WO2013073679A1 - Method for producing (2r,4r)-monatin polyvalent metal salt crystals - Google Patents

Method for producing (2r,4r)-monatin polyvalent metal salt crystals Download PDF

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WO2013073679A1
WO2013073679A1 PCT/JP2012/079826 JP2012079826W WO2013073679A1 WO 2013073679 A1 WO2013073679 A1 WO 2013073679A1 JP 2012079826 W JP2012079826 W JP 2012079826W WO 2013073679 A1 WO2013073679 A1 WO 2013073679A1
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monatin
polyvalent metal
amino acid
metal salt
producing
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PCT/JP2012/079826
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French (fr)
Japanese (ja)
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森　健一
杉山　雅一
美加 守屋
鈴木　俊一
秀美 藤井
恭平 藤原
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味の素株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C07K14/43—Sweetening agents, e.g. thaumatin, monellin
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom

Definitions

  • the present invention relates to a method for producing (2R, 4R) monatin polyvalent metal salt crystals.
  • the (2S, 4S) form of 4-hydroxy-4- (3-indolylmethyl) -2-aminoglutaric acid (hereinafter sometimes referred to as “monatin”) is native to the Transvalal region of northern South Africa. It is known to be an amino acid derivative that is contained in the root bark of Schlerochitom licifolius, has a sweetness several hundred times that of sucrose, and is useful as a sweetener (Patent Document 1).
  • Non-patent Documents 1 to 3, Patent Documents 2 and 3 Various reports have been made on methods for producing monatin (Non-patent Documents 1 to 3, Patent Documents 2 and 3), and there have been some examples of methods for producing optically active monatin. Since a multi-step process is required and the yield is not high, it is not necessarily an industrially suitable production method.
  • Patent Document 4 discloses a method of obtaining a crystal by epimerizing (2R, 4R) monatin from (2S, 4R) monatin.
  • the obtained crystals are (2R, 4R) monatin potassium salt crystals, the stability is not always satisfactory depending on the formulation.
  • the present invention includes the following contents. [1] Contacting (2S, 4R) monatin with one or more enzymes capable of producing (2R, 4R) monatin from aldehyde or (2S, 4R) monatin in an aqueous solution containing polyvalent metal ions And (2R, 4R) monatin polyvalent metal salt crystals, and the method for producing (2R, 4R) monatin polyvalent metal salt crystals.
  • a method for producing a (2R, 4R) monatin polyvalent metal salt crystal comprising obtaining a 2R, 4R) monatin polyvalent metal salt crystal.
  • Enzymes capable of producing (2R, 4R) monatin from (2S, 4R) monatin are L-amino acid aminotransferase, D-amino acid aminotransferase, and racemase
  • (2R, 4R) monatin Method for producing valent metal salt crystal are described by the enzymes capable of producing (2R, 4R) monatin from (2S, 4R) monatin.
  • the crystal has intrinsic X-ray diffraction peaks at diffraction angles (2 ⁇ ⁇ 0.2 °, CuK ⁇ ) at 4.9 °, 16.8 °, 18.0 °, 24.6 °, [1]
  • [17] Amount of (2R, 4R) monatin produced by simultaneously isomerizing (2S, 4R) monatin to (2R, 4R) monatin and crystallization of (2R, 4R) monatin polyvalent metal salt
  • the method further includes the step of adding an organic solvent to the aqueous solution after the formation of the (2R, 4R) monatin to promote precipitation of the (2R, 4R) monatin polyvalent metal salt crystal, [1] to [17]
  • One or more enzymes capable of producing (2R, 4R) monatin from aldehyde or (2S, 4R) monatin act on an aqueous solution containing (2S, 4R) monatin in the presence of a polyvalent metal ion.
  • FIG. 1 is a diagram showing isomerization of (2R, 4R) monatin to (2R, 4R) monatin by racemase.
  • FIG. 2 is a diagram showing the generation of (2R, 4R) monatin from (2S, 4R) monatin via 4R-IHOG by one aminotransferase. Aminotransferase does not have a stereospecific catalytic activity at the 2-position, thus generating 4R-IHOG from (2S, 4R) monatin, and then reversibly (2R, 4R) monatin from 4R-IHOG. Can be generated.
  • 4R-IHOG 4R- (Indol-3-yl-methyl) -4-hydroxy-2-oxoglutaric acid.
  • FIG. 1 is a diagram showing isomerization of (2R, 4R) monatin to (2R, 4R) monatin by racemase.
  • FIG. 2 is a diagram showing the generation of (2R, 4R) monatin
  • FIG. 3 is a diagram showing the production of (2R, 4R) monatin from (2S, 4R) monatin via 4R-IHOG by two aminotransferases. The first aminotransferase produces 4R-IHOG from (2S, 4R) monatin, and then the second aminotransferase produces (2R, 4R) monatin from 4R-IHOG.
  • FIG. 4 is a diagram showing the production of (2R, 4R) monatin from (2S, 4R) monatin via 4R-IHOG by two aminotransferases (L-amino acid aminotransferase and D-amino acid aminotransferase). .
  • L-amino acid aminotransferase produces 4R-IHOG from (2S, 4R) monatin
  • D-amino acid aminotransferase produces (2R, 4R) monatin from 4R-IHOG.
  • keto acid or L-amino acid or D-amino acid
  • racemase capable of converting L-amino acid to D-amino acid to the reaction system, thereby causing side reaction by L-amino acid aminotransferase (keto acid ⁇ L-amino acid) ), And side reactions (D-amino acids ⁇ keto acids) by D-amino acid aminotransferase.
  • FIG. 5 is a diagram showing a preferred example of the reaction shown in FIG.
  • PA L-amino acid aminotransferase
  • D-Ala ⁇ PA D-amino acid aminotransferase
  • FIG. 7 is a graph showing intrinsic X-ray diffraction peaks of the ((2R, 4R) monatin) 2 magnesium salt crystal obtained in Example 1.
  • FIG. 8 is a diagram showing the change over time of (2S, 4R) monatin and (2R, 4R) monatin in the reaction solution.
  • FIG. 9 is a diagram showing the change over time of (2S, 4R) monatin and (2R, 4R) monatin in the reaction supernatant after addition of magnesium salt.
  • FIG. 10 is a graph showing the change over time of (2S, 4R) monatin and (2R, 4R) monatin in the reaction solution.
  • the upper graph is a graph showing the change over time of (2S, 4R) monatin (left) and (2R, 4R) monatin (right) in the reaction solution under the conditions with and without preferential crystallization.
  • the middle graph shows the changes over time of (2S, 4R) monatin (left) and (2R, 4R) monatin (right) in the whole reaction solution or supernatant when only the enzyme reaction was performed (no preferential crystallization). Show.
  • the lower part shows the change over time of (2S, 4R) monatin (left) and (2R, 4R) monatin (right) in the whole reaction solution or supernatant when the enzyme reaction and preferential crystallization are performed simultaneously.
  • FIG. 11 is a graph showing intrinsic X-ray diffraction peaks of the ((2R, 4R) monatin) 2 magnesium salt crystal obtained in Example 11.
  • FIG. 12 is a diagram showing an intrinsic X-ray diffraction peak of the ((2R, 4R) monatin) 2 magnesium salt crystal obtained in Reference Example 3.
  • FIG. 13 is a diagram showing an intrinsic X-ray diffraction peak of the ((2R, 4R) monatin) 2 magnesium salt crystal obtained in Reference Example 4.
  • FIG. 14 is a diagram showing an intrinsic X-ray diffraction peak of the ((2R, 4R) monatin) 2 magnesium salt crystal obtained in Reference Example 5.
  • the present invention provides a method for producing (2R, 4R) monatin polyvalent metal salt crystals.
  • one or more enzymes capable of producing (2R, 4R) monatin from aldehyde or (2S, 4R) monatin in an aqueous solution containing a polyvalent metal ion are converted into (2S, 4R).
  • Contacting the monatin to precipitate the (2R, 4R) monatin polyvalent metal salt crystals is converted into (2S, 4R).
  • the method of the invention comprises (a) isomerizing (2S, 4R) monatin to produce (2R, 4R) monatin (isomerization) and (b) producing (2R, 4R) Precipitating (2R, 4R) monatin polyvalent metal salt crystals by contact with monatin and polyvalent metal ions (crystallization).
  • the method of the present invention may comprise recovering the resulting crystals.
  • (A) and (b) can be performed separately or simultaneously (in other words, in parallel), but are preferably performed simultaneously. By simultaneously performing (a) and (b), the amount of (2R, 4R) monatin produced can be increased.
  • the polymer When performing (a) and (b) separately, the polymer may be removed (eg, centrifuged, filtered) from the reaction solution obtained in (a) in order to avoid crystallization inhibition by the polymer. Good.
  • a seed crystal may be added to the solution.
  • precipitation of (2R, 4R) monatin polyvalent metal salt crystals can be promoted by adding an organic solvent to the aqueous solution after the formation of (2R, 4R) monatin.
  • the crystals precipitated in (b) can be easily obtained as wet crystals by subjecting them to a separation step such as a filtration step.
  • the obtained wet crystals may be washed. Dry crystals can be obtained by drying the wet crystals. Details of these matters will be described later.
  • the (2S, 4R) monatin used in the present invention can be obtained by a method known per se, for example, one produced by a chemical synthesis method or an enzymatic method can be used.
  • a chemical synthesis method any method can be used as long as (2S, 4R) monatin can be produced.
  • the methods described in US Pat. No. 7,064,219 and Patent Document 4 are used.
  • an enzymatic method any method can be used as long as (2S, 4R) monatin can be produced.
  • (2R) IHOG may be obtained enzymatically, oximated and reduced, and (2S, 4R) monatin obtained by column purification and crystallization.
  • the (2S, 4R) monatin that is a material in the method of the present invention only needs to contain at least a small amount of (2S, 4R) monatin, and is in a crystalline state, a powder state, a solution state (aqueous solution, organic solution, Any of water / organic solvent mixed solution) may be used.
  • a reaction solution containing (2S, 4R) monatin [a reaction solution used to produce (2S, 4R) monatin] is obtained as (2S , 4R)
  • An aqueous solution containing monatin may be used in the method of the present invention.
  • the (2S, 4R) monatin may also be provided in the aqueous solution used in the method of the present invention in the form of a salt, hydrate, or salt hydrate.
  • the salt include salts of monovalent metals (eg, potassium, sodium) and nonmetals (eg, ammonium), and salts of polyvalent metals (eg, divalent metals, trivalent metals described later).
  • Examples of hydrates include monohydrate, dihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, and nine water Japanese products are listed.
  • aldehyde used in the present invention will be described in detail.
  • examples of the aldehyde include aliphatic aldehydes and aromatic aldehydes, and aromatic aldehydes are preferable.
  • aliphatic aldehydes include those having 1 to 7 carbon atoms such as formaldehyde, acetaldehyde, propionaldehyde, n-butyraldehyde, 1-butyraldehyde, n-barrel aldehyde, capronaldehyde, n-heptylaldehyde, acrolein, methacrolein and the like. Saturated or unsaturated aldehydes can be used.
  • aromatic aldehydes examples include benzaldehyde, salicylaldehyde, m-hydroxybenzaldehyde, p-hydroxybenzaldehyde, o-nitrobenzaldehyde, p-nitrobenzaldehyde, 5-nitrosalicylaldehyde, 3,5-dichlorosalicylaldehyde, anisaldehyde, o-vanillin, vanillin, furfural, pyridoxal, 5-phosphate pyridoxal, and the like can be used.
  • aromatic aldehyde, pyridoxal, 5-nitrosalicylaldehyde, and 3,5-dichlorosalicylaldehyde are particularly preferable.
  • Aldehydes can be used in the range of 0.01 to 1 molar equivalent, more preferably 0.05 to 0.5 molar equivalent, relative to monatin present in the system.
  • an enzyme capable of producing (2R, 4R) monatin from (2S, 4R) monatin used in the present invention will be described in detail.
  • Examples of the enzyme capable of producing (2R, 4R) monatin from (2S, 4R) monatin include racemase, aminotransferase, amino acid dehydrogenase and the like, and racemase and aminotransferase are preferable. These may be used alone or in combination of two or more.
  • the racemase used in the present invention will be described in detail (FIG. 1).
  • the racemase is not particularly limited as long as it is an enzyme having an activity of converting (2S, 4R) monatin to (2R, 4R) monatin.
  • An enzyme having such activity is sometimes called epimerase.
  • racemase as long as the enzyme of another name also has such activity, it is called racemase and is included in the scope of the present invention.
  • Those skilled in the art can appropriately obtain a racemase having such activity. For example, those skilled in the art can obtain a racemase having such activity by using a predetermined screening method.
  • Examples of such screening methods include 20 mM (2S, 4R) monatin [or (2R, 4R) monatin], 100 mM Tris-HCl (pH 8.0), 50 ⁇ M pyridoxal phosphate, enzyme solution (purified enzyme, crude enzyme). Etc.) is allowed to react at 30 ° C. for 16 hours.
  • the protein specified by the amino acid sequence of SEQ ID NO: 2 is a racemase obtained by such a screening method.
  • a protein containing an amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2 and having an activity of converting (2S, 4R) monatin to (2R, 4R) monatin is used.
  • the amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2 for example, 70% or more, preferably 80% or more, more preferably 85% or more, relative to the amino acid sequence of SEQ ID NO: 2, Even more preferred is an amino acid sequence showing 90% or more, particularly preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity.
  • Racemase also includes (a) a protein comprising the amino acid sequence of SEQ ID NO: 2, (b) mutation of one or several amino acid residues (eg, substitution, deletion, addition or insertion) in the amino acid sequence of SEQ ID NO: 2. And a protein having an amino acid sequence and having an activity of converting (2S, 4R) monatin to (2R, 4R) monatin.
  • the mutation of one or several amino acid residues may be introduced into one region in the amino acid sequence, but may be introduced into a plurality of different regions.
  • the term “one or several” indicates a range that does not significantly impair the three-dimensional structure and activity of the protein.
  • the number indicated by the term “one or several” in the case of protein is, for example, 1 to 150, preferably 1 to 100, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5.
  • Such a mutation may be caused by a naturally occurring mutation (mutant or variant) based on individual differences, species differences, and the like of organisms (eg, microorganisms, animals) carrying a gene encoding racemase.
  • the position of the amino acid residue to be mutated in the amino acid sequence is obvious to those skilled in the art. Specifically, a person skilled in the art compares 1) the amino acid sequences of a plurality of proteins having the same type of activity (eg, the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of another racemase), and 2) is relatively conserved. Region, and relatively unconserved region, then 3) from the relatively conserved region and the relatively unconserved region, respectively, play an important role in function Since it is possible to predict a region that cannot play an important role in the region and function to be obtained, the correlation between structure and function can be recognized. Therefore, those skilled in the art can specify the position of the amino acid residue to be mutated in the racemase amino acid sequence.
  • amino acid residue substitution may be a conservative substitution.
  • conservative substitution refers to substitution of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art.
  • such families include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having uncharged polar side chains (Eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chain Amino acids (eg, threonine, valine, isoleucine), amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine), amino acids having side groups containing hydroxyl groups (eg, alcoholic, phenolic) ( Example, serine, thread Nin, tyrosine), and amino acids (e.g.
  • the conservative substitution of amino acids is a substitution between aspartic acid and glutamic acid, a substitution between arginine and lysine and histidine, a substitution between tryptophan and phenylalanine, and between phenylalanine and valine.
  • substitution, leucine, isoleucine and alanine substitution, and glycine and alanine substitution are substitutions.
  • Racemase is encoded by DNA that hybridizes under stringent conditions with a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and converts (2S, 4R) monatin to (2R, 4R) monatin.
  • a protein having activity is preferred.
  • “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. From the viewpoint that the polynucleotides having the same identity are hybridized and the polynucleotides having lower identity are not hybridized, the identity between the polynucleotides is preferably 70% or more, 80% The above is more preferable, 90% or more is further preferable, 95% or more is particularly preferable, and 98% or more is particularly preferable.
  • such conditions include hybridization at about 45 ° C. in 6 ⁇ SSC (sodium chloride / sodium citrate), followed by 50 ⁇ 0.2 ⁇ SSC in 0.1% SDS. One or more washings at ⁇ 65 ° C. may be mentioned.
  • SSC sodium chloride / sodium citrate
  • the racemase used in the present invention may have a purification tag attached to its N-terminal part or C-terminal part.
  • a purification tag attached to its N-terminal part or C-terminal part.
  • the purification tag include histidine (His) tag, calmodulin-binding peptide (CBP), strept-tag (Strep-tag) II, and FLAG.
  • Racemase can be obtained from racemase-producing bacteria or by cell-free synthesis.
  • racemase-producing bacteria include bacteria that naturally produce racemase and transformants that express racemase.
  • Racemase can be used as a purified enzyme, a crude enzyme, or an immobilized enzyme.
  • this transformant can be prepared by preparing an expression vector for racemase and then introducing the expression vector into a host.
  • the host for expressing racemase include Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria (eg, Corynebacterium glutamicum), and Bacillus bacteria (eg, Various prokaryotic cells such as Bacillus subtilis, Saccharomyces cerevisiae (eg, Saccharomyces cerevisiae), Pichia bacteria (eg, Pichia stipitis) (E.g., Aspergillus oryzae) It can be used cells.
  • Examples of preferred hosts are E. coli. E. coli. As a host, E. coli. When using E. coli, it is more preferable to use an expression vector into which a polynucleotide encoded by the DNA sequence of SEQ ID NO: 1 has been inserted.
  • Racemase is preferably 0.001 to 1000 U / ml, more preferably 0.1 to 100 U / ml with respect to monatin present in the reaction system (1 U is an activity to isomerize 1 ⁇ mol of monatin per minute. Show).
  • the pH of the reaction system in the isomerization reaction is preferably pH 5.0 to 11.0, more preferably pH 6.0 to 10.0, and still more preferably pH 7.0 to 9.0.
  • the concentration of (2S, 4R) monatin [or (2R, 4R) monatin] added to the reaction system is preferably 10 mM to 3.0 M, more preferably 100 mM to 1.0 M.
  • monatin may be added to the reaction system before starting the reaction, but may be added intermittently or continuously after starting the reaction.
  • the reaction temperature is not particularly limited as long as the isomerization reaction proceeds, but is preferably 15 ° C to 60 ° C, and more preferably 25 ° C to 42 ° C.
  • the reaction time is preferably 1 to 120 hours, more preferably 1 to 24 hours.
  • Pyridoxal phosphate (PLP) is not necessarily added to the reaction system, but the concentration of PLP when added is not particularly limited as long as the isomerization reaction proceeds, but is preferably 10 to 100 ⁇ M.
  • a salt such as NaCl or KCl may be added to stabilize the racemase in a liquid such as a racemase reaction solution or a buffer used for purification or dialysis of the racemase.
  • a salt such as NaCl or KCl
  • the (2R, 4R) monatin concentration in the reaction solution is preferably 20 mM to 3M, and more preferably 50 mM to 1M.
  • the magnesium source added to the reaction system is preferably 10 mM to 3M, more preferably 25 mM to 1M.
  • the temperature is preferably 0 to 60 ° C, more preferably 10 to 40 ° C.
  • the time is preferably 3 hours to 1 week, more preferably 24 hours to 60 hours.
  • the centrifugal supernatant of the reaction solution or the ultrafiltered filtrate may be used, or the amount of seed crystals to be added may be increased.
  • aminotransferase used in the present invention there are both conversion activity from 4R-IHOG to (2S, 4R) monatin (2S stereoselective activity) and conversion activity from 4R-IHOG to (2R, 4R) monatin (2R stereoselective activity). What you have can be used.
  • the conversion activity of the aminotransferase can be reversible. Enzymes having both activities may be used without purification. Moreover, you may use together, after refine
  • the aminotransferase will be further described with reference to FIGS.
  • the aminotransferase may convert (2S, 4R) monatin to (2R, 4R) monatin via 4R-IHOG.
  • the first aminotransferase converts (2S, 4R) monatin to 4R-IHOG
  • the second aminotransferase converts 4R-IHOG to (2R, 4R) monatin. May be.
  • aminotransferase Those skilled in the art can appropriately obtain such aminotransferase. It is known that wild-type or mutant aminotransferases can isomerize a given compound by a reversible aminotransferase reaction via other intermediate compounds. In such cases, the aminotransferase has apparent racemase activity. For example, aspartate aminotransferase is known to be able to acquire apparent racemase activity (see, for example, Kochhar, Sunil et al., European Journal of Biochemistry (1992), 203 (3), 563-9). Therefore, in the present invention, an aminotransferase capable of acting on (2S, 4R) monatin and exhibiting apparent racemase activity can be used. The amount of aminotransferase added to the reaction system is not particularly limited as long as the reaction can proceed appropriately.
  • aminotransferases having activity to convert 4R-IHOG to (2S, 4R) monatin (2S stereoselective activity) include L-amino acid aminotransferase and L-aspartate aminotransferase. . Specific examples include the enzymes described in International Publication 2012/050125.
  • aminotransferase second aminotransferase having the activity of converting 4R-IHOG to (2R, 4R) monatin (2R stereoselective activity
  • D-amino acid aminotransferase examples include the enzymes described in International Publication No. 03/056026, International Publication No.
  • first aminotransferase is an L-amino acid aminotransferase and the second aminotransferase is a D-amino acid aminotransferase (FIG. 4).
  • the amount of the first and second aminotransferases added to the reaction system is not particularly limited as long as the reaction can proceed appropriately.
  • a protein containing an amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4 and having an activity of converting 4R-IHOG to (2S, 4R) monatin is used. be able to.
  • the amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4 for example, 70% or more, preferably 80% or more, more preferably 85% or more, relative to the amino acid sequence of SEQ ID NO: 4, Even more preferred is an amino acid sequence showing 90% or more, particularly preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity.
  • a protein containing an amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 6 and having an activity of converting 4R-IHOG to (2R, 4R) monatin is used. be able to.
  • amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 6 for example, 70% or more, preferably 80% or more, more preferably 85% or more, relative to the amino acid sequence of SEQ ID NO: 6, Even more preferred is an amino acid sequence showing 90% or more, particularly preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity.
  • Amino acid sequence identity can be determined by the method described above.
  • L-amino acid aminotransferase is also (a ′) a protein comprising the amino acid sequence of SEQ ID NO: 4, (b ′) mutation of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 4 (eg, substitution, deletion) , Addition or insertion), and a protein having an activity of converting 4R-IHOG to (2S, 4R) monatin.
  • D-amino acid aminotransferase is also (a) a protein comprising the amino acid sequence of SEQ ID NO: 6, (b) mutation of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 6 (eg, substitution, deletion, addition) Or a protein having an activity of converting 4R-IHOG into (2R, 4R) monatin.
  • the mutation of one or several amino acid residues may be introduced into one region in the amino acid sequence, but may be introduced into a plurality of different regions.
  • the term “one or several” is similar to that described above. As described above, the position of the amino acid residue to be mutated in the amino acid sequence is obvious to those skilled in the art. In addition, when an amino acid residue is mutated by substitution, the substitution of the amino acid residue may be a conservative substitution as described above.
  • L-amino acid aminotransferase is encoded by DNA that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 and converts 4R-IHOG to (2S, 4R) monatin.
  • a protein having the activity of This activity of L-amino acid aminotransferase can be reversible.
  • D-amino acid aminotransferase is encoded by DNA that hybridizes with a base sequence complementary to the base sequence represented by SEQ ID NO: 5 under stringent conditions, and converts 4R-IHOG to (2R, 4R) monatin.
  • a protein having the activity of This activity of D-amino acid aminotransferase can be reversible. Stringent conditions are as described above.
  • the aminotransferase used in the present invention may have a purification tag added to the N-terminal part or C-terminal part thereof.
  • the purification tag include histidine (His) tag, calmodulin-binding peptide (CBP), strept-tag (Strep-tag) II, and FLAG.
  • L-amino acid aminotransferase and D-amino acid aminotransferase When both L-amino acid aminotransferase and D-amino acid aminotransferase are used, a small amount of keto acid (or L-amino acid or D-amino acid) and racemase that can convert L-amino acid to D-amino acid are added to the reaction system. By doing so, a side reaction by L-amino acid aminotransferase (keto acid ⁇ L-amino acid) and a side reaction by D-amino acid aminotransferase (D-amino acid ⁇ keto acid) can be coupled (FIG. 4).
  • keto acid or L-amino acid or D-amino acid
  • racemase that can convert L-amino acid to D-amino acid
  • amino acid in the L-amino acid or D-amino acid
  • examples of the “amino acid” in the L-amino acid or D-amino acid include natural ⁇ -amino acids such as alanine, glutamic acid, asparagine, cysteine, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, Examples include tyrosine, valine, aspartic acid, arginine, histidine, and lysine.
  • Keto acid is a keto acid produced from the above-mentioned L-amino acid or D-amino acid by the action of amino acid aminotransferase.
  • racemases that can convert L-amino acids to D-amino acids are known, and such racemases can be used in the present invention.
  • the keto acid is pyruvate
  • the L-amino acid is L-alamine
  • the D-amino acid is D-alanine (FIG. 5).
  • glutamic acid racemase is used as the racemase
  • the keto acid is ⁇ -ketoglutaric acid
  • the L-amino acid is L-glutamic acid
  • D-amino acid is D-glutamic acid (FIG. 6).
  • the concentration of keto acid eg, pyruvic acid, ⁇ -ketoglutaric acid
  • concentration of L-amino acid eg, L-alanine, L-glutamic acid
  • D-amino acid D-alanine, D-glutamic acid
  • concentration and the amount of racemase are not particularly limited as long as the reaction can proceed appropriately.
  • the concentration of (2S, 4R) monatin added to the reaction system is preferably 10 mM to 3.0M, more preferably 300 mM to 1.0M. In this case, monatin may be added to the reaction system before starting the reaction, but may be added intermittently or continuously after starting the reaction.
  • the reaction pH is preferably from 5.0 to 11.0, more preferably from 6.0 to 10.0, and even more preferably from 7.0 to 9.0.
  • the reaction temperature is not particularly limited as long as the aminotransferase reaction proceeds, but is preferably 10 ° C to 60 ° C, more preferably 15 ° C to 42 ° C.
  • the reaction time is not particularly limited, but is preferably 1 to 180 hours, and more preferably 1 to 76 hours.
  • Pyridoxal phosphate (PLP) is not necessarily added to the reaction system, but when added, the concentration of PLP is not particularly limited as long as the aminotransferase reaction proceeds, but is preferably 10 to 100 ⁇ M.
  • the aminotransferase both L-amino acid aminotransferase and D-amino acid aminotransferase may be used.
  • Each concentration in this case is not particularly limited as long as the reaction proceeds, but is preferably 0.01 to 10 mg / ml, and more preferably 0.1 to 5 mg / ml.
  • a small amount of keto acid (or L-amino acid or D-amino acid) and racemase capable of converting L-amino acid to D-amino acid may be added to the reaction system.
  • the keto acid concentration is preferably 1 to 100 mM, more preferably 10 to 50 mM.
  • the concentration of D-amino acid is preferably 1 to 100 mM, more preferably 20 to 50 mM.
  • the concentration of racemase is not particularly limited as long as the reaction proceeds, but it is preferably 0.1 to 100 ⁇ g / ml, more preferably 1 to 10 ⁇ g / ml.
  • the (2R, 4R) monatin concentration in the reaction solution is preferably 20 mM to 3M, and more preferably 50 mM to 1M.
  • the magnesium source added to the reaction system may be an amount sufficient for the monatin present in the system, but is preferably 10 mM to 1.5 M, and more preferably 25 mM to 500 mM.
  • the temperature is preferably 0 to 60 ° C, more preferably 10 to 40 ° C.
  • the time is preferably 3 hours to 1 week, more preferably 24 hours to 60 hours.
  • the centrifugal supernatant of the reaction solution or the ultrafiltered filtrate may be used, or the amount of seed crystals to be added may be increased.
  • (2R, 4R) monatin when (2R, 4R) monatin is produced from (2S, 4R) monatin by the action of an aldehyde catalyst, the isomerization reaction and (2R, 4R) monatin polyvalent metal salt crystallization are simultaneously advanced.
  • (2R, 4R) monatin can be removed from the reaction system as a salt crystal, so that the equilibrium state is inclined in the direction of (2R, 4R) monatin and the amount of (2R, 4R) monatin produced can be increased. it can.
  • the concentration of (2S, 4R) monatin added to the reaction system is preferably 50 mM to 3M, more preferably 100 mM to 1M. In this case, monatin may be added to the reaction system before starting the reaction, but may be added intermittently or continuously after starting the reaction.
  • the reaction pH is preferably pH 5.0 to 10.0, and more preferably pH 6.0 to 7.0 in order to suppress reverse aldol degradation.
  • the reaction temperature is not particularly limited as long as the aminotransferase reaction proceeds, but is preferably 10 ° C to 60 ° C, more preferably 15 ° C to 42 ° C.
  • the reaction time is not particularly limited, but is preferably 10 to 240 hours, more preferably 120 to 240 hours.
  • Pyridoxal phosphate (PLP) is not necessarily added to the reaction system, but when added, the concentration of PLP is not particularly limited as long as the aminotransferase reaction proceeds, but is preferably 10 to 100 ⁇ M.
  • PLP Pyridoxal phosphate
  • As the aminotransferase both L-amino acid aminotransferase and D-amino acid aminotransferase may be used.
  • Each concentration in this case is not particularly limited as long as the reaction proceeds, but is preferably 0.01 to 10 mg / ml, and more preferably 0.1 to 10 mg / ml.
  • a small amount of keto acid (or L-amino acid or D-amino acid) and racemase capable of converting L-amino acid to D-amino acid may be added to the reaction system.
  • the keto acid concentration is preferably 1 to 100 mM, more preferably 10 to 50 mM.
  • the concentration of D-amino acid is preferably 1 to 100 mM, more preferably 20 to 50 mM.
  • the concentration of racemase is not particularly limited as long as the reaction proceeds, but it is preferably 0.1 to 100 ⁇ g / ml, more preferably 1 to 10 ⁇ g / ml.
  • aminotransferase and racemase may be added to the reaction system before starting the reaction, but may be added intermittently or continuously after starting the reaction in order to compensate for inactivation or inhibition during the reaction.
  • the magnesium source added to the reaction system may be a sufficient amount with respect to monatin present in the system, but is preferably 25 mM to 1.5 M, and more preferably 50 to 500 mM.
  • the magnesium source may be added to the reaction system before the start of the reaction, but may be added intermittently or continuously after the start of the reaction in order to avoid deactivation or inhibition of the enzyme.
  • the enzyme source may be isolated or immobilized by a dialysis membrane or the like.
  • an inert gas such as argon gas or nitrogen gas may be blown into the reaction solution.
  • the amount of seed crystals may be increased.
  • (2R, 4R) monatin When (2R, 4R) monatin is produced from (2S, 4R) monatin by enzymatic isomerization reaction, a certain equilibrium state is reached in aqueous solution only by enzymatic reaction, but in this production method, enzymatic isomerization is achieved.
  • the (2R, 4R) monatin polyvalent metal salt By simultaneously proceeding the reaction and preferential crystallization of (2R, 4R) monatin polyvalent metal salt with polyvalent metal ions, the (2R, 4R) monatin polyvalent metal salt is converted into a reaction system before the isomerization reaction reaches equilibrium.
  • the amount of (2R, 4R) monatin polyvalent metal salt that can be removed outside and can be produced by enzymatic isomerization reaction can also be increased.
  • Aminotransferase can be obtained from, for example, an aminotransferase-producing bacterium or by synthesis in a cell-free system.
  • Examples of the aminotransferase-producing bacteria include bacteria that naturally produce aminotransferases and transformants that express aminotransferases.
  • the aminotransferase can be used as a purified enzyme, a crude enzyme, or an immobilized enzyme.
  • this transformant When a transformant that expresses aminotransferase is used as an aminotransferase-producing bacterium, this transformant can be prepared by preparing an expression vector for aminotransferase and then introducing the expression vector into a host.
  • a host for expressing aminotransferase for example, prokaryotic cells and eukaryotic cells as described above can be used. Examples of preferred hosts are E. coli. E. coli.
  • an aqueous solution is used as a solvent, and examples thereof include water and a buffer solution containing no organic solvent, and water and a buffer solution containing a small amount of an organic solvent.
  • the buffer solution include Tris buffer solution, phosphate buffer solution, carbonate buffer solution, borate buffer solution, and acetate buffer solution.
  • the organic solvent an organic solvent miscible with water is used, but alcohols such as methanol, ethanol, propanol, and isopropanol are particularly preferable. Two or more different organic solvents may be mixed and used.
  • the proportion of the organic solvent in the aqueous solution is preferably 10% by volume or less, more preferably 5% by volume or less, and still more preferably 2% by volume or less.
  • the polyvalent metal ion used in the present invention is not particularly limited, but can be generated by adding a polyvalent metal salt to an aqueous solution.
  • the polyvalent metal salt added to the reaction system for the production of the polyvalent metal ion is an element having a valence of 2 or more in the periodic table as long as it can form a salt with monatin.
  • the divalent metal salt include alkaline earth metal salts such as magnesium, calcium, strontium and barium; transition metal salts such as iron, nickel, copper and zinc.
  • metal salts, such as aluminum, are mentioned. These may be used alone or in combination of two or more.
  • divalent metal salts are preferable, alkaline earth metal salts are more preferable, magnesium salts, calcium salts, strontium salts, and barium salts are more preferable, magnesium salts, calcium salts, and barium salts are still more preferable, magnesium salts, calcium A salt is particularly preferred.
  • Polyvalent metal salts can be non-hydrates, even hydrates (eg, monohydrate, dihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate , Heptahydrate, octahydrate, nonahydrate).
  • Examples of a simple method for obtaining the polyvalent metal salt used in the present invention include inorganic polyvalent metal compounds such as calcium hydroxide, magnesium hydroxide, calcium carbonate, magnesium carbonate, and calcium acetate, magnesium acetate, oxalic acid.
  • Examples of the method include treating organic polyvalent metal compounds such as calcium, magnesium oxalate, calcium lactate, and magnesium lactate by various methods such as neutralization and salt exchange.
  • the polyvalent metal ion may be a sufficient amount with respect to the monatin present in the system, and is, for example, in the range of 0.4 to 0.6 molar equivalent, more preferably 0.45 to 0.55 molar equivalent. Can be used.
  • the temperature in the method of the present invention is preferably 0 to 50 ° C, more preferably 25 to 40 ° C.
  • the time in the method of the present invention is preferably 5 hours to 1 week, more preferably 10 hours to 48 hours.
  • the pH in the method of the present invention is preferably 4 to 10, more preferably 5 to 9, and still more preferably 6 to 8. Adjustment of pH can be performed using an acid and an alkali.
  • the acid used is not particularly limited, and an organic acid such as acetic acid or an inorganic acid such as hydrochloric acid or sulfuric acid can be used.
  • the alkali is not particularly limited, and alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and organic bases such as ammonia and amines can be used.
  • Precipitation of (2R, 4R) monatin polyvalent metal salt crystals can be promoted by adding an organic solvent to the aqueous solution after the (2R, 4R) monatin is produced.
  • the addition of the organic solvent is also preferably performed after the (2R, 4R) monatin dissolved in the aqueous solution reaches the saturation amount.
  • the organic solvent added to the aqueous solution to promote the precipitation of the (2R, 4R) monatin polyvalent metal salt crystal is not particularly limited as long as it is an organic solvent miscible with water.
  • methanol, ethanol, n- examples include propanol, isopropanol, n-butanol, t-butanol, sec-butanol, propylene glycol, acetonitrile, and THF.
  • (2R, 4R) monatin polyvalent metal salt crystals can be obtained.
  • ((2R, 4R) monatin) 2 divalent metal salt crystal is preferable and ((2R, 4R) monatin) 2 alkaline earth metal salt crystal is preferable from the viewpoint that human intake is allowed and it is easy to prepare.
  • ((2R, 4R) monatin) 2 magnesium salt crystals, ((2R, 4R) monatin) 2 calcium salt crystal, ((2R, 4R) monatin) 2 strontium salt crystals, ((2R, 4R) monatin) 2 barium salt crystals are more preferred, ((2R, 4R) monatin) 2 magnesium salt crystals, ((2R, 4R) monatin) 2 calcium salt crystals, ((2R, 4R) monatin) 2 barium salt crystals are even more preferred. , ((2R, 4R) monatin) 2 magnesium salt crystals, ((2R, 4R) monatin) 2 calcium salt crystals are particularly Masui.
  • Crystal precipitation can be obtained by subjecting an aqueous solution containing (2R, 4R) monatin and a magnesium source to standing or stirring crystallization by the method described above.
  • concentration of the (2R, 4R) monatin crystal in the solvent is not particularly limited as long as supersaturation is applied and crystals are precipitated, but 0.1 wt% to 60 wt% is preferable. From the viewpoint of obtaining a viscosity of a solution suitable for production, it is more preferably 1 wt% to 50 wt%, and further preferably 5 wt% to 45 wt%.
  • the melting temperature is not particularly limited as long as the crystals continue to dissolve, but is preferably 15 to 40 ° C.
  • Precipitated crystals can be easily obtained wet crystals by subjecting them to a separation step such as a filtration step.
  • the crystal washing is not particularly limited as long as crystal solvent exchange does not occur, but water can be used.
  • the crystal solvent exchange does not occur, it is miscible with water such as methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, sec-butanol, propylene glycol, acetonitrile, THF, acetone, DMF, etc. It may also contain a possible solvent or inorganic salt.
  • the wet crystal thus obtained can be converted into a dry crystal by subjecting it to a known drying step.
  • the drying equipment used in the drying process is not particularly limited, and a temperature range in which (2R, 4R) monatin does not dissolve the 2 magnesium salt can be used, and vacuum drying, airflow drying, hot air drying, etc. can be used. .
  • the (2R, 4R) monatin polyvalent metal salt crystal obtained by the method of the present invention is obtained by known separation and purification means, for example, concentration, concentration under reduced pressure, solvent extraction, crystallization, recrystallization, transfer dissolution, activated carbon treatment, ion exchange. Further purification can be achieved by combining treatments such as chromatography using a resin or a synthetic adsorption resin as required.
  • the (2R, 4R) monatin polyvalent metal salt crystals obtained by the method of the present invention can be converted to other salts such as potassium salts, sodium salts, calcium salts, and other salts or free forms such as ammonium salts. .
  • a method for converting the (2R, 4R) monatin polyvalent metal salt into a free form or other salts methods known to those skilled in the art can be used.
  • 2R, 4R) monatin polyvalent metal salt is dissolved or suspended in water, alcohol, or a mixed solvent thereof, and neutralized with an acid such as hydrochloric acid or sulfuric acid.
  • the free crystals obtained above are dissolved in an aqueous alkali metal solution such as sodium hydroxide, potassium hydroxide or calcium hydroxide, an aqueous ammonia solution or the like, and the solvent is distilled off under reduced pressure ( 2R, 4R) Method of crystallizing monatin salt, or adding an aqueous solution of alkali metal such as sodium hydroxide, potassium hydroxide or calcium hydroxide, or aqueous ammonia solution to the resin eluent containing educts, and distilling the solvent under reduced pressure Or a method of crystallizing (2R, 4R) monatin salt.
  • an aqueous alkali metal solution such as sodium hydroxide, potassium hydroxide or calcium hydroxide, an aqueous ammonia solution or the like
  • various additives that can be used as oral products such as beverages, foods, pharmaceuticals, quasi drugs, and feeds can be used to the extent that the effects of the present invention are not impaired.
  • the monatin polyvalent metal salt crystal of the present invention can be used for oral products such as beverages, foods, pharmaceuticals, quasi drugs, and feeds.
  • the dosage form is not particularly limited, and examples thereof include powder, granules, cubes, pastes, and liquids.
  • the water content and magnesium content of the obtained crystals [((2R, 4R) monatin) 2 magnesium salt] were analyzed by a moisture measurement method and a cation analysis method by ion chromatography. Details of the water measurement method and the cation analysis method performed are shown below.
  • the mother liquor was cooled to 10 ° C. and stirred for 5 hours to crystallize (2S, 4R) monatin potassium salt dihydrate.
  • the precipitated crystals were separated by filtration (wet crystals 32.74 g) and dried under reduced pressure to obtain 9.88 g (15.68 mmol) of the desired (2S, 4R) monatin potassium salt dihydrate (HPLC) Purity: 55.5%).
  • the solubility was higher than that of (2S, 4R) monatin potassium salt.
  • the solubility of (2S, 4R) monatin magnesium salt and (2R, 4R) monatin magnesium salt in water (H 2 O) was measured.
  • the solubility was measured in the same manner as described above.
  • For the (2S, 4R) monatin magnesium salt first, an equal amount of magnesium chloride was added to the (2S, 4R) monatin potassium salt, and then ethanol was further added. Next, the obtained slurry was filtered, and the wet crystals were dried under reduced pressure to prepare (2S, 4R) monatin magnesium salt.
  • (2R, 4R) monatin magnesium salt For the (2R, 4R) monatin magnesium salt, first, an equal amount of magnesium chloride was added to the (2R, 4R) monatin potassium salt, and then methanol was further added. Next, the obtained slurry was filtered, and the wet crystals were dried under reduced pressure to prepare (2R, 4R) monatin magnesium salt. As a result, the solubility of (2S, 4R) monatin magnesium salt and (2R, 4R) monatin magnesium salt was 14.5 wt% and 1.6 wt%, respectively. The solubility was significantly lower than that of the (2S, 4R) monatin magnesium salt.
  • Example 1 Synthesis of (2R, 4R) monatin 1.25 g (3.42 mmol) of (2S, 4R) monatin potassium salt dihydrate was dissolved in 5 mL of water, and 0.05216 g (0.0428 mmol) of salicylaldehyde was dissolved. And 0.3476 g (1.71 mmol) of magnesium chloride hexahydrate were added. After 42 hours at 65 ° C., seed crystals [((2R, 4R) monatin) 2 magnesium salt crystals having the same crystal form as that obtained in Reference Example 3] were added, and the mixture was further stirred for 191 hours.
  • the obtained slurry solution was cooled to room temperature, filtered, washed with 1 g of water, and then wet crystals were dried under reduced pressure at 40 ° C. to obtain 0.446 g of (2R, 4R) monatin.
  • the obtained crystals, mother liquor, and washings were analyzed by HPLC to carry out yield and quality analysis. From the intrinsic X-ray diffraction peak, it was confirmed that ((2R, 4R) monatin) 2 magnesium salt crystals having the same crystal form as that obtained in Reference Example 3 were obtained.
  • Example 2 E.E. Expression of racemase in E. coli (1) Construction of a racemase expression plasmid A DNA sequence having an NdeI recognition sequence at the 5 ′ end of the DNA sequence of racemase Rac39 and an XhoI recognition sequence at the 3 ′ end, and OptiGene Codon Optimization Analysis of GenScript. E. A synthetic DNA (SEQ ID NO: 1) with optimized gene expression efficiency in E. coli was designed and synthesized.
  • the synthetic DNA was subjected to restriction enzyme treatment with NdeI and XhoI, and ligated with pET-22b (Novagen) similarly treated with NdeI and XhoI. With this ligation solution E. coli was transformed and the target plasmid was extracted from the ampicillin resistant strain. These plasmids were named pET-22-Rac39-His. In these plasmids, racemase (Rac39-His) with His-tag added at the C-terminus is expressed.
  • E.E. Purification of His-tagged racemase from E. coli expression strain The constructed expression plasmid pET-22-Rac39-His was transformed into E. coli. E. coli BL21 (DE3) was introduced, and the transformant was inoculated into 100 ml of Overnight Express Instant Medium (Novagen) containing 100 mg / l of ampicillin, and shaken for 16 hours using a 500 ml Sakaguchi flask. The shaking temperature was 25 ° C.
  • bacterial cells were collected from 200 ml of the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl, and 10 mM imidazole, and subjected to ultrasonic disruption. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
  • the obtained soluble fraction was subjected to His-tag protein purification column His TALON superflow 5 ml Cartridge (Clontech) equilibrated with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, and Imidazole 10 mM, and was adsorbed on the carrier.
  • Example 3 Isomerization reaction using (2S, 4R) monatin or (2R, 4R) monatin as a substrate
  • a purified racemase was used for 15 minutes under the following conditions. The reaction was performed in 0.1 ml using an Eppendorf tube. After completion of the reaction, an equal amount of a reaction stop solution 200 mM Na citrate solution (pH 4.5) was added to the sample. HPLC was used for the analysis.
  • Example 4 E.I. Expression of amino acid aminotransferase in E. coli (1) Construction of L-amino acid aminotransferase expression plasmid NdeI at the 5 ′ end of the DNA sequence (SEQ ID NO: 3) of L-amino acid aminotransferase (AJ1616LAT) obtained from the conserved strain AJ1616 Bacillus altitudinis The recognition sequence was given a XhoI recognition sequence at the 3 ′ end. This DNA sequence was subjected to restriction enzyme treatment with NdeI and XhoI, and ligated with pET-22b (Novagen) similarly treated with NdeI and XhoI. With this ligation solution E.
  • SEQ ID NO: 3 L-amino acid aminotransferase expression plasmid NdeI at the 5 ′ end of the DNA sequence (SEQ ID NO: 3) of L-amino acid aminotransferase (AJ1616LAT) obtained from the conserved strain AJ16
  • LAT-His L-amino acid aminotransferase
  • D-amino acid aminotransferase expression plasmid DNA obtained by adding NdeI recognition sequence to the 5 'end of the DNA sequence of D-amino acid aminotransferase selected from in silico and XhoI recognition sequence to the 3' end was obtained from GenScript. For OptimumGene Codon Optimization Analysis. A synthetic DNA (SEQ ID NO: 5) with optimized gene expression efficiency in E. coli was obtained. This synthetic DNA was treated with restriction enzymes with NdeI and XhoI, and ligated with pET-22b (Novagen) similarly treated with NdeI and XhoI. With this ligation solution E. coli was transformed and the target plasmid was extracted from the ampicillin resistant strain. This plasmid was named pET-22-DAT-His. This plasmid expresses D-amino acid aminotransferase (DAT-His) with His-tag added at the C-terminus.
  • DAT-His D-amin
  • E.E. Purification of His-tagged L-amino acid aminotransferase from E. coli expression strain The constructed expression plasmid pET-22-LAT-His was transformed into E. coli.
  • E. coli BL21 (DE3) was introduced, and the transformant was inoculated into 160 ml of Overnight Express Instant Medium (Novagen) containing ampicillin 100 mg / l and shaken for 16 hours using a 500 ml Sakaguchi flask. The shaking temperature was 37 ° C.
  • E.E. Purification of His-tagged D-amino acid aminotransferase from E. coli expression strain The constructed expression plasmid pET-22-DAT-His was obtained from E. coli.
  • E. coli BL21 (DE3) was introduced, and the transformant was inoculated into 160 ml of Overnight Express Instant Medium (Novagen) containing ampicillin 100 mg / l and shaken for 16 hours using a 500 ml Sakaguchi flask. The shaking temperature was 30 ° C.
  • Example 5 (2S, 4R) Monatin Production Reaction of (2R, 4R) Monatin Using purified L-amino acid aminotransferase and D-amino acid aminotransferase, the reaction was performed for 76 hours under the following conditions. The reaction was carried out at 20 ml using a 50 ml Falcon tube with stirring. 990 ⁇ l of TE buffer was added to 10 ⁇ l of sample, ultrafiltered using Amicon Ultra 0.5 ml 10k (Millipore), and the filtrate was analyzed. HPLC was used for the analysis.
  • (2S, 4R) monatin 300 mM, pyruvate 10 mM, D-Ala 20 mM, PLP 50 ⁇ M, L-amino acid aminotransferase solution 3 mg / ml, D-amino acid aminotransferase solution 3 mg / ml, alanine racemase solution (Unitika) 0 0.002 mg / ml, pH 7 (KOH), 25 ° C.
  • (2S, 4R) monatin 128 mM and (2R, 4R) monatin 178 mM were produced (FIG. 8). Therefore, it was demonstrated that (2R, 4R) monatin can be produced from (2S, 4R) monatin by using aminotransferase and racemase.
  • Example 6 from the reaction mixture ((2R, 4R) monatin) were dissolved the reaction solution of magnesium sulfate heptahydrate 150mM according to obtain Example 5 2 magnesium salt crystals, obtained by centrifugation supernatant Was ultrafiltered using Amicon Ultra 15 ml 10k (Millipore). To the obtained filtrate, 0.001 g of seed crystals [(2R, 4R) monatin) 2 magnesium salt crystals having the same crystal form as that obtained in Reference Example 5] was added, and the mixture was stirred at 25 ° C. for 24 hours. It was carried out at 7.5 ml using a 15 ml falcon tube.
  • the obtained slurry solution was centrifuged, and the obtained crystal was washed with a small amount of water, and then the wet crystal was dried under reduced pressure at 40 ° C. to obtain 0.181 g of monatin (2R, 4R).
  • the obtained crystals, mother liquor, and washings were analyzed by HPLC to carry out yield and quality analysis. As a result, it was confirmed that ((2R, 4R) monatin) 2 magnesium salt crystals were obtained.
  • the HPLC analysis conditions were as described above (HPLC analysis conditions).
  • Example 7 (2R, 4R) monatin production reaction with a ((2R, 4R) monatin) using 2 magnesium salt preferential crystallization synchronized purified L- amino acid aminotransferase and D- amino acids aminotransferase, the following The reaction was performed for 69 hours under the conditions. The reaction was performed in 20 ml using a 50 ml Falcon tube while shaking at 100 rpm. 990 ⁇ l of TE buffer was added to 10 ⁇ l of sample, ultrafiltered using Amicon Ultra 0.5 ml 10k (Millipore), and the filtrate was analyzed. HPLC was used for the analysis. The HPLC analysis conditions were as described in Example 5.
  • (2S, 4R) monatin 400 mM, pyruvate 10 mM, D-Ala 20 mM, PLP 50 ⁇ M, L-amino acid aminotransferase solution 5 mg / ml, D-amino acid aminotransferase solution 5 mg / ml, alanine racemase solution (Unitika) 0 .0015 mg / ml, pH 7 (KOH), 25 ° C.
  • the reaction solution was adjusted to pH 6.5 with sulfuric acid and bubbled with argon gas.
  • L-amino acid aminotransferase solution 3 mg / ml, D-amino acid aminotransferase solution 5 mg / ml, and alanine racemase solution (Unitika) 0.0015 mg / ml were added to 2.8 ml of the reaction solution to make a final volume of 3 ml.
  • the L-amino acid aminotransferase solution and the D-amino acid aminotransferase solution were substituted with Tris-HCl (pH 7.0) 20 mM.
  • (2R, 4R) monatin in the reaction solution was 253 mM (middle in FIG. 10), whereas it was 292 mM when the enzyme reaction and preferential crystallization were performed simultaneously (FIG. 10). 10 bottom).
  • (2R, 4R) monatin in the reaction supernatant was 111 mM (lower part in FIG. 10), and crystals were precipitated. Therefore, it has been demonstrated that the (2R, 4R) monatin production reaction and the ((2R, 4R) monatin) 2- magnesium salt preferential crystallization increase the amount of (2R, 4R) monatin accumulated in the reaction solution. It was done.
  • the obtained slurry solution was centrifuged, and the obtained crystal was washed with a small amount of water, and then the wet crystal was dried under reduced pressure at 40 ° C. to obtain 0.216 g of (2R, 4R) monatin.
  • the obtained crystals, mother liquor, and washings were analyzed by HPLC to carry out yield and quality analysis. As a result, it was confirmed that ((2R, 4R) monatin) 2 magnesium salt crystals were obtained.
  • the HPLC analysis conditions are as described above (HPLC analysis conditions).
  • Example 8 Isomerization reaction from (2S, 4R) monatin to (2R, 4R) monatin using racemase
  • An isomerization reaction using racemase Rac39 prepared in the same manner as in Example 2 was performed on a 50 ml scale.
  • Example 9 (2R, 4R) Monatin Preferential Crystallization from Enzyme Reaction Solution 0.45 g (2.2 mmol) of magnesium chloride hexahydrate was dissolved in 45 ml of the enzyme reaction solution obtained in Example 8, and the pressure was reduced. Concentrated under. The obtained concentrated solution (7.7 g) was filtered through a 0.2 ⁇ filter, and the filtrate was seed crystal [having the same crystal form as the crystal obtained in Reference Example 5 ((2R, 4R) monatin) 2 magnesium salt crystal 2.4 mg was added and the mixture was stirred at 25 ° C. for 24 hours.
  • Example 10 Promotion of preferential crystallization by adding an organic solvent to the reaction solution
  • a 300 ml four-necked flask was purged with argon, and 58.0 g of water, 20.0 g (68.4 mmol) of (2S, 4R) monatin free form. ), 1.58 g (30.7 mmol) of magnesium hydroxide, and 0.850 g (6.96 mmol) of salicylaldehyde, and the mixture was heated and stirred at 65 ° C. for 24 hours.
  • Example 11 Promotion of Preferential Crystallization by Addition of Organic Solvent to Reaction Solution
  • a 1000 ml four-necked flask was purged with argon, water 290.7 g, (2S, 4R) monatin free body 100.0 g (342.1 mmol) ), 9.26 g (154.0 mmol) of magnesium hydroxide, 3.47 g (17.1 mmol) of magnesium chloride hexahydrate, 4.26 g (34.5 mmol) of salicylaldehyde, and heated and stirred at 65 ° C. for 24 hours. did.
  • (2R, 4R) monatin having good sweetness characteristics and excellent storage stability by allowing aldehyde or racemase to act on a solution containing (2S, 4R) monatin in the presence of a polyvalent metal ion.
  • An efficient method for producing a polyvalent metal salt can be provided.
  • it is significant that various foods, various pharmaceuticals, and various products containing (2R, 4R) monatin polyvalent metal salt can be provided.

Abstract

The present invention provides a method for efficiently producing (2R,4R)-monatin polyvalent metal salt crystals which have good sweetness characteristics and a high storage stability. More specifically, the present invention provides a method for producing (2R,4R)-monatin polyvalent metal salt crystals, said method comprising treating a (2S,4R)-monatin-containing aqueous solution in the presence of a polyvalent metal ion with one or more kinds of enzymes capable of synthesizing (2R,4R)-monatin from an aldehyde or (2S,4R)-monatin to give (2R,4R)-monatin polyvalent metal salt crystals. The aldehyde may be preferably an aromatic aldehyde. The one or more kinds of enzymes may be preferably racemase or one or more kinds of aminotransferases. The polyvalent metal may be preferably a divalent alkaline earth metal.

Description

（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法(2R, 4R) Monatin Multivalent Metal Salt Crystal Production Method
　本発明は、（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法に関する。 The present invention relates to a method for producing (2R, 4R) monatin polyvalent metal salt crystals.
　４－ヒドロキシ－４－（３－インドリルメチル）－２－アミノグルタル酸（以下「モナティン」と称することがある）の（２Ｓ，４Ｓ）体は、南アフリカ北部トランスバール（Ｎｏｒｔｈｅｒｎ　Ｔｒａｎｓｖａａｌ）地方に自生する植物シュレロチトン　イリシホリアス（Ｓｃｈｌｅｒｏｃｈｉｔｏｍ　ｉｌｉｃｉｆｏｌｉｕｓ）の根皮に含まれ、ショ糖の数百倍の甘味を有し、甘味料として有用なアミノ酸誘導体であることが知られている（特許文献１）。 The (2S, 4S) form of 4-hydroxy-4- (3-indolylmethyl) -2-aminoglutaric acid (hereinafter sometimes referred to as “monatin”) is native to the Transvalal region of northern South Africa. It is known to be an amino acid derivative that is contained in the root bark of Schlerochitom licifolius, has a sweetness several hundred times that of sucrose, and is useful as a sweetener (Patent Document 1).
　モナティンの製造方法については、種々の報告がなされており（非特許文献１～３、特許文献２、３）、光学活性なモナティンの製造方法についてもいくつか検討された例はあるが、製造に多工程を要し収率も高くはないため、必ずしも工業的に適した製造方法とはいい難かった。 Various reports have been made on methods for producing monatin (Non-patent Documents 1 to 3, Patent Documents 2 and 3), and there have been some examples of methods for producing optically active monatin. Since a multi-step process is required and the yield is not high, it is not necessarily an industrially suitable production method.
　特許文献４には、（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンをエピ化して結晶を得る方法が開示されている。しかしながら、得られた結晶は、（２Ｒ，４Ｒ）モナティンカリウム塩結晶であるため、処方次第では安定性が必ずしも満足いくものではなかった。 Patent Document 4 discloses a method of obtaining a crystal by epimerizing (2R, 4R) monatin from (2S, 4R) monatin. However, since the obtained crystals are (2R, 4R) monatin potassium salt crystals, the stability is not always satisfactory depending on the formulation.
特開昭６４－２５７５７号公報Japanese Patent Laid-Open No. 64-25757 米国特許第５，９９４，５５９号公報US Pat. No. 5,994,559 国際公開第０３／０５９８６５号パンフレットWO03 / 059865 pamphlet 米国特許第７，３９６，９４１号公報US Pat. No. 7,396,941
　良好な甘味特性を有する、保存安定性に優れた、（２Ｒ，４Ｒ）モナティン多価金属塩結晶の効率的な製造方法を提供することである。 It is to provide an efficient production method of (2R, 4R) monatin polyvalent metal salt crystals having good sweetness characteristics and excellent storage stability.
　本発明者らは、鋭意検討を重ねた結果、（２Ｓ，４Ｒ）モナティンを含有する水溶液に対して、多価金属イオン存在下、アルデヒドまたは（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素の１種または２種以上を作用させることによって、上記課題が達成できることを見出し、本発明を完成させるに至った。 As a result of intensive studies, the present inventors obtained (2R, 4R) monatin from an aldehyde or (2S, 4R) monatin in the presence of a polyvalent metal ion in an aqueous solution containing (2S, 4R) monatin. The inventors have found that the above problems can be achieved by acting one or more enzymes that can be produced, and have completed the present invention.
　すなわち、本発明は、以下の内容を含むものである。
〔１〕多価金属イオンを含有する水溶液中で、アルデヒドまたは（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素の１種または２種以上に（２Ｓ，４Ｒ）モナティンを接触させて、（２Ｒ，４Ｒ）モナティン多価金属塩結晶を析出させることを含む、（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔２〕（２Ｓ，４Ｒ）モナティンを含有する水溶液に対して、多価金属イオン存在下、アルデヒド、または、ラセマーゼ、アミノトランスフェラーゼからなる群から選ばれる１種または２種以上を作用させて、（２Ｒ，４Ｒ）モナティン多価金属塩結晶を得ることを含む、（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔３〕該アルデヒドが芳香族アルデヒドである、〔１〕または〔２〕の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔４〕（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素がラセマーゼである、〔１〕の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔５〕ラセマーゼが配列番号２のアミノ酸配列を含む、〔４〕の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔６〕（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素がアミノトランスフェラーゼである、〔１〕の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔７〕（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素がＬ－アミノ酸アミノトランスフェラーゼおよびＤ－アミノ酸アミノトランスフェラーゼである〔１〕の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔８〕（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素がＬ－アミノ酸アミノトランスフェラーゼ、Ｄ－アミノ酸アミノトランスフェラーゼ、およびラセマーゼである、〔１〕の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔９〕Ｌ－アミノ酸アミノトランスフェラーゼが配列番号４のアミノ酸配列を含み、かつＤ－アミノ酸アミノトランスフェラーゼが配列番号６のアミノ酸配列を含む、〔７〕または〔８〕の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔１０〕該多価金属が二価のアルカリ土類金属である、〔１〕～〔９〕のいずれかに記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔１１〕該アルカリ土類金属がマグネシウムである、〔１０〕の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔１２〕水溶液のｐＨが４～１１である、〔１〕～〔１１〕のいずれかに記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔１３〕水溶液中に存在する有機溶媒が５体積％以下である、〔１〕～〔１２〕のいずれかに記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔１４〕該結晶が、回折角度（２θ±０．２°、ＣｕＫα）として、８．９°、１１．２°、１５．０°、１７．８°、２２．５°に、固有Ｘ線回折ピークを有する、〔１〕～〔１３〕のいずれかに記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔１５〕該結晶が、回折角度（２θ±０．２°、ＣｕＫα）として、４．９°、１６．８°、１８．０°、２４．６°に、固有Ｘ線回折ピークを有する、〔１〕～〔１３〕のいずれかに記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔１６〕（２Ｒ，４Ｒ）モナティン多価金属塩結晶を回収することを含む、〔１〕～〔１５〕のいずれかに記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔１７〕（２Ｓ，４Ｒ）モナティンの（２Ｒ，４Ｒ）モナティンへの異性化、および（２Ｒ，４Ｒ）モナティン多価金属塩の晶析を同時に行うことにより、（２Ｒ，４Ｒ）モナティンの生成量を増加せしめる、〔１〕～〔１６〕のいずれかに記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。
〔１８〕（２Ｒ，４Ｒ）モナティンの生成後に有機溶媒を水溶液中に添加して、（２Ｒ，４Ｒ）モナティン多価金属塩結晶の析出を促進する工程をさらに含む、〔１〕～〔１７〕のいずれかに記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 That is, the present invention includes the following contents.
[1] Contacting (2S, 4R) monatin with one or more enzymes capable of producing (2R, 4R) monatin from aldehyde or (2S, 4R) monatin in an aqueous solution containing polyvalent metal ions And (2R, 4R) monatin polyvalent metal salt crystals, and the method for producing (2R, 4R) monatin polyvalent metal salt crystals.
[2] By reacting an aqueous solution containing (2S, 4R) monatin with one or more selected from the group consisting of aldehyde, racemase and aminotransferase in the presence of a polyvalent metal ion, A method for producing a (2R, 4R) monatin polyvalent metal salt crystal, comprising obtaining a 2R, 4R) monatin polyvalent metal salt crystal.
[3] The process for producing a (2R, 4R) monatin polyvalent metal salt crystal according to [1] or [2], wherein the aldehyde is an aromatic aldehyde.
[4] The process for producing a (2R, 4R) monatin polyvalent metal salt crystal according to [1], wherein the enzyme capable of producing (2R, 4R) monatin from (2S, 4R) monatin is racemase.
[5] The process for producing a (2R, 4R) monatin polyvalent metal salt crystal according to [4], wherein the racemase comprises the amino acid sequence of SEQ ID NO: 2.
[6] The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to [1], wherein the enzyme capable of producing (2R, 4R) monatin from (2S, 4R) monatin is an aminotransferase.
[7] (2R, 4R) monatin polyvalent metal salt crystal of [1], wherein the enzymes capable of producing (2R, 4R) monatin from (2S, 4R) monatin are L-amino acid aminotransferase and D-amino acid aminotransferase Manufacturing method.
[8] Enzymes capable of producing (2R, 4R) monatin from (2S, 4R) monatin are L-amino acid aminotransferase, D-amino acid aminotransferase, and racemase, [2] (2R, 4R) monatin Method for producing valent metal salt crystal.
[9] The (2R, 4R) monatin multivalent of [7] or [8], wherein the L-amino acid aminotransferase comprises the amino acid sequence of SEQ ID NO: 4 and the D-amino acid aminotransferase comprises the amino acid sequence of SEQ ID NO: 6 A method for producing metal salt crystals.
[10] The process for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of [1] to [9], wherein the polyvalent metal is a divalent alkaline earth metal.
[11] The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to [10], wherein the alkaline earth metal is magnesium.
[12] The process for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of [1] to [11], wherein the pH of the aqueous solution is 4 to 11.
[13] The process for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of [1] to [12], wherein the organic solvent present in the aqueous solution is 5% by volume or less.
[14] The crystal has intrinsic X-rays at diffraction angles (2θ ± 0.2 °, CuKα) of 8.9 °, 11.2 °, 15.0 °, 17.8 °, 22.5 °. The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of [1] to [13], which has a diffraction peak.
[15] The crystal has intrinsic X-ray diffraction peaks at diffraction angles (2θ ± 0.2 °, CuKα) at 4.9 °, 16.8 °, 18.0 °, 24.6 °, [1] A method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of [13].
[16] The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of [1] to [15], comprising recovering the (2R, 4R) monatin polyvalent metal salt crystal.
[17] Amount of (2R, 4R) monatin produced by simultaneously isomerizing (2S, 4R) monatin to (2R, 4R) monatin and crystallization of (2R, 4R) monatin polyvalent metal salt The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of [1] to [16], wherein
[18] The method further includes the step of adding an organic solvent to the aqueous solution after the formation of the (2R, 4R) monatin to promote precipitation of the (2R, 4R) monatin polyvalent metal salt crystal, [1] to [17] The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of the above.
　（２Ｓ，４Ｒ）モナティンを含有する水溶液に対して、多価金属イオン存在下、アルデヒドまたは（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素の１種または２種以上を作用させることによって、良好な甘味特性を有する、保存安定性に優れた、（２Ｒ，４Ｒ）モナティン多価金属塩の効率的な製造方法を提供できるようになった。 One or more enzymes capable of producing (2R, 4R) monatin from aldehyde or (2S, 4R) monatin act on an aqueous solution containing (2S, 4R) monatin in the presence of a polyvalent metal ion. Thus, it is possible to provide an efficient method for producing a (2R, 4R) monatin polyvalent metal salt having good sweetness characteristics and excellent storage stability.
図１は、ラセマーゼによる、（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンの異性化を示す図である。FIG. 1 is a diagram showing isomerization of (2R, 4R) monatin to (2R, 4R) monatin by racemase. 図２は、１種のアミノトランスフェラーゼによる、４Ｒ－ＩＨＯＧを介した（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンの生成を示す図である。アミノトランスフェラーゼは、２位の立体特異的な触媒活性を有さず、それ故、（２Ｓ，４Ｒ）モナティンから４Ｒ－ＩＨＯＧを生成し、次いで、４Ｒ－ＩＨＯＧから（２Ｒ，４Ｒ）モナティンを可逆的に生成し得る。４Ｒ－ＩＨＯＧ：４Ｒ－（インドール－３－イル－メチル）－４－ヒドロキシ－２－オキソグルタル酸。FIG. 2 is a diagram showing the generation of (2R, 4R) monatin from (2S, 4R) monatin via 4R-IHOG by one aminotransferase. Aminotransferase does not have a stereospecific catalytic activity at the 2-position, thus generating 4R-IHOG from (2S, 4R) monatin, and then reversibly (2R, 4R) monatin from 4R-IHOG. Can be generated. 4R-IHOG: 4R- (Indol-3-yl-methyl) -4-hydroxy-2-oxoglutaric acid. 図３は、２種のアミノトランスフェラーゼによる、４Ｒ－ＩＨＯＧを介した（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンの生成を示す図である。第１のアミノトランスフェラーゼが、（２Ｓ，４Ｒ）モナティンから４Ｒ－ＩＨＯＧを生成し、次いで、第２のアミノトランスフェラーゼが、４Ｒ－ＩＨＯＧから（２Ｒ，４Ｒ）モナティンを生成する。FIG. 3 is a diagram showing the production of (2R, 4R) monatin from (2S, 4R) monatin via 4R-IHOG by two aminotransferases. The first aminotransferase produces 4R-IHOG from (2S, 4R) monatin, and then the second aminotransferase produces (2R, 4R) monatin from 4R-IHOG. 図４は、２種のアミノトランスフェラーゼ（Ｌ－アミノ酸アミノトランスフェラーゼおよびＤ－アミノ酸アミノトランスフェラーゼ）による、４Ｒ－ＩＨＯＧを介した（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンの生成を示す図である。Ｌ－アミノ酸アミノトランスフェラーゼが、（２Ｓ，４Ｒ）モナティンから４Ｒ－ＩＨＯＧを生成し、次いで、Ｄ－アミノ酸アミノトランスフェラーゼが、４Ｒ－ＩＨＯＧから（２Ｒ，４Ｒ）モナティンを生成する。ケト酸（あるいはＬ－アミノ酸またはＤ－アミノ酸）、およびＬ－アミノ酸をＤ－アミノ酸に変換し得るラセマーゼを反応系に添加することにより、Ｌ－アミノ酸アミノトランスフェラーゼによる副反応（ケト酸→Ｌ－アミノ酸）、およびＤ－アミノ酸アミノトランスフェラーゼによる副反応（Ｄ－アミノ酸→ケト酸）をカップリングさせることができる。FIG. 4 is a diagram showing the production of (2R, 4R) monatin from (2S, 4R) monatin via 4R-IHOG by two aminotransferases (L-amino acid aminotransferase and D-amino acid aminotransferase). . L-amino acid aminotransferase produces 4R-IHOG from (2S, 4R) monatin, then D-amino acid aminotransferase produces (2R, 4R) monatin from 4R-IHOG. Addition of keto acid (or L-amino acid or D-amino acid) and racemase capable of converting L-amino acid to D-amino acid to the reaction system, thereby causing side reaction by L-amino acid aminotransferase (keto acid → L-amino acid) ), And side reactions (D-amino acids → keto acids) by D-amino acid aminotransferase. 図５は、図４に示される反応の好ましい例を示す図である。ＰＡ（あるいはＬ－ＡｌａまたはＤ－Ａｌａ）、およびアラニンラセマーゼを反応系に添加することにより、Ｌ－アミノ酸アミノトランスフェラーゼによる副反応（ＰＡ→Ｌ－Ａｌａ）、およびＤ－アミノ酸アミノトランスフェラーゼによる副反応（Ｄ－Ａｌａ→ＰＡ）をカップリングさせることができる。ＰＡ：ピルビン酸；Ａｌａ：アラニン。FIG. 5 is a diagram showing a preferred example of the reaction shown in FIG. By adding PA (or L-Ala or D-Ala) and alanine racemase to the reaction system, side reaction by L-amino acid aminotransferase (PA → L-Ala) and side reaction by D-amino acid aminotransferase ( D-Ala → PA) can be coupled. PA: pyruvic acid; Ala: alanine. 図６は、図４に示される反応の好ましい例を示す図である。α－ＫＧ（あるいはＬ－ＧｌｕまたはＤ－Ｇｌｕ）、およびグルタミン酸ラセマーゼを反応系に添加することにより、Ｌ－アミノ酸アミノトランスフェラーゼによる副反応（α－ＫＧ→Ｌ－Ｇｌｕ）、およびＤ－アミノ酸アミノトランスフェラーゼによる副反応（Ｄ－Ｇｌｕ→α－ＫＧ）をカップリングさせることができる。α－ＫＧ：α－ケトグルタル酸；Ｇｌｕ：グルタミン酸。FIG. 6 shows a preferred example of the reaction shown in FIG. By adding α-KG (or L-Glu or D-Glu) and glutamate racemase to the reaction system, a side reaction by L-amino acid aminotransferase (α-KG → L-Glu), and D-amino acid aminotransferase Side reaction (D-Glu → α-KG) can be coupled. α-KG: α-ketoglutaric acid; Glu: glutamic acid. 図７は、実施例１で得られた（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶の固有Ｘ線回折ピークを示す図である。FIG. 7 is a graph showing intrinsic X-ray diffraction peaks of the ((2R, 4R) monatin) 2 magnesium salt crystal obtained in Example 1. 図８は、反応液中の（２Ｓ，４Ｒ）モナティンおよび（２Ｒ，４Ｒ）モナティンの経時的変化を示す図である。ＳＲ－Ｍｏｎａｔｉｎ：（２Ｓ，４Ｒ）モナティン；ＲＲ－Ｍｏｎａｔｉｎ：（２Ｒ，４Ｒ）モナティン（以下同様）。FIG. 8 is a diagram showing the change over time of (2S, 4R) monatin and (2R, 4R) monatin in the reaction solution. SR-Monatin: (2S, 4R) monatin; RR-Monatin: (2R, 4R) monatin (hereinafter the same). 図９は、マグネシウム塩添加後の反応液上清中の（２Ｓ，４Ｒ）モナティンおよび（２Ｒ，４Ｒ）モナティンの経時的変化を示す図である。FIG. 9 is a diagram showing the change over time of (2S, 4R) monatin and (2R, 4R) monatin in the reaction supernatant after addition of magnesium salt. 図１０は、反応液中の（２Ｓ，４Ｒ）モナティンおよび（２Ｒ，４Ｒ）モナティンの経時的変化を示す図である。上段は、優先晶析無しおよび有りの条件下における、反応液中の（２Ｓ，４Ｒ）モナティン（左）および（２Ｒ，４Ｒ）モナティン（右）の経時的変化を示す図である。中段は、酵素反応のみ（優先晶析なし）を行った場合における、反応液全体または上清中の（２Ｓ，４Ｒ）モナティン（左）および（２Ｒ，４Ｒ）モナティン（右）の経時的変化を示す。下段は、酵素反応と優先晶析を同時に行った場合における、反応液全体または上清中の（２Ｓ，４Ｒ）モナティン（左）および（２Ｒ，４Ｒ）モナティン（右）の経時的変化を示す。FIG. 10 is a graph showing the change over time of (2S, 4R) monatin and (2R, 4R) monatin in the reaction solution. The upper graph is a graph showing the change over time of (2S, 4R) monatin (left) and (2R, 4R) monatin (right) in the reaction solution under the conditions with and without preferential crystallization. The middle graph shows the changes over time of (2S, 4R) monatin (left) and (2R, 4R) monatin (right) in the whole reaction solution or supernatant when only the enzyme reaction was performed (no preferential crystallization). Show. The lower part shows the change over time of (2S, 4R) monatin (left) and (2R, 4R) monatin (right) in the whole reaction solution or supernatant when the enzyme reaction and preferential crystallization are performed simultaneously. 図１１は、実施例１１で得られた（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶の固有Ｘ線回折ピークを示す図である。FIG. 11 is a graph showing intrinsic X-ray diffraction peaks of the ((2R, 4R) monatin) 2 magnesium salt crystal obtained in Example 11. 図１２は、参考例３で得られた（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶の固有Ｘ線回折ピークを示す図である。FIG. 12 is a diagram showing an intrinsic X-ray diffraction peak of the ((2R, 4R) monatin) 2 magnesium salt crystal obtained in Reference Example 3. 図１３は、参考例４で得られた（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶の固有Ｘ線回折ピークを示す図である。FIG. 13 is a diagram showing an intrinsic X-ray diffraction peak of the ((2R, 4R) monatin) 2 magnesium salt crystal obtained in Reference Example 4. 図１４は、参考例５で得られた（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶の固有Ｘ線回折ピークを示す図である。FIG. 14 is a diagram showing an intrinsic X-ray diffraction peak of the ((2R, 4R) monatin) 2 magnesium salt crystal obtained in Reference Example 5.
　本発明は、（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法を提供する。本発明の方法は、多価金属イオンを含有する水溶液中で、アルデヒドまたは（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素の１種または２種以上に（２Ｓ，４Ｒ）モナティンを接触させて、（２Ｒ，４Ｒ）モナティン多価金属塩結晶を析出させることを含む。換言すれば、（２Ｓ，４Ｒ）モナティンを含有する水溶液に対して、多価金属イオン存在下、アルデヒドまたは（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素の１種または２種以上を作用させて、（２Ｒ，４Ｒ）モナティン多価金属塩結晶を得ることを含む方法である。 The present invention provides a method for producing (2R, 4R) monatin polyvalent metal salt crystals. In the method of the present invention, one or more enzymes capable of producing (2R, 4R) monatin from aldehyde or (2S, 4R) monatin in an aqueous solution containing a polyvalent metal ion are converted into (2S, 4R). Contacting the monatin to precipitate the (2R, 4R) monatin polyvalent metal salt crystals. In other words, one or two enzymes capable of producing (2R, 4R) monatin from aldehyde or (2S, 4R) monatin in the presence of a polyvalent metal ion in an aqueous solution containing (2S, 4R) monatin. It is a method including obtaining a (2R, 4R) monatin polyvalent metal salt crystal by allowing more than one species to act.
　反応プロセスの観点からは、本発明の方法は、（ａ）（２Ｓ，４Ｒ）モナティンを異性化して（２Ｒ，４Ｒ）モナティンを生成すること（異性化）、および（ｂ）生成した（２Ｒ，４Ｒ）モナティンと多価金属イオンとの接触により（２Ｒ，４Ｒ）モナティン多価金属塩結晶を析出させること（晶析）を含む。本発明の方法は、得られた結晶を回収することを含んでいてもよい。（ａ）および（ｂ）は、別々または同時に（換言すれば、並行して）行うことができるが、同時に行うことが好ましい。（ａ）および（ｂ）を同時に行うことにより、（２Ｒ，４Ｒ）モナティンの生成量を増加させることができる。（ａ）および（ｂ）を別々に行う場合には、高分子による晶析阻害を避けるため、（ａ）で得られた反応液から高分子を除去（例、遠心分離、ろ過）してもよい。晶析の際、種晶を溶液中に添加することを含んでいてもよい。また、（２Ｒ，４Ｒ）モナティンの生成後に有機溶媒を水溶液中に添加することにより、（２Ｒ，４Ｒ）モナティン多価金属塩結晶の析出を促進することもできる。（ｂ）において析出してきた結晶は、ろ過工程等の分離工程に付すことにより、容易に湿結晶を得ることができる。得られた湿結晶は洗浄してもよい。湿結晶を乾燥させることにより、乾燥結晶を得ることができる。これらの事項についての詳細については後述する。 From the point of view of the reaction process, the method of the invention comprises (a) isomerizing (2S, 4R) monatin to produce (2R, 4R) monatin (isomerization) and (b) producing (2R, 4R) Precipitating (2R, 4R) monatin polyvalent metal salt crystals by contact with monatin and polyvalent metal ions (crystallization). The method of the present invention may comprise recovering the resulting crystals. (A) and (b) can be performed separately or simultaneously (in other words, in parallel), but are preferably performed simultaneously. By simultaneously performing (a) and (b), the amount of (2R, 4R) monatin produced can be increased. When performing (a) and (b) separately, the polymer may be removed (eg, centrifuged, filtered) from the reaction solution obtained in (a) in order to avoid crystallization inhibition by the polymer. Good. In the crystallization, a seed crystal may be added to the solution. Moreover, precipitation of (2R, 4R) monatin polyvalent metal salt crystals can be promoted by adding an organic solvent to the aqueous solution after the formation of (2R, 4R) monatin. The crystals precipitated in (b) can be easily obtained as wet crystals by subjecting them to a separation step such as a filtration step. The obtained wet crystals may be washed. Dry crystals can be obtained by drying the wet crystals. Details of these matters will be described later.
　本発明に使用される（２Ｓ，４Ｒ）モナティンは、自体公知の方法により入手でき、例えば、化学合成法または酵素法により製造されたものを使用することができる。化学合成法としては、（２Ｓ，４Ｒ）モナティンが生成しうる限り任意の方法を用いることができるが、例えば、米国特許第７，０６４，２１９号公報および特許文献４に記載されている方法を用いて、（４Ｒ）－４－ヒドロキシ－４－（３－インドリルメチル）－２－ヒドロキシイミノグルタミン酸の還元液を晶析して（２Ｓ，４Ｒ）モナティンを取得してもよい。酵素法としては、（２Ｓ，４Ｒ）モナティンが生成しうる限り任意の方法を用いることができるが、例えば、米国特許第７，２９７，８００号公報および米国特許第７，０６４，２１９号公報に記載されている方法を組み合わせて、（４Ｒ）ＩＨＯＧを酵素的に取得し、オキシム化・還元し、カラム精製・晶析により、（２Ｓ，４Ｒ）モナティンを取得してもよい。本発明の方法における材料である（２Ｓ，４Ｒ）モナティンとしては、（２Ｓ，４Ｒ）モナティンを少なくとも少量なりとも含有していればよく、結晶状態、粉体状態、溶液状態（水溶液、有機溶液、水・有機溶剤混合溶液）のいずれでも構わない。それゆえ、精製された（２Ｓ，４Ｒ）モナティンを用いても、（２Ｓ，４Ｒ）モナティンを含有する反応液〔（２Ｓ，４Ｒ）モナティンを製造するために用いられた反応液〕を、（２Ｓ，４Ｒ）モナティンを含有する水溶液として、本発明の方法に用いてもよい。
　（２Ｓ，４Ｒ）モナティンはまた、塩、水和物、または塩の水和物の形態で、本発明の方法に用いられる水溶液中に提供されてもよい。塩としては、一価の金属（例、カリウム、ナトリウム）および非金属（例、アンモニウム）の塩、ならびに後述する多価金属（例、後述する二価金属、三価金属）の塩が挙げられる。水和物としては、例えば、一水和物、二水和物、三水和物、四水和物、五水和物、六水和物、七水和物、八水和物、九水和物が挙げられる。 The (2S, 4R) monatin used in the present invention can be obtained by a method known per se, for example, one produced by a chemical synthesis method or an enzymatic method can be used. As a chemical synthesis method, any method can be used as long as (2S, 4R) monatin can be produced. For example, the methods described in US Pat. No. 7,064,219 and Patent Document 4 are used. It is also possible to obtain (2S, 4R) monatin by crystallizing a reducing solution of (4R) -4-hydroxy-4- (3-indolylmethyl) -2-hydroxyiminoglutamic acid. As an enzymatic method, any method can be used as long as (2S, 4R) monatin can be produced. For example, in US Pat. No. 7,297,800 and US Pat. No. 7,064,219 By combining the methods described, (2R) IHOG may be obtained enzymatically, oximated and reduced, and (2S, 4R) monatin obtained by column purification and crystallization. The (2S, 4R) monatin that is a material in the method of the present invention only needs to contain at least a small amount of (2S, 4R) monatin, and is in a crystalline state, a powder state, a solution state (aqueous solution, organic solution, Any of water / organic solvent mixed solution) may be used. Therefore, even when purified (2S, 4R) monatin is used, a reaction solution containing (2S, 4R) monatin [a reaction solution used to produce (2S, 4R) monatin] is obtained as (2S , 4R) An aqueous solution containing monatin may be used in the method of the present invention.
The (2S, 4R) monatin may also be provided in the aqueous solution used in the method of the present invention in the form of a salt, hydrate, or salt hydrate. Examples of the salt include salts of monovalent metals (eg, potassium, sodium) and nonmetals (eg, ammonium), and salts of polyvalent metals (eg, divalent metals, trivalent metals described later). . Examples of hydrates include monohydrate, dihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, and nine water Japanese products are listed.
　本発明に使用されるアルデヒドについて詳述する。アルデヒドとしては、脂肪族アルデヒドまたは芳香族アルデヒドが挙げられるが、芳香族アルデヒドが好ましい。 The aldehyde used in the present invention will be described in detail. Examples of the aldehyde include aliphatic aldehydes and aromatic aldehydes, and aromatic aldehydes are preferable.
　脂肪族アルデヒドとしては、例えば、ホルムアルデヒド、アセトアルデヒド、プロピオンアルデヒド、ｎ－ブチルアルデヒド、１－ブチルアルデヒド、ｎ－バレル　アルデヒド、カプロンアルデヒド、ｎ－ヘプチルアルデヒド、アクロレイン、メタクロレイン等の炭素数１～７の飽和または不飽和アルデヒドを用いることができる。 Examples of aliphatic aldehydes include those having 1 to 7 carbon atoms such as formaldehyde, acetaldehyde, propionaldehyde, n-butyraldehyde, 1-butyraldehyde, n-barrel aldehyde, capronaldehyde, n-heptylaldehyde, acrolein, methacrolein and the like. Saturated or unsaturated aldehydes can be used.
　芳香族アルデヒドとしては、例えば、ベンズアルデヒド、サリチルアルデヒド、ｍ－ヒドロキシベンズアルデヒド、ｐ－ヒドロキシベンズアルデヒド、ｏ－ニトロベンズアルデヒド、ｐ－ニトロベンズアルデヒド、５－ニトロサリチルアルデヒド、３，５－ジクロロサリチルアルデヒド、アニスアルデヒド、ｏ－バニリン、バニリン、フルフラール、ピリドキサール、５－リン酸ピリドキサール等を用いることができる。芳香族アルデヒドとしては、特に、ピリドキサール、５－ニトロサリチルアルデヒド、３，５－ジクロロサリチルアルデヒドが好ましい。 Examples of aromatic aldehydes include benzaldehyde, salicylaldehyde, m-hydroxybenzaldehyde, p-hydroxybenzaldehyde, o-nitrobenzaldehyde, p-nitrobenzaldehyde, 5-nitrosalicylaldehyde, 3,5-dichlorosalicylaldehyde, anisaldehyde, o-vanillin, vanillin, furfural, pyridoxal, 5-phosphate pyridoxal, and the like can be used. As the aromatic aldehyde, pyridoxal, 5-nitrosalicylaldehyde, and 3,5-dichlorosalicylaldehyde are particularly preferable.
　アルデヒドは、系に存在するモナティンに対して０．０１～１モル当量、より好ましくは０．０５～０．５モル当量の範囲で使用することができる。 Aldehydes can be used in the range of 0.01 to 1 molar equivalent, more preferably 0.05 to 0.5 molar equivalent, relative to monatin present in the system.
　次に、本発明に使用される（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素について詳述する。（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素は、例えば、ラセマーゼ、アミノトランスフェラーゼ、アミノ酸デヒドロゲナーゼ等が挙げられ、ラセマーゼ、アミノトランスフェラーゼが好ましい。これらは１種または２種以上を使用しても良い。 Next, an enzyme capable of producing (2R, 4R) monatin from (2S, 4R) monatin used in the present invention will be described in detail. Examples of the enzyme capable of producing (2R, 4R) monatin from (2S, 4R) monatin include racemase, aminotransferase, amino acid dehydrogenase and the like, and racemase and aminotransferase are preferable. These may be used alone or in combination of two or more.
　本発明に使用されるラセマーゼについて詳述する（図１）。ラセマーゼは、（２Ｓ，４Ｒ）モナティンを（２Ｒ，４Ｒ）モナティンに変換する活性を有する酵素である限り特に限定されない。このような活性を有する酵素はエピメラーゼと呼ばれることもある。本発明では、別名の酵素もこのような活性を有する限り、ラセマーゼと呼称され本発明の範囲に含まれる。当業者は、このような活性を有するラセマーゼを適宜取得することができる。例えば、当業者は、所定のスクリーニング法を用いることにより、このような活性を有するラセマーゼを得ることができる。このようなスクリーニング法としては、例えば、２０ｍＭ　（２Ｓ，４Ｒ）モナティン〔または（２Ｒ，４Ｒ）モナティン〕、１００ｍＭ　Ｔｒｉｓ－ＨＣｌ（ｐＨ８．０）、５０μＭ　ピリドキサールリン酸、酵素液（精製酵素、クルード酵素など）を含有する反応液を、３０℃で１６時間反応させることが挙げられる。配列番号２のアミノ酸配列で特定されるタンパク質は、このようなスクリーニング法により得られたラセマーゼである。 The racemase used in the present invention will be described in detail (FIG. 1). The racemase is not particularly limited as long as it is an enzyme having an activity of converting (2S, 4R) monatin to (2R, 4R) monatin. An enzyme having such activity is sometimes called epimerase. In the present invention, as long as the enzyme of another name also has such activity, it is called racemase and is included in the scope of the present invention. Those skilled in the art can appropriately obtain a racemase having such activity. For example, those skilled in the art can obtain a racemase having such activity by using a predetermined screening method. Examples of such screening methods include 20 mM (2S, 4R) monatin [or (2R, 4R) monatin], 100 mM Tris-HCl (pH 8.0), 50 μM pyridoxal phosphate, enzyme solution (purified enzyme, crude enzyme). Etc.) is allowed to react at 30 ° C. for 16 hours. The protein specified by the amino acid sequence of SEQ ID NO: 2 is a racemase obtained by such a screening method.
　ラセマーゼとしては、配列番号２のアミノ酸配列に対して有意なアミノ酸配列同一性を示すアミノ酸配列を含み、かつ（２Ｓ，４Ｒ）モナティンを（２Ｒ，４Ｒ）モナティンに変換する活性を有するタンパク質を用いることができる。配列番号２のアミノ酸配列に対して有意なアミノ酸配列同一性を示すアミノ酸配列としては、配列番号２のアミノ酸配列に対して、例えば７０％以上、好ましくは８０％以上、より好ましくは８５％以上、さらにより好ましくは９０％以上、特に好ましくは９５％以上、９６％以上、９７％以上、９８％以上または９９％以上のアミノ酸配列同一性を示すアミノ酸配列が挙げられる。 As a racemase, a protein containing an amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2 and having an activity of converting (2S, 4R) monatin to (2R, 4R) monatin is used. Can do. As the amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2, for example, 70% or more, preferably 80% or more, more preferably 85% or more, relative to the amino acid sequence of SEQ ID NO: 2, Even more preferred is an amino acid sequence showing 90% or more, particularly preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity.
　アミノ酸配列の同一性は、例えばＫａｒｌｉｎおよびＡｌｔｓｃｈｕｌによるアルゴリズムＢＬＡＳＴ（Ｐｒｏ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，９０，５８７３（１９９３））やＰｅａｒｓｏｎによるＦＡＳＴＡ（ＭｅｔｈｏｄｓＥｎｚｙｍｏｌ．，１８３，６３（１９９０））を用いて決定することができる。このアルゴリズムＢＬＡＳＴに基づいて、ＢＬＡＳＴＰとよばれるプログラムが開発されているので（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ参照）、これらのプログラムをデフォルト設定で用いて、アミノ酸配列の同一性を計算してもよい。また、アミノ酸配列の同一性としては、例えば、株式会社ゼネティックスのソフトウェアＧＥＮＥＴＹＸ　Ｖｅｒ７．０．９を使用し、ＯＲＦにコードされるポリペプチド鎖全長を用いて、Ｕｎｉｔ　Ｓｉｚｅ　ｔｏ　Ｃｏｍｐａｒｅ＝２の設定でＭａｒｃｈｉｎｇ　ｃｏｕｎｔをｐｅｒｃｅｎｔａｇｅ計算させた際の数値を用いてもよい。アミノ酸配列の同一性として、これらの計算で導き出される値のうち、最も低い値を採用してもよい。 The identity of amino acid sequences is determined using, for example, the algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) and FASTA by Pearson (Methods Enzymol., 183, 63 (1990)). Can be determined. Based on this algorithm BLAST, programs called BLASTP have been developed (see http://www.ncbi.nlm.nih.gov), and these programs are used with default settings, and amino acid sequence identity. May be calculated. As the identity of amino acid sequences, for example, using GENETYX software GENETYX Ver 7.0.9, using the full length of the polypeptide chain encoded by ORF, the setting of Unit Size to Compare = 2 is set. You may use the numerical value at the time of carrying out percentage calculation of count. As the amino acid sequence identity, the lowest value among the values derived by these calculations may be adopted.
　ラセマーゼはまた、（ａ）配列番号２のアミノ酸配列を含むタンパク質、（ｂ）配列番号２のアミノ酸配列において１または数個のアミノ酸残基の変異（例、置換、欠失、付加または挿入）を有するアミノ酸配列を含み、かつ（２Ｓ，４Ｒ）モナティンを（２Ｒ，４Ｒ）モナティンに変換する活性を有するタンパク質であってもよい。１または数個のアミノ酸残基の変異は、アミノ酸配列中の１つの領域に導入されてもよいが、複数の異なる領域に導入されてもよい。用語「１または数個」は、タンパク質の立体構造や活性を大きく損なわない範囲を示すものである。タンパク質の場合における用語「１または数個」が示す数は、例えば、１～１５０個、好ましくは１～１００個、より好ましくは１～５０個、１～３０個、１～２０個、１～１０個または１～５個である。このような変異は、ラセマーゼをコードする遺伝子を保持する生物（例、微生物、動物）の個体差および種差等に基づく天然に生じる変異（ｍｕｔａｎｔまたはｖａｒｉａｎｔ）に起因していてもよい。 Racemase also includes (a) a protein comprising the amino acid sequence of SEQ ID NO: 2, (b) mutation of one or several amino acid residues (eg, substitution, deletion, addition or insertion) in the amino acid sequence of SEQ ID NO: 2. And a protein having an amino acid sequence and having an activity of converting (2S, 4R) monatin to (2R, 4R) monatin. The mutation of one or several amino acid residues may be introduced into one region in the amino acid sequence, but may be introduced into a plurality of different regions. The term “one or several” indicates a range that does not significantly impair the three-dimensional structure and activity of the protein. The number indicated by the term “one or several” in the case of protein is, for example, 1 to 150, preferably 1 to 100, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5. Such a mutation may be caused by a naturally occurring mutation (mutant or variant) based on individual differences, species differences, and the like of organisms (eg, microorganisms, animals) carrying a gene encoding racemase.
　アミノ酸配列において変異を導入すべきアミノ酸残基の位置は、当業者に自明である。具体的には、当業者は、１）同種の活性を有する複数のタンパク質のアミノ酸配列（例、配列番号２のアミノ酸配列、および他のラセマーゼのアミノ酸配列）を比較し、２）相対的に保存されている領域、および相対的に保存されていない領域を明らかにし、次いで、３）相対的に保存されている領域および相対的に保存されていない領域から、それぞれ、機能に重要な役割を果たし得る領域および機能に重要な役割を果たし得ない領域を予測できるので、構造・機能の相関性を認識できる。したがって、当業者は、ラセマーゼのアミノ酸配列において変異を導入すべきアミノ酸残基の位置を特定できる。 The position of the amino acid residue to be mutated in the amino acid sequence is obvious to those skilled in the art. Specifically, a person skilled in the art compares 1) the amino acid sequences of a plurality of proteins having the same type of activity (eg, the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of another racemase), and 2) is relatively conserved. Region, and relatively unconserved region, then 3) from the relatively conserved region and the relatively unconserved region, respectively, play an important role in function Since it is possible to predict a region that cannot play an important role in the region and function to be obtained, the correlation between structure and function can be recognized. Therefore, those skilled in the art can specify the position of the amino acid residue to be mutated in the racemase amino acid sequence.
　アミノ酸残基が置換により変異される場合、アミノ酸残基の置換は、保存的置換であってもよい。用語「保存的置換」とは、所定のアミノ酸残基を、類似の側鎖を有するアミノ酸残基で置換することをいう。類似の側鎖を有するアミノ酸残基のファミリーは、当該分野で周知である。例えば、このようなファミリーとしては、塩基性側鎖を有するアミノ酸（例、リジン、アルギニン、ヒスチジン）、酸性側鎖を有するアミノ酸（例、アスパラギン酸、グルタミン酸）、非荷電性極性側鎖を有するアミノ酸（例、アスパラギン、グルタミン、セリン、スレオニン、チロシン、システイン）、非極性側鎖を有するアミノ酸（例、グリシン、アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファン）、β位分岐側鎖を有するアミノ酸（例、スレオニン、バリン、イソロイシン）、芳香族側鎖を有するアミノ酸（例、チロシン、フェニルアラニン、トリプトファン、ヒスチジン）、ヒドロキシル基（例、アルコール性、フェノール性）含有側鎖を有するアミノ酸（例、セリン、スレオニン、チロシン）、および硫黄含有側鎖を有するアミノ酸（例、システイン、メチオニン）が挙げられる。好ましくは、アミノ酸の保存的置換は、アスパラギン酸とグルタミン酸との間での置換、アルギニンとリジンとヒスチジンとの間での置換、トリプトファンとフェニルアラニンとの間での置換、フェニルアラニンとバリンとの間での置換、ロイシンとイソロイシンとアラニンとの間での置換、およびグリシンとアラニンとの間での置換であってもよい。 When an amino acid residue is mutated by substitution, the amino acid residue substitution may be a conservative substitution. The term “conservative substitution” refers to substitution of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art. For example, such families include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having uncharged polar side chains (Eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain Amino acids (eg, threonine, valine, isoleucine), amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine), amino acids having side groups containing hydroxyl groups (eg, alcoholic, phenolic) ( Example, serine, thread Nin, tyrosine), and amino acids (e.g. having sulfur-containing side chains, cysteine, methionine) and the like. Preferably, the conservative substitution of amino acids is a substitution between aspartic acid and glutamic acid, a substitution between arginine and lysine and histidine, a substitution between tryptophan and phenylalanine, and between phenylalanine and valine. Substitution, leucine, isoleucine and alanine substitution, and glycine and alanine substitution.
　ラセマーゼは、配列番号１で表される塩基配列に相補的な塩基配列とストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされ、かつ（２Ｓ，４Ｒ）モナティンを（２Ｒ，４Ｒ）モナティンに変換する活性を有するタンパク質が好ましい。「ストリンジェントな条件」とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいう。同一性を有するポリヌクレオチド同士がハイブリダイズし、それより低い同一性を示すポリヌクレオチド同士がハイブリダイズしない条件であるという観点で、ポリヌクレオチド同士の同一性としては、７０％以上が好ましく、８０％以上がより好ましく、９０％以上がさらに好ましく、９５％以上が殊更好ましく、９８％以上が特に好ましい。具体的には、このような条件としては、６×ＳＳＣ（塩化ナトリウム／クエン酸ナトリウム）中、約４５℃でのハイブリダイゼーション、続いて、０．２×ＳＳＣ、０．１％ＳＤＳ中、５０～６５℃での１または２回以上の洗浄が挙げられる。 Racemase is encoded by DNA that hybridizes under stringent conditions with a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and converts (2S, 4R) monatin to (2R, 4R) monatin. A protein having activity is preferred. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. From the viewpoint that the polynucleotides having the same identity are hybridized and the polynucleotides having lower identity are not hybridized, the identity between the polynucleotides is preferably 70% or more, 80% The above is more preferable, 90% or more is further preferable, 95% or more is particularly preferable, and 98% or more is particularly preferable. Specifically, such conditions include hybridization at about 45 ° C. in 6 × SSC (sodium chloride / sodium citrate), followed by 50 × 0.2 × SSC in 0.1% SDS. One or more washings at ˜65 ° C. may be mentioned.
　本発明で用いられるラセマーゼは、そのＮ末端部またはＣ末端部に精製用タグが付加されていてもよい。精製用タグを利用することで、ラセマーゼを簡便に精製することができる。精製用タグとしては、例えば、ヒスチジン（Ｈｉｓ）タグ、カルモジュリン結合ペプチド（ＣＢＰ）、ストレプトタグ（Ｓｔｒｅｐ－ｔａｇ）　ＩＩ、ＦＬＡＧが挙げられる。 The racemase used in the present invention may have a purification tag attached to its N-terminal part or C-terminal part. By using the purification tag, racemase can be easily purified. Examples of the purification tag include histidine (His) tag, calmodulin-binding peptide (CBP), strept-tag (Strep-tag) II, and FLAG.
　ラセマーゼは、ラセマーゼの産生菌から、または無細胞系による合成により、入手できる。ラセマーゼの産生菌としては、例えば、ラセマーゼを天然で産生している菌、およびラセマーゼを発現する形質転換体が挙げられる。ラセマーゼは、精製酵素、粗酵素、または固定化酵素として用いることができる。 Racemase can be obtained from racemase-producing bacteria or by cell-free synthesis. Examples of racemase-producing bacteria include bacteria that naturally produce racemase and transformants that express racemase. Racemase can be used as a purified enzyme, a crude enzyme, or an immobilized enzyme.
　ラセマーゼの産生菌として、ラセマーゼを発現する形質転換体が用いられる場合、この形質転換体は、ラセマーゼの発現ベクターを作製し、次いで、この発現ベクターを宿主に導入することにより作製できる。ラセマーゼを発現させるための宿主としては、例えばエシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）等のエシェリヒア属細菌、コリネバクテリウム属細菌（例、コリネバクテリウム・グルタミカム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ　ｇｌｕｔａｍｉｃｕｍ））、およびバチルス属細菌（例、バチルス・ズブチリス（Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ））をはじめとする種々の原核細胞、サッカロマイセス属細菌（例、サッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ））、ピヒア属細菌（例、ピヒア・スティピティス（Ｐｉｃｈｉａ　ｓｔｉｐｉｔｉｓ））、アスペルギルス属細菌（例、アスペルギルス・オリゼ（Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ））をはじめとする種々の真核細胞を用いることができる。好ましい宿主の例は、Ｅ．ｃｏｌｉである。宿主として、Ｅ．ｃｏｌｉを用いる場合、配列番号１のＤＮＡ配列によりコードするポリヌクレオチドが挿入された発現ベクターの使用もより好ましい。 When a transformant that expresses racemase is used as a racemase-producing bacterium, this transformant can be prepared by preparing an expression vector for racemase and then introducing the expression vector into a host. Examples of the host for expressing racemase include Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria (eg, Corynebacterium glutamicum), and Bacillus bacteria (eg, Various prokaryotic cells such as Bacillus subtilis, Saccharomyces cerevisiae (eg, Saccharomyces cerevisiae), Pichia bacteria (eg, Pichia stipitis) (E.g., Aspergillus oryzae) It can be used cells. Examples of preferred hosts are E. coli. E. coli. As a host, E. coli. When using E. coli, it is more preferable to use an expression vector into which a polynucleotide encoded by the DNA sequence of SEQ ID NO: 1 has been inserted.
　異性化反応の反応条件に関しては、当業者であれば簡単な予備的試験によって好適な条件を設定することができる。ラセマーゼは、反応系に存在するモナティンに対して、０．００１～１０００Ｕ／ｍｌが好ましく、０．１～１００Ｕ／ｍｌがより好ましい（１Ｕは、１分間に１μｍｏｌのモナティンを異性化する活性であることを示す）。異性化反応における反応系のｐＨは、ｐＨ５．０～１１．０が好ましく、ｐＨ６．０～１０．０がより好ましく、ｐＨ７．０～９．０が更に好ましい。反応系に添加される（２Ｓ，４Ｒ）モナティン〔または（２Ｒ，４Ｒ）モナティン〕の濃度は、１０ｍＭ～３．０Ｍが好ましく、１００ｍＭ～１．０Ｍがより好ましい。この場合、モナティンは、反応開始前に反応系に添加されてもよいが、反応開始後に断続的あるいは継続的に添加されてもよい。反応温度は、当該異性化反応が進行する温度である限り特に限定されないが、１５℃～６０℃が好ましく、２５℃～４２℃がより好ましい。反応時間は、１～１２０時間が好ましく、１～２４時間がより好ましい。ピリドキサールリン酸（ＰＬＰ）は、必ずしも反応系に添加する必要はないが添加する場合のＰＬＰの濃度は、異性化反応が進行する限り特に限定されないが、１０～１００μＭが好ましい。また、ラセマーゼの反応液あるいはラセマーゼの精製または透析に用いる緩衝液等の液体中でラセマーゼを安定化させるために、ＮａＣｌまたはＫＣｌなどの塩を添加してもよい。ラセマーゼを精製または透析する場合、目的の溶液に５０ｍＭ～５００ｍＭのＮａＣｌまたはＫＣｌを添加することが好ましい。 As for the reaction conditions of the isomerization reaction, those skilled in the art can set suitable conditions by a simple preliminary test. Racemase is preferably 0.001 to 1000 U / ml, more preferably 0.1 to 100 U / ml with respect to monatin present in the reaction system (1 U is an activity to isomerize 1 μmol of monatin per minute. Show). The pH of the reaction system in the isomerization reaction is preferably pH 5.0 to 11.0, more preferably pH 6.0 to 10.0, and still more preferably pH 7.0 to 9.0. The concentration of (2S, 4R) monatin [or (2R, 4R) monatin] added to the reaction system is preferably 10 mM to 3.0 M, more preferably 100 mM to 1.0 M. In this case, monatin may be added to the reaction system before starting the reaction, but may be added intermittently or continuously after starting the reaction. The reaction temperature is not particularly limited as long as the isomerization reaction proceeds, but is preferably 15 ° C to 60 ° C, and more preferably 25 ° C to 42 ° C. The reaction time is preferably 1 to 120 hours, more preferably 1 to 24 hours. Pyridoxal phosphate (PLP) is not necessarily added to the reaction system, but the concentration of PLP when added is not particularly limited as long as the isomerization reaction proceeds, but is preferably 10 to 100 μM. In addition, a salt such as NaCl or KCl may be added to stabilize the racemase in a liquid such as a racemase reaction solution or a buffer used for purification or dialysis of the racemase. When the racemase is purified or dialyzed, it is preferable to add 50 mM to 500 mM NaCl or KCl to the target solution.
　ラセマーゼを作用させた（２Ｓ，４Ｒ）モナティンと（２Ｒ，４Ｒ）モナティンを含む反応液から（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩を晶析する場合、当業者であれば簡単な予備的試験によって好適な条件を設定することができる。反応液中の（２Ｒ，４Ｒ）モナティン濃度としては、２０ｍＭ～３Ｍが好ましく、５０ｍＭ～１Ｍがより好ましい。反応系に添加されるマグネシウム源は、１０ｍＭ～３Ｍが好ましく、２５ｍＭ～１Ｍがより好ましい。温度は、０～６０℃が好ましく、１０～４０℃がより好ましい。時間は３時間～１週間が好ましく、２４時間～６０時間がより好ましい。高分子による晶析阻害を避けるため、反応液の遠心上清あるいは限外ろ過したろ液を用いる、あるいは添加する種晶の量を増やしてもよい。 Racemase allowed to act (2S, 4R) monatin and (2R, 4R) monatin from the reaction solution containing ((2R, 4R) monatin) To crystallize 2 magnesium salt, simple preliminary tests by those skilled in the art A suitable condition can be set. The (2R, 4R) monatin concentration in the reaction solution is preferably 20 mM to 3M, and more preferably 50 mM to 1M. The magnesium source added to the reaction system is preferably 10 mM to 3M, more preferably 25 mM to 1M. The temperature is preferably 0 to 60 ° C, more preferably 10 to 40 ° C. The time is preferably 3 hours to 1 week, more preferably 24 hours to 60 hours. In order to avoid crystallization inhibition by the polymer, the centrifugal supernatant of the reaction solution or the ultrafiltered filtrate may be used, or the amount of seed crystals to be added may be increased.
　次に、本発明に使用されるアミノトランスフェラーゼについて詳述する。アミノトランスフェラーゼとしては、４Ｒ－ＩＨＯＧから（２Ｓ，４Ｒ）モナティンへの変換活性（２Ｓ立体選択活性）と、４Ｒ－ＩＨＯＧから（２Ｒ，４Ｒ）モナティンへの変換活性（２Ｒ立体選択活性）とを共に有しているものを使用することができる。アミノトランスフェラーゼの変換活性は、可逆的であり得る。各活性を共に有する酵素を未精製のまま使用しても構わない。また、各活性を有する酵素をそれぞれ精製してから併用しても構わない。本発明では、別名の酵素もこのような活性を有する限り、アミノトランスフェラーゼと呼称され、本発明に包含される。
　アミノトランスフェラーゼについて、さらに図２、３に基づいて説明する。図２に示されるように、アミノトランスフェラーゼは、４Ｒ－ＩＨＯＧを介して（２Ｓ，４Ｒ）モナティンを（２Ｒ，４Ｒ）モナティンに変換してもよい。あるいは、図３に示されるように、第１アミノトランスフェラーゼが、（２Ｓ，４Ｒ）モナティンを４Ｒ－ＩＨＯＧに変換し、次いで、第２アミノトランスフェラーゼが、４Ｒ－ＩＨＯＧを（２Ｒ，４Ｒ）モナティンに変換してもよい。 Next, the aminotransferase used in the present invention will be described in detail. As aminotransferases, there are both conversion activity from 4R-IHOG to (2S, 4R) monatin (2S stereoselective activity) and conversion activity from 4R-IHOG to (2R, 4R) monatin (2R stereoselective activity). What you have can be used. The conversion activity of the aminotransferase can be reversible. Enzymes having both activities may be used without purification. Moreover, you may use together, after refine | purifying each enzyme which has each activity. In the present invention, as long as an enzyme of another name also has such an activity, it is called an aminotransferase and is included in the present invention.
The aminotransferase will be further described with reference to FIGS. As shown in FIG. 2, the aminotransferase may convert (2S, 4R) monatin to (2R, 4R) monatin via 4R-IHOG. Alternatively, as shown in FIG. 3, the first aminotransferase converts (2S, 4R) monatin to 4R-IHOG, and then the second aminotransferase converts 4R-IHOG to (2R, 4R) monatin. May be.
　当業者は、このようなアミノトランスフェラーゼを適宜取得することができる。野生型または変異アミノトランスフェラーゼは、他の中間化合物を介した可逆的なアミノトランスフェラーゼ反応により、所定の化合物を異性化できることが知られている。このような場合、アミノトランスフェラーゼは、見かけ上のラセマーゼ活性を有する。例えば、アスパラギン酸アミノトランスフェラーゼは、見かけ上のラセマーゼ活性を獲得できることが知られている（例、Ｋｏｃｈｈａｒ，Ｓｕｎｉｌら、Ｅｕｒｏｐｅａｎ　Ｊｏｕｒｎａｌ　ｏｆ　Ｂｉｏｃｈｅｍｉｓｔｒｙ（１９９２），２０３（３），５６３－９を参照）。したがって、本発明では、（２Ｓ，４Ｒ）モナティンに作用して見かけ上のラセマーゼ活性を発揮できるアミノトランスフェラーゼを用いることができる。反応系に添加されるアミノトランスフェラーゼの量は、反応を適宜進行させることができる限り特に限定されない。 Those skilled in the art can appropriately obtain such aminotransferase. It is known that wild-type or mutant aminotransferases can isomerize a given compound by a reversible aminotransferase reaction via other intermediate compounds. In such cases, the aminotransferase has apparent racemase activity. For example, aspartate aminotransferase is known to be able to acquire apparent racemase activity (see, for example, Kochhar, Sunil et al., European Journal of Biochemistry (1992), 203 (3), 563-9). Therefore, in the present invention, an aminotransferase capable of acting on (2S, 4R) monatin and exhibiting apparent racemase activity can be used. The amount of aminotransferase added to the reaction system is not particularly limited as long as the reaction can proceed appropriately.
　４Ｒ－ＩＨＯＧから（２Ｓ，４Ｒ）モナティンへの変換活性（２Ｓ立体選択活性）を有するアミノトランスフェラーゼ（第１アミノトランスフェラーゼ）としては、例えば、Ｌ－アミノ酸アミノトランスフェラーゼ、Ｌ－アスパラギン酸アミノトランスフェラーゼが挙げられる。具体的には、例えば、国際公開２０１２／０５０１２５に記載の酵素が挙げられる。
　４Ｒ－ＩＨＯＧから（２Ｒ，４Ｒ）モナティンへの変換活性（２Ｒ立体選択活性）を有するアミノトランスフェラーゼ（第２アミノトランスフェラーゼ）としては、例えば、Ｄ－アミノ酸アミノトランスフェラーゼが挙げられる。Ｄ－アミノ酸アミノトランスフェラーゼとしては、例えば、国際公開第０３／０５６０２６号、国際公開第２００９／０８８９４９号、国際公開第２０１２/１４７６７４号に記載の酵素が挙げられる。
　第１のアミノトランスフェラーゼが、Ｌ－アミノ酸アミノトランスフェラーゼであり、第２のアミノトランスフェラーゼが、Ｄ－アミノ酸アミノトランスフェラーゼである場合が好ましい（図４）。反応系に添加される第１および第２のアミノトランスフェラーゼの量は、反応を適宜進行しうる限り特に限定されない。 Examples of aminotransferases (first aminotransferases) having activity to convert 4R-IHOG to (2S, 4R) monatin (2S stereoselective activity) include L-amino acid aminotransferase and L-aspartate aminotransferase. . Specific examples include the enzymes described in International Publication 2012/050125.
Examples of the aminotransferase (second aminotransferase) having the activity of converting 4R-IHOG to (2R, 4R) monatin (2R stereoselective activity) include D-amino acid aminotransferase. Examples of the D-amino acid aminotransferase include the enzymes described in International Publication No. 03/056026, International Publication No. 2009/088949, International Publication No. 2012/147474.
It is preferred that the first aminotransferase is an L-amino acid aminotransferase and the second aminotransferase is a D-amino acid aminotransferase (FIG. 4). The amount of the first and second aminotransferases added to the reaction system is not particularly limited as long as the reaction can proceed appropriately.
　Ｌ－アミノ酸アミノトランスフェラーゼとしては、配列番号４のアミノ酸配列に対して有意なアミノ酸配列同一性を示すアミノ酸配列を含み、かつ４Ｒ－ＩＨＯＧを（２Ｓ，４Ｒ）モナティンに変換する活性を有するタンパク質を用いることができる。配列番号４のアミノ酸配列に対して有意なアミノ酸配列同一性を示すアミノ酸配列としては、配列番号４のアミノ酸配列に対して、例えば７０％以上、好ましくは８０％以上、より好ましくは８５％以上、さらにより好ましくは９０％以上、特に好ましくは９５％以上、９６％以上、９７％以上、９８％以上または９９％以上のアミノ酸配列同一性を示すアミノ酸配列が挙げられる。
　Ｄ－アミノ酸アミノトランスフェラーゼとしては、配列番号６のアミノ酸配列に対して有意なアミノ酸配列同一性を示すアミノ酸配列を含み、かつ４Ｒ－ＩＨＯＧを（２Ｒ，４Ｒ）モナティンに変換する活性を有するタンパク質を用いることができる。配列番号６のアミノ酸配列に対して有意なアミノ酸配列同一性を示すアミノ酸配列としては、配列番号６のアミノ酸配列に対して、例えば７０％以上、好ましくは８０％以上、より好ましくは８５％以上、さらにより好ましくは９０％以上、特に好ましくは９５％以上、９６％以上、９７％以上、９８％以上または９９％以上のアミノ酸配列同一性を示すアミノ酸配列が挙げられる。アミノ酸配列の同一性は、上述した方法により決定することができる。 As the L-amino acid aminotransferase, a protein containing an amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4 and having an activity of converting 4R-IHOG to (2S, 4R) monatin is used. be able to. As the amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4, for example, 70% or more, preferably 80% or more, more preferably 85% or more, relative to the amino acid sequence of SEQ ID NO: 4, Even more preferred is an amino acid sequence showing 90% or more, particularly preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity.
As the D-amino acid aminotransferase, a protein containing an amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 6 and having an activity of converting 4R-IHOG to (2R, 4R) monatin is used. be able to. As the amino acid sequence showing significant amino acid sequence identity to the amino acid sequence of SEQ ID NO: 6, for example, 70% or more, preferably 80% or more, more preferably 85% or more, relative to the amino acid sequence of SEQ ID NO: 6, Even more preferred is an amino acid sequence showing 90% or more, particularly preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity. Amino acid sequence identity can be determined by the method described above.
　Ｌ－アミノ酸アミノトランスフェラーゼはまた、（ａ’）配列番号４のアミノ酸配列を含むタンパク質、（ｂ’）配列番号４のアミノ酸配列において１または数個のアミノ酸残基の変異（例、置換、欠失、付加または挿入）を有するアミノ酸配列を含み、かつ４Ｒ－ＩＨＯＧを（２Ｓ，４Ｒ）モナティンに変換する活性を有するタンパク質であってもよい。
　Ｄ－アミノ酸アミノトランスフェラーゼはまた、（ａ）配列番号６のアミノ酸配列を含むタンパク質、（ｂ）配列番号６のアミノ酸配列において１または数個のアミノ酸残基の変異（例、置換、欠失、付加または挿入）を有するアミノ酸配列を含み、かつ４Ｒ－ＩＨＯＧを（２Ｒ，４Ｒ）モナティンに変換する活性を有するタンパク質であってもよい。
　１または数個のアミノ酸残基の変異は、アミノ酸配列中の１つの領域に導入されてもよいが、複数の異なる領域に導入されてもよい。用語「１または数個」は、上述したものと同様である。アミノ酸配列において変異を導入すべきアミノ酸残基の位置は、上述したとおり、当業者に自明である。また、アミノ酸残基が置換により変異される場合、アミノ酸残基の置換は、上述したような保存的置換であってもよい。 L-amino acid aminotransferase is also (a ′) a protein comprising the amino acid sequence of SEQ ID NO: 4, (b ′) mutation of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 4 (eg, substitution, deletion) , Addition or insertion), and a protein having an activity of converting 4R-IHOG to (2S, 4R) monatin.
D-amino acid aminotransferase is also (a) a protein comprising the amino acid sequence of SEQ ID NO: 6, (b) mutation of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 6 (eg, substitution, deletion, addition) Or a protein having an activity of converting 4R-IHOG into (2R, 4R) monatin.
The mutation of one or several amino acid residues may be introduced into one region in the amino acid sequence, but may be introduced into a plurality of different regions. The term “one or several” is similar to that described above. As described above, the position of the amino acid residue to be mutated in the amino acid sequence is obvious to those skilled in the art. In addition, when an amino acid residue is mutated by substitution, the substitution of the amino acid residue may be a conservative substitution as described above.
　Ｌ－アミノ酸アミノトランスフェラーゼは、配列番号３で表される塩基配列に相補的な塩基配列とストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされ、かつ４Ｒ－ＩＨＯＧを（２Ｓ，４Ｒ）モナティンに変換する活性を有するタンパク質が好ましい。Ｌ－アミノ酸アミノトランスフェラーゼの本活性は、可逆的であり得る。
　Ｄ－アミノ酸アミノトランスフェラーゼは、配列番号５で表される塩基配列に相補的な塩基配列とストリンジェントな条件下でハイブリダイズするＤＮＡによりコードされ、かつ４Ｒ－ＩＨＯＧを（２Ｒ，４Ｒ）モナティンに変換する活性を有するタンパク質が好ましい。Ｄ－アミノ酸アミノトランスフェラーゼの本活性は、可逆的であり得る。
　ストリンジェントな条件は、上述したとおりである。 L-amino acid aminotransferase is encoded by DNA that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 3 and converts 4R-IHOG to (2S, 4R) monatin. A protein having the activity of This activity of L-amino acid aminotransferase can be reversible.
D-amino acid aminotransferase is encoded by DNA that hybridizes with a base sequence complementary to the base sequence represented by SEQ ID NO: 5 under stringent conditions, and converts 4R-IHOG to (2R, 4R) monatin. A protein having the activity of This activity of D-amino acid aminotransferase can be reversible.
Stringent conditions are as described above.
　本発明で用いられるアミノトランスフェラーゼは、そのＮ末端部またはＣ末端部に精製用タグが付加されていてもよい。精製用タグを利用することで、アミノトランスフェラーゼを簡便に精製することができる。精製用タグとしては、例えば、ヒスチジン（Ｈｉｓ）タグ、カルモジュリン結合ペプチド（ＣＢＰ）、ストレプトタグ（Ｓｔｒｅｐ－ｔａｇ）　ＩＩ、ＦＬＡＧが挙げられる。 The aminotransferase used in the present invention may have a purification tag added to the N-terminal part or C-terminal part thereof. By using the purification tag, the aminotransferase can be easily purified. Examples of the purification tag include histidine (His) tag, calmodulin-binding peptide (CBP), strept-tag (Strep-tag) II, and FLAG.
　Ｌ－アミノ酸アミノトランスフェラーゼおよびＤ－アミノ酸アミノトランスフェラーゼの双方が用いられる場合、ケト酸（あるいはＬ－アミノ酸またはＤ－アミノ酸）、およびＬ－アミノ酸をＤ－アミノ酸に変換し得るラセマーゼを反応系に少量添加することにより、Ｌ－アミノ酸アミノトランスフェラーゼによる副反応（ケト酸→Ｌ－アミノ酸）、およびＤ－アミノ酸アミノトランスフェラーゼによる副反応（Ｄ－アミノ酸→ケト酸）をカップリングさせることができる（図４）。Ｌ－アミノ酸またはＤ－アミノ酸における「アミノ酸」としては、例えば、天然のα－アミノ酸である、アラニン、グルタミン酸、アスパラギン、システイン、グルタミン、イソロイシン、ロイシン、メチオニン、フェニルアラニン、プロリン、セリン、スレオニン、トリプトファン、チロシン、バリン、アスパラギン酸、アルギニン、ヒスチジン、およびリジンが挙げられる。ケト酸は、上述したＬ－アミノ酸またはＤ－アミノ酸からアミノ酸アミノトランスフェラーゼの作用により生成するケト酸である。Ｌ－アミノ酸をＤ－アミノ酸に変換し得るラセマーゼとしては、種々のものが知られているので、本発明では、このようなラセマーゼを用いることができる。好ましくは、ラセマーゼとしてアラニンラセマーゼが用いられる場合、ケト酸はピルビン酸であり、Ｌ－アミノ酸はＬ－アラミンであり、Ｄ－アミノ酸はＤ－アラニンである（図５）。また、ラセマーゼとしてグルタミン酸ラセマーゼが用いられる場合、ケト酸はα－ケトグルタル酸であり、Ｌ－アミノ酸はＬ－グルタミン酸であり、Ｄ－アミノ酸はＤ－グルタミン酸である（図６）。反応系に添加されるケト酸（例、ピルビン酸、α－ケトグルタル酸）の濃度、Ｌ－アミノ酸（例、Ｌ－アラニン、Ｌ－グルタミン酸）の濃度、Ｄ－アミノ酸（Ｄ－アラニン、Ｄ－グルタミン酸）の濃度、およびラセマーゼ（例、アラニンラセマーゼ、グルタミン酸ラセマーゼ）の量は、反応を適宜進行させることができる限り特に限定されない。 When both L-amino acid aminotransferase and D-amino acid aminotransferase are used, a small amount of keto acid (or L-amino acid or D-amino acid) and racemase that can convert L-amino acid to D-amino acid are added to the reaction system. By doing so, a side reaction by L-amino acid aminotransferase (keto acid → L-amino acid) and a side reaction by D-amino acid aminotransferase (D-amino acid → keto acid) can be coupled (FIG. 4). Examples of the “amino acid” in the L-amino acid or D-amino acid include natural α-amino acids such as alanine, glutamic acid, asparagine, cysteine, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, Examples include tyrosine, valine, aspartic acid, arginine, histidine, and lysine. Keto acid is a keto acid produced from the above-mentioned L-amino acid or D-amino acid by the action of amino acid aminotransferase. Various racemases that can convert L-amino acids to D-amino acids are known, and such racemases can be used in the present invention. Preferably, when alanine racemase is used as the racemase, the keto acid is pyruvate, the L-amino acid is L-alamine, and the D-amino acid is D-alanine (FIG. 5). When glutamic acid racemase is used as the racemase, the keto acid is α-ketoglutaric acid, the L-amino acid is L-glutamic acid, and the D-amino acid is D-glutamic acid (FIG. 6). The concentration of keto acid (eg, pyruvic acid, α-ketoglutaric acid) added to the reaction system, the concentration of L-amino acid (eg, L-alanine, L-glutamic acid), D-amino acid (D-alanine, D-glutamic acid) ) Concentration and the amount of racemase (eg, alanine racemase, glutamate racemase) are not particularly limited as long as the reaction can proceed appropriately.
　アミノトランスフェラーゼを作用させて（２Ｓ，４Ｒ）モナティンを（２Ｒ，４Ｒ）モナティンに異性化させる場合、当業者であれば簡単な予備的試験によって好適な条件を設定することができる。反応系に添加される（２Ｓ，４Ｒ）モナティンの濃度は、１０ｍＭ～３．０Ｍが好ましく、３００ｍＭ～１．０Ｍがより好ましい。この場合、モナティンは、反応開始前に反応系に添加されてもよいが、反応開始後に断続的あるいは継続的に添加されてもよい。反応ｐＨは、ｐＨ５．０～１１．０が好ましく、ｐＨ６．０～１０．０がより好ましく、ｐＨ７．０～９．０が更に好ましい。反応温度は、当該アミノトランスフェラーゼ反応が進行する温度である限り特に限定されないが、１０℃～６０℃が好ましく、１５℃～４２℃がより好ましい。反応時間は、特に限定されないが、１～１８０時間が好ましく、１～７６時間がより好ましい。ピリドキサールリン酸（ＰＬＰ）は、必ずしも反応系に添加する必要はないが添加する場合、ＰＬＰの濃度はアミノトランスフェラーゼ反応が進行する限り特に限定されないが、１０～１００μＭが好ましい。アミノトランスフェラーゼは、Ｌ－アミノ酸アミノトランスフェラーゼとＤ－アミノ酸アミノトランスフェラーゼの双方を用いてもよい。この場合のそれぞれの濃度は、反応が進行する限り特に限定されないが、０．０１～１０ｍｇ／ｍｌが好ましく、０．１～５ｍｇ／ｍｌがより好ましい。ケト酸（あるいはＬ－アミノ酸またはＤ－アミノ酸）、およびＬ－アミノ酸をＤ－アミノ酸に変換し得るラセマーゼを反応系に少量添加してもよい。この場合、ケト酸の濃度は１～１００ｍＭが好ましく、１０～５０ｍＭがより好ましい。Ｄ－アミノ酸の濃度は１～１００ｍＭが好ましく、２０～５０ｍＭがより好ましい。ラセマーゼの濃度は反応が進行する限り特に限定されないが、０．１～１００μｇ／ｍｌが好ましく、１～１０μｇ／ｍｌがより好ましい。 When aminotransferase is allowed to act to isomerize (2S, 4R) monatin to (2R, 4R) monatin, those skilled in the art can set suitable conditions by a simple preliminary test. The concentration of (2S, 4R) monatin added to the reaction system is preferably 10 mM to 3.0M, more preferably 300 mM to 1.0M. In this case, monatin may be added to the reaction system before starting the reaction, but may be added intermittently or continuously after starting the reaction. The reaction pH is preferably from 5.0 to 11.0, more preferably from 6.0 to 10.0, and even more preferably from 7.0 to 9.0. The reaction temperature is not particularly limited as long as the aminotransferase reaction proceeds, but is preferably 10 ° C to 60 ° C, more preferably 15 ° C to 42 ° C. The reaction time is not particularly limited, but is preferably 1 to 180 hours, and more preferably 1 to 76 hours. Pyridoxal phosphate (PLP) is not necessarily added to the reaction system, but when added, the concentration of PLP is not particularly limited as long as the aminotransferase reaction proceeds, but is preferably 10 to 100 μM. As the aminotransferase, both L-amino acid aminotransferase and D-amino acid aminotransferase may be used. Each concentration in this case is not particularly limited as long as the reaction proceeds, but is preferably 0.01 to 10 mg / ml, and more preferably 0.1 to 5 mg / ml. A small amount of keto acid (or L-amino acid or D-amino acid) and racemase capable of converting L-amino acid to D-amino acid may be added to the reaction system. In this case, the keto acid concentration is preferably 1 to 100 mM, more preferably 10 to 50 mM. The concentration of D-amino acid is preferably 1 to 100 mM, more preferably 20 to 50 mM. The concentration of racemase is not particularly limited as long as the reaction proceeds, but it is preferably 0.1 to 100 μg / ml, more preferably 1 to 10 μg / ml.
　アミノトランスフェラーゼを作用させた（２Ｓ，４Ｒ）モナティンと（２Ｒ，４Ｒ）モナティンを含む反応液から（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩を晶析する場合、当業者であれば簡単な予備的試験によって好適な条件を設定することができる。反応液中の（２Ｒ，４Ｒ）モナティン濃度としては、２０ｍＭ～３Ｍが好ましく、５０ｍＭ～１Ｍがより好ましい。反応系に添加されるマグネシウム源は、系に存在するモナティンに対して充分な量であればよいが、例えば、１０ｍＭ～１．５Ｍが好ましく、２５ｍＭ～５００ｍＭがより好ましい。温度は、０～６０℃が好ましく、１０～４０℃がより好ましい。時間は３時間～１週間が好ましく、２４時間～６０時間がより好ましい。高分子による晶析阻害を避けるため、反応液の遠心上清あるいは限外ろ過したろ液を用いる、あるいは添加する種晶の量を増やしてもよい。 Aminotransferase was allowed to act (2S, 4R) monatin and (2R, 4R) monatin from the reaction solution containing ((2R, 4R) monatin) To crystallize 2 magnesium salt, simple preliminary those skilled in the art Suitable conditions can be set by the test. The (2R, 4R) monatin concentration in the reaction solution is preferably 20 mM to 3M, and more preferably 50 mM to 1M. The magnesium source added to the reaction system may be an amount sufficient for the monatin present in the system, but is preferably 10 mM to 1.5 M, and more preferably 25 mM to 500 mM. The temperature is preferably 0 to 60 ° C, more preferably 10 to 40 ° C. The time is preferably 3 hours to 1 week, more preferably 24 hours to 60 hours. In order to avoid crystallization inhibition by the polymer, the centrifugal supernatant of the reaction solution or the ultrafiltered filtrate may be used, or the amount of seed crystals to be added may be increased.
　ラセマーゼ、あるいはアミノトランスフェラーゼを作用させて（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成させる場合、酵素反応のみでは水溶液中である一定の平衡状態に達する。しかし、本酵素反応と（２Ｒ，４Ｒ）モナティン多価金属塩晶析を同時に進行させることにより、（２Ｒ，４Ｒ）モナティンを塩結晶として反応系外に除くことができるため、平衡状態が（２Ｒ，４Ｒ）モナティンの方向に傾き、（２Ｒ，４Ｒ）モナティンの生成量を増加せしめることができる。また、アルデヒド触媒を作用させて（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成させる場合にも同様に異性化反応と（２Ｒ，４Ｒ）モナティン多価金属塩晶析を同時に進行させることにより、（２Ｒ，４Ｒ）モナティンを塩結晶として反応系外に除くことができるため、平衡状態が（２Ｒ，４Ｒ）モナティンの方向に傾き、（２Ｒ，４Ｒ）モナティンの生成量を増加せしめることができる。 When racemase or aminotransferase is allowed to act to produce (2R, 4R) monatin from (2S, 4R) monatin, the enzyme reaction alone reaches a certain equilibrium state in an aqueous solution. However, since the (2R, 4R) monatin polyvalent metal salt crystallization proceeds simultaneously with this enzyme reaction, (2R, 4R) monatin can be removed from the reaction system as a salt crystal. , 4R) in the direction of monatin, and the amount of (2R, 4R) monatin produced can be increased. Similarly, when (2R, 4R) monatin is produced from (2S, 4R) monatin by the action of an aldehyde catalyst, the isomerization reaction and (2R, 4R) monatin polyvalent metal salt crystallization are simultaneously advanced. (2R, 4R) monatin can be removed from the reaction system as a salt crystal, so that the equilibrium state is inclined in the direction of (2R, 4R) monatin and the amount of (2R, 4R) monatin produced can be increased. it can.
　アミノトランスフェラーゼを作用させて（２Ｓ，４Ｒ）モナティンを（２Ｒ，４Ｒ）モナティンに異性化させながら、同時に（２Ｒ，４Ｒ）モナティン多価金属塩晶析を行う場合、当業者であれば簡単な予備的試験によって好適な条件を設定することができる。反応系に添加される（２Ｓ，４Ｒ）モナティンの濃度は、５０ｍＭ～３Ｍが好ましく、１００ｍＭ～１Ｍがより好ましい。この場合、モナティンは、反応開始前に反応系に添加されてもよいが、反応開始後に断続的あるいは継続的に添加されてもよい。反応ｐＨは、ｐＨ５．０～１０．０が好ましく、逆アルドール分解を抑制するためにはｐＨ６．０～７．０がより好ましい。反応温度は、当該アミノトランスフェラーゼ反応が進行する温度である限り特に限定されないが、１０℃～６０℃が好ましく、１５℃～４２℃がより好ましい。反応時間は、特に限定されないが、１０～２４０時間が好ましく、１２０～２４０時間がより好ましい。ピリドキサールリン酸（ＰＬＰ）は、必ずしも反応系に添加する必要はないが、添加する場合、ＰＬＰの濃度はアミノトランスフェラーゼ反応が進行する限り特に限定されないが、１０～１００μＭが好ましい。アミノトランスフェラーゼは、Ｌ－アミノ酸アミノトランスフェラーゼとＤ－アミノ酸アミノトランスフェラーゼの双方を用いてもよい。この場合のそれぞれの濃度は、反応が進行する限り特に限定されないが、０．０１～１０ｍｇ／ｍｌが好ましく、０．１～１０ｍｇ／ｍｌがより好ましい。ケト酸（あるいはＬ－アミノ酸またはＤ－アミノ酸）、およびＬ－アミノ酸をＤ－アミノ酸に変換し得るラセマーゼを反応系に少量添加してもよい。この場合、ケト酸の濃度は１～１００ｍＭが好ましく、１０～５０ｍＭがより好ましい。Ｄ－アミノ酸の濃度は１～１００ｍＭが好ましく、２０～５０ｍＭがより好ましい。ラセマーゼの濃度は反応が進行する限り特に限定されないが、０．１～１００μｇ／ｍｌが好ましく、１～１０μｇ／ｍｌがより好ましい。この場合、アミノトランスフェラーゼやラセマーゼは反応開始前に反応系に添加されてもよいが、反応中の失活や阻害を補うため、反応開始後に断続的あるいは継続的に添加されてもよい。反応系に添加されるマグネシウム源は、系に存在するモナティンに対して充分な量であればよいが、例えば、２５ｍＭ～１．５Ｍが好ましく、５０～５００ｍＭがより好ましい。この場合、マグネシウム源は反応開始前に反応系に添加されてもよいが、酵素の失活や阻害を避けるため、反応開始後に断続的あるいは継続的に添加されてもよい。酵素による晶析阻害を避けるために、酵素源を透析膜などで隔離する、あるいは固定化してもよい。反応液中の酸化物による晶析阻害を抑制するため、反応液にアルゴンガスや窒素ガス等の不活性ガスを吹き込んでもよい。晶析阻害を抑制するためには、種晶の量を増やしてもよい。 When a (2R, 4R) monatin is isomerized to (2R, 4R) monatin by the action of aminotransferase and (2R, 4R) monatin polyvalent metal salt crystallization is simultaneously performed, a person skilled in the art can use a simple preliminary method. Suitable conditions can be set by a physical test. The concentration of (2S, 4R) monatin added to the reaction system is preferably 50 mM to 3M, more preferably 100 mM to 1M. In this case, monatin may be added to the reaction system before starting the reaction, but may be added intermittently or continuously after starting the reaction. The reaction pH is preferably pH 5.0 to 10.0, and more preferably pH 6.0 to 7.0 in order to suppress reverse aldol degradation. The reaction temperature is not particularly limited as long as the aminotransferase reaction proceeds, but is preferably 10 ° C to 60 ° C, more preferably 15 ° C to 42 ° C. The reaction time is not particularly limited, but is preferably 10 to 240 hours, more preferably 120 to 240 hours. Pyridoxal phosphate (PLP) is not necessarily added to the reaction system, but when added, the concentration of PLP is not particularly limited as long as the aminotransferase reaction proceeds, but is preferably 10 to 100 μM. As the aminotransferase, both L-amino acid aminotransferase and D-amino acid aminotransferase may be used. Each concentration in this case is not particularly limited as long as the reaction proceeds, but is preferably 0.01 to 10 mg / ml, and more preferably 0.1 to 10 mg / ml. A small amount of keto acid (or L-amino acid or D-amino acid) and racemase capable of converting L-amino acid to D-amino acid may be added to the reaction system. In this case, the keto acid concentration is preferably 1 to 100 mM, more preferably 10 to 50 mM. The concentration of D-amino acid is preferably 1 to 100 mM, more preferably 20 to 50 mM. The concentration of racemase is not particularly limited as long as the reaction proceeds, but it is preferably 0.1 to 100 μg / ml, more preferably 1 to 10 μg / ml. In this case, aminotransferase and racemase may be added to the reaction system before starting the reaction, but may be added intermittently or continuously after starting the reaction in order to compensate for inactivation or inhibition during the reaction. The magnesium source added to the reaction system may be a sufficient amount with respect to monatin present in the system, but is preferably 25 mM to 1.5 M, and more preferably 50 to 500 mM. In this case, the magnesium source may be added to the reaction system before the start of the reaction, but may be added intermittently or continuously after the start of the reaction in order to avoid deactivation or inhibition of the enzyme. In order to avoid crystallization inhibition by the enzyme, the enzyme source may be isolated or immobilized by a dialysis membrane or the like. In order to suppress crystallization inhibition by oxides in the reaction solution, an inert gas such as argon gas or nitrogen gas may be blown into the reaction solution. In order to suppress crystallization inhibition, the amount of seed crystals may be increased.
　酵素的異性化反応により（２Ｓ、４Ｒ）モナティンから（２Ｒ、４Ｒ）モナティンを生成される場合、酵素反応のみでは水溶液中である一定の平衡状態に達するが、本製法においては、酵素的異性化反応と多価金属イオンによる（２Ｒ、４Ｒ）モナティン多価金属塩の優先晶析を同時に進行させることによって、異性化反応が平衡に達する前に（２Ｒ、４Ｒ）モナティン多価金属塩を反応系外に除くことができ、酵素的異性化反応によって生成しうる（２Ｒ、４Ｒ）モナティン多価金属塩の量を増加せしめることもできる。 When (2R, 4R) monatin is produced from (2S, 4R) monatin by enzymatic isomerization reaction, a certain equilibrium state is reached in aqueous solution only by enzymatic reaction, but in this production method, enzymatic isomerization is achieved. By simultaneously proceeding the reaction and preferential crystallization of (2R, 4R) monatin polyvalent metal salt with polyvalent metal ions, the (2R, 4R) monatin polyvalent metal salt is converted into a reaction system before the isomerization reaction reaches equilibrium. The amount of (2R, 4R) monatin polyvalent metal salt that can be removed outside and can be produced by enzymatic isomerization reaction can also be increased.
　アミノトランスフェラーゼは、例えば、アミノトランスフェラーゼの産生菌から、または無細胞系による合成により、入手できる。アミノトランスフェラーゼの産生菌としては、例えば、アミノトランスフェラーゼを天然で産生している菌、およびアミノトランスフェラーゼを発現する形質転換体が挙げられる。アミノトランスフェラーゼは、精製酵素、粗酵素、または固定化酵素として用いることができる。 Aminotransferase can be obtained from, for example, an aminotransferase-producing bacterium or by synthesis in a cell-free system. Examples of the aminotransferase-producing bacteria include bacteria that naturally produce aminotransferases and transformants that express aminotransferases. The aminotransferase can be used as a purified enzyme, a crude enzyme, or an immobilized enzyme.
　アミノトランスフェラーゼの産生菌として、アミノトランスフェラーゼを発現する形質転換体が用いられる場合、この形質転換体は、アミノトランスフェラーゼの発現ベクターを作製し、次いで、この発現ベクターを宿主に導入することにより作製できる。アミノトランスフェラーゼを発現させるための宿主としては、例えば、上述したような、原核細胞および真核細胞を用いることができる。好ましい宿主の例は、Ｅ．ｃｏｌｉである。 When a transformant that expresses aminotransferase is used as an aminotransferase-producing bacterium, this transformant can be prepared by preparing an expression vector for aminotransferase and then introducing the expression vector into a host. As a host for expressing aminotransferase, for example, prokaryotic cells and eukaryotic cells as described above can be used. Examples of preferred hosts are E. coli. E. coli.
　本発明の方法では、溶媒として水溶液が用いられ、例えば、有機溶媒を含まない水および緩衝液、ならびに少量の有機溶媒を含む水および緩衝液が挙げられる。緩衝液としては、Ｔｒｉｓ緩衝液、リン酸緩衝液、炭酸緩衝液、ホウ酸緩衝液、酢酸緩衝液が挙げられる。有機溶媒としては、水と混和する有機溶媒が使用されるが、特にメタノール、エタノール、プロパノール、イソプロパノール等のアルコールが好ましい、有機溶媒は異なる２種以上のものを混合して用いてもよい。水溶液中に占める有機溶媒の割合は、１０体積％以下が好ましく、５体積％以下がより好ましく、２体積％以下が更に好ましい。触媒としてアルデヒドを用いる場合には、有機溶媒として少量の有機溶媒（例、アルデヒドのみ）を含む水溶液を、反応液として用いてもよい。一方、触媒として酵素を用いる場合には、有機溶媒を含まない水または緩衝液を、反応液として用いてもよい。 In the method of the present invention, an aqueous solution is used as a solvent, and examples thereof include water and a buffer solution containing no organic solvent, and water and a buffer solution containing a small amount of an organic solvent. Examples of the buffer solution include Tris buffer solution, phosphate buffer solution, carbonate buffer solution, borate buffer solution, and acetate buffer solution. As the organic solvent, an organic solvent miscible with water is used, but alcohols such as methanol, ethanol, propanol, and isopropanol are particularly preferable. Two or more different organic solvents may be mixed and used. The proportion of the organic solvent in the aqueous solution is preferably 10% by volume or less, more preferably 5% by volume or less, and still more preferably 2% by volume or less. When using an aldehyde as a catalyst, you may use the aqueous solution containing a small amount of organic solvents (for example, only an aldehyde) as an organic solvent as a reaction liquid. On the other hand, when an enzyme is used as a catalyst, water or a buffer solution not containing an organic solvent may be used as a reaction solution.
　本発明に使用される多価金属イオンは、特に限定されないが、多価金属塩を水溶液中に添加することにより生成させることができる。多価金属イオンの生成のために反応系に添加される多価金属塩は、周期律表において２以上の価数を有する元素であって、モナティンと塩を形成しうるものでありさえすれば、特に制限されない。人体摂取が許容されるものであるものが好ましい。具体的に、二価金属塩としては、例えば、マグネシウム、カルシウム、ストロンチウム、バリウム等のアルカリ土類金属塩；鉄、ニッケル、銅、亜鉛等の遷移金属塩等が挙げられ、三価金属としては、例えば、アルミニウム等の金属塩が挙げられる。これらは１種または、２種以上を併用しても構わない。中でも、二価金属塩が好ましく、アルカリ土類金属塩がより好ましく、マグネシウム塩、カルシウム塩、ストロンチウム塩、バリウム塩が更に好ましく、マグネシウム塩、カルシウム塩、バリウム塩が更に一層好ましく、マグネシウム塩、カルシウム塩が特に好ましい。多価金属塩は、非水和物であっても、水和物（例、一水和物、二水和物、三水和物、四水和物、五水和物、六水和物、七水和物、八水和物、九水和物）であってもよい。
　本発明で使用される多価金属塩を得る簡便な方法としては、例えば、水酸化カルシウム、水酸化マグネシウム、炭酸カルシウム、炭酸マグネシウム等の無機多価金属化合物、ならびに酢酸カルシウム、酢酸マグネシウム、シュウ酸カルシウム、ショウ酸マグネシウム、乳酸カルシウム、乳酸マグネシウム等の有機多価金属化合物を、中和、塩交換等の各種方法により処理する方法が挙げられる。 The polyvalent metal ion used in the present invention is not particularly limited, but can be generated by adding a polyvalent metal salt to an aqueous solution. The polyvalent metal salt added to the reaction system for the production of the polyvalent metal ion is an element having a valence of 2 or more in the periodic table as long as it can form a salt with monatin. There is no particular restriction. Those that are acceptable for human consumption are preferred. Specific examples of the divalent metal salt include alkaline earth metal salts such as magnesium, calcium, strontium and barium; transition metal salts such as iron, nickel, copper and zinc. For example, metal salts, such as aluminum, are mentioned. These may be used alone or in combination of two or more. Among them, divalent metal salts are preferable, alkaline earth metal salts are more preferable, magnesium salts, calcium salts, strontium salts, and barium salts are more preferable, magnesium salts, calcium salts, and barium salts are still more preferable, magnesium salts, calcium A salt is particularly preferred. Polyvalent metal salts can be non-hydrates, even hydrates (eg, monohydrate, dihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate , Heptahydrate, octahydrate, nonahydrate).
Examples of a simple method for obtaining the polyvalent metal salt used in the present invention include inorganic polyvalent metal compounds such as calcium hydroxide, magnesium hydroxide, calcium carbonate, magnesium carbonate, and calcium acetate, magnesium acetate, oxalic acid. Examples of the method include treating organic polyvalent metal compounds such as calcium, magnesium oxalate, calcium lactate, and magnesium lactate by various methods such as neutralization and salt exchange.
　多価金属イオンは、系に存在するモナティンに対して充分な量であればよいが、例えば、０．４～０．６モル当量、より好ましくは０．４５～０．５５モル当量の範囲で使用することができる。 The polyvalent metal ion may be a sufficient amount with respect to the monatin present in the system, and is, for example, in the range of 0.4 to 0.6 molar equivalent, more preferably 0.45 to 0.55 molar equivalent. Can be used.
　本発明の方法における温度は、０～５０℃が好ましく、２５～４０℃がより好ましい。本発明の方法における時間は、５時間～１週間が好ましく、１０時間～４８時間がより好ましい。 The temperature in the method of the present invention is preferably 0 to 50 ° C, more preferably 25 to 40 ° C. The time in the method of the present invention is preferably 5 hours to 1 week, more preferably 10 hours to 48 hours.
　本発明の方法におけるｐＨは、４～１０が好ましく、５～９がより好ましく、６～８が更に好ましい。ｐＨの調整は酸およびアルカリを用いて行うことができる。用いられる酸は特に限定されず、酢酸などの有機酸、または塩酸、硫酸などの無機酸を使用することができる。アルカリも特に限定されず、水酸化ナトリウム、水酸化カリウム等のアルカリ金属水酸化物、アンモニア、アミン等の有機塩基を使用することができる。 The pH in the method of the present invention is preferably 4 to 10, more preferably 5 to 9, and still more preferably 6 to 8. Adjustment of pH can be performed using an acid and an alkali. The acid used is not particularly limited, and an organic acid such as acetic acid or an inorganic acid such as hydrochloric acid or sulfuric acid can be used. The alkali is not particularly limited, and alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and organic bases such as ammonia and amines can be used.
　（２Ｒ，４Ｒ）モナティンの生成後に有機溶媒を水溶液中に添加することにより、（２Ｒ，４Ｒ）モナティン多価金属塩結晶の析出を促進することもできる。有機溶媒の添加は、水溶液中に溶解している（２Ｒ，４Ｒ）モナティンが飽和量に到達した後に行うこともまた好ましい。（２Ｒ，４Ｒ）モナティン多価金属塩結晶の析出を促進するために水溶液中に添加される有機溶媒は、水と混和する有機溶媒である限り特に限定されず、例えば、メタノール、エタノール、ｎ－プロパノール、イソプロパノール、ｎ－ブタノール、ｔ－ブタノール、ｓｅｃ－ブタノール、プロピレングリコール、アセトニトリル、ＴＨＦが挙げられる。 Precipitation of (2R, 4R) monatin polyvalent metal salt crystals can be promoted by adding an organic solvent to the aqueous solution after the (2R, 4R) monatin is produced. The addition of the organic solvent is also preferably performed after the (2R, 4R) monatin dissolved in the aqueous solution reaches the saturation amount. The organic solvent added to the aqueous solution to promote the precipitation of the (2R, 4R) monatin polyvalent metal salt crystal is not particularly limited as long as it is an organic solvent miscible with water. For example, methanol, ethanol, n- Examples include propanol, isopropanol, n-butanol, t-butanol, sec-butanol, propylene glycol, acetonitrile, and THF.
　本発明の方法によって、（２Ｒ，４Ｒ）モナティン多価金属塩結晶を得ることができる。中でも、人体摂取を許容され、容易に調製しやすいという観点で、（（２Ｒ，４Ｒ）モナティン）２二価金属塩結晶が好ましく、（（２Ｒ，４Ｒ）モナティン）２アルカリ土類金属塩結晶がより好ましく、（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶、（（２Ｒ，４Ｒ）モナティン）２カルシウム塩結晶、（（２Ｒ，４Ｒ）モナティン）２ストロンチウム塩結晶、（（２Ｒ，４Ｒ）モナティン）２バリウム塩結晶が更に好ましく、（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶、（（２Ｒ，４Ｒ）モナティン）２カルシウム塩結晶、（（２Ｒ，４Ｒ）モナティン）２バリウム塩結晶が更に一層好ましく、（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶、（（２Ｒ，４Ｒ）モナティン）２カルシウム塩結晶が特に好ましい。 By the method of the present invention, (2R, 4R) monatin polyvalent metal salt crystals can be obtained. Among these, ((2R, 4R) monatin) 2 divalent metal salt crystal is preferable and ((2R, 4R) monatin) 2 alkaline earth metal salt crystal is preferable from the viewpoint that human intake is allowed and it is easy to prepare. more preferably, ((2R, 4R) monatin) 2 magnesium salt crystals, ((2R, 4R) monatin) 2 calcium salt crystal, ((2R, 4R) monatin) 2 strontium salt crystals, ((2R, 4R) monatin) 2 barium salt crystals are more preferred, ((2R, 4R) monatin) 2 magnesium salt crystals, ((2R, 4R) monatin) 2 calcium salt crystals, ((2R, 4R) monatin) 2 barium salt crystals are even more preferred. , ((2R, 4R) monatin) 2 magnesium salt crystals, ((2R, 4R) monatin) 2 calcium salt crystals are particularly Masui.
　本発明の（２Ｒ，４Ｒ）モナティン多価金属塩結晶のうち（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶について詳述する。 Of the (2R, 4R) monatin polyvalent metal salt crystals of the present invention, ((2R, 4R) monatin) 2 magnesium salt crystals will be described in detail.
　上述の方法により（２Ｒ，４Ｒ）モナティンとマグネシウム源を含有する水溶液を静置または攪拌晶析に付することにより、結晶析出を得ることができる。溶媒中の（２Ｒ，４Ｒ）モナティン結晶濃度としては、過飽和が掛かっており結晶が析出しさえすれば特に制限は無いが０．１ｗｔ％～６０ｗｔ％が好ましい。製造に適した溶解液の粘度が得られるという観点から１ｗｔ％～５０ｗｔ％がより好ましく、５ｗｔ％～４５ｗｔ％が更に好ましく。溶解させる温度は結晶が溶解し続けさえすれば特に制限はないが、１５～４０℃が好ましい。 Crystal precipitation can be obtained by subjecting an aqueous solution containing (2R, 4R) monatin and a magnesium source to standing or stirring crystallization by the method described above. The concentration of the (2R, 4R) monatin crystal in the solvent is not particularly limited as long as supersaturation is applied and crystals are precipitated, but 0.1 wt% to 60 wt% is preferable. From the viewpoint of obtaining a viscosity of a solution suitable for production, it is more preferably 1 wt% to 50 wt%, and further preferably 5 wt% to 45 wt%. The melting temperature is not particularly limited as long as the crystals continue to dissolve, but is preferably 15 to 40 ° C.
　析出してきた結晶は、ろ過工程等の分離工程に付すことにより、容易に湿結晶を得ることができる。結晶洗浄に際しては、結晶溶媒交換を起しさえしなければ特に制限はないが、水を使用することができる。また、結晶溶媒交換を起こしさえしなければ、メタノール、エタノール、ｎ－プロパノール、イソプロパノール、ｎ－ブタノール、ｔ－ブタノール、ｓｅｃ－ブタノール、プロピレングリコール、アセトニトリル、ＴＨＦ、アセトン、ＤＭＦ，等の水と混和しうる溶媒や無機塩等も含んでいても構わない。 Precipitated crystals can be easily obtained wet crystals by subjecting them to a separation step such as a filtration step. The crystal washing is not particularly limited as long as crystal solvent exchange does not occur, but water can be used. In addition, as long as the crystal solvent exchange does not occur, it is miscible with water such as methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, sec-butanol, propylene glycol, acetonitrile, THF, acetone, DMF, etc. It may also contain a possible solvent or inorganic salt.
　こうして得られた湿結晶は、公知の乾燥工程に付することにより、乾燥結晶に導くことができる。乾燥工程に使用される乾燥設備は特に制限はなく、（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩が溶解しない程度の温度域が使用でき、減圧乾燥または気流乾燥、温風乾燥、等を使用できる。 The wet crystal thus obtained can be converted into a dry crystal by subjecting it to a known drying step. The drying equipment used in the drying process is not particularly limited, and a temperature range in which (2R, 4R) monatin does not dissolve the 2 magnesium salt can be used, and vacuum drying, airflow drying, hot air drying, etc. can be used. .
　本発明の方法で得られる（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶は、回折角度（２θ±０．２°、ＣｕＫα）として、８．９°、１１．２°、１５．０°、１７．８°、２２．５°又は４．９°、１６．８°、１８．０°、２４．６°に、固有Ｘ線回折ピークを有する。 As obtained by the method of the present invention ((2R, 4R) monatin) 2 magnesium salt crystals, the diffraction angle (2θ ± 0.2 °, CuKα) , 8.9 °, 11.2 °, 15.0 °, It has intrinsic X-ray diffraction peaks at 17.8 °, 22.5 °, or 4.9 °, 16.8 °, 18.0 °, 24.6 °.
　本発明の方法で得られる（２Ｒ，４Ｒ）モナティン多価金属塩結晶は、公知の分離精製手段、例えば、濃縮、減圧濃縮、溶媒抽出、晶出、再結晶、転溶、活性炭処理、イオン交換樹脂又は合成吸着樹脂等を用いたクロマトグラフィーなどの処理を必要に応じて組み合せることにより更に精製させることができる。 The (2R, 4R) monatin polyvalent metal salt crystal obtained by the method of the present invention is obtained by known separation and purification means, for example, concentration, concentration under reduced pressure, solvent extraction, crystallization, recrystallization, transfer dissolution, activated carbon treatment, ion exchange. Further purification can be achieved by combining treatments such as chromatography using a resin or a synthetic adsorption resin as required.
　本発明の方法で得られる（２Ｒ，４Ｒ）モナティン多価金属塩結晶は、カリウム塩、ナトリウム塩、カルシウム塩等のアルカリ金属塩、アンモニウム塩等の他の塩または遊離体に変換することができる。
　（２Ｒ，４Ｒ）モナティン多価金属塩を遊離体または他の塩に変換する方法としては、当業者に公知の方法を用いることができる。例えば、遊離体を得る方法としては、（２Ｒ，４Ｒ）モナティン多価金属塩を水、アルコール、又はこれらの混合溶媒等に溶解或いは懸濁させ、塩酸、硫酸等の酸で中和した後、（２Ｒ，４Ｒ）モナティンの遊離体を晶析する方法、或いは該塩を水に溶解し、強酸性イオン交換樹脂等により分解、脱塩し、遊離体を溶離液中に分離し、溶媒を減圧留去するか、遊離体を晶析する方法等を挙げることができる。
　他の塩に変換する場合、前記で得られた遊離体結晶を水酸化ナトリウム、水酸化カリウム、水酸化カルシウム等のアルカリ金属水溶液、アンモニア水溶液等に溶解し、溶媒を減圧留去するか、（２Ｒ，４Ｒ）モナティン塩を晶析する方法、または遊離体を含む樹脂溶離液に水酸化ナトリウム、水酸化カリウム、水酸化カルシウム等のアルカリ金属水溶液、或いはアンモニア水溶液等を添加し、溶媒を減圧留去するか、（２Ｒ，４Ｒ）モナティン塩を晶析する方法等を挙げることができる。 The (2R, 4R) monatin polyvalent metal salt crystals obtained by the method of the present invention can be converted to other salts such as potassium salts, sodium salts, calcium salts, and other salts or free forms such as ammonium salts. .
As a method for converting the (2R, 4R) monatin polyvalent metal salt into a free form or other salts, methods known to those skilled in the art can be used. For example, as a method for obtaining a free form, (2R, 4R) monatin polyvalent metal salt is dissolved or suspended in water, alcohol, or a mixed solvent thereof, and neutralized with an acid such as hydrochloric acid or sulfuric acid. (2R, 4R) Method of crystallizing monatin free form, or dissolving the salt in water, decomposing and desalting with a strong acidic ion exchange resin, etc., separating the free form in the eluent, and reducing the solvent under reduced pressure The method of distilling off or crystallizing a free body etc. can be mentioned.
When converting to other salts, the free crystals obtained above are dissolved in an aqueous alkali metal solution such as sodium hydroxide, potassium hydroxide or calcium hydroxide, an aqueous ammonia solution or the like, and the solvent is distilled off under reduced pressure ( 2R, 4R) Method of crystallizing monatin salt, or adding an aqueous solution of alkali metal such as sodium hydroxide, potassium hydroxide or calcium hydroxide, or aqueous ammonia solution to the resin eluent containing educts, and distilling the solvent under reduced pressure Or a method of crystallizing (2R, 4R) monatin salt.
　その他、各種食品素材の他に、本発明の効果を阻害しない程度に飲料、食品、医薬品、医薬部外品、飼料等の経口製品として使用可能な各種添加剤を使用することができる。 In addition to various food materials, various additives that can be used as oral products such as beverages, foods, pharmaceuticals, quasi drugs, and feeds can be used to the extent that the effects of the present invention are not impaired.
　本発明のモナティン多価金属塩結晶は、飲料、食品、医薬品、医薬部外品、飼料等の経口製品に使用することができる。その剤型は、特に限定されず、例えば、粉末、顆粒、キューブ、ペースト、液体などが挙げられる。 The monatin polyvalent metal salt crystal of the present invention can be used for oral products such as beverages, foods, pharmaceuticals, quasi drugs, and feeds. The dosage form is not particularly limited, and examples thereof include powder, granules, cubes, pastes, and liquids.
　以下の実施例により、本発明を詳細に説明するが、これらの実施例により本発明が限定されるものではない。 The present invention will be described in detail by the following examples, but the present invention is not limited by these examples.
（ＨＰＬＣ分析条件）
　実施例１では、ＨＰＬＣ分析を実施した場合、当該実施例に示す条件にてＨＰＬＣ分析を実施した。
　検出器：紫外吸光光度計（測定波長：２１０ｎｍ）
　カラム温度：４０℃
　カラム　　：ＣＡＰＣＥＬＬＰＡＫ　Ｃ１８　Ｔｙｐｅ　ＭＧＩＩ，内径３ｍｍ，
　　　　　　　長さ２５ｃｍ，粒径５μｍ、資生堂（株）
　移動相　　：Ａ液　２０ｍＭ燐酸二水素カリウム水溶液：アセトニトリル＝１００：５
　　　　　　　Ｂ液　２０ｍＭ燐酸二水素カリウム水溶液：アセトニトリル＝６０：４０
　グラジエントプログラム：以下の表１を参照 (HPLC analysis conditions)
In Example 1, when HPLC analysis was performed, HPLC analysis was performed under the conditions shown in the example.
Detector: UV absorption photometer (measurement wavelength: 210 nm)
Column temperature: 40 ° C
Column: CAPCELLPAK C18 Type MGII, inner diameter 3 mm,
Length 25cm, particle size 5μm, Shiseido Co., Ltd.
Mobile phase: A solution 20 mM potassium dihydrogen phosphate aqueous solution: acetonitrile = 100: 5
B liquid 20 mM potassium dihydrogen phosphate aqueous solution: Acetonitrile = 60: 40
Gradient program: see Table 1 below
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　流量　　　：毎分０．４５ｍＬ
　注入量　　：１０μＬ
　分析時間　：６０分 Flow rate: 0.45 mL per minute
Injection volume: 10 μL
Analysis time: 60 minutes
　得られた結晶〔（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩〕の含水量およびマグネシウム含量は、水分測定法、イオンクロマトグラフィーによるカチオン分析法により解析した。実施した水分測定法、カチオン分析法の詳細を以下に示す。 The water content and magnesium content of the obtained crystals [((2R, 4R) monatin) 2 magnesium salt] were analyzed by a moisture measurement method and a cation analysis method by ion chromatography. Details of the water measurement method and the cation analysis method performed are shown below.
（水分測定法）
　測定装置：平沼水分自動測定装置　ＡＱＶ－２０００　（平沼産業（株）製）
　測定条件：滴定液＝Ｈｙｄｒａｎａｌ　Ｃｏｍｐｏｓｉｔｅ　５Ｋ（Ｒｉｅｄｅｌ　ｄｅ　Ｈａｅｎ社製） (Moisture measurement method)
Measuring device: Hiranuma automatic moisture measuring device AQV-2000 (Hiranuma Sangyo Co., Ltd.)
Measurement conditions: titrant = Hydranal Composite 5K (Riedel de Haen)
（カチオン分析法）
　装置：東ソーＩＣ２００１
　カラム：ＴＳＫｇｅｌ　ＳｕｐｅｒＩＣ－Ｃａｔｉｏｎ（４．６×１５０ｍｍ）
　ガードカラム：ＴＳＫｇｅｌ　ＳｕｐｅｒＩＣ－Ｃａｔｉｏｎ（１ｃｍ）
　サプレスゲル：ＴＳＫｇｅｌ　ＴＳＫｓｕｐｐｒｅｓｓＩＣ－Ｃ
　カラム温度：４０℃
　溶離液流量：０．７ｍｌ／ｍｉｎ．
　サンプル注入量：３０μｌ
　検出：電気伝導度
　溶離液組成：２．２ｍＭメタンスルホン酸＋１．０ｍＭ　１８－クラウン－６－エーテル＋０．５ｍＭヒスチジン混合水溶液 (Cation analysis method)
Device: Tosoh IC2001
Column: TSKgel SuperIC-Cation (4.6 × 150 mm)
Guard column: TSKgel SuperIC-Cation (1 cm)
Suppress Gel: TSKgel TSKsuppressIC-C
Column temperature: 40 ° C
Eluent flow rate: 0.7 ml / min.
Sample injection volume: 30 μl
Detection: Electrical conductivity Eluent composition: 2.2 mM methanesulfonic acid +1.0 mM 18-crown-6-ether +0.5 mM histidine mixed aqueous solution
〔粉末Ｘ線回折測定方法〕
１）サンプル結晶０．５ｇを採取し、メノウ乳鉢で６０秒間すりつぶした。得られた粉末をガラスプレートにセットし、上から圧力をかけて平らにした。直ちに粉末Ｘ線回折装置にセットし、以下の条件で測定した。
２）Ｃｕ－Ｋα線による粉末Ｘ線回折の測定は、スペクトリス株式会社製Ｘ線回折装置ＰＷ３０５０を用い、管球：Ｃｕ、管電流：３０ｍＡ、管電圧：４０ｋＶ、サンプリング幅：０．０２０°、走査速度：３°／ｍｉｎ、波長：１．５４０５６Å、測定回折角範囲（２θ）：４～３０°の条件で測定した。
測定プログラム：Ｘ’ＰＥＲＴ　ＤＡＴＡ　ＣＯＬＬＥＣＴＩＯＮ
解析プログラム：Ｘ’ＰＥＲＴ　Ｈｉｇｈ　Ｓｃｏｒｅ
３）得られたデータをエクセルにてグラフ化し、４～３０°の範囲の特徴的鋭角極大ピークを読み取った。本方法の回折角の誤差は±０．２°である。 [Powder X-ray diffraction measurement method]
1) 0.5 g of sample crystals were collected and ground in an agate mortar for 60 seconds. The obtained powder was set on a glass plate and flattened by applying pressure from above. The sample was immediately set on a powder X-ray diffractometer and measured under the following conditions.
2) Measurement of powder X-ray diffraction by Cu-Kα ray was performed using an X-ray diffractometer PW3050 manufactured by Spectris Co., Ltd., tube: Cu, tube current: 30 mA, tube voltage: 40 kV, sampling width: 0.020 ° The measurement was performed under the conditions of scanning speed: 3 ° / min, wavelength: 1.54056 mm, measurement diffraction angle range (2θ): 4 to 30 °.
Measurement program: X'PERT DATA COLLECTION
Analysis program: X'PERT High Score
3) The obtained data was graphed with Excel, and a characteristic acute maximum peak in the range of 4 to 30 ° was read. The error of the diffraction angle of this method is ± 0.2 °.
参考例１：（２Ｓ，４Ｒ）モナティンの合成
　還元反応濃縮液１０１．４０ｇ（モナティン３６．６２ｇ、１２５．２８ミリモル含有、（２Ｓ，４Ｒ）：（２Ｒ，４Ｒ）＝３２：６８）にエタノール１４９．００ｇを加えた後、種晶として（２Ｒ，４Ｒ）モナティンカリウム塩一水和物０．２５ｇを添加し、５６℃、４時間攪拌し、（２Ｒ，４Ｒ）モナティンカリウム塩一水和物の優先晶析を行った。析出した結晶をろ過により分離し（湿結晶３１．２７ｇ）、母液２２５．８０ｇを得た（モナティン２２．４１ｇ、７６．６８ミリモル含有、（２Ｓ，４Ｒ）：（２Ｒ，４Ｒ）＝５３：４７）。この母液を１０℃に冷却した後、５時間攪拌し、（２Ｓ，４Ｒ）モナティンカリウム塩二水和物の晶析を行った。析出した結晶をろ過により分離し（湿結晶３２．７４ｇ）、減圧乾燥後、目的とする（２Ｓ，４Ｒ）モナティンカリウム塩二水和物９．８８ｇ（１５．６８ミリモル）を得た（ＨＰＬＣ純度：５５．５％）。この粗結晶９．３５ｇを水２５．３７ｇに溶解し、その溶解液にエタノール５８．９９ｇを加え２５℃にて５時間攪拌し、（２Ｓ，４Ｒ）モナティンカリウム塩二水和物の精晶析を行った。析出した結晶をろ過により分離し（湿結晶４．４９ｇ）、減圧乾燥後、目的とする（２Ｓ，４Ｒ）モナティンカリウム塩二水和物３．７５ｇ（９．６２ミリモル）を得た（ＨＰＬＣ純度：９６．０％）。
　再度、上述操作を繰り返し、ＨＰＬＣ純度１００％の（２Ｓ，４Ｒ）モナティンカリウム塩二水和物を得た。 Reference Example 1: Synthesis of (2S, 4R) monatin 101.40 g of the reduction reaction concentrate (monatin 36.62 g, containing 125.28 mmol, (2S, 4R) :( 2R, 4R) = 32: 68) and ethanol 149 After adding 0.000 g, 0.25 g of (2R, 4R) monatin potassium salt monohydrate is added as a seed crystal and stirred at 56 ° C. for 4 hours, and (2R, 4R) monatin potassium salt monohydrate is added. Preferential crystallization of the product was performed. The precipitated crystals were separated by filtration (31.27 g of wet crystals) to obtain 225.80 g of mother liquor (22.41 g of monatin, containing 76.68 mmol, (2S, 4R) :( 2R, 4R) = 53: 47. ). The mother liquor was cooled to 10 ° C. and stirred for 5 hours to crystallize (2S, 4R) monatin potassium salt dihydrate. The precipitated crystals were separated by filtration (wet crystals 32.74 g) and dried under reduced pressure to obtain 9.88 g (15.68 mmol) of the desired (2S, 4R) monatin potassium salt dihydrate (HPLC) Purity: 55.5%). 9.35 g of this crude crystal was dissolved in 25.37 g of water, 59.99 g of ethanol was added to the solution, and the mixture was stirred at 25 ° C. for 5 hours to obtain a crystal of (2S, 4R) monatin potassium salt dihydrate. Analysis was performed. The precipitated crystals were separated by filtration (4.49 g wet crystals) and dried under reduced pressure to obtain 3.75 g (9.62 mmol) of the desired (2S, 4R) monatin potassium salt dihydrate (HPLC) (Purity: 96.0%).
The above operation was repeated again to obtain (2S, 4R) monatin potassium salt dihydrate having an HPLC purity of 100%.
参考例２：モナティン金属塩の溶解度の測定
　（２Ｓ，４Ｒ）モナティンカリウム塩および（２Ｒ，４Ｒ）モナティンカリウム塩の水（Ｈ２Ｏ）に対する溶解度を測定した。溶解度の測定は、水１ｇに（２Ｓ，４Ｒ）モナティンカリウム塩１ｇ又は（２Ｒ，４Ｒ）モナティンカリウム塩１ｇを添加して２５℃で撹拌後、スラリーをフィルターろ過し、得られたろ液をＨＰＬＣ分析した。その結果、（２Ｓ，４Ｒ）モナティンカリウム塩および（２Ｒ，４Ｒ）モナティンカリウム塩の溶解度は、それぞれ、２０．１ｗｔ％および３７．８ｗｔ％であり、（２Ｒ，４Ｒ）モナティンカリウム塩の溶解度は、（２Ｓ，４Ｒ）モナティンカリウム塩の溶解度よりも高かった。
　次いで、（２Ｓ，４Ｒ）モナティンマグネシウム塩および（２Ｒ，４Ｒ）モナティンマグネシウム塩の水（Ｈ２Ｏ）に対する溶解度を測定した。溶解度の測定は、上述した方法と同様に行った。（２Ｓ，４Ｒ）モナティンマグネシウム塩については、先ず、（２Ｓ，４Ｒ）モナティンカリウム塩に等量の塩化マグネシウムを添加後、エタノールを更に添加した。次いで、得られたスラリーをろ過し、湿結晶を減圧乾燥することにより、（２Ｓ，４Ｒ）モナティンマグネシウム塩を調製した。（２Ｒ，４Ｒ）モナティンマグネシウム塩については、先ず、（２Ｒ，４Ｒ）モナティンカリウム塩に等量の塩化マグネシウムを添加後、メタノールを更に添加した。次いで、得られたスラリーをろ過し、湿結晶を減圧乾燥することにより、（２Ｒ，４Ｒ）モナティンマグネシウム塩を調製した。
　その結果、（２Ｓ，４Ｒ）モナティンマグネシウム塩および（２Ｒ，４Ｒ）モナティンマグネシウム塩の溶解度は、それぞれ、１４．５ｗｔ％および１．６ｗｔ％であり、（２Ｒ，４Ｒ）モナティンマグネシウム塩の溶解度は、（２Ｓ，４Ｒ）モナティンマグネシウム塩の溶解度よりも著しく低かった。 Reference Example 2: Measurement of solubility of monatin metal salt The solubility of (2S, 4R) monatin potassium salt and (2R, 4R) monatin potassium salt in water (H 2 O) was measured. The solubility was measured by adding 1 g of (2S, 4R) monatin potassium salt or 1 g of (2R, 4R) monatin potassium salt to 1 g of water, stirring at 25 ° C., filtering the slurry, and filtering the obtained filtrate. HPLC analysis. As a result, the solubility of (2S, 4R) monatin potassium salt and (2R, 4R) monatin potassium salt was 20.1 wt% and 37.8 wt%, respectively. The solubility was higher than that of (2S, 4R) monatin potassium salt.
Next, the solubility of (2S, 4R) monatin magnesium salt and (2R, 4R) monatin magnesium salt in water (H 2 O) was measured. The solubility was measured in the same manner as described above. For the (2S, 4R) monatin magnesium salt, first, an equal amount of magnesium chloride was added to the (2S, 4R) monatin potassium salt, and then ethanol was further added. Next, the obtained slurry was filtered, and the wet crystals were dried under reduced pressure to prepare (2S, 4R) monatin magnesium salt. For the (2R, 4R) monatin magnesium salt, first, an equal amount of magnesium chloride was added to the (2R, 4R) monatin potassium salt, and then methanol was further added. Next, the obtained slurry was filtered, and the wet crystals were dried under reduced pressure to prepare (2R, 4R) monatin magnesium salt.
As a result, the solubility of (2S, 4R) monatin magnesium salt and (2R, 4R) monatin magnesium salt was 14.5 wt% and 1.6 wt%, respectively. The solubility was significantly lower than that of the (2S, 4R) monatin magnesium salt.
参考例３：（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶の調製
　（２Ｒ，４Ｒ）モナティンカリウム塩結晶１０ｇ（２８．５ミリモル）を水２０ｍｌに溶解し、室温にて５０１ｍM塩化マグネシウム水溶液２８．３ｍLを添加した。２５℃で１８時間攪拌後モナティン溶解液にメタノール８０ｇを加え室温で６時間攪拌した。析出した結晶をろ別し、８０％メタノール５０ｇで２時間スラリー洗浄し、濾過後４０℃で減圧乾燥を行った。乾燥結晶を４４℃７８％の恒温恒湿器で２４時間保存し、さらに４０℃で減圧乾燥し（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶８．８ｇを得た。 Reference Example 3: ((2R, 4R) monatin) Preparation of 2 magnesium salt crystals (2R, 4R) Mona Tin potassium salt crystals 10g of (28.5 mmol) was dissolved in water 20 ml, magnesium 501mM chloride at room temperature an aqueous solution 28 .3 mL was added. After stirring at 25 ° C. for 18 hours, 80 g of methanol was added to the monatin solution, followed by stirring at room temperature for 6 hours. The precipitated crystals were separated by filtration, washed with slurry with 50 g of 80% methanol for 2 hours, filtered and dried under reduced pressure at 40 ° C. The dried crystals were stored for 24 hours in a constant temperature and humidity chamber at 44 ° C. and 78%, and further dried under reduced pressure at 40 ° C. ((2R, 4R) monatin) to obtain 8.8 g of 2 magnesium salt crystals.
１Ｈ－ＮＭＲ（ｉｎ　Ｄ２Ｏ）
１．９４－２．０１（１Ｈ，ｑ），２．５７－２．６１（１Ｈ，ｑ），２．９９－３．０３（１Ｈ，ｄ），３．１９－３．２３（１Ｈ，ｄ），３．５４－３．５７（１Ｈ，ｑ），７．０５－７．１７（３Ｈ，ｍ），７．４０－７．４２（１Ｈ，ｍ），７．６４－７．６６（１Ｈ，ｍ）
ＥＳＩ－ＭＳ：２９３．１（Ｍ+Ｈ）＋、２９１．１（Ｍ-Ｈ）－ 1 H-NMR (in D 2 O)
1.94-2.01 (1H, q), 2.57-2.61 (1H, q), 2.99-3.03 (1H, d), 3.19-3.23 (1H, d ), 3.54-3.57 (1H, q), 7.05-7.17 (3H, m), 7.40-7.42 (1H, m), 7.64-7.66 (1H) , M)
ESI-MS: 293.1 (M + H) + , 291.1 (M−H) −
水分含量：１０．８ｗｔ％
マグネシウム含量：３．６ｗｔ％
固有Ｘ線回折ピーク（２θ±０．２°、ＣｕＫα）：８．９°、１１．２°、１５．０°、１７．８°、２２．５°（図１２） Water content: 10.8wt%
Magnesium content: 3.6 wt%
Intrinsic X-ray diffraction peak (2θ ± 0.2 °, CuKα): 8.9 °, 11.2 °, 15.0 °, 17.8 °, 22.5 ° (FIG. 12)
参考例４：（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶の調製
　（２Ｒ，４Ｒ）モナティンカリウム塩結晶１２０ｇ（３４５ミリモル）を水１５０ｍｌに溶解し、６０℃にて硫酸マグネシウム４．１５ｇ（３４．５ミリモル）を添加した。更に硫酸マグネシウム１６．６１ｇ（１３８ミリモル）水溶液（水１００ｍｌ）を６．４時間かけて添加した。添加終了後、析出した結晶をろ別し、水１００ｍｌで洗浄して湿結晶を得た（２０４．７ｇ）。湿結晶を４０℃で減圧乾燥しマグネシウム塩結晶１０５ｇを得た。さらに少量混在する硫酸カリウムを除くため乾燥結晶１０５ｇに水４００ｍｌを添加し２５℃で１．５時間攪拌した。得られたスラリーをろ別し、水３００ｍｌで洗浄して湿結晶を得た（１５３．９ｇ）。湿結晶を４０℃で減圧乾燥し（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶８５．７ｇを得た。 Reference Example 4: ((2R, 4R) monatin) Preparation of 2 magnesium salt crystals (2R, 4R) Mona Tin potassium salt crystals 120 g (345 mmol) was dissolved in water 150 ml, magnesium sulfate at 60 ° C. 4.15 g ( 34.5 mmol) was added. Further, 16.61 g (138 mmol) of magnesium sulfate (100 ml of water) was added over 6.4 hours. After completion of the addition, the precipitated crystals were separated by filtration and washed with 100 ml of water to obtain wet crystals (204.7 g). The wet crystals were dried under reduced pressure at 40 ° C. to obtain 105 g of magnesium salt crystals. Further, 400 ml of water was added to 105 g of the dried crystals and stirred at 25 ° C. for 1.5 hours in order to remove a small amount of potassium sulfate. The resulting slurry was filtered and washed with 300 ml of water to obtain wet crystals (153.9 g). The wet crystals were dried under reduced pressure at 40 ℃ ((2R, 4R) monatin) was obtained 2 magnesium salt crystals 85.7 g.
固有Ｘ線回折ピーク（２θ±０．２°、ＣｕＫα）：４．９°、１６．８°、１８．０°、２４．６°（図１３）
水分含量：６．０ｗｔ％
マグネシウム含量：３．６１ｗｔ％ Intrinsic X-ray diffraction peaks (2θ ± 0.2 °, CuKα): 4.9 °, 16.8 °, 18.0 °, 24.6 ° (FIG. 13)
Water content: 6.0wt%
Magnesium content: 3.61 wt%
参考例５：（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶の調製
　（２Ｒ，４Ｒ）モナティンフリー体結晶３０ｇ（１００ミリモル）を水３００ｍｌに分散し、６５℃にて水酸化マグネシウム３．２１ｇ（５５ミリモル）を添加した。６５℃で１時間攪拌した。析出した結晶（２７．２８ｇ）をろ別し、４０℃で４時間減圧乾燥を行ってマグネシウム塩結晶２２．２９ｇを得た。 Reference Example 5: ((2R, 4R) monatin) Preparation of 2 magnesium salt crystals (2R, 4R) Mona tin-free substance crystals 30 g (100 mmol) was dispersed in water 300 ml, the magnesium hydroxide at 65 ° C. 3.21 g (55 mmol) was added. Stir at 65 ° C. for 1 hour. The precipitated crystals (27.28 g) were filtered off and dried under reduced pressure at 40 ° C. for 4 hours to obtain 22.29 g of magnesium salt crystals.
水分含量：２１．２２ｗｔ％
マグネシウム含量：３．４５ｗｔ％
固有Ｘ線回折ピーク（２θ±０．２°、ＣｕＫα）：８．７°、１０．５°、１５．９°、１７．４°２１．０°、２５．６°（図１４） Water content: 21.22 wt%
Magnesium content: 3.45 wt%
Intrinsic X-ray diffraction peaks (2θ ± 0.2 °, CuKα): 8.7 °, 10.5 °, 15.9 °, 17.4 ° 21.0 °, 25.6 ° (FIG. 14)
実施例１：（２Ｒ，４Ｒ）モナティンの合成
　水　５ｍＬに（２Ｓ，４Ｒ）モナティンカリウム塩二水和物　１．２５ｇ（３．４２ｍｍｏｌ）を溶解し、サリチルアルデヒド０．０５２１６ｇ（０．０４２８ｍｍｏｌ）及び塩化マグネシウム６水和物０．３４７６ｇ（１．７１ｍｍｏｌ）を添加した。６５℃で４２時間後に種晶〔参考例３で得られた結晶と同じ結晶形を有する（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶〕を添加し、更に１９１時間撹拌した。得られたスラリー溶液を室温まで冷却してから濾過を行い、水１ｇで結晶洗浄を行った後、湿結晶を４０℃で減圧乾燥して（２Ｒ，４Ｒ）モナティン０．４４６ｇを取得した。得られた結晶、母液、洗液をＨＰＬＣで分析し、収率及び品質解析を実施した。固有Ｘ線回折ピークから、参考例３で得られた結晶と同じ結晶形の、（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶が得られたことが確認された。 Example 1: Synthesis of (2R, 4R) monatin 1.25 g (3.42 mmol) of (2S, 4R) monatin potassium salt dihydrate was dissolved in 5 mL of water, and 0.05216 g (0.0428 mmol) of salicylaldehyde was dissolved. And 0.3476 g (1.71 mmol) of magnesium chloride hexahydrate were added. After 42 hours at 65 ° C., seed crystals [((2R, 4R) monatin) 2 magnesium salt crystals having the same crystal form as that obtained in Reference Example 3] were added, and the mixture was further stirred for 191 hours. The obtained slurry solution was cooled to room temperature, filtered, washed with 1 g of water, and then wet crystals were dried under reduced pressure at 40 ° C. to obtain 0.446 g of (2R, 4R) monatin. The obtained crystals, mother liquor, and washings were analyzed by HPLC to carry out yield and quality analysis. From the intrinsic X-ray diffraction peak, it was confirmed that ((2R, 4R) monatin) 2 magnesium salt crystals having the same crystal form as that obtained in Reference Example 3 were obtained.
ＨＰＬＣ　ａｒｅａ純度（２１０ｎｍ）：９４．２％
１Ｈ－ＮＭＲ（ｉｎ　Ｄ２Ｏ）
１．９３－２．００（１Ｈ，ｄｄ），２．５７－２．６１（１Ｈ，ｄｄ），２．９９－３．０２（１Ｈ，ｄ），３．１９－３．２２（１Ｈ，ｄ）３．５５－３．５６（１Ｈ，ｄｄ），７．０４－７．１５（３Ｈ，ｍ），７．３９－７．４１（１Ｈ，ｍ），７．６４－７．６６（１Ｈ，ｄ） HPLC area purity (210 nm): 94.2%
1 H-NMR (in D 2 O)
1.93-2.00 (1H, dd), 2.57-2.61 (1H, dd), 2.99-3.02 (1H, d), 3.19-3.22 (1H, d ) 3.55-3.56 (1H, dd), 7.04-7.15 (3H, m), 7.39-7.41 (1H, m), 7.64-7.66 (1H, d)
水分含量：１４．７ｗｔ％
マグネシウム含量：３．４ｗｔ％
固有Ｘ線回折ピーク（２θ±０．２°、ＣｕＫα）：８．９°、１１．２°、１５．０°、１７．８°、２２．５°（図７） Water content: 14.7wt%
Magnesium content: 3.4 wt%
Intrinsic X-ray diffraction peak (2θ ± 0.2 °, CuKα): 8.9 °, 11.2 °, 15.0 °, 17.8 °, 22.5 ° (FIG. 7)
実施例２：Ｅ．ｃｏｌｉでのラセマーゼの発現
（１）ラセマーゼ発現プラスミドの構築
　ラセマーゼＲａｃ３９のＤＮＡ配列の５’末端にＮｄｅＩ認識配列を、３’末端にＸｈｏＩ認識配列を付与したＤＮＡ配列を、ＧｅｎＳｃｒｉｐｔ社のＯｐｔｉｍｕｍＧｅｎｅ　Ｃｏｄｏｎ　Ｏｐｔｉｍｉｚａｔｉｏｎ　Ａｎａｌｙｓｉｓに供し、Ｅ．ｃｏｌｉでの遺伝子発現効率が最適化された合成ＤＮＡ（配列番号１）を設計し、合成した。 Example 2: E.E. Expression of racemase in E. coli (1) Construction of a racemase expression plasmid A DNA sequence having an NdeI recognition sequence at the 5 ′ end of the DNA sequence of racemase Rac39 and an XhoI recognition sequence at the 3 ′ end, and OptiGene Codon Optimization Analysis of GenScript. E. A synthetic DNA (SEQ ID NO: 1) with optimized gene expression efficiency in E. coli was designed and synthesized.
　合成ＤＮＡをＮｄｅＩ、ＸｈｏＩで制限酵素処理し、同様にＮｄｅＩ、ＸｈｏＩで処理したｐＥＴ－２２ｂ（Ｎｏｖａｇｅｎ）とライゲーションした。このライゲーション溶液でＥ．ｃｏｌｉを形質転換し、アンピシリン耐性株の中から目的のプラスミドを抽出した。これらのプラスミドをｐＥＴ－２２－Ｒａｃ３９－Ｈｉｓと命名した。これらのプラスミドでは、Ｃ末端にＨｉｓ－ｔａｇが付与されたラセマーゼ（Ｒａｃ３９－Ｈｉｓ）が発現する。 The synthetic DNA was subjected to restriction enzyme treatment with NdeI and XhoI, and ligated with pET-22b (Novagen) similarly treated with NdeI and XhoI. With this ligation solution E. coli was transformed and the target plasmid was extracted from the ampicillin resistant strain. These plasmids were named pET-22-Rac39-His. In these plasmids, racemase (Rac39-His) with His-tag added at the C-terminus is expressed.
（２）Ｅ．ｃｏｌｉ発現株からのＨｉｓタグ付加ラセマーゼの精製
　構築した発現プラスミドｐＥＴ－２２－Ｒａｃ３９－ＨｉｓをＥ．ｃｏｌｉ　ＢＬ２１（ＤＥ３）に導入し、形質転換体をアンピシリン１００ｍｇ／ｌを含むＯｖｅｒｎｉｇｈｔ　Ｅｘｐｒｅｓｓ　Ｉｎｓｔａｎｔ　ＴＢ　Ｍｅｄｉｕｍ（Ｎｏｖａｇｅｎ）　１００ｍｌに白金耳接種し、５００ｍｌ容坂口フラスコを用いて１６時間振盪させた。振盪温度は２５℃で行った。培養終了後、２００ｍｌの得られた培養液より菌体を遠心分離により集め、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１０ｍＭにて洗浄、懸濁し、超音波破砕を行った。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
　得られた可溶性画分を、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１０ｍＭで平衡化したＨｉｓ－ｔａｇタンパク質精製カラムＨｉｓ　ＴＡＬＯＮ　ｓｕｐｅｒｆｌｏｗ　５ｍｌ　Ｃａｒｔｒｉｄｇｅ（Ｃｌｏｎｔｅｃｈ）に供して担体に吸着させた。担体に吸着しなかったタンパク質（非吸着タンパク質）をＴｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１０ｍＭを用いて洗い流した後、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１５０ｍＭを用いて、５ｍｌ／ｍｉｎの流速で吸着したタンパク質の溶出を行った。得られた画分を集めて、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＰＬＰ　１０μＭで希釈し、ラセマーゼ溶液とした。 (2) E.E. Purification of His-tagged racemase from E. coli expression strain The constructed expression plasmid pET-22-Rac39-His was transformed into E. coli. E. coli BL21 (DE3) was introduced, and the transformant was inoculated into 100 ml of Overnight Express Instant Medium (Novagen) containing 100 mg / l of ampicillin, and shaken for 16 hours using a 500 ml Sakaguchi flask. The shaking temperature was 25 ° C. After completion of the culture, bacterial cells were collected from 200 ml of the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl, and 10 mM imidazole, and subjected to ultrasonic disruption. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
The obtained soluble fraction was subjected to His-tag protein purification column His TALON superflow 5 ml Cartridge (Clontech) equilibrated with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, and Imidazole 10 mM, and was adsorbed on the carrier. After washing the protein not adsorbed on the carrier (non-adsorbed protein) with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, Imidazole 10 mM, Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, Imidazole 150 mM Was used to elute the adsorbed protein at a flow rate of 5 ml / min. The obtained fractions were collected and diluted with Tris-HCl (pH 7.6) 20 mM, PLP 10 μM to obtain a racemase solution.
実施例３：（２Ｓ，４Ｒ）モナティンまたは（２Ｒ，４Ｒ）モナティンを基質とした異性化反応
　精製したラセマーゼを用いて、以下の条件で１５分間反応を行った。反応はエッペンチューブを用いて０．１ｍｌで行った。反応終了後、サンプルに等量の反応停止液２００ｍＭ　クエン酸Ｎａ溶液（ｐＨ４．５）を加えた。分析にはＨＰＬＣを用いた。 Example 3: Isomerization reaction using (2S, 4R) monatin or (2R, 4R) monatin as a substrate A purified racemase was used for 15 minutes under the following conditions. The reaction was performed in 0.1 ml using an Eppendorf tube. After completion of the reaction, an equal amount of a reaction stop solution 200 mM Na citrate solution (pH 4.5) was added to the sample. HPLC was used for the analysis.
　反応条件：（２Ｓ，４Ｒ）モナティンまたは（２Ｒ，４Ｒ）モナティン　２０ｍＭ、ＰＬＰ　５０μＭ、Ｔｒｉｓ－ＨＣｌ（ｐＨ８．０）　１００ｍＭ、ラセマーゼ精製酵素　０．７２ｍｇ／ｍｌ、２５℃、１２０ｒｐｍ。 Reaction conditions: (2S, 4R) monatin or (2R, 4R) monatin 20 mM, PLP 50 μM, Tris-HCl (pH 8.0) 100 mM, racemase purified enzyme 0.72 mg / ml, 25 ° C., 120 rpm.
　（２Ｓ，４Ｒ）モナティンおよび（２Ｒ，４Ｒ）モナティンの定量は、ＨＰＬＣ分析により行った。分析条件は、以下に示す通りである。
　　　移動相：２０ｍＭ　ＫＨ２ＰＯ４／アセトニトリル＝１００／５
　　　流速：１．０ｍｌ／ｍｉｎ
　　　カラム温度：４０℃
　　　検出：ＵＶ　２８０ｎｍ
　　　カラム：ＣＡＰＣＥＬＬ　ＰＡＫ　ＭＧＩＩ、４．６×１５０ｍｍ、３μｍ（資生堂） Quantification of (2S, 4R) monatin and (2R, 4R) monatin was performed by HPLC analysis. The analysis conditions are as shown below.
Mobile phase: 20 mM KH 2 PO 4 / acetonitrile = 100/5
Flow rate: 1.0 ml / min
Column temperature: 40 ° C
Detection: UV 280nm
Column: CAPCELL PAK MGII, 4.6 × 150 mm, 3 μm (Shiseido)
　その結果、Ｒａｃ３９の使用により、（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成する活性（０．１４Ｕ／ｍｇ）、および（２Ｒ，４Ｒ）モナティンから（２Ｓ，４Ｒ）モナティンを生成する活性（０．０９Ｕ／ｍｇ）が検出された（１Ｕは、１分間に１μｍｏｌのモナティンを異性化する活性であることを示す）。以上より、ラセマーゼを用いて（４Ｒ）モナティンを２位において異性化できることが実証された。 As a result, the use of Rac39 generates (2R, 4R) monatin from (2S, 4R) monatin (0.14 U / mg) and (2R, 4R) monatin produces (2S, 4R) monatin. Activity (0.09 U / mg) was detected (1 U indicates activity to isomerize 1 μmol monatin per minute). From the above, it was demonstrated that (4R) monatin can be isomerized at the 2-position using racemase.
実施例４：Ｅ．ｃｏｌｉでのアミノ酸アミノトランスフェラーゼの発現
（１）Ｌ－アミノ酸アミノトランスフェラーゼ発現プラスミドの構築
　保存菌株ＡＪ１６１６　Ｂａｃｉｌｌｕｓ　ａｌｔｉｔｕｄｉｎｉｓより取得したＬ－アミノ酸アミノトランスフェラーゼ（ＡＪ１６１６ＬＡＴ）のＤＮＡ配列（配列番号３）の５’末端にＮｄｅＩ認識配列を、３’末端にＸｈｏＩ認識配列を付与した。
　このＤＮＡ配列を、ＮｄｅＩ、ＸｈｏＩで制限酵素処理し、同様にＮｄｅＩ、ＸｈｏＩで処理したｐＥＴ－２２ｂ（Ｎｏｖａｇｅｎ）とライゲーションした。このライゲーション溶液でＥ．ｃｏｌｉを形質転換し、アンピシリン耐性株の中から目的のプラスミドを抽出した。このプラスミドをｐＥＴ－２２－ＬＡＴ－Ｈｉｓと命名した。このプラスミドでは、Ｃ末端にＨｉｓ－ｔａｇが付与されたＬ－アミノ酸アミノトランスフェラーゼ（ＬＡＴ－Ｈｉｓ）が発現する。 Example 4: E.I. Expression of amino acid aminotransferase in E. coli (1) Construction of L-amino acid aminotransferase expression plasmid NdeI at the 5 ′ end of the DNA sequence (SEQ ID NO: 3) of L-amino acid aminotransferase (AJ1616LAT) obtained from the conserved strain AJ1616 Bacillus altitudinis The recognition sequence was given a XhoI recognition sequence at the 3 ′ end.
This DNA sequence was subjected to restriction enzyme treatment with NdeI and XhoI, and ligated with pET-22b (Novagen) similarly treated with NdeI and XhoI. With this ligation solution E. coli was transformed and the target plasmid was extracted from the ampicillin resistant strain. This plasmid was named pET-22-LAT-His. In this plasmid, L-amino acid aminotransferase (LAT-His) with His-tag added at the C-terminus is expressed.
（２）Ｄ－アミノ酸アミノトランスフェラーゼ発現プラスミドの構築
　ｉｎ　ｓｉｌｉｃｏから選抜したＤ－アミノ酸アミノトランスフェラーゼのＤＮＡ配列の５’末端にＮｄｅＩ認識配列を、３’末端にＸｈｏＩ認識配列を付与したＤＮＡを、ＧｅｎＳｃｒｉｐｔ社のＯｐｔｉｍｕｍＧｅｎｅ　Ｃｏｄｏｎ　Ｏｐｔｉｍｉｚａｔｉｏｎ　Ａｎａｌｙｓｉｓに供し、Ｅ．ｃｏｌｉでの遺伝子発現効率が最適化された合成ＤＮＡ（配列番号５）を得た。
　この合成ＤＮＡをＮｄｅＩ、ＸｈｏＩで制限酵素処理し、同様にＮｄｅＩ、ＸｈｏＩで処理したｐＥＴ－２２ｂ（Ｎｏｖａｇｅｎ）とライゲーションした。このライゲーション溶液でＥ．ｃｏｌｉを形質転換し、アンピシリン耐性株の中から目的のプラスミドを抽出した。このプラスミドをｐＥＴ－２２－ＤＡＴ－Ｈｉｓと命名した。このプラスミドでは、Ｃ末端にＨｉｓ－ｔａｇが付与されたＤ－アミノ酸アミノトランスフェラーゼ（ＤＡＴ－Ｈｉｓ）が発現する。 (2) Construction of D-amino acid aminotransferase expression plasmid DNA obtained by adding NdeI recognition sequence to the 5 'end of the DNA sequence of D-amino acid aminotransferase selected from in silico and XhoI recognition sequence to the 3' end was obtained from GenScript. For OptimumGene Codon Optimization Analysis. A synthetic DNA (SEQ ID NO: 5) with optimized gene expression efficiency in E. coli was obtained.
This synthetic DNA was treated with restriction enzymes with NdeI and XhoI, and ligated with pET-22b (Novagen) similarly treated with NdeI and XhoI. With this ligation solution E. coli was transformed and the target plasmid was extracted from the ampicillin resistant strain. This plasmid was named pET-22-DAT-His. This plasmid expresses D-amino acid aminotransferase (DAT-His) with His-tag added at the C-terminus.
（３）Ｅ．ｃｏｌｉ発現株からのＨｉｓタグ付加Ｌ－アミノ酸アミノトランスフェラーゼの精製
　構築した発現プラスミドｐＥＴ－２２－ＬＡＴ－ＨｉｓをＥ．ｃｏｌｉ　ＢＬ２１（ＤＥ３）に導入し、形質転換体をアンピシリン１００ｍｇ／ｌを含むＯｖｅｒｎｉｇｈｔ　Ｅｘｐｒｅｓｓ　Ｉｎｓｔａｎｔ　ＴＢ　Ｍｅｄｉｕｍ（Ｎｏｖａｇｅｎ）　１６０ｍｌに白金耳接種し、５００ｍｌ容坂口フラスコを用いて１６時間振盪させた。振盪温度は３７℃で行った。培養終了後、８００ｍｌの得られた培養液より菌体を遠心分離により集め、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１０ｍＭにて洗浄、懸濁し、超音波破砕を行った。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
　得られた可溶性画分を、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１０ｍＭで平衡化したＨｉｓ－ｔａｇタンパク質精製カラムＨｉｓ　ＴＡＬＯＮ　ｓｕｐｅｒｆｌｏｗ　５ｍｌ　Ｃａｒｔｒｉｄｇｅ（Ｃｌｏｎｔｅｃｈ）に供して担体に吸着させた。担体に吸着しなかったタンパク質（非吸着タンパク質）をＴｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１０ｍＭを用いて洗い流した後、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１５０ｍＭを用いて、５ｍｌ／ｍｉｎの流速で吸着したタンパク質の溶出を行った。得られた画分を集めて、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭで希釈し、Ｌ－アミノ酸アミノトランスフェラーゼ溶液とした。 (3) E.E. Purification of His-tagged L-amino acid aminotransferase from E. coli expression strain The constructed expression plasmid pET-22-LAT-His was transformed into E. coli. E. coli BL21 (DE3) was introduced, and the transformant was inoculated into 160 ml of Overnight Express Instant Medium (Novagen) containing ampicillin 100 mg / l and shaken for 16 hours using a 500 ml Sakaguchi flask. The shaking temperature was 37 ° C. After completion of the culture, cells were collected from 800 ml of the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl, and 10 mM imidazole, and subjected to ultrasonic disruption. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
The obtained soluble fraction was subjected to His-tag protein purification column His TALON superflow 5 ml Cartridge (Clontech) equilibrated with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, and Imidazole 10 mM, and was adsorbed on the carrier. After washing the protein not adsorbed on the carrier (non-adsorbed protein) with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, Imidazole 10 mM, Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, Imidazole 150 mM Was used to elute the adsorbed protein at a flow rate of 5 ml / min. The obtained fractions were collected and diluted with 20 mM Tris-HCl (pH 7.6) to obtain an L-amino acid aminotransferase solution.
（４）Ｅ．ｃｏｌｉ発現株からのＨｉｓタグ付加Ｄ－アミノ酸アミノトランスフェラーゼの精製
　構築した発現プラスミドｐＥＴ－２２－ＤＡＴ－ＨｉｓをＥ．ｃｏｌｉ　ＢＬ２１（ＤＥ３）に導入し、形質転換体をアンピシリン１００ｍｇ／ｌを含むＯｖｅｒｎｉｇｈｔ　Ｅｘｐｒｅｓｓ　Ｉｎｓｔａｎｔ　ＴＢ　Ｍｅｄｉｕｍ（Ｎｏｖａｇｅｎ）　１６０ｍｌに白金耳接種し、５００ｍｌ容坂口フラスコを用いて１６時間振盪させた。振盪温度は３０℃で行った。培養終了後、８００ｍｌの得られた培養液より菌体を遠心分離により集め、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１０ｍＭにて洗浄、懸濁し、超音波破砕を行った。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
　得られた可溶性画分を、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１０ｍＭで平衡化したＨｉｓ－ｔａｇタンパク質精製カラムＨｉｓ　ＴＡＬＯＮ　ｓｕｐｅｒｆｌｏｗ　５ｍｌ　Ｃａｒｔｒｉｄｇｅ（Ｃｌｏｎｔｅｃｈ）に供して担体に吸着させた。担体に吸着しなかったタンパク質（非吸着タンパク質）をＴｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１０ｍＭを用いて洗い流した後、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭ、ＮａＣｌ　３００ｍＭ、Ｉｍｉｄａｚｏｌｅ　１５０ｍＭを用いて、５ｍｌ／ｍｉｎの流速で吸着したタンパク質の溶出を行った。得られた画分を集めて、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）　２０ｍＭで希釈し、Ｄ－アミノ酸アミノトランスフェラーゼ溶液とした。 (4) E.E. Purification of His-tagged D-amino acid aminotransferase from E. coli expression strain The constructed expression plasmid pET-22-DAT-His was obtained from E. coli. E. coli BL21 (DE3) was introduced, and the transformant was inoculated into 160 ml of Overnight Express Instant Medium (Novagen) containing ampicillin 100 mg / l and shaken for 16 hours using a 500 ml Sakaguchi flask. The shaking temperature was 30 ° C. After completion of the culture, cells were collected from 800 ml of the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl, and 10 mM imidazole, and subjected to ultrasonic disruption. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
The obtained soluble fraction was subjected to His-tag protein purification column His TALON superflow 5 ml Cartridge (Clontech) equilibrated with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, and Imidazole 10 mM, and was adsorbed on the carrier. After washing the protein not adsorbed on the carrier (non-adsorbed protein) with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, Imidazole 10 mM, Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, Imidazole 150 mM Was used to elute the adsorbed protein at a flow rate of 5 ml / min. The obtained fractions were collected and diluted with 20 mM Tris-HCl (pH 7.6) to obtain a D-amino acid aminotransferase solution.
実施例５：（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンの生成反応
　精製したＬ－アミノ酸アミノトランスフェラーゼとＤ－アミノ酸アミノトランスフェラーゼを用いて、以下の条件で７６時間反応を行った。反応は攪拌をしながら５０ｍｌ容ファルコンチューブを用いて２０ｍｌで行った。サンプル１０μｌにＴＥバッファー９９０μｌを加え、アミコンウルトラ０．５ｍｌ　１０ｋ（Ｍｉｌｌｉｐｏｒｅ）を用いて限外ろ過し、ろ液を分析した。分析にはＨＰＬＣを用いた。 Example 5: (2S, 4R) Monatin Production Reaction of (2R, 4R) Monatin Using purified L-amino acid aminotransferase and D-amino acid aminotransferase, the reaction was performed for 76 hours under the following conditions. The reaction was carried out at 20 ml using a 50 ml Falcon tube with stirring. 990 μl of TE buffer was added to 10 μl of sample, ultrafiltered using Amicon Ultra 0.5 ml 10k (Millipore), and the filtrate was analyzed. HPLC was used for the analysis.
　反応条件：（２Ｓ，４Ｒ）モナティン　３００ｍＭ、ピルビン酸　１０ｍＭ、Ｄ－Ａｌａ　２０ｍＭ、ＰＬＰ　５０μＭ、Ｌ－アミノ酸アミノトランスフェラーゼ溶液　３ｍｇ／ｍｌ、Ｄ－アミノ酸アミノトランスフェラーゼ溶液　３ｍｇ／ｍｌ、アラニンラセマーゼ溶液（ユニチカ）　０．００２ｍｇ／ｍｌ、ｐＨ７（ＫＯＨ）、２５℃。 Reaction conditions: (2S, 4R) monatin 300 mM, pyruvate 10 mM, D-Ala 20 mM, PLP 50 μM, L-amino acid aminotransferase solution 3 mg / ml, D-amino acid aminotransferase solution 3 mg / ml, alanine racemase solution (Unitika) 0 0.002 mg / ml, pH 7 (KOH), 25 ° C.
　（２Ｓ，４Ｒ）モナティンおよび（２Ｒ，４Ｒ）モナティンの定量は、ＨＰＬＣ分析により行った。分析条件は、以下に示す通りである。
　　　移動相：Ａ　２０ｍＭ　ＫＨ２ＰＯ４／アセトニトリル＝１００／１０
　　　　　　　Ｂ　アセトニトリル
　　　流速：１．０ｍｌ／ｍｉｎ
　　　カラム温度：４０℃
　　　検出：ＵＶ　２８０ｎｍ
　　　カラム：ＣＡＰＣＥＬＬ　ＰＡＫ　Ｃ１８　ＴＹＰＥ　ＭＧＩＩ　３μｍ
　　　　　　　４．６ｍｍ×１５０ｍｍ（資生堂） Quantification of (2S, 4R) monatin and (2R, 4R) monatin was performed by HPLC analysis. The analysis conditions are as shown below.
Mobile phase: A 20 mM KH 2 PO 4 / acetonitrile = 100/10
B Acetonitrile Flow rate: 1.0 ml / min
Column temperature: 40 ° C
Detection: UV 280nm
Column: CAPCELL PAK C18 TYPE MGII 3 μm
4.6mm x 150mm (Shiseido)
　その結果、反応７６時間後に（２Ｓ，４Ｒ）モナティン　１２８ｍＭ、（２Ｒ，４Ｒ）モナティン　１７８ｍＭが生成した（図８）。したがって、アミノトランスフェラーゼとラセマーゼを用いることで（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成できることが実証された。 As a result, 76 hours after the reaction, (2S, 4R) monatin 128 mM and (2R, 4R) monatin 178 mM were produced (FIG. 8). Therefore, it was demonstrated that (2R, 4R) monatin can be produced from (2S, 4R) monatin by using aminotransferase and racemase.
実施例６：反応液からの（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶の取得
　実施例５に記載の反応液に硫酸マグネシウム７水和物　１５０ｍＭを溶解し、遠心分離により得られた上清をアミコンウルトラ１５ｍｌ　１０ｋ（Ｍｉｌｌｉｐｏｒｅ）を用いて限外ろ過した。得られたろ液に種晶〔参考例５で得られた結晶と同じ結晶形を有する（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶〕を０．００１ｇ添加し、２５℃で２４時間撹拌した。１５ｍｌ容ファルコンチューブを用いて７．５ｍｌで行った。サンプリングした反応液、ならびに反応液を遠心分離して得られた上清１０μｌにＴＥバッファー９９０μｌを加え、アミコンウルトラ０．５ｍｌ　１０ｋ（Ｍｉｌｌｉｐｏｒｅ）を用いて限外ろ過し、ろ液を分析した。分析にはＨＰＬＣを用いた。ＨＰＬＣ分析条件は実施例５に記載の通りに行った。 Example 6: from the reaction mixture ((2R, 4R) monatin) were dissolved the reaction solution of magnesium sulfate heptahydrate 150mM according to obtain Example 5 2 magnesium salt crystals, obtained by centrifugation supernatant Was ultrafiltered using Amicon Ultra 15 ml 10k (Millipore). To the obtained filtrate, 0.001 g of seed crystals [(2R, 4R) monatin) 2 magnesium salt crystals having the same crystal form as that obtained in Reference Example 5] was added, and the mixture was stirred at 25 ° C. for 24 hours. It was carried out at 7.5 ml using a 15 ml falcon tube. 990 μl of TE buffer was added to 10 μl of the sampled reaction solution and the supernatant obtained by centrifuging the reaction solution, ultrafiltered using Amicon Ultra 0.5 ml 10 k (Millipore), and the filtrate was analyzed. HPLC was used for the analysis. The HPLC analysis conditions were as described in Example 5.
　その結果、２４時間で反応液上清中の（２Ｒ，４Ｒ）モナティンは１７４ｍＭから８１ｍＭに減少し、結晶が析出していた（図９）。 As a result, (2R, 4R) monatin in the reaction solution supernatant decreased from 174 mM to 81 mM in 24 hours, and crystals were precipitated (FIG. 9).
　得られたスラリー溶液を遠心分離し、得られた結晶を少量の水で洗浄した後、湿結晶を４０℃で減圧乾燥して（２Ｒ，４Ｒ）モナティン０．１８１ｇを取得した。得られた結晶、母液、洗液をＨＰＬＣで分析し、収率及び品質解析を実施した。その結果、（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶が得られたことが確認された。ＨＰＬＣ分析条件は上記（ＨＰＬＣ分析条件）に記載の通りに行った。 The obtained slurry solution was centrifuged, and the obtained crystal was washed with a small amount of water, and then the wet crystal was dried under reduced pressure at 40 ° C. to obtain 0.181 g of monatin (2R, 4R). The obtained crystals, mother liquor, and washings were analyzed by HPLC to carry out yield and quality analysis. As a result, it was confirmed that ((2R, 4R) monatin) 2 magnesium salt crystals were obtained. The HPLC analysis conditions were as described above (HPLC analysis conditions).
（２Ｒ，４Ｒ）モナティン含量：７３．８ｗｔ％
（２Ｓ，４Ｒ）モナティン含量：３．２７ｗｔ％
マグネシウム含量：３．６１ｗｔ％
カリウム含量：０．５００ｗｔ％ (2R, 4R) monatin content: 73.8 wt%
(2S, 4R) monatin content: 3.27 wt%
Magnesium content: 3.61 wt%
Potassium content: 0.500 wt%
実施例７：（２Ｒ，４Ｒ）モナティンの生成反応と（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩優先晶析の同時化
　精製したＬ－アミノ酸アミノトランスフェラーゼとＤ－アミノ酸アミノトランスフェラーゼを用いて、以下の条件で６９時間反応を行った。反応は１００ｒｐｍで振とうしながら５０ｍｌ容ファルコンチューブを用いて２０ｍｌで行った。サンプル１０μｌにＴＥバッファー９９０μｌを加え、アミコンウルトラ０．５ｍｌ　１０ｋ（Ｍｉｌｌｉｐｏｒｅ）を用いて限外ろ過し、ろ液を分析した。分析にはＨＰＬＣを用いた。ＨＰＬＣ分析条件は実施例５に記載の通りに行った。 Example 7: (2R, 4R) monatin production reaction with a ((2R, 4R) monatin) using 2 magnesium salt preferential crystallization synchronized purified L- amino acid aminotransferase and D- amino acids aminotransferase, the following The reaction was performed for 69 hours under the conditions. The reaction was performed in 20 ml using a 50 ml Falcon tube while shaking at 100 rpm. 990 μl of TE buffer was added to 10 μl of sample, ultrafiltered using Amicon Ultra 0.5 ml 10k (Millipore), and the filtrate was analyzed. HPLC was used for the analysis. The HPLC analysis conditions were as described in Example 5.
　反応条件：（２Ｓ，４Ｒ）モナティン　４００ｍＭ、ピルビン酸　１０ｍＭ、Ｄ－Ａｌａ　２０ｍＭ、ＰＬＰ　５０μＭ、Ｌ－アミノ酸アミノトランスフェラーゼ溶液　５ｍｇ／ｍｌ、Ｄ－アミノ酸アミノトランスフェラーゼ溶液　５ｍｇ／ｍｌ、アラニンラセマーゼ溶液（ユニチカ）　０．００１５ｍｇ／ｍｌ、ｐＨ７（ＫＯＨ）、２５℃。 Reaction conditions: (2S, 4R) monatin 400 mM, pyruvate 10 mM, D-Ala 20 mM, PLP 50 μM, L-amino acid aminotransferase solution 5 mg / ml, D-amino acid aminotransferase solution 5 mg / ml, alanine racemase solution (Unitika) 0 .0015 mg / ml, pH 7 (KOH), 25 ° C.
　その結果、反応６９時間後に（２Ｓ，４Ｒ）モナティン　１７９ｍＭ、（２Ｒ，４Ｒ）モナティン　２１１ｍＭが生成した（図１０）。 As a result, 69 hours after the reaction, (2S, 4R) monatin 179 mM and (2R, 4R) monatin 211 mM were produced (FIG. 10).
　この反応液を硫酸でｐＨ６．５に調整し、アルゴンガスでバブリングした。反応液２．８ｍｌにＬ－アミノ酸アミノトランスフェラーゼ溶液　３ｍｇ／ｍｌ、Ｄ－アミノ酸アミノトランスフェラーゼ溶液　５ｍｇ／ｍｌ、アラニンラセマーゼ溶液（ユニチカ）　０．００１５ｍｇ／ｍｌを添加し、終容量３ｍｌとした。Ｌ－アミノ酸アミノトランスフェラーゼ溶液とＤ－アミノ酸アミノトランスフェラーゼ溶液は、Ｔｒｉｓ－ＨＣｌ（ｐＨ７．０）　２０ｍＭでバッファー置換したものを用いた。優先晶析を同時に行う実験区では、この反応液に硫酸マグネシウム７水和物　１５０ｍＭを溶解し、種晶〔参考例５で得られた結晶と同じ結晶形を有する（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶〕０．００４ｇを添加した。反応には１５ｍｌ容ファルコンチューブを用い、２５℃でさらに８８時間攪拌しながら反応を行った。サンプリングした反応液、ならびに反応液を遠心分離して得られた上清１０μｌにＴＥバッファー９９０μｌを加え、アミコンウルトラ０．５ｍｌ　１０ｋ（Ｍｉｌｌｉｐｏｒｅ）を用いて限外ろ過し、ろ液を分析した。分析にはＨＰＬＣを用いた。ＨＰＬＣ分析条件は実施例５に記載の通りに行った。 The reaction solution was adjusted to pH 6.5 with sulfuric acid and bubbled with argon gas. L-amino acid aminotransferase solution 3 mg / ml, D-amino acid aminotransferase solution 5 mg / ml, and alanine racemase solution (Unitika) 0.0015 mg / ml were added to 2.8 ml of the reaction solution to make a final volume of 3 ml. The L-amino acid aminotransferase solution and the D-amino acid aminotransferase solution were substituted with Tris-HCl (pH 7.0) 20 mM. In the experimental zone where preferential crystallization is performed simultaneously, 150 mM magnesium sulfate heptahydrate is dissolved in this reaction solution, and seed crystals [having the same crystal form as that obtained in Reference Example 5 ((2R, 4R) monatin) 2 Magnesium salt crystals] were added. The reaction was carried out using a 15 ml Falcon tube at 25 ° C. with stirring for an additional 88 hours. 990 μl of TE buffer was added to 10 μl of the sampled reaction solution and the supernatant obtained by centrifuging the reaction solution, ultrafiltered using Amicon Ultra 0.5 ml 10 k (Millipore), and the filtrate was analyzed. HPLC was used for the analysis. The HPLC analysis conditions were as described in Example 5.
　その結果、酵素反応のみでは反応液中の（２Ｒ，４Ｒ）モナティンは２５３ｍＭであった（図１０中段）のに対し、酵素反応と優先晶析を同時に行った場合には２９２ｍＭであった（図１０下段）。また、酵素反応と優先晶析を同時に行った場合には、反応液上清中の（２Ｒ，４Ｒ）モナティンは１１１ｍＭであり（図１０下段）、結晶が析出していた。したがって、（２Ｒ，４Ｒ）モナティンの生成反応と（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩優先晶析の同時化によって、反応液中の（２Ｒ，４Ｒ）モナティンの蓄積量が向上することが実証された。 As a result, in the enzyme reaction alone, (2R, 4R) monatin in the reaction solution was 253 mM (middle in FIG. 10), whereas it was 292 mM when the enzyme reaction and preferential crystallization were performed simultaneously (FIG. 10). 10 bottom). When the enzyme reaction and the preferential crystallization were performed simultaneously, (2R, 4R) monatin in the reaction supernatant was 111 mM (lower part in FIG. 10), and crystals were precipitated. Therefore, it has been demonstrated that the (2R, 4R) monatin production reaction and the ((2R, 4R) monatin) 2- magnesium salt preferential crystallization increase the amount of (2R, 4R) monatin accumulated in the reaction solution. It was done.
　得られたスラリー溶液を遠心分離し、得られた結晶を少量の水で洗浄した後、湿結晶を４０℃で減圧乾燥して（２Ｒ，４Ｒ）モナティン０．２１６ｇを取得した。得られた結晶、母液、洗液をＨＰＬＣで分析し、収率及び品質解析を実施した。その結果、（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶が得られたことが確認された。ＨＰＬＣ分析条件は上記（ＨＰＬＣ分析条件）に記載の通りである。 The obtained slurry solution was centrifuged, and the obtained crystal was washed with a small amount of water, and then the wet crystal was dried under reduced pressure at 40 ° C. to obtain 0.216 g of (2R, 4R) monatin. The obtained crystals, mother liquor, and washings were analyzed by HPLC to carry out yield and quality analysis. As a result, it was confirmed that ((2R, 4R) monatin) 2 magnesium salt crystals were obtained. The HPLC analysis conditions are as described above (HPLC analysis conditions).
（２Ｒ，４Ｒ）モナティン含量：５８．７ｗｔ％
（２Ｓ，４Ｒ）モナティン含量：２．６ｗｔ％
マグネシウム含量：２．２ｗｔ％
カリウム含量：１．８ｗｔ％
水分含量：１８．０ｗｔ％ (2R, 4R) monatin content: 58.7 wt%
(2S, 4R) Monatin content: 2.6 wt%
Magnesium content: 2.2 wt%
Potassium content: 1.8wt%
Water content: 18.0wt%
実施例８：ラセマーゼを用いた（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンへの異性化反応
　実施例２と同様に調整したラセマーゼＲａｃ３９を用いた異性化反応を５０ｍｌスケールで行った。Ｔｒｉｓ－ＨＣｌバッファー　１００ｍＭ、（２Ｓ，４Ｒ）モナティン　１００ｍＭ、ＰＬＰ　５０μｍ、Ｒａｃ３９　０．０１６Ｕ／ｍｌからなる反応液を調製し、３３℃にて１２０ｒｐｍで振とうしながら６５時間反応を行った。反応６５時間目のサンプルを一部採取して、クエン酸ナトリウム溶液（ｐＨ４．５）を添加して反応を停止し、反応停止後の反応液を遠心して上清をＨＰＬＣ分析に供したところ、４０．１ｍＭの（２Ｒ，４Ｒ）モナティンが蓄積していた。 Example 8: Isomerization reaction from (2S, 4R) monatin to (2R, 4R) monatin using racemase An isomerization reaction using racemase Rac39 prepared in the same manner as in Example 2 was performed on a 50 ml scale. A reaction solution consisting of Tris-HCl buffer 100 mM, (2S, 4R) monatin 100 mM, PLP 50 μm, Rac39 0.016 U / ml was prepared, and the reaction was carried out for 65 hours while shaking at 120 rpm at 33 ° C. A part of the sample at 65 hours after the reaction was collected, the reaction was stopped by adding a sodium citrate solution (pH 4.5), the reaction solution after the reaction was stopped, and the supernatant was subjected to HPLC analysis. 40.1 mM (2R, 4R) monatin had accumulated.
実施例９：酵素反応液からの（２Ｒ，４Ｒ）モナティン優先晶析
　実施例８で得られた酵素反応液４５ｍｌに塩化マグネシウム６水和物０．４５ｇ（２．２ミリモル）を溶解し、減圧下で濃縮した。得られた濃縮液７．７ｇを０．２μのフィルターでろ過し、ろ過液に種晶［参考例５で得られた結晶と同じ結晶形を有する（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶］２．４ｍｇを添加し２５℃で２４ｈｒ撹拌した。得られたスラリー溶液から濾過を行い、水０．５ｇで結晶洗浄を行った後、湿結晶を４０℃で減圧乾燥して（２Ｒ，４Ｒ）モナティン０．４１ｇを取得した。得られた結晶、母液、洗液をＨＰＬＣで分析し、収率及び品質解析を実施した。その結果、（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶が得られたことが確認された。 Example 9: (2R, 4R) Monatin Preferential Crystallization from Enzyme Reaction Solution 0.45 g (2.2 mmol) of magnesium chloride hexahydrate was dissolved in 45 ml of the enzyme reaction solution obtained in Example 8, and the pressure was reduced. Concentrated under. The obtained concentrated solution (7.7 g) was filtered through a 0.2 μ filter, and the filtrate was seed crystal [having the same crystal form as the crystal obtained in Reference Example 5 ((2R, 4R) monatin) 2 magnesium salt crystal 2.4 mg was added and the mixture was stirred at 25 ° C. for 24 hours. Filtration was performed from the obtained slurry solution, and the crystals were washed with 0.5 g of water, and then the wet crystals were dried under reduced pressure at 40 ° C. to obtain 0.42 g of (2R, 4R) monatin. The obtained crystals, mother liquor, and washings were analyzed by HPLC to carry out yield and quality analysis. As a result, it was confirmed that ((2R, 4R) monatin) 2 magnesium salt crystals were obtained.
ＨＰＬＣ　ａｒｅａ純度（２１０ｎｍ）：９１．５％
水分含量：１６．３ｗｔ％
マグネシウム含量：３．７２ｗｔ％ HPLC area purity (210 nm): 91.5%
Water content: 16.3 wt%
Magnesium content: 3.72 wt%
実施例１０：反応液中への有機溶媒の添加による優先晶析の促進
　３００ｍｌ四つ口フラスコをアルゴン置換し、水　５８．０ｇ、（２Ｓ，４Ｒ）モナティンフリー体　２０．０ｇ（６８．４ｍｍｏｌ）、水酸化マグネシウム　１．５８ｇ（３０．７ｍｍｏｌ）、サリチルアルデヒド　０．８５０ｇ（６．９６ｍｍｏｌ）を添加し、６５℃で２４時間加熱撹拌した。続いて種晶［参考例５で得られた結晶と同じ結晶形を有する（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶］　１９ｍｇを添加した後にメタノール１９．３ｇを１時間かけて添加し、４８時間加熱撹拌した。さらにメタノール３８．７ｇを１時間かけて添加し２４時間加熱撹拌を続けた。得られたスラリー溶液は２５℃に冷却し、ろ過を行い５０％メタノール１０．０ｇで結晶洗浄を行った。得られた湿結晶を４０℃にて減圧乾燥し（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶１９．０ｇ（５４．５ｍｍｏｌ）を取得した。固有Ｘ線回折ピークから、参考例４で得られた結晶と同じ結晶形の、（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶が得られたことが確認された。 Example 10: Promotion of preferential crystallization by adding an organic solvent to the reaction solution A 300 ml four-necked flask was purged with argon, and 58.0 g of water, 20.0 g (68.4 mmol) of (2S, 4R) monatin free form. ), 1.58 g (30.7 mmol) of magnesium hydroxide, and 0.850 g (6.96 mmol) of salicylaldehyde, and the mixture was heated and stirred at 65 ° C. for 24 hours. Subsequently, seed crystal [having the same crystal form as the crystal obtained in Reference Example 5 ((2R, 4R) monatin) 2 magnesium salt crystal] After adding 19 mg, 19.3 g of methanol was added over 1 hour, and 48 Stir for hours. Further, 38.7 g of methanol was added over 1 hour, and stirring was continued for 24 hours. The obtained slurry solution was cooled to 25 ° C., filtered, and crystal washed with 10.0 g of 50% methanol. The resulting wet crystals were dried under reduced pressure at 40 ℃ ((2R, 4R) monatin) were obtained 2 magnesium salt crystals 19.0 g (54.5 mmol). From the intrinsic X-ray diffraction peak, it was confirmed that a ((2R, 4R) monatin) 2 magnesium salt crystal having the same crystal form as the crystal obtained in Reference Example 4 was obtained.
（２Ｒ，４Ｒ）モナティン収率　７９．７％
（２Ｒ，４Ｒ）モナティン含量　８３．７ｗｔ％
固有Ｘ線回折ピーク（２θ±０．２°、ＣｕＫα）：４．９°、１６．８°、１８．０°、２４．６° (2R, 4R) monatin yield 79.7%
(2R, 4R) monatin content 83.7wt%
Intrinsic X-ray diffraction peak (2θ ± 0.2 °, CuKα): 4.9 °, 16.8 °, 18.0 °, 24.6 °
参考例６：（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶からの（２Ｒ，４Ｒ）モナティンフリー体結晶の調製
　実施例１０で得られた（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶１５ｇ（４６．３ミルモル）を水２８５ｍｌに分散し、１０℃を保持したまま１Ｍ硫酸２４．２ｇを添加し１８時間撹拌した。生成したスラリーを分離し、得られた湿結晶２９．３ｇを４０℃の減圧下で乾燥し、（２Ｒ，４Ｒ）モナティンフリー体結晶１３．１ｇを取得した。 Reference Example 6: ((2R, 4R) monatin) (2R, 4R) from 2 magnesium salt crystals obtained in Preparation Example 10 of Mona tin-free substance crystals (2R, 4R) monatin) 2 magnesium salt crystals 15 g ( 46.3 mmol) was dispersed in 285 ml of water, and 24.2 g of 1M sulfuric acid was added while maintaining the temperature at 10 ° C., followed by stirring for 18 hours. The produced slurry was separated, and 29.3 g of the obtained wet crystals were dried under reduced pressure at 40 ° C. to obtain 13.1 g of (2R, 4R) monatin-free crystals.
純度：９９％
マグネシウム：１００ｐｐｍ以下 Purity: 99%
Magnesium: 100ppm or less
参考例７：（２Ｒ，４Ｒ）モナティンフリー体結晶からの（２Ｒ，４Ｒ）モナティンカリウム塩結晶の調製
　参考例６で得られた（２Ｒ，４Ｒ）モナティンフリー体結晶１０ｇ（３３．９ミリモル）を水１０ｇに分散し、５０％水酸化カルウム水溶液３．９ｇ（３３．９ミリモル）を添加して溶解した。４０℃を保持したままメタノール５８ｇを３時間かけて添加後、２５℃で１．５時間撹拌し生成したスラリーを分離した。得られた湿結晶１３．１ｇを４０℃の減圧下で乾燥し、（２Ｒ，４Ｒ）モナティンカリウム塩結晶１０．２ｇを取得した。 Reference Example 7: Preparation of (2R, 4R) monatin potassium salt crystals from (2R, 4R) monatin free crystals 10 g (33.9) of (2R, 4R) monatin free crystals obtained in Reference Example 6 Mmol) was dispersed in 10 g of water and dissolved by adding 3.9 g (33.9 mmol) of a 50% aqueous solution of calcium hydroxide. While maintaining 40 ° C., 58 g of methanol was added over 3 hours, followed by stirring at 25 ° C. for 1.5 hours to separate the resulting slurry. 13.1 g of the obtained wet crystal was dried under reduced pressure at 40 ° C. to obtain 10.2 g of (2R, 4R) monatin potassium salt crystal.
純度：９９％
水分含量：６．１ｗｔ％
カリウム含量：１２．５ｗｔ％ Purity: 99%
Water content: 6.1 wt%
Potassium content: 12.5wt%
実施例１１：反応液中への有機溶媒の添加による優先晶析の促進
　１０００ｍｌ四つ口フラスコをアルゴン置換し、水　２９０．７ｇ、（２Ｓ，４Ｒ）モナティンフリー体　１００．０ｇ（３４２．１ｍｍｏｌ）、水酸化マグネシウム　９．２６ｇ（１５４．０ｍｍｏｌ）、塩化マグネシウム６水和物　３．４７ｇ（１７．１ｍｍｏｌ）、サリチルアルデヒド　４．２６ｇ（３４．５ｍｍｏｌ）を添加し、６５℃で２４時間加熱撹拌した。続いて種晶［参考例３で得られた結晶と同じ結晶形を有する（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶］５．５０ｇ（１７．１ｍｍｏｌ）を添加した後に２－プロパノール３３．５ｇを６時間かけて添加し、その後９時間加熱撹拌した。続いて２－プロパノール６３．４ｇを３時間かけて添加しその後３０時間加熱撹拌した。さらに、２－プロパノール１９３．８ｇを２時間かけて添加しその後２２時間加熱撹拌を続けた。得られたスラリー溶液は４時間かけて２５℃に冷却し、ろ過を行い５０％２－プロパノール５０．０ｇで結晶洗浄を行った。得られた湿結晶を４０℃にて減圧乾燥し（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶１０２．９ｇ（２９２．４ｍｍｏｌ）を取得した。固有Ｘ線回折ピークから、参考例４で得られた結晶と同じ結晶形の、（（２Ｒ，４Ｒ）モナティン）２マグネシウム塩結晶が得られたことが確認された。 Example 11: Promotion of Preferential Crystallization by Addition of Organic Solvent to Reaction Solution A 1000 ml four-necked flask was purged with argon, water 290.7 g, (2S, 4R) monatin free body 100.0 g (342.1 mmol) ), 9.26 g (154.0 mmol) of magnesium hydroxide, 3.47 g (17.1 mmol) of magnesium chloride hexahydrate, 4.26 g (34.5 mmol) of salicylaldehyde, and heated and stirred at 65 ° C. for 24 hours. did. Then [having the same crystalline form as obtained in Reference Example 3 crystal ((2R, 4R) monatin) 2 magnesium salt crystals] seed crystals 5.50 g (17.1 mmol) After addition of 2-propanol 33.5g Was added over 6 hours and then heated and stirred for 9 hours. Subsequently, 63.4 g of 2-propanol was added over 3 hours, and then heated and stirred for 30 hours. Further, 193.8 g of 2-propanol was added over 2 hours, and the heating and stirring were continued for 22 hours. The resulting slurry solution was cooled to 25 ° C. over 4 hours, filtered, and washed with 50.0 g of 50% 2-propanol. The resulting wet crystals were dried under reduced pressure at 40 ℃ ((2R, 4R) monatin) were obtained 2 magnesium salt crystals 102.9g (292.4mmol). From the intrinsic X-ray diffraction peak, it was confirmed that a ((2R, 4R) monatin) 2 magnesium salt crystal having the same crystal form as the crystal obtained in Reference Example 4 was obtained.
（２Ｒ，４Ｒ）モナティン収率　８１．４％
（２Ｒ，４Ｒ）モナティン含量　８３．０ｗｔ％
固有Ｘ線回折ピーク（２θ±０．２°、ＣｕＫα）：４．９°、１６．８°、１８．０°、２４．６°（図１１） (2R, 4R) monatin yield 81.4%
(2R, 4R) monatin content 83.0wt%
Intrinsic X-ray diffraction peaks (2θ ± 0.2 °, CuKα): 4.9 °, 16.8 °, 18.0 °, 24.6 ° (FIG. 11)
　（２Ｓ，４Ｒ）モナティンを含有する溶液に対して、多価金属イオン存在下、アルデヒドまたはラセマーゼを作用させることによって、良好な甘味特性を有する、保存安定性に優れた、（２Ｒ，４Ｒ）モナティン多価金属塩の効率的な製造方法を提供できるようになった。これにより、（２Ｒ，４Ｒ）モナティン多価金属塩を含有する、各種食品、各種医薬品、各種製品を提供できるようになったことは意義深い。 (2R, 4R) monatin having good sweetness characteristics and excellent storage stability by allowing aldehyde or racemase to act on a solution containing (2S, 4R) monatin in the presence of a polyvalent metal ion. An efficient method for producing a polyvalent metal salt can be provided. Thus, it is significant that various foods, various pharmaceuticals, and various products containing (2R, 4R) monatin polyvalent metal salt can be provided.

Claims (18)

  1. 　多価金属イオンを含有する水溶液中で、アルデヒドまたは（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素の１種または２種以上に（２Ｓ，４Ｒ）モナティンを接触させて、（２Ｒ，４Ｒ）モナティン多価金属塩結晶を析出させることを含む、（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 Contacting (2S, 4R) monatin with one or more enzymes capable of producing (2R, 4R) monatin from aldehyde or (2S, 4R) monatin in an aqueous solution containing a polyvalent metal ion; A method for producing a (2R, 4R) monatin polyvalent metal salt crystal, comprising precipitating a (2R, 4R) monatin polyvalent metal salt crystal.
  2. 　（２Ｓ，４Ｒ）モナティンを含有する水溶液に対して、多価金属イオン存在下、アルデヒド、または、ラセマーゼ、アミノトランスフェラーゼからなる群から選ばれる１種または２種以上を作用させて、（２Ｒ，４Ｒ）モナティン多価金属塩結晶を得ることを含む、（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 One or two or more selected from the group consisting of aldehyde, racemase, and aminotransferase are allowed to act on an aqueous solution containing (2S, 4R) monatin in the presence of a polyvalent metal ion, and (2R, 4R). ) A method for producing a (2R, 4R) monatin polyvalent metal salt crystal comprising obtaining a monatin polyvalent metal salt crystal.
  3. 　該アルデヒドが芳香族アルデヒドである、請求項１または２記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to claim 1 or 2, wherein the aldehyde is an aromatic aldehyde.
  4. 　（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素がラセマーゼである、請求項１記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to claim 1, wherein the enzyme capable of producing (2R, 4R) monatin from (2S, 4R) monatin is racemase.
  5. 　ラセマーゼが配列番号２のアミノ酸配列を含む、請求項４記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to claim 4, wherein the racemase comprises the amino acid sequence of SEQ ID NO: 2.
  6. 　（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素がアミノトランスフェラーゼである、請求項１記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to claim 1, wherein the enzyme capable of producing (2R, 4R) monatin from (2S, 4R) monatin is an aminotransferase.
  7. 　（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素がＬ－アミノ酸アミノトランスフェラーゼおよびＤ－アミノ酸アミノトランスフェラーゼである、請求項１記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The (2R, 4R) monatin polyvalent metal salt crystal according to claim 1, wherein the enzymes capable of producing (2R, 4R) monatin from (2S, 4R) monatin are L-amino acid aminotransferase and D-amino acid aminotransferase. Production method.
  8. 　（２Ｓ，４Ｒ）モナティンから（２Ｒ，４Ｒ）モナティンを生成し得る酵素がＬ－アミノ酸アミノトランスフェラーゼ、Ｄ－アミノ酸アミノトランスフェラーゼ、およびラセマーゼである、請求項１記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The (2R, 4R) monatin polyvalent metal according to claim 1, wherein the enzymes capable of producing (2R, 4R) monatin from (2S, 4R) monatin are L-amino acid aminotransferase, D-amino acid aminotransferase, and racemase. A method for producing a salt crystal.
  9. 　Ｌ－アミノ酸アミノトランスフェラーゼが配列番号４のアミノ酸配列を含み、かつＤ－アミノ酸アミノトランスフェラーゼが配列番号６のアミノ酸配列を含む、請求項７または８記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 9. The (2R, 4R) monatin polyvalent metal salt crystal of claim 7 or 8, wherein the L-amino acid aminotransferase comprises the amino acid sequence of SEQ ID NO: 4, and the D-amino acid aminotransferase comprises the amino acid sequence of SEQ ID NO: 6. Production method.
  10. 　該多価金属が二価のアルカリ土類金属である、請求項１～９のいずれか一項に記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of claims 1 to 9, wherein the polyvalent metal is a divalent alkaline earth metal.
  11. 　該アルカリ土類金属がマグネシウムである、請求項１０記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to claim 10, wherein the alkaline earth metal is magnesium.
  12. 　水溶液のｐＨが４～１１である、請求項１～１１のいずれか一項に記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of claims 1 to 11, wherein the pH of the aqueous solution is 4 to 11.
  13. 　水溶液中に存在する有機溶媒が５体積％以下である、請求項１～１２のいずれか一項に記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of claims 1 to 12, wherein the organic solvent present in the aqueous solution is 5% by volume or less.
  14. 　該結晶が、回折角度（２θ±０．２°、ＣｕＫα）として、８．９°、１１．２°、１５．０°、１７．８°、２２．５°に、固有Ｘ線回折ピークを有する、請求項１～１３のいずれか一項に記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The crystal has intrinsic X-ray diffraction peaks at diffraction angles (2θ ± 0.2 °, CuKα) at 8.9 °, 11.2 °, 15.0 °, 17.8 °, 22.5 °. The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of claims 1 to 13,
  15. 　該結晶が、回折角度（２θ±０．２°、ＣｕＫα）として、４．９°、１６．８°、１８．０°、２４．６°に、固有Ｘ線回折ピークを有する、請求項１～１３のいずれか一項に記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The crystal has intrinsic X-ray diffraction peaks at 4.9 °, 16.8 °, 18.0 °, and 24.6 ° as diffraction angles (2θ ± 0.2 °, CuKα). 14. A method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of items 1 to 13.
  16. 　（２Ｒ，４Ｒ）モナティン多価金属塩結晶を回収することを含む、請求項１～１５のいずれか一項に記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The method for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of claims 1 to 15, comprising recovering the (2R, 4R) monatin polyvalent metal salt crystal.
  17. 　（２Ｓ，４Ｒ）モナティンの（２Ｒ，４Ｒ）モナティンへの異性化、および（２Ｒ，４Ｒ）モナティン多価金属塩の晶析を同時に行うことにより、（２Ｒ，４Ｒ）モナティンの生成量を増加せしめる、請求項１～１６のいずれか一項に記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 By isomerizing (2S, 4R) monatin to (2R, 4R) monatin and crystallization of (2R, 4R) monatin polyvalent metal salt simultaneously, the amount of (2R, 4R) monatin produced is increased. A process for producing a (2R, 4R) monatin polyvalent metal salt crystal according to any one of claims 1 to 16.
  18. 　（２Ｒ，４Ｒ）モナティンの生成後に有機溶媒を水溶液中に添加して、（２Ｒ，４Ｒ）モナティン多価金属塩結晶の析出を促進する工程をさらに含む、請求項１～１７のいずれか一項に記載の（２Ｒ，４Ｒ）モナティン多価金属塩結晶の製造方法。 The method according to any one of claims 1 to 17, further comprising the step of adding an organic solvent into the aqueous solution after the formation of (2R, 4R) monatin to promote precipitation of (2R, 4R) monatin polyvalent metal salt crystals. A method for producing a (2R, 4R) monatin polyvalent metal salt crystal described in 1.
PCT/JP2012/079826 2011-11-17 2012-11-16 Method for producing (2r,4r)-monatin polyvalent metal salt crystals WO2013073679A1 (en)

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