WO2013073679A1 - Procédé de production de cristaux de sel métallique polyvalent de (2r,4r)-monatine - Google Patents

Procédé de production de cristaux de sel métallique polyvalent de (2r,4r)-monatine 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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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.

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Abstract

Cette invention concerne un procédé de production efficace de cristaux de sel métallique polyvalent de (2R,4R)-monatine, ayant de bonnes caractéristiques édulcorantes et une stabilité élevée au stockage. Plus spécifiquement, cette invention concerne un procédé de production de cristaux de sel métallique polyvalent de (2R,4R)-monatine, ledit procédé comprenant le traitement d'une solution aqueuse contenant une (2S,4R)-monatine en présence d'un ion métallique polyvalent avec un ou plusieurs types d'enzymes capables de synthétiser une (2R,4R)-monatine à partir d'un aldéhyde ou une (2S,4R)-monatine pour obtenir des cristaux de sel métallique polyvalent de (2R,4R)-monatine. L'aldéhyde est de préférence un aldéhyde aromatique. Ledit ou lesdits types d'enzymes peuvent être de préférence une racémase ou un ou plusieurs types d'aminotransférases. De préférence, le métal polyvalent est un métal alcalino-terreux divalent.
PCT/JP2012/079826 2011-11-17 2012-11-16 Procédé de production de cristaux de sel métallique polyvalent de (2r,4r)-monatine WO2013073679A1 (fr)

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WO2003045914A1 (fr) * 2001-11-30 2003-06-05 Ajinomoto Co., Inc. Cristaux de sels stereoisomeres non naturels de monatine et leur utilisation
WO2003056026A1 (fr) * 2001-12-27 2003-07-10 Ajinomoto Co., Inc. Procede de production de derives d'acide glutamique
JP2004222657A (ja) * 2003-01-24 2004-08-12 Ajinomoto Co Inc ２ｒ−モナティンの製造方法
JP2005154291A (ja) * 2003-11-21 2005-06-16 Ajinomoto Co Inc グルタミン酸誘導体の有機アミン塩及びその利用
WO2005082850A1 (fr) * 2004-02-27 2005-09-09 Ajinomoto Co., Inc. Procédé pour la fabrication de monatine
JP2007284349A (ja) * 2004-07-27 2007-11-01 Ajinomoto Co Inc モナティンまたはその塩の製造方法
JP2008214345A (ja) * 2007-02-08 2008-09-18 Ajinomoto Co Inc 光学活性化合物の製造方法
JP2008538932A (ja) * 2005-04-26 2008-11-13 カーギル，インコーポレイティド モナティンの立体異性体及びそれらの前駆体の製造のためのポリペプチド及び生合成経路
JP2010042014A (ja) * 2002-12-09 2010-02-25 Ajinomoto Co Inc 変異型ｄ−アミノトランスフェラーゼおよびこれを用いた光学活性グルタミン酸誘導体の製造方法
WO2012050125A1 (fr) * 2010-10-14 2012-04-19 味の素株式会社 Procédé de production de monatine
WO2012147674A1 (fr) * 2011-04-25 2012-11-01 味の素株式会社 Procédé de production de monatine
WO2012169661A1 (fr) * 2011-06-08 2012-12-13 味の素株式会社 Cristal de sel métallique polyvalent de monatine

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003045914A1 (fr) * 2001-11-30 2003-06-05 Ajinomoto Co., Inc. Cristaux de sels stereoisomeres non naturels de monatine et leur utilisation
WO2003056026A1 (fr) * 2001-12-27 2003-07-10 Ajinomoto Co., Inc. Procede de production de derives d'acide glutamique
JP2010042014A (ja) * 2002-12-09 2010-02-25 Ajinomoto Co Inc 変異型ｄ−アミノトランスフェラーゼおよびこれを用いた光学活性グルタミン酸誘導体の製造方法
JP2004222657A (ja) * 2003-01-24 2004-08-12 Ajinomoto Co Inc ２ｒ−モナティンの製造方法
JP2005154291A (ja) * 2003-11-21 2005-06-16 Ajinomoto Co Inc グルタミン酸誘導体の有機アミン塩及びその利用
WO2005082850A1 (fr) * 2004-02-27 2005-09-09 Ajinomoto Co., Inc. Procédé pour la fabrication de monatine
JP2007284349A (ja) * 2004-07-27 2007-11-01 Ajinomoto Co Inc モナティンまたはその塩の製造方法
JP2008538932A (ja) * 2005-04-26 2008-11-13 カーギル，インコーポレイティド モナティンの立体異性体及びそれらの前駆体の製造のためのポリペプチド及び生合成経路
JP2008214345A (ja) * 2007-02-08 2008-09-18 Ajinomoto Co Inc 光学活性化合物の製造方法
WO2012050125A1 (fr) * 2010-10-14 2012-04-19 味の素株式会社 Procédé de production de monatine
WO2012147674A1 (fr) * 2011-04-25 2012-11-01 味の素株式会社 Procédé de production de monatine
WO2012169661A1 (fr) * 2011-06-08 2012-12-13 味の素株式会社 Cristal de sel métallique polyvalent de monatine

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