Mutant nitrile hydratase, nucleic acid encoding the mutant nitrile hydratase, vector and transformant containing the nucleic acid, method for producing the mutant nitrile hydratase, and method for producing an amide compound.

Abstract

A mutant nitrile hydratase mutated from a nitrile hydratase, said nitrile hydratase being derived from Pseudonocardia thermophila and having an α subunit having 90% or more sequence identity with SEQ ID NO: 1 and a β subunit having 90% or more sequence identity with SEQ ID NO: 2, by the substitution of an amino acid residue, which is at at least one position selected from the group consisting of the 23rd, 105th, 142nd, 145th, 152nd, 153rd, 180th and 188th positions from the N-terminus of the α subunit and the 89th, 122nd, 149th, 153rd, 189th, 207th, 232nd, 233rd, and 50th positions from the N-terminus of the β subunit, into a specific amino acid residue.

Description

Technical Field

[0001] 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.

Background Art

[0002] Nitrile hydratase is an enzyme having nitrile hydrating activity that converts the nitrile group of various compounds into an amide group by hydration, and is used in an industrial production process of an amide compound utilizing an enzyme reaction.

[0003] It is known that acrylonitrile, which is used as a raw material for producing acrylamide, an amide compound, may contain various impurities (for example, acrolein, which is a by-product). Patent Document 1 describes that acrolein usually occurs as a by-product during the production of acrylonitrile, and this forms an insoluble polymer as a side reaction.

[0004] Patent Document 2 describes that, as an example of an impurity contained in acrylonitrile, acrolein inhibits the synthesis reaction of acrylamide by nitrile hydratase. Patent Document 2 describes that by using an ion exchange resin or the like to reduce the concentration of acrolein in the raw material acrylonitrile to 1 ppm or less, inhibition by acrolein against the catalytic action of nitrile hydratase can be prevented.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

[0006] However, the method described in 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 does not reduce inhibition of nitrile hydratase activity even in the presence of impurities. Furthermore, from the viewpoint of reducing manufacturing costs, there is a need for the development of a technology that can reduce inhibition of nitrile hydratase activity without requiring processes for removing impurities or additives to trap impurities in the reaction system.

[0007] Therefore, this disclosure provides a novel mutant nitrile hydratase technology that reduces the inhibition of amide compound formation reactions using nitrile hydratase by impurities, even in the presence of impurities. [Means for solving the problem]

[0008] This disclosure includes the following aspects: <1> In a nitrile hydratase having an α-subunit with more than 90% sequence identity to the amino acid sequence represented by SEQ ID NO: 1, and a β-subunit with more than 90% sequence identity to the amino acid sequence represented by SEQ ID NO: 2, A mutant nitrile hydratase comprising a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) below, (a) In the α subunit, substitution from Ala to Gly, corresponding to the 23rd amino acid residue from the N-terminus of SEQ ID NO: 1, (b) In the α subunit, substitution from the amino acid residue Val to Met, corresponding to the 105th position from the N-terminus of SEQ ID NO: 1, (c) In the α subunit, the amino acid residue Leu corresponding to the 142nd position from the N-terminus of SEQ ID NO: 1 is substituted with Cys. (d) In the α subunit, substitution from the amino acid residue Glu to Asp, corresponding to the 145th position from the N-terminus of SEQ ID NO: 1, (e) In the α subunit, a substitution of Gly from amino acid residue Pro, corresponding to the 152nd N-terminus of Sequence ID No. 1, (f) In the α subunit, substitution of the amino acid residue Pro corresponding to the 153rd N-terminus of SEQ ID NO. 1 with Arg or Val, (g) In the α subunit, the amino acid residue Gly to Val, corresponding to the 180th N-terminus of SEQ ID NO: 1, (h) In the β subunit, the amino acid residue Glu to Leu corresponds to the 89th position from the N-terminus of SEQ ID NO: 2. (i) In the β subunit, substitution of the amino acid residue Ala, corresponding to the 122nd N-terminus of Sequence ID No. 2, with Ile, Leu, or Thr. (j) In the β subunit, the amino acid residue Thr to Gln corresponds to the 149th position from the N-terminus of SEQ ID NO: 2. (k) In the β subunit, substitution of the amino acid residue Lys, corresponding to the 153rd N-terminus of Sequence ID No. 2, with Val, Arg, or Pro. (l) In the β subunit, substitution from Cys to Ser at the amino acid residue corresponding to the 189th N-terminus of Sequence ID No. 2, (m) In the β subunit, substitution of the amino acid residue Glu, corresponding to the 207th N-terminus of Sequence ID No. 2, with Pro, Ser, or Gly. (n) In the β subunit, substitution of the amino acid residue Ala, corresponding to the 232nd N-terminus of Sequence ID No. 2, with Trp, Ser, Pro, Phe, Ile, Arg, Cys, or Gly. (o) In the β subunit, substitution of the amino acid residue Ala, corresponding to the 233rd position from the N-terminus of Sequence ID No. 2, with Glu or Gln. (p) In the α subunit, the amino acid residue Thr to Lys, corresponding to the 188th position from the N-terminus of SEQ ID NO: 1, (q) Substitution of the amino acid residue Glu to Gly in the β subunit, corresponding to the 50th amino acid residue from the N-terminus of Sequence ID No. 2. <2> The above includes at least two amino acid residue substitutions selected from the group consisting of (a) to (q), <1> The variant nitrile hydratase described above. <3> The above includes at least one amino acid residue substitution selected from the group consisting of (i), (g), (j), and (n). <1> or <2> The variant nitrile hydratase described above. <4> The above includes at least one amino acid residue substitution selected from the group consisting of (i), (g), (l), and (m). <1> or <2> The variant nitrile hydratase described above. <5> The above includes at least one amino acid residue substitution selected from the group consisting of (g), (p), and (q). <1> or <2> The variant nitrile hydratase described above. <6> The amino acid sequence of the α subunit further includes at least one amino acid residue substitution selected from the group consisting of (A1) to (A6) below, <1> or <2> The mutant nitrile hydratase described above, (A1) In the α subunit, the amino acid residue Arg to Val corresponds to the 20th N-terminus of SEQ ID NO: 1. (A2) In the α subunit, the amino acid residue Gln to Arg, corresponding to the 141st N-terminus of Sequence ID No. 1, (A3) In the α subunit, the amino acid residue Glu, corresponding to the 185th position from the N-terminus of Sequence ID No. 1, is substituted with Ala. (A4) In the α subunit, the amino acid residue Ala to Arg is substituted for the 187th amino acid residue from the N-terminus of Sequence ID No. 1. (A5) In the α subunit, the amino acid residue Pro, corresponding to the 200th N-terminus of SEQ ID NO: 1, (A6) Substitution of the amino acid residue Val to Leu in the α subunit, corresponding to the 204th amino acid residue from the N-terminus of SEQ ID NO: 1. <7> The amino acid sequence of the β subunit further comprises at least one amino acid residue substitution selected from the group consisting of (B1) to (B9) below. <1> or <2> The mutant nitrile hydratase described above, (B1) In the β subunit, the amino acid residue Thr to Ile corresponds to the 40th position from the N-terminus of SEQ ID NO: 2. (B2) In the β subunit, the amino acid residue Arg to Pro, corresponding to the 42nd position from the N-terminus of Sequence ID No. 2, (B3) In the β subunit, the amino acid residue Thr to Cys, corresponding to the 76th N-terminus of SEQ ID NO: 2, (B4) In the β subunit, the amino acid residue Thr to His, corresponding to the 83rd position from the N-terminus of Sequence ID No. 2, (B5) In the β subunit, substitution of the amino acid residue Leu, corresponding to the 88th N-terminus of Sequence ID No. 2, with Arg, Pro, or Ser. (B6) In the β subunit, substitution of the amino acid residue Arg corresponding to the 146th N-terminus of Sequence ID No. 2 with Gln or Thr, (B7) In the β subunit, substitution of the amino acid residue Arg corresponding to the 160th N-terminus of Sequence ID No. 2 to Gln or Ile, (B8) In the β subunit, the amino acid residue Glu to Arg, corresponding to the 108th N-terminus of Sequence ID No. 2, (B9) In the β subunit, substitution from the amino acid residue Tyr to Cys, corresponding to the 176th amino acid residue from the N-terminus of SEQ ID NO: 2. <8> The aforementioned <1> ~ <7> A nucleic acid encoding a variant nitrile hydratase as described in any one of the following. <9> The aforementioned <8> A vector containing the nucleic acid described above. <10> The expression vector is the aforementioned <9> The vector shown. <11> The aforementioned <10> A transformant containing the expression vector described above. <12> The aforementioned <11> The transformant described above is cultured in a culture medium, and From at least one of the cultured transformants and the culture medium, the above <1> ~ <7> To recover the mutant nitrile hydratase described in any one of the following, A method for producing mutant nitrile hydratase, including the above. <13> The mutant nitrile hydratase obtained by the production method described in <12>. <14> A method for producing an amide compound, comprising contacting the mutant nitrile hydratase described in any one of <1> to <7> with a nitrile compound.

Advantages of the Invention

[0009] According to the present disclosure, it is possible to provide a technique related to a novel mutant nitrile hydratase in which inhibition by impurities against an amide compound formation reaction using nitrile hydratase is reduced even in the presence of impurities.

Embodiments for Carrying Out the Invention

[0010] Hereinafter, embodiments of the present disclosure will be described. These descriptions and examples are illustrative of the embodiments and do not limit the scope of the embodiments.

[0011] In the numerical ranges described stepwise in the present disclosure, the upper limit value or lower limit value described in one numerical range may be replaced with the upper limit value or lower limit value of the numerical range described in other stepwise descriptions. Further, in the numerical ranges described in the present disclosure, the upper limit value or lower limit value of the numerical range may be replaced with the value shown in the examples.

[0012] In the present disclosure, each component may contain a plurality of corresponding substances. When referring to the amount of each component in the composition in the present disclosure, when there are a plurality of substances corresponding to each component in the composition, unless otherwise specified, it means the total amount of the plurality of substances present in the composition.

[0013] In the present disclosure, the term "step" includes not only an independent step but also the case where it cannot be clearly distinguished from other steps, as long as the intended purpose of the step is achieved. In the present disclosure, the numerical range indicated by using "~" indicates a range including the numerical values described before and after "~" as the minimum value and the maximum value, respectively. In this disclosure, the amount of each component in the composition means the total amount of any multiple substances present in the composition, unless otherwise specified, if there are multiple substances corresponding to each component in the composition.

[0014] In this disclosure, "enzyme activity" refers to nitrile hydration activity, which converts nitrile groups to amide groups. In this disclosure, descriptions of nucleotide sequences may focus on the sequence on one strand even when the nucleic acid strand containing the nucleotide sequence forms a double helix. However, in the case of the other strand of the double helix, such descriptions should be interpreted and applied to the complementary sequence.

[0015] In this disclosure, three-letter abbreviations represent amino acid residues unless otherwise specified. For example, "Ile" represents an isoleucine residue.

[0016] <Variant Nitrile Hydratase> This disclosure provides a mutant nitrile hydratase having an α-subunit with 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1 and a β-subunit with 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 2, wherein the mutant nitrile hydratase includes a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) below: (a) In the α subunit, substitution from Ala to Gly, corresponding to the 23rd amino acid residue from the N-terminus of SEQ ID NO: 1, (b) In the α subunit, substitution from the amino acid residue Val to Met, corresponding to the 105th position from the N-terminus of SEQ ID NO: 1, (c) In the α subunit, the amino acid residue Leu corresponding to the 142nd position from the N-terminus of SEQ ID NO: 1 is substituted with Cys. (d) In the α subunit, substitution from the amino acid residue Glu to Asp, corresponding to the 145th position from the N-terminus of SEQ ID NO: 1, (e) In the α subunit, a substitution of Gly from amino acid residue Pro, corresponding to the 152nd N-terminus of Sequence ID No. 1, (f) In the α subunit, substitution of the amino acid residue Pro corresponding to the 153rd N-terminus of SEQ ID NO. 1 with Arg or Val, (g) In the α subunit, the amino acid residue Gly to Val, corresponding to the 180th N-terminus of SEQ ID NO: 1, (h) In the β subunit, the amino acid residue Glu to Leu corresponds to the 89th position from the N-terminus of SEQ ID NO: 2. (i) In the β subunit, substitution of the amino acid residue Ala, corresponding to the 122nd N-terminus of Sequence ID No. 2, with Ile, Leu, or Thr. (j) In the β subunit, the amino acid residue Thr to Gln corresponds to the 149th position from the N-terminus of SEQ ID NO: 2. (k) In the β subunit, substitution of the amino acid residue Lys, corresponding to the 153rd N-terminus of Sequence ID No. 2, with Val, Arg, or Pro. (l) In the β subunit, substitution from Cys to Ser at the amino acid residue corresponding to the 189th N-terminus of Sequence ID No. 2, (m) In the β subunit, substitution of the amino acid residue Glu, corresponding to the 207th N-terminus of Sequence ID No. 2, with Pro, Ser, or Gly. (n) In the β subunit, substitution of the amino acid residue Ala, corresponding to the 232nd N-terminus of Sequence ID No. 2, with Trp, Ser, Pro, Phe, Ile, Arg, Cys, or Gly. (o) In the β subunit, substitution of the amino acid residue Ala, corresponding to the 233rd position from the N-terminus of Sequence ID No. 2, with Glu or Gln. (p) In the α subunit, the amino acid residue Thr to Lys, corresponding to the 188th position from the N-terminus of SEQ ID NO: 1, (q) Substitution of the amino acid residue Glu to Gly in the β subunit, corresponding to the 50th amino acid residue from the N-terminus of Sequence ID No. 2.

[0017] 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 to date and is a novel variant.

[0018] Unlike wild-type Pseudonocardia thermophylla-derived nitrile hydratase and conventional mutant nitrile hydratases, the mutant nitrile hydratase of this disclosure exhibits reduced inhibition by impurities in the presence of impurities (e.g., acrolein) in the presence of impurities, and shows excellent enzymatic activity in the synthesis of amide compounds from nitrile compounds.

[0019] Furthermore, the reduction in reaction inhibition by impurities possessed by the mutant nitrile hydratase of this disclosure is particularly pronounced when impurities are present at high concentrations (e.g., 1000 ppm or more). Therefore, with the mutant nitrile hydratase of this disclosure, it is possible to ensure that the reaction for synthesizing amide compounds proceeds sufficiently even when using raw materials that have not been sufficiently purified in the industrial production process of amide compounds using nitrile hydratase. Thus, with the mutant nitrile hydratase of this disclosure, it is possible to dramatically improve the efficiency of industrial amide compound production compared to using conventional enzymes.

[0020] The mutant nitrile hydratase of this disclosure has an α subunit having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and a β subunit having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, and is a mutant nitrile hydratase that includes a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) described above.

[0021] The amino acid sequence represented by Sequence ID No. 1 is the amino acid sequence of the α-subunit of nitrile hydratase derived from wild-type Pseudonocardia thermophila. The amino acid sequence represented by Sequence ID No. 2 is the amino acid sequence of the β-subunit of nitrile hydratase derived from wild-type Pseudonocardia thermophila.

[0022] An α-subunit having more than 90% sequence identity with the amino acid sequence represented by Sequence ID No. 1 means that, when compared to the α-subunit (also referred to as the wild-type α-subunit) found in nitrile hydratase derived from wild-type Pseudonocardia thermophila, it has an amino acid sequence that is more than 90% identical to the wild-type α-subunit. Similarly, a β-subunit that has more than 90% sequence identity with the amino acid sequence represented by Sequence ID No. 2 means that, when compared to the β-subunit (also called the wild-type β-subunit) found in nitrile hydratase derived from wild-type Pseudonocardia thermophila, it has an amino acid sequence that is more than 90% identical to the amino acid sequence of the wild-type β-subunit.

[0023] The mutant nitrile hydratase may have an α-subunit that has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, may have an α-subunit that has 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, or may have an α-subunit that has 98% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1. The mutant nitrile hydratase may have a β-subunit that has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, a β-subunit that has 95% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, or a β-subunit that has 98% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1.

[0024] Mutant nitrile hydratases have a basic structural unit in which the α-subunit and β-subunit associate as a dimer, and these dimers further associate to form tetramers. The 111th amino acid residue from the N-terminus of the α-subunit, a cysteine ​​residue, is post-translationally modified to cysteinesulfinic acid (Cys-SOOH), and the 113th amino acid residue from the N-terminus, a cysteine ​​residue, is post-translationally modified to cysteinesulfenic acid (Cys-SOH). Through these modified amino acid residues, the polypeptide chain of the α-subunit and the cobalt atom bind, forming the active site.

[0025] (Location of mutation) The mutant nitrile hydratases of this disclosure include a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) described above.

[0026] The mutant nitrile hydratases of this disclosure only need to contain at least one amino acid residue substitution selected from the group consisting of (a) to (q), and may contain a combination of multiple types. The mutant nitrile hydratases of this disclosure may further include substitutions of amino acid residues other than those in the group consisting of (a) to (q), to the extent that impurity-induced inhibition of amide compound formation reactions using nitrile hydratases is reduced.

[0027] From the viewpoint of further reducing the inhibition of amide compound formation reactions using nitrile hydratase by impurities, the mutant nitrile hydratase of this disclosure preferably contains at least two amino acid residue substitutions selected from the group consisting of (a) to (q).

[0028] The mutant nitrile hydratase of this disclosure is more preferably to include the amino acid residue substitution in (i) above, from the viewpoint of further reducing inhibition by impurities in amide compound formation reactions using nitrile hydratase.

[0029] The mutant nitrile hydratase of this disclosure may contain three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, or seventeen amino acid residue substitutions selected from the group consisting of (a) to (q) above. The mutant nitrile hydratases of this disclosure may contain 16 or fewer amino acid residue substitutions selected from the group consisting of (a) to (q) above, including 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer.

[0030] If the mutant nitrile hydratase of this disclosure contains a combination of two amino acid residue substitutions from the group consisting of (a) to (q), 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 in the amide compound formation reaction using nitrile hydratase, the following combinations may be included. In the following examples of combinations, only the mutation position is described for the amino acid residue substitutions from the group consisting of (a) to (q). For example, the combination of (j)β149 and (n)β232 refers to a combination in the (j)β subunit in which the amino acid residue Thr, corresponding to the 149th amino acid residue from the N-terminus of SEQ ID NO: 2, is substituted to Gln, and in the (n)β subunit in which the amino acid residue Ala, corresponding to the 232nd amino acid residue from the N-terminus of SEQ ID NO: 2, is substituted to Trp, Ser, Pro, Phe, Ile, Arg, Cys, or Gly. If there are multiple candidate amino acid residues after the mutation, as in (n) above, the following combination may be any of the candidates. This also applies to the examples of combinations described later when three or more amino acid residue substitutions are combined.

[0031] (j)β149 and (n)β232 combination, (i) The combination of β122 and (g) α180, (l)β189 and (m)β207 combination, (o)β233 and (h)β89 combination, (b) A combination of α105 and (c) α142, (c) A combination of α142 and (g) α180. (i) The combination of β122 and (n) β232, (i) The combination of β122 and (j) β149, (q)β50 ​​and (h)β89 combination, (p)α188 and (l)β189 combination, (p)α188 and (q)β50 ​​combination

[0032] If the mutant nitrile hydratase of this disclosure contains a combination of three amino acid residue substitutions from the group consisting of (a) to (q), the combination of the three amino acid residue substitutions is not particularly limited. However, for example, from the viewpoint of further reducing the inhibition of amide compound formation reactions using nitrile hydratase by impurities, the following combinations may be included. In the following examples of combinations, only the mutation position of the amino acid residue substitutions from the group consisting of (a) to (q) is described in a simplified manner. (i) A combination of β122, (n)β232, and (g)α180. (i) The combination of β122, (j) β149, and (n) β232, (m)β207 and (b)α105 and (g)α180 combination, (m)β207, (l)β189, and (g)α180 combination (m)β207, (n)β232, and (g)α180 combination (g)α180, (p)α188, and (q)β50 ​​combinations, (i) The combination of β122, (j) β149, and (m) β207, (i) The combination of β122, (l) β189, and (m) β207, (q) The combination of β50, (a) α23, and (b) α105.

[0033] If the mutant nitrile hydratase of this disclosure contains a combination of four amino acid residue substitutions from the group consisting of (a) to (q), the combination of the three amino acid residue substitutions is not particularly limited. However, for example, from the viewpoint of further reducing the inhibition of amide compound formation reactions using nitrile hydratase by impurities, the following combinations may be included. In the following examples of combinations, only the mutation positions of the amino acid residue substitutions from the group consisting of (a) to (q) are described in a simplified manner. (i) a combination of β122, (j) β149, (n) β232, and (g) α180. (i) A combination of β122, (m)β207, (n)β232, and (g)α180. (i) A combination of β122, (l) β189, (m) β207, and (g) α180. (p)α188 and (q)β50 ​​and (a)α23 and (l)β189 combinations, (p)α188 and (q)β50 ​​and (g)α180 and (o)β233 combinations, (a) A combination of α23, (d) α145, (e) α152, and (o) β233. (b) A combination of α105, (f) α153, (h) β89, and (n) β232. (q) The combination of β50, (d) α145, (e) α152, and (o) β233.

[0034] In addition, of course, substitutions may be made by combining five or more amino acid residue substitutions from the group consisting of (a) to (q) above.

[0035] When the mutant nitrile hydratase of this disclosure contains at least one amino acid residue substitution selected from the group consisting of (i), (g), (l), and (m), it not only suppresses reaction inhibition by the impurities but also tends to increase the thermal stability of the nitrile hydratase. Conventional nitrile hydratases tended to have decreased catalytic activity at temperatures above room temperature (20°C). Therefore, when raw materials with low solubility at room temperature were used in the reaction, the yield of the resulting amide compound was low, cooling equipment was required during production, and improvements were needed from the standpoint of production costs. In contrast, the mutant nitrile hydratase of the present disclosure having the above configuration exhibits higher thermal stability and maintains higher catalytic activity compared to conventional nitrile hydratases, even at temperatures above room temperature (e.g., 30°C), for example. As a result, the mutant nitrile hydratase of the present disclosure having the above configuration can raise the temperature of the reaction system even when raw materials with low solubility at room temperature are used in the reaction. Therefore, compared to conventional nitrile hydratases, it is easier to obtain a higher yield of the resulting amide compound, and a cooling device is not required during production, resulting in the secondary effect of being superior from the standpoint of production cost.

[0036] The mutant nitrile hydratases of this disclosure may further include, from the viewpoint of reducing impurity-induced inhibition of amide compound formation reactions using nitrile hydratases, at least one amino acid residue substitution selected from the group consisting of (A1) to (A6) below in the amino acid sequence of the α-subunit: (A1) In the α subunit, the amino acid residue Arg to Val corresponds to the 20th N-terminus of SEQ ID NO: 1. (A2) In the α subunit, the amino acid residue Gln to Arg, corresponding to the 141st N-terminus of Sequence ID No. 1, (A3) In the α subunit, the amino acid residue Glu, corresponding to the 185th position from the N-terminus of Sequence ID No. 1, is substituted with Ala. (A4) In the α subunit, the amino acid residue Ala to Arg is substituted for the 187th amino acid residue from the N-terminus of Sequence ID No. 1. (A5) In the α subunit, the amino acid residue Pro, corresponding to the 200th N-terminus of SEQ ID NO: 1, (A6) Substitution of the amino acid residue Val to Leu in the α subunit, corresponding to the 204th amino acid residue from the N-terminus of SEQ ID NO: 1.

[0037] The mutant nitrile hydratase of this disclosure may further include, from the viewpoint of reducing impurity-induced inhibition of amide compound formation reactions using nitrile hydratase, at least one amino acid residue substitution selected from the group consisting of (B1) to (B9) below in the amino acid sequence of the β subunit: (B1) In the β subunit, the amino acid residue Thr to Ile corresponds to the 40th position from the N-terminus of SEQ ID NO: 2. (B2) In the β subunit, the amino acid residue Arg to Pro, corresponding to the 42nd position from the N-terminus of Sequence ID No. 2, (B3) In the β subunit, the amino acid residue Thr to Cys, corresponding to the 76th N-terminus of SEQ ID NO: 2, (B4) In the β subunit, the amino acid residue Thr to His, corresponding to the 83rd position from the N-terminus of Sequence ID No. 2, (B5) In the β subunit, substitution of the amino acid residue Leu, corresponding to the 88th N-terminus of Sequence ID No. 2, with Arg, Pro, or Ser. (B6) In the β subunit, substitution of the amino acid residue Arg corresponding to the 146th N-terminus of Sequence ID No. 2 with Gln or Thr, (B7) In the β subunit, substitution of the amino acid residue Arg corresponding to the 160th N-terminus of Sequence ID No. 2 to Gln or Ile, (B8) In the β subunit, the amino acid residue Glu to Arg, corresponding to the 108th N-terminus of Sequence ID No. 2, (B9) In the β subunit, substitution from the amino acid residue Tyr to Cys, corresponding to the 176th amino acid residue from the N-terminus of SEQ ID NO: 2.

[0038] The mutant nitrile hydratase may include substitutions other than the amino acid residue substituent group consisting of (a) to (q), the amino acid residue substitution group consisting of (A1) to (A6), and the amino acid residue substitution group consisting of (B1) to (B9) (hereinafter referred to as "other amino acid residue substitutions"), to the extent that the effects of this disclosure are achieved.

[0039] In the mutant nitrile hydratase, the total number of substitutions selected from the amino acid residue substituent group consisting of (a) to (q), the amino acid residue substitution group consisting of (A1) to (A6), and the amino acid residue substitution group consisting of (B1) to (B9) is not particularly limited as long as it is 1 or more, for example, it may be 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0040] The mutant nitrile hydratase of this disclosure may, for example, contain at least one amino acid residue substitution selected from the group consisting of (i), (g), (j), and (n) above, or may contain the amino acid residue substitutions of (i), (j), and (n), or may contain all of the amino acid residue substitutions of (i), (g), (j), and (n) above, from the viewpoint of further reducing inhibition by impurities to amide compound formation reactions using nitrile hydratase.

[0041] As one embodiment, for example, the mutant nitrile hydratase of the present disclosure may contain at least one amino acid residue substitution selected from the group consisting of (i), (g), (l), and (m), from the viewpoint of further reducing inhibition by impurities in amide compound formation reactions using nitrile hydratase and from the viewpoint of having excellent thermal stability, it may contain the amino acid residue substitutions of (i), (l), and (m), or it may contain all of the amino acid residue substitutions of (i), (g), (l), and (m).

[0042] As one embodiment, for example, the mutant nitrile hydratase of the present disclosure may contain at least one amino acid residue substitution selected from the group consisting of (g), (p), and (q), may contain the amino acid residue substitutions of (p) and (q), or may contain all of the amino acid residue substitutions of (g), (p), and (q), from the viewpoint of further reducing the inhibition of amide compound formation reactions using nitrile hydratase by impurities.

[0043] Examples of combinations of two amino acid residue substitutions selected from the group consisting of (a) to (q), the group consisting of (A1) to (A6), and the group consisting of (B1) to (B9) are shown below, but the disclosure is not limited thereto. In the table, the numbers in the mutation position column represent the position of the amino acid sequence from the N-terminus in the corresponding subunit.

[0044] [Table 1-1]

[0045] [Table 1-2]

[0046] Examples of combinations of three amino acid residue substitutions selected from the group consisting of (a) to (q), the group consisting of (A1) to (A6), and the group consisting of (B1) to (B9) are shown below, but the disclosure is not limited thereto. In the table below, the numbers in the column for mutation site represent the position of the amino acid sequence from the N-terminus in the corresponding subunit.

[0047] [Table 2-1]

[0048] [Table 2-2]

[0049] Examples of combinations of four amino acid residue substitutions selected from the group consisting of (a) to (q), the group consisting of (A1) to (A6), and the group consisting of (B1) to (B9) are shown below, but the disclosure is not limited thereto. In the table below, the number in the column for the mutation site represents the position of the amino acid sequence in the corresponding subunit from the N-terminus.

[0050] [Table 3]

[0051] Examples of combinations of five amino acid residue substitutions selected from the group consisting of (a) to (q), the group consisting of (A1) to (A6), and the group consisting of (B1) to (B9) are shown below, but the disclosure is not limited thereto. In the table below, the numbers in the column for mutation site represent the position of the amino acid sequence from the N-terminus in the corresponding subunit.

[0052] [Table 4]

[0053] [Nitrile hydratase] The mutant nitrile hydratase of this disclosure has an α subunit with 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1 and a β subunit with 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 2, and includes a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) above, but the origin of the nitrile hydratase is not limited. The source of the nitrile hydratase may be, for example, nitrile hydratase derived from wild-type Pseudonocardia thermophila, or it may be a modified nitrile hydratase obtained by modifying wild-type Pseudonocardia thermophila nitrile hydratase.

[0054] (Modified nitrile hydratase) Modified nitrile hydratases also include modified nitrile hydratases obtained by modifying wild-type Pseudonocardia thermophila-derived nitrile hydratases with one or more modifications selected from the group consisting of (i) to (v) below: (i) Substitution of one or more amino acid residues with other amino acid residues, (ii) Deletion of one or more amino acid residues other than the amino acid residue substitutions in (a) to (q) above, (iii) Insertion of amino acid residues, (iv) Addition of amino acid residues to either the N-terminus or C-terminus or both of the amino acid sequence of the α subunit. (v) Addition of amino acid residues to either the N-terminus or C-terminus, or both, of the amino acid sequence of the β subunit.

[0055] In the α-subunit of the modified 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 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 1. The number of substituted amino acid residues may also be 0. In the β-subunit of the modified 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 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 1. The number of substituted amino acid residues may also be 0.

[0056] In the α-subunit of the modified nitrile hydratase, the number of deleted amino acid residues may be, for example, 1 to 10, 1 to 7, 1 to 4, 1 to 2, or 1. The number of deleted amino acid residues may also be 0. In the β-subunit of the modified nitrile hydratase, the number of deleted amino acid residues may be, for example, 1 to 10, 1 to 7, 1 to 4, 1 to 2, or 1. The number of deleted amino acid residues may also be 0.

[0057] In the α-subunit of the modified nitrile hydratase, the number of inserted amino acid residues is, for example, 1 to 10, 1 to 7, 1 to 4, 1 to 2, or 1. The number of inserted amino acid residues may also be 0. In the β-subunit of the modified nitrile hydratase, the number of inserted amino acid residues may be, for example, 1 to 10, 1 to 7, 1 to 4, 1 to 2, or 1. The number of inserted amino acid residues may also be 0.

[0058] In the α-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 such as terminal addition, the total number of substituted, deleted, or inserted amino acid residues may be 0. In the β-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 such as terminal addition, the total number of substituted, deleted, or inserted amino acid residues may be 0.

[0059] In the α-subunit of the modified nitrile hydratase, the number of terminally added amino acid residues is, for example, 1 to 60, 1 to 40, 1 to 20, 1 to 10, 1 to 5, 1 to 3, or 1 per end. The number of added amino acid residues may also be 0. In the β-subunit of the modified nitrile hydratase, the number of terminally added amino acid residues is, for example, 1 to 60, 1 to 40, 1 to 20, 1 to 10, 1 to 5, 1 to 3, or 1 per end. The number of added amino acid residues may also be 0. The terminal amino acid residues may be, for example, secretory signal sequences. The terminal 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.

[0060] The similarity between modified nitrile hydratase and wild-type Pseudonocardia thermophylla-derived nitrile hydratase can also be expressed by sequence identity. The amino acid sequence of the α-subunit of the modified nitrile hydratase has, for example, 90% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, or 99% or more sequence identity with the amino acid sequence of the α-subunit of the wild-type Pseudonocardia thermophila-derived nitrile hydratase represented by Sequence ID No. 1.

[0061] The amino acid sequence of the β-subunit of the modified nitrile hydratase has, for example, 90% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, or 99% or more sequence identity with the amino acid sequence of the β-subunit of the wild-type Pseudonocardia thermophila-derived nitrile hydratase represented by Sequence ID No. 2.

[0062] Sequence alignment can be performed using ClustalW(1.83) with initial parameters (including gap-open penalty: 10 and gap-extension penalty: 0.05). Sequence identity is calculated using the full length of the nitrile hydratase.

[0063] When the amino acid sequence of a modified nitrile hydratase is aligned with the amino acid sequence of wild-type Pseudonocardia thermophylla-derived nitrile hydratase, depending on the manner of modification of the modified nitrile hydratase, the distance from the N-terminus of the subunit may differ even for corresponding amino acid residues in the alignment, as defined above. In this disclosure, in such cases, the Xth amino acid residue from the N-terminus of the modified nitrile hydratase refers to the amino acid residue that aligns with the Xth amino acid residue from the N-terminus of the same subunit of wild-type Pseudonocardia thermophila nitrile hydratase. For example, in this disclosure, "the 23rd amino acid residue from the N-terminus of the α subunit," "the 23rd Ala residue from the N-terminus of the α subunit," or "the Ala residue which is the 23rd amino acid residue from the N-terminus of the α subunit" may be located at a position other than the 23rd from the N-terminus of the α subunit in the amino acid sequence of the modified nitrile hydratase. For example, the 23rd amino acid residue from the N-terminus of the α-subunit of a modified nitrile hydratase (which is not necessarily Ala) may, as a result of amino acid residue insertion, correspond to the Ala residue that is the 24th amino acid residue from the N-terminus of the α-subunit of wild-type Pseudonocardia thermophila nitrile hydratase. In such cases, "the 23rd amino acid residue from the N-terminus of the α-subunit," "the 23rd Ala residue from the N-terminus of the α-subunit," or "the Ala residue that is the 23rd amino acid residue from the N-terminus of the α-subunit" in this disclosure refers to the 24th amino acid residue from the N-terminus of the α-subunit in the modified nitrile hydratase.

[0064] In one embodiment, the amino acid sequence of the modified nitrile hydratase may be the amino acid sequence of the modified nitrile hydratase described in International Publication No. 2010 / 055666 and International Publication No. 2018 / 124247 or a similar sequence.

[0065] The method for producing the mutant nitrile hydratase of this disclosure is not particularly limited, but for example, a vector can be prepared that contains a nucleic acid represented by a nucleotide sequence encoding the amino acid sequence of the mutant nitrile hydratase of this disclosure, and the mutant nitrile hydratase can be produced using this vector. Details are described below.

[0066] <Nucleic acid> The nucleic acids of this disclosure encode the amino acid sequence of the mutant nitrile hydratase of this disclosure. More specifically, the nucleic acids of this disclosure have a nucleotide sequence that encodes the amino acid sequence of the mutant nitrile hydratase of this disclosure. Methods for synthesizing the nucleotide sequence encoding the amino acid sequence of the mutant nitrile hydratase of this disclosure include introducing mutation sites into the nucleotide sequence encoding the corresponding wild-type Pseudonocardia thermophila-derived nitrile hydratase, and chemically synthesizing the entire nucleotide sequence including the mutation sites. Methods for inducing gene mutations using the nucleotide sequence encoding wild-type Pseudonocardia thermophylla-derived nitrile hydratase as a template include, for example, site-directed mutagenesis (Kramer, W. and Frita, HJ, Methods in Enzymology, vol.154, p.350 (1987)), recombinant PCR (PCR Technology, Stockton Press (1989)), chemical synthesis of specific nucleic acids, hydroxyamine treatment of the gene, ultraviolet irradiation of strains containing the gene, treatment with chemical agents such as nitrosoguanidine or nitrite, and the use of commercially available mutagenesis kits. The nucleic acid in this disclosure may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0067] The gene encoding wild-type Pseudonocardia thermophylla-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, and 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 has, at least, a nucleotide substitution at the amino acid residue substitution positions (a) to (q) in the nucleotide sequence shown in SEQ ID NO: 3 or the nucleotide sequence shown in SEQ ID NO: 4, corresponding to at least one amino acid residue substitution selected from the group of amino acid residue substitutions (a) to (q).

[0068] <Vector> The vectors of this disclosure include the nucleic acids of this disclosure. Examples of vectors in this disclosure include known vectors into which the mutant nitrile hydratase disclosed herein has been introduced. The vectors in this disclosure may be phage vectors or plasmid vectors.

[0069] <Expression Vector> The vector of this disclosure is preferably an expression vector. The expression vector may be any expression vector comprising a nucleic acid represented by a nucleotide sequence encoding the amino acid sequence of the mutant nitrile hydratase of the present disclosure. Using the expression vector of the present disclosure, any host cell can be transformed to obtain a transformant or cell line, and then the transformant or cell line can be cultured to produce the mutant nitrile hydratase of the present disclosure.

[0070] The expression vector may optionally include, in addition to the nucleotide sequence encoding the mutant nitrile hydratase of this disclosure, nucleotide sequences constituting other regions (hereinafter also simply referred to as "other regions"). Other regions include, for example, regulatory regions required by the transformant to produce mutant nitrile hydratase, and regions necessary for autonomous replication.

[0071] Examples of regulatory regions necessary for producing the mutant nitrile hydratase include promoter sequences (including operator sequences that control transcription), ribosome-binding sequences (SD sequences), and transcription termination sequences.

[0072] Examples of promoter sequences include the trp promoter from the tryptophan operon of E. coli, the lac promoter from the lactose operon, and the PL and PR promoters from lambdafage. Furthermore, artificially designed and modified sequences, such as the tac and trc promoters, can also be used.

[0073] The sequence order of the regulatory region on the expression vector is not particularly limited, but it is preferable, for example, that the promoter sequence and the ribosome-binding sequence be located upstream of the gene encoding the mutant nitrile hydratase of this disclosure, 5' end. Furthermore, it is preferable that the transcription termination sequence be located downstream of the gene encoding the mutant nitrile hydratase, 3' end. Additionally, the α-subunit and β-subunit genes of the mutant nitrile hydratase may be expressed as independent cistrons via this regulatory region, or as a polycistron via a common regulatory region.

[0074] The expression vector may further include a nucleotide sequence encoding a select gene that can serve as a selection marker, from the viewpoint of facilitating the selection of the transformant.

[0075] 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 non-expression directly affects the activation of nitrile hydratase. A representative example is the protein involved in nitrile hydratase activation (nitrile hydratase activating protein) derived from Pseudonocardia thermophila, described in Japanese Patent Publication No. 11-253168. The sequence of the nitrile hydratase activating protein is shown in Sequence ID No. 36.

[0076] Methods for transforming expression vectors into desired host cells, and methods for producing nitrile hydratase in the transformed cells, etc., can be based on general methods and host cells known in the fields of molecular biology, biotechnology, and genetic engineering, as described in, for example, "Molecular Cloning 3rd Edition" (J. Sambrook et al.; Cold Spring Harbor Laboratory Press, 2001).

[0077] <Transformed> The transformants of this disclosure include the expression vector of this disclosure. According to the transformant of this disclosure, a mutant nitrile hydratase is produced in which the inhibition of amide compound formation reactions using nitrile hydratase by impurities is reduced, even in the presence of impurities. The transformants of this disclosure can be prepared by known methods. For example, one method involves constructing an expression vector containing a nucleotide sequence encoding a mutant nitrile hydratase and, if necessary, other regions as described above, and then transforming a desired host cell with the expression vector. Specifically, general methods known in the fields of molecular biology, biotechnology, and genetic engineering, as described in Sambrook, J., et al., "Molecular Cloning A Laboratory Manual, 3rd Edition", Cold Spring Harbor Laboratory Press, (2001), can be used.

[0078] Transformants can be created not only by incorporating an expression vector into host cells, but also by introducing silent mutations, such as changing infrequently used codons in host cells to more frequently used codons, as needed. This may increase the production of the mutant nitrile hydratase-derived protein incorporated into the expression vector.

[0079] Regarding the introduction of silent mutations, there are no particular restrictions on the method of introducing silent mutations, the site of mutation, the type of nucleotide to be changed, etc., as long as the method involves adjusting the codons of the nitrile hydratase gene and the signal sequence for secreting the nitrile hydratase gene outside the cell on the expression vector in accordance with the frequency of codon use in the host cell.

[0080] <Method for producing mutant nitrile hydratase> The method for producing mutant nitrile hydratase according to the present disclosure may also include culturing the aforementioned transformant of the present disclosure in a culture medium, and recovering the mutant nitrile hydratase according to the present disclosure from at least one of the cultured transformant and the culture medium.

[0081] (Method for culturing transformed organisms) The culture conditions for transformants obtained by transformation with an expression vector are the same as those for the host cells before transformation, and known conditions can be used. Examples of host cells include prokaryotes (such as Escherichia coli, Bacillus subtilis, and Actinomycetes), yeasts, and filamentous fungi. Any culture medium containing appropriate amounts of carbon source, nitrogen source, inorganic matter, and other nutrients can be used, whether it be a synthetic or natural medium. As for the culture medium components, known components used in culture media can be used. For example, organic nutrients such as meat extract, yeast extract, malt extract, peptone, NZ amine, and potato; carbon sources such as glucose, maltose, sucrose, starch, and organic acids; nitrogen sources such as ammonium sulfate, urea, and ammonium chloride; inorganic nutrients such as phosphates, magnesium, potassium, and iron; and vitamins can be used in appropriate combinations. Furthermore, when culturing transformants transformed with an expression vector containing a selection marker, for example, if the selection marker is drug-resistant, a culture medium containing the corresponding drug should be used, and if the selection marker is nutritionally dependent, a culture medium that does not contain the corresponding nutrients should be used. The pH of the culture medium can be appropriately selected within the range of pH 4 to pH 8. The culture of the transformants can be carried out using conventional culture methods such as shaking culture, aerated stirring culture, continuous culture, or fed-batch culture of a liquid medium containing the transformants. The culture conditions can be appropriately selected depending on the type of transformant, culture medium, and culture method, and there are no particular restrictions as long as the conditions allow the transformant to grow and produce mutant nitrile hydratase. The culture temperature is preferably 20°C to 45°C, more preferably 24°C to 37°C, and the culture is carried out aerobically. The culture period should be between 1 and 7 days, until the content of the protein with the desired mutant nitrile hydratase activity reaches its maximum.

[0082] For example, when the host cell is E. coli, LB medium or M9 medium are commonly used as the culture medium for the transformants, but more preferably, such a medium is used in which Fe ions and Co ions are added as components at a concentration of 0.1 μg / mL or more. After inoculating the transformants into the medium, they should be grown at an appropriate culture temperature (generally 20°C to 50°C).

[0083] (Recovery process for mutant nitrile hydratase) A method for producing mutant nitrile hydratase preferably includes a step of recovering mutant nitrile hydratase from at least one of the cultured transformants and the culture medium after cultivation.

[0084] After culturing the transformed transformants, the method for recovering the mutant nitrile hydratase can be the same as that commonly used in this field. If mutant nitrile hydratase is secreted outside the transformant, a crude enzyme solution containing the mutant nitrile hydratase can be easily obtained by centrifugation, filtration, etc., of the culture of the transformant. If mutant nitrile hydratase accumulates in the transformants, the cultured transformants can be recovered by centrifugation or other means, the recovered transformants can be suspended in a buffer solution, and the cell membranes of the transformants can be disrupted by known methods such as lysozyme treatment, freeze-thaw cycle, or sonication to recover the crude enzyme solution containing mutant nitrile hydratase.

[0085] Cationic polymers may be added to the crude enzyme solution containing the recovered mutant nitrile hydratase, and a filter aid may be added before filtration. The filtered enzyme solution containing the mutant nitrile hydratase can be further concentrated by ultrafiltration or other methods, and used as concentrated mutant nitrile hydratase (concentrated enzyme) after adding preservatives, etc. Alternatively, after concentrating the mutant nitrile hydratase, it may be converted into a powder (powdered enzyme) by spray drying or other methods. If separation and purification are required for the crude enzyme solution containing the recovered mutant nitrile hydratase, separation and purification can be performed by appropriately combining known chromatographic separation methods such as salting out with ammonium sulfate, precipitation with organic solvents such as alcohol, membrane separation by dialysis and ultrafiltration, or ion exchange chromatography, reverse-phase high-speed chromatography, affinity chromatography, and gel filtration chromatography.

[0086] <Method for producing amide compounds> The method for producing amide compounds according to the present disclosure comprises contacting a mutant nitrile hydratase according to the present disclosure with a nitrile compound. The mutant nitrile hydratase according to the present disclosure catalyzes a reaction for synthesizing amide compounds from nitrile compounds. According to the method for producing amide compounds according to the present disclosure, even in the presence of impurities, inhibition by impurities on the amide compound formation reaction using nitrile hydratase is reduced.

[0087] First, transformants or cell lines producing the mutant nitrile hydratase of this disclosure are cultured, and the resulting culture medium, cells, or cell-treated product is brought into contact with a nitrile compound in a medium. This brings the mutant nitrile hydratase of this disclosure into contact with the nitrile compound, and the mutant nitrile hydratase converts the nitrile compound to the corresponding amide compound. The cell-treated product, as used herein, refers to extracts or grinds from the transformants, post-isolated products such as crude enzyme preparations obtained by separating the nitrile hydratase-active fraction from these extracts or grinds, or enzyme purifies obtained by further purification, as well as immobilized products obtained by immobilizing the transformants, their extracts, grinds, or post-isolated products using appropriate means. The contact temperature described above is preferably within a temperature range in which the mutant nitrile hydratase of this disclosure does not become inactive, more preferably 0°C to 60°C, and more preferably 15°C to 40°C. For example, a culture medium in which a transformant or cell line producing the mutant nitrile hydratase of this disclosure has been cultured may be added directly to an aqueous solution containing a nitrile compound, or the culture medium may be centrifuged to separate the bacterial cells, and the bacterial cells may be added to the aqueous solution containing the nitrile compound. The pH of the aqueous solution during the reaction is preferably 7 to 9, and 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 regarding the culture of the transformant are described in the section on the method for producing mutant nitrile hydratase.

[0088] Specific examples of the nitrile compounds described above are not particularly limited as long as they are compounds that can act as substrates for the mutant nitrile hydratase of this disclosure, but include aliphatic saturated nitriles 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. In the method disclosed herein, the nitrile groups in the nitrile compound are converted to amide groups by hydration, so for example, acrylonitrile can be converted to acrylamide.

[0089] Next, without inactivating the mutant nitrile hydratase of this disclosure, activated carbon is added to the medium to purify the resulting amide compound without terminating the synthesis reaction of the amide compound from the nitrile compound using the mutant nitrile hydratase of this disclosure as a catalyst.

[0090] The reaction for producing amide compounds from nitrile compounds by contact with the mutant nitrile hydratase described herein may be carried out under neutral to basic conditions, such as pH 7-9, to match the optimal pH of the nitrile hydratase. The pH can be adjusted as needed by using basic substances such as ammonia or sodium hydroxide in a buffer solution. [Examples]

[0091] The embodiments will be described in more detail by the following examples, but the present invention is not limited in any way by the following examples. In the following examples, "mutation site" refers to the position of the difference in amino acid sequence between the α subunit having the amino acid sequence shown in SEQ ID NO: 1 and the β subunit having the amino acid sequence shown in SEQ ID NO: 2 and the nitrile hydratase derived from wild-type Pseudonocardia thermophylla.

[0092] (Acquisition of nitrile hydratase (C1) from wild-type Pseudonocardia thermophylla for comparison) 10 mL of LB liquid medium was prepared in a 30 mL test tube and sterilized by autoclaving at 121°C for 20 minutes. Ampicillin was added to this liquid medium to a final concentration of 100 μg / mL, and then the cell line MT-10822 described in Patent Document 1, Example 3 was inoculated onto a platinum loop and cultured at 37°C at 300 rpm for approximately 20 hours. Subsequently, 1 mL of the resulting culture was transferred to a suitable centrifuge tube, and the bacterial cells were separated by centrifugation (15000 rpm × 5 minutes). Next, plasmid pPT-DB1 was prepared from the isolated bacterial cells using alkaline SDS extraction. The prepared plasmid was used to transform competent cells of E. coli HB101 (manufactured by Toyobo Co., Ltd.) to obtain transformants (C1). Transformants (C1) produce nitrile hydratase (C1), which is a nitrile hydratase derived from wild-type Pseudonocardia thermophila.

[0093] (Obtaining mutant nitrile hydratase (C2) for comparison) Site-directed mutagenesis was performed using the "LA PCR in vitro mutagenesis Kit" (hereinafter also referred to as the mutagenesis kit) manufactured by Takara Shuzo Co., Ltd., in accordance with the method described in Comparative Example 2 of International Publication No. 2010 / 055666. Specifically, the wild-type Pseudonocardia thermophila-derived nitrile hydratase expression plasmid pPT-DB1 was used as a template, and transformants expressing a mutant nitrile hydratase in which the 92nd amino acid residue from the N-terminus of the α-subunit of wild-type Pseudonocardia thermophila-derived nitrile hydratase, Asp, was replaced with Glu. This transformant produces a comparative mutant nitrile hydratase (C2). Hereinafter, primers containing the mutant sequence to be introduced, as shown in Sequence ID No. 5, will also be referred to as mutation primers.

[0094] (Obtaining the mutant nitrile hydratase (1) of this disclosure) To obtain transformants expressing a mutant nitrile hydratase in which the 23rd amino acid residue from the N-terminus of SEQ ID NO: 1, Ala, is replaced with Gly in the α-subunit, mutational induction was performed using Takara Shuzo's "LA PCR in vitro mutagenesis Kit" (hereinafter referred to as the mutation induction kit). PCR was performed using the wild-type Pseudonocardia thermophila-derived nitrile hydratase expression plasmid pPT-DB1 as a template. PCR reaction No. 1 was performed using a 50 μL system containing 50 pmol of the mutation primers and M13 primer M4 (sequence described in SEQ ID NO: 33) shown in Table 5 (composition according to the conditions described in the mutation introduction kit). The reaction consisted of 25 cycles of thermal denaturation (98°C) for 15 seconds, annealing (55°C) for 30 seconds, and extension (72°C) for 120 seconds. PCR reaction No. 2 was performed using a 50 μL system (composition according to the conditions described in the mutagenesis kit) containing 50 pmol each of MUT4 primer (sequence described in SEQ ID NO: 34) and M13 primer RV (sequence described in SEQ ID NO: 35), following the same procedure as for PCR reaction No. 1.

[0095] Analysis of the DNA amplification product was performed by agarose electrophoresis (agarose concentration 1.0 wt%) using 5 μL each of the reaction completion solutions from PCR reactions No. 1 and No. 2, and the presence of amplified DNA products was confirmed. Using a Microcon100 (Takara Shuzo Co., Ltd.), excess primers and dNTPs were removed from each PCR reaction solution, and then TE was added to prepare 50 μL of each solution. A total of 47.5 μL of annealing solution (composition according to the conditions described in the mutagenesis kit) was prepared, containing 0.5 μL of each TE solution, and subjected to thermal denaturation treatment (98°C) for 10 minutes. Subsequently, the solution was cooled at a constant rate to 37°C over 60 minutes, and then held at 37°C for 15 minutes to perform the annealing treatment. 0.5 μL of TaKaRa LA Taq was added to the annealed solution and the mixture was heated at 72°C for 3 minutes to complete the heteroduplex. To this, 50 pmol each of the aforementioned M13 primer M4 and M13 primer RV were added to bring the total volume to 50 μL. Then, PCR reaction No. 3 was performed by repeating the following conditions for 25 cycles: heat denaturation (98°C) for 15 seconds, annealing (55°C) for 30 seconds, and extension reaction (72°C) for 120 seconds.

[0096] Analysis of the DNA amplification product was performed by agarose electrophoresis using 5 μL of the reaction completion solution from PCR reaction No. 3 (using Sigma-type VII low-melting-point agarose; agarose concentration 0.8 wt%), and the presence of approximately 2 kb of amplified DNA product was confirmed. Next, a DNA fragment of approximately 2 kb was excised from the agarose gel, and this agarose fragment (approximately 0.1 g) was finely crushed and suspended in 1 mL of TE solution. After that, the gel was incubated at 55°C for 1 hour to completely melt the agarose. The DNA fragments were purified from this molten solution by phenol / chloroform extraction and ethanol precipitation, and finally dissolved in 10 μL of TE. Purified amplified DNA fragments of approximately 2kb were cleaved with restriction enzymes EcoRI and HindIII. Subsequently, the restriction enzyme-treated solutions were purified by phenol / chloroform extraction and ethanol precipitation, and the DNA fragments were finally dissolved in 10 μL of TE. Similarly, the nitrile hydratase expression plasmid pPT-DB1 was cleaved using EcoRI and HindIII, and agarose gel electrophoresis was performed (using Sigma-45 type VII low-melting-point agarose; agarose concentration 0.7%) to excise only a DNA fragment of approximately 2.7Kb from the agarose gel. The cut agarose piece (approximately 0.1 g) was finely crushed and suspended in 1 mL of TE solution. Then, it was incubated at 55°C for 1 hour to completely melt the agarose. The DNA fragments were purified from this molten solution by phenol / chloroform extraction and ethanol precipitation, and finally dissolved in 10 μL of TE. The DNA fragments of approximately 2kb and 2.7kb obtained in this manner were ligated using a DNA ligation kit (Takara Shuzo Co., Ltd.). Subsequently, competent cells of E. coli HB101 (Toyobo Co., Ltd.) were transformed to obtain transformant (1). Plasmids were prepared from the above cells by alkaline SDS extraction, and the nucleotide sequence of the nitrile hydratase gene portion was determined using a DNA sequencer. Plasmid pPT-DB1 was confirmed to have a mutation in which Ala, the 23rd amino acid residue from the N-terminus of the α subunit, is replaced with Gly. Transformant (1) produces mutant nitrile hydratase (1) with the mutation described in Table 5.

[0097] (Obtaining the variant nitrile hydratases (2) to (17) of this disclosure) To obtain transformants (2) to (17) expressing mutant nitrile hydratases with amino acid residue substitutions as shown in Table 5, plasmids were obtained in the same manner as for transformant (1), except that primers 6 to 21 were used instead of primer 5. Transformants (2) to (17) produce nitrile hydratases (2) to (17) with the mutations described in Table 5.

[0098] (Obtaining the mutant nitrile hydratases (18) to (28) of this disclosure) To obtain a transformant (18) expressing a mutant nitrile hydratase with the amino acid residue substitutions shown in Table 6, the plasmid was obtained in the same manner as for transformant (1), except that the plasmid was obtained from transformant (9) instead of pPT-DB1 as the template, and the mutation primer of Sequence ID No. 18 was used as the mutation primer. Transformant (18) produces nitrile hydratase (18) with the mutations described in Table 6. Similarly, nitrile hydratase (19) was obtained from the plasmid obtained from transformant (9) and the mutation primer of SEQ ID NO: 15, nitrile hydratase (20) was obtained from the plasmid obtained from transformant (13) and the mutation primer of SEQ ID NO: 11, nitrile hydratase (21) was obtained from the plasmid obtained from transformant (10) and the mutation primer of SEQ ID NO: 18, nitrile hydratase (22) was obtained from the plasmid obtained from transformant (9) and the mutation primer of SEQ ID NO: 11, and nitrile hydratase (23) was obtained from the plasmid obtained from transformant (16) and the mutation primer of SEQ ID NO: 21. Nitrile hydratase (24) was obtained from the plasmid obtained from transformant (1) and the mutation primer of SEQ ID NO: 7; nitrile hydratase (25) was obtained from the plasmid obtained from transformant (6) and the mutation primer of SEQ ID NO: 19; nitrile hydratase (26) was obtained from the plasmid obtained from transformant (17) and the mutation primer of SEQ ID NO: 22; nitrile hydratase (27) was obtained from the plasmid obtained from transformant (5) and the mutation primer of SEQ ID NO: 23; and nitrile hydratase (28) was obtained from the plasmid obtained from transformant (7) and the mutation primer of SEQ ID NO: 24.

[0099] (Acquisition of the mutant nitrile hydratase described herein (29) to (37)) To obtain a transformant (29) expressing a mutant nitrile hydratase with amino acid residue substitutions as shown in Table 7, the plasmid was obtained in the same manner as for transformant (1), except that the plasmid was obtained from transformant (18) instead of pPT-DB1 as the template, and the mutation primer of Sequence ID No. 11 was used as the mutation primer. Transformant (29) produces nitrile hydratase (29) with the mutations described in Table 7. Similarly, nitrile hydratase (30) was obtained from the plasmid obtained from transformant (21) and the mutation primer of SEQ ID NO: 13, nitrile hydratase (31) was obtained from the plasmid obtained from transformant (20) and the mutation primer of SEQ ID NO: 6, nitrile hydratase (32) was obtained from the plasmid obtained from transformant (20) and the mutation primer of SEQ ID NO: 16, nitrile hydratase (33) was obtained from the plasmid obtained from transformant (20) and the mutation primer of SEQ ID NO: 18, and the plasmid obtained from the transformant was obtained from the plasmid obtained from transformant (9) and the mutation primer of SEQ ID NO: 16. Nitrile hydratase (34) was obtained from the mutation primer of SEQ ID NO: 17, plasmid obtained from the transformant (23), nitrile hydratase (35) was obtained from the mutation primer of SEQ ID NO: 11, plasmid obtained from the transformant (15), plasmid obtained from the transformant obtained from the mutation primer of SEQ ID NO: 25, nitrile hydratase (36) was obtained from the mutation primer of SEQ ID NO: 26, plasmid obtained from the transformant (8), plasmid obtained from the transformant obtained from the mutation primer of SEQ ID NO: 27, and nitrile hydratase (37) was obtained from the mutation primer of SEQ ID NO: 28.

[0100] (Obtaining the mutant nitrile hydratase described herein (38)-(46)) To obtain a transformant (38) expressing a mutant nitrile hydratase with the amino acid residue substitutions shown in Table 8, the plasmid was obtained in the same manner as for transformant (1), except that a plasmid was obtained from transformant (30) instead of pPT-DB1 as the template, and the mutation primer of Sequence ID No. 11 was used as the mutation primer. Transformant (38) produces nitrile hydratase (38) with the mutations described in Table 8. Similarly, nitrile hydratase (39) was obtained from the plasmid obtained from transformant (29) and the mutation primer of SEQ ID NO: 17, nitrile hydratase (40) from the plasmid obtained from transformant (34) and the mutation primer of SEQ ID NO: 11, nitrile hydratase (41) from the plasmid obtained from transformant (35) and the mutation primer of SEQ ID NO: 19, nitrile hydratase (42) from the plasmid obtained from transformant (8) and the mutation primer of SEQ ID NO: 22, nitrile hydratase (42) from the plasmid obtained from transformant and the mutation primer of SEQ ID NO: 32, and nitrile hydratase (42) from the plasmid obtained from transformant (40) and the mutation primer of SEQ ID NO: 18. Nitrile hydratase (43) was obtained from a plasmid obtained from a transformant (40) and the mutation primer of SEQ ID NO: 29 (44); nitrile hydratase (45) was obtained from a plasmid obtained from a transformant

[0101] In Tables 5 to 10, the column "Template" shows the number of the transformant from which the plasmid was obtained, the column "Mutation Primer" shows the sequence number of the primer used as the mutation primer, the column "Pre-mutation Amino Acid Remains" shows the pre-mutation amino acid residues at the mutation site described in the column "Mutation Site" (i.e., amino acid residues in wild-type Pseudonocardia thermophila-derived nitrile hydratase (C1)), and the column "Post-mutation Amino Acid Remains" shows the post-mutation amino acid residues at the mutation site described in the column "Mutation Site".

[0102] [Examples 1-1 to 1-17, 2-1 to 2-11, 3-1 to 3-9, 4-1 to 4-9, B2 to B7, C1 to C5, Comparative Examples 1, 2-1, 2-2, 3 to 4, and Reference Examples 1 to 3]

[0103] <Evaluation of the degree of suppression of reaction inhibition by impurities> Using transformants that produce nitrile hydratase of each number, the amide compound formation reaction in the presence of impurities was carried out as described below. 5 mL of LB liquid medium containing 40 μg / mL ferric sulfate heptahydrate and 10 μg / mL cobalt chloride dihydrate was prepared in a test tube and sterilized by autoclaving at 121°C for 20 minutes. Ampicillin was added to this medium to a final concentration of 100 μg / mL. Then, each transformant was inoculated in a loopful and cultured at 37°C at 200 rpm for approximately 20 hours. After the culture period, 1000 μl of the medium was taken and the cells were collected by centrifugation. These cells were suspended in 780 μL of 50 mM Tris-HCl aqueous solution (pH 8.0) containing 1000 ppm by mass of acrolein. 20 μL of acrylonitrile was added to this suspension and the mixture was reacted for 60 minutes with gentle stirring at the temperatures shown in Tables 5 to 10.

[0104] After the reaction was complete, the amount of amide compound (acrylamide) contained in the reaction solution was quantitatively analyzed using HPLC under the following analytical conditions.

[0105] The amount of amide compound obtained using each numbered nitrile hydratase in the presence of impurities (X) was compared with the amount of amide compound obtained in Comparative Example 1 using wild-type Pseudonocardia thermophila-derived nitrile hydratase (C1) in the presence of impurities (Y). This allowed for the relative magnitude of the enzymatic activity of each mutant nitrile hydratase in generating amide compounds from nitrile compounds, and this was defined as the degree of inhibition of reaction inhibition by impurities (X / Y). For example, in Comparative Example 1, since the results from the same Comparative Example 1 are being compared, the value of the degree of inhibition of reaction inhibition by impurities is 1.000. In Reference Example 1, the amount of amide compound obtained using nitrile hydratase (1) in the absence of impurities was compared with the amount of amide compound obtained in Example 1-1 using nitrile hydratase (1) in the presence of impurities. In Reference Example 2, the amount of amide compound obtained using nitrile hydratase (C1) in the absence of impurities was compared with the amount of amide compound obtained in Comparative Example 1 using nitrile hydratase (C1) in the presence of impurities. In Reference Example 3, the amount of amide compound obtained using nitrile hydratase (C2) in the absence of impurities was compared with the amount of amide compound obtained in Comparative Example 2-1 using nitrile hydratase (C2) in the presence of impurities.

[0106] The results are shown in each table. The reaction and analysis were performed at least three times for each nitrile hydratase used, and data variability due to dispensing procedures was corrected.

[0107] <Analysis conditions> Analytical instrument: HPLC manufactured by JASCO Corporation Column: YMCPackODS-A (150 x 6.00 mm) Analysis temperature: 40℃ Mobile phase: 3% acetonitrile, 10 mM phosphate

[0108] [Table 5]

[0109] [Table 6]

[0110] [Table 7]

[0111] [Table 8]

[0112] [Table 9]

[0113] [Table 10]

[0114] As shown in Table 5, when comparing Comparative Example 1, in which the reaction was carried out in the presence of impurities, with Reference Example 2, in which the reaction was carried out without impurities, the enzyme activity of wild-type nitrile hydratase (C1) was reduced compared to the case without impurities, indicating that the reaction was inhibited by the impurities. Furthermore, the mutant nitrile hydratase (C2) used for the comparative example also showed the same inhibition as (C1). Similarly, when comparing Comparative Example 2-1, in which the reaction was carried out in the presence of impurities, with Reference Example 3, in which the reaction was carried out without the addition of impurities, it was found that the enzyme activity was reduced compared to the case without impurities, indicating that the reaction was inhibited by the impurities. In contrast, the mutant nitrile hydratase of this disclosure exhibits a smaller difference in enzyme activity between when impurities are added to the reaction and when no impurities are added, compared to the wild-type nitrile hydratase (C1) and the mutant nitrile hydratase (C2) used for comparative examples. More specifically, the difference between the value of "degree of suppression of reaction inhibition by impurities" in Example 1-1 and the value of "degree of suppression of reaction inhibition by impurities" in Reference Example 1 (2.013 - 1.125 = 0.888) is smaller than the difference between the value of "degree of suppression of reaction inhibition by impurities" in Comparative Example 1 and the value of "degree of suppression of reaction inhibition by impurities" in Reference Example 2 (1.994 - 1.000 = 0.994). Furthermore, the difference between the value of "degree of suppression of reaction inhibition due to impurities" in Example 1-1 and the value of "degree of suppression of reaction inhibition due to impurities" in Reference Example 1 (2.013 - 1.125 = 0.888) is smaller than the difference between the value of "degree of suppression of reaction inhibition due to impurities" in Comparative Example 2-1 and the value of "degree of suppression of reaction inhibition due to impurities" in Reference Example 3 (2.263 - 0.960 = 1.303). In other words, the mutant nitrile hydratase of this disclosure reduces the inhibition by impurities in amide compound formation reactions using nitrile hydratase, even in the presence of impurities.

[0115] As shown in Tables 5 to 10, the mutant nitrile hydratases of each example, in which amino acid residues belonging to the group (a) to (q) above were substituted from the wild-type Pseudonocardia thermophila-derived nitrile hydratase, show reduced inhibition by impurities in the amide compound formation reaction using nitrile hydratase, even in the presence of impurities, compared to the wild-type Pseudonocardia thermophila-derived nitrile hydratase of Comparative Example 1 and the modified nitrile hydratase of Comparative Example 2-1.

[0116] As shown in Table 9, the mutant nitrile hydratases of each example, in which amino acid residue substitutions belonging to the group (a) to (q) above were made from wild-type Pseudonocardia thermophila-derived nitrile hydratase, showed reduced inhibition by impurities in amide compound formation reactions using nitrile hydratase, even in the presence of impurities of less than 1000 ppm (e.g., less than 10 ppm).

[0117] As shown in Table 10, the mutant nitrile hydratases of each example in which amino acid residue substitutions belonging to the group consisting of (i), (g), (l), and (m) were made to wild-type Pseudonocardia thermophila-derived nitrile hydratase did not decrease in enzyme activity even under reaction conditions of 30°C, which is higher than room temperature (20°C), and efficiently produced amide compounds. In other words, the mutant nitrile hydratases of each example in which amino acid residue substitutions belonging to the group consisting of (i), (g), (l), and (m) were made to wild-type Pseudonocardia thermophila-derived nitrile hydratase exhibited excellent heat resistance.

[0118] The disclosure of Japanese Patent Application No. 2022-106069 is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

Claims

1. A mutant nitrile hydratase having an α-subunit with 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 1, and a β-subunit with 90% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 2, and comprising a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) below, A mutant nitrile hydratase exhibits reduced inhibition of the amide compound formation reaction by impurities compared to wild-type nitrile hydratase having an α-subunit consisting of the amino acid sequence represented by SEQ ID NO: 1 and a β-subunit consisting of the amino acid sequence represented by SEQ ID NO:

2. (a) In the α subunit, substitution from Ala to Gly, corresponding to the 23rd amino acid residue from the N-terminus of SEQ ID NO: 1, (b) In the α subunit, substitution from the amino acid residue Val to Met, corresponding to the 105th position from the N-terminus of SEQ ID NO: 1, (c) In the α subunit, the amino acid residue Leu corresponding to the 142nd position from the N-terminus of SEQ ID NO: 1 is substituted with Cys. (d) In the α subunit, substitution from the amino acid residue Glu to Asp, corresponding to the 145th position from the N-terminus of SEQ ID NO: 1, (e) In the α subunit, substitution of amino acid residue Pro to Gly corresponding to the 152nd position from the N-terminus of SEQ ID NO: 1, (f) In the α subunit, a substitution from the amino acid residue Pro to Arg corresponding to the 153rd position from the N-terminus of SEQ ID NO: 1, (g) In the α subunit, substitution from the amino acid residue Gly to Val, corresponding to the 180th N-terminus of SEQ ID NO: 1, (h) In the β subunit, the amino acid residue Glu to Leu, corresponding to the 89th position from the N-terminus of SEQ ID NO: 2, (i) In the β subunit, substitution from Ala to Ile of the amino acid residue corresponding to the 122nd position from the N-terminus of SEQ ID NO: 2, (j) In the β subunit, substitution from the amino acid residue Thr to Gln, corresponding to the 149th amino acid residue from the N-terminus of SEQ ID NO:

2. (k) In the β subunit, the amino acid residue Lys to Arg, corresponding to the 153rd position from the N-terminus of SEQ ID NO: 2, (l) In the β subunit, substitution from Cys to Ser of the amino acid residue corresponding to the 189th position from the N-terminus of SEQ ID NO: 2, (m) In the β subunit, the amino acid residue Glu corresponding to the 207th position from the N-terminus of SEQ ID NO: 2 is substituted with Gly. (n) In the β subunit, substitution from the amino acid residue Ala to Trp, corresponding to the 232nd position from the N-terminus of Sequence ID No. 2, (o) In the β subunit, substitution from Ala to Glu, corresponding to the 233rd amino acid residue from the N-terminus of SEQ ID NO: 2 (p) In the α subunit, the amino acid residue Thr to Lys, corresponding to the 188th position from the N-terminus of SEQ ID NO: 1, (q) Substitution of the amino acid residue Glu to Gly in the β subunit, corresponding to the 50th position from the N-terminus of Sequence ID No.

2.

2. The mutant nitrile hydratase according to claim 1, comprising at least two amino acid residue substitutions selected from the group consisting of (a) to (q).

3. A mutant nitrile hydratase according to claim 1 or claim 2, comprising at least one amino acid residue substitution selected from the group consisting of (i), (g), (j), and (n).

4. A mutant nitrile hydratase according to claim 1 or claim 2, comprising at least one amino acid residue substitution selected from the group consisting of (i), (g), (l), and (m).

5. A mutant nitrile hydratase according to claim 1 or claim 2, comprising at least one amino acid residue substitution selected from the group consisting of (g), (p), and (q).

6. The mutant nitrile hydratase according to claim 1 or claim 2, wherein the amino acid sequence of the α subunit further comprises at least one amino acid residue substitution selected from the group consisting of (A1) to (A6) below. (A1) In the α subunit, the amino acid residue Arg to Val, corresponding to the 20th position from the N-terminus of Sequence ID No. 1, (A2) In the α subunit, the amino acid residue Gln to Arg, corresponding to the 141st position from the N-terminus of SEQ ID NO: 1, (A3) In the α subunit, the amino acid residue Glu corresponding to the 185th position from the N-terminus of SEQ ID NO: 1 is substituted with Ala. (A4) In the α subunit, the amino acid residue Ala to Arg corresponds to the 187th position from the N-terminus of SEQ ID NO:

1. (A5) In the α subunit, from the amino acid residue Pro corresponding to the 200th N-terminus of Sequence ID No. 1 to Ala, (A6) Substitution of the amino acid residue Val to Leu in the α subunit, corresponding to the 204th amino acid residue from the N-terminus of Sequence ID No.

1.

7. The mutant nitrile hydratase according to claim 1 or claim 2, wherein the amino acid sequence of the β subunit further comprises at least one amino acid residue substitution selected from the group consisting of (B1) to (B9) below. (B1) In the β subunit, the amino acid residue Thr to Ile, corresponding to the 40th position from the N-terminus of SEQ ID NO: 2, (B2) In the β subunit, the amino acid residue Arg corresponding to the 42nd position from the N-terminus of SEQ ID NO: 2 is substituted with Pro. (B3) In the β subunit, the amino acid residue Thr to Cys, corresponding to the 76th position from the N-terminus of SEQ ID NO: 2, (B4) In the β subunit, the amino acid residue Thr to His, corresponding to the 83rd position from the N-terminus of SEQ ID NO: 2, (B5) In the β 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 β subunit, substitution of the amino acid residue Arg corresponding to the 146th position from the N-terminus of SEQ ID NO: 2 to Gln or Thr, (B7) In the β subunit, substitution of the amino acid residue Arg corresponding to the 160th N-terminus of SEQ ID NO: 2 with Gln or Ile, (B8) In the β subunit, the amino acid residue Glu corresponding to the 108th position from the N-terminus of SEQ ID NO: 2 is substituted with Arg. (B9) Substitution of the amino acid residue Tyr to Cys in the β subunit, corresponding to the 176th amino acid residue from the N-terminus of Sequence ID No.

2.

8. A nucleic acid encoding a mutant nitrile hydratase according to claim 1 or claim 2.

9. A vector comprising the nucleic acid described in claim 8.

10. The vector according to claim 9, which is an expression vector.

11. A transformant comprising the expression vector described in claim 10.

12. The transformant according to claim 11 is cultured in a culture medium, and To recover the mutant nitrile hydratase according to claim 1 or claim 2 from at least one of the cultured transformant and the culture medium, A method for producing mutant nitrile hydratase, including the above.

13. A variant nitrile hydratase obtained by the manufacturing method described in claim 12.

14. A method for producing an amide compound, comprising contacting a mutant nitrile hydratase described in Claim 1 or Claim 2 with a nitrile compound.

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