Glucose epimerase

Abstract

The purpose of the present invention is to provide a glucose epimerase having high specificity to glucose and high temperature stability.　The present technology provides a glucose epimerase containing a polypeptide depicted in any one of (1) to (3): (1) a polypeptide which comprises the amino acid sequence depicted in any one of SEQ ID NOs: 1 to 5; (2) a polypeptide which has glucose epimerase activity and which is obtained by substituting, adding, inserting, or deleting one or several amino acid residues in the amino acid sequence depicted in any one of SEQ ID NOs: 1 to 5; and (3) a polypeptide which has glucose epimerase activity and which has, in the amino acid sequence depicted in any one of SEQ ID NOs: 1 to 5, 90% or more sequence identity to the amino acid sequence depicted in any one of SEQ ID NOs: 1 to 5.

Description

Glucose epimerase

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

[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, is excellent in heat resistance, and is suitable for industrial-scale use.

[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 Patent Application Laid-Open No. 2020-137485, Japanese Patent Application Laid-Open No. 2019-033702

[0005] Glucose epimerase, which is a kind of enzyme having the activity of epimerizing sugars, is an enzyme that catalyzes the epimerization of glucose and mannose. Mannose is difficult to be utilized by the living body as glucose, and the catalytic ability to convert glucose to mannose is expected to contribute to the reduction of calories derived from glucose. In addition, mannose in livestock feed is known to exhibit an inhibitory effect on the infection of harmful bacteria such as Salmonella. Thus, glucose epimerase is an enzyme that can be expected to contribute to various applications.

[0006] However, some of the glucose epimerases currently known have low specificity for glucose, and these react with galactose and cellobiose to produce by-products other than mannose. On the other hand, glucose epimerases with high specificity for glucose have poor temperature stability. Thus, there are still many applications where the glucose epimerases currently known are difficult to use, and further development of glucose epimerases is desired.

[0007] Therefore, the main objective of this technology is to provide glucose epimerases with various substrate specificities and characteristics.

[0008] This technology first provides a glucose epimerase comprising a polypeptide as shown in any of the following (1) to (3): (1) a polypeptide consisting of an amino acid sequence as shown in any of SEQ ID NOs: 1 to 5; (2) a polypeptide having glucose epimerase activity, wherein one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence as shown in any of SEQ ID NOs: 1 to 5; and (3) a polypeptide having glucose epimerase activity, wherein the amino acid sequence as shown in any of SEQ ID NOs: 1 to 5 has a sequence identity of 90% or more to the amino acid sequence as shown in any of SEQ ID NOs: 1 to 5. This technology also provides a glucose epimerase comprising a polypeptide as shown in any of the following (1) to (3) and derived from a microorganism of the phylum Bacteroidota. (1) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 1; (2) A polypeptide having glucose epimerase activity, wherein one or more amino acid residues in the amino acid sequence shown in Sequence ID No. 1 are substituted, added, inserted, deleted, or inverted; and (3) A polypeptide having glucose epimerase activity, wherein the amino acid sequence shown in Sequence ID No. 1 has a sequence identity of 90% or more to the amino acid sequence shown in Sequence ID No. 1. This technology also provides glucose epimerase derived from a microorganism of the genus Spirosoma, comprising any of the polypeptides shown in (1) to (3) below. (1) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 2; (2) A polypeptide having glucose epimerase activity, wherein one or more amino acid residues in the amino acid sequence shown in Sequence ID No. 2 are substituted, added, inserted, deleted, or inverted; and (3) A polypeptide having glucose epimerase activity, wherein the amino acid sequence shown in Sequence ID No. 2 has a sequence identity of 90% or more to the amino acid sequence shown in Sequence ID No. 2. This technology also provides glucose epimerase derived from a microorganism of the genus Lewinella, comprising any polypeptide shown in (1) to (3) below.(1) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 3, (2) A polypeptide having glucose epimerase activity in which one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in Sequence ID No. 3, and (3) A polypeptide having glucose epimerase activity in which the amino acid sequence shown in Sequence ID No. 3 has a sequence identity of 90% or more to the amino acid sequence shown in Sequence ID No. 3. This technology also provides glucose epimerase derived from a microorganism of the genus Spirochete (Sphaerochaeta), comprising any of the polypeptides shown in (1) to (3) below: (1) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 4, (2) A polypeptide having glucose epimerase activity in which one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in Sequence ID No. 4, and (3) A polypeptide having glucose epimerase activity in which the amino acid sequence shown in Sequence ID No. 4 has a sequence identity of 90% or more to the amino acid sequence shown in Sequence ID No. 4.

[0009] This technology provides DNA comprising any of the following polynucleotides: (1) a polynucleotide encoding glucose epimerase according to this technology; (2) a polynucleotide consisting of any of the base sequences shown in SEQ ID NOs: 6 to 10; and (3) a polynucleotide encoding a polypeptide having glucose epimerase activity, comprising a sequence equivalent to any of the base sequences shown in SEQ ID NOs: 6 to 10. 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.

[0010] This technology also provides a method for producing glucose epimerase, comprising: a culture step of culturing one or more microorganisms selected from the phylum Bacteroidota, the genus Spirosoma, the genus Lewinella, and the genus Sphaerochaeta, or a transformant related to this technology; and a recovery step of recovering glucose epimerase related to this technology from the culture obtained in the culture step.

[0011] This technology further provides a method for producing mannose, comprising the step of reacting glucose with glucose epimerase according to this technology. This technology also provides a method for producing food and beverages, comprising the step of reacting glucose-containing food and beverages and / or their raw materials with glucose epimerase according to this technology. In addition, this technology provides a method for reducing glucose in food and beverages, comprising the step of reacting glucose-containing food and beverages and / or their raw materials with glucose epimerase according to this technology. This technology also provides food and beverages obtained using glucose epimerase according to this technology. This technology also provides an enzyme preparation for mannose production and / or glucose reduction, comprising glucose epimerase according to this technology.

[0012] In addition, this technology provides the use of any polypeptide shown in (1) to (3) above as a glucose epimerase. This technology also provides the use of any polypeptide shown in (1) to (3) above for the production of mannose. This technology also provides the use of any polypeptide shown in (1) to (3) above for reducing glucose in food and beverages.

[0013] 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.

[0014] <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.

[0015] <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.

[0016] <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.

[0017] <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.

[0018] <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 NOs. 1-5 in this technology) or nucleotide sequence (SEQ ID NOs. 6-10 in this technology), but in which the difference does not substantially affect the function of the protein (glucose epimerase activity in this technology). Therefore, in this technology, enzymes having polypeptide chains consisting of equivalent amino acid sequences, or enzymes encoded by polynucleotides consisting of equivalent nucleotide sequences, exhibit glucose epimerase activity.

[0019] <Mannose> Mannose is a type of monosaccharide and is the 2-position epimer of glucose. Because mannose is not easily metabolized in the body, most of the mannose taken orally is excreted in the urine. Mannose has been reported to have effects such as treating urinary tract infections, suppressing tumors, and improving obesity by improving the gut microbiota.

[0020] This is a photograph showing the SDS-PAGE results for glucose epimerases 1-5 in the example. This is a graph showing the optimal pH for glucose epimerases 1-4 in the example. This is a graph showing the optimal temperature for glucose epimerases 1-5 in the example. This is a graph showing the temperature stability for glucose epimerases 1-5 in the example. This is a graph showing the mannose production rate of glucose epimerases 1-5 and the talose production rate of glucose epimerase 3 in the example. This is a photograph showing the TLC analysis results for glucose epimerases 1-5 in the example. This is a graph showing the relative enzyme activity values ​​(%) of glucose epimerase 1 and glucose epimerase 2 under high-concentration mannose conditions (conditions using 15 mM mannose as a substrate) in the example, with the enzyme activity value set to 100%.

[0021] 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.

[0022] 1. Glucose Epimerase The glucose epimerase related to this technology will be described in detail below.

[0023] [Amino Acid Sequence] The glucose epimerase relating to this technology comprises a polypeptide as shown in any of the following (1) to (3): (1) a polypeptide consisting of an amino acid sequence shown in any of SEQ ID NOs: 1 to 5; (2) a polypeptide having glucose epimerase activity, wherein one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in any of SEQ ID NOs: 1 to 5; and (3) a polypeptide having glucose epimerase activity, wherein the amino acid sequence shown in any of SEQ ID NOs: 1 to 5 has a sequence identity of 90% or more to the amino acid sequence shown in any of SEQ ID NOs: 1 to 5.

[0024] 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.

[0025] In the polypeptide of (3) above, the sequence identity with respect to the amino acid sequence shown in any of SEQ ID NOs: 1 to 5 is preferably 90% or more, but may be 70% or more, 80% or more, 85% or more, etc., as long as the function and effects of this technology are not impaired, and preferably 90% or more, more preferably 93% or more, even more preferably 95% or more, even more preferably 96% or more, and particularly preferably 97% or more, 98% or more, or 99% or more.

[0026] Here, in the polypeptide of (3) above, the sequence identity for each amino acid sequence shown in any of SEQ ID NOs: 1 to 5 is the sequence identity calculated by comparing it with the amino acid sequence shown in any of SEQ ID NOs: 1 to 5. 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.

[0027] 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 at positions 272 (histidine residue) and 404 (histidine residue) of the amino acid sequence. The active site of SEQ ID NO: 2 is presumed to be at positions 273 (histidine residue) and 404 (histidine residue) of the amino acid sequence. The active site of SEQ ID NO: 3 is presumed to be at positions 282 (histidine residue) and 421 (histidine residue) of the amino acid sequence. The active site of SEQ ID NO: 4 is presumed to be at positions 276 (histidine residue) and 408 (histidine residue) of the amino acid sequence. The active site of SEQ ID NO: 5 is presumed to be at positions 273 (histidine residue) and 404 (histidine residue) of the amino acid sequence.

[0028] 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.

[0029] In the polypeptide of (2) or (3) above, if an amino acid substitution is introduced to any of SEQ ID NOs: 1 to 5, 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.

[0030] It is preferable that the glucose epimerase containing the polypeptide of (2) or (3) has glucose epimerase activity equal to or greater than that of the glucose epimerase containing the polypeptide of (1). Specifically, the relative activity of the glucose 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 glucose epimerase containing the polypeptide of (1).

[0031] In addition to being used as glucose 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 mannose and for reducing glucose in food and beverages, as will be described later.

[0032] [Origin] The glucose epimerase related to this technology is not particularly limited in its origin, as long as it contains the polypeptide described above. Examples include glucose epimerases derived from microorganisms of the phylum Bacteroidota, genus Spirosoma, genus Lewinella, and genus Sphaerochaeta. Examples of microorganisms of the phylum Bacteroidota include Fibrisoma limi. Examples of microorganisms of the genus Spirosoma include Spirosoma validum. Examples of microorganisms of the genus Lewinella include Lewinella cohaerens. Examples of microorganisms of the genus Sphaerochaeta include Sphaerochaeta halotolerans.

[0033] Here, "glucose epimerase derived from one or more microorganisms selected from the phylum Bacteroidota, genus Spirosoma, genus Lewinella, and genus Sphaerochaeta" means glucose epimerase produced by one or more microorganisms (which may be wild-type or mutant strains) classified from the phylum Bacteroidota, genus Spirosoma, genus Lewinella, and genus Sphaerochaeta, or glucose epimerase obtained by genetic engineering using glucose epimerase genes. Therefore, recombinants produced by host microorganisms into which a glucose epimerase gene (or a modified version thereof) obtained from one or more microorganisms selected from the phylum Bacteroidota, genus Spirosoma, genus Lewinella, and genus Sphaerochaeta has been introduced also fall under the category of "glucose epimerase derived from one or more microorganisms selected from the phylum Bacteroidota, genus Spirosoma, genus Lewinella, and genus Sphaerochaeta."

[0034] The strains of one or more microorganisms selected from the phylum Bacteroidota, genus Spirosoma, genus Lewinella, and genus Sphaerochaeta 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.

[0035] More specifically, glucose epimerase containing a polypeptide consisting of the amino acid sequence shown in Sequence ID No. 1 (hereinafter also referred to as "glucose epimerase 1") is a glucose epimerase derived from microorganisms of the phylum Bacteroidota.

[0036] The glucose epimerase containing the polypeptide consisting of the amino acid sequence shown in Sequence ID No. 2 (hereinafter also referred to as "glucose epimerase 2") is a glucose epimerase derived from microorganisms of the genus Spirosoma.

[0037] The glucose epimerase containing the polypeptide with the amino acid sequence shown in Sequence ID No. 3 (hereinafter also referred to as "glucose epimerase 3") is a glucose epimerase derived from microorganisms of the genus Lewinella. The amino acid sequence including the signal sequence is shown in Sequence ID No. 11.

[0038] The glucose epimerase containing the polypeptide consisting of the amino acid sequence shown in Sequence ID No. 4 (hereinafter also referred to as "glucose epimerase 4") is a glucose epimerase derived from microorganisms of the genus Spirochete (Sphaerochaeta).

[0039] Furthermore, the glucose epimerase related to this technology may be an artificial protein artificially designed based on the amino acid sequences described in (1) to (3) above.

[0040] [Enzymatic Chemical Properties] (1) Action The glucose epimerase related to this technology is an enzyme that produces mannose using glucose as a substrate. In addition, the glucose epimerase related to this technology may also have other actions, as long as it has glucose epimerase activity.

[0041] The conversion rate from glucose to mannose in the glucose epimerase according to this technology is not particularly limited, but is, for example, 15% or more, preferably 18% or more, and more preferably 20% or more. The upper limit of the conversion rate from glucose to mannose in the glucose epimerase according to this technology is also not particularly limited and may be 100%. Specifically, the conversion rate of glucose to mannose by glucose epimerase 1 was 23.5% at 37°C and 24 hours of reaction; the conversion rate of glucose to mannose by glucose epimerase 2 was 20.6% at 37°C and 24 hours of reaction; the conversion rate of glucose to mannose by glucose epimerase 3 was 19.2% at 37°C and 72 hours of reaction; the conversion rate of glucose to mannose by glucose epimerase 4 was 21.3% at 37°C and 72 hours of reaction; and the conversion rate of glucose to mannose by glucose epimerase containing a polypeptide consisting of the amino acid sequence shown in Sequence ID No. 5 (hereinafter also referred to as "glucose epimerase 5") was 23.7% at 37°C and 72 hours of reaction.

[0042] (2) Substrate specificity The glucose epimerases related to this technology produce D-mannose when D-glucose is used as a substrate. Glucose epimerases 1, 2, and 5 have low catalytic activity towards D-galactose and cellobiose, and did not act as substrates under the conditions of the examples described later. Low catalytic activity towards D-galactose and cellobiose can reduce the formation of other by-products.

[0043] On the other hand, glucose epimerase 3 also uses lactose, cellobiose, and maltose as substrates, and to a small extent, D-galactose as a substrate to produce talose (the conversion rate from D-galactose to talose is 2.9%). Glucose epimerase 4 also uses lactose and cellobiose as substrates, but its catalytic activity for maltose is low, and it did not act as a substrate under the conditions of the examples described later.

[0044] (3) Optimal pH The glucose epimerase according to the present technology has an optimal pH in the range of pH 4.5 to 8.5. Specifically, glucose epimerase 1 has an optimal pH in the range of pH 5.0 to 6.0. Also, glucose epimerase 1 has an activity of 80% or more of the activity at the optimal pH at pH 5.0 to 7.0. Glucose epimerase 2 has an optimal pH in the range of pH 4.5 to 6.0. Also, glucose epimerase 2 has an activity of 80% or more of the activity at the optimal pH at pH 4.5 to 7.0. Glucose epimerases 3 and 4 have an optimal pH in the range of pH 7.0 to 8.5. Also, glucose epimerases 3 and 4 have an activity of 80% or more of the activity at the optimal pH at pH 7.0 to 8.0.

[0045] The optimal pH is the value measured using 50 mM acetate buffer (pH 3.0 to 6.0) and 50 mM phosphate buffer (pH 7.0 to 10.0).

[0046] (4) Optimal temperature The optimal temperature of the glucose epimerase according to the present technology is 30 to 50°C. Specifically, the optimal temperature of glucose epimerases 1 and 2 is 40°C. Also, glucose epimerases 1 and 2 have an activity of 80% or more of the activity at the optimal temperature at 40 to 50°C. The optimal temperature of glucose epimerases 3 and 4 is 30 to 40°C. Also, glucose epimerase 3 has an activity of 80% or more of the activity at the optimal temperature at 30 to 40°C. Also, glucose epimerase 4 has an activity of 80% or more of the activity at the optimal temperature at 20 to 40°C. The optimal temperature of glucose epimerase 5 is 40 to 50°C. Also, glucose epimerase 5 has an activity of 80% or more of the activity at the optimal temperature at 30°C to 50°C.

[0047] The optimal temperature of glucose epimerases 1 and 2 is the value measured using 50 mM acetate buffer pH 6.0. The optimal temperature of glucose epimerases 3 to 5 is the value measured using 50 mM phosphate buffer pH 8.0.

[0048] (5) When thermostable glucose epimerases 1 to 3 and 5 are treated at 4°C to 50°C for 20 minutes, the residual activity is 80% or more. When glucose epimerase 4 is treated at 4°C to 20°C for 20 minutes, the residual activity is 80% or more.

[0049] (6) Molecular weight The molecular weight of the glucose epimerase according to the present technology is 45 to 55 kDa. As specific examples, the molecular weight of glucose epimerase 1 is 49.9 kDa, the molecular weight of glucose epimerase 2 is 49.1 kDa, the molecular weight of glucose epimerase 3 is 51.7 kDa (the molecular weight when including the signal sequence is 54.6 kDa), the molecular weight of glucose epimerase 4 is 49.5 kDa, and the molecular weight of glucose epimerase 5 is 49.4 kDa.

[0050] The molecular weight is the value measured by SDS-PAGE.

[0051] 2. DNA encoding glucose 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 glucose epimerase according to the present technology, (2) A polynucleotide consisting of the base sequence shown in any of SEQ ID NOs: 6 to 10, and (3) A polynucleotide containing a sequence equivalent to the base sequence shown in any of SEQ ID NOs: 6 to 10 and encoding a polypeptide having glucose epimerase activity

[0052] The "polynucleotide encoding the glucose epimerase according to the present technology" in (1) above refers to a polynucleotide that can obtain the glucose epimerase according to the present technology when expressed, including not only a polynucleotide having a base sequence corresponding to the amino acid sequence of the glucose 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.

[0053] Specific examples of polynucleotides encoding glucose epimerase related to this technology are shown in SEQ ID NO: 6 (genomic DNA sequence encoding the amino acid sequence of SEQ ID NO: 1), SEQ ID NO: 7 (genomic DNA sequence encoding the amino acid sequence of SEQ ID NO: 2), SEQ ID NO: 8 (genomic DNA sequence encoding the amino acid sequence of SEQ ID NO: 3), SEQ ID NO: 9 (genomic DNA sequence encoding the amino acid sequence of SEQ ID NO: 4), SEQ ID NO: 10 (genomic DNA sequence encoding the amino acid sequence of SEQ ID NO: 5), and SEQ ID NO: 12 (genomic DNA sequence encoding the amino acid sequence of SEQ ID NO: 11).

[0054] The DNA relating to this technology also includes DNA containing polynucleotides that, when compared with the base sequence of the polynucleotide encoding glucose epimerase relating to this technology as described in (1) above, have the same function as the protein encoded by it, but have a partially different base sequence (hereinafter also referred to as "equivalent sequence").

[0055] Specific examples of equivalent sequences include polynucleotides encoding polypeptides having glucose epimerase activity, which consist of a base sequence that includes one or more base substitutions, additions, insertions, deletions, or inversions based on the base sequence of a polynucleotide encoding glucose epimerase related to this technology (for example, any sequence from sequence numbers 6 to 10).

[0056] 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.

[0057] Furthermore, specific examples of equivalent sequences include sequences having sequence identity of, 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, with respect to the base sequence of the polynucleotide encoding glucose epimerase related to this technology (for example, any sequence from Sequence ID No. 6 to 10).

[0058] 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.

[0059] In this technology, DNA that hybridizes under stringent conditions with DNA having a complementary base sequence to DNA having a base sequence shown in any of sequence numbers 6 to 10 is also included in the DNA related to this technology, as long as it encodes a polypeptide having glucose epimerase activity.

[0060] 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.

[0061] 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.

[0062] [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.

[0063] Furthermore, the DNA related to this technology can also be isolated from microorganisms that produce polypeptides having glucose 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.

[0064] 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.

[0065] 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).

[0066] 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).

[0067] 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.

[0068] To confirm whether the obtained DNA encodes the target polypeptide, the determined base sequence can be compared with the base sequence described in any of SEQ ID NOs: 6 to 10. Alternatively, the amino acid sequence estimated from the determined base sequence can be compared with the amino acid sequence described in SEQ ID NOs: 1 or 2.

[0069] [Uses of DNA related to this technology] The DNA related to this technology can be used as a probe for identifying the gene encoding glucose epimerase related to this technology, the DNA encoding glucose epimerase related to this technology, or as a primer for amplifying or mutating the DNA encoding glucose epimerase related to this technology.

[0070] The DNA related to this technology can be used to prepare glucose epimerase related to this technology. A genetic engineering preparation method using the DNA related to this technology makes it possible to obtain glucose epimerase related to this technology in a more homogeneous state. Furthermore, this method is also suitable for preparing large quantities of glucose epimerase related to this technology. If the DNA related to this technology is a polynucleotide DNA that does not contain a start codon, glucose 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.

[0071] The DNA related to this technology can also be used as an experimental tool for elucidating the mechanism of action of glucose epimerase related to this technology, or as a tool for designing or creating mutants (modified versions) of glucose epimerase related to this technology.

[0072] 3. Expression cassette and recombinant vector The expression cassette and recombinant vector relating to this technology include the DNA relating to this technology as described above.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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 methods 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 5. Method for Producing Glucose Epimerase The method for producing glucose 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.

[0086] (1) Culture process The culture process is a process of culturing a microorganism that produces glucose epimerase or a transformant related to this technology. Examples of microorganisms that produce glucose epimerase include microorganisms of the phylum Bacteroidota, microorganisms of the genus Spirosoma, microorganisms of the genus Lewinella, and microorganisms of the genus Sphaerochaeta. The microorganism used may be a wild-type strain or a mutant strain (for example, a mutant strain induced by ultraviolet irradiation).

[0087] 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.

[0088] 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.

[0089] The culture temperature should be set appropriately within a range in which the microorganism or transformant can grow and produce glucose epimerase. For example, the culture temperature can be set to 15 to 37°C.

[0090] The culture time can range from approximately 12 to 48 hours. The culture process can be terminated when the glucose epimerase reaches its peak yield.

[0091] (2) Recovery process The recovery process is a process of recovering glucose 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 recovery methods used in enzyme production can be freely used in combination.

[0092] Specifically, for example, if glucose 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 glucose epimerase. Alternatively, by selecting an appropriate expression cassette or expression vector and host, the expressed glucose epimerase can be secreted into the culture medium.

[0093] The water-soluble fraction containing glucose 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 glucose epimerase in the water-soluble fraction. Concentration can be performed, for example, by vacuum concentration, membrane concentration, salting out, or fractional precipitation using hydrophilic organic solvents (e.g., methanol, ethanol, acetone, etc.). Furthermore, the purification of glucose epimerase can be performed by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

[0094] The purified glucose epimerase can be powdered and distributed to the market as needed by methods such as freeze-drying, vacuum drying, or spray-drying.

[0095] 6. Method for Producing Mannose The method for producing mannose according to this technology includes a step of reacting glucose with glucose 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.

[0096] (1) Raw material preparation process The raw material preparation process is the process of preparing glucose, which will be used as a raw material. The glucose 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.

[0097] (2) Enzyme action step The enzyme action step is a step in which the glucose used as a raw material is treated with the glucose epimerase related to this technology. The glucose epimerase related to this technology may be used alone or in combination of two or more types. Details of the glucose epimerase related to this technology are as described above and will not be explained here.

[0098] Furthermore, as long as the function and effects of this technology are not impaired, it may be used in combination with other enzymes other than glucose epimerase related to this technology, depending on the purpose.

[0099] When using two or more enzymes in combination, the two or more enzymes may be added to the glucose 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.

[0100] Other enzymes related to this technology besides glucose 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.

[0101] 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.

[0102] When the glucose epimerase according to this technology is applied to the glucose used as a raw material, the pH can be set to, for example, pH 4.5 to 10.0, preferably pH 5.0 to 9.0, and more preferably pH 5.0 to 8.5.

[0103] The temperature at which the glucose epimerase according to this technology is applied to the glucose raw material can be set to, for example, 4°C to 60°C, preferably 10°C to 50°C, and more preferably 20°C to 45°C.

[0104] When the glucose epimerase according to this technology is applied to the glucose 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.

[0105] The amount of glucose epimerase relative to glucose 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 glucose epimerase per 1 g of glucose 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 glucose epimerase per 1 g of glucose 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.

[0106] In this technology, glucose epimerase activity is defined as the amount of enzyme that produces 1 nmol of glucose per minute when treated with a 10 mmol / L mannose solution (glucose epimerase 1 and 2: 0.5 mol / L acetate buffer, pH 6.0; glucose epimerase 3-5: 50 mM phosphate buffer, pH 8.0) as a substrate at 37°C, with 1 unit (1 U) being defined.

[0107] (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.

[0108] 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.

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

[0110] The recovered mannose 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.

[0111] 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 glucose in food and beverages according to this technology, include a step of applying glucose 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.

[0112] (1) Raw Material Preparation Process The raw material preparation process is the process of preparing glucose-containing food or 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 or beverage. In this technology, raw materials containing glucose (glucose-containing raw materials) are particularly preferred. Furthermore, the form of the raw materials is not particularly limited, and examples include liquid, powder, slurry, etc.

[0113] (2) Enzyme Action Process The enzyme action process is a process in which glucose epimerase related to this technology is applied to glucose-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 mannose production method described above, so the explanation is omitted here.

[0114] (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 mannose production method described above, so the explanation is omitted here.

[0115] (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.

[0116] 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.

[0117] 8. Food and beverages: Food and beverages relating to this technology are characterized by being obtained using the glucose epimerase relating to this technology. Details of the glucose epimerase relating to this technology are as described above and will therefore not be explained here.

[0118] (1) Amount of glucose contained in food and beverages The amount of glucose contained in food and beverages used in this technology is not particularly limited, but it is preferable that it is reduced compared to the glucose content of food and beverages produced without using the glucose epimerase related to this technology. Specifically, the glucose content of 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 glucose content of food and beverages produced without using the glucose epimerase related to this technology.

[0119] The glucose reduction rate of food and beverages produced using this technology compared to food and beverages produced without using glucose epimerase is, for example, 2% or more, preferably 3% or more, more preferably 4% or more, even more preferably 5% or more, 10% or more, 15% or more, or 20% or more.

[0120] (2) Amount of mannose contained in food and beverages The amount of mannose contained in food and beverages used in this technology is not particularly limited, but it is preferable that it is increased compared to the mannose content in food and beverages produced without using the glucose epimerase according to this technology. Specifically, the mannose content 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 mannose content in food and beverages produced without using the glucose epimerase according to this technology.

[0121] (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.

[0122] 9. Enzyme preparations for mannose production and / or glucose reduction, glucose epimerase enzyme preparations The enzyme preparations for mannose production and / or glucose reduction related to this technology are enzyme preparations containing glucose epimerase related to this technology. Furthermore, the glucose epimerase enzyme preparations related to this technology are enzyme preparations containing any of the polypeptides shown in (1) to (3) above as active ingredients. That is, the glucose epimerase enzyme preparations related to this technology are enzyme preparations containing the glucose epimerase mentioned above. Details of the glucose epimerase related to this technology and the polypeptides shown in (1) to (3) are as described above and will not be explained here.

[0123] (1) Glucose epimerase content The glucose epimerase content in the enzyme preparation for mannose production and / or glucose reduction related to this technology, and in the glucose epimerase enzyme preparation, can be freely set as long as the effects of this technology are not impaired. The lower limit of the glucose 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 glucose in the composition to be acted upon (e.g., food and beverages).

[0124] The upper limit of the glucose 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 glucose in the composition to be acted upon (e.g., food and beverages).

[0125] (2) Other Components The enzyme preparations for mannose production and / or glucose reduction, and glucose epimerase enzyme preparations related to this technology may be used in combination with other components, as long as the action and effects of this technology are not impaired. Other components may include, for example, excipients commonly used in formulation (starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol, etc.), buffers (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 residues (components derived from the culture medium, contaminating proteins, bacterial components, etc.) produced in the glucose epimerase production method mentioned above. Furthermore, known or future-discovered functional components can be used in combination as appropriate for the purpose.

[0126] (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.).

[0127] 10. Method for producing talose Among the glucose epimerases related to this technology, there are enzymes that can produce talose using D-galactose (Gal) as a substrate, and therefore it is possible to produce talose using such glucose epimerases. Specifically, glucose epimerase 3 is an example of an enzyme that can use D-galactose (Gal) as a substrate.

[0128] The method for producing talose includes a step of reacting D-galactose with glucose epimerase related 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 carried out as needed. Note that the enzyme reaction step, raw material preparation step, enzyme inactivation step, and recovery step in the method for producing talose are the same as the enzyme reaction step, raw material preparation step, enzyme inactivation step, and recovery step in the method for producing mannose described above, so a detailed explanation is omitted here.

[0129] 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.

[0130] 1. Selection of Microorganisms: To find novel glucose epimerases, a screening was conducted using glucose epimerase activity as an indicator from a library of bacterial strains. As a result, glucose epimerase 1 from a Bacteroidota phylum microorganism, glucose epimerase 2 from a Spirosoma genus microorganism, glucose epimerase 3 from a Lewinella genus microorganism, glucose epimerase 4 from a Spirochete genus microorganism, and glucose epimerase 5 from an unknown microorganism were selected as promising candidates.

[0131] 2. Sequence Verification: The amino acid sequences (SEQ ID NOs: 1-5) of glucose epimerases 1-5 were determined from the genomic DNA information obtained by genomic DNA analysis using a next-generation sequencer.

[0132] 3. Construction of the Expression System: Synthetic nucleic acids obtained by adding His-tags to the selected amino acid sequences (SEQ ID NOs: 1-5) were inserted into the cloning site of the expression vector to construct a glucose 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 sequences were incorporated.

[0133] 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.

[0134] 5. Purification of glucose 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 glucose 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. Glucose epimerase activity (epimerylation of mannose) was confirmed in a portion of each obtained fraction using the method described later, and the fraction with activity 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 glucose epimerase 1 was 49.9 kDa, glucose epimerase 2 was 49.1 kDa, glucose epimerase 3 was 54.6 kDa (51.7 kDa when the signal sequence was removed), glucose epimerase 4 was 49.5 kDa, and glucose epimerase 5 was 49.4 kDa.

[0135] 6. To measure glucose epimerase activity, 10 μL of either a 50 mM acetate buffer (pH 6.0) solution containing 15 mM mannose (glucose epimerase 1 and 2) or a 50 mM phosphate buffer (pH 8.0) solution (glucose epimerase 3-5) was mixed with 5 μL of enzyme solution. The mixture was reacted at 37°C for 60 minutes. 300 μL of the color developer from a glucose assay kit (Fujifilm Wako Pure Chemical Industries, Ltd. "Glucose Assay Kit-WST") was added to 2 μL of the reaction mixture, and the mixture was reacted at 37°C for 5 minutes. The absorbance at 505 nm was then measured. The presence of glucose epimerase activity was confirmed by observing glucose production. Specifically, the enzyme activity that promotes the epimerization of 1 nmol of mannose per minute was defined as 1 unit (U), and the enzyme activity value was measured accordingly.

[0136] 7. Characterization of glucose epimerase (1) Optimal pH The pH of the substrate solution was adjusted to pH 3, 4, 5, 6, 7, 8, 9, or 10 with 50 mM acetate buffer or 50 mM Tris-HCl buffer. The activity was measured in the same manner as the activity measurement method described above, except that the enzyme reaction was carried out at each pH. The activity at the pH condition showing the maximum activity of the purified enzyme was set to 100%, and the relative amount of activity at each pH condition was calculated as relative activity (%). The results are shown in Figure 2. As shown in Figure 2, the optimal pH for glucose epimerase 1 was pH 5.0 to 6.0, the optimal pH for glucose epimerase 2 was pH 4.5 to 6.0, and the optimal pH for glucose epimerases 3 and 4 was pH 7.0 to 8.5.

[0137] (2) The activity was measured in the same manner as the activity measurement method described above, except that the optimal enzyme reaction temperature was changed to 10°C, 20°C, 30°C, 40°C, 50°C, or 60°C. The relative amount of 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 3. As shown in Figure 3, the optimal temperature for glucose epimerase 1 and 2 was 40°C, the optimal temperature for glucose epimerase 3 and 4 was 30-40°C, and the optimal temperature for glucose epimerase 5 was 40-50°C.

[0138] (3) The temperature-stable purified enzymes were subjected to temperature treatment by incubation at 4°C, 20°C, 30°C, 40°C, 50°C, or 60°C for 20 minutes. The enzyme activity values ​​of each enzyme solution before and after temperature treatment were measured using the activity measurement method described above. The relative value of the enzyme activity value after temperature treatment, with the enzyme activity value before temperature treatment set to 100%, was derived as the residual activity (%). The results are shown in Figure 4. As shown in Figure 4, glucose epimerases 1-3 and 5 showed high residual activity of 80% or more from 4°C to 50°C. In addition, glucose epimerase 4 showed high residual activity of 80% or more from 4°C to 20°C.

[0139] (4) Mannose production rate, talose production rate (glucose epimerase 3) 50 μL of a 50 mM acetate buffer (pH 6.0) solution (glucose epimerase 1 and 2) or a 50 mM phosphate buffer (pH 8.0) solution (glucose epimerase 3-5) 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 (glucose epimerase 1 and 2) or 72 hours (glucose epimerase 3-5). The mannose production rate was confirmed by HPLC analysis under the following conditions. The results are shown in Figure 5. The mannose production rate of glucose epimerase 1 was 23.5%, the mannose production rate of glucose epimerase 2 was 20.6%, the mannose production rate of glucose epimerase 3 was 19.2%, the mannose production rate of glucose epimerase 4 was 21.3%, and the mannose production rate of glucose epimerase 5 was 23.7%.

[0140] [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)

[0141] A 50 μL solution of 50 mM phosphate buffer (pH 8.0) containing 50 mM galactose as a substrate was mixed with 10 μL of crude enzyme solution. The mixture was reacted at 37°C for 72 hours, and the talose production rate was confirmed by HPLC analysis under the above conditions. The results are shown in Figure 5. The talose production rate of glucose epimerase 3 was 2.9%.

[0142] (5) 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. 5 μL of the reaction solution was subjected to TLC analysis to confirm the reaction catalyzed by glucose epimerase 1 or glucose epimerase 2. 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 6. As shown in Figure 6, glucose epimerases 1, 2, and 5 used D-mannose (Man) as a substrate, while not using D-galactose (Gal), D-fructose (Fru), D-xylose (Xyl), lactose (Lac), cellobiose (Cel), maltose (Mal), sucrose (Suc), or D-mannitol as substrates. Glucose epimerase 3 also used lactose (Lac), cellobiose (Cel), and maltose (Mal) as substrates, and produced talose in small amounts using D-galactose (Gal) as a substrate (see Figure 5). Glucose epimerase 4 used lactose (Lac) and cellobiose (Cel) as substrates, but not maltose (Mal). Furthermore, as shown in Figure 5, which shows the HPLC analysis results during the measurement of mannose production rates, glucose epimerases 1-5 also used D-glucose as a substrate.

[0143] Enzymes containing polypeptides with the amino acid sequences shown in SEQ ID NOs: 1-5 belong to the AGE family epimerases. However, many enzymes belonging to the AGE family epimerases do not act on glucose. Surprisingly, this technology demonstrated that enzymes containing polypeptides with the amino acid sequences shown in SEQ ID NOs: 1-5 do act on glucose. Furthermore, it was demonstrated that enzymes containing polypeptides with the amino acid sequences shown in SEQ ID NOs: 1, 2, and 5 exhibit high specificity for glucose.

[0144] 8. Application Considerations (1) Materials The materials used in the application considerations are shown in Table 1 below.

[0145]

[0146] (2) Method 50 μL of vegetable juice shown in Table 1 and 10 μL of glucose epimerase 1 enzyme solution were mixed and reacted at 37°C for 48 to 72 hours. The mannose production rate was confirmed by HPLC analysis under the same conditions as for (4) mannose production rate. Similarly, 50 μL of fruit juice shown in Table 1, adjusted to pH 6, and 10 μL of glucose epimerase 1 or glucose epimerase 2 enzyme solution were mixed, and the same reaction and analysis were performed. Furthermore, 50 μL of fruit juice shown in Table 1, adjusted to pH 8, and 10 μL of glucose epimerase 1 to 5 enzyme solution were mixed, and the same reaction and analysis were performed.

[0147] (3) Results The results are shown in Table 2 below.

[0148]

[0149] (4) Discussion As shown in Table 2, it was confirmed that the glucose in the beverage was reduced when glucose epimerases 1-5 were applied to the beverage.

[0150] 9. Using a 50 mM acetate buffer (pH 6.0) solution containing 15 mM to 750 mM mannose as the glucose epimerase activity substrate under high-concentration mannose conditions, the activity was measured in the same manner as the activity measurement method described above, and the relative value (%) of the enzyme activity was derived, with the enzyme activity value under conditions using 15 mM mannose as the substrate set to 100%.

[0151] The results are shown in Figure 7. As shown in Figure 7, glucose epimerase 1 and glucose epimerase 2 showed high activity even under higher mannose concentrations.

[0152] 10. Mannose production rate under high-concentration glucose conditions A 50 μL solution of 50 mM acetate buffer (pH 6.0) containing 500 mM glucose or 2.78 M (equivalent to 50% w / w) glucose (glucose epimerase 1 and 2) or a 50 mM phosphate buffer (pH 8.0) solution (glucose epimerase 3-5) was mixed with 10 μL of crude enzyme solution, and the reaction was carried out at 37°C for 48 to 96 hours. The mannose production rate was confirmed by HPLC analysis under the same conditions as described in (4) Mannose production rate above. The results are shown in Table 3.

[0153]

[0154] As shown in Table 3, even under high-concentration glucose conditions equivalent to 50% (w / w), the mannose production rates were 16.5% for glucose epimerase 1, 12.1% for glucose epimerase 2, 15.5% for glucose epimerase 3, 15.6% for glucose epimerase 4, and 15.7% for glucose epimerase 5. Both glucose epimerases were confirmed to be usable for mannose production using high-concentration glucose solutions as raw materials.

[0155] 11. Mannose production rate under high-concentration glucose and high-temperature conditions A 50 μL solution of 50 mM acetate buffer (pH 6.0) containing 2.78 M (equivalent to 50% w / w) glucose (glucose epimerase 1 and 2) or a 50 mM phosphate buffer (pH 8.0) solution (glucose epimerase 3-5) was mixed with 10 μL of crude enzyme solution, and the reaction was carried out at 50°C for 72 hours. The mannose production rate was confirmed by HPLC analysis under the same conditions as described in (4) above. The results are shown in Table 4.

[0156]

[0157] As shown in Table 4, even under high temperature conditions of 50°C and high glucose concentration equivalent to 50% (w / w), the mannose production rate of glucose epimerase 1 was 15.0%, glucose epimerase 2 was 10.3%, glucose epimerase 3 was 13.9%, glucose epimerase 4 was 14.2%, and glucose epimerase 5 was 16.7%. This means that even under higher temperature conditions of 50°C, the mannose production rate can be maintained at more than 85% of that under 37°C conditions. It was confirmed that both glucose epimerases can be used for mannose production using high-concentration glucose solutions as raw materials under higher temperature conditions.

Claims

1. Glucose epimerase comprising a polypeptide as shown in any of (1) to (3) below: (1) a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 to 5; (2) a polypeptide having glucose epimerase activity, wherein one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in any of SEQ ID NOs: 1 to 5; and (3) a polypeptide having glucose epimerase activity, wherein the amino acid sequence shown in any of SEQ ID NOs: 1 to 5 has a sequence identity of 90% or more to the amino acid sequence shown in any of SEQ ID NOs: 1 to 5.

2. Glucose epimerases comprising any of the following polypeptides (1) to (3) and derived from Bacteroidota microorganisms: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, (2) a polypeptide having glucose 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 glucose 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.

3. Glucose epimerases comprising a polypeptide as shown in any of (1) to (3) below, and derived from a microorganism of the genus Spirosoma: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, (2) a polypeptide having glucose epimerase activity, wherein one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 are substituted, added, inserted, deleted, or inverted, and (3) a polypeptide having glucose epimerase activity, wherein the amino acid sequence shown in SEQ ID NO: 2 has a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO:

2.

4. Glucose epimerases comprising a polypeptide as shown in any of (1) to (3) below, and derived from a microorganism of the genus Lewinella: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3, (2) a polypeptide having glucose epimerase activity, wherein one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 3 are substituted, added, inserted, deleted, or inverted, and (3) a polypeptide having glucose epimerase activity, wherein the amino acid sequence shown in SEQ ID NO: 3 has a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO:

3.

5. Glucose epimerases comprising a polypeptide as described in any of (1) to (3) below, and derived from a microorganism of the genus Sphaerochaeta: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, (2) a polypeptide having glucose epimerase activity, wherein one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 4 are substituted, added, inserted, deleted, or inverted, and (3) a polypeptide having glucose epimerase activity, wherein the amino acid sequence shown in SEQ ID NO: 4 has a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO:

4.

6. DNA comprising any of the following polynucleotides (1) to (3): (1) a polynucleotide encoding glucose epimerase as described in claim 1; (2) a polynucleotide consisting of any of the base sequences shown in SEQ ID NOs: 6 to 10; and (3) a polynucleotide encoding a polypeptide having glucose epimerase activity, comprising a sequence equivalent to any of the base sequences shown in SEQ ID NOs: 6 to 10.

7. An expression cassette or recombinant vector comprising the DNA described in claim 6.

8. A transformant obtained by transforming a host with the expression cassette or recombinant vector described in claim 7.

9. A method for producing glucose epimerase, comprising: a culture step of culturing one or more microorganisms selected from the phylum Bacteroidota, the genus Spirosoma, the genus Lewinella, and the genus Sphaerochaeta, or the transformant described in claim 8; and a recovery step of recovering the glucose epimerase described in claim 1 from the culture obtained in the culture step.

10. A method for producing mannose, comprising the step of reacting glucose with the glucose epimerase described in claim 1.

11. A method for producing food and beverages, comprising the step of reacting glucose-containing food and beverages and / or raw materials thereof with the glucose epimerase described in claim 1.

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

13. Food and beverages obtained using the glucose epimerase described in claim 1.

14. An enzyme preparation for mannose production and / or glucose reduction, comprising the glucose epimerase described in claim 1.

15. Use of polypeptides as glucose epimerases as shown in any of the following (1) to (3): (1) a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 to 5; (2) a polypeptide having glucose epimerase activity, wherein one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in any of SEQ ID NOs: 1 to 5; and (3) a polypeptide having glucose epimerase activity, wherein the amino acid sequence shown in any of SEQ ID NOs: 1 to 5 has a sequence identity of 90% or more to the amino acid sequence shown in any of SEQ ID NOs: 1 to 5.

16. Use of polypeptides shown in any of (1) to (3) below for the production of mannose: (1) a polypeptide consisting of an amino acid sequence shown in any of SEQ ID NOs: 1 to 5; (2) a polypeptide having glucose epimerase activity, wherein one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in any of SEQ ID NOs: 1 to 5; and (3) a polypeptide having glucose epimerase activity, wherein the amino acid sequence shown in any of SEQ ID NOs: 1 to 5 has a sequence identity of 90% or more to the amino acid sequence shown in any of SEQ ID NOs: 1 to 5.

17. Use of polypeptides shown in any of the following (1) to (3) for reducing glucose in food and beverages: (1) a polypeptide consisting of an amino acid sequence shown in any of SEQ ID NOs: 1 to 5; (2) a polypeptide having glucose epimerase activity, in which one or more amino acid residues are substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in any of SEQ ID NOs: 1 to 5; and (3) a polypeptide having glucose epimerase activity, in which the amino acid sequence shown in any of SEQ ID NOs: 1 to 5 has a sequence identity of 90% or more to the amino acid sequence shown in any of SEQ ID NOs: 1 to 5.

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