Method for producing glucuronic acid

Abstract

Provided is a method for producing glucuronic acid or a glucuronic acid derivative more easily, at lower cost, and with reduced environmental impact in comparison to existing methods.　The method for producing glucuronic acid includes a step for causing a flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity to act on glucose in the presence of a mediator to generate glucuronic acid.

Description

Technical Field

[0001] The present invention relates to a method for producing glucuronic acid using flavin - bound glucose dehydrogenase, a method for producing a glucuronic acid derivative, and a catalyst for producing glucuronic acid or a glucuronic acid derivative.

Background Art

[0002] Glucuronic acid (chemical formula: C 6 H 10 O 7 ) is a typical uronic acid derived from glucose. Glucuronic acid has a detoxifying effect such as conjugating harmful substances in the body and excreting them in urine. Currently in Japan, glucuronic acid and its intramolecular ester glucuronolactone are used in pharmaceutical or quasi - drug beverage products.

[0003] As existing methods for producing glucuronic acid, methods using enzymes that oxidize the hydroxymethyl group at the 6 - position of glucose, such as glucose oxidase modified from galactose oxidase (Patent Document 1), aldehyde dehydrogenase (Patent Document 2), and alcohol dehydrogenase (Patent Document 3), have been reported. However, these enzymes have problems such as low specific activity of the enzyme and low oxidation specificity for the hydroxymethyl group of glucose, and produce glucose oxides such as gluconic acid in addition to glucuronic acid.

[0004] Also, as methods for producing glucuronic acid using enzymes with saccharides other than glucose as substrates, a method for producing glucuronic acid by reacting α - 1,4 - polyglucuronic acid prepared by oxidizing starch with a bacterium of the genus Paenibacillus or a glycoside - binding hydrolase derived from the same bacterium (Patent Document 4), a method for producing glucuronic acid using an enzyme that hydrolyzes oxidized trehalose as a substrate (Patent Document 5), and a method for producing glucuronic acid using myo - inositol oxygenase with myo - inositol as a substrate (Patent Document 6) have been reported. However, problems with these production methods include complicated substrate preparation and high substrate prices.

[0005] In addition, as a method for producing glucuronic acid from glucose by fermentation, a production method using Pseudogluconobacter saccharoketogenes Rh47-3 strain (Patent Document 7) has been reported. However, compared with the enzymatic method, the fermentation method has a problem that impurities increase in the culture solution, resulting in a lower purity of the target product, glucuronic acid, and requiring advanced purification. Also, as a method for producing glucuronic acid without using an enzyme, a method (Non-Patent Document 1) is known in which starch is converted to oxidized starch with nitric acid and then hydrolyzed with sulfuric acid to produce glucuronic acid. However, this method has a problem that a large amount of reagents with a high environmental load such as nitric acid and sulfuric acid must be used.

[0006] When glucuronic acid binds to other compounds via a glycosidic bond, that is, glucuronidation occurs, it is known that the resulting glucuronide has increased water solubility and enhanced physiological activity compared to other compounds. To utilize this property, methods for glucuronidating compounds have been studied so far. As an enzymatic method, for example, a method has been reported in which UDP-glucuronosyltransferase present in vivo is used to transfer glucuronic acid to an arbitrary substrate for glucuronidation (Non-Patent Document 2). Also, as a chemical synthesis method, for example, a method (Non-Patent Document 3) has been reported in which 2,3,4-tri-o-acetyl-α-D-glucuronic acid methyl is used as a glucuronic acid donor and reacted with trimethylsilyl triflate as a Lewis acid catalyst to impart glucuronic acid for glucuronidation to a target having a hydroxyl group to be glucuronidated. However, problems with both methods include the high cost of the reagents used in the reaction, the low yield of the target glucuronide, and the need for purification for removing by-products and the like after the reaction.

[0007] On the one hand, flavin - bound glucose dehydrogenase (flavin - bound GDH, EC 1.1.5.9) is an enzyme that uses flavin as a coenzyme and catalyzes the reaction of dehydrogenating (oxidizing) the hydroxy group at the 1 - position of glucose. Flavin - bound GDH derived from the genus Aspergillus is not affected by dissolved oxygen, has low reactivity towards maltose and galactose, and has high substrate specificity for glucose, so it is used for measuring blood glucose concentration.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Non - Patent Documents

[0009]

Non - Patent Document 1

Non - Patent Document 2

Non - Patent Document 3

Summary of the Invention

[0010] An object of the present invention is to provide a method for producing glucuronic acid or a glucuronic acid derivative that is simple, low-cost, and has a reduced environmental load as compared with existing methods. [Means for Solving the Problems]

[0011] The present inventors intensively studied a method for directly producing glucuronic acid from glucose, which is an inexpensive raw material. As a result, among flavin-bound GDHs, which are enzymes that oxidize glucose to glucono-1,5-lactone, there are those that have specificity for the oxidation of the hydroxymethyl group at the 6-position of glucose. When an enzyme having the glucose-6-dehydrogenase activity is allowed to act on glucose, it was found that the hydroxymethyl group at the 6-position of glucose is oxidized to specifically produce glucuronic acid. Further, when the enzyme is allowed to act on a glucose derivative such as a glucoside, it was found that the hydroxymethyl group at the 6-position of the glucose skeleton is oxidized to specifically produce a glucuronic acid derivative.

[0012] That is, the present invention relates to the following [1] to

[11] . [1] A method for producing glucuronic acid, comprising a step of allowing a flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity to act on glucose in the presence of a mediator to produce glucuronic acid. [2] A method for producing a glucuronic acid derivative, comprising a step of allowing a flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity to act on a glucose derivative in the presence of a mediator to produce a glucuronic acid derivative. [3] The method for producing a glucuronic acid derivative according to [2], wherein the glucose derivative is an amino sugar or an N-acetylated product thereof, a glucoside, or a glucose analog. [4] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of [1] to [3], wherein the flavin-binding glucose dehydrogenase is any one of the following proteins (i) to (iii): (i) A protein having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 (ii) A protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose-6-dehydrogenase activity (iii) A protein having an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose-6-dehydrogenase activity. [5] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of [1] to [4], wherein the flavin-binding glucose dehydrogenase has the following properties (1) to (8): (1) Action: Catalyzes the reaction of dehydrogenating (oxidizing) the hydroxymethyl group at the 6-position of glucose using flavin as a coenzyme (2) Solubility: Water-soluble (3) pH stability: Stable at least between pH 5.5 and 8.7 (4) Thermal stability: Stable at least at 35 °C (5) Substrate specificity: When the reactivity with glucose is taken as 100%, the reactivity with maltose, xylose, and galactose is 2.0% or less (6) Km value (for glucose): 30 mM or more (7) Molecular weight: 64 to 66 kDa (calculated from the amino acid sequence after signal removal) (8) Glucose oxidase activity: Not detectable. [6] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of [1] to [5], wherein the flavin - bound glucose dehydrogenase is derived from a microorganism belonging to the genus Colletotrichum, Glomerella, Diaporthe, Kusicidea, Acremonium, Lasiosphaeris, Fusarium or Phaeomoniopsis. [7] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of [1] to [6], wherein a recombinant microorganism into which a gene encoding a flavin - bound glucose dehydrogenase is introduced is used. [8] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to [7], wherein the gene encoding the flavin - bound glucose dehydrogenase is a gene consisting of any one of the following DNAs (a) to (e): (a) DNA having the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 (b) DNA having a base sequence in which one to several bases are deleted, substituted or added in the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37, and encoding a protein having glucose - 6 - dehydrogenase activity (c) DNA having a base sequence having 80% or more sequence identity to the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37, and encoding a protein having glucose - 6 - dehydrogenase activity (d) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37, and encoding a protein having glucose - 6 - dehydrogenase activity (e) DNA encoding the following protein (i), (ii) or (iii) (i) A protein having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 (ii) A protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, and having glucose-6-dehydrogenase activity (iii) A protein having an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, and having glucose-6-dehydrogenase activity. [9] Furthermore, a method for producing glucuronic acid or a method for producing a glucuronic acid derivative according to any one of [1] to [8], which comprises allowing an oxidase to act thereon.

[10] A flavin-binding glucose dehydrogenase having glucose-6-dehydrogenase activity, which is any one of the following proteins (i) to (iii): (i) A protein having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 (ii) A protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, and having glucose-6-dehydrogenase activity (iii) A protein having an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, and having glucose-6-dehydrogenase activity.

[11] A catalyst for producing glucuronic acid or a glucuronic acid derivative, which comprises a flavin-binding glucose dehydrogenase protein having glucose-6-dehydrogenase activity [Advantages of the Invention]

[0013] According to the present invention, glucuronic acid can be specifically and directly produced from glucose, which is an inexpensive raw material. Therefore, it is simpler compared with the existing methods, and the manufacturing cost is significantly reduced. In addition, since the reaction proceeds at normal temperature and pressure without using strong acids such as nitric acid and sulfuric acid, the danger during manufacturing can be avoided, and the environmental load can also be suppressed. Furthermore, since glucuronic acid derivatives can be specifically and directly produced from glucose derivatives, it is simpler compared with the existing glucuronidation methods, and the manufacturing cost is reduced. In addition, effects such as enhancing the water solubility of glucose derivatives can be expected by the oxidation of hydroxymethyl groups. [Brief Description of the Drawings]

[0014]

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Mode for Carrying Out the Invention

[0015] As used herein, a flavin-linked glucose dehydrogenase having glucose-6-dehydrogenase activity (hereinafter sometimes referred to as "flavin-linked GDH") refers to an enzyme that uses flavin as a coenzyme and catalyzes the reaction of dehydrogenating (oxidizing) the hydroxymethyl group at the 6-position of glucose. The flavin-linked GDH of the present invention selectively acts on the 6-position of glucose and specifically oxidizes the hydroxymethyl group at the 6-position of glucose to a carboxyl group. Therefore, when the enzyme is allowed to act on glucose, glucuronic acid is specifically produced. Further, when the enzyme is allowed to act on a glucose derivative such as a glucoside, the hydroxymethyl group at the 6-position of the glucose skeleton is specifically oxidized, so that a glucuronic acid derivative is specifically produced from the glucose derivative. Glucose-6-dehydrogenase activity can be confirmed by allowing glucose-6-dehydrogenase to act on glucose, analyzing the reaction product by thin-layer chromatography or HPLC, and comparing it with a glucuronic acid standard in which the 6-position of glucose is oxidized. The flavin-linked GDH of the present invention substantially does not have glucose-1-dehydrogenase activity and substantially does not produce gluconic acid from the substrate glucose. Here, "substantially" means that the production of gluconic acid cannot be confirmed as a result of thin-layer chromatography or HPLC analysis of the reaction product.

[0016] The flavin-binding GDH of the present invention is not particularly limited as long as it is an enzyme having glucose-6-dehydrogenase activity, but is preferably any of the following proteins (i) to (iii). (i) A protein having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 (ii) A protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose-6-dehydrogenase activity (iii) A protein having an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose-6-dehydrogenase activity

[0017] In the amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, the number of amino acid residue deletions, substitutions or insertions is not limited as long as it exhibits enzyme activity equivalent to that of a protein having the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, but is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 8.

[0018] In this specification, the sequence identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably 99% or more. The percentage of such sequence identity can be calculated using publicly available or commercially available software having an algorithm that compares a reference sequence as a query sequence. As an example, BLAST, FASTA, or GENETYX (Genetics Co., Ltd.) etc. can be used.

[0019] The amino acid sequences represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 are based on the genomic information of Colletotrichum plurivorum MAFF305790, Fungus_F5126, Colletotrichum_sp., Colletotrichum gloeosporioides, Colletotrichum orbiculare, Colletotrichum tofieldiae, Colletotrichum godetiae MAFF240289, Glomerella sp. RD057037, Diaporthe helianthi, Khuskia oryzae, Acremonium strictum, Khuskia oryzae, Lasiosphaeris hirsute, Diaporthaceae sp., Colletotrichum tanaceti, Fusarium langsethiae, Phialemoniopsis curvata, respectively, in that order. Further, SEQ ID NOs: 36 and 38 are sequences in which the sequences presumed to be the secretion signals of SEQ ID NOs: 32 and 34 are replaced with the signal sequence of GDH derived from Aspergillus oryzae. The amino acid sequence represented by SEQ ID NO: 4 or 6 is identical to SEQ ID NO: 2 described in Patent No. 6455714 for known amino acid sequences and SEQ ID NO: 1 described in Patent No. 5435180. The amino acid sequences represented by SEQ ID NO: 8, 10, 12, 18, 20, 22, 24, 26, 28, 30, 32, and 34 are amino acid sequences registered in publicly known databases, but there are no reported examples of proteins having these amino acid sequences exhibiting glucose-6-dehydrogenase activity.

[0020] The flavin-binding GDH of the present invention preferably has the following properties (1) to (8). Examples of flavins include flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), with FAD being preferred. (1) Action: Catalyzes the dehydrogenation (oxidation) reaction of the hydroxymethyl group at the 6-position of glucose, using flavin as a coenzyme. (2) Solubility: Water-soluble (3) pH stability: Stable at least between pH 5.5 and 8.6 This enzyme has a residual enzyme activity of 80% or more after treatment at 30°C for 1 hour, provided that the pH is at least between pH 5.5 and 8.6. The pH stability is preferably pH 4.3 - 9.3, pH 5.5 - 8.7, pH 3.2 - 9.3, pH 5.5 - 9.3, pH 4.4 - 9.3, pH 4.0 - 9.6, pH 4.4 - 9.3, pH 5.0 - 9.3, pH 3.3 - 9.6, pH 5.5 - 8.6, pH 4.3 - 9.6, pH 5.0 - 9.6, pH 4.0 - 8.6, pH 4.9 - 8.7, pH 3.3 - 9.9, pH 4.4 - 9.8, pH 4.0 - 8.8, pH 4.4 - 9.8, pH 4.0 - 9.6. Note that even at the same pH, the residual activity may vary depending on the type of buffer solution. (4) Thermal stability: Stable at least at 35°C This enzyme has a residual enzyme activity of 80% or more after treatment for 60 minutes in 100 mM potassium phosphate buffer (pH 6.0, 7.0, or 8.0) or 100 mM Tris-HCl buffer (pH 8.0), provided that the temperature is at least 35°C. Preferably, it is stable up to 40°C, up to 45°C, or up to 50°C. (5) Substrate specificity: When the activity against glucose is set to 100%, the activity against maltose, xylose, and galactose is 2.0% or less. This enzyme has low activity against maltose, xylose, and galactose and high substrate specificity for glucose. When the activity against 50 mM D-glucose is set to 100%, the activity against 50 mM maltose, D-xylose, and D-galactose is 2.0% or less, preferably 0.3% or less, 0.9% or less, or 0.2% or less. (6) Km value (for glucose): 30 mM or more The Km value for D-glucose is preferably 150 - 300 mM, 50 - 120 mM, or 30 - 80 mM. (7) Molecular weight: 64 - 66 kDa (calculated from the amino acid sequence after signal removal) The molecular weight of this enzyme is calculated from the amino acid sequence in the form where the signal peptide sequence of the enzyme is predicted by the signal sequence prediction site (SignalP-5.0, http: / / www.cbs.dtu.dk / services / SignalP / ) and the predicted signal part is removed. (8) Glucose oxidase activity: Not detectable

[0021] As described above, the flavin-binding GDH of the present invention acts specifically on the 6-position of glucose, so it has low activity against xylose. Therefore, this enzyme can be used for the measurement of glucose and is useful as an enzyme for a biosensor for glucose measurement.

[0022] As the microorganism from which the flavin-binding GDH of the present invention is derived, microorganisms belonging to the genus Colletotrichum (e.g., Colletotrichum plurivorum, Colletotrichum sp. (RD056779), Colletotrichum gloeosporioides, Colletotrichum orbiculare, Colletotrichum tofieldiae, Colletotrichum godetiae, Colletotrichum tanaceti), the genus Glomerella (e.g., Glomerella sp. RD057037), the genus Diaporthe (e.g., Diaporthe helianthi), the genus Khuskia (e.g., Khuskia oryzae), the genus Acremonium (e.g., Acremonium strictum), the genus Lasiosphaeris (e.g., Lasiosphaeris hirsute), the genus Fusarium (e.g., Fusarium langsethiae), the genus Phialemoniopsis (e.g., Phialemoniopsis curvata), etc. can be mentioned. The flavin-binding GDH of the present invention may be any of an enzyme derived from the above microorganism (wild strain or mutant strain), a recombinant enzyme obtained by a genetic engineering method using the gene encoding the flavin-binding GDH of the present invention, and a synthetic enzyme obtained by chemical synthesis. Preferably, it is a recombinant enzyme.

[0023] The gene encoding the flavin-binding GDH of the present invention is preferably a gene consisting of any of the following DNAs (a) to (e). (a) DNA having the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 (b) DNA having a base sequence in which 1 to several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 and encoding a protein having glucose-6-dehydrogenase activity DNA encoding a protein having a base sequence with at least 80% sequence identity to the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 and having glucose-6-dehydrogenase activity DNA encoding a protein having glucose-6-dehydrogenase activity and hybridizing under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 (e) DNA encoding the following protein (i), (ii) or (iii) (i) A protein having the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 (ii) A protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose-6-dehydrogenase activity (iii) A protein having an amino acid sequence with at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose-6-dehydrogenase activity

[0024] In the nucleotide sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37, the one to several nucleotides, the one to several nucleotides are preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2. In addition, the deletion of a nucleotide means the deletion or disappearance of a nucleotide, the substitution of a nucleotide means that a nucleotide is replaced by another nucleotide, and the addition of a nucleotide means that a nucleotide is added. "Addition" includes the addition of nucleotides to one or both ends of the sequence and the insertion of another nucleotide between the nucleotides in the sequence.

[0025] In this specification, the sequence identity of the nucleotide sequence is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more. The sequence identity of the nucleotide sequence can be determined using the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 1993, 90: 5873-5877). Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed (J. Mol. Biol., 1990, 215, p. 403-410). Alternatively, the homology analysis (Search homology) program of the genetic information processing software Genetyx may be used. Specific methods of these analysis methods are known (see www.ncbi.nlm.nih.gov).

[0026] As used herein, stringent conditions refer to conditions under which highly identical nucleotide sequences hybridize to each other, while nucleotide sequences with lower identity do not hybridize. The "stringent conditions" can be appropriately changed depending on the desired level of identity. The higher the stringency, the more likely only sequences with higher identity will hybridize. For example, as stringent conditions, those described in Molecular Cloning: A Laboratory Manual (Second Edition, J. Sambrook et al., 1989) can be mentioned. That is, conditions such as incubating with a probe at 65°C for 8 to 16 hours in a solution containing 6×SSC (composition of 1×SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5× Denhardt's, and 100 mg / mL herring sperm DNA for hybridization can be mentioned.

[0027] The preparation of flavin-binding GDH using the gene encoding flavin-binding GDH of the present invention can be achieved, for example, by introducing an expression vector containing the gene encoding flavin-binding GDH of the present invention into a host cell such as a microorganism and culturing the resulting transformant to produce the flavin-binding GDH of the present invention. The transformant may retain the gene encoding flavin-binding GDH of the present invention in the form of a vector, or may retain the gene in the genome. The gene encoding flavin-binding GDH of the present invention can be prepared in an isolated state using any method commonly used in the art, such as the PCR method, with reference to the gene sequence information disclosed herein.

[0028] The type of vector is not particularly limited, and examples include vectors commonly used for protein production, such as plasmids, cosmids, phages, viruses, YACs, BACs, etc. Among them, plasmid vectors are preferred, and commercially available plasmid vectors for protein expression, such as pET and pBIC, can be preferably used. The procedure for introducing a gene into a plasmid vector is well known in the art.

[0029] Examples of host microorganisms to be transformed for expressing the flavin-binding GDH of the present invention include bacteria, yeasts, and filamentous fungi belonging to the genus Escherichia, Rhodococcus, Streptomyces, Bacillus, Brevibacillus, Staphylococcus, Enterococcus, Listeria, Saccharomyces, Pichia, Shizosaccharomyces, Kluyveromyces, Aspergillus, Penicillium, and Trichoderma.

[0030] The medium and culture conditions used for culturing the transformant can be appropriately selected by those skilled in the art according to the type of the transformant. For example, it can be carried out under aerobic conditions such as aeration stirring and shaking using a medium containing a carbon source, an inorganic nitrogen source or an organic nitrogen source, inorganic salts, and other necessary organic micronutrients that can be assimilated by the microorganism. The medium may be any of a synthetic medium, a natural medium, a semi-synthetic medium, or a commercially available medium. The medium is preferably a liquid medium. The pH of the medium is preferably in the range of, for example, pH 5 to pH 9, and the pH may be adjusted during the culture in consideration of productivity. For example, the culture temperature is preferably in the range of 10°C to 40°C, and the culture period is preferably in the range of 2 days to 14 days.

[0031] After culturing, the culture can be used, but preferably, it is used after performing a separation operation such as centrifugation to obtain a culture supernatant. Alternatively, microbial cells are obtained, the microbial cells are disrupted by any method, and after obtaining a supernatant from the disrupted solution, it is used. Here, the culture may be a culture solution, microbial cells, or a processed product thereof (lyophilized cells, acetone-dried cells, etc.). It may also be an immobilized enzyme or immobilized cells immobilized by any method. For the purification of the flavin-binding GDH of the present invention produced by the transformant, known purification methods can be used. For example, a purified enzyme can be obtained by combining purification operations such as ultrafiltration, salting out, solvent precipitation, heat treatment, dialysis, ion exchange chromatography, hydrophobic chromatography, gel filtration, affinity chromatography, etc.

[0032] The method for producing glucuronic acid of the present invention has a step of reacting glucose with the flavin-binding GDH of the present invention in the presence of a mediator to produce glucuronic acid. In this step, the form of the flavin-binding GDH is not particularly limited and may be a crude enzyme, a purified enzyme, or a microorganism containing the flavin-binding GDH. The microorganism containing the flavin-binding GDH is preferably a recombinant microorganism into which a gene encoding the flavin-binding GDH has been introduced. The microorganism containing the flavin-binding GDH may be alive or dead, and also includes processed products of the microbial cells as described above. The production of glucuronic acid is usually carried out in an aqueous medium. Examples of the aqueous medium include water, buffer solutions, monohydric alcohols, dihydric alcohols, etc.

[0033] The glucose as the substrate is usually D-glucose. In this step, the substrate concentration is preferably about 10 mM to 2 M.

[0034] As the mediator used in this step, a chemical substance having excellent electron donating and accepting ability can be used. The mediator is also referred to as an electron transfer agent, an electron acceptor, or a redox mediator. Examples of mediators include osmium compounds (e.g., osmium(II)-2,2'-bipyridine complex), quinone compounds (e.g., benzoquinone, 1,4-naphthoquinone, vitamin K3 (menadione)), phenolic compounds (tert-butylhydroquinone, hydroquinone, 4-aminophenol, butylhydroxyanisole, eugenol, catechol, guaiacol, pyrogallol, vanillin, n-propyl gallate), phenazine compounds (e.g., phenazine methosulfate, 1-methoxy-5-methylphenazinium methyl sulfate, methylene blue), ferricyanides (e.g., potassium ferricyanide), flavonoids (quercetin dihydrate, hesperidin), etc. Among them, tert-butylhydroquinone and 1-methoxy-5-methylphenazinium methyl sulfate are preferred. The amount of the mediator used can be appropriately set depending on its type, but usually, about 0.5 mM to 50 mM is preferred.

[0035] The conditions for allowing the flavin-bound GDH of the present invention to act on glucose are not particularly limited as long as the flavin-bound GDH is not inactivated. This step proceeds under normal temperature and pressure. Also, since the reaction proceeds under neutral to alkaline conditions, there is no need to use a strong acid during the reaction process. The reaction temperature is usually 10°C to 60°C, preferably 20°C to 40°C, and the reaction time is usually 30 minutes to 72 hours, preferably 1 hour to 48 hours, more preferably 3 hours to 24 hours. Also, the reaction pH is preferably pH 5.0 to pH 9.0.

[0036] The amount of the flavin-bound GDH of the present invention used is preferably about 1 U / mL to 50 U / mL as the final concentration.

[0037] In this step, from the viewpoint of re-oxidizing the mediator, it is preferable to further allow an oxidase to act. Examples of the oxidase include phenol oxidases such as laccase (EC 1.10.3.2) and peroxidase (EC 1.11.1.7). From the perspective of removing reactive oxygen species in the reaction system, it is preferable to use catalase (EC 1.11.1.6). The usage amount of these enzymes is preferably about 0.25 U / mL to 500 U / mL as the final concentration.

[0038] In this way, glucuronic acid is specifically generated from glucose as the substrate. On the other hand, gluconic acid is not substantially generated. Substantially means that when analyzing the reaction products, the generation of gluconic acid cannot be confirmed even by methods such as gluconic acid measurement kits, thin-layer chromatography, and HPLC. Therefore, according to this step, the purification load of glucuronic acid can be reduced. When isolating and purifying glucuronic acid, known isolation and purification methods can be used. The generated glucuronic acid can be used in pharmaceuticals, quasi-drugs, foods, etc. in the state of glucuronic acid or its intramolecular ester glucuronolactone.

[0039] The method for producing the glucuronic acid derivative of the present invention includes a step of reacting a glucose derivative with the flavin-bound GDH of the present invention in the presence of a mediator to produce a glucuronic acid derivative. The flavin-bound GDH is the same as described above. The production of the glucuronic acid derivative is usually carried out in an aqueous medium such as water, buffer solution, monohydric alcohol, and dihydric alcohol.

[0040] In this specification, the glucose derivative is selected from glucose analogs in which the hydroxyl groups constituting glucose, such as amino sugars and their N-acetylated products, glucosides, and polyols, are substituted with hydrogen. Glucose is preferably D-glucose. Examples of amino sugars and their N-acetylated products include glucosamine, N-acetylglucosamine, etc. Glucoside is a general term for glycosides in which the hemiacetal hydroxyl group of glucose is ether-bonded to another compound, or glycosides in which glucose and a saturated hydrocarbon such as octane are thioether-bonded with a sulfur atom in between. Other compounds include aglycones, monosaccharides, disaccharides, polysaccharides with three or more sugars, etc. Aglycones are non-sugar moieties and examples include saturated hydrocarbons such as alcohols, phenols, phenylpropanoids, octanes, etc. Preferred glucosides are cellobiose, arbutin, piceid, methyl glucoside, octylthioglucoside. Glucose analogs include 1,5-anhydro-D-glucitol, 2-deoxy-D-glucose, etc.

[0041] In this step, the substrate concentration is preferably about 10 mM to 1000 mM.

[0042] The mediator used in this step is the same as those described above. The amount of mediator used is preferably about 0.5 mM to 50 mM.

[0043] The conditions for reacting the flavin-bound GDH of the present invention with a glucose derivative are not particularly limited as long as the flavin-bound GDH is not inactivated. This step also proceeds at normal temperature and pressure, and the reaction proceeds under neutral to alkaline conditions. The reaction temperature is usually 10°C to 60°C, preferably 20°C to 40°C, and the reaction time is usually 30 minutes to 72 hours, preferably 1 hour to 48 hours, more preferably 3 hours to 24 hours. Also, the reaction pH is preferably pH 5.0 to pH 9.0.

[0044] The amount of the flavin-bound GDH of the present invention used is preferably 1 U / mL to 50 U / mL as the final concentration.

[0045] Also in this step, from the viewpoint of re-oxidizing the mediator, it is preferable to further act on an oxidase. Examples of the oxidase include phenol oxidases such as laccase (EC 1.10.3.2), peroxidase (EC 1.11.1.7). Also, from the viewpoint of removing active oxygen, it is preferable to use catalase (EC 1.11.1.6). The usage amount of these enzymes is preferably about 0.25 U / mL to 500 U / mL as the final concentration.

[0046] In this way, the hydroxymethyl group at the 6-position of the glucose skeleton of the glucose derivative as the substrate is oxidized to a carboxyl group, and a glucuronic acid derivative is specifically produced. The production rate of glucuronide is almost 100% based on the analysis result by thin layer chromatography. The produced glucuronic acid derivative can be used as a reference substance for glucuronic acid conjugates produced in vivo, a raw material for pharmaceuticals, foods or cosmetics, a surfactant, etc.

[0047] The catalyst for producing glucuronic acid or a glucuronic acid derivative of the present invention contains a flavin-binding glucose dehydrogenase having glucose-6-dehydrogenase activity, and is an enzyme catalyst for producing glucuronic acid or a glucuronic acid derivative from glucose or a glucose derivative. The catalyst for producing glucuronic acid or a glucuronic acid derivative may contain, in addition to the flavin-binding GDH of the present invention, for example, an excipient, a suspending agent, a buffer, a stabilizer, a preservative, physiological saline, etc.

Example

[0048] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[0049] (Method for measuring glucose dehydrogenase (GDH) activity) 1.00 mL of 100 mM potassium phosphate buffer (pH 6.0), 1.00 mL of 1 M D-glucose solution, 0.14 mL of 3 mM 2,6-dichlorophenol indophenol (hereinafter referred to as "DCIP"), 0.20 mL of 3 mM 1-methoxy-5-methylphenazinium methyl sulfate (hereinafter referred to as "1-m-PMS") and 0.61 mL of ultrapure water were mixed, incubated at 37 °C for 10 minutes, then 0.05 mL of the enzyme solution was added to start the reaction. For 5 minutes from the start of the reaction, the decrease in absorbance per minute (ΔA600) at 600 nm with the progress of the enzymatic reaction was measured, and the enzyme activity was calculated from the linear part according to the following formula. At this time, the enzyme activity was defined as 1 U for the amount of enzyme that reduces 1 μmol of DCIP per minute at 37 °C and pH 6.0.

[0050] Glucose dehydrogenase (GDH) activity (U / mL) = (-(ΔA600 - ΔA600blank) × 3.0 × dilution factor of enzyme) / (10.8 × 1.0 × 0.05) In the formula, 3.0 is the volume (mL) of the reaction reagent + enzyme solution, 10.8 is the molar absorption coefficient of DCIP at pH 6.0, 1.0 is the optical path length (cm) of the cell, 0.05 is the volume (mL) of the enzyme solution, and ΔA600blank represents the decrease in absorbance per minute at 600 nm when the reaction is started by adding the diluted enzyme solution instead of the enzyme solution.

[0051] [Example 1] (Obtaining flavin-binding glucose dehydrogenase CpGDH) As a result of searching for GDH-producing bacteria, GDH activity was confirmed in the culture supernatant of Colletotrichum plurivorum MAFF305790.

[0052] (1) Bacterial cell culture A liquid medium consisting of 2% (w / v) dextrin (FUJIFILM Wako Pure Chemical Corporation), 1% (w / v) polypeptone (FUJIFILM Wako Pure Chemical Corporation), 0.5% (w / v) potassium dihydrogen phosphate (Nacalai Tesque), 0.05% (w / v) magnesium sulfate heptahydrate (Nacalai Tesque) and water was prepared. 10 mL of it was put into a large test tube and autoclaved at 121 °C for 20 minutes. The cooled liquid medium was inoculated with the above GDH-producing bacteria and cultured with shaking at 25 °C for 72 hours, and then the wet bacterial cells were collected using a sieve.

[0053] (2) Isolation of total RNA After freezing 200 mg of the wet bacterial cells obtained in (1) at -80 °C, 100 μg of total RNA was extracted using ISOGEN II (Nippon Gene).

[0054] (3) Preparation of cDNA library A cDNA library was prepared from the RNA obtained in (2) by reverse transcription reaction using reverse transcriptase and an oligo dT primer with an adapter sequence. The reaction reagents used were the SMARTer RACE cDNA Amplification kit (Takara Bio Inc.), and the reaction conditions were carried out according to the protocol described in the instruction manual.

[0055] (4) Cloning of GDH gene Using the cDNA library obtained in (3) as a template, PCR was performed using a primer pair for obtaining the GDH gene. As a result, a PCR product that seemed to be the internal sequence of the GDH gene was confirmed. The primer pair was designed for obtaining various GDH genes based on a plurality of GDH sequences that had already been elucidated by the inventors. The PCR product was purified and its nucleotide sequence was determined.

[0056] Based on the determined nucleotide sequence, primers for elucidating the upstream and downstream sequences of the GDH gene were designed. Using these primers, the full-length GDH (hereinafter referred to as "CpGDH") gene derived from the Colletotrichum plurivorum MAFF305790 GDH strain was elucidated by the 5' RACE method and the 3' RACE method.

[0057] The optimized sequence of the elucidated CpGDH gene sequence according to the codon frequency of Aspergillus oryzae is shown in SEQ ID NO: 1. Furthermore, the amino acid sequence predicted from the gene sequence is shown in SEQ ID NO: 2.

[0058] (5) Preparation of an expression plasmid vector containing the CpGDH gene A plasmid vector was prepared using an improved promoter of an amylase system derived from Aspergillus oryzae described in a publicly known document 1 (Hidetaka Mine, Chemistry and Biology, 38, 12, 831-838, 2000). First, using the cDNA library obtained in (3) as a template, a PCR product containing the CpGDH gene was obtained. Next, using the PCR product as a template, a CpGDH gene for vector insertion was prepared.

[0059] Finally, the prepared CpGDH gene was ligated downstream of the promoter to produce a plasmid vector capable of expressing the gene. The prepared expression plasmid vector was introduced into Escherichia coli JM109 strain for transformation. The obtained transformant was cultured, and a plasmid vector was extracted from the collected cells using illustra plasmidPrep Midi Flow Kit (GE Healthcare). When the sequence analysis of the insert in the plasmid vector was performed, a nucleotide sequence containing the CpGDH gene was confirmed.

[0060] (6) Obtaining transformants Using the plasmid vector extracted in (5), a recombinant mold (Aspergillus oryzae) producing CpGDH was prepared according to the methods described in publicly known document 2 (Biosci. Biotech. Biochem., 61(8), 1367-1369, 1997) and publicly known document 3 (Gene manipulation technology for Aspergillus sake, Katsuya Gomi, Brewing Society of Japan, 494-502, 2000). The obtained recombinant strain was purified on a Czapek-Dox solid medium. As the host to be used, Aspergillus oryzae NS4 strain was used. This strain was bred at the Brewing Research Institute in 1997 (Heisei 9) as described in publicly known document 2, and currently, those available for sale at the National Research Institute of Brewing are obtainable.

[0061] (7) Confirmation of CpGDH derived from recombinant mold A liquid medium consisting of dextrin (FUJIFILM Wako Pure Chemical Corporation) 2% (w / v), polypeptone (FUJIFILM Wako Pure Chemical Corporation) 1% (w / v), potassium dihydrogen phosphate (Nacalai Tesque) 0.5% (w / v), magnesium sulfate heptahydrate (Nacalai Tesque) 0.05% (w / v) and water was prepared. 10 mL of it was placed in a large test tube (22 mm × 200 mm) and autoclaved at 121°C for 20 minutes. The transformed cells obtained in (6) were inoculated into the cooled liquid medium and cultured with shaking at 30°C for 120 hours. After the culture was completed, it was centrifuged to collect the supernatant, and when the GDH activity was measured by the above-described GDH activity measurement method, the CpGDH activity of the present invention was confirmed.

[0062] (8) Purification of CpGDH 150 mL of the liquid medium described in (7) was placed in a 500 mL Sakaguchi flask and autoclaved at 121°C for 20 minutes. The transformed cells obtained in (6) were inoculated into the cooled liquid medium and cultured with shaking at 30°C for 72 hours. After the culture was completed, the culture solution was filtered through a filter cloth, the collected filtrate was centrifuged to collect the supernatant, and further filtered through a membrane filter (10 μm, Advantec) to collect the culture supernatant.

[0063] Regarding the collected culture supernatant, contaminating proteins were removed and purified using a TOYOPEARL DEAE-650S (Tosoh Corporation) column. After the purified sample was concentrated using an ultrafiltration membrane with a molecular weight cut-off of 10,000, it was replaced with water to obtain purified CpGDH. When the purified CpGDH was subjected to SDS-polyacrylamide gel electrophoresis, it was confirmed that a single band was shown.

[0064] (Obtaining FGDH and CsGDH) The gene sequence of 1872 bp described in SEQ ID NO: 1 of Patent No. 6455714 and the gene sequence of 1908 bp described in SEQ ID NO: 2 of Patent No. 5435180 were synthesized and expressed in Aspergillus oryzae NS4 strain by the same method as in Example 1, and then purified. The purified enzyme derived from SEQ ID NO: 1 of Patent No. 6455714 was designated as FGDH, and the purified enzyme derived from SEQ ID NO: 2 of Patent No. 5435180 was designated as CsGDH. The gene sequence and amino acid sequence of FGDH are described in SEQ ID NOs: 3 and 4, respectively, and the gene sequence and amino acid sequence of CsGDH are described in SEQ ID NOs: 5 and 6, respectively.

[0065] [Example 2] (Examination of Enzymatic and Chemical Properties of Each GDH) The properties of CpGDH, FGDH, and CsGDH obtained in Example 1 were examined. (1) Measurement of Absorption Spectrum For each GDH obtained in Example 1, the absorption spectra at 300 - 600 nm before and after the addition of D - glucose were measured using a plate reader (SpectraMax Plus384, Molecular Devices). As a result, since the absorption maxima observed around wavelengths 360 - 380 nm and around wavelengths 450 - 460 nm disappeared upon the addition of D - glucose, it was revealed that all of the GDHs are flavin - binding proteins.

[0066] (2) Measurement of Glucose Oxidase (GOD) Activity 0.2 mL of 1 M potassium phosphate buffer (pH 7.0), 2.0 mL of 1 M D - glucose, 0.2 mL of 25 mM 4 - aminoantipyrine, 0.2 mL of 420 mM phenol, 0.2 mL of 1 mg / mL peroxidase (from Wasabia japonica, manufactured by Fujifilm Wako Pure Chemical Corporation), and 0.2 mL of ultrapure water were mixed. 0.1 mL of the mixed solution was placed in a 96 - well plate and incubated at 25°C for 5 minutes. 0.1 mL of each GDH obtained in Example 1 was added to initiate the reaction. The change in absorbance at 500 nm with the progress of the enzyme reaction was measured using the plate reader for 5 minutes from the start of the reaction to examine the GOD activity. Incidentally, for the control, water or GOD from Aspergillus niger (manufactured by Nacalai Tesque) was added instead of GDH to initiate the reaction. As a result, a change in absorbance at 500 nm was confirmed in the control with the addition of GOD from Aspergillus niger, but no change in absorbance was observed for the GDH of the present invention, similar to the control with the addition of water. Therefore, it was confirmed that all GDHs are dehydrogenases that do not utilize oxygen as an electron acceptor.

[0067] (3) Substrate specificity According to the above - mentioned GDH activity measurement method, using D - glucose, maltose, D - xylose, or D - galactose at a final concentration of 50 mM as the substrate, the activity of each GDH with respect to each substrate was measured. The results are shown in Table 1.

[0068]

Table 1

[0069] When the activity with respect to D - glucose was set to 100% for all GDHs, the activities with respect to maltose, D - xylose, or D - galactose were 0.3%, 0.4%, or 0.2% or less, and all were 2.0% or less.

[0070] (4) Km value for D - glucose For each GDH, according to the above activity measurement method, the activity was measured by changing the concentration of D-glucose as the substrate, and the Michaelis constant (Km) was determined from the Hanes-Woolf plot. The activity measurement was carried out at 25 °C. The results are shown in Table 2.

[0071] [Table 2]

[0072] As a result, the Km values of CpGDH, CsGDH, and FGDH were 196 mM, 50 mM, and 52 mM, respectively. Since the Km value is likely to vary depending on the measurement method and the plot used for calculation, the Km of CpGDH is considered to be in the range of 150 mM to 300 mM, the Km of FGDH is in the range of 30 mM to 80 mM, and the Km of CsGDH is in the range of 30 mM to 80 mM.

[0073] (5) Thermal stability Each GDH was prepared at 6 U / mL and treated at each temperature for 60 minutes in 100 mM potassium phosphate buffer (pH 8.0). As a result, the temperature range at which the residual enzyme activity was 80% or more when the enzyme activity before treatment was set to 100% was up to 40 °C for CpGDH, up to 40 °C for FGDH, and up to 45 °C for CsGDH (see Figure 1).

[0074] (6) Stable pH range The stable pH of each GDH was examined. Each GDH was prepared at 6 U / mL, and buffer solutions were added so that the final concentrations were 100 mM sodium citrate phosphate buffer (pH 2.2 - 7.0), 100 mM potassium acetate buffer (pH 3.0 - 6.0), 100 mM potassium phosphate buffer (pH 6.0 - 8.0), 100 mM Tris-HCl buffer (pH 7.0 - 9.0), and 100 mM glycine-NaOH buffer (pH 9.0 - 10.0). After treatment at 30 °C for 1 hour at each pH, the residual activity of each GDH was measured. As a result, the pH ranges in which the residual enzyme activity values of each GDH were 80% or more when the enzyme activity value before treatment was taken as 100% were pH 4.3 - 9.3 for CpGDH, pH 5.5 - 8.7 for FGDH, and pH 3.2 - 9.3 for CsGDH (see Figure 2). From the above, it was found that the GDH of the present invention is stable at least in the range of pH 5.5 - 8.7. Note that the residual activity may differ depending on the type of buffer solution even at the same pH.

[0075] [Example 3] (Analysis of Glucose Oxidase Obtained in the Presence of 1-m-PMS) A reaction system using purified CpGDH and 1-m-PMS as mediators was constructed to obtain glucose oxidase. The preparation method and various analysis methods are described below.

[0076] (1) Preparation of Glucose Oxidase 0.04 mL of 1 M potassium phosphate buffer (pH 7.0), 0.01 mL of 2 M D-glucose solution, 0.02 mL of 0.2 M 1-m-PMS solution, 0.02 mL of 10,000 U / mL catalase (from bovine liver, manufactured by Fujifilm Wako Pure Chemical Corporation), 0.08 mL of 90 U / mL CpGDH, FGDH, or CsGDH, and 0.23 mL of ultrapure water were mixed and inverted and mixed overnight at room temperature. Then, the air in the reaction vessel was replaced and inverted and mixed for several more hours to obtain a reaction product containing glucose oxidase.

[0077] (2) Thin-Layer Chromatography Analysis of Glucose Oxidase (1) The reaction product obtained was used as a sample for thin-layer chromatography (hereinafter referred to as TLC) analysis. 0.001 mL of the sample was dropped and dried on a silica gel plate (Merck Millipore, Silica gel60 F254), and developed with acetonitrile / ultrapure water (70:30) for 10 minutes. After drying the silica gel plate, concentrated sulfuric acid / ethanol (5:95) was sprayed and then heated. As a result, in the CpGDH, FGDH, and CsGDH reaction products, spots were confirmed at the same position as the glucuronic acid standard, and no spots were confirmed at the same position as the D-glucose standard and the gluconic acid standard. On the other hand, as a comparative example, GDH derived from Aspergillus terreus (Patent Document 8), which is a commonly well-known GDH, was purified and the same test was conducted. As a result, no spots were detected at the same position as the glucuronic acid standard and the glucose standard, and a spot was detected at the same position as the gluconic acid standard. From this, it was confirmed that while general GDH oxidizes the 1-position of glucose to produce gluconic acid, CpGDH, FGDH, and CsGDH oxidize the 6-position of glucose to produce glucuronic acid.

[0078] (3) Examination of glucose oxidation under various conditions (1) Among the conditions described in (1), the conditions in which the buffer solution used was changed to 1M sodium phosphate buffer (pH 7.0 or 8.0) or 1M potassium phosphate buffer (pH 8.0), the amount of D-glucose added was changed to twice the amount, or the addition amount of 1-m-PMS was changed to 0.0025 mL or 0.005 mL. Reaction products containing glucose oxide were prepared and subjected to TLC analysis. As a result, under any conditions, in the CpGDH, FGDH, and CsGDH reaction products, spots were confirmed at the same position as the glucuronic acid standard, and no spots were confirmed at the same position as the D-glucose standard and the gluconic acid standard.

[0079] (4) High-performance liquid chromatography analysis of glucose oxide To remove 1-m-PMS from the CpGDH reaction product obtained in (1), 2 - 20 mg of powdered activated carbon (Futamura Chemical Co., Ltd., Taiko Activated Carbon SG) washed with ultrapure water after crushing was added, and the mixture was allowed to stand at room temperature for 5 minutes. Since 1-m-PMS was adsorbed onto the activated carbon by this treatment, the supernatant was recovered by centrifugation at 4°C, 8,000 × g for 1 minute to obtain a CpGDH reaction product from which 1-m-PMS had been removed. After fluorescently labeling the supernatant with the GlyScope ABEE labeling kit (J-Chemical Co., Ltd.), HPLC analysis was performed using a sugar analysis column, Horenpak C18 (J-Chemical Co., Ltd.). As a result, no peak of the substrate D-glucose was observed, and a peak was confirmed at the same position as the glucuronic acid standard (see Figure 4). Note that the fluorescent labeling and HPLC analysis were carried out according to the protocol described in the kit manual. As the HPLC analyzer, the SIL-10A series (Shimadzu Corporation) was used, and as the detector, a fluorescence detector RF-10AXL (excitation wavelength / fluorescence wavelength = 305 / 360 nm, Shimadzu Corporation) was used.

[0080] (5) Analysis of glucose oxidase using a nuclear magnetic resonance (NMR) apparatus (4) The CpGDH reaction product from which 1-m-PMS had been removed was preliminarily frozen at -40°C and freeze-dried using a freeze dryer (EYELA, FREEZE DRYER FD-1). As a result of analyzing the obtained freeze-dried sample with a nuclear magnetic resonance apparatus, a peak indicating that the hydroxymethyl group at the 6th position of glucose had been converted to a carboxyl group was obtained, confirming that glucuronic acid was selectively produced from glucose.

[0081] (6) Analysis using a gluconic acid measurement kit In order to confirm whether gluconic acid was contained in the CpGDH reaction product from which 1-m-PMS obtained in (4) had been removed, activity measurement was carried out using an F-kit D-gluconic acid / glucono-δ-lactone (J.K. International Co., Ltd.). As a result of evaluation according to the protocol described in the instruction manual, the accurate gluconic acid concentration could be measured for the gluconic acid standard used as a control, but gluconic acid was not detected in the CpGDH reaction product.

[0082] (7) Analysis using a glucuronic acid measurement kit In order to measure the glucuronic acid concentration contained in the CpGDH reaction product from which 1-m-PMS obtained in (4) had been removed, analysis was carried out using a glucuronic acid measurement kit (Megazyme, K-URONIC). As a result of evaluation according to the protocol described in the instruction manual, it was confirmed that glucuronic acid was generated.

[0083] [Example 4] (Analysis of glucose oxidase obtained in the presence of tert-butylhydroquinone) A reaction system was constructed using purified CpGDH and tert-butylhydroquinone (hereinafter referred to as TBHQ) as a mediator to obtain glucose oxidase. The preparation method and analysis method thereof are described below.

[0084] (1) Preparation of glucose oxidase using TBHQ 0.02 mL of 1M sodium phosphate buffer (pH 8.0), 0.01 mL of 1M D-glucose solution, 0.002 mL of 1M TBHQ (Tokyo Chemical Industry Co., Ltd.), 0.005 mL of 10 U / mL laccase (Sigma-Aldrich Co., Ltd., derived from Aspergillus), 0.04 mL of 90 U / mL CpGDH, and 0.123 mL of ultrapure water were mixed and inverted and mixed overnight at room temperature in the dark to obtain a reaction product containing glucose oxidase. Also, a reaction product containing glucose oxidase was obtained in the same manner under the condition that the laccase concentration was changed to 1 / 5 of the above composition.

[0085] (2) TLC analysis of glucose oxidase As a result of performing TLC analysis in the same manner as in (2) of Example 3, spots were confirmed at the same position as the glucuronic acid standard also in the CpGDH reaction product using TBHQ as a mediator, and nothing was detected at the same positions as the glucose standard and the glucuronic acid standard.

[0086] (3) Examination of Glucose Oxidation in the Presence of Peroxidase and Catalase A reaction solution was prepared and reacted by mixing 0.02 mL of 1M sodium phosphate buffer (pH 8.0), 0.01 mL of 1M D-glucose solution, 0.002 mL of 1M TBHQ (Tokyo Chemical Industry Co., Ltd.), 0.01 mL of 10,000 U / mL catalase (Fuji Film Wako Pure Chemical Corporation, derived from bovine liver), 0.005 mL of 100 U / mL horseradish peroxidase (Fuji Film Wako Pure Chemical Corporation, derived from horseradish), 0.04 mL of 90 U / mL CpGDH, and 0.113 mL of ultrapure water. As a result of performing TLC analysis on the reaction product, spots were confirmed at the same position as the glucuronic acid standard as in (1), and nothing was detected at the same positions as the glucose standard and the glucuronic acid standard.

[0087] (4) Analysis Using a Gluconic Acid Measurement Kit As a result of analyzing using a D-gluconic acid / glucono-δ-lactone measurement kit (F-kit) in the same manner as in (6) of Example 3, it was shown that CpGDH did not produce gluconic acid even in the reaction system using TBHQ as a mediator.

[0088] (5) Analysis Using a Glucuronic Acid Measurement Kit As a result of analyzing using a glucuronic acid measurement kit (K-URONIC) in the same manner as in (7) of Example 3, it was confirmed that the same amount of glucuronic acid as the glucose used in the reaction was produced in the reaction system using TBHQ as a mediator.

[0089] [Example 5] (Analysis of Glucose Oxide Obtained in the Presence of Butylhydroxyanisole) A reaction system using purified CpGDH and butylhydroxyanisole as mediators was constructed to obtain glucose oxidase. The preparation method and analysis method are described below.

[0090] (1) Preparation of Glucose Oxidase Using Butylhydroxyanisole Mix 0.02 mL of 1M sodium phosphate buffer (pH 8.0), 0.01 mL of 1M D-glucose solution, 0.02 mL of 0.1M butylhydroxyanisole (FUJIFILM Wako Pure Chemical Corporation), 0.01 mL of 10,000 U / mL catalase (FUJIFILM Wako Pure Chemical Corporation, derived from bovine liver), 0.005 mL of 10 U / mL laccase (Sigma-Aldrich, derived from Aspergillus sp.), 0.04 mL of 90 U / mL CpGDH, and 0.095 mL of ultrapure water, and mix by inverting overnight at room temperature in the dark to obtain a reaction product containing glucose oxidase.

[0091] (2) TLC Analysis of Glucose Oxidase As a result of performing TLC analysis in the same manner as in (2) of Example 3, spots were confirmed at the same position as the glucuronic acid standard in the CpGDH reaction product prepared using butylhydroxyanisole as a mediator, and nothing was detected at the same positions as the glucose standard and the gluconic acid standard.

[0092] [Example 6] (Analysis of Various Substrate Oxides) Oxides such as glycosides were obtained using purified CpGDH. The preparation method and various analysis methods are described below.

[0093] (1) Preparation of Substrate Oxide The reaction solution consisting of 100 mM potassium phosphate buffer (pH 8.0) at the final concentration, 13 - 100 mM D-(+)-cellobiose as the substrate, α-albutin, β-albutin, pseude, N-acetyl-d-glucosamine, 10 mM 1-m-PMS as the mediator, and 500 U / mL catalase (from FUJIFILM Wako Pure Chemical Corporation, derived from bovine liver) as the scavenger of reactive oxygen species was added with CpGDH to a final concentration of 14 U / mL, and mixed by inverting overnight at room temperature. Then, the air in the reaction vessel was replaced, and the mixture was further mixed by inverting for several hours at room temperature to obtain the substrate oxide containing glycoside oxide and the like.

[0094] (2) TLC analysis of the substrate oxide As a result of performing TLC analysis in the same manner as in (2) of Example 3, a new spot was detected at a position different from that before the reaction (see Figure 5). This indicates that CpGDH oxidizes the glucose residue in the glycoside.

[0095] [Example 7] (Obtaining CglGDH, CoGDH, CtoGDH, CgoGDH, GsGDH and DhGDH) Instead of Colletotrichum plurivorum MAFF305790, Colletotrichum gloeosporioides, Colletotrichum orbiculare, Colletotrichum tofieldiae, Colletotrichum godetiae MAFF240289, Glomerella sp. RD057037 or Diaporthe helianthi were used and each was expressed in Aspergillus oryzae NS4 strain in the same manner as in Example 1 and purified. The purified enzyme derived from Colletotrichum gloeosporioides was designated as CglGDH, the purified enzyme derived from Colletotrichum orbiculare was designated as CoGDH, the purified enzyme derived from Colletotrichum tofieldiae was designated as CtoGDH, the purified enzyme derived from Colletotrichum godetiae MAFF240289 was designated as CgoGDH, the purified enzyme derived from Glomerella sp. RD057037 was designated as GsGDH, and the purified enzyme derived from Diaporthe helianthi was designated as DhGDH. Their respective gene sequences were described in SEQ ID NO: 7, 9, 11, 13, 15, 17, and the amino acid sequences were described in SEQ ID NO: 8, 10, 12, 14, 16, 18.

[0096] (Examination of Enzymatic Chemical Properties) (1) Measurement of Glucose-6-Dehydrogenase Activity Glucose oxidase was prepared using CglGDH, CoGDH, CtoGDH, CgoGDH, GsGDH and DhGDH under the conditions described in Example 4, and TLC analysis was performed. As a result, spots of these glucose oxidases were confirmed at the same position as the glucuronic acid standard, and nothing was detected at the same position as the glucose standard and the gluconic acid standard (see Figure 6).

[0097] (2) Substrate Specificity The activity of each GDH against each substrate was measured in the same manner as in Example 2. The results are shown in Table 3.

[0098] [Table 3]

[0099] When the activity of any GDH against D - glucose was set as 100%, the activity against maltose or D - xylose was 0.2% or less than 0.9%, and all were 2.0% or less.

[0100] (3) Km value for D - glucose The Michaelis constant (Km) was determined in the same manner as in Example 2. The results are shown in Table 4.

[0101]

Table 4

[0102] Since the Km value is likely to vary depending on the measurement method and the plot for calculation, the Km of CglGDH is considered to be 400 mM - 900 mM, the Km of CoGDH is 250 mM - 650 mM, the Km of CtoGDH is 400 mM - 900 mM, the Km of CgoGDH is 200 mM - 450 mM, the Km of GsGDH is 250 mM - 650 mM, and the Km of DhGDH is 60 mM - 140 mM.

[0103] (4) Thermal stability Each GDH was prepared at 6 U / mL in the same manner as in Example 2 and treated at each temperature for 60 minutes in 100 mM potassium phosphate buffer (pH 8.0). As a result, the temperature range in which the residual enzyme activity was 80% or more when the enzyme activity before treatment was set as 100% was up to 40°C for CglGDH, CtoGDH, CgoGDH, and GsGDH, up to 45°C for CoGDH, and up to 50°C for DhGDH (see Figure 7).

[0104] (5) Range of stable pH As a result of examining the stable pH of each GDH in the same manner as in Example 2, the pH ranges in which the residual enzyme activity values of each GDH were 80% or more when the enzyme activity value before treatment was set to 100% were pH 5.5 to 9.3 for CglGDH, pH 4.4 to 9.3 for CoGDH, pH 4.0 to 9.6 for CtoGDH, pH 4.4 to 9.3 for CgoGDH, pH 5.0 to 9.3 for GsGDH, and pH 3.3 to 9.6 for DhGDH (see Fig. 8). From the above, it was found that the GDH of the present invention is stable at least in the range of pH 5.5 to 9.3.

[0105] [Example 8] (Acquisition of Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH, CtaGDH, FlaGDH, PcGDH, Fla_A.oGDH, and Pc_A.oGDH) Instead of Colletotrichum plurivorum MAFF305790, sequence information with high amino acid sequence identity to CpGDH was obtained from the publicly available genomic data of Khuskia oryzae, Acremonium strictum, Lasiosphaeris hirsute, Diaporthaceae sp., Colletotrichum tanaceti, Fusarium langsethiae, and Phialemoniopsis curvata. After optimizing for the codon frequency of Aspergillus oryzae, each was expressed and purified in Aspergillus oryzae NS4 strain in the same manner as in Example 1. In addition, sequences in which the signal sequence portions of GDHs derived from Fusarium langsethiae and Phialemoniopsis curvata were replaced with the signal sequence of GDH derived from Aspergillus oryzae were each expressed and purified in Aspergillus oryzae NS4 strain in the same manner as in Example 1. Purified enzymes derived from Khuskia oryzae were designated as Ko37GDH and Ko38GDH, those from Acremonium strictum as AsGDH, those from Lasiosphaeris hirsute as LhGDH, those from Diaporthaceae sp. as DsGDH, those from Colletotrichum tanaceti as CtaGDH, those from Fusarium langsethiae as FlaGDH, those from Phialemoniopsis curvata as PcGDH, a purified enzyme in which the putative signal sequence portion of the enzyme from Fusarium langsethiae was replaced with the signal sequence of GDH from Aspergillus oryzae as Fla_A.oGDH, and a purified enzyme in which the putative signal sequence portion of the enzyme from Phialemoniopsis curvata was replaced with the signal sequence of GDH from Aspergillus oryzae as Pc_A.oGDH. The gene sequences optimized for the codon frequencies of Aspergillus oryzae of Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH, CtaGDH, FlaGDH, PcGDH, Fla_A.oGDH, and Pc_A.oGDH are set forth in SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37, and the amino acid sequences are set forth in SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38, respectively.

[0106] (Examination of Enzymatic Chemical Properties) (1) Measurement of Glucose-6-Dehydrogenase Activity Glucose oxidase was prepared using Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH, CtaGDH, FlaGDH, PcGDH, Fla_A.oGDH, and Pc_A.oGDH under the conditions described in Example 4, and TLC analysis was performed. As a result, spots of these glucose oxidases were confirmed at the same position as the glucuronic acid standard, and nothing was detected at the same position as the glucose standard and the gluconic acid standard (see Figure 9).

[0107] (2) Substrate Specificity The activity of each GDH against each substrate was measured in the same manner as in Example 2. The results are shown in Table 5.

[0108] [Table 5]

[0109] When the activity against D-glucose was set to 100%, the activity against maltose or D-xylose was 0.5% or less, and all were 2.0% or less for any of the GDHs.

[0110] (3) Km value for D-glucose The Michaelis constant (Km) was determined in the same manner as in Example 2. The results are shown in Table 6.

[0111] [Table 6]

[0112] Since the Km value is likely to vary depending on the measurement method and the plot used for calculation, the Km of Ko37GDH is considered to be 25 mM to 60 mM, the Km of AsGDH is 25 mM to 60 mM, the Km of Ko38GDH is 700 mM to 1,500 mM, the Km of DsGDH is 150 mM to 300 mM, the Km of CtaGDH is 110 mM to 240 mM, the Km of FlaGDH is 900 mM to 1,600 mM, and the Km of Fla_A.oGDH is 600 mM to 1,200 mM.

[0113] (4) Thermal stability Each GDH was prepared at 6 U / mL in the same manner as in Example 2 and treated at each temperature for 60 minutes in each 100 mM buffer shown in Table 7. As a result, the temperature range at which the residual enzyme activity was 80% or more when the enzyme activity before treatment was set to 100% was up to 35°C for Ko37GDH, up to 40°C for DsGDH, up to 45°C for Ko38GDH, LhGDH, FlaGDH, and Fla_A.oGDH, and up to 50°C for AsGDH, CtaGDH, PcGDH, and Pc_A.oGDH (see Figure 10).

[0114]

Table 7

[0115] (5) Stable pH range The stable pH of each GDH was examined in the same manner as in Example 2. As a result, when the enzyme activity value before treatment was set to 100%, the pH ranges in which the residual enzyme activity values of each GDH were 80% or more were pH 5.5 to 8.6 for Ko37GDH, pH 4.3 to 9.6 for AsGDH, pH 5.0 to 9.6 for Ko38GDH, pH 4.0 to 8.6 for LhGDH, pH 4.9 to 8.7 for DsGDH, pH 3.3 to 9.9 for CtaGDH, pH 4.4 to 9.8 for FlaGDH, pH 4.0 to 8.8 for PcGDH, pH 4.4 to 9.8 for Fla_A.oGDH, and pH 4.0 to 9.6 for Pc_A.oGDH (see Fig. 11).

[0116] [Example 9] (Analysis of picic acid oxide using a nuclear magnetic resonance apparatus (NMR)) Picic oxide was obtained using purified CpGDH. The preparation method and 1 1H-NMR analysis and 13 13C-NMR analysis results are described below.

[0117] (1) Preparation of substrate oxide CpGDH was added to a reaction solution consisting of 20 mM potassium phosphate buffer (pH 7.0) at a final concentration, 50 mM equivalent of picic powder as a substrate, 10 mM TBHQ as a mediator, 200 U / mL catalase (from Fujifilm Wako Pure Chemical Corporation, derived from bovine liver) as an active oxygen scavenger, and 2 U / mL laccase (from Sigma-Aldrich, derived from Aspergillus) at a final concentration of 14 U / mL. While adding 1N NaOH to keep the pH in the reaction system at 6.5 - 7.0, the mixture was stirred while aerating at room temperature to obtain picic oxide.

[0118] (2) 1H-NMR analysis and 1 13C-NMR analysis of picic oxide 13 13C-NMR analysis For the picic acid oxide obtained in (1), purification was performed using a C18 column. After removing impurities, its structure was identified by nuclear magnetic resonance analysis in the same manner as in Example 3. On the other hand, picic acid was analyzed in the same manner as a comparative example. 1 From the results of 1H-NMR, 13 13C-NMR, it was confirmed that the hydroxymethyl group at the C6 position of the glucose residue of picic acid had become a carboxyl group, and it was confirmed that the other structures were common (see Figures 12 and 13). From this, it was shown that CpGDH specifically oxidizes the glucose residue in picic acid.

[0119] [Example 10] (1) Large-scale preparation of glucuronic acid CpGDH was added to a reaction solution consisting of 20 mM sodium phosphate buffer (pH 7.0) at the final concentration, 2 M glucose as a substrate, 10 mM guaiacol as a mediator, and 2.1 U / mL laccase (from Sigma-Aldrich, derived from Aspergillus) to a final concentration of 60 U / mL. While adding 1N NaOH so that the pH in the reaction system became 6.5 - 7.0, aeration was carried out at room temperature with stirring to obtain glucuronic acid.

[0120] (2) TLC analysis of glucuronic acid As a result of performing TLC analysis in the same manner as in (2) of Example 3, a dark spot was confirmed at the same position as the standard glucuronic acid, and the spot of glucose could not be confirmed. From this, it was shown that under the conditions of (1), all of the glucose used as a substrate could be converted into glucuronic acid. [Example 11] (Large-scale preparation of picic acid oxide) Using purified CpGDH, a large amount of picic acid oxide was obtained. The preparation method is described below.

[0121] (1) Preparation of picic acid oxide (addition of polyethylene glycol and ethanol) CpGDH was added to a reaction solution consisting of 20 mM sodium phosphate buffer (pH 7.0), 200 mM equivalent of picayde powder as a substrate, 10 mM TBHQ as a mediator, 500 U / mL catalase (from Fujifilm Wako Pure Chemical Corporation, derived from bovine liver) as a scavenger of reactive oxygen species, 10% (v / v) polyethylene glycol (molecular weight: 200, from Nacalai Tesque), 10% (v / v) ethanol, and 50 mU / mL laccase (from Sigma-Aldrich, derived from Aspergillus) to a final concentration of 20 U / mL. While adding 1N NaOH to adjust the pH in the reaction system to 6.5 - 7.0, the mixture was stirred at room temperature with aeration for 165 hours. As a result, it was confirmed that almost all of the picayde, which was the substrate, had been oxidized.

[0122] (2) Analysis of picayde oxide As a result of performing TLC analysis on the picayde oxide prepared under the conditions of (1), spots were confirmed at the same positions as the picayde oxide analyzed by NMR in Example 9. Also, nothing was detected at the spots at the same positions as the picayde standard analyzed in Example 9. From this, it was confirmed that under the conditions of (1), all of the picayde, which was the substrate, had become picayde oxide.

[0123] (3) Preparation of picayde oxide (with ethanol addition) CpGDH was added to a reaction solution consisting of 20 mM sodium phosphate buffer (pH 7.0), 50 mM equivalent of picayde powder as a substrate, 10 mM TBHQ as a mediator, 500 U / mL catalase (from Fujifilm Wako Pure Chemical Corporation, derived from bovine liver) as a scavenger of reactive oxygen species, 10% (v / v) ethanol, and 0.13 mU / mL laccase (from Sigma-Aldrich, derived from Aspergillus) to a final concentration of 20 U / mL. While adding 1N NaOH to adjust the pH in the reaction system to 6.5 - 7.0, the mixture was stirred at room temperature with aeration overnight to obtain picayde oxide. Also, picayde oxide could be obtained in the same manner under the conditions where the final concentration of ethanol was changed to 20% or 30%.

[0124] (4) Analysis of picetannic oxide (3) The TLC analysis of the picetannic oxide prepared under the conditions of (3) showed that spots were confirmed at the same positions as those of the picetannic oxide analyzed by NMR in Example 9. Also, nothing was detected at the spots at the same positions as those of the picetannic standard analyzed in Example 9. From this, it was confirmed that under the conditions of (3), all of the substrate picetannic acid had been converted into picetannic oxide.

[0125] (5) Preparation of picetannic oxide (alkaline conditions) A culture supernatant containing CtaGDH was added to a reaction solution consisting of 20 mM glycine-NaOH buffer (pH 10.0) at a final concentration, 50 mM equivalent of picetannic acid powder as a substrate, and 10 mM TBHQ as a mediator so that the final concentration was 20 U / mL, and the mixture was stirred for 1 hour while aerating at room temperature to obtain picetannic oxide.

[0126] (6) Analysis of picetannic oxide (5) The TLC analysis of the picetannic oxide prepared under the conditions of (5) showed that spots were confirmed at the same positions as those of the picetannic oxide analyzed by NMR in Example 9. Also, nothing was detected at the spots at the same positions as those of the picetannic acid analyzed in Example 9. Also, among the conditions described in (5), when the glycine-NaOH buffer (pH 10) was changed to a sodium phosphate buffer (pH 7.0) and 3.2 mU / mL of laccase (from Sigma-Aldrich, derived from Aspergillus) was added, it was confirmed by TLC analysis that picetannic acid as the substrate remained even after reacting for the same time. From this, it was confirmed that under the alkaline conditions of (5), the substrate picetannic acid was rapidly converted into picetannic oxide in its entirety compared to the neutral condition.

[0127] (7) Preparation of picetannic oxide (natural oxidation of mediator) The reaction solution consisting of 20 mM glycine-NaOH buffer (pH 10.0), 50 mM equivalent of picacid powder as the substrate, and 10 mM TBHQ as the mediator was added with CtaGDH to a final concentration of 20 U / mL, stirred for 1 hour while aerating at room temperature, and picacid oxide was obtained.

[0128] (8) Analysis of picacid oxide (7) The result of TLC analysis of the picacid oxide prepared under the conditions of (7) showed that spots were confirmed at the same positions as the picacid oxide analyzed by NMR in Example 9. Also, nothing was detected in the spots at the same positions as the picacid analyzed in Example 9. From this, it was confirmed that under the conditions of (7), all of the substrate picacid had been rapidly converted into picacid oxide. Also, since all of the substrate picacid was converted into picacid oxide even without the addition of laccase, it was confirmed that the mediator was rapidly and automatically re-oxidized under alkaline conditions.

[0129] [Example 12] (Analysis of arbutin oxide) Purified CpGDH was used to obtain arbutin oxide, and its glucuronic acid residues were analyzed.

[0130] (1) Preparation of arbutin oxide The reaction solution consisting of 20 mM sodium phosphate buffer (pH 7.0), 50 mM arbutin as the substrate, 10 mM TBHQ as the mediator, 500 U / mL catalase (from Fujifilm Wako Pure Chemical Corporation, derived from bovine liver) as an active oxygen scavenger, and 30 mU / mL laccase (from Sigma-Aldrich, derived from Aspergillus sp.) was added with CtaGDH to a final concentration of 20 U / mL, and 1N NaOH was added while aerating at room temperature to keep the pH in the reaction system at 6.5 - 7.0, and stirred for 48 - 72 hours to obtain arbutin oxide.

[0131] (2) Sugar cleavage of arbutin oxide For the albutin oxide prepared under the condition of (1), trifluoroacetic acid was added so that the final concentration became 4 M, and the mixture was heated at 100 °C for 3 hours. Then, isopropanol was added to the treatment solution so that the final concentration became 80% (v / v), and the mixture was allowed to stand at room temperature for 24 hours to dry. The dried sample was dissolved in ultrapure water and used for the analysis of the cleaved sugar moiety.

[0132] (3) Analysis of sugar residues cleaved from albutin oxide For the sugar residues derived from the albutin oxide prepared under the condition of (2), as a result of performing TLC analysis in the same manner as in (2) of Example 3, spots were confirmed at the same position as the glucuronic acid standard, and nothing was detected at the same positions as the glucose standard and the glucuronic acid standard. From this, it was confirmed that even in albutin, the glucose residue was converted to glucuronic acid by using CpGDH.

[0133] [Example 13] (Solubility of pisidic oxide) The solubility of pisidic oxide in water was confirmed.

[0134] (1) Solubility of pisidic oxide in water As a result of dissolving pisidic powder in water so that the final concentration became 2 mM, the whole amount did not dissolve completely and powder remained. On the other hand, the pisidic oxide prepared in (1) of Example 11 was purified by a C18 column, and as a result of confirming the solubility of the obtained purified pisidic oxide in water, the added pisidic oxide completely dissolved even under the condition where the final concentration was about 200 mM. From this, it was confirmed that the conversion of the glucose residue of pisidic to a glucuronic acid residue improved the solubility in water by at least 100 times or more.

Claims

1. A method for producing glucuronic acid, comprising the step of reacting glucose with a flavin - binding glucose dehydrogenase having glucose - 6 - dehydrogenase activity, which is any one of the following proteins (i) to (iii) in the presence of a mediator to produce glucuronic acid: (i) A protein having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 (ii) A protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose - 6 - dehydrogenase activity (iii) A protein having an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose - 6 - dehydrogenase activity.

2. A method for producing a glucuronic acid derivative, comprising the step of reacting a glucose derivative with a flavin - binding glucose dehydrogenase having glucose - 6 - dehydrogenase activity, which is any one of the following proteins (i) to (iii) in the presence of a mediator to produce a glucuronic acid derivative: (i) A protein having an amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 (ii) A protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose - 6 - dehydrogenase activity (iii) A protein having an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose - 6 - dehydrogenase activity.

3. The method for producing a glucuronic acid derivative according to claim 2, wherein the glucose derivative is an amino sugar or its N-acetylated product, a glucoside, or a glucose analog.

4. The method for producing glucuronic acid or a glucuronic acid derivative according to any one of claims 1 to 3, wherein the flavin-bound glucose dehydrogenase has the following properties (1) to (8): (1) Action: Catalyzes the reaction of dehydrogenating (oxidizing) the hydroxymethyl group at the 6-position of glucose using flavin as a coenzyme. (2) Solubility: Water-soluble (3) pH stability: Stable at least between pH 5.5 and 8.7 (4) Thermal stability: Stable at least at 35 °C (5) Substrate specificity: When the reactivity with glucose is set to 100%, the reactivity with maltose, xylose, and galactose is 2.0% or less. (6) Km value (for glucose): 30 mM or more (7) Molecular weight: 64 to 66 kDa (calculated from the amino acid sequence after signal removal) (8) Glucose oxidase activity: Not detectable.

5. The method for producing glucuronic acid or a glucuronic acid derivative according to any one of claims 1 to 4, wherein the flavin-bound glucose dehydrogenase is derived from a microorganism belonging to the genus Colletotrichum, Glomerella, Diaporthe, Kusicella, Acremonium, Lasiosphaeris, Fusarium, or Phaeomoniopsis.

6. The method for producing glucuronic acid or a glucuronic acid derivative according to any one of claims 1 to 5, wherein a recombinant microorganism into which a gene encoding a flavin-bound glucose dehydrogenase consisting of any one of the following DNAs (a) to (c), (e) is introduced is used: (a) DNA having the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37 (b) DNA having a nucleotide sequence in which one to several nucleotides are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17, and encoding a protein having glucose-6-dehydrogenase activity DNA encoding a protein having a base sequence with 90% or more sequence identity to the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 and having glucose-6-dehydrogenase activity DNA which hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37 and encodes a protein having glucose-6-dehydrogenase activity DNA encoding the protein of any one of the following (i), (ii) or (iii) (i) A protein having the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 (ii) A protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose-6-dehydrogenase activity (iii) A protein having an amino acid sequence with 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 and having glucose-6-dehydrogenase activity.

7. The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of claims 1 to 6, further comprising the action of an oxidase.

8. A catalyst composition for producing glucuronic acid or a glucuronic acid derivative, which is used to produce glucuronic acid or a glucuronic acid derivative from glucose or a glucose derivative and contains a flavin-binding glucose dehydrogenase protein having glucose-6-dehydrogenase activity, which is any one of the following (i) to (iii): (i) A protein having the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 A protein having an amino acid sequence in which one to several amino acid residues are deleted, substituted or inserted in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, and having glucose-6-dehydrogenase activity A protein having an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, and having glucose-6-dehydrogenase activity.

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