WO2015119281A1 - Method for producing phenyl propane-based compound intermediate using enzymes - Google Patents

Method for producing phenyl propane-based compound intermediate using enzymes Download PDF

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WO2015119281A1
WO2015119281A1 PCT/JP2015/053552 JP2015053552W WO2015119281A1 WO 2015119281 A1 WO2015119281 A1 WO 2015119281A1 JP 2015053552 W JP2015053552 W JP 2015053552W WO 2015119281 A1 WO2015119281 A1 WO 2015119281A1
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compound
glutathione
enzyme
gene
hydroxy
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Japanese (ja)
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ゆかり 大田
勇二 秦田
長谷川　良一
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国立研究開発法人海洋研究開発機構
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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
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12P11/00—Preparation of sulfur-containing organic compounds
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01018—Glutathione transferase (2.5.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to a method for specifically producing a glutathione-added phenylpropane compound, which is an intermediate of a compound having a phenylpropane structure, from a carbonylphenylpropane compound using an enzyme, and an enzyme therefor.
  • Lignin is an amorphous polymer that exists as a component of plant vascular cell walls. It is a complex condensation of phenylpropane-based structural units, and the main feature of its chemical structure is the inclusion of methoxy groups. ing. Lignin has a function of sticking woody plant cells to each other and strengthening the tissue, and 18 to 36% in wood and 15 to 25% in herbs. Therefore, in order to effectively use wood, various attempts have been made to decompose lignin and obtain useful compounds.
  • phenylpropane compounds for example, coumaric acid, cinnamic acid, caffeic acid (3,4-dihydroxycinnamic acid), eugenol, anethole, coniferyl alcohol, sinapil alcohol, ferulic acid and the like are known. Yes.
  • phenylpropane compounds are a group of useful compounds that become pharmaceuticals and functional foods such as perfumes, fragrances, essential oils, bactericides, anesthetics and antioxidants, and synthetic intermediates thereof.
  • a method of gasifying lignin contained in lignocellulosic materials such as wood and rice straw by high-temperature and high-pressure treatment see Patent Documents 1 and 2 below, the description of which is incorporated herein by reference
  • pressurization A method for nonspecifically reducing the molecular weight by a physicochemical method such as a hydrothermal method (see Patent Document 3 below, the description of which is incorporated herein by reference) is known.
  • a physicochemical method such as a hydrothermal method
  • a phenylpropane compound having a unit structure possessed by lignin and having 3 carbon side chains directly bonded to the benzene ring skeleton is further reduced in molecular weight.
  • the carbon side chain directly bonded to the benzene ring skeleton such as guaiacol (guaiacol), syringol, etc.
  • the carbon side chain directly bonded to the benzene ring skeleton such as vanillin, syringaldehyde, etc.
  • the decomposition product contains a wide variety of components, and it is very difficult to obtain only the phenylpropane-based compound, and it cannot be specifically produced.
  • these physicochemical methods are employed, a lot of energy and special equipment are required to implement these methods.
  • Non-Patent Document 1 it is described as “Pseudomonas genus bacteria”, Brevundimonas genus bacteria (see Patent Document 6 below, the description of this document is here) And Pseudomonas bacteria (see Non-Patent Document 2 below, the description of which is incorporated herein by reference).
  • Patent Documents 4 to 6 and Non-Patent Documents 1 and 2 include a model compound of lignin by a sphingobium genus, a brebandimonas genus bacterium, a pseudomonas genus bacterium, and a ⁇ -aryl ether cleaving enzyme produced by these microorganisms.
  • a model compound of lignin by a sphingobium genus a brebandimonas genus bacterium, a pseudomonas genus bacterium, and a ⁇ -aryl ether cleaving enzyme produced by these microorganisms.
  • Non-patent document 3 uses a ⁇ -aryl ether-type degrading enzyme derived from a sphingobium bacterium, and uses guaiacylglycerol- ⁇ -guay.
  • a carbonyl group is formed by acting on the carbon atom at the C ⁇ position of the guaiacylglycerol- ⁇ -guayacyl ether and the alcoholic hydroxy group bonded to the carbon atom.
  • a phenylpropane compound can be produced through an enzyme sequential reaction in three stages of the reaction. It is listed.
  • Patent Documents 5 to 6 and Non-Patent Documents 1 to 3 are methods for producing a phenylpropane-based compound from a lignin model compound using a microorganism or its production enzyme.
  • these documents do not describe that a phenylpropane compound was obtained from natural biomass by allowing microorganisms or enzymes to act.
  • the enzyme described in Patent Document 6 is an enzyme that cleaves a ⁇ -aryl ether type bond in a compound having a ⁇ -aryl ether type bond and a free phenolic hydroxyl group in the arylglycerol moiety.
  • the activity with respect to a compound having no free phenolic hydroxyl group in the arylglycerol moiety is 1/100 of the activity with respect to the compound having a free phenolic hydroxyl group in the arylglycerol moiety, which is not sufficient.
  • Non-Patent Document 3 is a guaiacylglycerol- ⁇ -guaiacyl ether in which four kinds of optical isomers ( ⁇ S, ⁇ R; ⁇ R, ⁇ S; ⁇ R, ⁇ R; ⁇ S, ⁇ S) exist. Only ⁇ S and ⁇ S, ⁇ S guaiacylglycerol- ⁇ -guaiacyl ether can be used as starting materials, but ⁇ S, ⁇ R and ⁇ R, ⁇ R cannot be used as starting materials. As a result, there is a problem that the yield of the obtained phenylpropane-based compound is lowered.
  • Non-Patent Document 3 Furthermore, with respect to the substrate on which the enzyme described in Non-Patent Document 3 acts, only decomposition of a compound having a free phenolic hydroxyl group is known, and no compound having a free phenolic hydroxyl group is described in Non-Patent Document 3. .
  • An object of the present invention is to provide a method for specifically and efficiently producing a glutathione-added phenylpropane-based compound, which is an intermediate of a phenylpropane-based compound, from a carbonylphenylpropane-based compound, which is used in the production of the compound having the same.
  • the inventors of the present invention have accumulated intensive research, deciphered the genome sequence information of Novosphingobium sp. MBES04 strain, which is a lignin-degrading microorganism isolated from deep-sea sediments, and obtained lignin from the information.
  • MBES04 strain which is a lignin-degrading microorganism isolated from deep-sea sediments, and obtained lignin from the information.
  • glutathione-added phenyl is obtained by allowing the carbonylphenylpropane-based compound to act on the enzyme of the following (2) derived from a microorganism belonging to the genus Novosphingobium in the presence of reduced glutathione.
  • a method for producing a glutathione-added phenylpropane-based compound including a step of obtaining a propane-based compound.
  • the enzyme of (2) is C10G0076 having the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing or C10G0077 having the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.
  • the microorganism belonging to the genus Novosphingobium is Novosphingobium sp. MBES04 (accession number: NITE P-01797).
  • the glutathione S-transferase has a molecular weight of 31 kDa that can be confirmed by SDS-PAGE; an optimum pH is 8.5 to 9.5; and an optimum temperature is 30 to 35 ° C. It is.
  • the glutathione S-transferase has a molecular weight of 31 kDa which can be confirmed by SDS-PAGE; an optimum pH of 7 to 8; and an optimum temperature of 25 to 30 ° C.
  • the glutathione S-transferase is a glutathione S-transfer having the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing.
  • the glutathione S-transferase is glutathione S-transfer having the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.
  • a glutathione-added phenylpropane compound that is an intermediate of the phenylpropane compound can be specifically and efficiently produced from the compound.
  • FIG. 1 is a diagram schematically showing a relationship between an embodiment of an enzyme group used in a method for producing a phenylpropane-based compound and an optical isomer of an acting substrate.
  • FIG. 2 is a diagram showing the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0069 gene as a query.
  • FIG. 3 shows the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0093 gene as a query.
  • FIG. 4 shows the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0076 gene as a query.
  • FIG. 1 is a diagram schematically showing a relationship between an embodiment of an enzyme group used in a method for producing a phenylpropane-based compound and an optical isomer of an acting substrate.
  • FIG. 2 is a diagram showing the results of a DELTA-BLAST search using the amino acid sequence encoded by the
  • FIG. 5 shows the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0077 gene as a query.
  • FIG. 6 shows the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0078 gene as a query.
  • FIG. 7 shows the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0075 gene as a query.
  • FIG. 8 is a diagram showing the result of evaluating the optimum temperature of C10G0069.
  • FIG. 9 is a diagram showing the results of evaluating the optimum pH of C10G0069.
  • FIG. 10 is a diagram showing a result of evaluating the optimum temperature of C10G0093.
  • FIG. 11 is a diagram showing the results of evaluating the optimum pH of C10G0093.
  • FIG. 12 is a diagram showing the result of evaluating the optimum temperature of C10G0076.
  • FIG. 13 is a graph showing the results of evaluating the optimum pH of C10G0076.
  • FIG. 14 is a diagram showing the results of evaluating the optimum temperature of C10G0077.
  • FIG. 15 is a diagram showing the results of evaluating the optimum pH of C10G0077.
  • glutathione-added phenylpropane is produced by allowing the carbonylphenylpropane-based compound to act on the enzyme of the following (2) derived from a microorganism belonging to the genus Novosphingobium in the presence of reduced glutathione. It is a manufacturing method of a glutathione addition phenylpropane type compound including the process of obtaining a type compound.
  • 3-hydroxy-1- (4-hydroxy) to which glutathione has been added by the action of the enzyme (2) derived from a Novosphingobium microorganism by the carbonylphenylpropane compound 3-methoxyphenyl) -1-propanone, 3-hydroxy-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-propanone, 3-hydroxy-1- (4-hydroxyphenyl) -1-propanone Phenylpropane compounds such as are produced.
  • the phenylpropane compound is not particularly limited as long as it is a compound having a phenylpropane structure.
  • R represents an alkyl group, an alkoxy group or a hydrogen atom having 1 or 2 or more carbon atoms of 1 to 5.
  • R represents an alkyl group, an alkoxy group or a hydrogen atom having 1 or 2 or more carbon atoms of 1 to 5.
  • R represents an alkyl group, an alkoxy group or a hydrogen atom having 1 or 2 or more carbon atoms of 1 to 5.
  • R represents an alkyl group, an alkoxy group or a hydrogen atom having 1 or 2 or more carbon atoms of 1 to 5.
  • R represents an alkyl group, an alkoxy group or a hydrogen atom having 1 or 2 or more carbon atoms of 1 to 5.
  • R represents an alkyl group, an alkoxy group or a hydrogen atom having 1 or 2 or more carbon atoms of 1 to 5.
  • R represents an alkyl group, an
  • 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone has a structure of the following formula (I).
  • 3-Hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone can be analyzed qualitatively and quantitatively, for example, by reverse phase HPLC.
  • the reverse-phase HPLC conditions are, for example, using an octadecylsilyl group-modified silica gel column (ODS column), eluent A (2 mM ammonium acetate, 0.05% V / V formic acid) and eluent B (100% V / V). Methanol), the column temperature was set to 40 ° C., the flow rate was set to 1.2 ml / min, and a mixed solution of eluent A 90% V / V and eluent B 10% V / V was fed for 1 minute.
  • ODS column octadecylsilyl group-modified silica gel column
  • eluent A (2 mM ammonium acetate, 0.05% V / V formic acid
  • 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone can be detected as a peak near a retention time of 4.5 minutes by using a UV detector (270 nm). If a standard product of 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone is used, it can be quantified by a calibration curve method or an internal standard method.
  • a carbonylphenylpropane compound is used as a starting material.
  • a carbonylphenylpropane-based compound is not particularly limited as long as it has an arylglycerol- ⁇ -aryl ether type bond, the carbon atom at the C ⁇ position is carbonyl; and a phenyl group is bonded to the carbon atom at the C ⁇ position.
  • an enzyme that acts on a carbon atom at the C ⁇ position of guaiacylglycerol- ⁇ -guaiacyl ether to form a carbonyl group at this site preferably a sphingobium species (Sphingobium spp.
  • LigD, LigL, LigN and LigO encoded by ligD gene (YP_004833998), ligL gene (AB491221), ligN gene (AB491222) and ligO gene (YP_004836720) derived from SYK-6 strain, and Novosf Examples include compounds obtained by allowing C10G0069 and C10G0093 described in SEQ ID NOS: 1 and 2 of the sequence listing derived from Novosphingium sp. MBES04 strain to act on biomass containing lignin, lignin-related substances, or both .
  • Specific examples of the carbonylphenylpropane-based compound include a compound represented by the following formula (F), but are not limited thereto. (F)
  • the lignin is not particularly limited as long as it is commonly known in the art.
  • the lignin is present in plant vascular bundles, and mainly comprises three types of phenylpropanoids represented by the following formulas (B) to (D) as structural units. It is known to have a complex polymerized dendritic structure. (B) (C) (D)
  • the lignin-related substance is not particularly limited as long as it is a substance derived from lignin.
  • substances that are model compounds of lignin for example, Guaiacylglycerol- ⁇ -guayacyl ether and the like.
  • Biomass is not particularly limited as long as it contains lignin, lignin-related substances, or both, and examples thereof include natural products such as grass and trees, those obtained by processing these natural products, agricultural waste, and the like. It is done.
  • Biomass containing lignin and / or lignin-related substances can be, for example, solid, suspended, liquid, etc. depending on the presence or absence of pretreatment.
  • a suspension obtained by adding pulverized lignin-containing biomass to a liquid can be used.
  • the lignin-containing biomass may be a lignin extract.
  • the lignin extract for example, powdered lignin-containing biomass is 0.1% W / V to 50% W / V, preferably 1% W / V to 20% W / V. It is suspended in a solvent suitable for extraction of lignin to form a suspension, and this suspension is at 10 ° C. to 150 ° C., preferably 20 ° C. to 130 ° C., more preferably 20 ° C.
  • a solvent suitable for extraction of lignin is not particularly limited, and examples thereof include water, low molecular alcohols such as dioxane, methanol and isopropanol, dimethylformamide, and the like, preferably water and dioxane.
  • an enzyme derived from a microorganism belonging to the genus Novosphingobium is used to obtain a glutathione-added phenylpropane compound from a carbonylphenylpropane compound.
  • the microorganism of the genus Novosphingobium is not particularly limited as long as it is a gram-negative bacillus having a size of 0.3 to 0.8 ⁇ 2 to 3 ⁇ m, belonging to the genus Novosphingobium, also known as the genus Sphingomonas.
  • Novosphingobium microorganisms that are known to decompose various aromatic compounds.
  • a preferred specific example of the microorganisms belonging to the genus Novosphingobium used in the production method of the present invention is Novosphingium sp. MBES04 (hereinafter also referred to as MBES04 strain).
  • the MBES04 strain is designated as “Novosphingium sp. It has been deposited with the date of receipt dated January 30, 2014 in 2-5-8) Kazusa Kamashishi, Kisarazu City. In addition, Novosfingobium sp. MBES04 has been transferred from domestic deposit to international deposit on February 9, 2015 at the Patent Microbiology Deposit Center of the National Institute of Product Evaluation Technology (Received) Number: NITE ABP-01797).
  • Glutathione S-transferase which has the enzyme (2) Gst and the multidomain of PRK15113 and maiA on the N-terminal side and acts on veratrylglycerol- ⁇ -guayacyl ether, is 1- (4-hydroxy-3- Methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one reductively cleaves the arylglycerol- ⁇ -aryl ether type bond at the ⁇ -position carbon atom to give a ⁇ -position carbon atom Has the activity of producing 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone with glutathione added thereto.
  • Gst is an amino acid sequence commonly found in glutathione S-transferase with PSSM-Id of 2223698.
  • PRK15113 is an amino acid sequence commonly found in glutathione S-transfer; Provisional with PSSM-Id of 185068.
  • maiA is an amino acid sequence often found in maleilacetoacetate isomerase with PSSM-Id of 233333.
  • the E-value of Gst is 4.21e-38 for C10G0076 and 6.35e-11 for C10G0077.
  • the E-value of PRK15113 is 3.10e-16 for C10G0076 and 4.58e-03 for C10G0077.
  • the MaiA E-value is 8.01e-17 for C10G0076 and 1.01e-05 for C10G0077.
  • “having a multi-domain” means having a multi-domain amino acid sequence on the N-terminal side or C-terminal side in addition to having the amino acid sequence of the multi-domain itself, It means having an amino acid sequence similar to a domain.
  • Enzyme (2) is an aryl at the ⁇ -position carbon atom of 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one in the presence of glutathione.
  • the enzyme can produce (-3-methoxyphenyl) -1-propanone, this presumption does not change the technical scope of the present invention.
  • the enzyme (2) acts on veratrylglycerol- ⁇ -guayacyl ether in the presence of reduced glutathione to catalyze the addition of glutathione to the ⁇ -position carbon atom. From this, enzyme (2) can also show a catalytic activity also with respect to the structural unit which does not have a free phenolic hydroxyl group in the arylglycerol part often seen in natural lignin. This is a heterogeneous catalytic activity to be noticed that can never be obtained with the enzyme described in Non-Patent Document 6.
  • enzyme (2) are C10G0076 described in SEQ ID NO: 3 in the Sequence Listing and C10G0077 described in SEQ ID NO: 4 in the Sequence Listing.
  • C10G0076 and C10G0077 catalyze glutathione addition cleavage of ⁇ -O-4 bond in the presence of reduced glutathione.
  • these enzymes do not exhibit such catalytic activity in the absence of reduced glutathione. Therefore, in the production method of the present invention, the enzyme reaction is carried out in the presence of reduced glutathione.
  • the molecular weight of C10G0076 is 31 kDa by SDS-PAGE; the optimum pH is 8.5 to 9.5; and the optimum temperature is 30 to 35 ° C.
  • the molecular weight of C10G0077 is 31 kDa by SDS-PAGE; the optimum pH is 7-8; and the optimum temperature is 25-30 ° C.
  • the molecular weight represents the molecular weight of the E. coli recombinant protein.
  • the optimal pH and optimal temperature as used in this specification are the range of pH and temperature which show the value about 80% or more from the highest value of a target product among the results measured in the Example mentioned later.
  • the enzyme (2) used in the production method of the present invention may be glutathione S-transferase having Gst and a multidomain of PRK15113 and maiA on the N-terminal side and acting on veratrylglycerol- ⁇ -guayacyl ether.
  • glutathione S-transferase having Gst and a multidomain of PRK15113 and maiA on the N-terminal side and acting on veratrylglycerol- ⁇ -guayacyl ether.
  • those having the above-described physicochemical properties of C10G0076 or C10G0077 are preferable.
  • a preferred embodiment of the enzyme (2) has, for example, Gst having an amino acid sequence similar to the amino acid sequence described in SEQ ID NO: 3 or 4 in the sequence listing, and a multi-domain of PRK15113 and maiA on the N-terminal side, and Examples include glutathione® S-transferase which acts on veratrylglycerol- ⁇ -guayacyl ether.
  • Examples of such an enzyme include Gst, which includes an amino acid sequence having one to several amino acid deletions, substitutions or additions in the amino acid sequence set forth in SEQ ID NO: 3 or 4 in the sequence listing, and PRK15113 on the N-terminal side. And maiA multidomain, and 90% or more identity with glutathione S-transferase acting on veratrylglycerol- ⁇ -guayacyl ether and the amino acid sequence set forth in SEQ ID NO: 3 or 4 in the sequence listing Examples include Gst containing an amino acid sequence and glutathione S-transferase having a multi-domain of PRK15113 and maiA on the N-terminal side and acting on veratrylglycerol- ⁇ -guayacyl ether.
  • the range of “1 to several” in “deletion, substitution or addition of 1 to several amino acids” of the amino acid sequence has Gst and a multi-domain of PRK15113 and maiA on the N-terminal side, and although it is not particularly limited as long as it acts on veratrylglycerol- ⁇ -guayacyl ether, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19 or 20, preferably 1, 2, 3, 4 or 5.
  • amino acid deletion means deletion or disappearance of an amino acid residue in the sequence
  • amino acid substitution means that an amino acid residue in the sequence is replaced with another amino acid residue
  • “Addition of amino acid” means that a new amino acid residue is added to the sequence.
  • a specific embodiment of “deletion, substitution or addition of one to several amino acids” includes an embodiment in which one to several amino acids are replaced with another chemically similar amino acid.
  • a case where a certain hydrophobic amino acid is substituted with another hydrophobic amino acid a case where a certain polar amino acid is substituted with another polar amino acid having the same charge, and the like can be mentioned.
  • Such chemically similar amino acids are known in the art for each amino acid.
  • Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine.
  • Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.
  • Examples of positively charged (basic) amino acids include arginine, histidine, and lysine.
  • Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • the “identity” in the amino acid sequence is particularly limited as long as it has Gst and a multi-domain of PRK15113 and maiA on the N-terminal side and acts on veratrylglycerol- ⁇ -guayacyl ether.
  • the identity between sequences when aligned with the amino acid sequence shown in SEQ ID NO: 3 or 4 in the sequence listing is 90% or more, preferably 95% or more, more preferably 97% or more. Yes, more preferably 98% or more, still more preferably 99% or more.
  • the method for determining the identity of the amino acid sequence is not particularly limited. For example, using a commonly known method, the amino acid sequence described in SEQ ID NO: 3 or 4 in the sequence listing is aligned with the target amino acid sequence, It is calculated
  • the molecular weight of C10G0076 or C10G0077 is 31 kDa by SDS-PAGE. Accordingly, Gst having an amino acid sequence similar to the amino acid sequence described in SEQ ID NO: 3 or 4 in the sequence listing, multi-domain of PRK15113 and maiA on the N-terminal side, and veratrylglycerol- ⁇ -guayacyl ether
  • the molecular weight of glutathione -S-transferase that acts on C is a molecular weight that approximates the molecular weight of C10G0076 or C10G0077.
  • SDS-PAGE preferably has a molecular weight of 26-36 kDa, more preferably 28-34 kDa, more preferably 30 It is more preferably ⁇ 32 kDa, and still more preferably around 31 kDa.
  • the method for obtaining the enzyme (2) is not particularly limited.
  • an enzyme extract obtained from a microorganism belonging to the genus Novosphingobium or a fraction thereof is used as a substrate for 1- (4-hydroxy-3-methoxyphenyl).
  • ) -3-Hydroxy-2- (2-methoxyphenoxy) propan-1-one by acting on 3-hydroxy-1 in which the base mass is reduced or glutathione is added to the ⁇ -position carbon atom of the product
  • the enzyme (2) can be obtained.
  • the Novosphingobium microorganism for example, in the deep sea (the sea below 200 m below the surface), using the enzyme action, substrate (optical isomer) specificity, physicochemical properties, molecular weight, etc. of C10G0076 or C10G0077 as indicators. And a screening method for examining enzyme activity in a culture supernatant obtained by culturing a microorganism of the genus Novosphingobium inhabiting a coniferous submerged tree.
  • the amino acid sequence described in SEQ ID NO: 3 or 4 of the sequence listing is encoded by genetic engineering. You may create from the gene which has a base sequence.
  • the gene which consists of a base sequence which codes the amino acid sequence of sequence number 3 and 4 of a sequence table is each described in sequence number 9 and 10 of a sequence table.
  • the enzyme (2) is obtained by using a transformant prepared by introducing a vector containing the base sequence shown in SEQ ID NO: 9 or 10 and a foreign gene such as a drug resistance gene or a heterologous base sequence. be able to.
  • a glutathione-added phenylpropane compound can be obtained by allowing the enzyme (2) to act on the carbonylphenylpropane compound.
  • acting enzyme (2) means, for example, 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propane possessed by enzyme (2) It means that the arylglycerol- ⁇ -aryl ether type bond at the ⁇ -position carbon atom of the 1-one is reductively cleaved.
  • C10G0076 and C10G0077 which are specific examples of the enzyme (2), are selectively used depending on the optical isomer of the compound serving as the substrate.
  • a schematic representation of the relationship between these enzymes and acting optical isomers is shown in FIG.
  • the following combinations (1) to (3) can be considered as combinations of these enzymes.
  • the pH and temperature at which C10G0076 and C10G0077 are active may be set. preferable. These enzymes may be used simultaneously or separately.
  • C10G0076 may be allowed to act after C10G0076 is allowed to act in the presence of reduced glutathione, and C10G0076 may be allowed to act after C10G0077 is allowed to act.
  • a buffering agent in the working system In order to suppress fluctuations in pH during the action of the enzyme (2), it is preferable to add a buffering agent in the working system, and in order to promote the reductive cleavage action of the enzyme (2), a weakly alkaline or alkaline buffering agent. It is more preferable to add.
  • the enzyme (2) can be used in combination with other enzymes.
  • a ligD gene (YP_004833998) derived from Sphingobium sp. SYK-6 strain as an enzyme that oxidizes the carbon atom at the C ⁇ position of guaiacylglycerol- ⁇ -guaiacyl ether.
  • C10G0078 and C10G0075 described in SEQ ID NOS: 5 and 6 in the Sequence Listing but are not limited thereto.
  • the concentration and abundance of each component such as a starting material, a buffer, an enzyme, and reduced glutathione when the enzyme is allowed to act are not particularly limited and can be set as appropriate. Further, in order to increase the contact frequency between the starting material and the enzyme, these mixtures may be stirred or shaken.
  • the action time is not particularly limited as long as the production of the target substance is recognized, and is, for example, several hours to several days, and can be appropriately set depending on the enzyme titer and the like. Specifically, it is about 1 hour to 36 hours or more.
  • HPLC analysis conditions of 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one can be employed.
  • 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one is analyzed qualitatively and quantitatively by, for example, reverse phase HPLC. it can.
  • the reverse-phase HPLC conditions are, for example, using an octadecylsilyl group-modified silica gel column (ODS column), eluent A (2 mM ammonium acetate, 0.05% V / V formic acid) and eluent B (100% V / V). Methanol), the column temperature was set to 40 ° C., the flow rate was set to 1.2 ml / min, and a mixed solution of eluent A 90% V / V and eluent B 10% V / V was fed for 1 minute. Thereafter, the eluent B is fed for 7 minutes in a gradient of 10% V / V to 95% V / V.
  • 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone can be detected as a peak near a retention time of 6.5 minutes by using a UV detector (270 nm). If a standard product of 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one is used, it can be quantified by a calibration curve method or an internal standard method. Is possible.
  • the method for confirming a glutathione-added phenylpropane compound employs, for example, HPLC analysis conditions for 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone in which glutathione is added to the ⁇ -position carbon atom it can.
  • 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone in which glutathione is added to the carbon atom at the ⁇ -position is the above 1- (4-hydroxy-3-methoxyphenyl)-
  • it can be detected as a peak with a retention time of 3.4 to 3.5 minutes.
  • the target substance produced in the action system can be used as it is without any special treatment.
  • a commonly known aromatic compound such as a solid phase extraction method or a chromatogram method is purified. If the solvent is distilled off and dried, it can be obtained as a solid.
  • the first specific embodiment of the production method of the present invention is as follows, for example.
  • the lignin-containing biomass is suspended in a solvent suitable for dissolving lignin such as dioxane and water, and kept at 20 ° C. to 80 ° C. for several hours to several days.
  • the solid content is removed from the suspension after the incubation to obtain a lignin extract.
  • the lignin extract is evaporated to dryness, and the resulting dried product is dissolved in an organic solvent such as ethyl acetate or DMF or a water-containing organic solvent to obtain a lignin extract.
  • Lignin extract enzyme that oxidizes carbon atom at the C ⁇ position of guaiacylglycerol- ⁇ -guaiacyl ether; 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propane
  • the second specific embodiment of the production method of the present invention is as follows, for example.
  • a reaction solution containing the above lignin extract; an enzyme that oxidizes the carbon atom at the C ⁇ position of guaiacylglycerol- ⁇ -guaiacyl ether; a buffering agent; and an NAD salt is prepared, and several times at pH 7-10, 20-40 ° C.
  • the 3rd specific aspect of the manufacturing method of this invention is as follows, for example.
  • a reaction solution containing the above lignin extract; an enzyme that oxidizes the carbon atom at the C ⁇ position of guaiacylglycerol- ⁇ -guaiacyl ether; a buffering agent; and an NAD salt is prepared, and several times at pH 7-10, 20-40 ° C.
  • the glutathione-added phenylpropane compound obtained by the production method of the present invention includes, for example, 3-hydroxy-1- (4-hydroxy-3-) in which glutathione is further added to the carbon atom at the ⁇ -position such as C10G0078 and C10G0075 described above. It can be used as a phenylpropane-based compound by acting on an enzyme that removes the glutathione of methoxyphenyl) -1-propanone, or as a raw material for resins, adhesives, resist materials, pharmaceuticals, etc., or an intermediate thereof.
  • the compound when 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone is finally obtained, the compound is 1- (4-hydroxy-3-methoxyphenyl)- Via 1,3-propanediol, it can be converted into coniferyl alcohol, which is industrially useful as a raw material for pharmaceuticals, fragrances, food materials and the like.
  • glutathione S-transfer which has a multi-domain of maiA and maiA and acts on veratrylglycerol- ⁇ -guayacyl ether.
  • a preferred embodiment of the enzyme of the present invention is a glutathione S-transferase having a molecular weight of 31 kDa that can be confirmed by SDS-PAGE; an optimum pH of 8.5 to 9.5; and an optimum temperature of 30 to 35 ° C. And the molecular weight that can be confirmed by SDS-PAGE is 31 kDa; the optimum pH is 7 to 8; and the optimum temperature is 25 to 30 ° C., which is glutathione S-transfer.
  • a specific embodiment of the present invention is glutathione S-transfer having the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing, and glutathione S-transfer having the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.
  • Example 1 Detection of MBES04 strain gene
  • LB medium Becton Dickinson
  • 5 mM magnesium sulfate was inoculated with Novosphingobium MBES04 strain and cultured with shaking at 30 ° C. and 120 rpm for 24 hours.
  • MBES04 strain cells were obtained from the obtained culture broth by centrifugation at 8000 rpm for 5 minutes.
  • Total DNA was extracted from the obtained cells using NucleoSpin Plant II Midi kit (manufactured by Takara Bio Inc.). An 8 kb long mate pair library was created using the extracted DNA, and the sequence was analyzed using the genome sequencer GS FLX system (Roche Diagnostics). As a result of sequence analysis, base sequences related to sequence data having 142,389 reads, an average length of 441.66 bp and a total number of bases of 62,888,162 were obtained.
  • the entire nucleotide sequence contained in the 39 SuperContigs obtained was converted into the gene region encoded by the entire nucleotide sequence contained in the 39 SuperContigs, ie, the protein.
  • the region corresponding to the open reading frame to be encoded was estimated.
  • the SYK-6 strain binds to the carbon atom at the C ⁇ position of Compound I, that is, the first stage reaction when metabolizing ligacylglycerol- ⁇ -guaiacyl ether (Compound I), which is a lignin dimer model compound.
  • Enzymes that catalyze the oxidation of hydroxy groups include LigD, LigL, LigN, and LigO (see Non-Patent Documents 5 and 6) and the ligD gene (YP_004833998), ligL gene (AB491122), ligN gene (AB491222), and The ligO gene (YP_004836720) is known.
  • Table 1 shows information on the homology between the above 6 genes derived from MBES04 and ligD, ligL, ligN and ligO derived from SYK-6.
  • the hit with the gene sequence having the highest sequence match was found to be 71% identical to c01g1162
  • JDR-2 CP001656.1
  • PP1Y strain with 75% identity to c01g1324 gene (FR856862.1), a gene presumed to encode short-chain dehydrogenase / reductase at positions 1154509 to 1155339; Novosphingobium sp. With a 78% consistency with the c10g0069 gene ) Positions 1241135 to 1242028 on the genome of the PP1Y strain (FR856862.1) A gene presumed to encode short-chain dehydrogenase / reductase in the above; on the genome of Novosphingobium sp.
  • PP1Y strain (FR8566862.1) with 76% identity to c10g0080 gene A gene presumed to encode short-chain dehydrogenase / reductase at positions 1251645 to 12525546;
  • Novosphingobium aromaticivorans strain DSM124 with 80% identity to the c10g0093 gene In the position of 844288-844180 on the genome (CP000248.1) 1266103 on the genome of Novosphingobium sp.
  • PP1Y strain (FR8566862.1) with 77% identity to the c10g0094 gene, which is presumed to encode the short-chain dehydrogenase / reductase It was a gene presumed to encode short-chain dehydrogenase / reductase at the position of No. 1266694.
  • the second stage reaction when SYK-6 strain metabolizes Compound I that is, 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propane-1 -Enzymes that catalyze the reaction of cleaving the ether bond at the ⁇ -O-4 position of ONE (compound II) include ligF gene (YP_004833997) (see Non-Patent Document 7) and ligE gene (YP_004833998) (Non-Patent Document 8). Is known).
  • the ligG gene (YP_004833999) (refer nonpatent literature 9) is known as an enzyme which catalyzes the 3rd step reaction which removes glutathione from the glutathione containing intermediate produced by the 2nd step reaction.
  • genes having homology to the ligE gene, the ligF gene, and the ligG gene derived from the SYK-6 strain are represented by Coverage 50% ⁇ and Identity 25% ⁇ .
  • the BLAST homology search was performed with Simality 50% ⁇ , E-value ⁇ e5 as threshold values. As a result, it was found that the c10g0076 gene had 68% identity in ligF, the c10g0077 gene had 80% identity in ligE, and the c10g0078 gene had 71% identity in ligG.
  • BLASTn homology search (Coverage 50% ⁇ ) was performed using the base sequences of 3 genes from the MBES04 strain: c10g0076 gene, c10g0077 gene and c10g0078 gene as a query, the most successful gene sequence was c10g0076 It encodes a glutathione - S-transferase-like protein located at positions 1247655 to 1248181 on the genome of Novosphingium sp.
  • PP1Y strain (FR8566862.1) with 82% identity to the gene Presumed gene; Novosphingobium species with 80% consistency for c10g0078 gene sp.) a gene presumed to encode a glutathione S-transferase-like protein located at positions 1284451 to 1249198 on the genome of the PP1Y strain (FR856862.1); Novosphingo with 78% identity to the c10g0079 gene It was a gene presumed to encode a glutathione S-transferase family protein located at positions 1249325 to 1250064 on the genome of Novosphingium sp. PP1Y strain (FR856862.1).
  • amino acid sequences encoded by the c10g0076 gene and the c10g0077 gene have PRK15113 and maiA multidomains on the N-terminal side. Furthermore, it was found that the amino acid sequence encoded by the c10g0078 gene has PRK10387 and maiA multidomains on the N-terminal side.
  • the c10g0076 gene, c10g0077 gene, and c10g0078 gene are genes close to each other. Furthermore, the c10g0075 gene adjacent to the c10g0076 gene encodes a protein presumed to be Glutathione S-transferase in a direction opposite to the direction encoded by the three genes c10g0076, c10g0077, and c10g0078.
  • a BLASTn search Crossage 50% ⁇
  • each protein encoded by 6 genes of c01g1162 gene, c01g1324 gene, c10g0069 gene, c10g0080 gene, c10g0093 gene, and c10g0094 gene is expressed as C01G1162, C01G1324, C10G0069, C10G0080, C10G0093, and C10G0094; and c10g0075 gene,
  • Each protein encoded by the four genes c10g0076, c10g0077, and c10g0078 was designated as C10G0075, C10G0076, C10G0077, and C10G0078.
  • a PCR reaction was performed using the plasmid pRSETA (manufactured by Invitrogen) as a template and primers A and B shown in Table 2.
  • the primer set shown in Table 2 (C and D, E and F, G and H, I and J, K and L, M and N, O and P, Q and R, S and T, and U and V) PCR was carried out using the genomic DNA of MBES04 strain as a template according to the conditions shown below to obtain cDNA obtained by amplifying the above gene fragments.
  • PCR conditions 1 ⁇ PCR buffer (containing MgCl 2 ) 200 ⁇ m dNTPs, 0.6 ⁇ m 27f, 0.6 ⁇ m 1525r, 1.4 U of LA Taq DNA (manufactured by Polymerase Takara Bio Inc.) Thermal cycler temperature condition 97 ° C 2 minutes, [97 ° C 30 seconds, 60 ° C 1 minute, 72 ° C 90 seconds] x 30 cycles, 72 ° C 5 minutes
  • a plasmid was prepared from the obtained transformant, and it was confirmed by sequence analysis that the nucleotide sequence of the ligated fragment on the plasmid completely matched the gene sequence on the genome of MBES04 strain.
  • Base sequence analysis was performed using BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit, v3.1 (manufactured by Applied Biosystems), ABI 3730, XL DNA Analyzer (Applied Biosystems).
  • BL21DE3pLysE manufactured by Life Technology
  • Recombinant enzyme was produced using each transformant, and the resulting recombinant enzyme was purified using Ni-agarose carrier.
  • the obtained compound I was erithro form: threo form ⁇ 3: 1 under these conditions.
  • the RS display was determined with reference to the optical rotation data described in Non-Patent Document 11.
  • Compound I was added to Tris-HCl buffer (pH 7.5) to a final concentration of 5 mM and NAD + (added in the form of sodium salt) to a final concentration of 10 mM. Subsequently, the purified C01G1162, C01G1324, C10G0069, C10G0080, C10G0093 and C10G0094 obtained in Example 3 were added to this solution one by one and incubated at room temperature for 16 hours to obtain a reaction solution. Thereafter, the change of Compound I in the reaction solution was analyzed by HPLC under the following conditions.
  • reaction solution was purified by solid phase extraction (OASISSWAX; manufactured by Waters), and reverse-phase UPLC-time-of-flight accurate mass spectrometry (ACCUITYUUPLC H- Class, XevoG2 QTOF, manufactured by Waters).
  • OASISSWAX solid phase extraction
  • ACCUITYUUPLC H- Class, XevoG2 QTOF reverse-phase UPLC-time-of-flight accurate mass spectrometry
  • Reversed-phase HPLC conditions (reversed-phase UPLC-time-of-flight accurate mass spectrometry conditions) Column; ACQUITY UPLC BEH C18 Column, 130 ⁇ , 1.7 ⁇ m (Waters), 2.1 mm d. ⁇ 100 mm L.
  • Mass analysis conditions (reverse phase UPLC-time-of-flight accurate mass analysis conditions) Detection mass range 100-1000 Da, data acquisition scan interval 0.1 second, desolvation gas temperature 500 ° C, ion source ESI negative mode ion source temperature 150 ° C, cone voltage 30V
  • the molecular weight of the product compound was 318 and the estimated composition formula was C 17 H 18 O 6 .
  • the product compound is Compound II in which a dehydrogenation reaction occurs at the C ⁇ position of Compound I and the alcohol residue (hydroxy group) is changed to a carbonyl group. It was estimated.
  • Example 5 Characteristics of Compound I Degrading Enzyme
  • Compound II was synthesized according to the method described in Non-Patent Document 10. In addition, HPLC analysis of Compound II was performed under the conditions of elution in the order of (1) ⁇ R form and (2) ⁇ S form using Chiral Pack IE3 manufactured by Daicel Corporation as follows.
  • the synthesized compound II was ⁇ R form: ⁇ S form ⁇ 1: 1 under these conditions.
  • the RS display was determined with reference to the optical rotation data described in Non-Patent Document 11.
  • the synthesized compound II was subjected to reverse-phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4. As a result, it was found that the compound I was well separated from the compound I decomposition product obtained when C10G0069 and C10G0093 were allowed to act on compound I. Consistent mass chromatography and spectra were obtained. From this, it was found that C10G0069 and C10G0093 are proteins having a dehydrogenase activity that oxidizes the hydroxy group at the C ⁇ position of Compound I and changes it to a carbonyl group.
  • reaction solution 100 ⁇ l containing 10 mg / l C10G0069, 10 mM compound I, 20 mM sodium NAD and 50 mM (pH 9.2) N-Cyclohexyl-2-aminoethanesulfonic acid-NaOH (CHES) buffer (pH 9.5),
  • the optimum temperature of C10G0069 was evaluated by reacting at a temperature of 5-45 ° C. for 30 minutes. As a result, as shown in FIG. 8, the optimum temperature was around 10 to 15 ° C.
  • a reaction solution (100 ⁇ l) containing 10 mg / l C10G0069, 10 mM compound I, 20 mM sodium NAD, and 50 mM buffer (MES, MOPS, TAPS, CHES, and CAPS) adjusted to pH 6.0 to 10.4 was added at 15 ° C.
  • the optimal pH of C10G0069 was evaluated by reacting for 30 minutes. As a result, as shown in FIG. 9, the optimum pH was around 8.5 to 9.5.
  • the optimum temperature and optimum pH are based on values obtained by analyzing the concentration of Compound II produced in the reaction solution after the reaction by reverse phase HPLC under the conditions described in Example 4.
  • reaction solution 100 ⁇ l containing 5 mg / l C10G0093, 10 mM compound I, 20 mM NAD sodium and 50 mM (pH 9.2) CHES buffer (pH 9.5) is reacted at a temperature of 5 to 45 ° C. for 30 minutes.
  • CHES buffer pH 9.5
  • a reaction solution (100 ⁇ l) containing 5 mg / l C10G0093, 10 mM compound I and 20 mM sodium NAD and 50 mM buffer (MES, MOPS, TAPS, CHES and CAPS) adjusted to pH 6.0 to 10.4 was prepared at 30 ° C.
  • the optimum pH of C10G0093 was evaluated by reacting for 30 minutes. As a result, as shown in FIG. 11, the optimum pH was around 8.5 to 10.5.
  • the optimum temperature and optimum pH are based on values obtained by analyzing the concentration of Compound II produced in the reaction solution after the reaction by reverse phase HPLC under the conditions described in Example 4.
  • Compound I has four types of optical isomers: ⁇ S, ⁇ R (SR); ⁇ R, ⁇ S (RS); ⁇ R, ⁇ R (RR); ⁇ S, ⁇ S (SS).
  • SR ⁇ S
  • RS ⁇ R
  • RR ⁇ R
  • SS ⁇ S, ⁇ S
  • Chiral HPLC analysis condition column Chiral Pack IE-3 (manufactured by Daicel), 4.6 mm d. ⁇ 250 mm L.
  • Eluent (A) Water, (B) Acetonitrile, liquid delivery: 0-10 minutes 20% V / V (B), 10-15 minutes 20-30% V / V (B), 15-30 minutes 30% V / V (B), column temperature: 40 ° C., flow rate: 1.0 ml / min, detection photodiode array detector UV200-500 nm (PDA model 2998, manufactured by Waters) and killer (system engineering)
  • Example 6 Identification of Compound II Degrading Enzyme
  • Compound I or Compound II was added to Tris-HCl buffer (pH 7.5) to a final concentration of 5 mM and reduced glutathione to a final concentration of 10 mM, and then purified C10G0075, C10G0076, obtained in Example 3, C10G0077 and C10G0078 were added to each protein and incubated at room temperature for 16 hours to obtain a reaction solution. Thereafter, the change of Compound I or Compound II in the reaction solution was analyzed by reverse phase HPLC under the conditions described in Example 4.
  • Example 7 Degradation characteristics of compound II degrading enzyme
  • the reaction conditions when C10G0076 acts on Compound II were examined.
  • C10G0076 catalyzed the cleavage of ⁇ -O-4 linked glutathione in the presence of reduced glutathione, but showed no activity in the absence of reduced glutathione.
  • the molecular weight was shown to be 31 kDa by SDS-PAGE.
  • a reaction solution (100 ⁇ l) containing 5 mg / l C10G0076, 5 mM Compound II, 10 mM reduced glutathione and 100 mM (pH 8.9) CHES buffer (pH 9.5) is reacted at a temperature of 15 to 45 ° C. for 30 minutes.
  • the optimum temperature of C10G0076 was evaluated. As a result, the optimum temperature was around 30 to 35 ° C. as shown in FIG. Further, a reaction solution (100 ⁇ l) containing 5 mg / l C10G0076, 5 mM Compound II, 10 mM reduced glutathione, and 50 mM buffer (MES, MOPS, TAPS, CHES, and CAPS) adjusted to pH 5.6 to 10.4, The optimum pH of C10G0076 was evaluated by reacting at 30 ° C. for 30 minutes. As a result, as shown in FIG. 13, the optimum pH was around 8.5 to 9.5. The optimum temperature and the optimum pH are based on values obtained by analyzing the concentration of guaiacol produced in the reaction solution after the reaction by reverse-phase HPLC under the conditions described in Example 4 (retention time around 5.5 minutes).
  • C10G0077 catalyzed the cleavage of ⁇ -O-4 linked glutathione in the presence of reduced glutathione, but showed no activity in the absence of reduced glutathione.
  • the molecular weight was shown to be 31 kDa by SDS-PAGE.
  • a reaction solution (100 ⁇ l) containing 5 mg / l C10G0077, 5 mM Compound II, 10 mM reduced glutathione and 100 mM (pH 8.9) MOPS buffer (pH 7.5) is reacted at a temperature of 5 to 35 ° C. for 30 minutes. Thus, the optimum temperature of C10G0077 was evaluated.
  • the optimum temperature was around 25-30 ° C. as shown in FIG. Furthermore, a reaction solution (100 ⁇ l) containing 5 mg / l C10G0077, 5 mM Compound II, 10 mM reduced glutathione, and 50 mM buffer (MES, MOPS, TAPS, CHES, and CAPS) adjusted to pH 5.6 to 10.4, 20
  • the optimum pH of C10G0077 was evaluated by reacting at 30 ° C. for 30 minutes. As a result, as shown in FIG. 15, the optimum pH was around 7-8.
  • the optimum temperature and optimum pH are based on values obtained by analyzing the concentration of guaiacol produced in the reaction solution after the reaction by reverse phase HPLC under the conditions described in Example 4.
  • Compound II has two optical isomers, a ⁇ R form and a ⁇ S form.
  • changes in the optical isomer composition of Compound II occurring before and after the reaction were analyzed by HPLC using a chiral column, Chiral Pack IE-3.
  • C10G0076 selectively acts on the ⁇ S form
  • C10G0077 selectively acts on the ⁇ R form.
  • C10G0076 and C10G0077 have reactivity with non-phenolic lignin model dimer compounds of Compound II, indicating that veratrylglycerol- ⁇ -guaiacyl ether (1- (3,4-dimethoxyphenyl) -2- Using (2-methoxyphenoxy) propane-1,3-diol; Compound V), confirmation was made as follows. Normal lignin has many non-phenolic structural units. Therefore, having the activity of decomposing compound V means that the enzyme has the activity of decomposing natural lignin.
  • N, N′-dimethylformamide (DMF) was used as a solvent instead of a phase transfer catalyst, and the aldol reaction conditions of HCHO were set to K 2 CO 2.
  • An improved method was used, such as in a 3- EtOH system.
  • the NMR spectrum of the product was consistent with Compound V data published by USDA. When the 1 H-NMR spectrum was calculated using the integral value of ⁇ -proton, the ratio of erythro isomer to threo isomer was about 1.1: 1.
  • C10G0076 0.1 ⁇ g, 1 mM Compound V and 2 mM reduced glutathione, 100 mM (pH 9.5) N-Cyclohexyl-2-aminoethanesulfonic acid-NaOH (CHES) buffer (pH 9.5) and 10% V / V DMF
  • the mixed solution was reacted at 30 ° C. for 15 minutes.
  • the reaction solution after the reaction was subjected to reverse phase HPLC analysis, decrease in Compound V and formation of guaiacol were observed. This revealed that C10G0076 has reactivity with non-phenolic lignin model dimer compounds.
  • Example 8. Identification of Compound III Generating Enzyme Using Compound II as a substrate, the reaction was carried out with the purified protein of C10G0075, C10G0076 and C10G0077 obtained in Example 3, or the combination of the purified protein of C10G0076, C10G0077 and C10G0078. As a result, it was found that compound III was produced from the reaction product of both combinations. Compound III showed a retention time consistent with the metabolite (3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone) obtained when Compound I was added to the culture medium of MBES04 strain. .
  • the reaction solution was purified by a solid phase extraction method (OASIS WAX; manufactured by Waters) and subjected to reverse phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4.
  • OASIS WAX solid phase extraction method
  • UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4.
  • the analysis result of Compound III was This agreed well with the analysis result of 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone, which was the main product obtained when the MBES04 strain was cultured in a medium containing Compound I.
  • C10G0076 specifically recognizes ⁇ S form + C10G0075
  • C10G0076 specifically recognizes ⁇ S form + C10G0078
  • C10G0077 specifically recognizes ⁇ R form + C10G0075
  • C10G0077 specifically recognizes ⁇ R form + C10G0078
  • Example 9 Molecular weight of Compound III-producing enzyme
  • the molecular weight of C10G0078 was shown to be 33 kDa by SDS-PAGE.
  • the molecular weight of C10G0075 was shown to be 27 kDa by SDS-PAGE.
  • Example 10 Lignin degradation using Compound I degrading enzyme, Compound II degrading enzyme and Compound III producing enzyme (1)]
  • a compound having a phenylpropane structure was produced from the Casiogakuzu extract by the following procedure. 50 g of dried Casiogakuzu powder was suspended in 300 mL of dioxane and immersed for 6 days at room temperature. The solid content was removed by filtration from the obtained suspension with a filter paper to obtain a filtrate. The filtrate was dried under reduced pressure with an evaporator to obtain a casidioxane extract. Casidioxane extract was dissolved in DMF to a final concentration of 10% W / V. Using the obtained Casiogakuzu extract, a reaction solution having the following composition was prepared and reacted at room temperature for 24 hours.
  • reaction solution 50 ⁇ L was purified by a solid phase extraction method (OASISSWAX; manufactured by Waters) and dried under nitrogen gas. The residue after drying was dissolved in 0.5 mL of 20% V / V acetonitrile and subjected to reverse phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4.
  • OASISSWAX solid phase extraction method
  • Example 11 Lignin degradation using Compound I degrading enzyme, Compound II degrading enzyme and Compound III producing enzyme (2)] Using the Casiogakuzu extract prepared in Example 10, a reaction solution having the following composition was prepared and reacted at room temperature for 16 hours.
  • any one of the combination purified enzymes C10G0069 protein concentration: 0.41 mg / mL shown in Table 3 1/20 volume Purified enzyme C10G0093 (protein concentration: 0.16 mg / mL) 1/20 volume purified enzyme C10G0076 (protein concentration: 0.29 mg / mL) 1/20 volume purified enzyme C10G0077 (protein concentration: 0.28 mg / mL) 1/20 volume purified enzyme C10G0075 (protein concentration: 0.62 mg / mL) 1/20 volume purified enzyme C10G0078 (protein concentration: 0.10 mg / mL) 1/20 volume reduced glutathione 5 mM NAD sodium salt 5mM
  • reaction solution 50 ⁇ L was purified by a solid phase extraction method (OASISSWAX; manufactured by Waters) and dried under nitrogen gas. The residue after drying was dissolved in 0.5 mL of 20% V / V acetonitrile and subjected to reverse phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4.
  • OASISSWAX solid phase extraction method
  • Example 12 Lignin degradation using Compound I-degrading enzyme, Compound II-degrading enzyme and Compound III-producing enzyme (3)] 50 g of dried inarawa powder was suspended in a mixed solution of 300 mL of dioxane and 15 mL of water and immersed for 6 days at room temperature. After soaking, insoluble matters were filtered off from the suspension with a filter paper to obtain a filtrate. The filtrate was dried under reduced pressure with an evaporator and then dried with a desiccator to obtain 2.65 g of a solid. 50 ml of ethyl acetate and 70 ml of pure water were added to the solid to dissolve the solid. Furthermore, it melt
  • reaction solution 50 ⁇ L was purified by a solid phase extraction method (OASISSWAX; manufactured by Waters) and dried under nitrogen gas. The residue after drying was dissolved in 0.5 mL of 20% V / V acetonitrile and subjected to reverse phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4.
  • OASISSWAX solid phase extraction method
  • Example 13 Lignin degradation using Compound I degrading enzyme, Compound II degrading enzyme and Compound III producing enzyme (4)]
  • a compound having a phenylpropane structure from a shiitake mushroom waste bed was produced by the following procedure. 10 g of dried shiitake mushroom bed powder was suspended in 100 mL of ion-exchanged water and immersed at room temperature for 3 hours. The solid content was filtered off from the obtained suspension with a filter paper to obtain a filtrate (water washing solution 1). The residue was suspended in 100 mL of isopropanol and immersed for 3 hours at room temperature. After soaking, the solid content was filtered off with a filter paper to obtain a filtrate (isopro solution 1 after washing with water).
  • Washing solution 1 after washing with water, 25 mL of isopropyl solution 1 and 90% isopropyl solution 1 were taken out and dried under reduced pressure while heating to 45 ° C. with an evaporator. 469, 55 and 247 mg of solids were obtained from Wash Solution 1, Washed Isopro Solution 1 and 90% Isopro Solution 1, respectively.
  • the solid substance obtained from Washing Solution 1 was dissolved in 2 mL (234.4 mg / mL) of water, and after washing, obtained from Isopro Solution 1 (27.4 mg / mL) and 90% Isopro Solution 1 (123.5 mg / mL). Each solid obtained was dissolved in 2 mL of DMF. Using these three kinds of dissolved extracts, a reaction solution having the following composition was prepared and reacted at room temperature for 24 hours.
  • Reaction solution composition (4) Shiitake waste bed extract (one of the above three extracts) 1/20 volume (The concentration of the dry solid contained in the reaction solution is 11.7 mg / mL when the washing solution 1 is used, 1.37 mg / mL when the isopropyl solution 1 is used after washing with water, and 6 when the 90% isopropyl solution 1 is used.
  • reaction solution 50 ⁇ L was purified by a solid phase extraction method (OASISSWAX; manufactured by Waters) and dried under nitrogen gas. The residue after drying was dissolved in 0.5 mL of 20% V / V acetonitrile and subjected to reverse phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4.
  • OASISSWAX solid phase extraction method
  • Non-Patent Documents 4 to 12 described above are as follows. The description of the documents is incorporated herein by reference: Non-Patent Document 4: Besemer J et al .; Nucleic Acids Research, 2001, vol 29, p2607 Non-Patent Document 5: Masai, E .; S. Kubota. Y. Katayama, S .; Kawai, M .; Yamazaki, and N.K. Morohoshi. 1993. Biosci. Biotechnol. Biochem. 57: 1655-1659.
  • Non-Patent Document 6 Sato Y, Moriuchi H, Hisyama S, Otsuka Y, Oshima K, Kasai D, Nakamura M, Ohara S, Katayama Y, Fukuda M, Masai E. Appl Environ Microbiol. 2009; 75 (16): 5195-201.
  • Non-Patent Document 7 Masai, E .; Y. Katayama, S .; Kubota, S .; Kawai, M .; Yamazaki, and N.K. Morohoshi. 1993. FEBS Lett. 323: 135-140.
  • Non-Patent Document 8 Masai, E .; Y.
  • Non-Patent Document 9 Masai E, Ichimura A, Sato Y, Miyauchi K, Katayama Y, Fukuda M. et al. J Bacteriol. 2003 Mar; 185 (6): 1768-75.
  • Non-Patent Document 10 Hosoya et al .: Journal of the Wood Society of Japan 26 (2) 118-21, 1980
  • Non-Patent Document 11 Hishiyama et al: Tetrahedron Letters 53, 842-845, 2012
  • Non-Patent Document 12 K. Itoh: Mokuzai Gakashishi vol. 38, No. 6, 579-584 (1992)
  • sequences listed in the sequence listing are as follows: [SEQ ID NO: 1] C10G0069 MTQVKGRTAFITGGGSGVALGQAKVFARAGCKVAIADIRQDHLDEAMAWFEAENAKGANYEVMAVKLDITDREAYAKVADEVEAKLGPVELLFNTAGVSHFGAIQDATYDDWDWQIDVNLRGVINGVRTFVPRMIERGNGGHVVNTASMSAFVALKGTGIYCTTKMAVRGLTETLALDLEEHGIGVSLLCPGAVNTNIHEALLTRPKHLADTGYYQAGPEMFAHLKNVIECGMEPETLANHVLKAVEENQLYVLAYPEFRKPLEDIHARVMAALANPEDDPDYDRRVAHGVPGGEAKEEEKTA [SEQ ID NO: 2] C10G0093 MQDLPGKTAFVTGGASGIGLGIAKALLGAGMNVAIADIRQDHLDDAAAELDGGDKVLALQLDVTDRAAFAAAADATEAKFGKIHILCNNAGVAVVGPTD
  • Glutathione-added 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone is obtained as an intermediate of -1-propanone.
  • 3-Hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone can be converted into various industrially useful compounds, for example, raw materials for resins, adhesives, resist materials, pharmaceuticals, etc. It can be used as a raw material for a production method of 1- (4-hydroxy-3-methoxyphenyl) -1,3-propanediol and the like that can be expected to be used as

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Abstract

The objective of the present invention is to provide a method which, relative to prior methods, specifically and efficiently produces a glutathione-added phenyl propane-based compound, which is an intermediate of a phenyl propane-based compound, from a carbonyl phenyl propane-based compound, said intermediate to be utilized when producing a compound having a phenyl propane structure from natural biomass containing lignins by exerting enzyme action. The abovementioned objective is solved by glutathione S-transferase derived from micro-organisms of the genus Novosphingobium, and a method for producing a glutathione-added phenyl propane-based compound, said method including a step for obtaining the glutathione-added phenyl propane-based compound by exerting the action of said enzyme on a carbonyl phenyl propane-based compound in the presence of reduced glutathione.

Description

酵素を利用したフェニルプロパン系化合物中間体の製造方法Method for producing phenylpropane-based compound intermediate using enzyme 関連出願の相互参照Cross-reference of related applications
本出願は、２０１４年２月１０日出願の日本特願２０１４－０２３８４４号の優先権を主張し、その全記載はここに開示として援用される。 This application claims the priority of Japanese Patent Application No. 2014-023844 filed on Feb. 10, 2014, the entire description of which is incorporated herein by reference.
本発明は、酵素を利用してカルボニルフェニルプロパン系化合物からフェニルプロパン構造を有する化合物の中間体であるグルタチオン付加フェニルプロパン系化合物を特異的に生産する方法及びそのための酵素に関する。 The present invention relates to a method for specifically producing a glutathione-added phenylpropane compound, which is an intermediate of a compound having a phenylpropane structure, from a carbonylphenylpropane compound using an enzyme, and an enzyme therefor.
リグニンは植物の維管束細胞壁成分として存在する無定形高分子物質であって、フェニルプロパン系の構成単位が複雑に縮合したものであり、メトキシ基を含有することが化学構造上の大きな特徴になっている。リグニンは木質化した植物細胞を相互に膠着し、組織を強化する働きをしており、木材中に１８～３６％、草本中には１５～２５％存在する。そこで、木材を有効利用するために、リグニンを分解し、有用化合物を得ようとする試みが種々なされている。 Lignin is an amorphous polymer that exists as a component of plant vascular cell walls. It is a complex condensation of phenylpropane-based structural units, and the main feature of its chemical structure is the inclusion of methoxy groups. ing. Lignin has a function of sticking woody plant cells to each other and strengthening the tissue, and 18 to 36% in wood and 15 to 25% in herbs. Therefore, in order to effectively use wood, various attempts have been made to decompose lignin and obtain useful compounds.
一方、フェニルプロパン系化合物としては、例えば、クマル酸、ケイ皮酸、コーヒー酸（３，４－ジヒドロキシケイ皮酸）、オイゲノール、アネトール、コニフェリルアルコール、シナピルアルコール、フェルラ酸などが知られている。フェニルプロパン系化合物は、工業分野においては、香水、香料、精油、殺菌剤、麻酔薬、抗酸化剤などの医薬品や機能性食品及びそれらの合成中間体となる有用な化合物群である。 On the other hand, as phenylpropane compounds, for example, coumaric acid, cinnamic acid, caffeic acid (3,4-dihydroxycinnamic acid), eugenol, anethole, coniferyl alcohol, sinapil alcohol, ferulic acid and the like are known. Yes. In the industrial field, phenylpropane compounds are a group of useful compounds that become pharmaceuticals and functional foods such as perfumes, fragrances, essential oils, bactericides, anesthetics and antioxidants, and synthetic intermediates thereof.
例えば、木材、イナワラなどのリグノセルロース物質に含まれるリグニンを、高温高圧処理によりガス化する方法（下記特許文献１及び２を参照、該文献の記載はここに開示として援用される）、加圧熱水法（下記特許文献３を参照、該文献の記載はここに開示として援用される）などの物理化学的な方法によって、非特異的に低分子化する方法が知られている。しかしながら、これらの手法では、リグニンから特定の化合物を製造することは非常に困難である。これらの方法を採用すると、リグニンの持つ単位構造であり、ベンゼン環骨格に直接結合する炭素側鎖の炭素数が３個であるフェニルプロパン系化合物はさらに低分子化される。すなわち、グアイアコール（グアヤコール）、シリンゴールなどのベンゼン環骨格に直接結合する炭素側鎖の炭素数が０個のフェノール類や、バニリン、シリンガアルデヒドなどのベンゼン環骨格に直接結合する炭素側鎖の炭素数が１個のフェニルメタン化合物などに非特異的に変換される。また、分解生成物は多種多様な成分が混在しており、フェニルプロパン系化合物のみを取得することは非常に困難であり、特異的に製造することができない。また、これらの物理化学的な方法を採用する場合、これらの方法を実施するためには多くのエネルギーや特別な装置が必要である。 For example, a method of gasifying lignin contained in lignocellulosic materials such as wood and rice straw by high-temperature and high-pressure treatment (see Patent Documents 1 and 2 below, the description of which is incorporated herein by reference), pressurization A method for nonspecifically reducing the molecular weight by a physicochemical method such as a hydrothermal method (see Patent Document 3 below, the description of which is incorporated herein by reference) is known. However, with these techniques, it is very difficult to produce a specific compound from lignin. When these methods are employed, a phenylpropane compound having a unit structure possessed by lignin and having 3 carbon side chains directly bonded to the benzene ring skeleton is further reduced in molecular weight. In other words, the carbon side chain directly bonded to the benzene ring skeleton such as guaiacol (guaiacol), syringol, etc., and the carbon side chain directly bonded to the benzene ring skeleton such as vanillin, syringaldehyde, etc. Non-specifically converted to a phenylmethane compound having one carbon. In addition, the decomposition product contains a wide variety of components, and it is very difficult to obtain only the phenylpropane-based compound, and it cannot be specifically produced. In addition, when these physicochemical methods are employed, a lot of energy and special equipment are required to implement these methods.
そこで、リグニンを生物学的に処理してリグニン分解物を得ようとする試みがなされている。例えば、リグノセルロース物質に白色腐朽菌を接種及び培養することによってリグニンを分解する方法が知られている（下記特許文献４を参照、該文献の記載はここに開示として援用される）。 Therefore, attempts have been made to biologically treat lignin to obtain a lignin degradation product. For example, a method of degrading lignin by inoculating and cultivating a white rot fungus on a lignocellulosic material is known (see Patent Document 4 below, which is incorporated herein by reference).
ところで、天然リグニン中にはβ－アリールエーテル型結合が約５０％存在していることから、β－アリールエーテル型結合を分解し得るか否かは、天然リグニンの分解において重要な意義をもつ。このβ－アリールエーテル型結合の特異的な分解酵素を生産する微生物としては、スフィンゴビウム（Ｓｐｈｉｎｇｏｂｉｕｍ）属細菌（下記特許文献５及び非特許文献１を参照、該文献の記載はここに開示として援用される；ただし、非特許文献１には「シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属細菌」として記載されている）、ブレバンディモナス（Ｂｒｅｖｕｎｄｉｍｏｎａｓ）属細菌（下記特許文献６を参照、該文献の記載はここに開示として援用される）及びシュードモナス属細菌（下記非特許文献２を参照、該文献の記載はここに開示として援用される）が知られている。 By the way, since about 50% of β-aryl ether type bonds exist in natural lignin, whether or not β-aryl ether type bonds can be decomposed is important in the decomposition of natural lignin. Examples of microorganisms that produce a specific degrading enzyme of this β-aryl ether type bond include bacteria belonging to the genus Sphingobium (see Patent Document 5 and Non-Patent Document 1 below), the description of which is disclosed herein. However, in Non-Patent Document 1, it is described as “Pseudomonas genus bacteria”, Brevundimonas genus bacteria (see Patent Document 6 below, the description of this document is here) And Pseudomonas bacteria (see Non-Patent Document 2 below, the description of which is incorporated herein by reference).
特許文献４～６及び非特許文献１～２には、スフィンゴビウム属細菌、ブレバンディモナス属細菌及びシュードモナス属細菌並びにこれらの微生物によって生産されるβ－アリールエーテル切断酵素によって、リグニンのモデル化合物であるグアイアシルグリセロール－β－グアイアシルエーテル又は３－ヒドロキシ－２－（２－メトキシフェノキシ）－１－（３－メトキシ－４－ヒドロキシフェニル）－１－プロパノンのβ－アリールエーテル切断によってフェニルプロパン系化合物を生産する方法が記載されている。 Patent Documents 4 to 6 and Non-Patent Documents 1 and 2 include a model compound of lignin by a sphingobium genus, a brebandimonas genus bacterium, a pseudomonas genus bacterium, and a β-aryl ether cleaving enzyme produced by these microorganisms. By cleavage of the guaiacylglycerol-β-guaiacyl ether or 3-hydroxy-2- (2-methoxyphenoxy) -1- (3-methoxy-4-hydroxyphenyl) -1-propanone which is A method of producing a series compound is described.
また、下記非特許文献３（該文献の記載はここに開示として援用される）には、スフィンゴビウム属細菌由来のβ－アリールエーテル型結合分解酵素を用いて、グアイアシルグリセロール－β－グアイアシルエーテルからフェニルプロパン系化合物を生産しようとする場合、グアイアシルグリセロール－β－グアイアシルエーテルのＣα位の炭素原子及び該炭素原子に結合するアルコール性ヒドロキシ基に作用してカルボニル基を形成する第１の反応；生じたカルボニル化合物内に存在するβ－Ｏ－４位のエーテル結合を開裂させる第２の反応；並びに、第２の反応で生じたグルタチオン含有中間体からグルタチオンを除去する第３の反応の３段階の酵素逐次反応を経ることによって、フェニルプロパン系化合物を製造することができることが記載されている。 Non-patent document 3 below (the description of which is incorporated herein by reference) uses a β-aryl ether-type degrading enzyme derived from a sphingobium bacterium, and uses guaiacylglycerol-β-guay. In the case of producing a phenylpropane compound from an acyl ether, a carbonyl group is formed by acting on the carbon atom at the Cα position of the guaiacylglycerol-β-guayacyl ether and the alcoholic hydroxy group bonded to the carbon atom. A second reaction that cleaves the ether bond at the β-O-4 position present in the resulting carbonyl compound; and a third reaction that removes glutathione from the glutathione-containing intermediate generated in the second reaction. It is noted that a phenylpropane compound can be produced through an enzyme sequential reaction in three stages of the reaction. It is listed.
特開２０１２－１４０３４６号公報JP 2012-140346 A 特開２０１２－５０９２４号公報JP 2012-50924 A 特開２０１０－２３９９１３号公報JP 2010-239913 A 特開昭５０－４６９０３号公報JP 50-46903 A 特開平５－３３６９７６号公報JP-A-5-336976 特開２００２－３４５５７号公報JP 2002-34557 A
特許文献４に記載の方法によれば、微生物の作用によってリグニンを分解し得る可能性がある。しかし、特許文献４に記載の白色腐朽菌や白色腐朽菌が産生するリグニン分解酵素をリグノセルロース物質に作用させた場合、これらのリグニン分解反応の特異性が非常に低いことから、構造に統一性がない生成物ができるばかりか、主として生成物による重合反応が進行する。したがって、特許文献４に記載の方法は、反応生成物が工業原料としての利用性に欠けるという問題を有する。 According to the method described in Patent Document 4, there is a possibility that lignin can be degraded by the action of microorganisms. However, when the white rot fungi described in Patent Document 4 and the lignin-degrading enzyme produced by white rot fungi are allowed to act on lignocellulosic substances, the specificity of these lignin degradation reactions is very low, so the structure is unified. In addition to producing a product having no slag, a polymerization reaction mainly proceeds with the product. Therefore, the method described in Patent Document 4 has a problem that the reaction product lacks utility as an industrial raw material.
特許文献５～６及び非特許文献１～３に記載の方法は、リグニンのモデル化合物から、微生物やその生産酵素を利用して、フェニルプロパン系化合物を製造する方法である。しかし、これらの文献には、天然のバイオマスから、微生物や酵素を作用させることによって、フェニルプロパン系化合物を得たとする記載はない。 The methods described in Patent Documents 5 to 6 and Non-Patent Documents 1 to 3 are methods for producing a phenylpropane-based compound from a lignin model compound using a microorganism or its production enzyme. However, these documents do not describe that a phenylpropane compound was obtained from natural biomass by allowing microorganisms or enzymes to act.
特に、特許文献６に記載の酵素は、β－アリールエーテル型結合を有し、かつ、アリールグリセロール部分に遊離フェノール性水酸基を有する化合物におけるβ－アリールエーテル型結合を切断する酵素である。しかし、アリールグリセロール部分に遊離フェノール性水酸基を有しない化合物に対する活性はアリールグリセロール部分に遊離フェノール性水酸基を有する化合物に対する活性の１００分の１であり、到底十分な活性とはいえない。 In particular, the enzyme described in Patent Document 6 is an enzyme that cleaves a β-aryl ether type bond in a compound having a β-aryl ether type bond and a free phenolic hydroxyl group in the arylglycerol moiety. However, the activity with respect to a compound having no free phenolic hydroxyl group in the arylglycerol moiety is 1/100 of the activity with respect to the compound having a free phenolic hydroxyl group in the arylglycerol moiety, which is not sufficient.
また、特許文献６に記載の酵素を培養によって生産するためには、誘導物質を添加するなど、煩雑な培養条件設定が必要である。該酵素を一般的な組み換え宿主を使用して大量生産するための酵素遺伝子情報は得られていない。さらに、ブレバンディモナス属細菌由来のβ－アリールエーテル型結合分解酵素が作用する１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オン及びその誘導体である１－（４－ベンジロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンには２種の光学異性体が存在するところ、各光学異性体に対する酵素活性については特許文献６においてなんら記載がない。 Moreover, in order to produce the enzyme described in Patent Document 6 by culturing, complicated culturing conditions such as adding an inducer are required. Enzyme gene information for mass production of the enzyme using a general recombinant host has not been obtained. Furthermore, 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one on which a β-aryl ether-type decomposing enzyme derived from a genus Brevundimonas acts And its derivative 1- (4-benzyloxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one has two optical isomers. There is no description in Patent Document 6 regarding the enzyme activity for isomers.
非特許文献３に記載の酵素は、４種類の光学異性体（αＳ，βＲ；αＲ，βＳ；αＲ，βＲ；αＳ，βＳ）が存在するグアイアシルグリセロール－β－グアイアシルエーテルのうち、αＲ，βＳ体及びαＳ，βＳ体のグアイアシルグリセロール－β－グアイアシルエーテルのみを出発原料とすることができるが、αＳ，βＲ体及びαＲ，βＲ体を出発原料とすることはできない。その結果として、得られるフェニルプロパン系化合物の収量が低くなるという問題がある。さらに非特許文献３に記載の酵素が作用する基質については、遊離フェノール性水酸基を有する化合物の分解のみが知られており、遊離フェノール性水酸基を有しない化合物については非特許文献３において記載がない。 The enzyme described in Non-Patent Document 3 is a guaiacylglycerol-β-guaiacyl ether in which four kinds of optical isomers (αS, βR; αR, βS; αR, βR; αS, βS) exist. Only βS and αS, βS guaiacylglycerol-β-guaiacyl ether can be used as starting materials, but αS, βR and αR, βR cannot be used as starting materials. As a result, there is a problem that the yield of the obtained phenylpropane-based compound is lowered. Furthermore, with respect to the substrate on which the enzyme described in Non-Patent Document 3 acts, only decomposition of a compound having a free phenolic hydroxyl group is known, and no compound having a free phenolic hydroxyl group is described in Non-Patent Document 3. .
そこで、本発明が解決しようとする課題は、特許文献５～６及び非特許文献１～３に記載の方法と比較して、酵素を作用させることにより、リグニンを含む天然バイオマスからフェニルプロパン構造を有する化合物を生産するに際して利用される、カルボニルフェニルプロパン系化合物からフェニルプロパン系化合物の中間体であるグルタチオン付加フェニルプロパン系化合物を特異的かつ効率的に生産する方法を提供することにある。 Therefore, the problem to be solved by the present invention is that, compared with the methods described in Patent Documents 5 to 6 and Non-Patent Documents 1 to 3, a phenylpropane structure is obtained from natural biomass containing lignin by acting an enzyme. An object of the present invention is to provide a method for specifically and efficiently producing a glutathione-added phenylpropane-based compound, which is an intermediate of a phenylpropane-based compound, from a carbonylphenylpropane-based compound, which is used in the production of the compound having the same.
本発明者らは、上記課題を解決するために鋭意研究を積み重ね、深海沈木から単離したリグニン分解微生物であるノボスフィンゴビウム・エスピー　ＭＢＥＳ０４株のゲノム配列情報を解読し、その情報からリグニンを含む天然バイオマスからフェニルプロパン構造を有する化合物を特異的に生産することに関与する一連の酵素の遺伝子群を取得することに成功した。さらに取得した遺伝子群から組換え酵素群を作製し、リグニン含有バイオマスを基質としてこれらの酵素群の分解様式を解析した結果、フェニルプロパン系化合物を特異的かつ高収率で生産することができる酵素６種を見出した。そのうち、グルタチオン・Ｓ－転移酵素であるＣ１０Ｇ００７６及びＣ１０Ｇ００７７は、それぞれ相違する１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンの光学異性体に作用し、しかもアリールグリセロール部分に遊離フェノール性水酸基を有しない化合物であっても活性が非常に高いという驚くべき知見を得た。本発明はこのような成功例や知見に基づいて完成するに至った発明である。 In order to solve the above-mentioned problems, the inventors of the present invention have accumulated intensive research, deciphered the genome sequence information of Novosphingobium sp. MBES04 strain, which is a lignin-degrading microorganism isolated from deep-sea sediments, and obtained lignin from the information. We succeeded in obtaining a gene cluster of a series of enzymes involved in the specific production of compounds having a phenylpropane structure from natural biomass. Furthermore, as a result of producing a recombinant enzyme group from the acquired gene group and analyzing the degradation mode of these enzyme groups using lignin-containing biomass as a substrate, an enzyme capable of producing a phenylpropane compound in a specific and high yield We found 6 species. Among them, glutathione S-transferases C10G0076 and C10G0077 have different optical properties of 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one. A surprising finding was obtained that even a compound that acts on an isomer and does not have a free phenolic hydroxyl group in the arylglycerol moiety has a very high activity. The present invention has been completed based on such successful examples and knowledge.
したがって、本発明によれば、カルボニルフェニルプロパン系化合物に、還元型グルタチオンの存在下で、ノボスフィンゴビウム（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ）属微生物に由来する下記（２）の酵素を作用させることにより、グルタチオン付加フェニルプロパン系化合物を得る工程を含む、グルタチオン付加フェニルプロパン系化合物の製造方法が提供される。
（２）Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用する、ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ Therefore, according to the present invention, glutathione-added phenyl is obtained by allowing the carbonylphenylpropane-based compound to act on the enzyme of the following (2) derived from a microorganism belonging to the genus Novosphingobium in the presence of reduced glutathione. Provided is a method for producing a glutathione-added phenylpropane-based compound including a step of obtaining a propane-based compound.
(2) Glutathione S-transfer having a multi-domain of PRK15113 and maiA on the N-terminal side and acting on veratrylglycerol-β-guayacyl ether
好ましくは、本発明の製造方法において、前記（２）の酵素が、配列表の配列番号３に記載のアミノ酸配列を有するＣ１０Ｇ００７６又は配列表の配列番号４に記載のアミノ酸配列を有するＣ１０Ｇ００７７である。 Preferably, in the production method of the present invention, the enzyme of (2) is C10G0076 having the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing or C10G0077 having the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.
本発明の別の側面によれば、ノボスフィンゴビウム（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ）属微生物に由来する、Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用する、ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅが提供される。 According to another aspect of the present invention, Gst derived from a microorganism belonging to the genus Novosphingobium, having a multi-domain of PRK15113 and maiA on the N-terminal side, and veratrylglycerol-β-guayacyl ether A glutathione S-transferase is provided that acts on
好ましくは、本発明の酵素において、前記ノボスフィンゴビウム属微生物が、ノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．）　ＭＢＥＳ０４（受託番号：ＮＩＴＥ　Ｐ－０１７９７）である。 Preferably, in the enzyme of the present invention, the microorganism belonging to the genus Novosphingobium is Novosphingobium sp. MBES04 (accession number: NITE P-01797).
好ましくは、本発明の酵素において、前記ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅは、ＳＤＳ－ＰＡＧＥで確認できる分子量が３１ｋＤａであり；最適ｐＨが８．５～９．５であり；かつ、最適温度が３０～３５℃である。 Preferably, in the enzyme of the present invention, the glutathione S-transferase has a molecular weight of 31 kDa that can be confirmed by SDS-PAGE; an optimum pH is 8.5 to 9.5; and an optimum temperature is 30 to 35 ° C. It is.
好ましくは、本発明の酵素において、前記ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅは、ＳＤＳ－ＰＡＧＥで確認できる分子量が３１ｋＤａであり；最適ｐＨが７～８であり；かつ、最適温度が２５～３０℃である。 Preferably, in the enzyme of the present invention, the glutathione S-transferase has a molecular weight of 31 kDa which can be confirmed by SDS-PAGE; an optimum pH of 7 to 8; and an optimum temperature of 25 to 30 ° C.
好ましくは、本発明の酵素において、前記ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅは、配列表の配列番号３に記載のアミノ酸配列を有するｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅである。 Preferably, in the enzyme of the present invention, the glutathione S-transferase is a glutathione S-transfer having the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing.
好ましくは、本発明の酵素において、前記ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅは、配列表の配列番号４に記載のアミノ酸配列を有するｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅである。 Preferably, in the enzyme of the present invention, the glutathione S-transferase is glutathione S-transfer having the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.
本発明の製造方法や酵素によれば、農業廃棄物や木材などの天然物に由来するリグニンやリグニン関連物質を含むバイオマスからフェニルプロパン構造を有する有用化合物を製造するに際して利用される、カルボニルフェニルプロパン系化合物からフェニルプロパン系化合物の中間体であるグルタチオン付加フェニルプロパン系化合物を特異的かつ効率的に製造することができる。 According to the production method and enzyme of the present invention, carbonylphenylpropane used for producing a useful compound having a phenylpropane structure from biomass containing lignin derived from natural products such as agricultural waste and wood and lignin-related substances. A glutathione-added phenylpropane compound that is an intermediate of the phenylpropane compound can be specifically and efficiently produced from the compound.
図１は、フェニルプロパン系化合物の製造方法で用いられる酵素群の一態様と、作用する基質の光学異性体との関係を模式化した図である。FIG. 1 is a diagram schematically showing a relationship between an embodiment of an enzyme group used in a method for producing a phenylpropane-based compound and an optical isomer of an acting substrate. 図２は、ｃ１０ｇ００６９遺伝子がコードするアミノ酸配列をクエリーとしてＤＥＬＴＡ－ＢＬＡＳＴサーチを行った結果を示した図である。FIG. 2 is a diagram showing the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0069 gene as a query. 図３は、ｃ１０ｇ００９３遺伝子がコードするアミノ酸配列をクエリーとしてＤＥＬＴＡ－ＢＬＡＳＴサーチを行った結果を示した図である。FIG. 3 shows the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0093 gene as a query. 図４は、ｃ１０ｇ００７６遺伝子がコードするアミノ酸配列をクエリーとしてＤＥＬＴＡ－ＢＬＡＳＴサーチを行った結果を示した図である。FIG. 4 shows the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0076 gene as a query. 図５は、ｃ１０ｇ００７７遺伝子がコードするアミノ酸配列をクエリーとしてＤＥＬＴＡ－ＢＬＡＳＴサーチを行った結果を示した図である。FIG. 5 shows the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0077 gene as a query. 図６は、ｃ１０ｇ００７８遺伝子がコードするアミノ酸配列をクエリーとしてＤＥＬＴＡ－ＢＬＡＳＴサーチを行った結果を示した図である。FIG. 6 shows the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0078 gene as a query. 図７は、ｃ１０ｇ００７５遺伝子がコードするアミノ酸配列をクエリーとしてＤＥＬＴＡ－ＢＬＡＳＴサーチを行った結果を示した図である。FIG. 7 shows the results of a DELTA-BLAST search using the amino acid sequence encoded by the c10g0075 gene as a query. 図８は、Ｃ１０Ｇ００６９の最適温度を評価した結果を示した図である。FIG. 8 is a diagram showing the result of evaluating the optimum temperature of C10G0069. 図９は、Ｃ１０Ｇ００６９の最適ｐＨを評価した結果を示した図である。FIG. 9 is a diagram showing the results of evaluating the optimum pH of C10G0069. 図１０は、Ｃ１０Ｇ００９３の最適温度を評価した結果を示した図である。FIG. 10 is a diagram showing a result of evaluating the optimum temperature of C10G0093. 図１１は、Ｃ１０Ｇ００９３の最適ｐＨを評価した結果を示した図である。FIG. 11 is a diagram showing the results of evaluating the optimum pH of C10G0093. 図１２は、Ｃ１０Ｇ００７６の最適温度を評価した結果を示した図である。FIG. 12 is a diagram showing the result of evaluating the optimum temperature of C10G0076. 図１３は、Ｃ１０Ｇ００７６の最適ｐＨを評価した結果を示した図である。FIG. 13 is a graph showing the results of evaluating the optimum pH of C10G0076. 図１４は、Ｃ１０Ｇ００７７の最適温度を評価した結果を示した図である。FIG. 14 is a diagram showing the results of evaluating the optimum temperature of C10G0077. 図１５は、Ｃ１０Ｇ００７７の最適ｐＨを評価した結果を示した図である。FIG. 15 is a diagram showing the results of evaluating the optimum pH of C10G0077.
以下、本発明の詳細について説明する。
本発明の製造方法は、カルボニルフェニルプロパン系化合物に、還元型グルタチオンの存在下で、ノボスフィンゴビウム（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ）属微生物に由来する下記（２）の酵素を作用させることにより、グルタチオン付加フェニルプロパン系化合物を得る工程を含む、グルタチオン付加フェニルプロパン系化合物の製造方法である。
（２）Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用する、ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ Details of the present invention will be described below.
In the production method of the present invention, glutathione-added phenylpropane is produced by allowing the carbonylphenylpropane-based compound to act on the enzyme of the following (2) derived from a microorganism belonging to the genus Novosphingobium in the presence of reduced glutathione. It is a manufacturing method of a glutathione addition phenylpropane type compound including the process of obtaining a type compound.
(2) Glutathione S-transfer having a multi-domain of PRK15113 and maiA on the N-terminal side and acting on veratrylglycerol-β-guayacyl ether
本発明の製造方法では、カルボニルフェニルプロパン系化合物が、ノボスフィンゴビウム属微生物に由来する上記（２）の酵素の作用を受けることにより、グルタチオンが付加した３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノン、３－ヒドロキシ－１－（４－ヒドロキシ－３、５－ジメトキシフェニル）－１－プロパノン、３－ヒドロキシ－１－（４－ヒドロキシフェニル）－１－プロパノンなどのフェニルプロパン系化合物が生成される。 In the production method of the present invention, 3-hydroxy-1- (4-hydroxy) to which glutathione has been added by the action of the enzyme (2) derived from a Novosphingobium microorganism by the carbonylphenylpropane compound. -3-methoxyphenyl) -1-propanone, 3-hydroxy-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-propanone, 3-hydroxy-1- (4-hydroxyphenyl) -1-propanone Phenylpropane compounds such as are produced.
フェニルプロパン系化合物は、フェニルプロパン構造を有する化合物であれば特に限定されず、例えば、下記一般式（Ａ）
Figure JPOXMLDOC01-appb-C000001
 　　　　　　　　（Ａ）
（式中、Ｒは１個又は２個以上の炭素数が１～５であるアルキル基、アルコキシ基又は水素原子を示す。）
で表わされる、１位の位置にカルボニル基を有し、かつ、３位及びフェニル基の４位にヒドロキシ基を有する化合物であり、より具体的には３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノン、３－ヒドロキシ－１－（４－ヒドロキシ－３、５－ジメトキシフェニル）－１－プロパノン又は３－ヒドロキシ－１－（４－ヒドロキシフェニル）－１－プロパノンであり、好ましくは３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンである。 The phenylpropane compound is not particularly limited as long as it is a compound having a phenylpropane structure. For example, the following general formula (A)
Figure JPOXMLDOC01-appb-C000001
 (A)
(In the formula, R represents an alkyl group, an alkoxy group or a hydrogen atom having 1 or 2 or more carbon atoms of 1 to 5.)
And a compound having a carbonyl group at the 1-position and a hydroxy group at the 3-position and the 4-position of the phenyl group, and more specifically 3-hydroxy-1- (4-hydroxy- 3-methoxyphenyl) -1-propanone, 3-hydroxy-1- (4-hydroxy-3,5-dimethoxyphenyl) -1-propanone or 3-hydroxy-1- (4-hydroxyphenyl) -1-propanone And preferably 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone.
これらのうち、例えば、３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンは、下記式（Ｉ）の構造からなる。
Figure JPOXMLDOC01-appb-C000002
 　　　　　　　（Ｉ） Among these, for example, 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone has a structure of the following formula (I).
Figure JPOXMLDOC01-appb-C000002
 (I)
３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンは、例えば、逆相ＨＰＬＣにより、定性及び定量的に分析できる。逆相ＨＰＬＣの条件は、例えば、オクタデシルシリル基修飾シリカゲルカラム（ＯＤＳカラム）を用いて、溶離液Ａ（２ｍＭ　酢酸アンモニウム、０．０５％Ｖ／Ｖ　ギ酸）及び溶離液Ｂ（１００％Ｖ／Ｖ　メタノール）を用いて、カラム温度を４０℃、流速を１．２ｍｌ／ｍｉｎと設定して、溶離液Ａ　９０％Ｖ／Ｖ及び溶離液Ｂ　１０％Ｖ／Ｖの混合溶液を１分間送液した後、溶離液Ｂを１０％Ｖ／Ｖ～９５％Ｖ／Ｖのグラジェントで７分間送液する。この条件により、３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンは、ＵＶ検出器（２７０ｎｍ）を用いることにより、保持時間４．５分付近のピークとして検出できる。３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンの標準品を用いれば、検量線法や内標準法などにより、定量することが可能である。 3-Hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone can be analyzed qualitatively and quantitatively, for example, by reverse phase HPLC. The reverse-phase HPLC conditions are, for example, using an octadecylsilyl group-modified silica gel column (ODS column), eluent A (2 mM ammonium acetate, 0.05% V / V formic acid) and eluent B (100% V / V). Methanol), the column temperature was set to 40 ° C., the flow rate was set to 1.2 ml / min, and a mixed solution of eluent A 90% V / V and eluent B 10% V / V was fed for 1 minute. Thereafter, the eluent B is fed for 7 minutes in a gradient of 10% V / V to 95% V / V. Under these conditions, 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone can be detected as a peak near a retention time of 4.5 minutes by using a UV detector (270 nm). If a standard product of 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone is used, it can be quantified by a calibration curve method or an internal standard method.
本発明の製造方法では、カルボニルフェニルプロパン系化合物を出発原料として使用する。カルボニルフェニルプロパン系化合物は、アリールグリセロール－β－アリールエーテル型結合を有し、Ｃα位の炭素原子がカルボニルであり；かつ、Ｃα位の炭素原子にフェニル基が結合した化合物であれば特に限定されず、例えば、グアイアシルグリセロール－β－グアイアシルエーテルのＣα位の炭素原子に作用して、この部位でカルボニル基を形成させる酵素、好ましくは後述する実施例に記載のスフィンゴビウム・スピーシーズ（Ｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．）ＳＹＫ－６株由来のｌｉｇＤ遺伝子(ＹＰ＿００４８３３９９８)、ｌｉｇＬ遺伝子（ＡＢ４９１２２１）、ｌｉｇＮ遺伝子（ＡＢ４９１２２２）及びｌｉｇＯ遺伝子（ＹＰ＿００４８３６７２０）がコードするＬｉｇＤ、ＬｉｇＬ、ＬｉｇＮ及びＬｉｇＯ、並びにノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．）　ＭＢＥＳ０４株由来の配列表の配列番号１及び２に記載のＣ１０Ｇ００６９及びＣ１０Ｇ００９３などを、リグニン、リグニン関連物質又はこの両方を含むバイオマスに作用させて得られる化合物が挙げられる。カルボニルフェニルプロパン系化合物の具体例として、下記式（Ｆ）の化合物が挙げられるが、これに限定されない。
Figure JPOXMLDOC01-appb-C000003
 （Ｆ） In the production method of the present invention, a carbonylphenylpropane compound is used as a starting material. A carbonylphenylpropane-based compound is not particularly limited as long as it has an arylglycerol-β-aryl ether type bond, the carbon atom at the Cα position is carbonyl; and a phenyl group is bonded to the carbon atom at the Cα position. For example, an enzyme that acts on a carbon atom at the Cα position of guaiacylglycerol-β-guaiacyl ether to form a carbonyl group at this site, preferably a sphingobium species (Sphingobium spp. sp.) LigD, LigL, LigN and LigO encoded by ligD gene (YP_004833998), ligL gene (AB491221), ligN gene (AB491222) and ligO gene (YP_004836720) derived from SYK-6 strain, and Novosf Examples include compounds obtained by allowing C10G0069 and C10G0093 described in SEQ ID NOS: 1 and 2 of the sequence listing derived from Novosphingium sp. MBES04 strain to act on biomass containing lignin, lignin-related substances, or both . Specific examples of the carbonylphenylpropane-based compound include a compound represented by the following formula (F), but are not limited thereto.
Figure JPOXMLDOC01-appb-C000003
 (F)
リグニンは、当業界において通常知られるものであれば特に限定されないが、例えば、植物の維管束に存在し、主として下記式（Ｂ）～（Ｄ）として示す３種のフェニルプロパノイドを構成単位として複雑に重合した樹枝状構造を有するものとして知られているものである。
Figure JPOXMLDOC01-appb-C000004
 　　　　（Ｂ）
Figure JPOXMLDOC01-appb-C000005
 　　　　（Ｃ）
Figure JPOXMLDOC01-appb-C000006
 　　　　（Ｄ） The lignin is not particularly limited as long as it is commonly known in the art. For example, the lignin is present in plant vascular bundles, and mainly comprises three types of phenylpropanoids represented by the following formulas (B) to (D) as structural units. It is known to have a complex polymerized dendritic structure.
Figure JPOXMLDOC01-appb-C000004
 (B)
Figure JPOXMLDOC01-appb-C000005
 (C)
Figure JPOXMLDOC01-appb-C000006
 (D)
リグニン関連物質は、リグニンから誘導される物質であれば特に限定されず、例えば、リグニンの分解物やリグニンを処理して得られる物質に加えて、リグニンのモデル化合物とされている物質、例えば、グアイアシルグリセロール－β－グアイアシルエーテルなどを含む。バイオマスは、リグニン、リグニン関連物質又はこの両方を含むものであれば特に限定されず、例えば、草や木などの天然物、これら天然物に処理を加えて得られるもの、農業廃棄物などが挙げられる。 The lignin-related substance is not particularly limited as long as it is a substance derived from lignin. For example, in addition to substances obtained by treating lignin degradation products and lignin, substances that are model compounds of lignin, for example, Guaiacylglycerol-β-guayacyl ether and the like. Biomass is not particularly limited as long as it contains lignin, lignin-related substances, or both, and examples thereof include natural products such as grass and trees, those obtained by processing these natural products, agricultural waste, and the like. It is done.
リグニン及び／又はリグニン関連物質を含むバイオマス（以下、リグニン含有バイオマスともよぶ。）は、前処理の有無などによって、例えば、固体状、懸濁状、液体状などであり得る。例えば、リグニン含有バイオマスを粉砕したものを液体に加えて得られる懸濁液とすることもできる。 Biomass containing lignin and / or lignin-related substances (hereinafter also referred to as lignin-containing biomass) can be, for example, solid, suspended, liquid, etc. depending on the presence or absence of pretreatment. For example, a suspension obtained by adding pulverized lignin-containing biomass to a liquid can be used.
また、リグニン含有バイオマスは、リグニン抽出物であってもよい。リグニン抽出物としては、例えば、リグニン含有バイオマスの粉末化したものを、０．１％Ｗ／Ｖ～５０％Ｗ／Ｖ、好ましくは１％Ｗ／Ｖ～２０％Ｗ／Ｖとなるように、リグニンの抽出に適した溶媒中に懸濁して懸濁液とし、この懸濁液を１０℃～１５０℃、好ましくは２０℃～１３０℃、より好ましくは２０℃～８０℃で、数時間～数日間、好ましくは１時間～６日間の抽出処理に供し、次いで抽出処理液から固形分を除いた液体状のリグニン抽出物、又は液体状のリグニン抽出物から溶媒を留去し、乾固することにより得られる固体状のリグニン抽出物などが挙げられる。 The lignin-containing biomass may be a lignin extract. As the lignin extract, for example, powdered lignin-containing biomass is 0.1% W / V to 50% W / V, preferably 1% W / V to 20% W / V. It is suspended in a solvent suitable for extraction of lignin to form a suspension, and this suspension is at 10 ° C. to 150 ° C., preferably 20 ° C. to 130 ° C., more preferably 20 ° C. to 80 ° C., for several hours to several Subject to extraction treatment for 1 day, preferably 1 hour to 6 days, and then remove the solvent from the liquid lignin extract obtained by removing solids from the extraction treatment liquid or the liquid lignin extract and dry to dryness. And a solid lignin extract obtained by the above.
リグニンの抽出に適した溶媒は特に限定されず、例えば、水、ジオキサン、メタノール、イソプロパノールなどの低分子アルコール類、ジメチルホルムアミドなどが挙げられ、好ましくは水及びジオキサンである。 A solvent suitable for extraction of lignin is not particularly limited, and examples thereof include water, low molecular alcohols such as dioxane, methanol and isopropanol, dimethylformamide, and the like, preferably water and dioxane.
本発明の製造方法では、カルボニルフェニルプロパン系化合物からグルタチオン付加フェニルプロパン系化合物を得るために、ノボスフィンゴビウム属微生物に由来する酵素を用いる。ノボスフィンゴビウム属微生物は、ノボスフィンゴビウム（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ）属、別名としてスフィンゴモナス（Ｓｐｈｉｎｇｏｍｏｎａｓ）属に属する、０．３～０．８×２～３μｍのグラム陰性桿菌であれば特に限定されないが、例えば、各種芳香族化合物を分解することが知られているノボスフィンゴビウム属微生物である。本発明の製造方法において用いられるノボスフィンゴビウム属微生物の好ましい具体例は、ノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．）　ＭＢＥＳ０４（以下、ＭＢＥＳ０４株ともよぶ。）である。 In the production method of the present invention, an enzyme derived from a microorganism belonging to the genus Novosphingobium is used to obtain a glutathione-added phenylpropane compound from a carbonylphenylpropane compound. The microorganism of the genus Novosphingobium is not particularly limited as long as it is a gram-negative bacillus having a size of 0.3 to 0.8 × 2 to 3 μm, belonging to the genus Novosphingobium, also known as the genus Sphingomonas. For example, Novosphingobium microorganisms that are known to decompose various aromatic compounds. A preferred specific example of the microorganisms belonging to the genus Novosphingobium used in the production method of the present invention is Novosphingium sp. MBES04 (hereinafter also referred to as MBES04 strain).
ＭＢＥＳ０４株は、微生物の識別の表示を「Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．ＭＢＥＳ０４」とし、かつ、受託番号を「ＮＩＴＥ　Ｐ－０１７９７」として、独立行政法人製品評価技術基盤機構の特許微生物寄託センター（〒２９２－０８１８　千葉県木更津市かずさ鎌足２－５－８）に２０１４年１月３０日付けの受領日により寄託されている。また、ノボスフィンゴビウム・スピーシーズ　ＭＢＥＳ０４は、独立行政法人製品評価技術基盤機構の特許微生物寄託センターにおいて、２０１５年２月　９日付けの受領日により、国内寄託から国際寄託へ移管されている（受領番号：ＮＩＴＥ　ＡＢＰ－０１７９７）。 The MBES04 strain is designated as “Novosphingium sp. It has been deposited with the date of receipt dated January 30, 2014 in 2-5-8) Kazusa Kamashishi, Kisarazu City. In addition, Novosfingobium sp. MBES04 has been transferred from domestic deposit to international deposit on February 9, 2015 at the Patent Microbiology Deposit Center of the National Institute of Product Evaluation Technology (Received) Number: NITE ABP-01797).
酵素（２）Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用する、ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅは、１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンのβ位の炭素原子におけるアリールグリセロール－β－アリールエーテル型結合を還元的に切断して、β位の炭素原子にグルタチオンが付加された３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンを生成する活性を有する。Ｇｓｔは、ＰＳＳＭ－Ｉｄが２２２３６９８である、ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅによく見出されるアミノ酸配列である。ＰＲＫ１５１１３は、ＰＳＳＭ－Ｉｄが１８５０６８である、ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ；Ｐｒｏｖｉｓｉｏｎａｌによく見出されるアミノ酸配列である。ｍａｉＡは、ＰＳＳＭ－Ｉｄが２３３３３３である、ｍａｌｅｙｌａｃｅｔｏａｃｅｔａｔｅ　ｉｓｏｍｅｒａｓｅ中によく見出されるアミノ酸配列である。ＧｓｔのＥ－ｖａｌｕｅは、Ｃ１０Ｇ００７６に対して４．２１ｅ－３８であり、Ｃ１０Ｇ００７７に対して６．３５ｅ－１１である。ＰＲＫ１５１１３のＥ－ｖａｌｕｅは、Ｃ１０Ｇ００７６に対して３．１０ｅ－１６であり、Ｃ１０Ｇ００７７に対して４．５８ｅ－０３である。ｍａｉＡのＥ－ｖａｌｕｅは、Ｃ１０Ｇ００７６に対して８．０１ｅ－１７であり、Ｃ１０Ｇ００７７に対して１．０１ｅ－０５である。なお、本明細書で「マルチドメインを有する」とは、マルチドメインそのもののアミノ酸配列を有することに加えて、マルチドメインの一部のアミノ酸配列をＮ末端側又はＣ末端側などに有すること、マルチドメインと近似するアミノ酸配列を有することなどを意味する。 Glutathione S-transferase, which has the enzyme (2) Gst and the multidomain of PRK15113 and maiA on the N-terminal side and acts on veratrylglycerol-β-guayacyl ether, is 1- (4-hydroxy-3- Methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one reductively cleaves the arylglycerol-β-aryl ether type bond at the β-position carbon atom to give a β-position carbon atom Has the activity of producing 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone with glutathione added thereto. Gst is an amino acid sequence commonly found in glutathione S-transferase with PSSM-Id of 2223698. PRK15113 is an amino acid sequence commonly found in glutathione S-transfer; Provisional with PSSM-Id of 185068. maiA is an amino acid sequence often found in maleilacetoacetate isomerase with PSSM-Id of 233333. The E-value of Gst is 4.21e-38 for C10G0076 and 6.35e-11 for C10G0077. The E-value of PRK15113 is 3.10e-16 for C10G0076 and 4.58e-03 for C10G0077. The MaiA E-value is 8.01e-17 for C10G0076 and 1.01e-05 for C10G0077. In this specification, “having a multi-domain” means having a multi-domain amino acid sequence on the N-terminal side or C-terminal side in addition to having the amino acid sequence of the multi-domain itself, It means having an amino acid sequence similar to a domain.
１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オン、β位の炭素原子にグルタチオンが付加された３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノン及びベラトリルグリセロール－β－グアイアシルエーテルの構造を下記（Ｆ）、（Ｈ）及び（Ｊ）に示す。
Figure JPOXMLDOC01-appb-C000007
 （Ｆ）
Figure JPOXMLDOC01-appb-C000008
 （Ｈ）
Figure JPOXMLDOC01-appb-C000009
 （Ｊ） 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one, 3-hydroxy-1- (4 with glutathione added to the carbon atom at the β-position The structures of -hydroxy-3-methoxyphenyl) -1-propanone and veratrylglycerol-β-guayacyl ether are shown in (F), (H) and (J) below.
Figure JPOXMLDOC01-appb-C000007
 (F)
Figure JPOXMLDOC01-appb-C000008
 (H)
Figure JPOXMLDOC01-appb-C000009
 (J)
酵素（２）は、グルタチオンの存在下で、１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンのβ位の炭素原子におけるアリールグリセロール－β－アリールエーテル型結合に作用して、結果的にアリールエーテルと還元型グルタチオンとを置換することにより、β位の炭素原子にグルタチオンを付加させた３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンを生成させることができる酵素であると推測し得るが、この推測によって本発明の技術的範囲が変動するものではない。 Enzyme (2) is an aryl at the β-position carbon atom of 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one in the presence of glutathione. A 3-hydroxy-1- (4-hydroxy) in which glutathione is added to a carbon atom at the β-position by acting on a glycerol-β-aryl ether type bond and consequently substituting the aryl ether with reduced glutathione. Although it can be presumed that the enzyme can produce (-3-methoxyphenyl) -1-propanone, this presumption does not change the technical scope of the present invention.
また、酵素（２）は、還元型グルタチオンの存在下で、ベラトリルグリセロール－β－グアイアシルエーテルに作用して、β位の炭素原子にグルタチオンを付加することを触媒する。このことから、酵素（２）は、天然のリグニンに多く見られるアリールグリセロール部分に遊離フェノール性水酸基を有しない構成単位に対しても触媒活性を示し得る。これは非特許文献６に記載の酵素では決して得ることができない、着目すべき異質な触媒活性である。 The enzyme (2) acts on veratrylglycerol-β-guayacyl ether in the presence of reduced glutathione to catalyze the addition of glutathione to the β-position carbon atom. From this, enzyme (2) can also show a catalytic activity also with respect to the structural unit which does not have a free phenolic hydroxyl group in the arylglycerol part often seen in natural lignin. This is a heterogeneous catalytic activity to be noticed that can never be obtained with the enzyme described in Non-Patent Document 6.
酵素（２）の具体的な例は、配列表の配列番号３に記載のＣ１０Ｇ００７６及び配列表の配列番号４に記載のＣ１０Ｇ００７７である。 Specific examples of the enzyme (2) are C10G0076 described in SEQ ID NO: 3 in the Sequence Listing and C10G0077 described in SEQ ID NO: 4 in the Sequence Listing.
Ｃ１０Ｇ００７６及びＣ１０Ｇ００７７の理化学的性質は以下のとおりである。すなわち、Ｃ１０Ｇ００７６及びＣ１０Ｇ００７７は還元型グルタチオンの存在下で、β－Ｏ－４結合のグルタチオン添加開裂を触媒する。一方、これらの酵素は、還元型グルタチオンの非存在下では、このような触媒活性を示さない。そこで、本発明の製造方法では、還元型グルタチオンの存在下で酵素反応が実施される。Ｃ１０Ｇ００７６の分子量はＳＤＳ－ＰＡＧＥで３１ｋＤａであり；最適ｐＨは８．５～９．５であり；及び、最適温度は３０～３５℃である。Ｃ１０Ｇ００７７の分子量はＳＤＳ－ＰＡＧＥで３１ｋＤａであり；最適ｐＨは７～８であり；及び、最適温度は２５～３０℃である。なお、本明細書では、分子量は大腸菌組換えタンパク質についての分子量を表わす。また、本明細書でいう最適ｐＨ及び最適温度は、後述する実施例にて測定した結果のうち、目的産物の最も高い値から概ね８０％程度以上の値を示すｐＨ及び温度の範囲である。 The physicochemical properties of C10G0076 and C10G0077 are as follows. That is, C10G0076 and C10G0077 catalyze glutathione addition cleavage of β-O-4 bond in the presence of reduced glutathione. On the other hand, these enzymes do not exhibit such catalytic activity in the absence of reduced glutathione. Therefore, in the production method of the present invention, the enzyme reaction is carried out in the presence of reduced glutathione. The molecular weight of C10G0076 is 31 kDa by SDS-PAGE; the optimum pH is 8.5 to 9.5; and the optimum temperature is 30 to 35 ° C. The molecular weight of C10G0077 is 31 kDa by SDS-PAGE; the optimum pH is 7-8; and the optimum temperature is 25-30 ° C. In the present specification, the molecular weight represents the molecular weight of the E. coli recombinant protein. Moreover, the optimal pH and optimal temperature as used in this specification are the range of pH and temperature which show the value about 80% or more from the highest value of a target product among the results measured in the Example mentioned later.
１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンにおいて、β位の炭素原子が不斉炭素であることから、βＳ（Ｓ）；βＲ（Ｒ）という２種類の光学異性体が存在する。これらのうち、Ｃ１０Ｇ００７６は、１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンのＳ体に特異的に反応し、Ｃ１０Ｇ００７７はそのＲ体に特異的に反応する。 In 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one, the β-position carbon atom is an asymmetric carbon, so βS (S) There are two types of optical isomers, βR (R). Of these, C10G0076 specifically reacts with the S form of 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one, and C10G0077 Reacts specifically to R form.
本発明の製造方法で用いられる酵素（２）は、Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用するｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅであれば特に限定されないが、例えば、上記したＣ１０Ｇ００７６又はＣ１０Ｇ００７７の理化学的性質を有するものであれば好ましい。酵素（２）の好ましい態様は、例えば、配列表の配列番号３又は４に記載のアミノ酸配列と類似するアミノ酸配列を有する、Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用するｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅが挙げられる。 The enzyme (2) used in the production method of the present invention may be glutathione S-transferase having Gst and a multidomain of PRK15113 and maiA on the N-terminal side and acting on veratrylglycerol-β-guayacyl ether. Although not particularly limited, for example, those having the above-described physicochemical properties of C10G0076 or C10G0077 are preferable. A preferred embodiment of the enzyme (2) has, for example, Gst having an amino acid sequence similar to the amino acid sequence described in SEQ ID NO: 3 or 4 in the sequence listing, and a multi-domain of PRK15113 and maiA on the N-terminal side, and Examples include glutathione® S-transferase which acts on veratrylglycerol-β-guayacyl ether.
このような酵素としては、例えば、配列表の配列番号３又は４に記載のアミノ酸配列において１から数個のアミノ酸の欠失、置換又は付加を有するアミノ酸配列を含む、Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用するｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅや配列表の配列番号３又は４に記載のアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含むＧｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用するｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅが挙げられる。 Examples of such an enzyme include Gst, which includes an amino acid sequence having one to several amino acid deletions, substitutions or additions in the amino acid sequence set forth in SEQ ID NO: 3 or 4 in the sequence listing, and PRK15113 on the N-terminal side. And maiA multidomain, and 90% or more identity with glutathione S-transferase acting on veratrylglycerol-β-guayacyl ether and the amino acid sequence set forth in SEQ ID NO: 3 or 4 in the sequence listing Examples include Gst containing an amino acid sequence and glutathione S-transferase having a multi-domain of PRK15113 and maiA on the N-terminal side and acting on veratrylglycerol-β-guayacyl ether.
ここで、アミノ酸配列の「１から数個のアミノ酸の欠失、置換又は付加」における「１から数個」の範囲は、Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用する範囲であれば特に限定されないが、例えば、１、２、３、４、５、６、７、８、９、１０、１１、１２、１３、１４、１５、１６、１７、１８、１９又は２０個、好ましくは１、２、３、４又は５程度を意味する。また、「アミノ酸の欠失」とは配列中のアミノ酸残基の欠落若しくは消失を意味し、「アミノ酸の置換」は配列中のアミノ酸残基が別のアミノ酸残基に置き換えられていること、及び「アミノ酸の付加」とは配列中に新たなアミノ酸残基が付け加えられていることをそれぞれ意味する。 Here, the range of “1 to several” in “deletion, substitution or addition of 1 to several amino acids” of the amino acid sequence has Gst and a multi-domain of PRK15113 and maiA on the N-terminal side, and Although it is not particularly limited as long as it acts on veratrylglycerol-β-guayacyl ether, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19 or 20, preferably 1, 2, 3, 4 or 5. In addition, “amino acid deletion” means deletion or disappearance of an amino acid residue in the sequence, “amino acid substitution” means that an amino acid residue in the sequence is replaced with another amino acid residue, and “Addition of amino acid” means that a new amino acid residue is added to the sequence.
「１から数個のアミノ酸の欠失、置換又は付加」の具体的な態様としては、１から数個のアミノ酸が別の化学的に類似したアミノ酸で置き換えられた態様がある。例えば、ある疎水性アミノ酸を別の疎水性アミノ酸に置換する場合、ある極性アミノ酸を同じ電荷を有する別の極性アミノ酸に置換する場合などを挙げることができる。このような化学的に類似したアミノ酸は、アミノ酸毎に当該技術分野において知られている。具体例を挙げると、非極性（疎水性）アミノ酸としては、アラニン、バリン、イソロイシン、ロイシン、プロリン、トリプトファン、フェニルアラニン、メチオニンなどが挙げられる。極性（中性）アミノ酸としては、グリシン、セリン、スレオニン、チロシン、グルタミン、アスパラギン、システインなどが挙げられる。陽電荷をもつ（塩基性）アミノ酸としては、アルギニン、ヒスチジン、リジンなどが挙げられる。また、負電荷をもつ（酸性）アミノ酸としては、アスパラギン酸、グルタミン酸などが挙げられる。 A specific embodiment of “deletion, substitution or addition of one to several amino acids” includes an embodiment in which one to several amino acids are replaced with another chemically similar amino acid. For example, a case where a certain hydrophobic amino acid is substituted with another hydrophobic amino acid, a case where a certain polar amino acid is substituted with another polar amino acid having the same charge, and the like can be mentioned. Such chemically similar amino acids are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
また、アミノ酸配列における「同一性」（Ｉｄｅｎｔｉｔｙ）は、Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用する範囲であれば特に限定されないが、例えば、配列表の配列番号３又は４に記載のアミノ酸配列とアラインメントした場合の配列間の同一性が９０％以上であり、好ましくは９５％以上であり、より好ましくは９７％以上であり、さらに好ましくは９８％以上であり、なおさらに好ましくは９９％以上である。アミノ酸配列の同一性を求める方法は特に限定されないが、例えば、通常知られる方法を利用して、配列表の配列番号３又は４に記載のアミノ酸配列と対象となるアミノ酸配列とをアラインメントし、両者の配列の一致率を計算することにより求められる。 The “identity” in the amino acid sequence is particularly limited as long as it has Gst and a multi-domain of PRK15113 and maiA on the N-terminal side and acts on veratrylglycerol-β-guayacyl ether. However, for example, the identity between sequences when aligned with the amino acid sequence shown in SEQ ID NO: 3 or 4 in the sequence listing is 90% or more, preferably 95% or more, more preferably 97% or more. Yes, more preferably 98% or more, still more preferably 99% or more. The method for determining the identity of the amino acid sequence is not particularly limited. For example, using a commonly known method, the amino acid sequence described in SEQ ID NO: 3 or 4 in the sequence listing is aligned with the target amino acid sequence, It is calculated | required by calculating the coincidence rate of arrangement | sequence.
Ｃ１０Ｇ００７６又はＣ１０Ｇ００７７の分子量はＳＤＳ－ＰＡＧＥで３１ｋＤａである。そこで、配列表の配列番号３又は４に記載のアミノ酸配列と類似するアミノ酸配列を有する、Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用するｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅの分子量は、Ｃ１０Ｇ００７６又はＣ１０Ｇ００７７の分子量と近似する分子量であり、例えば、ＳＤＳ－ＰＡＧＥで、２６～３６ｋＤａであることが好ましく、２８～３４ｋＤａであることがより好ましく、３０～３２ｋＤａであることがさらに好ましく、３１ｋＤａ付近であることがなおさらに好ましい。 The molecular weight of C10G0076 or C10G0077 is 31 kDa by SDS-PAGE. Accordingly, Gst having an amino acid sequence similar to the amino acid sequence described in SEQ ID NO: 3 or 4 in the sequence listing, multi-domain of PRK15113 and maiA on the N-terminal side, and veratrylglycerol-β-guayacyl ether The molecular weight of glutathione -S-transferase that acts on C is a molecular weight that approximates the molecular weight of C10G0076 or C10G0077. For example, SDS-PAGE preferably has a molecular weight of 26-36 kDa, more preferably 28-34 kDa, more preferably 30 It is more preferably ~ 32 kDa, and still more preferably around 31 kDa.
酵素（２）を取得する方法は特に限定されないが、例えば、ノボスフィンゴビウム属微生物から得た酵素抽出液やその分画液を用いて、基質として１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンに作用させることにより、基質量の減少や生成物であるβ位の炭素原子にグルタチオンを付加させた３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンの存在を確認することで、酵素（２）を得ることができる。具体的には、Ｃ１０Ｇ００７６又はＣ１０Ｇ００７７の酵素作用、基質（光学異性体）特異性、理化学的性質、分子量などを指標として、ノボスフィンゴビウム属微生物、例えば、深海（水面下２００ｍ以下の海）において、腐食が進んだ針葉樹沈木に生息するノボスフィンゴビウム属微生物を培養した培養上清中の酵素活性を調べるといったスクリーニング法を挙げることができる。また、例えば、配列表の配列番号３又は４の記載を参照して、物理化学的に合成してもよいし、遺伝子工学的に配列表の配列番号３又は４に記載のアミノ酸配列をコードする塩基配列を有する遺伝子から作成してもよい。なお、配列表の配列番号３及び４に記載のアミノ酸配列をコードする塩基配列からなる遺伝子は、それぞれ配列表の配列番号９及び１０に記載したものである。例えば、配列番号９又は１０に記載の塩基配列と薬剤耐性遺伝子などの外来遺伝子又は異種塩基配列とを含むベクターを導入して作製した形質転換体を利用することにより、酵素（２）を取得することができる。 The method for obtaining the enzyme (2) is not particularly limited. For example, an enzyme extract obtained from a microorganism belonging to the genus Novosphingobium or a fraction thereof is used as a substrate for 1- (4-hydroxy-3-methoxyphenyl). ) -3-Hydroxy-2- (2-methoxyphenoxy) propan-1-one by acting on 3-hydroxy-1 in which the base mass is reduced or glutathione is added to the β-position carbon atom of the product By confirming the presence of-(4-hydroxy-3-methoxyphenyl) -1-propanone, the enzyme (2) can be obtained. Specifically, in the Novosphingobium microorganism, for example, in the deep sea (the sea below 200 m below the surface), using the enzyme action, substrate (optical isomer) specificity, physicochemical properties, molecular weight, etc. of C10G0076 or C10G0077 as indicators. And a screening method for examining enzyme activity in a culture supernatant obtained by culturing a microorganism of the genus Novosphingobium inhabiting a coniferous submerged tree. Further, for example, it may be synthesized physicochemically with reference to the description of SEQ ID NO: 3 or 4 in the sequence listing, or the amino acid sequence described in SEQ ID NO: 3 or 4 of the sequence listing is encoded by genetic engineering. You may create from the gene which has a base sequence. In addition, the gene which consists of a base sequence which codes the amino acid sequence of sequence number 3 and 4 of a sequence table is each described in sequence number 9 and 10 of a sequence table. For example, the enzyme (2) is obtained by using a transformant prepared by introducing a vector containing the base sequence shown in SEQ ID NO: 9 or 10 and a foreign gene such as a drug resistance gene or a heterologous base sequence. be able to.
カルボニルフェニルプロパン系化合物に酵素（２）を作用させることにより、グルタチオン付加フェニルプロパン系化合物が得られる。ここで、「酵素（２）を作用させる」とは、例えば、酵素（２）が有する、１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンのβ位の炭素原子におけるアリールグリセロール－β－アリールエーテル型結合を還元的に切断する作用を発揮させることを意味する。 A glutathione-added phenylpropane compound can be obtained by allowing the enzyme (2) to act on the carbonylphenylpropane compound. Here, “acting enzyme (2)” means, for example, 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propane possessed by enzyme (2) It means that the arylglycerol-β-aryl ether type bond at the β-position carbon atom of the 1-one is reductively cleaved.
酵素（２）の具体例であるＣ１０Ｇ００７６及びＣ１０Ｇ００７７は、上記したとおり、基質となる化合物の光学異性体によって使い分けられる。これらの酵素と作用する光学異性体との関係を模式化したものを図１とする。例えば、これらの酵素の組み合わせとしては、以下の（１）～（３）の組み合わせが考えられる。
（１）Ｃ１０Ｇ００７６
（２）Ｃ１０Ｇ００７７
（３）Ｃ１０Ｇ００７６及びＣ１０Ｇ００７７ As described above, C10G0076 and C10G0077, which are specific examples of the enzyme (2), are selectively used depending on the optical isomer of the compound serving as the substrate. A schematic representation of the relationship between these enzymes and acting optical isomers is shown in FIG. For example, the following combinations (1) to (3) can be considered as combinations of these enzymes.
(1) C10G0076
(2) C10G0077
(3) C10G0076 and C10G0077
酵素（２）をカルボニルフェニルプロパン系化合物に作用させるに際しては、例えば、Ｃ１０Ｇ００７６及びＣ１０Ｇ００７７を活性化するための還元型グルタチオンの存在下で、Ｃ１０Ｇ００７６及びＣ１０Ｇ００７７が活性を示すｐＨ及び温度に設定することが好ましい。また、これらの酵素は同時又は別々に用いてもよい。例えば、還元型グルタチオンの存在下でＣ１０Ｇ００７６を作用させた後にＣ１０Ｇ００７７を作用させてもよく、Ｃ１０Ｇ００７７を作用させた後にＣ１０Ｇ００７６を作用させてもよい。酵素（２）の作用時にｐＨの変動を抑えるために、作用系内に緩衝剤を添加することが好ましく、酵素（２）の還元的切断作用を促進するために、弱アルカリ性又はアルカリ性の緩衝剤を添加することがより好ましい。 When the enzyme (2) is allowed to act on the carbonylphenylpropane-based compound, for example, in the presence of reduced glutathione for activating C10G0076 and C10G0077, the pH and temperature at which C10G0076 and C10G0077 are active may be set. preferable. These enzymes may be used simultaneously or separately. For example, C10G0076 may be allowed to act after C10G0076 is allowed to act in the presence of reduced glutathione, and C10G0076 may be allowed to act after C10G0077 is allowed to act. In order to suppress fluctuations in pH during the action of the enzyme (2), it is preferable to add a buffering agent in the working system, and in order to promote the reductive cleavage action of the enzyme (2), a weakly alkaline or alkaline buffering agent. It is more preferable to add.
後述する実施例に記載があるとおり、酵素（２）は他の酵素とともに組み合わせて用いることができる。そのような酵素の例として、グアイアシルグリセロール－β－グアイアシルエーテルのＣα位の炭素原子を酸化する酵素としてスフィンゴビウム・スピーシーズ（Ｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．）ＳＹＫ－６株に由来するｌｉｇＤ遺伝子(ＹＰ＿００４８３３９９８)、ｌｉｇＬ遺伝子（ＡＢ４９１２２１）、ｌｉｇＮ遺伝子（ＡＢ４９１２２２）及びｌｉｇＯ遺伝子（ＹＰ＿００４８３６７２０）がコードするアミノ酸配列を有する酵素、並びにノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．）　ＭＢＥＳ０４株由来の配列表の配列番号１及び２に記載のＣ１０Ｇ００６９及びＣ１０Ｇ００９３など；１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンのβ位の炭素原子におけるアリールグリセロール－β－アリールエーテル型結合を還元的に切断する酵素としてｌｉｇＦ遺伝子（ＹＰ＿００４８３３９９７）及びｌｉｇＥ遺伝子（ＹＰ＿００４８３３９９８）がコードするアミノ酸配列を有する酵素など；β位の炭素原子に還元部位を有する３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンの該還元部位を脱離する酵素としてｌｉｇＧ遺伝子（ＹＰ＿００４８３３９９９）がコードするアミノ酸配列を有する酵素、並びに配列表の配列番号５及び６に記載のＣ１０Ｇ００７８及びＣ１０Ｇ００７５などが挙げられるが、これらに限定されない。 As described in the examples described later, the enzyme (2) can be used in combination with other enzymes. As an example of such an enzyme, a ligD gene (YP_004833998) derived from Sphingobium sp. SYK-6 strain as an enzyme that oxidizes the carbon atom at the Cα position of guaiacylglycerol-β-guaiacyl ether. , An enzyme having an amino acid sequence encoded by the ligL gene (AB491221), the ligN gene (AB491222), and the ligO gene (YP_004836720), as well as SEQ ID NO: 1 of the sequence listing derived from Novosphingium sp. MBES04 strain C10G0069 and C10G0093 and the like described in 2; 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) An enzyme having an amino acid sequence encoded by the ligF gene (YP_004833997) and the ligE gene (YP_004833998) as an enzyme that reductively cleaves an arylglycerol-β-aryl ether type bond at the β-position carbon atom of pan-1-one; Amino acid sequence encoded by the ligG gene (YP_004833999) as an enzyme that eliminates the reduction site of 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone having a reduction site at the β-position carbon atom And C10G0078 and C10G0075 described in SEQ ID NOS: 5 and 6 in the Sequence Listing, but are not limited thereto.
酵素を作用させる際の出発物質、緩衝剤、酵素、還元型グルタチオンなどの各成分の濃度や存在量は特に限定されず、適宜設定できる。また、出発物質と酵素との接触頻度を高めるために、これらの混合物を撹拌や振とうなどしてもよい。作用時間は目的物質の生成が認められる時間であれば特に限定されず、例えば、数時間～数日間であり、酵素の力価などによって適宜設定することができる。具体的には、１時間～３６時間程度又はそれ以上の時間である。 The concentration and abundance of each component such as a starting material, a buffer, an enzyme, and reduced glutathione when the enzyme is allowed to act are not particularly limited and can be set as appropriate. Further, in order to increase the contact frequency between the starting material and the enzyme, these mixtures may be stirred or shaken. The action time is not particularly limited as long as the production of the target substance is recognized, and is, for example, several hours to several days, and can be appropriately set depending on the enzyme titer and the like. Specifically, it is about 1 hour to 36 hours or more.
カルボニルフェニルプロパン系化合物の確認方法は、例えば、１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンのＨＰＬＣ分析条件を採用できる。具体的には、１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンは、例えば、逆相ＨＰＬＣにより、定性及び定量的に分析できる。逆相ＨＰＬＣの条件は、例えば、オクタデシルシリル基修飾シリカゲルカラム（ＯＤＳカラム）を用いて、溶離液Ａ（２ｍＭ　酢酸アンモニウム、０．０５％Ｖ／Ｖ　ギ酸）及び溶離液Ｂ（１００％Ｖ／Ｖ　メタノール）を用いて、カラム温度を４０℃、流速を１．２ｍｌ／ｍｉｎと設定して、溶離液Ａ　９０％Ｖ／Ｖ及び溶離液Ｂ　１０％Ｖ／Ｖの混合溶液を１分間送液した後、溶離液Ｂを１０％Ｖ／Ｖ～９５％Ｖ／Ｖのグラジェントで７分間送液する。この条件により、３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンは、ＵＶ検出器（２７０ｎｍ）を用いることにより、保持時間６．５分付近のピークとして検出できる。１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンの標準品を用いれば、検量線法や内標準法などにより、定量することが可能である。グルタチオン付加フェニルプロパン系化合物の確認方法は、例えば、β位の炭素原子にグルタチオンが付加された３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンのＨＰＬＣ分析条件を採用できる。例えば、β位の炭素原子にグルタチオンが付加された３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンは、上記の１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンのＨＰＬＣ分析条件によって、保持時間３．４～３．５分付近のピークとして検出できる。 As a method for confirming the carbonylphenylpropane-based compound, for example, HPLC analysis conditions of 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one can be employed. Specifically, 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one is analyzed qualitatively and quantitatively by, for example, reverse phase HPLC. it can. The reverse-phase HPLC conditions are, for example, using an octadecylsilyl group-modified silica gel column (ODS column), eluent A (2 mM ammonium acetate, 0.05% V / V formic acid) and eluent B (100% V / V). Methanol), the column temperature was set to 40 ° C., the flow rate was set to 1.2 ml / min, and a mixed solution of eluent A 90% V / V and eluent B 10% V / V was fed for 1 minute. Thereafter, the eluent B is fed for 7 minutes in a gradient of 10% V / V to 95% V / V. Under these conditions, 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone can be detected as a peak near a retention time of 6.5 minutes by using a UV detector (270 nm). If a standard product of 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one is used, it can be quantified by a calibration curve method or an internal standard method. Is possible. The method for confirming a glutathione-added phenylpropane compound employs, for example, HPLC analysis conditions for 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone in which glutathione is added to the β-position carbon atom it can. For example, 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone in which glutathione is added to the carbon atom at the β-position is the above 1- (4-hydroxy-3-methoxyphenyl)- Depending on the HPLC analysis conditions of 3-hydroxy-2- (2-methoxyphenoxy) propan-1-one, it can be detected as a peak with a retention time of 3.4 to 3.5 minutes.
作用系において生成された目的物質は、特別な処理を加えることなくそのままの状態で使用することができるが、例えば、作用後に固相抽出法やクロマトグラム法などの通常知られる芳香族化合物を精製する手段を採用して精製したものとすることができ、さらに溶媒を留去し乾燥させれば固体状のものとして得られる。 The target substance produced in the action system can be used as it is without any special treatment. For example, after the action, a commonly known aromatic compound such as a solid phase extraction method or a chromatogram method is purified. If the solvent is distilled off and dried, it can be obtained as a solid.
本発明の製造方法では、本発明の目的を達成し得る限り、上記した酵素作用工程の前段若しくは後段又は工程中に、種々の工程や操作を加入することができる。 In the production method of the present invention, as long as the object of the present invention can be achieved, various steps and operations can be added before, after, or during the enzyme action step.
本発明の製造方法の第１の具体的態様は、例えば、以下のとおりである。
リグニン含有バイオマスをジオキサンや水などのリグニンを溶解するのに適した溶媒中に懸濁し、２０℃～８０℃にて数時間～数日間保温する。保温後の懸濁液から固形分を除去して、リグニン抽出液を得る。リグニン抽出液を乾固し、得られた乾固物を酢酸エチルやＤＭＦなどの有機溶媒や含水有機溶媒に溶解してリグニン抽出物とする。リグニン抽出物；グアイアシルグリセロール－β－グアイアシルエーテルのＣα位の炭素原子を酸化する酵素；１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンのβ位の炭素原子におけるアリールグリセロール－β－アリールエーテル型結合を還元的に切断する酵素（酵素（２））；β位の炭素原子にグルタチオンが付加した３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンの該グルタチオンを脱離する酵素；緩衝剤；ＮＡＤ塩；及び還元型グルタチオンを含む反応液を調製し、ｐＨ７～１０、２０～４０℃で、数時間～数十時間反応させる。反応後の反応液から固相抽出法により精製し、不活性ガス下で乾固して、固体状のフェニルプロパン系化合物を得る。 The first specific embodiment of the production method of the present invention is as follows, for example.
The lignin-containing biomass is suspended in a solvent suitable for dissolving lignin such as dioxane and water, and kept at 20 ° C. to 80 ° C. for several hours to several days. The solid content is removed from the suspension after the incubation to obtain a lignin extract. The lignin extract is evaporated to dryness, and the resulting dried product is dissolved in an organic solvent such as ethyl acetate or DMF or a water-containing organic solvent to obtain a lignin extract. Lignin extract; enzyme that oxidizes carbon atom at the Cα position of guaiacylglycerol-β-guaiacyl ether; 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propane An enzyme that reductively cleaves an arylglycerol-β-aryl ether type bond at the β-position carbon atom of the 1-one (enzyme (2)); 3-hydroxy-1- with glutathione added to the β-position carbon atom A reaction solution containing (4-hydroxy-3-methoxyphenyl) -1-propanone, the enzyme that eliminates the glutathione; buffer; NAD salt; and reduced glutathione, was prepared, and the pH was 7 to 10, and 20 to 40 ° C. Let react for several hours to several tens of hours. The reaction solution after the reaction is purified by solid phase extraction and dried under an inert gas to obtain a solid phenylpropane compound.
本発明の製造方法の第２の具体的態様は、例えば、以下のとおりである。
上記リグニン抽出物；グアイアシルグリセロール－β－グアイアシルエーテルのＣα位の炭素原子を酸化する酵素；緩衝剤；及びＮＡＤ塩を含む反応液を調製し、ｐＨ７～１０、２０～４０℃で、数時間～数十時間反応させて得た反応液に、１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンのβ位の炭素原子におけるアリールグリセロール－β－アリールエーテル型結合を還元的に切断する酵素（酵素（２））；β位の炭素原子にグルタチオンを付加した３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンの該グルタチオンを脱離する酵素；及び還元型グルタチオンを添加して、さらにｐＨ７～１０、２０～４０℃で、数時間～数十時間反応させる。反応後の反応液から固相抽出法により精製し、不活性ガス下で乾固して、固体状のフェニルプロパン系化合物を得る。 The second specific embodiment of the production method of the present invention is as follows, for example.
A reaction solution containing the above lignin extract; an enzyme that oxidizes the carbon atom at the Cα position of guaiacylglycerol-β-guaiacyl ether; a buffering agent; and an NAD salt is prepared, and several times at pH 7-10, 20-40 ° C. The carbon atom at the β-position of 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one That reductively cleaves arylglycerol-β-aryl ether type bonds in enzyme (enzyme (2)); 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) in which glutathione is added to the β-position carbon atom An enzyme that removes glutathione from 1-propanone; and reduced glutathione are added, and the mixture is further reacted at pH 7 to 10, 20 to 40 ° C. for several hours to several tens of hours. Let me respond. The reaction solution after the reaction is purified by solid phase extraction and dried under an inert gas to obtain a solid phenylpropane compound.
本発明の製造方法の第３の具体的態様は、例えば、以下のとおりである。
上記リグニン抽出物；グアイアシルグリセロール－β－グアイアシルエーテルのＣα位の炭素原子を酸化する酵素；緩衝剤；及びＮＡＤ塩を含む反応液を調製し、ｐＨ７～１０、２０～４０℃で、数時間～数十時間反応させ、次いで１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オンのβ位の炭素原子におけるアリールグリセロール－β－アリールエーテル型結合を還元的に切断する酵素（酵素（２））及び還元型グルタチオンを添加して、さらにｐＨ７～１０、２０～４０℃で、数時間～数十時間反応させ、次いでβ位の炭素原子にグルタチオンを付加した３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンの該グルタチオンを脱離する酵素を添加して、さらにｐＨ７～１０、２０～４０℃で、数時間～数十時間反応させる。反応後の反応液から固相抽出法により精製し、不活性ガス下で乾固して、固体状のフェニルプロパン系化合物を得る。 The 3rd specific aspect of the manufacturing method of this invention is as follows, for example.
A reaction solution containing the above lignin extract; an enzyme that oxidizes the carbon atom at the Cα position of guaiacylglycerol-β-guaiacyl ether; a buffering agent; and an NAD salt is prepared, and several times at pH 7-10, 20-40 ° C. Reaction for hours to tens of hours, followed by arylglycerol-β at the carbon atom at the β-position of 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propan-1-one -An enzyme (enzyme (2)) that reductively cleaves an aryl ether type bond and reduced glutathione are added, and further reacted at pH 7 to 10, 20 to 40 ° C for several hours to several tens of hours, and then β-position Of 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone with glutathione added to the carbon atom of the enzyme to eliminate the glutathione Then, the mixture is further reacted at pH 7 to 10, 20 to 40 ° C. for several hours to several tens of hours. The reaction solution after the reaction is purified by solid phase extraction and dried under an inert gas to obtain a solid phenylpropane compound.
本発明の製造方法によって得られたグルタチオン付加フェニルプロパン系化合物は、例えば、さらに上記したＣ１０Ｇ００７８やＣ１０Ｇ００７５などのβ位の炭素原子にグルタチオンを付加した３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンの該グルタチオンを脱離する酵素に作用させてフェニルプロパン系化合物として、樹脂、接着剤、レジスト材料、医薬品などの原料又はその中間体として利用することができる。具体的には、最終的に３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンが得られる場合、該化合物は、１－（４－ヒドロキシ－３－メトキシフェニル）－１，３－プロパンジオールを経由して、医薬品、香料、食品材料などの原料として産業上有用なコニフェリルアルコールなどへと転換することができる。 The glutathione-added phenylpropane compound obtained by the production method of the present invention includes, for example, 3-hydroxy-1- (4-hydroxy-3-) in which glutathione is further added to the carbon atom at the β-position such as C10G0078 and C10G0075 described above. It can be used as a phenylpropane-based compound by acting on an enzyme that removes the glutathione of methoxyphenyl) -1-propanone, or as a raw material for resins, adhesives, resist materials, pharmaceuticals, etc., or an intermediate thereof. Specifically, when 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone is finally obtained, the compound is 1- (4-hydroxy-3-methoxyphenyl)- Via 1,3-propanediol, it can be converted into coniferyl alcohol, which is industrially useful as a raw material for pharmaceuticals, fragrances, food materials and the like.
本発明の別の側面によれば、ノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．）　ＭＢＥＳ０４（ＮＩＴＥ　Ｐ－０１７９７）などのノボスフィンゴビウム（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ）属微生物に由来する、Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用する、ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅが提供される。 According to another aspect of the present invention, Gst derived from microorganisms belonging to the genus Novosphingium such as Novosphingium sp. MBES04 (NITE P-01797) and PRK15113 on the N-terminal side. And glutathione S-transfer which has a multi-domain of maiA and maiA and acts on veratrylglycerol-β-guayacyl ether.
本発明の酵素の好ましい態様は、ＳＤＳ－ＰＡＧＥで確認できる分子量が３１ｋＤａであり；最適ｐＨが８．５～９．５であり；かつ、最適温度が３０～３５℃であるｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ、及びＳＤＳ－ＰＡＧＥで確認できる分子量が３１ｋＤａであり；最適ｐＨが７～８であり；かつ、最適温度が２５～３０℃であるｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅである。 A preferred embodiment of the enzyme of the present invention is a glutathione S-transferase having a molecular weight of 31 kDa that can be confirmed by SDS-PAGE; an optimum pH of 8.5 to 9.5; and an optimum temperature of 30 to 35 ° C. And the molecular weight that can be confirmed by SDS-PAGE is 31 kDa; the optimum pH is 7 to 8; and the optimum temperature is 25 to 30 ° C., which is glutathione S-transfer.
本発明の具体的態様は、配列表の配列番号３に記載のアミノ酸配列を有するｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ、及び配列表の配列番号４に記載のアミノ酸配列を有するｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅである。 A specific embodiment of the present invention is glutathione S-transfer having the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing, and glutathione S-transfer having the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.
以下、本発明を実施例によりさらに詳細に説明するが、本発明はこれら実施例に限定されるものではなく、本発明の課題を解決し得る限り、本発明は種々の態様をとることができる。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various modes as long as the problems of the present invention can be solved. .
［実施例１．ＭＢＥＳ０４株遺伝子の検出］
５ｍＭ　硫酸マグネシウムを添加したＬＢ培地（ベクトンディッキンソン社製）に、ノボスフィンゴビウム（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ）　ＭＢＥＳ０４株を接種し、３０℃、１２０ｒｐｍで２４時間振とう培養を行った。得られた培養液から８０００ｒｐｍ、５分間の遠心操作によりＭＢＥＳ０４株菌体を得た。 [Example 1. Detection of MBES04 strain gene]
LB medium (Becton Dickinson) supplemented with 5 mM magnesium sulfate was inoculated with Novosphingobium MBES04 strain and cultured with shaking at 30 ° C. and 120 rpm for 24 hours. MBES04 strain cells were obtained from the obtained culture broth by centrifugation at 8000 rpm for 5 minutes.
得られた菌体からＮｕｃｌｅｏＳｐｉｎ　ＰｌａｎｔＩＩ　Ｍｉｄｉキット（タカラバイオ社製）を用いて総ＤＮＡを抽出した。抽出したＤＮＡを用いて、８ｋｂ長のメイトペアライブラリーを作成し、ゲノムシークエンサー　ＧＳ　ＦＬＸシステム（ロシュ・ダイアグノティクス社製）を用いて配列の解析を行った。配列解析の結果、リード数１４２，３８９、平均長４４１．６６ｂｐ　総塩基数６２，８８８，１６２の配列データに係る塩基配列を得た。 Total DNA was extracted from the obtained cells using NucleoSpin Plant II Midi kit (manufactured by Takara Bio Inc.). An 8 kb long mate pair library was created using the extracted DNA, and the sequence was analyzed using the genome sequencer GS FLX system (Roche Diagnostics). As a result of sequence analysis, base sequences related to sequence data having 142,389 reads, an average length of 441.66 bp and a total number of bases of 62,888,162 were obtained.
得られた塩基配列を解析ソフトウエアＮｅｗｂｌｅｒ　２．６（ロシュ・ダイアグノティクス社製）を用いてアセンブルした結果、平均長６４３４．５ｂｐを持つ７８３個のコンティグが形成された。続いて、８ｋｂのメイトペアライブラリーの両端シーケンス情報から、各コンティグの配列を精査して得られたメイトペア情報に基づきＳｃａｆｆｏｌｄを生成させたところ、総塩基数５，５９６，３０６から成る３７個のＳｕｐｅｒＣｏｎｔｉｇが得られた。さらにサンガー法を用いてゲノム配列の一部を再解析し、ＳｕｐｅｒＣｏｎｔｉｇを３９個とした。得られた３９個のＳｕｐｅｒＣｏｎｔｉｇに含まれる全塩基配列を、ＧｅｎｅＭａｒｋＳ法（非特許文献４を参照）を用いて、３９個のＳｕｐｅｒＣｏｎｔｉｇに含まれる全塩基配列中にコードされる遺伝子領域、すなわちタンパク質をコードするオープンリーディングフレームに相当する領域を推定した。 As a result of assembling the obtained base sequence using analysis software Newbler 2.6 (Roche Diagnostics), 783 contigs having an average length of 6434.5 bp were formed. Subsequently, Scaffold was generated based on the mate pair information obtained by examining the sequence of each contig from the both-end sequence information of the 8 kb mate pair library. As a result, 37 scaffolds consisting of 5,596,306 total bases were generated. SuperContig was obtained. Furthermore, a part of the genome sequence was reanalyzed using the Sanger method, and 39 SuperContigs were set. Using the GeneMarkS method (see Non-Patent Document 4), the entire nucleotide sequence contained in the 39 SuperContigs obtained was converted into the gene region encoded by the entire nucleotide sequence contained in the 39 SuperContigs, ie, the protein. The region corresponding to the open reading frame to be encoded was estimated.
［実施例２．リグニン分解酵素の探索］
ＳＹＫ－６株がリグニン二量体モデル化合物であるグアイアシルグリセロール－β－グアイアシルエーテル（化合物Ｉ）を代謝する際の第一段階の反応、すなわち、化合物ＩのＣα位の炭素原子に結合するヒドロキシ基の酸化を触媒する酵素として、ＬｉｇＤ、ＬｉｇＬ、ＬｉｇＮ及びＬｉｇＯ（非特許文献５及び６を参照）並びにそれらをコードするｌｉｇＤ遺伝子(ＹＰ＿００４８３３９９８)、ｌｉｇＬ遺伝子（ＡＢ４９１２２１）、ｌｉｇＮ遺伝子（ＡＢ４９１２２２）及びｌｉｇＯ遺伝子（ＹＰ＿００４８３６７２０）が知られている。 [Example 2. Search for lignin-degrading enzymes]
The SYK-6 strain binds to the carbon atom at the Cα position of Compound I, that is, the first stage reaction when metabolizing ligacylglycerol-β-guaiacyl ether (Compound I), which is a lignin dimer model compound. Enzymes that catalyze the oxidation of hydroxy groups include LigD, LigL, LigN, and LigO (see Non-Patent Documents 5 and 6) and the ligD gene (YP_004833998), ligL gene (AB491122), ligN gene (AB491222), and The ligO gene (YP_004836720) is known.
そこで、実施例１により得られたＭＢＥＳ０４株のゲノム配列情報から検出された全遺伝子をクエリーとして、ＳＹＫ－６株由来のｌｉｇＤ遺伝子、ｌｉｇＬ遺伝子、ｌｉｇＮ遺伝子及びｌｉｇＯ遺伝子に相同性を示す遺伝子を、閾値：Ｃｏｖｅｒａｇｅ　５０％＜；Ｉｄｅｎｔｉｔｙ　２５％＜；Ｓｉｍｉｌａｒｉｔｙ　５０％＜；及びＥ－ｖａｌｕｅ　＜ｅ５に基づいてＢＬＡＳＴ相同性検索（ｂｌａｓｔ　２．２.２６； Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ）を行った。 Thus, using all the genes detected from the genome sequence information of MBES04 strain obtained in Example 1 as a query, genes showing homology to the ligD gene, ligL gene, ligN gene and ligO gene from SYK-6 strain, Threshold: Coverage 50% <; Identity 25% <; Similarity 50% <; and E-value <e5, BLAST homology search (blast 2.2.26; National Center for Biotechnology Information) was performed.
その結果、ＭＢＥＳ０４株のゲノム配列情報から、Ｓｈｏｒｔ－ｃｈａｉｎ　ｄｅｈｙｄｒｏｇｅｎａｓｅ／ｒｅｄｕｃｔａｓｅとアノテーションされる配列を有するｃ０１ｇ１１６２遺伝子、ｃ０１ｇ１３２４遺伝子、ｃ１０ｇ００６９遺伝子、ｃ１０ｇ００８０遺伝子、ｃ１０ｇ００９３遺伝子及びｃ１０ｇ００９４遺伝子の計６つの遺伝子が見出された。 As a result, from the genome sequence information of the MBES04 strain, a total of six genes, c01g1162 gene, c01g1324 gene, c10g0069 gene, c10g0080 gene, c10g0093 gene, and c10g0094 gene having a sequence annotated as Short-chain dehydrogenase / reductase were found. It was.
ＭＢＥＳ０４株由来の上記６遺伝子とＳＹＫ－６株由来のｌｉｇＤ、ｌｉｇＬ、ｌｉｇＮ及びｌｉｇＯとの相同性に関する情報を表１に示した。
Figure JPOXMLDOC01-appb-T000010
 Table 1 shows information on the homology between the above 6 genes derived from MBES04 and ligD, ligL, ligN and ligO derived from SYK-6.
Figure JPOXMLDOC01-appb-T000010
ＭＢＥＳ０４株由来の６遺伝子：ｃ０１ｇ１１６２遺伝子、ｃ０１ｇ１３２４遺伝子、ｃ１０ｇ００６９遺伝子、ｃ１０ｇ００８０遺伝子、ｃ１０ｇ００９３遺伝子及びｃ１０ｇ００９４遺伝子の塩基配列をクエリーとして相同性検索（ＢＬＡＳＴｎ、ｈｔｔｐ：／／ｂｌａｓｔ.ｎｃｂｉ.ｎｌｍ.ｎｉｈ.ｇｏｖ／Ｂｌａｓｔ.ｃｇｉ？ＰＲＯＧＲＡＭ＝ｂｌａｓｔｎ＆ＰＡＧＥ＿ＴＹＰＥ＝ＢｌａｓｔＳｅａｒｃｈ＆ＬＩＮＫ＿ＬＯＣ＝ｂｌａｓｔｈｏｍｅ、Ｃｏｖｅｒａｇｅ　５０％＜）を行ったところ、最も配列一致性の高い遺伝子配列としてヒットしたものは、ｃ０１ｇ１１６２遺伝子に対しては７１％の一致性でパエニバシラス・スピーシーズ（Ｐａｅｎｉｂａｃｉｌｌｕｓ　ｓｐ.）ＪＤＲ－２株のゲノム上（ＣＰ００１６５６.1）の４９３５５６２～４９３６３００番の位置にあるｓｈｏｒｔ－ｃｈａｉｎ　ｄｅｈｙｄｒｏｇｅｎａｓｅ／ｒｅｄｕｃｔａｓｅをコードすると推定される遺伝子；ｃ０１ｇ１３２４遺伝子に対しては７５％の一致性でノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ.）ＰＰ１Ｙ株のゲノム上（ＦＲ８５６８６２．１）の１１５４５０９～１１５５３３９番の位置にあるｓｈｏｒｔ－ｃｈａｉｎ　ｄｅｈｙｄｒｏｇｅｎａｓｅ／ｒｅｄｕｃｔａｓeをコードすると推定される遺伝子；ｃ１０ｇ００６９遺伝子に対しては７８％の一致性でノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ.）ＰＰ１Ｙ株のゲノム上（ＦＲ８５６８６２．１）の１２４１１３５～１２４２０２８番の位置にあるｓｈｏｒｔ－ｃｈａｉｎ　ｄｅｈｙｄｒｏｇｅｎａｓｅ／ｒｅｄｕｃｔａｓeをコードすると推定される遺伝子；ｃ１０ｇ００８０遺伝子に対しては７６％の一致性でノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ.）ＰＰ１Ｙ株のゲノム上（ＦＲ８５６８６２．１）の１２５１６４５～１２５２５４６番の位置にあるｓｈｏｒｔ－ｃｈａｉｎ　ｄｅｈｙｄｒｏｇｅｎａｓｅ／ｒｅｄｕｃｔａｓeをコードすると推定される遺伝子；ｃ１０ｇ００９３遺伝子に対しては８０％の一致性でノボスフィンゴビウム・アロマチシボランス（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ ａｒｏｍａｔｉｃｉｖｏｒａｎｓ）ＤＳＭ　１２４４４株のゲノム上（ＣＰ０００２４８．１）の８４３２８８～８４４１８０番の位置にあるｓｈｏｒｔ－ｃｈａｉｎ　ｄｅｈｙｄｒｏｇｅｎａｓｅ／ｒｅｄｕｃｔａｓeをコードすると推定される遺伝子；ｃ１０ｇ００９４遺伝子に対しては７７％の一致性でノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ.）ＰＰ１Ｙ株のゲノム上（ＦＲ８５６８６２．１）の１２６６１０３～１２６６９７４番の位置にあるｓｈｏｒｔ－ｃｈａｉｎ　ｄｅｈｙｄｒｏｇｅｎａｓｅ／ｒｅｄｕｃｔａｓeをコードすると推定される遺伝子であった。 6 genes from MBES04 strain: homology search (BLASTn, http://blast.ncbi.nilm.nih.gov/) using the base sequences of c01g1162 gene, c01g1324 gene, c10g0069 gene, c10g0080 gene, c10g0093 gene and c10g0094 gene as queries Blast.cgi? PROGRAM = blastn & PAGE_TYPE = BlastSearch & LINK_LOC = blastome, Coverage 50% <), the hit with the gene sequence having the highest sequence match was found to be 71% identical to c01g1162 On the genome of strains (Paenibacillus sp.) JDR-2 (CP001656.1) A gene presumed to encode short-chain dehydrogenase / reductase at positions 4935562 to 4936300; on the genome of Novosphingium sp. PP1Y strain with 75% identity to c01g1324 gene (FR856862.1), a gene presumed to encode short-chain dehydrogenase / reductase at positions 1154509 to 1155339; Novosphingobium sp. With a 78% consistency with the c10g0069 gene ) Positions 1241135 to 1242028 on the genome of the PP1Y strain (FR856862.1) A gene presumed to encode short-chain dehydrogenase / reductase in the above; on the genome of Novosphingobium sp. PP1Y strain (FR8566862.1) with 76% identity to c10g0080 gene A gene presumed to encode short-chain dehydrogenase / reductase at positions 1251645 to 12525546; Novosphingobium aromaticivorans strain DSM124 with 80% identity to the c10g0093 gene In the position of 844288-844180 on the genome (CP000248.1) 1266103 on the genome of Novosphingobium sp. PP1Y strain (FR8566862.1) with 77% identity to the c10g0094 gene, which is presumed to encode the short-chain dehydrogenase / reductase It was a gene presumed to encode short-chain dehydrogenase / reductase at the position of No. 1266694.
上記６遺伝子のうち、ｃ１０ｇ００６９遺伝子及びｃ１０ｇ００９３遺伝子がコードするアミノ酸配列を用いてＤＥＬＴＡ－ＢＬＡＳＴサーチ（Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎ、Ｄｏｍａｉｎ Ｅｎｈａｎｃｅｄ　Ｌｏｏｋｕｐ　Ｔｉｍｅ　Ａｃｃｅｌｅｒａｔｅｄ　ＢＬＡＳＴ）（ｈｔｔｐ：//ｂｌａｓｔ.ｎｃｂｉ.ｎｌｍ.ｎｉｈ.ｇｏｖ/Ｂｌａｓｔ.ｃｇｉ？ＰＡＧＥ＿ＴＹＰＥ＝ＢｌａｓｔＳｅａｒｃｈ＆ＰＲＯＧＲＡＭ＝ｂｌａｓｔｐ＆ＢＬＡＳＴ＿ＰＲＯＧＲＡＭＳ＝ｄｅｌｔａＢｌａｓｔ）を行った。その結果、上記２遺伝子がコードするタンパク質は、いずれもＲｏｓｓｍａｎｎ－ｆｏｌｄ　ＮＡＤ（Ｐ）（＋）結合タンパク質スーパーファミリーに属し、かつ、ＰＲＫ０６１９４のマルチドメインを有する、Ｓｈｏｒｔ－ｃｈａｉｎ　ｄｅｈｙｄｒｏｇｅｎａｓｅ/ｒｅｄｕｃｔａｓｅであることがわかった（図２及び図３を参照）。 Among the above 6 genes, DELTA-BLAST search using the amino acid sequences encoded by the c10g0069 gene and the c10g0093 gene (National Center for Biotechnology Information, Domain EnhancedLookuptimeAcceleratedBLAST.cht. gov / Blast.cgi? PAGE_TYPE = BlastSearch & PROGRAM = blastp & BLAST_PROGRAMS = deltaBlast). As a result, the proteins encoded by the above two genes are all short-chain dehydrogenase / reductase belonging to the Rossmann-fold NAD (P) (+) binding protein superfamily and having PRK06194 multidomain. Okay (see FIGS. 2 and 3).
一方、ＳＹＫ－６株が化合物Ｉを代謝する際の第２段階の反応、すなわち、１－（４－ヒドロキシ－３－メトキシフェニル）－３－ヒドロキシ－２－（２－メトキシフェノキシ）プロパン－１－オン（化合物ＩＩ）のβ－Ｏ－４位のエーテル結合を開裂させる反応を触媒する酵素として、ｌｉｇＦ遺伝子（ＹＰ＿００４８３３９９７）（非特許文献７を参照）及びｌｉｇＥ遺伝子（ＹＰ＿００４８３３９９８）（非特許文献８を参照）が知られている。また、上記第２段階の反応で生じたグルタチオン含有中間体からグルタチオンを除去する第３段階の反応を触媒する酵素として、ｌｉｇＧ遺伝子（ＹＰ＿００４８３３９９９）（非特許文献９を参照）が知られている。 On the other hand, the second stage reaction when SYK-6 strain metabolizes Compound I, that is, 1- (4-hydroxy-3-methoxyphenyl) -3-hydroxy-2- (2-methoxyphenoxy) propane-1 -Enzymes that catalyze the reaction of cleaving the ether bond at the β-O-4 position of ONE (compound II) include ligF gene (YP_004833997) (see Non-Patent Document 7) and ligE gene (YP_004833998) (Non-Patent Document 8). Is known). Moreover, the ligG gene (YP_004833999) (refer nonpatent literature 9) is known as an enzyme which catalyzes the 3rd step reaction which removes glutathione from the glutathione containing intermediate produced by the 2nd step reaction.
そこで、実施例１により検出されたＭＢＥＳ０４株のゲノム配列情報をクエリーとして、ＳＹＫ－６株由来のｌｉｇＥ遺伝子、ｌｉｇＦ遺伝子及びｌｉｇＧ遺伝子に相同性を示す遺伝子を、Ｃｏｖｅｒａｇｅ　５０％＜、Ｉｄｅｎｔｉｔｙ　２５％＜、Ｓｉｍｉｌａｒｉｔｙ　５０％＜、 Ｅ－ｖａｌｕｅ　＜ｅ５を閾値としてＢＬＡＳＴ相同性検索した。その結果、ｃ１０ｇ００７６遺伝子がｌｉｇＦに６８％、ｃ１０ｇ００７７遺伝子がｌｉｇＥに８０％、及びｃ１０ｇ００７８遺伝子がｌｉｇＧに７１％のＩｄｅｎｔｉｔｙ（一致性）を示すことがわかった。 Therefore, using the genome sequence information of the MBES04 strain detected in Example 1 as a query, genes having homology to the ligE gene, the ligF gene, and the ligG gene derived from the SYK-6 strain are represented by Coverage 50% <and Identity 25% <. The BLAST homology search was performed with Simality 50% <, E-value <e5 as threshold values. As a result, it was found that the c10g0076 gene had 68% identity in ligF, the c10g0077 gene had 80% identity in ligE, and the c10g0078 gene had 71% identity in ligG.
ＭＢＥＳ０４株由来の３遺伝子：ｃ１０ｇ００７６遺伝子、ｃ１０ｇ００７７遺伝子及びｃ１０ｇ００７８遺伝子の塩基配列をクエリーとしてＢＬＡＳＴｎ相同性検索（Ｃｏｖｅｒａｇｅ　５０％＜）を行ったところ、最も一致性の高い遺伝子配列としてヒットしたものは、ｃ１０ｇ００７６遺伝子に対しては８２％の一致性でノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ.）ＰＰ１Ｙ株のゲノム上（ＦＲ８５６８６２．１）の１２４７６５５～１２４８３８１番の位置にあるｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ様タンパク質をコードすると推定される遺伝子；ｃ１０ｇ００７８遺伝子に対しては８０％の一致性でノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ.）ＰＰ１Ｙ株のゲノム上（ＦＲ８５６８６２．１）の１２４８４５１～１２４９１９８番の位置にあるｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ様タンパク質をコードすると推定される遺伝子；ｃ１０ｇ００７９遺伝子に対しては７８％の一致性でノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ.）ＰＰ１Ｙ株のゲノム上（ＦＲ８５６８６２．１）の１２４９３２５～１２５００６４番の位置にあるｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅファミリータンパク質をコードすると推定される遺伝子であった。 When a BLASTn homology search (Coverage 50% <) was performed using the base sequences of 3 genes from the MBES04 strain: c10g0076 gene, c10g0077 gene and c10g0078 gene as a query, the most successful gene sequence was c10g0076 It encodes a glutathione － S-transferase-like protein located at positions 1247655 to 1248181 on the genome of Novosphingium sp. PP1Y strain (FR8566862.1) with 82% identity to the gene Presumed gene; Novosphingobium species with 80% consistency for c10g0078 gene sp.) a gene presumed to encode a glutathione S-transferase-like protein located at positions 1284451 to 1249198 on the genome of the PP1Y strain (FR856862.1); Novosphingo with 78% identity to the c10g0079 gene It was a gene presumed to encode a glutathione S-transferase family protein located at positions 1249325 to 1250064 on the genome of Novosphingium sp. PP1Y strain (FR856862.1).
また、ｃ１０ｇ００７６遺伝子、ｃ１０ｇ００７７遺伝子及びｃ１０ｇ００７８遺伝子がコードするアミノ酸配列を用いてＤＥＬＴＡ－ＢＬＡＳＴサーチ（Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎ、Ｄｏｍａｉｎ Ｅｎｈａｎｃｅｄ　Ｌｏｏｋｕｐ　Ｔｉｍｅ　Ａｃｃｅｌｅｒａｔｅｄ　ＢＬＡＳＴ）（ｈｔｔｐ：//ｂｌａｓｔ.ｎｃｂｉ.ｎｌｍ.ｎｉｈ.ｇｏｖ/Ｂｌａｓｔ.ｃｇｉ？ＰＡＧＥ＿ＴＹＰＥ＝ＢｌａｓｔＳｅａｒｃｈ＆ＰＲＯＧＲＡＭ＝ｂｌａｓｔｐ＆ＢＬＡＳＴ＿ＰＲＯＧＲＡＭＳ＝ｄｅｌｔａＢｌａｓｔ）を行った（図４～６を参照）。その結果、上記３遺伝子がコードするアミノ酸配列は、いずれもＧｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅと推定されるタンパク質に保存されているマルチドメインであるＧｓｔ（ＣＯＧ０６２５）配列を有することが示唆された。また、ｃ１０ｇ００７６遺伝子及びｃ１０ｇ００７７遺伝子がコードするアミノ酸配列は、Ｎ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有することがわかった。さらに、ｃ１０ｇ００７８遺伝子がコードするアミノ酸配列は、Ｎ末端側にＰＲＫ１０３８７及びｍａｉＡのマルチドメインを有することがわかった。 In addition, using the amino acid sequences encoded by the c10g0076 gene, c10g0077 gene, and c10g0078 gene, the DELTA-BLAST search (National Center for Biotechnology Information, Domain EnhancedLooknuptime.Blast.BLAST.BLAST.BLAST.BLAST.BLAST). /Blast.cgi?PAGE_TYPE=BlastSearch&PROGRAM=blastp&BLAST_PROGRAMS=deltaBlast) (see FIGS. 4 to 6). As a result, it was suggested that the amino acid sequences encoded by the three genes all have a Gst (COG0625) sequence that is a multidomain conserved in a protein presumed to be Glutathione S-transferase. In addition, it was found that the amino acid sequences encoded by the c10g0076 gene and the c10g0077 gene have PRK15113 and maiA multidomains on the N-terminal side. Furthermore, it was found that the amino acid sequence encoded by the c10g0078 gene has PRK10387 and maiA multidomains on the N-terminal side.
ｃ１０ｇ００７６遺伝子、ｃ１０ｇ００７７遺伝子及びｃ１０ｇ００７８遺伝子は、互いに近接した遺伝子である。さらにｃ１０ｇ００７６遺伝子に隣接したｃ１０ｇ００７５遺伝子は、Ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅと推定されるタンパク質を、ｃ１０ｇ００７６遺伝子、ｃ１０ｇ００７７遺伝子及びｃ１０ｇ００７８遺伝子の３遺伝子がコードする向きとは反対向きにコードするものであった。ｃ１０ｇ００７５遺伝子の塩基配列をクエリーとしてＢＬＡＳＴｎ検索（Ｃｏｖｅｒａｇｅ５０％＜）を行ったところ、最も一致性の高い遺伝子配列としてヒットしたものは、７９％の一致性でノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ.）ＰＰ１Ｙ株のゲノム上（ＦＲ８５６８６２．１）の１２４６７４２～１２４７３７１番の位置にあるｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅをコードすると推定される遺伝子であった。また、このｃ１０ｇ００７５遺伝子がコードするアミノ酸配列をクエリーとしてＤＥＬＴＡ－ＢＬＡＳＴサーチを行ったところ、ｃ１０ｇ００７５遺伝子がコードするタンパク質は、Ｇｓｔ、ＰＲＫ１１７５２、ＰＲＫ１５１１３及びｍａｉＡといった、Ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅと推定されるタンパク質に保存されたマルチドメイン配列を有していることが示唆された（図７を参照）。 The c10g0076 gene, c10g0077 gene, and c10g0078 gene are genes close to each other. Furthermore, the c10g0075 gene adjacent to the c10g0076 gene encodes a protein presumed to be Glutathione S-transferase in a direction opposite to the direction encoded by the three genes c10g0076, c10g0077, and c10g0078. When a BLASTn search (Coverage 50% <) was performed using the base sequence of the c10g0075 gene as a query, the most highly consistent gene sequence was found to be 79% consistent with Novosphingium sp. It was a gene presumed to encode glutathione S-transfer at positions 1467742 to 1247371 on the genome of the PP1Y strain (FR856862.1). Further, when a DELTA-BLAST search was performed using the amino acid sequence encoded by this c10g0075 gene as a query, the protein encoded by the c10g0075 gene was conserved in proteins presumed to be Glutathione S-transfer, such as Gst, PRK11752, PRK15113, and maiA. It was suggested to have a multidomain sequence (see FIG. 7).
［実施例３．形質転換体を利用した酵素生産］
Ｓｈｏｒｔ－ｃｈａｉｎ　ｄｅｈｙｄｒｏｇｅｎａｓｅ／ｒｅｄｕｃｔａｓｅとアノテーションされるＭＢＥＳ０４株由来のｃ０１ｇ１１６２遺伝子、ｃ０１ｇ１３２４遺伝子、ｃ１０ｇ００６９遺伝子、ｃ１０ｇ００８０遺伝子、ｃ１０ｇ００９３遺伝子及びｃ１０ｇ００９４遺伝子の６遺伝子にコードされる各タンパク質；並びに、ｃ１０ｇ００７５遺伝子、ｃ１０ｇ００７６遺伝子、ｃ１０ｇ００７７遺伝子及びｃ１０ｇ００７８遺伝子の４遺伝子にコードされる各タンパク質が化合物Ｉの代謝に関与することを確認するために、大腸菌を宿主として、これらの遺伝子がコードするタンパク質を組換え生産する形質転換体を作製した。 [Example 3. Enzyme production using transformants]
Proteins encoded by 6 genes, c01g1162 gene, c01g1324 gene, c10g0069 gene, c10g0080 gene, c10g0093 gene, and c10g0093 gene, and c10g0075 gene, c10g0076 gene, c10g0076 gene, and c10g0076 gene, which are annotated as Short-chain dehydrogenase / reductase; In order to confirm that each protein encoded by the gene and c10g0078 gene is involved in the metabolism of Compound I, a transformant that produces recombinantly encoded proteins encoded by E. coli was prepared. did.
ここで、ｃ０１ｇ１１６２遺伝子、ｃ０１ｇ１３２４遺伝子、ｃ１０ｇ００６９遺伝子、ｃ１０ｇ００８０遺伝子、ｃ１０ｇ００９３遺伝子及びｃ１０ｇ００９４遺伝子の６遺伝子にコードされる各タンパク質をＣ０１Ｇ１１６２、Ｃ０１Ｇ１３２４、Ｃ１０Ｇ００６９、Ｃ１０Ｇ００８０、Ｃ１０Ｇ００９３及びＣ１０Ｇ００９４と表記し；並びに、ｃ１０ｇ００７５遺伝子、ｃ１０ｇ００７６遺伝子、ｃ１０ｇ００７７遺伝子及びｃ１０ｇ００７８遺伝子の４遺伝子にコードされる各タンパク質をＣ１０Ｇ００７５、Ｃ１０Ｇ００７６、Ｃ１０Ｇ００７７及びＣ１０Ｇ００７８と表記することにした。 Here, each protein encoded by 6 genes of c01g1162 gene, c01g1324 gene, c10g0069 gene, c10g0080 gene, c10g0093 gene, and c10g0094 gene is expressed as C01G1162, C01G1324, C10G0069, C10G0080, C10G0093, and C10G0094; and c10g0075 gene, Each protein encoded by the four genes c10g0076, c10g0077, and c10g0078 was designated as C10G0075, C10G0076, C10G0077, and C10G0078.
プラスミドｐＲＳＥＴＡ（インビトロジェン社製）を鋳型とし表２に示すプライマーＡとＢを用いてＰＣＲ反応を行った。一方、表２に示すプライマーセット（ＣとＤ、ＥとＦ、ＧとＨ、ＩとＪ、ＫとＬ、ＭとＮ、ＯとＰ、ＱとＲ、ＳとＴ、及びＵとＶ）を用いて、ＭＢＥＳ０４株のゲノムＤＮＡを鋳型として下記に示す条件に従ってＰＣＲ反応を行い、上記各遺伝子断片を増幅したｃＤＮＡを得た。
Figure JPOXMLDOC01-appb-T000011
 A PCR reaction was performed using the plasmid pRSETA (manufactured by Invitrogen) as a template and primers A and B shown in Table 2. On the other hand, the primer set shown in Table 2 (C and D, E and F, G and H, I and J, K and L, M and N, O and P, Q and R, S and T, and U and V) PCR was carried out using the genomic DNA of MBES04 strain as a template according to the conditions shown below to obtain cDNA obtained by amplifying the above gene fragments.
Figure JPOXMLDOC01-appb-T000011
ＰＣＲ条件
１× ＰＣＲ ｂｕｆｆｅｒ （ＭｇＣｌ２含有）２００ μｍ ｄＮＴＰｓ, ０．６ μｍ ２７ｆ, ０.６ μｍ １５２５ｒ, １．４ Ｕ ｏｆ ＬＡ Ｔａｑ ＤＮＡ (ポリメラーゼタカラバイオ社製)
サーマルサイクラー温度条件９７℃ ２分、［９７℃３０秒、６０℃１分、７２℃９０秒］×３０サイクル、７２℃ ５分  PCR conditions 1 × PCR buffer (containing MgCl 2 ) 200 μm dNTPs, 0.6 μm 27f, 0.6 μm 1525r, 1.4 U of LA Taq DNA (manufactured by Polymerase Takara Bio Inc.)
Thermal cycler temperature condition 97 ° C 2 minutes, [97 ° C 30 seconds, 60 ° C 1 minute, 72 ° C 90 seconds] x 30 cycles, 72 ° C 5 minutes
表２に示すプライマーセットを用いたＰＣＲ反応により増幅された、ｐＲＳＥＴＡを鋳型として得られたＤＮＡ断片とＭＢＥＳ０４株のゲノムＤＮＡを鋳型として得られたＤＮＡ断片の各１種とを混合し、次いでＩｎ　ｆｕｓｉｏｎ　ＨＤクローニングキット（タカラバイオ社製）を用いて連結することにより組換えプラスミドを得た。得られた組換えプラスミドを、遺伝子毎に、大腸菌（Ｓｔｅｌｌaｒ　Ｃｏｍｐｅｔｅｎｔ　ｃｅｌｌ）に導入することにより、形質転換した。 A DNA fragment amplified by PCR using the primer set shown in Table 2 and mixed with each of the DNA fragments obtained using pRSETA as a template and each DNA fragment obtained using genomic DNA of MBES04 strain as a template, and then In A recombinant plasmid was obtained by ligation using a fusion HD cloning kit (manufactured by Takara Bio Inc.). The obtained recombinant plasmid was transformed into each gene by introducing it into E. coli (Stellar Competent cell).
得られた形質転換体からプラスミドを調製し、プラスミド上の連結断片の塩基配列がＭＢＥＳ０４株のゲノム上の遺伝子配列と完全に一致することを配列解析により確認した。塩基配列解析はＢｉｇＤｙｅ（登録商標）　Ｔｅｒｍｉｎａｔｏｒ　ｖ３．１　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ、ｖ３．１（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社製）ＡＢＩ　３７３０　ＸＬ　ＤＮＡ　Ａｎａｌｙｚｅｒ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社製）を用いた。続いて調製した各プラスミドを用いて、ＢＬ２１ＤＥ３ｐＬｙｓＥ（ライフテクノロジー社製）を形質転換した。各形質転換体を用いて組換え酵素を生産させ、得られた組換え酵素をＮｉ－アガロース担体を用いて精製した。 A plasmid was prepared from the obtained transformant, and it was confirmed by sequence analysis that the nucleotide sequence of the ligated fragment on the plasmid completely matched the gene sequence on the genome of MBES04 strain. Base sequence analysis was performed using BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit, v3.1 (manufactured by Applied Biosystems), ABI 3730, XL DNA Analyzer (Applied Biosystems). Subsequently, BL21DE3pLysE (manufactured by Life Technology) was transformed with each of the prepared plasmids. Recombinant enzyme was produced using each transformant, and the resulting recombinant enzyme was purified using Ni-agarose carrier.
［実施例４．化合物Ｉ分解酵素の同定］
化合物Ｉの合成は、非特許文献１０に記載の方法に準じて行った。得られた合成物のＮＭＲスペクトルは、ＵＳＤＡ（ＵＳ　Ｆｏｒｅｓｔ　Ｐｒｏｄｕｃｔｓ　Ｌａｂｏｒａｔｏｒｙ）が公開している、化合物Ｉのデータと一致した。また、化合物ＩのＨＰＬＣ分析は、株式会社ダイセル製のキラルパックＩＥ３を用いて、以下のとおりに、（１）αＳ、βＲ、（２）αＲ、βＳのｅｒｉｔｈｒｏ体；（３）αＲ、βＲ、（４）αＳ、βＳのｔｈｒｅｏ体の順で溶出する条件で行った。 [Example 4. Identification of Compound I Degrading Enzyme]
Compound I was synthesized according to the method described in Non-Patent Document 10. The NMR spectrum of the synthesized product was in agreement with the data of Compound I published by USDA (US Forest Products Laboratory). In addition, HPLC analysis of Compound I was performed using a Chiralpak IE3 manufactured by Daicel Corporation as follows: (1) erythro form of αS, βR, (2) αR, βS; (3) αR, βR, (4) It was carried out under the condition of eluting in order of αS and βS threo form.
キラルＨＰＬＣ分析条件（化合物Ｉの光学異性体分析条件）
カラム；キラルパックＩＥ－３（ダイセル社製）、４．６ ｍｍ Ｉ.ｄ. ｘ ２５０ ｍｍ Ｌ.溶離液；（Ａ）水、（Ｂ）アセトニトリル、送液：２０％Ｖ／Ｖ（Ｂ）、カラム温度；４０℃、流速：１．０ｍｌ／ｍｉｎ、検出　フォトダイオードアレイ検出器 ＵＶ２００-５００ｎｍ（ＰＤＡ　モデル２９９８、ウォーターズ社製）及び旋光度検出器（キラライザー、システムエンジニアリング社製） Chiral HPLC analysis conditions (Condition I optical isomer analysis conditions)
Column: Chiralpak IE-3 (manufactured by Daicel), 4.6 mm Id. X 250 mm L. Eluent: (A) Water, (B) Acetonitrile, liquid feed: 20% V / V (B) Column temperature: 40 ° C., flow rate: 1.0 ml / min, detection Photodiode array detector UV200-500 nm (PDA model 2998, manufactured by Waters) and optical rotation detector (Chiralizer, manufactured by System Engineering)
得られた化合物Ｉは、この条件で、ｅｒｉｔｈｒｏ体：ｔｈｒｅｏ体≒３：１であった。なお、上記のＲＳ表示は、非特許文献１１に記載の旋光度データを参考に決定した。 The obtained compound I was erithro form: threo form≈3: 1 under these conditions. The RS display was determined with reference to the optical rotation data described in Non-Patent Document 11.
化合物Ｉを終濃度５ｍＭ、ＮＡＤ＋（ナトリウム塩の形で添加）を終濃度１０ｍＭになるようにトリス塩酸緩衝液（ｐＨ７．５）に添加した。次いで、この溶液に、実施例３で得られた精製済のＣ０１Ｇ１１６２、Ｃ０１Ｇ１３２４、Ｃ１０Ｇ００６９、Ｃ１０Ｇ００８０、Ｃ１０Ｇ００９３及びＣ１０Ｇ００９４を各一種ずつ添加し、室温で１６時間インキュベートして、反応液を得た。その後、下記条件で反応液中の化合物Ｉの変化をＨＰＬＣ分析した。 Compound I was added to Tris-HCl buffer (pH 7.5) to a final concentration of 5 mM and NAD + (added in the form of sodium salt) to a final concentration of 10 mM. Subsequently, the purified C01G1162, C01G1324, C10G0069, C10G0080, C10G0093 and C10G0094 obtained in Example 3 were added to this solution one by one and incubated at room temperature for 16 hours to obtain a reaction solution. Thereafter, the change of Compound I in the reaction solution was analyzed by HPLC under the following conditions.
逆相ＨＰＬＣ条件
カラム；Ｘｂｒｉｄｇｅ　ＯＳＴ　Ｃ１８（ウォーターズ社製）、４．６ｍｍ－Ｉ．ｄ．×１００ｍｍ－Ｌ．溶離液；（Ａ）［２ｍＭ　酢酸アンモニウム、０．０５％Ｖ／Ｖ　ギ酸］、（Ｂ）メタノール、送液；０－１ｍｉｎ　１０％Ｖ／Ｖ（Ｂ）、１－８ｍｉｎ　１０％Ｖ／Ｖ－９０％Ｖ／Ｖ（Ｂ）、カラム温度；４０℃、流速；１．２ｍｌ／ｍｉｎ、検出；フォトダイオードアレイ検出器　ＵＶ２００－５００ｎｍ（ＰＤＡ　モデル２９９８、ウォーターズ社製） Reversed phase HPLC condition column; Xbridge OST C18 (Waters), 4.6 mm-I. d. × 100mm-L. Eluent: (A) [2 mM ammonium acetate, 0.05% V / V formic acid], (B) methanol, liquid feed: 0-1 min 10% V / V (B), 1-8 min 10% V / V- 90% V / V (B), column temperature; 40 ° C., flow rate; 1.2 ml / min, detection; photodiode array detector UV 200-500 nm (PDA model 2998, manufactured by Waters)
反応液のＨＰＬＣ分析の結果、Ｃ１０Ｇ００６９又はＣ１０Ｇ００９３を作用させた場合にのみ、化合物Ｉが減少し、新たな化合物が生成されていることが分かった。 As a result of HPLC analysis of the reaction solution, it was found that only when C10G0069 or C10G0093 was allowed to act, Compound I decreased and a new compound was produced.
ここで生成した化合物を同定するために、反応液を固相抽出法（ＯＡＳＩＳ ＷＡＸ ；ウォーターズ社製）にて精製し、以下の条件で逆相ＵＰＬＣ－飛行時間型精密質量分析（ＡＣＣＵＩＴＹ ＵＰＬＣ Ｈ―Ｃｌａｓｓ, ＸｅｖoＧ２　ＱＴＯＦ,ウォーターズ社製）に供した。 In order to identify the compound produced here, the reaction solution was purified by solid phase extraction (OASISＳWAX; manufactured by Waters), and reverse-phase UPLC-time-of-flight accurate mass spectrometry (ACCUITYＵUPLC H- Class, XevoG2 QTOF, manufactured by Waters).
逆相ＨＰＬＣ条件（逆相ＵＰＬＣ－飛行時間型精密質量分析条件）
カラム；ＡＣＱＵＩＴＹ　ＵＰＬＣ　ＢＥＨ　Ｃ１８　Ｃｏｌｕｍｎ，１３０オングストローム，　１．７　μｍ、（ウォーターズ社製）、２．１　ｍｍ　Ｉ．ｄ．×１００　ｍｍ　Ｌ．溶離液；（Ａ）［２ｍＭ　酢酸アンモニウム、０．０５％Ｖ／Ｖ　ギ酸］、（Ｂ）９５％Ｖ／Ｖ　アセトニトリル、送液：０－５分　５％Ｖ／Ｖ－９５％Ｖ／Ｖ（Ｂ）、５－７分　９５％Ｖ／Ｖ（Ｂ）、カラム温度；４０℃、流速；０．４ｍｌ／ｍｉｎ Reversed-phase HPLC conditions (reversed-phase UPLC-time-of-flight accurate mass spectrometry conditions)
Column; ACQUITY UPLC BEH C18 Column, 130 Å, 1.7 μm (Waters), 2.1 mm d. × 100 mm L. Eluent: (A) [2 mM ammonium acetate, 0.05% V / V formic acid], (B) 95% V / V acetonitrile, liquid delivery: 0-5 minutes 5% V / V-95% V / V ( B), 5-7 minutes 95% V / V (B), column temperature; 40 ° C., flow rate; 0.4 ml / min
質量分析条件（逆相ＵＰＬＣ－飛行時間型精密質量分析条件）
検出質量範囲１００―１０００　Ｄａ、データ取得スキャン間隔０．１秒、デソルベーションガス温度　５００℃、イオンソース　ＥＳＩネガティブモード　イオンソース温度１５０℃、コーン電圧３０Ｖ Mass analysis conditions (reverse phase UPLC-time-of-flight accurate mass analysis conditions)
Detection mass range 100-1000 Da, data acquisition scan interval 0.1 second, desolvation gas temperature 500 ° C, ion source ESI negative mode ion source temperature 150 ° C, cone voltage 30V
逆相ＵＰＬＣ－飛行時間型精密質量分析の結果、生成化合物の分子量は３１８であり、推定組成式はＣ１７Ｈ１８Ｏ６であることがわかった。化合物ＩのＭＳスペクトルと生成化合物のＭＳスペクトルとを比較した結果、生成化合物は化合物ＩのＣα位で脱水素反応が起こり、アルコール残基（ヒドロキシ基）がカルボニル基へと変化した化合物ＩＩであることが推定された。 As a result of reversed-phase UPLC-time-of-flight accurate mass spectrometry, it was found that the molecular weight of the product compound was 318 and the estimated composition formula was C 17 H 18 O 6 . As a result of comparing the MS spectrum of Compound I with the MS spectrum of the product compound, the product compound is Compound II in which a dehydrogenation reaction occurs at the Cα position of Compound I and the alcohol residue (hydroxy group) is changed to a carbonyl group. It was estimated.
［実施例５．化合物Ｉ分解酵素の特性］
化合物ＩＩの合成は、非特許文献１０に記載の方法に準じて行った。また、化合物ＩＩのＨＰＬＣ分析は、株式会社ダイセル製のキラルパックＩＥ３を用いて、以下のとおりに、（１）βＲ体、（２）βＳ体の順で溶出する条件で行った。 [Example 5. Characteristics of Compound I Degrading Enzyme]
Compound II was synthesized according to the method described in Non-Patent Document 10. In addition, HPLC analysis of Compound II was performed under the conditions of elution in the order of (1) βR form and (2) βS form using Chiral Pack IE3 manufactured by Daicel Corporation as follows.
キラルＨＰＬＣ分析条件（化合物ＩＩの光学異性体分析条件）
カラム；キラルパックＩＥ－３（ダイセル社製）、４．６　ｍｍ　Ｉ．ｄ．×２５０　ｍｍ　Ｌ．溶離液；（Ａ）水、（Ｂ）アセトニトリル、送液：３０％Ｖ／Ｖ（Ｂ）、カラム温度；４０℃、流速：１．０ｍｌ／ｍｉｎ、検出　フォトダイオードアレイ検出器　ＵＶ２００－５００ｎｍ（ＰＤＡ　モデル２９９８、ウォーターズ社製）及びキラライザー（システムエンジニアリング社製） Chiral HPLC analysis conditions (optical isomer analysis conditions of Compound II)
Column; Chiralpak IE-3 (manufactured by Daicel), 4.6 mm d. × 250 mm L. Eluent: (A) Water, (B) Acetonitrile, liquid supply: 30% V / V (B), column temperature: 40 ° C., flow rate: 1.0 ml / min, detection photodiode array detector UV 200-500 nm (PDA Model 2998, manufactured by Waters) and killer (system engineering)
合成した化合物ＩＩは、この条件で、βＲ体：βＳ体≒１：１であった。なお、上記のＲＳ表示は、非特許文献１１に記載の旋光度データを参考に決定した。 The synthesized compound II was βR form: βS form≈1: 1 under these conditions. The RS display was determined with reference to the optical rotation data described in Non-Patent Document 11.
合成した化合物ＩＩを実施例４に記載の条件で、逆相ＵＰＬＣ－飛行時間型精密質量分析に供したところ、化合物ＩにＣ１０Ｇ００６９及びＣ１０Ｇ００９３を作用させた場合に得られた化合物Ｉ分解物と良く一致するマスクロマト及びスペクトルを得た。このことより、Ｃ１０Ｇ００６９及びＣ１０Ｇ００９３は化合物ＩのＣα位のヒドロキシ基を酸化し、カルボニル基へと変化させるデヒドロゲナーゼ活性を持つタンパク質であることが分かった。 The synthesized compound II was subjected to reverse-phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4. As a result, it was found that the compound I was well separated from the compound I decomposition product obtained when C10G0069 and C10G0093 were allowed to act on compound I. Consistent mass chromatography and spectra were obtained. From this, it was found that C10G0069 and C10G0093 are proteins having a dehydrogenase activity that oxidizes the hydroxy group at the Cα position of Compound I and changes it to a carbonyl group.
化合物ＩにＣ１０Ｇ００６９が作用する場合の反応条件を調べた。Ｃ１０Ｇ００６９はＮＡＤ＋が共存した場合に、Ｃα位のヒドロキシ基を酸化した。しかし、ＮＡＤ＋が存在しないときには活性を示さなかった。またＮＡＤＨ存在下では、化合物ＩのＣα位のヒドロキシ基を酸化しなかったが、化合物ＩＩのカルボニル残基を還元して、ヒドロキシ基を与えた。ＮＡＤＰ＋、ＮＡＤＰＨ存在下では活性を示さなかった。分子量はＳＤＳ－ＰＡＧＥで３４ｋＤａであることが示された。また、１０ｍｇ／ｌ　Ｃ１０Ｇ００６９、１０ｍＭ　化合物Ｉ、２０ｍＭ　ＮＡＤナトリウム及び５０ｍＭ　（ｐＨ９．２）Ｎ－Ｃｙｃｌｏｈｅｘｙｌ－２－ａｍｉｎｏｅｔｈａｎｅｓｕｌｆｏｎｉｃ　ａｃｉｄ－ＮａＯＨ（ＣＨＥＳ）緩衝液（ｐＨ９．５）を含む反応液（１００μｌ）を、５～４５℃の温度で３０分間反応させることによってＣ１０Ｇ００６９の最適温度を評価した。その結果、図８に示すとおり、最適温度は１０～１５℃付近であった。さらに、１０ｍｇ／ｌ　Ｃ１０Ｇ００６９、１０ｍＭ　化合物Ｉ、２０ｍＭ　ＮＡＤナトリウム及びｐＨ６．０～１０．４に調製した５０ｍＭ　緩衝液（ＭＥＳ、ＭＯＰＳ、ＴＡＰＳ、ＣＨＥＳ及びＣＡＰＳ）を含む反応液（１００μｌ）を、１５℃で３０分間反応させることによってＣ１０Ｇ００６９の最適ｐＨを評価した。その結果、図９に示すとおり、最適ｐＨは８．５～９．５付近であった。なお、最適温度及び最適ｐＨは、反応後の反応液中に生成した化合物ＩＩの濃度を実施例４に記載の条件の逆相ＨＰＬＣにより分析した値に基づく。 Reaction conditions when C10G0069 acts on Compound I were examined. C10G0069 oxidized the hydroxy group at the Cα position when NAD + coexisted. However, it did not show activity in the absence of NAD +. In the presence of NADH, the hydroxy group at the Cα position of Compound I was not oxidized, but the carbonyl residue of Compound II was reduced to give a hydroxy group. No activity was shown in the presence of NADP + and NADPH. The molecular weight was shown to be 34 kDa by SDS-PAGE. Also, a reaction solution (100 μl) containing 10 mg / l C10G0069, 10 mM compound I, 20 mM sodium NAD and 50 mM (pH 9.2) N-Cyclohexyl-2-aminoethanesulfonic acid-NaOH (CHES) buffer (pH 9.5), The optimum temperature of C10G0069 was evaluated by reacting at a temperature of 5-45 ° C. for 30 minutes. As a result, as shown in FIG. 8, the optimum temperature was around 10 to 15 ° C. Further, a reaction solution (100 μl) containing 10 mg / l C10G0069, 10 mM compound I, 20 mM sodium NAD, and 50 mM buffer (MES, MOPS, TAPS, CHES, and CAPS) adjusted to pH 6.0 to 10.4 was added at 15 ° C. The optimal pH of C10G0069 was evaluated by reacting for 30 minutes. As a result, as shown in FIG. 9, the optimum pH was around 8.5 to 9.5. The optimum temperature and optimum pH are based on values obtained by analyzing the concentration of Compound II produced in the reaction solution after the reaction by reverse phase HPLC under the conditions described in Example 4.
化合物ＩにＣ１０Ｇ００９３Ｃが作用する場合の反応条件を調べた。Ｃ１０Ｇ００９３はＮＡＤ＋が共存した場合に、Ｃα位のヒドロキシ基を酸化したが、ＮＡＤ＋が存在しないときには活性を示さなかった。またＮＡＤＨ存在下では、化合物ＩのＣα位のヒドロキシ基を酸化しなかったが、化合物ＩＩのカルボニル残基を還元してヒドロキシ基を与えた。ＮＡＤＰ＋、ＮＡＤＰＨ存在下でも活性を示さなかった。分子量はＳＤＳ－ＰＡＧＥで３４ｋＤａであることが示された。また、５ｍｇ／ｌ　Ｃ１０Ｇ００９３、１０ｍＭ　化合物Ｉ、２０ｍＭ　ＮＡＤナトリウム及び５０ｍＭ　（ｐＨ９．２）ＣＨＥＳ緩衝液（ｐＨ９．５）を含む反応液（１００μｌ）を、５～４５℃の温度で３０分間反応させることによってＣ１０Ｇ００９３の最適温度を評価した。その結果、図１０に示すとおり、最適温度は２５～３０℃付近であった。さらに、５ｍｇ／ｌ　Ｃ１０Ｇ００９３、１０ｍＭ　化合物Ｉ及び２０ｍＭ　ＮＡＤナトリウム及びｐＨ６．０～１０．４に調製した５０ｍＭ　緩衝液（ＭＥＳ、ＭＯＰＳ、ＴＡＰＳ、ＣＨＥＳ及びＣＡＰＳ）を含む反応液（１００μｌ）を、３０℃で３０分間反応させることによってＣ１０Ｇ００９３の最適ｐＨを評価した。その結果、図１１に示すとおり、最適ｐＨは８．５～１０．５付近であった。なお、最適温度及び最適ｐＨは、反応後の反応液中に生成した化合物ＩＩの濃度を実施例４に記載の条件の逆相ＨＰＬＣにより分析した値に基づく。 Reaction conditions when C10G0093C acts on Compound I were examined. C10G0093 oxidized the hydroxy group at the Cα position when NAD + coexisted, but showed no activity when NAD + was not present. In the presence of NADH, the hydroxy group at the Cα position of Compound I was not oxidized, but the carbonyl residue of Compound II was reduced to give a hydroxy group. No activity was shown even in the presence of NADP + and NADPH. The molecular weight was shown to be 34 kDa by SDS-PAGE. In addition, a reaction solution (100 μl) containing 5 mg / l C10G0093, 10 mM compound I, 20 mM NAD sodium and 50 mM (pH 9.2) CHES buffer (pH 9.5) is reacted at a temperature of 5 to 45 ° C. for 30 minutes. Was used to evaluate the optimum temperature of C10G0093. As a result, as shown in FIG. 10, the optimum temperature was around 25-30 ° C. Furthermore, a reaction solution (100 μl) containing 5 mg / l C10G0093, 10 mM compound I and 20 mM sodium NAD and 50 mM buffer (MES, MOPS, TAPS, CHES and CAPS) adjusted to pH 6.0 to 10.4 was prepared at 30 ° C. The optimum pH of C10G0093 was evaluated by reacting for 30 minutes. As a result, as shown in FIG. 11, the optimum pH was around 8.5 to 10.5. The optimum temperature and optimum pH are based on values obtained by analyzing the concentration of Compound II produced in the reaction solution after the reaction by reverse phase HPLC under the conditions described in Example 4.
化合物Ｉには、αＳ，βＲ（ＳＲ）；αＲ，βＳ（ＲＳ）；αＲ，βＲ（ＲＲ）；αＳ，βＳ（ＳＳ）という４種類の光学異性体が存在する。これらのうち、Ｃ１０Ｇ００６９及びＣ１０Ｇ００９３が基質として作用する化合物Ｉの光学異性体を調べるために、反応の前後に起こる化合物Ｉの光学異性体組成の変化及び産物である化合物ＩＩの光学異性体組成を、以下に示す条件でキラルＨＰＬＣ分析することとした。 Compound I has four types of optical isomers: αS, βR (SR); αR, βS (RS); αR, βR (RR); αS, βS (SS). Among these, in order to investigate the optical isomer of Compound I in which C10G0069 and C10G0093 act as substrates, the change in the optical isomer composition of Compound I that occurs before and after the reaction and the optical isomer composition of Compound II, which is the product, The chiral HPLC analysis was performed under the following conditions.
キラルＨＰＬＣ分析条件
カラム；キラルパックＩＥ－３（ダイセル社製）、４．６　ｍｍ　Ｉ．ｄ．×２５０　ｍｍ　Ｌ．溶離液；（Ａ）水、（Ｂ）アセトニトリル、送液：０－１０分　２０％Ｖ／Ｖ（Ｂ）、１０－１５分　２０－３０％Ｖ／Ｖ（Ｂ）、１５－３０分　３０％Ｖ／Ｖ（Ｂ）、カラム温度；４０℃、流速：１．０ｍｌ／ｍｉｎ、検出　フォトダイオードアレイ検出器　ＵＶ２００－５００ｎｍ（ＰＤＡ　モデル２９９８、ウォーターズ社製）及びキラライザー（システムエンジニアリング社製） Chiral HPLC analysis condition column: Chiral Pack IE-3 (manufactured by Daicel), 4.6 mm d. × 250 mm L. Eluent: (A) Water, (B) Acetonitrile, liquid delivery: 0-10 minutes 20% V / V (B), 10-15 minutes 20-30% V / V (B), 15-30 minutes 30% V / V (B), column temperature: 40 ° C., flow rate: 1.0 ml / min, detection photodiode array detector UV200-500 nm (PDA model 2998, manufactured by Waters) and killer (system engineering)
キラルＨＰＬＣ分析の結果、Ｃ１０Ｇ００６９は、化合物ＩのＲＳ及びＲＲの２つの光学異性体に特異的に反応すること；Ｃ１０Ｇ００９３はＳＲ及びＳＳの２つの光学異性体に特異的に反応することがわかった。 Chiral HPLC analysis showed that C10G0069 reacts specifically with the two optical isomers of compound I, RS and RR; C10G0093 reacts specifically with the two optical isomers, SR and SS. .
また、化合物ＩＩをＮＡＤＨ存在下で還元する際に生じる化合物Ｉの光学異性体の大部分（感度補正無のＨＰＬＣクロマト面積値で、＞９０％の光学純度）は、Ｃ１０Ｇ００６９の場合にはαＲ体であり、ＳＣ１０Ｇ００９３の場合にはαＳ体であった。 Further, most of the optical isomers of Compound I produced when reducing Compound II in the presence of NADH (> 90% optical purity by HPLC chromatographic area value without sensitivity correction) are αR forms in the case of C10G0069. In the case of SC10G0093, it was αS form.
［実施例６．化合物ＩＩ分解酵素の同定］
化合物Ｉ又は化合物ＩＩを終濃度５ｍＭ、還元型グルタチオンを終濃度１０ｍＭになるように、トリス塩酸緩衝液（ｐＨ７．５）に添加し、次いで実施例３で得られた精製済のＣ１０Ｇ００７５、Ｃ１０Ｇ００７６、Ｃ１０Ｇ００７７及びＣ１０Ｇ００７８をタンパク質毎に添加し、室温で１６時間インキュベートして反応液を得た。その後、実施例４に記載の条件で反応液中の化合物Ｉ又は化合物ＩＩの変化を逆相ＨＰＬＣで分析した。 [Example 6. Identification of Compound II Degrading Enzyme]
Compound I or Compound II was added to Tris-HCl buffer (pH 7.5) to a final concentration of 5 mM and reduced glutathione to a final concentration of 10 mM, and then purified C10G0075, C10G0076, obtained in Example 3, C10G0077 and C10G0078 were added to each protein and incubated at room temperature for 16 hours to obtain a reaction solution. Thereafter, the change of Compound I or Compound II in the reaction solution was analyzed by reverse phase HPLC under the conditions described in Example 4.
その結果、化合物Ｉは変化しなかったが、化合物ＩＩはＣ１０Ｇ００７６又はＣ１０Ｇ００７７と反応させた場合にのみ、別の２つの化合物へ変化することがわかった。逆相ＨＰＬＣ分析では、化合物ＩＩから生じた一方の物質はグアヤコールであることが、グアヤコール標準品とのリテンションタイム及びＵＶ吸収スペクトルとの比較からわかった。反応後に生じた他方の化合物の構造を推定するために、反応液を、固相抽出法（ＯＡＳＩＳ ＷＡＸ ；ウォーターズ社製）にて精製した後に、実施例４に記載の条件で逆相ＵＰＬＣ－飛行時間型精密質量分析に供した。 As a result, it was found that Compound I was not changed, but Compound II was changed to another two compounds only when reacted with C10G0076 or C10G0077. In reverse phase HPLC analysis, it was found from comparison of retention time and UV absorption spectrum with guaiacol standard that one of the substances generated from compound II was guaiacol. In order to estimate the structure of the other compound produced after the reaction, the reaction solution was purified by a solid phase extraction method (OASISＳWAX; manufactured by Waters), and then reverse-phase UPLC-flight under the conditions described in Example 4 The sample was subjected to time-type accurate mass spectrometry.
逆相ＵＰＬＣ－飛行時間型精密質量分析（ネガティブモード）の結果、Ｃ１０Ｇ００７６又はＣ１０Ｇ００７７と化合物ＩＩとの反応によって生じた物質から、ｍ/z５００．２のシグナルが得られた。これは、化合物ＩにＳＹＫ－６株のＬｉｇＥ及びＬｉｇＦを作用させたときに生じると報告されている３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノン（化合物ＩＩＩ）のグルタチオン付加体の分子量５０１．１からプロトンが１つ脱離した値にほぼ相当する。すなわち、Ｃ１０Ｇ００７６及びＣ１０Ｇ００７７は、ＳＹＫ－６株のＬｉｇＥ及びＬｉｇＦと同様に、化合物ＩＩのβ－Ｏ－４結合を切断する活性を持つと判断された。 As a result of reversed-phase UPLC-time-of-flight accurate mass spectrometry (negative mode), a signal of m / z 500.2 was obtained from the substance produced by the reaction of C10G0076 or C10G0077 with Compound II. This is reported to occur when SYK-6 strains LigE and LigF are allowed to act on Compound I. 3-Hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone (Compound III) This roughly corresponds to a value obtained by eliminating one proton from the molecular weight 501.1 of the glutathione adduct. That is, C10G0076 and C10G0077 were determined to have the activity of cleaving the β-O-4 bond of Compound II, similar to SYE-6 strain LigE and LigF.
［実施例７．化合物ＩＩ分解酵素の分解特性］
化合物ＩＩにＣ１０Ｇ００７６が作用する場合の反応条件を調べた。Ｃ１０Ｇ００７６は、還元型グルタチオンが共存した場合にβ－Ｏ－４結合のグルタチオン添加開裂を触媒したが、還元型グルタチオンが存在しない場合には活性を示さなかった。分子量はＳＤＳ－ＰＡＧＥで３１ｋＤａであることが示された。また、５ｍｇ／ｌ　Ｃ１０Ｇ００７６、５ｍＭ　化合物ＩＩ、１０ｍＭ　還元型グルタチオン及び１００ｍＭ　（ｐＨ８．９）ＣＨＥＳ緩衝液（ｐＨ９．５）を含む反応液（１００μｌ）を、１５～４５℃の温度で３０分間反応させることによってＣ１０Ｇ００７６の最適温度を評価した。その結果、図１２に示すとおり、最適温度は３０～３５℃付近であった。さらに、５ｍｇ／ｌ　Ｃ１０Ｇ００７６、５ｍＭ　化合物ＩＩ及び１０ｍＭ　還元型グルタチオン及びｐＨ５．６～１０．４に調製した５０ｍＭ　緩衝液（ＭＥＳ、ＭＯＰＳ、ＴＡＰＳ、ＣＨＥＳ及びＣＡＰＳ）を含む反応液（１００μｌ）を、３０℃で３０分間反応させることによってＣ１０Ｇ００７６の最適ｐＨを評価した。その結果、図１３に示すとおり、最適ｐＨは８．５～９．５付近であった。なお、最適温度及び最適ｐＨは、反応後の反応液中に生成したグアヤコールの濃度を実施例４に記載の条件（保持時間５．５分付近）の逆相ＨＰＬＣにより分析した値に基づく。 [Example 7. Degradation characteristics of compound II degrading enzyme]
The reaction conditions when C10G0076 acts on Compound II were examined. C10G0076 catalyzed the cleavage of β-O-4 linked glutathione in the presence of reduced glutathione, but showed no activity in the absence of reduced glutathione. The molecular weight was shown to be 31 kDa by SDS-PAGE. In addition, a reaction solution (100 μl) containing 5 mg / l C10G0076, 5 mM Compound II, 10 mM reduced glutathione and 100 mM (pH 8.9) CHES buffer (pH 9.5) is reacted at a temperature of 15 to 45 ° C. for 30 minutes. Thus, the optimum temperature of C10G0076 was evaluated. As a result, the optimum temperature was around 30 to 35 ° C. as shown in FIG. Further, a reaction solution (100 μl) containing 5 mg / l C10G0076, 5 mM Compound II, 10 mM reduced glutathione, and 50 mM buffer (MES, MOPS, TAPS, CHES, and CAPS) adjusted to pH 5.6 to 10.4, The optimum pH of C10G0076 was evaluated by reacting at 30 ° C. for 30 minutes. As a result, as shown in FIG. 13, the optimum pH was around 8.5 to 9.5. The optimum temperature and the optimum pH are based on values obtained by analyzing the concentration of guaiacol produced in the reaction solution after the reaction by reverse-phase HPLC under the conditions described in Example 4 (retention time around 5.5 minutes).
化合物ＩＩにＣ１０Ｇ００７７が作用する場合の反応条件を調べた。Ｃ１０Ｇ００７７は、還元型グルタチオンが共存した場合にβ－Ｏ－４結合のグルタチオン添加開裂を触媒したが、還元型グルタチオンが存在しない場合には活性を示さなかった。分子量はＳＤＳ－ＰＡＧＥで３１ｋＤａであることが示された。また、５ｍｇ／ｌ　Ｃ１０Ｇ００７７、５ｍＭ　化合物ＩＩ、１０ｍＭ　還元型グルタチオン及び１００ｍＭ　（ｐＨ８．９）ＭＯＰＳ緩衝液（ｐＨ７．５）を含む反応液（１００μｌ）を、５～３５℃の温度で３０分間反応させることによってＣ１０Ｇ００７７の最適温度を評価した。その結果、図１４に示すとおり、最適温度は２５～３０℃付近であった。さらに、５ｍｇ／ｌ　Ｃ１０Ｇ００７７、５ｍＭ　化合物ＩＩ及び１０ｍＭ　還元型グルタチオン及びｐＨ５．６～１０．４に調製した５０ｍＭ　緩衝液（ＭＥＳ、ＭＯＰＳ、ＴＡＰＳ、ＣＨＥＳ及びＣＡＰＳ）を含む反応液（１００μｌ）を、２０℃で３０分間反応させることによってＣ１０Ｇ００７７の最適ｐＨを評価した。その結果、図１５に示すとおり、最適ｐＨは７～８付近であった。なお、最適温度及び最適ｐＨは、反応後の反応液中に生成したグアヤコールの濃度を実施例４に記載の条件の逆相ＨＰＬＣにより分析した値に基づく。 The reaction conditions when C10G0077 acts on Compound II were examined. C10G0077 catalyzed the cleavage of β-O-4 linked glutathione in the presence of reduced glutathione, but showed no activity in the absence of reduced glutathione. The molecular weight was shown to be 31 kDa by SDS-PAGE. A reaction solution (100 μl) containing 5 mg / l C10G0077, 5 mM Compound II, 10 mM reduced glutathione and 100 mM (pH 8.9) MOPS buffer (pH 7.5) is reacted at a temperature of 5 to 35 ° C. for 30 minutes. Thus, the optimum temperature of C10G0077 was evaluated. As a result, the optimum temperature was around 25-30 ° C. as shown in FIG. Furthermore, a reaction solution (100 μl) containing 5 mg / l C10G0077, 5 mM Compound II, 10 mM reduced glutathione, and 50 mM buffer (MES, MOPS, TAPS, CHES, and CAPS) adjusted to pH 5.6 to 10.4, 20 The optimum pH of C10G0077 was evaluated by reacting at 30 ° C. for 30 minutes. As a result, as shown in FIG. 15, the optimum pH was around 7-8. The optimum temperature and optimum pH are based on values obtained by analyzing the concentration of guaiacol produced in the reaction solution after the reaction by reverse phase HPLC under the conditions described in Example 4.
化合物ＩＩには、βＲ体、βＳ体という２種類の光学異性体が存在する。これらのうち、Ｃ１０Ｇ００７６及びＣ１０Ｇ００７７が基質として作用する光学異性体を調べるために、反応の前後に起こる化合物ＩＩの光学異性体組成の変化を、キラルカラム　キラルパックＩＥ－３を用いてＨＰＬＣ分析した。その結果、Ｃ１０Ｇ００７６はβＳ体に選択的に作用し、Ｃ１０Ｇ００７７はβＲ体に選択的に作用することがわかった。 Compound II has two optical isomers, a βR form and a βS form. Among these, in order to investigate optical isomers in which C10G0076 and C10G0077 act as substrates, changes in the optical isomer composition of Compound II occurring before and after the reaction were analyzed by HPLC using a chiral column, Chiral Pack IE-3. As a result, it was found that C10G0076 selectively acts on the βS form, and C10G0077 selectively acts on the βR form.
また、Ｃ１０Ｇ００７６及びＣ１０Ｇ００７７が、化合物ＩＩの非フェノール性リグニンモデル２量体化合物に対する反応性を持つことを、ベラトリルグリセロール－β－グアイアシルエーテル（１－（３，４－ジメトキシフェニル）－２－（２－メトキシフェノキシ）プロパン－１，３－ジオール；化合物Ｖ)を用いて、以下のとおりに確認した。なお、通常のリグニンは非フェノール性の構成単位が多い。そこで、化合物Ｖを分解する活性を有するということは、その酵素は天然のリグニンを分解する活性を有する蓋然性がある。 Also, C10G0076 and C10G0077 have reactivity with non-phenolic lignin model dimer compounds of Compound II, indicating that veratrylglycerol-β-guaiacyl ether (1- (3,4-dimethoxyphenyl) -2- Using (2-methoxyphenoxy) propane-1,3-diol; Compound V), confirmation was made as follows. Normal lignin has many non-phenolic structural units. Therefore, having the activity of decomposing compound V means that the enzyme has the activity of decomposing natural lignin.
化合物Ｖの合成は、非特許文献１０及び非特許文献１２を参考にして、相関移動触媒を用いる代わりにＮ，Ｎ’－ジメチルホルムアミド（ＤＭＦ）を溶媒とし、ＨＣＨＯのアルドール反応条件をＫ２ＣＯ３－ＥｔＯＨ系で行うなどに改良した方法を用いた。生成物のＮＭＲスペクトルは、ＵＳＤＡが公開している化合物Ｖのデータと一致した。１Ｈ－ＮＭＲスペクトルで、β－プロトンの積分値を用いて計算したところ、ｅｒｉｔｈｒｏ体：ｔｈｒｅｏ体の比率は約１．１：１であった。 For the synthesis of Compound V, referring to Non-Patent Document 10 and Non-Patent Document 12, N, N′-dimethylformamide (DMF) was used as a solvent instead of a phase transfer catalyst, and the aldol reaction conditions of HCHO were set to K 2 CO 2. An improved method was used, such as in a 3- EtOH system. The NMR spectrum of the product was consistent with Compound V data published by USDA. When the 1 H-NMR spectrum was calculated using the integral value of β-proton, the ratio of erythro isomer to threo isomer was about 1.1: 1.
Ｃ１０Ｇ００７６　０．１μｇ、１ｍＭ　化合物Ｖ及び２ｍＭ　還元型グルタチオンを含む、１００ｍＭ（ｐＨ９．５）Ｎ－Ｃｙｃｌｏｈｅｘｙｌ－２－ａｍｉｎｏｅｔｈａｎｅｓｕｌｆｏｎｉｃ　ａｃｉｄ－ＮａＯＨ（ＣＨＥＳ）緩衝液（ｐＨ９．５）及び１０％Ｖ／Ｖ　ＤＭＦの混合溶液を、３０℃にて１５分間反応させた。反応後の反応液を逆相ＨＰＬＣ分析に供したところ、化合物Ｖの減少とグアヤコールの生成が見られた。これにより、Ｃ１０Ｇ００７６が非フェノール性リグニンモデル２量体化合物に対する反応性を持つことが明らかとなった。非フェノール性リグニンモデル２量体化合物である化合物Ｖに対する反応の効率は、Ｃ１０Ｇ００７６を用いた場合、フェノール性のリグニンモデル２量体化合物である化合物ＩＩに対する反応効率の１６．２％に相当した。 C10G0076 0.1 μg, 1 mM Compound V and 2 mM reduced glutathione, 100 mM (pH 9.5) N-Cyclohexyl-2-aminoethanesulfonic acid-NaOH (CHES) buffer (pH 9.5) and 10% V / V DMF The mixed solution was reacted at 30 ° C. for 15 minutes. When the reaction solution after the reaction was subjected to reverse phase HPLC analysis, decrease in Compound V and formation of guaiacol were observed. This revealed that C10G0076 has reactivity with non-phenolic lignin model dimer compounds. When C10G0076 was used, the efficiency of the reaction with Compound V, which is a non-phenolic lignin model dimer compound, was 16.2% of the reaction efficiency with respect to Compound II, which was a phenolic lignin model dimer compound.
一方、Ｃ１０Ｇ００７７　１．０μｇ、１ｍＭ　化合物Ｖ及び２ｍＭ　還元型グルタチオンを含む、１００ｍＭ　３－Ｍｏｒｐｈｏｌｉｎｏｐｒｏｐａｎｅｓｕｌｆｏｎｉｃ　ａｃｉｄ－ＮａＯＨ（ＭＯＰＳ）緩衝液（ｐＨ８．０）及び１０％Ｖ／Ｖ　ＤＭＦの混合溶液を、２０℃にて１５分間反応させた。反応後の反応液を逆相ＨＰＬＣ分析に供したところ、Ｃ１０Ｇ００７７を用いた場合も、化合物Ｖの減少とグアヤコールの生成が見られた。これにより、Ｃ１０Ｇ００７７が非フェノール性リグニンモデル２量体化合物に対する反応性を持つことが明らかとなった。非フェノール性リグニンモデル２量体化合物である化合物Ｖに対する反応の効率は、Ｃ１０Ｇ００７７を用いた場合、フェノール性のリグニンモデル２量体化合物である化合物ＩＩに対する反応効率の３８０％に相当した。 On the other hand, a mixed solution of 100 mM 3-Morpholinopropanolic-NaOH (MOPS) buffer (pH 8.0) and 10% V / V DMF containing 1.0 μg of C10G0077, 1 mM compound V and 2 mM reduced glutathione at 20 ° C. For 15 minutes. When the reaction solution after the reaction was subjected to reverse phase HPLC analysis, the decrease in Compound V and the formation of guaiacol were also observed when C10G0077 was used. This revealed that C10G0077 has reactivity with non-phenolic lignin model dimer compounds. When C10G0077 was used, the efficiency of the reaction with Compound V, which was a non-phenolic lignin model dimer compound, corresponded to 380% of the reaction efficiency with respect to Compound II, which was a phenolic lignin model dimer compound.
［実施例８．化合物ＩＩＩ生成酵素の同定］
化合物ＩＩを基質として、実施例３で得られたＣ１０Ｇ００７５、Ｃ１０Ｇ００７６及びＣ１０Ｇ００７７の精製タンパク質；又はＣ１０Ｇ００７６、Ｃ１０Ｇ００７７及びＣ１０Ｇ００７８の精製タンパク質の組み合わせで反応させた。その結果、両方の組み合せの反応物から、化合物ＩＩＩが生成されたことがわかった。化合物ＩＩＩは化合物ＩをＭＢＥＳ０４株の培養液に添加した際に得られる代謝物（３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノン）と一致するリテンションタイムを示した。反応液を固相抽出法（ＯＡＳＩＳ ＷＡＸ ；ウォーターズ社製）にて精製し、実施例４に記載の条件で逆相ＵＰＬＣ－飛行時間型精密質量分析に供したところ、化合物ＩＩＩの分析結果は、化合物Ｉを含む培地でＭＢＥＳ０４株を培養した際に得られた主生成物である３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンの分析結果とよく一致した。 [Example 8. Identification of Compound III Generating Enzyme]
Using Compound II as a substrate, the reaction was carried out with the purified protein of C10G0075, C10G0076 and C10G0077 obtained in Example 3, or the combination of the purified protein of C10G0076, C10G0077 and C10G0078. As a result, it was found that compound III was produced from the reaction product of both combinations. Compound III showed a retention time consistent with the metabolite (3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone) obtained when Compound I was added to the culture medium of MBES04 strain. . The reaction solution was purified by a solid phase extraction method (OASIS WAX; manufactured by Waters) and subjected to reverse phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4. As a result, the analysis result of Compound III was This agreed well with the analysis result of 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone, which was the main product obtained when the MBES04 strain was cultured in a medium containing Compound I.
Ｃ１０Ｇ００７５及びＣ１０Ｇ００７８が基質として反応する化合物に光学異性体特異性があることを、実施例３で得られた精製酵素を以下のように組み合わせて、化合物ＩＩの変換反応を行うことによって確認した。酵素反応は、下記（１）～（４）の組み合せ酵素（反応液１００μＬあたり、精製酵素Ｃ１０Ｇ００７５：０．５μｇ、精製酵素Ｃ１０Ｇ００７６：０．５μｇ、精製酵素Ｃ１０Ｇ００７７：０．５μｇ、精製酵素Ｃ１０Ｇ００７８：０．５μｇを使用）、１ｍＭ　化合物ＩＩ及び１０ｍＭ 還元型グルタチオンを含む、１００ｍＭ　ＭＯＰＳ緩衝液（ｐＨ８．０）の混合溶液中で、室温にて１６時間反応させた。
（１）Ｃ１０Ｇ００７６（βＳ体を特異的に認識）＋Ｃ１０Ｇ００７５
（２）Ｃ１０Ｇ００７６（βＳ体を特異的に認識）＋Ｃ１０Ｇ００７８
（３）Ｃ１０Ｇ００７７（βＲ体を特異的に認識）＋Ｃ１０Ｇ００７５
（４）Ｃ１０Ｇ００７７（βＲ体を特異的に認識）＋Ｃ１０Ｇ００７８ It was confirmed by carrying out the conversion reaction of Compound II that the compounds reacted with C10G0075 and C10G0078 have optical isomer specificity by combining the purified enzymes obtained in Example 3 as follows. The enzyme reaction was performed by combining the following enzymes (1) to (4) (purified enzyme C10G0075: 0.5 μg, purified enzyme C10G0076: 0.5 μg, purified enzyme C10G0077: 0.5 μg, purified enzyme C10G0078: 0 per 100 μL of reaction solution) Reaction was carried out at room temperature for 16 hours in a mixed solution of 100 mM MOPS buffer (pH 8.0) containing 1 mM Compound II and 10 mM reduced glutathione.
(1) C10G0076 (specifically recognizes βS form) + C10G0075
(2) C10G0076 (specifically recognizes βS form) + C10G0078
(3) C10G0077 (specifically recognizes βR form) + C10G0075
(4) C10G0077 (specifically recognizes βR form) + C10G0078
反応後の基質と生成物の変化をキラルＨＰＬＣ分析にて検出した。その結果、（１）、（２）及び（３）の組み合わせでは反応が進行し、３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンの生成が見られた。しかし、（４）の組み合わせでは、３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンの生成が見られなかった。したがって、Ｃ１０Ｇ００７８はβＳ体のみを特異的に認識するが、Ｃ１０Ｇ００７５はβＲ体及びβＳ体の２種の光学異性体の両方から３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンを生成できることがわかった。 Changes in the substrate and product after the reaction were detected by chiral HPLC analysis. As a result, the reaction proceeded with the combination of (1), (2) and (3), and the production of 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone was observed. However, in the combination of (4), formation of 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone was not observed. Therefore, C10G0078 specifically recognizes only the βS form, whereas C10G0075 recognizes 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1 from both of the two optical isomers, the βR form and the βS form. -It was found that propanone can be produced.
［実施例９．化合物ＩＩＩ生成酵素の分子量］
Ｃ１０Ｇ００７８の分子量はＳＤＳ－ＰＡＧＥで３３ｋＤａであることが示された。Ｃ１０Ｇ００７５の分子量はＳＤＳ－ＰＡＧＥで２７ｋＤａであることが示された。 [Example 9. Molecular weight of Compound III-producing enzyme]
The molecular weight of C10G0078 was shown to be 33 kDa by SDS-PAGE. The molecular weight of C10G0075 was shown to be 27 kDa by SDS-PAGE.
［実施例１０．化合物Ｉ分解酵素、化合物ＩＩ分解酵素及び化合物ＩＩＩ生成酵素を用いたリグニン分解（１）］
カシオガクズ抽出液からフェニルプロパン構造を有する化合物を以下の手順で製造した。
乾燥カシオガクズ粉末５０ｇをジオキサン３００ｍＬに懸濁し、室温で６日間浸漬した。得られた懸濁液からろ紙で固形分をろ過して除去し、ろ液を得た。ろ液をエバポレーターで減圧乾固し、カシジオキサン抽出物を得た。カシジオキサン抽出物が１０％Ｗ／Ｖ終濃度となるようにＤＭＦに溶解した。得られたカシオガクズ抽出液を用いて、以下の組成の反応液を作成し、室温で２４時間反応させた。 [Example 10. Lignin degradation using Compound I degrading enzyme, Compound II degrading enzyme and Compound III producing enzyme (1)]
A compound having a phenylpropane structure was produced from the Casiogakuzu extract by the following procedure.
50 g of dried Casiogakuzu powder was suspended in 300 mL of dioxane and immersed for 6 days at room temperature. The solid content was removed by filtration from the obtained suspension with a filter paper to obtain a filtrate. The filtrate was dried under reduced pressure with an evaporator to obtain a casidioxane extract. Casidioxane extract was dissolved in DMF to a final concentration of 10% W / V. Using the obtained Casiogakuzu extract, a reaction solution having the following composition was prepared and reacted at room temperature for 24 hours.
反応液（０．１ｍＬ）組成（１）：
カシオガクズ抽出液　１/２０容量
(反応液に含まれる乾燥固形物の濃度は５．０ｍｇ／ｍＬ)
ＣＨＥＳ緩衝液　ｐＨ９．２　５０ｍＭ
精製酵素Ｃ１０Ｇ００６９（タンパク質濃度：0.41mg/mL）　１/２０容量（５μｌ）
精製酵素Ｃ１０Ｇ００９３（タンパク質濃度：0.16mg/mL）　１/２０容量　
精製酵素Ｃ１０Ｇ００７６（タンパク質濃度：0.29mg/mL）　１/２０容量　
精製酵素Ｃ１０Ｇ００７８（タンパク質濃度：0.10mg/mL）　１/２０容量　
還元型グルタチオン　５ｍＭ
ＮＡＤナトリウム塩　５ｍＭ Reaction liquid (0.1 mL) composition (1):
Casiogakuzu extract 1/20 volume
(The concentration of the dry solid contained in the reaction solution is 5.0 mg / mL)
CHES buffer pH 9.2 50 mM
Purified enzyme C10G0069 (protein concentration: 0.41 mg / mL) 1/20 volume (5 μl)
Purified enzyme C10G0093 (protein concentration: 0.16 mg / mL) 1/20 volume
Purified enzyme C10G0076 (protein concentration: 0.29 mg / mL) 1/20 volume
Purified enzyme C10G0078 (protein concentration: 0.10 mg / mL) 1/20 volume
Reduced glutathione 5 mM
NAD sodium salt 5mM
反応後、反応液５０μＬを固相抽出法（ＯＡＳＩＳ ＷＡＸ ；ウォーターズ社製）にて精製し、窒素ガス下にて乾固した。乾固後の残渣を、０．５ｍＬの２０％Ｖ／Ｖ　アセトニトリルに溶解し、実施例４に記載の条件で逆相ＵＰＬＣ－飛行時間型精密質量分析に供した。 After the reaction, 50 μL of the reaction solution was purified by a solid phase extraction method (OASISＳWAX; manufactured by Waters) and dried under nitrogen gas. The residue after drying was dissolved in 0.5 mL of 20% V / V acetonitrile and subjected to reverse phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4.
逆相ＵＰＬＣ－飛行時間型精密質量分析の結果、添加酵素の作用により、リテンションタイム２．４分にｍ／ｚ１９５．１のシグナルが得られた。本リテンションタイムとマスシグナルの値は、化合物ＩＩＩのそれらとよく一致した。その生成量は既知濃度の化合物ＩＩＩにおけるシグナル強度から１．１μｇ／ｍＬと算出された。またリテンションタイム２．５分にｍ／ｚ２２５．１のシグナルが得られた。その生成量は、既知濃度の化合物ＩＩＩにおけるシグナル強度で計算した場合、０．８μｇ／ｍＬであった。リテンションタイム２．４分におけるマススペクトルと２．５分におけるマススペクトルとを比較したところ、２．５分に検出された化合物（化合物ＩＶ）は化合物ＩＩＩ中の水素の１つがメトキシ基に置換された化合物であると予測された。 As a result of reversed-phase UPLC-time-of-flight accurate mass spectrometry, a signal of m / z 195.1 was obtained at a retention time of 2.4 minutes due to the action of the added enzyme. The retention time and mass signal values agreed well with those of Compound III. The amount produced was calculated to be 1.1 μg / mL from the signal intensity at a known concentration of Compound III. A signal of m / z 225.1 was obtained at a retention time of 2.5 minutes. The amount produced was 0.8 μg / mL as calculated by the signal intensity at a known concentration of Compound III. When the mass spectrum at the retention time of 2.4 minutes was compared with the mass spectrum at 2.5 minutes, the compound (compound IV) detected at 2.5 minutes had one of the hydrogens in compound III replaced with a methoxy group. It was predicted that
［実施例１１．化合物Ｉ分解酵素、化合物ＩＩ分解酵素及び化合物ＩＩＩ生成酵素を用いたリグニン分解（２）］
実施例１０で調製したカシオガクズ抽出液を用いて、以下の組成の反応液を作成し、室温で１６時間反応させた。 [Example 11. Lignin degradation using Compound I degrading enzyme, Compound II degrading enzyme and Compound III producing enzyme (2)]
Using the Casiogakuzu extract prepared in Example 10, a reaction solution having the following composition was prepared and reacted at room temperature for 16 hours.
反応液（０．１ｍＬ）組成（２）：
カシオガクズ抽出液　１/２０容量
(反応液に含まれる乾燥固形物の濃度は５．０ｍｇ／ｍＬ)
ＴＡＰＳ緩衝液　ｐＨ８．５　１００ｍＭ
下記精製酵素のうち、いずれか表３に示す組み合わせ
精製酵素Ｃ１０Ｇ００６９（タンパク質濃度：0.41mg/mL）　１/２０容量　
精製酵素Ｃ１０Ｇ００９３（タンパク質濃度：0.16mg/mL）　１/２０容量
精製酵素Ｃ１０Ｇ００７６（タンパク質濃度：0.29mg/mL）　１/２０容量
精製酵素Ｃ１０Ｇ００７７（タンパク質濃度：0.28mg/mL）　１/２０容量
精製酵素Ｃ１０Ｇ００７５（タンパク質濃度：0.62mg/mL）　１/２０容量
精製酵素Ｃ１０Ｇ００７８（タンパク質濃度：0.10mg/mL）　１/２０容量
還元型グルタチオン　５ｍＭ
ＮＡＤナトリウム塩　５ｍＭ Reaction liquid (0.1 mL) composition (2):
Casiogakuzu extract 1/20 volume
(The concentration of the dry solid contained in the reaction solution is 5.0 mg / mL)
TAPS buffer pH 8.5 100 mM
Among the following purified enzymes, any one of the combination purified enzymes C10G0069 (protein concentration: 0.41 mg / mL) shown in Table 3 1/20 volume
Purified enzyme C10G0093 (protein concentration: 0.16 mg / mL) 1/20 volume purified enzyme C10G0076 (protein concentration: 0.29 mg / mL) 1/20 volume purified enzyme C10G0077 (protein concentration: 0.28 mg / mL) 1/20 volume purified enzyme C10G0075 (protein concentration: 0.62 mg / mL) 1/20 volume purified enzyme C10G0078 (protein concentration: 0.10 mg / mL) 1/20 volume reduced glutathione 5 mM
NAD sodium salt 5mM
反応後、反応液５０μＬを固相抽出法（ＯＡＳＩＳ ＷＡＸ ；ウォーターズ社製）にて精製し、窒素ガス下にて乾固した。乾固後の残渣を、０．５ｍＬの２０％Ｖ／Ｖ　アセトニトリルに溶解し、実施例４に記載の条件で逆相ＵＰＬＣ－飛行時間型精密質量分析に供した。 After the reaction, 50 μL of the reaction solution was purified by a solid phase extraction method (OASISＳWAX; manufactured by Waters) and dried under nitrogen gas. The residue after drying was dissolved in 0.5 mL of 20% V / V acetonitrile and subjected to reverse phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4.
逆相ＵＰＬＣ－飛行時間型精密質量分析の結果、添加酵素の作用により、生成された化合物ＩＩＩの生成量を表３に示した。表３に示されるように、６種の酵素を添加した場合に化合物ＩＩＩが最も高い濃度で生成された。
Figure JPOXMLDOC01-appb-T000012
 As a result of reversed-phase UPLC-time-of-flight accurate mass spectrometry, the amount of compound III produced by the action of the added enzyme is shown in Table 3. As shown in Table 3, Compound III was produced at the highest concentration when 6 enzymes were added.
Figure JPOXMLDOC01-appb-T000012
［実施例１２．化合物Ｉ分解酵素、化合物ＩＩ分解酵素及び化合物ＩＩＩ生成酵素を用いたリグニン分解（３）］
乾燥イナワラ粉末５０ｇを、ジオキサン３００ｍＬ及び水１５ｍＬの混合溶液に懸濁し、室温で６日間浸漬した。浸漬後、懸濁液からろ紙で不溶物をろ別し、ろ液を得た。ろ液をエバポレーターで減圧乾固、その後デシケーターにて乾燥させ、２．６５ｇの固形物を得た。固形物に酢酸エチル５０ｍｌ及び純水７０ｍＬを添加し、固形物を溶解させた。さらに固形物が１０％Ｗ/ＶとなるようにＤＭＦに溶解した。得られたイナワラ抽出物ＤＭＦ溶液を用いて、以下の組成の反応液を作成し、室温で２４時間反応させた。 [Example 12. Lignin degradation using Compound I-degrading enzyme, Compound II-degrading enzyme and Compound III-producing enzyme (3)]
50 g of dried inarawa powder was suspended in a mixed solution of 300 mL of dioxane and 15 mL of water and immersed for 6 days at room temperature. After soaking, insoluble matters were filtered off from the suspension with a filter paper to obtain a filtrate. The filtrate was dried under reduced pressure with an evaporator and then dried with a desiccator to obtain 2.65 g of a solid. 50 ml of ethyl acetate and 70 ml of pure water were added to the solid to dissolve the solid. Furthermore, it melt | dissolved in DMF so that a solid substance might be 10% W / V. Using the obtained Inawara extract DMF solution, a reaction solution having the following composition was prepared and reacted at room temperature for 24 hours.
反応液（０．１ｍＬ）組成（３）：
イナワラ抽出物ＤＭＦ液　１/２０容量
(反応液に含まれる乾燥固形物の濃度は５．０ｍｇ／ｍＬ)
ＣＨＥＳ緩衝液　ｐＨ９．２　５０ｍＭ
精製酵素Ｃ１０Ｇ００６９（タンパク質濃度：0.41mg/mL）　１/２０容量　
精製酵素Ｃ１０Ｇ００９３（タンパク質濃度：0.16mg/mL）　１/２０容量　
精製酵素Ｃ１０Ｇ００７５（タンパク質濃度：0.62mg/mL）　１/２０容量　
精製酵素Ｃ１０Ｇ００７６（タンパク質濃度：0.29mg/mL）　１/２０容量　
精製酵素Ｃ１０Ｇ００７７（タンパク質濃度：0.28mg/mL）　１/２０容量　
還元型グルタチオン　５ｍＭ
ＮＡＤナトリウム塩　５ｍＭ Reaction liquid (0.1 mL) composition (3):
Inawara extract DMF solution 1/20 volume
(The concentration of the dry solid contained in the reaction solution is 5.0 mg / mL)
CHES buffer pH 9.2 50 mM
Purified enzyme C10G0069 (protein concentration: 0.41 mg / mL) 1/20 volume
Purified enzyme C10G0093 (protein concentration: 0.16 mg / mL) 1/20 volume
Purified enzyme C10G0075 (protein concentration: 0.62 mg / mL) 1/20 volume
Purified enzyme C10G0076 (protein concentration: 0.29 mg / mL) 1/20 volume
Purified enzyme C10G0077 (protein concentration: 0.28 mg / mL) 1/20 volume
Reduced glutathione 5 mM
NAD sodium salt 5mM
反応後、反応液５０μＬを固相抽出法（ＯＡＳＩＳ ＷＡＸ ；ウォーターズ社製）にて精製し、窒素ガス下にて乾固した。乾固後の残渣を、０．５ｍＬの２０％Ｖ／Ｖ　アセトニトリルに溶解し、実施例４に記載の条件で逆相ＵＰＬＣ－飛行時間型精密質量分析に供した。 After the reaction, 50 μL of the reaction solution was purified by a solid phase extraction method (OASISＳWAX; manufactured by Waters) and dried under nitrogen gas. The residue after drying was dissolved in 0.5 mL of 20% V / V acetonitrile and subjected to reverse phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4.
逆相ＵＰＬＣ－飛行時間型精密質量分析の結果、添加酵素の作用により、化合物ＩＩＩ及び化合物ＩＶが生成されていることが確認された。その生成量はそれぞれ８．４μｇ／ｍＬ及び０．７μｇ／ｍＬであった。 As a result of reversed-phase UPLC-time-of-flight accurate mass spectrometry, it was confirmed that Compound III and Compound IV were produced by the action of the added enzyme. The production amounts were 8.4 μg / mL and 0.7 μg / mL, respectively.
［実施例１３．化合物Ｉ分解酵素、化合物ＩＩ分解酵素及び化合物ＩＩＩ生成酵素を用いたリグニン分解（４）］
シイタケ廃菌床からのフェニルプロパン構造を有する化合物を以下の手順で製造した。乾燥シイタケ廃菌床粉末１０ｇをイオン交換水１００ｍＬに懸濁し、３時間室温で浸漬した。得られた懸濁液からろ紙で固形分をろ別し、ろ液（水洗液１）を得た。残渣をイソプロパノール１００ｍＬに懸濁し３時間室温で浸漬した。浸漬後、ろ紙で固形分をろ別し、ろ液（水洗後イソプロ液１）を得た。一方、乾燥シイタケ廃菌床粉末１０ｇを９０％Ｖ／Ｖ　イソプロパノール水溶液１００ｍＬに懸濁し、３時間室温で浸漬した。ろ紙で固形分をろ過別し、ろ液（９０％イソプロ液１）を得た。 [Example 13. Lignin degradation using Compound I degrading enzyme, Compound II degrading enzyme and Compound III producing enzyme (4)]
A compound having a phenylpropane structure from a shiitake mushroom waste bed was produced by the following procedure. 10 g of dried shiitake mushroom bed powder was suspended in 100 mL of ion-exchanged water and immersed at room temperature for 3 hours. The solid content was filtered off from the obtained suspension with a filter paper to obtain a filtrate (water washing solution 1). The residue was suspended in 100 mL of isopropanol and immersed for 3 hours at room temperature. After soaking, the solid content was filtered off with a filter paper to obtain a filtrate (isopro solution 1 after washing with water). On the other hand, 10 g of dried shiitake mushroom bed powder was suspended in 100 mL of 90% V / V aqueous isopropanol solution and immersed at room temperature for 3 hours. The solid content was filtered off with a filter paper to obtain a filtrate (90% isopropyl solution 1).
水洗液１、水洗後イソプロ液１及び９０％イソプロ液１を２５ｍＬずつ抜き取り、エバポレーターで４５℃に加温しながら、減圧乾固した。水洗液１、水洗後イソプロ液１及び９０％イソプロ液１から、それぞれ４６９、５５及び２４７ｍｇの固形物を得た。水洗液１から得られた固形物は水２ｍＬ（２３４.４ｍｇ/ｍＬ）に溶解し、水洗後イソプロ液１(２７．４ｍｇ／ｍＬ)及び９０％イソプロ液１（１２３.５ｍｇ/ｍＬ）から得られた固形物はそれぞれＤＭＦ２ｍＬに溶解した。これらの３種の溶解抽出物を用いて、以下の組成の反応液を作成し、室温で２４時間反応させた。 Washing solution 1, after washing with water, 25 mL of isopropyl solution 1 and 90% isopropyl solution 1 were taken out and dried under reduced pressure while heating to 45 ° C. with an evaporator. 469, 55 and 247 mg of solids were obtained from Wash Solution 1, Washed Isopro Solution 1 and 90% Isopro Solution 1, respectively. The solid substance obtained from Washing Solution 1 was dissolved in 2 mL (234.4 mg / mL) of water, and after washing, obtained from Isopro Solution 1 (27.4 mg / mL) and 90% Isopro Solution 1 (123.5 mg / mL). Each solid obtained was dissolved in 2 mL of DMF. Using these three kinds of dissolved extracts, a reaction solution having the following composition was prepared and reacted at room temperature for 24 hours.
反応液組成（４）：
シイタケ廃菌床抽出液（上記３種の抽出物の内のいずれか）　１/２０容量
(反応液に含まれる乾燥固形物の濃度は水洗液１を用いた場合１１．７ｍｇ／ｍＬ、水洗後イソプロ液１を用いた場合１．３７ｍｇ／ｍＬ、９０％イソプロ液１を用いた場合６．１８ｍｇ／ｍＬ)
ＣＨＥＳ緩衝液　ｐＨ９．２　５０ｍＭ
精製酵素Ｃ１０Ｇ００６９（タンパク質濃度：0.41mg/mL）　１/２０容量　
精製酵素Ｃ１０Ｇ００９３（タンパク質濃度：0.16mg/mL）　１/２０容量
精製酵素Ｃ１０Ｇ００７５（タンパク質濃度：0.62mg/mL）　１/２０容量　
精製酵素Ｃ１０Ｇ００７６（タンパク質濃度：0.29mg/mL）　１/２０容量　
精製酵素Ｃ１０Ｇ００７７（タンパク質濃度：0.28mg/mL）　１/２０容量　
還元型グルタチオン　５ｍＭ
ＮＡＤナトリウム塩　５ｍＭ Reaction solution composition (4):
Shiitake waste bed extract (one of the above three extracts) 1/20 volume
(The concentration of the dry solid contained in the reaction solution is 11.7 mg / mL when the washing solution 1 is used, 1.37 mg / mL when the isopropyl solution 1 is used after washing with water, and 6 when the 90% isopropyl solution 1 is used. .18 mg / mL)
CHES buffer pH 9.2 50 mM
Purified enzyme C10G0069 (protein concentration: 0.41 mg / mL) 1/20 volume
Purified enzyme C10G0093 (protein concentration: 0.16 mg / mL) 1/20 volume Purified enzyme C10G0075 (protein concentration: 0.62 mg / mL) 1/20 volume
Purified enzyme C10G0076 (protein concentration: 0.29 mg / mL) 1/20 volume
Purified enzyme C10G0077 (protein concentration: 0.28 mg / mL) 1/20 volume
Reduced glutathione 5 mM
NAD sodium salt 5mM
反応後、反応液５０μＬを固相抽出法（ＯＡＳＩＳ ＷＡＸ ；ウォーターズ社製）にて精製し、窒素ガス下にて乾固した。乾固後の残渣を、０．５ｍＬの２０％Ｖ／Ｖ　アセトニトリルに溶解し、実施例４に記載の条件で逆相ＵＰＬＣ－飛行時間型精密質量分析に供した。 After the reaction, 50 μL of the reaction solution was purified by a solid phase extraction method (OASISＳWAX; manufactured by Waters) and dried under nitrogen gas. The residue after drying was dissolved in 0.5 mL of 20% V / V acetonitrile and subjected to reverse phase UPLC-time-of-flight accurate mass spectrometry under the conditions described in Example 4.
逆相ＵＰＬＣ－飛行時間型精密質量分析の結果、添加酵素の作用により、化合物ＩＩＩが生成されていることが確認された。その生成量は水洗液１を用いた場合は１．１μｇ／ｍＬ、水洗後イソプロ液１を用いた場合は０．６８μｇ／ｍＬ、及び９０％イソプロ液１を用いた場合は１．３７μｇ／ｍＬであった。化合物ＩＶの生成量は水洗液１を用いた場合は０．６μｇ／ｍＬ、水洗後イソプロ液１を用いた場合は０．３μｇ／ｍＬ、及び９０％イソプロ液１を用いた場合は０．７μｇ／ｍＬであった。 As a result of reversed-phase UPLC-time-of-flight accurate mass spectrometry, it was confirmed that Compound III was produced by the action of the added enzyme. The amount produced is 1.1 μg / mL when the washing solution 1 is used, 0.68 μg / mL when the isopropyl solution 1 is used after washing with water, and 1.37 μg / mL when the 90% isopropyl solution 1 is used. Met. The amount of compound IV produced is 0.6 μg / mL when Washing Solution 1 is used, 0.3 μg / mL when Isopro Solution 1 is used after washing with water, and 0.7 μg when 90% Isopro Solution 1 is used. / ML.
［参考文献］
上記した非特許文献４～１２は以下のとおりである、該文献の記載はここに開示として援用される：
非特許文献４：Ｂｅｓｅｍｅｒ　Ｊら；Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓｅａｒｃｈ、２００１、vol ２９、p２６０７
非特許文献５：Ｍａｓａｉ，Ｅ．，Ｓ．Ｋｕｂｏｔａ．Ｙ．Ｋａｔａｙａｍａ，Ｓ．Ｋａｗａｉ，Ｍ．Ｙａｍａｓａｋｉ，ａｎｄ　Ｎ．Ｍｏｒｏｈｏｓｈｉ．１９９３．Ｂｉｏｓｃｉ．　Ｂｉｏｔｅｃｈｎｏｌ．Ｂｉｏｃｈｅｍ．５７：１６５５－１６５９．
非特許文献６：Ｓａｔｏ　Ｙ，Ｍｏｒｉｕｃｈｉ　Ｈ，Ｈｉｓｈｉｙａｍａ　Ｓ，Ｏｔｓｕｋａ　Ｙ，Ｏｓｈｉｍａ　Ｋ，Ｋａｓａｉ　Ｄ，Ｎａｋａｍｕｒａ　Ｍ，Ｏｈａｒａ　Ｓ，Ｋａｔａｙａｍａ　Ｙ，Ｆｕｋｕｄａ　Ｍ，Ｍａｓａｉ　Ｅ．Ａｐｐｌ　Ｅｎｖｉｒｏｎ　Ｍｉｃｒｏｂｉｏｌ．２００９；７５（１６）：５１９５－２０１．
非特許文献７：Ｍａｓａｉ，Ｅ．，Ｙ．Ｋａｔａｙａｍａ，Ｓ．Ｋｕｂｏｔａ，Ｓ．Ｋａｗａｉ，　Ｍ．Ｙａｍａｓａｋｉ，ａｎｄ　Ｎ．Ｍｏｒｏｈｏｓｈｉ．１９９３．ＦＥＢＳ　Ｌｅｔｔ．３２３：１３５－１４０．
非特許文献８：Ｍａｓａｉ，Ｅ．，Ｙ．Ｋａｔａｙａｍａ，Ｓ．Ｋａｗａｉ，Ｓ．Ｎｉｓｈｉｋａｗａ，Ｍ．Ｙａｍａｓａｋｉ，ａｎｄ　Ｎ．Ｍｏｒｏｈｏｓｈｉ．１９９１．Ｊ．Ｂａｃｔｅｒｉｏｌ．１７３：７９５０－７９５５．
非特許文献９：Ｍａｓａｉ　Ｅ，Ｉｃｈｉｍｕｒａ　Ａ，Ｓａｔｏ　Ｙ，Ｍｉｙａｕｃｈｉ　Ｋ，Ｋａｔａｙａｍａ　Ｙ，Ｆｕｋｕｄａ　Ｍ．Ｊ　Ｂａｃｔｅｒｉｏｌ．２００３　Ｍａｒ；１８５（６）：１７６８－７５．
非特許文献１０：細谷ら：木材学会誌２６（２）１１８－２１、１９８０
非特許文献１１：Ｈｉｓｈｉｙａｍａ　ｅｔ　ａｌ：Ｔｅｔｒａｈｅｄｒｏｎ　Ｌｅｔｔｅｒｓ５３,８４２－８４５、２０１２
非特許文献１２：Ｋ.Ｉｔｏｈ： Ｍｏｋｕｚａｉ Ｇａｋｋａｉｓｈｉ ｖｏｌ. ３８,Ｎｏ.６, ５７９－５８４（１９９２） [References]
Non-Patent Documents 4 to 12 described above are as follows. The description of the documents is incorporated herein by reference:
Non-Patent Document 4: Besemer J et al .; Nucleic Acids Research, 2001, vol 29, p2607
Non-Patent Document 5: Masai, E .; S. Kubota. Y. Katayama, S .; Kawai, M .; Yamazaki, and N.K. Morohoshi. 1993. Biosci. Biotechnol. Biochem. 57: 1655-1659.
Non-Patent Document 6: Sato Y, Moriuchi H, Hisyama S, Otsuka Y, Oshima K, Kasai D, Nakamura M, Ohara S, Katayama Y, Fukuda M, Masai E. Appl Environ Microbiol. 2009; 75 (16): 5195-201.
Non-Patent Document 7: Masai, E .; Y. Katayama, S .; Kubota, S .; Kawai, M .; Yamazaki, and N.K. Morohoshi. 1993. FEBS Lett. 323: 135-140.
Non-Patent Document 8: Masai, E .; Y. Katayama, S .; Kawai, S .; Nishikawa, M .; Yamazaki, and N.K. Morohoshi. 1991. J. et al. Bacteriol. 173: 7950-7955.
Non-Patent Document 9: Masai E, Ichimura A, Sato Y, Miyauchi K, Katayama Y, Fukuda M. et al. J Bacteriol. 2003 Mar; 185 (6): 1768-75.
Non-Patent Document 10: Hosoya et al .: Journal of the Wood Society of Japan 26 (2) 118-21, 1980
Non-Patent Document 11: Hishiyama et al: Tetrahedron Letters 53, 842-845, 2012
Non-Patent Document 12: K. Itoh: Mokuzai Gakashishi vol. 38, No. 6, 579-584 (1992)
配列表に記載の配列は以下のとおりである：
［配列番号１］Ｃ１０Ｇ００６９
MTQVKGRTAFITGGGSGVALGQAKVFARAGCKVAIADIRQDHLDEAMAWFEAENAKGANYEVMAVKLDITDREAYAKVADEVEAKLGPVELLFNTAGVSHFGAIQDATYDDWDWQIDVNLRGVINGVRTFVPRMIERGNGGHVVNTASMSAFVALKGTGIYCTTKMAVRGLTETLALDLEEHGIGVSLLCPGAVNTNIHEALLTRPKHLADTGYYQAGPEMFAHLKNVIECGMEPETLANHVLKAVEENQLYVLAYPEFRKPLEDIHARVMAALANPEDDPDYDRRVAHGVPGGEAKEEEKTA
［配列番号２］Ｃ１０Ｇ００９３
MQDLPGKTAFVTGGASGIGLGIAKALLGAGMNVAIADIRQDHLDDAAAELDGGDKVLALQLDVTDRAAFAAAADATEAKFGKIHILCNNAGVAVVGPTDMATFADWDWVMGVNVGGTINGIVTMLPRMLKHGEGGHIVNTASMSALVPAPGTTIYSSGKAAVTSMMECMRPELESRGIVCSAFCPGAVQSNIAEAGRTRPDALAETGYAEADKGRQAGGSFFHLYQTKEEVGERVLTGILNDELYILTHAEFLIGVQERGEATTAAVQVQLPENEEYKNTFGVLFRNSAITQEIDRQKALRAAQMSEAGVA
［配列番号３］Ｃ１０Ｇ００７６
MLTLYSFGPGANSLKPLLALYEKGLEFTPRFVDPTKFEHHEEWFKKINPRGQVPALDHDGNVITESTVICEYLEDAFPDAPRLRPTDPVQIAEMRVWTKWVDEYFCWCVSTIGWERGIGPMARALSDEEFEEKVKRIPIPEQQAKWRSARAGFPKEVLDEEMRKIRVSIDRLEKRLSESTWLAGEDYTLADICNFAIANGMEKGFDDIVNTAATPNLVAWIERINARPACIEMFAKSKSEFAARKPFAKSEEQAQA
［配列番号４］Ｃ１０Ｇ００７７
MAKDNRITLYDLQLASGCTISPFVWRTKYALAHKGFDMDIVPGGFTGIAERTGGRSERAPVIVDDGKWVLDSWKIAEYLDETYPDRPMLFEGPSMKVLTKFLDAWLWKTIIAPWFRCYILDYHDLSLPQDHAYVRESRETMFLGGQKLEDVQAGREDRLPHVPPLLEPLRQLLRDTPWLGGATPNYADYTALAIFLWTGSVCTTPPLTEDDPLRDWLDRGFDLYGGLGRHPGMHTLFGLKLREGDPEPFDRTGLGIEPAPVNQGSAEPATAS
［配列番号５］Ｃ１０Ｇ００７８
MYQIPGCPFSERVELLLDLKGLGDVLVDHEIDISKPRPDWLLKKTRGTTSLPALELENGETLKESMVIMRYIEDRFPEVPVARQDPYEHALEAMLCATDGAYTGAGYRMILNRDKARREELKAEVDAQYARLDDFLRHYSPDGVYLFDRFGWAEVAFAPMFKRLWFLEYYEDYEVPQNLTRVLLWREATLAEPVVQARHGHRELMTLYYDYTQGGGNGRLPEGRSVSSFTLDPPWRARPMPPRDKWGQGASDAELGLIPGITVRSDTVPA
［配列番号６］Ｃ１０Ｇ００７５
MLEELDANYTLRPISLTNREQKEDWYLARNPNGRIPTLIDHEVDAGNGGFAVFESGAILIYLAEKFGRFLPADTMGRSRAIQWVMWQMSGLGPMMGQATVFNRYFEPRLPEVIDRYTRESRRLFEVMDTHLADNEFLAGDYSIADIACFPWVRGHDWACIDMEGLPHLQRWFETIGERPAVQRGLLLPEPPKADEMAEKTTRQGKNILA
［配列番号７］ｃ１０ｇ００６９遺伝子
ATGACACAGGTAAAGGGACGCACCGCGTTCATCACTGGCGGCGGTTCGGGCGTGGCGCTCGGCCAGGCCAAGGTTTTCGCCAGGGCTGGCTGCAAGGTCGCCATTGCCGACATCCGCCAGGATCACCTCGACGAGGCCATGGCCTGGTTCGAGGCCGAGAACGCCAAGGGCGCGAACTACGAGGTGATGGCGGTCAAACTCGACATCACCGACCGCGAGGCTTACGCCAAGGTCGCCGACGAGGTGGAAGCGAAGCTGGGGCCCGTCGAACTGCTGTTCAACACCGCCGGGGTCTCGCACTTCGGCGCGATCCAGGATGCCACTTACGACGACTGGGACTGGCAGATCGACGTCAACCTGCGCGGTGTGATCAACGGCGTGCGCACCTTCGTGCCGCGCATGATCGAGCGCGGCAACGGCGGCCACGTGGTCAACACCGCCTCGATGTCGGCCTTCGTGGCGCTCAAGGGCACGGGCATCTACTGCACCACCAAGATGGCGGTGCGCGGGCTGACCGAGACTCTGGCCCTCGATCTGGAAGAGCATGGCATCGGCGTGTCGCTGCTGTGCCCGGGCGCGGTCAACACCAACATTCACGAAGCGCTGCTGACCCGCCCCAAGCATCTGGCGGACACCGGCTACTACCAGGCCGGGCCGGAGATGTTCGCGCATCTCAAGAACGTGATCGAATGCGGCATGGAGCCCGAGACGCTGGCGAACCACGTCCTGAAGGCCGTGGAGGAGAACCAGCTTTACGTTCTCGCCTATCCGGAATTCCGCAAGCCGCTGGAAGACATCCACGCGCGCGTCATGGCCGCCCTCGCCAATCCCGAGGACGATCCCGACTACGACCGGCGCGTGGCGCACGGCGTACCGGGCGGCGAGGCCAAGGAGGAGGAAAAGACGGCATGA
［配列番号８］ｃ１０ｇ００９３遺伝子
ATGCAGGATCTACCGGGGAAGACCGCCTTTGTGACCGGCGGCGCCAGCGGGATTGGCCTGGGGATAGCCAAGGCCCTGCTGGGGGCGGGCATGAACGTTGCTATCGCCGACATTCGCCAGGACCATCTCGATGACGCCGCAGCCGAACTGGACGGCGGCGACAAGGTGCTCGCGCTCCAGCTCGACGTCACCGATCGCGCTGCCTTTGCCGCCGCCGCCGATGCCACCGAGGCCAAGTTCGGCAAGATCCACATCCTGTGCAACAACGCAGGGGTTGCGGTTGTCGGCCCCACCGACATGGCGACTTTCGCCGACTGGGACTGGGTCATGGGCGTGAACGTGGGCGGCACGATCAACGGCATCGTCACCATGCTGCCGCGCATGTTGAAGCACGGCGAGGGCGGCCACATCGTCAACACCGCCTCGATGTCCGCGCTCGTGCCCGCGCCGGGCACCACGATCTATTCCTCGGGCAAAGCCGCCGTCACCTCGATGATGGAATGCATGCGCCCCGAACTGGAATCGCGCGGCATCGTGTGCTCGGCCTTCTGCCCAGGCGCGGTGCAGTCGAACATTGCCGAAGCCGGGCGCACCCGTCCCGACGCGCTGGCCGAGACCGGCTACGCCGAGGCCGACAAGGGGCGCCAGGCGGGGGGCAGTTTCTTCCACCTCTACCAGACCAAGGAAGAGGTCGGTGAGCGCGTGCTGACGGGCATCCTCAACGATGAACTCTACATCCTCACCCACGCCGAGTTCCTCATCGGCGTGCAGGAGCGCGGCGAGGCGACCACCGCGGCGGTGCAGGTCCAGTTGCCCGAGAACGAGGAGTACAAGAACACCTTCGGCGTGCTCTTCCGCAACTCGGCGATCACCCAGGAGATCGATCGGCAGAAGGCCCTGCGCGCCGCGCAGATGTCCGAAGCCGGCGTCGCGTAA
［配列番号９］ｃ１０ｇ００７６遺伝子
ATGTTGACGCTGTACAGCTTTGGCCCCGGAGCCAATTCGCTTAAGCCCCTGCTGGCCCTTTACGAGAAAGGCCTCGAATTTACGCCGCGCTTCGTCGATCCGACCAAGTTCGAGCATCACGAGGAATGGTTCAAGAAGATCAACCCGCGCGGCCAGGTTCCCGCGCTCGATCACGATGGCAACGTCATCACCGAATCGACGGTGATTTGCGAATACCTCGAAGACGCCTTCCCCGATGCGCCCCGGCTGCGTCCCACCGACCCTGTGCAGATCGCCGAGATGCGGGTCTGGACCAAGTGGGTGGACGAATACTTCTGCTGGTGCGTCTCCACCATCGGCTGGGAGCGCGGCATCGGTCCGATGGCCCGTGCCCTGTCGGACGAGGAGTTCGAGGAAAAGGTCAAACGCATCCCGATCCCTGAGCAGCAGGCCAAGTGGCGCAGCGCCCGCGCCGGCTTCCCCAAGGAGGTTCTGGACGAGGAAATGCGCAAGATCCGCGTCTCGATCGACCGTCTCGAAAAGCGCCTTTCCGAAAGCACCTGGCTGGCGGGCGAGGACTATACGCTTGCCGACATCTGCAACTTCGCCATCGCCAACGGCATGGAGAAGGGCTTTGATGACATCGTCAATACGGCCGCTACGCCCAACCTCGTCGCCTGGATCGAACGCATCAACGCGCGTCCCGCCTGCATCGAGATGTTCGCCAAGTCCAAGAGCGAGTTCGCCGCGCGCAAGCCCTTCGCCAAGAGCGAAGAGCAGGCACAGGCCTGA
［配列番号１０］ｃ１０ｇ００７７遺伝子
ATGGCCAAGGACAACCGCATAACGCTTTACGATCTGCAGCTTGCCTCGGGCTGCACGATCAGCCCCTTCGTGTGGCGCACCAAGTACGCGCTGGCGCACAAGGGCTTCGACATGGATATCGTGCCGGGCGGCTTTACCGGCATTGCCGAGCGCACGGGCGGGCGTTCCGAACGTGCGCCAGTGATCGTCGACGATGGCAAGTGGGTGCTCGACAGCTGGAAGATCGCCGAATACCTCGATGAGACTTATCCCGATCGCCCGATGCTGTTCGAGGGCCCTTCCATGAAGGTGCTCACCAAGTTCCTCGACGCCTGGCTATGGAAGACGATCATCGCGCCGTGGTTCCGCTGCTACATCCTCGACTACCATGATCTGTCGTTGCCGCAGGACCATGCCTACGTGCGCGAATCGCGCGAGACGATGTTCCTGGGCGGGCAGAAGCTGGAGGATGTGCAGGCGGGCCGCGAGGATCGGCTTCCGCACGTGCCGCCGTTGCTGGAGCCGCTGCGCCAGCTGCTGCGCGATACGCCCTGGCTGGGCGGTGCGACACCCAACTACGCGGACTACACCGCGCTCGCCATCTTCCTGTGGACCGGCTCTGTGTGCACCACGCCGCCGCTCACCGAGGATGACCCGTTACGCGACTGGCTCGACCGCGGCTTCGACCTTTACGGCGGGCTGGGGCGTCATCCGGGCATGCACACGCTGTTCGGCCTGAAGCTGCGCGAGGGCGATCCAGAGCCTTTCGACCGCACCGGCCTTGGCATCGAGCCCGCGCCGGTCAACCAGGGCTCGGCCGAGCCGGCTACGGCGAGCTGA
［配列番号１１］ｃ１０ｇ００７８遺伝子
ATGTACCAGATCCCGGGCTGCCCGTTCTCGGAGCGGGTGGAGTTGTTGCTCGATCTCAAGGGGCTGGGCGATGTCCTCGTCGACCACGAGATCGACATTTCCAAACCGCGCCCGGACTGGCTCCTGAAGAAGACGCGGGGGACGACTTCGCTTCCCGCGCTGGAGCTGGAGAACGGGGAGACCTTGAAGGAGAGCATGGTCATCATGCGCTACATCGAGGACCGCTTCCCCGAAGTTCCGGTCGCGCGCCAGGATCCCTACGAGCACGCCCTCGAGGCGATGCTGTGCGCGACCGATGGCGCCTACACCGGGGCGGGCTATCGCATGATCCTGAACCGGGACAAGGCCAGGCGCGAGGAATTGAAGGCCGAAGTCGATGCGCAGTACGCCCGGCTCGACGATTTCCTGCGGCACTACAGCCCGGATGGGGTCTACCTGTTCGACCGTTTCGGCTGGGCCGAAGTGGCCTTTGCGCCGATGTTCAAGCGGCTGTGGTTCCTCGAATACTACGAGGACTACGAGGTGCCGCAGAACCTGACGCGGGTGCTTCTGTGGCGCGAGGCGACACTGGCCGAACCTGTCGTGCAGGCGCGCCACGGCCACCGCGAGCTGATGACGCTCTACTACGACTACACCCAGGGCGGTGGAAACGGACGTCTGCCCGAAGGGCGCAGCGTCTCCAGCTTCACCCTCGATCCGCCGTGGCGCGCGCGGCCCATGCCGCCGCGTGACAAGTGGGGGCAGGGCGCCAGCGATGCCGAGCTCGGGCTGATCCCGGGCATTACCGTCCGTTCGGACACGGTGCCCGCCTGA
［配列番号１２］ｃ１０ｇ００７５遺伝子
ATGCTCGAGGAGCTGGACGCGAACTACACGTTGCGTCCGATCTCGCTGACCAACCGCGAGCAGAAGGAAGACTGGTATCTCGCCCGCAATCCCAACGGGCGTATCCCCACACTGATCGACCATGAGGTCGATGCCGGGAACGGCGGTTTTGCGGTGTTCGAATCGGGTGCGATCCTGATCTACCTTGCCGAGAAGTTCGGCCGTTTCCTGCCAGCCGACACGATGGGCCGCAGCCGCGCGATCCAGTGGGTGATGTGGCAGATGTCGGGCCTCGGCCCCATGATGGGACAGGCGACCGTCTTCAACCGCTACTTCGAGCCCAGGCTGCCCGAGGTCATCGACCGCTACACGCGCGAGAGCCGCCGCCTCTTCGAAGTGATGGACACGCACCTCGCCGACAACGAATTCCTCGCGGGCGACTATTCGATCGCCGACATCGCCTGCTTCCCGTGGGTGCGCGGGCATGACTGGGCCTGCATCGACATGGAGGGGCTGCCCCACCTGCAACGCTGGTTCGAGACCATCGGTGAGCGCCCGGCCGTCCAGCGCGGCCTGCTCTTGCCCGAACCGCCCAAGGCGGACGAGATGGCCGAGAAGACGACCCGCCAGGGCAAGAACATCCTGGCCTGA
［配列番号１３］プライマーＡ
TCTCGAGCTCGGATCC
［配列番号１４］プライマーＢ
CTGGTACCATGGAATTCG
［配列番号１５］プライマーＣ
GGATCCGAGCTCGAGATGATCAAGGGTATCGAAGG
［配列番号１６］プライマーＤ
CGAATTCCATGGTACCAGTCAGAACTCCTGTGCGG
［配列番号１７］プライマーＥ
GGATCCGAGCTCGAGATGACGAACTGGCTTATCAC
［配列番号１８］プライマーＦ
CGAATTCCATGGTACCAGTCAGACCTCGGCGAAG
［配列番号１９］プライマーＧ
GGATCCGAGCTCGAGATGACACAGGTAAAGGGACG
［配列番号２０］プライマーＨ
CGAATTCCATGGTACCAGTCATGCCGTCTTTTCCTC
［配列番号２１］プライマーＩ
GGATCCGAGCTCGAGATGGGAGAGACGACAAAAC
［配列番号２２］プライマーＪ
CGAATTCCATGGTACCAGTCAGGTGAGGTCGGC
［配列番号２３］プライマーＫ
GGATCCGAGCTCGAGATGCAGGATCTACCGGG
［配列番号２４］プライマーＬ
CGAATTCCATGGTACCGCAAGCTGTGTCATGC
［配列番号２５］プライマーＭ
GGATCCGAGCTCGAGATGACGGGCGGGG
［配列番号２６］プライマーＮ
CGAATTCCATGGTACCAGTCAGAGCGCGTTGGC
［配列番号２７］プライマーＯ
GGATCCGAGCTCGAGATGCTGGAACTGTGGACTTC
［配列番号２８］プライマーＰ
CGAATTCCATGGTACCAGGTAGGTGTGCTCATCGTTCA
［配列番号２９］プライマーＱ
GGATCCGAGCTCGAGATGTTGACGCTGTACAGCTTTG
［配列番号３０］プライマーＲ
CGAATTCCATGGTACCAGCTCCTCAGGCCTGTGC
［配列番号３１］プライマーＳ
GGATCCGAGCTCGAGATGGCCAAGGACAACC
［配列番号３２］プライマーＴ
CGAATTCCATGGTACCAGTCAGCTCGCCGTAGC
［配列番号３３］プライマーＵ
GGATCCGAGCTCGAGATGGCATGGGACGATG 
［配列番号３４］プライマーＶ
CGAATTCCATGGTACCAGGATGACGGTGTGCTTCAC The sequences listed in the sequence listing are as follows:
[SEQ ID NO: 1] C10G0069
MTQVKGRTAFITGGGSGVALGQAKVFARAGCKVAIADIRQDHLDEAMAWFEAENAKGANYEVMAVKLDITDREAYAKVADEVEAKLGPVELLFNTAGVSHFGAIQDATYDDWDWQIDVNLRGVINGVRTFVPRMIERGNGGHVVNTASMSAFVALKGTGIYCTTKMAVRGLTETLALDLEEHGIGVSLLCPGAVNTNIHEALLTRPKHLADTGYYQAGPEMFAHLKNVIECGMEPETLANHVLKAVEENQLYVLAYPEFRKPLEDIHARVMAALANPEDDPDYDRRVAHGVPGGEAKEEEKTA
[SEQ ID NO: 2] C10G0093
MQDLPGKTAFVTGGASGIGLGIAKALLGAGMNVAIADIRQDHLDDAAAELDGGDKVLALQLDVTDRAAFAAAADATEAKFGKIHILCNNAGVAVVGPTDMATFADWDWVMGVNVGGTINGIVTMLPRMLKHGEGGHIVNTASMSALVPAPGTTIYSSGKAAVTSMMECMRPELESRGIVCSAFCPGAVQSNIAEAGRTRPDALAETGYAEADKGRQAGGSFFHLYQTKEEVGERVLTGILNDELYILTHAEFLIGVQERGEATTAAVQVQLPENEEYKNTFGVLFRNSAITQEIDRQKALRAAQMSEAGVA
[SEQ ID NO: 3] C10G0076
MLTLYSFGPGANSLKPLLALYEKGLEFTPRFVDPTKFEHHEEWFKKINPRGQVPALDHDGNVITESTVICEYLEDAFPDAPRLRPTDPVQIAEMRVWTKWVDEYFCWCVSTIGWERGIGPMARALSDEEFEEKAKIRFSARAGFPKEVLDEKDI
[SEQ ID NO: 4] C10G0077
MAKDNRITLYDLQLASGCTISPFVWRTKYALAHKGFDMDIVPGGFTGIAERTGGRSERAPVIVDDGKWVLDSWKIAEYLDETYPDRPMLFEGPSMKVLTKFLDAWLWTDIIAPWFRCYILDYHDLSLPQDHAYVRESRETMFLGGDGTPVLY
[SEQ ID NO: 5] C10G0078
MYQIPGCPFSERVELLLDLKGLGDVLVDHEIDISKPRPDWLLKKTRGTTSLPALELENGETLKESMVIMRYIEDRFPEVPVARQDPYEHALEAMLCATDGAYTGAGYRMILNRDKARREELKAEVDAQYARLDDFLRHYSPDGVYLFDRFGWAEVAFAPMFKRLWFLEYVSEDMF
[SEQ ID NO: 6] C10G0075
MLEELDANYTLRPISLTNREQKEDWYLARNPNGRIPTLIDHEVDAGNGGFAVFESGAILIYLAEKFGRFLPADTMGRSRAIQWVMWQMSGLGPMMGQATVFNRYFEPRLPEVIDRYTRESRRLFEVMDTHLADNEFLAGDYSIADIACFPWVRGHDWACIDMEGLPNIQR
[SEQ ID NO: 7] c10g0069 gene

[SEQ ID NO: 8] c10g0093 gene

[SEQ ID NO: 9] c10g0076 gene

[SEQ ID NO: 10] c10g0077 gene

[SEQ ID NO: 11] c10g0078 gene

[SEQ ID NO: 12] c10g0075 gene

[SEQ ID NO: 13] Primer A
TCTCGAGCTCGGATCC
[SEQ ID NO: 14] Primer B
CTGGTACCATGGAATTCG
[SEQ ID NO: 15] Primer C
GGATCCGAGCTCGAGATGATCAAGGGTATCGAAGG
[SEQ ID NO: 16] Primer D
CGAATTCCATGGTACCAGTCAGAACTCCTGTGCGG
[SEQ ID NO: 17] Primer E
GGATCCGAGCTCGAGATGACGAACTGGCTTATCAC
[SEQ ID NO: 18] Primer F
CGAATTCCATGGTACCAGTCAGACCTCGGCGAAG
[SEQ ID NO: 19] Primer G
GGATCCGAGCTCGAGATGACACAGGTAAAGGGACG
[SEQ ID NO: 20] Primer H
CGAATTCCATGGTACCAGTCATGCCGTCTTTTCCTC
[SEQ ID NO: 21] Primer I
GGATCCGAGCTCGAGATGGGAGAGACGACAAAAC
[SEQ ID NO: 22] Primer J
CGAATTCCATGGTACCAGTCAGGTGAGGTCGGC
[SEQ ID NO: 23] Primer K
GGATCCGAGCTCGAGATGCAGGATCTACCGGG
[SEQ ID NO: 24] Primer L
CGAATTCCATGGTACCGCAAGCTGTGTCATGC
[SEQ ID NO: 25] Primer M
GGATCCGAGCTCGAGATGACGGGCGGGG
[SEQ ID NO: 26] Primer N
CGAATTCCATGGTACCAGTCAGAGCGCGTTGGC
[SEQ ID NO: 27] Primer O
GGATCCGAGCTCGAGATGCTGGAACTGTGGACTTC
[SEQ ID NO: 28] Primer P
CGAATTCCATGGTACCAGGTAGGTGTGCTCATCGTTCA
[SEQ ID NO: 29] Primer Q
GGATCCGAGCTCGAGATGTTGACGCTGTACAGCTTTG
[SEQ ID NO: 30] Primer R
CGAATTCCATGGTACCAGCTCCTCAGGCCTGTGC
[SEQ ID NO: 31] Primer S
GGATCCGAGCTCGAGATGGCCAAGGACAACC
[SEQ ID NO: 32] Primer T
CGAATTCCATGGTACCAGTCAGCTCGCCGTAGC
[SEQ ID NO: 33] Primer U
GGATCCGAGCTCGAGATGGCATGGGACGATG
[SEQ ID NO: 34] Primer V
CGAATTCCATGGTACCAGGATGACGGTGTGCTTCAC
本発明の製造方法や酵素によって、天然のリグニンやリグニン関連物質を含むバイオマスや該バイオマスを処理して得られるカルボニルフェニルプロパン系化合物から３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンの中間体としてグルタチオン付加３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンが得られる。３－ヒドロキシ－１－（４－ヒドロキシ－３－メトキシフェニル）－１－プロパノンは、種々の産業上有用な化合物に変換することができ、例えば、樹脂、接着剤、レジスト材料、医薬品などの原料として利用することが期待できる１－（４－ヒドロキシ－３－メトキシフェニル）－１，３－プロパンジオールなどの製造方法の原料として利用することができる。 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) from a carbonylphenylpropane-based compound obtained by treating biomass containing natural lignin or a lignin-related substance or the biomass with the production method or enzyme of the present invention. Glutathione-added 3-hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone is obtained as an intermediate of -1-propanone. 3-Hydroxy-1- (4-hydroxy-3-methoxyphenyl) -1-propanone can be converted into various industrially useful compounds, for example, raw materials for resins, adhesives, resist materials, pharmaceuticals, etc. It can be used as a raw material for a production method of 1- (4-hydroxy-3-methoxyphenyl) -1,3-propanediol and the like that can be expected to be used as

Claims (8)

  1. カルボニルフェニルプロパン系化合物に、還元型グルタチオンの存在下で、ノボスフィンゴビウム（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ）属微生物に由来する下記（２）の酵素を作用させることにより、グルタチオン付加フェニルプロパン系化合物を得る工程
    を含む、グルタチオン付加フェニルプロパン系化合物の製造方法。
    （２）Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用する、ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ     A step of obtaining a glutathione-added phenylpropane compound by allowing the carbonylphenylpropane compound to act on the enzyme of the following (2) derived from a microorganism belonging to the genus Novosphingium in the presence of reduced glutathione: And a method for producing a glutathione-added phenylpropane-based compound.
    (2) Glutathione S-transfer having a multi-domain of PRK15113 and maiA on the N-terminal side and acting on veratrylglycerol-β-guayacyl ether
  2. 前記（２）の酵素が、配列表の配列番号３に記載のアミノ酸配列を有するＣ１０Ｇ００７６又は配列表の配列番号４に記載のアミノ酸配列を有するＣ１０Ｇ００７７である、請求項１に記載の製造方法。 The production method according to claim 1, wherein the enzyme of (2) is C10G0076 having the amino acid sequence described in SEQ ID NO: 3 of the sequence listing or C10G0077 having the amino acid sequence described in SEQ ID NO: 4 of the sequence listing.
  3. ノボスフィンゴビウム（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ）属微生物に由来する、Ｇｓｔ並びにＮ末端側にＰＲＫ１５１１３及びｍａｉＡのマルチドメインを有し、かつ、ベラトリルグリセロール－β－グアイアシルエーテルに作用する、ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ。 Glutathione S-transfer, which is derived from a microorganism belonging to the genus Novosphingobium, has a multi-domain of Pst15113 and maiA on the N-terminal side and acts on veratrylglycerol-β-guayacyl ether.
  4. 前記ノボスフィンゴビウム属微生物が、ノボスフィンゴビウム・スピーシーズ（Ｎｏｖｏｓｐｈｉｎｇｏｂｉｕｍ　ｓｐ．）　ＭＢＥＳ０４（受託番号：ＮＩＴＥ　Ｐ－０１７９７）である、請求項３に記載のｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ。 The glutathione S-transferase according to claim 3, wherein the microorganism belonging to the genus Novosphingobium is Novosphingium sp. MBES04 (Accession number: NITE P-01797).
  5. 前記ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅは、ＳＤＳ－ＰＡＧＥで確認できる分子量が３１ｋＤａであり；最適ｐＨが８．５～９．５であり；かつ、最適温度が３０～３５℃である、請求項３に記載のｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ。 4. The glutathione S-transferase has a molecular weight of 31 kDa that can be confirmed by SDS-PAGE; an optimum pH of 8.5 to 9.5; and an optimum temperature of 30 to 35 ° C. glutathione S-transfer.
  6. 前記ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅは、ＳＤＳ－ＰＡＧＥで確認できる分子量が３１ｋＤａであり；最適ｐＨが７～８であり；かつ、最適温度が２５～３０℃である、請求項３に記載のｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ。 The glutathione S-transferase has a molecular weight of 31 kDa that can be confirmed by SDS-PAGE; an optimum pH of 7 to 8; and an optimum temperature of 25 to 30 ° C. .
  7. 前記ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅは、配列表の配列番号３に記載のアミノ酸配列を有する、請求項３に記載のｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ。 The glutathione S-transferase according to claim 3, wherein the glutathione S-transferase has the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing.
  8. 前記ｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅは、配列表の配列番号４に記載のアミノ酸配列を有する、請求項３に記載のｇｌｕｔａｔｈｉｏｎｅ Ｓ－ｔｒａｎｓｆｅｒａｓｅ。
     
          The glutathione S-transferase according to claim 3, wherein the glutathione S-transferase has the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.

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