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  • C12Y402/00—Carbon-oxygen lyases (4.2)
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  • C12Y402/01084—Nitrile hydratase (4.2.1.84)
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  • C12N15/09—Recombinant DNA-technology
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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
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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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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)
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  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes

Definitions

• the present disclosure relates to a mutant nitrile hydratase, a nucleic acid encoding the mutant nitrile hydratase, a vector and a transformant containing the nucleic acid, a method for producing the mutant nitrile hydratase, and a method for producing an amide compound.
• Nitrile hydratase is an enzyme with nitrile hydration activity that converts the nitrile groups of various compounds into amide groups through hydration, and is used in industrial processes for producing amide compounds using enzymatic reactions.
• acrylonitrile which is used as a raw material for the production of acrylamide, an amide compound, can contain various impurities (for example, acrolein, a by-product, etc.).
• impurities for example, acrolein, a by-product, etc.
• WO 2005/000014 describes that during the production of acrylonitrile, acrolein is usually generated as a by-product, which forms an insoluble polymer as a side reaction.
• Patent Document 2 describes that acrolein, as an example of an impurity contained in acrylonitrile, inhibits the synthesis reaction of acrylamide by nitrile hydratase. Patent Document 2 describes that inhibition of the catalytic action by nitrile hydratase by acrolein can be prevented by controlling the concentration of acrolein in the raw material acrylonitrile to 1 ppm or less using an ion exchange resin or the like.
• Patent Document 2 avoids inhibition of nitrile hydratase activity by acrolein by controlling the acrolein concentration in the raw material acrylonitrile to 1 ppm or less, and even in the presence of impurities, nitrile hydratase It does not reduce inhibition to activity.
• the present disclosure provides a technology related to a novel mutant nitrile hydratase that reduces inhibition by impurities of an amide compound production reaction using nitrile hydratase even in the presence of impurities.
• nitrile hydratase having A mutant nitrile hydratase comprising a substitution of at least one amino acid residue selected from the group consisting of the following (a) to (q), (a) In the ⁇ subunit, substitution of the amino acid residue Ala corresponding to the 23rd position from the N-terminus of SEQ ID NO: 1 with Gly; (b) in the ⁇ subunit, substitution of the amino acid residue Val corresponding to the 105th position from the N-terminus of SEQ ID NO: 1 with Met; (c) in the ⁇ subunit, substitution of the amino acid residue Leu corresponding to the 142nd position from the N-terminus of SEQ ID NO: 1 with Cys; (d) in the ⁇ subunit, substitution of the amino acid amino acid
• ⁇ 2> The mutant nitrile hydratase according to ⁇ 1>, which comprises at least two amino acid residue substitutions selected from the group consisting of (a) to (q).
• ⁇ 3> The mutant nitrile according to ⁇ 1> or ⁇ 2>, comprising at least one amino acid residue substitution selected from the group consisting of (i), (g), (j), and (n). hydratase.
• ⁇ 4> The mutant nitrile according to ⁇ 1> or ⁇ 2>, comprising at least one amino acid residue substitution selected from the group consisting of (i), (g), (l), and (m). hydratase.
• ⁇ 5> The mutant nitrile hydratase according to ⁇ 1> or ⁇ 2>, which comprises at least one amino acid residue substitution selected from the group consisting of (g), (p), and (q).
• ⁇ 6> The variant according to ⁇ 1> or ⁇ 2> above, wherein the amino acid sequence of the ⁇ subunit further comprises at least one amino acid residue substitution selected from the group consisting of (A1) to (A6) below.
• the amino acid sequence of the ⁇ subunit further comprises at least one amino acid residue substitution selected from the group consisting of (B1) to (B9) below.
• nitrile hydratase (B1) In the ⁇ subunit, substitution of the amino acid residue Thr corresponding to the 40th position from the N-terminus of SEQ ID NO: 2 with He; (B2) In the ⁇ subunit, substitution of the amino acid residue Arg corresponding to the 42nd position from the N-terminus of SEQ ID NO: 2 with Pro; (B3) In the ⁇ subunit, substitution of the amino acid residue Thr corresponding to the 76th position from the N-terminus of SEQ ID NO: 2 with Cys, (B4) In the ⁇ subunit, substitution of the amino acid residue Thr corresponding to the 83rd position from the N-terminus of SEQ ID NO: 2 with His; (B5) In the ⁇ subunit, substitution of the amino acid residue Leu corresponding to the
• ⁇ 8> A nucleic acid encoding the mutant nitrile hydratase according to any one of ⁇ 1> to ⁇ 7> above.
• ⁇ 9> A vector comprising the nucleic acid according to ⁇ 8> above.
• ⁇ 11> A transformant comprising the expression vector according to ⁇ 10> above.
• ⁇ 12> Cultivating the transformant according to ⁇ 11> above in a medium, and Recovering the mutant nitrile hydratase according to any one of ⁇ 1> to ⁇ 7> from at least one of the cultured transformant and the medium;
• a method for producing a mutant nitrile hydratase comprising: ⁇ 13> A mutant nitrile hydratase obtained by the production method described in ⁇ 12> above.
• ⁇ 14> A method for producing an amide compound, comprising bringing the mutant nitrile hydratase according to any one of ⁇ 1> to ⁇ 7> into contact with a nitrile compound.
• the upper limit or lower limit described in one numerical range may be replaced with the upper limit or lower limit of another numerical range described step by step.
• the upper limit or lower limit of the numerical range may be replaced with the values shown in the Examples.
• each component may contain multiple types of corresponding substances.
• the amount of each component in the composition in this disclosure if there are multiple types of substances corresponding to each component in the composition, unless otherwise specified, the amount of each component in the composition is means the total amount of substance.
• the term "step” is included not only in an independent step but also in the case where the intended purpose of the step is achieved even if the step cannot be clearly distinguished from other steps.
• a numerical range indicated using “ ⁇ ” indicates a range that includes the numerical values written before and after " ⁇ " as the minimum and maximum values, respectively.
• the amount of each component in the composition means the total amount of the multiple substances present in the composition. do.
• enzyme activity refers to nitrile hydration activity that converts nitrile groups to amide groups.
• the explanation of a nucleotide sequence may be made by focusing on the sequence on one strand even when the nucleic acid strand holding the nucleotide sequence forms a double strand. For the other strand of the duplex, the description of such sequences should translate to its complementary sequence.
• ⁇ Mutant nitrile hydratase> The present disclosure provides an ⁇ subunit having a sequence identity of 90% or more to the amino acid sequence represented by SEQ ID NO: 1, a ⁇ subunit having a sequence identity of 90% or more to the amino acid sequence represented by SEQ ID NO: 2, Provided is a mutant nitrile hydratase comprising a substitution of at least one amino acid residue selected from the group consisting of the following (a) to (q): (a) In the ⁇ subunit, substitution of the amino acid residue Ala corresponding to the 23rd position from the N-terminus of SEQ ID NO: 1 with Gly; (b) in the ⁇ subunit, substitution of the amino acid residue Val corresponding to the 105th position from the N-terminus of SEQ ID NO: 1 with Met; (c) in the ⁇ subunit, substitution of the amino acid residue Leu corresponding to the 142nd position from the N-terminus of SEQ ID NO: 1 with Cys; (d) in the ⁇ subunit
• a mutant nitrile hydratase containing a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) above has not been reported so far and is a novel mutant.
• the mutant nitrile hydratase of the present disclosure differs from the wild-type Pseudonocardia thermophila-derived nitrile hydratase and the conventional mutant nitrile hydratase, even in the presence of impurities (for example, acrolein, etc.). Also, inhibition by impurities of the amide compound production reaction using nitrile hydratase is reduced, and it exhibits excellent enzymatic activity for the synthesis reaction of amide compounds from nitrile compounds.
• impurities for example, acrolein, etc.
• the reduction in reaction inhibition caused by the impurities of the mutant nitrile hydratase of the present disclosure is particularly remarkable when the impurities are contained at a high concentration (for example, 1000 ppm or more). Therefore, according to the mutant nitrile hydratase of the present disclosure, in an industrial amide compound manufacturing process using nitrile hydratase, even when a raw material that has not been sufficiently purified is used, a reaction for synthesizing an amide compound is possible. It becomes possible to make sufficient progress. In this way, according to the mutant nitrile hydratase of the present disclosure, it is also possible to dramatically improve the efficiency of industrial production of amide compounds compared to the case of using conventional enzymes.
• the mutant nitrile hydratase of the present disclosure has an ⁇ subunit that has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and an ⁇ subunit that has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2.
• the mutant nitrile hydratase is a mutant nitrile hydratase, which has a ⁇ subunit with a specific function, and includes a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) described above.
• the amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence of the ⁇ subunit of wild-type Pseudonocardia thermophila-derived nitrile hydratase.
• the amino acid sequence represented by SEQ ID NO: 2 is the amino acid sequence of the ⁇ subunit of nitrile hydratase derived from wild-type Pseudonocardia thermophila.
• the ⁇ subunit having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 is the ⁇ subunit possessed by the nitrile hydratase derived from wild type Pseudonocardia thermophila (wild type ⁇ subunit This means that the ⁇ subunit has an amino acid sequence that is 90% or more identical to the amino acid sequence of the wild type ⁇ subunit.
• the ⁇ subunit having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 is the ⁇ subunit possessed by the nitrile hydratase derived from wild type Pseudonocardia thermophila (wild type ⁇ This means that the ⁇ subunit has an amino acid sequence that is 90% or more identical to the amino acid sequence of the wild type ⁇ subunit.
• the mutant nitrile hydratase has an ⁇ subunit that has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, and has a sequence identity of 95% or more with the amino acid sequence represented by SEQ ID NO: 1. It may have an ⁇ subunit having 98% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1.
• the mutant nitrile hydratase has a ⁇ subunit that has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 2, and has a sequence identity of 95% or more with the amino acid sequence represented by SEQ ID NO: 1. It may have a ⁇ subunit having 98% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1.
• the basic structural unit of mutant nitrile hydratase is a dimer in which an ⁇ subunit and a ⁇ subunit associate, and the dimers further associate to form a tetramer.
• the cysteine residue which is the 111th amino acid residue from the N-terminus of the ⁇ subunit, is converted to cysteine sulfinic acid (Cys-SOOH), and the cysteine residue, which is the 113th amino acid residue from the N-terminus, is converted to cysteine sulfenic acid (Cys-SOOH).
• -SOH are each subjected to post-translational modification, and the polypeptide chain of the ⁇ subunit and cobalt atom are bonded to each other via this modified amino acid residue to form an active center.
• the mutant nitrile hydratase of the present disclosure includes a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) above.
• the mutant nitrile hydratase of the present disclosure only needs to contain at least one amino acid residue substitution selected from the group consisting of (a) to (q) above, and may contain a combination of multiple types.
• the mutant nitrile hydratase of the present disclosure includes substitutions of amino acid residues other than the group consisting of (a) to (q) above, within a range that reduces inhibition by impurities of the amide compound production reaction using nitrile hydratase. It may further contain.
• the mutant nitrile hydratase of the present disclosure contains at least two amino acid residues selected from the group consisting of (a) to (q) above, from the viewpoint of further reducing inhibition by impurities of the amide compound production reaction using nitrile hydratase. It is preferable to include group substitution.
• the mutant nitrile hydratase of the present disclosure more preferably contains the amino acid residue substitution described in (i) above from the viewpoint of further reducing inhibition by impurities of the amide compound production reaction using nitrile hydratase.
• the mutant nitrile hydratase of the present disclosure has three or more selected from the group consisting of (a) to (q) above, from the viewpoint of further reducing inhibition by impurities of the amide compound production reaction using nitrile hydratase, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more or 17 amino acid residue substitutions.
• the mutant nitrile hydratase of the present disclosure has 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less selected from the group consisting of (a) to (q) above. It may contain no more than 1, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, or no more than 2 amino acid residue substitutions.
• the mutant nitrile hydratase of the present disclosure includes a combination of two amino acid residue substitutions from the group consisting of (a) to (q) above
• the combination of the two amino acid residue substitutions is not particularly limited, but for example, from the viewpoint of further reducing inhibition by impurities of the amide compound production reaction using nitrile hydratase, the following combinations may be included.
• the mutation positions are briefly described for the amino acid residue substitutions of the group consisting of (a) to (q) above.
• the above-mentioned combination of (j) ⁇ 149 and (n) ⁇ 232 is the substitution of amino acid residue Thr corresponding to the 149th position from the N-terminus of SEQ ID NO: 2 with Gln in the (j) ⁇ subunit
• (n ) refers to a combination of substitution of amino acid residue Ala corresponding to the 232nd position from the N-terminus of SEQ ID NO: 2 with Trp, Ser, Pro, Phe, He, He, Arg, Cys, or Gly in the ⁇ subunit.
• the following combination may be any of the candidates. This also applies to the exemplified description of combinations in cases where combinations of three or more types of amino acid residue substitutions are included, which will be described later.
• the mutant nitrile hydratase of the present disclosure includes a combination of three amino acid residue substitutions from the group consisting of (a) to (q) above
• the combination of the three amino acid residue substitutions is not particularly limited, but for example, from the viewpoint of further reducing inhibition by impurities of the amide compound production reaction using nitrile hydratase, the following combinations may be included.
• the mutation positions are briefly described for the amino acid residue substitutions of the group consisting of (a) to (q) above.
• the mutant nitrile hydratase of the present disclosure includes a combination of four amino acid residue substitutions from the group consisting of (a) to (q) above
• the combination of the three amino acid residue substitutions is not particularly limited, but for example, from the viewpoint of further reducing inhibition by impurities of the amide compound production reaction using nitrile hydratase, the following combinations may be included.
• only the mutation positions are briefly described for the amino acid residue substitutions of the group consisting of (a) to (q) above.
• substitutions may be made by combining five or more amino acid residue substitutions from the group consisting of (a) to (q) above.
• the mutant nitrile hydratase of the present disclosure contains at least one amino acid residue substitution selected from the group consisting of (i), (g), (l), and (m), reaction inhibition caused by the impurities is suppressed.
• the thermostability of nitrile hydratase tends to increase.
• Conventional nitrile hydratases tend to have reduced catalytic activity at temperatures above room temperature (20° C.). Therefore, when raw materials with low solubility at room temperature are used in the reaction, the yield of the amide compound obtained is small, or a cooling device is required during production, so improvements have been sought from the viewpoint of production costs.
• the mutant nitrile hydratase of the present disclosure having the above configuration for example, even at a temperature higher than room temperature (e.g. 20°C) (e.g. 30°C), compared to the conventional nitrile hydratase, High thermal stability, high catalytic activity, and easy to maintain.
• room temperature e.g. 20°C
• the mutant nitrile hydratase of the present disclosure having the above configuration can raise the temperature of the reaction system even when a raw material with low solubility at room temperature is used in the reaction, so it is superior to conventional nitrile hydratase.
• the yield of the amide compound obtained is likely to be high, and a cooling device is not required during production, resulting in a secondary effect of being superior in terms of production cost.
• the amino acid sequence of the ⁇ subunit is selected from the group consisting of (A1) to (A6) below, from the viewpoint of further reducing inhibition by impurities of the amide compound production reaction using nitrile hydratase.
• Optionally further comprises at least one amino acid residue substitution selected from: (A1) In the ⁇ subunit, substitution of the amino acid residue Arg corresponding to the 20th position from the N-terminus of SEQ ID NO: 1 with Val; (A2) In the ⁇ subunit, substitution of the amino acid residue Gln corresponding to the 141st position from the N-terminus of SEQ ID NO: 1 with Arg; (A3) In the ⁇ subunit, substitution of the amino acid residue Glu corresponding to the 185th position from the N-terminus of SEQ ID NO: 1 with Ala; (A4) In the ⁇ subunit, substitution of the amino acid residue Ala corresponding to the 187th position from the N-terminus of SEQ ID NO: 1 with Arg; (A5) In the ⁇ subunit, amino acid residues Pro to Ala correspond to the 200th position from the N-terminus of SEQ ID NO: 1, (A6) In the ⁇ subunit, substitution of amino acid residue Val corresponding to the 204th position from the N-terminus of
• the amino acid sequence of the ⁇ subunit is selected from the group consisting of (B1) to (B9) below, from the viewpoint of further reducing inhibition by impurities of the amide compound production reaction using nitrile hydratase.
• Optionally further comprises at least one amino acid residue substitution selected from: (B1) In the ⁇ subunit, substitution of the amino acid residue Thr corresponding to the 40th position from the N-terminus of SEQ ID NO: 2 with He; (B2) In the ⁇ subunit, substitution of the amino acid residue Arg corresponding to the 42nd position from the N-terminus of SEQ ID NO: 2 with Pro; (B3) In the ⁇ subunit, substitution of the amino acid residue Thr corresponding to the 76th position from the N-terminus of SEQ ID NO: 2 with Cys, (B4) In the ⁇ subunit, substitution of the amino acid residue Thr corresponding to the 83rd position from the N-terminus of SEQ ID NO: 2 with His; (B5) In the ⁇ subunit, substitution of the amino acid residue Leu corresponding to the 88th position from the N-terminus of SEQ ID NO: 2 with Arg, Pro or Ser; (B6) In the ⁇ subunit, substitution of the amino acid residue Arg corresponding to the 146th
• the mutant nitrile hydratase has an amino acid residue substitution group consisting of the above (a) to (q), and an amino acid residue substitution group consisting of the above (A1) to (A6), within the range where the effects of the present disclosure are achieved. , and substitutions other than the amino acid residue substitution group consisting of (B1) to (B9) (hereinafter referred to as "other amino acid residue substitutions").
• the amino acid residue substitution group consisting of the above (a) to (q), the amino acid residue substitution group consisting of the above (A1) to (A6), and the above (B1) to (B9) The total number of substitutions selected from the amino acid residue substitution group consisting of is not particularly limited as long as it is 1 or more, for example, 1-20, 1-15, 1-10, 1-8, 1-7, 1- It may be any of 6, 1-5, 1-4, 1-3, 1-2, or 1.
• the mutant nitrile hydratase of the present disclosure is, for example, from the above (i), (g), (j), and (n), from the viewpoint of further reducing inhibition by impurities of the amide compound production reaction using nitrile hydratase.
• the mutant nitrile hydratase of the present disclosure has the above-mentioned (i) from the viewpoint of further reducing inhibition by impurities of the amide compound production reaction using nitrile hydratase and from the viewpoint of excellent thermal stability.
• (g), (l), and (m) may include at least one amino acid residue substitution selected from the group consisting of (i), (l), and (m). It may contain all the amino acid residue substitutions of (i), (g), (l) and (m) above.
• the mutant nitrile hydratase of the present disclosure has the above-mentioned (g), (p), and (q) from the viewpoint of further reducing inhibition by impurities of the amide compound production reaction using nitrile hydratase.
• q) may include all amino acid residue substitutions.
• the numbers in the column of mutation position represent the position from the N-terminal side of the amino acid sequence in the corresponding subunit.
• the numbers in the column of mutation location represent the position from the N-terminal side of the amino acid sequence in the corresponding subunit.
• the numbers in the column of mutation location represent the position from the N-terminal side of the amino acid sequence in the corresponding subunit.
• the numbers in the column of mutation location represent the position from the N-terminal side of the amino acid sequence in the corresponding subunit.
• the mutant nitrile hydratase of the present disclosure has an ⁇ subunit that has a sequence identity of 90% or more to the amino acid sequence represented by SEQ ID NO: 1, and an ⁇ subunit that has a sequence identity of 90% or more to the amino acid sequence represented by SEQ ID NO: 2.
• the origin of nitrile hydratase is if it has a ⁇ subunit of Not restricted.
• nitrile hydratase may be, for example, nitrile hydratase derived from wild-type Pseudonocardia thermophila, or a modified nitrile hydratase obtained by modifying nitrile hydratase derived from wild-type Pseudonocardia thermophila. It may be tase.
• the modified nitrile hydratase includes a modified nitrile hydratase obtained by adding one or more modifications selected from the group consisting of the following (i) to (v) to the wild-type Pseudonocardia thermophila-derived nitrile hydratase.
• the number of substituted amino acid residues is, for example, 1 to 20, or 1 to 15, or 1 to 10, or 1 to 8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2, or 1.
• the number of substituted amino acid residues may be zero.
• the number of substituted amino acid residues is, for example, 1 to 20, or 1 to 15, or 1 to 10, or 1 to 8, or 1-7, or 1-6, or 1-5, or 1-4, or 1-3, or 1-2, or 1.
• the number of substituted amino acid residues may be zero.
• the number of amino acid residues deleted is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, Or it is 1.
• the number of deleted amino acid residues may be zero.
• the number of amino acid residues deleted is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, Or it is 1.
• the number of deleted amino acid residues may be zero.
• the number of inserted amino acid residues is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, or It is 1.
• the number of inserted amino acid residues may be zero.
• the number of inserted amino acid residues is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, or It is 1.
• the number of inserted amino acid residues may be zero.
• the total number of amino acid residues substituted, deleted, or inserted is, for example, 1 to 20, or 1 to 14, or 1 to 8, or 1 to 4, or 1 to 2, or 1. In cases where there is terminal addition, the total number of substituted, deleted or inserted amino acid residues may be zero. In the ⁇ subunit of the modified nitrile hydratase, the total number of substituted, deleted, or inserted amino acid residues is, for example, 1 to 20, or 1 to 14, or 1 to 8, or 1 to 4, or 1 to 2, or 1. In cases where there is terminal addition, the total number of substituted, deleted or inserted amino acid residues may be zero.
• the number of terminally added amino acid residues is, for example, 1 to 60, or 1 to 40, or 1 to 20, or 1 to 10 per terminal. , or 1 to 5, or 1 to 3, or 1.
• the number of added amino acid residues may be zero.
• the number of terminally added amino acid residues is, for example, 1 to 60, or 1 to 40, or 1 to 20, or 1 to 10 per terminal. , or 1 to 5, or 1 to 3, or 1.
• the number of added amino acid residues may be zero.
• the terminal additional amino acid residue may be, for example, a secretion signal sequence. Terminal additional amino acid residues may be present only at the N-terminus, only at the C-terminus, or at both the N-terminus and the C-terminus.
• the similarity between the modified nitrile hydratase and the wild-type Pseudonocardia thermophila-derived nitrile hydratase can also be expressed by sequence identity.
• the amino acid sequence of the ⁇ subunit of the modified nitrile hydratase is, for example, 90% or more different from the amino acid sequence of the ⁇ subunit of the wild-type Pseudonocardia thermophila-derived nitrile hydratase represented by SEQ ID NO: 1. have sequence identity, or have 95% or more sequence identity, or have 96% or more sequence identity, or have 97% or more sequence identity, or have 98% or more sequence identity; or have 99% or more sequence identity.
• the amino acid sequence of the ⁇ subunit of the modified nitrile hydratase is, for example, 90% or more different from the amino acid sequence of the ⁇ subunit of the wild-type Pseudonocardia thermophila-derived nitrile hydratase represented by SEQ ID NO: 2. have sequence identity, or have 95% or more sequence identity, or have 96% or more sequence identity, or have 97% or more sequence identity, or have 98% or more sequence identity; or have 99% or more sequence identity.
• Alignment of sequences can be performed with ClustalW (1.83) using initial parameters (including gap open penalty: 10, gap extension penalty: 0.05). Note that sequence identity is calculated based on the full length of nitrile hydratase.
• the amino acid sequence of the modified nitrile hydratase is aligned with the amino acid sequence of the wild-type Pseudonocardia thermophila-derived nitrile hydratase, depending on the manner of modification of the modified nitrile hydratase, corresponding amino acid residues may differ from each other in the alignment. Even if there are subunits, the distances from the N-terminus of the subunits defined above may differ. In the present disclosure, in such a case, for a modified nitrile hydratase, the X-th amino acid residue from the N-terminus of a subunit refers to the Refers to the amino acid residue corresponding to the Xth amino acid residue in terms of alignment.
• the 23rd amino acid residue from the N terminus of the ⁇ subunit may be located at a position other than the 23rd position from the N-terminus of the ⁇ subunit on the amino acid sequence of the modified nitrile hydratase.
• the 23rd amino acid residue (not necessarily Ala) from the N-terminus of the ⁇ subunit of the modified nitrile hydratase is It may correspond to the Ala residue, which is the 24th amino acid residue from the N-terminus of the ⁇ subunit of Tase.
• the 23rd amino acid residue from the N-terminus of the ⁇ -subunit refers to the 24th amino acid residue from the N-terminus of the ⁇ subunit in the modified nitrile hydratase.
• the amino acid sequence of the modified nitrile hydratase may be the amino acid sequence of the modified nitrile hydratase described in WO 2010/055666 and WO 2018/124247, or a sequence similar thereto.
• the method for producing the mutant nitrile hydratase of the present disclosure is not particularly limited, for example, a vector containing a nucleic acid represented by a nucleotide sequence encoding the amino acid sequence of the mutant nitrile hydratase of the present disclosure is prepared, and this vector , can produce mutant nitrile hydratase. Details will be explained below.
• Nucleic acids of the present disclosure encode amino acid sequences of mutant nitrile hydratases of the present disclosure. More specifically, the nucleic acids of the present disclosure have a nucleotide sequence encoding the amino acid sequence of a mutant nitrile hydratase of the present disclosure.
• a method for synthesizing a nucleotide sequence encoding the amino acid sequence of the mutant nitrile hydratase of the present disclosure a method of introducing a mutation point into a nucleotide sequence encoding the corresponding wild-type nitrile hydratase derived from Pseudonocardia thermophila; , a method of chemically synthesizing the entire nucleotide sequence including the mutation point, and the like.
• methods for generating mutations in genes using the nucleotide sequence encoding wild-type Pseudonocardia thermophila-derived nitrile hydratase as a template include site-directed mutagenesis (Kramer, W.
• Nucleic acids of the present disclosure may be deoxyribonucleic acids (DNA) or ribonucleic acids (RNA).
• the gene encoding wild-type Pseudonocardia thermophila-derived nitrile hydratase consists of the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4.
• the nucleotide sequence shown in SEQ ID NO: 3 corresponds to the amino acid sequence consisting of SEQ ID NO: 1
• the nucleotide sequence shown in SEQ ID NO: 4 corresponds to the amino acid sequence consisting of SEQ ID NO: 2.
• the nucleic acid encoding the mutant nitrile hydratase contains the above ( It has a nucleotide substitution corresponding to at least one amino acid residue substitution selected from the amino acid residue substitution groups a) to (q).
• Vectors of the present disclosure include nucleic acids of the present disclosure.
• Examples of vectors of the present disclosure include vectors in which the mutant nitrile hydratase of the present disclosure is introduced into known vectors.
• Vectors of the present disclosure may be phage vectors or plasmid vectors.
• the vectors of the present disclosure are expression vectors.
• the expression vector may be any expression vector as long as it contains a nucleic acid represented by a nucleotide sequence encoding the amino acid sequence of the mutant nitrile hydratase of the present disclosure.
• the mutant nitrile hydra of the present disclosure can be obtained. can produce tase.
• the expression vector may optionally contain, in addition to the nucleotide sequence encoding the mutant nitrile hydratase of the present disclosure, a nucleotide sequence constituting another region (hereinafter also simply referred to as "other region"). You can stay there.
• Other regions include, for example, a control region necessary for the transformant to produce a mutant nitrile hydratase, a region necessary for autonomous replication, and the like.
• control region necessary for producing the mutant nitrile hydratase examples include a promoter sequence (including an operator sequence that controls transcription), a ribosome binding sequence (SD sequence), a transcription termination sequence, etc. .
• promoter sequences include the trp promoter of the tryptophan operon derived from Escherichia coli, the lac promoter of the lactose operon, and the PL promoter and PR promoter derived from lambda phage. Furthermore, artificially designed and modified sequences such as the tac promoter and trc promoter can also be used.
• the sequence order of the control region on the expression vector is not particularly limited, but for example, the promoter sequence and ribosome binding sequence are located upstream on the 5' end side of the gene encoding the mutant nitrile hydratase of the present disclosure. This is desirable. Furthermore, the transcription termination sequence is desirably located downstream on the 3' end side of the gene encoding the mutant nitrile hydratase of the present disclosure. Further, the ⁇ subunit gene and ⁇ subunit gene of mutant nitrile hydratase may be expressed as independent cistrons by this control region, or may be expressed as a polycistrone by a common control region.
• the expression vector may further contain a nucleotide sequence encoding a selection gene that can serve as a selection marker.
• the expression vector may also express a gene encoding a protein involved in the activation of nitrile hydratase.
• the protein involved in the activation of nitrile hydratase is a protein whose expression or absence directly influences the activation of nitrile hydratase, and is described in JP-A No. 11-253168.
• a typical example is a protein involved in nitrile hydratase activation (nitrile hydratase activation protein) derived from Pseudonocardia thermophila, which is described in the publication.
• the sequence of the nitrile hydratase activation protein is shown in SEQ ID NO: 36.
• Transformants of the present disclosure include expression vectors of the present disclosure.
• a mutant nitrile hydratase is produced in which impurity-induced inhibition of the amide compound production reaction using nitrile hydratase is reduced even in the presence of impurities.
• the transformant of the present disclosure can be produced by a known method. For example, a method may be used in which an expression vector containing a nucleotide sequence encoding a mutant nitrile hydratase and, if necessary, other regions described above is constructed, and the expression vector is transformed into a desired host cell.
• the transformant not only integrates the expression vector into the host cell, but also introduces silent mutations, if necessary, so that codons that are used less frequently in the host cell become codons that are used more frequently. It can also be produced by Thereby, it may be possible to increase the amount of protein produced from the mutant nitrile hydratase incorporated into the expression vector.
• any method involves adjusting the codons of the nitrile hydratase gene on the expression vector and the signal sequence for secreting the nitrile hydratase gene to the outside of the cell in accordance with the codon usage frequency in the host cell.
• the method of silent mutagenesis, the mutation point, the type of nucleotide to be changed, etc. are not particularly limited.
• the method for producing the mutant nitrile hydratase of the present disclosure includes culturing the aforementioned transformant of the present disclosure in a medium, and producing the mutant nitrile hydratase of the present disclosure from at least one of the cultured transformant and the medium.
• the method for producing a mutant nitrile hydratase may include recovering the hydratase.
• the conditions for culturing the transformant obtained by transformation with the expression vector are the same as the culture conditions for the host cell before transformation, and known conditions can be used.
• host cells include prokaryotes (E. coli, Bacillus subtilis, actinomycetes, etc.), yeast, filamentous fungi, and the like.
• the medium either a synthetic medium or a natural medium can be used as long as it contains appropriate amounts of carbon sources, nitrogen sources, inorganic substances, and other nutrients.
• the culture medium components known components used for culture media can be used.
• organic nutritional sources such as meat extract, yeast extract, malt extract, peptone, NZ amines and potatoes; carbon sources such as glucose, maltose, sucrose, starch and organic acids; nitrogen sources such as ammonium sulfate, urea and ammonium chloride; Acid salts, inorganic nutritional sources such as magnesium, potassium, and iron, and vitamins can be used in appropriate combinations.
• organic nutritional sources such as meat extract, yeast extract, malt extract, peptone, NZ amines and potatoes
• carbon sources such as glucose, maltose, sucrose, starch and organic acids
• nitrogen sources such as ammonium sulfate, urea and ammonium chloride
• Acid salts inorganic nutritional sources such as magnesium, potassium, and iron, and vitamins
• culturing a transformant transformed with an expression vector containing a selection marker for example, if the selection marker is drug resistant, use a medium containing the corresponding drug, and if the selection marker is auxotrophic. If so, a medium that
• the transformant can be cultured using conventional culture methods such as shaking culture, aerated agitation culture, continuous culture, and fed-batch culture in a liquid medium containing the transformant.
• Culture conditions may be appropriately selected depending on the type of transformant, medium, and culture method, and are not particularly limited as long as the conditions allow the transformant to grow and produce mutant nitrile hydratase.
• the culture is carried out aerobically at a temperature of preferably 20°C to 45°C, more preferably 24°C to 37°C.
• the culture period may range from 1 day to 7 days until the content of the protein having the desired mutant nitrile hydratase activity is maximized.
• LB medium or M9 medium is generally used as a medium for culturing the transformant, but it is more preferable to add Fe ions and Co ions as components to such a medium.
• This is a medium containing 1 ⁇ g/mL or more.
• the method for producing a mutant nitrile hydratase preferably includes a step of recovering the mutant nitrile hydratase from at least one of the cultured transformant and the culture medium.
• a method commonly used in this field can be used to recover the mutant nitrile hydratase.
• a crude enzyme solution containing the mutant nitrile hydratase can be easily obtained by centrifuging, filtering, etc. the culture of the transformant.
• mutant nitrile hydratase accumulates in the transformant, the cultured transformant is collected by centrifugation, etc., the collected transformant is suspended in a buffer solution, and this suspension is treated with lysozyme.
• a crude enzyme solution containing the mutant nitrile hydratase may be recovered by disrupting the cell membrane of the transformant according to known methods such as freezing, thawing, and ultrasonic disruption.
• a cationic polymer may be added to the recovered crude enzyme solution containing the mutant nitrile hydratase, a filter aid may be added, and the solution may be filtered.
• the filtered enzyme solution containing mutant nitrile hydratase can be further concentrated by ultrafiltration or the like, and can be used as concentrated mutant nitrile hydratase (concentrated enzyme) by adding a preservative or the like.
• the mutant nitrile hydratase may be made into a powder (powdered enzyme) by a spray drying method or the like.
• the crude enzyme solution containing the recovered mutant nitrile hydratase requires separation and purification, for example, salting out using ammonium sulfate, organic solvent precipitation using alcohol, membrane separation using dialysis, ultrafiltration, etc.
• separation and purification can be carried out by appropriately combining known chromatographic separation methods such as ion exchange chromatography, reversed phase high performance chromatography, affinity chromatography, and gel filtration chromatography.
• the method for producing an amide compound of the present disclosure includes contacting a mutant nitrile hydratase of the present disclosure with a nitrile compound.
• the mutant nitrile hydratase of the present disclosure catalyzes a reaction that synthesizes an amide compound from a nitrile compound. According to the method for producing an amide compound of the present disclosure, even in the presence of impurities, inhibition of the amide compound production reaction using nitrile hydratase by impurities is reduced.
• a transformant or cell line that produces the mutant nitrile hydratase of the present disclosure is cultured, and the resulting culture solution, cells, or cell-treated product is brought into contact with a nitrile compound in a medium.
• the treated cell products herein include extracts and ground products from the transformants, crude enzyme preparations obtained by separating the nitrile hydratase active fractions of these extracts and ground products, and further purified enzyme preparations.
• the above-mentioned contact temperature is preferably within a temperature range at which the mutant nitrile hydratase of the present disclosure is not inactivated, more preferably 0°C to 60°C, more preferably 15°C to 40°C.
• a culture solution obtained by culturing a transformant or cell line that produces the mutant nitrile hydratase of the present disclosure may be directly added to an aqueous solution containing a nitrile compound, or the culture solution may be centrifuged to obtain bacterial cells. may be separated and the bacterial cells added to an aqueous solution containing a nitrile compound.
• the pH of the aqueous solution during the reaction is preferably 7 to 9, more preferably 7.5 to 8.5.
• the nitrile compound may be present in the aqueous solution at a concentration of, for example, 0.25% to 20.0% by volume, more preferably 2.0% to 5.0% by volume. Details of culturing the transformant are as described in the section on the method for producing mutant nitrile hydratase.
• nitrile compounds are not particularly limited as long as they are compounds that can act as substrates for the mutant nitrile hydratase of the present disclosure, but include fats exemplified by acetonitrile, propionitrile, succinonitrile, and adiponitrile. aliphatic unsaturated nitriles exemplified by acrylonitrile and methacrylonitrile; aromatic nitriles exemplified by benzonitrile and phthalodinitrile; and heterocyclic nitriles exemplified by 3-cyanopyridine and 2-cyanopyridine. etc.
• a nitrile group in a nitrile compound is converted to an amide group by hydration, so that, for example, acrylonitrile can be converted to acrylamide.
• the reaction for producing an amide compound from a nitrile compound by contacting with the mutant nitrile hydratase of the present disclosure is carried out under neutral to basic conditions, such as pH 7 to 9, in accordance with the optimum pH of the nitrile hydratase. It's okay.
• the pH can be adjusted, for example, by using a basic substance such as ammonia or sodium hydroxide in a buffer solution as necessary.
• a "mutation site” refers to a wild-type pseudomorph that has an ⁇ subunit having the amino acid sequence shown by SEQ ID NO: 1 and a ⁇ subunit having the amino acid sequence shown by SEQ ID NO: 2. This refers to the position of the difference in the amino acid sequence from nitrile hydratase derived from Cardia thermophila.
• plasmid pPT-DB1 was prepared from the isolated bacterial cells by an alkaline SDS extraction method.
• the prepared plasmid was transformed into E. coli HB101 competent cells (manufactured by Toyobo Co., Ltd.) to obtain a transformant (C1).
• the transformant (C1) produces nitrile hydratase (C1), which is a wild-type Pseudonocardia thermophila-derived nitrile hydratase.
• mutant nitrile hydratase (1) of the present disclosure In order to obtain a transformant expressing a mutant nitrile hydratase in which Ala, which is the 23rd amino acid residue from the N-terminus of SEQ ID NO: 1, was replaced with Gly in the ⁇ subunit, we used "LA PCR" manufactured by Takara Shuzo Co., Ltd. Mutagenesis was performed using an "in vitro mutagenesis kit” (hereinafter referred to as the mutagenesis kit). A PCR reaction was performed using the wild-type Pseudonocardia thermophila-derived nitrile hydratase expression plasmid pPT-DB1 as a template. PCR reaction no.
• 1 is a system with a total volume of 50 ⁇ L containing 50 pmol of the mutation primer with the sequence number shown in Table 5 and the M13 primer M4 (the sequence is shown in SEQ ID NO: 33) (the composition is according to the conditions described in the mutation introduction kit), and heat denatured.
• the reaction was carried out by repeating 25 cycles of the following conditions: (98°C) for 15 seconds, annealing (55°C) for 30 seconds, and extension reaction (72°C) for 120 seconds.
• PCR reaction no is a system with a total volume of 50 ⁇ L containing 50 pmol of the mutation primer with the sequence number shown in Table 5 and the M13 primer M4 (the sequence is shown in SEQ ID NO: 33) (the composition is according to the conditions described in the mutation introduction kit), and heat denatured.
• the reaction was carried out by repeating 25 cycles of the following conditions: (98°C) for 15 seconds, annealing (55°C) for 30 seconds, and extension reaction (72°C) for 120 seconds
• PCR reaction no. 1 and no When the DNA amplification product was analyzed by agarose electrophoresis (agarose concentration 1.0% by weight) using 5 ⁇ L of each of the reaction completion solutions in step 2, the presence of the amplified DNA product was confirmed. After removing excess primers and dNTPs from each PCR reaction solution using Microcon 100 (manufactured by Takara Shuzo Co., Ltd.), TE was added to prepare 50 ⁇ L of each solution. A total of 47.5 ⁇ L of annealing solution containing 0.5 ⁇ L of each of the TE solutions (the composition was according to the conditions described in the mutagenesis kit) was prepared, and heat denaturation treatment (98° C.) was performed for 10 minutes.
• agarose electrophoresis agarose concentration 1.0% by weight
• annealing treatment was performed by cooling to 37° C. at a constant rate over 60 minutes and then holding at 37° C. for 15 minutes.
• 0.5 ⁇ L of TaKaRa LA Taq was added to the solution after the annealing treatment, and heat treatment was performed at 72° C. for 3 minutes to complete a heteroduplex.
• 50 pmol each of the M13 primer M4 and the M13 primer RV were added to make the total volume 50 ⁇ L.
• PCR reaction No. 1 was performed by repeating 25 cycles of heat denaturation (98°C) for 15 seconds, annealing (55°C) for 30 seconds, and extension reaction (72°C) for 120 seconds. I did 3.
• PCR reaction no When the DNA amplification product was analyzed by agarose electrophoresis (using Sigma Type VII low melting point agarose; agarose concentration 0.8% by weight) using 5 ⁇ L of the reaction completed solution from step 3, the presence of an amplified DNA product of approximately 2 kb was detected. was confirmed. Subsequently, only a DNA fragment of approximately 2 Kb was excised from the agarose gel, and the agarose piece (approximately 0.1 g) was finely ground and suspended in 1 mL of TE solution. Thereafter, the mixture was kept at 55° C. for 1 hour to completely melt the agarose.
• agarose electrophoresis using Sigma Type VII low melting point agarose; agarose concentration 0.8% by weight
• This melted solution was subjected to phenol/chloroform extraction and ethanol precipitation to purify the DNA fragments, which were finally dissolved in 10 ⁇ L of TE.
• the purified approximately 2 kb amplified DNA fragment was digested with restriction enzymes EcoRI and HindIII. Thereafter, this restriction enzyme treatment solution was subjected to phenol/chloroform extraction and ethanol precipitation to purify the DNA fragments, which were finally dissolved in 10 ⁇ L of TE.
• the nitrile hydratase expression plasmid pPT-DB1 was cut with EcoRI and HindIII, and subjected to agarose gel electrophoresis (using Type VII low melting point agarose manufactured by Sigma; agarose concentration 0.7%).
• plasmid pPT-DB1 had a mutation that changed Ala, the 23rd amino acid residue from the N-terminus of the ⁇ subunit, to Gly.
• Transformant (1) produces mutant nitrile hydratase (1) having the mutations listed in Table 5.
• nitrile hydratase (19) was obtained from the plasmid obtained from transformant (9) and the mutation primer of SEQ ID NO: 15, and nitrile hydratase was obtained from the plasmid obtained from transformant (13) and the mutation primer of SEQ ID NO: 11.
• nitrile hydratase (30) was obtained from the plasmid obtained from the transformant (21) and the mutation primer of SEQ ID NO: 13 and nitrile hydratase (30) was obtained from the plasmid obtained from the transformant (20) and the mutation primer of SEQ ID NO: 6.
• Nitrile hydratase (33) was obtained from the plasmid obtained from the transformant (9) and the mutation primer of SEQ ID NO: 17.
• nitrile hydratase (35) obtained from the plasmid obtained from the transformant (23) and the mutation primer of SEQ ID NO: 11, and nitrile hydratase (35) obtained from the plasmid obtained from the transformant (15) and the mutation primer of SEQ ID NO: 25.
• nitrile hydratase (36) from the plasmid obtained from the transformant and the mutation primer of SEQ ID NO: 26, and the transformant obtained from the plasmid obtained from the transformant (8) and the mutation primer of SEQ ID NO: 27.
• Nitrile hydratase (37) was obtained from the obtained plasmid and the mutation primer of SEQ ID NO: 28.
• nitrile hydratase (39) was obtained from the plasmid obtained from the transformant (29) and the mutation primer of SEQ ID NO: 17, and nitrile hydratase was obtained from the plasmid obtained from the transformant (34) and the mutation primer of SEQ ID NO: 11.
• Nitrile hydratase (42) was obtained from the plasmid obtained from the transformant and the mutation primer of SEQ ID NO: 32, and the transformant was obtained from the plasmid obtained from the transformant and the mutation primer of SEQ ID NO: 32.
• Nitrile hydratase (43) was obtained from the plasmid obtained from (40) and the mutation primer of SEQ ID NO: 18, and nitrile hydratase (44) was obtained from the plasmid obtained from the transformant (40) and the mutation primer of SEQ ID NO: 29.
• Nitrile hydratase (45) was obtained from the plasmid obtained from the transformant (35) and the mutation primer of SEQ ID NO: 27, and the mutation primer of SEQ ID NO: 28 was obtained from the transformant (1).
• a plasmid obtained from a transformant obtained from the mutation primer of SEQ ID NO: 11 a plasmid obtained from a transformant obtained from the mutation primer of SEQ ID NO: 29, and a plasmid obtained from a transformant obtained from the mutation primer of SEQ ID NO: 30.
• Nitrile hydratase (46) was obtained from a plasmid obtained from a transformant obtained from the mutation primer and from the mutation primer of SEQ ID NO: 31, respectively.
• the number of the transformant from which the plasmid was obtained is in the item "template”, the sequence number of the primer used as the primer for mutation in the item “primer for mutation”, and the sequence number of the primer used as the primer for mutation in the item “amino acid residue before mutation”.
• the amino acid residue before mutation at the mutation position described in the item “Mutation site” that is, the amino acid residue in wild-type Pseudonocardia thermophila-derived nitrile hydratase (C1)
• the amino acid residue in the item "Amino acid residue after mutation The amino acid residues after mutation at the mutation positions described in the item “Mutation location” are shown.
• the amount of the amide compound (acrylamide) contained in the reaction solution was quantitatively analyzed using HPLC under the following analysis conditions.
• wild-type nitrile hydratase (C1) was found to contain impurities when comparing Comparative Example 1, in which the reaction was performed in the presence of impurities, and Reference Example 2, in which the reaction was performed in the absence of impurities. It was found that the enzyme activity was lower than when no additive was added, indicating that the reaction was inhibited by impurities.
• the mutant nitrile hydratase (C2) for comparative example is also the above-mentioned (C1).
• the mutant types of each Example in which amino acid residues belonging to the group consisting of (a) to (q) are substituted for the wild-type Pseudonocardia thermophila-derived nitrile hydratase are substituted for the wild-type Pseudonocardia thermophila-derived nitrile hydratase.
• the nitrile hydratase is capable of producing amide compounds using nitrile hydratase even in the presence of impurities. It can be seen that the inhibition of the reaction by impurities is reduced.
• amino acid residue substitutions belonging to the group consisting of (i), (g), (l), and (m) were made to the wild-type Pseudonocardia thermophila-derived nitrile hydratase. It can be seen that the enzyme activity of the mutant nitrile hydratase of each example does not decrease even under reaction conditions of 30° C., which is a temperature higher than room temperature (20° C.), and the amide compound is efficiently produced. That is, the mutations of each example in which amino acid residues belonging to the group consisting of (i), (g), (l), and (m) are substituted for wild-type Pseudonocardia thermophila-derived nitrile hydratase. It can be seen that type nitrile hydratase has excellent heat resistance.

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