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

Abstract

A mutant nitrile hydratase which is derived from Pseudonocardia thermophila and has an alpha subunit having a sequence identity of 90% or more with SEQ ID NO: 1 and a beta subunit having a sequence identity of 90% or more with SEQ ID NO: 2, the mutant nitrile hydratase contains a substitution in which an amino acid residue at at least one position selected from the group consisting of the 23rd position, the 105th position, the 142 position, the 145th position, the 152nd position, the 153rd position, the 180th position and the 188th position from the N-terminal of the alpha subunit and the 89th position, the 122nd position, the 149th position, the 153rd position, the 189th position, the 207th position, the 232 position, the 233th position and the 50th position from the N-terminal of the beta subunit is substituted by 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 Background Art

[0003] Nitrile hydratase is an enzyme having a nitrile hydrating activity that converts the nitrile group of various compounds into an amide group by hydration, and is used in industrial amide compound production processes utilizing an enzyme reaction.

[0004] Acrylonitrile is known to be used as a raw material for the production of acrylamide, which is an amide compound. Acrylonitrile may contain various impurities (for example, acrolein as a byproduct, etc.). Patent Document 1 describes that acrolein is generally produced as a byproduct in the production of acrylonitrile, and forms an insoluble polymer as a side reaction.

[0005] Patent Document 2 describes that acrolein, as an example of impurities contained in acrylonitrile, 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, the inhibition of the catalytic action of nitrile hydratase by acrolein can be prevented from occurring.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application Publication No. 2012-139233

[0009] Patent Document 2: Japanese Patent Application Publication No. 2012-29695 Summary of the invention

[0010] Problems to be solved by the invention

[0011] However, the method described in Patent Document 2 is a method for avoiding the inhibition of nitrile hydratase activity caused by acrolein by controlling the acrolein concentration in the raw material acrylonitrile to 1 ppm or less, rather than a method for reducing the inhibition of nitrile hydratase activity even in the presence of impurities. In addition, from the viewpoint of reducing production costs, it is required to develop a technology that can reduce the inhibition of nitrile hydratase activity without requiring a process for removing impurities, an additive for capturing impurities in the reaction system, etc.

[0012] Therefore, the present disclosure provides a technology related to a novel mutant nitrile hydratase that reduces the hindrance of an amide compound production reaction using the nitrile hydratase caused by the impurities even in the presence of the impurities.

[0013] Means for solving problems

[0014] This disclosure includes the following schemes.

[0015] <1> A mutant nitrile hydratase comprising an α subunit having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 1 and a β subunit having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 2, wherein the mutant nitrile hydratase comprises at least one amino acid residue substitution selected from the group consisting of (a) to (q) below:

[0016] (a) in the α subunit, the amino acid residue Ala corresponding to the 23rd amino acid residue from the N-terminus of SEQ ID NO: 1 is substituted with Gly;

[0017] (b) substitution of the amino acid residue Val corresponding to position 105 from the N-terminus of SEQ ID NO: 1 in the α subunit with Met;

[0018] (c) in the α subunit, the amino acid residue Leu corresponding to position 142 from the N-terminus of SEQ ID NO: 1 is substituted with Cys;

[0019] (d) substitution of the amino acid residue Glu corresponding to position 145 from the N-terminus of SEQ ID NO: 1 in the α subunit with Asp;

[0020] (e) substitution of the amino acid residue Pro corresponding to position 152 from the N-terminus of SEQ ID NO: 1 in the α subunit with Gly;

[0021] (f) substitution of the amino acid residue Pro corresponding to position 153 from the N-terminus of SEQ ID NO: 1 in the α subunit with Arg or Val;

[0022] (g) substitution of the amino acid residue Gly corresponding to position 180 from the N-terminus of SEQ ID NO: 1 in the α subunit with Val;

[0023] (h) substitution of the amino acid residue Glu corresponding to position 89 from the N-terminus of SEQ ID NO: 2 in the β subunit with Leu;

[0024] (i) substitution of the amino acid residue Ala corresponding to position 122 from the N-terminus of SEQ ID NO: 2 in the β subunit with Ile, Leu or Thr;

[0025] (j) in the β subunit, the amino acid residue Thr corresponding to position 149 from the N-terminus of SEQ ID NO: 2 is substituted with Gln;

[0026] (k) substitution of the amino acid residue Lys corresponding to position 153 from the N-terminus of SEQ ID NO: 2 in the β subunit with Val, Arg or Pro;

[0027] (l) in the β subunit, the amino acid residue Cys corresponding to position 189 from the N-terminus of SEQ ID NO: 2 is substituted with Ser;

[0028] (m) substitution of the amino acid residue Glu corresponding to position 207 from the N-terminus of SEQ ID NO: 2 in the β subunit with Pro, Ser or Gly;

[0029] (n) in the β subunit, the amino acid residue Ala corresponding to position 232 from the N-terminus of SEQ ID NO: 2 is substituted with Trp, Ser, Pro, Phe, Ile, Arg, Cys or Gly;

[0030] (o) substitution of the amino acid residue Ala corresponding to position 233 from the N-terminus of SEQ ID NO: 2 in the β subunit with Glu or Gln;

[0031] (p) substitution of Thr at the amino acid residue corresponding to position 188 from the N-terminus of SEQ ID NO: 1 in the α subunit with Lys;

[0032] (q) In the β subunit, the amino acid residue Glu corresponding to the 50th amino acid residue from the N-terminus of SEQ ID NO: 2 is substituted with Gly.

[0033] <2> The mutant nitrile hydratase according to <1> above, which contains at least two amino acid residue substitutions selected from the group consisting of (a) to (q).

[0034] <3> The mutant nitrile hydratase according to <1> or <2> above, comprising at least one amino acid residue substitution selected from the group consisting of (i), (g), (j), and (n) above.

[0035] <4> The mutant nitrile hydratase according to <1> or <2> above, which contains at least one amino acid residue substitution selected from the group consisting of (i), (g), (l), and (m) above.

[0036] <5> The mutant nitrile hydratase according to <1> or <2> above, comprising at least one amino acid residue substitution selected from the group consisting of (g), (p), and (q) above.

[0037] <6> The mutant nitrile hydratase according to <1> or <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 the following (A1) to (A6):

[0038] (A1) substitution of the amino acid residue Arg corresponding to the 20th position from the N-terminus of SEQ ID NO: 1 in the α subunit with Val;

[0039] (A2) substitution of the amino acid residue Gln corresponding to position 141 from the N-terminus of SEQ ID NO: 1 in the α subunit with Arg;

[0040] (A3) substitution of the amino acid residue Glu corresponding to position 185 from the N-terminus of SEQ ID NO: 1 in the α subunit with Ala;

[0041] (A4) substitution of the amino acid residue Ala corresponding to position 187 from the N-terminus of SEQ ID NO: 1 in the α subunit with Arg;

[0042] (A5) substitution of the amino acid residue Pro corresponding to the 200th position from the N-terminus of SEQ ID NO:1 in the α subunit with Ala;

[0043] (A6) In the α subunit, the amino acid residue Val corresponding to position 204 from the N-terminus of SEQ ID NO: 1 is substituted with Leu.

[0044] <7> The mutant nitrile hydratase according to <1> or <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 the following (B1) to (B9):

[0045] (B1) substitution of Thr in the β subunit, corresponding to the 40th amino acid residue from the N-terminus of SEQ ID NO: 2, with Ile;

[0046] (B2) substitution of the amino acid residue Arg corresponding to position 42 from the N-terminus of SEQ ID NO: 2 in the β subunit with Pro;

[0047] (B3) substitution of Thr at the amino acid residue corresponding to position 76 from the N-terminus of SEQ ID NO: 2 in the β subunit with Cys;

[0048] (B4) substitution of Thr with His as the amino acid residue corresponding to position 83 from the N-terminus of SEQ ID NO: 2 in the β subunit;

[0049] (B5) substitution of the amino acid residue Leu corresponding to position 88 from the N-terminus of SEQ ID NO: 2 in the β subunit with Arg, Pro or Ser;

[0050] (B6) substitution of the amino acid residue Arg corresponding to position 146 from the N-terminus of SEQ ID NO: 2 in the β subunit with Gln or Thr;

[0051] (B7) substitution of the amino acid residue Arg corresponding to position 160 from the N-terminus of SEQ ID NO: 2 in the β subunit with Gln or Ile;

[0052] (B8) substitution of the amino acid residue Glu corresponding to position 108 from the N-terminus of SEQ ID NO: 2 in the β subunit with Arg;

[0053] (B9) In the β subunit, the amino acid residue Tyr corresponding to position 176 from the N-terminus of SEQ ID NO: 2 is substituted with Cys.

[0054] <8> A nucleic acid encoding the mutant nitrile hydratase according to any one of <1> to <7> above.

[0055] <9> A vector comprising the nucleic acid described in <8> above.

[0056] <10> The vector according to <9> above, which is an expression vector.

[0057] <11> A transformant comprising the expression vector described in <10> above.

[0058] <12> A method for producing a mutant nitrile hydratase, the method comprising the following steps:

[0059] a step of culturing the transformant according to <11> above in a culture medium; and

[0060] A step of recovering the mutant nitrile hydratase according to any one of <1> to <7> above from at least one of the cultured transformant and the culture medium.

[0061] <13> A mutant nitrile hydratase obtained by the production method described in <12> above.

[0062] <14> A method for producing an amide compound, the method comprising the step of contacting the mutant nitrile hydratase according to any one of <1> to <7> above with a nitrile compound.

[0063] Effects of the Invention

[0064] According to the present disclosure, it is possible to provide a technology involving a novel mutant nitrile hydratase that reduces the inhibition of an amide compound production reaction using the nitrile hydratase caused by impurities even in the presence of impurities. DETAILED DESCRIPTION

[0065] Hereinafter, embodiments of the present disclosure will be described. These descriptions and examples are examples of the embodiments and do not limit the scope of the embodiments.

[0066] In the numerical range recorded in stages in this disclosure, the upper limit or lower limit recorded in one numerical range can be replaced by the upper limit or lower limit of the numerical range recorded in other stages. In addition, in the numerical range recorded in this disclosure, the upper limit or lower limit of the numerical range can be replaced by the value shown in the embodiment.

[0067] In the present disclosure, each component may contain a plurality of substances belonging to the component.

[0068] In the present disclosure, when the amount of each component in a composition is mentioned, if there are multiple substances belonging to each component in the composition, unless otherwise specified, it means the total amount of the multiple substances present in the composition.

[0069] The term "process" in the present disclosure does not refer to only an independent process, but is also included in the term as long as the intended purpose of the process can be achieved even if it cannot be clearly distinguished from other processes.

[0070] In the present disclosure, a numerical range expressed using "to" means a range including the numerical values ​​described before and after "to" as the minimum value and the maximum value, respectively.

[0071] In the present disclosure, when there are plural substances belonging to each component in the composition, the amount of each component in the composition refers to the total amount of the plural substances present in the composition unless otherwise specified.

[0072] In the present disclosure, "enzyme activity" refers to nitrile hydration activity that converts a nitrile group into an amide group.

[0073] Regarding the description of the nucleotide sequence in the present disclosure, even when the nucleic acid chains retaining the nucleotide sequence form a double helix, there is a case where the description is made with an eye on the sequence on one chain, and in the other chain of the double helix, such a description of the sequence should be read interchangeably with its complementary sequence and applied.

[0074] In the present disclosure, unless otherwise specified, three letters represent amino acid residues. For example, "Ile" represents an isoleucine residue.

[0075] <Mutated nitrile hydratase>

[0076] The present disclosure provides a mutant nitrile hydratase having an α subunit having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1 and a β subunit having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 2, wherein the mutant nitrile hydratase contains at least one amino acid residue substitution selected from the group consisting of the following (a) to (q):

[0077] (a) in the α subunit, the amino acid residue Ala corresponding to the 23rd amino acid residue from the N-terminus of SEQ ID NO: 1 is substituted with Gly;

[0078] (b) substitution of the amino acid residue Val corresponding to position 105 from the N-terminus of SEQ ID NO: 1 in the α subunit with Met;

[0079] (c) in the α subunit, the amino acid residue Leu corresponding to position 142 from the N-terminus of SEQ ID NO: 1 is substituted with Cys;

[0080] (d) substitution of the amino acid residue Glu corresponding to position 145 from the N-terminus of SEQ ID NO: 1 in the α subunit with Asp;

[0081] (e) substitution of the amino acid residue Pro corresponding to position 152 from the N-terminus of SEQ ID NO: 1 in the α subunit with Gly;

[0082] (f) substitution of the amino acid residue Pro corresponding to position 153 from the N-terminus of SEQ ID NO: 1 in the α subunit with Arg or Val;

[0083] (g) substitution of the amino acid residue Gly corresponding to position 180 from the N-terminus of SEQ ID NO: 1 in the α subunit with Val;

[0084] (h) substitution of the amino acid residue Glu corresponding to position 89 from the N-terminus of SEQ ID NO: 2 in the β subunit with Leu;

[0085] (i) substitution of the amino acid residue Ala corresponding to position 122 from the N-terminus of SEQ ID NO: 2 in the β subunit with Ile, Leu or Thr;

[0086] (j) in the β subunit, the amino acid residue Thr corresponding to position 149 from the N-terminus of SEQ ID NO: 2 is substituted with Gln;

[0087] (k) substitution of the amino acid residue Lys corresponding to position 153 from the N-terminus of SEQ ID NO: 2 in the β subunit with Val, Arg or Pro;

[0088] (l) in the β subunit, the amino acid residue Cys corresponding to position 189 from the N-terminus of SEQ ID NO: 2 is substituted with Ser;

[0089] (m) substitution of the amino acid residue Glu corresponding to position 207 from the N-terminus of SEQ ID NO: 2 in the β subunit with Pro, Ser or Gly;

[0090] (n) in the β subunit, the amino acid residue Ala corresponding to position 232 from the N-terminus of SEQ ID NO: 2 is substituted with Trp, Ser, Pro, Phe, Ile, Arg, Cys or Gly;

[0091] (o) substitution of the amino acid residue Ala corresponding to position 233 from the N-terminus of SEQ ID NO: 2 in the β subunit with Glu or Gln;

[0092] (p) substitution of Thr at the amino acid residue corresponding to position 188 from the N-terminus of SEQ ID NO: 1 in the α subunit with Lys;

[0093] (q) In the β subunit, the amino acid residue Glu corresponding to the 50th amino acid residue from the N-terminus of SEQ ID NO: 2 is substituted with Gly.

[0094] The mutant nitrile hydratase having a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) above is a novel mutant that has not been reported so far.

[0095] The mutant nitrile hydratase of the present disclosure is different from the nitrile hydratase derived from the wild-type Pseudonocardia Thermophila and the conventional mutant nitrile hydratase. In the presence of impurities, in the presence of impurities (e.g., acrolein, etc.), the hindrance to the amide compound production reaction using the nitrile hydratase caused by impurities is reduced, and the nitrile hydratase exhibits excellent enzyme activity in the reaction of synthesizing amide compounds from nitrile compounds.

[0096] In addition, the reduction in the above-mentioned reaction hindrance caused by impurities in the mutant nitrile hydratase of the present disclosure is particularly significant when the impurities are contained at a high concentration (e.g., 1000 ppm or more). Therefore, according to the mutant nitrile hydratase of the present disclosure, in the industrial amide compound production process using nitrile hydratase, even when using raw materials that are not fully purified, the reaction of synthesizing amide compounds can be fully carried out. In this way, according to the mutant nitrile hydratase of the present disclosure, the efficiency of industrial amide compound production can be greatly improved compared with the case of using conventional enzymes.

[0097] The mutant nitrile hydratase of the present disclosure is a mutant nitrile hydratase having an α subunit having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1, and a β subunit having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 2, and containing a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) described above.

[0098] The amino acid sequence shown in SEQ ID NO: 1 refers to the amino acid sequence of the α-subunit of nitrile hydratase derived from wild-type Pseudonocardia thermophila.

[0099] The amino acid sequence shown in SEQ ID NO: 2 refers to the amino acid sequence of the β subunit of nitrile hydratase derived from wild-type Pseudonocardia thermophila.

[0100] The α subunit having a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 1 means an α subunit having an amino acid sequence that is 90% or more identical to the amino acid sequence of the wild-type α subunit when compared with the α subunit of the nitrile hydratase derived from wild-type Pseudonocardia thermophila (also referred to as the wild-type α subunit).

[0101] Similarly, the β subunit having a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 2 means a β subunit having an amino acid sequence that is 90% or more identical to the amino acid sequence of the wild-type β subunit when compared with the β subunit of the nitrile hydratase derived from wild-type Pseudonocardia thermophila (also referred to as the wild-type β subunit).

[0102] The mutant nitrile hydratase has an α subunit having a sequence identity of more than 90% with the amino acid sequence shown in sequence number 1, may have an α subunit having a sequence identity of more than 95% with the amino acid sequence shown in sequence number 1, or may have an α subunit having a sequence identity of more than 98% with the amino acid sequence shown in sequence number 1.

[0103] The mutant nitrile hydratase has a β subunit having a sequence identity of more than 90% with the amino acid sequence shown in sequence number 2, may have a β subunit having a sequence identity of more than 95% with the amino acid sequence shown in sequence number 1, or may have a β subunit having a sequence identity of more than 98% with the amino acid sequence shown in sequence number 1.

[0104] For the mutant nitrile hydratase, the dimer formed by the association of the α subunit and the β subunit becomes its basic structural unit, and the dimer further associates to form a tetramer. The 111th amino acid residue from the N-terminus of the α subunit, i.e., the cysteine ​​residue, is translated into cysteine ​​sulfinic acid (Cys-SOOH), and the 113th amino acid residue from the N-terminus, i.e., the cysteine ​​residue, is translated into cysteine ​​sulfenic acid (Cys-SOH), and then they are modified, and the polypeptide chain of the α subunit is bonded to the cobalt atom through the modified amino acid residues to form an active center.

[0105] (Mutation location)

[0106] The mutant nitrile hydratase of the present disclosure contains a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) above.

[0107] The mutant nitrile hydratase of the present disclosure may contain at least one amino acid residue substitution selected from the group consisting of (a) to (q) above, or may contain a plurality of substitutions in combination.

[0108] The mutant nitrile hydratase of the present disclosure may further contain substitution of amino acid residues other than the group consisting of (a) to (q) as long as the inhibition of the amide compound production reaction using the nitrile hydratase due to impurities is reduced.

[0109] The mutant nitrile hydratase of the present disclosure preferably contains at least two amino acid residue substitutions selected from the group consisting of (a) to (q) above from the viewpoint of further reducing the inhibition of the amide compound production reaction using the nitrile hydratase due to impurities.

[0110] The mutant nitrile hydratase of the present disclosure preferably contains the amino acid residue substitution (i) described above from the viewpoint of further reducing the inhibition of the amide compound production reaction using the nitrile hydratase due to impurities.

[0111] The mutant nitrile hydratase of the present disclosure may contain 3 or more, 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 selected from the group consisting of the above (a) to the above (q), from the viewpoint of further reducing the inhibition of the amide compound production reaction using the nitrile hydratase caused by impurities.

[0112] The mutant nitrile hydratase of the present disclosure may contain 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less amino acid residue substitutions selected from the group consisting of the aforementioned (a) to the aforementioned (q).

[0113] In the case where the mutant nitrile hydratase combination of the present disclosure contains two amino acid residue substitutions from the group consisting of the aforementioned (a) to the aforementioned (q), the combination of the aforementioned two amino acid residue substitutions is not particularly limited, and for example, from the viewpoint of further reducing the hindrance to the amide compound production reaction using the nitrile hydratase caused by impurities, the following combination may be included. It should be noted that in the examples of the following combinations, the amino acid residue substitutions from the group consisting of the aforementioned (a) to the aforementioned (q) are simplified and only the mutation positions are recorded. For example, the combination of the aforementioned (j) β149 and (n) β232 refers to a substitution of the amino acid residue Thr corresponding to the 149th position from the N-terminal of sequence number 2 in the (j) β subunit to Gln, and a substitution of the amino acid residue Ala corresponding to the 232nd position from the N-terminal of sequence number 2 in the (n) β subunit to Trp, Ser, Pro, Phe, Ile, Arg, Cys or Gly. As in the aforementioned (n), when there are multiple candidates for the amino acid residue after mutation, the following combination may be any of the aforementioned candidates. In this regard, the same applies to the description of examples of combinations described below in the case where the combination contains three or more amino acid residue substitutions.

[0114] Combination of (j) β149 and (n) β232;

[0115] Combination of (i) β122 and (g) α180;

[0116] Combination of (l) β189 and (m) β207;

[0117] Combination of (o) β233 and (h) β89;

[0118] Combination of (b) α105 and (c) α142;

[0119] (c) combination of α142 and (g) α180;

[0120] A combination of (i) β122 and (n) β232;

[0121] A combination of (i) β122 and (j) β149;

[0122] Combination of (q) β50 and (h) β89;

[0123] Combination of (p) α188 and (l) β189;

[0124] Combination of (p)α188 and (q)β50.

[0125] When the mutant nitrile hydratase combination of the present disclosure contains three amino acid residue substitutions from the group consisting of the aforementioned (a) to the aforementioned (q), the combination of the aforementioned three amino acid residue substitutions is not particularly limited, and for example, from the viewpoint of further reducing the inhibition of the amide compound production reaction using the nitrile hydratase caused by impurities, the following combination may be included. It should be noted that in the following combination examples, with respect to the amino acid residue substitutions from the group consisting of the aforementioned (a) to the aforementioned (q), only the mutation position is described for simplicity.

[0126] Combination of (i) β122 and (n) β232 and (g) α180;

[0127] Combination of (i) β122 and (j) β149 and (n) β232;

[0128] Combination of (m) β207, (b) α105, and (g) α180;

[0129] Combination of (m) β207 and (l) β189 and (g) α180;

[0130] Combination of (m) β207, (n) β232, and (g) α180;

[0131] (g) combination of α180 and (p) α188 and (q) β50;

[0132] Combination of (i) β122 and (j) β149 and (m) β207;

[0133] Combination of (i) β122 and (l) β189 and (m) β207;

[0134] Combination of (q) β50 and (a) α23 and (b) α105.

[0135] When the mutant nitrile hydratase combination of the present disclosure contains four amino acid residue substitutions from the group consisting of the aforementioned (a) to the aforementioned (q), the combination of the aforementioned three amino acid residue substitutions is not particularly limited, and for example, from the viewpoint of further reducing the inhibition of the amide compound production reaction using the nitrile hydratase caused by impurities, the following combination may be included. It should be noted that in the following combination examples, with respect to the amino acid residue substitutions from the group consisting of the aforementioned (a) to the aforementioned (q), only the mutation positions are described for simplicity.

[0136] Combination of (i) β122 and (j) β149 and (n) β232 and (g) α180;

[0137] Combination of (i) β122 and (m) β207 and (n) β232 and (g) α180;

[0138] Combination of (i) β122 and (l) β189 and (m) β207 and (g) α180;

[0139] Combination of (p) α188 and (q) β50 and (a) α23 and (l) β189;

[0140] Combination of (p) α188 and (q) β50 and (g) α180 and (o) β233;

[0141] Combination of (a) α23 and (d) α145 and (e) α152 and (o) β233;

[0142] Combination of (b) α105 and (f) α153 and (h) β89 and (n) β232;

[0143] Combination of (q) β50 and (d) α145 and (e) α152 and (o) β233.

[0144] Furthermore, of course, substitution may be performed by combining five or more amino acid residues from the group consisting of the above-mentioned (a) to (q).

[0145] When the mutant nitrile hydratase of the present disclosure contains at least one amino acid residue selected from the group consisting of (i), (g), (l) and (m), in addition to the effect of suppressing the reaction inhibition caused by impurities, there is also a tendency for the thermal stability of the nitrile hydratase to be improved. Conventional nitrile hydratase tends to have reduced catalytic activity at temperatures above room temperature (20° C.). Therefore, when a low-soluble raw material is used for the reaction at room temperature, the yield of the obtained amide compound is small, or a cooling device is required during the production, and improvement is required from the viewpoint of production cost.

[0146] In contrast, the mutant nitrile hydratase of the present disclosure having the above-mentioned structure has high thermal stability and is easy to maintain high catalytic activity at a temperature (e.g., 30° C.) or higher than room temperature (e.g., 20° C.) compared to conventional nitrile hydratase. Therefore, the mutant nitrile hydratase of the present disclosure having the above-mentioned structure can increase the temperature of the reaction system even when a raw material with low solubility at room temperature is used for the reaction, so that the yield of the amide compound obtained is easy to become higher than that of conventional nitrile hydratase, and a cooling device is not required during production, so that a more excellent secondary effect can be obtained from the viewpoint of production cost.

[0147] In the mutant nitrile hydratase of the present disclosure, from the viewpoint of further reducing the inhibition of the amide compound production reaction using the nitrile hydratase caused by impurities, the amino acid sequence of the α subunit may further contain at least one amino acid residue substitution selected from the group consisting of the following (A1) to (A6):

[0148] (A1) substitution of the amino acid residue Arg corresponding to the 20th amino acid residue from the N-terminus of SEQ ID NO: 1 in the α subunit with Val;

[0149] (A2) substitution of the amino acid residue Gln corresponding to position 141 from the N-terminus of SEQ ID NO: 1 in the α subunit with Arg;

[0150] (A3) substitution of the amino acid residue Glu corresponding to position 185 from the N-terminus of SEQ ID NO: 1 in the α subunit with Ala;

[0151] (A4) substitution of the amino acid residue Ala corresponding to position 187 from the N-terminus of SEQ ID NO: 1 in the α subunit with Arg;

[0152] (A5) substitution of the amino acid residue Pro corresponding to the 200th position from the N-terminus of SEQ ID NO:1 in the α subunit with Ala;

[0153] (A6) In the α subunit, the amino acid residue Val corresponding to position 204 from the N-terminus of SEQ ID NO: 1 is substituted with Leu.

[0154] In the mutant nitrile hydratase of the present disclosure, from the viewpoint of further reducing the inhibition of the amide compound production reaction using the nitrile hydratase caused by impurities, the amino acid sequence of the β subunit may further contain at least one amino acid residue substitution selected from the group consisting of the following (B1) to (B9):

[0155] (B1) substitution of Thr in the β subunit, corresponding to the 40th amino acid residue from the N-terminus of SEQ ID NO: 2, with Ile;

[0156] (B2) substitution of the amino acid residue Arg corresponding to position 42 from the N-terminus of SEQ ID NO: 2 in the β subunit with Pro;

[0157] (B3) substitution of Thr at the amino acid residue corresponding to position 76 from the N-terminus of SEQ ID NO: 2 in the β subunit with Cys;

[0158] (B4) substitution of Thr with His as the amino acid residue corresponding to position 83 from the N-terminus of SEQ ID NO: 2 in the β subunit;

[0159] (B5) substitution of the amino acid residue Leu corresponding to position 88 from the N-terminus of SEQ ID NO: 2 in the β subunit with Arg, Pro or Ser;

[0160] (B6) substitution of the amino acid residue Arg corresponding to position 146 from the N-terminus of SEQ ID NO: 2 in the β subunit with Gln or Thr;

[0161] (B7) substitution of the amino acid residue Arg corresponding to position 160 from the N-terminus of SEQ ID NO: 2 in the β subunit with Gln or Ile;

[0162] (B8) substitution of the amino acid residue Glu corresponding to position 108 from the N-terminus of SEQ ID NO: 2 in the β subunit with Arg;

[0163] (B9) In the β subunit, the amino acid residue Tyr corresponding to position 176 from the N-terminus of SEQ ID NO: 2 is substituted with Cys.

[0164] The mutant nitrile hydratase may contain substitutions other than the amino acid residue substitution group consisting of the aforementioned (a) to (q), the amino acid residue substitution group consisting of the aforementioned (A1) to (A6), and the amino acid residue substitution group consisting of the aforementioned (B1) to (B9) (hereinafter referred to as "other amino acid residue substitutions") within the scope of exerting the effects of the present disclosure.

[0165] The total number of substitutions selected from the amino acid residue substitution group consisting of the above-mentioned (a) to (q), the amino acid residue substitution group consisting of the above-mentioned (A1) to (A6), and the amino acid residue substitution group consisting of the above-mentioned (B1) to (B9) in the mutant nitrile hydratase is not particularly limited as long as it is 1 or more, and for example, it can be any of 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.

[0166] 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), (j), and (n), may contain amino acid residue substitutions of (i), (j), and (n), or may contain all of (i), (g), (j), and (n), for example, from the viewpoint of further reducing the inhibition of the amide compound production reaction using the nitrile hydratase caused by impurities.

[0167] 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), may contain amino acid residue substitutions of (i), (l), and (m), or may contain all of (i), (g), (l), and (m) from the viewpoint of further reducing the inhibition of the amide compound production reaction using the nitrile hydratase caused by impurities and from the viewpoint of excellent thermal stability.

[0168] 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 amino acid residue substitutions of (p) and (q), or may contain all of (g), (p), and (q) substitutions from the viewpoint of further reducing the inhibition of the amide compound production reaction using the nitrile hydratase caused by impurities.

[0169] Examples of combinations of substitutions of two amino acid residues selected from the group consisting of the aforementioned (a) to the aforementioned (q), the group consisting of the aforementioned (A1) to (A6), and the group consisting of the aforementioned (B1) to (B9) are shown below, but the present disclosure is not limited thereto. It should be noted that in the table, the numbers in the column of the mutation position indicate the position from the N-terminal side of the amino acid sequence in the corresponding subunit.

[0170] [Table 1-1]

[0171]

[0172] [Table 1-2]

[0173]

[0174] Examples of combinations of substitutions of three amino acid residues selected from the group consisting of (a) to (q), (A1) to (A6), and (B1) to (B9) are shown below, but the present disclosure is not limited thereto. In the following table, the numbers in the column of the mutation site indicate the position from the N-terminal side of the amino acid sequence in the corresponding subunit.

[0175] [Table 2-1]

[0176]

[0177] [Table 2-2]

[0178]

[0179] Examples of combinations of substitutions of four amino acid residues selected from the group consisting of the aforementioned (a) to the aforementioned (q), the group consisting of the aforementioned (A1) to (A6), and the group consisting of the aforementioned (B1) to (B9) are shown below, but the present disclosure is not limited thereto. It should be noted that in the following table, the numbers in the column of the mutation site indicate the position from the N-terminal side of the amino acid sequence in the corresponding subunit.

[0180] [Table 3]

[0181]

[0182] Examples of combinations of substitutions of five amino acid residues selected from the group consisting of (a) to (q), (A1) to (A6), and (B1) to (B9) are shown below, but the present disclosure is not limited thereto. In the following tables, the numbers in the columns of mutation sites indicate the positions from the N-terminal side of the amino acid sequence in the corresponding subunit.

[0183] [Table 4]

[0184]

[0185] [Nitrile Hydratase]

[0186] The mutant nitrile hydratase of the present disclosure may be derived from any source as long as it comprises an α subunit having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1 and a β subunit having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 2, and contains a substitution of at least one amino acid residue selected from the group consisting of (a) to (q) described above.

[0187] The source of the nitrile hydratase may be, for example, a nitrile hydratase derived from a wild-type Pseudonocardia thermophila or a modified nitrile hydratase derived from a wild-type Pseudonocardia thermophila.

[0188] (Modified Nitrile Hydratase)

[0189] The modified nitrile hydratase also includes a modified nitrile hydratase obtained by subjecting a wild-type nitrile hydratase derived from Pseudonocardia thermophila to one or more modifications selected from the group consisting of the following (i) to (v):

[0190] (i) substitution of one or more amino acid residues with other amino acid residues;

[0191] (ii) deletion of one or more amino acid residues other than the substitutions of the amino acid residues in (a) to (q) described above;

[0192] (iii) insertion of amino acid residues;

[0193] (iv) adding amino acid residues to one or both of the N-terminus and the C-terminus of the amino acid sequence of the α subunit;

[0194] (v) adding amino acid residues to one or both of the N-terminus and the C-terminus of the amino acid sequence of the β subunit.

[0195] 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.

[0196] 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.

[0197] In the α-subunit of the modified nitrile hydratase, the number of deleted amino acid residues is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, or 1. The number of deleted amino acid residues may be zero.

[0198] In the modified β-subunit of nitrile hydratase, the number of deleted amino acid residues is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, or 1. The number of deleted amino acid residues may be zero.

[0199] In the α-subunit of the modified nitrile hydratase, the number of amino acid residues inserted is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, or 1. The number of amino acid residues inserted may be 0.

[0200] In the modified β-subunit of nitrile hydratase, the number of amino acid residues inserted is, for example, 1 to 10, or 1 to 7, or 1 to 4, or 1 to 2, or 1. The number of amino acid residues inserted may be 0.

[0201] 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 the case of terminal additions, the total number of substituted, deleted or inserted amino acid residues may be 0.

[0202] In the modified β-subunit of 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 the case of terminal additions, the total number of substituted, deleted or inserted amino acid residues may be 0.

[0203] In the α-subunit of the modified nitrile hydratase, the number of amino acid residues added to each terminal is, for example, 1 to 60, 1 to 40, 1 to 20, 1 to 10, 1 to 5, 1 to 3, or 1. The number of added amino acid residues may be 0.

[0204] In the modified nitrile hydratase β subunit, the number of amino acid residues added to the terminal is, for example, 1 to 60, or 1 to 40, or 1 to 20, or 1 to 10, or 1 to 5, or 1 to 3, or 1 per terminal. The number of added amino acid residues may also be 0.

[0205] The terminal additional amino acid residue may also be, for example, a secretion signal sequence. The terminal additional amino acid residue may be present only at the N-terminus, or only at the C-terminus, or at both the N-terminus and the C-terminus.

[0206] The similarity of the modified nitrile hydratase to the wild-type nitrile hydratase from Pseudonocardia thermophila can also be expressed by sequence identity.

[0207] The amino acid sequence of the α-subunit of the modified nitrile hydratase has, for example, 90% or more sequence identity, or 95% or more sequence identity, or 96% or more sequence identity, or 97% or more sequence identity, or 98% or more sequence identity, or 99% or more sequence identity with the amino acid sequence of the α-subunit of the nitrile hydratase derived from the wild-type thermophilic Pseudonocardia shown in SEQ ID NO: 1.

[0208] The amino acid sequence of the β subunit of the modified nitrile hydratase has, for example, 90% or more sequence identity, or 95% or more sequence identity, or 96% or more sequence identity, or 97% or more sequence identity, or 98% or more sequence identity, or 99% or more sequence identity with the amino acid sequence of the β subunit of the nitrile hydratase derived from the wild-type thermophilic Pseudonocardia shown in SEQ ID NO: 2.

[0209] Alignment between sequences can be performed using ClustalW (1.83) and initial parameters (including gap opening penalty: 10, gap extension penalty: 0.05). It should be noted that the sequence identity was calculated based on the full length of nitrile hydratase.

[0210] When the amino acid sequence of the modified nitrile hydratase is aligned with the amino acid sequence of the wild-type nitrile hydratase derived from Pseudonocardia thermophila, depending on the modification mode of the modified nitrile hydratase, there are cases where the distance from the N-terminus of the subunit specified as above is different even for the corresponding amino acid residues in the alignment.

[0211] In this disclosure, in such a case, the amino acid residue at position X from the N-terminus of the subunit of the modified nitrile hydratase refers to the amino acid residue corresponding to the amino acid residue at position X from the N-terminus of the subunit of the wild-type Pseudonocardia thermophila nitrile hydratase.

[0212] For example, the so-called "amino acid residue at position 23 from the N-terminus of the α subunit", "Ala residue at position 23 from the N-terminus of the α subunit" or "amino acid residue at position 23 from the N-terminus of the α subunit, i.e., Ala residue" in the present disclosure may be located at a position other than position 23 from the N-terminus of the α subunit in the amino acid sequence of the modified nitrile hydratase.

[0213] For example, as a result of the insertion of the amino acid residue, the amino acid residue at the 23rd position from the N-terminus of the α subunit of the modified nitrile hydratase (which may not necessarily be Ala) may correspond to the amino acid residue at the 24th position from the N-terminus of the α subunit of the nitrile hydratase derived from the wild-type Pseudonocardia thermophila, i.e., the Ala residue. In such a case, the "amino acid residue at the 23rd position from the N-terminus of the α subunit", "the Ala residue at the 23rd position from the N-terminus of the α subunit" or "the amino acid residue at the 23rd position from the N-terminus of the α subunit, i.e., the Ala residue" in the description in the present disclosure refers to the amino acid residue at the 24th position from the N-terminus of the α subunit in the modified nitrile hydratase.

[0214] As 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 sequence similar thereto.

[0215] The method for producing the mutant nitrile hydratase of the present disclosure is not particularly limited, and 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 can be prepared, and the mutant nitrile hydratase can be produced using the vector.

[0216] <Nucleic Acid>

[0217] The nucleic acid of the present disclosure encodes the amino acid sequence of the mutant nitrile hydratase of the present disclosure. More specifically, the nucleic acid of the present disclosure has a nucleotide sequence encoding the amino acid sequence of the mutant nitrile hydratase of the present disclosure.

[0218] Examples of methods for synthesizing a nucleotide sequence encoding the amino acid sequence of the mutant nitrile hydratase of the present disclosure include a method of introducing a mutation site into a nucleotide sequence encoding a corresponding wild-type nitrile hydratase derived from Pseudonocardia thermophila, and a method of chemically synthesizing a full-length nucleotide sequence containing the mutation site.

[0219] Examples of methods for mutating a gene using a nucleotide sequence encoding a nitrile hydratase derived from a wild-type Pseudonocardia thermophila as a template include site-specific mutagenesis (Kramer, W. and Frita, HJ, Methods in Enzymology, vol. 154, p. 350 (1987)), recombinant PCR (PCR Technology, Stockton Press (1989)), a method for chemically synthesizing a nucleic acid of a specific portion, a method for treating a gene with hydroxylamine, a method for irradiating a strain harboring the gene with ultraviolet light, a method for treating the gene with chemical agents such as nitrosoguanidine and nitrous acid, and a method for using a commercially available mutation introduction kit.

[0220] The nucleic acid of the present disclosure may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0221] The gene encoding the nitrile hydratase derived from the wild-type Pseudonocardia thermophila comprises the nucleotide sequence shown in SEQ ID NO:3 and the nucleotide sequence shown in SEQ ID NO:4.

[0222] The nucleotide sequence shown in sequence number 3 corresponds to the amino acid sequence consisting of sequence number 1, and the nucleotide sequence shown in sequence number 4 corresponds to the amino acid sequence consisting of sequence number 2.

[0223] The nucleic acid encoding the mutant nitrile hydratase has a nucleotide substitution corresponding to at least one amino acid residue substitution selected from the group of amino acid residue substitutions (a) to (q) at least at the amino acid residue substitution position (a) to (q) in the nucleotide sequence shown in SEQ ID NO: 3 or the nucleotide sequence shown in SEQ ID NO: 4.

[0224] <Carrier>

[0225] The vector of the present disclosure contains the nucleic acid of the present disclosure.

[0226] Examples of the vector of the present disclosure include vectors obtained by introducing the mutant nitrile hydratase of the present disclosure into known vectors. The vector of the present disclosure may be a phage vector or a plasmid vector.

[0227] <Expression vector>

[0228] The vector of the present disclosure is preferably an expression vector.

[0229] The expression vector may be an expression vector containing a nucleic acid represented by a nucleotide sequence encoding the amino acid sequence of the mutant nitrile hydratase of the present disclosure. By transforming any host cell with the expression vector of the present disclosure to obtain a transformant or a cell line, and then culturing the transformant or cell line, the mutant nitrile hydratase of the present disclosure can be produced.

[0230] The expression vector may contain, as necessary, a nucleotide sequence constituting other regions (hereinafter also referred to as "other regions") in addition to the nucleotide sequence encoding the mutant nitrile hydratase of the present disclosure.

[0231] Examples of the other regions include a regulatory region necessary for the transformant to produce the mutant nitrile hydratase and a region required for autonomous replication.

[0232] Examples of the regulatory region necessary for producing the mutant nitrile hydratase include a promoter sequence (including an operator sequence that regulates transcription), a ribosome binding sequence (SD sequence), and a transcription termination sequence.

[0233] Examples of promoter sequences include the trp promoter from the tryptophan operon of Escherichia coli, the lac promoter from the lactose operon, the PL promoter and the PR promoter from lambda phage, etc. In addition, artificially designed and modified sequences such as the tac promoter and the trc promoter can also be used.

[0234] The sequence order of the aforementioned regulatory region on the expression vector is not particularly limited. For example, it is desirable that the promoter sequence and the ribosome binding sequence are located upstream of the 5' end of the gene encoding the mutant nitrile hydratase of the present disclosure. In addition, it is desirable that the transcription termination sequence is located downstream of the 3' end of the gene encoding the mutant nitrile hydratase of the present disclosure. In addition, the α subunit gene and the β subunit gene of the mutant nitrile hydratase can be expressed in the form of independent cistrons using the regulatory region, or can be expressed in the form of multiple cistrons using a common regulatory region.

[0235] From the viewpoint of facilitating the screening of the transformants, the expression vector may further contain a nucleotide sequence encoding a selection gene that can serve as a selection marker.

[0236] The expression vector can express a gene encoding a protein involved in the activation of nitrile hydratase. The aforementioned protein involved in the activation of nitrile hydratase refers to a protein having a property of directly affecting the activation of nitrile hydratase whether the protein is expressed, and as a representative example, a protein involved in the activation of nitrile hydratase derived from thermophilic Pseudonocardia (nitrile hydratase activation protein) recorded in Japanese Patent Publication No. 11-253168 can be cited. It should be noted that the sequence of the aforementioned nitrile hydratase activation protein is shown in sequence number 36.

[0237] Regarding the method of transforming the expression vector into the desired host cell and the method of producing nitrile hydratase in the aforementioned transformant, conventional methods and host cells known in the fields of molecular biology, bioengineering, and genetic engineering, such as those described in "Molecular Cloning 3rd Edition" (J. Sambrook et al.; Cold Spring Harbor Laboratory Press, 2001), can be used.

[0238] <Transformants>

[0239] The transformant of the present disclosure contains the expression vector of the present disclosure.

[0240] According to the transformant of the present disclosure, a mutant nitrile hydratase can be produced in which the inhibition of the amide compound production reaction using the nitrile hydratase caused by the impurities is reduced even in the presence of the impurities.

[0241] The transformant of the present disclosure can be prepared by known methods. For example, a method of constructing an expression vector containing a nucleotide sequence encoding a mutant nitrile hydratase and the above-mentioned other regions (as needed) and transforming the expression vector into a desired host cell can be cited. Specifically, conventional methods known in the fields of molecular biology, bioengineering and genetic engineering described in Sambrook, J. et al., "Molecular Cloning A Laboratory Manual, 3rd Edition", Cold Spring Harbor Laboratory Press, (2001) can be used.

[0242] In addition to incorporating the expression vector into the host cell, the transformant can also be prepared by, if necessary, carrying out a step of introducing a silent mutation (changing a codon with a low usage frequency in the host cell to a codon with a high usage frequency). This can increase the production of the protein derived from the mutant nitrile hydratase after incorporation of the expression vector.

[0243] Regarding the introduction of silent mutations, as long as the codons of the nitrile hydratase gene located on the expression vector and the signal sequence for secreting the nitrile hydratase gene to the outside of the cell are adjusted according to the codon usage frequency in the host cell, the method for introducing silent mutations, the mutation site, the type of nucleotide to be changed, etc. are not particularly limited.

[0244] <Method for producing mutant nitrile hydratase>

[0245] The method for producing the mutant nitrile hydratase of the present disclosure may be a method for producing the mutant nitrile hydratase comprising the steps of: culturing the transformant of the present disclosure described above in a culture medium; and recovering the mutant nitrile hydratase of the present disclosure from at least one of the cultured transformant and the culture medium.

[0246] (Cultivation Method of Transformant)

[0247] The culture conditions for the transformants transformed with the expression vector are the same as those for the host cells before transformation, and known conditions can be used.

[0248] Examples of the host cell include prokaryotes (Escherichia coli, Bacillus subtilis, actinomycetes, etc.), yeast, and filamentous fungi.

[0249] As the culture medium, either a synthetic medium or a natural medium can be used as long as it contains appropriate amounts of a carbon source, a nitrogen source, inorganic substances and other nutrients.

[0250] As the medium components, known components used in the medium can be used. For example, organic nutrient sources 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 nutrient sources such as phosphate, magnesium, potassium and iron, and vitamins can be used in appropriate combination.

[0251] It should be noted that in the culture of transformants obtained by transformation using an expression vector containing a selection marker, for example, when the selection marker has drug resistance, a culture medium containing the corresponding drug is used, and when the selection marker is auxotrophic, a culture medium that does not contain the corresponding nutrient can be used.

[0252] The pH of the culture medium can be appropriately selected within the range of pH 4 to pH 8.

[0253] The transformant can be cultured using a liquid medium containing the transformant, a shaking culture, aeration stirring culture, continuous culture, fed-batch culture or other common culture methods.

[0254] The culture conditions may be appropriately selected depending on the types of the transformant, the medium, and the culture method, and are not particularly limited as long as the transformant can grow and produce the mutant nitrile hydratase.

[0255] The culture temperature is preferably 20°C to 45°C, more preferably 24°C to 37°C, and aerobic culture is performed.

[0256] The culture time may be 1 to 7 days until the content of the protein having the target mutant nitrile hydratase activity reaches a maximum.

[0257] For example, when the host cell is Escherichia coli, LB medium, M9 medium, etc. are generally used as a medium for culturing the transformant, and a medium containing 0.1 μg / mL or more of Fe ions and Co ions as components is more preferably used. After the transformant is inoculated into the medium, it can be grown at an appropriate culture temperature (generally 20° C. to 50° C.).

[0258] (Mutated nitrile hydratase recovery step)

[0259] 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.

[0260] The method for recovering the mutant nitrile hydratase after culturing the transformed transformant can be a method commonly used in the art.

[0261] When the mutant nitrile hydratase is secreted outside the transformant, a crude enzyme solution containing the mutant nitrile hydratase can be easily obtained by centrifuging or filtering the culture of the transformant.

[0262] When the mutant nitrile hydratase accumulates in the transformant, the transformant after culture is recovered by centrifugation or the like, and then the recovered transformant is suspended in a buffer solution, and the suspension is treated by a known method such as lysozyme treatment, freeze-thaw, ultrasonic disruption, etc. to destroy the cell membrane of the transformant, thereby recovering a crude enzyme solution containing the mutant nitrile hydratase.

[0263] The recovered crude enzyme solution containing mutant nitrile hydratase may be filtered by adding a cationic polymer and a filter aid. The filtered enzyme solution containing mutant nitrile hydratase may be further concentrated by ultrafiltration or the like, and a preservative or the like may be added to use it as a concentrated mutant nitrile hydratase (concentrated enzyme). In addition, after the mutant nitrile hydratase is concentrated, a powder of the mutant nitrile hydratase (powdered enzyme) may be prepared by spray drying or the like.

[0264] When separation and purification are required for the recovered crude enzyme solution containing the mutant nitrile hydratase, for example, salting out using ammonium sulfate or the like, precipitation using an organic solvent such as alcohol, membrane separation using dialysis and ultrafiltration, or known chromatographic separation methods such as ion exchange chromatography, reverse phase high performance chromatography, affinity chromatography, gel filtration chromatography, etc. can be appropriately combined to carry out separation and purification.

[0265] <Method for producing amide compound>

[0266] The method for producing an amide compound of the present disclosure includes a step of contacting the mutant nitrile hydratase of the present disclosure with a nitrile compound. The mutant nitrile hydratase of the present disclosure catalyzes a reaction of synthesizing 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, the hindrance to the amide compound production reaction using the nitrile hydratase caused by impurities is reduced.

[0267] First, a transformant or cell line producing the mutant nitrile hydratase of the present disclosure is cultured, and the obtained culture solution, cells or cell treated product is brought into contact with a nitrile compound in a medium. Thus, the mutant nitrile hydratase of the present disclosure comes into contact with the nitrile compound, and the mutant nitrile hydratase of the present disclosure converts the nitrile compound into the corresponding amide compound. The cell treated product here refers to an extract or crushed product from the transformant, a crude enzyme preparation obtained by separating the nitrile hydratase active fraction of these extracts or crushed products, or a post-separated product such as an enzyme purified product obtained by further purification, or an immobilized product obtained by immobilizing the transformant, the extract, crushed product or post-separated product of the transformant using appropriate means.

[0268] The above-mentioned contact temperature is preferably within the temperature range in which the mutant nitrile hydratase of the present disclosure is not inactivated, more preferably 0°C to 60°C, and more preferably 15°C to 40°C. For example, the culture solution obtained by culturing the transformant or cell line producing the mutant nitrile hydratase of the present disclosure can be directly added to the aqueous solution containing the nitrile compound, or the culture solution can be centrifuged to separate the bacterial cells, and the bacterial cells can be added to the aqueous solution containing the 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 can 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 on the culturing of the transformant are described in the section on the method for producing the mutant nitrile hydratase.

[0269] Specific examples of the nitrile compound are not particularly limited as long as they are compounds that can act as substrates for the mutant nitrile hydratase of the present disclosure, and examples thereof include aliphatic saturated nitriles such as acetonitrile, propionitrile, succinonitrile, and adiponitrile, aliphatic unsaturated nitriles such as acrylonitrile and methacrylonitrile, aromatic nitriles such as benzonitrile and phthalonitrile, and heterocyclic nitriles such as 3-cyanopyridine and 2-cyanopyridine.

[0270] In the method of the present disclosure, since the nitrile group in the nitrile compound is converted into an amide group by hydration, for example, acrylonitrile can be converted into acrylamide.

[0271] Next, activated carbon is added to the medium without inactivating the mutant nitrile hydratase of the present disclosure, thereby purifying the generated amide compound without completing the reaction of synthesizing an amide compound from a nitrile compound using the mutant nitrile hydratase of the present disclosure as a catalyst.

[0272] The reaction of producing an amide compound from a nitrile compound by contacting with the mutant nitrile hydratase of the present disclosure can be carried out under neutral to alkaline conditions such as pH 7 to 9, depending on the optimal pH of the nitrile hydratase. The pH can be adjusted by using an alkaline substance such as ammonia or sodium hydroxide in a buffer as needed.

[0273] Example

[0274] The embodiments are described in more detail by the following examples, but the present invention is not limited to the following examples. It should be noted that the "mutation site" in the following examples refers to the position of the difference in amino acid sequence from the wild-type Pseudonocardia thermophila nitrile hydratase comprising an α subunit having the amino acid sequence shown in SEQ ID NO: 1 and a β subunit having the amino acid sequence shown in SEQ ID NO: 2.

[0275] (Obtaining of nitrile hydratase (C1) from wild-type Pseudonocardia thermophila for comparison)

[0276] Prepare 10 mL of LB liquid culture medium in a 30 mL test tube, and sterilize the liquid culture medium by autoclave (121°C, 20 minutes). Add ampicillin to the liquid culture medium to a final concentration of 100 μg / mL, then inoculate a platinum loop of the cell line MT-10822 described in Patent Document 1 and Example 3, and culture at 37°C and 300 rpm for about 20 hours. Then, take 1 mL of the obtained culture solution into a suitable centrifuge tube, and separate the bacterial cells by centrifugation (15000 rpm×5 minutes).

[0277] Next, plasmid pPT-DB1 was prepared from the isolated bacterial cells by alkaline SDS extraction. The prepared plasmid was transformed into competent cells of Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) to obtain a transformant (C1). The transformant (C1) produced nitrile hydratase (C1) which is a nitrile hydratase derived from wild-type Pseudonocardia thermophila.

[0278] (Obtaining a mutant nitrile hydratase (C2) for comparison)

[0279] According to the method described in Comparative Example 2 of International Publication No. 2010 / 055666, site-specific mutagenesis was performed using the "LA PCR in vitro mutagenesis Kit" (hereinafter referred to as a mutagenesis kit) manufactured by Takara Shuzo Co., Ltd. Specifically, the plasmid pPT-DB1 expressing the nitrile hydratase derived from the wild-type thermophilic Pseudonocardia was used as a template to obtain a transformant expressing a mutant nitrile hydratase obtained by replacing the 92nd amino acid residue, i.e., Asp, from the N-terminus of the α subunit of the nitrile hydratase derived from the wild-type thermophilic Pseudonocardia with Glu. The transformant produced a mutant nitrile hydratase (C2) for comparison. Hereinafter, a primer containing a mutant sequence to be introduced such as the sequence of sequence number 5 is also referred to as a primer for mutation in the following text.

[0280] (Obtaining the mutant nitrile hydratase (1) of the present disclosure)

[0281] In order to obtain a transformant expressing a mutant nitrile hydratase in which the amino acid residue Ala at position 23 from the N-terminus of sequence number 1 in the α subunit is substituted with Gly, mutagenesis was performed using the "LA PCR in vitro mutagenesis Kit" (hereinafter referred to as the mutagenesis kit) manufactured by Takara Shuzo Co., Ltd. A PCR reaction was performed using the plasmid pPT-DB1 expressing the nitrile hydratase derived from the wild-type Pseudonocardia thermophila as a template.

[0282] PCR reaction No. 1 used a system with a total amount of 50 μL of 50 pmol of mutation primers containing the sequence numbers shown in Table 5 and M13 primer M4 (sequence recorded in sequence number 33) (the composition varies depending on the conditions described in the mutagenesis kit), and was performed by repeating 25 cycles of thermal denaturation (98°C) for 15 seconds, annealing (55°C) for 30 seconds, and extension reaction (72°C) for 120 seconds.

[0283] PCR reaction No. 2 was performed using a 50 μL system containing 50 pmol of each of MUT4 primer (sequence recorded in sequence number 34) and M13 primer RV (sequence recorded in sequence number 35) (the composition varies depending on the conditions described in the mutagenesis kit) using the same operations as PCR reaction No. 1.

[0284] The DNA amplification products were analyzed by agarose electrophoresis (agarose concentration: 1.0 wt %) using 5 μL of each of the reaction completion solutions of PCR reactions No. 1 and No. 2. As a result, the presence of amplified DNA products was confirmed.

[0285] Excess primers and dNTPs were removed from each PCR reaction solution using Microcon100 (manufactured by Takara Shuzo Co., Ltd.), and then TE was added to prepare 50 μL of each solution. A total of 47.5 μL of annealing solution (the composition varies depending on the conditions described in the mutagenesis kit) containing 0.5 μL of each TE solution was prepared, and a heat denaturation treatment (98° C.) was performed for 10 minutes, followed by cooling to 37° C. at a constant speed over 60 minutes, and then maintaining at 37° C. for 15 minutes to perform annealing treatment.

[0286] 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 double-stranded hybridization.

[0287] 50 pmol of each of the M13 primer M4 and the M13 primer RV were added thereto to make a total of 50 μL. Then, PCR reaction No. 3 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.

[0288] The DNA amplification product was analyzed by agarose electrophoresis (using type VII low melting point agarose manufactured by Sigma; agarose concentration: 0.8 wt %) using 5 μL of the reaction completion solution of PCR reaction No. 3. The presence of an amplified DNA product of about 2 kb was confirmed.

[0289] Next, only a DNA fragment of about 2 Kb was cut out from the agarose gel, and the agarose piece (about 0.1 g) was finely pulverized and suspended in 1 mL of TE solution, and then kept at 55°C for 1 hour to completely melt the agarose.

[0290] The melt was subjected to phenol / chloroform extraction and ethanol precipitation to purify the DNA fragment, and finally dissolved in 10 μL of TE.

[0291] The purified amplified DNA fragment of about 2 kb was cleaved with restriction enzymes EcoRI and HindIII, and the restriction enzyme-treated solution was subjected to phenol / chloroform extraction and ethanol precipitation to purify the DNA fragment, which was finally dissolved in 10 μL of TE.

[0292] Similarly, plasmid pPT-DB1 expressing nitrile hydratase was cleaved with EcoRI and HindIII, subjected to agarose gel electrophoresis (using type VII low melting point agarose manufactured by Sigma; agarose concentration: 0.7%), and only a DNA fragment of about 2.7 Kb was excised from the agarose gel.

[0293] The agarose pieces (about 0.1 g) were finely pulverized and suspended in 1 mL of TE solution, and then kept at 55°C for 1 hour to completely melt the agarose.

[0294] The melt was subjected to phenol / chloroform extraction and ethanol precipitation to purify the DNA fragment, and finally dissolved in 10 μL of TE.

[0295] The DNA fragments of about 2 kb and about 2.7 kb thus obtained were ligated using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.). Then, competent cells of Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) were transformed to obtain a transformant (1). In addition, a plasmid was prepared from the above-mentioned bacteria using an alkaline SDS extraction method, and the nucleotide sequence of the nitrile hydratase gene portion was determined using a DNA sequencer. It was confirmed that the plasmid pPT-DB1 had a mutation in which the amino acid residue at the 23rd position from the N-terminus of the α subunit, i.e., Ala, was changed to Gly. The transformant (1) produced a mutant nitrile hydratase (1) having the mutation described in Table 5.

[0296] (Obtaining the mutant nitrile hydratases (2) to (17) of the present disclosure)

[0297] In order to obtain transformants (2) to (17) expressing mutant nitrile hydratases having amino acid residue substitutions as shown in Table 5, plasmids were obtained in the same manner as for transformant (1), except that primers of SEQ ID NOs: 6 to 21 were used instead of the primer of SEQ ID NO: 5. Transformants (2) to (17) produced nitrile hydratases (2) to (17) having the mutations described in Table 5.

[0298] (Obtaining the mutant nitrile hydratase (18) to (28) of the present disclosure)

[0299] In order to obtain a transformant (18) expressing a mutant nitrile hydratase having an amino acid residue substitution as shown in Table 6, a plasmid was obtained from the transformant (9) instead of pPT-DB1 as a template and a mutagenesis primer of SEQ ID NO: 18 was used as a mutagenesis primer. The same operation as for the transformant (1) was performed to obtain a plasmid to obtain a transformant (18). The transformant (18) produced a nitrile hydratase (18) having the mutations described in Table 6.

[0300] Similarly, nitrile hydratase (19) was obtained from the plasmid obtained from transformant (9) and the mutation primer of sequence number 15; nitrile hydratase (20) was obtained from the plasmid obtained from transformant (13) and the mutation primer of sequence number 11; nitrile hydratase (21) was obtained from the plasmid obtained from transformant (10) and the mutation primer of sequence number 18; nitrile hydratase (22) was obtained from the plasmid obtained from transformant (9) and the mutation primer of sequence number 11; nitrile hydratase (23) was obtained from the plasmid obtained from transformant (16) and the mutation primer of sequence number 21. 23); nitrile hydratase (24) was obtained from the plasmid obtained from transformant (1) and the mutation primer of sequence number 7; nitrile hydratase (25) was obtained from the plasmid obtained from transformant (6) and the mutation primer of sequence number 19; nitrile hydratase (26) was obtained from the plasmid obtained from transformant (17) and the mutation primer of sequence number 22; nitrile hydratase (27) was obtained from the plasmid obtained from transformant (5) and the mutation primer of sequence number 23; nitrile hydratase (28) was obtained from the plasmid obtained from transformant (7) and the mutation primer of sequence number 24.

[0301] (Obtaining of mutant nitrile hydratase of the present disclosure (29) to (37))

[0302] In order to obtain a transformant (29) expressing a mutant nitrile hydratase having an amino acid residue substitution as shown in Table 7, a plasmid was obtained from the transformant (18) in place of pPT-DB1 as a template and a mutagenesis primer of SEQ ID NO: 11 was used as a mutagenesis primer. The transformant (29) was obtained in the same manner as the transformant (1), thereby obtaining a plasmid. The transformant (29) produced a nitrile hydratase (29) having the mutations described in Table 7.

[0303] Similarly, nitrile hydratase (30) was obtained from a plasmid obtained from transformant (21) and a primer for mutation of sequence number 13; nitrile hydratase (31) was obtained from a plasmid obtained from transformant (20) and a primer for mutation of sequence number 6; nitrile hydratase (32) was obtained from a plasmid obtained from transformant (20) and a primer for mutation of sequence number 16; nitrile hydratase (33) was obtained from a plasmid obtained from transformant (20) and a primer for mutation of sequence number 18; a transformant was obtained from a plasmid obtained from transformant (9) and a primer for mutation of sequence number 16, and a transformant was obtained from a plasmid obtained from the transformant. A nitrile hydratase (34) was obtained from a plasmid obtained from a transformant (23) and a primer for mutation of sequence number 17; a nitrile hydratase (35) was obtained from a plasmid obtained from a transformant (23) and a primer for mutation of sequence number 11; a transformant was obtained from a plasmid obtained from a transformant (15) and a primer for mutation of sequence number 25, and a nitrile hydratase (36) was obtained from a plasmid obtained from the transformant and a primer for mutation of sequence number 26; a transformant was obtained from a plasmid obtained from a transformant (8) and a primer for mutation of sequence number 27, and a nitrile hydratase (37) was obtained from a plasmid obtained from the transformant and a primer for mutation of sequence number 28.

[0304] (Obtaining the mutant nitrile hydratase of the present disclosure (38) to (46))

[0305] In order to obtain a transformant (38) expressing a mutant nitrile hydratase having an amino acid residue substitution as shown in Table 8, a plasmid was obtained from the transformant (30) in the same manner as the transformant (1), except that pPT-DB1 was used as a template and the primer for mutation of SEQ ID NO: 11 was used as a primer for mutation. The transformant (38) produced a nitrile hydratase (38) having the mutations described in Table 8.

[0306] Similarly, the following nitrile hydratases were obtained: nitrile hydratase (39) was obtained from a plasmid obtained from transformant (29) and a primer for mutation of sequence number 17; nitrile hydratase (40) was obtained from a plasmid obtained from transformant (34) and a primer for mutation of sequence number 11; nitrile hydratase (41) was obtained from a plasmid obtained from transformant (35) and a primer for mutation of sequence number 19; a transformant was obtained from a plasmid obtained from transformant (8) and a primer for mutation of sequence number 22, and nitrile hydratase (42) was obtained using a plasmid obtained from a transformant obtained from the transformant and a primer for mutation of sequence number 27, and a primer for mutation of sequence number 32; a plasmid obtained from a transformant (40) and a primer for mutation of sequence number 18 was obtained. nitrile hydratase (43) is obtained from the transformant (40); nitrile hydratase (44) is obtained from the plasmid obtained from the transformant (35) and the mutation primer of sequence number 27; nitrile hydratase (45) is obtained from the plasmid obtained from the transformant and the mutation primer of sequence number 28; a transformant is obtained from the plasmid obtained from the transformant (1) and the mutation primer of sequence number 11, a transformant is obtained from the plasmid obtained from the aforementioned transformant and the mutation primer of sequence number 29, a transformant is obtained using the plasmid obtained from the aforementioned transformant and the mutation primer of sequence number 30, and nitrile hydratase (46) is obtained using the plasmid obtained from the aforementioned transformant and the mutation primer of sequence number 31.

[0307] In Tables 5 to 10, the item "Template" shows the number of the transformant from which the plasmid was obtained, the item "Primer for mutation" shows the sequence number of the primer used as the primer for mutation, the item "Amino acid residue before mutation" shows the amino acid residue before mutation at the mutation position recorded in the item "Mutation site" (that is, the amino acid residue in the nitrile hydratase (C1) derived from the wild-type Pseudonocardia thermophila), and the item "Amino acid residue after mutation" shows the amino acid residue after mutation at the mutation position recorded in the item "Mutation site".

[0308] [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]

[0309] <Evaluation of the degree of inhibition of reaction inhibition caused by impurities>

[0310] Using the transformant producing the nitrile hydratase of each number, a reaction for producing an amide compound in the presence of impurities was carried out as shown below.

[0311] Prepare 5 mL of LB liquid culture medium containing 40 μg / mL of iron sulfate heptahydrate and 10 μg / mL of cobalt chloride dihydrate in a test tube and sterilize it by autoclave (121°C, 20 minutes). Add ampicillin to the culture medium to a final concentration of 100 μg / mL. Then, inoculate each transformant in the amount of one platinum loop and culture each at 37°C and 200 rpm for about 20 hours. Take 1000 μl of the culture medium after the culture is completed, centrifuge and recover the bacteria. Suspend the bacteria in 780 μL of 50 mM Tris hydrochloric acid aqueous solution (the Tris hydrochloric acid aqueous solution contains 1000 mass ppm of acrolein) (pH 8.0). Add 20 μL of acrylonitrile to the suspension and react it for 60 minutes while slowly stirring at the temperature shown in Tables 5 to 10.

[0312] After completion of the reaction, quantitative analysis of the amount of the amide compound (acrylamide) contained in the reaction solution was performed using HPLC under the following analysis conditions.

[0313] The "amount of amide compounds obtained by using the nitrile hydratase of each number in the presence of impurities (X)" is compared with the "amount of amide compounds in Comparative Example 1 obtained by using the wild-type thermophilic Pseudonocardia nitrile hydratase (C1) in the presence of impurities (Y)", and the relative magnitude of the enzyme activity of the mutant nitrile hydratase of each example in generating amide compounds from nitrile compounds is obtained, and this is taken as the inhibition degree of reaction inhibition caused by impurities (X / Y). For example, in Comparative Example 1, the results of the same Comparative Example 1 are compared, so the value of the inhibition degree of reaction inhibition caused by impurities is 1.000.

[0314] In Reference Example 1, "the amount of the amide compound obtained by using nitrile hydratase (1) in the absence of impurities" was compared with "the amount of the amide compound in Example 1-1 obtained by using nitrile hydratase (1) in the presence of impurities".

[0315] In Reference Example 2, "the amount of amide compound obtained by using nitrile hydratase (C1) in the absence of impurities" was compared with "the amount of amide compound in Comparative Example 1 obtained by using nitrile hydratase (C1) in the presence of impurities".

[0316] In Reference Example 3, "the amount of amide compound obtained by using nitrile hydratase (C2) in the absence of impurities" was compared with "the amount of amide compound in Comparative Example 2-1 obtained by using nitrile hydratase (C2) in the presence of impurities".

[0317] The results are shown in the tables. The reaction and analysis were performed three or more times for each nitrile hydratase, and data deviations due to the injection operation and the like were corrected.

[0318] 〈Analysis conditions〉

[0319] Analytical instrument: HPLC manufactured by JASCO Corporation

[0320] Column: YMC Pack ODS-A (150×6.00mm)

[0321] Analysis temperature: 40℃

[0322] Mobile phase: 3% acetonitrile, 10 mM phosphoric acid

[0323] [Table 5]

[0324]

[0325] [Table 6]

[0326]

[0327] [Table 7]

[0328]

[0329] [Table 8]

[0330]

[0331] [Table 9]

[0332]

[0333] [Table 10]

[0334]

[0335] As shown in Table 5, for the wild-type nitrile hydratase (C1), when the comparative example 1 in which the reaction was carried out in the presence of impurities was compared with the reference example 2 in which the reaction was carried out without adding impurities, the enzyme activity was reduced compared with the case where no impurities were added, and it was found that the reaction was inhibited by the impurities. In addition, for the mutant nitrile hydratase (C2) used for the comparative example, similarly to the above (C1), when the comparative example 2-1 in which the reaction was carried out in the presence of impurities was compared with the reference example 3 in which the reaction was carried out without adding impurities, the enzyme activity was reduced compared with the case where no impurities were added, and it was found that the reaction was inhibited by the impurities.

[0336] In contrast, the mutant nitrile hydratase of the present disclosure has a smaller difference in enzyme activity between the wild-type nitrile hydratase (C1) and the mutant nitrile hydratase (C2) used in the comparative example when the reaction is carried out with the addition of impurities and the reaction is carried out without the addition of impurities. More specifically, the difference between the value of "the degree of inhibition of reaction hindrance caused by impurities" in Example 1-1 and the value of "the degree of inhibition of reaction hindrance caused by impurities" in Reference Example 1 (2.013-1.125=0.888) is smaller than the difference between the value of "the degree of inhibition of reaction hindrance caused by impurities" in Comparative Example 1 and the value of "the degree of inhibition of reaction hindrance caused by impurities" in Reference Example 2 (1.994-1.000=0.994). In addition, the difference between the value of "the inhibition degree of reaction inhibition caused by impurities" in Example 1-1 and the value of "the inhibition degree of reaction inhibition caused by impurities" in Reference Example 1 (2.013-1.125=0.888) is smaller than the difference between the value of "the inhibition degree of reaction inhibition caused by impurities" in Comparative Example 2-1 and the value of "the inhibition degree of reaction inhibition caused by impurities" in Reference Example 3 (2.263-0.960=1.303). That is, for the mutant nitrile hydratase of the present disclosure, the inhibition of the amide compound production reaction using the nitrile hydratase caused by impurities is reduced even in the presence of impurities.

[0337] As shown in Tables 5 to 10, it can be seen that the mutant nitrile hydratase of each example obtained by replacing the amino acid residue belonging to the group consisting of (a) to (q) with the wild-type Pseudonocardia thermophila nitrile hydratase is less inhibited by impurities in the amide compound production reaction using the nitrile hydratase in the presence of impurities than the nitrile hydratase derived from the wild-type Pseudonocardia thermophila of Comparative Example 1 and the modified nitrile hydratase of Comparative Example 2-1.

[0338] As shown in Table 9, it can be seen that for the mutant nitrile hydratase of each example obtained by substituting the amino acid residue belonging to the group consisting of (a) to (q) with the wild-type Pseudonocardia thermophila nitrile hydratase, the inhibition of the amide compound production reaction using the nitrile hydratase caused by impurities is reduced even in the presence of impurities of less than 1000 ppm (for example, less than 10 ppm).

[0339] As shown in Table 10, it can be seen that the mutant nitrile hydratase of each example obtained by substituting the amino acid residue belonging to the group consisting of (i), (g), (l) and (m) above with respect to the wild-type Pseudonocardia thermophila nitrile hydratase does not reduce the enzyme activity under the reaction condition of 30°C which is a temperature higher than room temperature (20°C), and efficiently produces an amide compound. In other words, it can be seen that the mutant nitrile hydratase of each example obtained by substituting the amino acid residue belonging to the group consisting of (i), (g), (l) and (m) above with respect to the wild-type Pseudonocardia thermophila nitrile hydratase has excellent heat resistance.

[0340] 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 in this specification are incorporated herein by reference to the same extent as if they were specifically and individually described.

Claims

1. A mutant nitrile hydratase comprising an α subunit having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 1 and a β subunit having a sequence identity of 90% or more to the amino acid sequence of SEQ ID NO: 2, wherein the mutant nitrile hydratase comprises at least one amino acid residue substituted from the group consisting of (a) to (q): (a) in the α subunit, the amino acid residue Ala corresponding to the 23rd amino acid residue from the N-terminus of SEQ ID NO: 1 is substituted with Gly; (b) substitution of the amino acid residue Val corresponding to position 105 from the N-terminus of SEQ ID NO: 1 in the α subunit with Met; (c) in the α subunit, the amino acid residue Leu corresponding to position 142 from the N-terminus of SEQ ID NO: 1 is substituted with Cys; (d) substitution of the amino acid residue Glu corresponding to position 145 from the N-terminus of SEQ ID NO: 1 in the α subunit with Asp; (e) substitution of the amino acid residue Pro corresponding to position 152 from the N-terminus of SEQ ID NO: 1 in the α subunit with Gly; (f) substitution of the amino acid residue Pro corresponding to position 153 from the N-terminus of SEQ ID NO: 1 in the α subunit with Arg or Val; (g) substitution of the amino acid residue Gly corresponding to position 180 from the N-terminus of SEQ ID NO: 1 in the α subunit with Val; (h) substitution of the amino acid residue Glu corresponding to position 89 from the N-terminus of SEQ ID NO: 2 in the β subunit with Leu; (i) substitution of the amino acid residue Ala corresponding to position 122 from the N-terminus of SEQ ID NO: 2 in the β subunit with Ile, Leu or Thr; (j) in the β subunit, the amino acid residue Thr corresponding to position 149 from the N-terminus of SEQ ID NO: 2 is substituted with Gln; (k) substitution of the amino acid residue Lys corresponding to position 153 from the N-terminus of SEQ ID NO: 2 in the β subunit with Val, Arg or Pro; (l) in the β subunit, the amino acid residue Cys corresponding to position 189 from the N-terminus of SEQ ID NO: 2 is substituted with Ser; (m) substitution of the amino acid residue Glu corresponding to position 207 from the N-terminus of SEQ ID NO: 2 in the β subunit with Pro, Ser or Gly; (n) in the β subunit, the amino acid residue Ala corresponding to position 232 from the N-terminus of SEQ ID NO: 2 is substituted with Trp, Ser, Pro, Phe, Ile, Arg, Cys or Gly; (o) substitution of the amino acid residue Ala corresponding to position 233 from the N-terminus of SEQ ID NO: 2 in the β subunit with Glu or Gln; (p) substitution of Thr at the amino acid residue corresponding to position 188 from the N-terminus of SEQ ID NO: 1 in the α subunit with Lys; (q) In the β subunit, the amino acid residue Glu corresponding to the 50th amino acid residue from the N-terminus of SEQ ID NO: 2 is substituted with Gly. 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 . The mutant nitrile hydratase according to claim 1 , which comprises at least one amino acid residue substitution selected from the group consisting of (i), (g), (j), and (n).

4. The 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 . The mutant nitrile hydratase according to claim 1 , which comprises 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 contains at least one amino acid residue substitution selected from the group consisting of the following (A1) to (A6): (A1) substitution of the amino acid residue Arg corresponding to the 20th position from the N-terminus of SEQ ID NO: 1 in the α subunit with Val; (A2) substitution of the amino acid residue Gln corresponding to position 141 from the N-terminus of SEQ ID NO: 1 in the α subunit with Arg; (A3) substitution of the amino acid residue Glu corresponding to position 185 from the N-terminus of SEQ ID NO: 1 in the α subunit with Ala; (A4) substitution of the amino acid residue Ala corresponding to position 187 from the N-terminus of SEQ ID NO: 1 in the α subunit with Arg; (A5) substitution of the amino acid residue Pro corresponding to the 200th position from the N-terminus of SEQ ID NO:1 in the α subunit with Ala; (A6) In the α subunit, the amino acid residue Val corresponding to position 204 from the N-terminus of SEQ ID NO: 1 is substituted with Leu.

7. The mutant nitrile hydratase according to claim 1 or claim 2, wherein The amino acid sequence of the β subunit further contains at least one amino acid residue substitution selected from the group consisting of (B1) to (B9) below: (B1) substitution of Thr in the β subunit, corresponding to the 40th amino acid residue from the N-terminus of SEQ ID NO: 2, with Ile; (B2) substitution of the amino acid residue Arg corresponding to position 42 from the N-terminus of SEQ ID NO: 2 in the β subunit with Pro; (B3) substitution of Thr at the amino acid residue corresponding to position 76 from the N-terminus of SEQ ID NO: 2 in the β subunit with Cys; (B4) substitution of Thr with His as the amino acid residue corresponding to position 83 from the N-terminus of SEQ ID NO: 2 in the β subunit; (B5) substitution of the amino acid residue Leu corresponding to position 88 from the N-terminus of SEQ ID NO: 2 in the β subunit with Arg, Pro or Ser; (B6) substitution of the amino acid residue Arg corresponding to position 146 from the N-terminus of SEQ ID NO: 2 in the β subunit with Gln or Thr; (B7) substitution of the amino acid residue Arg corresponding to position 160 from the N-terminus of SEQ ID NO: 2 in the β subunit with Gln or Ile; (B8) substitution of the amino acid residue Glu corresponding to position 108 from the N-terminus of SEQ ID NO: 2 in the β subunit with Arg; (B9) In the β subunit, the amino acid residue Tyr corresponding to position 176 from the N-terminus of SEQ ID NO: 2 is substituted with Cys.

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

9. A vector comprising the nucleic acid according to claim 8.

10. The vector according to claim 9, which is an expression vector. A transformant comprising the expression vector according to claim 10.

12. A method for producing a mutant nitrile hydratase, the method comprising the following steps: a step of culturing the transformant according to claim 11 in a culture medium; and A step of recovering the mutant nitrile hydratase according to claim 1 or claim 2 from at least one of the cultured transformant and the culture medium.

13. A mutant nitrile hydratase obtained by the production method according to claim 12.

14. A method for producing an amide compound, comprising the step of contacting the mutant nitrile hydratase according to claim 1 or claim 2 with a nitrile compound.

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