcare Biosciences Co., Ltd.), pET vectors (Merck K.K.), and pBE-S (Takara Bio Inc.). The expression vector can be used in any combination with a host cell. For example, when using Escherichia coli as the host cell, examples include a combination of a pET vector and the DH5α strain, a combination of a pET vector and the BL21 (DE3) strain, or a combination of a pDR540 vector and the JM109 strain.

[0069] 4. Transformants The transformants related to this technology are obtained by transforming a host with the expression cassette or recombinant vector related to this technology described above.

[0070] The host used in the production of transformants according to this technology is not particularly limited as long as it has the following characteristics (1) to (4): (1) It is possible to introduce an expression cassette or recombinant vector; (2) The expression cassette or recombinant vector is stable; (3) It is capable of autonomous proliferation; and (4) It is capable of expressing the traits of the genes in the introduced expression cassette or recombinant vector. Suitable examples of such hosts include bacteria belonging to the Escherichia genus such as Escherichia coli, the Bacillus genus such as Bacillus subtilis, and the Pseudomonas genus such as Pseudomonas putida; filamentous fungi, yeast, etc. Animal cells, insect cells, plants, etc. may also be used.

[0071] Transformants of this technology can be obtained by introducing DNA related to this technology, an expression cassette related to this technology, or a recombinant vector related to this technology into a host. The site where the DNA related to this technology is introduced is not particularly limited as long as the target gene can be expressed, and may be on a plasmid or on the genome.

[0072] Specific methods for introducing the expression cassette or recombinant vector related to this technology include, for example, recombinant vector methods and genome editing methods. The conditions for introducing the expression cassette or recombinant vector into the host can be appropriately set according to the type of host, etc. If the host is bacteria, for example, methods using competent cells treated with calcium ions and electroporation can be used. If the host is yeast, for example, electroporation, spheroplast methods and lithium acetate methods can be used. If the host is animal cells, for example, electroporation, calcium phosphate methods and lipofection methods can be used. If the host is insect cells, for example, calcium phosphate methods, lipofection methods and electroporation methods can be used. If the host is plant cells, for example, electroporation, Agrobacterium methods, particle gun methods and PEG methods can be used.

[0073] Confirmation of whether the DNA related to this technology, the expression cassette related to this technology, or the recombinant vector related to this technology has been incorporated into a host can be performed by PCR, Southern hybridization, Northern hybridization, etc.

[0074] To confirm whether the DNA, expression cassette, or recombinant vector related to this technology has been incorporated into a host using PCR, for example, the genomic DNA, expression cassette, or recombinant vector can be isolated and purified from the transformant. The isolation and purification of the genomic DNA, expression cassette, or recombinant vector can be performed, for example, based on the lysate obtained by lysing the bacteria when the host is a bacterium. As a method of lysis, for example, treatment with a lytic enzyme such as lysozyme is performed, and if necessary, proteases, other enzymes, and surfactants such as sodium lauryl sulfate (SDS) are used in combination.

[0075] Furthermore, physical disruption methods such as freeze-thaw cycles and French press treatments may be combined. DNA isolation and purification from the lysate can be performed, for example, by deproteinization treatments using phenol and protease, ribonuclease treatment, alcohol precipitation, and by appropriately combining commercially available kits.

[0076] DNA cleavage can be performed according to standard methods, for example, using restriction enzyme therapy. For example, type II restriction enzymes that act on specific nucleotide sequences can be used. Binding of DNA to an expression cassette or expression vector can be performed, for example, using a DNA ligase.

[0077] Subsequently, using the separated and purified DNA as a template, primers specific to the DNA related to this technology are designed and PCR is performed. The amplified products obtained by PCR are subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide and SYBR Green solution, and the amplified products are detected as bands to confirm that transformation has occurred.

[0078] Furthermore, PCR can be performed using primers pre-labeled with fluorescent dyes or the like to detect the amplified product. In addition, a method may be employed in which the amplified product is bound to a solid phase such as a microplate and confirmed by fluorescence and enzymatic reactions.

[0079] 5. Method for Producing Galactose Epimerase The method for producing galactose epimerase according to this technology involves at least a culture step and a recovery step. In addition, it is possible to freely combine various steps commonly used in enzyme production as needed. The following describes each step in detail.

[0080] (1) Culture process The culture process is a process of culturing a microorganism that produces galactose epimerase or a transformant related to this technology. Examples of microorganisms that produce galactose epimerase include microorganisms of the class Anaerolineae. The microorganism used may be a wild-type strain or a mutant strain (for example, a mutant strain produced by ultraviolet irradiation).

[0081] The culture conditions in the culture process can be appropriately set considering the properties of the microorganism being cultured and the nutritional and physiological properties of the transformant being cultured. The microorganism or transformant can be cultured in solid or liquid media, but liquid culture is preferred. Furthermore, for industrial production, aerated and stirred culture is preferred.

[0082] As nutrients for the culture medium, any substance necessary for the growth of microorganisms or transformants can be used as appropriate. As a carbon source, any assimilated carbon compound is acceptable, such as glucose, sucrose, lactose, maltose, molasses, and pyruvic acid. As a nitrogen source, any assimilated nitrogen compound is acceptable, such as peptone, meat extract, yeast extract, casein hydrolysate, and soybean meal alkali extract. In addition to carbon and nitrogen sources, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, as well as specific amino acids and specific vitamins, may be used as needed.

[0083] The culture temperature should be set appropriately within a range in which the microorganism or transformant can grow and produce galactose epimerase. For example, the culture temperature can be set to 15 to 37°C.

[0084] The culture time can range from approximately 12 to 48 hours. The culture process can be terminated when the galactose epimerase reaches its maximum yield.

[0085] (2) Recovery process The recovery process is a process of recovering the galactose epimerase related to this technology from the culture obtained in the culture process. The recovery method in the recovery process is not particularly limited, and one or more general enzyme recovery methods can be freely used in combination.

[0086] Specifically, for example, if galactose epimerase expressed using a transformant is present inside the transformant, the transformant is separated from the culture supernatant using a common separation method such as centrifugation. The culture supernatant is removed and the separated transformant is recovered. The separated transformant is treated using mechanical methods such as sonication or French press, or enzymatic methods such as lysozyme, and solubilized using enzymes such as proteases or surfactants such as sodium lauryl sulfate (SDS) as needed to obtain a water-soluble fraction containing galactose epimerase. Alternatively, by selecting an appropriate expression cassette or expression vector and host, the expressed galactose epimerase can be secreted into the culture medium.

[0087] The water-soluble fraction containing galactose epimerase obtained in this way may be subjected to purification as is, or it may be subjected to purification after concentrating a predetermined amount of galactose epimerase in the water-soluble fraction. Concentration can be performed, for example, by vacuum concentration, membrane concentration, salting out, or fractional precipitation using a hydrophilic organic solvent (e.g., methanol, ethanol, acetone, etc.). Furthermore, the purification of galactose epimerase can be performed by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

[0088] The purified galactose epimerase can be powdered and distributed to the market as needed by methods such as freeze-drying, vacuum drying, or spray-drying.

[0089] 6. Method for Producing Taloss The method for producing taloss according to this technology includes a step of reacting galactose with galactose epimerase according to this technology (hereinafter also referred to as the "enzyme reaction step"). In addition, a raw material preparation step, an enzyme inactivation step, a recovery step, etc. may be performed as needed. Each step will be described in detail below.

[0090] (1) Raw material preparation process The raw material preparation process is the process of preparing galactose, which will be used as a raw material. The galactose used as a raw material may be in the form of a composition mixed with other components, as long as it does not impair the action or effect of this technology. Furthermore, the form of the raw material is not particularly limited and may be in the form of liquid, powder, slurry, etc.

[0091] (2) Enzyme Action Process The enzyme action process is a step in which the galactose used as a raw material is treated with the galactose epimerase related to this technology. The galactose epimerase related to this technology may be used alone or in combination of two or more types. Details of the galactose epimerase related to this technology have been described above, so an explanation will be omitted here.

[0092] Furthermore, as long as the action and effects of this technology are not impaired, it may be used in combination with other enzymes other than galactose epimerase related to this technology, depending on the purpose.

[0093] When using two or more enzymes in combination, the two or more enzymes may be added to the galactose raw material simultaneously, or they may be added separately. The order in which the two or more enzymes are added separately is not particularly limited.

[0094] Other enzymes related to this technology besides galactose epimerase include, for example, amylase (α-amylase, β-amylase, glucoamylase), invertase, fructosyltransferase, galactosidase (α-galactosidase, β-galactosidase), lactase, glucosidase (α-glucosidase, β-glucosidase), protease (acid protease, neutral protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, dextranase, transglutaminase, protein deamide enzyme, pullulanase, etc. These other enzymes may be included in combinations of multiple types.

[0095] The various conditions of the enzyme action process can be freely set, as long as they do not impair the effectiveness of this technology. For example, pH, temperature, action time, etc., can be set according to the physicochemical properties of the enzyme used, such as the optimal pH, stable pH range, optimal temperature, and temperature stability. The optimal reaction conditions can be determined through preliminary experiments. The following are examples of conditions for using each enzyme.

[0096] When the galactose epimerase according to this technology is applied to the galactose used as a raw material, the pH can be set to, for example, pH 4.5 to 10.0, preferably pH 5.0 to 7.0, more preferably pH 5.0 to 6.0, or pH 6.0 to 7.0.

[0097] The temperature at which the galactose epimerase according to this technology is applied to the galactose used as a raw material can be set to, for example, 4°C to 60°C, preferably 10°C to 50°C, and more preferably 30°C to 45°C.

[0098] When the galactose epimerase according to this technology is applied to the galactose used as a raw material, the application time can be set to, for example, 15 minutes to 120 hours, preferably 30 minutes to 72 hours, and more preferably 2 hours to 48 hours.

[0099] The amount of galactose epimerase relative to galactose in the enzymatic action step can be freely set as long as it does not impair the effectiveness of this technology. The lower limit of the amount of galactose epimerase per 1 g of galactose can be set to, for example, 0.001 U / g or more, 0.01 U / g or more, 0.1 U / g or more, 1 U / g or more, 10 U / g or more, 100 U / g or more, 1000 U / g or more, 10000 U / g or more, 100000 U / g or more, 100000 U / g or more, 100000 U / g or more, etc. The upper limit of the amount of galactose epimerase per 1 g of galactose can be set to, for example, 1,000,000 U / g or less, 100,000 U / g or less, 10000 U / g or less, 100 U / g or less, 1 U / g or less, 0.1 U / g or less, etc.

[0100] In this technology, galactose epimerase activity is defined as the amount of enzyme that produces 1 nmol of galactose per minute when treated with a 10 mmol / L talose solution (0.5 mol / L acetate buffer, pH 6) as a substrate at 37°C, with 1 unit (1 U) being defined.

[0101] (3) Enzyme Inactivation Step The enzyme inactivation step is a step in which the enzyme is inactivated after the enzyme action step described above. The enzyme inactivation step is not an essential step and can be performed as needed. Furthermore, if multiple enzymes are used in the enzyme action step described above, enzyme inactivation can be performed between the actions of the enzymes. For example, if multiple enzymes are used in the enzyme action step, it is possible to perform the enzyme inactivation step as appropriate in the middle of the enzyme action step.

[0102] The method for inactivating an enzyme is not particularly limited, and one or more general inactivation methods can be freely combined and used depending on the properties of the enzyme to be inactivated. For example, one method is to inactivate the enzyme by heating it above its enzyme inactivation temperature.

[0103] (4) Recovery Process The recovery process is the process of recovering the manufactured talose. Depending on the type of talose being manufactured, one or more general recovery methods used in talose manufacturing can be freely combined and used.

[0104] The recovered talose can be further processed, etc., depending on the form of the final product, to the extent that it does not impair the function or effect of this technology.

[0105] 7. Method for manufacturing food and beverages, and method for reducing sugars in food and beverages The method for manufacturing food and beverages according to this technology, and the method for reducing galactose in food and beverages according to this technology, include a step of applying galactose epimerase according to this technology to sugar-containing food and beverages (hereinafter also referred to as the "enzyme action step"). In addition, a raw material preparation step, an enzyme inactivation step, a recovery step, etc., may be performed as needed. Each step will be described in detail below.

[0106] (1) Raw Material Preparation Process The raw material preparation process is the process of preparing galactose-containing food and beverages or their raw materials to be used as raw materials. The raw materials that can be used in this technology are not particularly limited in terms of origin, type, etc., as long as they do not impair the effects of this technology, and can be freely selected according to the target food and beverage. In this technology, raw materials containing galactose (galactose-containing raw materials) are particularly preferred. Furthermore, the form of the raw materials is not particularly limited, and examples include liquid, powder, slurry, etc.

[0107] (2) Enzyme Action Process The enzyme action process involves applying galactose epimerase related to this technology to galactose-containing food and beverages and / or their raw materials. The details of the enzyme action process are the same as those of the enzyme action process in the method for producing talose described above, so the explanation is omitted here.

[0108] (3) Enzyme Inactivation Step The enzyme inactivation step is a step in which the enzyme is inactivated after the enzyme action step described above. The details of the enzyme inactivation step are the same as the enzyme inactivation step in the method of producing talose described above, so the explanation is omitted here.

[0109] (4) Recovery Process The recovery process is the process of recovering the manufactured food and beverages. Depending on the type of food and beverage being manufactured, one or more general recovery methods used in the manufacture of food and beverages can be freely combined and used.

[0110] The collected food and beverages may be further processed, etc., depending on the form of the final product, to the extent that the function and effect of this technology are not impaired.

[0111] 8. Food and beverages: Food and beverages relating to this technology are characterized by being obtained using the galactose epimerase relating to this technology. Details of the galactose epimerase relating to this technology are as described above and will therefore not be explained here.

[0112] (1) Amount of galactose contained in food and beverages The amount of galactose contained in food and beverages used in this technology is not particularly limited, but it is preferable that it is reduced compared to the galactose content in food and beverages manufactured without using the galactose epimerase related to this technology. Specifically, the galactose content in food and beverages related to this technology is, for example, 98% or less, preferably 97% or less, more preferably 96% or less, even more preferably 95% or less, 90% or less, or 85% or less compared to the galactose content in food and beverages manufactured without using the galactose epimerase related to this technology.

[0113] The galactose reduction rate of food and beverages according to this technology compared to food and beverages produced without using the galactose epimerase according to this technology is, for example, 2% or more, preferably 3% or more, more preferably 4% or more, even more preferably 5% or more, 10% or more, or 15% or more.

[0114] (2) Amount of talose contained in food and beverages The amount of talose contained in food and beverages used in this technology is not particularly limited, but it is preferable that it is increased compared to the amount of talose in food and beverages manufactured without using the galactose epimerase according to this technology. Specifically, the amount of talose contained in food and beverages according to this technology is, for example, 10% or more, preferably 15% or more, and more preferably 20% or more, compared to the amount of talose in food and beverages manufactured without using the galactose epimerase according to this technology.

[0115] (3) Types of food and beverages The types of food and beverages related to this technology are not particularly limited as long as they do not impair the effects of this technology, but beverages are preferred. Examples of beverages include vegetable juices and fruit juices. Other examples of foods and beverages include Japanese sweets (mochi, manju, daifuku, castella, anko, etc.), Western sweets (cream, cake, ice cream, etc.), soups, sauces, dressings, etc.

[0116] 9. Enzyme preparation for talose production and / or galactose reduction The enzyme preparation for talose production and / or galactose reduction related to this technology is an enzyme preparation containing the galactose epimerase related to this technology. Details of the galactose epimerase related to this technology are as described above and will not be explained here.

[0117] (1) Galactose epimerase content The galactose epimerase content in the enzyme preparation for talose production and / or galactose reduction related to this technology can be freely set as long as the effects of this technology are not impaired. The lower limit of the galactose epimerase content can be set to, for example, 0.001 U / g or more, 0.01 U / g or more, 0.1 U / g or more, 1 U / g or more, 10 U / g or more, 100 U / g or more, 1000 U / g or more, 10000 U / g or more, 100000 U / g or more, 1000000 U / g or more, 1000000 U / g or more, etc., per 1 g of galactose in the composition to be acted upon (e.g., food and beverages).

[0118] The upper limit of the galactose epimerase content is not particularly limited as long as it does not impair the effectiveness of this technology, but it can be set to, for example, 1,000,000 U / g or less, 100,000 U / g or less, 10,000 U / g or less, 1,000 U / g or less, 100 U / g or less, 10 U / g or less, 1 U / g or less, 1 U / g or less, 0.1 U / g or less, etc., per 1 g of galactose in the composition to be acted upon (e.g., food and beverages).

[0119] (2) Other Components The enzyme preparation for sugar reduction related to this technology may be used in combination with other components as long as it does not impair the action or effect of this technology. Other components may include, for example, excipients commonly used in formulation (starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol, etc.), buffering agents (phosphates, citrates, acetates, etc.), stabilizers (propylene glycol, ascorbic acid, etc.), suspending agents, pH adjusters, colorants, flavoring agents, disintegrants, lubricants, preservatives (phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc.), antiseptics (ethanol, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc.), physiological saline, the other enzymes mentioned above, and culture residue (components derived from the culture medium, contaminating proteins, bacterial components, etc.) produced in the galactose epimerase production method mentioned above. Furthermore, known or future-discovered functional components can be used in combination as appropriate for the purpose.

[0120] (3) Form of the enzyme preparation The form of the enzyme preparation of the present invention is not particularly limited, and examples include liquid, solid (powder, granules, etc.).

[0121] The present technology will be described in more detail below based on the following examples. The examples described below are representative examples of the present technology and should not be interpreted as narrowing the scope of the present technology.

[0122] 1. Selection of Microorganisms: In order to find a novel galactose epimerase, a screening was conducted using galactose epimerase activity as an indicator from a bacterial strain library. As a result, galactose epimerase 1 from a microorganism of the class Anaerolineae was selected as a promising candidate.

[0123] 2. Sequence Verification: The amino acid sequence of galactose epimerase 1 (SEQ ID NO: 1) was determined from the genomic DNA information obtained by genomic DNA analysis using a next-generation sequencer.

[0124] 3. Construction of the Expression System A synthetic nucleic acid obtained by adding a His-tag to the selected amino acid sequence (SEQ ID NO: 1) was inserted into the cloning site of the expression vector to construct a galactose epimerase expression vector. Escherichia coli (BL21(DE3)) was transformed with this expression vector. Sequence analysis of the obtained transformants was performed to confirm that the target sequence was incorporated.

[0125] 4. Culture of Transformants The transformed cells obtained were cultured in Difco LB broth liquid medium at 37°C and 200 rpm for 2 days. After culturing, the medium was centrifuged to collect the cells. The collected cells were suspended in 10 mM imidazole-HCl buffer (pH 8.0) containing 0.3 M NaCl, then lysed, and the lysate was centrifuged. The supernatant was then filtered through a membrane filter and collected.

[0126] 5. Purification of Galactose Epimerase The cell lysate recovered above was subjected to a HisTrap HP (I.D. × L = 0.7 × 2.5 cm; manufactured by Cytiva) equilibrated with 10 mM imidazole-HCl buffer (pH 8.0) containing 0.3 M NaCl. After washing the column with the same buffer, the galactose epimerase adsorbed to the column was eluted with 500 mM imidazole-HCl buffer (pH 8.0) containing 0.3 M NaCl, and the column was fractionated. Galactose epimerase activity (epimery of galactose) was confirmed in a portion of each obtained fraction using the method described later, and the active fraction was collected to obtain a primary purified solution. Purification was confirmed by SDS-PAGE. The results of SDS-PAGE are shown in Figure 1. The molecular weight of galactose epimerase 1 was 48.9 kDa.

[0127] 6. Characterization of Galactose Epimerase (1) Talose Production Rate 50 μL of 50 mM acetate buffer (pH 6.0) solution containing 50 mM galactose as a substrate was mixed with 10 μL of crude enzyme solution, and the reaction was carried out at 37°C for 48 hours. The talose production rate was confirmed by HPLC analysis under the following conditions. The results are shown in Figure 2. The talose production rate of galactose epimerase 1 was 14.7%.

[0128] [HPLC Conditions] Column: MCIgel CK08EC (Mitsubishi Chemical Corporation) Temperature: 75°C Elution: Ultrapure water Flow rate: 0.6 mL / min Detection: Differential refractive index system RID-10A (Shimadzu Corporation)

[0129] (2) Mannose production rate A 50 μL solution of 50 mM acetate buffer (pH 6.0) containing 50 mM glucose as a substrate was mixed with 10 μL of crude enzyme solution, and the reaction was carried out at 37°C for 24 hours. The mannose production rate was confirmed by HPLC analysis under the above conditions. The mannose production rate of galactose epimerase 1 was extremely low at 0.4%.

[0130] (3) Substrate specificity: 10 μL of a 20 mM acetate buffer (pH 6.0) solution containing 50 mM D-mannose (Man), D-galactose (Gal), D-fructose (Fru), D-xylose (Xyl), lactose (Lac), cellobiose (Cel), maltose (Mal), sucrose (Suc), or D-mannitol as a substrate was mixed with 2 μL of purified enzyme solution or ultrapure water, and the reaction was carried out at 37°C for 48 hours. The reaction catalyzed by galactose epimerase 1 was confirmed by subjecting 5 μL of the reaction solution to TLC analysis. A mixture of 1-butanol:2-propanol:water = 2:2:1 was used as the developing solvent. The product was detected by spraying with a prepared anisaldehyde solution containing acetic acid and sulfuric acid (Tokyo Chemical Industries, Ltd.) and then heating. The results are shown in Figure 3. As shown in Figure 3, galactose epimerase 1 uses D-mannose (Man), D-galactose (Gal), and lactose (Lac) as substrates, but does not use D-fructose (Fru), D-xylose (Xyl), cellobiose (Cel), maltose (Mal), sucrose (Suc), or D-mannitol as substrates. Furthermore, as shown in the HPLC analysis results from the mannose production rate measurement mentioned above, it was found that galactose epimerase 1 uses D-glucose as a substrate only in very small amounts.

[0131] (4) Optimal temperature A 10 mmol / L talose solution (0.5 mol / L acetate buffer, pH 6) was used as the substrate, and the enzyme reaction temperature was changed to 10°C, 20°C, 30°C, 40°C, 50°C, or 60°C, and the sample was treated for 1 hour. The relative activity at each temperature condition was calculated as relative activity (%), with the activity at the temperature condition showing the maximum activity of the purified enzyme being set to 100%. The results are shown in Figure 4. As shown in Figure 4, the optimal temperature for galactose epimerase 1 was 30-40°C.

[0132] (5) Optimal pH A 10 mmol / L talose solution (0.5 mol / L acetate buffer, pH 6) was used as the substrate, and the pH of the substrate solution was changed to pH 3, 4, 5, 6, 7, 8, 9, or 10 with 50 mM acetate buffer or 50 mM Tris-HCl buffer, and treated at 37°C for 1 hour. The activity at the pH condition that showed the maximum activity of the purified enzyme was taken as 100%, and the relative amount of activity at each pH condition was calculated as relative activity (%). The results are shown in Figure 5. As shown in Figure 5, the optimal pH for galactose epimerase 1 was pH 6 to 7.

Claims

1. Galactose epimerase comprising a polypeptide as shown in any of the following (1) to (3): (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, (2) a polypeptide having galactose epimerase activity, wherein one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 are substituted, added, inserted, deleted, or inverted, and (3) a polypeptide having galactose epimerase activity, wherein the amino acid sequence shown in SEQ ID NO: 1 has a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO:

1.

2. The galactose epimerase according to claim 1, which is derived from a microorganism of the class Anaerolineae.

3. DNA comprising any of the following (1) to (3): (1) a polynucleotide encoding the galactose epimerase described in claim 1; (2) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2; and (3) a polynucleotide comprising a sequence equivalent to the base sequence shown in SEQ ID NO: 2 and encoding a polypeptide having galactose epimerase activity.

4. An expression cassette or recombinant vector comprising the DNA described in claim 3.

5. A transformant obtained by transforming a host with the expression cassette or recombinant vector described in claim 4.

6. A method for producing galactose epimerase, comprising: a culture step of culturing a microorganism of the class Anaerolineae or a transformant according to claim 5; and a recovery step of recovering the galactose epimerase according to claim 1 from the culture obtained in the culture step.

7. A method for producing talose, comprising the step of reacting galactose with the galactose epimerase described in claim 1.

8. A method for producing food and beverages, comprising the step of reacting a galactose-containing food and / or raw material thereof with the galactose epimerase described in claim 1.

9. A method for reducing galactose in food and beverages, comprising the step of reacting galactose-containing food and / or its raw materials with the galactose epimerase described in claim 1.

10. Food and beverages obtained using the galactose epimerase described in claim 1.

11. An enzyme preparation for the production of talose and / or for galactose reduction, comprising the galactose epimerase described in claim 1.

12. Use of polypeptides as galactose epimerases as any of the following (1) to (3): (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, (2) a polypeptide having galactose epimerase activity, wherein one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in SEQ ID NO: 1, and (3) a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and having galactose epimerase activity.

13. Use of polypeptides shown in any of the following (1) to (3) for the production of talose: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, (2) a polypeptide having galactose epimerase activity, in which one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in SEQ ID NO: 1, and (3) a polypeptide having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and having galactose epimerase activity.

14. Use of polypeptides shown in any of the following (1) to (3) for reducing galactose in food and beverages: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, (2) a polypeptide having galactose epimerase activity in which one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in SEQ ID NO: 1, and (3) a polypeptide having 90% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1, and having galactose epimerase activity.

Images