WO2020196619A1 - Nouvelle l-acide aminé oxydase et procédé de production d'acide aminé d ou d'un dérivé correspondant - Google Patents

Nouvelle l-acide aminé oxydase et procédé de production d'acide aminé d ou d'un dérivé correspondant Download PDF

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WO2020196619A1
WO2020196619A1 PCT/JP2020/013326 JP2020013326W WO2020196619A1 WO 2020196619 A1 WO2020196619 A1 WO 2020196619A1 JP 2020013326 W JP2020013326 W JP 2020013326W WO 2020196619 A1 WO2020196619 A1 WO 2020196619A1
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amino acid
seq
acid sequence
protein
derivative
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創平 伊藤
祥吾 中野
優季 南野
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静岡県公立大学法人
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/50—Polycarboxylic acids having keto groups, e.g. 2-ketoglutaric acid

Definitions

  • the present invention relates to a novel L-amino acid oxidase, and more particularly to an L-amino acid oxidase capable of obtaining D-amino acid by dynamic optical resolution of DL-amino acid with high efficiency.
  • a dynamic optical resolution method has been reported as a method capable of synthesizing only D-form or L-form amino acids in high yield from racemic amino acids, amino acid derivatives or synthetic intermediates.
  • This dynamic optical resolution method is a splitting method using an enzyme, and since it can be carried out under normal pressure conditions, it is attracting attention as an environmentally friendly and clean synthetic method today.
  • D-amino acid oxidase is generally used for the synthesis of L-amino acids by the dynamic optical resolution method.
  • DAAO D-amino acid oxidase
  • Patent Document 1 and Non-Patent Documents 1 and 2 the optical resolution of L-amino acids and their derivatives using DAAO has been reported in many documents and is widely used industrially.
  • L-amino acid oxidase LAAO
  • LAAD L-amino acid deaminase
  • LAAD is widely used industrially for optically dividing only D-form from racemic amino acids, amino acid derivatives or synthetic intermediates by an enzymatic method, and a method combining racemase and acylase, Alternatively, a method in which racemase and amidase are combined is used.
  • LAAO is not used for the synthesis of D-amino acids
  • L-amino acid which is a substrate, is an important compound that constitutes the organism itself, and that hydrogen peroxide is generated by the enzymatic reaction of LAAO. It is speculated.
  • Non-Patent Document 3 LAAO derived from Aplysia californica (Jumbo sea slug) was expressed in Escherichia coli.
  • the expression level of LAAO derived from californica was very small, and remained at 0.2 mg per 1 L of the culture solution.
  • LAAO derived from snake venom on the market is very expensive at 225,000 yen at 10 mg even though it is a research reagent (Non-Patent Document 4).
  • DAAO in contrast to the industrial use of DAAO in the synthesis of L-amino acids, there is a problem that the use of LAAO in the synthesis of D-amino acids is difficult at this stage.
  • LAAD L-amino acid deaminase
  • PMS phenazinemethsulfate
  • phenylalanine racemase (EC number 5.1.1.11.1) has a problem that ATP is required for the reaction itself. Furthermore, most of these amino acid racems are PLP-type enzymes and are prone to side reactions and termination reactions. Then, in order to synthesize D-amino acid, it is necessary to match the stability, reactivity and substrate selectivity of a plurality of enzymes, and there is a problem that it is not suitable for industrial application.
  • the present invention has been made in view of the above points, and an object of the present invention is that L-amino acid oxidase itself can be produced in large quantities at low cost by a large amount of expression of a recombinant protein or the like, and L has a wide substrate selectivity. -The purpose is to artificially create an amino acid oxidase and provide it as a new L-amino acid oxidase.
  • Another object of the present invention is to provide a novel L-amino acid oxidase having high activity for racemic amino acids, amino acid derivatives or synthetic intermediates and enabling highly efficient optical resolution. is there.
  • Another object of the present invention is to provide a method for obtaining a D-amino acid, a D-amino acid derivative or a synthetic intermediate from a racemic amino acid, an amino acid derivative or a synthetic intermediate.
  • the present inventors have artificially designed an L-amino acid oxidase that can be expressed in large quantities in an Escherichia coli expression system, has wide substrate selectivity, and has high activity, and is a novel artificial L-amino acid oxidase (ArtLAAO). Succeeded in getting.
  • ArtLAAO novel artificial L-amino acid oxidase
  • the ArtLAAO of the present invention has an ee value of more than 99%, an extremely high optical purity, and a high yield for each of the plurality of DL-phenylalanine derivatives. We have found that it can be divided into phenylalanine derivatives, and have completed the present invention.
  • the novel L-amino acid oxidase of the present invention comprises (1) an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, and has L-amino acid oxidase activity.
  • An amino acid sequence having L-amino acid oxidase activity which comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.
  • It is a protein consisting of any of the amino acid sequences (1) to (4) of the amino acid sequence having 98% or more identity with respect to the amino acid sequence and having the L-amino acid oxidase activity.
  • This allows a large amount of recombinant protein to be expressed, and a novel L-amino acid oxidase having a wide substrate selectivity can be obtained.
  • the novel L-amino acid oxidase can perform high-efficiency optical resolution on racemic amino acids, amino acid derivatives, or synthetic intermediates.
  • the L-amino acid oxidase activity in the present invention includes not only L-amino acid oxidase activity but also L-amino acid derivative oxidase activity.
  • novel L-amino acid oxidase gene of the present invention contains (5) the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, and is a protein consisting of an amino acid sequence having L-amino acid oxidase activity. (6) From an amino acid sequence having L-amino acid oxidase activity, which comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.
  • a protein comprising an amino acid sequence having 90% or more identity with respect to the amino acid sequence represented by SEQ ID NO: 1 and having L-amino acid oxidase activity or (8) SEQ ID NO: 3
  • any of (5) to (8) of a protein comprising an amino acid sequence having 98% or more identity with respect to the amino acid sequence represented by SEQ ID NO: 5 and having L-amino acid oxidase activity It is a gene that encodes a protein. This allows a large amount of recombinant protein to be expressed, and a novel L-amino acid oxidase gene having a wide substrate selectivity can be obtained.
  • the recombinant vector of the present invention is a recombinant vector containing the gene encoding the above-mentioned L-amino acid oxidase protein or the above-mentioned L-amino acid oxidase gene.
  • the recombinant protein of L-amino acid oxidase of the present invention can be mass-produced in large quantities, and L-amino acid oxidase can be mass-produced easily and at low cost.
  • the host cell of the present invention contains the above-mentioned recombinant vector.
  • the L-amino acid oxidase of the present invention can be mass-produced easily and at low cost.
  • the method for producing a D-amino acid or a derivative thereof of the present invention comprises a step of causing the above-mentioned protein to act as an L-amino acid oxidase on the L-amino acid or its derivative to generate imino acid, and the resulting imino. It has a step of allowing a reducing agent to act on an acid.
  • the novel L-amino acid oxidase of the present invention is allowed to act on an L-amino acid or a derivative of an L-amino acid, the L-amino acid or a derivative thereof is oxidized to form an imino acid.
  • the L-amino acid or its derivative By acting a reducing agent on this imino acid, it is converted into a racemic amino acid or a derivative thereof.
  • the L-amino acid or its derivative reacts with the L-amino acid oxidase to form imino acid again, but the D-amino acid or its derivative remains without reacting with the L-amino acid oxidase. Therefore, the L-amino acid or its derivative is finally converted to the D-amino acid or its derivative.
  • the method for producing keto acid of the present invention is a step of causing the above-mentioned protein to act as L-amino acid oxidase on L-amino acid or a derivative thereof to generate imino acid, and hydrolyzing the produced imino acid. It has a step of making it.
  • the novel L-amino acid oxidase of the present invention is allowed to act on an L-amino acid or a derivative of an L-amino acid, the L-amino acid or a derivative thereof is oxidized to form imino acid. Since this imino acid is naturally hydrolyzed in the aqueous reaction solution, keto acid is produced.
  • Keto acid is a compound that is difficult to synthesize by organic synthesis, but in the present invention, it can be produced only by an enzymatic reaction with the novel L-amino acid oxidase described above, so that it can be produced at low cost.
  • the optical resolution method of the present invention includes a step of allowing the above-mentioned protein to act as L-amino acid oxidase on DL-amino acid or DL-amino acid derivative.
  • novel L-amino acid oxidase of the present invention is allowed to act on DL-amino acids and the like, they do not react with D-amino acids and the like, but L-amino acids and the like are oxidized to form imino acids as intermediates.
  • This imino acid is either (1) converted into a racemate again by the action of a racemization catalyst such as a reducing agent, or (2) hydrolyzed in a reaction system to form keto acid.
  • DL-amino acids or derivatives of DL-amino acids are DL-phenylalanine, DL-tryptophan, DL-methionine, DL-leucine, DL-glutamine, DL-glutamic acid, DL-tyrosine, DL-isoleucine, DL-arginine and them. It is preferably a compound selected from the group consisting of derivatives of. As a result, a suitable amino acid as a substrate is selected.
  • ammonia borane, sodium borohydride, lithium aluminum hydride and cyanotri are opposed to imino acid produced by the action of L-amino acid oxidase on DL-amino acid or DL-amino acid derivative. It has a step of acting a reducing agent selected from the group consisting of sodium borohydride.
  • a compound suitable as a racemization catalyst for racemizing the imino acid formed by oxidation of L-amino acid or the like is selected.
  • a novel L-amino acid oxidase having the following excellent effects, a method for producing D-amino acid or a derivative thereof using the same, a method for producing keto acid, and a novel L-amino acid oxidase
  • the optical resolution method used can be provided. (1) Since a large amount of expression is possible in an Escherichia coli expression system, L-amino acid oxidase can be obtained easily and at low cost. Therefore, it can be used industrially. (2) Since the substrate selectivity is wide, it can act regardless of the type of amino acid and is highly versatile. Furthermore, it can act with high activity not only on amino acids but also on amino acid derivatives. (3) Efficient dynamic optical resolution can be performed in one pot without being affected by the reducing agent that is a racemization catalyst coexisting in the reaction system, and a high-purity optically active substance can be produced in high yield. Obtainable.
  • Example 4 is a chiral HPLC chromatogram showing the results of dynamic optical resolution of DL-phenylalanine (DL-1a) in Example 4 (upper: control (before optical resolution), lower: after optical resolution by ArtLAAO).
  • DL-1b 2-fluoro-DL-phenylalanine
  • DL-1c 3-fluoro-DL-phenylalanine
  • the protein of the present invention is a protein consisting of any of the following amino acid sequences (1) to (4) and has L-amino acid oxidase activity.
  • An amino acid sequence containing the amino acid sequence represented by SEQ ID NO: 1 (ArtLAAO1), SEQ ID NO: 3 (ArtLAAO4) or SEQ ID NO: 5 (ArtLAAO5) and having L-amino acid oxidase activity (2) SEQ ID NO: 1 (ArtLAAO1).
  • An amino acid sequence having L-amino acid oxidase activity comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 3 (ArtLAAO4) or SEQ ID NO: 5 (ArtLAAO5).
  • an amino acid sequence containing 98% or more identity with respect to the amino acid sequence represented by SEQ ID NO: 5 (ArtLAAO5) and having L-amino acid oxidase activity an amino acid sequence containing 98% or more identity with respect to the amino acid sequence represented by SEQ ID NO: 5 (ArtLAAO5) and having L-amino acid oxidase activity.
  • the L-amino acid oxidase activity in the present invention means L-amino acid oxidase activity, L-amino acid derivative oxidase activity, or both.
  • the range of the number of amino acids deleted, substituted or added in "amino acids in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1" is defined as amino acids.
  • the deleted, substituted or added protein has the same characteristics as the protein consisting of the amino acid sequence of SEQ ID NO: 1 (ArtLAAO1), it is not particularly limited.
  • the range of the number of amino acids to be deleted, substituted or added is 1 from the viewpoint of having excellent properties such as its L-aminooxidase activity, wide substrate selectivity, and large-scale expression of recombinant proteins.
  • the number is preferably from 60 to, more preferably from 1 to 30, and even more preferably from 1 to 10.
  • the range of identity of the "amino acid sequence having 90% or more identity with respect to the amino acid sequence represented by SEQ ID NO: 1" is such that proteins having different identities are the amino acids of SEQ ID NO: 1. It is not particularly limited as long as it has the same characteristics as the protein consisting of the sequence (ArtLAAO1). Specifically, the identity with respect to the amino acid sequence represented by SEQ ID NO: 1 is 90% from the viewpoint of having excellent properties such as its L-aminooxidase activity, wide substrate selectivity, and large-scale expression of recombinant protein. The above is preferable, 95% or more is more preferable, and 98% or more is further preferable.
  • the number of amino acids deleted, substituted or added to "amino acids in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 5" is not particularly limited as long as the protein in which the amino acid is deleted, substituted or added has the same characteristics as the protein consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5 (ArtLAAO4 or ArtLAAO5).
  • the range of the number of amino acids to be deleted, substituted or added is 1 from the viewpoint of having excellent properties such as its L-aminooxidase activity, wide substrate selectivity, and large-scale expression of recombinant proteins. ⁇ 15 is preferred, 1-10 is more preferred, and 1-5 is even more preferred.
  • the range of identity of "amino acid sequence having 98% or more identity with respect to the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 5" is such that proteins having different identities are sequenced. It is not particularly limited as long as it has the same characteristics as the protein consisting of the amino acid sequence of No. 3 or SEQ ID NO: 5 (ArtLAAO4 or ArtLAAO5).
  • the identity with respect to the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 5 from the viewpoint of having excellent properties such as its L-aminooxidase activity, wide substrate selectivity, and large-scale expression of recombinant protein. Is preferably 98% or more, more preferably 98.5% or more, and further preferably 99% or more.
  • the protein of the present invention also includes a protein modified with a sugar chain or the like.
  • the type, position, etc. of the sugar chain added to the protein differ depending on the host cell used for producing the recombinant protein, but the protein obtained by using any host cell is also included.
  • the protein of the present invention is a novel artificial L-amino acid oxidase (ArtLAAO), and has an action of oxidizing L-amino acid or a derivative thereof to form imino acid as shown in Chemical Formula 1 below.
  • ArticleLAAO novel artificial L-amino acid oxidase
  • the resulting imino acid is racemized and converted to a racemate, so that the L-amino acid is finally converted to a D-amino acid.
  • the reducing agent is not present in the reaction system, the generated imino acid is hydrolyzed to form keto acid, so that keto acid is synthesized from the L-amino acid or its derivative. Therefore, the L-amino acid oxidase of the present invention is suitably used for optical resolution of DL-amino acid or a derivative thereof.
  • All of the ArtLAAOs of the present invention have a wide substrate selectivity, and have high activity not only on hydrophilic amino acids and hydrophobic amino acids but also on aromatic amino acids and their derivatives.
  • the substrate is not particularly limited as long as it is an L-amino acid or a derivative thereof that is oxidized by the novel L-amino acid oxidase of the present invention, but specifically, as an example, L-methionine, L-leucine, L-phenylalanine.
  • reaction substrate having particularly high activity examples include L-methionine or L-leucine for hydrophobic amino acids, L-phenylalanine or L-tryptophan for aromatic amino acids, and L-glutamine or L-glutamic acid for hydrophilic amino acids. Be done.
  • the ArtLAAO of the present invention has sufficient activity not only for natural L-amino acids but also for various derivatives of L-amino acids.
  • L-phenylalanine derivatives and L in which the hydrogen atom of the phenyl group portion of L-phenylalanine is substituted with a fluorine atom, a bromine atom, a nitro group, an amino group, a methoxy group, a trifluoromethyl group or the like, respectively.
  • the ArtLAAO of the present invention can easily convert an L-amino acid derivative into a D-amino acid derivative or a L-amino acid derivative into keto acid.
  • the characteristics of ArtLAAO1 consisting of the amino acid sequence represented by SEQ ID NO: 1 are as follows. (1) The optimum pH is 7 to 8. (2) The optimum temperature is 25 ° C to 35 ° C. (3) The isoelectric point (pI) is 5.01. (4) The molecular weight is 74.27 kDa.
  • the characteristics of ArtLAAO4 consisting of the amino acid sequence represented by SEQ ID NO: 3 are as follows. (1) The optimum pH is 6.5 to 7.0. (2) The isoelectric point (pI) is 4.9. (3) The molecular weight is 74.1 kDa.
  • the characteristics of ArtLAAO5 consisting of the amino acid sequence represented by SEQ ID NO: 5 are as follows. (1) The optimum pH is 6.0. (2) The isoelectric point (pI) is 5.1. (3) The molecular weight is 73.9 kDa.
  • the protein of the present invention can be produced according to a conventional method by expressing a protein using Escherichia coli or the like as a host using a gene encoding this protein. It is also possible to produce ArtLAAO by chemical synthesis, but unlike the conventional LAAO, the ArtLAAO of the present invention is a novel protein capable of achieving a large amount of recombinant protein expression, so gene recombination technology can be used. It is preferable to use and manufacture.
  • the gene encoding this protein is as follows ( 5) A gene encoding any of the proteins (8). (5) A protein comprising the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 and consisting of an amino acid sequence having L-amino acid oxidase activity, (6) SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.
  • a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the represented amino acid sequence and having L-amino acid oxidase activity, (7) the amino acid sequence represented by SEQ ID NO: 1.
  • the gene encoding this ArtLAAO protein can be obtained by a known method such as artificial gene synthesis.
  • a gene consisting of the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 can be used.
  • a recombinant vector can be obtained by inserting a DNA fragment encoding ArtLAAO obtained by artificial gene synthesis as an insert and inserting it downstream of the promoter of an appropriate expression vector.
  • the promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected depending on the type of host.
  • a transformant producing a protein having the L-amino acid oxidase activity of the present invention can be obtained.
  • the produced protein can be recovered by a predetermined method.
  • the expression vector either a vector capable of autonomously replicating in the host cell or a vector integrated into the chromosome of the host cell is used, but a plasmid vector which is a vector capable of autonomously replicating in the host cell, A phage vector, a virus vector, or the like is preferably used.
  • plasmid vectors for expressing Escherichia coli such as pET vector, pCold vector, pBR vector and pUC vector are preferably used.
  • the promoter is not particularly limited as long as it can be expressed in a host cell such as Escherichia coli, and examples thereof include promoters such as trp promoter, lac promoter, lacT7 promoter, PL promoter, and PR promoter.
  • the expression vector may contain a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selectable marker such as a drug resistance gene, a ribosome binding site, and the like.
  • any of microorganisms, animal cells, insect cells, plant cells, etc. can be used as long as a recombinant vector can be introduced and the target gene can be expressed.
  • the microorganism is not particularly limited, and examples thereof include microorganisms such as Escherichia (Escherichia coli), Bacillus, Streptomyces, Streptomyces, and Staphylococcus. Specific examples of Escherichia coli used include E. coli. Examples thereof include BL21 strain, BL21 (DE3) strain, JM109 strain, and HB101 strain of cori.
  • the method for introducing the recombinant vector into the host cell can be carried out based on any known method, for example, a method using electroporation or calcium ions.
  • the transformant into which the recombinant vector having the gene encoding the ArtLAAO protein has been introduced is cultured based on the usual medium and culture method used for culturing the host cell.
  • an expression vector containing a promoter that induces protein expression protein expression occurs by adding an inducer to the medium.
  • an inducer for example, when Escherichia coli transformed with an expression vector containing a lac promoter is used, it is linked downstream of the promoter by adding isopropyl- ⁇ -D-thiogalactopyranoside (IPTG). Protein is expressed.
  • the target protein can be obtained by recovering the protein of the present invention from a culture such as a cultured cell of a transformant or a culture solution.
  • the "culture” includes any of the culture medium or medium after the culture treatment, the cells after the culture, or crushed products thereof.
  • the expressed protein When the expressed protein is accumulated in the cells of the transformant, the expressed protein can be recovered by disrupting the cells or the like according to a known method.
  • the desired protein can be obtained by collecting the supernatant portion of the culture solution.
  • the recovered protein can be purified based on the existing protein purification method.
  • the novel L-amino acid oxidase (ArtLAAO) of the present invention can be expressed in large quantities in an Escherichia coli expression system.
  • L-amino acid oxidase can be produced easily and at low cost, so that L-amino acid oxidase can be industrially used.
  • the ArtLAAO of the present invention can be used for dynamic optical resolution for converting DL-amino acid or its derivative into D-amino acid or its derivative, and can realize industrial production of D-amino acid or D-amino acid derivative.
  • the dynamic optical resolution method using ArtLAAO of the present invention is performed in the following order. When ArtLAAO is allowed to act on DL-amino acids or derivatives thereof, ArtLAAO and D-amino acids do not react with each other, but L-amino acids and the like are oxidized to form imino acid as an intermediate (see Chemical Formula 1 above). ).
  • This imino acid is converted into a racemate again by the action of a racemization catalyst such as a reducing agent.
  • a racemization catalyst such as a reducing agent.
  • L-amino acids and the like react with L-amino acid oxidase again to form imino acids, but D-amino acids and the like remain without reacting.
  • DL-amino acids and the like are finally converted into D-amino acids and the like, and a high-purity optically active substance is obtained.
  • racemization of the generated imino acid is surely performed with respect to 1 mM (0.5 mM as a substrate) of the reaction raw material (DL-amino acid or its derivative).
  • the reducing agent coexists with 15 mM or more, more preferably 20 mM or more, and particularly preferably 30 mM or more.
  • the reducing agent used as the racemic catalyst include ammonia borane, sodium borohydride, lithium aluminum hydride, sodium cyanotrihydroborate, etc., and even when coexisting with ArtLAAO, their functions are not so great.
  • Ammonia borane is particularly preferable from the viewpoint that it does not affect each other and can maintain mutual reactivity.
  • the amount of ArtLAAO added to 1 mM (0.5 mM as a substrate) of the reaction raw material (DL-amino acid or a derivative thereof) is preferably 1 U or more, and more preferably 2 U or more.
  • the reaction temperature is preferably 20 ° C to 40 ° C, more preferably 25 ° C to 35 ° C, still more preferably 28 ° C to 32 ° C.
  • the reaction time varies depending on the amount of the reaction raw material, but is about 3 to 24 hours, and the timing of completion of the reaction can be determined by analyzing the reaction solution by chiral HPLC or the like.
  • an optically active substance having a high enantiomeric excess (ee) of more than 99% can be obtained, and a yield of more than 78% can be obtained.
  • Dynamic optical resolution is realized in which a highly pure optically active substance can be obtained in one pot with high efficiency such as showing the yield.
  • the ArtLAAO of the present invention can also be used for optical resolution to convert DL-amino acid or a derivative thereof into D-amino acid or a derivative thereof and keto acid. In this case, it is not necessary to add a reducing agent which is a racemization catalyst.
  • the optical resolution method using ArtLAAO of the present invention is performed in the following order. When ArtLAAO is allowed to act on DL-amino acid or a derivative thereof, ArtLAAO and D-amino acid and the like do not react with each other, but L-amino acid and the like are oxidized to form imino acid as an intermediate.
  • this imino acid is naturally hydrolyzed in an aqueous reaction solution and converted to keto acid.
  • the D-amino acid or the like By separating the D-amino acid or the like remaining without the reaction from the keto acid produced by the enzymatic reaction and hydrolysis, the D-amino acid or the like and the keto acid can be obtained, respectively.
  • the separation of D-amino acid and the like from keto acid can be carried out by, for example, a selective precipitation method in an acidic aqueous solution, an ion exchange chromatography method or the like.
  • Example 1 Design of artificial L-amino acid oxidase (ArtLAAO1)
  • ArtLAAO1 Design of artificial L-amino acid oxidase
  • AROD L-arginine oxidase
  • Example 2 2. Production of Artificial L-Amino Acid Oxide (ArtLAAO1)
  • a gene (SEQ ID NO: 2) encoding ArtLAAO1 consisting of 655-residue amino acids obtained in Example 1 was prepared by artificial gene synthesis. The gene sequence is shown in Table 3 below.
  • the prepared ArtLAAO1 gene was cloned downstream of the T7 promoter of the pET-28b vector (a product of Novagen) to obtain a recombinant vector containing the ArtLAAO1 gene.
  • the constructed ArtLAAO1 expression vector was introduced into an Escherichia coli BL21 (DE3) strain and transformed to obtain an ArtLAAO1 production strain.
  • the obtained ArtLAAO1 production strain was inoculated into 1 L of LB medium and mass-cultured. Specifically, shake culture is carried out under the condition of 37 ° C. for about 4 to 6 hours, and when the OD value at 600 nm reaches 0.4 to 0.6, IPTG (isopropyl- ⁇ -D-thiogalactopira) Noside) was added to a final concentration of 0.5 mM to induce the expression of ArtLAAO1 protein. After the addition of IPTG, the culture temperature was lowered to 23 ° C. and the cells were shake-cultured for 16 hours. The cells after culturing were collected, and the ArtLAAO1 protein expressed from the cells was collected. Specifically, the protein was recovered by the following method.
  • IPTG isopropyl- ⁇ -D-thiogalactopira
  • the culture broth was centrifuged at 6000 xg for 10 minutes to remove the LB liquid medium. After suspension in a buffer containing 60 mL of 20 mM Tris-HCl (pH 8.0) and 10 mM NaCl, the cells were disrupted with an ultrasonic homogenizer. This crushed solution was centrifuged at 11000 ⁇ g for 30 minutes, and the supernatant fraction was used as a crude enzyme solution. The crude enzyme solution was immediately adsorbed on a HisTrap TM HP column (a product of GE Healthcare Japan Co., Ltd.), washed with 30 mL of buffer, and then eluted with a buffer containing 70 and 300 mM imidazole.
  • HisTrap TM HP column a product of GE Healthcare Japan Co., Ltd.
  • the reaction solution for activity measurement shown in Table 4 below was prepared, and 10 ⁇ L of the recovered ArtLAAO1 was added to 90 ⁇ L of this reaction solution, and the activity of L-amino acid oxidase was measured. The expression level of ArtLAAO1 protein was confirmed from the cell specific activity.
  • Example 3 3. Measurement of activity of artificial L-amino acid oxidase (ArtLAAO1) 20 types of L-amino acids and 2 types of L-amino acid derivatives, and 2 types of DL-amino acids and 16 types of DL-amino acid derivatives were obtained in Example 2.
  • the specific activity of artificial L-amino acid oxidase (ArtLAAO1) was measured.
  • the method for measuring the L-amino acid oxidase activity in this example is as follows. The reaction solution for activity measurement shown in Table 4 was weighed in a microtube, and this was incubated in a water bath at 30 ° C. for 30 minutes.
  • Table 5 below shows the results of enzyme activity measurement for 20 types of L-amino acids, 2 types of DL-amino acids, and 18 types of amino acid derivatives.
  • the protein of SEQ ID NO: 1 showed activity against 13 different L-amino acids, 2 DL-amino acids and 18 amino acid derivatives. Therefore, it was clarified that the protein obtained in Example 2 is an enzyme capable of widely oxidizing L-amino acids and amino acid derivatives.
  • Example 4 Dynamic optical resolution by artificial L-amino acid oxidase (ArtLAAO1) (1) Next, for DL-phenylalanine and its derivatives, dynamic optical resolution (DKR) using ArtLAAO shown in Chemical Formula 2 below was attempted. In the following Chemical Formula 2, the flow in which the D-phenylalanine derivative is produced by the dynamic optical resolution of the DL-phenylalanine derivative using the novel L-amino acid oxidase (ArtLAAO) of the present invention in Example 4 is shown. There is. As DL-phenylalanine and its derivatives, 10 kinds of reaction raw materials (DL-1a to DL-1j) shown in Table 7 below were prepared. DKR is prepared at about 30 ° C.
  • Figures 1 to 6 show the results of optical resolution of DL-phenylalanine and its derivatives (DL-1a to DL-1f) using ArtLAAO1. As shown in FIGS. 1 to 6, it was found that the peak of L-form disappeared by the addition of ArtLAAO (+ ArtLAAO) and converted to D-form. Further, Table 8 below shows the enantiomeric excess (ee) and the yield calculated from the peak area values of the D-form and the L-form. Among the DL-phenylalanine derivatives, the ee value of 4-amino-DL-phenylalanine (DL-1g) was 88%, and the ee values of the other derivatives were all over 99%.
  • the yields of all the compounds were as high as 68 to 85%. From the above results, it was found that the DL-phenylalanine derivative can be converted into the D-phenylalanine derivative with high optical purity and high yield by the dynamic optical resolution method by using the ArtLAAO of the present invention.
  • Example 5 Dynamic optical resolution by artificial L-amino acid oxidase (ArtLAAO1) (2) Furthermore, for DL-tryptophan and its derivatives, and glycine derivatives, dynamic optical resolution (DKR) using ArtLAAO1 was attempted in the same manner as in Example 4. Eight kinds of reaction raw materials (DL-2a to DL-2h) shown in Table 9 below were prepared as DL-tryptophan and its derivatives and glycine derivatives, and DKR was performed. The analysis of DKR and its results was carried out in the same manner as in Example 4. Table 9 shows the analysis conditions and retention conditions of chiral HPLC for each compound.
  • Table 10 shows the enantiomeric excess calculated from the peak area values of D-form and L-form as a result of optical resolution of DL-tryptophan and its derivatives and glycine derivatives (DL-2a to DL-2h) using ArtLAAO1.
  • ee and yield are shown.
  • the ee value of DL-7-azatryptophan (DL-2g) was slightly low at 86%, the ee value of all the other derivatives exceeded 99%.
  • the yields of all the compounds were as high as 69 to 80%. From the above results, it was found that by using the ArtLAAO of the present invention, the DL-tryptophan derivative and the like can be converted into the D-tryptophan derivative and the like by the dynamic optical resolution method with high optical purity and high yield.
  • Example 6 Design of Artificial L-Amino Acid Oxide (ArtLAAO4) Based on the AROD homolog sequence shown in Table 1 above, in order to further obtain an enzyme having the same function as the artificial L-amino acid oxidase (ArtLAAO1) obtained in Examples 1 to 5. We tried to design a new artificial L-amino acid oxidase.
  • ArtLAAO4 Artificial L-Amino Acid Oxide
  • Example 7 Production of Artificial L-Amino Acid Oxide (ArtLAAO4)
  • a gene (SEQ ID NO: 4) encoding ArtLAAO4 consisting of 656-residue amino acids obtained in Example 6 was prepared by artificial gene synthesis. The gene sequence is shown in Table 12 below.
  • the prepared ArtLAAO4 gene was cloned downstream of the T7 promoter of the pET-28b vector (a product of Novagen) to obtain a recombinant vector containing the ArtLAAO4 gene.
  • the constructed ArtLAAO4 expression vector was introduced into an Escherichia coli BL21 (DE3) strain and transformed to obtain an ArtLAAO4 production strain.
  • the obtained ArtLAAO4 production strain was inoculated into 1 L of LB medium and mass-cultured. Specifically, shake culture is carried out under the condition of 37 ° C. for about 4 to 6 hours, and when the OD value at 600 nm reaches 0.4 to 0.6, IPTG (isopropyl- ⁇ -D-thiogalactopira) Noside) was added to a final concentration of 0.5 mM to induce the expression of ArtLAAO4 protein. After the addition of IPTG, the culture temperature was lowered to 23 ° C. and the cells were shake-cultured for 16 hours. The cells after culturing were recovered, and the ArtLAAO4 protein expressed from the cells was recovered. The same amount of protein as ArtLAAO1 described above could be recovered.
  • IPTG isopropyl- ⁇ -D-thiogalactopira
  • the protein was recovered by the following method.
  • the culture broth was centrifuged at 6000 xg for 10 minutes to remove the LB liquid medium.
  • the crude enzyme solution was immediately adsorbed on a HisTrap TM HP column (a product of GE Healthcare Japan Co., Ltd.), washed with 30 mL of buffer, and then eluted with a buffer containing 70 and 300 mM imidazole.
  • Example 8 Measurement of the activity of artificial L-amino acid oxidase (ArtLAAO4)
  • the specific activity of the artificial L-amino acid oxidase (ArtLAAO4) obtained in Example 7 was measured for 20 types of L-amino acids.
  • the measurement of the L-amino acid oxidase activity was carried out in the same manner as in Example 3 described above.
  • the enzyme activity measurement results for 20 types of L-amino acids of ArtLAAO4 are shown in Table 13 below.
  • the protein of SEQ ID NO: 3 showed activity against 11 different L-amino acids. Therefore, it was clarified that the protein obtained in Example 7 is an enzyme capable of widely oxidizing L-amino acids. Enzymatic parameters for 6 L-amino acids (L-methionine, L-leucine, L-phenylalanine, L-tryptophan, L-glutamine and L-glutamic acid) that showed particularly high activity among 20 types of L-amino acids It was determined. The results are shown in Table 14 below. As a result of the analysis, it was revealed that ArtLAAO4 has the highest enzymatic efficiency for L-methionine. On the other hand, ArtLAAO4 was found to have high activity against L-phenylalanine. From the above results, it was shown that the ArtLAAO of the present invention is an L-amino acid oxidase having a wide substrate selectivity.
  • Example 9 Design of Artificial L-Amino Acid Oxide (ArtLAAO5) AROD shown in Table 1 above to further obtain enzymes having the same functions as ArtLAAO1 obtained in Examples 1 to 5 and ArtLAAO4 obtained in Examples 6 to 8. An attempt was made to design a new artificial L-amino acid oxidase based on the homolog sequence.
  • ArtLAAO5 Artificial L-Amino Acid Oxide
  • Example 10 Production of Artificial L-Amino Acid Oxide (ArtLAAO5)
  • a gene (SEQ ID NO: 6) encoding ArtLAAO5 consisting of 655-residue amino acids obtained in Example 9 was prepared by artificial gene synthesis. The gene sequence is shown in Table 16 below.
  • the prepared ArtLAAO5 gene was cloned downstream of the T7 promoter of the pET-28b vector (a product of Novagen) to obtain a recombinant vector containing the ArtLAAO5 gene.
  • the constructed ArtLAAO5 expression vector was introduced into an Escherichia coli BL21 (DE3) strain and transformed to obtain an ArtLAAO5 production strain.
  • the obtained ArtLAAO5 production strain was inoculated into 1 L of LB medium and mass-cultured, and the ArtLAAO5 protein was recovered by the same method as in Example 7. The same amount of protein as ArtLAAO1 described above could be recovered.
  • the L-amino acid oxidase activity was measured by adding 10 ⁇ L of the recovered ArtLAAO5 protein to 90 ⁇ L of the activity measurement reaction solution shown in Table 4 above, and it was confirmed that the obtained ArtLAAO5 had L-amino acid oxidase activity.
  • Example 11 Measurement of activity of artificial L-amino acid oxidase (ArtLAAO5)
  • the specific activity of artificial L-amino acid oxidase (ArtLAAO5) obtained in Example 10 was measured for 20 kinds of L-amino acids.
  • the measurement of the L-amino acid oxidase activity was carried out in the same manner as in Example 3 described above.
  • the results of enzyme activity measurement for 20 types of L-amino acids of ArtLAAO5 are shown in Table 17 below.
  • the protein of SEQ ID NO: 5 showed activity against 8 different L-amino acids. Therefore, it was clarified that the protein obtained in Example 10 is an enzyme capable of widely oxidizing L-amino acids.
  • enzymatic parameters were determined for 5 L-amino acids (L-methionine, L-leucine, L-phenylalanine, L-glutamine and L-glutamic acid) that showed particularly high activity.
  • the results are shown in Table 18 below.
  • ArtLAAO5 has the highest enzymatic efficiency for L-leucine. From the above results, it was shown that the ArtLAAO of the present invention is an L-amino acid oxidase having a wide substrate selectivity.
  • Example 12 The heat resistance and durability of each of the three types of artificial L-amino acid oxidases (ArtLAAO1, ArtLAAO4 and ArtLAAO5) obtained in the above-mentioned examples were examined.
  • the heat resistance was tested as follows. An artificial L-amino acid oxidase enzyme solution was applied for 10 minutes in a heat block set at 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C, 75 ° C and 80 ° C, respectively. Incubated and ice bathed.
  • the reaction solutions for activity measurement shown in Table 19 below were prepared and incubated at 30 ° C. for 10 minutes in a water bath.
  • the durability was tested as follows. 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours in a heat block in which the enzyme solution of artificial L-amino acid oxidase was set at 30 ° C. Incubated for 168 hours.
  • the reaction solution for activity measurement shown in Table 19 was prepared and incubated at 30 ° C. for 10 minutes in a water bath. Weigh 90 ⁇ L of this activity measurement reaction solution into a quartz cell, add 10 ⁇ L of each enzyme solution after heat treatment, start the reaction, and immediately use an ultraviolet-visible spectrophotometer (UV-2450, Shimadzu Corporation) to measure 505 nm. The change in absorbance was measured to determine the residual enzyme activity. The results are shown in FIG. 7 (B).
  • ArtLAAO1 has no effect on the enzyme activity in the heat treatment at 30 to 35 ° C., but the enzyme activity is reduced to about half after the heat treatment at 40 to 45 ° C.
  • ArtLAAO4 and ArtLAAO5 maintained about 90% of the enzyme activity even after the heat treatment at 55 ° C., and were found to be excellent in heat resistance.
  • the enzyme activity of ArtLAAO1 decreases to about 10 to 20% in a few hours under the condition of 30 ° C., but the enzyme activity itself remains as it is for about 144 hours (6 days), so that it can withstand a long reaction. I understood.
  • ArtLAAO4 and ArtLAAO5 maintain about 30% of the enzyme activity even after 24 hours, and in particular, ArtLAAO4 maintains about 30% of the enzyme activity even after 168 hours (7 days), and it is clear that it is excellent in durability. It became.
  • the novel L-amino acid oxidase (ArtLAAO) of the present invention can be produced in large quantities at low cost because it can express a large amount of recombinant protein, and further, it has a wide substrate selectivity, and therefore D-amino acid. Alternatively, it is widely used for optical resolution of its derivatives. Therefore, ArtLAAO is useful for the synthesis of pharmaceutical raw materials and raw material intermediates.
  • SEQ ID NO: 1 Amino acid sequence of artificial L-amino acid oxidase (ArtLAAO1)
  • SEQ ID NO: 2 Nucleotide sequence of gene encoding artificial L-amino acid oxidase (ArtLAAO1)
  • SEQ ID NO: 3 Amino acid sequence of artificial L-amino acid oxidase (ArtLAAO4)
  • No. 4 Nucleotide sequence of the gene encoding artificial L-amino acid oxidase (ArtLAAO4)
  • SEQ ID NO: 6 Gene encoding artificial L-amino acid oxidase (ArtLAAO5) Base sequence

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Abstract

La présente invention peut produire une L-acide aminé oxydase à faible coût et en grande quantité par l'intermédiaire d'une expression élevée d'une protéine recombinée, et similaire, et fournit une nouvelle L-acide aminé oxydase (ArtLAAO) ayant une large sélectivité de substrat. Cette nouvelle L-acide aminé oxydase est une protéine comprenant l'une quelconque des séquences d'acides aminés (1) à (4) : (1) une séquence d'acides aminés comprenant une équence d'acides aminés représentée par SEQ ID NO : 1 SEQ ID NO : 3 ou SEQ ID NO; 5, et ayant une activité de L-acide aminé oxydase; (2) une séquence d'acides aminés comprenant une séquence d'acides aminés représentée par SEQ ID NO : 1 SEQ ID NO : 3 et SEQ ID NO: 5 avec un ou plusieurs acides aminés délétés, substitués ou ajoutés, et ayant une activité de L-acide aminé oxydase; (3) une séquence d'acides aminés comprenant une séquence d'acides aminés ayant au moins 90 % d'identité d'acides aminés avec la séquence d'acides aminés représentée par SEQ ID NO : 1, et ayant une activité de L-acide aminé oxydase; et (4) une séquence d'acides aminés comprenant une séquence d'acides aminés ayant au moins 98 % d'identité d'acides aminés avec la séquence d'acides aminés représentée par SEQ ID NO : 3 et SEQ ID NO: 5, et ayant une activité de L-acide aminé oxydase.
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CN115414382B (zh) * 2022-06-23 2024-03-26 南京邮电大学 级联放大气体治疗平台及其制备方法和应用
WO2024048720A1 (fr) * 2022-09-01 2024-03-07 花王株式会社 Procédé de recherche de lipase

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