WO2021149587A1

WO2021149587A1 - Method for producing glucuronic acid

Info

Publication number: WO2021149587A1
Application number: PCT/JP2021/001086
Authority: WO
Inventors: 昂洸藤井; 高史宅見; 俊雄荒木; 本田　通済
Original assignee: 池田食研株式会社
Priority date: 2020-01-23
Filing date: 2021-01-14
Publication date: 2021-07-29
Also published as: CN114981438A; JPWO2021149587A1

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

Method for producing glucuronic acid

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

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

As an existing method for producing glucuronic acid, an enzyme that oxidizes the hydroxymethyl group at the 6-position of glucose, for example, glucose oxidase modified from galactose oxidase (Patent Document 1), aldehyde dehydrogenase (Patent Document 2), alcohol A method using a dehydrogenase (Patent Document 3) has been reported. However, these enzymes have problems that the specific activity of the enzyme is low, the oxidation specificity of glucose to the hydroxymethyl group is low, and glucose oxides such as gluconic acid are produced in addition to glucuronic acid.

In addition, as a method for producing glucuronic acid using an enzyme using a saccharide other than glucose as a substrate, α-1,4-polyglucuronic acid produced by oxidizing starch is mixed with Paenivacillus bacteria or a glycoside-binding hydrolyzing enzyme derived from the same bacteria. A method for producing glucuronic acid by acting (Patent Document 4), a method for producing glucuronic acid using an enzyme that hydrolyzes trehalose oxide as a substrate (Patent Document 5), and myo-inositol oxygenase using myo-inositol as a substrate. A method for producing glucuronic acid using glucuronic acid (Patent Document 6) has been reported. However, the problems of these production methods are that the preparation of the substrate is complicated and the price of the substrate is high.

In addition, as a method for producing glucuronic acid from glucose by a fermentation method, a production method using Pseudogluconovator saccharoketogenes Rh47-3 strain has been reported (Patent Document 7), but the fermentation method is an enzyme method. In comparison, the increase in contaminants in the culture solution reduces the purity of the target product, glucuronic acid, and has the problem of requiring a high degree of purification.
Further, as a method for producing glucuronic acid without using an enzyme, a method for producing glucuronic acid by converting starch into oxidized starch with nitric acid and then hydrolyzing with sulfuric acid is known (Non-Patent Document 1). However, this method has a problem that a large amount of reagents having a large environmental load such as nitric acid and sulfuric acid must be used.

It is known that when glucuronic acid binds to another compound via a glycosidic bond, that is, when it is glucuronidated, the produced glucuronide becomes more water-soluble and has a stronger physiological action than other compounds. In order to utilize this property, methods for glucuronidating compounds have been studied so far, and as an enzymatic method, for example, UDP-glucuronidation transferase present in the living body is used to transfer glucuronic acid to an arbitrary substrate. A method for glucuronidation has been reported (Non-Patent Document 2). Further, as a chemical synthesis method, for example,

methyl

2,3,4-tri-o-acetyl-α-D-glucuronate as a glucuronic acid donor and trimethylsilyl triflate as a Lewis acid catalyst are used for a target having a hydroxyl group to be glucuronidated. A method of imparting glucuronic acid to glucuronidation by reacting (Non-Patent Document 3) has been reported. However, the problems of both methods are that the reagent used for the reaction is expensive, the yield of the target glucuronide is low, and purification for the purpose of removing by-products after the reaction is required. ..

On the other hand, flavin-linked glucose dehydrogenase (flavin-linked GDH, EC 1.1.5.9) catalyzes the reaction of dehydrogenating (oxidizing) the hydroxy group at the 1-position of glucose using flavin as a coenzyme. It is an enzyme. Flavin-bound GDH from the genus Aspergillus is not affected by dissolved oxygen, has low activity on maltose and galactose, and has high substrate specificity on glucose, and is therefore used for measuring blood glucose concentration.

International Publication No. 2003/072742 International Publication No. 2013/183610 Japanese Unexamined Patent Publication No. 5-68541 JP-A-2009-165415 JP-A-2002-153294 U.S. Pat. No. 7,923,231 International Publication No. 2008/139844 International Publication No. 2006/101239

An object of the present invention is to provide a method for producing a glucuronic acid or a glucuronic acid derivative, which is simpler, lower cost, and less environmentally friendly than the existing method.

The present inventor diligently studied a method for directly producing glucuronic acid from glucose, which is an inexpensive raw material, and found that 6 of glucose was added to flavin-bound GDH, which is an enzyme that oxidizes glucose to glucono-1,5-lactone. Some have specificity for oxidation to the hydroxymethyl group at the position, and when the enzyme having glucose-6-dehydrogenase activity is allowed to act on glucose, the hydroxymethyl group at the 6-position of glucose is oxidized to specifically produce glucuronic acid. Found to generate. It was also found that when the enzyme is allowed to act on a glucose derivative such as glucoside, the hydroxymethyl group at the 6-position of the glucose skeleton is oxidized to specifically produce a glucuronic acid derivative.

That is, the present invention relates to the following [1] to [11].
[1] A method for producing glucuronic acid, which comprises a step of reacting glucose with a flavin-binding glucose dehydrogenase having glucose-6-dehydrogenase activity in the presence of a mediator to produce glucuronic acid.
[2] A method for producing a glucuronic acid derivative, which comprises a step of allowing a flavin-binding glucose dehydrogenase having a glucose-6-dehydrogenase activity to act on the 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 a glucuronic acid derivative according to any one of [1] to [3], wherein the flavin-binding glucose dehydrogenase is a protein according to any one of (i) to (iii) below. Manufacturing method:
(I) Protein having the amino acid sequence shown 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) 1 to 1 in the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38. Proteins with deleted, substituted or inserted amino acid sequences and glucose-6-dehydrogenase activity (iii) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, has an amino acid sequence having 80% or more sequence identity with the amino acid sequence, and has glucose-6-dehydrogenase activity. Protein to have.
[5] The method for producing glucuronic acid or a method for producing a glucuronic acid derivative according to any one of [1] to [4], wherein the flavin-bound glucose dehydrogenase has the following properties (1) to (8).
(1) Action: Catalyzing the reaction of dehydrogenating (oxidizing) the hydroxymethyl group at position 6 of glucose using flavin as a coenzyme (2) Solubility: Water-soluble (3) pH stability: At least pH 5.5 ~ Stable between 8.7 (4) Thermal stability: Stable at least at 35 ° C. (5) Substrate specificity: When the action on glucose is 100%, the action on maltose, xylose and galactose is 2.0. % Or less (6) Km value (against glucose): 30 mM or more (7) Molecular weight: 64-66 kDa (calculation from amino acid sequence after signal removal)
(8) Glucose oxidase activity: could not be detected.
[6] Flavin-bound glucose dehydrogenases are derived from microorganisms belonging to the genera Collettricum, Glomerella, Diaporte, Cuskia, Acremonium, Laciospaeris, Fusarium or Fiamoniosis [1]-[5] ] The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of.
[7] The method for producing glucuronic acid according to any one of [1] to [6], which uses a recombinant microorganism into which a gene encoding a flavin-binding glucose dehydrogenase is introduced as a flavin-binding glucose dehydrogenase. A method for producing a glucuronic acid derivative.
[8] The method for producing glucuronic acid or the production of a glucuronic acid derivative according to [7], wherein the gene encoding the flavin-binding glucose dehydrogenase is a gene consisting of any of the following DNAs (a) to (e). Method:
(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) 1 to 1 in 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. DNA encoding a protein having a base sequence in which several bases have been deleted, substituted or added, and having glucose-6-dehydrogenase activity.
(C) For 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. DNA encoding a protein having a nucleotide sequence having 80% or more sequence identity and having glucose-6-dehydrogenase activity
(D) Complementary to the nucleotide 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. DNA that hybridizes with DNA consisting of a unique base sequence under stringent conditions and encodes a protein having glucose-6-dehydrogenase activity.
(E) DNA encoding the following protein (i), (ii) or (iii)
(I) A protein having the amino acid sequence shown 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) 1 to 1 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. Protein with a few amino acid residues deleted, substituted or inserted and having glucose-6-dehydrogenase activity (iii) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, has an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown, and has glucose-6-dehydrogenase activity. Protein to have.
[9] The method for producing glucuronic acid or a method for producing a glucuronic acid derivative according to any one of [1] to [8], which further causes an oxidase to act.
[10] A flavin-linked glucose dehydrogenase having glucose-6-dehydrogenase activity, which is one of the following proteins (i) to (iii):
(I) A protein having the amino acid sequence shown 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) 1 to 1 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. Protein with a few amino acid residues deleted, substituted or inserted and having glucose-6-dehydrogenase activity (iii) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, has an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown, and has glucose-6-dehydrogenase activity. Protein to have.
[11] A catalyst for producing a glucuronic acid or a glucuronic acid derivative containing a flavin-binding glucose dehydrogenase protein having glucose-6-dehydrogenase activity.

According to the present invention, since glucuronic acid can be specifically produced directly from glucose, which is an inexpensive raw material, it is simpler than the existing method and the production cost is significantly reduced. In addition, since the reaction proceeds at room temperature and normal pressure without using a strong acid such as nitric acid or sulfuric acid, it is possible to avoid danger during manufacturing and reduce the environmental load.
Furthermore, since the glucuronic acid derivative can be specifically produced directly from the glucose derivative, it is simpler than the existing glucuronidation method, and the production cost is reduced. In addition, the effect of increasing the water solubility of the glucose derivative can be expected by oxidizing the hydroxymethyl group.

The thermal stability of CpGDH, FGDH and CsGDH is shown. The pH stability of CpGDH, FGDH and CsGDH is shown. The TLC analysis result of glucose oxide is shown. The results of HPLC analysis of glucose oxide are shown. The TLC analysis result of the substrate oxide is shown. The TLC analysis result of glucose oxide is shown. The thermal stability of CglGDH, CoGDH, CtoGDH, CgoGDH, GsGDH and DhGDH is shown. The pH stability of CglGDH, CoGDH, CtoGDH, CgoGDH, GsGDH and DhGDH is shown. The TLC analysis result of glucose oxide is shown. The thermal stability of Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH and CtaGDH is shown. FlaGDH, PcGDH, Fla_A. oGDH and Pc_A. Shows the thermal stability of oGDH. The pH stability of Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH and CtaGDH is shown. FlaGDH, PcGDH, Fla_A. oGDH and Pc_A. Shows the pH stability of oGDH. The ^{results of 1} H-NMR analysis and ¹³ C-NMR analysis of Piceid are shown. The results of ¹ H-NMR analysis and ¹³ C-NMR analysis of piceid oxide are shown.

In the present specification, the flavin-linked glucose dehydrogenase having a glucose-6-dehydrogenase activity (hereinafter, may be referred to as "flavin-linked GDH") is a hydroxy at the 6-position of glucose using flavin as a coenzyme. An enzyme that catalyzes the reaction of dehydrogenating (oxidizing) a methyl group. The flavin-bound 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 the carboxy group. Therefore, when the enzyme is allowed to act on glucose, it specifically acts. Produces glucuronic acid. Further, when the enzyme is allowed to act on a glucose derivative such as 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 causes glucose-6-dehydrogenase to act on glucose, and the reaction product is analyzed by thin layer chromatography or HPLC. It can be confirmed by comparing with.
The flavin-bound GDH of the present invention has substantially no glucose-1-dehydrogenase activity and substantially does not produce gluconic acid from the substrate glucose. Here, "substantial" means that the production of gluconic acid cannot be confirmed as a result of thin layer chromatography or HPLC analysis of the reaction product.

The flavin-bound 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 the amino acid sequence shown 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) 1 to 1 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. Protein with a few amino acid residues deleted, substituted or inserted and having glucose-6-dehydrogenase activity (iii) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, has an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown, and has glucose-6-dehydrogenase activity. Protein

One to several in the amino acid sequence set forth in 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 in an amino acid sequence in which an amino acid residue is deleted, substituted or inserted is determined by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, It is not limited as long as it exhibits the same enzymatic activity as the protein having the amino acid sequence shown by 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, but 1 to 20 is preferable. From 10 to 10 is even more preferred, and 1 to 8 is even more preferred.

As used herein, with the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38. The sequence identity of is 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, still more preferably 99% or more. The identity percentage of such sequences can be calculated using publicly available or commercially available software with an algorithm that compares the reference sequence as a query sequence. As an example, BLAST, FASTA, GENETYX (Genetics) and the like can be used.

The amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 are in order Colletotrichum. plurivorum) MAFF305790, fan gas · F5126 (Fungus_F5126), Colletotrichum sp. (Colletotrichum_sp.), Colletotrichum Gros Eos polio Lee Death (Colletotrichum gloeosporioides), Colletotrichum-Obikyurare (Colletotrichum orbiculare), Colletotrichum-Tofi Erudi meet (Colletotrichum tofieldiae), Colletotrichum Godetiae MAFF240289, Glomerella sp. RD057037, Diaporte Helianthi, Diaporte herianthi, Kuskia oryzae Khuskia oryzae), Rashiosupaerisu-Hasutou (Lasiosphaeris hirsute), Deer Porta CEA et sp. (Diaporthaceae sp.), Colletotrichum-Tanaketi (Colletotrichum tanaceti), Fusarium Lang Setia et (Fusarium langsethiae), Fiamonioshisu-Karubata (Phialemoniopsis curvata ) Is based on the genomic information. In addition, SEQ ID NOs: 36 and 38 are sequences in which the sequence presumed to be the secretory signal of SEQ ID NOs: 32 and 34 is replaced with the signal sequence of GDH derived from Aspergillus oryzae.
The amino acid sequence represented by SEQ ID NO: 4 or 6 is the same as SEQ ID NO: 2 of the known amino acid sequence Patent No. 6455714 and SEQ ID NO: 1 of Patent No. 5435180, SEQ ID NOs: 8, 10, 12, 18, 20. , 22, 24, 26, 28, 30, 32, and 34 are amino acid sequences registered in a known database, and the protein having the amino acid sequence has glucose-6-dehydrogenase activity. There are no reports of this.

The flavin-bound GDH of the present invention preferably has the following properties (1) to (8). Examples of flavin include flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), preferably FAD.
(1) Action: Catalyze 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: At least pH 5.5 ~ Stable between 8.6 This enzyme has a residual enzyme activity of 80% or more after treatment at 30 ° C. for 1 hour at least between pH 5.5 and 8.6. The pH stability is preferably pH 4.3 to 9.3, pH 5.5 to 8.7, pH 3.2 to 9.3, pH 5.5 to 9.3, pH 4.4 to 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 6.6, 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.
Even if the pH is the same, the residual activity may differ depending on the type of buffer solution.
(4) Thermal stability: Stable at at least 35 ° C. This enzyme is 100 mM potassium phosphate buffer (pH 6.0, 7.0 or 8.0) and 100 mM Tris-HCl buffer (pH 8) at at least 35 ° C. .0) After 60 minutes of treatment, it has 80% or more residual enzyme activity. It is preferably stable up to 40 ° C, 45 ° C, or 50 ° C.
(5) Substrate specificity: When the action on glucose is 100%, the action on maltose, xylose and galactose is 2.0% or less. This enzyme has low action on maltose, xylose and galactose and has low action on glucose. High substrate specificity. When the action on D-glucose of 50 mM is 100%, the action on maltose, D-xylose and D-galactose of 50 mM is 2.0% or less, preferably 0.3% or less, 0. 0.9% or less or 0.2% or less.
(6) Km value (against glucose): 30 mM or more The Km value for D-glucose is preferably 150 to 300 mM, 50 to 120 mM, or 30 to 80 mM.
(7) Molecular weight: 64-66 kDa (calculation from amino acid sequence after signal removal)
For the molecular weight of this enzyme, the secretory signal sequence of the enzyme is predicted at the signal sequence prediction site (SignalP-5.0, http://www.cbs.dtu.dk/services/SignalP/), and the predicted signal part is removed. The molecular weight is calculated from the amino acid sequence in this form.
(8) Glucose oxidase activity: undetectable

As described above, the flavin-bound GDH of the present invention acts specifically on the 6-position of glucose, and therefore has low activity on xylose. Therefore, this enzyme can be used for measuring glucose and is useful as an enzyme for a biosensor for measuring glucose.

The derived microorganism flavin-binding GDH of the present invention, Colletotrichum (Colletotrichum) genus (e.g., Colletotrichum plurivorum, Colletotrichum sp. (RD056779), Colletotrichum gloeosporioides, Colletotrichum orbiculare, Colletotrichum tofieldiae, Colletotrichum godetiae, Colletotrichum tanaceti), Guromerera genus ( For example, Glomerella sp. RD057037), Diaporte (eg, Diaporte helianthi), Kuskia (eg, Khuskia oryzae), Acremonium (eg, Acremonium strictum), Laciosperishisaria (eg, Lasia). (For example, Flavin langsetiae), microorganisms belonging to the genus Colletotrichum (for example, Colletotrichum curvata) and the like can be mentioned.
The flavin-bound GDH of the present invention is an enzyme derived from the above-mentioned microorganism (wild strain or mutant strain), a recombinant enzyme obtained by a genetic engineering method using a gene encoding the flavin-bound GDH of the present invention, and chemical synthesis. It may be any of the synthetic enzymes obtained by. It is preferably a recombinant enzyme.

The gene encoding the flavin-bound 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 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) 1 to 1 in 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. DNA encoding a protein having a base sequence in which several bases have been deleted, substituted or added, and having glucose-6-dehydrogenase activity.
(C) For 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. DNA encoding a protein having a nucleotide sequence having 80% or more sequence identity and having glucose-6-dehydrogenase activity
(D) Complementary to the nucleotide 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. DNA that hybridizes with DNA consisting of a unique base sequence under stringent conditions and encodes a protein having glucose-6-dehydrogenase activity.
(E) DNA encoding the following protein (i), (ii) or (iii)
(I) A protein having the amino acid sequence shown 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) 1 to 1 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. Protein with a few amino acid residues deleted, substituted or inserted and having glucose-6-dehydrogenase activity (iii) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, has an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown, and has glucose-6-dehydrogenase activity. Protein

One to several 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. In the base sequence in which bases are deleted, substituted or added, 1 to several bases are preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and 1 or 2. Is even more preferable. In addition, deletion of a base means loss or disappearance of a base, substitution of a base means that a base has been replaced with another base, and addition of a base means that a base has been added. means. "Addition" includes the addition of a base to one or both ends of a sequence and the insertion of another base between the bases in the sequence.

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

In the present specification, the stringent condition means a condition in which base sequences having high identity hybridize with each other and base sequences having lower identity do not hybridize with each other. The "stringent condition" can be appropriately changed depending on the level of identity to be sought. The higher the stringent conditions, the more identical sequences will hybridize. For example, as stringent conditions, the conditions described in Molecular Cloning: A Laboratory Manual (Second Edition, J. Sambrook et.al, 1989) can be mentioned. That is, 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 × denhart and 100 mg / mL herring sperm DNA. Conditions include conditions for constant temperature at 65 ° C. for 8 to 16 hours together with the probe for hybridization.

To prepare a flavin-bound GDH using the gene encoding the flavin-bound GDH of the present invention, for example, an expression vector containing the gene encoding the flavin-bound GDH of the present invention is introduced into a host cell such as a microorganism. The flavin-bound GDH of the present invention can be produced by culturing the transformed transformant. The transformant may carry the gene encoding the flavin-bound GDH of the present invention in the state of a vector, or the gene may be held in the genome.
The gene encoding the flavin-bound GDH of the present invention shall be prepared in an isolated state by using an arbitrary method used in the art such as the PCR method with reference to the gene sequence information disclosed in the present specification. Can be done.

The type of vector is not particularly limited, and examples thereof include vectors usually used for protein production, such as plasmids, cosmids, phages, viruses, YAC, and BAC. Among them, a plasmid vector is preferable, and a commercially available plasmid vector for protein expression, for example, pET or pBIC can be preferably used. Procedures for introducing genes into plasmid vectors are well known in the art.

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

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, using a medium containing a carbon source, an inorganic nitrogen source or an organic nitrogen source, an inorganic salt, and other necessary organic micronutrient sources that can be assimilated by microorganisms, under aerobic conditions such as aeration, stirring, and shaking. Can be done. The medium may be 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 culturing in consideration of productivity. For example, the culture temperature is preferably 10 ° C. to 40 ° C., and the culture period is preferably in the range of 2 to 14 days.

After culturing, the culture can be used, but it is preferably used after obtaining a culture supernatant by performing a separation operation such as centrifugation. Alternatively, it is used after obtaining microbial cells, crushing the microbial cells by an arbitrary method, and obtaining a supernatant from the crushed solution. Here, the culture may be a culture solution, microbial cells or a processed product thereof (lyophilized cells, acetone-dried cells, etc.). Further, it may be an immobilized enzyme or an immobilized bacterial cell immobilized by any method.
A known purification method can be used for the purification of the flavin-bound GDH of the present invention produced by the transformant. 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, and affinity chromatography.

The method for producing glucuronic acid of the present invention comprises a step of allowing glucose of the present invention to act on the flavin-bound GDH of the present invention in the presence of a mediator to produce glucuronic acid. In this step, the form of flavin-bound GDH is not particularly limited, and may be a crude enzyme, a purified enzyme, or a microorganism containing flavin-bound GDH. The microorganism containing the flavin-bound GDH is preferably a recombinant microorganism into which a gene encoding the flavin-bound GDH has been introduced. The microorganism containing the flavin-bound GDH is not limited to life or death, and also includes a treated product of the above-mentioned microbial cells.
The production of glucuronic acid is usually carried out in an aqueous medium. Examples of the aqueous medium include water, a buffer solution, a monohydric alcohol, and a dihydric alcohol.

Glucose as a substrate is usually D-glucose. In this step, the substrate concentration is preferably about 10 mM to 2 M.

As the mediator used in this step, a chemical substance having an excellent electron transfer ability can be used. Mediators are also referred to as electron carriers, electron acceptors, and redox mediators.
Mediators include osmium compounds (eg, osmium (ll) -2,2'-bipyridine complex), quinone compounds (eg, 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 (eg, phenazinemethsulfate, 1-methoxy-5-methylphena) Examples thereof include diummethylsulfate (methylene blue), ferricyanide (for example, potassium ferricyanide), flavonoid (quercetin dihydrate, hesperidin) and the like. Of these, tert-butylhydroquinone and 1-methoxy-5-methylphenadium methylsulfate are preferable.
The amount of the mediator used can be appropriately set depending on the type thereof, but is usually preferably about 0.5 mM to 50 mM.

The conditions under which the flavin-bound GDH of the present invention is allowed to act on glucose are not particularly limited as long as the flavin-bound GDH is not inactivated. In this step, the reaction proceeds at room temperature and normal pressure. Moreover, since the reaction proceeds under neutral to alkaline conditions, it is not necessary to use a strong acid in 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. be.
The reaction pH is preferably pH 5.0 to pH 9.0.

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.

In this step, it is preferable to further act on an oxidase from the viewpoint of reoxidizing the mediator. Examples of the oxidase include phenol oxidases such as laccase (EC 1.10.3.2) and peroxidase (EC 1.11.1.7).
Further, from the viewpoint of removing active oxygen in the reaction system, it is preferable to use catalase (EC 1.11.1.6).
The amount of these enzymes used is preferably about 0.25 U / mL to 500 U / mL as the final concentration.

In this way, glucuronic acid is specifically produced from the substrate glucose. On the other hand, gluconic acid is substantially not produced. Substantial means that when the reaction product is analyzed, the production of gluconic acid cannot be confirmed even by a method such as a gluconic acid measurement kit, thin layer chromatography, or HPLC. Therefore, according to this step, the purification load of glucuronic acid can be reduced. When glucuronic acid is isolated / purified, a known isolation / purification method can be used.
The produced glucuronic acid can be used as glucuronic acid or in the form of glucuronolactone, which is an intramolecular ester thereof, in pharmaceuticals, quasi-drugs, foods and the like.

The method for producing a glucuronic acid derivative of the present invention comprises a step of allowing a glucose derivative to act on a flavin-bound GDH of the present invention in the presence of a mediator to produce a glucuronic acid derivative. The flavin-bound GDH is similar to that described above.
The production of the glucuronic acid derivative is also usually carried out in an aqueous medium such as water, a buffer solution, a monohydric alcohol or a dihydric alcohol.

In the present 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 replaced with hydrogen. The glucose is preferably D-glucose.
Examples of the amino sugar and its N-acetylated product include glucosamine and N-acetylglucosamine.
Glucoside is a general term for glycosides in which a hemiacetal hydroxy group of glucose is ether-bonded to another compound, or glycosides in which saturated hydrocarbons such as glucose and octane are thioether-bonded with a sulfur atom sandwiched between them.
Examples of other compounds include aglycones, monosaccharides, disaccharides, polysaccharides of trisaccharides or higher, and the like. Aglycone is a non-sugar portion, and examples thereof include saturated hydrocarbons such as alcohol, phenol, phenylpropanoid, and octane.
As the glucoside, cellobiose, arbutin, piceid, methylglucoside, and octylthioglucoside are preferable.
Glucose analogs include 1,5-anhydro-D-glucitol, 2-deoxy-D-glucose and the like.

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

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

The conditions under which the flavin-bound GDH of the present invention is allowed to act on the glucose derivative are not particularly limited as long as the flavin-bound GDH is not inactivated. In this step as well, the reaction proceeds under 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. be.
The reaction pH is preferably pH 5.0 to pH 9.0.

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.

Also in this step, it is preferable to further act on an oxidase from the viewpoint of reoxidizing the mediator. Examples of the oxidase include phenol oxidases such as laccase (EC 1.10.3.2.) And peroxidase (EC 1.11.1.1.7).
Moreover, it is preferable to use catalase (EC 1.11.1.6) from the viewpoint of removing active oxygen.
The final concentration of these enzymes is preferably about 0.25 U / mL to 500 U / mL.

In this way, the hydroxymethyl group at the 6-position of the glucose skeleton of the glucose derivative as a substrate is oxidized to a carboxy 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 standard substance for a glucuronic acid conjugate produced in vivo, a raw material for pharmaceuticals, foods or cosmetics, a surfactant and the like.

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

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

(Glucose dehydrogenase (GDH) activity measurement method)
100 mM potassium phosphate buffer (pH 6.0) 1.00 mL, 1MD-glucose solution 1.00 mL, 3 mM 2,6-dichlorophenol indophenol (hereinafter referred to as “DCIP”) 0.14 mL and 3 mM 1-methoxy-5 -Methylphenadium Methylsulfate (hereinafter referred to as "1-m-PMS") 0.20 mL and ultrapure water 0.61 mL are mixed, kept warm at 37 ° C for 10 minutes, and then 0.05 mL of enzyme solution is added for reaction. Started.
For 5 minutes from the start of the reaction, the amount of decrease in absorbance (ΔA600) at 600 nm with the progress of the enzyme reaction was measured, and the enzyme activity was calculated from the linear portion 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.

Glucose dehydrogenase (GDH) activity (U / mL) = (-(ΔA600-ΔA600 blank) × 3.0 × enzyme dilution ratio) / (10.8 × 1.0 × 0.05)
In the formula, 3.0 is the liquid volume (mL) of the reaction reagent + enzyme solution, 10.8 is the molar extinction coefficient of DCIP at pH 6.0, and 1.0 is the optical path length (cm) of the cell, 0.05. Is the amount of the enzyme solution (mL), and ΔA600blank is the amount of decrease in the absorbance at 600 nm per minute when the diluted solution of the enzyme is added instead of the enzyme solution and the reaction is started.

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

(1) Cell culture Dextrin (Fuji Film Wako Junyakusha) 2% (w / v), Polypeptone (Fuji Film Wako Junyakusha) 1% (w / v), Potassium dihydrogen phosphate (Nacalai Tesque) A liquid medium consisting of 0.5% (w / v), magnesium sulfate heptahydrate (Nacalai Tesque) 0.05% (w / v) and water was prepared, and 10 mL was placed in a thick test tube at 121 ° C. , Autoclaved for 20 minutes. The GDH-producing bacteria were inoculated into a cooled liquid medium, cultured with shaking at 25 ° C. for 72 hours, and then wet cells were collected using bleaching.

(2) Isolation of total RNA After freezing 200 mg of wet cells obtained in (1) at −80 ° C., 100 μg of total RNA was extracted using ISOGENII (Nippon Gene).

(3) Preparation of cDNA library From the RNA obtained in (2), a cDNA library was prepared by a reverse transcription reaction using a reverse transcriptase and an oligo dT primer with an adapter sequence. As the reaction reagent, SMARTer RACE cDNA Amplification kit (Takara Bio Inc.) was used, and the reaction conditions were carried out according to the protocol described in the manual.

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

Based on the determined nucleotide sequence, we designed primers to elucidate the upstream and downstream sequences of the GDH gene. Using these primers, the total length of the GDH (hereinafter referred to as "CpGDH") gene derived from the Colletotrichum plerivorum MAFF305790 GDH strain was elucidated by the 5'RACE method and the 3'RACE method.

The elucidated CpGDH gene sequence optimized for 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.

(5) Preparation of an expression plasmid vector containing the CpGDH gene Aspergillus described in Known Document 1 (Heterogeneous gene expression system of the genus Aspergillus, Toshiki Minetoki, Chemistry and Biology, 38, 12, 831-838, 2000). A plasmid vector was prepared using an improved amylase-based promoter derived from Orize. First, a PCR product containing the CpGDH gene was obtained using the cDNA library obtained in (3) as a template. Next, using the PCR product as a template, a CpGDH gene for vector insertion was prepared.

Finally, the CpGDH gene prepared downstream of the promoter was ligated to prepare a plasmid vector capable of expressing the gene. The prepared expression plasmid vector was introduced into Escherichia coli JM109 strain and transformed. The obtained transformant was cultured, and a plasmid vector was extracted from the collected bacterial cells using illustra plasmidPrep MidiFlow Kit (GE Healthcare). When the sequence of the insert in the plasmid vector was analyzed, the nucleotide sequence containing the CpGDH gene was confirmed.

(6) Acquisition of Transformant Using the plasmid vector extracted in (5), Known Document 2 (Bioscii. Biotech. Biochem., 61 (8), 1367-1369, 1997) and Known Document 3 (Sake Aspergillus) A recombinant mold (Aspergillus oryzae) that produces CpGDH was prepared according to the method described in (Katsuya Gomi, Jyokyo, 494-502, 2000). The resulting recombinant strain was purified in Czapek-Dox solid medium.
Aspergillus oryzae NS4 strain was used as the host to be used. As described in Known Document 2, this strain was bred at a brewing laboratory in 1997, and is currently available for sale at the Liquor Research Institute.

(7) Confirmation of CpGDH derived from recombinant mold Dextrin (Fuji Film Wako Pure Pharmaceutical Co., Ltd.) 2% (w / v), Polypeptone (Fuji Film Wako Pure Pharmaceutical Co., Ltd.) 1% (w / v), Potassium dihydrogen phosphate (w / v) Prepare a liquid medium consisting of 0.5% (w / v) of Nacalai Tesque, 0.05% (w / v) of magnesium sulfate heptahydrate (Nacalai Tesque), and water, and add 10 mL to a thick test tube (Nakarai Tesque). It was placed in (22 mm × 200 mm) and autoclaved at 121 ° C. for 20 minutes. The transformant obtained in (6) was inoculated into a cooled liquid medium and cultured with shaking at 30 ° C. for 120 hours. After completion of the culture, the supernatant was collected by centrifugation, and the GDH activity was measured by the above-mentioned GDH activity measuring method. As a result, the CpGDH activity of the present invention was confirmed.

(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 transformant obtained in (6) was inoculated into a cooled liquid medium and cultured with shaking at 30 ° C. for 72 hours. After completion of the culture, 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, Advantech) to collect the culture supernatant.

The collected culture supernatant was purified by removing contaminating proteins using a TOYOPEARL DEAE-650S (Tosoh Corporation) column. The purified sample was concentrated with an ultrafiltration membrane having a molecular weight cut off of 10,000 and then replaced with water to obtain purified CpGDH. When the purified CpGDH was subjected to SDS-polyacrylamide gel electrophoresis, it was confirmed that it showed a single band.

(Acquisition of FGDH and CsGDH)
The gene sequence 1872 bp described in SEQ ID NO: 1 of Japanese Patent No. 6455714 and the gene sequence 1908 bp described in SEQ ID NO: 2 of Japanese Patent No. 5435180 were synthesized and expressed in Aspergillus oryzae NS4 strain by the same method as in Example 1, respectively. 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 FGDH gene sequence and amino acid sequence are set forth in SEQ ID NOs: 3 and 4, and the CsGDH gene sequence and amino acid sequence are set forth in SEQ ID NOs: 5 and 6.

[Example 2]
(Examination of enzymatic chemistry of each GDH)
The properties of CpGDH, FGDH and CsGDH obtained in Example 1 were investigated.
(1) Measurement of Absorption Spectrum For each GDH obtained in Example 1, the absorption spectrum at 300-600 nm before and after the addition of D-glucose was measured using a plate reader (SpectraMax Plus384, Molecular Device Co., Ltd.). As a result, the absorption maximums observed near the wavelength of 360-380 nm and the wavelength of 450-460 nm disappeared by the addition of D-glucose, and it was clarified that all GDHs were flavin-bound proteins.

(2) Measurement of glucose oxidase (GOD) activity 1M potassium phosphate buffer (pH 7.0) 0.2 mL, 1M D-glucose 2.0 mL, 25 mM 4-aminoantipyrine 0.2 mL, 420 mM phenol 0.2 mL, 0.2 mL of 1 mg / mL peroxidase (derived from Fuji Film Wako Pure Chemical Industries, Ltd., Western Wasabi) and 0.2 mL of ultrapure water were mixed, 0.1 mL of the mixed solution was placed in a 96-well plate, and the mixture was kept warm at 25 ° C. for 5 minutes. 0.1 mL of each GDH obtained in Example 1 was added, and the reaction was started. For 5 minutes from the start of the reaction, the change in absorbance at 500 nm with the progress of the enzyme reaction was measured with the plate reader to examine the GOD activity. The control started the reaction by adding water or GOD derived from Aspergillus niger (Nacalai Tesque) instead of GDH. As a result, a change in absorbance at 500 nm was confirmed in the control to which GOD derived from Aspergillus niger was added, but no change in absorbance was observed in the GDH of the present invention as in the control to which water was added. Therefore, it was confirmed that all GDHs are dehydrogenases that do not utilize oxygen as an electron acceptor.

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

When the activity against D-glucose is 100%, the activity against maltose, D-xylose or D-galactose is 0.3%, 0.4% or 0.2% or less in any of the GDHs. Was less than 2.0%.

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

As a result, CpGDH was 196 mM, CsGDH was 50 mM, and FGDH was 52 mM. Since the Km value is likely to fluctuate depending on the measurement method and the calculated plot, it is considered that the Km of CpGDH is 150 mM to 300 mM, the Km of FGDH is 30 mM to 80 mM, and the Km of CsGDH is 30 mM to 80 mM.

(5) Thermal stability When each GDH was prepared at 6 U / mL and treated in 100 mM potassium phosphate buffer (pH 8.0) at each temperature for 60 minutes, the enzyme activity before treatment was set to 100%. The temperature range in which the residual enzyme activity of CpGDH is 80% or more is up to 40 ° C. for CpGDH, up to 40 ° C. for FGDH, and up to 45 ° C. for CsGDH (see FIG. 1).

(6) Stable pH range The stable pH of each GDH was examined. Each GDH is adjusted to 6 U / mL, and the final concentration is 100 mM sodium citrate buffer (pH 2.2-7.0), 100 mM potassium acetate buffer (pH 3.0-6.0), 100 mM potassium phosphate. Buffer solution (pH 6.0-8.0), 100 mM Tris-HCl buffer solution (pH 7.0-9.0), 100 mM glycine-NaOH buffer solution (pH 9.0-10.0). After addition and treatment at 30 ° C. for 1 hour at each pH, the residual activity of each GDH was measured. As a result, the pH range in which the residual enzyme activity value of each GDH is 80% or more when the enzyme activity value before treatment is 100% is pH 4.3 to 9.3 for CpGDH and pH 5.5 to 8. for FGDH. 7. The pH of CsGDH was 3.2 to 9.3 (see FIG. 2). From the above, it was found that the GDH of the present invention is stable in the range of at least pH 5.5 to 8.7. Even if the pH is the same, the residual activity may differ depending on the type of buffer solution.

[Example 3]
(Analysis of glucose oxide 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 oxide. The preparation method and various analysis methods are described below.

(1) Preparation of glucose oxide 1M potassium phosphate buffer (pH 7.0) 0.04 mL, 2MD-glucose solution 0.01 mL, 0.2M 1-m-PMS solution 0.02 mL, 10,000 U / mL Catalase (Fuji Film Wako Pure Chemical Industries, Ltd., derived from bovine liver) 0.02 mL, 90 U / mL CpGDH, FGDH or CsGDH 0.08 mL, ultrapure water 0.23 mL are mixed, inverted and mixed overnight at room temperature, and then in the reaction vessel. The air was replaced and the mixture was inverted and mixed for several hours to obtain a reaction product containing a glucose oxide.

(2) Thin-layer chromatography analysis of glucose oxide Using the reaction product obtained in (1) as a sample, thin-layer chromatography (hereinafter referred to as TLC) analysis was performed. 0.001 mL of the sample was added dropwise onto a silica gel plate (Merck Millipore, Silicon gel60 F254), dried, and developed with acetonitrile / ultrapure water (70:30) for 10 minutes. As a result of drying the silica gel plate, spraying concentrated sulfuric acid / ethanol (5:95) and then heating, spots were confirmed at the same positions as the glucuronic acid preparation in the CpGDH, FGDH and CsGDH reaction products, and D-glucose was observed. No spots were found at the same positions as the standard and gluconic acid standard. On the other hand, as a comparative example, GDH derived from Aspergillus terreus (Patent Document 8), which is a well-known GDH, was purified, and the same test was performed. No spots were detected, and spots were detected at the same positions as the gluconic acid preparation. From this, it was confirmed that general GDH oxidizes the 1-position of glucose to produce gluconic acid, whereas CpGDH, FGDH, and CsGDH oxidize the 6-position of glucose to produce glucuronic acid. Was done.

(3) Examination of Glucose Oxidation under Various Conditions Among the conditions described in (1), the buffer solution to be used is 1M sodium phosphate buffer (pH 7.0 or 8.0) or 1M potassium phosphate buffer (pH 8). Reaction containing glucose oxide under the condition changed to 0.0), the condition where the amount of D-glucose added was changed to double the amount, or the condition where the amount of 1-m-PMS added was changed to 0.0025 mL or 0.005 mL. As a result of preparing the products and performing TLC analysis, spots were confirmed at the same positions as the glucuronic acid preparations in the CpGDH, FGDH, and CsGDH reaction products under all conditions, and the D-glucose preparation and the gluconic acid were found. No spot was found at the same position as the standard.

(4) High Performance Liquid Chromatography Analysis of Glucose Oxide In order to remove 1-m-PMS from the CpGDH reaction product obtained in (1), powdered activated carbon washed with ultrapure water after crushing (Futamura Chemical Co., Ltd.) , Taiko activated carbon (SG) was added in an amount of 2 to 20 mg, and the mixture was allowed to stand at room temperature for 5 minutes. Since 1-m-PMS was adsorbed on 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 GlyScop ABEE labeling kit (J-Chemical, Inc.), HPLC analysis was performed using the sugar analysis column Honenpack C18 (J-Chemical, Inc.). -No glucose peak was observed, and a peak was confirmed at the same position as the glucuronic acid preparation (see FIG. 4). Fluorescent labeling and HPLC analysis are performed according to the protocol described in the kit manual. The HPLC analyzer is the SIL-10A series (Shimadzu Corporation), and the detector is the fluorescence detector RF-10AXL (excitation wavelength). / Fluorescent wavelength = 305/360 nm, Shimadzu Corporation) was used.

(5) Analysis of glucose oxide using a nuclear magnetic resonance (NMR) device The CpGDH reaction product from which 1-m-PMS was removed in (4) was pre-frozen at -40 ° C and freeze-dried (EYELA, FREEZE DRYER). It was freeze-dried using 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 6-position of glucose was converted to a carboxy group was obtained, so that glucuronic acid was selectively selected from glucose. It was confirmed that it was produced in Japan.

(6) Analysis using gluconic acid measurement kit F-kit D-gluconic acid to confirm whether gluconic acid is contained in the CpGDH reaction product from which 1-m-PMS obtained in (4) has been removed. / The activity was measured using gluconolactone (JK International Co., Ltd.). As a result of evaluation according to the protocol described in the instruction manual, accurate gluconic acid concentration could be measured in the gluconic acid preparation used as a control, but gluconic acid was not detected in the CpGDH reaction product.

(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) has been removed, a glucuronic acid measurement kit (Megazyme, K-) The analysis was performed using URONIC). As a result of evaluation according to the protocol described in the instruction manual, it was confirmed that glucuronic acid was produced.

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

(1) Preparation of glucose oxide using TBHQ 1M sodium phosphate buffer (pH 8.0) 0.02 mL, 1MD-glucose solution 0.01 mL, 1M TBHQ (Tokyo Kasei Kogyo Co., Ltd.) 0.002 mL, 10 U / mL Lacquerze (Sigma-Aldrich, derived from Aspergillus genus) 0.005 mL, 90 U / mL CpGDH 0.04 mL, ultrapure water 0.123 mL are mixed and mixed overnight at room temperature in a light-shielded state, and a reaction containing glucose oxide is mixed. Obtained the product. Further, among the above compositions, a reaction product containing glucose oxide was also obtained under the condition that the laccase concentration was changed to 1/5.

(2) TLC analysis of glucose oxide As a result of performing TLC analysis in the same manner as in (2) of Example 3, a spot was confirmed at the same position as the glucuronic acid standard in the CpGDH reaction product using TBHQ as a mediator. Nothing was detected at the same position as the glucose and gluconic acid preparations.

(3) Examination of glucose oxidation when peroxidase and catalase are added 1M sodium phosphate buffer (pH 8.0) 0.02mL, 1MD-glucose solution 0.01mL, 1M TBHQ (Tokyo Kasei Kogyo Co., Ltd.) 0.002mL, 10,000 U / mL catalase (Fuji Film Wako Pure Pharmaceutical Co., Ltd., derived from bovine liver) 0.01 mL, 100 U / mL Western wasabi peroxidase (Fuji Film Wako Pure Pharmaceutical Co., Ltd., derived from Western Wasabi) 0.005 mL, 90 U / mL CpGDH 0.04 mL , A reaction solution mixed with 0.113 mL of ultrapure water was prepared and reacted, and as a result of TLC analysis of the reaction product, a spot was confirmed at the same position as the glucuronic acid standard as in (1), and a glucose standard was confirmed. Nothing was detected at the same position as the product and the gluconic acid standard.

(4) Analysis using a gluconic acid measurement kit As a result of analysis using a D-gluconic acid / gluconolactone measurement kit (F-kit) in the same manner as in (6) of Example 3, a reaction system using TBHQ as a mediator. However, it was shown that CpGDH did not produce gluconic acid.

(5) Analysis using a glucuronic acid measurement kit As a result of analysis using a glucuronic acid measurement kit (K-URONIC) in the same manner as in (7) of Example 3, glucose used in the reaction in the reaction system using TBHQ as a mediator It was confirmed that the same amount of glucuronic acid as was produced.

[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 oxide. The preparation method and analysis method are described below.

(1) Preparation of glucose oxide using butyl hydroxyanisole 1M sodium phosphate buffer (pH 8.0) 0.02 mL, 1M D-glucose solution 0.01 mL, 0.1M butyl hydroxyanisole (Fuji Film Wako Pure Chemical Industries, Ltd.) 0.02 mL, 10,000 U / mL catalase (Fuji Film Wako Junyaku Co., Ltd., derived from bovine liver) 0.01 mL, 10 U / mL lacquerase (Sigma-Aldrich, derived from Aspergillus genus) 0.005 mL, 90 U / mL CpGDH0 .04 mL and 0.095 mL of ultrapure water were mixed and mixed by inversion overnight at room temperature and in a light-shielded state to obtain a reaction product containing glucose oxide.

(2) TLC analysis of glucose oxide As a result of performing TLC analysis in the same manner as in (2) of Example 3, a spot was found at the same position as the glucuronic acid preparation in the CpGDH reaction product prepared using butylhydroxyanisole as a mediator. It was confirmed that nothing was detected at the same position as the glucose and gluconic acid preparations.

[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.

(1) Preparation of substrate oxide D- (+)-cellobiose, α-albutin, β-albutin, piseide, N- with a final concentration of 100 mM potassium phosphate buffer (pH 8.0) and 13 to 100 mM as a substrate. The final concentration is 14 U / mL in a reaction solution consisting of acetyl-d-glucosamine, 10 mM 1-m-PMS as a mediator, and 500 U / mL catalase (from Fuji Film Wako Pure Chemical Industries, bovine liver) as a remover for active oxygen. CpGDH was added and mixed by inversion overnight at room temperature. Then, the air in the reaction vessel was replaced, and the mixture was further mixed by inversion at room temperature for several hours to obtain a substrate oxide containing a glycoside oxide or the like.

(2) TLC analysis of 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 FIG. 5). This indicates that CpGDH oxidizes glucose residues in glycosides.

[Example 7]
(Acquisition of CglGDH, CoGDH, CtoGDH, CgoGDH, GsGDH and DhGDH)
Instead of Colletotrichum plurivorum MAFF305790, Colletotrichum gloeosporioides, Colletotrichum orbiculare, Colletotrichum tofieldiae, using Colletotrichum godetiae MAFF240289, Glomerella sp. RD057037 or Diaporthe Helianthi, were each expressed in Aspergillus oryzae NS4 strain in the same manner as in Example 1, purified did. Colletotrichum gloeosporioides origin of the purified enzyme CglGDH, Colletotrichum orbiculare origin of the purified enzyme CoGDH, Colletotrichum tofieldiae origin of the purified enzyme CtoGDH, Colletotrichum godetiae MAFF240289 origin of the purified enzyme CgoGDH, Glomerella sp. RD057037 GsGDH the purified enzyme derived from, Diaporthe helianthi The derived purified enzyme was designated as DhGDH. The respective gene sequences are set forth in SEQ ID NOs: 7, 9, 11, 13, 15 and 17, and the amino acid sequences are set forth in SEQ ID NOs: 8, 10, 12, 14, 16 and 18.

(Examination of enzymatic chemical properties)
(1) Measurement of Glucose-6-Dehydrogenase Activity Glucose oxides were prepared using CglGDH, CoGDH, CtoGDH, CgoGDH, GsGDH and DhGDH under the conditions described in Example 4, and as a result of TLC analysis, these glucoses were obtained. A spot was found on the oxide 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 FIG. 6).

(2) Substrate specificity The activity of each GDH on each substrate was measured by the same method as in Example 2. The results are shown in Table 3.

When the activity against D-glucose was 100%, the activity against maltose or D-xylose was 0.2% or 0.9% or less, and both were 2.0% or less.

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

Since the Km value tends to fluctuate depending on the measurement method and the calculated plot, the Km of CglGDH is 400 mM to 900 mM, the Km of CoGDH is 250 mM to 650 mM, the Km of CtoGDH is 400 mM to 900 mM, and the Km of CgoGDH is 200 mM to 450 mM. The Km of GsGDH is considered to be 250 mM to 650 mM, and the Km of DhGDH is considered to be 60 mM to 140 mM.

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

(5) Stable pH range As a result of examining the stable pH of each GDH by the same method as in Example 2, the residual enzyme activity value of each GDH is 80% or more when the enzyme activity value before treatment is 100%. The pH range is 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, and pH 5. The pH was 0 to 9.3 and pH 3.3 to 9.6 at DhGDH (see FIG. 8). From the above, it was found that the GDH of the present invention is stable in the range of at least pH 5.5 to 9.3.

[Example 8]
(Acquisition of Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH, CtaGDH, FlaGDH, PcGDH, Fla_A.oGDH and Pc_A.oGDH)
Instead of Colletotrichum plurivorum MAFF305790, Khuskia oryzae, Acremonium strictum, Lasiosphaeris hirsute, Diaporthaceae sp., Colletotrichum tanaceti, Fusarium langsethiae, acquires high sequence information from the genome data of CpGDH amino acid sequence identity that is published in Phialemoniopsis curvata, After optimizing the codon frequency of Aspergillus oryzae, each was expressed and purified in Aspergillus oryzae NS4 strain by the same method as in Example 1. In addition, a sequence in which the signal sequence portion of GDH derived from Fusarium langsetsiae and Phialemoniopsis curvata was replaced with the signal sequence of GDH derived from Aspergillus oryzae was expressed and purified in the Aspergillus oryzae NS4 strain by the same method as in Example 1.
Purified enzymes derived from Khuskia oryzae are Ko37 GDH and Ko38 GDH, purified enzymes derived from Acremonium strikeum are AsGDH, purified enzymes derived from Lasiospheres hilsute are derived from LhGDH, diaporthease The purified enzyme derived from FlaGDH, the purified enzyme derived from Hiersutism curvata was replaced with PcGDH, and the deduced signal sequence portion of the enzyme derived from Fusarium langesethia was replaced with the signal sequence of GDH derived from Aspergillus oryzae. pc_A. A purified enzyme in which the estimated signal sequence portion of the enzyme derived from oGDH and Phialemoniopsis curvata was replaced with the signal sequence of GDH derived from Aspergillus oryzae. It was set to oGDH.
Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH, CtaGDH, FlaGDH, PcGDH, Fla_A. oGDH, Pc_A. The gene sequence optimized for the codon frequency of Aspergillus oryzae in oGDH is set forth in SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and the amino acid sequence is shown in SEQ ID NOs: 20, 22, 24. , 26, 28, 30, 32, 34, 36, 38, respectively.

(Examination of enzymatic chemical properties)
(1) Measurement of Glucose-6-Dehydrogenase Activity Under the conditions described in Example 4, Ko37GDH, AsGDH, Ko38GDH, LhGDH, DsGDH, CtaGDH, FlaGDH, PcGDH, Fla_A. oGDH and Pc_A. As a result of preparing glucose oxides using oGDH and performing TLC analysis, spots of these glucose oxides were confirmed at the same positions as the glucuronic acid preparations, and at the same positions as the glucose preparations and the gluconic acid preparations. Nothing was detected (see Figure 9).

(2) Substrate specificity The activity of each GDH on each substrate was measured by the same method as in Example 2. The results are shown in Table 5.

When the activity against D-glucose was 100%, the activity against maltose or D-xylose was 0.5% or 0.2% or less, and both were 2.0% or less.

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

Since the Km value tends to fluctuate depending on the measurement method and the calculated plot, the Km of Ko37GDH is 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, and the Km of DsGDH is 150 mM to. 300 mM, Km of CtaGDH is 110 mM to 240 mM, Km of FlaGDH is 900 mM to 1,600 mM, Fla_A. The Km of oGDH is considered to be 600 mM to 1,200 mM.

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

(5) Stable pH range As a result of examining the stable pH of each GDH by the same method as in Example 2, the residual enzyme activity value of each GDH is 80% or more when the enzyme activity value before treatment is 100%. The pH range of Ko37GDH is 5.5 to 8.6, AsGDH is pH 4.3 to 9.6, Ko38GDH is pH 5.0 to 9.6, LhGDH is pH 4.0 to 8.6, and DsGDH is pH 4. 9 to 8.7, pH 3.3 to 9.9 for CtaGDH, pH 4.4 to 9.8 for FlaGDH, pH 4.0 to 8.8 for PcGDH, Fla_A. pH 4.4-9.8 at oGDH, Pc_A. The pH was 4.0 to 9.6 in oGDH (see FIG. 11).

[Example 9]
(Analysis of Piceid Oxide Using Nuclear Magnetic Resonance Device (NMR))
The purified CpGDH was used to obtain the oxide of piceid. The preparation method, ^{1 1} H-NMR analysis and ¹³ C-NMR analysis results are described below.

(1) Preparation of substrate oxide A final concentration of 20 mM potassium phosphate buffer (pH 7.0), a piseide powder equivalent to 50 mM as a substrate, 10 mM TBHQ as a mediator, and 200 U / mL catalase (Fuji film) as a remover for active oxygen. CpGDH was added to a reaction solution consisting of Wako Junyakusha, derived from bovine liver) and 2 U / mL lacquerase (derived from Sigma-Aldrich, Aspergillus genus) so that the final concentration was 14 U / mL, and the pH in the reaction system was 6 While adding 1N of NaOH so as to have a pH of .5 to 7.0, the mixture was stirred while being aerated at room temperature to obtain a piseide oxide.

(2) ¹ H-NMR analysis and ¹³ C-NMR analysis of piceid oxide The piceid oxide obtained in (1) was purified using a C18 column to remove impurities, and then the same as in Example 3. The structure was identified by nuclear magnetic resonance analysis according to the above method. On the other hand, as a comparative example, the same analysis was performed on piceid.
^From the results of 1 H-NMR and ¹³ C-NMR, it was confirmed that the hydroxymethyl group at the C6 position of the glucose residue of piceid was a carboxy group, and that the other structures were common. (See FIGS. 12 and 13). This indicates that CpGDH specifically oxidizes glucose residues in piceid.

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

(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 a glucose spot could not be confirmed. From this, it was shown that under the condition (1), the entire amount of glucose used as a substrate could be converted into glucuronic acid.
[Example 11]
(Large amount of piceid oxide preparation)
A large amount of piceid oxide was obtained using purified CpGDH. The preparation method is described below.

(1) Preparation of piceid oxide (polyethylene glycol, ethanol addition)
Final concentration 20 mM sodium phosphate buffer (pH 7.0), 200 mM equivalent piseide powder as substrate, 10 mM TBHQ as mediator, 500 U / mL catalase as active oxygen remover (Fuji Film Wako Junyakusha, derived from bovine liver) ), 10% (v / v) polyethylene glycol (molecular weight: 200, Nacalai Tesque), 10% (v / v) ethanol, 50 mU / mL lacquerase (Sigma-Aldrich, derived from Aspergillus). CpGDH was added so that the concentration became 20 U / mL, and 1N NaOH was added so that the pH in the reaction system became 6.5 to 7.0, and the mixture was stirred for 165 hours while aerating at room temperature. It was confirmed that almost all of the substrate, Piseide, was oxidized.

(2) Analysis of Piceid Oxide As a result of TLC analysis of the piceid oxide prepared under the condition of (1), a spot was confirmed at the same position as the piceid oxide obtained by NMR analysis in Example 9. In addition, nothing was detected in the spot at the same position as the piceid standard analyzed in Example 9. From this, it was confirmed that, under the condition (1), the total amount of piceid, which is a substrate, is a piceid oxide.

(3) Preparation of piceid oxide (addition of ethanol)
Final concentration 20 mM sodium phosphate buffer (pH 7.0), 50 mM equivalent piseide powder as substrate, 10 mM TBHQ as mediator, 500 U / mL catalase as active oxygen remover (Fuji Film Wako Junyakusha, derived from bovine liver) ), 10% (v / v) ethanol, 0.13 mU / mL CpGDH was added to a reaction solution consisting of lacquerase (Sigma-Aldrich, derived from the genus Aspergillus) so that the final concentration was 20 U / mL, and the mixture was added to the reaction system. Piseido oxide was obtained by stirring overnight while aerating at room temperature while adding 1N NaOH so that the pH became 6.5 to 7.0. Further, the piceid oxide could be obtained in the same manner under the condition that the final concentration of ethanol was changed to 20% or 30%.

(4) Analysis of Piceid Oxide As a result of TLC analysis of the piceid oxide prepared under the condition of (3), a spot was confirmed at the same position as the piceid oxide obtained by NMR analysis in Example 9. In addition, nothing was detected in the spot at the same position as the piceid standard analyzed in Example 9. From this, it was confirmed that, under the condition (3), the total amount of piceid, which is a substrate, is a piceid oxide.

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

(6) Analysis of Piceid Oxide As a result of TLC analysis of the piceid oxide prepared under the condition of (5), a spot was confirmed at the same position as the piceid oxide obtained by NMR analysis in Example 9. In addition, nothing was detected in the spot at the same position as the piceid analyzed in Example 9. Further, among the conditions described in (5), the glycine-NaOH buffer solution (pH 10) was changed to a sodium phosphate buffer solution (pH 7.0), and 3.2 mU / mL laccase (Sigma-Aldrich, Aspergillus). Under the condition of adding (origin), it was confirmed by analysis with TLC that the substrate, piseide, remained even after the reaction for the same time. From this, it was confirmed that under the alkaline condition of (5), the total amount of piceid, which is a substrate, was rapidly converted to a piceid oxide as compared with the case of the neutral condition.

(7) Preparation of piceid oxide (mediator natural oxidation)
Add CtaGDH to a reaction solution consisting of a final concentration of 20 mM glycine-NaOH buffer (pH 10.0), a piceid powder equivalent to 50 mM as a substrate, and 10 mM TBHQ as a mediator so that the final concentration is 20 U / mL, and aerate at room temperature. The mixture was stirred for 1 hour to obtain piceid oxide.

(8) Analysis of Piceid Oxide As a result of TLC analysis of the piceid oxide prepared under the condition of (7), a spot was confirmed at the same position as the piceid oxide obtained by NMR analysis in Example 9. In addition, nothing was detected in the spot at the same position as the piceid analyzed in Example 9. From this, it was confirmed that, under the condition (7), the total amount of piceid, which is a substrate, was quickly converted into a piceid oxide. In addition, since the entire amount of piceid, which is a substrate, was converted to piceid oxide without the addition of laccase, it was confirmed that the mediator was rapidly and automatically reoxidized under alkaline conditions.

[Example 12]
(Analysis of arbutin oxide)
Arbutin oxide was obtained using purified CpGDH and its glucuronic acid residue was analyzed.

(1) Preparation of albutine oxide A final concentration of 20 mM sodium phosphate buffer (pH 7.0), 50 mM albutine as a substrate, 10 mM TBHQ as a mediator, and 500 U / mL catalase as a remover for active oxygen (Fuji Film Wako Jun). CtaGDH was added to a reaction solution consisting of 30 mU / mL lacquerase (derived from Sigma-Aldrich, Aspergillus) to a final concentration of 20 U / mL, and the pH in the reaction system was 6.5. While adding 1N NaOH so as to be ~ 7.0, the mixture was aerated at room temperature and stirred for 48 to 72 hours to obtain an arbutine oxide.

(2) Sugar cleavage of arbutin oxide With respect to the arbutin oxide prepared under the condition of (1), trifluoroacetic acid is added so that the final concentration becomes 4M, and the mixture is heated at 100 ° C. for 3 hours, and then the treated solution is finished. Isopropanol was added to a concentration of 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 analysis of the cleaved sugar moiety.

(3) Analysis of sugar residues cleaved from arbutin oxide As a result of performing TLC analysis on sugar residues derived from arbutin oxide prepared under the conditions of (2) in the same manner as in (2) of Example 3, glucuron A spot was confirmed at the same position as the acid standard, and nothing was detected at the same position as the glucose standard and the gluconic acid standard. From this, it was confirmed that the glucose residue was converted to glucuronic acid in arbutin by using CpGDH.

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

(1) Solubility of Piceid Oxide in Water As a result of dissolving the piceid powder in water so that the final concentration was 2 mM, the entire amount was not completely dissolved, and the powder remained. On the other hand, the piceid oxide prepared in (1) of Example 11 was purified on a C18 column, and the solubility of the obtained purified piceid oxide in water was confirmed. As a result, the final concentration was about 200 mM. However, the added piceid oxide was completely dissolved. From this, it was confirmed that the solubility in water was improved by at least 100 times or more by converting the glucose residue of piceid to the glucuronic acid residue.

Claims

A method for producing glucuronic acid, which comprises a step of reacting glucose with a flavin-binding glucose dehydrogenase having glucose-6-dehydrogenase activity in the presence of a mediator to produce glucuronic acid.
A method for producing a glucuronic acid derivative, which comprises a step of allowing a flavin-binding 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.
The method for producing a glucuronic acid derivative according to claim 2, wherein the glucose derivative is an amino sugar or an N-acetylated product thereof, a glucoside, or a glucose analog.
The method for producing glucuronic acid or a method for producing a glucuronic acid derivative according to any one of claims 1 to 3, wherein the flavin-binding glucose dehydrogenase is the protein according to any one of (i) to (iii) below:
(I) A protein having the amino acid sequence shown 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) 1 to 1 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. Protein with a few amino acid residues deleted, substituted or inserted and having glucose-6-dehydrogenase activity (iii) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, has an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown, and has glucose-6-dehydrogenase activity. Protein to have.
The method for producing glucuronic acid or a method for producing a glucuronic acid derivative according to any one of claims 1 to 4, wherein the flavin-bound glucose dehydrogenase has the following properties (1) to (8).
(1) Action: Catalyzing the reaction of dehydrogenating (oxidizing) the hydroxymethyl group at position 6 of glucose using flavin as a coenzyme (2) Solubility: Water-soluble (3) pH stability: At least pH 5.5 ~ Stable between 8.7 (4) Thermal stability: Stable at least at 35 ° C. (5) Substrate specificity: When the action on glucose is 100%, the action on maltose, xylose and galactose is 2.0. % Or less (6) Km value (against glucose): 30 mM or more (7) Molecular weight: 64-66 kDa (calculation from amino acid sequence after signal removal)
(8) Glucose oxidase activity: could not be detected.
Any one of claims 1 to 5 from which the flavin-bound glucose dehydrogenase is derived from a microorganism belonging to the genera Collettricum, Glomerella, Diaporte, Cuskia, Acremonium, Laciospaeris, Fuzarium or Fiamoniosis. The method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to the above item.
The method for producing glucuronic acid or a glucuronic acid derivative according to any one of claims 1 to 6, wherein a recombinant microorganism into which a gene encoding a flavin-binding glucose dehydrogenase is introduced is used as the flavin-binding glucose dehydrogenase. Production method.
The method for producing glucuronic acid or a method for producing a glucuronic acid derivative according to claim 7, wherein the gene encoding the flavin-binding glucose dehydrogenase is a gene consisting of any 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) 1 to 1 in 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. DNA encoding a protein having a base sequence in which several bases have been deleted, substituted or added, and having glucose-6-dehydrogenase activity.
(C) For 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. DNA encoding a protein having a nucleotide sequence having 80% or more sequence identity and having glucose-6-dehydrogenase activity
(D) Complementary to the nucleotide 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. DNA that hybridizes with DNA consisting of a unique base sequence under stringent conditions and encodes a protein having glucose-6-dehydrogenase activity.
(E) DNA encoding the following protein (i), (ii) or (iii)
(I) A protein having the amino acid sequence shown 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) 1 to 1 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. Protein with a few amino acid residues deleted, substituted or inserted and having glucose-6-dehydrogenase activity (iii) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, has an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown, and has glucose-6-dehydrogenase activity. Protein to have.
Further, the method for producing glucuronic acid or the method for producing a glucuronic acid derivative according to any one of claims 1 to 8, wherein an oxidase is allowed to act.
A flavin-bound glucose dehydrogenase having glucose-6-dehydrogenase activity, which is one of the following proteins (i) to (iii):
(I) A protein having the amino acid sequence shown 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) 1 to 1 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. Protein with a few amino acid residues deleted, substituted or inserted and having glucose-6-dehydrogenase activity (iii) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 , 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, has an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown, and has glucose-6-dehydrogenase activity. Protein to have.
A catalyst for producing glucuronic acid or a glucuronic acid derivative containing a flavin-binding glucose dehydrogenase protein having glucose-6-dehydrogenase activity.