WO2021149587A1 - Procédé de production d'acide glucuronique - Google Patents

Procédé de production d'acide glucuronique Download PDF

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WO2021149587A1
WO2021149587A1 PCT/JP2021/001086 JP2021001086W WO2021149587A1 WO 2021149587 A1 WO2021149587 A1 WO 2021149587A1 JP 2021001086 W JP2021001086 W JP 2021001086W WO 2021149587 A1 WO2021149587 A1 WO 2021149587A1
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glucose
glucuronic acid
amino acid
flavin
seq
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PCT/JP2021/001086
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Japanese (ja)
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昂洸 藤井
高史 宅見
俊雄 荒木
本田　通済
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池田食研株式会社
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Priority to JP2021573110A priority Critical patent/JPWO2021149587A1/ja
Priority to CN202180009959.XA priority patent/CN114981438A/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides

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  • 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.
  • 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.
  • 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.
  • ⁇ -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.
  • Patent Document 6 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.
  • Non-Patent Document 7 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.
  • a chemical synthesis method for example, methyl 2,3,4-tri-o-acetyl- ⁇ -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.
  • 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. ..
  • flavin-linked glucose dehydrogenase 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.
  • 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.
  • 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.
  • 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)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the enzyme when 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.
  • 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.
  • 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).
  • 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.
  • 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 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.
  • sequence identity is 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, still more preferably 99% or more.
  • 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.
  • 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).
  • flavin include flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), preferably FAD.
  • FAD flavin adenine dinucleotide
  • FMN flavin mononucleotide
  • Solubility Water-soluble
  • 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.
  • 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.
  • 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.
  • 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.
  • Molecular weight 64-66 kDa (calculation from amino acid sequence after signal removal)
  • 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.
  • Glucose oxidase activity undetectable
  • 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.
  • 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).
  • 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)
  • 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.
  • 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
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the culture temperature is preferably 10 ° C. to 40 ° C.
  • the culture period is preferably in the range of 2 to 14 days.
  • the culture 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.
  • 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.
  • 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.
  • 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.
  • the substrate concentration is preferably about 10 mM to 2 M.
  • 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).
  • 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.
  • ferricyanide for example, potassium ferricyanide
  • flavonoid quercetin dihydrate, hesperidin
  • 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.
  • 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.
  • an oxidase from the viewpoint of reoxidizing the mediator.
  • the oxidase include phenol oxidases such as laccase (EC 1.10.3.2) and peroxidase (EC 1.11.1.7).
  • phenol oxidases such as laccase (EC 1.10.3.2) and peroxidase (EC 1.11.1.7).
  • 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.
  • glucuronic acid is specifically produced from the substrate glucose.
  • 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.
  • 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.
  • 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.
  • 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.
  • glucoside cellobiose, arbutin, piceid, methylglucoside, and octylthioglucoside are preferable.
  • Glucose analogs include 1,5-anhydro-D-glucitol, 2-deoxy-D-glucose and the like.
  • 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.
  • 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.
  • 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.
  • oxidase examples include phenol oxidases such as laccase (EC 1.10.3.2.) And peroxidase (EC 1.11.1.1.7).
  • phenol oxidases such as laccase (EC 1.10.3.2.)
  • peroxidase EC 1.11.1.1.7
  • catalase EC 1.11.1.6
  • the final concentration of these enzymes is preferably about 0.25 U / mL to 500 U / mL.
  • 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.
  • 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.
  • 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.
  • DCIP 2,6-dichlorophenol indophenol
  • 1-m-PMS 1-methoxy-5 -Methylphenadium Methylsulfate
  • the amount of decrease in absorbance ( ⁇ A600) 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 ⁇ mol of DCIP per minute at 37 ° C. and pH 6.0.
  • 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
  • 1.0 is the optical path length (cm) of the cell, 0.05.
  • ⁇ A600blank 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.
  • a cDNA library was prepared by a reverse transcription reaction using a reverse transcriptase and an oligo dT primer with an adapter sequence.
  • a reverse transcriptase and an oligo dT primer with an adapter sequence.
  • 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.
  • 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.
  • CpGDH the total length of the GDH 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.
  • 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).
  • GE Healthcare illustra plasmidPrep MidiFlow Kit
  • 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.
  • the purified CpGDH was subjected to SDS-polyacrylamide gel electrophoresis, it was confirmed that it showed a single band.
  • Example 2 (Examination of enzymatic chemistry of each GDH) The properties of CpGDH, FGDH and CsGDH obtained in Example 1 were investigated.
  • 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.
  • GOD derived from Aspergillus niger Nacalai Tesque
  • 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.
  • 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%.
  • CpGDH was 196 mM
  • CsGDH was 50 mM
  • 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.
  • each GDH was 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.
  • 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.
  • TLC thin-layer chromatography
  • 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.
  • 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.
  • 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.
  • Example 4 Analysis of glucose oxide obtained in the presence of tert-butylhydroquinone
  • TBHQ tert-butylhydroquinone
  • 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.
  • 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.
  • 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.
  • 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
  • the Km of CgoGDH is 200 mM to 450 mM.
  • the Km of GsGDH is considered to be 250 mM to 650 mM
  • the Km of DhGDH is considered to be 60 mM to 140 mM.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • the Km of DsGDH is 150 mM to.
  • 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.
  • 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).
  • 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)
  • NMR Nuclear Magnetic Resonance Device
  • 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.
  • Example 10 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.
  • 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%.
  • 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.

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Abstract

L'invention concerne un procédé de production d'acide glucuronique ou de dérivé d'acide glucuronique plus facilement, à moindre coût, et avec un impact environnemental réduit par rapport aux procédés existants.　Le procédé de production d'acide glucuronique comprend une étape pour amener une glucose déshydrogénase liée à la flavine ayant une activité de glucose-6-déshydrogénase à agir sur le glucose en présence d'un médiateur pour générer de l'acide glucuronique.
PCT/JP2021/001086 2020-01-23 2021-01-14 Procédé de production d'acide glucuronique WO2021149587A1 (fr)

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WO2014002973A1 (fr) * 2012-06-29 2014-01-03 東洋紡株式会社 Nouvelle glucose déshydrogénase
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WO2017002896A1 (fr) * 2015-07-02 2017-01-05 池田食研株式会社 Glucose déshydrogénase de liaison à la flavine
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WO2011105241A1 (fr) * 2010-02-25 2011-09-01 富山県 Procédé d'obtention de conjugué d'acide glucuronique en utilisant saccharomyces cerevisiae
WO2014002973A1 (fr) * 2012-06-29 2014-01-03 東洋紡株式会社 Nouvelle glucose déshydrogénase
JP2016158578A (ja) * 2015-03-03 2016-09-05 池田食研株式会社 フラビン結合型グルコース脱水素酵素
WO2017002896A1 (fr) * 2015-07-02 2017-01-05 池田食研株式会社 Glucose déshydrogénase de liaison à la flavine
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